Application of copper ion chelator combined with NF-κB inhibitor in the preparation of tumor therapeutic drugs

Abstract

The present invention provides the use of a copper ion chelator combined with an NF‑κB inhibitor in the preparation of a tumor therapeutic drug. The present invention's research shows that copper ions can activate NF‑κB to mediate the carcinogenic function of CTR1. At the same time, we found that NF‑κB inhibitors such as QNZ inhibit inflammatory pathways while promoting the expression of CTR1 at the transcriptional level, thereby inducing copper ion enrichment, which can promote the potential proliferation ability and drug resistance of tumors by activating pathways such as PDK1‑AKT and MAPK. The combined use of copper ion chelators and QNZ can not only overcome the potential proliferation ability and drug resistance of tumors caused by copper ion enrichment caused by QNZ, but also reduce the dose-dependent toxicity of QNZ and the oncogene function of copper ions, and has a good synergistic effect.

Description

Technical Field

[0001] The present invention belongs to the technical field, and more specifically, relates to the application of a copper ion chelator combined with an NF-κB inhibitor in the preparation of tumor therapeutic drugs. Background Art

[0002] There are three main subtypes of breast cancer in terms of molecular classification: estrogen receptor (ER) and progesterone receptor (PR) positive, growth hormone receptor (HER2) positive and triple-negative breast cancer (ER / PR / HER2 are all negative, TNBC). In addition to surgery and conventional radiotherapy and chemotherapy, new targeted therapies based on precise molecular classification of breast cancer provide new clinical treatment strategies for breast cancer. For example, estrogen receptor antagonists Tamoxifen and Fulvestrant have been used to treat ER / PR-positive breast cancer; Trastuzumab has been used to treat HER2-positive breast cancer. It is worth noting that 15% of breast cancer patients are currently TNBC, which includes inflammatory breast cancer (IBC). As a rare, highly lethal and rapidly progressive subtype of highly invasive breast cancer, inflammatory breast cancer has no effective treatment so far, making it one of the hot topics in breast cancer research in recent years. Although there are many potential targets for the treatment of TNBC, such as the development of inhibitors for poly adenosine diphosphate ribose polymerase (PARP) associated with BRCA mutations, the development of inhibitors for phosphatidylinositol 3-kinase (PI3K) associated with PIK3CA mutations, and the development of inhibitors for immune checkpoint programmed cell death protein 1 (PD-1) / programmed cell death ligand 1 (PD-L1) and cell cycle regulator cyclin-dependent kinase 4 / 6 (CDK4 / 6), there is not enough evidence to show that these inhibitors can significantly improve the treatment of inflammatory breast cancer. Although JAK2 inhibitors can be used for targeted therapy of inflammatory breast cancer, the heterogeneity of inflammatory breast cancer has hindered the research on biomarkers and targeted therapy for inflammatory breast cancer. Therefore, there is an urgent need to develop new targets for the treatment of inflammatory breast cancer, which will also provide a strong theoretical and experimental basis for the precise treatment of inflammatory breast cancer.

[0003] Chronic inflammation plays a critical role in the occurrence and development of cancer, but the current research on the mechanism of chronic inflammation in the formation and development of breast cancer is still unclear. The inflammatory microenvironment plays an important role in the growth, metastasis, drug resistance and angiogenesis of breast cancer. Increased copper ion levels have been detected in tumor tissues and serum of breast cancer patients, suggesting that abnormal copper metabolism homeostasis in breast cancer patients may induce inflammatory responses and promote tumor development. Copper is an indispensable trace element in cell life activities. It can promote the expression of proinflammatory factors and then induce chronic diseases such as metabolic disorders, neurodegenerative diseases and cancer. A large number of studies have also confirmed that copper ions can activate the NF-κB signaling pathway and induce inflammatory responses. Recent studies have shown that Cu directly regulates the function of oncoproteins by binding and activating MEK1-ERK, PDK1-AKT and ULK1 / 2-autophagy signals and is closely related to carcinogenic pathways. However, whether other carcinogenic or tumor suppressor proteins are directly regulated by Cu, and whether and how other metals directly interact with carcinogenic pathways related to tumorigenesis, deserves further study. Since the copper transporter 1 (CTR1)-Cu axis plays an important role in tumorigenesis, promising cancer treatments targeting this pathway have been developed. Cu chelators such as tetrathiomolybdate (TTM) have been used in clinical trials for breast cancer, BRAF-V600E melanoma, and thyroid tumors. However, the nonspecific chelation function of Cu chelators with other metals and the important role of Cu in normal biological processes have greatly limited their clinical application. Since CTR1, as the main protein for copper ion transport, has been shown to be highly expressed in a variety of tumors, knocking down CTR1 can produce effective anti-tumor effects in melanoma, non-small cell lung cancer, and breast cancer. Therefore, new antibodies targeting CTR1, especially immune recognition of its N-terminal Cu binding region, are worth developing for precision tumor treatment. On the other hand, given the potential connection between the Cu-CTR1 axis and platinum chemotherapy, the combination of Cu chelators and platinum drugs is also an effective potential tumor intervention strategy.

[0004] Chronic inflammation promotes the occurrence and development of tumors and resistance to chemotherapy by inducing gene mutations, changing the expression and transformation of oncogenes and tumor suppressor genes, inhibiting cell apoptosis, and inducing angiogenesis. The transcription factor NF-κB family plays a vital role in immunity, inflammation, proliferation, apoptosis, and survival. NF-κB plays a major role in promoting the phenotype and invasion of inflammatory breast cancer. Current studies have found that the NF-κB signaling pathway plays a very critical role in the development of cancer. Its activation promotes the proliferation or anti-apoptosis of cancer cells, which makes NF-κB an important clinical treatment target for cancer. Therefore, inhibiting the cancer-promoting function of the NF-κB pathway has become a potential solution for targeted tumor treatment. The quinazoline derivative EVP4593 (QNZ) is widely used to inhibit the NF-κB signaling pathway. In mouse and rat liver cancer models and malignant glioma animal models, QNZ has shown significant anti-tumor effects. Although studies have reported that the use of non-targeted anti-inflammatory drugs in the early stage, including non-steroidal anti-inflammatory drugs (aspirin, celecoxib and ibuprofen), statins, glucocorticoids (dexamethasone), natural products with anti-inflammatory activity (polyphenols, curcumin, resveratrol), etc., can inhibit chronic inflammation, prevent the occurrence of tumors, and improve the killing effect of other therapies on tumor cells, but the single use of anti-inflammatory drugs has been shown to have limited efficacy in cancer treatment. In the prior art, clinical trials have used copper ion chelator TTM alone for breast cancer treatment, which has the disadvantages of high toxicity and lack of targeting indicators, while the use of NF-κB inhibitor QNZ alone for breast cancer treatment will promote the enrichment of copper ions, leading to breast tumor resistance, with the disadvantages of high toxicity and easy to produce resistance. There is no report on the use of copper ion chelator combined with NF-κB inhibitor in tumor treatment, especially breast cancer treatment. Summary of the invention

[0005] The purpose of the present invention is to overcome the above-mentioned defects and deficiencies in the prior art and provide an application of a copper ion chelator combined with an NF-κB inhibitor in the preparation of a tumor therapeutic drug.

[0006] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0007] The present invention provides a preclinical experimental basis for enhancing the therapeutic effect of inflammatory tumors by studying the mechanism of limited efficacy of anti-inflammatory drugs in cancer treatment. The present invention's research shows that copper ions can activate NF-κB to mediate the carcinogenic function of CTR1. It is also found that NF-κB inhibitors such as QNZ inhibit inflammatory pathways and promote the expression of CTR1 at the transcriptional level, thereby inducing copper ion enrichment, which can promote the potential proliferation ability and drug resistance of tumors by activating pathways such as PDK1-AKT and MAPK, that is, inhibiting NF-κB can significantly promote CTR1 transcriptional expression and copper ion uptake, thereby activating downstream ERK and AKT pathways, which may be the tumor's resistance to the treatment of NF-κB inhibitor QNZ. Through in vitro cytological experiments, in vivo animal experiments and organoid drug sensitivity experiments, it is verified that the combined use of copper ion chelators and QNZ can not only cause the potential proliferation ability and drug resistance of tumors by overcoming the copper ion enrichment caused by QNZ, but also reduce the dose-dependent toxicity of QNZ and the oncogene function of copper ions, and has a good synergistic effect on breast cancer. Therefore, the present invention proposes a new strategy for targeted treatment of breast cancer by combining copper ion chelators with NF-κB inhibitors.

[0008] On the other hand, copper ions can promote tumor proliferation and drug resistance by activating ERK and AKT pathways, so targeting copper ions can provide a new direction for tumor treatment. CTR1, as the main protein for transporting copper ions, has oncogene function and is regulated by different signaling pathways. The present study shows that copper ions can activate the NF-κB pathway associated with inflammation, and NF-κB activation mediates the carcinogenic function of CTR1, and copper ion chelators can inhibit the activation of inflammatory pathways. At the same time, it was found that the use of NF-κB inhibitor QNZ alone or by knocking down the IKKβ subunit alone to inhibit the inflammatory pathway will promote the expression of CTR1 transcripts, as well as cause copper ion uptake and downstream ERK and AKT pathway activation. Chronic inflammation is closely related to the occurrence, development and efficacy of cancer anticancer treatment, and inhibiting inflammation can be used as a potential solution for treating tumors. We point out that inhibiting inflammatory pathways can promote the phenomenon and potential mechanism of tumor proliferation and drug resistance by promoting the expression of CTR1 transcripts and activating the downstream pathways of copper ions. To this end, the present invention proposes to use a copper ion chelator while applying QNZ to inhibit the function of CTR1 in transporting copper ions, overcome QNZ resistance, and achieve the purpose of combined treatment of tumors, especially tumors related to inflammation.

[0009] Therefore, the present invention provides the use of a copper ion chelator combined with an NF-κB inhibitor in the preparation of a tumor therapeutic drug, wherein the tumor is a tumor expressing a CTR1 oncogene and having an activated NF-κB signaling pathway. In addition, the copper ion chelator can also be used alone to prepare a therapeutic drug for a tumor formed by the cancer-promoting function of the NF-κB pathway.

[0010] Furthermore, the copper ion chelating agent is tetrathiomolybdate (TTM).

[0011] Furthermore, the NF-κB inhibitor is a quinazoline derivative EVP4593 (QNZ) or IKK-16 (IKKInhibitor VII).

[0012] Furthermore, the tumor may be a tumor associated with inflammation, such as breast cancer, liver cancer, gastric cancer, nasopharyngeal cancer, etc.

[0013] Preferably, the tumor is breast cancer.

[0014] Preferably, the breast cancer is triple-negative breast cancer.

[0015] Further preferably, the triple-negative breast cancer is inflammatory breast cancer.

[0016] The present invention also provides a tumor treatment drug, comprising a copper ion chelator and an NF-κB inhibitor.

[0017] Furthermore, the copper ion chelating agent is tetrathiomolybdate.

[0018] Furthermore, the NF-κB inhibitor is a quinazoline derivative EVP4593 or IKK-16 (IKK Inhibitor VII).

[0019] Furthermore, the drug has copper ion chelator and NF-κB inhibitor as main active ingredients.

[0020] Furthermore, the medicine contains an effective dose of a copper ion chelator and an NF-κB inhibitor.

[0021] Preferably, the molar concentration ratio of the copper ion chelator to the NF-κB inhibitor is (6-20):(1-2).

[0022] Furthermore, the medicine also includes pharmaceutically acceptable excipients.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The present invention provides the use of a copper ion chelator combined with an NF-κB inhibitor in the preparation of a tumor therapeutic drug. The present invention's research shows that copper ions can activate NF-κB to mediate the carcinogenic function of CTR1. At the same time, we found that NF-κB inhibitors such as QNZ inhibit inflammatory pathways while promoting the expression of CTR1 at the transcriptional level, thereby inducing copper ion enrichment, which can promote the potential proliferation ability and drug resistance of tumors by activating pathways such as PDK1-AKT and MAPK. The combined use of copper ion chelators and QNZ can not only overcome the potential proliferation ability and drug resistance of tumors caused by copper ion enrichment caused by QNZ, but also reduce the dose-dependent toxicity of QNZ and the oncogene function of copper ions, and has a good synergistic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the result of the study on the carcinogenic function of CTR1 mediated by copper ions; Figure 1 A is the transcriptome sequencing result after breast cancer cells were treated with CuSO4, B is the activation of NF-κB pathway after breast cancer cells were treated with different concentrations of CuSO4 for 30 minutes, C is the transcriptional activity of NF-κB after cells were treated with CuSO4 and copper ion chelator TTM for 16 hours, D is the activation of NF-κB pathway in breast cancer cells with CTR1 knockdown after being treated with NF-κB activator LPS at different time points, E is the activation of NF-κB pathway in breast cancer cells with CTR1 knockdown after being treated with NF-κB activator TNFα at different time points, F is the expression of CTR1 after overexpression of IKKβ in breast cancer cells with CTR1 knockdown, G is the cell clone formation after overexpression of IKKβ in breast cancer cells with CTR1 knockdown, H is the statistical analysis of clone formation in G, I is the cell clone formation after overexpression of IKKβ in breast cancer cells with CTR1 knockdown, J is the statistical analysis of clone formation in I, and K is the cell proliferation after overexpression of IKKβ in breast cancer cells with CTR1 knockdown.

[0026] Figure 2 Consuming intracellular copper ions and inhibiting inflammatory pathways can synergistically inhibit the proliferation of breast cancer cells; Figure 2A shows the expression of CTR1 protein and transcript after breast cancer cells were treated with different concentrations of NF-κB inhibitor QNZ, B shows the membrane localization of CTR1 after cells were treated with NF-κB inhibitor QNZ, C shows the transcript of CTR1 after different breast cancer cells were treated with NF-κB inhibitor QNZ, D shows the expression of CTR1 and the activation of AKT and ERK1 / 2 pathways in breast cancer cells with IKKβ knockdown, E shows the transcript expression of CTR1 in breast cancer cells with IKKβ knockdown, F shows the growth of breast cancer cells with IKKβ knockdown treated with different concentrations of copper ion chelator TTM, G shows the clone formation of breast cancer cells with IKKβ knockdown treated with different concentrations of copper ion chelator TTM, and H shows the statistical analysis of clone formation in Figure G.

[0027] Figure 3 This study is about the synergistic effect of copper ion chelator TTM and QNZ on the proliferation and apoptosis of breast cancer cells. Figure 3 Figures A and B show the cell growth of different breast cancer cells after treatment with copper ion chelators TTM and QNZ alone or in combination, C and E show the cell proliferation of different breast cancer cells after treatment with copper ion chelators TTM and QNZ alone or in combination, D is the statistical analysis of cell proliferation in C, F is the statistical analysis of cell proliferation in E, G and I show the cell clone formation of different breast cancer cells after treatment with different concentrations of copper ion chelators TTM and QNZ alone or in combination, H is the heat map analysis of clone formation in G, J is the heat map analysis of clone formation in I, K and M show the cell apoptosis of different breast cancer cells after treatment with copper ion chelators TTM and QNZ alone or in combination, L is the statistical analysis of cell apoptosis in K, and N is the statistical analysis of cell apoptosis in M; CDI in A to B refers to drug synergistic effect, CDI<1 means that the drugs have synergistic effect, and CDI<0.7 means that the drug synergistic effect is significant; EdU in C and E indicates DNA replication ability, and DAPI indicates cell nucleus.

[0028] Figure 4 The copper ion chelator TTM and QNZ have a synergistic effect on the proliferation and apoptosis of breast cancer cells; Figure 4A to G in the figure show the cell growth of different breast cancer cells after being treated with copper ion chelators TTM and QNZ alone or in combination, H, J and L show the cell proliferation of different breast cancer cells after being treated with copper ion chelators TTM and QNZ alone or in combination, I is the statistical analysis of cell proliferation in H, K is the statistical analysis of cell proliferation in J, M is the statistical analysis of cell proliferation in L, N and P show the cell apoptosis of different breast cancer cells after being treated with copper ion chelators TTM and QNZ alone or in combination, O is the statistical analysis of cell apoptosis in N, and Q is the statistical analysis of cell apoptosis in P; in A to G, EdU indicates DNA replication ability, DAPI indicates cell nucleus, CDI refers to drug synergistic effect, CDI < 1 means that the drugs have synergistic effect, and CDI < 0.7 means that the drug synergistic effect is significant; in H to L, EdU indicates DNA replication ability, and DAPI indicates cell nucleus.

[0029] Figure 5 The copper ion chelator TTM and QNZ synergistically inhibited the growth of breast tumors in vivo and promoted the death of breast cancer organoids; Figure 5A shows the treatment of tumor-bearing mice with copper ion chelators TTM and QNZ alone or in combination at different time points, and the measurement of the subcutaneous tumor volume of the mice and statistical analysis. B shows the statistical analysis of the weight of the subcutaneous tumor of the tumor-bearing mice after treatment with copper ion chelators TTM and QNZ alone or in combination. C shows the statistical analysis of the weight of the tumor-bearing mice after treatment with copper ion chelators TTM and QNZ alone or in combination. D shows the growth of subcutaneous tumors in the tumor-bearing mice after treatment with copper ion chelators TTM and QNZ alone or in combination. E shows the detection of the growth of subcutaneous tumors in the tumor-bearing mice after treatment with copper ion chelators TTM and QNZ alone or in combination using the cell proliferation antibody Ki-67. The proliferation of tumor cells after treating tumor-bearing mice, F is the treatment of C57 mice with copper ion chelators TTM and QNZ alone or in combination at different time points, and the measurement of the subcutaneous tumor volume of mice and statistical analysis, G is the statistical analysis of the weight of subcutaneous tumors of C57 mice after treatment with copper ion chelators TTM and QNZ alone or in combination, H is the statistical analysis of the weight of C57 mice after treatment with copper ion chelators TTM and QNZ alone or in combination, I is the growth of subcutaneous tumors in C57 mice after treatment with copper ion chelators TTM and QNZ alone or in combination, J is the use of cell proliferation antibody p-Histon H3 detects the proliferation of tumor cells in C57 mice after treatment with copper ion chelators TTM and QNZ alone or in combination. K is the apoptosis of organoids after treatment with luminal breast cancer organoids at 20, 30 μM copper ion chelator TTM and 5 μM QNZ alone or in combination. L is the viability of organoids after treatment with luminal breast cancer organoids at 10, 20 μM copper ion chelator TTM and 2.5, 5 μM QNZ alone or in combination. M is the apoptosis of TNBC organoids after treatment with copper ion chelator TTM and QNZ alone or in combination. N is the viability of TNBC organoids after treatment with copper ion chelator TTM and QNZ alone or in combination. In K~N, PHO is phenylarsine oxide, which serves as a positive control for reduced organoid viability. Live represents organoid survival, Dead represents organoid death, and BF represents imaging of organoids under white light. DETAILED DESCRIPTION

[0030] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0031] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0032] Example 1 Study on the Carcinogenic Function of CTR1 Mediated by Copper Ions

[0033] (1) Study on the activation of NF-κB pathway by copper ions: Breast cancer cells were treated with 100 μM CuSO4 for 24 hours, and the cells were collected and total RNA was extracted for transcriptome sequencing, and the sequencing results were analyzed. Breast cancer T-47D cells were cultured in serum-free medium for 16 hours, and the cells were treated with different concentrations of CuSO4 for 30 minutes to detect the activation of the NF-κB pathway. Luciferase reporter plasmids indicating NF-κB transcriptional activity were expressed in HEK293T cells, and the cells were treated with 50 μM CuSO4 and 50 μM copper ion chelator TTM for 16 hours, and the transcriptional activity of NF-κB was detected by chemiluminescence.

[0034] (2) NF-κB activation mediates the oncogenic function of CTR1: pLKO.1-shCTR1-tet on was packaged into lentivirus and used to infect breast cancer cells MDA-MB-231 (MDA-MB-231shCTR1-tet on), and then screened with 1 μg / ml puromycin to form stable cell clones. The cells were then treated with 1 μg / ml doxycycline (DOX) for 72 h. The cells were treated with NF-κB activators LPS and TNFα at different time points to detect the activation of the NF-κB pathway. An IKKβ overexpressing cell line was constructed in the MDA-MB-231shCTR1-tet on cell line, and a stable cell clone was formed after selection with 100 μg / ml hygromycin. The cells were then treated with 1 μg / ml doxycycline (DOX) for 72 hours, and 1000 breast cancer cells constructed above were inoculated in each well of a 6-well plate. After the cells were cultured with complete medium until the cell clones were visible to the naked eye, the cells were fixed and stained with crystal violet to observe the cell clone formation and perform statistical analysis. At the same time, 1.2% agarose and 2X cell culture medium were mixed in a 1:1 ratio and added to a 6-well plate. After solidification, 0.35% agarose medium containing the above-constructed cells was added, and the cells were cultured for 2 to 3 weeks after solidification. The number and size of cell clones formed in each group were stained and compared, and statistical analysis was performed.

[0035] The inflammatory microenvironment plays an important role in breast cancer growth, metastasis, drug resistance and angiogenesis. A large number of studies have also confirmed that copper ions can activate the NF-κB signaling pathway and induce inflammatory responses. CTR1, as the main transporter of copper ions, is highly expressed in a variety of tumors including breast cancer. The present invention found through transcriptome sequencing that copper ions can activate the inflammatory pathway in breast cancer cells ( Figure 1 A), and protein immunoblotting and dual luciferase assays verified that copper ions can activate the NF-κB signaling pathway ( Figure 1 B~C). It was later found that the reduction of copper ion levels in breast cancer cells with knockdown of CTR1 could inhibit the activation of NF-κB ( Figure 1 D~E), indicating that the function of NF-κB is mediated by copper ions. At the same time, through the clone formation experiment ( Figure 1 F~H) and soft agar test ( Figure 1 I~J) and cell proliferation-toxicity experiments ( Figure 1 K), we found that NF-κB activation mediates the oncogenic function of CTR1.

[0036] Example 2 Depletion of intracellular copper ions and inhibition of inflammatory pathways synergistically inhibit the proliferation of breast cancer cells

[0037] (1) Inhibition of inflammatory pathways promotes CTR1 transcript expression: After treating breast cancer cells MDA-MB-231 and T-47D with different concentrations of the NF-κB inhibitor QNZ for 16 hours, the cells were collected and the CTR1 protein expression was detected by western blotting. HEK293T cells were treated with 10 μM QNZ, and the membrane localization of CTR1 was detected by immunofluorescence. Breast cancer cells MDA-MB-231 and T-47D were treated with 10 μM QNZ for 16 hours, and the cells were collected and the CTR1 transcript expression was detected by fluorescence quantitative PCR. pLenti-shIKKβ was packaged into lentivirus and infected breast cancer cells MDA-MB-231 and T-47D, and 1 μg / ml puromycin was used to screen to form stable cell clones. The cells were collected and the expression of CTR1 protein and transcripts was detected by western blotting and fluorescence quantitative PCR.

[0038] (2) Consuming intracellular copper ions and inhibiting inflammatory pathways synergistically inhibit the proliferation of breast cancer cells: Treat the cells constructed in the above (1) with different concentrations of copper ion chelator TTM for a certain time point, and use cell proliferation-toxicity reagent (CCK-8) to detect the proliferation of breast cancer cells. 2000 cells constructed in the above (1) were inoculated in each well of a 6-well plate, and the cells were treated with TTM for 24 hours, and the medium containing TTM was discarded. The cells were cultured with complete medium until cell clones were visible to the naked eye, and the cells were fixed and stained with crystal violet to observe the cell clone formation, and statistical analysis was performed.

[0039] Increased copper ion levels have been detected in tumor tissues and serum of breast cancer patients, suggesting that abnormal copper metabolism homeostasis in breast cancer patients may induce inflammatory responses and promote tumor development. QNZ is widely used to inhibit the NF-κB signaling pathway. The present invention uses protein immunoblotting, immunofluorescence and fluorescence quantitative PCR experiments to find that the use of QNZ to inhibit the inflammatory pathway promotes the expression and membrane localization of CTR1 at the transcriptional level, thereby promoting the function of copper ion transport ( Figure 2A~C). The main kinase activated by NF-κB contains two kinase subunits IKKα and IKKβ. The present invention also found through protein immunoblotting and fluorescence quantitative PCR experiments that knocking down IKKβ kinase inhibited the activation of the NF-κB signaling pathway, which promoted the expression of CTR1 at the transcriptional level, and promoted the activation of the AKT pathway related to cell survival and the ERK1 / 2 pathway related to cell proliferation ( Figure 2 D~E), enhancing the survival ability of tumor cells. CTR1 is an important protein for transporting copper ions. Its increased expression indicates an increase in the level of intracellular copper ions. In order to study whether copper ion chelators can antagonize the side effects of tumor resistance caused by inhibiting inflammatory pathways in the treatment of breast cancer, the present invention found through cell proliferation-toxicity experiments and clone formation experiments that the use of copper ion chelator TTM to consume intracellular copper ions and inhibit inflammatory pathways can synergistically inhibit the proliferation of breast cancer cells ( Figure 2 F~H).

[0040] In summary, through in vitro mechanism studies, the results of the present invention show that inhibition of NF-κB can significantly promote CTR1 transcriptional expression and copper ion uptake, thereby activating downstream ERK and AKT pathways. This result may be an important reason why breast tumors develop resistance to treatment with the NF-κB inhibitor QNZ.

[0041] Example 3 Study on the synergistic effect of copper ion chelator TTM and QNZ on the proliferation and apoptosis of breast cancer cells

[0042] Combined with the studies in Examples 1 and 2, it is shown that the reason why breast tumors develop resistance to the treatment of the NF-κB inhibitor QNZ may be that the inhibition of NF-κB can significantly promote the transcriptional expression of CTR1 and the uptake of copper ions, thereby activating the downstream ERK and AKT pathways. Therefore, the present invention uses the copper ion chelator TTM to inhibit the increase in intracellular copper ion levels caused by QNZ treatment. The specific method is as follows:

[0043] (1) Cell proliferation-toxicity assay to detect the synergistic inhibitory effect of copper ion chelators TTM and QNZ on breast cancer cells: After treating breast cancer cells MDA-MB-231 and T-47D with 30 μM TTM and 8 μM QNZ alone or in combination for 24 hours, the growth of breast cancer cells was detected using cell proliferation-toxicity detection reagent (CCK-8) and statistical analysis was performed.

[0044] (2) EdU cell proliferation detection: Synergistic inhibitory effect of copper ion chelators TTM and QNZ on breast cancer cells: After breast cancer cells MDA-MB-231 were treated with 30 μM TTM and 10 μM QNZ alone or in combination for 12 hours, and breast cancer cells T-47D were treated with 100 μM TTM and 5 μM QNZ alone or in combination for 12 hours, the growth of breast cancer cells was detected using EdU cell proliferation detection kit (Ribobio, C10310-3) and statistical analysis was performed.

[0045] (3) Clone formation experiment to detect the synergistic inhibitory effect of copper ion chelators TTM and QNZ on breast cancer cells: 1000 breast cancer cells were seeded in each well of a 6-well plate. After the cells were treated with TTM and QNZ alone or in combination for 24 hours, the drug-containing culture medium was discarded and the cells were cultured with complete culture medium until cell clones were visible to the naked eye. The cells were fixed and stained with crystal violet to observe the cell clone formation and perform statistical analysis.

[0046] (4) Detection of the synergistic killing effect of copper ion chelators TTM and QNZ on breast cancer cells: After treating breast cancer MDA-MB-231 cells with 35 μM TTM and 8 μM QNZ alone or in combination for 24 hours, and after treating breast cancer T-47D cells with 100 μM TTM and 10 μM QNZ alone or in combination for 24 hours, the cells were collected and the cell apoptosis was detected using the Annexin V-FITC / PI apoptosis kit (Vazyme, A211-01).

[0047] In terms of cell experiments, the present invention found through cell proliferation-toxicity experiments, EdU cell proliferation detection, clone formation experiments and cell apoptosis experiments that TTM and QNZ significantly inhibited the proliferation of breast cancer cells and promoted cell apoptosis, with a good synergistic effect ( Figure 3 A~N). The present invention also confirmed this finding in different breast cancer cell lines ( Figure 4 A~Q).

[0048] Example 4 Copper ion chelator TTM and QNZ synergistically inhibit breast tumor growth in vivo and promote breast cancer organoid death

[0049] (1) Effect of the copper ion chelator TTM combined with QNZ on the tumor-bearing mouse model. 6×10 6 breast cancer cells MDA-MB-231, and wait until the tumor volume reaches 50 to 100 cubic millimeters (mm 3), QNZ (1 mg / kg) and TTM (5 mg / kg) were injected intraperitoneally every day, either alone or in combination, and the weight of tumor-bearing mice and the size of subcutaneous tumors were measured every 2 to 3 days. 6 Mouse breast cancer cells E0771, when the tumor volume reaches 50-100 mm3 (mm 3 ), QNZ (0.4 mg / kg) and TTM (5 mg / kg) were injected intraperitoneally every day, either alone or in combination. The weight of the tumor-bearing mice and the size of the subcutaneous tumor were measured every 2 to 3 days. When the subcutaneous tumor of the control tumor-bearing mice grew to a certain size, the mice were euthanized and the weight of the tumor was weighed. Using the formula (length × width 2 The tumor volume was calculated by statistic analysis.

[0050] (2) Immunohistochemical analysis of the growth of the tumor tissue in (1). The tumor tissue obtained in (1) was fixed, embedded and sliced ​​for immunohistochemistry, and the growth of the tumor tissue was detected using the Ki-67 antibody that indicates cell proliferation.

[0051] (3) Effect of the combined use of copper ion chelator TTM and QNZ on breast cancer organoids. Fresh human ER-positive and triple-negative breast cancer tissues were dissociated and cultured for organoids. After organoids were formed, 2000 to 3000 organoids were inoculated in 96-well transparent plates and black ELISA plates, respectively. Luminal breast cancer organoids were treated with 10μM, 20μM or 30μM TTM and 2.5μM, 5μM QNZ alone or in combination, and TNBC breast cancer organoids were treated with 20μM TTM and 10μM QNZ alone or in combination for 48h. The survival of organoids was detected using the LIVE / DEAD cell viability assay kit (Invitrogen, L3224) and the CellTiter-Glo luminescent cell viability assay kit (Promega, G7570).

[0052] The results are as follows Figure 5 As shown, the mouse subcutaneous tumor experiment showed that the combination of TTM and QNZ can effectively inhibit the growth of breast cancer tumors in vivo ( Figure 5 A~J). And through organoid staining and vitality detection experiments, the present invention found that the combination of TTM and QNZ significantly promoted the death of breast cancer organoids ( Figure 5 K~N). Therefore, copper ion chelator TTM and QNZ can synergistically inhibit the growth of breast tumors in vivo and promote the death of breast cancer organoids. The combination of copper ion chelator and NF-κB inhibitor can be used as a targeted therapy for breast cancer.

[0053] In addition, according to the therapeutic mechanism of the present invention, those skilled in the art can reasonably expect that the combined use of other NF-κB inhibitors (such as IKK-16) and copper ion chelators in the treatment of breast cancer also has a synergistic effect.

Claims

1. Use of a copper ion chelator combined with an NF-κB inhibitor in the preparation of a drug for treating triple-negative breast cancer, characterized in that: The copper ion chelator is tetrathiomolybdate, and the NF-κB inhibitor is quinazoline derivative EVP4593.

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