Effects Of Bufalin On ACHN Cells And Mechanism Study

BackgroundRenal cell carcinoma(RCC)is one of prevalent malignant tumors,accounting for 2%of adult malignancies;its incidence is steadily rising by approximately 2%per year.Although the rate of early diagnosis of RCC has increased with the development of diagnostic techniques in recent years,up to 30%of patients are diagnosed with metastasized RCC,and one-third of local RCCs eventually recur and metastasize after surgical resection.Currently,surgery still plays a dominant role in the first-line treatment of RCC.However,metastatic RCC(mRCC)cannot be treated with radical surgery and is generally not sensitive to chemotherapy or radiotherapy.Combined treatment(cytoreductive nephrectomy,cytokine therapy,targeted therapeutic agent treatment,etc.)may prolong survival.Despite the improvements in most treatment modalities,mRCC patients still have extremely poor outcomes,with an overall median survival of<1 year.Hence,there is an urgent need to enhance our understanding of the biological signaling pathways involved in RCC and to identify novel therapeutic agents.Bufalin,an active component of the traditional Chinese drug Chan Su,has been reported to have anti-tumor effects on various types of cancers,including breast,lung,liver,prostate,and cervical cancer.These studies have suggested that bufalin is a potential therapeutic agent for combined chemotherapy.However,the effect of bufalin on RCC has not yet been thoroughly evaluated.In this study,we sought to illustrate the anti-tumor effects and potential basic mechanisms of bufalin in the human RCC ACHN cell line.ObjectiveTo explore the biological behavior changes and investigate the possible mechanism caused by Bufalin on ACHN cells.So as to seek a therapeutic agent for Renal cell carcinoma.Method1.Biological behavior changes caused by Bufalin on ACHN cells1.1 CCK-8 assay was used to detect the effects of proliferation in ACHN cells1.2 Colony-forming assay was used to detect the changes of colony-forming in ACHN cells1.3 Cell apoptosis and cell-cycle analyse were used to assess the effects of Bufalin on apoptosis and cell cycle progression in ACHN cells.1.4 Wound-healing assay and cell migration and invasion assay were used to assess the ability of metastasis in ACHN cells2.Study of the mechanism of the effect of Bufalin on AVHN cells2.1 PCRThe relevant mRNA expressions of Akt/E-cadherin and N-cadherin were determined by QT-PCR.2.2 Immunofluorescence staining assayThe immunofluorescence staining was used to confirmed the location and expression of E-cadherin in ACHN cells after treated with bufalin2.3 Western blotThe relevant protein expression of cell cycle,metastasis and PI3K/Akt/mTOR signali-ng pathwaywere determined by Western blot.Result1.Biological behavior changes caused by Bufalin on ACHN cells1.1 Bufalin suppressed ACHN cell proliferation and the IC50 values of bufalin for ACHN cells at 12,24,48,and 72 h were 228.08 ± 9.64,29.41 ± 2.60,10.49 ±0.79,and 6.7 ± 0.97 nM,respectively.1.2 The numbers and sizes of colonies formed were significantly reduced in a dose-dependent manner,from 233.00 ± 12.12 to 183.67±5.69,94.33±5.86,and 65.33 ± 4.51 after treatment with bufalin(concentrations of 0,5,10 and 20 nM).1.3 Bufalin induces G2/M phase arrest in ACHN cells.The percentage of ACHN cells in the G2/M phase increased from 7.86%± 0.96%to 15.49%± 3.51%,23.38%± 2.98%,and 37.07%± 7.05%after treatment with bufalin(concentrations of 0,5,10,and 20 nM,respectively)for 24 h.1.4 Bufalin inhibits ACHN cell migration and invasion.bufalin decreased the migration speed of ACHN cells in a dose-dependent manner and bufalin significantly reduced ACHN cell invasion through Matrigel and migration through the membrane in the bottom chamber.2.Study of the mechanism of the effect of Bufalin on ACHN cells2.1 Treated with 20nM Bufalin for 24h,QT-PCR showed that Bufalin up-regulated the expression of E-cadherin and down-regulated of N-cadherin,however there were no significant changes in Akt mRNA expression.2.2 The immunofluorescence staining confirmed the upregulation of E-cadherin in ACHN cells treated with bufalin.2.3 Bufalin induced G2/M phase arrest in ACHN cells by up-regulating the expression of p21,but down-regulating of cyclin Bland CDK1.After 24 h of treatment with bufalin,the expressions level of N-cadherin and HIF-la decreased,whereas that of E-cadherin increased in a dose-dependent manner.There were no significant changes in Akt protein expression.However,phospho-Akt(Ser473)and phospho-mTOR levels were reduced.ConclusionIn conclusion,to our knowledge,our study is the first to show that bufalin induces RCC ACHN cell cycle arrest and suppresses metastasis via disruption of the PI3K/Akt/mTOR signaling pathway.Our results indicate that bufalin is a potential therapeutic agent for RCC.