Arsenic Sulfide Reverses Cisplatin Resistance In Non-small Cell Lung Cancer Through Targeting PD-L1

ObjectivesGlobally,with an estimated 19.3 million new cancer cases and 10.0 million deaths,lung cancer continued to be the second most commonly diagnosed cancer type and the primary cause of cancer-related deaths in both men and women in 2020.The reason for high mortality is that only 15%of patients are diagnosed in the early stage.Despite the occurrences of new treatments,most patients are in advanced stage at the time of diagnosis,so the 5-year survival rate is only about 15%.Non-small cell lung cancer(NSCLC)accounts for nearly 85%of all lung cancer diagnoses.At present,the treatment of advanced NSCLC is becoming more and more individualized,which needs to be customized for each patient according to the identification of driving gene mutations.Specific tyrosine kinase inhibitors(TKIs),such as EGFR TKIs and ALK TKIs,produced higher response rates,longer progression free survival,and lower toxicity rates in patients with these gene alterations compared with chemotherapy.However,chemotherapy is still an indispensable treatment for patients with advanced NSCLC without sensitive gene mutations.Platinum-based chemotherapy,particularly cisplatin(DDP),has been demonstrated to be efficient therapeutic treatment for NSCLC.Acquired drug resistance developed during clinical treatment is a large barrier that negatively impacts the survival rate of patients.Programmed cell death ligand 1(PD-L1),also known as B7-H1 or CD274,has been found to be widely overexpressed in various types of cancer cells,including NSCLC,and is believed to play an important role for cancer cells to escape from immune surveillance.Programmed cell death 1(PD-1),also known as CD279,is predominantly expressed on activated T cells.Binding of PD-L1 to PD-1 inhibits T cell effector function by inducing exhaustion of T cells,resulting in an immunosuppressive state.There have been several monoclonal antibodies targeting PD-1/PD-L1 axis approved by the US Food and Drug Administration(FDA)and have achieved great success in treating NSCLC.It has been found that PD-L1 is involved in chemotherapy resistance in several cancer cell lines.The expression of PD-L1 in tumor tissue of chemotherapy resistant non-small cell lung cancer patients was significantly higher than that of chemotherapy sensitive patients.The expression of PD-L1 in cisplatin resistant non-small cell lung cancer cell line was significantly higher than its corresponding ordinary cell line.And upregulation of PD-L1 after chemotherapy is correlated with poor prognosis in patients with NSCLC.Arsenic sulfide(As4S4),the active ingredient of the traditional Chinese medicine realgar,has attracted much research attention in recent years.As4S4 has antitumor activities in several cancers.During my master’s period of study,we found that the antitumor activities of As4S4 were correlated with its ability to active p53-dependent pathway.Many researches have showed that AS4S4 can play the role as chemotherapy sensitization.Recent studies investigated the role of p53 in PD-L1 regulation and found that p53 was negatively correlated with PD-L1 expression.Furthermore,p53 could regulate PD-L1 via miR-34a.Based on the above research,we conducted this study to verify the relationship between PD-L1 and DDP resistance and identify whether As4S4 could decrease the PD-L1 expression and reverse the DDP resistance in NSCLC.MethodsWe first used the MTT assay to assess the effects of serial concentrations of DDP on cell proliferation in A549/DDP,H1299/DDP cells(DDP-resistance)and their parental A549,H1299 cells.The human lung adenocarcinoma cell lines A549(p53 wide type),A549/DDP(p53 wide type)and H1299(p53 deficient)were obtained from Institute of Basic Medical Sciences,Chinese Academy of Medical Sciences.H1299 cells were treated with gradually increasing concentrations of DDP to produce the DDPresistance H1299/DDP cells.The basic mRNA and protein expression levels of PD-L1 was detected using RT-qPCR and western blot assay in above cells.Annexin V-fluorescein isothiocyanate(FITC)/propidium iodide(PI)double staining was used to quantify the apoptotic rate of A549/DDP cells treated with As4S4,DDP or the combination of As4S4 and DDP.In order to investigate whether As4S4 synergistically facilitated the sensitivity of A549/DDP and H1299/DDP to DDP,As4S4 at a non-lethal dose was used.PD-L1 siRNA was transfected into in A549/DDP and H1299/DDP cells using RFect to downregulate the expression of PD-L1.IFN-y was used to increase the expression of PD-Ll in A549 and H1299 cells.The expression of p53 and PD-L1 proteins was measured by western blotting analysis.The levels of miR-34a-5p,miR-34a-3p and PD-L1 in cells were measured by RT-qPCR assay.A549/DDP cells were pretreated with 30 μM PFT-α(a p53 specific inhibitor at a concentration that did not cause significant cytotoxicity in cells)for 30 minutes prior to addition of As4S4.To verify the synergistic effect of As4S4 and DDP in vivo,we established xenograft mouse models.A549/DDP cells were subcutaneously implanted into BALB/c nude mice,and the mice were treated with DDP with or without As4S4.The method of injection was intraperitoneal injection for 3 weeks.ResultsA549/DDP and H1299/DDP cells showed high resistance to the DDP challenge compared to their parental cells.Higher protein expression of PD-L1 was observed in A549/DDP and H1299/DDP cells compared with A549 and H1299 cells using western blot assay.Increased mRNA level of PD-L1 was detected using RT-qPCR in DDPresistant cells.Western blot assay indicated that PD-L1 was successfully downregulated in A549/DDP and H1299/DDP cells by specific PD-L1 siRNA.The depletion of PD-L1 inhibited DDP resistance in A549/DDP and H1299/DDP cells.Treatment with IFN-y(10 ng/mL)increased the protein expression of PD-L1 in a time-dependent manner in A549 and H1299 cells.Co-treatment of IFN-γ and DDP significantly enhanced DDP resistance in A549 and H1299 cells.In order to investigate whether As4S4 synergistically facilitated the sensitivity of A549/DDP and H1299/DDP to DDP,AS4S4 at a non-lethal dose was used.Specially,1.5 μM As4S4 and 2.0 μM As4S4 did not yield measurable impact on cell viability in A549/DDP and H1299/DDP cells,respectively.But 1.5 μM As4S4 clearly enhanced the sensitivity of A549/DDP cells to DDP when treated for 48 hours.We next examined whether As4S4 could enhance DDP-induced apoptosis using flow cytometry.As4S4 increased DDP-induced apoptosis in A549/DDP cells.But co-treatment of As4S4(2.0μM)and DDP did not exhibit synergistic inhibition effect in H1299/DDP cells.Western blot analysis showed that treatment of As4S4(1.5μM)upregulated p53 expression and downregulated PD-L1 expression in A549/DDP cells in a timedependent manner.Real-time qPCR analysis showed that AS4S4(1.5 μM)increased miR-34a-5p level in a time-dependent manner and had no influence on miR-34a-3p level in A549/DDP cells.We observed that As4S4 had no influence on expression ofp53 and PD-L1,as well as miR-34a level in H1299/DDP cells.Inhibition of p53 by PFT-αpartially restored the protein levels of p53 and PD-L1,which were changed by AS4S4 treatment.Flow cytometry analysis revealed that PFT-α alone did not cause apoptosis and had no influence on apoptosis rate induced by DDP in A549/DDP cells.But pretreatment with PFT-α suppressed apoptosis rate induced by co-treatment of As4S4 and DDP compared to non-treated control in A549/DDP cells.Furthermore,the upregulation of miR-34a-5p level induced by As4S4 was significantly decreased by additional PFT-α.DDP significantly inhibited tumor growth,and the effi cacy was significantly enhanced by additional As4S4 treatment,whereas the anti-tumor activity of As4S4 alone was dismal in xenograft mouse models.DDP treatment alone had no apparent influence on expression of p53 in tumor tissue.The expression of p53 was significantly increased after treatment with As4S4.DDP treatment upregulated PD-L1 expression in xenograft mouse models.Upregulation of PD-L1 induced by DDP in the xenograft models was inhibited by treatment of As4S4.ConclusionsUpregulation of PD-L1 was correlated with DDP resistance in NSCLC cells.DDP treatment could upregulate PD-L1 expression in vivo.As4S4 could upregulate p53 expression and downregulate PD-L1 expression in NSCLC cells with wide type p53.Mechanistic analyses indicated that As4S4 might change miR-34a and PD-L1 levels by regulating the expression of p53,and sensitize NSCLC cells to DDP.SignificancePD-L1 could be a potential indicator to predict the chemosensitivity of patients with NSCLC.Targeting PD-L1 might be a promising strategy for the reversal of chemoresistance of lung cancer.Furthermore,As4S4 might be a potential agent to treat chemotherapy refractory NSCLC.