CN117679427A

CN117679427A - Application of pharmaceutical composition in preparation of drug resistant drugs for treating tumors

Info

Publication number: CN117679427A
Application number: CN202311835082.7A
Authority: CN
Inventors: 刘秀均; 蔡豪; 盛唯瑾; 郑艳波; 李毅; 甄永苏
Original assignee: Institute of Medicinal Biotechnology of CAMS
Current assignee: Institute of Medicinal Biotechnology of CAMS
Priority date: 2023-12-28
Filing date: 2023-12-28
Publication date: 2024-03-12

Abstract

The invention provides an application of a pharmaceutical composition in preparing medicines for treating tumor drug resistance, wherein the pharmaceutical composition consists of dipyridamole, ubenimex and dexamethasone. The invention verifies the effect of reversing drug resistance of the pharmaceutical composition in lung cancer, breast cancer or pancreatic cancer cells and cisplatin-resistant, doxorubicin-resistant, gemcitabine-resistant or gefitinib-resistant cells. The invention also verifies that the pharmaceutical composition achieves reversing tumor resistance by inhibiting P-gp expression and P-gp ATPase activity, increasing intracellular drug content, enhancing the damaging effect of chemotherapeutics on DNA by reducing the expression of DNA repair protein ERCC1, enhancing apoptosis by increasing ROS level while reducing HIF-1 alpha protein to prevent drug resistance caused by downstream drug resistance protein induced by HIF-1 alpha, enhancing the apoptosis induction effect of P53 protein, and inhibiting the anti-apoptosis effect of Stat3 protein and Bcl-2 protein.

Description

Application of pharmaceutical composition in preparation of drug resistant drugs for treating tumors

Technical Field

The invention relates to the technical field of anti-tumor medicaments, in particular to application of a pharmaceutical composition in the treatment of anti-drug-resistant tumors.

Background

Malignant tumors are a global problem that is severely threatening the health of human life. Treatment regimens for tumor patients are often accompanied by post-operative personalized combination chemotherapy or combination targeted therapy, and the like. However, the therapeutic effect is often poor, mainly due to the multi-drug resistance (multiple drug resistance, MDR) generated during the treatment, resulting in a poor prognosis for tumor patients. Tumor cell multidrug resistance is one of the major factors impeding tumor therapeutic success, and is an important factor that leads to tumor therapeutic difficulties and recurrence.

There are a number of mechanisms by which MDR occurs, of which the transport of proteins present on the cell membrane, nuclear membrane, is the predominant mechanism, and these proteins can pump drugs out of the cell by way of ATP water explanation and release energy, causing tumor cells to produce MDR. At present, more MDR-related proteins are P-glycoprotein (P-gp)/ABCB 1, multi-drug resistance-related protein 1 (MRP 1)/ABCC 1, breast cancer drug resistance protein (BCRP)/ABCG 2 and the like, and the jet pumps play a key role in multi-drug resistance of various tumors. Hypoxia inducible factor (hypoxia inducible factor, HIF) is a global regulator of hypoxia response, involved in the regulation of tumor angiogenesis, cell proliferation, cell metastasis and infiltration. The MDR1 gene is sensitive to hypoxia, when the tissue cells of the organism are in a hypoxia state, the MDR1 gene expression is obviously increased, and the HIF can obviously raise wild type p53 in a hypoxia environment, has no effect on mutant p53, prevents the cells from apoptosis as expected, and increases the transcription of the MDR1 gene. In addition, after tumor cells are resistant, intracellular DNA repair is increased, and DNA damaged by the drugs can be recovered and division can be continued. ERCC1 (precision repair 1,endonuclease non-catalytic subunit) is one of the most studied drug-resistant biomarkers so far, and the product of this gene plays a role in the nucleotide excision repair pathway, and is necessary for repairing DNA damage formed by ultraviolet light-induced or cisplatin and other electrophilic compounds. ERCC1 expression and drug resistance are linearly related, and drug resistant cells express more ERCC1 than sensitive cells.

The chemotherapy drugs commonly used in clinic, such as cisplatin, gemcitabine, doxorubicin and the like, and the small molecule targeting drugs, such as gefitinib and the like, are first-line drugs for lung cancer, pancreatic cancer, breast cancer and the like, and are also very easy to cause tumor resistance. Comprehensive existing researches on various drug resistance related pathways or proteins show that tumor drug resistance is related to various mechanisms, is a complex process of multiple genes, multiple factors and multiple steps, and can improve drug resistance state without changing the expression of a certain cell pathway or protein. Therefore, the combination of multiple drugs with definite mechanisms and the comprehensive intervention of disease processes, which act on multiple targets, multiple paths and different links, will be a new trend for the treatment of complex diseases and the research and development of drugs in the future.

Chinese patent application No. CN 102198139A discloses a novel broad-spectrum pharmaceutical composition for high-efficiency, low-toxicity and comprehensive targeted therapy consisting of dipyridamole, ubenimex and dexamethasone. However, there has been no study in the prior art between this pharmaceutical composition and the treatment of resistant tumors.

Disclosure of Invention

The invention aims to provide a novel technical scheme for overcoming the treatment of drug-resistant tumors.

The invention adopts the concept that the proliferation inhibition effect of the pharmaceutical composition on different drug-resistant tumor cell lines is inspected through in vitro drug effect evaluation, the drug resistance improvement effect of the pharmaceutical composition on drug-resistant tumor cells is inspected, and a plurality of pairs of tumor cell lines comprising drug resistance and sensitivity are selected, wherein the tumor cell lines comprise AsPC-1 and AsPC-1/GEM (human pancreatic cancer gemcitabine resistant lines); h460 H460/DDP (human lung carcinoma cisplatin-resistant strain); MCF-7, MCF-7/ADM (human breast cancer doxorubicin resistant strain); a549, a549/GR (gefitinib resistant strain for human lung cancer); a549, A549/DDP (cisplatin resistant strain of human lung cancer) and the like. On the other hand, a nude mice transplantation tumor model of human lung cancer DDP drug-resistant H460/DDP cells is selected for in vivo efficacy experiment to test the efficacy of the drug composition, the DDP and the drug composition and the DDP combined for carrying out related action mechanism research on human drug-resistant lung cancer transplantation tumor.

Based on this, the invention provides the application of a pharmaceutical composition in preparing medicines for treating tumor resistance, wherein the pharmaceutical composition consists of a component A, a component B and a component C,

the component A is dipyridamole, a derivative of dipyridamole which is acceptable in pharmacy, an analogue of dipyridamole which is acceptable in pharmacy or a pharmaceutically acceptable salt thereof;

the component B is ubenimex, a pharmaceutically acceptable derivative of ubenimex, a pharmaceutically acceptable analogue of ubenimex or a pharmaceutically acceptable salt thereof;

the component C is dexamethasone, a pharmaceutically acceptable derivative of dexamethasone, a pharmaceutically acceptable analogue of dexamethasone or a pharmaceutically acceptable salt thereof.

As a preferred embodiment, the mass ratio of component A, component B and component C is 100:20:1 as the mass ratio of dipyridamole, ubenimex and dexamethasone.

The pharmaceutical composition of the present invention may be described in chinese patent application CN 102198150A, CN 102198139a, or in Yan-Bo Zheng et al, DBDx-based drug combinations show highly potent therapeutic efficacy against human pancreatic CANCER xenografts in athymic mice, CANCER bio & heat, 17jun 2020.

In the present invention, the drug is an antitumor drug.

Such tumors include, but are not limited to, lung, breast or pancreatic cancer.

In the invention, the mechanism of realizing anti-tumor drug resistance based on the pharmaceutical composition is that P-gp protein expression in cells is significantly down-regulated, ATPase activity is inhibited, HIF-1 alpha protein expression and ERCC1 protein expression are down-regulated, so that the application of the anti-tumor drug resistance is suitable for drugs which are expressed as P-gp protein over-expression, HIF-1 alpha protein over-expression and/or ERCC1 protein over-expression in tumor cells, ATPase activity is enhanced, and hypoxia of tumor microenvironment leads to changes of important molecular signal paths such as HIF-1 alpha paths, apoptosis paths including Bcl-2 paths, including but not limited to cisplatin, gemcitabine, doxorubicin or gefitinib.

Based on this, the tumor resistance of the present invention includes, but is not limited to, tumor cisplatin resistance, tumor doxorubicin resistance, tumor gemcitabine resistance, or tumor gefitinib resistance.

Thus, the pharmaceutical composition of the present invention achieves reversal of tumor resistance by inhibiting P-gp expression and P-gp ATPase activity, thereby increasing intracellular drug content.

The pharmaceutical composition reduces the expression of excision repair complementary protein ERCC1, and reduces DNA repair capacity to reverse tumor resistance.

The pharmaceutical composition of the invention achieves reversion of tumor resistance by increasing ROS level to enhance apoptosis, decreasing HIF-1 alpha protein to prevent resistance caused by downstream drug resistant proteins induced by HIF-1 alpha.

The pharmaceutical composition of the invention also realizes reversing tumor resistance by enhancing the apoptosis induction effect of the P53 protein and inhibiting the anti-apoptosis effect of the Stat3 protein and the Bcl-2 protein.

The invention discovers that the pharmaceutical composition plays a role on a plurality of targets related to a tumor drug resistance mechanism for the first time, realizes low toxicity, safety and effective reversion of tumor drug resistance, and simultaneously realizes high-efficiency inhibition capability on drug-resistant tumors by combined application of low-dose chemotherapeutic drugs and the pharmaceutical composition. The medicine composition is suggested to be a brand new therapy for improving drug-resistant tumor treatment in the future.

Drawings

FIG. 1 shows comparison of ROS content and expression levels of three drug-resistance related proteins HIF-1α, ERCC1, P-gp in DDP-sensitive and DDP-resistant cells in example 1; wherein, ROS in two pairs of cancer cell lines were detected using ROS detection kit (scale bar 100 μm). B. ROS fluorescence intensity was quantified for each cell line by flow cytometry. C. Protein levels of HIF-1. Alpha., ERCC1 and P-gp were analyzed by Western blotting. D. Semi-quantitative HIF-1α, ERCC1, and P-gp expression were performed by imagene J software (tumor cells on the left side of each histogram and drug resistant cells on the right side), P < 0.05, P < 0.01, and P < 0.001;

FIG. 2 shows the expression levels of three drug-resistance-associated proteins HIF-1. Alpha., ERCC1, P-gp in two pairs of DDP-sensitive and drug-resistant cells according to the fluorescent test in example 1; wherein, green represents immunofluorescence images of HIF-1α, ERCC1 and P-gp staining, red represents immunofluorescence images of cell membranes (scale bar 50 μm) after treatment for b.24 hours or 48 hours, cytotoxicity of DDP to two pairs of cell lines was assessed by MTT analysis, P < 0.05, < 0.01 and < 0.001 (each group of bar graphs corresponds to H460/DDP 24H, H460/DDP 48H, H460 24H, a549/DDP 48H, a549 48H, respectively from left to right);

FIG. 3 is a graph of the cytotoxicity of a pharmaceutical composition and DDP using H460/DDP cells in example 2; wherein, the pharmaceutical compositions with different concentrations are tested for the growth inhibition effect on H460/DDP cells for 24 hours or 48 hours by MTT method (24 hours on the left side and 48 hours on the right side of each bar graph). MTT assay for growth inhibition of H460/DDP cells by DDP at a concentration of 4. Mu.g/mL or 8. Mu.g/mL in combination with different concentrations of the pharmaceutical composition (each set of bar graphs corresponds to DDP 4 (. Mu.g/mL) +DBDx 24H, DDP 4 (. Mu.g/mL) +DBDx 48H, DDP 8 (. Mu.g/mL) +DBDx 24H, DDP 8 (. Mu.g/mL) +DBDx 48H, respectively from left to right). C. After the indicated duration, the growth inhibitory effect of a constant concentration of 20. Mu.g/mL or 40. Mu.g/mL of the pharmaceutical composition and of DDP of different concentrations on H460/DDP cells was evaluated by MTT method (each set of bar graphs corresponds to DBDx 20 (. Mu.g/mL) +DDP 24H, DBDx 20 (. Mu.g/mL) +DDP 48H, DBDx 40 (. Mu.g/mL) +DDP 24H, DBDx 40 (. Mu.g/mL) +DDP 48H, respectively from left to right).

FIG. 4 is a graph showing the resistance reversal ability and synergistic therapeutic effect of drug combinations and DDP using H460/DDP cells in example 2; wherein, A. Combination index value of pharmaceutical composition and DDP combination in H460/DDP cells. CI values were calculated using cell viability in C. The two-drug interaction coefficients (coefficient of drug in interaction, CDI) were used to evaluate the two-drug interaction properties. CDI is calculated according to the following formula: cdi=ab/AxB. The calculation was performed based on the number of living cells (absorbance value), AB is the ratio of the combination of two drugs to the control group, and A or B is the ratio of the individual drug groups to the control group. Such as CDI<1, the two medicines are proved to have synergistic action and CDI<The synergy of the two medicines is very remarkable at 0.7; if cdi=1, then the two drugs actThe mass is added; such as CDI>1, the two drugs have antagonistic action. B. The pharmaceutical compositions of DDP, 20. Mu.g/mL in H460/DDP cells were combined with DDP, and the IC50 values of 40. Mu.g/mL with DDP after various treatment times (24H on the left side and 48H on the right side of each bar graph). C. In H460/DDP cells, the drug resistance reversal index of the combination of 20. Mu.g/mL of the pharmaceutical composition with DDP and 40. Mu.g/mL with DDP at different treatment times (24H on the left side and 48H on the right side of each histogram) was calculated as follows: drug resistance reversal index = IC _50DDP ÷IC _{50 pharmaceutical composition+DDP} . D. Proliferation inhibition of the pharmaceutical composition and DDP combination was evaluated by colony formation assay. The quantitative data for diameter are shown as mean ± standard deviation, with ddp4+ pharmaceutical composition 40 μg/mL group compared to other groups, P < 0.05, P < 0.01 and P < 0.001. The DDP4+ pharmaceutical composition 20 μg/mL group was compared with the control group and the single drug group, ^## p < 0.01 ^### P＜0.001；

FIG. 5 is a graph showing the effect of drug composition and DDP combination in example 4 on ROS and cisplatin content in H460/DDP cells; wherein, A. Fluorescence images of ROS levels in H460/DDP cells (scale bar 100 μm) were treated with DDP4 μg/mL, pharmaceutical compositions (20 and 40 μg/mL), combinations of pharmaceutical compositions and DDP (20+4 μg/mL and 40+4 μg/mL) for 12 hours. B. The ROS fluorescence intensity in a was quantified by flow cytometry. C. According to P-gp-Glo ^TM The kit detects the P-gp ATPase inhibition intensity of the pharmaceutical composition and verapamil, and compares the basic group with other groups, wherein P is less than 0.05, P is less than 0.01 and P is less than 0.001.D. The concentration of DDP in H460/DDP cells was determined by ICP-MS method for 12 hours by co-culturing DDP 4. Mu.g/mL, the pharmaceutical composition and DDP 4. Mu.g/mL. Data are shown as mean ± standard deviation, DDP4 group compared to other groups, P < 0.05, P < 0.01 and P < 0.001;

FIG. 6 is a graph showing the effect of drug combination and DDP on expression of drug resistance-associated proteins in example 4; wherein, 4 mug/mL of DDP, pharmaceutical compositions (20 and 40 mug/mL) and the expression of HIF-1 alpha, ERCC1 and P-gp proteins in H460/DDP cells after 24 hours of combined action of the pharmaceutical compositions with DDP were studied by Western blotting. B. Expression of semi-quantitative HIF-1. Alpha., ERCC1 and P-gp in Panel A (each histogram corresponds to the control group from left to right, respectively)DDP 4. Mu.g/mL, DBDx 20. Mu.g/mL, DBDx 40. Mu.g/mL, DDP 4. Mu.g/mL+DBDx 20. Mu.g/mL). Comparison of control group with other groups P<0.05、**P<0.01 and P<0.001. Comparing the DDP4 mug/mL group, the drug combination group and the combined drug group, ^# P＜0.05、 ^## p < 0.01 ^### P < 0.001. Effects of hif-1 a and ERCC1 protein expression on survival status of lung adenocarcinoma patients, P < 0.05, P < 0.01, and P < 0.001;

FIG. 7 is an apoptosis-inducing assay for combination of pharmaceutical compositions and DDP in example 5; wherein, after incubation with DDP, pharmaceutical composition and combination of pharmaceutical composition and DDP for 24 hours, H460/DDP cell fluorescence image. Green and red fluorescence represent JC-1 monomer and J-aggregate, respectively (scale bar 200 μm). B. Apoptosis rate of H460/DDP cells treated with the same protocol of Panel A for 24 hours was assessed by flow cytometry. C. Panel B quantification of apoptosis Rate DDP 4. Mu.g/mL+40. Mu.g/mL of the pharmaceutical composition was compared to the other groups, ^* P<0.05、 ^** P<0.01、 ^*** P<0.001.DDP 4. Mu.g/mL + pharmaceutical composition 20. Mu.g/mL group was compared with the control group and the single drug group, ^# P＜0.05、 ^## p < 0.01 ^### P＜0.001。

FIG. 8 is a graph showing the effect of combination of a pharmaceutical composition and DDP on apoptosis-related pathways in example 5; wherein, P53, stat3, P-Stat3 and Bcl proteins expression in H460/DDP cells treated with DDP, pharmaceutical compositions and combinations of pharmaceutical compositions and DDP for 24 hours were studied using Western blotting. B. The expression of P53, stat3, P-Stat3 and Bcl2 in panel A was semi-quantitatively detected (each set of histograms corresponds to control, DDP 4. Mu.g/mL, DBDx 20. Mu.g/mL, DBDx 40. Mu.g/mL, DDP 4. Mu.g/mL+DBDx 20. Mu.g/mL, respectively from left to right). The control group was compared with the other groups, ^* P<0.05、 ^** P<0.01、 ^*** P<0.001. the DDP 4. Mu.g/mL group was compared with the pharmaceutical composition group and the combination group, ^# P＜0.05、 ^## p < 0.01 ^### P＜0.001；

FIG. 9 is an anti-tumor effect of the pharmaceutical composition of example 6 and DDP in combination in an H460/DDP tumor implantation model; wherein, A is schematic diagram of tumor treatment. B. Images of H460/DDP solid tumors on day 20 post-cell implantation. C. Tumor weights were recorded on day 20 after cell implantation, P < 0.05, P < 0.01 and P < 0.001 compared to the other groups. D. The inhibition rate of the last DDP 4mg/kg, the pharmaceutical composition 121mg/kg and the combination therapy of the pharmaceutical composition and DDP (121+4mg/kg) on tumor growth is less than 0.05, less than 0.01 and less than 0.001 compared with the other groups;

FIG. 10 shows the change in tumor volume and body weight of nude mice in example 6; wherein, tumor growth curves of H460/DDP xenograft tumors in each individual BALB/c nude mice of the four treatment groups. B. All tumor volume data are shown in panel B. C. Average body weight of BALB/C nude mice for each treatment group, P <0.05, < P <0.01, and P <0.001;

FIG. 11 is a pathological tissue analysis of the tumor nude mouse model in example 6;

FIG. 12 is a graph showing changes in HIF-1. Alpha. Expression levels in tumor tissues of each treatment group in a H460/DDP tumor-transplanted nude mouse model of example 7; wherein, the expression level of HIF-1 alpha in H460/DDP xenograft tumor pathological sections treated with DDP 4mg/kg, pharmaceutical composition 121mg/kg and pharmaceutical composition and DDP combination (121+4mg/kg).

(scale bar 100 μm) semi-quantitative expression of HIF-1α in Panel A. The combination group is compared with the other groups,

* P <0.05, < P <0.01 and P <0.001. The 121. Mu.g/mL group of the pharmaceutical composition was compared with the control group and the DDP group,

^# P＜0.05、 ^## p <0.01 ^### P＜0.001；

FIG. 13 is a graph showing the change in P-gp expression level in tumor tissues of each treatment group of H460/DDP tumor-transplanted nude mouse model in example 7; wherein, the expression level of P-gp in tumor sections (scale bar 100 μm). B. Semi-quantification of P-gp expression of Panel A. Combination group compared with other groups, P <0.05、**P<0.01 and P<0.001. The DBDx 121. Mu.g/mL group was compared with the control group and the DDP group, ^# P<0.05、 ^## P<0.01 and ^### P<0.001；

FIG. 14 is a table of ERCC1 in tumor tissue of each treatment group of H460/DDP tumor-transplanted nude mouse model in example 7A change in the achievement level; wherein, A. The expression level of ERCC1 in tumor sections (scale bar 100 μm). B. Semi-quantification of ERCC1 expression of panel a. Combination group compared with other groups, P<0.05、**P<0.01 and P<0.001. The DBDx 121. Mu.g/mL group was compared with the control group and the DDP group, ^# P<0.05、 ^## P<0.01 and ^### P<0.001；

Detailed Description

The following examples serve to illustrate the technical solution of the invention without limiting it.

The present invention relates to the following medicaments or pharmaceutical compositions:

pharmaceutical composition mother liquor: weighing 20mg of dipyridamole Mo Biaozhun product powder, dissolving in 1mL of dimethyl sulfoxide, dissolving 4mg of ubenimex standard product powder in 0.2mL of DMSO, dissolving 0.2mg of dexamethasone standard product powder in 0.01mL of DMSO, and fully and uniformly mixing the three to obtain a medicinal composition mother solution with the concentration of 20mg/mL, wherein the mother solution, the three parts are separately packaged and frozen at-70 ℃ to avoid repeated freeze thawing, and are uniformly mixed immediately before use.

The DDP freeze-dried powder (DDP for injection) is a Qilu pharmaceutical product and is prepared by using physiological saline.

The invention relates to the following biological materials:

AsPC-1, asPC-1/GEM (gemcitabine resistant strain of human pancreatic cancer); h460 H460/DDP (human lung carcinoma cisplatin-resistant strain); MCF-7, MCF-7/ADM (human breast cancer doxorubicin resistant strain); a549, a549/GR (gefitinib resistant strain for human lung cancer); a549 and a549/DDP (cisplatin resistant strain for human lung cancer) are stored in a tumor room of a medical biotechnology institute in a passage way, drug resistant cell strains are cultured in a culture medium containing corresponding drugs to maintain drug resistance, and the culture medium of the cultured cells is RPMI1640 (10% FBS) culture medium and is purchased from Hyclone company.

BALB/c female nude mice, 8 weeks old, weighing 18-22g, purchased from St Bei Fu (Beijing) laboratory animal technologies Co., ltd.

Anti-HIF-1α (sc-13515), anti-P-gp (sc-55510), anti-ERCC1 (sc-17809), anti-Stat3 (sc-8019), anti-Phospho-Stat3 (sc-8059) and Anti-P53 (sc-126) were purchased from Santa Cruz; anti-Bcl-2 (# 15017) was purchased from CST company; anti-GAPDH nude mouse monoclonal antibodies were purchased from Protein tech; HRP-labeled goat anti-nude mouse IgG secondary antibody, HRP-labeled goat anti-rabbit IgG secondary antibody, and immunohistochemical secondary antibody kit were purchased from Peking China fir gold bridge biotechnology Co., ltd; alexa Fluor 488-labeled goat anti-nude mouse IgG secondary antibodies and Alexa Fluor 488-labeled goat anti-rabbit IgG secondary antibodies were purchased from bi-cloud days.

In the present invention, "%" for explaining the concentration is "% by mass", unless otherwise specified: "is mass ratio.

EXAMPLE 1 investigation of changes in ROS content and expression levels of three proteins, HIF-1. Alpha., P-gp and ERCC1, in DDP-resistant lung cancer cells

DDP, a DNA damaging chemotherapeutic agent, also promotes the increase in intracellular free radical content, particularly ROS (reactive oxygen species ), thereby inducing death of various cancer cells. P-gp is a key transporter that actively transports DDP out of cancer cells and leads to drug resistance. Expression of P-gp has also been reported to be positively regulated by HIF-1α. HIF-1 a in tumor cells is typically activated under hypoxic conditions of the cells and alters downstream signaling pathways through transcription to promote proliferation and metastatic activity of the tumor cells. ERCC1 is used as the main regulator of DNA repair to reduce the DNA damage effect of DDP.

According to the dual functions of ROS in inhibiting tumor cell activity and increasing intracellular HIF-1 alpha expression, the ROS detection kit is used for detecting the ROS level in DDP sensitive lung cancer cells and DDP drug resistant lung cancer cells. The method comprises the following steps: H460/DDP cells well grown were digested with trypsin at 2X 10 ⁵ The cells were seeded at a density per well in a six-well plate, the original medium was removed after 24h in a carbon dioxide incubator, 2mL of the drug-containing medium containing the drug composition or DDP, and the DDP containing both the DDP and the drug composition (DDP concentrations in both the combination and single drug group dosing regimen were the same) were added to the six-well plate, the drug-containing medium was discarded after 12h in the carbon dioxide incubator, washed twice with RPMI1640 medium without serum, ROS assay diluted to 1 μm was added to the six-well plate, 1mL per well, and incubated in the carbon dioxide incubator for 20min. After washing twice with serum-free RPMI1640 medium, the sample was subjected to fluorescence microscopyThe content of ROS in the cells was observed with a mirror, or the cells were quantitatively detected by a flow cytometer after being digested with trypsin.

As shown in FIG. 1A, the green fluorescent signals of A549/DDP cells and H460/DDP cells were stronger than those of A549 cells and H460 cells, respectively, indicating that the ROS content in DDP-resistant lung cancer cells was higher.

The fluorescence intensity of ROS analyzed by flow cytometry also showed that DDP-resistant lung cancer cells had slightly higher ROS levels than DDP-sensitive cells, as shown in FIG. 1B.

Measurement of expression levels of HIF-1 a in cells by western blotting: tumor cells well grown were digested with trypsin at 5X 10 per well ⁵ The density of cells was seeded in six well plates and cultured in a carbon dioxide incubator for 24 hours. Removing the original culture medium after 24 hours, adding the pharmaceutical composition or DDP and the pharmaceutical composition-containing culture medium, removing the pharmaceutical composition-containing culture medium after 2mL per well for 24 hours, placing a six-well plate on ice to keep a low-temperature state for enzymolysis prevention, washing twice with precooled PBS, collecting cells in a centrifuge tube by using a cell scraper, lysing the cells at 0 ℃ for 30min by using RIPA lysate containing protease inhibitors to release proteins, centrifuging at 12000rpm for 15min at 4 ℃ by using a low-temperature centrifuge, taking the supernatant protein solution, discarding the precipitate, quantifying the concentration of the protein solution by using a BCA protein quantification kit, adjusting the loading buffer to 1X according to the volume of 1:4 by using 5X loading buffer and the protein solution, and adjusting the concentration of all samples to the lowest concentration level by using 1X loading buffer, wherein the concentration is noted to ensure the lowest loading amount. Finally, all samples are placed in a water bath at 100 ℃ for 5min for inactivation so as to prevent protein degradation. And adding an equal volume of sample into the gel loading hole, and then performing electrophoresis by using an electrophoresis apparatus of 200mA, and stopping electrophoresis when the bromophenol blue indicator approaches the front edge (1-2 cm) of the gel. The gel, PVDF membrane and sponge were clamped with a membrane transfer clamp and transferred using a membrane transfer instrument 150v for 2h. Then, PVDF membrane was blocked with 5% skimmed milk for 1h, the target band region was cut out, and placed in an antibody incubation box, and antibodies such as P-gp, HIF-1α, ERCC1, P53, stat3, P-Stat3, bcl2 and GAPDH were used, respectively (1:1000 dilution was performed) Release) was incubated overnight at 4 ℃. The membrane was washed three times with TBST solution for 10min each time, conjugated with the corresponding HRP-labeled goat anti-nude mouse IgG secondary antibody, HRP-labeled goat anti-rabbit IgG secondary antibody, incubated for 1h at room temperature, washed three times with TBST solution for 5min each time, and finally imaged by exposure to light using a gel imager.

Measurement of expression levels of HIF-1 a in cells by immunofluorescence experiments: the healthy tumor cells in growth state were digested with trypsin at 2X 10 ⁴ The density of each well was plated in a 24-well plate and cultured in a carbon dioxide incubator for 24 hours. After 24h, the cells were fixed for 20min at room temperature using 4% paraformaldehyde and then blocked for one hour at room temperature using 5% milk. After blocking, cells were stained with PBS for 15min 3 times each for 5min, stained with lipophilic cell membrane dye di probe, washed with PBS 3 times each for 5min, incubated overnight at 4 ℃ using P-gp, HIF-1α, ERCC1 (1:100 dilution) respectively, washed with PBST 3 times each for 5min, incubated for one hour at room temperature with the corresponding Alexa Fluor 488-labeled goat anti-nude IgG secondary antibody, alexa Fluor 488-labeled goat anti-rabbit IgG secondary antibody, washed with PBST 3 times each for 5min, and observed by fluorescence microscopy.

As shown in FIGS. 1C and D, HIF-1. Alpha., P-gp and ERCC1 were expressed at higher levels in DDP-resistant cells than in sensitive cells.

As shown in the spatial distribution of the fluorescent images of FIG. 2A, the nuclei in DDP-resistant cells had higher HIF-1. Alpha. Expression levels. It has been reported that resistance to chemotherapeutic drugs is achieved by the production of low levels of ROS in cancer stem cells. In addition, the expression of P-gp and ERCC1 proteins was also characterized by Western blotting and immunofluorescence experiments. FIG. 2A shows that both P-gp and ERCC1 proteins are significantly upregulated in DDP resistant cells.

The MTT assay was used to verify that DDP resistant cells had better viability than sensitive cells after 24 hours and 48 hours of treatment with DDP: adherent tumor cells well grown were trypsinized, prepared with RPMI1640 (10% fbs) medium to 5000 cells/100 μl of cell-containing medium, and inoculated in 96-well plates at 100 μl per well and cultured in a carbon dioxide incubator for 24 hours. Sucking out original culture medium, adding 100 μl of each well of the culture medium containing the pharmaceutical composition, DDP or both into 96-well plate, culturing in carbon dioxide incubator for 24 hr or 48 hr, adding 10 μl MTT solution into each well, continuously culturing for four hr, discarding the solution in 96-well plate, adding 150 μl DMSO into each well, shaking uniformly for 20min, measuring absorbance at 570nm wavelength of each well with enzyme-labeled instrument, and calculating survival rate of each administration group with absorbance value of control group.

The results are shown in FIG. 2B: there was a significant statistical difference in cell survival between the two pairs of DDP-resistant lung cancer cells and sensitive lung cancer cells. H460/DDP cells had a cell viability of greater than 60% even after 24 hours treatment with 32. Mu.g/mL DDP.

EXAMPLE 2 investigation of the Effect of pharmaceutical compositions and DDP combinations on H460/DDP cell growth in vitro

Cytotoxicity of the pharmaceutical composition, DDP single or combination, on DDP-resistant lung cancer cells H460/DDP was determined using the MTT method as in example 1 for 24 hours and 48 hours.

The results showed that the cell viability of H460/DDP cells at low concentrations (20. Mu.g/mL) was almost 100%. After 24 hours and 48 hours of treatment with the pharmaceutical composition alone, the cell viability of the H460/DDP cells was almost 100% at low concentrations (20. Mu.g/mL), and was higher than 60% even at high concentrations (80. Mu.g/mL) (FIG. 3A), indicating that the pharmaceutical composition had low cytotoxicity to the H460/DDP cells.

FIG. 3B shows that H460/DDP cells significantly decreased cell viability following combination treatment at the same concentration of DDP and at a concentration of pharmaceutical composition between 20 μg/mL and 40 μg/mL. Thus, it is assumed that 20 μg/mL and 40 μg/mL of the pharmaceutical composition are appropriate combined concentrations with DDP. FIG. 3C is a graph of H460/DDP cells treated with a 20 μg/mL and 40 μg/mL constant concentration pharmaceutical composition in combination with a range of concentrations of DDP to evaluate synergistic inhibition, which results show significant dose and time dependent toxicity, greater than either the pharmaceutical composition or the DDP alone regimen.

Example 3 investigation of the Effect of a combination of a pharmaceutical composition and a plurality of anti-tumor Agents on the growth of a plurality of tumor-resistant cells in vitro

The effect of the combination of the pharmaceutical composition with other drugs on the growth of drug-resistant cells in vitro was evaluated by half inhibitory concentration IC 50:

1. effects of DDP in combination with pharmaceutical compositions of different concentrations on H460/DDP

IC50 values of DDP, DDP+20. Mu.g/mL DBDx, and DDP+40. Mu.g/mL DBDx in H460/DDP cells after 24H and 48H of treatment were examined, respectively. As a result, as shown in FIG. 4B, it was found that the IC50 values of the two dosing regimens of DDP and pharmaceutical composition 40. Mu.g/mL+DDP were 34.91 and 8.77. Mu.g/mL, respectively, at 24 hours and 13.23 and 0.49. Mu.g/mL, respectively, at 48 hours.

The resistance reversal ability of the combination of the pharmaceutical composition and DDP is quantitatively determined by calculating a resistance reversal index. The higher the resistance reversal index value, the stronger the ability to reduce resistance.

Drug resistance reversal index = IC ₅₀ _DDP ÷IC _{50 pharmaceutical composition+DDP}

FIG. 4C shows that DBDx 40 μg/mL+DDP has a resistance reversal index value above 10 at 48 hours, indicating strong resistance to reversal.

The synergy between the pharmaceutical composition and DDP was analyzed using a Combination Index (CI). In accordance with CI theory, ci=1 represents the pharmacodynamic additive effect, CI >1 represents the pharmacodynamic antagonistic effect, and CI <1 represents the pharmacodynamic synergistic effect. The Chou-TAlay plot of fig. 4A shows that all CI values for DBDx 20 μg/ml+ddp and DBDx 40 μg/ml+ddp are well below 1, indicating significant synergy.

2. Investigation of effects of combination of pharmaceutical composition DBDx and gemcitabine on AsPC-1 gemcitabine resistant cells, combination of pharmaceutical composition and cisplatin on H460 resistant tumor treatment cells, combination of pharmaceutical composition and gefitinib on A549 gefitinib resistant cells, combination of pharmaceutical composition and doxorubicin on MCF-7 resistant cells

The effect of DBDx on AsPC-1 cells, asPC-1 gemcitabine resistant cells, H460 cisplatin resistant cells, A549 cells and A549 gefitinib resistant cells, MCF-7 and MCF-7 doxorubicin resistant cells were first evaluated in the same manner.

Next, IC50 of 2. Mu.g/mL of DBDx+gemcitabine resistant cells (AsPC-1/GEM) to AsPC-1 cells and AsPC-1 gemcitabine resistant cells (AsPC-1/GEM), 4. Mu.g/mL of DBDx+cisplatin to H460 cells and H460/DDP cells, 8. Mu.g/mL of DBDx+doxorubicin resistant cells (MCF-7/ADM) to MCF-7 and MCF-7 doxorubicin resistant cells (MCF-7/ADM), 10. Mu.g/mL of DBDx+gefitinib to A549 cells and A549 gefitinib resistant cells (A549/GR) after 48H of treatment were examined, and the results are shown in Table 1:

TABLE 1 IC50 of different regimens for multiple cells and resistant cells

The results show that compared with single administration of gemcitabine, cisplatin, doxorubicin or gefitinib, the composition has obviously reduced IC50 for various tumor drug-resistant cells when being combined with the pharmaceutical composition DBDx, and the pharmaceutical composition DBDx and various medicaments are proved to have obvious drug resistance reversing capability for the drug-resistant cells when being combined.

Example 4 investigation of intracellular ROS and drug resistance related proteins HIF-1α, P-gp and ERCC1 changes under the Combined action of DBDx and DDP

It is known that a substantial increase in ROS can break the balance between low levels of ROS and DDP resistance, thereby enhancing apoptosis and restoring sensitivity of tumor cells to DDP.

FIG. 5A shows that fluorescence images after 12 hours of treatment with DDP 4 μg/mL, DBDx (20 and 40 μg/mL), DDP+DBDx combinations (20+4 μg/mL and 40+4 μg/mL), respectively, show that cells of the combination group show stronger green signals, indicating that the pharmaceutical composition can enhance intracellular ROS production when combined with DDP compared to other treatment groups.

Quantitative analysis of ROS signaling by flow cytometry also revealed an amplification of ROS production by DBDx in the combination group, with the median fluorescent signal intensity of the combination of the pharmaceutical composition and DDP (40+4. Mu.g/mL) being almost 2 times that of the DDP 4. Mu.g/mL treatment group (FIG. 5B).

Furthermore, ATPase activity of P-gp after treatment with a range of concentrations of pharmaceutical composition DBDx, the inhibition of ATPase by the pharmaceutical composition increased with increasing DDP concentration (FIG. 5C). Then, after treating drug-resistant cells H460/DDP for 12H with a combination of DDP (4. Mu.g/mL) and a series of concentrations of DBDx, the intracellular concentration of DDP in the H460/DDP cells was detected. Figure 5D shows that intracellular DDP concentrations were higher in the combination group cells than in the DDP single drug group and increased with increasing concentration of the pharmaceutical composition.

These results indicate that the pharmaceutical composition DBDx can increase the intracellular DDP content by inhibiting P-gp expression and P-gp ATPase activity, and simultaneously reduce the expression of DNA repair protein ERCC1 so as to enhance the damage effect of DDP on DNA.

The expression level of HIF-1. Alpha. Protein was studied in various treated H460/DDP cells by Western blotting in the same manner as in example 1.

As shown in FIG. 6A, HIF-1. Alpha. Protein was significantly down-regulated after 24 hours of treatment with DBDx alone or in combination with DDP, in contrast to other studies in which HIF-1. Alpha. Expression levels increased with increasing ROS levels. This suggests that DBDx may increase ROS levels to enhance apoptosis while reducing HIF-1. Alpha. Protein to prevent resistance by downstream related drug resistant proteins induced by HIF-1. Alpha.

Accordingly, the expression of the key transporter P-gp and the DNA repair protein ERCC1 was observed using Western blotting, and the DBDx group and co-administration group treatments significantly reduced the expression of P-gp and ERCC1, similar to HIF-1α (FIG. 6A).

These results indicate that the pharmaceutical composition DBDx can increase the intracellular DDP content by inhibiting P-gp expression, and simultaneously reduce the expression of DNA repair protein ERCC1 so as to enhance the damage effect of DDP on DNA. The expression levels of HIF-1α, P-gp and ERCC1 in the combination showed statistically significant differences compared to the control and DDP groups (FIG. 6B).

Analysis of expression data of HIF-1. Alpha. And ERCC1 clinical samples from 500 lung adenocarcinoma patients in the TCGA database, and evaluation of the effects of HIF-1. Alpha. And ERCC1 on prognosis. As shown in FIG. 6C, overexpression of HIF-1α and ERCC1 results in shorter survival times in lung adenocarcinoma patients. This suggests that better prognosis for lung adenocarcinoma patients may be obtained by inhibiting the expression of HIF-1 a and ERCC 1.

EXAMPLE 5 investigation of the Effect of the combination of pharmaceutical compositions DBDx and DDP on H460/DDP apoptosis

DDP achieves a significant tumor-inhibiting effect in the treatment of cancer patients by inducing apoptosis of a variety of tumor cells. To examine the effect of the combination of DBDx and DDP on apoptosis, H460/DDP cells were treated with DDP 4 μg/mL, DBDx (20 and 40 μg/mL), DBDx and DDP combinations (20+4 μg/mL, and 40+4 μg/mL), respectively, for 24 hours, and then the change in Mitochondrial Membrane Potential (MMP) of the cells of each dosing group was detected by JC-1 probe. The method comprises the following steps:

tumor cells in good growth state were digested with trypsin, plated in six well plates at a density of 20 ten thousand cells per well, and cultured in a carbon dioxide incubator for 24 hours. After 24h the original medium was removed, 2ml of drug-containing medium containing the pharmaceutical composition, DDP or both was added per well, 24h the drug-containing medium was removed, the cells were washed three times with PBS, incubated with JC-1 solution for 20min at 37 ℃, then washed twice with pre-chilled JC-1 fuel buffer, fluorescence was observed with a fluorescence microscope and photographed.

The results are shown in FIG. 7A: changes in MMP are often used as an indicator of early apoptosis, where green fluorescence indicates that MMP is at low levels and JC-1 exists as a monomer. Red fluorescence indicates that MMPs are at high levels, JC-1 aggregates in the mitochondrial matrix, and cells are in a healthy state. FIG. 7A shows that cells treated with DBDx (20 and 40. Mu.g/mL) showed a medium green signal, which means that DBDx can induce early apoptosis compared to control and DDP treated groups. In contrast, the combined treatment of DBDx and DDP (20+4. Mu.g/mL and 40+4. Mu.g/mL) significantly enhanced the green signal in the cells, indicating that the apoptosis-inducing effect of DDP was restored by the pharmaceutical composition, and that DBDx and DDP synergistically increased the early apoptosis rate of the cells.

The apoptosis rate of the cells was quantitatively analyzed in H460/DDP cells using flow cytometry. The results showed that apoptosis rates after 24 hours were significantly higher for the DBDx and DDP combinations (20+4. Mu.g/mL and 40+4. Mu.g/mL) than for the other treatment groups, reaching 35% and 40% above, respectively (FIGS. 7B and 7C).

Further examine the underlying mechanism of apoptosis inducing effect in the combination treatment group: the expression levels of signal transduction and transcriptional activator 3 (Stat 3), P-Stat3 and apoptosis-related proteins P53 and Bcl-2 were studied using western blotting. Among them, stat3 is considered to be an important regulatory protein that promotes tumor cell survival, apoptosis and proliferation, and Bcl2 expression is closely related to Stat 3. P53 is critical for inhibiting tumor progression and plays an important role in cancer cell apoptosis by disrupting DNA. P53 has been reported to enhance apoptosis by binding to B cell lymphoma 2 (Bcl 2). Bcl-2 as an anti-apoptotic protein can inhibit the apoptosis-inducing effects of many cytotoxins.

The results are shown in FIG. 8A, where the combination of high concentrations of DBDx and DBDx+DDP increased P53 expression and decreased Stat3, P-Stat3 and Bcl-2 expression compared to the control group. FIG. 8B shows that there are significant statistical differences in the expression levels of P53, stat3, P-Stat3 and Bcl2 in drug-resistant tumor cells treated in the combination of the dosing group and the control group.

The results demonstrate that the combination of the pharmaceutical composition DBDx and DDP enhances the apoptosis-inducing effect of P53 and inhibits the anti-apoptotic effects of Stat3 and Bcl-2.

Example 6 investigation of synergistic anti-tumor Effect of pharmaceutical composition and DDP combination on H460/DDP nude mice tumor transplantation model

Evaluation of the synergistic therapeutic effect of the pharmaceutical compositions DBDx and DDP in combination in the H460/DDP model: the H460/DDP tumor cells with good growth state are digested by trypsin every 1×10 ⁷ The H460/DDP cells were suspended in 200. Mu.L PBS and then subcutaneously injected into the right side underarm of nude mice until the average volume of the nude mice subcutaneously transplanted tumor exceeded 100mm ³ At this time, 24 nude mice were equally distributed to four treatment groups, receiving oral treatment with DBDx or intraperitoneal injection of DDP, respectively. The four treatment groups were: physiological saline, DDP 4mg/kg, DBDx 121mg/kg, DDP+DBDx (4 mg/kg+121 mg/kg), wherein DBDx is orally administered 1 time per day, 10 times per day, and DDP is administered by intraperitoneal injection, once every 5 days, twice per day. Detecting the change condition of the weight and the tumor volume of each nude mouse every two days, and detecting the specific length (a) and the specific width (b) of the tumor body by using a vernier caliper, wherein the calculation formula of the tumor volume is as follows V＝(a×b ² ) 2, to average tumor volume over 2000mm ³ At this time, the nude mice were euthanized, and tumors and individual organs of each nude mouse were collected, weighed and tumor inhibition was calculated.

Average tumor size of nude mice in three treatment groups reaches 100mm ³ Followed by oral administration of DBDx and intraperitoneal administration of DDP (FIG. 9A). All tumors were collected after the last dose to confirm the therapeutic effect of the different protocols, as shown in fig. 9B.

All tumors were weighed (fig. 9C) and tumor inhibition rates were calculated for the four treatment groups using tumor weights. The results showed that the tumor inhibition rate of the combination of DBDx and DDP (121+4mg/kg) was 70.5% (FIG. 9D), which is significantly higher than that of the 121mg/kg group (29.51%) and DDP 4mg/kg group (36.82%) of the pharmaceutical composition. The results show that the combination of DBDx and DDP has good synergy.

Then, the antitumor efficacy of the different treatment groups was evaluated by tumor growth curves, respectively (fig. 10A). The tumor growth of the nude mice given DDP was faster and there was no statistical difference from the control nude mice. This suggests that DDP chemotherapy fails to inhibit tumor growth of DDP-resistant lung cancer. DBDx treatment showed a weaker tumor growth inhibition in nude mice, mainly due to the weaker induction of apoptosis in DDP resistant tumors. Administration of DBDx in combination with DDP almost completely inhibited tumor growth, which was clearly associated with a decrease in DDP resistance protein and repair of the apoptotic effects of DDP following DBDx combination. Significant statistical differences in tumor volumes were observed between the combination and DBDx groups, the combination and DDP groups (fig. 10B).

The body weight of the nude mice was monitored during the treatment period to evaluate the side effects of the various treatments. The body weight of the treated group was not affected compared to the control group (fig. 10C).

In addition, after the last administration, major organs of nude mice including heart, liver, spleen, lung, kidney, intestine and bone and tumor were collected, tumor and tissue organs were fixed with 10% formaldehyde fixing solution for 12 hours, bone tissue was decalcified with 5% nitric acid solution additionally, then dehydrated, dehydrated and transparentized with transparent agent xylene, and the permeabilized tissue and tumor were embedded with paraffin. The H & E stained chips were used after slicing, oven drying, dewaxing and the observations are shown in fig. 11.

The results show that no significant damage was observed in the drug compositions DBDx and DDP combined treatment stained slides compared to the control group.

EXAMPLE 7 investigation of the Effect of the combination of pharmaceutical compositions DBDx and DDP on the expression of HIF-1 alpha, P-gp and ERCC 1-resistance-associated proteins in tumor tissue of H460/DDP nude mouse tumor transplantation model

Tumor tissue slices of four experimental groups are dewaxed, the dewaxed tumor tissue slices are washed three times by PBS, endogenous catalytic enzymes are removed by a citrate buffer solution for antigen retrieval treatment, firstly, the citrate buffer solution is heated to boiling, the tissue slices are placed in the boiling saline solution for 5min, and the slices are washed 3 times by PBS after the citrate solution is naturally cooled. Dripping endogenous peroxidase blocking agent on each tissue slice for incubation for 10min, washing the slices with PBS for 3min each time, incubating the three antibodies of P-gp, HIF-1 alpha and ERCC1 for 2h at 37 ℃ in total for 3 batches of four groups of tissue slices respectively, washing the slices with PBS for 3 times after 2h, carrying out coupling reaction with the corresponding enhancing enzyme marked goat anti-nude mouse IgG secondary antibody or enhancing enzyme marked goat anti-rabbit IgG secondary antibody for 30min each time, washing the tissue slices with PBS for 3min each time, dripping DAB color development liquid onto the tissue slices for combining with the secondary antibody, washing residual dye liquid immediately after the color development of the tissue slices, and finally counterstaining cell nuclei with hematoxylin, photographing with a microscope and analyzing pictures with Image J software.

To verify the effect of DBDx on DDP resistance-associated protein, HIF-1 a protein expression levels in tumor sections were observed by immunohistochemical assay, and as shown in fig. 12A, the combination of pharmaceutical composition and pharmaceutical composition + DDP significantly reduced HIF-1 a expression in H460/DDP tumor model compared to control and DDP-administered groups. Semi-quantitative analysis of HIF-1α expression indicated a significant statistical difference between the pharmaceutical composition or combination and the control or DDP group (fig. 12B).

The result shows that the pharmaceutical composition DBDx can obviously inhibit the expression of HIF-1 alpha of an H460/DDP tumor model, inhibit the proliferation and drug resistance of tumors, and the like.

The expression level of P-gp protein in tumor sections was observed by immunohistochemical assay, as shown in FIG. 13A, and the pharmaceutical composition and combination of pharmaceutical composition and DDP significantly reduced the expression of P-gp in H460/DDP tumor model compared to the control group and DDP-administered group. Semi-quantitative analysis of P-gp expression indicated a significant statistical difference between the pharmaceutical composition or combination group and the control or DDP group (fig. 13B).

The results demonstrate that the pharmaceutical composition DBDx can significantly inhibit the expression of P-gp and improve the accumulation of DDP in cells.

Expression levels of ERCC1 protein in tumor sections were observed by immunohistochemical assay. Results as shown in fig. 14A, the combination of DBDx and dbdx+ddp significantly reduced ERCC1 expression in the H460/DDP tumor model compared to the control and DDP-dosed groups. Semi-quantitative analysis of ERCC1 expression indicated a significant statistical difference between the DBDx or combination group and the control or DDP group (fig. 14B).

The results demonstrate that DBDx can significantly inhibit ERCC1 expression, thereby reducing ERCC1 repair of DNA damage.

In summary, by comparing a plurality of groups of cancer cells and drug-resistant cells thereof (including DDP drug-resistant cells, gemcitabine drug-resistant cells, doxorubicin drug-resistant cells and gefitinib drug-resistant cells), the present invention has an increased intracellular hypoxia status of drug-resistant cells compared to sensitive cells, and therefore, drug-resistant cells show up-regulation of HIF-1α expression, which is a key transcription regulator of cells to hypoxia response. The increase in intracellular hypoxia may be a significant feature of drug resistance, however, a substantial increase in the degree of hypoxia induces apoptosis in tumor cells. Thus, the discovery of increased intracellular hypoxia in drug resistant cells may motivate the discovery of potential mechanisms and better understanding of resistance problems.

On the basis, the invention combines the cell tolerance dose antitumor drug and the drug composition, DDP further promotes the increase of the intracellular hypoxia degree (ROS) so as to induce the massive generation of HIF-1 alpha to form stronger drug resistance, activates downstream channels to promote cell proliferation and the like, however, the drug composition can effectively inhibit the generation of HIF-1 alpha in tumor cells, thereby avoiding further aggravation of drug resistance, and enhancing the hypoxia state so as to promote apoptosis of tumor cells.

In vitro cell experiments show that the enhanced intracellular hypoxia level and the great reduction of HIF-1 alpha can enhance the cytotoxicity and the biological activity of the antitumor drug. Meanwhile, the pharmaceutical composition DBDx obviously reduces the expression of P-gp and inhibits the transport activity of P-gp, thereby increasing the content of the medicine in cells; the pharmaceutical composition can also obviously reduce critical DNA repair speed-limiting step protein ERCC1, and under the synergistic effect of the three aspects, the pharmaceutical composition shows extremely strong drug resistance reversing capability of cisplatin-resistant non-small cell lung cancer cells, gemcitabine-resistant metastatic pancreatic adenocarcinoma cells, cisplatin-resistant large cell lung cancer cells, doxorubicin-resistant breast cancer cells and gefitinib-resistant non-small cell lung cancer cells. As a demonstration, the efficacy of the synergistic treatment of the pharmaceutical composition DBDx was further verified on in vivo nude mouse tumor models. The results demonstrate that the new drug combination methods identified have substantial benefits in overcoming the major problems associated with multi-tumor drug resistant cancer chemotherapy.

Therefore, the pharmaceutical composition DBDx is expected to be applied to the following fields: the pharmaceutical composition DBDx is used for tumor treatment alone; the drug combination of the DBDx and the anti-tumor drug can prevent the drug resistance of the anti-tumor drug and realize the attenuation and synergy; after tumor drug resistance, the drug combination of the drug composition DBDx and the anti-tumor drug can reverse drug resistance. Based on the characteristic of the comprehensive effect of the drug composition DBDx, the drug composition is also expected to be expanded to drug resistance treatment of wide drugs.

Claims

1. The application of the pharmaceutical composition in preparing medicines for treating tumor resistance comprises a component A, a component B and a component C,

2. Use according to claim 1, characterized in that the mass ratio of component a, component B and component C is 100:20:1 in terms of the mass ratio of dipyridamole, ubenimex and dexamethasone.

3. The use according to claim 1, characterized in that the tumor comprises lung cancer, breast cancer or pancreatic cancer.

4. Use according to claim 1, characterized in that the tumor resistance comprises tumor cisplatin resistance, tumor doxorubicin resistance, tumor gemcitabine resistance or tumor gefitinib resistance.

5. Use according to claim 1, characterized in that the pharmaceutical composition achieves reversal of tumor resistance by inhibiting P-gp expression and P-gp ATPase activity, thereby increasing the intracellular drug content.

6. The use according to claim 1, characterized in that the pharmaceutical composition reduces the expression of the excision repair complementary protein ERCC1, and the reduction of DNA repair capacity effects reversal of tumor resistance.

7. Use according to claim 1, characterized in that the pharmaceutical composition achieves reversal of tumor resistance by increasing ROS levels to enhance apoptosis, decreasing HIF-1 a protein expression to prevent HIF-1 a induced downstream drug resistance protein induced resistance.

8. Use according to claim 1, characterized in that the pharmaceutical composition achieves reversal of tumor resistance by enhancing the apoptosis-inducing effect of P53 protein and inhibiting the anti-apoptotic effect of Stat3 protein and Bcl-2 protein.