Compositions and methods for treating drug-resistant cancer

Abstract

The present invention provides combinations and methods for treating temozolomide (TMZ)-resistant cancer patients, comprising a combination of TMZ and an isoform-selective HDAC8 inhibitor, e.g., BMX, in a relative ratio effective to overcome TMZ resistance by enhancing TMZ-mediated cytotoxic effects by downregulating the β-catenin / c-Myc / SOX2 signaling pathway and upregulating WT-p53-mediated MGMT inhibition.

Description

[Technical Field]

[0001] cross reference This non-provisional application claims priority under 35 U.S.C. §119(a) to U.S. Provisional Patent Application No. 63 / 194,585, filed May 28, 2021, the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to new compositions and methods for treating drug-resistant cancers, particularly TMZ-resistant cancers. [Background technology]

[0003] Glioblastoma multiforme (GBM) is one of the most aggressive tumors, with an aggressive pattern and a high recurrence rate, and is a World Health Organization grade IV astrocytoma [1]. Despite multimodality treatment with surgery and radiotherapy combined with chemotherapy, the prognosis for GBM patients remains poor, with a median survival time of less than 15 months and treatment resistance [2-4].

[0004] Colon cancer, or colorectal cancer (CRC), is one of the most common malignancies and the third leading cause of cancer deaths worldwide. While standard treatments for colon cancer or CRC are well-established and well-researched, they remain clinically challenging and associated with high mortality rates. Because the disease presents few warning signs, patients are often diagnosed with advanced disease at initial evaluation, resulting in a 5-year survival rate of approximately 10% [5-6]. Standard treatments for CRC include surgery, radiation therapy, and / or chemotherapy, with oxaliplatin (Oxp) and its prodrug, capecitabine, widely used in clinical practice [7-8]. Unfortunately, with this type of DNA-crosslinking agent treatment, recurrence remains common within the first few years, even after completing a full treatment cycle [9].

[0005] Temozolomide (TMZ) is an imidazotetrazine lipophilic prodrug of the alkylating agent dacarbazine, which has good blood-brain barrier permeability. TMZ undergoes spontaneous nonenzymatic conversion at physiological pH to the reactive compound 5-(3-methyltriazen-1-yl)-imidazole-4-carboxamide (MTIC)

[10] . MTIC's cytotoxicity is thought to be due to DNA alkylation (methylation), primarily occurring at the O6 and N7 positions of guanine. Since its first FDA approval in 2005, TMZ has been widely used as standard chemotherapy for newly diagnosed glioblastoma multiforme (GBM). Beyond GBM, TMZ has proven to be as effective as dacarbazine. For this reason, TMZ is also used "off-label" in patients with malignant melanoma after standard treatment. Additionally, numerous clinical trials are underway to demonstrate the efficacy of TMZ in other indications, such as brain metastases, lymphoma, neuroendocrine tumors, pituitary tumors, Ewing's sarcoma, primitive neuroectodermal tumors, refractory leukemia, lung cancer, and other tumors.

[11] TMZ is a well-tolerated treatment for elderly, pediatric, or palliative care patients and can be used as a single agent or as a first-line or x-line treatment adjuvant to radiation or chemotherapy. However, drug resistance develops in TMZ-treated patients due to overexpression of O6-methylguanine methyltransferase (MGMT). This represents a significant and clinically significant obstacle that must be overcome to successfully treat GBM.

[0006] However, fewer than 50% of patients respond to TMZ because of overexpression of O6-methylguanine methyltransferase (MGMT), which reverses methylation of the O6 position of guanine, thereby repairing DNA in GBM cells and preventing chemotherapy [12-14]. Comparison of MGMT protein levels between newly diagnosed and TMZ-treated CRC patients supports the possibility that a reduction in MGMT promotes the efficacy of TMZ treatment [15-17]. In addition to promoter methylation, MGMT is also regulated by various transcription factors, such as p53, Sp1, NF-κB, CEBP, and AP-18. Among these, p53 downregulates MGMT transcription by directly interacting with the MGMT promoter [18,19]. Therefore, in addition to MGMT promoter methylation, p53 may regulate MGMT expression, leading to TMZ resistance. Thus, to overcome TMZ resistance, additional mechanisms regulating MGMT must be identified. Summary of the Invention [Problem to be solved by the invention]

[0007] Therefore, it is desirable to develop new and better therapies or treatments for drug-resistant cancers, particularly TMB-resistant GBM or CRC. [Means for solving the problem]

[0008] Thus, the present invention provides new methods for the treatment of drug-resistant cancers, such as TMZ-resistant GBM or CRC.

[0009] In the present invention, it has been unexpectedly found that compound X, such as BMX, can enhance TMZ-mediated cytotoxicity effects on GBM-R cell lines and CRC cell lines HT29, HCT116, and RKO. Thus, the present invention provides a new method for treating drug-resistant cancer, particularly TMZ-resistant GBM or CRC, in patients, comprising administering a combination of BMX and TMZ to the patient.

[0010] In the present invention, we have demonstrated that the histone deacetylase 8 inhibitor (HDAC8i), BMX (NBM-TL-BMX-OS01), exhibits significant antiproliferative effects in colorectal cancer cells, human umbilical endothelial cells, lung cancer cells, and glioblastoma cells, and also exhibits tumor-suppressing ability in animal xenograft models [20, 21]. However, we unexpectedly discovered that BMX can overcome drug resistance in cancer cells. In one example, BMX was able to overcome GBM-R cells by enhancing TMZ-mediated cytotoxicity by downregulating the β-catenin / c-Myc / SOX2 signaling pathway and upregulating p53-mediated MGMT inhibition. High HDAC8 expression in human GBM tissues and GBM-R cell lines correlated with MGMT levels, and the combination of BMX and TMZ induced wild-type (WT)-p53-mediated apoptosis in GBM-R cell lines through WT-p53-mediated MGMT inhibition. Furthermore, the combination of BMX and TMZ suppressed cell proliferation and GSC phenotype activity in GBM-R cell lines via the β-catenin / c-Myc / cyclin D1 / SOX2 signaling pathway.

[0011] In one example of the present invention, we demonstrated that the combination of BMX and TMZ induces cell cycle arrest, senescence, autophagy, and apoptosis in CRC cells through upregulation of p53 / p21 / Puma / Bax, which is impaired by the crosstalk of downregulation of the Wnt / β-catenin / cyclin D1 / c-Myc / p62 pathway. Therefore, BMX may be a promising strategy for optimal personalized treatment of TMZ-resistant GBM or CRC patients with wild-p53.

[0012] Thus, in one embodiment, the present invention provides a combination for treating TMZ-resistant cancer, comprising TMZ and a compound of formula A: [ka] [In the formula, R 1 is hydrogen, alkyl, alkenyl, C5-C6 cycloalkyl, a 5- or 6-membered unsaturated carbocyclic ring or a 5- or 6-membered heterocyclic ring, or (CH2)mR 4 and; X is C, -O-, -N- or -S-; Y is —O—, —NH, or —O—C1-C4 alkyl; n is an integer from 0 to 10, m is an integer from 0 to 5; R 2 and R 3 are independently C1-C6 alkyl, R 4 is C5-C6 cycloalkyl, or halogen, -CF3, -OR 7 or -NR 7 R 8 is a 5- or 6-membered unsaturated carbocyclic or heterocyclic ring optionally substituted by 7 and R 8 are independently hydrogen or C1-C6 alkyl; R 5is OH, NH2 or C5-C6 cycloalkyl, a 5- or 6-membered unsaturated carbocyclic or heterocyclic ring, where cycloalkyl, carbocyclic and heterocyclic rings are selected from the group consisting of halogen, NH2, NO2, C1-C6 alkoxy, C 1-6 Alkylthio, OR 7 '', NR 7 R 8 or optionally substituted by CF3, R 6 is H or hydroxy or C2-C 10 C1-C optionally substituted with alkenyl 10 alkyl or R 1 together to form -C2H2-] or a pharmaceutically acceptable salt, stereoisomer, enantiomer, prodrug or solvate thereof, TMZ and Compound A are combined in a relative ratio that effectively overcomes TMZ resistance, providing a combination.

[0013] In one embodiment of the invention, compound A is [ka] The compound BMX has the structure:

[0014] According to the present invention, TMZ resistance is overcome by enhancing TMZ-mediated cytotoxic effects by downregulating the β-catenin / c-Myc / SOX2 signaling pathway and upregulating WT-p53-mediated MGMT inhibition.

[0015] In the present invention, TMZ and compound A, such as BMX, are administered separately or sequentially.

[0016] In one example of the invention, the cancer is glioblastoma multiforme (GBM) or colorectal cancer (CRC).

[0017] In another aspect, the present invention provides a method for treating TMZ-resistant cancer in a patient, the method comprising administering to the patient a therapeutically effective amount of a combination according to the present invention.

[0018] In one example of the present invention, the cancer is GBM or CRC.

[0019] In a further aspect, the present invention provides a method for optimal personalized treatment of drug-resistant cancer in a patient, comprising determining expression of WT-p53 in the patient, and, if expression of WT-p53 is present in the patient, administering to the patient a therapeutically effective amount of BMX or a combination thereof.

[0020] In one embodiment of the invention, the drug is TMZ.

[0021] In one particular embodiment of the invention, the drug-resistant cancer is a TMZ-resistant cancer, in particular GMB or CRC.

[0022] In the present invention, it was confirmed that BMX is effective in enhancing the inhibition of WT-p53 cancer cells.

[0023] In a further aspect, the present invention provides use of a combination of BMX and TMZ for producing a medicament or kit for treating TMZ-resistant cancer.

[0024] The present invention is further illustrated by the following examples, however, it should be understood that the following examples are intended for illustrative purposes only and should not be construed as limiting the invention in any way. [Brief explanation of the drawings]

[0025] The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred.

[0026] [Figure 1] Figure 1 provides a pathway analysis of genes potentially related to HDAC8 using bioinformatics tools. Here, shRNA HDAC8 was entered into the CLUE database and CPs and PCLs with scores above 90 were selected (A). The target genes were entered into the CPDB pathway analysis database (B) for further experiments. (C) The top 10 pathways for selecting CPs and PCLs (with scores above 90) for shRNA HDAC8 are as follows: VEGF; PI3K-Akt signaling pathway; JAK-STAT pathway and regulation; signaling pathway; MAPK signaling pathway - Homo sapiens (human); apoptosis; autophagy; HIF-1 signaling pathway; TNF-related weak inducer of apoptosis (TWEAK) signaling pathway; and Wnt signaling pathway. VEGF; PI3K-Akt signaling pathway; JAK-STAT pathway and regulation; signal transduction pathway; MAPK signaling pathway - Homo sapiens (human); apoptosis; autophagy; HIF-1 signaling pathway; TNF-related weak inducer of apoptosis (TWEAK) signaling pathway; Wnt signaling pathway.

[0027] [Figure 2]Figure 2 shows that BMX inhibited the growth and proliferation of GBM cells (U87MG and A172), and that the combination of BMX and TMZ inhibited the growth and proliferation of GBM-R cells (U87MG-R and A172-R). (A) Chemical structure of BMX. (B) Cell viability of GBM and GBM-R cell lines after treatment with 0.5 μM, 10 μM, 15 μM, 30 μM, or 50 μM BMX. (C) Cell viability of GBM and GBM-R cell lines after treatment with 0.25 μM, 50 μM, 100 μM, 200 μM, 400 μM, or 800 μM TMZ. (D) Viability of GBM and GBM-R cells after 24-hour treatment with 10 μM BMX with or without various concentrations of TMZ (0.25 μM, 50 μM, 100 μM, 200 μM, 400 μM, or 800 μM). (E) Viability of GBM and GBM-R cells after 24-hour treatment with 50 μM TMZ with or without various concentrations of BMX (0.5 μM, 10 μM, 15 μM, 30 μM, or 50 μM). (F) Viability of GBM-R cells after 24, 48, and 72-hour treatment with 50 μM TMZ with or without 10 μM BMX. (G) 14-day clonogenic assay of GBM and GBM-R cell lines with BMX (0 μM, 5 μM, or 10 μM) with or without TMZ (50 μM). Data are presented as the mean ± SEM from three experiments. *p<0.05 compared to controls (A172 and U87MG), #p<0.05 compared to (A172-R and U87MG-R).

[0028] [Figure 3]Figure 3 shows that BMX enhanced TMZ-mediated cytotoxicity by targeting the Wnt / β-catenin / GSK3β pathway and suppressing cell proliferation in GBM-R cells. (A) The activation status of GSK-3β and β-catenin in GBM-R cells after 48 hours of treatment with 5 μM or 10 μM BMX, with or without 50 μM TMZ. (B) The c-Myc and cyclin D1 protein levels in GBM-R cells after 48 hours of treatment with 5 μM or 10 μM BMX, with or without 50 μM TMZ. (C) The changes in the phosphorylation status of β-catenin (Ser33 / 37 / 41) and c-Myc and cyclin D1 protein expression in GBM-R cells after treatment with 10 μM BMX and 50 μM TMZ, with or without 10 μM MG132.

[0029] [Figure 4] Figure 4 shows that the combination of BMX and TMZ enhanced the TMZ-mediated cytotoxic effect by promoting TMZ-mediated apoptosis in GBM-R cells. (A) Cell cycle distribution of GBM (U87MG and A172) and GBM-R (U87MG-R and A172-R) cells treated with BMX with or without TMZ for 48 hours. (B) Histogram showing the percentage of cells in G0 / G1, S, and G2 / M phases. (C) Bar graph of the percentage of subG1. (D) Annexin V / PI apoptosis assay of GBM (U87MG and A172) and GBM-R (U87MG-R and A172-R) cells treated with BMX with or without TMZ for 48 hours. (E) Histogram showing the percentage of apoptotic cells.

[0030] [Figure 5]Figure 5 shows that the combination of BMX and TMZ enhanced the TMZ-mediated cytotoxicity effect through WT-p53-mediated MGMT inhibition in GBM-R cells. (A) Expression patterns of WT-p53 and MGMT in GBM (U87MG and A172) and GBM-R (U87MG-R and A172-R) cell lines. (B) Protein changes of WT-p53, MGMT, P21, Bax, Bcl-2, Puma, and cleaved caspase-3 in U87MG-R and A172-R cells treated with 5 μM or 10 μM BMX with or without 50 μM TMZ for 48 hours. (C) Treatment of GBM (U87MG and A172) and GBM-R (U87MG-R and A172-R) cells with 5 or 10 μM BMX, with or without 50 μM TMZ, for 48 h reduced MGMT levels and increased WT-p53 and Phospho-WT-p53 levels (ser 15). β-Actin was used as an internal control.

[0031] [Figure 6] Figure 6 shows that the combination of BMX and TMZ reduced GSC formation in GBM-R cells. (A) Expression of CSC-associated genes (CD133, CD44, and SOX2) between parental and resistant daughter cell lines. (B) Changes in CD133, CD44, and SOX2 protein levels after 48 hours of treatment with 5 μM and 10 μM BMX, with or without 50 μM TMZ, in U87MG-R and A172-R cells. (C) Immunohistochemical staining of HDAC8 and CSC-associated genes (CD133 and CD44) in human primary GBM (from the same patient before concurrent radiotherapy and chemotherapy) and recurrent GBM tumor tissue (after concurrent radiotherapy and chemotherapy) obtained by surgical biopsy.

[0032] [Figure 7] FIG. 7 provides a working model of the mechanism of the combination of BMX and TMZ to overcome TMZ resistance in GBM-R cells.

[0033] [Figure 8] FIG. 8 shows the genetic characteristics of GBM cell lines. (A) Expression of HDAC8 (Abcam company) in GBM cells by Western blot.

[0034] [Figure 9] Figure 9 shows that BMX was a potent semisynthetic HDAC8 inhibitor. (B) Examination of MGMT methylation-paired GBMs through big data analysis. Wild-type and mutant (mutant) are labeled in blue and red, respectively (one-tailed t-test, *: p<0.05). (A) HDAC8 expression levels stimulated by different doses of BMX (0-10 μM) in the presence or absence of TMZ (50 μM) were determined using qRT-PCR assay. (B) HDAC8 expression levels stimulated by BMX (0-10 μM) with or without TMZ (50 μM) were determined using Western blotting.

[0035] [Figure 10]Figure 10 shows that BMX, and BMX and TMZ together inhibited the growth and proliferation of U87MG, U87MG-R, A172, and A172-R cells. (A) Cell viability of GBM and GBM-R cell lines after treatment with the indicated concentrations of BMX (0.5 μM, 10 μM, 15 μM, 30 μM, or 50 μM) for 24, 48, and 72 hours. (B) Cell viability of GBM and GBM-R cell lines after treatment with the indicated concentrations of TMZ (0.25 μM, 50 μM, 100 μM, 200 μM, 400 μM, or 800 μM) for 24, 48, and 72 hours. (C) Cell viability of GBM and GBM-R cell lines after treatment with 10 μM BMX with or without different concentrations (0.25 μM, 50 μM, 100 μM, 200 μM, 400 μM, or 800 μM) of TMZ for 24, 48, and 72 hours. (D) Cell viability of GBM and GBM-R cell lines after treatment with 50 μM TMZ with or without different concentrations (0.5 μM, 10 μM, 15 μM, 30 μM, or 50 μM) of BMX for 24, 48, and 72 hours. (E) Cell viability of GBM-R cell lines after treatment with 50 μM TMZ with or without 10 μM BMX for 24, 48, and 72 hours.

[0036] [Figure 11] Figure 11 shows the in vitro cytotoxicity of BMX in GBM cells. (A) U87 and U87R cells, and (B) A172 and A172R cells were treated with TMZ (50 and 100 μM). The inhibitory effect of TMZ on GBM cells was determined using a clonogenic assay. *p<0.05, **p<0.01, ***p<0.001.

[0037] [Figure 12] FIG. 12 shows drug and gene profiles in glioblastoma, including HDAC8 expression levels in two wild-type p53 (WT-p53) parental GBM cell lines (A172 and U87MG) and two TMZ-resistant GBM cell lines.

[0038] [Figure 13]Figure 13 shows that the combination of BMX, TMZ, oxaliplatin, and doxorubicin inhibited cell proliferation in CRC cells. (A) The proliferation of HT29, HCT116, and RKO cells treated with BMX, TMZ, Oxp, Dox, BMX + TMZ, BMX + Oxp, or BMX + Dox at various drug concentrations and treatment times was assayed using the CCK-8 method. (B) Colony-forming assays of HT29, HCT116, and RKO cells treated with BMX, TMZ, Oxp, BMX + TMZ, and BMX + Oxp resulted in quantification of clones and statistical graphs. (C) Cell cycle analysis of HT29, HCT116, and RKO cells after 48 hours of treatment with different concentrations of BMX or BMX in combination with TMZ, as well as the percentage of cells in each cell cycle phase. (D) Apoptosis analysis after 48 hours of treatment with different concentrations of BMX or BMX in combination with TMZ, and the apoptosis rates of cells in HT29, HCT116, and RKO cells. All results are shown as the mean ± SD from three independent experiments. *p<0.05, **p<0.01, ***p<0.001 compared to control (HT29 cells); #p<0.05, ##p<0.01, ###p<0.001 compared to control (HCT116 cells); †p<0.05, ††p<0.01, †††p<0.001 compared to control (RKO cells).

[0039] [Figure 14]Figure 14 shows that the apoptosis and autophagy effects induced by BMX and the combination of BMX and TMZ were mediated by p53-mediated MGMT inhibition. (A) Western blot analysis of the expression of p53, p53 Lys382 acetylation, p53 Ser15 phosphorylation, p21, p16, MGMT, γH2AX, E2F1, and E2F3 in HT29, HCT116, and RKO cells treated with various concentrations of BMX (5 μM and 10 μM) and various concentrations of BMX and TMZ in combination for 48 hours. (B) Expression of cleaved caspase-3, cleaved caspase-7, cleaved caspase-8, cleaved caspase-9, and cleaved PARP proteins in HT29, HCT116, and RKO cells treated with various concentrations of BMX (5 μM and 10 μM) and various concentrations of BMX and TMZ in combination for 48 hours. (C) Expression of Bax, Bcl-2, BID, Bim, Bak, and Puna proteins in HT29, HCT116, and RKO cells treated with various concentrations of BMX (5 μM and 10 μM) and various concentrations of BMX in combination with TMZ for 48 h. GAPDH was used as a loading control.

[0040] [Figure 15] Figure 15 shows that BMX, or a combination of BMX and TMZ, induces cellular senescence in HT29, HCT116, and RKO cells. Senescence-associated β-galactosidase (SAβ-gal) staining of the combination of BMX and TMZ. Cells were treated with 10 μM BMX + TMZ (50 μM) for 48 hours, and the cells were stained with SAβ-gal (blue cytoplasmic staining). Scale bar, 50 μm. Quantification of SAβ-gal activity. All results are shown as the mean ± SD from three independent experiments. *p<0.05, **p<0.01, ***p<0.001 compared to control (HT29 cells); #p<0.05, ##p<0.01, ###p<0.001 compared to control (HCT116 cells); †p<0.05, ††p<0.01, †††p<0.001 compared to control (RKO cells).

[0041] [Figure 16] Figure 16 shows that the combination of BMX and TMZ reduced CSC formation in HT29, HCT116, and RKO cells. Changes in CD133, CD44, and SOX2 protein levels after 48 hours of treatment of HT29, HCT116, and RKO cells with 5 μM and 10 μM BMX, with or without 50 μM TMZ. GAPDH was used as a loading control.

[0042] [Figure 17] Figure 17 shows that BMX enhanced TMZ-mediated cytotoxicity by targeting the Wnt / β-catenin / GSK3β pathway in CRC cells. (A) GSK-3β, β-catenin activation status, c-Myc, and cyclin D1 in HT29, HCT116, and RKO cells after 48 hours of treatment with 5 μM or 10 μM BMX with or without 50 μM TMZ. (B) The expression of GSK-3β, β-catenin activation status, c-Myc, and cyclin D1 is upregulated by 50 μM TMZ and BMX with or without MG132 in T29, HCT116, and RKO cells. (C) The expression of p53 and MGMT is upregulated by 50 μM TMZ and BMX with or without MG132 in T29, HCT116, and RKO cells. GAPDH was used as a loading control.

[0043] [Figure 18]Figure 18 shows that autophagy was involved in the induction of cell death by BMX alone and the combination of BMX and TMZ. (A) Western blot analysis was performed to assess the expression of LC3 and P62 / SQSTM1 in HT29, HCT116, and RKO cells treated with BMX (5 μM and 10 μM) with or without 50 μM TMZ. (B) P62 / SQSTM1 expression was downregulated by BMX with or without 50 μM TMZ and MG132 in HT29, HCT116, and RKO cells. (C) Pretreatment with BAF and VAD reduced cell apoptosis in HT29, HCT116, and RKO cells exposed to BMX (5 μM and 10 μM) with or without 50 μM TMZ for 48 hours. (D) The effects of VAD and BAF on BMX (5 μM and 10 μM) with or without 50 μM TMZ induced the expression of cleaved caspase-3, cleaved PARP, P62, and LC3. GAPDH was used as a loading control. DETAILED DESCRIPTION OF THE INVENTION

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0045] The present invention provides a new method for treating patients with TMZ-resistant cancers (eg, GBM and CRC) using a combination of TMZ and Compound A.

[0046] Compound A is a novel small molecule isoform-selective HDAC8 inhibitor. Compound A is disclosed in U.S. Patent No. 7,994,357, the contents of which are incorporated herein by reference in their entirety. Compound A has the structure of Formula A, or a pharmaceutically acceptable salt, stereoisomer, enantiomer, prodrug or solvate thereof:

[0047] [ka] [In the formula, R 1 is hydrogen, alkyl, alkenyl, C5-C6 cycloalkyl, a 5- or 6-membered unsaturated carbocyclic ring or a 5- or 6-membered heterocyclic ring, or (CH2)mR 4 and; X is C, -O-, -N- or -S-; Y is -O-, -NH or -O-C1-C4 alkyl; n is an integer from 0 to 10; m is an integer from 0 to 5; R 2 and R 3 are independently C1-C6 alkyl; R 4 is halogen, -CF3, -OR 7 or -NR 7 R 8 C-C cycloalkyl or a 5- or 6-membered unsaturated carbocyclic or heterocyclic ring optionally substituted by R 7 and R 8 are independently hydrogen or C1-C6 alkyl; R 5 is OH, NH2 or C5-C6 cycloalkyl, a 5- or 6-membered unsaturated carbocyclic or heterocyclic ring, where cycloalkyl, carbocyclic and heterocyclic rings are selected from the group consisting of halogen, NH2, NO2, C1-C6 alkoxy, C 1-6 Alkylthio, OR 7 ", NR 7 R 8 or optionally substituted by CF3, R 6 is H or hydroxy or C2-C 10 C1-C optionally substituted by alkenyl 10 alkyl or R 1 together to form -C2H2-].

[0048] In one particular embodiment of the present invention, compound A is BMX, which is derived from the semisynthesis of osthole and plays a novel role in learning and memory, as reported by Yang YC et al.

[22] : [ka]

[0049] BMX is known to be an isoform-selective HDAC8 inhibitor with the least toxicity and the ability to cross the blood-brain barrier

[22] .

[0050] As used herein, the term "temozolomide" or "TMZ" refers specifically to a drug sold under the trade name "Temodar" and used to treat some brain tumors, such as glioblastoma multiforme or malignant astrocytoma. TMZ has the following structure: [ka]

[0051] Temozolomide (TMZ) is an alkylating agent used in the treatment of some cancers, such as second-line treatment for astrocytoma and first-line treatment for glioblastoma multiforme. The combination of olaparib and temozolomide has also been found to show substantial clinical activity against recurrent small cell lung cancer.

[0052] As used herein, the terms "glioblastoma multiforme," "glioblastoma," or "GBM" refer to brain cancer that arises from normal brain cells or from a pre-existing low-grade astrocytoma. There is no known way to prevent GBM. Treatment usually involves surgery, followed by chemotherapy and radiation therapy. Drug therapy with temozolomide (TMZ) is often used as part of chemotherapy.

[0053] As used herein, the terms "colon cancer," "colorectal cancer," or "CRC," also known as intestinal or rectal cancer, refer to cancer originating in the colon or rectum (part of the large intestine). Signs and symptoms may include blood in the stool, changes in bowel habits, weight loss, and fatigue. Standard treatment for CRC is surgery, radiation therapy, and / or chemotherapy, with oxaliplatin (Oxp) and its prodrug, capecitabine, being widely used in clinical practice. Unfortunately, recurrence under treatment with this type of DNA cross-linking agent remains common within the first few years, even after completing a full cycle.

[0054] In the present invention, we found that BMX overcomes TMZ resistance by enhancing TMZ-mediated cytotoxic effects through downregulating the β-catenin / c-Myc / SOX2 signaling pathway and upregulating WT-p53-mediated MGMT inhibition. Our results indicate that BMX or its combination with TMZ holds promise for optimal personalized treatment of TMZ-resistant WT-p53 GBM or CRC cells.

[0055] The present invention is further described by the following examples, which are provided for purposes of illustration and not limitation. [Example]

[0056] 1.1 Materials and Methods

[0057] 1.1.1 Cell culture and reagents

[0058] Four GBM cell lines, U87, U87R, A172, and A172R, were used in this study. The human GBM cell lines U87-MG (ATCC HTB-14; GBM of unknown origin) and A172 (ATCC CRL-1620; ATCC) were provided by the American Type Culture Collection (ATCC; Manassas, VA, USA). U87R and A172R cells were obtained from Dr. Tsung-I Hsu and Dr. Jian-Ying Chung (The Ph.D. Program for Neural Regenerative Medicine, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan). These cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and 50 μM TMZ for at least 60 days. TMZ resistance in U87R and A172R cells was confirmed using a colony formation assay (Figure 11). Cells were cultured in DMEM supplemented with 10% FBS, 100 U / mL penicillin, and 100 mg / mL streptomycin (all Gibco; Thermo Fisher Scientific, Waltham, MA, USA) and maintained in a humidified incubator at 37°C and 5% CO NBM-BMX (BMX), (E)-2-(4-methoxybenzyloxy)-3-prenyl-4-methoxy-N-hydroxycinamide, was provided by NatureWise Biotech & Medicals Corporation (Taipei, Taiwan).

[0059] 1.1.2 Cell proliferation and colony formation assays

[0060] We plated 3,000 GBM cells per well in a 96-well plate and allowed them to adhere overnight. To verify the responsiveness of cell lines to BMX and TMZ monotherapy, cells were treated with different doses of BMX or TMZ for 24, 48, and 72 hours. To confirm the responsiveness of cells to the BMX-TMZ combination, cells were treated with different doses of TMZ (0–800 μg / mL) with or without BMX (10 μM) for 24, 48, and 72 hours, or with different doses of BMX (0–50 μM) with or without TMZ (50 μM) for 24, 48, and 72 hours. After treatment, absorbance values ​​were measured at the indicated time points using a CCK8 kit (Targetmol, Shanghai, China). Results are reported as the mean ± standard deviation of at least three replicates.

[0061] A172, A172-R, U87MG, and U87MG-R cells were seeded onto 6 cm culture dishes (1000 cells / dish) and incubated for 14 days. Cells were washed three times with phosphate-buffered saline, fixed in 4% paraformaldehyde for 30 minutes, and stained with 0.1% crystal violet for 20 minutes at 25°C. Colonies were carefully washed with tap water and then counted and analyzed, with colonies defined as at least 50 cells. Results are expressed as the mean colony count ± SE from three independent experiments.

[0062] 1.1.3 Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

[0063] The ABI Prism® 7700 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) was used for quantitative analysis of mRNA expression. 5Cells (800 cells / ml) were seeded into 6-well plates, and total RNA was extracted using the Tissue Total RNA Mini Kit (Geneaid, Taipei, Taiwan). A 10 ng sample of total RNA was transcribed into cDNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Gene expression was quantified using Fast SYBR Green Master Mix (Applied Biosystems) according to the manufacturer's instructions, with 18s as the internal reference. All procedures were performed according to the manufacturer's protocol. Thermal cycling conditions were as follows: 50°C for 2 minutes, 95°C for 10 minutes, and 40 cycles of 95°C for 15 seconds and 60°C for 1 second. Each sample was analyzed in triplicate. Threshold cycle (Ct) values ​​were calculated using StepOnePlus (Applied Biosystems) software. The relative expression of each mRNA was calculated using the 2-(ΔCt) method. The primer sequences for HDAC8 were as follows: HDAC8 forward 5'-GCGTGATTTCCAGCACATAA-3' (SEQ ID NO: 1), HDAC8 reverse 5'-ATACTTGACCGGGGTCATCC-3' (SEQ ID NO: 2). 18s forward 5'-TCAAGTGCAGTGCAACAACTC-3' (SEQ ID NO: 3), 18s reverse 5'-AGAGGACAGGGTGGAGTAATCA-3' (SEQ ID NO: 4).

[0064] 1.1.4 Flow cytometry analysis of DNA cell cycle

[0065] For DNA cell cycle analysis, after 48 h of treatment with different doses of BMX (0–10 μM) in the presence or absence of TMZ (50 μM), cells were harvested by trypsinization, washed twice with phosphate-buffered saline, and fixed with methanol. Cells were then washed again and subjected to RNase A at a final concentration of 0.05 mg / mL (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany) and incubated with 10 μg / mL propidium iodide (PI; Sigma-Aldrich; Merck Millipore) for 15 min at 4°C in the dark. Cell cycle analysis was performed using a fluorescence-activated cell sorting (FACS) flow cytometer (Attune NxT Flow Cytometer, Thermo Fisher Scientific).

[0066] 1.1.5 Flow cytometry analysis of apoptosis

[0067] To analyze cell apoptosis in the presence or absence of TMZ (50 μM) at different doses of BMX (0–10 μM), FITC-labeled annexin V / PI staining was performed using the CF® 488A Annexin V and PI Apoptosis Kit (Fremont, CA, USA) according to the manufacturer's instructions. Flow cytometric analysis of PI and annexin was performed 48 hours after treatment. A total of 10,000 nuclei were measured using a FACS flow cytometer (Attune NxT Flow Cytometer, Thermo Fisher Scientific).

[0068] 1.1.6 Immunohistochemical staining

[0069] Immunohistochemical staining was performed on 4-μm-thick paraffin sections. Sections were dewaxed, hydrated, and stored overnight at 4°C. Standard avidin-biotin complex methods were used for antibodies against CD133 (AP1802a, Abgent, San Diego, CA, USA), P62 (ab56416, Abcam, Cambridge, MA, USA), and LC3II (AP1802a, Abgent). After allowing sections to return to room temperature, biotinylated secondary antibodies and horseradish-labeled streptavidin were added. The samples were then incubated in an oven at 37°C. DAB development, hematoxylin counterstaining, gradient alcohol dehydration, and xylene clearing were then performed. All samples were then sealed with neutral rubber. Human brain tissue: The ethical guidelines for this study were approved by the Institutional Review Board of Kaohsiung Medical University Hospital (No. KMUHIRB-F(I)-20200024). Informed consent was obtained from all subjects participating in this study.

[0070] 1.1.7 Western blot analysis

[0071] Cells were harvested and lysed in RIPA lysis buffer (EMD Millipore, Billerica, MA, USA, 10x RIPA buffer) containing protease inhibitors. Protein concentration was determined using a protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA). SDS loading buffer was mixed with the protein samples. Proteins (20 μg / lane) were separated using 8%–12% SDS-PAGE and transferred to PVDF membranes, which were blocked with 5% bovine serum albumin in Tris-buffered saline (TBS)-Tween 20 (0.5%; TBS-T) for 1 h at room temperature and incubated with primary antibodies overnight at 44°C, followed by horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at room temperature. After extensive washing with TBS-T, HRP signals were detected with a chemical HRP substrate. The antibodies used are listed in Table 3. The signal of each target protein was visualized by incubation with enhanced chemiluminescence reagents and exposure to X-ray film.

[0072] 1.1.8 Statistical analysis

[0073] Data are presented as mean ± standard deviation. Statistical analysis was performed using one-way analysis of variance. Data were compared using Student's t-test. The statistical significance level was * p<0.05, ** p<0.01, *** p was set at <0.001. [Table 1]

[0074] 1.1.9 Predicting the possible mechanisms of HDAC8 inhibitors through a multi-database platform Indirect process: Using CLUE, the binding scores of shRNA HDAC8 were calculated from their 1 million profiles and ranked by similarity with allelic compounds and gene perturbations. Criteria were filtered based on a positive score above 90, and the targeted genes in each case were collected as shRNA HDAC8 regulators of biological function. This gene list was input into the CPDB platform for enrichment analysis to obtain clear pathway information. Direct process: Based on BMX-L1000 gene expression data, both up- and down-regulated genes that respond to the biological function of drugs in HepG2 cells were listed. Significant differentially expressed genes (DEGs) were defined by a ±1.5-fold change and a p-value of <0.05 when BMX (1 μM) was compared with the DMSO control. In this way, the DEGs were used as input to search the CPDB for pathway analysis. To narrow down the prioritized pathways, we focused on two outcomes and selected common elements.

[0075] 1.2 Results:

[0076] 1.2.1 Pathway analysis of possible expression profiles of HDAC8 inhibitors through bioinformatics tools

[0077] To explore the possible mechanisms of HDAC8 inhibitor and gene involvement, we used the Connectivity Map (C-Map) and Library of Integrated Network-Based Cellular Signatures Unified Environment (CLUE) system database (https: / / clue.io / ) and the ConsensusPathDB (CPDB) platform (http: / / cpdb.molgen.mpg.de / ). We utilized two bioinformatics processes: direct analysis and indirect analysis (Figure 1A). For direct analysis, HepG2 cells were treated with BMX in L1000 plates, which respond to the biological function of BMX (Figure 1A, right). Significant differentially expressed genes with a 1.5-fold change (1583 up-regulated and 900 down-regulated) were used to search the CPDB platform, revealing possible pathways (p-value < 0.05). Next, we analyzed the inhibitory function of HDAC8 through an indirect approach, a pattern-matching algorithm, in the CLUE platform. Using the shRNA HDAC8 signature as a simulation of BMX treatment (an HDAC8 inhibitor), we then accessed CLUE, which calculated over one million profiles matching similar signature patterns from 19,811 small molecule compounds or gene perturbations (e.g., 18,493 shRNAs, 3,462 overexpression constructs), and then generated a connectivity score. A positive score indicated a similar mechanism between the query signature and the instance signature, while a negative score indicated an opposite function. According to our criteria, we selected compounds (CPs), knockdown genes (KDs), overexpressed genes (OEs), and perturbation classes (PCLs) with a connectivity score above 90. CLUE classified similarly functional compounds or genes from the same family into specific groups that could hypothesize a mechanism of action. However, this big data system did not provide detailed route information.For this reason, we combined the CPDB platform for complementary analysis from shHDAC8- and BMX-treated cells (Figure 1A, left). These different bioinformatics pipelines likely yielded several mechanisms / pathways, and we intersected these two datasets to filter potential pathways. The Wnt signaling pathway was one of the top mechanisms revealed through our multi-database platform (Figure 1B).

[0078] 1.2.2 BMX enhanced TMZ-mediated cytotoxic effects and inhibited the growth and proliferation of GBM-R cells

[0079] To investigate whether HDAC8 correlates with therapy-resistant GBM, we examined the expression levels of HDAC8 in two parental GBM cell lines (A172 and U87MG, which express wild-type p53 (WT-p53) (Figure 12)) and two TMZ-resistant GBM cell lines (A172-R and U87MG-R, which express WT-p53 mutants). HDAC8 overexpression was detected in both GBM-R cell lines (Figures 8A and 8B).

[0080] In this example, NBM-BMX (provided by NatureWise Biotech & Medicals Corporation; BMX was used in this study) was used as an HDAC8 inhibitor to mimic the effect of shRNA HDAC8 for further experiments. The structure of BMX (397.46 Da) is shown in Figure 2A. BMX was confirmed to be an HDAC8 inhibitor by treating four cell lines with BMX and detecting BMX-induced inhibition of HDAC8 mRNA and protein expression (Figures 9A and 9B).

[0081] It is believed that BMX can enhance the sensitivity of both GBM cells and GBM-R cells to TMZ-mediated cytotoxic effects. In this example, it was found that there is a combination effect between BMX and TMZ when treating GBM and GBM-R in A172 / A172-R and U87MG / U87MG-R cells. MTT assays were performed to evaluate cell proliferation and cell viability at different concentrations for the BMX alone group, TMZ alone group, and combination group at 24, 48, and 72 hours. In each single treatment group, the results suggested that the cytotoxic effect in each group increased in a time-dependent manner (Figure 10A). These results indicated that the IC of BMX alone was significantly higher than that of TMZ alone. 50 The IC values ​​were 21.00 ± 2.34 μM / >52.64 ± 3.62 μM in A172 / A172-R cells and 29.84 ± 2.32 μM / >68.13 ± 4.69 μM in U87MG / U87MG-R cells (Figure 2B), suggesting that BMX alone inhibits GBM cell proliferation but not GBM-R cell proliferation. 50The values ​​were 73.48 ± 3.65 μM / 80.99 ± 1.68 μM for A172 / U87MG cells and 595.07 ± 23.42 μM / 302.51 ± 15.24 μM for A172-R / U87MG-R cells, confirming the reliability of the results for GBM-R cells (Figure 2C). In the combination treatment group, 10 μM BMX was used in combination with different doses of TMZ (Figure 2D), and 50 μM TMZ (the same maintenance concentration in the GBM-R cell line) was combined with different doses of BMX (Figure 2E) to determine the doses of BMX and TMZ that could most potentiate the TMZ-mediated cytotoxic effect in GBM-R cells. The data revealed that 50 μM TMZ and 10 μM BMX exerted the highest cytotoxic effect in both GBM-R cell lines. We used this combination in a time-dependent manner and observed a cytotoxic effect at 48 hours (Figure 2F; 10 μM BMX at 48 hours: 0.88-fold, 0.77-fold, and 0.63-fold; BMX and TMZ: 0.74-fold, 0.56-fold, and 0.47-fold). Clonogenic assays also revealed that 10 μM BMX and 50 μM TMZ, but not BMX alone, suppressed GBM-R cells (Figure 2G). Taken together, these data suggest that this combined treatment inhibits the growth and proliferation of GBM (U87MG and A172) and GBM-R (U87MG-R and A172-R) cells, and that the combination of 10 μM BMX and 50 μM TMZ exerted the strongest cytotoxic effect (inhibition of cell proliferation and cell viability) on GBM-R cells. Nevertheless, BMX alone still moderately reduced cell viability, indicating a partial pharmacological cytotoxic effect in A172R / U87R compared with TMZ alone, which had no inhibitory effect. Therefore, in further experiments, combined treatment with BMX and TMZ was compared with BMX alone.

[0082] 1.2.3 BMX enhanced TMZ-mediated cytotoxic effects by targeting the Wnt / β-catenin / GSK3β pathway in GBM-R cells

[0083] The mechanism was investigated by enhancing TMZ-mediated cytotoxicity in GBM-R cells. Based on pathway analysis, we hypothesized that the canonical Wnt signaling (also known as Wnt / β-catenin) pathway is involved in the proliferation of GBM-R cells. The genetic background of each cell line indicated the absence of mutations in Wnt genes, such as adenomatous polyposis coli and β-catenin (CTNNB1). To detect the status of β-catenin, we used phospho-β-catenin (Ser33 / Ser37 / Thr41) as the active form of β-catenin. GSK3β (S9) was used to phosphorylate β-catenin, resulting in its degradation. The results showed that in U87R and A172R cells, 10 μM BMX and 50 μM TMZ directly reduced the protein levels of β-catenin and phospho-β-catenin (Ser33 / Ser37 / Thr41) through phosphorylation by GSK-3β, whereas BMX alone only slightly reduced these levels. Furthermore, the phosphorylation level of GSK-3β (S9) was also reduced, indicating that GSK-3β activity increased and phosphorylated β-catenin (Fig. 3A). The effects of BMX on proliferation markers c-Myc and cyclin D1 were also observed to be reduced by BMX in the presence or absence of TMZ (Fig. 3B).

[0084] To verify that β-catenin protein levels are reduced by proteolysis, GBM-R cells were treated with the proteasome inhibitor MG132. The results revealed that MG132 reversed β-catenin degradation and increased c-Myc and cyclin D1 expression under the conditions of 10 μM BMX and 50 μM TMZ (Figure 3C). These results demonstrate that BMX enhances GSK3β activity through downregulation of Ser9 phosphorylation, thereby enhancing β-catenin phosphorylation at Ser33 / Ser37 / Thr41 and inducing proteolysis. Taken together, these data demonstrate that 10 μM BMX and 50 μM TMZ enhance TMZ-mediated cytotoxicity, partially via the Wnt / β-catenin / GSK3β pathway, and thus reduce GBM-R cell proliferation.

[0085] 1.2.4 BMX enhanced TMZ-mediated cytotoxic effects by promoting TMZ-mediated apoptosis in GBM-R cells.

[0086] To investigate whether BMX can induce cell cycle arrest, we analyzed the effects of BMX (5 μM and 10 μM) alone and in combination with 50 μM TMZ on the cell cycle in A172-R and U87MG-R cell lines. Results showed that 10 μM BMX alone induced G0 / G1 cell cycle arrest in A172-R cells (70.34%) and U87MG-R cells (77.95%). Next, 5 and 10 μM BMX in combination with 50 μM TMZ not only increased the amount of G0 / G1 cell cycle arrest but also caused sub-G1 arrest (apoptosis) in both GBM-R cell lines (Figure 4A-C).

[0087] Flow cytometry revealed that the combination of BMX and TMZ induced a dose-dependently high percentage of apoptotic cells (21.7% / 25.95%) in the A172-R / U87MG-R cell lines (Figure 4D). Furthermore, late apoptosis was also predominant after treatment with 10 μM BMX and 50 μM TMZ (Figure 4E). Thus, while BMX alone could induce cell cycle arrest and only inhibit cell proliferation without inducing apoptosis, the combination of BMX and TMZ promoted TMZ-mediated apoptosis and led to enhanced cytotoxicity in GBM-R cells.

[0088] 1.2.5 BMX enhanced TMZ-mediated cytotoxicity through WT-p53-mediated MGMT inhibition in GBM-R cells

[0089] Since the combination of BMX and TMZ can promote TMZ-mediated apoptosis, we speculated that BMX may enhance TMZ-mediated apoptosis through WT-p53-mediated MGMT inhibition. We first examined WT-p53 and MGMT levels in A172 / A172-R and U87MG / U87MG-R cells and confirmed that TMZ resistance was associated with WT-p53 and MGMT (Figure 5A). Bioinformatics analysis also suggested that only 33% of patients had p53 mutations, while the rest were p53 WT (Table 2). Next, we evaluated the TCGA and DriverDB databases to compare the overall survival rates between p53 mutations and p53 WT. Colony formation assays (Figure 11) revealed that p53 WT cases exhibited a poorer prognosis in GBM patients compared with mutated cases.

[0090] We investigated the proapoptotic signaling pathway in WT-p53-mediated apoptosis. We also examined the ability of TMZ to repair MGMT. Results revealed that proapoptotic markers, such as P21, Bax / Bcl2, and Puma, were elevated, whereas MGMT levels were reduced after treatment with BMX with or without 50 μM TMZ. However, cleaved caspase-3 was only observed after treatment with 10 μM BMX and 50 μM TMZ (Figure 5B). To determine whether apoptosis is induced by BMX alone, TMZ alone, or their combination in WT-p53-mediated MGMT inhibition, A172-R and U87MG-R cells were treated with 50 μM TMZ with or without 5 and 10 μM BMX. TMZ alone was only able to moderately suppress MGMT expression without increasing WT-p53 and DNA damage markers (WT-p53-ser15). However, the combination of 10 μM BMX and 50 μM TMZ significantly reduced MGMT expression, and the expression levels of WT-p53 and a DNA damage marker (WT-p53-ser15) were also elevated, indicating that MGMT was negatively regulated by WT-p53-mediated apoptosis (Figure 5C).

[0091] Furthermore, by evaluating the scatter plots of p53 WT and mutant cells (Figure 8B), we found that GBM p53 WT cells had hypermethylated MGMT and reduced MGMT mRNA and protein expression. Furthermore, TMZ alone was unable to induce WT-p53-mediated apoptosis in GBM-R cells. However, these data suggest that the combination of BMX and TMZ may enhance TMZ-mediated cytotoxicity through WT-p53-mediated MGMT inhibition in GBM-R cells. BMX alone could moderately reduce MGMT levels but did not induce WT-p53-mediated apoptosis in GBM-R cells. [Table 2]

[0092] 1.2.6 The combination of BMX and TMZ reduced GSC formation in GBM-R cells

[0093] Because GSC markers are central to GBM resistance, we examined the levels of GSC markers in all cell lines; high expression levels of CD133, CD44, and SOX2 were detected in A172-R and U87MG-R cells, indicating that TMZ resistance is partly associated with GSC markers (Figure 6A). Furthermore, treatment with 10 μM BMX and 50 μM TMZ significantly reduced the expression levels of CD133, CD44, and SOX2 in both GBM-R cell lines (Figure 6B). Therefore, the combination of BMX and TMZ could enhance the TMZ-mediated cytotoxic effect by attenuating GSC markers and converting the stem cell phenotype in GBM-R cells.

[0094] We also examined HDAC8 and GSC markers in TMZ-resistant GBM human tissues through immunohistochemistry (Figure 6C), revealing that HDAC8 and GSC are closely associated with TMZ resistance in GBM.

[0095] 1.3. Conclusion

[0096] Although preclinical studies have shown that HDACs have antitumor effects on gliomas, previous studies have not addressed or predicted their potential for the treatment of chemotherapy-resistant GBM. In this study, we found for the first time that BMX, a novel isotype-selective HDAC8 inhibitor, can enhance TMZ-mediated cytotoxicity not only by downregulating the β-catenin / c-Myc / SOX2 pathway to inhibit stemness, but also by upregulating WT-p53-mediated MGMT inhibition and inducing apoptosis in TMZ-resistant GBM cells. Furthermore, we found that the inverse correlation between WT-p53 / MGMT reversal and the Wnt / β-catenin / GSKβ signaling pathway may be involved in the oncogenic role of GBM and TMZ-resistant GBM.

[0097] Based on the above results, we propose the following working model (see Figure 7).

[0098] First, the β-catenin / c-Myc / cyclin D1 / SOX2 signaling pathway (right pathway) in TMZ-resistant GBM. Our previous study and bioinformatics analysis in this study suggested that the Wnt / β-catenin / GSK3β pathway may influence therapy selection for GBM

[17] . In this study, we demonstrated that BMX, both in the absence and presence of TMZ (thin and thick lines), enhanced GSK3β activity by downregulating Ser9 phosphorylation, thereby enhancing β-catenin phosphorylation at Ser33 / Ser37 / Thr41 and inducing β-catenin proteolysis. β-catenin degradation was confirmed by the proteasome inhibitor MG132. Undegraded β-catenin translocates into the nucleus and binds to TCL4, activating downstream target genes, such as c-Myc and cyclin D1, to induce cell proliferation and cell cycle continuation. Both BMX alone (thin line) and BMX and TMZ (thick line) suppressed the expression of c-Myc and cyclin D1 and induced cell cycle arrest. However, BMX alone was unable to induce sub-G1 cell cycle arrest. Only the combination of BMX and TMZ induced significant cell cycle arrest and progression to sub-G1, indicating that it induced late apoptosis in GBM-R cells (dotted line in the lower right of Figure 7).

[0099] Furthermore, GSCs play an important role in treatment resistance in GBM. GSCs are characterized by their self-renewal capacity both in vitro and in vivo through high expression of neural stem cell markers, such as CD133 and CD44, and transcription factors, such as SOX2

[23] . In the present study, we found that BMX, both without and with TMZ, attenuates not only CD133 and CD44 but also SOX2, thereby suppressing stemness by downregulating the expression of GSCs

[24] . As previously reported, c-Myc is also required for maintaining CSCs in glioma

[24] . We conclude that BMX alone and the combination of BMX and TMZ suppressed cell proliferation by enhancing TMZ-mediated cytotoxicity via the β-catenin / c-Myc / cyclin D1 / SOX2 signaling pathway in GBM-R cells.

[0100] Furthermore, in TMZ-resistant GBM, WT-p53 mediated MGMT inhibition (left pathway in Figure 7). The mechanism of action of TMZ in GBM is DNA damage caused by methylation of guanine at the O6 position. MGMT reverses methylation to repair DNA in GBM cells, thereby conferring GBM resistance. Although MGMT-independent pathways also play an important role in TMZ resistance [25-27], the MGMT-dependent pathway remains considered the primary pathway for TMZ resistance. In this study, GBM-R cell lines (A172-R and U87MG-R) expressed high levels of MGMT protein, thus confirming that the MGMT-dependent pathway is indeed the primary mechanism of TMZ resistance in these cell lines. It can be speculated that BMX suppressed MGMT expression in MGMT-dependent GBM-R cell lines, thereby attenuating MGMT's ability to repair DNA damage. In GBM-R cell lines, BMX alone moderately reduced MGMT expression, whereas TMZ alone did not (Figures 5B and 5C). However, the combination of BMX and TMZ significantly reduced MGMT protein levels and enhanced TMZ-mediated apoptosis in an MGMT-dependent manner.

[0101] As shown in Figure 7, this model involves two major signaling pathways. The right-hand pathway: the β-catenin / c-Myc / cyclin D1 / Sox 2 signaling pathway. Treatment of GBM-R cell lines with BMX alone (thin line) or BMX plus TMZ (thick line) reduced GSK3β (S9) and active β-catenin. Subsequently, c-Myc and cyclin D1 were also reduced, inducing cell cycle arrest and attenuating stem cell activity. However, only BMX plus TMZ (dashed line) could induce apoptosis. The left-hand pathway: WT-p53 mediated MGMT inhibition. Treatment of GBM-R cell lines with BMX alone or BMX plus TMZ, both with BMX alone (thin line) and with BMX plus TMZ (thick line), increased WT-p53 and downregulated MGMT levels, cell cycle arrest, and stemness. However, WT-p53 and a DNA damage marker (WT-p53-ser15, not shown) increased after activation of cell cycle arrest markers (P21) and pro-apoptotic markers (BAX / Bcl2, and Puma), indicating significant DNA damage and apoptosis only in the presence of BMX and TMZ (thick lines). Red indicates upregulation. Green indicates downregulation.

[0102] In this example, we found that BMX had a potent effect in inhibiting HDACs and reduced MGMT through the restoration of WT-p53 (all P53 lanes in Figures 5A-C). In this study, we showed that BMX alone (thin line on the left) moderately increased WT-p53 levels and moderately downregulated MGMT expression, leading to the maintenance of DNA repair capacity (Figure 5B). We also speculated that HDAC inhibition may reduce MGMT expression through the reactivation of WT-p53. BMX alone moderately increased WT-p53 levels and moderately downregulated MGMT expression, leading to the maintenance of DNA repair. Furthermore, BMX alone induced a cell cycle arrest marker (P21). In this study, the combination of BMX and TMZ (thick line in the left part) induced extensive DNA damage through overexpression of WT-p53 (and Ser15) and downregulation of MGMT expression, ultimately leading to WT-p53-mediated apoptosis (Figure 5C). This combination (compared to BMX alone) also increased the expression of cell cycle arrest markers (P21), proapoptotic proteins (Bax / Bcl2 and Puma), and cleaved caspase-3 expression for WT-p53-mediated apoptosis. Taken together, these results suggest that while BMX alone (thin line in the left part of Figure 7) only partially induced WT-p53-mediated MGMT inhibition, the combination of BMX and TMZ (thick line in the left part of Figure 7) enhanced the TMZ cytotoxic effect in GBM-R cell lines, overcoming TMZ resistance.

[0103] In conclusion, we unexpectedly found that BMX overcomes TMZ resistance by downregulating the β-catenin / c-Myc / SOX2 signaling pathway and enhancing TMZ-mediated cytotoxicity by upregulating WT-p53-mediated MGMT inhibition. These findings suggest that the combination of BMX and TMZ holds promise for optimal personalized treatment of TMZ-resistant WT-p53 GBM cells.

[0104] Example 2

[0105] 2.1. Materials and Methods

[0106] 2.1.1 Cell lines and cell culture

[0107] Three CRC cell lines, HT29, HCT116, and RKO, were used in this study. The American Type Culture Collection (ATCC; Manassas, VA, USA) provided the human CRC cell lines HT29 (ATCC HTB-38; mutant TP53, p.R273H; APC frameshift, p.E1554fs; wild-type β-catenin), HCT116 (ATCC CCL-247; wild-type TP53; wild-type APC; deleted β-catenin, p.S45del), and RKO (ATCC CRL-2577; wild-type TP53; wild-type APC; wild-type β-catenin). The three CRC cell lines were cultured under adherent conditions maintained at 37°C in a cell incubator containing 5% CO2. HCT-116 and HT-29 cell lines were cultured in McCoy's 5A medium supplemented with 10% fetal bovine serum (Gibco; Thermo Fischer Scientific, Grand Island, NY, USA), 1% penicillin, and 1% streptomycin. RKO cells were cultured in MEM medium supplemented with 10% FBS, 1% penicillin, 1% streptomycin, and 1% sodium pyruvate. Cell cultures were passaged every 3 days by trypsinization. BM-BMX (BMX), (E)-2-(4-methoxybenzyloxy)-3-prenyl-4-methoxy-N-hydroxycinamide, was provided by NatureWise Biotech & Medicals Corporation (Taipei, Taiwan).

[0108] 2.1.2 Cell proliferation assay

[0109] We plated 4000 CRC cells per well in a 96-well plate and allowed them to adhere overnight. To verify the responsiveness of cell lines to BMX and TMZ monotherapy, cells were treated with different doses of BMX or TMZ for 24, 48, and 72 hours. To confirm the responsiveness of cells to the BMX-TMZ combination, cells were treated with different doses of TMZ (0-1000 μg / mL) with or without BMX (5 μM) for 24, 48, and 72 hours, or with different doses of BMX (0-10 μM) with or without TMZ (50 μM) for 24, 48, and 72 hours. After treatment, absorbance values ​​were measured at the indicated time points using a CCK8 kit (Targetmol, Shanghai, China). Results are reported as the mean ± standard deviation of at least three replicates.

[0110] 2.1.3 Flow cytometry analysis of DNA cell cycle

[0111] Cells were treated with different doses of BMX (0–10 μM) in the presence or absence of TMZ (50 μM) for 48 hours. Untreated cells served as a negative control. All samples were run in triplicate in at least three independent experiments. Flow cytometry analysis of propidium iodide (PI) was performed. For DNA cell cycle analysis, cells were trypsinized, centrifuged, washed with phosphate-buffered saline (PBS), and fixed with methanol. Cells were then washed again and incubated with PI working solution (10 μg / mL PI and 20 mg / mL RNase A) in the dark at 37°C for 15 minutes. PI fluorescence of 10,000 individual nuclei was calculated using a flow cytometer (Attune NxT Flow Cytometer, Thermo Fisher Scientific). The fractions of cells in G0 / G1, S, G2 / M, and sub-G0 / G1 phases were analyzed using Attune NxT flow cytometry software and determined as the mean peak fluorescence intensity for each histogram.

[0112] 2.1.4 Flow cytometry analysis of apoptosis

[0113] Apoptosis induction in different doses of BMX (0–10 μM) in the presence or absence of TMZ (50 μM) was assayed by detecting membrane externalization of phosphatidylserine using the CF® 488A Annexin V and PI Apoptosis Kit (Fremont, CA, USA) according to the manufacturer's instructions.

[16] All samples were then immediately analyzed by flow cytometry.

[0114] 2.1.5 Quantitative Real-Time RT-PCR

[0115] Cells (2 × 10 ) were transfected using a Tissue Total RNA Mini Kit (Geneaid, Taipei, Taiwan) according to the manufacturer's instructions. 5 RNA was extracted from 1000 cells (8000 cells). RNA concentration and purity were examined at 260-280 nm using a NanoDrop® spectrophotometer (Thermo Scientific, Waltham, MA, USA). Subsequently, cDNA synthesis was performed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems), also according to the manufacturer's instructions. qPCR reactions were performed using the Power SYBR Green PCR Master Mix (Applied Biosystems) according to the manufacturer's recommendations in a 7500 Real-time PCR System (Applied Biosystems) with 18 s as an internal standard. Threshold cycle (Ct) values ​​were calculated using StepOnePlus (Applied Biosystems) software. Relative expression of each mRNA was calculated using the 2-(ΔCt) method. The primer sequences for HDAC8 were as follows: HDAC8 forward 5'-GCGTGATTTCCAGCACATAA-3' (SEQ ID NO: 1), HDAC8 reverse 5'-ATACTTGACCGGGGTCATCC-3' (SEQ ID NO: 2). 18s forward 5'-TCAAGTGCAGTGCAACAACTC-3' (SEQ ID NO: 3), 18s reverse 5'-AGAGGACAGGGTGGAGTAATCA-3' (SEQ ID NO: 4).

[0116] 2.1.6 Colony formation assay

[0117] For the anchorage-dependent growth assay, 1,000 cells were resuspended in medium and seeded into 6-well plates. Medium containing various concentrations of BMX (0-10 μM) alone, in the presence or absence of TMZ (50 μM), was replaced every 2-3 days. After 14 days, the medium was removed, and the cells were washed, fixed with 4% paraformaldehyde for 30 minutes, and stained with 0.1% crystal violet for 20 minutes at 25°C. After lysing the stained cells with dimethyl sulfoxide (DMSO), the crystal violet intensity was quantified by absorbance at 570 nm. Results are expressed as the average colony count ± SE from three independent experiments.

[0118] 2.1.7 Senescence-associated (SA) β-galactosidase (SA-β-gal) assay

[0119] SA expression of β-gal activity was measured using a Senescence Detection Kit (CS0030-1KT; Sigma-Aldrich; Merck Millipore, Darmstadt, Germany). Briefly, cells were treated with different doses of BMX (0–10 μM) in the presence or absence of TMZ (50 μM) for 48 h, washed with PBS, fixed with fixative solution for 0.5 h at room temperature, and then incubated with SA-β-gal staining solution overnight at 37°C. SA-β-gal activity was examined by X-gal (5-bromo-4-chloro-3-indolyl β-D-galactoside) staining at pH 6.0. Blue-stained senescent cells were photographed. Randomly selected fields (n = 3) were analyzed by light microscopy to quantify the percentage of senescent cells.

[0120] 2.1.8 Western blot analysis

[0121] Western blot analysis was used to examine the expression levels of the indicated proteins in the test cell lines under various concentrations of BMX (0–10 μM) in the presence or absence of TMZ (50 μM) or OXP (5 μM), as well as SAHA, VPA, or PCI-34051. SDS-PAGE and Western blot analysis were performed on the prepared lysates as previously outlined

[16] . Acetyl-histone H3 (Lys9 / Lys14), acetyl-histone H4 (Lys8), P53, acetyl-P53 (Lys382), phospho-P53 (Ser15), P21, P16, MGMT, phospho-H2AX (S139), E2F1, E2F3, cleaved caspase-3, cleaved caspase-8, cleaved caspase-7, cleaved caspase-9, PARP, Bax, Bcl-2, Bid, Bim, Bak, Puma, β-catenin, phospho-β-catenin (Ser33 / 37 / 41), GSK3β, phospho-GSK3β (Ser Specific primary antibodies against c-Myc, cyclin D1, p62, LC3B, CD133, CD44, SOX-2, and HDAC8 were used for detection, with GAPDH, α-tubulin, or β-actin used as internal controls. After incubation with the primary antibodies, the cells were then incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody, and the HRP signal was detected using a chemical HRP substrate. The antibodies used by the inventors are listed in Table 4. The signal for each target protein was visualized by incubation with an enhanced chemiluminescence reagent and exposure to X-ray film.

[0122] [Table 3-1] [Table 3-2]

[0123] 2.1.9 Statistical analysis

[0124] Data are presented as mean ± standard deviation. Statistical analysis was performed using one-way analysis of variance. Data were compared using Student's t-test. The statistical significance level was* p<0.05, ** p<0.01, *** p was set at <0.001.

[0125] 2.2.Results

[0126] 2.2.1 Optimizing the combination of BMX and TMZ in three CRC cell lines

[0127] To examine the effects of BMX or TMZ on CRC cell growth, three human colorectal cancer cell lines, HT29 (p53 mutant), HCT116 (p53 wild-type), and RKO (p53 wild-type), were used. They were treated with BMX (0.313, 0.625, 1.25, 2.5, 5, and 10 μM) or TMZ (25, 50, 100, 200, 400, 800, and 1,000 μM) separately for 24, 48, and 72 hours. The results showed that CRC cell viability was significantly inhibited in a dose-dependent manner. The half-maximal inhibitory concentration (IC50) values ​​of BMX or TMZ alone in HT-29, HCT-116, and RKO cells were calculated (Table 5). Using a clonogenic assay, which represents in vivo tumorigenicity, TMZ was effective against tumorsphere formation in clonogenic assays of HT29, HCT116, and RKO cells, with IC50 values ​​of TMZ of 359.45±50.43, 137.66±22.73, and 244.01±29.42 μM, respectively. The results showed that BMX and TMZ inhibited the basic cell growth of three types of colorectal cancer cells, including HT-29, HCT-116, and RKO, at three different incubation times.

[0128] [Table 4]

[0129] To evaluate whether BMX improves the chemosensitivity of TMZ, BMX and TMZ were administered together to HT-29, HCT-116, and RKO cells. The combination of BMX (5 μM) and TMZ (25, 50, 100, 200, and 400 μM) showed greater cell growth inhibition than either BMX or TMZ administered individually. The combination of BMX (5 μM) and TMZ (25, 50, 100, 200, and 400 μM) showed greater cell growth inhibition than either BMX or TMZ administered individually. Subsequently, we examined the time-dependent inhibition of cell proliferation by TMZ and BMX in combination with 50 μM TMZ at different concentrations (0.313, 0.625, 1.25, 2.5, 5, and 10 μM). Notably, BMX reduced the IC50 of TMZ in HT-29, HCT-116, and RKO cells (Table 5). These findings suggested that BMX inhibits CRC cell proliferation and improves TMZ chemosensitivity. 50 μM TMZ and 5 μM BMX showed the highest cytotoxic effect in HT-29, HCT-116, and RKO cells. We used this combination in a time-dependent manner and observed a cytotoxic effect at 48 hours. This finding suggested that BMX improves TMZ chemosensitivity. The combination of BMX and TMZ inhibited cell proliferation in a time-dependent manner. Therefore, all subsequent experiments were performed using 50 μM TMZ in combination with different concentrations of BMX (2.5, 5, and 10 μM) for 48 hours.

[0130] We next examined colony formation in the presence of BMX alone or in combination with TMZ. In a typical sequential fashion, we found that this inhibitory effect increased when BMX was combined with 50 μM TMZ. By increasing the TMZ concentration (150 μM), BMX could potentially be reduced to 1–2 μM rather than 5–10 μM. Taken together, these results demonstrate that the combined use of BMX and TMZ synergistically inhibits CRC cancer cell proliferation and colony formation. Therefore, all subsequent experiments were performed using 50 μM TMZ in combination with different concentrations of BMX (2.5, 5, and 10 μM) for 48 hours.

[0131] 2.2.2 Comparison of the effects of the combination of BMX and TMZ on CRC with conventional drugs

[0132] Cell cycle arrest is one of the major causes of cell proliferation inhibition. To evaluate the possible mechanism by which BMX or the combination treatment inhibited cell growth, cell cycle profiles were assayed using flow cytometry. As shown in Figure 13, the combination treatment significantly induced G2 / M arrest in HT29 and HCT116 cells, and showed a much stronger effect on G2 / M arrest than any other single drug. 50 μM TMZ and 2.5, 5, and 10 μM BMX not only increased the amount of G0 / G1 cell cycle arrest, but also caused sub-G1 arrest (apoptosis) in the RKO cell line.

[0133] The synergistic effect of BMX and TMZ after 48 hours of treatment was measured by Annexin V binding in three types of CRC cell lines. Treatment with BMX and TMZ induced a significant increase in the percentage of apoptotic cells compared with each drug individually. BMX increased the early apoptotic cells to 23.78%, 49.34%, and 59.18% in HT29, HCT116, and RKO cells, and also increased late apoptosis. With combined treatment, the late apoptotic population in HT29, HCT116, and RKO cells increased from 1.08% to 10.36%, from 3.67% to 19.37%, and from 0.32% to 16.48% after 48 hours of incubation.

[0134] 2.2.3 Apoptosis induced by BMX and the combination of BMX and TMZ is mediated by p53-mediated MGMT inhibition

[0135] The p53 pathway has been reported to be involved in chemotherapy-induced apoptosis in various cancer cells

[28] . BMX has been shown to activate p53, leading to cell death mediated by the β-catenin pathway

[16] . To clarify whether the anticancer effects of BMX and TMZ are due to DNA damage, we examined DNA damage and corresponding p53 pathway markers in three CRC cell lines with different p53 phenotypes. Considering the basal protein expression status of markers including p53, acetyl-p53 (Lyx382), p53 (Ser15), p21, p16, MGMT, γ-H2AX, E2F1, E2F3, and GAPDH in HT29, HCT116, and RKO cells, BMX alone may not only enhance p53 expression but also regulate other important genes that interfere with cell growth. Treatment with BMX alone or in combination with TMZ dose-dependently increased the levels of p53 phosphorylation (Ser15) and γ-H2AX phosphorylation (Ser139) in HT29, HCT116, and RKO cells. In HT29, HCT116, and RKO cells, p53 acetylation at Lys382 increased in a time-dependent manner, and the expression of p53 downstream targets p21 and p16 was enhanced. As shown by Western blotting of p53 wild-type and mutant cells, CRC p53 wild-type cells exhibited hypermethylation of MGMT and similarly reduced MGMT protein expression. Furthermore, the combination of BMX and TMZ significantly reduced E2F3 expression (Figure 14A). Interestingly, histone H3 acetylation was also increased by BMX or BMX and TMZ. This suggests that BMX affects the activity of histone acetyltransferases and / or HDACs in cells, leading to the acetylation of proteins including p53. Combining BMX with TMZ can increase the expression of p21 and p16 and the phosphorylation of γH2AX through enhanced p53 expression and activation of MGMT inhibition mediated by p53 function (Figure 14A).

[0136] The balance between proapoptotic (stress or death) signals and antiapoptotic molecules, including Bcl-2 and Bid, Bax, or poma, is the main reason why apoptotic responses are triggered through caspase-dependent pathways

[29] . Caspase cleavage, shown in Figure 14B, indicated that the activities of caspase-7, caspase-8, caspase-9, and caspase-3 were not significantly altered at low concentrations of BMX, but were highly upregulated in a dose-dependent manner when combined with TMZ in HT29 cells, ultimately contributing to PARP cleavage and apoptosis. The apoptotic protein expression levels of cleaved caspase-3, caspase-7, caspase-9, and caspase-PARP were found to be significantly increased in a concentration-dependent manner after 10 μM BMX treatment in HCT116 and RKO cell lines. Furthermore, we investigated the proapoptotic signaling pathway in p53 wild-type cell-mediated apoptosis. The results revealed that BMX treatment reduced the levels of the anti-apoptotic protein Bcl-2 and increased the levels of the pro-apoptotic proteins Bax, Bim, and Puma. However, BMX treatment did not upregulate the pro-apoptotic Bcl-2 family proteins Bak and Bid. In addition, the synergistic effect of BMX and TMZ was superior to that of BMX alone (Figure 14C). The combination of TMZ and BMX produced more senescent cells than each treatment alone, especially in p53 wild-type cells, such as HCT116 and RKO (Figure 15). Because CD133, CD44, and SOX2 are highly associated with drug resistance in CSCs and are used as phenotypic markers of CSCs, including CRC, treatment with BMX and TMZ significantly dose-dependently reduced the expression levels of CD133, CD44, and SOX2 in HT29, HCT116, and RKO (Figure 16). Therefore, the combination of BMX and TMZ could enhance the TMZ-mediated cytotoxic effect by attenuating CSC markers and converting the stemness phenotype of CRC cells.Therefore, the above results indicated that the combined treatment of BMX and TMZ in CRC cells activated the caspase-dependent signaling pathway and induced cell apoptosis.

[0137] 2.4 BMX enhanced TMZ-mediated cytotoxicity by targeting the Wnt / β-catenin / GSK3β pathway in CRC cells.

[0138] Next, we investigated the mechanism by which BMX potentiates the TMZ-mediated cytotoxic effect on Wnt / β-catenin activity in three CRC cell lines. As shown in Figure 3A, BMX treatment increased the expression levels of β-catenin, phospho-β-catenin (Ser33 / Ser37 / Thr41), and phospho-GSK-3β (Ser9) proteins in the three CRC cell lines, whereas phospho-β-catenin (Ser33 / Ser37 / Thr41) and phospho-GSK-3β (Ser9) levels were decreased. Combined treatment with 5 μM BMX and TMZ directly reduced the protein levels of β-catenin in the three cell lines, and reduced the protein levels of phospho-β-catenin (S33 / S37 / T41) through phosphorylation by GSK-3β. Furthermore, we further investigated the effects of BMX on the proliferation markers c-Myc and cyclin D1 and found that both BMX with and without TMZ could reduce the proliferation markers c-Myc and cyclin D1 (Figure 17A). These results demonstrated that combined treatment with 5 μM BMX and TMZ enhanced GSK3β activity through downregulation of Ser9 phosphorylation, resulting in enhanced phosphorylation of β-catenin at Ser33 / Ser37 / Thr41, inducing its proteolysis (Figure 17B). In addition, application of MG132 reversed β-catenin degradation and increased MGMT expression under the treatment with 5 μM BMX and 50 μM TMZ (Figure 17C). Taken together, these data revealed that BMX and TMZ enhanced TMZ-mediated cytotoxicity, in part through the Wnt / β-catenin / GSK3β pathway, thus reducing CRC cell proliferation.

[0139] 2.5 Autophagy played a key role in cell death induced by BMX and the combination of BMX and TMZ.

[0140] Lipidated LC3 and the autophagy substrate P62 are commonly used as markers for assessing autophagosomes and autophagy

[17] . Treatment with BMX or the combination of BMX and TMZ also increased the expression of P62 and LC3-II, the processed form of LC3, in a concentration-dependent manner (Figure 18A). β-catenin negatively regulates P62 expression [17,30]. To verify that the decrease in P62 protein levels was caused by β-catenin protein degradation, we applied the proteasome inhibitor MG132 to cells treated with BMX or the combination of BMX and TMZ. As expected, BMX-induced β-catenin degradation was reversed, and P62 expression was also suppressed by MG132 application (Figure 18B). Upon β-catenin protein degradation by the combination treatment, P62 was no longer inhibited, subsequently triggering the downstream autophagy pathway (Figure 18B). To determine the role of autophagy in cell death induced by BMX or the combination of BMX and TMZ, we used BAF, a protein biosynthesis inhibitor that blocks the late stages of autophagy, and Z-VAD-FMK (carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone), a cell-permeable pan-caspase inhibitor, before the addition of BMX or the combination of BMX and TMZ. We found that Z-VAD-FMK suppressed early apoptosis induced by BMX and TMZ treatment in three cell lines. In addition, pretreatment with BAF A1 reduced cell death induced by BMX or the combination of BMX and TMZ, as determined by flow cytometry, which was consistent with the reduced expression of cleaved caspase-3, caspase-7, caspase-8, and caspase-9 induced by BMX or the combination of BMX and TMZ. Interactions between different autophagy / apoptosis-related proteins and the corresponding signaling pathways have been identified, implying that crosstalk exists between the two pathways.To investigate the role of apoptosis in 6c-induced autophagy, we treated cells with BAF or Z-VAD-FMK before adding BMX or the combination of BMX and TMZ. As shown in Figure 18D, Z-VAD-FMK and BAF suppressed early apoptosis, but BAF suppressed caspase-3 activation induced by the combination of BMX and TMZ without interfering with LC3I / II in all cell lines. However, Z-VAD-FMK inhibited caspase-3 activation induced by the combination of BMX and TMZ in p53 mutant cell lines, but with interference with LC3I / II. Taken together, these results emphasize the importance of stimulating autophagy during cell death.

[0141] 2.3. Conclusion

[0142] Conventional radiochemotherapy for CRC treatment is sometimes ineffective, partly because CRC patients do not respond to this therapy regimen and / or suffer from severe drug toxicity. This study demonstrated that the combination of BMX and TMZ exhibits specific, efficient, and synergistic antiproliferative and apoptotic effects in HCC cells, particularly HCT116 and RKO. In conclusion, BMX and TMZ produce the best synergistic effects, and the mechanism is currently the most important link. Furthermore, we conclude that BMX, a specific HDAC8i, inhibits cell proliferation and induces cell cycle arrest, cellular senescence, autophagy, and apoptosis, resulting in cell death, in combination with temozolomide (TMZ).

[0143] Furthermore, the combination of BMX and TMZ was found to induce synergistic apoptotic cell death through caspase-3 cleavage and PARP activation. In this study, we demonstrated that the combination of BMX and TMZ induced enhanced phospho-p53 (ser15) expression and increased DNA damage, such as γ-H2AX foci. The increased expression of phospho-p53 (ser15) may be due to the increased total p53 expression reported in our previous studies [16, 17]. Additionally, this study revealed that BMX may have an HDAC-dependent synergistic effect with TMZ on CRC cell viability.

[0144] Considering the above, high expression of HDAC8 in human GBM tissues and GBM-R cell lines correlates with MGMT levels. The combination of BMX and TMZ induced WT-p53-mediated apoptosis in GBM-R cell lines through WT-p53-mediated MGMT inhibition. Furthermore, the combination of BMX and TMZ also suppressed cell proliferation and GSC phenotype activity via the β-catenin / c-Myc / cyclin D1 / SOX2 signaling pathway in GBM-R cell lines. Therefore, BMX may be a promising strategy for precise, personalized treatment of GBM patients with WT-p53 and TMZ resistance.

[0145] Overall, as an indication of a synergistic mechanism, the present invention demonstrates that the combination of BMX and TMZ is effective in inducing CRC cell death by upregulating p53 / p21 / E2F3 / Bax and downregulating the Wnt / β-catenin / cyclin D1 / c-Myc / p62 pathway. Therefore, the combination of BMX and TMX was found to have promising effects on cell death, including the induction of apoptosis and autophagy. The results in the Examples indicate that the combination of BMX and TMZ may play a role by helping to understand HDAC8-dependent synergy in CRC cell death. These findings suggest a clinically important new mechanism for resistance to combination chemotherapy regimens.

[0146] While this specification contains many details, these should not be construed as limitations on the scope of the invention or the claims, but rather as descriptions of features characteristic of particular embodiments or examples of the invention. Certain features that are described herein in the context of separate embodiments or examples may also be practiced in combination in a single embodiment. Furthermore, the present invention includes the following aspects. [Aspect 1] 1. A combination for treating TMZ-resistant cancer in a patient, comprising temozolomide (TMZ) and a compound of formula A: [ka] [In the formula, R 1 is hydrogen, alkyl, alkenyl, C 5 -C 6 cycloalkyl, a 5- or 6-membered unsaturated carbocyclic or 5- or 6-membered heterocyclic ring, or (CH 2 )mR 4 and; X is C, -O-, -N- or -S-; Y is -O-, -NH or -OC 1 -C 4 is alkyl; n is an integer from 0 to 10; m is an integer from 0 to 5; R 2 and R 3 independently, C 1 -C 6 is alkyl; R 4 is C 5 -C 6 Cycloalkyl, or halogen, -CF 3 , -OR 7 or -NR 7 R 8 is a 5- or 6-membered unsaturated carbocyclic or heterocyclic ring optionally substituted by 7 and R 8 are independently hydrogen or C 1 -C 6 is alkyl; R 5 OH, NH 2 , or C 5 -C 6 Cycloalkyl, 5- or 6-membered unsaturated carbocyclic or heterocyclic rings, where the cycloalkyl, carbocyclic and heterocyclic rings are substituted with halogen, NH 2 , NO 2 、C 1 -C 6 Alkoxy, C 1-6 Alkylthio, OR 7 '', NR 7 R 8 or CF 3 and R 6 is H or hydroxy or C 2 -C 10 C optionally substituted by alkenyl 1 -C 10 alkyl or R 1 Together with -C 2 H 2 -becomes] or a pharmaceutically acceptable salt, stereoisomer, enantiomer, prodrug or solvate thereof, A combination agent, wherein TMZ and Compound A are combined in a relative ratio that effectively overcomes TMZ resistance. [Aspect 2] Compound A is

change

[0147] References 1. Louis, D.N.; Perry, A.; Reifenberger, G.; von Deimling, A.; Figarella-Branger, D.; Cavenee, W.K.; Ohgaki, H.; Wiestler, O.D.; Kleihues, P.; Ellison, D.W. The 2016 World Health Organization Classification of Tumors of the Central Nervous System: a summary. Acta Neuropathol 2016, 131, 803-820, doi:10.1007 / s00401-016-1545-1. 2. Park, J.K.; Hodges, T.; Arko, L.; Shen, M.; Iacono, D.D.; McNabb, A.; Bailey, N.O.; Kreisl, T.N.; Iwamoto, F.M.; Sul, J., et al. Scale to Predict Survival After Surgery for Recurrent Glioblastoma Multiforme. Journal of Clinical Oncology 2010, 28, 3838-3843, doi:10.1200 / jco.2010.30.0582. 3. Stupp, R.; Mason, W.P.; van den Bent, M.J.; Weller, M.; Fisher, B.; Taphoorn, M.J.; Belanger, K.; Brandes, A.A.; Marosi, C.; Bogdahn, U., et al. Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med 2005, 352, 987-996, doi:10.1056 / NEJMoa043330. 4. Stupp, R.; Hegi, M.E.; Mason, W.P.; van den Bent, M.J.; Taphoorn, M.J.; Janzer, R.C.; Ludwin, S.K.; Allgeier, A.; Fisher, B.; Belanger, K., et al. Effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase III study: 5-year analysis of the EORTC-NCIC trial. Lancet Oncol 2009, 10, 459-466, doi:10.1016 / s1470-2045(09)70025-7. 5. Siegel, R. L., Miller, K. D., and Jemal, A. (2015) Cancer statistics, 2015. CA Cancer J Clin 65, 5-29 6. Vecchione, L., Gambino, V., Raaijmakers, J., Schlicker, A., Fumagalli, A., Russo, M., Villanueva, A., Beerling, E., Bartolini, A., Mollevi, DG, El-Murr, N., Chiron, M., Calvet, L., Nicolazzi, C., Combeau, C., Henry , C. , Simon , IM , Tian , S. , in 't Veld , S. , D'Ario , G. , Mainardi , S. , Beijersbergen , RL , Lieftink , C. , Linn , S. , Rumpf-Kienzl , C. , Delorenzi , M. , Wessels , L. , Salazar , R. , Di Nicolantonio , F. , Bardelli , A. , van http: / / dx.doi.org / 10.1037 / 0033-295X.101.1.13 Rheenen, J., Medema, RH, Tejpar, S., & Bernards, R. (2016). Cell 165, 317–3307. Xie , Y.-H. , Chen , Y.-X. , and Fang , J.-Y. (2020) Comprehensive review of targeted therapy for colorectal cancer. Signal Transduction and Targeted Therapy 5 , 22 8. Floudas, C. S., Brar, G., Mabry-Hrones, D., Duffy, A. G., Wood, B., Levy, E., Krishnasamy, V., Fioravanti, S., Bonilla, C. M., Walker, M., Morelli, M. P., Kleiner, D. E., Steinberg, S. M., Figg, W. D., Greten, T. F., and Xie, C. (2019) A Pilot Study of the PD-1 Targeting Agent AMP-224 Used With Low-Dose Cyclophosphamide and Stereotactic Body Radiation Therapy in Patients With Metastatic Colorectal Cancer. Clin Colorectal Cancer 18, e349-e360. 9. Martino-Echarri, E., Henderson, B. R., and Brocardo, M. G. (2014) Targeting the DNA replication checkpoint by pharmacologic inhibition of Chk1 kinase: a strategy to sensitize APC mutant colon cancer cells to 5-fluorouracil chemotherapy. Oncotarget 5, 9889-9900. 10. Temozolomide [US Prescribing Information]. Whitehouse Station, NJ; Merck & Co., Inc.; 2019Newlands ES, Stevens MFG, Wedge SR et.al, Temozolomide: a review of its discovery, chemical properties, pre-clinical development and clinical trials. Cancer Treat Rev. 1997; 23(1):35-61. 11. Tatar Z, Thivat E, Planchat E Temozolomide and unusual indications: review of literature. Cancer Treat Rev. 2013; 39(2):125-35. 12. Alonso, M.M.; Gomez-Manzano, C.; Bekele, B.N.; Yung, W.K.; Fueyo, J. Adenovirus-based strategies overcome temozolomide resistance by silencing the O6-methylguanine-DNA methyltransferase promoter. Cancer Res 2007, 67, 11499-11504, doi:10.1158 / 0008-5472.Can-07-5312. 13. Le Rhun, E.; Preusser, M.; Roth, P.; Reardon, D.A.; van den Bent, M.; Wen, P.; Reifenberger, G.; Weller, M. Molecular targeted therapy of glioblastoma. Cancer Treat Rev 2019, 80, 101896, doi:10.1016 / j.ctrv.2019.101896. 14. Cabrini, G.; Fabbri, E.; Lo Nigro, C.; Dechecchi, M.C.; Gambari, R. Regulation of expression of O6-methylguanine-DNA methyltransferase and the treatment of glioblastoma (Review). Int J Oncol 2015, 47, 417-428, doi:10.3892 / ijo.2015.3026. 15. Wiewrodt, D., Nagel, G., Dreimuller, N., Hundsberger, T., Perneczky, A., and Kaina, B. (2008) MGMT in primary and recurrent human glioblastomas after radiation and chemotherapy and comparison with p53 status and clinical outcome. Int J Cancer 122, 1391-1399 16. Tsai, C. Y., Ko, H. J., Chiou, S. J., Lai, Y. L., Hou, C. C., Javaria, T., Huang, Z. Y., Cheng, T. S., Hsu, T. I., Chuang, J. Y., Kwan, A. L., Chuang, T. H., Huang, C. F., Loh, J. K., and Hong, Y. R. (2021) NBM-BMX, an HDAC8 Inhibitor, Overcomes Temozolomide Resistance in Glioblastoma Multiforme by Downregulating the beta-Catenin / c-Myc / SOX2 Pathway and Upregulating p53-Mediated MGMT Inhibition. Int J Mol Sci 22 17. Chu, C. W., Ko, H. J., Chou, C. H., Cheng, T. S., Cheng, H. W., Liang, Y. H., Lai, Y. L., Lin, C. Y., Wang, C., Loh, J. K., Cheng, J. T., Chiou, S. J., Su, C. L., Huang, C. F., and Hong, Y. R. (2019) Thioridazine Enhances P62-Mediated Autophagy and Apoptosis Through Wnt / β-Catenin Signaling Pathway in Glioma Cells. Int J Mol Sci 2019, 20, doi:10.3390 / ijms20030473. 18. Bocangel, D.; Sengupta, S.; Mitra, S.; Bhakat, K.K. p53-Mediated down-regulation of the human DNA repair gene O6-methylguanine-DNA methyltransferase (MGMT) via interaction with Sp1 transcription factor. Anticancer Res 2009, 29, 3741-3750. 19. Natsume, A.; Ishii, D.; Wakabayashi, T.; Tsuno, T.; Hatano, H.; Mizuno, M.; Yoshida, J. IFN-beta down-regulates the expression of DNA repair gene MGMT and sensitizes resistant glioma cells to temozolomide. Cancer Res 2005, 65, 7573-7579, doi:10.1158 / 0008-5472.Can-05-0036. 20. Yang, H.-Y., Hsu, Y.-F., Chiu, P.-T., Ho, S.-J., Wang, C.-H., Chi, C.-C., Huang, Y.-H., Lee, C.-F., Li, Y.-S., Ou, G., and Hsu, M.-J. (2013) Anti-cancer activity of an osthole derivative, NBM-T-BMX-OS01: targeting vascular endothelial growth factor receptor signaling and angiogenesis. PloS one 8, e81592-e81592. 21. Chen, T. J., Zhou, Y. F., Ning, J. J., Yang, T., Ren, H., Li, Y., Zhang, S., and Chen, M. W. (2015) NBM-T-BMX-OS01, an Osthole Derivative, Sensitizes Human Lung Cancer A549 Cells to Cisplatin through AMPK-Dependent Inhibition of ERK and Akt Pathway. Cellular Physiology and Biochemistry 36, 893-906. 22. Yang, Y.C.; Chen, C.N.; Wu, C.I.; Huang, W.J.; Kuo, T.Y.; Kuan, M.C.; Tsai, T.H.; Huang, J.S.; Huang, C.Y. NBM-T-L-BMX-OS01, Semisynthesized from Osthole, Is a Novel Inhibitor of Histone Deacetylase and Enhances Learning and Memory in Rats. Evid Based Complement Alternat Med 2013, 2013, 514908, doi:10.1155 / 2013 / 514908. 23. Lathia, J.D.; Mack, S.C.; Mulkearns-Hubert, E.E.; Valentim, C.L.; Rich, J.N. Cancer stem cells in glioblastoma. Genes Dev 2015, 29, 1203-1217, doi:10.1101 / gad.261982.115. 24. Wang, J.; Wang, H.; Li, Z.; Wu, Q.; Lathia, J.D.; McLendon, R.E.; Hjelmeland, A.B.; Rich, J.N. c-Myc is required for maintenance of glioma cancer stem cells. PLoS One 2008, 3, e3769, doi:10.1371 / journal.pone.0003769. 25. Gaspar, N.; Marshall, L.; Perryman, L.; Bax, D.A.; Little, S.E.; Viana-Pereira, M.; Sharp, S.Y.; Vassal, G.; Pearson, A.D.; Reis, R.M., et al. MGMT-independent temozolomide resistance in pediatric glioblastoma cells associated with a PI3-kinase-mediated HOX / stem cell gene signature. Cancer Res 2010, 70, 9243-9252, doi:10.1158 / 0008-5472.Can-10-1250. 26. Yi, G.Z.; Huang, G.; Guo, M.; Zhang, X.; Wang, H.; Deng, S.; Li, Y.; Xiang, W.; Chen, Z.; Pan, J., et al. Acquired temozolomide resistance in MGMT-deficient glioblastoma cells is associated with regulation of DNA repair by DHC2. Brain 2019, 142, 2352-2366, doi:10.1093 / brain / awz202. 27. Yang, W.B.; Chuang, J.Y.; Ko, C.Y.; Chang, W.C.; Hsu, T.I. Dehydroepiandrosterone Induces Temozolomide Resistance Through Modulating Phosphorylation and Acetylation of Sp1 in Glioblastoma. Mol Neurobiol 2019, 56, 2301-2313, doi:10.1007 / s12035-018-1221-7. 28. Jaamaa, S., Af Hallstrom, T. M., Sankila, A., Rantanen, V., Koistinen, H., Stenman, U. H., Zhang, Z., Yang, Z., De Marzo, A. M., Taari, K., Ruutu, M., Andersson, L. C., and Laiho, M. (2010) DNA damage recognition via activated ATM and p53 pathway in nonproliferating human prostate tissue. Cancer Res 70, 8630-8641. 29. Bogenberger, J. M., Kornblau, S. M., Pierceall, W. E., Lena, R., Chow, D., Shi, C. X., Mantei, J., Ahmann, G., Gonzales, I. M., Choudhary, A., Valdez, R., Camoriano, J., Fauble, V., Tiedemann, R. E., Qiu, Y. H., Coombes, K. R., Cardone, M., Braggio, E., Yin, H., Azorsa, D. O., Mesa, R. A., Stewart, A. K., and Tibes, R. (2014) BCL-2 family proteins as 5-Azacytidine-sensitizing targets and determinants of response in myeloid malignancies. Leukemia 28, 1657-1665. 30. Aberle, H., Bauer, A., Stappert, J., Kispert, A., and Kemler, R. (1997) beta-catenin is a target for the ubiquitin-proteasome pathway. Embo j 16, 3797-3804

Claims

1. 1. A combination for treating TMZ-resistant cancer in a patient, comprising temozolomide (TMZ) and [Chemical Formula 1] or a pharmaceutically acceptable salt, enantiomer or solvate thereof, A combination agent in which TMZ and the compound BMX are combined in a relative ratio that effectively overcomes TMZ resistance.

2. 2. The combination of claim 1, wherein TMZ resistance is overcome by enhancing TMZ-mediated cytotoxic effects.

3. The combination of claim 2, wherein the enhanced TMZ-mediated cytotoxic effect is due to downregulating the β-catenin / c-Myc / SOX2 signaling pathway and upregulating WT-p53-mediated MGMT inhibition.

4. 2. The combination of claim 1, wherein TMZ and the compound BMX are administered separately or sequentially.

5. 2. The combination of claim 1, wherein the cancer is glioblastoma multiforme (GBM) or colorectal cancer (CRC).

6. A medicament comprising the combination of any one of claims 1 to 4 for use in a method for the optimal individualized treatment of drug-resistant cancers with WT-p53 expression in patients.

7. The pharmaceutical composition according to claim 6, wherein the drug is TMZ.

8. The pharmaceutical composition of claim 6, wherein the drug-resistant cancer is TMZ-resistant GMB or CRC.

9. A medicine or kit for a patient with TMZ-resistant cancer, comprising the combination agent according to any one of claims 1 to 4.

10. The medicament or kit according to claim 9, wherein the cancer is GBM or CRC.

Images