ABSTRACT
The gene encoding MAP30 protein was cloned from bitter melon and recombinant MAP30 was expressed and purified. The human hepatoma G2.2.15 cells were exposed to different concentrations of MAP30. MTT assay was used to evaluate the cytotoxicity of the drugs and real-time PCR and Southern hybridization were applied to quantify extracellular HBV DNA and replicative intermediates intracellular and cccDNA in nucleus. HBsAg and HBeAg were assessed by enzyme-linked immunosorbent assay (ELISA). The results showed that exposure of HepG2.2.15 cells to MAP30 resulted in inhibition of HBV DNA replication and HBsAg secretion. After exposed to three different concentrations of MAP30 for 2, 4, 6, and 8 days respectively, the inhibition rates of extracellular HBV DNA, HBsAg, and HBeAg of each concentration decreased significantly (P < 0.05). After 9 days of treatment, the inhibition rates of extracellular HBV DNA of the different concentrations differed greatly (P < 0.001). The MAP30 could inhibit the production of HBV (P < 0.01) dose-dependently. The expression of HBsAg was significantly decreased by MAP30 dose-dependently (P < 0.001) and time-dependently (P < 0.001). Lower dose of MAP30 (8.0 microg/ml) could inhibit the expression of HBsAg and HBeAg.
Subject(s)
Antiviral Agents/pharmacology , Hepatitis B virus/drug effects , Ribosome Inactivating Proteins, Type 2/pharmacology , Cell Line, Tumor , DNA Replication/drug effects , DNA, Viral/antagonists & inhibitors , Dose-Response Relationship, Drug , Humans , Momordica charantia/chemistry , Plant Proteins/therapeutic use , Recombinant Proteins , Ribosome Inactivating Proteins, Type 2/isolation & purificationABSTRACT
Trichosanthin (TCS), is purified from the Chinese medicine, exerts antitumor activities by inducing apoptosis in many different tumor cell lines. The cDNA of trichosanthin was cloned and TCS was purified. The results showed that the proliferation of MCG803 cells were significantly suppressed by TCS in a dose-dependent manner at the concentration ranging from 20 to 100 microg/ml. The result of sequencing analysis indicates we obtained the TCS whole length gene. MTT assay was adopted to measure the growth inhibition ratio of MCG803 cells treated with TCS and apoptosis was assayed by agarose gel electrophoresis. DNA agarose gel electrophoresis showed a gradient, which confirmed that TCS could induce MCG803 cells apoptosis. The proportion of the periodic tumor cells were altered by TCS. Sub-G(1) curves were displayed by flow cytometry analysis. Results of Northern and Western blots showed that the transcription and expression of P21, was gradually up-regulated as treatment time increased. On the contrary, the transcription and expression of p53, was down-regulated. These data provided powerful evidences for the first time that recombinant TCS can induce the apoptosis of the MCG803 cells.
Subject(s)
Apoptosis/drug effects , Trichosanthin/pharmacology , Antineoplastic Agents, Phytogenic/pharmacology , Cell Line, Tumor , Cell Proliferation , Dose-Response Relationship, Drug , Humans , Recombinant Proteins , Stomach Neoplasms , Transcription, Genetic/drug effects , Tumor Suppressor Protein p53/genetics , rho GTP-Binding Proteins/geneticsABSTRACT
MAP30, an attractive protein isolated from bitter melon, has been previously found to have the anti-tumor and anti-HIV activities. In this study, MAP30 was cloned and expressed and the effects of the recombinant protein on cell proliferation and apoptosis of human colorectal carcinoma LoVo cells were investigated. The results showed that the proliferation of LoVo cells were significantly suppressed by MAP30 in time- and dose-dependent manners at the concentration ranging from 0.67 to 4.67 muM. The apoptotic nuclei of LoVo cells induced by MAP30 were obviously observed, and the genomic degradation was detected by single-cell gel electrophoresis (comet assay). Nuclear condensation and boundary aggregation or split, apoptotic bodies were seen by fluorescence and electron microscopy. The proportion of the periodic tumor cells was altered by MAP30. Sub-G1 curves were displayed by a flow cytometry analysis. Results of northern and western blots showed that the transcription and expression of Bax, a member of pro-apoptotic proteins, were gradually up-regulated as treated time increased. On the contrary, the transcription and expression of Bcl-2, an anti-apoptotic member, were down-regulated. These data provided powerful evidences for the first time that recombinant MAP30 can induce the apoptosis of the human colorectal carcinoma LoVo cells.
