Effects Of Novel Cyclic Tetrapeptide HDACIs On Proliferation And Migration Of Breast Cancer Cells

Histone deacetylases (HDACs) are a class of endogenous proteases responsible for the epigenetic modification of histone. It has been revealed that abnormal accumulation or hyper-activation of HDACs is statistically related to malignancy and poor prognosis. Accordingly, HDAC inhibitors (HDACIs), which are capable of up-regulating tumor suppressor genes via interacting with HDACs, exhibit significant anticancer potential. Due to their high efficiency and high selectivity against cancer cells, HDACIs are considered as promising anticancer candidates. Herein, biological evaluations were conducted to identify high efficient individuals among the previously synthesized peptide HDACIs, followed by investigation of the signaling pathways and the regulation mechanisms underlying HDACIs-induced cell death via the application of the selected compound.In multiple cancer cell lines, experiments on protein expression were conducted, cell biological features were measured and structure-function relationship was interpreted to confirm CTS203(cyc/o(-L-Asu(NHOH)-Am3c6c-L-Phe-D-Pro-)) to be a qualified HDACIs. Furthermore, cell morphology, cell cycle distribution and the expression of particular regulators were examined, and the results indicated that cell cycle arrest and apoptosis are two major time-dependent cytotoxic effects of CTS203. Stick to the increasing exposure time, the expression of cyclin D1was gradually decreased whereas the expression of p21increased, both contributing to cell cycle arrest. The accumulation of cytochrome c or the activation of caspase-3sebquently multiplied, indicating apoptosis has taken its place. The expressions of caspase-8and caspase-9simultaneously varied, indicating the initiation of mitochondrial apoptotic pathway.Then we focus on the investigation of the mechanism underlying time-dependent cytotoxicity of CTS203. The immunofluorescence images sampled at specific time points indicated that during the first few hours of exposure, the participation of autophagy attenuated CTS203-induced cytotoxicity. Examines of the schedule for autophagosomes formation and the expression of autophagy-associated proteins further confirmed that CTS203-induced autophagy was cyto-protective. Therefore, the inhibition of autophagy resulted in a strengthened cytotoxic effect as well as a shorten response time. In addition, the measurement of apoptotic factors revealed that after inducing the cleavage of Beclin-1, it is the co-operation of caspase-9and caspase-8that responsible for the amplification of apoptotic signals, thus lead to the promotion of cytotoxicity.The inhibitory effects of CTS203on cancer cell migration and colony formation were then investigated. As revealed by wound healing assay and soft agar assay, CTS203effectively limited the capabilities of migration and colony formation against MCF-7cells. Measurement of migration-associated regulators and docking study subsequently revealed the mechanisms underlying CTS203-induced migration inhibition. Specifically, variations of protein expression indicated that CTS203induced cell migration inhibition by up-regulating the expression of RECK and the ratio of TIMP-1/MMP-2, which would lead to attenuated extracellular activity of MMP-2; docking results revealed that CTS203has a potential to complex with MMP-2and could make the enzyme disabled by forming MMP-2-CTS203complexes.In summary, an effective anticancer candidate CTS203which is multifunctional in anticancer treatment was screened out. The enhanced cytotoxic effect achieved by combination treatment of CTS203and3-MA implied the synergism between CTS203and other anticancer agents as well as the benefits of combination treatment. Furthermore, MMP-2was acknowledged as a possible target of CTS203, which shed new light on the development of selective inhibitors of MMP-2and the investigation of mechanisms underlying HDACIs-induced migration inhibition.