低剂量砷联合Cyclopamine协同抑制非雄激素依赖的前列腺癌PC3细胞生长的实验研究

发布时间：2018-11-25 12:24

【摘要】：目的：前列腺癌（Prostatic cancer,Pca）是西方国家最常见的男性恶性肿瘤，而近年我国该病发病率亦逐年上升，大部分患者最终会进展为非雄激素依赖的Pca，但目前尚无公认可以有效的治疗非雄激素依赖Pca的手段。因此，，寻求可以有效治疗非雄激素依赖前列腺癌的新的治疗手段是目前临床迫切需要解决的问题。三氧化二砷（Arsenic trioxide，As203，ATO)已被证实对于治疗复发性或难治性急性早幼粒细胞白血病（APL）具有良好疗效。但某些二期临床试验结果发现单独用ATO治疗一些实体瘤，如转移性肾细胞癌或转移性黑色素瘤，其疗效相对有限。故寻找一种辅助药物，与ATO联合使用，使低浓度的ATO发挥高浓度的作用效果，并降低潜在的毒副作用，很有可能是一种改进ATO治疗某些实体肿瘤的可行有效的方法。Hedgehog（Hh）信号通路晚期前列腺癌中呈高表达。最近研究表明ATO能够通过抑制Hh信号通路下游GLI蛋白而抑制尤文肉瘤和生神经管细胞瘤的增殖；而Smo阻滞剂Cyclopamine（CYC）亦可通过对Hh信号通路的阻滞作用而抑制前列腺癌等肿瘤的生长。故本实验选取非雄激素依赖前列腺癌PC3细胞为研究对象，观察低剂量三氧化二砷联合Cyclopamine对PC3细胞的抑制作用及其可能的分子作用机制。方法：1.运用免疫荧光及DAPI染色鉴定PC3细胞及PC3细胞内HH-GLI信号通路的表达情况。2.采用MTT比色法检测不同浓度ATO及Cyclopamine单独或联合使用对不同的时间点PC3细胞生长的抑制。3.应用裸鼠致瘤实验进一步验证ATO联合Cyclopamine对PC3细胞生长的抑制效果。4.Real-time PCR法检测ATO联合Cyclopamine对PC3细胞内Hh信号通路中重要分子的mRNA的表达的影响。5.检测ATO联合Cyclopamine对PC3细胞内细胞凋亡及细胞周期的影响。结果：1.免疫荧光剂DAPI染色结果显示肿瘤增殖抗原Ki67、前列腺特异性膜抗原PSAM及HH-GLI1信号通路中重要的核内转录因子GLI1均在PC3细胞中呈高表达2.MTT结果显示ATO或Cyclopamine单独使用均能抑制PC3细胞的生长，呈现明显的剂量及时间依赖性；二者联合使用则优于单剂量、甚至二倍剂量的ATO或Cyclopamine，经Chou’s公式分析二者可显著协同抑制PC3细胞生长。3.动物实验进一步显示ATO联合Cyclopamine使用较单剂量组更能明显抑制PC3细胞移植瘤的生长(p 0.05)。4.Real-time PCR分析结果显示ATO联合Cyclopamine使用较单剂量组更明显下调Hh信号通路中GLI1蛋白mRNA的表达(p 0.05)。5.流式细胞术检测结果显示，ATO联合Cyclopamine使用具有轻微诱导凋亡作用，且较单剂量组能更明显阻滞PC3细胞于S期(p 0.01)。结论：低剂量砷联合Cyclopamine能协同抑制非雄激素依赖前列腺癌PC3细胞的生长，其机制与二者联合使用可更明显下调PC3细胞中Hh信号通路中GLI1蛋白、改变细胞周期及轻微诱导凋亡作用有关。故低剂量砷联合Cyclopamine有可能成为潜在的治疗非雄激素依赖前列腺癌的有效治疗方式。
[Abstract]:Objective: prostate cancer (Prostatic cancer,Pca) is the most common male malignant tumor in western countries. In recent years, the incidence of prostate cancer in China has been increasing year by year. Most of the patients will eventually develop into androgen dependent Pca,. However, there is no recognized effective treatment of non-androgen-dependent Pca. Therefore, it is urgent to seek a new treatment for androgen-dependent prostate cancer. Arsenic trioxide (Arsenic trioxide,As203,ATO) has been proved to be effective in the treatment of recurrent or refractory acute promyelocytic leukemia (APL). However, some secondary clinical trials showed that the efficacy of ATO alone in the treatment of some solid tumors, such as metastatic renal cell carcinoma or metastatic melanoma, was relatively limited. Therefore, to find a kind of auxiliary drug, combined with ATO, so that low concentration of ATO can play a high concentration effect, and reduce the potential side effects. It may be a feasible and effective method to improve ATO in the treatment of some solid tumors. Hedgehog (Hh) signaling pathway is highly expressed in advanced prostate cancer. Recent studies have shown that ATO can inhibit the proliferation of Ewing's sarcoma and neurotuboma by inhibiting the downstream GLI protein of Hh signaling pathway. Smo blocker Cyclopamine (CYC) can inhibit the growth of prostate cancer and other tumors by blocking Hh signaling pathway. Therefore, the inhibitory effect of low dose arsenic trioxide combined with Cyclopamine on PC3 cells and its possible molecular mechanism were observed in non-androgen dependent prostate cancer (PC3) cells. Method 1: 1. Immunofluorescence and DAPI staining were used to identify the expression of HH-GLI signal pathway in PC3 cells and PC3 cells. 2. MTT colorimetric assay was used to detect the inhibitory effects of different concentrations of ATO and Cyclopamine on the growth of PC3 cells at different time points. The inhibitory effect of ATO combined with Cyclopamine on the growth of PC3 cells was further verified by tumorigenic assay in nude mice. The effect of ATO combined with Cyclopamine on the expression of mRNA in Hh signaling pathway in PC3 cells was detected by 4.Real-time PCR assay. 5. The effects of ATO combined with Cyclopamine on apoptosis and cell cycle of PC3 cells were detected. Results: 1. Immunofluorescence DAPI staining of tumor proliferative antigen Ki67, Both prostate-specific membrane antigen (PSAM) and GLI1, an important intracellular transcription factor in HH-GLI1 signaling pathway, were highly expressed in PC3 cells. The results showed that ATO or Cyclopamine alone could inhibit the growth of PC3 cells. There were obvious dose-dependent and time-dependent. The combination of the two groups was superior to the single dose, even the double dose of ATO or Cyclopamine, could significantly inhibit the growth of PC3 cells by Chou's formula analysis. Animal experiments further showed that ATO combined with Cyclopamine could significantly inhibit the growth of PC3 cell xenografts compared with single dose group (p0.05). 4.Real-time PCR analysis showed that ATO combined with Cyclopamine was more down-regulated than that of single dose group. Expression of GLI1 protein mRNA in Hh signaling pathway (p0.05). The results of flow cytometry showed that ATO combined with Cyclopamine could induce apoptosis slightly and block PC3 cells in S phase more obviously than single dose group (p0.01). Conclusion: low dose arsenic combined with Cyclopamine can synergistically inhibit the growth of PC3 cells with non-androgen dependent prostate cancer, and the combination of both can down-regulate the GLI1 protein in Hh signaling pathway in PC3 cells. Changes in cell cycle and slight induction of apoptosis are involved. Therefore, low dose arsenic combined with Cyclopamine may be a potential therapy for androgen-dependent prostate cancer.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.25

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