The Apoptosis-inducing Effects On Hela Cell Of A Novel Synthetic Nucleoside 2'-O-methylguanosine (P6)

Objective: To investigate the antitumor effects of a novel synthetic nucleoside—P6 on human cervical carcinoma cell line—Hela cells. Simultaneously, its effects on the normal activated lymphocytes were also investigated in order to evaluate its side effects. The current study also intends to explore the possible mechanisms by which P6 exerts its antitumor effects.Methods:I. Cell culture: In all the experiments conducted in this study, the Hela cells were cultured in RPMI 1640 supplemented with 2% FBS at 37°C 5%CO₂ in vitro for 168 hours. The activated mouse lymphocytes were cultured in RPMI 1640 supplemented with 15% FBS at 37°C 5%CO₂ in vitro for 72 hours. All the cells in our experiments were treated with 0μg/mL, 50μg/mL, 100μg/mL and 150μg/mL P6 respectively.II. Activation of lymphocytes: The lymphocytes isolated from mouse spleen were activated by ConA at 2.5μg/mL and cultured for 72 hours in the presence or absence of P6.III. Microscopy and MTT assay: Hela cells and the activated lymphocytes were cultured for the given period in the presence of P6 at the concentrations ranging from 0 to 150μg/mL. The morphological alterations of the cells were examined by microscopy and their proliferation was assayed by MTT assay.IV. Detection of DNA fragmentation: DNA was extracted from Hela cells and the activated lymphocytes were cultured for the given period in thepresence of P6 at the concentrations ranging from 0 to 150jj,g/mL. The fragmentation of DNA was detected by use of agarose gel electrophoresis.V. RT-PCR analysis: Total mRNA were extracted from Hela cells and the activated lymphocytes cultured for the given period in the presence of P6 at the concentrations ranging from 0 to 150ug/mL. The transcription of Bax gene and Bcl-2 gene were then assayed by RT-PCR.VI. Flow cytometry: After incubation for the given period in the presence of P6 at the concentrations ranging from 0 to 150ng/mL, Hela cells and the activated lymphocytes were then stained with PI and the cell cycles were assayed by flow cytometry.VII. Chemiluminescence assay: By the end of culture, Hela cells were collected and lysed. The activity of the released caspase-3 was measured by chemiluminescence assay.Results:I. P6 can inhibit the proliferation of Hela cells: Microscopy and MTT assay showed that the proliferation of Hela cells was inhibited in the presence of P6. This inhibitory effect of P6, as indicated by flow cytometry, associates with the arrest of Hela cells in S-phase of the cell cycle.II. P6 can induce apoptosis in Hela cells: P6 may act as an apoptosis-inducing agent, because it was found that P6 can induce DNA fragmentation, activate csapase-3, down-regulate the expression of Bcl-2 gene, and increase the cells in sub-Gl phase of the cell cycle.III. P6 can inhibit the proliferation of activated lymphocytes, and can induce apoptosis in them: Similar findings were made in P6-treated mouse T cells, including inhibition of T-cell proliferation, arrest of T-cells in S-phase or sub-Gl phase of the cell cycle, up-regulation of Bax gene expression and down-regulation of Bcl-2 gene expression.Conclusions:I. Synthetic P6 can inhibit the proliferation of Hela cells by arrest the cells in S-phase of the cell cycle, and this inhibitory effect is independent of P6 concentration.II. Like natural P6, synthetic P6 may also act as an apoptosis-inducing agent by virtue of its ability to up-regulate the activation of caspase-3, regulate the expression of Bcl-2 gene, and arrest the cells in sub-Gl phase of the cell cycle.III. Synthetic P6 can inhibit the proliferation of activated lymphocyte in a dose-dependent manner. This inhibition of lymphocyte's proliferation results from the arrest of the lymphocyte in S-phase of the cell cycle.IV. Synthetic P6 can induce the apoptosis of activated lymphocytes by virtue of its ability to regulate the expression of Bax gene and Bcl-2 gene, and arrest the cells in sub-Gl phase of the cell cycle. All these suggest that immunosuppression might be one of the side effects if it is used in the treatment of tumors.V. Evidently, it is necessary to make more detailed studies such as the studies using animal models of various tumors before it undergoes clinical trails for its use in tumor treatment.