ABSTRACT
A series of chemotherapeutic drugs that induce DNA damage, such as cisplatin (DDP), are standard clinical treatments for ovarian cancer, testicular cancer, and other diseases that lack effective targeted drug therapy. Drug resistance is one of the main factors limiting their application. Sensitizers can overcome the drug resistance of tumor cells, thereby enhancing the antitumor activity of chemotherapeutic drugs. In this study, we aimed to identify marketable drugs that could be potential chemotherapy sensitizers and explore the underlying mechanisms. We found that the alcohol withdrawal drug disulfiram (DSF) could significantly enhance the antitumor activity of DDP. JC-1 staining, propidium iodide (PI) staining, and western blotting confirmed that the combination of DSF and DDP could enhance the apoptosis of tumor cells. Subsequent RNA sequencing combined with Gene Set Enrichment Analysis (GSEA) pathway enrichment analysis and cell biology studies such as immunofluorescence suggested an underlying mechanism: DSF makes cells more vulnerable to DNA damage by inhibiting the Fanconi anemia (FA) repair pathway, exerting a sensitizing effect to DNA damaging agents including platinum chemotherapy drugs. Thus, our study illustrated the potential mechanism of action of DSF in enhancing the antitumor effect of DDP. This might provide an effective and safe solution for combating DDP resistance in clinical treatment.
Subject(s)
Female , Male , Humans , Cisplatin/pharmacology , Disulfiram/pharmacology , Testicular Neoplasms/drug therapy , Fanconi Anemia/drug therapy , Alcoholism/drug therapy , Drug Resistance, Neoplasm , Cell Line, Tumor , Substance Withdrawal Syndrome/drug therapy , Apoptosis , Antineoplastic Agents/therapeutic use , Cell ProliferationABSTRACT
ObjectiveTo observe the effect of Feiyanning prescription （FYN） on cisplatin （DDP） resistance in non-small cell lung cancer （NSCLC） and explore the underlying mechanism. MethodCell counting kit-8 （CCK-8） assay was used to detect the proliferation of A549 and A549/DDP （DDP-resistant） cells treated by DDP （0， 2.0， 4.0， 6.0， 8.0， 10.0 mg⋅L-1） and the proliferation of A549/DDP cells treated by FYN （0， 100， 200， 300， 400， 500， 600 mg⋅L-1）. Based on immunofluorescence staining and Western blot （WB）， the expression of epithelial mesenchymal transition （EMT）-related proteins in A549 and A549/DDP groups was observed. A549/DDP cells were classified into control group， FYN group （200 mg⋅L-1）， DDP group （6.0 mg⋅L-1）， and combination group ［FYN （200 mg⋅L-1） + DDP （6.0 mg⋅L-1）］ and respectively treated with corresponding drugs. Then， invasion ability of each group was examined by transwell assay， and the expression of EMT-related proteins in each group by WB. Moreover， real-time fluorescence quantitative polymerase chain reaction（Real-time PCR） and immunofluorescence staining were separately applied to detect the mRNA and protein expression of drug resistance-related factors in each group， respectively. ResultCompared with A549 group， A549/DDP group showed high resistance to DDP （P<0.01）， low expression of E-cadherin， and high protein expression of Vimentin， N-cadherin， and Snail （P<0.05， P<0.01）. As compared with the control group， FYN inhibited the proliferation of A549/DDP cells in a concentration-dependent manner （P<0.01）， and the FYN group， DDP group， and combination group demonstrated low invasion ability （P<0.01）. In addition， the invasion ability in the combination group was particularly lower than that in the DDP group （P<0.01）. The expression of E-cadherin protein was higher and the protein expression of N-cadherin， Vimentin， and Snail was lower in the in FYN group than in the control group （P<0.01）. The protein expression of E-cadherin， N-cadherin， and Vimentin was lower and the expression of Snail was higher in the DDP group than in the control group （P<0.05，P<0.01）. The protein expression of E-cadherin， N-cadherin， Vimentin， and Snail in the combination group decreased as compared with that in the control group （P<0.01）. Compared with the DDP alone， the combination raised the expression of E-cadherin and lowered the protein expression of N-cadherin， Vimentin， and Snail （P<0.01）. The protein and mRNA expression of lung resistance-related protein （LRP） and multidrug resistance 1 （MDR1） was lower and the protein and mRNA expression of topoisomerase Ⅱα （TOPO Ⅱα） was higher in the FYN group than in the control group （P<0.01）. The protein and mRNA expression of LRP， MDR1， and TOPO Ⅱα was higher in the DDP group than in the control group （P<0.01）. The expression of LRP protein and mRNA showed no significant variation， but the protein and mRNA expression of MDR1 and TOPO Ⅱα increased in the combination group compared with those in the control group （P<0.01）. Compared with the DDP group， FYN group and combination group showed low protein and mRNA expression of LRP and MDR1 and high protein and mRNA expression of TOPO Ⅱα （P<0.01）. Compared with FYN， the combination elevated the protein and mRNA expression of LRP， MDR1， and TOPO Ⅱα （P<0.01）. ConclusionFYN prescription can reverse the DDP resistance of NSCLC by modulating EMT.
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@#Objective To investigate the effects of long non-coding RNA(LncRNA) growth arrest specific transcript 5(GAS5) negatively regulating nucleophosmin 1(NPM1) on cisplatin(DDP) resistance of gastric cancer cells.Methods The normal human gastric mucosa cell line GES-1 and human gastric cancer cell lines BG3-823,MGC-803 and AGS were selected as the research objects,of which the level of LncRNA GAS5 in each cell was measured by qRT-PCR.The drug resistance of AGS cells to DDP(AGS/DDP) was induced,and the experiment was divided into control group,empty plasmid group(BC group),GAS5 nonsense interference group(pLJM-GAS5 NC group) and GAS5 overexpression group(pLJM-GAS5 group).MTT method was used to determine the effect of DDP on the proliferation of AGS and AGS/DDP cells;and the levels of NPM1,multidrug resistance 1(MDR1),excision repair cross complementation group 1(ERCC1),multidrug resistance-associated protein 1(MRP1) and N-cadherin in AGS and AGS/DDP cells were measured by Western blot.Results Compared with the normal gastric mucosa GES-1 cells,the level of LncRNA GAS5 in BG3-823 and AGS cells decreased significantly,and among them,the level of LncRNA GAS5 in AGS cells was the lowest,so AGS cells were used for the follow-up experiments.Compared with the control group,the level of LncRNA GAS5 in AGS cells of BC group and pLJM-GAS5 NC group decreased significantly,while the levels of NPM1,MDRl,ERCC1,MRP1 and N-cadherin increased significantly;compared with BC group and pLJM-GAS5 NC group,the level of LncRNA GAS5 in AGS/DDP cells of pLJM-GAS5 group increased significantly,while the levels of NPM1,MDR1,ERCC1,MRP1 and N-cadherin decreased significantly;after treatment with DDP of the same concentration(except 0 μmol/L),compared with the control group,the inhibition rate of AGS/DDP cell proliferation in BC group and pLJM-GAS5 NC group decreased significantly;compared with BC group and pLJM-GAS5 NC group,the inhibition rate of AGS/DDP cell proliferation in pLJM-GAS5group was significantly higher.The semi inhibitory concentration(IC_(50)) of DDP on AGS/DDP cells in pLJM-GAS5 group for 48 h was(65.38±5.04) μmol/L,which was significantly lower than(120.74±4.17) μmol/L and(120.24±4.29) μmol/L in BC group and pLJM-GAS5 NC group.Conclusion Up-regulating the level of LncRNA GAS5 in AGS/DDP cells can reverse the drug resistance of AGS/DDP cells,which may be related to the down-regulation of NPM1expression
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Because the early symptoms of ovarian cancer are not typical and there is a lack of effective screening methods， most patients are diagnosed at an advanced stage， which seriously endangers the health of modern women. Platinum-based chemotherapy after tumor reduction is the first choice for patients with advanced and recurrent ovarian cancer， but almost all patients with recurrent ovarian cancer will eventually develop platinum resistance. Therefore， the search for natural， safe， and effective chemotherapeutic sensitizers has become an urgent and important topic in the study of ovarian cancer. With the increasingly extensive application of traditional Chinese medicine （TCM） in the treatment of cancer， the research on Chinese herbal monomers is also deepening， and the mechanisms of Chinese herbal monomers in intervening in cisplatin （DDP）-induced resistance of ovarian cancer is becoming increasingly clearer. Based on the research status of Chinese herbal monomers available in many Chinese and English databases， it was found that Chinese herbal monomers were involved in the reversal of DDP-induced resistance of ovarian cancer via many routes， mainly through increasing the intracellular drug concentration， reversing the blocked apoptosis， correcting the abnormal intracellular signaling pathway， enhancing DNA damage and inhibiting DNA repair， regulating intracellular autophagy， and suppressing epithelial mesenchymal transition （EMT）. Chinese herbal monomers weaken the resistance of ovarian cancer to DDP from multiple targets and enhance the toxicity of DDP to ovarian cancer cells in vitro and transplanted tumors in vivo. Therefore， Chinese herbal monomers are expected to become natural sensitizers for ovarian cancer chemotherapy with DDP. However， the current studies on Chinese herbal monomers are still confined to the single experimental type， and their action mechanisms and toxic and side effects remain to be further clarified. The application of Chinese herbal monomers for sensitizing DDP chemotherapy still needs to be verified by multi-target， multi-level experimental studies and large-scale clinical studies in the future.
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@#Objective: To study the effects of vitamin C (VC) on reversing cisplatin (DDP) resistance in oral squamous cell carcinoma (OSCC) and the mechanism. Methods: Human OSCC CAL27 cells were cultured in vitro and DDP-resistant CAL27 cell line (CAL27/ DDP) was screened by increasing concentration gradient method. Plate clone formation assay, CCK-8, Wound healing assay, Annexin V-FITC/PI staining flow cytometry were used to determine the effects of DDP alone or in combination with VC on colony formation, proliferation, migration and apoptosis of CAL27/DDP cells. Western blotting was used to detect the expression level of P-gp protein in CAL27 cells, CAL27/DDP cells and VC treated CAL27/DDP cells. Results: The inhibition concentration (IC50) of DDP increased significantly in CAL27/DDP cells as compared with that in CAL27 cells (P<0.05), indicating CAL27/DDP was DDP-resistant.After the combination with VC, the IC50 of DDP on CAL27/DDP cells was significantly reduced compared with that of DDP alone (P<0.05). DDP combined with VC significantly inhibited the migration of CAL27/DDP cells (P<0.01), and promoted the apoptosis rate (P<0.01). The expression level of P-gp protein in CAL27/DDP cells was increased compared with that in CAL27 cells (P<0.05), but decreased after VC intervention (P<0.05). Conclusion: VC can reverse DDP-resistance in OSCC cells by inhibiting P-gp protein expression.
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@#[Abstract] Objective: To investigate the effect of apatinib (APA) combined with cisplatin (DDP) on the proliferation, invasion and migration capacity of gastric carcinoma (GC) cells and its molecular mechanism. Methods: Cancer and para-cancerous tissue samples resected from 50 GC patients, who were surgically treated in Wuwei People's Hospital from January 2016 to June 2019, were collected for this study; in addition, GC cell lines MGC803 and SGC7901 were also collected. qPCR was used to detect the HMGA2 expression in tissues and mRNA expressions of molecules related to cell proliferation, migration and invasion in GC cell lines. MGC803 and SGC7901 cells were transfected with pcHMGA2 by liposome transfection technology. After treatment with DDP and APA at different concentrations, the cells were divided into NC, pcHMGA2, pcHMGA2+DDP and pcHMGA2+DDP+APA groups. Protein expression of HMGA2 in GC cells was detected by Western blotting, and proliferation, migration and invasion of the cells were detected by MTT and Transwell assay, respectively. Results: The mRNA expression of HMGA2 in GC tissues was higher than that in para-cancerous tissues (P<0.05), and the survival rate of GC patients in the high expression group was significantly reduced (P<0.01). DDP significantly inhibited the proliferation, invasion and migration of MGC803 and SGC7901 cells (all P<0.01); the proliferation, invasion and migration of MGC803 and SGC7901 cells in DDP+APA group significantly decreased (all P<0.01) as compared with DDP group; APA significantly enhanced the inhibitory effect of DDP on HMGA2 expression in GC cells (P<0.01); APA enhanced the anticancer activity of DDP against GC by down-regulating HMGA2 expression. Conclusion: APA promotes the anticancer activity of DDP against GC, and its molecular mechanism is the promotion of the inhibitory effect of DDP on HMGA2 expression.
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@# Objective: To investigate the expression profile and function of FANCF gene (a key gene in FA/BRCA pathway) in both cisplatin (DDP)-resistant and DDP-sensitive human triple-negative breast cancer cell lines and to analyze its correlation with DDP-resistance in breast cancer. Methods: The DDP-resistant breast cancer MDA-MB-231 cell line (MDA-MB-231/DDP) was established by induction of gradient DDP. The expression of FANCF gene in both sensitive and resistant cell lines was knocked-down by RNAi interference technology and the knockdown efficiency was validated at both RNA and protein level. The cell viability of MDA-MB-231 cells and MDA-MB-231/DDP cells was determined by the CCK8 assay; Flow cytometry was used to examine the cell cycle distribution and apoptosis; the mRNAand protein expressions of FANCF gene were examined by using qRT-PCR and western blotting, respectively. Results: The resistance index of MDA-MB-231/DDP cells was 13.5 after 3-month induction. The mRNA and protein expressions of FANCF were significantly increased in MDA-MB-231/DDP cells (all P<0.01). Cell cycle analysis indicated that the DDP treatment significantly induced G0/G1 arrest and decreased the cell proportion in phase S and G2/M. siRNA-mediated knockdown of FANCF could not only be able to increase sensitivity of MDA-MB-231 to DDP but also promote the cell apoptosis (all P<0.01). Conclusion: FANCF attributes to the occurrence of DDP-resistance through anti-apoptosis effect, which might be served as a potential treatment target for drug-resistant human breast cancer.
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Aim To study the growth inhibitory effect of the conjugate ( ovarian cancer specific targeting peptide and cisplatin, OSTP-DDP ) that targeting ovarian cancer cells A2780. Methods Using chemical method to syn-thesize OSTP-DDP, ovarian cancer cells A2780 were cul-tured in vitro, using CCK-8 method ( Cell Counting Kit-8) to detect the growth inhibitory effect of ovarian cancer A2780 cells, which were disposed by OSTP-DDP and DDP. Annexin V-FITC was used to detect the cycle and apoptosis effect of ovarian cancer A2780 cells which were disposed by OSTP-DDP and DDP. Results According to the mass spectrometry and the high performance liquid chromatography ( HPLC ) analysis, OSTP-DDP was proved to synthesize successfully. CCK-8 assay showed that both OSTP-DDP and DDP could play the growth in-hibitory effect and showed a concentration-dependent manner when cells were treated in different concentrations (10,20,40,80,160,320μmol·L-1 ) respectively after 24 h, 48 h, 72 h. And the effect of OSTP-DDP was stronger than DDP (P<0. 05), indicated OSTP-DDP had targeted cytostatic effect. The result of the flow cytometry showed that cell cycle was mostly arrested in G1 phase after 72h treated by OSTP-DDP and DDP, the inhibitory effect of OSTP-DDP was stronger than DDP (P<0. 05). The apop-tosis effect of OSTP-DDP was stronger than DDP ( P <0. 01),suggested that OSTP-DDP had a stronger targeting apoptosis-inducing effect. Conclusion OSTP-DDP has the targeting growth inhibitory effect on the ovarian cancer cell A2780, OSTP as a chemotherapeutic drug targeting vector has a great prospect to treat ovarian cancer.
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Objective To compare the short-term therapeutic effect, adverse reaction and influence on immune function between chrono-chemotherapy and routine-chemotherapy with docetaxel +cisplatin(DP)combined with concurrent chemoradiotherapy with cisplatin(DDP)in locally advanced(Ⅲ,Ⅳa stage)nasopharyngeal carcinoma(NPC).Methods 70 cases of newly diagnosed locally advanced NPC were randomly divided into group A and group B,35 cases in each group.Two groups of patients were treated with two cycles of neoadjuvant chemotherapy with DP combined with concurrent chemoradiotherapy with DDP.Both nereoadjuvant chemotherapy and concurrent chemotherapy of patients in group A were administrated with chrono-chemotherapy.Both nereoadjuvant chemotherapy and concurrent chemotherapy of patients in group B were administrated with routine-chemotherapy.Therapeutic effect,adverse reactions and other indicators were observed in both groups.Results After 3 months,there was no sifnificant difference of short term total efficacy between two groups in nasopharyngeal primary tumor(35 cases vs.34 cases),cervical lymph node(32 cases vs.31 cases).The incidence of adverse reactions of marrow toxicity(WBC,Hb,PLT)in group A was lower than group B(P<0.05),the incidence of digestive tract(nausea and vomiting,diarrhea)in group A was lower than group B(P<0.05),while there was no significant difference in oral mucositis between two groups(P<0.05).The high creatinine incidence in group A was lower than group B(P<0.05).There was no significant difference in high blood urea nitrogen and high transaminase incidences between two groups.After treatment, the T lymphocytes ( CD3 +, CD4 +, CD8 +, CD4 +/CD8 +) had no change in group A,the T lymphocytes(CD3 +,CD4 +)in group B decreased significantly compared with pre-treatment(P<0.05),and were lower than group A(P<0.05).Conclusion Adverse reactions and the degree of decline in cellular immune function of chrono-chemotherapy are lower than routine-chemotherapy in locally advanced NPC.
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0.05). No significant differences were detected in the median time of remission, median survival time and 1-, 2-year survival rates between the groups. Moreover, no significant differences were detected in the grade Ⅲ～Ⅳ leukopenia, grade Ⅲ～Ⅳ thrombocytopenia, grade Ⅲ～Ⅳ nausea and vomiting and grade Ⅲ～Ⅳ constipation between the groups. Conclusions:The response rate of the MVP regimen is slightly lower than the HMVP regimen, but the HMVP regimen is not noticeably superior. It may increase the toxicity such as leukopenia, nausea/vomiting and constipation, as wellas being more expensive. In short, MVP regimen should be selected between the regimens in the chemotherapy of advanced NSCLC.