PEITC-containing pharmaceutical composition and application thereof in cancer treatment

Abstract

The invention provides an application of PEITC for preparing a preparation or a composition, and the preparation or the composition is used for (a) changing mutant p53 to restore wild type activity of the mutant p53, (b) inhibiting tumor cell proliferation caused by the mutant p53, (c) inducing apoptosis of mutant p53 tumor cells, and / or (d) preventing or treating diseases caused by p53 mutation. The invention also provides application of the p53 gene detection reagent, a kit comprising the PEITC and the p53 gene detection reagent, and a method for non-therapeutically inhibiting tumor cells in vitro.

Description

[0001] This application is a divisional application of an invention patent application with an application date of April 18, 2017, application number 201780037010.4, and invention name “Pharmaceutical composition containing PEITC and its application in cancer treatment”. Technical Field

[0002] The present invention relates to the field of medicine, and more specifically to a pharmaceutical composition containing PEITC and application thereof in the prevention and treatment of cancer and other diseases caused by p53 mutation. Background Art

[0003] This patent application is a follow-up patent of CN200510040865.1, CN200610126892.5, CN200910052231.6, CN201310205609.8, CN201310352414.6, CN201310364101.2 and CN201410346419.2 as well as US8039511B2, US8410170B2, EP06817815.1, CA2630262 and JP5308160.

[0004] Cancer is a major disease that threatens human health. According to the Global Cancer Report 2014, there were 14 million new cancer cases and 8.2 million deaths in 2012 worldwide.

[0005] Although significant achievements have been made in the treatment and prevention of tumors in recent years due to advances in epidemiology, early diagnosis and further improvements in treatment methods, tumors are still one of the major diseases that threaten people's lives. Discovering new, highly effective and low-toxic anti-cancer and cancer prevention drugs remains an important topic in drug research.

[0006] Fraumeni syndrome (LFS) is an autosomal dominant genetic disease caused by germline mutations in the p53 gene. Patients with this disease have a significantly increased risk of developing cancer. Therefore, targeting mutant p53 for treatment opens up a new avenue for tumor prevention.

[0007] However, research on cancer treatment and prevention targeting mutant p53 has just begun. With the continuous discovery and application of many small molecule drug screening methods, there are reports on new compounds targeting p53, especially the compound APR-246 targeting mutant p53 has entered Phase II clinical trials, which brings new hope for cancer treatment.

[0008] However, artificially designed small molecule drugs generally adopt a single-target screening method. Since single-target drugs are only effective against one mutated gene, once the growth channel is blocked by the single-target drug, the tumor cells will actively choose other pathways to cooperate with their own growth. As long as other pathways are successfully found, the single-target drug will lose its effect. This is the most troublesome problem of drug resistance in cancer treatment. Once drug resistance occurs, it means a poor prognosis for cancer treatment. Breaking through the drug resistance of targeted drug therapy is one of the research focuses of the global medical community. To solve drug resistance, effective multi-target inhibitors must be found.

[0009] In summary, there is an urgent need in the art to develop cancer treatment and prevention drugs that are less likely to develop drug resistance and target mutant p53. Summary of the invention

[0010] The purpose of the present invention is to provide a pharmaceutical composition containing PEITC and its application in the prevention and treatment of cancer and other diseases caused by p53 mutation, especially in the prevention and treatment of cancer targeting mutant p53.

[0011] In the first aspect of the present invention, a use of PEITC is provided for preparing a preparation or composition for (a) changing (or activating) mutant p53, (b) inhibiting the proliferation of mutant p53 tumor cells, (c) inducing apoptosis of mutant p53 tumor cells, and / or (d) preventing or treating diseases caused by p53 mutations.

[0012] In another preferred embodiment, the mutant p53 has a mutation at a site selected from the group consisting of R175, C176, Y220, P223, C242, G245, R248, R249, R273, V274, P278, R282 or a combination thereof.

[0013] In another preferred embodiment, the mutant p53 is selected from the following group: mutant p53 R175 , mutant p53 C176 , mutant p53 C242 .

[0014] In another preferred embodiment, the mutant p53 is a mutant p53 R175 .

[0015] In another preferred embodiment, the mutant p53 is a mutant p53 R175H .

[0016] In another preferred embodiment, the change (or activation) includes inducing the mutant p53 to regain a conformation or function similar to that of wild-type p53.

[0017] In another preferred embodiment, the wild-type-like refers to that the conformation or function of the changed (or activated) mutant p53 is similar to that of the wild-type p53 by ≥90%, preferably ≥95%, more preferably ≥99%, and most preferably ≥99.9%.

[0018] In another preferred embodiment, the functions include activating phosphorylated ATM / CHK2, delaying S and G2 / M phases, and / or inducing cell apoptosis.

[0019] In another preferred embodiment, the PEITC includes naturally occurring PEITC or artificially synthesized PEITC.

[0020] In another preferred example, the naturally occurring PEITC includes PEITC from natural food sources.

[0021] In another preferred embodiment, the natural food includes cruciferous plants.

[0022] In another preferred embodiment, the cruciferous plant is selected from the following group:

[0023] Greens, radishes, Chinese cabbage, watercress, broccoli, carrots, kale, horseradish, mustard, cauliflower, raspberries, or a combination.

[0024] In another preferred embodiment, the PEITC is extracted from cruciferous plants.

[0025] In another preferred embodiment, the composition is a pharmaceutical composition.

[0026] In another preferred embodiment, the pharmaceutical composition is used to prevent and / or treat cancer.

[0027] In another preferred embodiment, the cancer is selected from the following group:

[0028] Breast cancer, pancreatic cancer, liver cancer, prostate cancer, cervical cancer, ovarian cancer, oral cancer, esophageal cancer, gastric cancer, colorectal cancer, nasopharyngeal cancer, lung cancer, bladder cancer, soft tissue sarcoma, brain tumor, lymphocytic tumor, osteogenic sarcoma, or a combination thereof.

[0029] In another preferred embodiment, the dosage forms of the pharmaceutical composition include injections, suppositories, implants, ointments, solutions and oral dosage forms.

[0030] In another preferred embodiment, the oral dosage forms include tablets, capsules, films, oral liquids and granules.

[0031] In another preferred embodiment, the dosage form of the pharmaceutical composition includes a sustained-release dosage form and a non-sustained-release dosage form.

[0032] In another preferred embodiment, the pharmaceutical composition may also contain other anti-tumor active ingredients.

[0033] In another preferred embodiment, the pharmaceutical composition may further contain active ingredients Nutlin, MG132, and / or Zn 2+

[0034] In another preferred embodiment, the pharmaceutical composition is in unit dosage form, and the content of PEITC in each unit dosage form is about 0.1 to 1 (or 0.25-1, or 0.5-1) of the daily dose, wherein the daily dose is 5-500 mg, preferably 20-200 mg, and more preferably 60-180 mg.

[0035] In a second aspect of the present invention, a use of a p53 gene detection reagent is provided for preparing a diagnostic reagent or a diagnostic kit, wherein the diagnostic reagent or the diagnostic kit is used to (a) determine the therapeutic effect of PEITC, and / or (b) determine whether a tumor patient is suitable for treatment with PEITC.

[0036] In another preferred embodiment, the reagent comprises a protein chip, a nucleic acid chip, or a combination thereof.

[0037] In another preferred embodiment, the judgment includes auxiliary judgment and / or pre-treatment judgment.

[0038] In another preferred embodiment, the diagnostic reagent or diagnostic kit detects a p53 gene mutation selected from the following group:

[0039] p53 R175 、p53 C176 、p53 C242 、p53 Y220 、p53 P223 、p53 C242 、p53 G245 、p53 R248 、p53 R249 、p53 R273 、p53 V274 、p53 P278 、p53 R282 or a combination thereof.

[0040] In another preferred embodiment, the diagnostic reagent or diagnostic kit detects the R→H mutation at position 175 of the p53 gene.

[0041] In another preferred embodiment, the diagnostic reagent or diagnostic kit is used to detect a sample selected from the following group: a surgically removed tissue sample, a paraffin section tissue sample, a biopsy puncture tissue sample, a blood sample, or a combination thereof.

[0042] In another preferred embodiment, the p53 gene mutation detection reagent is selected from the following group: p53 gene, p53 protein, p53 protein-specific antibody, or a combination thereof.

[0043] In another preferred embodiment, the p53 protein or its specific antibody is coupled with or carries a detectable marker.

[0044] In another preferred embodiment, the detectable marker is selected from the following group: a chromophore, a chemiluminescent group, a fluorophore, an isotope or an enzyme.

[0045] In a third aspect of the present invention, a kit is provided, comprising:

[0046] (a) container A, and PEITC or a drug containing PEITC in container A;

[0047] (b) container B, and a p53 gene detection reagent in container B;

[0048] (c) Instructions.

[0049] In another preferred embodiment, the description states the following:

[0050] (i) If the p53 of the tumor cells in the test subject is mutant, it indicates that the PEITC treatment effect is better, and / or the tumor patient is suitable for treatment with PEITC;

[0051] In another preferred embodiment, the description further states the following:

[0052] (ii) If the p53 of the tumor cells in the test subject is wild type, it indicates that the PEITC treatment effect is poor and / or the tumor patient is not suitable for treatment with PEITC.

[0053] In a fourth aspect of the present invention, a method for non-therapeutic inhibition of tumor cells in vitro is provided, the method comprising the steps of:

[0054] (i) providing a tumor cell, detecting the mutation of the p53 gene of the tumor cell, and if the p53 of the tumor cell is a mutant, performing step (ii);

[0055] (ii) culturing the tumor cells in the presence of PEITC.

[0056] In another preferred embodiment, the concentration of PEITC is 1-100 μM, preferably 4-50 μM, and more preferably 5-20 μM.

[0057] In the fifth aspect of the present invention, a pharmaceutical composition is provided, wherein the pharmaceutical composition comprises an active ingredient (a) PEITC, an active ingredient (b) Nutlin, and a pharmaceutically acceptable carrier.

[0058] In another preferred embodiment, the pharmaceutical composition may further contain Zn 2+ , or MG132.

[0059] In another preferred embodiment, the pharmaceutical composition is used to (a) change (or activate) mutant p53, (b) inhibit the proliferation of mutant p53 tumor cells, (c) induce apoptosis of mutant p53 tumor cells, and / or (d) prevent or treat diseases caused by p53 mutations.

[0060] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 PEITC inhibited cell proliferation and induced apoptosis in p53 mutant DU145 cells.

[0062] Figure 1 A shows the results of measuring cell proliferation rate using the WST-1 method, including DU145 cells treated with DMSO (control) or PEITC for 3 days.

[0063] Figure 1 B shows the effect of PEITC on cell apoptosis. It includes DU145 cells treated with DMSO (control) or PEITC for 3 days. Detection of histone-associated DNA fragments indicates cell apoptosis.

[0064] Figure 2 PEITC was shown to inhibit mutant p53 R175 Dependent cell proliferation and induction of apoptosis.

[0065] Figure 2 a Human tumor cell lines harboring p53 hotspot gene mutations and wild-type p53 were treated with DMSO (control) or PEITC for 3 days.

[0066] Figure 2 b shows the results of measuring cell proliferation rate using the WST-1 method, including SK-BR-3 cells and A549 cells transfected with siRNA and treated with DMSO or PEITC for 3 days.

[0067] Figure 2c shows the effect of PEITC on cell apoptosis. These included untransfected or siRNA-transfected SK-BR-3 cells and A549 cells treated with DMSO or 4 μM PEITC for 3 days. Apoptosis was detected by Annexin-V staining using a BD lsrfortessa instrument.

[0068] Figure 2 d shows the results of measuring cell proliferation rate by WST-1 method, including H1299 cells transfected with pcDNA3, pcDNA3-p53R175, pcDNA3-p53R273 or pcDNA3-wtp53 treated with DMSO or PEITC for 3 days.

[0069] Figure 2 e shows the effect of PEITC on cell apoptosis. H1299 cells transfected with pcDNA3, pcDNA3-p53R175, pcDNA3-p53R273, or pcDNA3-wtp53 were treated with DMSO or 8 μM PEITC for 3 days. Apoptosis was detected by Annexin-V staining using a BD lsrfortessa instrument.

[0070] Figure 3 PEITC induced mutant p53 R175 The protein changes to a "wild-type-like" conformation.

[0071] Figure 3 a shows the ELISA method using PAb240 antibody (mutant) and PAb1620 antibody (wild type) to determine the effect of PEITC on recombinant purified GST-p53 R175H The influence of the conformation.

[0072] Figure 3 b and Figure 3 c shows the results of immunofluorescence assay. SK-BR-3 cells were treated with DMSO or 4 μM PEITC for 6 h. Immunofluorescence of cells was performed using PAb240 and PAb1620 antibodies. A549 cell line was used as a control to show that the conformation of the wild-type p53 gene is not altered by PEITC. H1299 cell line was used as an anti-p53 antibody control. The threshold for all assays was set at 20 μM. *** indicates p ≤ 0.0001 compared with PAB240 and PAB1620.

[0073] Figure 3 d shows the results of immunoprecipitation of mutant p53 protein in SK-BR-3 cell lysate using PAb240 antibody and detection with p53 antibody (fl393).

[0074] Figure 4 PEITC can activate mutant p53 R175 The protein revives its wild-type transcriptional activation function.

[0075] Figure 4 a shows that PEITC induces mutant p53 protein to bind to chromatin. SK-BR-3 cells were treated with PEITC for 4 hours, and the chromatin-bound and nuclear soluble fractions were analyzed by immunoblotting. Histone H3 and topoisomerase IIB were used as markers for chromatin and nuclear soluble fractions, respectively.

[0076] Figure 4 b shows the results of RNA extraction and detection of gene expression levels using TaqMan gene expression assay kits. SK-BR-3, H1299, and A549 cells were treated with DMSO or 4 μM PEITC for 4 h, and p53-regulated genes were amplified by qRT-PCR.

[0077] Figure 4 c shows the results of RNA extraction and detection of gene expression levels using TaqMan gene expression assay kits. Among them, SK-BR-3 cells transfected with p53 siRNA or NS siRNA were treated with DMSO or 4 μM PEITC for 4 hours, and p53-regulated genes were amplified by qRT-PCR.

[0078] Figure 4 d shows the results of luciferase assay, in which SK-BR-3, HOP92, AU565, H1299, and MEF [(10)3 / 175 and (10)3 / 273] cells were transfected with plasmid 16451 and treated with PEITC (4 or 6 μM) for 24 hours, and then luciferase assay was performed.

[0079] Figure 4 e shows the results of Western blotting to determine protein levels. After SK-BR-3 cells were treated with 4 μM PEITC or 20 μM etoposide for 4 hours, the expression level of p21 was analyzed by Western blot. A549 cells were treated with 4 μM PEITC for 4 hours as a control. Protein levels were determined by Western blotting using p53 DO-1 and GAPDH antibodies.

[0080] Figure 5 The results of gene expression level detection are shown. Among them, PEITC induced the expression of p21, a typical p53 target gene, in DU145 cells. DU145 cells were treated with DMSO (control) or PEITC for 4 hours. RNA was extracted and the expression level of the gene was detected using the TaqMan gene expression kit.

[0081] Figure 6 showed that ITCs restored the transcriptional activation function of p53.

[0082] Figure 6 A shows the results of immunoblotting analysis of p53 and p21 expression levels. SCC114 cells were treated with different concentrations of DMSO (control) or ITCs for 24 hours. 40 mg of total cell protein was extracted and measured on polyacrylamide gel electrophoresis and detected with p21 antibody. The blot was stripped and probed again with p53 (DO-1) antibody and anti-GAPDH antibody.

[0083] Figure 6 B shows the results of chromatin immunoprecipitation (CHIP), in which the lysate of SCC-114 cells treated with PEITC for 24 hours was immunoprecipitated with anti-p53 antibody (DO-1), and then PCR was performed for p53 recognition elements p21, PUMA, and MDM2.

[0084] Figure 7 The results showed that p53 protein was degraded by proteasome after PEITC treatment in SK-BR-3 and A549 cells.

[0085] Figure 7 a shows the p53 protein degradation in SK-BR-3 cells treated with different concentrations of phenylethyl isothiocyanate and inhibitors (10 μM Nutlin-3 or 20 μM MG132) for 4 h.

[0086] Figure 7 b shows the p53 protein degradation in SK-BR-3 cells treated with PEITC (4 μM or 8 μM), 20 μM MG132, or both for 4 h.

[0087] Figure 7 c shows the p53 protein degradation in SK-BR-3 cells treated with PEITC (4 μM or 8 μM), 10 μM MG132, or both for 4 h.

[0088] Figure 7 d shows the p53 protein degradation after A549 cells were treated with different concentrations of phenylethyl isothiocyanate and inhibitors (10 μM Nutlin-3 or 20 μM MG132) for 24 hours. Cells were collected and cell lysates were prepared. The lysates were subjected to SDS-PAGE electrophoresis and detected with p53 DO-1 antibody.

[0089] Figure 7e shows the degradation of p53 protein after SK-BR-3 cells were treated with different concentrations of PEITC or DMSO for 4 hours. Cells were collected to prepare soluble and insoluble fractions. 30 μg of soluble or insoluble lysate was subjected to SDS-PAGE electrophoresis and detected with p53 DO-1 antibody.

[0090] Figure 8 It was shown that after PEITC treatment of SK-BR-3 cells, p53 R175 Proteins are autophagic.

[0091] Figure 8 a shows the cell autophagy after SK-BR-3 cells were treated with PEITC (4 μM or 8 μM), CHQ (50 μM), or both for 4 hours. The lysates were subjected to SDS-PAGE electrophoresis and detected with p53 DO-1 antibody.

[0092] Figure 8 b shows the autophagy of SK-BR-3 cells transfected with ATG5 siRNA or NS siRNA. 30 μg of lysate was subjected to SDS-PAGE electrophoresis and then probed with anti-ATG5 antibody. The blot was stripped and re-bound with anti-GAPDH antibody.

[0093] Figure 8 c shows the autophagy of SK-BR-3 cells transfected with ATG5 siRNA or NS siRNA after treatment with DMSO or PEITC for 4 h. Protein levels were measured by Western blotting using p53DO-1 and GAPDH antibodies.

[0094] Figure 8 d shows the results of measuring cell proliferation rate by WST-1 method. SK-BR-3 cells transfected with ATG5 siRNA or NS siRNA were treated with DMSO or PEITC for 3 days.

[0095] Figure 8 e shows the effect of PEITC on cell apoptosis. SK-BR-3 cells transfected with ATG5 siRNA or NS siRNA were treated with DMSO or PEITC for 3 days. Detection of histone-related DNA fragments indicates cell apoptosis.

[0096] Fig. 9 The effects of zinc and redox agents on the induction of p53 by PEITC were shown R175 Effects of protein reactivation.

[0097] Fig. 9a shows the effect of zinc on PEITC activity. SK-BR-3 cells were treated with PEITC, zinc or both. Cell proliferation rate was determined by WST-1 method. PEITC activity was expressed as IC50 value of growth inhibition rate.

[0098] Fig. 9 b shows the effect of zinc or zinc and PEITC co-treatment on the recombinant purified GST-p53 determined by ELISA using PAb240 antibody (mutant) and PAb1620 antibody (wild type). R175H The influence of the conformation.

[0099] Fig. 9 c shows the effect of PEITC on the reduction of glutathione in SK-BR-3 cells. SK-BR-3 cells were treated with PEITC (4 μM or 8 μM) or DMSO for 4 h. The ratio of reducing agent GSH to oxidizing agent GSSG was detected using the GSH / GSSG GLO glutathione kit.

[0100] Fig. 9 d shows the effect of NAC on PEITC activity. SK-BR-3 cells were treated with different concentrations of PEITC or PEITC combined with 3 μM NAC for 3 days. Cell proliferation rate was measured by WST-1 method.

[0101] Fig. 9 e shows the results of measuring cell proliferation rate by WST-1 method, in which SK-BR-3 cells were treated with PEITC alone or in combination with 2 mM ATZ or 500 units of PEG catalase for 3 days.

[0102] Fig. 9 f shows the effect of each component on cell apoptosis. Among them, DMSO, ATZ, NAC or PEITC were treated alone, or PEITC and ATZ or NAC were co-treated in untransfected or siRNA-transfected SK-BR-3 cells for 3 days. Apoptosis was detected by Annexin-V staining using BD lsrfortessa instrument.

[0103] Fig.10 PEITC induced H2AX foci, activated ATM and Chk2, arrested G2 / M and S phases, and induced apoptosis. SK-BR-3 cells and A549 cells were treated with PEITC or DMSO for 3 days and stained with anti-γ–H2AX antibody.

[0104] Fig.10 a shows a merged image of cells stained with anti-γ-H2AX antibody (green) and DAPI (blue), where the threshold for all detections was set at 20 μM.

[0105] Fig.10b shows the percentage of cells with γ-H2AX foci (≤10 or >10, as indicated).

[0106] Fig.10 c shows the results of immunoblot analysis of SK-BR-3 cells and A549 cells treated with PEITC or DMSO for 4 hours. The immunoblot was performed using anti-pATM S1981 and anti-pCHK2 Thr68 antibodies. After stripping the blot, the anti-ATM and anti-CHK2 antibodies were re-bound.

[0107] Fig.10 d and Fig.10 e shows the results of flow cytometric analysis of SK-BR-3 (d) or A549 (e) cells treated with PEITC, 10 μM Nutlin-3 or both for 24 h.

[0108] Fig.10 f shows the results of apoptosis detection by Annexin-V staining using BD Isolate Fortessa instrument, including SK-BR-3 cells and A549 cells treated with 4 μM PEITC, 10 μM Nutlin-3 or both for 24 hours.

[0109] Fig.11 We show that ITCs restore the cell cycle arrest function of p53 mutant protein.

[0110] Fig.11 A and Fig.11 B shows flow cytometric analysis of SCC003 cells treated with BITC (5 μM or 10 μM) or DMSO (control).

[0111] Fig.11 C and Fig.11 D shows flow cytometric analysis of SCC003 cells treated with PEITC (5 μm or 10 μM) or DMSO (control).

[0112] Fig.11 E and Fig.11 F shows flow cytometric analysis of SCC114 cells treated with BITC (5 μM or 10 μM) or DMSO (control).

[0113] Fig.11 G and Fig.11 H shows flow cytometric analysis of SCC114 cells treated with PEITC (5 μm or 10 μM) or DMSO (control).

[0114] Fig.12 ITCs were shown to induce mutant p53-dependent cell cycle arrest.

[0115] Fig.12 A shows the results of immunoblotting analysis of SCC114 cells in the DMSO control group (untransfected), nonspecific siRNA (N) group, and mtp53 siRNA (P) group after 24, 48, and 72 hours of culture. Among them, the expression of p53 protein in the cells of the mtp53 siRNA transfection group was reduced after transfection, while the control group did not.

[0116] Fig.12 B shows the results of the WST-1 assay to measure the proliferation rate of siRNA-transfected cells, in which mutant p53 cells were able to proliferate after treatment with PEITC / BITC.

[0117] Fig.12 C. Fig.12 D. Fig.12 E shows the results of flow cytometry analysis, wherein SCC114 cells transfected with DMSO control group (untransfected), nonspecific siRNA (N) group, and mtp53 siRNA (P) group were treated with BITC (5 μM or 10 μM) or DMSO (control) and then analyzed by flow cytometry.

[0118] Fig.12 F. Fig.12 G. Fig.12 H shows the results of flow cytometry analysis, wherein SCC114 cells transfected with DMSO control group (untransfected), nonspecific (scrambled) siRNA (N) group, and mtp53 siRNA (P) group were treated with PEITC (5 μM or 10 μM) or DMSO (control) and then analyzed by flow cytometry.

[0119] Fig.13 Showing that PEITC induces mutant p53 in vivo R175 Protein reactivation and inhibition of tumor growth in nude mice.

[0120] Fig.13 a shows typical images of mouse mammary fat pad (upper panel) and H&E staining (lower panel). The threshold for all detections was defined as 20 μM.

[0121] Fig.13 b shows the tumor volume measurement results. The tumor was measured with a vernier caliper and the tumor volume was calculated. The tumor volume calculation formula is: L x W 2 x 0.523 (**, p≤0.009, *, p≤0.03) (n=7).

[0122] Fig.13 c shows the weekly changes in animal body weight.

[0123] Fig.13d shows the distribution of animals according to the mean number of tumor cells per tissue section in the control and PEITC groups (***, p≤0.00026) (n=7).

[0124] Fig.13 e shows representative images of Ki67 (**, p≤0.007) and p53 (*, p≤0.033) staining (upper panel) and positive cell counts (lower panel) in transplanted tumor tissues (n=7). ** indicates Ki67 p≤0.007, and * indicates p53 p≤0.033. Results are shown as mean values. The threshold for all detections was defined as 200 μM.

[0125] Fig.13 f shows the results of immunoblotting analysis of the expression level of p53 in nude mouse transplanted tumors in the PEITC group and the control group. The blots are representative images of 12 tumor tissue lysates in each group.

[0126] Fig.13 g shows the qRT-PCR results of p53-regulated genes in animals of the PEITC and control groups (n=4).

[0127] Fig.13 h shows immunoblotting of p21 and Bax in SK-BR-3 tumor xenografts.

[0128] Fig.14 It was shown that PEITC inhibited the growth of DU145 cell xenografts in nude mice.

[0129] Fig.14 A shows a representative image of the lateral aspect of a mouse.

[0130] Fig.14 B shows the tumor volume measurement results. The tumor was measured with a vernier caliper and the tumor volume was calculated. The formula is L×W 2 ×0.523 (**, P≤0.003; ***, P≤0001) (n=7).

[0131] Fig.14 C shows the weekly changes in animal body weight (g).

[0132] In each figure, WT indicates wild type, WB indicates immunoblotting, WCL indicates whole cell lysate, and AIN-93M indicates AIN-93M standard diet. DETAILED DESCRIPTION

[0133] After extensive and in-depth research, the inventors unexpectedly discovered for the first time that PEITC can restore the wild-type activity of mutant p53 (activate the activity of quasi-wild-type p53), inhibit the proliferation of tumor cells caused by p53 mutations and induce their apoptosis. Experiments have shown that PEITC can induce the transformation of mutant p53, especially mutant p53 R175 、p53 P223L 、p53 V274F 、p53 R248 The wild-type p53-like conformation or function is restored, thereby restoring and activating wild-type p53 targets, such as phosphorylating ATM / CHK2, blocking S and G2 / M phases, and inducing cell apoptosis. The present invention is completed on this basis.

[0134] The inventors have found that:

[0135] 1. PEITC can restore the wild-type conformation and transcriptional activation function of mutant p53 with missense mutations at hotspot mutation points R175, C176, Y220, P223, C242, G245, R248, R249, R273, V274, P278, and R282, and induce apoptosis of cells expressing the above hotspot mutant p53.

[0136] 2. PEITC can inhibit mutant p53 R175 Breast cancer cells SK-BR-3, AU565, and mutant p53 expressing R248 Expression of oral cancer cell SCC114 and mutant p53 P223L or p53 V274F Proliferation of prostate cancer cells DU145 expressing phenotype.

[0137] 3. In vivo animal experiments have shown that PEITC can inhibit the growth of xenograft tumors of breast cancer cells SK-BR-3 and prostate cancer cells DU145.

[0138] 4. In the expression of mutant p53 R175 Breast cancer cells SK-BR-3 and AU565 express mutant p53 P223L or p53 V274F Prostate cancer cells DU145 and expressing mutant p53 R248 In oral cancer cells SCC114, PEITC can convert the mutant conformation into a wild-type conformation, revive the transcriptional activation function, activate wild-type p53 targets such as phosphorylation of ATM / CHK2, block the S and G2 / M phases, and induce cell apoptosis.

[0139] 4. PEITC can increase the expression of mutant p53 in breast cancer cells SK-BR-3 and AU565 R175Protein sensitivity to proteasomal and autophagic degradation.

[0140] 5. Zinc ions can enhance PEITC-induced breast cancer cells SK-BR-3 and AU565 mutant p53 R175 The resurrection effect.

[0141] 6. Redox changes affect reactivation of p53 R175 and growth inhibition, and restoring p53 R175 The conformation is not important.

[0142] the term

[0143] As used herein, the term "wild-type p53-like conformation and / or function" means that the mutant p53 substantially possesses the conformation and / or function of wild-type p53, and the conformation and / or function of the altered (activated) mutant p53 is ≥90% similar to that of wild-type p53, preferably ≥95%, more preferably ≥99%, and most preferably ≥99.9%. Preferably, the wild-type p53-like conformation and / or function is the conformation and / or function of human wild-type p53.

[0144] As used herein, the term "altering mutant p53" refers to altering (reactivating) mutant p53 to restore its wild-type activity and / or configuration (activating pseudo-wild-type p53 activity), inhibiting the proliferation of tumor cells caused by p53 mutations and inducing their apoptosis.

[0145] p53 gene

[0146] As used herein, the terms "p53" and "p53 gene" are used interchangeably and refer to the tumor suppressor gene p53, and mutations in the p53 gene are very common in human cancers. Most p53 gene mutations are missense mutations, which can be further subdivided into contact mutations (directly destroying the binding of p53 to DNA) and conformational mutations (destroying the conformation of p53). Both of these mutations cause the inactivation of normal wild-type p53. Studies have shown that certain specific small molecules can achieve tumor suppressor function by changing (activating) mutant p53 and are used for the treatment of cancer.

[0147] The p53 gene is located on human chromosome 17p13.1. The 53kD nuclear phosphorylated protein composed of 393 amino acids encoded by it is called p53 protein. The wild-type p53 protein is extremely unstable, with a half-life of only a few minutes, and has transactivation function and a broad spectrum of tumor suppression. The mutant p53 protein has increased stability and a prolonged half-life, and can be detected by immunohistochemistry.

[0148] Under normal conditions, the activity level of wild-type (wtp53) in cells is strictly controlled, and the protein half-life is very short. HDM2 / MDM2, as the most important p53 negative regulatory molecule, degrades p53 through transcriptional inhibition and the exercise of E3 function. At the same time, hdm2 / mdm2 is a target gene of p53. The negative feedback mechanism formed by p53-HDM2 / MDM2 keeps the activity of wtp53 at a low level in cells. As an important network node molecule of the signaling pathway in the body, p53 is now known to regulate more than 150 genes, forming a sophisticated and complex p53 regulatory network, which plays an important role in maintaining genome stability.

[0149] As one of the most important tumor suppressor genes, p53 is closely related to the occurrence and development of cancer. About 50% of human cancers have p53 mutations. Among all p53 mutations, missense mutations caused by point mutations are dominant, accounting for about 80% of the total. Among these p53 missense mutations, the proportion of point mutations occurring in the DBD region is as high as 97%. In fact, every amino acid in the DBD region of p53 can undergo point mutations to form corresponding mutants, but mutations at the following 12 sites appear frequently in cancer and are closely related to the progression of cancer. They are called hotspot mutations. They are: R175, C176, Y220, P223, C242, G245, R248, R249, R273, V274, P278, and R282.

[0150] PEITC

[0151] As used in this article, PEITC (Phenethyl isothiocyanate) is rich in watercress and other cruciferous vegetables. Animal experiments have shown that PEITC has a chemopreventive effect on cancer, and epidemiological studies also support the effectiveness of such compounds in preventing human cancer. In fact, a Phase I clinical study of PEITC for the prevention of lung cancer in healthy people has been completed, and a Phase II clinical study is underway. PEITC-induced oxidative stress contributes to cell apoptosis, however, the exact mechanism and molecular target of this effect are not yet clear.

[0152] Existing studies have shown that PEITC and cisplatin, when used in combination, induce tumor cell apoptosis through the p53 pathway. However, the above studies did not clarify the molecular mechanism of inducing apoptosis, and it is not clear which hotspot mutations in the p53 DBD region are at work.

[0153] The inventors surprisingly found that PEITC can change (reactivate) mutant p53 in vitro and in vivo, revealing a new mechanism of action of natural food-derived compounds. In cells containing mutant p53, PEITC has an effect on the more common "hotspot" mutations in the p53 mutant sequence, such as: R175 、p53 R248 、p53 P223 and p53 V274 The inhibitory activity is strong, especially against p53, one of the most common "hotspot" mutations. R175 Mechanistic studies have shown that PEITC induces mutant p53 by restoring its wild-type conformation and transcriptional activation function. R175 Specifically, in cells treated with PEITC, altered mutant p53 R175 By activating wild-type p53 targets, namely phosphorylating ATM / CHK2, PEITC delays the S and G2 / M phases and induces apoptosis. Further mechanistic studies have shown that this growth inhibitory effect of PEITC is affected to some extent by zinc ion concentration and cellular redox state. In addition, PEITC inhibits p53 through proteasome and autophagy. R175 The mutant was sensitive to degradation in a concentration-dependent manner. R175 The changes in p53 expression in the mitochondria and mitochondria increase its sensitivity to degradation pathways, which is likely to contribute to its anti-cancer activity. Studies on the inhibition of tumor growth in xenograft mouse models further confirmed that the natural food-derived compound PEITC can also resurrect mutant p53 in vivo. R175 These unprecedented findings demonstrate that mutant p53 can be altered by natural food-derived compounds, which has important implications for cancer prevention and treatment.

[0154] The main advantages of the present invention include:

[0155] (a) PEITC is the only natural food-derived compound that targets mutant P53 for tumor treatment.

[0156] (b) Since PEITC is a food-derived compound, it is very safe. Moreover, PEITC can be used not only as a drug for treating tumors, but also as a health product for preventing tumors;

[0157] (c) Even if taken for a long time, PEITC is not easy to cause drug resistance, and for tumor patients who have already developed resistance to other anti-tumor drugs, the use of PEITC treatment can still achieve good results;

[0158] (d) PEITC can be used alone or in combination with other anti-tumor drugs;

[0159] (e) PEITC can also play a good therapeutic effect on other diseases caused by P53 mutations;

[0160] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0161] General Materials and Methods

[0162] Cell lines

[0163] HOP92, OVCAR3, and SW620 were purchased from NCIDTP, DCDT Tumor Repository, Fredrick, Maryland. H1299, HT29, A549, MDA-MB-231, AU565, SK-BR-3, DU145, SCC003, SCC016, SCC114, SCC122, and MCF7 were from Tissue Culture Source Resource, Georgetown University, Washington, DC. All cell lines were mycoplasma negative and cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS). Normal colon cells CCD841 purchased from ATCC were cultured in Eagle's basal medium containing 10% FBS. 3T3 Balb / c fibroblasts (p53 + / + ), then cultured in Dulbecco's modified Eagle's medium containing 10% FBS. (10)3(p53 - / - ) Mouse embryonic fibroblasts (MEFs) and (10)3-derived p53 mutant MEFs [(10)3 / 175 and (10)3 / 273] were cultured in Dulbecco's modified Eagle's medium containing 10% FBS and 400 μg / mL G418. MEF (10)3 and its derived human p53 residue R175 and R273 mutant cell lines were gifts from Dr. Darren R. Carpizo.

[0164] Cell proliferation assay

[0165] As described above, the effect of PEITC on tumor cell proliferation was determined by the WST-1 assay (Roche). Briefly, an appropriate amount of PEITC was taken and diluted with DMSO so that 10 μl of the drug stock solution contained the desired drug concentration and a DMSO concentration of 1% in a final volume of 1 ml of SK-BR-3 cells (40,000 cells / ml). The SK-BR-3 cell culture containing PEITC was added to a 96-well microplate, with 4,000 cells per well. 1% DMSO was used as a control, and the culture medium without cells was used as a blank control. The culture plates were incubated at 37°C for 3 days, followed by the addition of the WST-1 reagent and incubation for 2 hours. The OD was then measured using a microplate reader (Bio-Rad). 450 OD of cells treated with PEITC 450 OD of cells in the DMSO control group 450 The percentage of cell proliferation was calculated by the ratio of the numerical values. The effect of PEITC on the proliferation of the following cells was determined by a similar experimental method: H1299, HOP92, AU565, OVCAR3, SW620, HT29, A549, MCF7, CCD841, SK-BR-3, DU145, SCC003, SCC016, SCC114, and SCC122 cells transfected with p53 siRNA or NS siRNA.

[0166] Cell transfection

[0167] p53 siRNA was purchased from SMARTpool (Thermo Scientific / Dharmacon, Lafayette, CO, USA). siRNA was transfected using Lipofectamine 2000 according to the manufacturer's instructions (Invitrogen). Briefly, before transfection, cells were cultured in 10 cm dishes for 24 hours, and the cell confluence was 50-60%. siRNA (0.430 nmol) and 43 μL Lipofectamine 2000 were taken and mixed with 1 mL of Opti-MEM (Invitrogen). The mixture was added to the cell culture medium and cultured for 6 hours. After 24 hours, a second similar transfection was performed. 72 hours after the initial transfection, cells were harvested, lysates were prepared or treated with specified concentrations of PEITC or DMSO, and tumor cell proliferation was measured using WST-1 reagent (Roche) as described above. For ATG5 knockdown experiments in SK-BR-3 cells, ATG5 siRNA (Santa Cruz Biotechnology) transfection was performed as described above, but a single transfection was performed for 6 hours. After 24 hours, transfected cells were harvested to prepare lysates or treated with PEITC for 4 hours to prepare lysates.

[0168] pcDNA3, pcDNA3-wtp53, pcDNA3-p53 were transfected with Lipofectamine 2000 according to the manufacturer's instructions (Invitrogen). R175 and pcDNA3-p53 R273 Plasmid. Briefly, cells were cultured in 10 cm dishes for 24 h before transfection, with a confluency of 50-60%. Plasmid (14 μg) and 43 μL Lipofectamine 2000 were mixed with 1 mL of Opti-MEM (Invitrogen). The mixture was added to the cell culture medium and cultured for 6 h. After 24 h, transfected cells were treated with PEITC and subjected to WST-1 assay or Annexin V staining as described above. Transfected cells were stored in RPMI 1640 medium containing 10% FBS and 400 μg / mL G418.

[0169] ELISA

[0170] pGeX4T1-mutp53 was cultured in LB medium containing 100 μg / mL ampicillin at 37°C. R175H E. coli cells (BL21DE3) transformed with plasmids were cultured until the OD value at 600nm was 0.4. At the same temperature, 0.5mM isopropyl-1-thio-β-galactoside (IPTG) was added and the flask was shaken for 3 hours to induce the expression of recombinant protein. After bacterial precipitation, lysis buffer (250mM Tris-HCl, pH7.5, 1mM EDTA, 150mM NaCl, 1% Triton X-100, 0.5% Nonidet P-40, 0.1% Tween 20, 0.2% SDS, 1M DTT and protease inhibitors) was added to lyse the cells, and the cells were frozen and thawed three times, and then ultrasonically disrupted (three cycles, one minute each). After ultrasonic disruption, the solution became clear after centrifugation at 18500×g for 30 minutes at 4°C. The supernatant was transferred to a new tube and stored. Resuspend the precipitate with sodium lauryl sarcosyl buffer (lysis buffer + 2% sodium lauryl sarcosyl), and then use probe ultrasonic disruption (three cycles, one minute each). The supernatant obtained in the above two steps was diluted with 1x PBS at a ratio of 1:1, and incubated with glutathione-agarose beads (σ, G4510) at 4°C with constant rotation for 2 hours. After washing with PBS several times, the protein was eluted with elution buffer (100mM Tris-HCl, pH 8.0, 10mM GSH (σ, G4251), 300mM NaCl, 1mM dithiothreitol (DTT) and protease inhibitors). Take another 25ng of recombinant GST-mutant p53 R175H, diluted with 100mM Tris-Cl, 300mM NaCl buffer at pH 7.5. Treated with DMSO or 4μM PEITC and incubated on ice for 1 hour. The obtained protein samples were added to the ELISA plate and incubated at 4°C for 2 hours. Washed with 1xPBST (containing 0.05% Tween-20) and blocked with 5% skim milk at 4°C for 2 hours. After washing, mouse primary antibody (PAB240 or PAB1620) diluted 1:1000 in 1xPBST was added and incubated at 4°C overnight. Washed with 1xPBST, horseradish peroxidase-labeled anti-mouse secondary antibody was added for reaction at 4°C for 1 hour. Washed with 1xPBST, substrate (SuperSignal ELISA Pico Chemiluminescent Substrate, Thermo Scientific) was added, and the chemiluminescent intensity was measured at 450nM. Purified GST and purified wild-type p53 (Thermo Scientific) were used as controls. To determine the effect of ZnCl2, 25 ng of recombinant GST-mutant p53 was also added. R175H The cells were diluted with 100 mM Tris-Cl, 300 mM NaCl buffer at pH 7.5, treated with 2.5 μM ZnCl2 or 4 μM PEITC alone or in combination, and incubated on ice for 1 hour. ELISA assays were then performed as described above.

[0171] Annexin V staining

[0172] Annexin V staining was performed according to the manufacturer's instructions (Biolegend). Briefly, cells to be tested were treated with PEITC or DMSO as a control, and the cells were collected 3 days later, washed once with 1x PBS, and resuspended in 0.5 mL Annexin V binding buffer. The cells were collected by centrifugation, and 5 μL of fluorescent dye bound to Annexin V was added to the remaining buffer, incubated at room temperature for 15 minutes under light-proof conditions, followed by the addition of 0.5 mL Annexin V binding buffer and 5 μL of 0.1 μg / mL PI staining solution. The cells were then analyzed using a BD LSRFORTESSA flow cytometer (BD Biosciences). In order to clarify the effect of Nutlin-3 treatment on cell-induced apoptosis, cells to be tested were treated with specified concentrations of PEITC, Nutlin-3 alone, or both, or DMSO for 24 or 72 hours. As described above, the treated cells were stained with Annexin V. To determine the effect of reducing agent NAC or PEG-catalase on PEITC-induced apoptosis, cells were treated with PEITC alone, reducing agent or oxidizing agent, or PEITC combined with reducing agent or oxidizing agent. Treated cells were stained with Annexin V as described above. Cells transfected with ATG5 siRNA or NS siRNA were treated with DMSO or the indicated concentrations of PEITC for 72 hours. To detect apoptosis, a cell death detection ELISA combined with a photometric enzyme assay (Roche) was used to quantify cytoplasmic histone-associated DNA fragments in apoptotic cells.

[0173] Immunofluorescence staining

[0174] Cells were treated with PEITC (4 or 6 μM) or 1% DMSO as a control for 6 hours in four-well slides (Lab-Tek). Cells were washed twice with 1x PBS and fixed with formaldehyde (3.7%) for 15 minutes at room temperature (RT). Fixed cells were treated with 0.5% Triton X-100 (Sigma) for 5 minutes at room temperature. Cells were washed four times with 1x PBS containing 0.5% Tween-20 and blocked with 10% goat serum (Sigma) at 4°C overnight. After washing cells four times with 0.1% Tween-20, they were incubated with mouse primary antibodies PAB240 (1:300, Calbiochem) or PAB1620 (1:300, Calbiochem) that can distinguish between mutant and wild-type p53 overnight at 4°C. After washing four times with 0.1% Tween-20, cells were incubated with Alexa Fluor 488-labeled goat anti-mouse IgG (1:400, Invitrogen) at room temperature for 2 h. Cells were washed four times with 0.1% Tween-20 and nuclei were stained with Prolong Gold Anti-Fade reagent (Invitrogen) containing DAPI. Coverslips were placed and cells were treated for 24 h at room temperature in the dark. Immunofluorescence analysis was performed using a Zeiss LSM 510NLO with a Plan-Apochromat 63×1.4-aperture oil objective and an Axiovert 200M inverted laser scanning microscope. Images were acquired with a Photomultiplier Tubes (PMT) detector and analyzed with Image J software. Fluorescence staining intensity was quantified using Metamorph software.

[0175] To determine the effect of PEITC on γ-H2AX foci formation in tumor cells, cells transfected with p53 siRNA or NS siRNA were treated with 4 μM PEITC or 1% DMSO as a control for 3 days at 37°C. As described above, mouse anti-γ-H2AX monoclonal antibody (1:300, Upstate) was used as the primary antibody, cells were fixed with formaldehyde, and γ-H2AX foci were detected by immunostaining.

[0176] Co-immunoprecipitation

[0177] The cells to be tested were treated with the indicated concentrations of PEITC or 1% DMSO as a control for 6 hours. To prepare cell lysates, the harvested cells were washed once with 1x PBS, and the cell pellets were resuspended in lysis buffer (20mM Tris-Cl (pH 8.0), 137mM sodium chloride, 10% glycerol, 1% NP-40, 2mM EDTA) containing protease inhibitors (Roche Molecular Biochemicals) and incubated on ice for 30 minutes. The cell suspension was centrifuged at 18500×g and 4°C for 10 minutes, and the supernatant was collected. The supernatant was diluted with lysis buffer, and an appropriate amount of proteinG Agarose (Roche) was added to 200μg of the lysate and gently shaken at 4°C for 1 hour. The mouse PAB240 antibody (2μg, Calbiochem) was added to the pre-purified lysate and gently shaken for 2 hours at 4°C. Then proteinG Agarose- was added to the suspension and incubated at 4°C for 2 hours. The precipitate was washed four times with lysis buffer supplemented with protease inhibitors, and the immunoprecipitate was eluted by boiling in Laemmli buffer and then subjected to 4-12% SDS-PAGE electrophoresis. The immunoprecipitated p53 protein was detected by immunoblotting using FL393 (Santa Cruz Biotechnology) as the primary antibody. The secondary antibody was peroxidase-labeled anti-mouse IgG (1:2000, GE healthcare). Protein blots were detected using the ECLPrime Western Blot detection kit according to the manufacturer's instructions (Amersham). As a control, the blot was cleared and then re-detected with anti-p53 (DO-1) antibody (1:1000, Santa Cruz Biotechnology) or anti-GAPDH antibody (1:2000, Novus Biologicals). The density of the p53 band in the samples treated with PEITC relative to the DMSO control sample was determined using Gene Tools software.

[0178] Preparation of lysate and Western blot analysis

[0179] Different lysis buffers were used to prepare soluble, insoluble and whole cell lysate fractions. Cells were washed twice with 1x PBS and collected for preparation of lysate (soluble). RIPA buffer (10 mM sodium phosphate (pH 7.2), 300 mM NaCl, 0.1% SDS, 1% Nonidet P-40, 1% deoxycholate, 2 mM EDTA) was added to the cells and placed in an ice bath for 30 minutes. The cell suspension was then centrifuged at 18500×g at 4°C for 10 minutes and the supernatant was collected unless otherwise mentioned. The precipitate was the insoluble fraction. The insoluble fraction was dissolved in a lysis buffer containing 2% SDS (65 mM Tris-HCl (pH 8.0), 150 mM NaCl, 2% SDS, 50 mM DTT). As described above, the harvested cell pellet was dissolved in 2% SDS lysis buffer to prepare the whole cell lysate fraction. The fractions were collected by centrifugation at 18500×g and 4°C for 10 minutes. Then 30-50 μg of the lysate was subjected to 4–12% SDS / PAGE electrophoresis. The proteins were transferred to PVDF membranes and detected by ECL Prime Western blot according to the manufacturer's instructions (Amersham). Antibodies to p21, Bax, ATM, pATMS1981, CHK2, pCHK2 Thr68, and p53 (DO-1) were purchased from Santa Cruz Biotechnology, and GAPDH antibodies were purchased from Novus Biologicals. Antibodies to ATG5 (1:1000, cell signaling) were donated by Dr. Shivendra Singh.

[0180] Chromatin isolation

[0181] The cells to be tested were treated with the specified concentration of PEITC or DMSO as a control for 4 hours. The cells were digested with trypsin and collected by centrifugation at 500×g for 5 minutes. The cell pellet was washed once with ice-cold PBS and transferred to a 1.5 mL microcentrifuge tube and centrifuged at 500×g for 2 minutes. The cell pellet was stored at -80°C and then separated into the nuclear soluble fraction and the chromatin-bound protein fraction of the chromatin according to the manufacturer's instructions (Subcellular protein fractionation kit, Thermo Scientific). 10 μg of soluble nuclear extracts and chromatin-bound nuclear extract proteins from cell samples treated with PEITC or DMSO were subjected to 4-12% SDS-PAGE electrophoresis and the proteins were transferred to PVDF membranes. p53 content was determined by Western blotting using p53 (DO-1) antibody (1:1000, Santa Cruz Biotechnology). Histone H3 and TopoII B were used as internal standards for chromatin and soluble nuclear fractions. Histone H3 polyclonal antibody was purchased from (ThermoScientific) and mouse anti-topoii B monoclonal antibody was purchased from (Santa Cruz Biotechnology).

[0182] RNA extraction and quantitative RT-PCR

[0183] The cells to be tested were extracted with Qiagen RNA extraction kit, and cDNA was synthesized with a high-capacity RNA-cDNA conversion kit (Applied Biosystems, Invitrogen). The gene expression level was quantitatively determined by TaqMan RT-PCR (qRT-PCR) (Applied Biosystems, Invitrogen). The results were normalized with GAPDH, and the mean value and the standard deviation of three repeated measurements were expressed. RNA from xenograft tumor tissues was also extracted with a QIAGEN kit, and then qRT-PCR was performed, and the gene expression level was determined by GAPDH normalization. The change in the expression level of each tumor gene between the PEITC-treated group and the control group was calculated and expressed as the mean value and standard deviation.

[0184] Glutathione level measurement

[0185] The levels of reduced glutathione (GSH) and oxidized glutathione (GSSG) were determined using the GSH / GSSG-GLO Glutathione Assay Kit (Promega). Briefly, cells were treated with PEITC or DMSO as a control for 4 h, and glutathione was measured according to the manufacturer's instructions (Promega).

[0186] Detection of luciferase activity of transduced genes

[0187] The WWP-Luc (p21 / WAF1 promoter) plasmid donated by Bert Vogelstein (Addgene plasmid #16451) encoding the p21 promoter sequence that binds to the specific site of the p53 WT protein was transfected into the cells to be tested and treated with 4 or 6 μM PEITC for 24 hours. According to the manufacturer's instructions (Luciferase assay, Promega), the luciferase activity of the transduced gene in the cell lysate was detected.

[0188] Cell cycle analysis

[0189] The cells to be tested were treated with PEITC, Nutlin-3, or both for 24 or 72 hours, and DMSO was used as a control. The cells were collected and analyzed by flow cytometry for cell cycle detection. Briefly, the cells were washed with PBS without calcium and magnesium ions, trypsinized for 5 minutes, and the cells were collected and centrifuged at 190×g and 4°C for 3 minutes. The collected cells were washed once with PBS, resuspended in 1 ml of 70% ethanol, and stored at –20°C overnight. Then, after centrifugation at 420×g for 10 minutes, the precipitated cell pellets were collected, washed once with 1 ml of ice-cold PBS, and resuspended in 1 ml of freshly prepared PI staining solution (PBS containing 0.1% Triton X-100, 0.05μg / mL propidium iodide, and 0.1mg / mL RNase (Sigma)). The cell suspension was first placed in the dark at room temperature for 30 minutes, and then at 4°C for 30 minutes. Samples were detected by Becton Dickinson FACS and data analysis was performed using the Mod Fit program (Verity Software House).

[0190] ATM and CHK2 detection after PEITC treatment

[0191] The cells to be tested were treated with DMSO, ATZ, NAC, PEG-catalase, or PEITC alone or with ATZ, NAC, or PEG-catalase and PEITC for 4 hours. The cells were then collected after centrifugation at 1600×g, 4°C for 10 minutes, washed once with PBS, resuspended in RIPA buffer containing a mixture of protease and phosphatase inhibitors (10 mM sodium phosphate (pH 7.2) 300 mM NaCl, 0.1% SDS, 1% Nonidet P-40, 1% deoxycholate, and 2 mM EDTA), placed on ice for 30 minutes, centrifuged at 18500×g, 4°C for 10 minutes, and the supernatant was collected. 200 μg of the lysate was subjected to 4-12% SDS / PAGE electrophoresis. After electrophoresis, the proteins were transferred to a PVDF membrane and detected by Western blotting with anti-pATM Ser1981 antibody (1:500) (Santa Cruz Biotechnology) or anti-pCHK2 Thr68 antibody (1:500) (Santa Cruz Biotechnology). The secondary antibody was peroxidase-labeled anti-mouse IgG (1:1000, GE Healthcare). The blots were developed using the ECL Prime Western Blot Detection Kit according to the manufacturer's instructions (Amersham). As a control, the blots were stripped and re-detected with anti-ATM antibody (1:500, Santa Cruz Biotechnology) or Chk2 antibody (Santa Cruz Biotechnology).

[0192] Mouse xenograft model

[0193] Twenty female athymic nu / nu BALB / C mice (CAnN.Cg-Foxn1nu / Crl, 4-6 weeks old) were purchased from Charles River Laboratories (Wilmington, MA). All in vivo studies and tumor collection were in accordance with the procedures and guidelines of the Institutional Animal Care and Use Committee (IACUC). Mice were weighed and placed in polycarbonate cages (five / cage, with the same mean weight and variance per cage) for one week. Mice had ad libitum access to water and AIN-93M diet. After one week of isolation, mice were randomly assigned to either an AIN-93M diet control group or an AIN-93M diet group containing PEITC (5 μmol PEITC / g diet, 10 mice / group). Both diets were provided by Research Diets (New Brunswick, NJ). The number of animals used was determined based on previous experimental results, which have demonstrated that the results obtained with the number of animals used in the SK-BR-3 cell xenograft mouse model are statistically significant. The concentration of PEITC in the feed was also based on the results of the previous mouse bioassay. As mentioned above, PEITC in the feed was extracted with ethyl acetate and then 1,2-benzenedithiols were added to react with ITC for cyclization. The measured concentration was 4.97±0.16μmol / g. The difference in PEITC between samples was calculated using a one-way ANOVA, which showed no statistical significance (α=0.05).

[0194] In the human breast cancer xenograft mouse model, the control group and the PEITC group mice were fed with 2x10 6 Tumor cells (suspended in 50 μL Matrigel) were injected into the left and right mammary fat pads of mice (“cancer chemoprevention” setting). No mice died during the experiment. Tumor size was measured externally with a vernier caliper every week to assess tumor formation and growth for 10 bioassay cycles. According to the formula L x W 2 The tumor volume was calculated by x 0.523.

[0195] In the in vitro transplanted tumor mouse model of prostate cancer, due to the particularity of the tumor location and the small size of the mouse tumor, it is not easy to measure from the outside. Also due to the small size, the volume measurements of some mice's tumors are still uneven after several weeks. For this reason, the final tumor volume calculation only adopts the animal tumor data (n=7) that conforms to the growth pattern and has no abnormal fluctuations. No abnormal data was analyzed using GraphPad software. The rest of the operations are consistent with the human breast cancer xenograft tumor model.

[0196] After all animals were sacrificed, tumors were collected and stained with hematoxylin and eosin (H&E) as described below. During the experiment, the animals were weighed once a week. S Cyclization reaction, ITC concentration in the blood collected at autopsy was measured. The concentration of PEITC in the blood of animals in the treatment group and control group was 1.13±0.15μM (n=3) and 0.37±0.03μM (n=2), respectively. Since the animals in each group were fed different diets, the experiment could not be conducted in a blinded manner; however, tumor sample collection and histopathological analysis were completed in a blinded manner. Pathologists detected the tumor incidence in H&E-stained tissue sections in a blinded manner.

[0197] Histopathological analysis, immunohistochemistry, Western blot, and qRT-PCR analysis of transplanted tumors

[0198] Pathological examination of H&E-stained tissue sections was performed to determine whether they were tumorous. Immunohistochemistry was performed according to the standards of Histopathology and Tissue Shared Resources of Georgetown University. Briefly, the tissues were cut into 5 μm slices, dewaxed with xylene and dehydrated with graded alcohols, and then the tissue sections were immersed in 10 nM citrate buffer (pH 6) containing 0.05% Tween at 98°C for 20 minutes for heat-induced antigen retrieval (HIER). Immunohistochemical staining was performed using Dako's horseradish peroxidase-labeled polymer (k4001, k4003) according to the manufacturer's instructions. Briefly, the sections were treated with 3% hydrogen peroxide and 10% normal goat serum for 10 minutes each, and then the primary antibody p53 (DO-7) (1:800, Dako) was added and placed at room temperature (RT) for 1 hour, or the Ki-67 (1:15, Novus Biologicals) antibody was added and placed at 4°C overnight. The sections were then exposed to the appropriate HRP-labeled polymer for 30 minutes and DAB colorimetric solution (Dako) for 5 minutes. The sections were then counterstained with hematoxylin (Fisher, Harris Modified Hematoxylin), blued in 1% ammonium hydroxide, dehydrated, and mounted with Acrymount. Sections that were not incubated with the primary antibody were used as negative controls. The sections were examined under an Olympus BX61 microscope at 200 times magnification. Representative images of the entire tumor tissue section were photographed using a DP70 camera and processed using DP70 software. Images were analyzed using Image J software. In addition, due to the small size of the tumor, four sections were used to determine the number of cells for each tumor analysis. Twenty pictures were taken from different areas of each antibody-stained section to calculate the total number of cells. The data for each tumor are expressed as the average of the total number of cells stained with different antibodies.

[0199] Because the amount of tumor tissue obtained, especially from mice supplemented with PEITC, was limited, tumors were randomly grouped for western blotting and qRT-PCR analysis. For Western blot, lysates of SK-BR-3 xenografts (n = 12) in each group were homogenized at a ratio of 20 w / v of tumor tissue and lysis buffer (Pierce). As described above, 25 μg of lysate was subjected to 4–12% SDS / PAGE electrophoresis, and the proteins were transferred to PVDF membranes and subjected to Western blotting with p53 (DO-1) antibody.

[0200] Statistical analysis

[0201] Two-tailed t-tests were used to test statistical differences in tumor volume and biological endpoints. P values ​​≤ 0.05 were considered statistically significant. All statistical analyses were two-sided.

[0202] Example 1

[0203] Inhibitory effect of PEITC on proliferation of mutant p53 cells

[0204] 1. PEITC inhibits the proliferation of prostate cancer cells DU145 by acting on mutant p53

[0205] The effect of PEITC on the proliferation of prostate cancer cell line DU145 was studied by WST-1 assay. To this end, DU145 cells were treated with different concentrations of PEITC for 72 hours. Afterwards, the OD450 values ​​of each group of cells were detected and the corresponding IC50 was calculated. The results showed that PEITC reduced the proliferation of DU145 cell line (IC 50 s=~8μM)( Figure 1 A).

[0206] 2. PEITC acts on breast cancer cells SK-BR-3 and AU565 mutant p53 R175 Inhibition of proliferation WST-1 test studies have shown that PEITC can reduce the proliferation of various cells expressing mutant p53. R175 The inhibitory effect was strongest in mutant human breast cancer cells SK-BR-3, AU565 and human non-small cell lung cancer HOP92. These tumor cells have an IC of PEITC 50 Compared with other hotspot mutation cells, it decreased by about 2.5-5 times ( Figure 2 a). However, no significant proliferation inhibition was observed when cells expressing wild-type p53 were treated with PEITC.

[0207] 3. PEITC acts on mutant p53 in oral cancer cells SCC114R248 Inhibit its proliferation

[0208] Similarly, the results of the WST-1 assay performed in the oral cancer cell lines SCC003, SCC016, SCC114, and SCC122 showed that the greatest inhibition rate was observed after PEITC treatment in SCC114 cells expressing R248 mutant p53, but this phenomenon was not found in SCC003 cells expressing wild-type p53.

[0209] The above results indicate that PEITC inhibits the proliferation of tumor cell lines by targeting mutant p53.

[0210] Example 2

[0211] PEITC inhibits tumor cell proliferation and induces apoptosis in a mutant p53-dependent manner

[0212] 1. PEITC with p53 R175 or p53 R248 Inhibits proliferation and induces apoptosis of breast cancer cells SK-BR-3 in a cytokine-dependent manner

[0213] In order to determine whether PEITC inhibits tumor cell proliferation by regulating p53 R175 The transformation was mediated by the change of wild-type p53 activity. The WST-1 assay after transfection with p53 siRNA showed that p53 R175 SK-BR-3 cells expressing reduced Figure 2 b) The sensitivity of PEITC to inhibit proliferation was significantly reduced, while cells transfected with NS siRNA remained highly sensitive. However, no significant difference in proliferation was observed in A549 cells in the same experiment ( Figure 2 b). The above results indicate that PEITC-induced proliferation inhibition is dependent on mutant p53 protein.

[0214] To determine whether PEITC induces apoptosis of tumor cells by changing the mutant p53 protein, flow cytometry was used to further detect tumor cell lines expressing different mutant p53 proteins. The experimental results showed that PEITC induced apoptosis of tumor cells in comparison with MDA-MB-231 (p53 R280 ) or A549 cells ( Figure 2 c) Compared with the control, the percentage of apoptotic cells in SK-BR-3 cells treated with 4 μM PEITC increased by about 3 times ( Figure 2 c) It is noteworthy that p53 was treated with PEITC or DMSO R175 There was no significant difference in apoptosis rate between cells expressing reduced SK-BR-3 ( Figure 2c), which demonstrates that PEITC is dependent on mutant p53 to induce apoptosis.

[0215] 2.PEITC with p53 P223 or p53 V274 Inhibits proliferation and induces apoptosis of prostate cancer cell line DU145 in a cytokine-dependent manner

[0216] In the prostate cancer cell line DU145, the results of cell death enzyme-linked immunosorbent assay combined with photometric enzyme assay showed that the apoptosis rate of DU145 cells treated with 8 μM PEITC increased by about 2 times compared with the control cells treated with DMSO ( Figure 1 B)

[0217] The results in this example indicate that PEITC inhibits the proliferation of tumor cells and induces apoptosis of tumor cells by mediating the changes of mutant p53.

[0218] Example 3

[0219] PEITC restores the “wild-type-like” conformation and transcriptional activation function of mutant p53

[0220] 1. PEITC acts on mutant p53 in breast cancer cells SK-BR-3 R175H , restoring its “wild-type-like” conformation and transcriptional activation function

[0221] Since PEITC is dependent on mutant p53 to induce apoptosis, it is reasonable to infer that it may do so by restoring the wild-type function of p53. To this end, an enzyme-linked immunosorbent assay (ELISA) with anti-p53 antibodies with specific conformations was used to measure the effect of PEITC on p53. R175 The results showed that glutathione-S-transferase (GST)-p53 was incubated with PEITC. R175H In the recombinant, the PAB1620 (specific to the wild type) fraction increased by about 2.8 times, while the PAB240 (specific to the mutant) fraction decreased by about 2.6 times ( Figure 3 a). Immunofluorescence assay of SK-BR-3 cells treated with PEITC showed that the fluorescence intensity of PAB1620 antibody increased by about 2 times, while the fluorescence intensity of PAB240 antibody decreased ( Figure 3 b and Figure 3 c). Importantly, SK-BR-3 cells treated with PEITC ( Figure 3 d) p53 R175 Immunoprecipitation showed that the immunoreactivity of PAB240 was reduced by more than 95%. These results demonstrate that PEITC induces p53 R175 of a “wild-type-like” conformation.

[0222] Can p53 be restored? R175 The ability to bind to DNA is crucial for the suppression of p53 mutant tumors. Therefore, we further investigated whether PEITC could enrich p53 in nuclear chromatin. R175 . Figure 4 a The nuclear chromatin-bound fraction of SK-BR-3 cells treated with PEITC showed p53 R175 Consistent with this, 4 μM PEITC increased the expression of p53 target genes in SK-BR-3 cells, especially p21, MDM2, PUMA, NOXA, BCL2, BAX ( Figure 4 b) A549, H1299 or p53 were treated with PEITC R175 In SK-BR-3 cells, where expression was reduced, no significant changes were observed. This suggests that the target of p53 induced by PEITC is p53 R175 Dependent ( Figure 4 b and Figure 4 c).

[0223] 2. PEITC acts on mutant p53 in prostate cancer cells DU145 P223L or p53 V274F , restoring its “wild-type-like” conformation and transcriptional activation function

[0224] In the prostate cancer study, since PEITC was able to induce apoptosis in DU145 cells expressing mutant p53 protein, we inferred that it was also through the pathway of restoring the wild-type function of p53. Therefore, we studied its effect on the expression of the typical p53 target gene p21. The results showed that DU145 cells treated with PEITC (8μM) indeed enhanced the expression of p21 ( Figure 5 ). This result indicates that PEITC can restore the "wild-type-like" conformation and transcriptional activation function of p53 mutants.

[0225] 3. PEITC acts on mutant p53 in oral cancer cells SCC114 R248 , restoring its “wild-type-like” conformation and transcriptional activation function

[0226] Similarly, in oral cancer SCC114 cells, after treatment with PEITC, as the drug concentration increased, the expression level of mutant P53 protein gradually decreased, while the expression level of P21 protein gradually increased ( Figure 6 A). Chromatin immunoprecipitation experiments further confirmed that SCC114 cells treated with PEITC could restore the ability of mutant P53 protein to bind to specific sites in the promoters of genes p21, PUMA and MDM2. ( Figure 6 B).

[0227] In order to provide more evidence that PEITC can restore the transcriptional activation function of p53, the present invention conducted a luciferase reporter gene detection test. The results showed that the luciferase activity of cells treated with 4 μM PEITC increased by about 2-2.5 times ( Figure 4 d). This proves that PEITC (4 μM) can induce the expression of p21 gene in SK-BR-3 cells, while the DNA damaging agent, etoposide, cannot do so ( Figure 4 e) This suggests that this induction is mutant p53 dependent.

[0228] All the results in this example show that PEITC can restore the "wild-type-like" conformation of mutant p53 protein and obtain transcriptional activation function.

[0229] Example 4

[0230] PEITC enhances the expression of p53 in breast cancer SK-BR-3 cells R175 Protein degradation via proteasome and autophagy

[0231] Examples 1-3 show that PEITC (≥10 μM) selectively degrades mutant p53 protein rather than wild-type p53 protein. R175 To a "wild-type" state, wild-type p53 is regulated by MDM2, so the restored p53 R175 The decreased stability of p53 may be due to the accumulation of ubiquitinated proteins in the insoluble fraction due to MDM2-dependent proteasome degradation. To verify this, SK-BR-3 cells were treated with PEITC, the proteasome inhibitor MG132, or the specific MDM2 inhibitor Nutlin-3 alone or in combination. The results showed that neither MG132 nor Nutlin-3 could prevent the p53 R175 The reduction ( Figure 7 a). Compared with cells treated with PEITC or MG132 alone, p53 significantly accumulated in both the soluble and insoluble fractions of whole cell lysates (WCL) of SK-BR-3 cells treated with 4 or 8 μM PEITC and 20 μM MG132 ( Figure 7 b). Similarly, p53 was significantly increased in SK-BR-3 whole cell lysates (WCL) treated with 4 or 8 μM PEITC and 20 μM Nutlin ( Figure 7 c), while no difference in p53 accumulation was observed in A549 ( Figure 7 d). p53 is present in whole cell lysates from SK-BR-3 cells treated with 8 μM PEITC alone ( Figure 7 b) To observe p53 R175At higher concentrations (≥8 μM), p53 R175 The accumulation of Figure 7 e). The above results indicate that altered p53 R175 The decreased stability is due to degradation by the proteasome.

[0232] Since proteins may also be cleared through the autophagy process, the present invention further studied the effect of PEITC on autophagy in SK-BR-3 cells. The results showed that compared with cells treated with PEITC alone, the combined treatment of SK-BR-3 cells with 8μM PEITC and 50μM autophagy inhibitor chloroquine (CHQ) significantly increased the content of p53 in WCL. However, no significant difference was observed between cells treated with 4μM PEITC and 50μM CHQ and cells treated with PEITC alone, indicating that higher concentrations of PEITC can induce the occurrence of autophagy ( Figure 8 a). Since the formation of autophagy requires the participation of autophagy protein 5 (ATG5), we transfected SK-BR-3 cells with ATG5 siRNA or NS siRNA to detect the sensitivity of cells to PEITC. The results showed that compared with cells transfected with NS siRNA, SK-BR-3 cells with reduced ATG5 expression showed higher levels of p53 when treated with 8μM or 16μM concentration of PEITC. R175 ( Figure 8 b), while there was no significant change in the PEITC treatment at concentrations ranging from 2 μM to 4 μM ( Figure 8 c). This indicates that SK-BR-3 cells with reduced ATG5 expression have the ability to resist the anti-malignant tumor cell proliferation effect of PEITC, while NS SiRNA transfected cells still maintain high sensitivity ( Figure 8 d and Figure 8 e), suggesting that autophagy negatively controls cell growth in these cells.

[0233] The experimental results in this example demonstrate that PEITC degrades mutant p53 in cells through two different pathways. R175 Protein, respectively: MDM2-dependent proteasome degradation pathway and degradation of p53 through autophagy R175 protein.

[0234] Example 5

[0235] Zinc ions enhance the induction of p53 in breast cancer SK-BR-3 cells by PEITC R175 Resurrection ability

[0236] The proper folding conformation of the wild-type p53 protein requires zinc ions. R175The mutant p53 was expressed by co-treatment with zinc chloride (ZnCl2) and PEITC in the optimal concentration range (10-20 μM). R175 SK-BR-3 cells, and found that zinc chloride enhanced the inhibitory effect of PEITC on tumor cell proliferation by about 3.3 times ( Fig. 9 a), but no such effect was found with ZnCl2 alone. Similarly, GST-p53 was incubated with PEITC (4 μM) and ZnCl2 (2.5 μM). R175H This resulted in a significant increase in PAB1620, while there was no significant change in PAB240 ( Fig. 9 b) These results demonstrate that zinc ions help PEITC to reconstitute p53 R175 of a “wild-type-like” conformation.

[0237] Example 6

[0238] Redox changes alter p53 R175 and inhibiting tumor cell proliferation, and restoring p53 R175 Conformation is not important

[0239] Studies have shown that PEITC induces the production of reactive oxygen species by inactivating the glutathione antioxidant system in cancer cells. Redox changes affect the conformation of wild-type p53 protein. The results of glutathione expression level detection showed that compared with the DMSO control group, the glutathione level in SK-BR-3 cells treated with PEITC (4 or 8 μM) decreased ( Fig. 9 c). The combined treatment of SK-BR-3 cells with PEITC and a reducing agent (3 mM N-acetylcysteine ​​(NAC) or 500 units of PEG-catalase) weakened the inhibitory effect of PEITC on tumor cell proliferation and induction of apoptosis, while the catalase-specific inhibitor 3-amino-1,2,4-triazole (ATZ) enhanced the inhibitory effect of PEITC on tumor cell proliferation and apoptosis ( Fig. 9 d, Fig. 9 e and Fig. 9 f) p53 was treated with PEITC alone or in combination with ATZ or NAC R175 There was no significant difference in apoptosis in SK-BR-3 cells with reduced expression of Fig. 9 f) These results suggest that redox changes have an important role in altering p53 R175 and inhibiting tumor cell proliferation, and restoring p53 R175 The conformation is not important.

[0240] The results in this example demonstrate that zinc ions and redox changes affect PEITC-induced p53 R175The resurrection has important consequences.

[0241] Example 7

[0242] Effects of PEITC on cell cycle and ATM / CHK2 activation

[0243] 1. PEITC revives mutant p53 in breast cancer cells SK-BR-3 R175 transactivation function, thereby activating the DNA damage response of cell apoptosis

[0244] The effect of PEITC on DNA damage repair was evaluated by subjecting SK-BR-3 cells to oxidative stress. The results showed that the γ-H2AX foci in SK-BR-3 cells treated with 4 μM PEITC increased by about 1.8 times compared with the DMSO control group, which represents the accumulation of DNA double-strand breaks (DSB's), while no difference was detected in A549 cells by the same experiment ( Fig.10 a and Fig.10 b) This suggests that the accumulation of DSB's is related to p53 R175 Related.

[0245] To further investigate the mutant p53 R175 After being changed to "wild-type-like" p53, does it lose its ability to inhibit the ATM / checkpoint kinase 2 (CHK2) pathway? Fig.10 The results show that pATM-S1981 and pCHK2 / Thr68 in SK-BR-3 cells treated with 4 μM PEITC and DMSO control cells are lacking in p53 R175H ATM / CHK2 inhibition leads to reactivation of the DNA damage response. The results of PEITC treatment of A549 cells without pATM-S1981 and pCHK2-Thr68 are consistent with the γ–H2AX focus data ( Fig.10 a and Fig.10 b). This suggests that restoration of DNA damage repair by PEITC depends on the redox state of the cell.

[0246] Altered mutant p53 R175 The experiment on the effect on cell cycle progression found that treatment of SK-BR-3 cells with 4 μM PEITC for 24 hours significantly delayed the G2 / M and S phases ( Fig.10 d); This indicates that PEITC inhibits cell proliferation, delaying not only the G2 / M phase but also the S phase. A549 cells treated with 4 μM PEITC for 24 hours had a delayed G1 phase. This indicates that the delay in cell cycle progression is related to p53 R175In addition, compared with the single application of PEITC or Nutlin-3, the SK-BR-3 cells co-treated with 10μM Nutlin-3 and 4μM PEITC for 24 and 72 hours showed that the number of S phase cells increased significantly ( Fig.10 d) The number of apoptotic cells increased significantly ( Fig.10 f), indicating the synergistic effect of Nutlin-3. Nutlin-3 alone or in combination with PEITC treatment of A549 cells for 24 hours significantly delayed the G1 phase; 72 hours, delayed the G1 and G2 / M phases ( Fig.10 e). Demonstrate that the observed effects of Nutlin-3 are specific to wild-type p53.

[0247] In conclusion, the above results indicate that PEITC plays an important role in restoring mutant p53 R175 The transactivation function of mitochondria revives the DNA damage response of cell apoptosis.

[0248] 2. PEITC acts on mutant p53 in oral cancer cells SCC114 R248 Induces G1 cell cycle arrest

[0249] In oral cancer cells, SCC114 cells expressing mutant P53 were able to restore the process of cells entering the S phase from the G1 phase after being treated with different concentrations of PEITC (P<0.05) ( Fig.11 E, 11F, 11G, 11H). However, for SCC003 cells expressing wild-type P53, there was no statistically significant difference between the experimental group and the control group (P>0.05) ( Fig.11 A, 11B, 11C, 11D). The experimental results showed that PEITC-induced G1 cell cycle arrest depends on p53 R248 In addition, the inventors further analyzed the cell cycle by transfecting SCC114 cells with siRNA to determine whether mutant p53 plays a role in restoring G1 phase cycle arrest. Flow cytometry and immunoblot analysis showed that when the mutant P53 gene was silenced by siRNA, cell cycle arrest no longer occurred ( Fig.12 ).

[0250] The experimental results of this example prove that PEITC induces G1 phase arrest in tumor cells by acting on mutant p53 protein; at the same time, PEITC restores DNA damage repair in cells by acting on ATM / CHK2 kinase.

[0251] Example 8

[0252] PEITC inhibits xenograft tumor growth by altering mutant p53 proteins at different hotspots

[0253] 1. PEITC inhibits the growth of human breast cancer SK-BR-3 xenograft tumors

[0254] The results of the human breast cancer animal model test showed that compared with the control diet group, the tumor growth of mice in the PEITC diet group (5μmol / gAIN-93M) was significantly inhibited (p<0.05) ( Fig.13 a and Fig.13 b). The reduction in tumor volume in the control group after six weeks can be explained by the non-invasive phenotype of SK-BR-3 cells. There was no difference in body weight between the two groups ( Fig.13 c) These results indicate that PEITC exhibits antitumor activity in the SK-BR-3 xenograft tumor model.

[0255] Further histological examination showed that the number of tumor cells in the PEITC group was significantly reduced ( Fig.13 a and Fig.13 d). At the same time, the number of cells stained for Ki67 and p53 mutations in the tumor was also significantly reduced ( Fig.13 e) To evaluate whether PEITC can reactivate mutant p53 in vivo R175H , the expression level of p53 protein in tumor tissues was detected. The results showed that although there may be inherent characteristics of the experimental mouse model, p53 in the PEITC group R175H There were differences in the levels of mutant p53 in the PEITC group, but compared with the control group, the mutant p53 protein level in the PEITC group was significantly reduced ( Fig.13 f). In addition, the mRNA and protein expressions of p53-regulated genes p21 and Bax in the PEITC group were increased ( Fig.13 g and Fig.13 h). These results provide evidence that PEITC inhibits the growth of SK-BR-3 xenograft tumors and reactivates mutant p53 in vivo.

[0256] 2. PEITC inhibits the growth of human prostate cancer DU145 xenograft tumors

[0257] The results of the prostate cancer animal model experiment showed that compared with the control group, statistically significant tumor growth inhibition was observed in the group of animals fed PEITC (p<0.05) ( Fig.14 A and 14B). During the bioassay period, no significant differences in body weight were observed between the two groups of animals ( Fig.14 C) These results demonstrate that PEITC has antitumor activity in the DU145 xenograft tumor model.

[0258] The results of this example show that PEITC can change p53 R175H , inhibiting the growth of xenograft tumors in vivo.

[0259] discuss

[0260] Changing mutant p53 through transactivation pathways provides a promising direction for targeted tumor therapy. The changes in mutant p53 have been verified in mouse models of different tumors. According to the literature, there have been reports of artificially designed small molecules restoring mutant p53 reverse transcription. However, there have been no reports of studies on natural food-derived compounds changing mutant p53. The research of the present invention shows that PEITC selectively degrades mutant p53 protein without affecting the wild type. Specifically, PEITC changes mutant p53 R175 , inhibiting cell proliferation and inducing apoptosis, leading to the selective elimination of such cells. This is a new mechanism for the induction of apoptosis by a natural food-derived compound. It is very interesting to study the interaction between PEITC and a series of different genes in cells that ultimately lead to cell death. Although p53 regulates the anti-apoptotic regulatory factor BCL2 family by transactivating multiple related and unrelated factors, PEITC, in addition to affecting BCL2, also restores p53 by inducing the expression of multiple apoptosis-initiating targets, including BH3-only members. R175 Importantly, BCL2 could not inhibit PEITC-induced apoptosis.

[0261] The research results of the present invention show that the altered p53 R175 It can bind to zinc through proper folding, and ZnCl2 significantly increased PAB1620, which is unique to wild-type p53, suggesting that ZnCl2 enhances the anti-cell proliferation activity of PEITC.

[0262] PEITC may exacerbate PEITC-induced oxidative stress by increasing reactive oxygen species in mutant p53 cells. R175H The conformation of p53 was not affected, but increasing oxidative stress helped to restore p53 R175 ATZ enhanced the anti-proliferation ability of PEITC, while PEG-catalase or NAC played an inhibitory role, providing evidence for the above conclusions from both positive and negative aspects.

[0263] The in vivo study of the present invention confirmed that PEITC from natural food sources can induce apoptosis of tumor cells by changing the mutant p53 protein of experimental mice. The analysis results showed that the ITC concentration in the blood samples of mice fed with PEITC was 1.13±0.15μM, which is consistent with the results of human pharmacokinetic studies - the peak concentration of PEITC in the plasma of volunteers who consumed about 50 grams of raw watercress (approximately equivalent to 40 mg of PEITC) was about 1μM.

[0264] The present invention's research, in a "chemoprevention setting", confirmed the inhibitory effect of PEITC on tumors. Specifically, before the injection of mutant p53 cells and tumor formation, the experimental animals were fed a feed containing PEITC. At the same time, the injected mutant p53 cells were divided into two types: "start-up stage" or "already cancerous". The results showed that in mice fed a feed containing PEITC, mutant p53 in the body was degraded, proliferation was inhibited, and tumor volume was significantly reduced. In addition, the increase in p53 target gene mRNA provides p53 in mice fed a feed containing PEITC. R175H Evidence of the Resurrection.

[0265] The medical community has conducted a lot of research, trying to find small molecule compounds from chemical libraries that may be used as tumor treatment drugs by changing mutant p53. However, there are few studies targeting natural food-derived compounds for prevention.

[0266] By targeting p53 R175 The present invention illustrates a new mechanism of PEITC for preventing and treating cancer. Mutations in the p53 gene may occur at different stages of cancer, such as in the early stages of breast cancer (DCIS, Ductal carcinoma in situ) and liver cancer, and in the late stages of pancreatic cancer, hepatocellular carcinoma, prostate cancer, etc. As far as breast cancer is concerned, the DCIS stage is the pre-stage of developing into invasive breast cancer. The effect of PEITC on the p53 mutant gene at this stage can be fully used for the prevention and early intervention of breast cancer. Restoring p53 R175 The discovery of "wild-type-like" conformations and functions, combined with the unique safety of natural food-derived compounds, has opened up new avenues for targeted cancer prevention and treatment.

[0267] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A use of PEITC, characterized in that: Used to prepare a preparation or pharmaceutical composition, the preparation or pharmaceutical composition is used for (a) inhibiting the proliferation of mutant p53 breast cancer cells, (c) inducing apoptosis of mutant p53 breast cancer cells, and the pharmaceutical composition also contains the active ingredient Zn 2+ ; The mutant p53 has a mutation at a site selected from the group consisting of: R175; The Zn 2+ The concentration is 10-20 μM; The concentration of PEITC is 1-100 μM.

2. The use according to claim 1, characterized in that The concentration of PEITC is 4-50 μM.

3. The use according to claim 1, characterized in that The mutant p53 is a mutant p53 R175H .

4. The use according to claim 1, characterized in that The concentration of PEITC is 5-20 μM.

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