| Objective:Piperlongumine(PL)is an alkaloid isolated from long Indian pepper that has been found to induce the death of cancer cells by promoting the production of reactive oxygen species(ROS).Despite its potent cytotoxicity,several animal studies have shown that PL alone has limited antitumor efficacy.Recent studies have shown that ROS can promote the expression of programmed cell death ligand 1(PD-L1),so we speculated that PL may limit the anti-tumor effect of PL by affecting the expression of the immune checkpoint molecule PD-L1.In this study,we investigated the antitumor effect of PL on hep G2 and Bel-7402 hepatocellular carcinoma cell lines,and whether PL-induced ROS interferes with the expression of immune checkpoint molecules,and further explored the reasons and mechanisms for the limited antitumor effect of PL in hepatocellular carcinoma.Finally,we verified the anti-tumor mechanism of PL and the therapeutic effect of PL combined with PD-L1 m Ab through in vivo experiments.This study is expected to provide new ideas for the treatment of liver cancer.Methods:In this study,hep G2 and Bel-7402 cell lines were selected to explore the effect of PL on liver cancer.The following experimental steps were followed:1.Detection of ROS level.Flow cytometry was used to detect the intracellular ROS levels in different treatment groups.2.Detection of cell proliferation.CCK8 assay was used to detect the proliferation of HCC cells in different treatment groups.3.Detection of cell migration ability.The migration of cells in different treatment groups was detected by wound-healing assay and Transwell assay.The expression of migration-related proteins E-cadherin,N-cadherin and Vimentin in cells of different treatment groups was detected by Western-blot.4.Detection of cell apoptosis.Flow cytometry was used to detect the apoptosis of HCC cells in different groups.Hoechst 33342 nuclear staining was used to observe the apoptosis of cells in different treatment groups.Western-blot was used to detect the expression of apoptosis related proteins Bcl-2 and PARP and oxidative stress related protein CHOP.5.The expression of PD-L1,CMTM6 and Tim-3.Western-blot was used to detect the expression of PD-L1,CMTM6 and Tim-3 in different treatment groups.The m RNA expression levels of CMTM6 and PD-L1 were detected by RT-q PCR.The expression of PD-L1 was further detected by flow cytometry.6.Detection of the NF-κB and STAT3expression.The phosphorylation of PD-L1 related to the regulatory transcription factor NF-κB and STAT3,we therefore detect their expression by Western-blot.7.Effects of PARP inhibitors on the expression of PD-L1,CMTM6 and HIF-1α.Western-blot was possessed for the detection of PARP inhibitor olaparib on the expression of PARP,HIF-1α,PD-L1and CMTM6.The effects of olaparib,a PARP inhibitor,on HIF-1αand PD-L1 m RNA levels were detected by RT-q PCR.8.Establishment of liver cancer model in BALB/c mice.2x10~6 well-grown mouse hepatoma cells Hepa1-6 were injected into the left armpit of 30mice,the tumor formation of the mice was observed every 3 days,and the weight changes of the mice were recorded.On day 7,the obvious tumor mass was observed and palpated,and the mice were randomly divided into four groups:one group was injected with PBS,one group was injected with 3.5 mg/kg PL,one group was injected with 200μg PD-L1monoclonal antibody,and the other group was injected with 200μg of PD-L1 monoclonal antibody and 3.5 mg/kg of PL,the tumor tissue was removed after 14 days,and the tumor volume was measured.9.The expression of PD-L1,HIF-1αand PARP in mouse tumor tissue was detected by flow cytometry.10.The frequency of T lymphocyte subsets in peripheral blood and mouse tumor tissue was detected by flow cytometry.Results:1.The results showed that with the increase of PL concentration,the intracellular ROS level increased significantly,while NAC could completely reverse the ROS level,the difference was statistically significant(P<0.0001).2.The results of cell proliferation experiment showed that with the increase of PL concentration,the proliferation ability of hepatoma cells decreased significantly(P<0.001),and could be completely inhibited by NAC,the difference was statistically significant(P<0.001).3.The results of cell migration experiment showed that:with the increase of PL concentration,the migration ability of hepatoma cells was significantly inhibited(P<0.001).And NAC could partially reverse this effect,and the difference was statistically significant(P<0.01).Western blotting results showed that with the increase of PL concentration,the expression of migration-related protein E-cadherin was significantly up-regulated,while N-cadherin and Vimentin were significantly decreased,and the difference was statistically significant(P<0.001);However,NAC could partially reverse the expression of E-cadherin and Vimentin,and the difference was statistically significant(P<0.001).4.Apoptosis test results showed that:with the increase of PL concentration,the percentage of apoptotic liver cancer cells increased,and the difference was statistically significant(P<0.01);The results of Hoechst 33342 nuclear staining showed that with the increase of PL concentration,the number of cells with nuclear pyknosis and nuclear fragmentation increased significantly,and NAC could partially reverse this effect;correspondingly,we found that the expression of apoptosis-related protein Bcl-2 was significantly decreased with the increased PL concentration,and the expression of Cleaved PARP and oxidative stress-related protein CHOP was significantly increased;NAC could partially reverse their expression with a statistically significant difference(P<0.01).5.Western blotting results showed that PL promoted the expressions of PD-L1,CMTM6,and Tim-3(P<0.01);NAC could partially reverse the expressions of PD-L1,CMTM6 and Tim-3,and the difference was statistically significant(P<0.01).The results of RT-q PCR experiment showed that compared with the blank control group,the m RNA levels of PD-L1 and HIF-1αin the PL-treated group were significantly increased,while the m RNA levels of PD-L1 and HIF-1αin the PL and olaparib co-treated group were significantly decreased,and the difference was statistically significant(P<0.0001).The results of flow cytometry showed that compared with the control group and the experimental group pre-added with NAC,the level of PD-L1 was significantly increased,and the difference was statistically significant(P<0.0001).6.The immunoblotting results of NF-κB and STAT3 showed that PL inhibited the expression of p-NF-κB and p-STAT3,and the difference was statistically significant(P<0.01).7.The results of western blotting showed that PL promoted the expression of HIF-1α(P<0.01),and NAC could significantly reverse this effect(P<0.001);the protein expression levels of PD-L1,HIF-1αand CMTM6were significantly decreased if olaparib was used for 1 h pretreatment before adding PL,and the difference was statistically significant(P<0.001).The results of RT-q PCR experiment showed that compared with the control group,the m RNA levels of PD-L1 and HIF-1αin the PL treatment group were significantly increased,while the m RNA levels of PD-L1 and HIF-1αin the combined PL and olaparib treatment group were significantly decreased,and the difference was statistically significant(P<0.0001).8.The results of the BALB/c mouse tumorigenesis experiment showed that on the 7th day after the injection of mouse liver cancer cells Hepa1-6,the tumor mass was successfully touched the armpit of the mouse,and the tumor tissue was peeled off on the 21st day.The results showed that tumor necrosis occurred in the combined PL and PD-L1 monoclonal antibody treatment group,and the tumor volume was further reduced compared to sole PL or PD-L1monoclonal antibody treatment group,but the results were not statistically significant.9.Flow cytometry results showed that the expressions of PD-L1,HIF-1αand PARP in the PL-treated group were significantly higher than those in the PBS control group(P<0.001).10.the frequency of T lymphocyte subsets in peripheral blood and tumor tissue was detected by flow cytometry:the results showed that PL treatment could significantly increase the frequency of CD8~+T cells in peripheral blood(P<0.01),but at the same time significantly inhibited the tumor infiltration of CD8~+T cells in tissues;PL combined with PD-L1monoclonal antibody has a certain recovery effect on the reduction of CD8~+T cell infiltration in tumor tissues caused by PL treatment.It is worth noting that both PL therapy and PD-L1 monoclonal antibody therapy can significantly promote the infiltration of CD4~+T cells in tumors,and PL combined with PD-L1 monoclonal antibody further promotes the infiltration of CD4~+T cells in tumors(P<0.0001).Further analysis found that PL treatment or PD-L1 monoclonal antibody treatment had no significant effect on Treg infiltration in tumors.Conclusion:1.PL can inhibit the proliferation and migration of liver cancer cells,and promote the apoptosis of liver cancer cells,which provides a new direction for the treatment of liver cancer.2.PL can promote the expression of immune checkpoint molecules(PD-L1,Tim-3),and may promote the expression of PD-L1/CMTM6 through the ROS/PARP/HIF-1αpathway.This deeply revealed the mechanism by which PL exerts limited antitumor effects in liver cancer.3.PL combined with PD-L1 monoclonal antibody therapy can restore the reduction of CD8~+T cell infiltration in tumor tissue caused by PL and significantly increase the level of CD4~+T cell infiltration,and enhance effect on the anti-tumor effect of PL to a certain extent. |
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