TRPC3 Mediates Chemo-Resistance In Non-Small Cell Lung Cancer Cells

Objective: Lung cancer has ranked the highest mortality rate of cancers in the world.Non-small cell lung cancer(NSCLC)accounts for about 80% of all lung cancers,and its incidence and fatality rate have increased year by year in the past 50 years,among which the incidence of male lung cancer patients accounts for the first place of malignant tumors,and it has become a major disease endangering life and health.Transient Receptor Potential(TRP)channels are non-selective cation channels located on cell membranes.An important problem in tumor chemotherapy is the generation of multidrug resistance(MDR).Increased efflux of anticancer drugs,resulting in reduced intracellular drug accumulation,is currently thought to be the main cause of chemo-resistance.In contrast,the transmembrane transporters responsible for drug efflux are mainly from the ATPbinding cassette transporters(ABC transporters).Among them,ABCB1,ABCC1 and ABCG2 are closely associated with the acquisition of MDR to tumor chemotherapeutic drugs.In contrast,ABCB1(MDR1 or P-gp)is one of the most representative ABC transporters.Studies have shown that many TRP ion channels are associated with tumor chemo-resistance,including TRPV2,TRPC5 and TRPC6,however,little is known about the interaction between TRP ion channels and ABC transporters,and the regulation of their synergy in tumors.Therefore,the purpose of this study was to determine the coexpression of TRP ion channels in ABC transporters in human NSCLC Taxol-resistant cell lines,to clarify the interaction between TRP ion channels and ABC transporters in resistant cell lines,to further identify chemo-resistance pathways by studying the changes in chemo-resistance phenotypes regulated by TRP ion channels and ABC transporters,and to target TRP ion channels to find The study was conducted to identify small molecules that increase the sensitivity of drug-resistant NSCLC to Taxol.By focusing on TRP ion channels and their related regulatory pathways,the study may provide a new idea for the treatment of chemo-resistance in NSCLC and lay the foundation for subsequent studies on the regulation of cellular chemo-resistance by TRP ion channels.Methods:(1)Human NSCLC Taxol-resistant cell line A549/Taxol was constructed by the interval method of increasing drug concentration gradient.(2)Western blot was performed to detect the expression of ABC transporters in A549 and A549/Taxol to determine the drug resistance pathway of A549/Taxol.Detection of its expression on the cell membrane was followed by immunofluorescence to further determine the expression of these ABC transporters.(3)Real-time fluorescence quantitative PCR was performed to determine the expression of TRP ion channels in A549 and A549/Taxol.The TRP ion channels that were significantly expressed in A549/Taxol were identified and further determined by Western blot and immunohistochemical staining.(4)Correlation analysis was performed to determine the correlation between the highly expressed ABC transporters and the highly expressed TRP ion channel in A549/Taxol and the relationship between them.(5)Select specific inhibitors/agonists based on the TRP ion channels highly expressed in A549/Taxol and examine their effect on cell proliferation.Applications in combination drug administration were also identified.(6)Determine whether the inhibitor/agonist affects cell fate,including cell cycle,apoptosis,and cellular senescence,via this highly expressed TRP ion channel by calcium imaging.(7)Combine the above findings to determine the possible mechanisms by which the TRP ion channel affects cell fate.Results:(1)The construction of drug-resistant cell lines was determined by cell proliferation assays.Taxol inhibited the proliferation of A549 and A549/Taxol with IC50 values of 6.11 μM,88.58 μM and a resistance multiple of 14.5.(2)Western blot and immunofluorescence revealed that ABCB1 expression was significantly elevated in A549/Taxol;while ABCG2 expression did not change significantly in A549/Taxol;ABCC1 was not expressed in either cell.(3)The results of real-time fluorescence quantitative PCR,Western blot,and immunofluorescence revealed that TRPC3 expression was significantly elevated in A549/Taxol.(4)Correlation analysis showed a significant correlation between TRPC3 and ABCB1.Knockdown of ABCB1 in drugresistant cells was followed by a decrease in TRPC3 expression.Correspondingly,the knockdown of TRPC3 was followed by a similar reduction in ABCB1 expression.(5)The generic suppressor of TRP ion channel 2-APB has an inhibitory effect on the proliferation of both A549 and A549/Taxol cells with IC50 values of 105.36 μM and18.45 μM,respectively,indicating that A549/Taxol was more sensitive to 2-APB.The combination of Taxol + 2-APB has a synergistic inhibitory effect on A549/Taxol.(6)2-APB down-regulated TRPC3 expression in A549/Taxol cells.2-APB,a universal inhibitor of TRPC3 channels,and Pyr3,a specific inhibitor,both significantly impaired cell proliferation in A549/Taxol.(7)The results of the apoptosis assay showed that 2-APB did not affect apoptosis produced by A549,but could promote apoptosis in A549/Taxol,which was further enhanced after the combination of Taxol and 2-APB.Cell cycle assays showed that 2-APB blocked the A549 cell cycle in the G1 phase.2-APB blocked the A549/Taxol cell cycle in the S phase.(8)2-APB caused cell senescence in A549/Taxol,while 2-APB action on A549 did not cause cell senescence.This cell senescence trend was more pronounced in A549/Taxol knockdown by TRPC3.Knockdown of TRPC3 significantly increased the mitochondrial oxygen consumption rate(OCR).A549/Taxol with the knockdown of TRPC3 had increased ATP conversion rate compared to A549/Taxol without knockdown of TRPC3.(9)2-APB activated the Erk1/2 signaling pathway of A549 thereby blocking the cell cycle and inhibiting cell proliferation.2-APB activated the Erk1/2-Akt signaling pathway of A549/Taxol thereby inhibiting intracellular calcium signaling,blocking the cell cycle,inhibiting cell proliferation,and promoting apoptosis and senescence.Conclusion:(1)Expression of TRPC3 ion channel and multidrug resistance protein ABCB1 was upregulated in drug-resistant cell lines A549/Taxol.(2)ABCB1 was significantly associated with TRPC3 in Taxol-resistant NSCLC cells,and there was a dynamic reciprocal regulation of ABCB1 and TRPC3 expression and distribution.(3)Targeted inhibition of TRPC3 and combination of Taxol,cum Taxol,and 2-APB had a synergistic inhibitory effect on Taxol-resistant cell lines A549/Taxol.(4)2-APB,a universal inhibitor of TRP channels,regulates apoptosis and cell cycle and promotes cell senescence in A549/Taxol by inhibiting TRPC3.(5)The combination of Taxol and 2-APB had a synergistic inhibitory effect on A549/Taxol.(6)2-APB affects the cell fate of Taxol-resistant cell lines A549/Taxol by activating the Erk1/2-Akt pathway.