The Action Mechanism Underlying The Effects Of A Novel Src Kinase Inhibitor BRP800 On The Lung Adenocarcinoma Cell Line H1299

Objective: To investigate the proliferation and apoptosis effects of a novel Src inhibitor BRP800 on the lung adenocarcinoma cell line H1299 ,and its possible mechanisms.Methods: (1) In vitro, human lung adenocarcinoma cell line H1299 were treated with various concentrations of BRP800 for 24h. The absorbance value was detected and growth inhibition rate was calculated by MTT assay. (2) The changes of cell morphology were observed by inverted microscope. (3) Cell cycle and apoptosis were assessed by flow cytometry; (4) The expression level of Src kinase and Akt were determined by Western Blot analysis.The expression level of cell-cycle related proteins and apoptotic-related proteins were observed by Western blot analysis.(5) The mRNA expressions of CyclinD3 and CDK4 were determined by reverse transcriplase polymerase chain reaction(RT-PCR).Results: (1)MTT assay indicated that the inhibitory effects of BRP800 and Dasatinib on the proliferation of H1299 cell were found in a dose-and-time dependent manner. There was a significant difference between BRP800 and Dasatinib group (P<0.05).(2)The changes of cell morphology were observed by inverted microscope. BRP800 could decrease the cell-cell adhesion and cell activity of H1299. Cell density in culture was significantly decreased by BRP800 at 30 nM within 24 hour(.3)Flow cytometry showed that the H1299 cell cycle was arrested by BRP800 at 24 hours. The ratio of G0/G1 phase was increased and the ratio of S and G2/M phase were decreased. But BRP800 was not able to induce the apoptosis of H1299 cells .(4)We evaluated the effect of BRP800 on key regulatory molecules of cell cycle, including Cyclins A, B1,D1, D2, D3, E, CDKs 4, 6, 9 and CKIs p21, p27, as well as p53,phospho-Rb. After 24 hour treatment, BRP800 repressed the expression of cyclin D1, D3, cyclin E. At the same time, BRP800 significantly decreased the expression of CDK4 and CDK6 in a concentration-dependent manner. There were the same effects on the expressions of CyclinD3 and CDK4. Cyclins and CDKs play a important role in G1 phase and S phase of cell cycle. Consistent with the cell cycle change, the p21 and p27 proteins were dramatically increased upon BRP800 treatment. P53 is a DNA damage checkpoint in G1 phase. If there has any damage, P53 protein will block the cell cycle (it can stop DNA replication) to provide sufficient time for DNA damage repair. We found that the p53 protein was significantly increased even at 3nM of BRP800. pRb is a common rate-limiting substrate in the G1 phase of the Cyclin-CDK complex-mediated phosphorylation .So it is a center control point component of G1 phase and S phase of cell cycle. We also found that treatment with BRP800 resulted in a significant reduction in Rb phosphorylation. (5) To further confirm that BRP800 failed to induce H1299 cells apoptosis, we checked the activation of initiative apoptosis enzymes (caspase-8 and -9) and executive apoptosis enzyme caspase-3 by immunoblotting assay after BRP800 treatment. Compared with the control group, BRP800 had no effect on the activation of the caspase cascade signals or the cleavage of PARP proteins. we further checked the effects of BRP800 on Bcl-2 and Mcl-1.Western blot analyses showed that BRP800 had no effect on the expression of Bcl-2 or Mcl-1 protein.(6) To further determine the mechanisms of action underlying BRP800-mediated anti-proliferation activity on human lung adenocarcinoma cell line H1299, Src activity was measured by its specific antibodies. BRP800 caused complete or near-complete inhibition of Src activity, as measured by phosphorylation at Y416. However, BRP800 displayed little effect on total Src expression. Because AKT is one of the downstream molecules of Src, We also examined the activation of the total and phosphorylated Akt following BRP800 treatment. Inhibition of Akt phosphorytion was detected after 10 nM of BRP800 treatment, although it still expressed even with 300 nM. (7) Reverse transcription PCR showed that the mRNA of CDK4 and Cyclin D3 were decreased in a dose-dependent manner which was consistent with the change of their protein changes. Conclusion: (1) BRP800 could effectively inhibit the growth of the lung adenocarcinoma cell line H1299 in a dose-and-time dependent manner. (2)As if the cell morphology, BRP800 could decrease the cell-cell adhesion, reduce the cell density and inhabit the cell activity. (3) BRP800 could also induce a cell cycle arrest of H1299 cell at the phase of G0/G1 , but failed to induce cell apoptosis. (4) Western blot also could confirm that BRP800 arrest cell cycle at the phase of G0/G1. (5) BRP800 could inhibit the activation of Src kinase. The molecular mechanism of cell growth inhibition of BRP800 may be related to blocking the Src/PI3K / AKT kinase cascade signaling pathway.