低剂量砷剂联合Cyclopamine对前列腺癌细胞株PC-3生长影响机制的初步研究
摘要
前列腺癌(Prostatic cancer,Pca)为西方国家最常见的恶性肿瘤,近十年我国Pca的检出率也明显上升,已位居泌尿系统肿瘤的第三位,其大多数都依赖于雄激素,但14—30个月后,几乎所有患者病变都将发展为激素非依赖性Pca,目前尚没有公认的有效的治疗非雄依赖Pca的手段和药物。
自1996年哈医大首先报道静滴三氧化二砷(Arsenic trioxide,As_2O_3)治疗复发的急性早幼粒细胞白血病(APL)达到很高的完全缓解率以来,国内外学者对其抗肿瘤机制进行了广泛而深入的研究,已证实As_2O_3对多数肿瘤(包括实体瘤)有效,但对于非雄依赖的Pca及基因和端粒酶方面的研究甚少,故我们拟进一步探讨As_2O_3对前列腺癌非雄细胞系周期阻滞、凋亡及端粒酶活性的影响。
同时,近年来的研究发现,Hedgehog(Hh)通路的激活在内胚层肿瘤包括的发生、发展中起重要作用,有学者称Hh信号通路可能是我们所了解的PCa发病的更可靠的机理,环杷明(Cyclopamine)是Hh通路的拮抗剂,本实验拟进一步印证Cyclopamine对前列腺癌非雄依赖细胞系PC-3细胞的影响及作用机制,为以后的联合用药及动物实验奠定基础。
砷剂是一个剧毒化合物,Cyclopamine亦有导致动物畸形的报道,其用药的安全性是人们首要关注的问题之一,若通过前期试验进一步证实As_2O_3或Cyclopamine均可引起PCa非雄依赖的PC-3细胞的凋亡和周期抑制,并且分别或是均能抑制端粒酶及信号通路的活性,那么将As_2O_3同Cyclopamine联合应用,是否可以通过降低剂量而降低毒性和潜在副作用,同时又在前列腺癌非雄依赖细胞的研究中有着更好的抗肿瘤作用,从而成为更有效、更安全的非雄依赖的前列腺癌的中西医结合的治疗手段,需要实验进一步的探讨。
本实验共分为四个部分:
第一部分:三氧化二砷对前列腺癌PC-3细胞系细胞周期及端粒酶活性的影响
我们应用体外细胞生长抑制试验(MTT比色法)研究对PC-3细胞生长的影响;流式细胞仪检测细胞周期分布情况及凋亡情况;TRAP-ELASA方法检测药物作用前后端粒酶的活性。MTT结果发现,6μmol/l As_2O_3对PC-3细胞即具有抑制作用,随着浓度的增加和作用时间的延长,抑制作用进一步增强,作用48小时,IC50为16.21μmol/L。18μmol/L的As_2O_3与PC-3细胞共培养48h后,电镜下部分癌细胞出现凋亡形态学改变,经6μmol/L、12μmol/L、18μmol/L、24μmol /L的As_2O_3作用48h后,细胞凋亡率逐渐增加,在流式图上可见到代表凋亡的亚G1峰,且24μmol/L组、18μmol/L组、12μmol/L组的As_2O_3组细胞凋亡率明显高于6μmol/组(P<0.05)。四组浓度处理后的PC-3细胞凋亡率分别为4.8%,15.2%, 19.6% ,29.7%,同正常组比较有显著性差异(P<0.01),G0/G1期细胞比例明显增加,而S期和G2/M期细胞明显减少。PC-3细胞端粒酶活性随着作用时间及作用浓度的增加而降低,具有时间剂量效应关系,但当浓度增加至18μmol/L以上时,随着浓度的增加,对端粒酶活性的抑制无显著性差异(P>0.05)。
第二部分:三氧化二砷对前列腺癌DU-145细胞周期阻滞及凋亡的影响
DU-145也是非雄依赖的前列腺癌细胞系,研究结果发现,As_2O_3对DU-145细胞生长具有抑制作用,2μmol/L的As_2O_3作用24h即对DU-145细胞具有抑制作用,当浓度增加至6μmol/L以上时,抑制作用更为明显,作用48小时,IC50为8.02μmol /L,作用72小时IC50为6.21μmol /L;电镜观察DU-145细胞超微结构发现:8.0μmol/L的As_2O_3与DU-145细胞共培养48h后,部分癌细胞出现凋亡形态学改变,晚期可产生凋亡小体。流式细胞仪检测细胞凋亡及细胞周期的分布发现经6、8、10μmol/L的As_2O_3作用48h后, DU-145细胞凋亡率明显增加,与空白对照组比较有显著性差异(P<0.01), G0/G1期细胞比例明显下降,而G2/M期细胞比例明显增加,和对照组相比有显著性差异(P<0.01),在48小时2μmol/LAs_2O_3作用组未见代表细胞凋亡的亚G1峰,而在8μmol/L作用组,在正常二倍体峰前面可看到一个明显的代表凋亡的亚G1峰;As_2O_3处理DU-145细胞基因组DNA电泳结果发现:不同浓度的As_2O_3处理DU-145细胞48h后,基因组DNA凝胶电泳呈现明显的梯状图谱,这是核小体间DNA成180-200bp整倍断裂的结果。
第三部分:Cyclopamine对非雄依赖的前列腺癌PC-3细胞的影响
通过MTT检测Cyclopamine对细胞生长增殖的影响,BrdU掺入检测PC-3细胞DNA合成的情况,流式检测Cyclopamine对PC-3细胞周期的影响, TRAP-ELISA法细胞检测端粒酶的活性。实验结果显示:Cyclopamine可以通过抑制Hh信号转导通路而明显抑制PC-3细胞的增殖,在4-8μmol/L有明显的抑制作用; BrdU掺入结果PC-3细胞未加Cyclopamine时BrdU掺入阳性细胞平均约占细胞总数的79%±5.6,加入2μmol/L的Cyclopamine作用48小时后PC-3细胞BrdU掺入阳性细胞平均约占细胞总数的49%±8.3,低于对照组79 %±5.6 (P<0.05 ),加入4μmol/L的Cyclopamine作用48小时后PC-3细胞BrdU掺入阳性细胞平均约占细胞总数的30%±6.8,明显低于对照组(P<0.01),但加入Tomatidine(Cyclopamine类似物)作用48小时后BrdU掺入阳性细胞平均约占细胞总数的72%±6.9,与对照组相比差异无显著性(P>0.05);流式细胞仪检测细胞周期的结果为:4μmol/L的Cyclopamine作用48 h后,PC-3细胞进入S期比例由空白对照组的34.8%±1.13下降为13.7%±0.18,G1期的细胞比例由空白对照组的46.8%±1.32下降为34.8%±0.78, G2期的细胞比例由空白对照组的19.7%±0.49上升为52.4%±0.56, Cyclopamine处理组在流式图上还出现了明显的凋亡峰;Cyclopamine对PC-3细胞端粒酶活性在低浓度和高浓度时均无明显抑制作用,并且随着作用时间的延长,亦无抑制作用。
第四部分:低剂量砷剂联合Cyclopamine对前列腺癌细胞株PC-3生长影响的研究
通过MTT检测不同浓度砷剂联合Cyclopamine对PC-3细胞生长的影响,电镜观察低剂量砷剂(6μmol/L)和Cyclopamine(2μmol/L)对PC-3细胞形态和超微结构的影响,以流式细胞仪检测细胞凋亡,TRAP-ELISA检测端粒酶活性,BrdU掺入方法检测低剂量砷剂和Cyclopamine对PC-3细胞DNA合成的影响, RT-PCR法检测GLI1的表达。实验结果显示:低剂量砷剂联合Cyclopamine可以明显抑制PC-3细胞的增殖,作用48h可见细胞凋亡;流式细胞图发现经6μmol/L的As_2O_3和2μmol/L的Cyclopamine共同作用48h后,在流式图上可见到代表凋亡的亚G1峰,联合用药组的细胞凋亡率明显高于6μmol/L的As_2O_3组和2μmol/L的Cyclopamine组(P<0.01),其凋亡率与高浓度As_2O_3组(18μmol/L)的凋亡率无显著性差别(P>0.05),高于4μmol/L的Cyclopamine组(P<0.05);PC-3细胞对照组BrdU掺入阳性细胞平均约占细胞总数的79%±2.33,低剂量砷剂和Cyclopamine联合用药组作用PC-3细胞48h后,BrdU掺入阳性细胞数平均约占细胞总数的26%±3.82,与单药作用组分别作用有显著性差异(P<0.01),与4μmol/L的Cyclopamine作用组及18μmol/L的As_2O_3作用组相比则无显著性差异(P>0.05);6μmol/L的As_2O_3作用PC-3细胞48h后可以抑制端粒酶的活性,吸光度A值为0.792±0.011,而联合用药组作用PC-3细胞48h后亦可抑制端粒酶的活性,吸光度A值为0.769±0.045,两组之间无显著性差异(P>0.05);但与对照组相比(A值为0.892±0.021)均有显著性差异(P<0.05);RT-PCR结果显示,在DNALadder约292bp处出现明暗相间的分子条带,即为目的基因GLI1;而在约579 bp处出现粗细、明暗均较均一的条带,此即内参GAPDH,说明PC-3细胞GLI1的表达较强,与2μmol/L的Cyclopamine共同作用48h后,GLI1的表达有所减弱,随着其浓度的增加,在4μmol/L组GLI1的表达进一步减弱,与对照组相比均有显著性差异(P<0.05);而联合用药组尽管对GLI1的表达也有抑制,与对照组相比有显著性差异(P<0.05),但与2μmol/L的Cyclopamine单独用药组相比,抑制作用无增强或减弱,统计学分析无显著性差异(P>0.05),进一步的研究发现,无论是低剂量砷剂还是高剂量砷剂,其对GLI1的表达均无影响。
结论:
一.As_2O_3可明显抑制前列腺癌非雄依赖的PC-3细胞的增殖,其途径可能与以下两方面机制有关,一是诱导细胞凋亡,抑制细胞周期,二是下调细胞的端粒酶活性,但对Hh信号通路的关键因子GLI1的活性无影响。
二.As_2O_3对前列腺癌非雄依赖的DU-145细胞系也有明显的抑制作用,且随着药物浓度增加和作用时间延长,凋亡细胞明显增加,出现G2/M期阻滞和DNA的断裂。
三.Cyclopamine可以通过抑制Hh信号转导通路而明显抑制PC-3细胞的增殖和DNA的合成,降低GLI1的表达,抑制细胞的周期,诱导细胞的凋亡,但对细胞端粒酶活性无抑制作用。
四.低剂量砷剂(6μmol/L)和Cyclopamine(2μmol/L)的联合应用可以抑制PC-3细胞的增殖,并诱导细胞凋亡,细胞凋亡率明显高于6μmol/L的As_2O_3组和2μmol/L的Cyclopamine组,与高浓度As_2O_3组( 18μmol/L )的凋亡率无显著性差别,但明显高于4μmol/L的Cyclopamine组;其对端粒酶的活性和GLI1的表达均有抑制作用,但与单药作用组相比无显著性差异,二者的联合应用,有协同作用,可以通过降低药物剂量,降低潜在的可能的毒副作用,并取得同样甚至优于高剂量单药作用同样的抑制PC-3细胞的效果。
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 (As_2O_3 )can get a fairly higher effective rate to recurrent APL, experts has been studying its mechanism intensively which testified that As_2O_3 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 As_2O_3 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 As_2O_3or 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 As_2O_3 and Cyclopamine ?
Our experiment is composed of four parts:
Part I: The effect of As_2O_3 on cell-cycle and telomerase activity of PC-3 cell lines
MTT assay is used to study the influence of As_2O_3 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 As_2O_3 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 As_2O_3, some apoptopic changes were found under the electronic microscope. Cultured 48 hours together with the As_2O_3 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 As_2O_3 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 As_2O_3 on cell-cycle and apoptosis of DU-145 cell lines
DU-145 is also cell lines of androgen-independent Pca., and from the stduy we can see that As_2O_3 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 As_2O_3. 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 As_2O_3 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 As_2O_3 ,DNA ladder chart can be seen,which is the result of 180~200bp 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 As_2O_3 and cyclopamine in androgen-independent prostate cancer cell lines PC-3.
Similar approaches have been taken to determine the dose effect of As_2O_3 in combination with Cyclopamine in cell growth. In order to establish whether the combined effects of As_2O_3 and Cyclopamine are synergistic, the cells were treated with low lose As_2O_3 (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 As_2O_3 with Cyclopamine could significantly inhibit PC-3 cell proliferation. Apoptosis appears after 48 hours treatment of 6μM As_2O_3 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 As_2O_3 and Cyclopamine causes similar rate of apoptotic cell death as the treatment with high concentration (18μM) As_2O_3. 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 As_2O_3 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 As_2O_3 and Cyclopamine treatment exhibits no significant difference as compared with 4μM Cyclopamine or 18μM As_2O_3 treatment( P>0.05 ) . Both the treatment of PC-3 cells with 6μM As_2O_3 and combination of As_2O_3 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 As_2O_3 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, As_2O_3 treatment had no effect on the expression level of GLI1 within a wide range of concentration.
Conclusions
1. As_2O_3 significantly decreases the proliferation of androgen- independent prostate cancer PC-3 cells. As_2O_3 may target two separate signaling pathways: 1. As_2O_3 may induce cell apoptosis and inhibit cell cycle.
2. As_2O_3 may down-regulate telomerase activity. However As_2O_3 has no effect on the function of GLI1 which is the essential and ultimate effectors of Hh signaling pathway.
2. As_2O_3 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 As_2O_3 (6μM) and Cyclopamine( 2μM) has synergistic anti-proliferative effects on PC-3cells. Combined cyclopamine and As_2O_3 treatment caused higher rate of inhibition on cell proliferation and a significant increase in apoptotic death as compared with the treatment of 6μM As_2O_3, 2μM or 4μM cyclopamine alone. The apoptotic death rate in the combined treatment group is similar to that in high dose As_2O_3 ( 18μmol/L ) treatment group. No additive inhibitory effect on telomerase and GLI1 has been observed in combined treatment. Combination therapy with As_2O_3 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.
自1996年哈医大首先报道静滴三氧化二砷(Arsenic trioxide,As_2O_3)治疗复发的急性早幼粒细胞白血病(APL)达到很高的完全缓解率以来,国内外学者对其抗肿瘤机制进行了广泛而深入的研究,已证实As_2O_3对多数肿瘤(包括实体瘤)有效,但对于非雄依赖的Pca及基因和端粒酶方面的研究甚少,故我们拟进一步探讨As_2O_3对前列腺癌非雄细胞系周期阻滞、凋亡及端粒酶活性的影响。
同时,近年来的研究发现,Hedgehog(Hh)通路的激活在内胚层肿瘤包括的发生、发展中起重要作用,有学者称Hh信号通路可能是我们所了解的PCa发病的更可靠的机理,环杷明(Cyclopamine)是Hh通路的拮抗剂,本实验拟进一步印证Cyclopamine对前列腺癌非雄依赖细胞系PC-3细胞的影响及作用机制,为以后的联合用药及动物实验奠定基础。
砷剂是一个剧毒化合物,Cyclopamine亦有导致动物畸形的报道,其用药的安全性是人们首要关注的问题之一,若通过前期试验进一步证实As_2O_3或Cyclopamine均可引起PCa非雄依赖的PC-3细胞的凋亡和周期抑制,并且分别或是均能抑制端粒酶及信号通路的活性,那么将As_2O_3同Cyclopamine联合应用,是否可以通过降低剂量而降低毒性和潜在副作用,同时又在前列腺癌非雄依赖细胞的研究中有着更好的抗肿瘤作用,从而成为更有效、更安全的非雄依赖的前列腺癌的中西医结合的治疗手段,需要实验进一步的探讨。
本实验共分为四个部分:
第一部分:三氧化二砷对前列腺癌PC-3细胞系细胞周期及端粒酶活性的影响
我们应用体外细胞生长抑制试验(MTT比色法)研究对PC-3细胞生长的影响;流式细胞仪检测细胞周期分布情况及凋亡情况;TRAP-ELASA方法检测药物作用前后端粒酶的活性。MTT结果发现,6μmol/l As_2O_3对PC-3细胞即具有抑制作用,随着浓度的增加和作用时间的延长,抑制作用进一步增强,作用48小时,IC50为16.21μmol/L。18μmol/L的As_2O_3与PC-3细胞共培养48h后,电镜下部分癌细胞出现凋亡形态学改变,经6μmol/L、12μmol/L、18μmol/L、24μmol /L的As_2O_3作用48h后,细胞凋亡率逐渐增加,在流式图上可见到代表凋亡的亚G1峰,且24μmol/L组、18μmol/L组、12μmol/L组的As_2O_3组细胞凋亡率明显高于6μmol/组(P<0.05)。四组浓度处理后的PC-3细胞凋亡率分别为4.8%,15.2%, 19.6% ,29.7%,同正常组比较有显著性差异(P<0.01),G0/G1期细胞比例明显增加,而S期和G2/M期细胞明显减少。PC-3细胞端粒酶活性随着作用时间及作用浓度的增加而降低,具有时间剂量效应关系,但当浓度增加至18μmol/L以上时,随着浓度的增加,对端粒酶活性的抑制无显著性差异(P>0.05)。
第二部分:三氧化二砷对前列腺癌DU-145细胞周期阻滞及凋亡的影响
DU-145也是非雄依赖的前列腺癌细胞系,研究结果发现,As_2O_3对DU-145细胞生长具有抑制作用,2μmol/L的As_2O_3作用24h即对DU-145细胞具有抑制作用,当浓度增加至6μmol/L以上时,抑制作用更为明显,作用48小时,IC50为8.02μmol /L,作用72小时IC50为6.21μmol /L;电镜观察DU-145细胞超微结构发现:8.0μmol/L的As_2O_3与DU-145细胞共培养48h后,部分癌细胞出现凋亡形态学改变,晚期可产生凋亡小体。流式细胞仪检测细胞凋亡及细胞周期的分布发现经6、8、10μmol/L的As_2O_3作用48h后, DU-145细胞凋亡率明显增加,与空白对照组比较有显著性差异(P<0.01), G0/G1期细胞比例明显下降,而G2/M期细胞比例明显增加,和对照组相比有显著性差异(P<0.01),在48小时2μmol/LAs_2O_3作用组未见代表细胞凋亡的亚G1峰,而在8μmol/L作用组,在正常二倍体峰前面可看到一个明显的代表凋亡的亚G1峰;As_2O_3处理DU-145细胞基因组DNA电泳结果发现:不同浓度的As_2O_3处理DU-145细胞48h后,基因组DNA凝胶电泳呈现明显的梯状图谱,这是核小体间DNA成180-200bp整倍断裂的结果。
第三部分:Cyclopamine对非雄依赖的前列腺癌PC-3细胞的影响
通过MTT检测Cyclopamine对细胞生长增殖的影响,BrdU掺入检测PC-3细胞DNA合成的情况,流式检测Cyclopamine对PC-3细胞周期的影响, TRAP-ELISA法细胞检测端粒酶的活性。实验结果显示:Cyclopamine可以通过抑制Hh信号转导通路而明显抑制PC-3细胞的增殖,在4-8μmol/L有明显的抑制作用; BrdU掺入结果PC-3细胞未加Cyclopamine时BrdU掺入阳性细胞平均约占细胞总数的79%±5.6,加入2μmol/L的Cyclopamine作用48小时后PC-3细胞BrdU掺入阳性细胞平均约占细胞总数的49%±8.3,低于对照组79 %±5.6 (P<0.05 ),加入4μmol/L的Cyclopamine作用48小时后PC-3细胞BrdU掺入阳性细胞平均约占细胞总数的30%±6.8,明显低于对照组(P<0.01),但加入Tomatidine(Cyclopamine类似物)作用48小时后BrdU掺入阳性细胞平均约占细胞总数的72%±6.9,与对照组相比差异无显著性(P>0.05);流式细胞仪检测细胞周期的结果为:4μmol/L的Cyclopamine作用48 h后,PC-3细胞进入S期比例由空白对照组的34.8%±1.13下降为13.7%±0.18,G1期的细胞比例由空白对照组的46.8%±1.32下降为34.8%±0.78, G2期的细胞比例由空白对照组的19.7%±0.49上升为52.4%±0.56, Cyclopamine处理组在流式图上还出现了明显的凋亡峰;Cyclopamine对PC-3细胞端粒酶活性在低浓度和高浓度时均无明显抑制作用,并且随着作用时间的延长,亦无抑制作用。
第四部分:低剂量砷剂联合Cyclopamine对前列腺癌细胞株PC-3生长影响的研究
通过MTT检测不同浓度砷剂联合Cyclopamine对PC-3细胞生长的影响,电镜观察低剂量砷剂(6μmol/L)和Cyclopamine(2μmol/L)对PC-3细胞形态和超微结构的影响,以流式细胞仪检测细胞凋亡,TRAP-ELISA检测端粒酶活性,BrdU掺入方法检测低剂量砷剂和Cyclopamine对PC-3细胞DNA合成的影响, RT-PCR法检测GLI1的表达。实验结果显示:低剂量砷剂联合Cyclopamine可以明显抑制PC-3细胞的增殖,作用48h可见细胞凋亡;流式细胞图发现经6μmol/L的As_2O_3和2μmol/L的Cyclopamine共同作用48h后,在流式图上可见到代表凋亡的亚G1峰,联合用药组的细胞凋亡率明显高于6μmol/L的As_2O_3组和2μmol/L的Cyclopamine组(P<0.01),其凋亡率与高浓度As_2O_3组(18μmol/L)的凋亡率无显著性差别(P>0.05),高于4μmol/L的Cyclopamine组(P<0.05);PC-3细胞对照组BrdU掺入阳性细胞平均约占细胞总数的79%±2.33,低剂量砷剂和Cyclopamine联合用药组作用PC-3细胞48h后,BrdU掺入阳性细胞数平均约占细胞总数的26%±3.82,与单药作用组分别作用有显著性差异(P<0.01),与4μmol/L的Cyclopamine作用组及18μmol/L的As_2O_3作用组相比则无显著性差异(P>0.05);6μmol/L的As_2O_3作用PC-3细胞48h后可以抑制端粒酶的活性,吸光度A值为0.792±0.011,而联合用药组作用PC-3细胞48h后亦可抑制端粒酶的活性,吸光度A值为0.769±0.045,两组之间无显著性差异(P>0.05);但与对照组相比(A值为0.892±0.021)均有显著性差异(P<0.05);RT-PCR结果显示,在DNALadder约292bp处出现明暗相间的分子条带,即为目的基因GLI1;而在约579 bp处出现粗细、明暗均较均一的条带,此即内参GAPDH,说明PC-3细胞GLI1的表达较强,与2μmol/L的Cyclopamine共同作用48h后,GLI1的表达有所减弱,随着其浓度的增加,在4μmol/L组GLI1的表达进一步减弱,与对照组相比均有显著性差异(P<0.05);而联合用药组尽管对GLI1的表达也有抑制,与对照组相比有显著性差异(P<0.05),但与2μmol/L的Cyclopamine单独用药组相比,抑制作用无增强或减弱,统计学分析无显著性差异(P>0.05),进一步的研究发现,无论是低剂量砷剂还是高剂量砷剂,其对GLI1的表达均无影响。
结论:
一.As_2O_3可明显抑制前列腺癌非雄依赖的PC-3细胞的增殖,其途径可能与以下两方面机制有关,一是诱导细胞凋亡,抑制细胞周期,二是下调细胞的端粒酶活性,但对Hh信号通路的关键因子GLI1的活性无影响。
二.As_2O_3对前列腺癌非雄依赖的DU-145细胞系也有明显的抑制作用,且随着药物浓度增加和作用时间延长,凋亡细胞明显增加,出现G2/M期阻滞和DNA的断裂。
三.Cyclopamine可以通过抑制Hh信号转导通路而明显抑制PC-3细胞的增殖和DNA的合成,降低GLI1的表达,抑制细胞的周期,诱导细胞的凋亡,但对细胞端粒酶活性无抑制作用。
四.低剂量砷剂(6μmol/L)和Cyclopamine(2μmol/L)的联合应用可以抑制PC-3细胞的增殖,并诱导细胞凋亡,细胞凋亡率明显高于6μmol/L的As_2O_3组和2μmol/L的Cyclopamine组,与高浓度As_2O_3组( 18μmol/L )的凋亡率无显著性差别,但明显高于4μmol/L的Cyclopamine组;其对端粒酶的活性和GLI1的表达均有抑制作用,但与单药作用组相比无显著性差异,二者的联合应用,有协同作用,可以通过降低药物剂量,降低潜在的可能的毒副作用,并取得同样甚至优于高剂量单药作用同样的抑制PC-3细胞的效果。
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 (As_2O_3 )can get a fairly higher effective rate to recurrent APL, experts has been studying its mechanism intensively which testified that As_2O_3 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 As_2O_3 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 As_2O_3or 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 As_2O_3 and Cyclopamine ?
Our experiment is composed of four parts:
Part I: The effect of As_2O_3 on cell-cycle and telomerase activity of PC-3 cell lines
MTT assay is used to study the influence of As_2O_3 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 As_2O_3 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 As_2O_3, some apoptopic changes were found under the electronic microscope. Cultured 48 hours together with the As_2O_3 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 As_2O_3 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 As_2O_3 on cell-cycle and apoptosis of DU-145 cell lines
DU-145 is also cell lines of androgen-independent Pca., and from the stduy we can see that As_2O_3 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 As_2O_3. 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 As_2O_3 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 As_2O_3 ,DNA ladder chart can be seen,which is the result of 180~200bp 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 As_2O_3 and cyclopamine in androgen-independent prostate cancer cell lines PC-3.
Similar approaches have been taken to determine the dose effect of As_2O_3 in combination with Cyclopamine in cell growth. In order to establish whether the combined effects of As_2O_3 and Cyclopamine are synergistic, the cells were treated with low lose As_2O_3 (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 As_2O_3 with Cyclopamine could significantly inhibit PC-3 cell proliferation. Apoptosis appears after 48 hours treatment of 6μM As_2O_3 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 As_2O_3 and Cyclopamine causes similar rate of apoptotic cell death as the treatment with high concentration (18μM) As_2O_3. 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 As_2O_3 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 As_2O_3 and Cyclopamine treatment exhibits no significant difference as compared with 4μM Cyclopamine or 18μM As_2O_3 treatment( P>0.05 ) . Both the treatment of PC-3 cells with 6μM As_2O_3 and combination of As_2O_3 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 As_2O_3 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, As_2O_3 treatment had no effect on the expression level of GLI1 within a wide range of concentration.
Conclusions
1. As_2O_3 significantly decreases the proliferation of androgen- independent prostate cancer PC-3 cells. As_2O_3 may target two separate signaling pathways: 1. As_2O_3 may induce cell apoptosis and inhibit cell cycle.
2. As_2O_3 may down-regulate telomerase activity. However As_2O_3 has no effect on the function of GLI1 which is the essential and ultimate effectors of Hh signaling pathway.
2. As_2O_3 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 As_2O_3 (6μM) and Cyclopamine( 2μM) has synergistic anti-proliferative effects on PC-3cells. Combined cyclopamine and As_2O_3 treatment caused higher rate of inhibition on cell proliferation and a significant increase in apoptotic death as compared with the treatment of 6μM As_2O_3, 2μM or 4μM cyclopamine alone. The apoptotic death rate in the combined treatment group is similar to that in high dose As_2O_3 ( 18μmol/L ) treatment group. No additive inhibitory effect on telomerase and GLI1 has been observed in combined treatment. Combination therapy with As_2O_3 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.
引文
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