Study On The Effect And Underlying Mechanism Of Gigantol On The Apoptosis Of Breast Cancer Cells Induced By Cisplatin

Nowadays,cancer is one of the main causes of death in the global population.Breast cancer(BC)is the most common female malignant tumor today.In the global cancer statistics in 2018,BC is the most commonly diagnosed cancer among women in the world,and it is also the main cause of death in women due to cancer.BC is the second most commonly diagnosed cancer with an incidence rate,following lung cancer.However,the incidence of BC has been on the rise in recent years.According to the global cancer report published by the World Health Organization in 2020,the incidence of BC has exceeded that of lung cancer.At present,the commonly used chemotherapy drugs include traditional platinum drugs such as cisplatin(DDP).Although DDP can effectively treat many types of cancers including BC,due to the long-term use of DDP in the treatment of cancer,the organs toxicity,such as nephrotoxicity,liver toxicity and ototoxicity,usually brings many side effects to patients.Natural plant-derived compounds are widely used and are not only well tolerated but also cost-effective.Recent studies have shown that many active ingredients of natural medicines have the ability to enhance the efficacy of DDP or anti-DDP resistance on various malignant tumors,and have less toxic effects.Gigantol is a bibenzyl compound in Dendrobium medicinal materials.Recent studies have shown that it has the activity of inhibiting tumors,and there is no research on the effect of gigantol-assisted DDP or other chemotherapeutics.Based on the prediction and analysis of network pharmacology,this study explored the effect and mechanism of gigantol on BC cell apoptosis induced by DDP,and provides new research directions and important experimental basis for antitumor activity of gigantol and its similar structural components.ObjectiveThrough network pharmacology and molecular docking to predict the potential targets of gigantol on BC,and through experiments to study the effect of gigantol on DDP-induced BC cell apoptosis,and preliminarily explore and verify the possible mechanism of gigantol on BC cell apoptosis induced by DDP through molecular biology methods.Methods(1)Based on the 2D molecular structure of gigantol,using network pharmacology research methods,gather the potential targets of gigantol for BC,establish a target interaction map,and conduct enrichment analysis of target genes to screen potential signaling pathway.Using molecular docking to dock the 3D structure of gigantol with the related proteins of the signaling pathway.(2)The MTT method was used to detect the proliferation activity of gigantol(2.5,5,10,20,40,80,160,320 μM)on MDA-MB-468 and MCF-7 cells for 24,48,72 h.Selected a certain concentration of gigantol for subsequent investigation of the combination effect.(3)Used the MTT method to detect the IC50 of DDP on MDA-MB-468 and MCF-7 cells for 48 h,and detected the effects of different concentrations of gigantol(20,40,60 μM)combined with DDP on BC cells for 48 h.Calculated the IC50 of DDP for 48 h in combination with gigantol,and compared with the IC50 of DDP-alone for 48 h.(4)The colony formation experiment was used to detect the effect of different concentrations of gigantol(40,60μM)combined with DDP on clonogenic ability of BC cells.(5)Observed the cell morphology and nuclear status of BC cells treated with 60 μM gigantol alone,10 μM DDP alone,60 μM gigantol+10 μM DDP for 48 h under the fluorescent microscope after staining with Hoechst 33342 or ordinary inverted microscope.(6)Cells were stained with Annexin V-FITC/PI,and flow cytometry was used to detect apoptosis rate of BC cells treated with 60 μM gigantol alone,10 μM DDP alone,60 μM gigantol+10 μM DDP for 48 h.(7)Western blot was used to detect the expression apoptosis-related factors(Bax,Bcl-2 Caspase 9,Caspase 7,PARP,Cleaved-Caspase 9,Cleaved-Caspase 7,Cleaved-PARP)of BC cells treated with 60 μM gigantol alone,10 μM DDP alone,60 μM gigantol+10 μM DDP for 48 h.(8)Western blot was used to detect the protein of the PI3K/Akt/mTOR signal pathway in BC cells treated with 60 μM gigantol alone,10 μM DDP alone,60 μM gigantol+10 μM DDP for 48 h.The 10 μM LY294002(PI3K/Akt pathway inhibitor)alone,and 10 μM LY294002+10μM DDP was administered for 48 h as the comparison groups to preliminarily explore the mechanism of gigantol on the apoptosis of BC cells induced by DDP.(9)Western blot was used to detect the expression apoptosis-related factors(Bax,Bcl-2,Caspase 9,Caspase 7,PARP,Cleaved-Caspase 9,Cleaved-Caspase 7,Cleaved-PARP)of BC cells treated with 60 μM gigantol alone,10 μM DDP alone,60 μM gigantol+10 μM DDP,10μM LY294002 alone,and 10 μM LY294002+10 μM DDP for 48 h,to preliminarily verify the mechanism that gigantol enhanced apoptosis of BC cells induced by DDP.Results(1)There were 97 potential targets of gigantol on breast cancer.GO analysis results showed that the potential targets are mainly related to kinase activity.The signaling pathway enriched by KEGG pathway enrichment analysis was PI3K/Akt signaling pathway.Through molecular docking,the 3D structure of gigantol can be docked in the active pockets with the 3D protein structure of PI3K,Akt and the downstream mTOR.(2)The inhibitory effect of gigantol on the proliferation of MDA-MB-468 and MCF-7 cells was concentration-dependent and time-dependent.(3)Compared with DDP alone,the combination of gigantol and DDP for MDA-MB-468 cells and MCF-7 cells exerted stronger ability to inhibit proliferation.The IC50 of DDP treating on MDA-MB-468 cells for 48 h was 12.29 μM,and the IC50 of DDP treating on MCF-7 cells for 48 h was 22.66 μM.When DDP combined with 40,60 μM gigantol,the IC50 of DDP on MDA-MB-468 cells decreased significantly to 3.73 and 2.92 μM at 48 h.When DDP combined with 20,40,60 μM gigantol,the IC50 of DDP on the MCF-7 cells at 48 h decreased significantly to 19.62,17.12 and 8.36 μM.(4)Compared with the DDP alone group,(40,60 μM)gigantol significantly enhanced the ability of DDP to induce the decrease in the number of MDA-MB-468 and MCF-7 cell colonies.(5)In the cell morphology observation experiment,the cells of the control group were intact and in an adherent state.In the gigantol alone group or DDP alone group,a small number of cells became smaller and the cytoplasm was more concentrated.More cells in the combination of gigantol and DDP group showed vacuolated cytoplasm,and floated off.In addition,the results of Hoechst 33342 staining showed that,compared with the control group,a number of cells treated with gigantol or DDP showed intensely stained blue fluorescence.Treated with gigantol and DDP in combination,more cells showed dense blue fluorescence,and the nuclei were concentrated and broken.(6)Flow cytometry studies showed that gigantol significantly enhanced the apoptosis of MDA-MB-468 and MCF-7 cells induced by DDP.The apoptosis rate of MDA-MB-468 cells treated with DDP alone for 48 h was 25.50 ± 0.89%,gigantol significantly enhanced the cell apoptosis rate induced by DDP to 31.0±1.92%(P<0.01);The apoptosis rate of MCF-7 cells treated with DDP alone for 48 h was 17.74 ± 0.53%,gigantol significantly enhanced the cell apoptosis rate induced by DDP to 25.18 ± 0.45%(P<0.001).(7)Compared with the control group,DDP alone could reduce the expression of Bcl-2 in MDA-MB-468 and MCF-7 cells(P<0.01),increase the expression of Bax and activate the Cleaved-Caspase 9,Cleaved-Caspase 7 and Cleaved-PARP(P<0.05 or P<0.01),gigantol could enhance the decrease of Bcl-2 expression induced by DDP(P<0.05 or P<0.01),increase the expression of Bax(P<0.05 or P<0.01),and increase the activation of Cleaved-Caspase 9,Cleaved-Caspase 7 and Cleaved-PARP(P<0.01).(8)Compared with the control group,gigantol alone or LY294002 alone could significantly down-regulate the expression of p-PI3K,p-Akt and p-mTOR(P<0.01).Compared with DDP alone group,the expression of p-PI3K,p-Akt and p-mTOR was significantly down-regulated in gigantol combined with DDP group or LY294002 combined with DDP group(P<0.05 or P<0.01).(9)In MDA-MB-468 cells and MCF-7 cells,compared with DDP alone,gigantol enhanced DDP-induced decrease in Bcl-2 expression(P<0.01),and increased expression of Bax(P<0.01)and activated the Cleaved-Caspase 9,Cleaved-Caspase 7 and Cleaved-PARP(P<0.01),LY294002 combined with DDP also significantly reduced the expression of Bcl-2(P<0.01),increased the expression of Bax and activated the Cleaved-Caspase 9,Cleaved-Caspase 7 and Cleaved-PARP(P<0.01).These results preliminarily showed that gigantol has a similar effect to LY294002 to enhance DDP-induced BC cells apoptosis by inhibiting the PI3K/Akt/mTOR signaling pathway.ConclusionThis study is for the first time to use network pharmacology and molecular docking methods to investigate the mechanism of gigantol on BC,indicated that gigantol can act on BC through the PI3K/Akt/mTOR signaling pathway.Gigantol induced the expression of apoptosis-related factor Bcl-2 to decrease,and the expression of Bax to increase and Cleaved-Caspase 9,Cleaved-Caspase 7 and Cleaved-PARP to active in BC cells.Gigantol may enhance DDP-induced breast cancer cell apoptosis by down-regulating the expression of p-PI3K,p-Akt and p-mTOR.This result preliminarily showed that in the study of BC in vitro,gigantol can enhance the apoptosis of breast cancer cells induced by DDP by inhibiting the PI3K/Akt/mTOR signaling pathway.