The Effects Of Creatine Phosphate Sodium On Proliferation Of Human Ovarian Cancer Cell Line SKOV3 And Its Mechanisms

Objective: Cardiotoxicity is a frequent side effect of some anticancer drugs.The anthracycline class of cytotoxic antibiotics are the most famous. As a high energic phosphate compound, Creatine Phosphate Sodium(CP)supplied the energy when cardiac cell received ischemia or hypoxia and protected cell membrane in order to avoid the injury from oxygen free radical.Now CP has been applicated in heart medical and surgery apartment . Some scholar think that CP may increase the growth of tumor cell .To study the effects of CP on the proliferation of human ovarian cancer cell line SKOV3 and its mechanisms.The goal is to provide the theoretic basis for clinical therapy.Methods: 1 human ovarian cancer cell line SKOV3 was incubated in culture medium in vitro, using MTT (tetrzolium-based colorimetric assay) assay to detect the growth rate among different CP concentration groups (1,6,12mmol/L) and different time groups (24,48,72h). 2 Apoptosis and distribution of cell cycle were examined by flow cytometry. 3 According to the result of MTT, establishing control and experimental group, extracting total RNA of each group cell, assessing the integrality and content of RNA, the level of CyclinD1mRNA,Bcl-2mRNA expression was examined by semiquantitative Reverse transcription polymerase chain reaction(RT-PCR) technique in the SKOV3 cells treated before and after with CP. 4 The expression of CyclinD1,Bcl-2 protein was semi-quantitately examined by flow cytometry in SKOV3 cells treated before and after with CP.Results: 1 MTT assay results:CP (1,6,12 mmol/L) could inhibit the proliferation of SKOV3 in vitro. After treated with CP(1 mmol/L) for 24h,48h and 72h, compared with control group ,the OD values of CP-treated group decreased, but there was no significant difference between control group and CP(1 mmol/L) group(P>0.05). After treated with CP(6,12 mmol/L) for 24h,48h and 72h, compared with control group ,the OD values of CP-treated groups decreased, and there was significant difference between control group and CP(6,12 mmol/L) groups(P<0.05).Furthmore , with the increasing concentration of CP and prolonging of treatment time, the inhibition ratio increased gradually, that was ,CP inhibited the proliferation of SKOV3 cells significantly in dose-and time-dependent manner. The highest inhibition ratio was in the group with the concentration of 12mmol/L and 72h. 2 When cells were harvested for the analysis on distribution of cell cycle and apoptosis by flow cytometry, the results were: After treatment with 1,6,12mmol/L CP for 72h, the number of cells in G0/G1 phase increased gradually, while the number of cells in S phase decreased grudually. That was, CP could induce an arrest of cell cycle in G0/G1 phase by a dose-dependent manner. In addition, after being treated with 1,6,12mmol/L CP for 72h, the typical apoptotic peak could not be observed with the increasing concentration of CP,and the apoptotic percentage was no significant difference between control group and every treatment group(P>0.05). 3 RT-PCR detection results:There was different depression of the CyclinD1mRNA expression in different concentration groups of SKOV3 cells after being treated CP. After treated with CP(1 mmol/L) for 72h, compared with control group,the expression of CyclinD1mRNA decreased, but there was no significant difference between control group and CP(1 mmol/L) group(P>0.05). After treated with CP(6,12mmol/L) for 72h, compared with control group,the expression of CyclinD1mRNA decreased, and there was significant difference between control group and CP(6,12 mmol/L) group(P<0.05). CP obviously repressed the CyclinD1mRNA expression in SKOV3 at 12 mmol/L after treating 72h. After treated with CP(1,6,12 mmol/L) for 72h, compared with control group,the expression of Bcl-2mRNA decreased, but there was no significant difference between control group and CP groups(P>0.05). 4 FCM assay results:The expression of CyclinD1,Bcl-2 protein had been examined in SKOV3 cells being treated with 0,1,6,12mmol/L CP for 72h, the FI-value of CyclinD1 in SKOV3 was 1,0.988,0.863,0.718, the FI-value of Bcl-2 in SKOV3 was 1,0.977,0.947,0.912, respectively. To the FI values of CyclinD1 protein, there was no significant difference between control group and CP(1 mmol/L) group(P>0.05). To the FI values of CyclinD1 protein, there was significant difference between control group and CP(6,12 mmol/L) group(P<0.05). The expression of CyclinD1 protein was significantly inhibited after treatment with CP of 12mmol/L,72h. To the FI values of Bcl-2 protein, there was no significant difference between control group and CP (1,6,12 mmol/L) group(P>0.05). There was consistent with the RT-PCR results.Conclusions:1 We comfirmed that CP had the capability of inhibiting the proliferation of SKOV3 cells in vitro .2 CP could not induce cellular apoptosis. Within a certain drug concentration, the mRNA and protein expression of Bcl-2 was no significant difference between control group and CP groups.3 We comfirmed that CP induced an arrest of cell cycle in G0/G1 phase in SKOV3 cells. Within a certain drug concentration, CP could inhibit the mRNA and protein expression of CyclinD1 in SKOV3 cells. Cell cycle arrest maybe one of the mechanisms of its anti-tumor.4 This research proved that CP could not increase the growth of human ovarian cancer cell line SKOV3 in vitro. This privide a new theoretic way for clinical therapy.