Effects Of High Voltage Electric Field On NSCLC Sensibility To Chemotherapy And The Mechanisms

Background:Lung cancer is one of the most frequently diagnosed cancers and is the leading cause of cancer-related death worldwide.Non-small-cell lung cancer(NSCLC),a heterogeneous class of tumours,represents approximately 85% of all new lung cancer diagnoses.Due to the hallmarks of lung cancer,such as invasion and metastasis,most NSCLC re-emerge and metastasis after surgery.Chemotherapy plays great role in the NSCLC treatment,however,multidrug resistance(MDR)often contributes to chemotherapy failure.The efflux of intracellular drugs plays an important role in tumor MDR.To investigate the mechanism of the efflux of intracellular drugs,and to put efforts on inhibiting the drug efflux,thus enhancing the effects of chemotherapy on NSCLC.Studies suggested that ATP-binding cassette G2(ABCG2)contributes to the efflux of intracellular drugs.The vacuolar ATPases(V-ATPases)are a family of proton pumps that couple ATP hydrolysis to proton transport into intracellular compartments and across the plasma membrane.V-ATPase involves in the maintaining of cancer cells pH homeostasis thus influencing the intracellular drug centration and downregulating the viability of drugs.High voltage electric field could cause reversed and unreversed electroporation on cancer cells,thus leading to intracellular homeostasis and eventually cell death.Moreover,studies reported that high voltage electric field could interfere the expression of cellular proteins in cancer cells and inhibit the viability of membrane proteins.This study aims at confirming whether high voltage electric field treatment could enhance the effect of chemotherapy on adenocarcinoma A549 cells,and finding its potential mechanisms.Objective:To investigate the role of high voltage electric field on cell viability,adhesion,and metastasis of A549 cells,and to confirm whether high voltage electric field could enhance the effect of chemotherapy by interfering ABCG2 and V-ATPase expression and activities Methods:1.Given gradient strength high voltage electric field on A549 cells,then the A549 cells were divided into control group,500 V/cm group,1000 V/cm group,and 1500 V/cm group.Later,A549 cell viability was measured by CCK8 kit,cell adhesion ability and metastatic ability were measured by adhesion assay and scratch test.2.According to the results from the first part of the research,the appropriate voltage of electric field was set.Meanwhile,finding the appropriate concentration of Doxorubicin,docetaxel,cisplatin,etoposide,gemcitabine and paclitaxel on A549 cell line.Then combining the high voltage electric field and drugs on A549 cells,then evaluating the cell viability comparing the group only treated with drugs,thus confirming whether co-treatment could enhance the effect of chemotherapy.Moreover,Doxorubicin was used for further investigation by measuring the apoptotic index and cell viabilities,and ABCG2 expression after treatments.3.After finding the role on high voltage electric field on ABCG2 expression,we used the A549 cell lines with ABCG2 overexpression or knockdown,for further confirming whether high voltage electric field could interfere ABCG2 expression thus enhancing A549’s sensitivity to Doxorubicin.The cells were divided into electric group,electric+ Doxorubicin group,ABCG2 siRNA+ Doxorubicin group,and ABCG2 overexpression+electric+ Doxorubicin group,then the cell viability,Caspase9,Caspase 3,Bcl2 and Bax were respectively measured.4.To investigate the role of high voltage electric field on V-ATPase activity,and confirm whether high voltage electric field enhanced the anti-cancer activity of Doxorubicin by regulating V-ATPase.Then the cells were divided into control group,500 V/cm group,1000 V/cm group and 1500 V/cm group,then these cell viabilities were measured.Later,the cells were divided Doxorubicin group,electric+ Doxorubicin,bafilomycin A1+electric+ Doxorubicin,then the Caspase9,Caspase3,Bcl2,and Bax were measured Results:1.The cell viabilities treated with the 500 V/cm,1000 V/cm,and 1500 V/cm were respectively decreased to 97.4±6.7%,95.2±5.4% and 67.2±4.9%,compared with the control group.Moreover,the cell adhesion abilities were decreased to 87.3±7.8%,67.4±6.9%,and 57.2±4.2%,and scratch distances were increased to 137.1±8.9%,159.2±11.3%,and 196.4±14.5%,compared with control group.2.The appropriate concentration of chemotherapeutic drugs was selected: Doxorubicin 2.5 μM,docetaxel 2 μM,cisplatin 2 μM,etoposide 1 μM,gemcitabine 0.15 μM,and paclitaxel 0.08 μM.Co-treatment of high voltage electric field and drugs showed better anti-cancer effects than single-used chemotherapeutic drugs.Moreover,high voltage electric field and Doxorubicin co-treatment significantly upregulated the Caspase9 and Caspase3 activities,increased Bax expression and decreased Bcl2 levels.In addition,both the ABCG2 protein and mRNA concentration were downregulated after high voltage electric field treatment.3.Using RT-PCR and Western blot to confirm the ABCG2 overexpression and knockdown rates.Then we found ABCG2 overexpression effectively reversed the high voltage electric field treatment-enhanced anti-cancer activity of Doxorubicin.However,ABCG siRNA treatment promoted the high voltage electric field treatment-enhanced anti-cancer activity of Doxorubicin.4.500 V/cm high voltage electric field exerted little effect on V-ATPase activity,but 1000 V/cm and 1500 V/cm high voltage electric field significantly inhibited the V-ATPase activity.Bafilomycin A1 and Doxorubicin co-treatment group significantly inhibited the cell viability compared with the Doxorubicin group.Furthermore,we found bafilomycin A1 treatment had no effect on Caspase3 and Caspase9 activities,and Bax and Bcl2 expression.However,co-treatment of bafilomycin A1 and Doxorubicin significantly upregulated the Caspase3 and Caspase9 activities,and promoted the Bax expression and inhibited the Bcl2 expression compared with the Doxorubicin single treated group.Conclusion:1.High voltage electric field treatment could inhibited the adenocarcinoma A549 cell adhesion and metastasis.2.1000 V/cm high voltage electric field treatment had no significant effect on A549 cell viability,but dramatically enhanced the cell’s sensitivity to chemotherapeutic drugs.3.High voltage electric field treatment specifically or non-specifically inhibited the expression of ABCG2,and regulation of ABCG2 expression could reverse or enhance the anti-cancer activity of high voltage electric field.These results indicated that electric field induced A549 cell’s enhanced sensitivity to Doxorubicin by regulation of ABCG2 expression.4.High voltage electric field treatment inhibited the V-ATPase activity.In addition,inhibiting V-ATPase activity significantly enhance the anti-cancer effect of electric field.These results indicated that electric field induced A549 cell’s enhanced sensitivity to Doxorubicin by regulation of V-ATPase activity.