Mechanistic Study Of Short Peptide In Experimental Hepatocarcinoma Model

Objective: To explore the anticancer effect of a newly synthesized short peptide CMS024-02 on human hepatocarcinoma BEL-7402 model and to investigate its possible mechanism from cellular, subcellular and molecular aspects. Methods: The anticancer effects of CMS024-02 (320, 160, 80μg/kg/d) were speculated in a human hepatocarcinoma BEL-7402 transplant model in nude mice by the changes of tumor weight, volume, animal living status, blood and bone marrow smear.The negative effects of CMS024-02 on DNA synthesis ability, cellular metabolic activity and cytoxicity to cultured BEL-7402 cells were evaluated by BrdU incorporation, MTT metabolism, and LDH releasing, respectively. The senescence- inducing effect of CMS024-02 on BEL-7402 cells in vitro was assayed by β -galactosidase staining method. The DNA ladder phenomena were observed by agarose electrophoresis method in CMS024-02 treated BEL-7402 cells.PCNA expression and DNA strand breakage in tissue sections from CMS024-02 (160μg/kg/d) treated group was observed by immunohistochemistry method and TUNEL method. Apoptosis and necrosis inducing effects were observed by light and electron microscope.AFM ( atomic force microscopy) was employed to observe the effect of CMS024-02 on DNA structure extracted from BEL-7402 cells in vitro. Genetic expression profile of BEL-7402 transplante mice were explored by Human Genome Focus Genechip.RT-PCR, Western Blot, immunohistochemistry, or ELSIA method was used to observe the effect of CMS024-02 on multiple gene and protein expression, such asPTEN> Akt> MDM2, NF-kB, COX-2, p27, p21 and p53 in BEL-7402 transplanted nude mice tissues.Results: CMS024-02 showed an significant (P<0.05) inhibitive effects on the proliferation of hepatocarcinoma tissues assayed by weight and volume reduction, A significant 57.60% and 58.24% inhibitory rate were observed in 160|j.g/kg/d and 320ug/kg/d dosage group respectively. Compared with that of the cyclophosphamide group (20mg/kg/d), the weight of nude mice in different dosage CMS024-02 groups were increased together with better life quality. CMS024-02 shows no inhibitory effect on bone marrow of nude mice, and the same in quantity, shape and ratio of blood cells of nude mice.CMS024-02 could inhibit DNA synthesis, depress cellular metabolic activity and cause cytoxic effect on BEL-7402 in vitro. CMS024-02 also could induce BEL-7402 cells to a senescent phenotype and the occurance of DNA ladder. CMS024-02 could depress PCNA expression level in BEL-7402 hepatoma transplant tissues, and induce apoptosis. Results of HE staining and electron microscopy shows that CMS024-02 could induce apoptosis and necrosis of BEL-7402 cells in the nude mice model.Result of AFM observation suggested that lug/mLCMS024-02 might induce DNA aggregation;and Genechip analysis shows that CMS024-02 could affect multiple genes' expression level that may be associated with cancer. Results of RT-PCR, Western Blot, immunohistochemistry, ELSIA and enzyme activity assay show that CMS024-02 could affect the equilibrium between oncogene and tumor suppressor gene. The mRNA and protein expression of tumor suppressor gene PTE^ p21> p27 were ascended, but the p53 protein showed no significant changes. The mRNA and protein level of oncogene Akt and COX-2 were decreased, while mRNA and nuclear protein expression of oncogene MDM2both were decreased;p-PTEN and p-Akt protein expression were all decreased;The phosphatase activity of PTEN was increased accompanied by the decrease of Akt kinase activity. Although CMS024-02 showed no effect on mRNA expression of NF-kB p50 and p65 subunit, it could inhibit their protein expression. Conclusion: CMS024-02 could significantly inhibit the growth of human hepatocarcinoma BEL-7402 in a nude mouse xenographt model, and shows no significant side effect. CMS024-02 could inhibit the activation of PI3K/Akt pathway by activating PTEN phosphatase activity and increasing its product in tumor cells. CMS024-02 could sequester MDM2 to the nuclei and might prevent the degradation of p53. It could also inhibit the protein expression of NF-kB p50> p65 subunit in together with inhibition the activation of one of its downstream genes - COX-2. In conclusion, all those changes caused by CMS024-02 may explain the anti-proliferation and death-inducing effects of hepatocarcinoma cells.