CN114280308A

CN114280308A - Application of Smad3 protein as novel anti-cisplatin drug-resistant target of ovarian cancer

Info

Publication number: CN114280308A
Application number: CN202111354419.3A
Authority: CN
Inventors: 洪波; 孙飞龙
Original assignee: Hefei Institutes of Physical Science of CAS
Current assignee: Hefei Institutes of Physical Science of CAS
Priority date: 2021-11-16
Filing date: 2021-11-16
Publication date: 2022-04-05

Abstract

The invention discloses application of Smad3 protein as a novel anti-cisplatin drug-resistant target of ovarian cancer. At present, the relation between Smad3 protein and ovarian cancer and the relation between cis-platinum resistance mechanism in ovarian cancer are not researched. In order to define the cisplatin-resistant mechanism of ovarian cancer patients and provide more defined treatment strategies and medicines for ovarian cancer patients, the invention obtains a new molecular target Smad3 protein for regulating and controlling cisplatin sensitivity, and shows that the Smad3 protein is applied as a new cisplatin-resistant target of ovarian cancer. The invention adopts the combination of disulfiram and cisplatin to treat ovarian cancer, and the treatment effect is better than that of single cisplatin.

Description

Application of Smad3 protein as novel anti-cisplatin drug-resistant target of ovarian cancer

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to application of Smad3 protein serving as a new target point in enhancing cisplatin sensitivity of ovarian cancer, a combined pharmaceutical composition capable of inhibiting Smad3 protein, and a medicine for treating ovarian cancer, wherein the medicine comprises the combined pharmaceutical composition.

Background

Chemotherapy is currently the main strategy for cancer treatment, and drug resistance is a major challenge. Resistance is generally divided into primary resistance, which refers to the insensitivity of the tumor to initial treatment, and acquired resistance, which refers to resistance after tumor treatment. Chemotherapy resistance is caused by a number of reasons, including decreased intracellular accumulation of chemotherapeutic agents, inactivation of apoptosis, increased repair of DNA damage, cancer stem cell characteristics, expression of stress response partners, and the like. In addition, tumor microenvironments including immune cells, fibroblasts, vascular endothelial cells, etc. are also considered factors affecting drug resistance.

Cisplatin, which has been approved for cancer therapy for more than 50 years since its discovery, requires one to two water molecules to exert its anti-cancer effect, and it reacts with water molecules to bind to the double helix structure of DNA, forming intra-and inter-strand linkages, and the formation of adductor muscles further causes the bending of the DNA double helix, thereby inhibiting DNA replication in tumor cells. Cisplatin, as a first-line chemotherapeutic drug for various cancer treatments, is resistant and still one of the reasons for hindering cancer treatment.

Disulfiram (Disulfiam/DSF) is a drug approved by the Food and Drug Administration (FDA) for treating alcohol addiction, and is found to have an anticancer effect. Specifically, disulfiram is rapidly converted in vivo to two different metabolites: methyl-n, n-diethylthiocarbamate sulfoxide (Me-DTC-SO) and copper bisdiethyldithiocarbamate (CuET), Me-DTC-SO being believed to have aldehyde dehydrogenase (ALDH) inhibitory activity for the treatment of alcohol addiction, whereas CuET has been demonstrated to have anti-cancer activity. However, a recent study shows that the target of CuET is the linker nucleoprotein localization protein 4(NPL4) of p97 seprase, and the CuET inhibits the p 97-dependent protein degradation by directly binding and immobilizing NPL4 in cytoplasm, thereby triggering a heat shock reaction and playing an anticancer role. To date, there are many reports on the anticancer mechanism of DSF, including inhibition of proteasome, induction of oxidative stress, reduction of angiogenesis, inhibition of tumor-associated macrophages, etc., indicating that DSF can effectively kill tumor cells.

Currently, several studies have shown that disulfiram enhances the sensitivity of cisplatin to kill cancer cells in breast, bladder, lung and cervical cancer cell lines. Ovarian cancer is one of the common gynecological malignant tumors, and the mortality rate is the first gynecological malignant tumor. Platinum-based chemotherapy is the primary treatment for ovarian cancer, however, resistance to drugs in therapy has severely hampered the treatment of ovarian cancer. Therefore, the discovery of drugs and new targets capable of overcoming ovarian cancer drug resistance is urgently needed, and a new strategy is provided for clinical treatment of ovarian cancer.

Disclosure of Invention

In view of the above, the invention provides an application of Smad3 protein as a new target point in enhancing cisplatin sensitivity of ovarian cancer, and the inventors find that the expression level of Smad3 protein can affect the cisplatin sensitivity of tumor cells, the key target point of the disulfiram for enhancing the cisplatin sensitivity is Smad3 protein, and the combined use of disulfiram and cisplatin can inhibit the expression of Smad3 protein and further inhibit the expression of Vimentin, so that the cisplatin sensitivity of ovarian cancer cells is enhanced, and a new strategy is provided for clinical treatment of ovarian cancer.

In order to achieve the purpose, the invention provides the following technical scheme:

the invention firstly provides application of Smad3 protein serving as a novel target point in enhancing cisplatin sensitivity of ovarian cancer.

The invention further provides a combined pharmaceutical composition for enhancing the cisplatin sensitivity of ovarian cancer, which comprises disulfiram and cisplatin.

In a further scheme, the molar ratio of disulfiram to cisplatin is 1: 2.5-10.

In a further scheme, the molar ratio of disulfiram to cisplatin is 1: 2.5.

in a further scheme, the molar ratio of disulfiram to cisplatin is 1: 5.

in a further scheme, the molar ratio of disulfiram to cisplatin is 1: 10.

the invention further provides a medicament for treating ovarian cancer, which comprises the combined pharmaceutical composition of any one of the preceding items.

In a further scheme, the ovarian cancer treatment drug is an ovarian cancer drug for overcoming cisplatin drug resistance.

In a further scheme, the medicine for treating ovarian cancer also comprises pharmaceutically acceptable auxiliary materials.

Compared with the prior art, the invention has the following beneficial effects:

the invention discovers that the sensitivity of ovarian cancer tumor cells to cisplatin can be influenced by the expression level of Smad3 protein, the combined use of disulfiram and cisplatin can inhibit the expression of Smad3 protein, further inhibit the expression of Vimentin Vimentin, and finally enhance the sensitivity of cancer cells to cisplatin by inhibiting epithelial-mesenchymal transition (EMT), namely, the key target of the disulfiram for enhancing the sensitivity of cisplatin is Smad3 protein.

Drawings

Figure 1 is a total protein heatmap in the proteomic analysis in example 1;

FIG. 2 is a Wien plot and differential protein thermographic analysis of the proteomic analysis in example 1;

FIG. 3 is a KEGG functional enrichment and protein interaction network in proteomics analysis in example 1;

FIG. 4 is the results of the expression amounts of Smad3 protein in four experimental groups in example 1;

FIG. 5 shows the results of Western Blot test in example 2;

FIG. 6 is the cell viability analysis of the experimental group of human ovarian cancer cells SKOV3 in example 3 after treatment;

FIG. 7 is the cell viability analysis of the experimental group of human ovarian cancer cells HeyA8 in example 3 after treatment;

FIG. 8 is the cell viability analysis of the experimental group of human ovarian cancer cells OV90 in example 3 after treatment;

FIG. 9 is the effect of the four experimental groups on the cell cycle in example 4;

FIG. 10 is a graph showing the effect of PI/Annexin V double staining method on apoptosis in example 4 in four groups;

FIG. 11 shows the tumor volume changes in the mouse experimental mice in example 5;

FIG. 12 is the change in body weight of experimental mice in example 5;

FIG. 13 is the change in the weight of tumor taken out from the back of the mouse in the experiment in example 5;

FIG. 14 is a photograph of a tumor taken out from the back of the mouse experimental mouse in example 5.

Detailed Description

In order that the invention may be more fully understood, reference will now be made to the specific embodiments illustrated. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

Interpretation of terms

The term "combination" or "use in combination" as used herein means that two or more active substances may be administered to a subject each as a single formulation simultaneously, or each as a single formulation sequentially in any order.

In order to make the technical solutions disclosed in the present invention more clearly understood by those skilled in the art, the technical solutions of the present invention will be further described below with reference to specific embodiments. It is to be understood that materials, reagents and the like used in the following examples are commercially available unless otherwise specified; the test methods employed are, unless otherwise specified, conventional in the art.

Example 1 proteomics analysis of molecular targets modulating cisplatin sensitivity

Preparing materials: human ovarian carcinoma cells SKOV3, purchased from ATCC (the American Type Culture Collection) in USA.

The analysis method comprises the following steps: after the frozen human ovarian cancer cells SKOV3 were recovered, the cells were cultured in RPMI-1640 medium supplemented with 10% (volume fraction) fetal bovine serum and 1% (volume fraction) penicillin/streptomycin under 37 deg.C、5％CO₂(ii) a And when the cell fusion degree reaches 80% -90%, subculturing the cells.

When the fusion degree of the tumor cells reaches 80% -90%, collecting the cells in a culture bottle after trypsinization, centrifuging for 5min at 1100r/min, absorbing supernatant after centrifugation, adding 2mL of culture medium, mixing the cells uniformly, taking 10 mu L of cell suspension from the cell suspension, adding into a 1.5mL EP tube, simultaneously adding 10 mu L of trypan blue (for detecting whether the cells survive) for mixing uniformly, taking 10 mu L of mixed solution from the cell suspension, adding into another new EP tube, adding 10 mu L of trypan blue for mixing uniformly, taking 10 mu L of the mixed solution from the cell suspension, calculating the total number of the cells under a microscope by using a cell counting plate, and calculating the required cell amount.

After counting at 5X 10 per well⁵Culturing the cells in 10cm culture dishes, setting four groups of test groups, namely a control group, a disulfiram single-drug treatment group, a cisplatin single-drug treatment group and a two-drug combined treatment group, wherein each group is repeated three times; after incubation at 37 ℃ for 24 hours in an incubator, the control group was dosed with one-thousandth of DMSO (group A), the concentration of the cisplatin-single-agent treatment group was 25. mu.M (group B), the concentration of the disulfiram-single-agent treatment group was 10. mu.M (group C), and the concentration of the two-agent combination group was 25. mu.M cisplatin + 10. mu.M disulfiram (group D).

After the medicine is treated for 24 hours, cells are collected by trypsinization, the cells are centrifuged for 5 minutes at 1100r/min, the supernatant is discarded, 2mL of cold PBS solution is added for cleaning, the cells are centrifuged for 5 minutes at 1100r/min, the supernatant is discarded, 1mL of PBS is added for suspension, then the cells are transferred into a 1.5mL EP tube, the cells are centrifuged for 5 minutes at 1200r/min by a 4 ℃ low-temperature centrifuge, the supernatant is discarded, and the cells are stored at-80 ℃;

the amount of protein expressed in each experimental group was determined by mass spectrometry, and the change in the fold expression (fold) of protein in other groups compared to the control group and the p-value measured by T-test were calculated. Further analyzing the data and drawing by using an R language, and respectively drawing a volcanic chart (ggplot), a heat map (pheatmap), a wien chart (venn), KEGG functional enrichment (enrichKEGG) and a protein interaction network (STRINGDb) by using corresponding R packets; specifically, a total protein heatmap was first constructed using the expression levels of all proteins in each group; volcano plots were then plotted to determine the expression of a differential protein set to | log (folding) | 1 and p-value <0.05 in the other three groups (group B, C, D) compared to the control group (group a); using the data of the volcano map difference comparison to take intersection to draw a Weinn map, obtaining the proteins of the D group which are all up-regulated or all down-regulated relative to the other three groups (group A, B, C), and using the difference proteins after intersection to draw a heat map, wherein the difference proteins are set as | log (fold change) | >1.5 and p-value < 0.05; and drawing a KEGG functional enrichment and protein interaction network by using the intersected differential protein, analyzing the molecular target for regulating and controlling the cis-platinum sensitivity, and finally verifying the molecular target by a Western Blot experiment.

And (4) analyzing results: the data obtained by R language analysis are used for carrying out total protein heat map analysis on four groups of experimental data, the experimental results are shown in figure 1, and the results show that two groups of proteins have obvious protein expression difference, one group is the protein with high expression in the other group and low expression in the D group (combined group), and the other group is the protein with low expression in the other group and low expression in the D group, so that disulfiram can regulate and control the sensitivity of cisplatin by regulating and controlling certain molecular targets.

And (3) performing volcano map analysis on the data, performing wien map analysis and differential protein heat map analysis after intersection extraction, wherein the experimental result is shown in fig. 2, and the result shows that compared with the other three groups, 73 proteins with remarkably low expression (down regulation) exist in the group D, and 61 proteins with remarkably high expression (up regulation) exist in the group D.

The KEGG functional enrichment and protein interaction network is drawn for analysis, the experimental result is shown in figure 3, the KEGG result shows that the difference protein after intersection is taken and is obviously accumulated in a TNF signal path, the TNF signal path contains a key factor NF-kB, and the NF-kB directly or indirectly influences the expression of genes in a TGF-beta signal path according to literature reports; the results of protein interaction networks indicate that the Smad3 protein interacts with a variety of proteins, and Smad3, as a key factor of the classical pathway of signaling by the TGF- β family, plays an important role in the TGF- β signaling pathway.

The expression level of the Smad3 protein in the four experimental groups is further analyzed, the experimental results are shown in FIG. 4, and the results show that compared with the other three groups, the Smad3 protein is remarkably low in the combined group and higher in the cisplatin single-drug treatment group, which means that disulfiram promotes the sensitivity of tumor cells to cisplatin by inhibiting the expression of Smad 3. The document reports that the SMAD2/3 dimer forms a heterotrimeric complex with SMAD4, which translocates into the nucleus and activates or inhibits transcription of different genes that regulate cell growth, motility, epithelial-mesenchymal transition, and the like.

Example 2 validation of the cellular mechanisms and key targets for disulfiram to modulate cisplatin sensitivity

In order to verify the cellular mechanism and key targets of the disulfiram for regulating cis-platinum sensitivity, Western Blot is adopted to detect a marker protein Smad3, an apoptosis marker protein clear-PAPR and an EMT marker protein (Vimentin) of a TGF-beta signaling pathway.

The specific method comprises the following steps:

The specific method comprises the following steps: the drug treatment and the steps before treatment were the same as in example 1;

the cells were seeded in six-well plates, 4 experimental groups were set up as in example 1, and the cells were treated with the drug 24 hours after growth. After the drug treatment for 12 hours, observing the growth condition of cells in a six-hole plate by a microscope, sucking off the supernatant of the six-hole plate, adding 1mL PBS in each hole, sucking off the PBS, adding 80 μ L of prepared protein lysate in each hole, scraping adherent cells at the bottom by a cell scraper, transferring the cell lysate to an EP tube, placing a sample on ice, vortexing the sample on a vortexing instrument once every 5min for about 25s each time, vortexing the sample twice, standing the vortexed samples for 5min, centrifuging the vortexed samples for 10min at 13000r/min by a precooled 4 ℃ centrifuge, transferring the supernatants to a new EP tube, and storing the fresh EP tube at a refrigerator at-80 ℃ after marking. Protein samples were quantified using the BCA kit from the bio-technologies corporation, cloudband, specifically, the instructions were followed, and two replicates were set for each sample to be tested. And after sample addition, incubating the transparent 96-well plate for about 20 minutes at room temperature, detecting a light absorption value by using an enzyme-labeling instrument, processing obtained data in an Excel table, and calculating the protein content of the sample required to be added during electrophoresis. The laboratory uses 40% methylene bisacrylamide, prepares separation gel and concentrated gel according to the formula (the separation gel is 10% and the concentrated gel is 4% due to the protein strip to be measured), firstly adds the separation gel, then uses isopropanol to seal, firstly absorbs the isopropanol and then adds the concentrated gel after the separation gel is solidified (about 25min), inserts a comb, and waits for the concentrated gel to be solidified. Protein samples with a total volume of 20 mul are prepared according to the protein amount of 50 mug, corresponding deionized water and protein loading buffer are added according to the quantitative result of BCA, metal bath is carried out at 100 ℃ for 5min, and centrifugation is carried out at 13000r/min for 1 min. Assembling an electrophoresis tank, adding a proper electrophoresis solution into the tank, loading, and recording the loading sequence. The voltage is adjusted to 80V electrophoresis for 30min, after the electrophoresis is concentrated to a line, the voltage is adjusted to 120V electrophoresis for about 1 hour, and the electrophoresis is stopped when the target strip completely runs out. Preparing PVDF membrane, filter paper, sponge and the like before electrophoresis, soaking the PVDF membrane in methanol for about 30s, and then soaking the PVDF membrane, the filter paper, the sponge and a splint in cold membrane transferring liquid. The gel block is cut off after preparation, the membrane starts to be rotated after assembly, the periphery of the membrane rotating groove is surrounded by ice blocks for cooling, the membrane is rotated at 100V for 1 hour, and the electrophoresis liquid is recovered. After the membrane is transferred, cutting a notch at the upper right corner as a mark, preparing 5% defatted milk by using a TBST solution, transferring a PVDF membrane into the milk, sealing the PVDF membrane on a shaker at room temperature for two hours, simultaneously recovering the membrane transfer liquid and storing the membrane transfer liquid in a refrigerator at 4 ℃, washing the membrane for 2 times by using the TBST after the sealing is finished, soaking the membrane in the TBST, preparing a primary antibody (PARP: BSA: 1: 1000; Vimentin: BSA: 1: 1000; Smad 3: 1: 1000; beta-actin: BSA: 1: 5000) by using 5% BSA, transferring the membrane to a preservative film by using a tweezers, transferring some TSBT on the membrane by using a gun to keep the membrane moist, cutting required strips according to a Marker strip (positive and negative marking incubation), respectively adding a target strip and a primary antibody solution into an antibody box, and standing the shaker at 4 ℃. Recovering primary antibody in the morning the next day, and storing the recovered primary antibody at 4 ℃. The membrane was washed three times with TBST for 15min each. The corresponding secondary antibody was prepared according to the species of the primary antibody and incubated for two hours at room temperature on a shaker. After completion, the membrane was washed three times with TBST, 15min each time, and the last TBST was not poured off (wet membrane). Preparing on-machine color development, developing by using an ECL luminescence kit, and storing data.

The experimental results are as follows: western Blot results As shown in FIG. 5, the expression level of clear-PARP protein was significantly increased after 12 hours of the two-drug combination group treatment, compared with the other three groups, which means that disulfiram enhances the ability of cisplatin to induce apoptosis in cancer cells. Compared with the other three groups, the expression level of Smad3 protein is remarkably reduced after the two-drug combination group is treated for 12 hours, and the expression level of Vimentin is remarkably reduced. The experimental results show that after the disulfiram is combined with cisplatin, the expression of Smad3 is targeted and inhibited, the expression of Vimentin is further inhibited, and finally the drug resistance of cisplatin is reversed by inhibiting the EMT process.

Example 3 detection of killing ability of disulfiram and cisplatin to ovarian cancer cells

Preparing materials: human ovarian cancer cells SKOV3, HeyA8, OV90, all purchased from ATCC (the American Type Culture Collection) in USA, were cultured by the following Culture method after recovering the frozen human ovarian cancer cells SKOV3, HeyA8, OV 90:

SKOV 3: culturing in RPMI-1640 medium containing 10% fetal calf serum and 1% penicillin/streptomycin at 37 deg.C under 5% CO₂(ii) a And when the cell fusion degree reaches 80% -90%, subculturing the cells.

② OV 90: culturing in RPMI-1640 medium containing 10% fetal calf serum and 1% penicillin/streptomycin at 37 deg.C under 5% CO₂(ii) a And when the cell fusion degree reaches 80% -90%, subculturing the cells.

③ HeyA 8: culturing with DMEM medium containing 10% fetal calf serum and 1% penicillin/streptomycin at 37 deg.C under 5% CO₂(ii) a And when the cell fusion degree reaches 80% -90%, subculturing the cells.

The specific method comprises the following steps: when the fusion degree of the tumor cells reaches 80% -90%, collecting the cells in a culture bottle after trypsinization, centrifuging for 5min at 1100r/min, absorbing the supernatant after centrifugation, adding 2mL of culture medium, uniformly mixing the cells, taking 10 mu L of cell suspension from the cell suspension, adding 10 mu L of trypan blue (for detecting whether the cells survive) into a 1.5mL EP tube, uniformly mixing, taking 10 mu L of mixed solution, adding 10 mu L of trypan blue into another new EP tube, uniformly mixing, taking 10 mu L of mixed solution, calculating the total number of the cells by using a cell counting plate under a microscope, and calculating the required cell amount.

The original cell suspension was diluted with medium in a new centrifuge tube and seeded into 96-well plates at 100 μ L per well (3000 cells/well). After the cells are planted for 24 hours, sucking the supernatant, adding 100 mu L of inhibitors (disulfiram and cisplatin) with different concentrations or contrast solvents (the concentration of each inhibitor is diluted by a culture medium in a gradient way, and each concentration is set to be 3 times, putting the mixture into an incubator, continuously culturing the mixture for 72 hours, adding 20 mu L of CellTiter-Glo Luminescent assay substrate into each well, gently mixing the mixture uniformly, waiting for reaction for 5-10min, and detecting the mixture by using an Envision PerkinElmer multi-labeled microplate detector. Processing data with Excel table after detection, calculating the percentage of the average value of the cell treated by inhibitor under different concentrations in the control group, and drawing the half Inhibitory Concentration (IC) of two drugs by processing the obtained result with PRISM5₅₀) Curve line.

According to the obtained semi-inhibitory concentration curve of the drugs, the appropriate concentration of the two drugs is selected to carry out a two-drug combined experiment to determine the capability of the drugs to kill cells in a synergistic manner. The steps are the same as above, after the 96-well plate is planted with cells and cultured for 24 hours, four groups of test groups (a control group, a disulfiram single-drug treatment group, a cisplatin single-drug treatment group and a two-drug combined treatment group) are set, each group is repeated three times, the two drugs are referred to a semi-inhibitory concentration curve, and the appropriate concentration is selected for multiple tests, and the cells are treated for 72 hours. After the treatment was completed, cell proliferation was measured by CellTiter-Glo luminescence assay (Promega, Madison, Wis., USA). The data processing method is the same as above.

And (4) analyzing results: the results are shown in fig. 6-8, and indicate that disulfiram treatment can significantly down-regulate the cell viability of ovarian cancers SKOV3, HeyA8, OV90, and that the cell viability is dose-dependently reduced with increasing disulfiram concentration; cisplatin treatment can down-regulate cell viability of ovarian cancers SKOV3, HeyA8 and OV90 at higher concentration, and the cell viability is reduced in a dose-dependent manner along with the increase of the concentration of cisplatin, which indicates that the three cells are not sensitive to cisplatin and are drug-resistant cells. After selecting proper single-drug concentration, the cell viability in the drug combination treatment group is remarkably reduced compared with the single-drug treatment group, which indicates that the disulfiram remarkably increases the sensitivity of ovarian cancer cells to cisplatin.

Example 4 detection of the Effect of the combination of two drugs on the cell cycle by PI Monostain assay

The specific method comprises the following steps: the procedure before cell counting was the same as in example 1;

after counting at 5X 10 per well⁵Culturing the cells in six-hole plates with four holes, and setting four groups of test groups after culturing for 24 hours, wherein the test groups are respectively a control group, a disulfiram single-drug treatment group, a cisplatin single-drug treatment group and a two-drug combined treatment group;

after the medicine is treated for 24 hours, the four groups are respectively digested by pancreatin to collect cells, centrifuged at 1100r/min for 5min, and the supernatant is discarded; adding 2mL of cold PBS solution into the tube, centrifuging for 5min at 1100r/min, discarding the supernatant, adding 4mL of cold 70% ethanol dropwise to fix cells on a vortex apparatus while oscillating at the minimum rotation speed, storing in a refrigerator at-20 ℃ for at least 24 hours, and detecting on-machine;

centrifuging the fixed cells for 5min at 1100r/min, discarding the supernatant, adding 5mL of PBS solution, centrifuging for 5min at 1100r/min, discarding the supernatant, adding 500. mu.L of PBS solution, mixing the cells uniformly, transferring the cells to a flow tube, adding 500. mu.L of PI/RNase staining buffer (BD 7Pharmingen, San Diago, CA, USA) into the cell suspension, staining, tapping the bottom of the mixed cells uniformly, keeping out of the sun at room temperature, reacting for 30min, and detecting by using a flow cytometer (BD Biosciences, San Jose, CA, United States). The data obtained were processed by ModFit (BD biosciences) software.

And (4) analyzing results: the influence of four groups of experimental groups on the cell cycle is detected by adopting a PI single staining method and using a flow cytometer, the experimental result is shown in figure 9, and the result shows that compared with a control group, the cisplatin blocks the cell cycle at the G1 stage, the cell cycle process is prevented, the cell replication is inhibited, and the disulfiram enhances the blocking effect of the cisplatin on the cell cycle.

Meanwhile, the PI/Annexin V double staining method is adopted to determine the apoptosis, and specific experimental materials and methods are as follows.

Preparing materials: human ovarian carcinoma cells SKOV3, purchased from ATCC (the American Type Culture Collection) in USA;

the specific method comprises the following steps: the procedure before dosing was the same as for the single dyeing method in example 4 for the four experimental groups. After four groups of medicines are treated for 48 hours, the cells are collected by trypsinization, centrifuged for 5 minutes at 1100r/min, the supernatant is discarded, 2ml of cold PBS solution is added for washing twice, centrifuged for 5 minutes at 1100r/min, the supernatant is discarded, the 10 XBinding buffer stock solution is diluted into a proper amount of 1 XBinding buffer by pure water, and the 1 XBinding buffer is added into the cells in the tube to prepare 1 × 10⁶Cell suspension of individual cells/ml and mix well. From this, 100. mu.L to 1.5mL of EP tubes were pipetted, and the control group (DMSO-treated group) was divided into four groups, namely a negative control group (no reagent added), a PI single-stained group (PI only), an Annexin V single-stained group (Annexin V only), and a double-stained control group (PI and Annexin V co-stained). In four control groups (DMSO-treated groups), 5. mu.l of the corresponding reagent was added, and in each of the other three experimental groups (two single-drug-treated groups and two-drug-combined group), 5. mu.l of Annexin V and 5. mu.l of PI were added, mixed, transferred to a flow tube, and reacted at room temperature in the dark for 20 minutes. Then 400. mu.l of 1 XBinding buffer is added into each tube, and after mixing, the apoptosis is detected by a flow cytometer. The data were further processed with FlowJo V10 software (FlowJo LLC, Ashland, Oregon, USA)

And (4) analyzing results: the PI/Annexin V double staining method is adopted, the flow cytometry is used for detecting the influence of four groups of experimental groups on the apoptosis, the experimental result is shown in figure 10, and the result shows that compared with a control group, the two-drug combined group can obviously promote the apoptosis, the disulfiram enhances the induction effect of the cisplatin on the apoptosis, promotes the early-withering and late-withering processes of cancer cells, and obviously reduces the number of the living cells.

Example 5 testing of the Effect of the combination of disulfiram and cisplatin on tumor growth in vivo

Preparing materials: human ovarian carcinoma cells SKOV3 (purchased from ATCC, usa), athymic nude mice (female).

The specific method comprises the following steps: the animal experiment scheme is approved by the ethical committee of experimental animals of the Chinese academy of fertilizer-combining substance science research institute, 4-week-old athymic female nude mice are purchased from Nanjing Wintoli, and the nude mice are firstly raised for one week in an animal house of a special pathogen-free animal (namely SPF level) of the Chinese academy of fertilizer-combining substance science research institute according to the ethical requirements of the animals so as to adapt to the environment;

collecting SKOV3 cells with good growth condition, centrifuging at 1100r/min for 5min, removing supernatant, adding 3mL PBS to dilute the cells into cell suspension, counting by a cell counting plate, adding 3mL matrix glue, mixing well, and storing on ice;

before the tumor of the mouse, the mouse is anesthetized by 2.5 percent avermectin anesthetic, and 200 mu L of anesthetic is injected into the abdominal cavity of the mouse per 20g according to the weight of the mouse; after confirming that the mice were anesthetized, 200. mu.L of cell suspension (2X 10 cells) was injected subcutaneously on both sides of the back⁶Individual cells); after the injection is finished, the mice are waited to wake up, and all the mice are kept breeding when normal;

when the tumor diameter increased to 4 to 5mm, all mice were randomly divided into four groups, dosing experiments were started every day, and changes in mouse body weight, tumor volume, and mouse status were recorded; the medicine is administrated by intraperitoneal injection, and is divided into four groups: a control group (DMSO + PBS), a cisplatin monomer group (5mg/kg), a disulfiram monomer group (25mg/kg), and a two-drug combination group (5mg/kg cisplatin +25mg/kg disulfiram); the drug is administered every two days, the tumor size and body weight of the mice are measured using an electronic balance and a vernier caliper, and the mental state of the mice is observed, the tumor size is calculated according to the following formula: volume (length × width)/2;

stopping administration after continuous administration for 12 days, continuously measuring and observing the mouse for 4 days, after the experiment is finished, taking eyeball blood from the mouse until the mouse dies, sending the blood to a hospital, detecting relevant indexes such as heart, liver and the like, and detecting whether the mouse is normal or not; the tumor on the back of the mouse was removed, weighed on an analytical balance, fixed with 4% paraformaldehyde for 24 hours, and stored in a refrigerator at 4 ℃ with 70% ethanol.

And (4) analyzing results: the change in tumor volume of the mice is shown in fig. 11, and the tumor volume of the mice in the two-drug combination group is significantly reduced compared to the control group, indicating that disulfiram significantly enhances the sensitivity of ovarian cancer cells to cisplatin in vivo; the body weight change of the mice is shown in fig. 12, and the body weights of the four groups of mice are not significantly different, which means that the mice do not have serious side effect reaction at this administration dose, the weight of the tumor taken out of the back of the mice is shown in fig. 13, and the photograph of the tumor taken out of the back is shown in fig. 14.

Example 6 determination of optimum concentrations of the combination of disulfiram and cisplatin

Preparing materials: the same as in example 3.

The specific method comprises the following steps: the same as in example 3.

The synergy index value (CI) was calculated using the formula: CI-D1/DX 1+ D2/DX, wherein DX1 and DX2 are the doses of the drugs whose growth inhibition rates reached specific values when the two drugs were used alone, and D1 and D2 are the doses of the two drugs alone when the two drugs were combined to achieve the same growth inhibition rate as the drug combination treatment; CI <1 indicates that the two drugs are synergistic in combination, while CI >1 indicates that there is no synergy in the two drugs in combination.

And (4) analyzing results: synergy index evaluation experiments were performed on SKOV3 cells, and the results are shown in table 1, which indicated that 50 μ M cisplatin +10 μ M disulfiram had the best synergy efficiency, when CI was 0.209. Synergy index evaluation experiments were performed on HeyA8 cells and the results are shown in table 2 and indicate that 12.5 μ M cisplatin +1.25 μ M disulfiram had the best synergy efficiency when CI was 0.513. Synergy index evaluation experiments were performed on OV90 cells, and the results are shown in table 3, which indicated that 25 μ M cisplatin +10 μ M disulfiram had the best synergy efficiency, when CI was 0.284.

TABLE 1 evaluation of synergy index for human ovarian carcinoma SKOV3 cells disulfiram and cisplatin

TABLE 2 evaluation of synergy index for human ovarian cancer HeyA8 cells disulfiram and cisplatin

TABLE 3 evaluation of synergy index for human ovarian carcinoma OV90 cells disulfiram and cisplatin

In conclusion, the disulfiram treatment can reverse the drug resistance of the ovarian cancer cells to cisplatin, enhance the sensitivity of the ovarian cancer cells to cisplatin, enhance the killing capacity of the cisplatin to the ovarian cancer cells, block the cell cycle process and enhance the apoptosis of the ovarian cancer cells. The reason is that after the disulfiram is combined with cisplatin, the expression of Smad3 is targeted and inhibited, the expression of Vimentin Vimentin is further inhibited, and finally the resistance of the cisplatin is reversed by inhibiting the EMT process. It is also demonstrated by mouse experiments that disulfiram significantly enhances the sensitivity of cancer cells to cisplatin in vivo.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

Application of Smad3 protein as a novel target point in enhancing cisplatin sensitivity of ovarian cancer.
2. A pharmaceutical combination composition for enhancing cisplatin sensitivity in ovarian cancer, comprising disulfiram and cisplatin.
3. The combination pharmaceutical composition according to claim 2, wherein the molar ratio of disulfiram to cisplatin is 1: 2.5-10.
4. The combination pharmaceutical composition according to claim 3, wherein the molar ratio of disulfiram to cisplatin is 1: 2.5.
5. the combination pharmaceutical composition according to claim 3, wherein the molar ratio of disulfiram to cisplatin is 1: 5.
6. the combination pharmaceutical composition according to claim 3, wherein the molar ratio of disulfiram to cisplatin is 1: 10.
7. a medicament for the treatment of ovarian cancer comprising a combination pharmaceutical composition according to any one of claims 2 to 6.
8. The agent for treating ovarian cancer according to claim 7, wherein the agent for treating ovarian cancer is an agent for treating ovarian cancer that overcomes resistance to cisplatin.
9. The ovarian cancer treatment drug of claim 7, further comprising a pharmaceutically acceptable excipient.