| Objective: By exploring the inhibition of cervical cancer Hela cells in vitro by Chlorpromazine(CPZ) and of the inhibiting mechanism, and by studying the effects of Chlorpromazine combined with anti-cancer agent Cisplatin on human cervical cancer cell Hela in vitro, this paper tries to obtain the anticancer therapeutic effect of Chlorpromazine, and to evaluate safety and the feasibility of Chlorpromazine treatment, so as to provide a new way in the medical treatment of cervical carcinoma.Methods: Human Hela cervical carcinoma cells are taken as study object and are divided into three groups: Chlorpromazine group, Cisplatin group and combination group. 1 Effect of CPZ of different concentrations on the proliferation of Hela cells with different functioning time(24h, 48h, 72h)and OD values were measured by MTT colorimetric method. Forty-eight hours after Chlorpromazine's functioning, flow cytometer (FCM) was used to detect the changes of percentage and cell cycle of apoptosis cells,and to measure the amount of Bax and Bcl-2 protein and the analysis of changes of DNA-fragment were analyzed by DNA gelatum electrophoresis. 2 Forty-eight hours after combined functioning of Chlorpromazine and Cisplatin, the proliferation of Hela cells and OD values were measured by MTT colorimetric method; the HE staining was performed,and the morphological changes of Hela cells were observed by light microscopy; The immunohistochemical technique (S-P) was also used to identify the amount of P53 in Hela cells.Results: 1 Results of MTT indicate that CPZ, to certain extent, can inhibit the proliferation of Hela cells (compared with control group, P<0.01) and the inhibition enhances dramatically as concentration of CPZ is raised and functioning time is prolonged. The effect was presented in a dose- and time-dependent manner. When the concentration of CPZ was respectively 10-7mol/L, 10-6mol/L, 10-5mol/L, and 10-4mol/L the inhibition rate of Hela cells growth was 22.74%, 41.69%, 51.39% and 67.45% respectively, which was significantly different from the control group (same amount of culture solution was added) (P<0.01). When the concentration of Cisplatin was respectively 0.2μmol/L, 0.5μmol/L, 1.0μmol/L and 2.0μmol/L, the inhibition rate was 43.88%, 48.59%, 61.49% and 71.52% respectively. When Chlorpromazine and Cisplatin were used in combition, the inhibition rate increased to 49.70%, 63.52%, 70.42% and 76.15% respectively, much higher than those when Cisplatin only was used.2 Analysis of FCM results: the results of FCM test on cell cycle distribution and apoptosis of Hela celles indicate that the number of cells in G0/G1 phase increased gradually, while the number of cells in S phase and G2/M phase decreased gradually 48h after Chlorpromazine of 10-7, 10-6, 10-5 and 10-4mol /L CPZ were applied. That is, CPZ could hold cell cycle of Hela cells in G1 phase on a dose-dependent basis. In addition, 48h after Chlorpromazine of 10-7, 10-6, 10-5 mol /L was applied, a typical apoptotic peak was observed and the apoptosis increased gradually as Chlorpromazine concentration was raised, and the apoptotic percentage was 5.03%, 9.35% and 23.85% respectively. Analysis on expression of Bax and Bcl-2 by FCM showed that treating Hela cells with 10-7, 10-6, 10-5 mol /L CPZ respectively for 48h resulted in an increase of FI values of Bax and a decrease of FI values of Bcl-2 as Chlorpromazine concentration increased. To each kind of protein, its FI values differ dramatically between the Chlorpromazine-treated group and control group(P<0.01).3 The DNA gelaum electrophoresis showed that: forty-eight hours after treating Hela cells with Chlorpromazine of 10-7, 10-6, 10-5 and 10-4mol /L respectively, a typical DNA ladder was observed in every treatment group, while not in the control group.4 HE staining results showed that Hela cells gradually became round as Chlorpromazine concentration increased. lots of kyan-cellular nucleus, pink endochylema and apoptosis cells after karyopycnosis, thickness, collapse were observed . In Cisplatin group, the apoptotic cells were observed when the concentration was 0.5μmol/L, while the apoptotic cells were observed when the concentration was 0.2μmol/L in combination group.5 Analysis of P53 protein expression by immunohistochemical staining(S-P): P53 protein was expressed mainly in cell nucleus. In Chlorpromazine group, the expression of P53 protein showed a tendency of increasing; the optical density value of nuclear P53 protein was 0.1240±0.0551 when the concentration was 10-7mol/L; and when the concentration increased to 10-5mol/L, the OD value was 0.2020±0.0626. There was a significant difference compared with the control group (P < 0.01). In Cisplatin group, the expression of P53 protein went on increasing. When the concentration was 0.5μmol/L, the OD value was 0.2260±0.0691(P<0.01). There was a significant difference compared with the control group (P < 0.01). In combination group, the expression of P53 protein and the OD value increased markedly. That is when least concentration of Chlorpromazine(10-7mol/L)and Cisplatin(0.2μmol/L)are applied, the OD value reached 0.2560±0.0305. There was an exrtremely significant difference compared with the control group (P < 0.01) There existed a statistical significance compared with the Cisplatin group (P < 0.01) in which Hela cells were treated with Cisplatin of same concentration.Conclusions: 1 With certain dose and treating time, Chlorpromazine can inhibit proliferation of Hela cells in a dose- and time-dependent manner, and it can induce Hela cellular apoptosis. 2 The coordinative effect was observed through combining Chlorpromazine with Cisplatin. Cisplatin, if combined with Chlorpromazine, can further inhibit the proliferation of Hela cells in vitro and dramatically increase the expression of P53 protein. |
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