Activation of peroxisome proliferator-activated receptor γ inhibits cell growth via apoptosis and arrest of the cell cycle in human colorectal cancer
Activation of peroxisome proliferator-activated receptor γ inhibits cell growth via apoptosis and arrest of the cell cycle in human colorectal cancer
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DOI：
10.1111/j.1443-9573.2007.00290.x
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发表时间：
2007-05-01
影响因子：
3.5
通讯作者：
Wang, Ya Dong
中科院分区：
文献类型：
--
作者：
Lin, Mao Song;chen, We Chang;Wang, Ya Dong
目的：研究过氧化物酶体增殖物激活受体γ（peroxisome proliferator-activated receptor，PPAR γ）在大肠癌HT-29细胞中的表达及其配体对细胞生长的影响，探讨选择性配体激活PPAR γ是否能诱导大肠癌HT-29细胞凋亡和细胞周期阻滞。采用逆转录聚合酶链反应（RT-PCR）和蛋白质印迹法（Western blot）检测细胞中过氧化物酶体增殖物激活受体γ（PPARgamma）mRNA和蛋白质的表达。HT-29细胞用两种特异性PPAR γ配体处理：罗格列酮和15-d-PGJ（2）。分别采用MTT法和软琼脂集落形成实验检测罗格列酮和15-d-PGJ（2）激活的PPAR γ对细胞贴壁依赖性和非贴壁依赖性生长的影响。通过TUNEL染色和流式细胞术（FCM）测定，通过CaspSCREEN（TM）流式细胞术细胞凋亡检测试剂盒（BioVision，Palo Alto，USA）测量细胞凋亡。此外，在罗格列酮和15-d-PGJ（2）处理48 h前后，通过免疫细胞化学染色法测定caspase-3的表达。结果：HT-29细胞中存在PPAR γ mRNA和蛋白的表达。MTT法显示，用0、0.1、1或10 μ mol/L的PPAR γ激活剂罗格列酮或15-d-PGJ（2）处理这些细胞0、24、48或72 h，以剂量和时间依赖的方式抑制锚定依赖性细胞生长。在细胞生长过程中罗格列酮处理导致集落形成减少，并且在细胞培养物中该作用不能立即逆转。罗格列酮和15 d-PGJ（2）处理48 h后，TUNEL染色显示阳性细胞DNA断裂。流式细胞仪检测显示，10 μ mol/L罗格列酮或15-d-PGJ（2）作用24 h和48 h后，细胞凋亡率分别为14.8 ± 0.8%和28.5 ± 1.3%或15 ± 0.7%和40 ± 1.2%。而未经处理的细胞凋亡率分别为3.8 +/-0.4%和8.8 +/-0.4%。与这些结果一致，与对照组相比，罗格列酮或15-d-PGJ（2）处理的细胞中caspase-3表达的阳性率显著增加。为了探索细胞周期的调节是否参与了PPAR γ配体对细胞生长的影响，使用PI染色的FCM进行了评估。10 μ mol/L罗格列酮或15-d-PGJ（2）作用HT-29细胞24 h和48 h后，G（0）/G（1）期细胞比例增加。结论：HT-29细胞表达PPARgamma，PPARgamma激活可通过诱导细胞凋亡和抑制细胞周期而抑制细胞生长。PPAR-gamma有望成为结肠癌治疗的新靶点。
OBJECTIVE: To investigate the expression of peroxisome proliferator-activated receptors (PPAR)gamma and the effects of PPAR gamma ligands on cells growth in colorectal cancer (CRC) cell line HT-29, and to explore whether the activation of PPARy by its selective ligand can induce apoptosis and the arrest of the cell cycle in these cells.METHODS: A CRC cell line, HT-29, was used in this study. PPAR gamma mRNA and the protein expressions were measured by reverse transcriptase-polymerase chain reaction and Western blot. The HT-29 cells were treated with two specific PPAR gamma ligands: rosiglitazone and 15-d-PGJ(2). The effects of PPAR gamma activated by rosiglitazone and 15-d-PGJ(2) on the anchorage-dependent and anchorage-independent growth of the cells were assessed by methylthiazolyl terazolium (MTT) and soft agar colony assay, respectively. Apoptosis was measured by TUNEL staining and flow cytometry (FCM) assay by CaspSCREEN (TM) Flowcytometric Apoptosis Detection Kit (BioVision, Palo Alto, USA). Furthermore, the caspase-3 expression was determined by a immunocytochemical staining method before and after treatment with rosiglitazone and 15-d-PGJ(2), for 48 h. The cell cycles were measured by flow cytometric analysis using propidium iodide (PI).RESULTS: PPAR gamma mRNA and protein expressions were observed in the HT-29 cells. The MTT assay showed that treatment of these cells with 0, 0.1, 1 or 10 mu mol/L PPAR gamma activators rosiglitazone or 15-d-PGJ(2) for 0, 24, 48 or 72 h resulted in the inhibition of anchorage-dependent cell growth in a dosage- and time-dependent way. Rosiglitazone treatment during cell growth resulted in the reduction of colony formation and the effects were not immediately reversible in the cell culture. TUNEL staining showed DNA fragmentation in positive cells after treatment with rosiglitazone and 15d-PGJ(2) for 48 h. In addition, FCM showed that the apoptosis rates were 14.8 +/- 0.8% and 28.5 +/- 1.3% or 15 +/- 0.7% and 40 +/- 1.2% after the cells were incubated with 10 mu mol/L rosiglitazone or 15-d-PGJ(2) for 24 h and 48 h, while the apoptosis rates of cells without treatment were 3.8 +/- 0.4% and 8.8 +/- 0.4%, respectively Consistent with these results, the positivity rates of caspase-3 expression in cells treated with rosiglitazone or 15-d-PGJ(2) increased significantly when compared with the control group. To explore whether the regulation of the cell cycle was involved in the effect of PPAR gamma ligands on cell growth, FCM using PI staining was assessed. The ratio of G(0)/G(1), phase cells increased after incubated with 10 mu mol/L rosiglitazone or 15-d-PGJ(2) for 24 h and 48 h.CONCLUSIONS: Our results showed that PPAR gamma was expressed in HT-29 cells and PPAR gamma activation could inhibit cell growth through inducing apoptosis and suppressing the cell cycle. PPAR gamma may be considered as a new therapeutic target for colon cancer in humans.