Songgang Li, Jiyu Li, Yingbin Liu, Yiyu Qin, Zhiwei Quan
Department of General Surgery, Xinhua Hospital, School of Medicine, Shanghai Jiao Tong University, 1665 Kongjiang Road, Shanghai 200092, PR China.
Hepatogastroenterology. 2009 Sep-Oct;56(94-95):1253-60.
BACKGROUND/AIMS: As one of the mostly aggressive and fatal malignancy, gallbladder carcinoma has been known to be resistant to many anticancer drugs. Although it is under active investigation, it is still difficult to achieve satisfactory effect for most chemo-drugs on this tumor. It has previously reported that somatostatin could increase the chemosensitivity of gallbladder carcinoma cells (GBC-SD) and reduce the therapeutic dose of Doxorubicin in killing GBC-SD cells. SST could enhance the chemosensitivity of gallbladder carcinoma to Doxorubincin (DOX) by transient arresting cell cycle to S phase. We tried to clarify the mechanism by which SST utilized to enhance the chemosensitivity of GBC-SD cells to DOX. We further investigated whether the enhanced chemosensitivity of GBC-SD cells to DOX in the presence of SST is via apoptosis or cell cycle regulation. In addition, we also looked into related factors involved in cell cycle regulation and apoptosis.
Twenty-four hours after somatostatin treatment, doxorubicin was gradually added and the growth curve of GBC-SD cells was determined according to MTT test. Cell apoptosis was measured by flow cytometry (FCM) using Annexin V/ Propidium Iodide Binding. Cell cycle was also examined by FCM. The somatostatin receptor (SSTR) subtypes in GBC-SD cells were identified using immunocytochemistry and RT-PCR assay. The expressions of p53, Bax and phosphorylated RB (pRB) protein were examined using western blotting assay.
When GBC-SD cells were treated with SST alone, no significant cell growth inhibition and cell apoptosis were observed. SST could induce a transient S phase arrest in GBC-SD cells. The mRNA expression of SSTR1, 2, 3, 4, 5 were all detected in GBC-SD cells, whereas only SSTR1, 2, 3 were detected in GBC-SD cells using immunocytochemistry assay. After GBC-SD cells were treated with SST for 24h, the expression level of p53 and Bax protein in GBC-SD cells was similar to that of the control group, however up-regulated pRB protein expression was observed (p < 0.05).
Our results suggested that the synergistic inhibitory effect of somatostatin and doxorubicin co-treatment on GBC-SD cells was not due to SST induced apoptosis concerning the expression of p53 and Bax protein. Our data clearly showed all 5 SST receptor subtypes expressed in GBC-SD cells by RT-PCR and 3 SST receptors by immunocytochemistry. Accumulated evidence has been proved the relationship between cell cycle regulation and RB protein phosphorylation. In the chemosensitized GBC-SD cell line treated with SST, phosphorylated RB and cell cycle arrest were simultaneously manifested. We reasoned that somatostatin might enhance the chemosensitivity of GBC-SD cells to doxorubin through arresting the cell cycle at S phase, but not P53 and Bax protein induced cell apoptosis.
背景/目的：胆囊癌作为最具侵袭性和致命性的恶性肿瘤之一，已知对多种抗癌药物耐药。尽管目前正在积极研究，但大多数化疗药物对该肿瘤仍难以取得满意疗效。此前有报道称，生长抑素可增加胆囊癌细胞（GBC-SD）的化疗敏感性，并降低阿霉素杀死GBC-SD细胞的治疗剂量。生长抑素可通过使细胞周期短暂停滞于S期来增强胆囊癌对阿霉素（DOX）的化疗敏感性。我们试图阐明生长抑素用于增强GBC-SD细胞对DOX化疗敏感性的机制。我们进一步研究了在生长抑素存在下GBC-SD细胞对DOX增强的化疗敏感性是否通过凋亡或细胞周期调控实现。此外，我们还研究了参与细胞周期调控和凋亡的相关因素。
生长抑素处理24小时后，逐渐加入阿霉素，并根据MTT试验测定GBC-SD细胞的生长曲线。采用膜联蛋白V/碘化丙啶结合法通过流式细胞术（FCM）检测细胞凋亡。也通过FCM检测细胞周期。采用免疫细胞化学和RT-PCR检测法鉴定GBC-SD细胞中的生长抑素受体（SSTR）亚型。采用蛋白质印迹法检测p53、Bax和磷酸化RB（pRB）蛋白的表达。
单独用生长抑素处理GBC-SD细胞时，未观察到明显的细胞生长抑制和细胞凋亡。生长抑素可诱导GBC-SD细胞短暂停滞于S期。在GBC-SD细胞中检测到SSTR1、2、3、4、5的mRNA表达，而采用免疫细胞化学检测法仅在GBC-SD细胞中检测到SSTR1、2、3。GBC-SD细胞经生长抑素处理24小时后，GBC-SD细胞中p53和Bax蛋白的表达水平与对照组相似，但观察到pRB蛋白表达上调（p<0.05）。
我们的结果表明，生长抑素和阿霉素联合处理对GBC-SD细胞的协同抑制作用并非由于生长抑素诱导的与p53和Bax蛋白表达相关的凋亡。我们的数据清楚地表明，通过RT-PCR在GBC-SD细胞中表达了所有5种生长抑素受体亚型，通过免疫细胞化学检测到3种生长抑素受体。越来越多的证据证实了细胞周期调控与RB蛋白磷酸化之间的关系。在用生长抑素处理的化疗增敏GBC-SD细胞系中，磷酸化RB和细胞周期停滞同时出现。我们推断，生长抑素可能通过使细胞周期停滞于S期来增强GBC-SD细胞对阿霉素的化疗敏感性，而不是通过P53和Bax蛋白诱导细胞凋亡。
