| Prostatic cancer (Pca) is the most common malignant tumor in western country. Now its morbidity increasing obviously in China in recent 10 years and has reached to the top 3 in the urinary system tumor. Most of Pca depends on the androgen ,but 14-30 months later, almost all of them will develop to androgen-independent Pca. Till now, there is no recognized effective treatment and medicine.After the first report from Medical University of Haerbin in 1996 that the Arsenic trioxide (As2O3 )can get a fairly higher effective rate to recurrent APL, experts has been studying its mechanism intensively which testified that As2O3 are effective to most of the tumors,but the study of telomerase activity and gene of independent-androgen Pca is very few, so we hereby discuss the influence of As2O3 on the cell-cycle block,apoptosis and telomerase activity of the independent-androgen Pca cell lines.Meanwhile, recent research found that the activation of Hh path played an important role in the initiation and development of the endodermal tumors. Some scholars even asserted that Hh signal path might be the most reliable mechanism of the Pca till now. Cyclopamine is the antagonist of Hh path ,the experiment here is to prove furthurly the effect and mechanism of Cyclopamine on the PC-3 cell line independent-androgen Pca and to lay a foundation for future combined use of medicine and animal experiment.Arsenic agent is a highly poisonous compound and Cyclopamine is also reported to have the possibility of leading to the deformity,their security as a medicine take the first priority of people's concern. If we can prove through former experiment that both As2O3or Cyclopamine can cause the apoptosis and block of cell-cycle of PC-3 cell, and they can respectively or both can inhibit the activity of telomerase and Hh signal path, can we achieve a better aiti-tumor effect and reduce the possible side effect while combining As2O3 and Cyclopamine ?Our experiment is composed of four parts:Part I: The effect of As2O3 on cell-cycle and telomerase activity of PC-3 cell linesMTT assay is used to study the influence of As2O3 on PC-3 cell's growth; the flow cytometric(FCM) to check the cell-cycle and apoptosis , and the TRAP-ELASA method to detect the activity of telomerase .From the result of MTT, we found that 6μmol/L As2O3 can inhabit the growth of PC-3 cell, and the inhibition strengthened with the increasing of the concentration and time. The effecting 48hours of IC50 is 16.21μmol/L. After incubated 48 hours together with 18μmol/L As2O3, some apoptopic changes were found under the electronic microscope. Cultured 48 hours together with the As2O3 of 6μmol/L,12μmol/L,18μmol/L and 24μmol /L respectively, the apoptosis rate of PC-3 cell increased accordingly, and the sub-G1 peak can be seen in the FCM.The apoptopic rate of group 24μmol /L , 18μmol/L and 12μmol/L of As2O3 obviously is higher than group 6μmol/L (P<0.05), the treated PC-3 cell apoptopic rate were 4.8%,15.2%, 19.59% ,29.67% respectively, which are quite different from normal group (P<0.01), the proportion of G0/G1 increased and the cells of S phase and G2/M phase decreased obviously. The activity of telomerase of PC-3 decreased with the increasing of the time and concentration, but when concentration is up to 18μmol/L, as the increasing of concentration, the inhibition of telomerase activity has no difference (P>0.05).Part II: The effect of As2O3 on cell-cycle and apoptosis of DU-145 cell linesDU-145 is also cell lines of androgen-independent Pca., and from the stduy we can see that As2O3 inhibits the growth of DU-145 cell even at 2μmol/L and 24 hours, and it is getting more obvious when concentrated up to 6μmol/L;effecting 48 hours with the cells , the IC50 is 8.02μmol /L, the IC50 of 72 hours is 6.21μmol /L;With the electronic microscope to observe the ultramicro structure, we can find some apoptopic changes after incubated 48 hours together with 18μmol/L As2O3. From the study of the cell-cycle in the FCM,the apoptopic rate increased apperantly, after 48 hours with 6,8,10μmol/L As2O 3,compared with the negative control, and there is a significant difference (P<0.01),.The ratio of G0/G1 phase decreased greatly and that of G2/M phase cell increased obviously. Compared with the control group, the difference is obvious(P<0.01). In the group of 2μmol/L of As2O3 while 48 hours, there is no peak of subG1 which represents the deceasing of cell. But in the group of 8μmol/L,there can be found one obvious peak of sub- G1 before the normal duplicate-peak.From the result electrochromatography of DU-145 cell treated by different concentrations of As2O3 ,DNA ladder chart can be seen,which is the result of 180200bp times fracture of the DNA.Part III: The effect of cyclopamine on androgen-independent prostate cancer cell lines PC-3.To investigate the role of Hedgehog specific inhibitor cyclopamine in cell proliferation, several techniques has been applied. MTT assay, BrdU incorporation, flow cytofluorometric analysis and TRAP-ELISA are carried out to monitor cell growth rates, DNA synthesis, cell cycle progression and telomerase activity. The data presented in this study suggests that cyclopamine significantly inhibits androgen-independent prostate cancer PC-3 cells growth through blocking Hedgehog signaling pathway at a concentration of 4-8μM. Without cyclopamine treatment, BrdU incorporation rate in PC-3 cells is 79%±5.6. Treatment of PC-3 cells with 2μM cyclopamine for 48 hours led to a significant reduction of BrdU incorporation (P<0.05) compared with untreated control cells (49%±8.3 versus 79%±5.6). Similarly, treatment of PC-3 cells with 4μM Cyclopamine for 48 hours led to a significant further reduction of BrdU incorporation (P<0.05) (30%±6.8 versus 79%±5.6). In contrast, treatment of PC-3 cells with Tomatidine for 48 hours causes no significant change of BrdU incorporation (P>0.05) (72%±6.9 versus 79%±5.6). The data from flow cytofluorometric analysis indicates that the treatment of PC-3 cells with 4μM cyclopamine for 48 hours reduces the ratio of cells in S phase from 34.8%±1.13 to 13.7%±0.18,, increase the ratio of cells in G2 phase from 19.7%±0.49 to 52.4%±0.56. In addition, induction of apoptosis has been indicated in cyclopamine treatment group with apoptosis peak in flow cytofluorometric analysis. Cyclopamine has no significant inhibitory effect on the activity of telomerase in PC-3 cells as shown in dose response curve and time course.Part IV: Investigation of synergy between low dose As2O3 and cyclopamine in androgen-independent prostate cancer cell lines PC-3.Similar approaches have been taken to determine the dose effect of As2O3 in combination with Cyclopamine in cell growth. In order to establish whether the combined effects of As2O3 and Cyclopamine are synergistic, the cells were treated with low lose As2O3 (6μM) and cyclopamine (2μM). Cell structures and morphology are examined with electron microscopy. MTT assays, BrdU incorporation, flow cytofluorometric analysis and TRAP- ELISA are carried out to monitor cell growth rates, DNA synthesis, cell cycle progression and telomerase activity. RT-PCR is used to monitor the expression of GLI1. The results from present studies indicate that the combined use of low dose As2O3 with Cyclopamine could significantly inhibit PC-3 cell proliferation. Apoptosis appears after 48 hours treatment of 6μM As2O3 in combination with 2μM Cyclopamine as determined by flow cytofluorometric analysis with sub-G1 apoptotic cells. The combination of these two drugs induces a significant additive cell apoptotic effect (P<0.01) compared to individual drug used alone at the same concentration. In addition, the combination treatment of As2O3 and Cyclopamine causes similar rate of apoptotic cell death as the treatment with high concentration (18μM) As2O3. The apoptotic death rate of both treatment group is significantly higher than that of the group treated with 4μM Cyclopamine (p<0.05). The combination of As2O3 and Cyclopamine treatment significantly attenuates BrdU incorporation in PC-3 cells to 26%±3.82 as compared to 79%±2.33 in untreated control cells as well as with individual treatment(P<0.01). The combination of As2O3 and Cyclopamine treatment exhibits no significant difference as compared with 4μM Cyclopamine or 18μM As2O3 treatment( P>0.05 ) . Both the treatment of PC-3 cells with 6μM As2O3 and combination of As2O3 with Cyclopamine cause significant inhibitory effect on the activity of telomerase compared with control untreated group after 48 hours. There is no significant difference between these two groups(P>0.05). The absorbance A which functions as a indicator for telomerase activity is 0.792±0.011 in the treatment with 6μM As2O3 group, 0.769±0.045 in combined treatment group versus 0.892±0.021 in control untreated group(P<0.05);To determine the importance of the Hedgehog pathways to AIPC cell growth , RT-PCR is used to detect the expression of GLI1 gene. A band corresponding to molecular weight of 292bp has been amplified with GLI1 gene specific primers. The primer set for GAPDH is used to amplify GAPDH gene which is used as an internal loading control. An even distribution of GADPH fragment with 579bp through the entire samples is observed and suggested equal loading of input. Strong expression of GLI1 has been detected in PC-3 cells. Treatment with Cyclopamine for 48 hours causes significant down-regulation of GLI1 expression in a dose dependent manner. However, combination treatment causes no further reduction on GLI1 expression compared to the treatment with 2μM Cyclopamine alone. In contrast, As2O3 treatment had no effect on the expression level of GLI1 within a wide range of concentration.Conclusions1. As2O3 significantly decreases the proliferation of androgen- independent prostate cancer PC-3 cells. As2O3 may target two separate signaling pathways: 1. As2O3 may induce cell apoptosis and inhibit cell cycle.2. As2O3 may down-regulate telomerase activity. However As2O3 has no effect on the function of GLI1 which is the essential and ultimate effectors of Hh signaling pathway.2. As2O3 also significantly inhibits the proliferation of androgen- independent prostate cancer DU-145 cells. With increasing concentration and duration of the drug treatment, cell apoptosis increases significantly as indicated by DNA fragmentation and cell arrests at G2/M phase.3. Cyclopamine significantly decreases the proliferation and DNA synthesis in PC-3 cells through inhibiting Hh signaling pathway. It decreases GLI1 transcription activity, inhibits cell cycle, and induces cell apoptosis. However, cyclopamine has no inhibitory effect on the function of telomerase.4. The combination treatment of low dose As2O3 (6μM) and Cyclopamine( 2μM) has synergistic anti-proliferative effects on PC-3cells. Combined cyclopamine and As2O3 treatment caused higher rate of inhibition on cell proliferation and a significant increase in apoptotic death as compared with the treatment of 6μM As2O3, 2μM or 4μM cyclopamine alone. The apoptotic death rate in the combined treatment group is similar to that in high dose As2O3 ( 18μmol/L ) treatment group. No additive inhibitory effect on telomerase and GLI1 has been observed in combined treatment. Combination therapy with As2O3 and cyclopamine has synergistic effects and may enable enhanced anticancer efficacy with low doses and no greater toxicity, thus improving the treatment of prostate cancer. |
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