| Ovarian cancer,one of the three most common Ovarian cancers in the female reproductive system,is by far the cause of the highest mortality of all gynecological malignancies,causing a severe threat to women’s lives and health.Because of the hidden malignancy of ovarian cancer,many ovarian cancers are diagnosed at an advanced stage,resulting in a low overall survival rate for ovarian cancer patients.The current treatment methods for ovarian cancer are mainly chemotherapy and surgery In the initial treatment of ovarian cancer patients,the successful effect of surgery is an independent factor affecting the prognosis of patients.For patients with advanced stage,initial surgery emphasizes satisfactory tumor cell reduction.In addition to surgical treatment,platinum combined with paclitaxel is another gold standard for the treatment of ovarian cancer.The effective rate of first-line platinum-containing chemotherapy for ovarian cancer is 70%,and even if a complete response is achieved,there are still 50%～70%of patients will relapse.Although the treatment of ovarian cancer has been updated in recent years,there is no uniform treatment for recurrent ovarian cancer.The initial treatment of ovarian cancer is standardized,while the treatment of recurrent ovarian cancer is individualized.The main treatment methods include second-line chemotherapy,secondary tumor cell reduction,targeted therapy,endocrine therapy,and radiotherapy.According to the time of discontinuation of platinum drugs,the recurrence of ovarian cancer can be divided into platinum sensitive relapse(platinum drugs>6 months discontinuation and disease recurrence)and platinum resistant relapse(platinum drugs discontinuation<6 months relapse).For patients with platinum-resistant recurrence,the guidelines recommend alternative non-platinum chemotherapy monotherapy(docetaxel,etoposide,gemcitabine,liposomal doxorubicin,paclitaxel,topotecan),targeted therapy(bevacizumab,olapary),non-platinum chemotherapy,and bevacizumab combination therapy.For first-line purple shirt drugs combined with platinum regimen chemotherapy,second-line docetaxel monotherapy patients,there is a potential for drug resistance.The mechanism of multidrug resistance(MDR)is involved in a variety of intracellular drug transmembrane carriers and signaling pathways.Among them,the most widely studied mechanisms with clear clinical significance include:① Drug target mutation;②Reduction in drug intake or inflow;③Activation of compensatory cell survival signaling pathway;④DNA damage repair pathway increased;⑤The expression of drug efflux pump was increased.Juliano and Ling proposed "classical MDR" in 1976,which refers to reducing the concentration of drug-resistant cells through the efflux pump action of transmembrane transporters.P-glycoproteins(P-glycoprotein P-gp),also known as ABC transporter B family member 1(ATP-binding cassette subfamily B member 1,ABCB1);or multidrug resistance protein 1(Multidrug resistance protein 1,MDR1),Additional multidrug resistance-associated protein-1(Multidrug resistance-associated protein 1,MRP1),ABCC and breast cancer resistance proteins Breast cancer resistance protein,BCRP,ABCG2)are also members of the superfamily most often involved in drug-resistant chemotherapy.According to researchers,most of these transmembrane transporters are ABC transporters.More than 100 species have been identified,with a ABCB1 relative molecular mass of 170×103,a ATP dependent membrane protein consisting of 1280 amino acid residues.in addition to cancer cells,ABCB1 are highly expressed on the apical surface of epithelial cells,such as colon,hepatobiliary duct,proximal convoluted tubules,pancreatic tubules,adrenal gland,placenta(blood-placental barrier),testis(blood testis barrier),and cerebral capillaries(blood-brain barrier).Anatomically,ABCB1 acts as an efflux transporter that limits cellular uptake of drugs from the blood into the brain and from the intestinal lumen into the intestinal cells.On the other hand,ABCB1 promote drug excretion into bile and urine in hepatocytes and epithelial cells,respectively.ABCB1 consists of two homologous nucleotide binding regions(nucleotide binding domain,NBD)and two transmembrane regions(transmembrane domain,TMD)connected by ligon regions.Each transmembrane domain consists of 6 transmembrane helices comprising 12 transmembrane helical efflux pumps combined with hydrophobic drug substrates.Its hydrophilic region contains ATP binding sites that bind two ATP molecules.The efflux of drug substrates results in ATP hydrolysis to ADP and inorganic phosphates,allowing the transmembrane domain to bind another efflux substrate.This continuous circulation leads to a low intracellular concentration of substrate drugs,which leads to drug resistance in tumor cells.ABCB1 anticancer drug substrates include taxanes(paclitaxel,docetaxel),anthracyclines(doxorubicin,rubicin),vincristine alkaloids(vincristine,vincristine),phloretinoids(etoposide,teniposide),and tyrosine kinase inhibitors(tyrosine kinase inhibitors,TKIs)such as lapatinib,erlotinib,sunitinib,etc.Overexpression of ABCB1 is associated with a variety of cancers,in addition to solid tumors,it also includes hematological malignanciesFor ABCB1,there are many ways to reverse cancer resistance:the main results are as follows:① Application of chemical reversal agents;②To change the way and system of clinical drug use in release;③Combined with other drugs to improve drug effectiveness or reduce drug toxicity;④Reverse resistance at gene level;⑤Rapid and efficient application of nanocarrier system to tumor;⑥Development of new drugs.ABCB1 inhibitors have undergone three generations of development,the first generation of inhibitors are mainly found in accidental conditions,including verapamil,quinidine,amiodarone and so on.In the process of application,they are gradually replaced by organs such as the kidney and gradually replaced by the second generation of inhibitors.Compared with the first-generation inhibitors,the second-generation inhibitors will enhance the activity of the drug and have a better therapeutic effect on tumors.The main clinical use is dextral verapamil,PSC833 and so on.However,the second generation inhibitor has serious damage to the detoxification function of the liver,and the long-term application will cause the metabolic disorder of the drug and endanger the health of the body.Third-generation ABCB1 inhibitors such as dofequidar,zosuquidar,etc.,these drugs have stronger effects and fewer pharmacokinetic interactions,but when used in combination,they can cause some chemotherapy toxicity.Although the current research on tumor inhibitors is in-depth,there is still no relevant report of MDR reversal agents applied in clinical practice.Targeted drugs such as TKIs play an important role in tumor treatment,but TKIs and traditional anticancer drugs rarely have a synergistic effect.Because the main side effects of TKIs and traditional anticancer drugs do not overlap,at the same time,TKIs can bind to the ATP binding site of tyrosine kinase,thereby inhibiting the function of the ABC transport pump.Studies have found that afatinib,erlotinib,lapatinib and other clinically commonly used drugs inhibit the function of ABCB1 by acting on their target protein ABCB1,thereby increasing the accumulation of substrate anticancer drugs in MDR cells to increase The anti-tumor effects of traditional anti-cancer drugs in vivo and in vitroApatinib(YN968D1)is a small molecule tyrosine kinase inhibitor independently developed by my country.It acts on vascular endothelial growth factor receptor 2(VEGFR-2)to selectively block signal transduction.Play the role of anti-angiogenesis,and then control tumor occurrence.In December 2014,apatinib was approved for the treatment of advanced gastric cancer that failed second-line chemotherapy.At present,a number of clinical studies have been carried out in lung cancer,liver cancer.esophageal cancer,osteosarcoma and other tumor types,especially its combined chemotherapy,targeted or immunotherapy to explore the sensitization of combined therapy and the reversal of drug resistance related clinical research is ongoing Achieve initial results.Clinical studies have shown that apatinib has a positive effect in the treatment of ovarian cancer.In summary,the related research of apatinib in the treatment of drug-resistant advanced solid tumors has gradually become a focus of clinical research.So far,there is no research on the synergistic mechanism of apatinib and docetaxel in the treatment of ovarian cancer.This project intends to study the effect of apatinib on ABCB 1 mediated resistance to docetaxel in ovarian cancer through clinical,in vivo,and in vitro,and to explore the treatment of apatinib combined with docetaxel in metastatic ovarian cancer The synergistic mechanism of action provides new treatment ideas for patients with recurrent and metastatic ovarian cancerPart Ⅰ Efficacy and safety of apatinib combined with docetaxel compared with docetaxel alone in the treatment of metastatic recurrent ovarian cancerPurpose:Efficacy and safety studies discussion of treatment of second-line and second-line above metastatic recurrent ovarian cancer between apatinib mesylate combined with docetaxel.Methods:Clinically,47 patients with platinum-resistant recurrent and metastatic ovarian cancer were collected and divided into 2 groups according to the treatment plan:chemotherapy alone group(24 cases),apatinib combined with chemotherapy group(23 cases).Specific administration method:patients in the chemotherapy-only treatment group were treated with docetaxel single-agent chemotherapy,and the apatinib combined chemotherapy group was treated with docetaxel and apatinib combined treatment to compare the efficacy and disadvantages of these two groups of ovarian cancer patients When the reaction occurred,the blood concentration of docetaxel was monitored,and the relationship between the blood concentration and the efficacy of the two groups of patients was studied and analyzedResults:1.The ORR and DCR of the apatinib combined with chemotherapy group were better than the chemotherapy group alone.Among the chemotherapy group,there were 0 cases of CR,4 cases of PR,7 cases of SD,31 cases of PD,the objective remission rate was 16.67%,disease control rate was 45.83%;among the combined chemotherapy group with apatinib mesylate,there were 0 cases of CR,11 cases of PR,8 cases of SD,4 cases of PD,the objective remission rate was 47.83%,disease control rate was 82.61%,there were statistical significance of objective remission rate comparative differences between two groups(P=0.022),there were statistical significance of disease control rate comparative differences between two groups(P=0.009).2.The median PFS of the apatinib combined with chemotherapy group was better than that of the chemotherapy group alone.Median PFS of chemotherapy group is 3.2 months(range from 1.8～5.1),median PFS of combined chemotherapy group with apatinib mesylate is 8.8 months(range from 7.5～10.1),PFS of combined chemotherapy group with apatinib mesylate is significantly higher than chemotherapy group,under inspection of log-rank,there was statistical significance of differences(χ2=17.22,P<0.01)3.The median OS in theapatinib combined with chemotherapy group was better than that in the chemotherapy group alone.The median OS in the chemotherapy alone group was 10(8.44-11.56)months,and the median OS in the chemotherapy combined with apatinib group was 16.7(14.84-18.56)months.The survival time of the chemotherapy combined with apatinib group was longer than that in the chemotherapy alone group.Long,after log-rank test,there is a significant difference between the two(x2=44.35,P<0.01)Common adverse reactions in the apatinib combined with chemotherapy group were hypertension,hand-foot syndrome,and proteinuria.Compared with the chemotherapy alone group,the common adverse reactions in the chemotherapy combined with apatinib group were hypertension(52.17%),hand-foot syndrome(34.78%),proteinuria(30.43%),which were statistically significant,and the remaining adverse reactions There was no significant difference between the two groups4.The AUC of docetaxel is positively correlated with the efficacy.There were liner relation between AUC and therapeutic evaluation.χ2=6.224,P=0.013.Pearson relevant results shows that,R=-0.368,P=0.011,which reveals that there were liner relation between AUC and therapeutic evaluation,there were positive correlation between AUC and therapeutic evaluationConclusion:Apatinib combined with docetaxel in the treatment of platinum-resistant recurrent and metastatic ovarian cancer has obvious advantages over chemotherapy alone in both the short-term and long-term effects,and the safety is better,only hypertension and hand-foot syndrome The incidence of protein Niuria is slightly higher than that of the single chemotherapy group.Apatinib combined with docetaxel has a significant therapeutic effect,which provides support for wide clinical applicationPart Ⅱ The role of docetaxel resistance in ovarian cancer involved with level of apatinib mesylate reversed ABCB1Purpose:To construct a drug-resistant tumor-bearing nude mouse animal model to verify whether apatinib can synergize drug-resistant ovarian cancer cells on the anti-tumor effect of the ABCB 1 transporter substrate chemotherapeutic drug docetaxel.The synergistic anti-tumor effect of tinib and docetaxel on ovarian cancer in vivo provides laboratory evidence for improving the treatment of ovarian cancerMethods:1.Construction of ovarian cancer drug-resistant BALB/c tumor-bearing nude mice animal model,a)control group,no drug intervention;b)docetaxel group intraperitoneal injection of 8 mg/kg docetaxel;c)apatinib group:intraperitoneal injection of 100 mg/kg apatinib;d)apatinib combined with docetaxel group:intraperitoneal injection of 8 mg/kg docetaxel+100 mg/kg apatinib2.LC-MS/MS method was used to detect the concentration of docetaxel in tissue and plasma.3.Apoptosis was detected by TUNEL assay4.Western blot was used to detect the changes of p-GP protein levels in cells5.Qrt-pcr was used to detect the mRNA expression of ABCB1 transporter.Results:1.Apatinib enhances the inhibitory effect of docetaxel on the growth of ES-2/D cell xenograft tumors.There was no significant difference in initial body weight of mice in each group(F=0.347,p=0.792),and the balance between groups was good.The mice in the docetaxel group were all lower than the other three groups on the 20th day,and the differences were statistically significant(P<0.01).There are significant differences in the volume of transplanted tumors between different treatment groups.The tumor volume of mice in the control group,apatinib group,and docetaxel group increased significantly(t=60.96,P<0.01;t=22.15,P<0.01;t=10.81,P<0.01),while the tumor volume of mice in the apatinib combined with docetaxel group was smaller than the initial volume,the difference was statistically significant(t=7.45,p=0.001).The tumor suppression rate in the control group was 1.89%;the tumor suppression rate in the apatinib alone treatment group was 82.00%;the tumor suppression rate in the docetaxel treatment group was 66.54%;and the apatinib combined with docetaxel was used The tumor inhibition rate of mice in the group was 111.46%,and the tumor inhibition rate of mice in the combined treatment group was higher than that in the single treatment group(P<0.05),and the difference was statistically significant2.Apatinib increases the concentration of docetaxel in mouse tumor tissues.The difference in the concentration of docetaxel and apatinib in tumors is statistically significant(t=12.78,P<0.01),but there is no significant difference in the concentration in plasma,kidney,liver,lung and heart(t=1.56,p=0.148;t=0.028,p=0.978;t=0.544,p=0.598;t=0.436,p=0.672;t=0.1 18,p=0.909).3.Apatinib potentiates docetaxel-induced ES-2/D cell apoptosis.After TUNEL staining,the immunofluorescence results found that,compared with the control group,docetaxel group and apatinib group,the combined use of docetaxel and apatinib will cause obvious nuclear fragmentation,which is more obvious.Many green fluorescent TUNEL stained positive cells4.Apatinib down-regulates the expression of ABCB1 transporter mRNA in mouse tumor tissues.The qRT-PCR experiment was used to detect the expression level of ABCB1 transporter mRNA in the tumor tissues of four groups of mice,the control group,apatinib group,docetaxel group and docetaxel combined with apatinib group mice The expression level of ABCB1 transporter mRNA in tumor tissues decreased sequentially.One-way analysis of variance showed that the expression levels of different treatment groups were significantly different(F=174.19,P<0.01).Pairwise comparison with Turkey test showed that the expression levels of ABCB1 transporter mRNA in each treatment group were different All were statistically significant(P<0.01).Therefore,the docetaxel combined with apatinib administration group can significantly reduce the expression of ABCB1 transporter in tumor tissues at the mRNA level.5.Docetaxel combined with apatinib reduced the expression of P glycoprotein in tumors.The results of qRT-PCR showed that the gene expression level of MDR1 in tumor tissues increased significantly after docetaxel treatment(P<0.01),and the gene expression levels of BCRP and MRP2 in intestinal tissues did not change significantly after drug treatment.Western blot results showed that the P-gp expression level in tumor tissues was significantly increased after docetaxel treatment(P<0.01).Apatinib combined with or without docetaxel had an effect on intestinal tissue MDR1,MRP2,and BCRP.There was no effect on the expression of(P>0.05)Conclusion:1.In this part of the animal experiment study,apatinib increased the inhibitory effect of docetaxel on the growth of ovarian cancer tumor tissue2.The synergistic anti-tumor effect of apatinib and docetaxel on ovarian cancer in vivo is mainly through increasing the local concentration of docetaxel in the tumor,inducing tumor tissue cell apoptosis,inhibiting tumor tissue ABCB1 gene and reducing P-gp expression achieve.For patients who are resistant to docetaxel,it provides experimental evidence for the effectiveness of apatinib combined with docetaxel.Part Ⅲ The role of docetaxel resistance in ovarian cancer involved with level of apatinib mesylate reversed ABCB1Purpose:The effect of apatinib on ABCB1-mediated docetaxel chemotherapy for ovarian cancer MDR and its mechanism were verified in vitro.This part mainly uses cytology experiments to further explore the therapeutic effect of apatinib combined with docetaxel in drug-resistant ovarian cancer and its synergistic mechanism,and provide references for clinical drug-resistant ovarian cancer patients.Methods:1.MTT method to determine the concentration of apatinib used in in vitro experiments,MTT method to detect cell proliferation inhibition rate.2.Apoptosis test to detect the effect of apatinib on the apoptosis of ovarian cancer cells.3.Drug accumulation test to detect the effect of rhodamine 123 accumulation in ovarian cancer cells.4.To study the effect of apatinib on the process of ABCB 1 transport and excretion through in vitro transport experiments.5.The Pgp-GloTM kit detects the effect of apatinib on ATPase.6.Westeron blot to detect the effect of apatinib on the expression of ABCB1 transporter in drug-resistant ovarian cancer cells.7.qRT-PCR to detect the effects of different concentrations of apatinib,verapamil and lapatinib on the expression of MDR 1.8.Westeron blot to detect the protein expression of NF-κ B signaling pathway,and immunofluorescence method to detect the effect of apatinib on the nuclear translocation of the P65 subunit of NF-κ B.Results:1.Preliminary experiment on the concentration of apatinib at the cellular level.The three concentrations of apatinib and chemotherapy drugs used in ovarian cancer sensitive ES-2 cells and ovarian cancer resistant ES-2/D cells are selected as 0.1,0.3,0.5 μ M and 0.5,1.0,respectively.2.0 μ M2.Apatinib can increase the anti-tumor effect of docetaxel on ES2/D cells in a concentration-dependent manner.The IC50 of the sensitive cell line ES-2 with the increase of the concentration of apatinib was not statistically significant(F=2.75,P=0.112);the resistant cell line ES-2/D increased with the apatinib The IC50 of the combined concentration increased,and the difference was statistically significant(F=12103,P<0.01)3.Apatinib concentration-dependently increases the apoptotic effect of docetaxel-induced ES-2/D cells.There was no significant difference in the apoptosis rate of ES-2/D cells induced under different concentrations((F=0.081,P=0.968).Apatinib and docetaxel have no significant difference in the apoptosis rate of ES-2/D cells The main effects were statistically significant(F=509.40,P<0.01;F=717.92,P<0.01).When combined with apatinib,the apoptotic rate of ES-2/D cells showed increasing With the increase of the concentration of metaxetine,the rate of apoptosis also increased The apoptosis rate of different concentrations of apatinib combined with docetaxel was significantly different from that of the same concentration of docetaxel alone(P<0.01);4.Apatinib increases the accumulation of Rh123 in ES-2/D cells.There was no statistically significant difference in the accumulation of Rhodamine 123 in the different concentrations of apatinib experimental group and verapamil positive control group in ES-2 cells(F=0.624,P=0.729).The concentration of apatinib is directly proportional to the accumulation of Rh123 in ES-2/D cells.The higher the concentration of apatinib,the greater the accumulation of Rh123(P<0.05),but the accumulation of Rh123 in ES-2 cells is higher.Low,no effect on cells.5.Apatinib enhances the activity of ABCBI transporter ATPase.Under the influence of different concentrations of apatinib,the activity of ABCB1 transporter ATPase is significantly different(F=124.195,P<0.01);under the influence of different concentrations of docetaxel,the activity of ABCB1 transporter ATPase is significantly different(F=165.79,P<0.01);Compared with the control group,both apatinib and docetaxel can enhance the activity of ATPase(P<0.05).6.Apatinib reduces the expression of MDR1 gene mRNA in ES-2/D cells.The expression level of ABCB1 in ES-2/D cells was significantly higher than that in ES-2 cells(P<0.05),and apatinib was inversely proportional to the expression level of ABCB1 in ES-2/D cells.The higher the concentration of apatinib High,the lower the expression level of ABCB1.However,the expression of ABCBI gene in ES-2/D cells was not affected by verapamil and lapatinib,and the difference was not significant(P>0.05).7.Apatinib reduces the expression of ABCB1 protein in ES-2/D cells.With the gradual increase in the concentration of apatinib,the greater its inhibitory effect on the ABCB1 transporter of drug-resistant ES-2/D cells,the greater the ABCB1 expression level in the cells,which is the same in the case of lapatinib.The concentration of lapatinib has no effect on the expression level of ABCB1 transporter.8.Apatinib down-regulates the expression of ABCB 1 transporter by inhibiting the NF-κB pathway.To detect the effect of apatinib administration on the NF-κB pathway in ES-2/D cells,similar to the effect of PDTC,an inhibitor of the NF-κB pathway,and apatinib significantly down-regulated the cytoplasm of ES-2/D cells And the expression level of NF-κB p65 in the nucleus.Apatinib can inhibit the activation of NF-κB signaling pathway,thereby reducing the expression level of ABCB1 transporter in ES-2/D cells.Conclusion:1.This part of the study confirmed that apatinib can significantly enhance the cytotoxicity of docetaxel in ES-2/D resistant cells.2.Both apatinib and docetaxel can enhance the activity of ATPase,suggesting that both are substrates of ABCB1.3.Apatinib significantly inhibited the expression of P-gp protein in ES-2/D resistant cells4.Apatinib inhibited the expression of ABCB1 from both the mRNA level and the protein level.5.Apatinib can significantly inhibit the activation of the NF-κB pathway,thereby inhibiting the expression of ABCB1 transporter at the protein level. |
