The Regulatory Mechanism Of PI3K/Akt And Ubiquitin Ligase Cbl-b In Bufalin-induced Human Gastric Cancer Cell Apoptosis

ObjectiveGastric cancer is the second most common cancer in the world,and still remains one of the major causes of cancer death in China.Although 5-year survival rate of early gastric cancer is over 90%after surgery,but the prognosis for advanced gastric cancer that can't undergo surgery is poor,with a 5-year survival rate of less than 10%.About 2/3 patients with gastric cancer is often diagnosed at an advanced stage in China,and they can only accept chemoradiotherapy.Although various chemotherapeutic agents, such as epirubicin,fluorouracil,docetaxel and oxaliplatin,have been used for advanced gastric cancer and the survival has improved,but the efficacy of chemotherapy is limited.Bufalin is one of the major active components of Chan Su,a traditional Chinese medicine.Other and our studies showed that bufalin induced apoptosis in some human leukemia and solid cancer cell lines.It has been reported that bufalin downregulated Bcl-2,c-myc,WT-1 and activated mitogen-activated protein kinase(MAPK) singling pathway during cell apoptotic progression.However,the exact mechanism of bufalin-induced apoptosis is still less clear.Many studies showed that phosphatidylinositol 3-kinase(PI3K)/Akt signaling pathway played a critical role in controlling the balance between cell survival and apoptosis.Activated Akt(p-Akt) phosphorylated Bad,Caspase-9 and I-kappaB kinase (IKK),thus promoting survival and blocking apoptosis in some cancer cells.Some cytotoxic drugs induced cancer cell apoptosis by inhibiting the PI3K/Akt pathway.But the effect of bufalin on PI3K/Akt pathway during apoptosis of tumor cells,especially in gastric cancer cells,remains unknown. In this study,we investigated the mechanism of proteins related to apoptosis,such as Bcl-2 family,Survivin and Caspase-3,and PI3K/Akt singling pathway in bufalin-induced gastric cancer MGC803 cell apoptosis,we also investigated the regulatory mechanism of ubiquitin ligase Cbl-b,an upstream regulator of PI3K,in bufalin-induced apoptosis.Materials and Methods1.The effect of bufalin on MGC803 cell proliferation was measured using MTT assay.2.Cell cycle phase distribution and hypodiploid DNA was determined by flow cytometry with PI.3.Morphological analysis was performed by cytospin preparation with Wright-Giemsa stain.The slides were observed under a light microscope and the mitotic index was determined.4.Mitochondrial transmembrane potential was determined by flow cytometry with Rhodamine 123.5.Expression of Bcl-2,Bax,Survivin,Caspase-3,Akt,p-Akt and Cbl-b was analyzed by Western blot.6.Statistical analysis.All values are expressed as means±SEM.The differences of the results between two groups were evaluated by Student's t-test.The data from three or more groups were evaluated by one-way analysis of variance(ANOVA) followed by SNK test.P＜0.05 was considered to be statistically significant.Results1.MTT assay showed that bufalin inhibited MGC803 cell proliferation in timeand dose-dependent manner.The inhibiting concentration of 50%cell growth(IC₅₀) at 24,48 and 72 h was 96.03nmol/L,29.72nmol/L and 12.31nmol/L,respectively.An increase in G₂/M phase was observed after the cells exposed to 20nmol/L bufalin for 24h and the percentage of cells in G₂/M phase was 41.13±4.85%.The morphological analysis demonstrated that most of the cells were in a binucleate state without cell division and some were in metaphase after exposure to 20nmol/L Bufalin for 24 h, while the mitotic index increased from 3.57±0.32%to 15.97±1.96%(P＜0.01) compare with the untreated control group.When the cells were treated with 80nmol/L bufalin for 24h,sub-G1 peak(representing apoptosis) emerged and the percentage of sub-G1 phase was 18.76±2.42%.The morphological analysis demonstrated that the apoptotic cells became rounded in shape and their nuclei exhibited a fragmented morphology, forming apoptotic bodies.2.After exposure to 80nmol/L bufalin for 12 and 24 h,the mitochondrial transmembrane potential was much lower than that of the untreated control cells and the fluorescence intensity of Rhodamine 123 decreased from 2124.32±86.03 to 1502.44±84.09 and 1129.43±113.68(P＜0.01),respectively.After exposure to 20nmol/L and 80nmol/L bufalin for 24 h,the expression of Bax was increased to 1.27 and 1.80 times of the untreated control cells and Bcl-2 was decreased to 75%and 82%of the control cells.The radio of Bax/Bcl-2 was increased to 1.69 and 2.20 times of the untreated control cells,respectively.Meanwhile,the expression of Survivin was increased to 2.21 and 2.27 times of the untreated control cells.The cleavage of Caspase-3 was observed when the cells were treated with 80nmol/L bufalin for 24 h.3.After 80nmol/L bufalin treatment for 1-16 h,Akt phosphorylation increased slightly at 1 h and 8 h and then was inhibited completely at 16 h.The total Akt level was unchanged during treatment.MGC803 cells were pretreated with PI3K specific inhibitor LY294002(25μmol/L) and then exposed to 80nmol/L bufalin for 24 h,MTT assay demonstrated that the cell proliferation reduced from 51.26±4.10%to 39.89±3.10%(P=0.01) and the percentage of apoptofic cells increased from 18.46±2.76%to 31.40±4.77%(P=0.015) compare with treatment with bufalin alone. LY294002 treatment alone reduced cell proliferation and induced apoptosis very slightly.Western blot analysis showed that the upregulation of Survivin by bufalin was suppressed partially when the cells were pretreated with LY294002.In contrast,the ERK inhibitor PD98059 had no significant effect both on cell proliferation inhibition and apoptosis induced by bufalin in MGCS03 cells.Taken together,these results indicate that the PI3K/Akt pathway,but not ERK pathway,plays a crucial role in bufalin-induced apoptosis in MGC803 cells.4.Treatment with 80nmol/L bufalin for 1～16 h,the expression of Cbl-b was unchanged at 1h and 8h,and at 16h the expression of Cbl-b was 2.83 times over the untreated control cells.The time of upregulation of Cbl-b was identical with that of downregulation of p-Akt.Pretreatment with 25μmol/L LY294002 for 1h and then exposure to 80nmol/L bufalin for 1h and 8h,Akt phosphorylation was inhibited and the expression of Cbl-b reduced to 51%and 44%of the untreated control cells.5.Pretreatrnent with 2μmol/CsA for 30min,an inhibitor of calcium mobilization, and then exposure to 80nmol/L bufalin for 16h,CsA reversed bufalin-induced upregulation of Cbl-b and CsA alone did not affect the expression of Cbl-b.These results imply calcium mobilization by bufalin maybe is one of the reasons of increase of Cbl-b.To further certify the hypothesis,the expression of Cbl-b was determined after the cells were exposed to calcium ionophore A23187 and/or CsA.The expression of Cbl-b was 2.40 times over the untreated control cells after the cells were exposed to 1μmol/L A23187 for 16h.After the cells were exposed to 1μmol/L A23187 and 2μmol/L CsA for 16h,the expression of Cbl-b was 1.83 times over the untreated control cells.These results indicated CsA partially reversed A23187-induced upregulation of Cbl-b.Conclusion1.Bufalin inhibited MGC803 cell proliferation.It induced M phase arrest of cell cycle at low concentration,but induced apoptosis at high concentration in MGC803 cells.Bufalin-induced apoptosis accompanied with the collapse of mitochondrial transmembrane potential,the upregulation of Bax and Survivin,the downregulation of Bcl-2 and the activation of Caspase-3 in MGC803 cells.2.Bufalin inhibited activation of Akt during MGC803 cell apoptotic progression and LY294002 significantly enhanced bufalin-induced proliferation inhibition and apoptosis in MGC803 cells.LY294002 partially reversed bufalin-induced upregulation of Survivin.3.Bufalin upregulated Cbl-b during MGC803 cell apoptotic progression and CsA partially reversed bufalin-induced upregulation of Cbl-b.4.The expression of Cbl-b was upregulated by calcium mobilization in gastric cancer MGC803 cells.