塞来昔布对胰腺癌的放疗增敏作用及其机制研究

Radiosensitizing effect of celecoxib on human pancreatic carcinoma

目的研究环氧合酶-2选择性抑制制塞来昔布联合放射治疗对胰腺癌的作用,并探讨其作用机制。方法克隆形成实验和裸鼠移植瘤模型观察塞来昔布、放疗和两者联合对胰腺癌细胞SW1990体内外增殖的影响。Western印迹法检测细胞增殖相关蛋白表达。原位缺口末端标记法(TUNEL)研究胰腺癌细胞凋亡。RT-PCR检测凋亡相关基因表达的变化。体外血管生成和体外侵袭力测定方法检测塞来昔布、放疗及两者联合对胰腺癌细胞新生血管生成和细胞侵袭力的影响,RT-PCR、明胶酶谱和反式酶谱方法检测基质金属蛋白酶(MMP)-2、MMP-9及其组织抑制剂(TIMP)-1、TIMP-2的表达。结果塞来昔布在体内外均有放射增敏作用。胰腺癌细胞增殖细胞核抗原(PCNA)蛋白表达水平在塞来昔布组和联合放疗组均降低,联合组较塞来昔布组有进一步降低。塞来昔布诱导胰腺癌细胞凋亡,单独放疗并不能诱导SW1990细胞发生凋亡,两者联合凋亡细胞数明显增加。塞来昔布下调bcl-2 mRNA表达,而放疗诱导bcl-2 mRNA表达上调,联合组bcl-2的表达最低。单剂量放疗时,裸鼠胰腺癌移植瘤的生长延迟时间为22 d,联合塞来昔布为38 d。单独放疗并不能抑制体外胰腺癌细胞的新生血管生成和侵袭,塞来昔布在体外抑制胰腺癌新生血管形成和侵袭,塞来昔布与放疗联合其抑制作用较单用塞来昔布无明显增强。塞来昔布抑制胰腺癌细胞合成、分泌和激活MMP-2、MMP-9,但对TIMP- 1、TIMP-2的合成、分泌和激活无明显影响。结论COX-2选择性抑制剂塞来昔布对胰腺癌放疗有增敏作用,其机制涉及诱导细胞凋亡,并通过抑制胰腺癌细胞新生血管生成和细胞侵袭,间接对胰腺癌的放疗起增敏作用。

Objective To investigate the sensitizing effects and the mechanisms of selective cyclooxygenase-2（COX-2） inhibitor celecoxib on radiotherapy of pancreatic cancer. Methods Radiosensitization of celecoxib in pancreatic cancer cell SW1990 in vivo and in vitro were investigated by colony forming assay and xenograft tumor model. Expressions of proliferating cell nuclear antigen （PCNA） and Cyclin D1 were assessed by Western Blot. Effect on apoptosis was studied by TUNEL. Expression of bcl-2 and bax was assayed by RT-PCR. Expression and secretion of matrix metalloproteinases（MMPs） and tissue inhibitors of metalloproteinases（TIMPs） were assessed by RT-PCR and zymography. Results Celecoxib enhanced the effect of radiotherapy on pancreatic cancer in vitro and in vivo. TUNEL demonstrated a significant increase of apoptotic cells in vitro after treatment with celecoxib alone or combined with radiation, but no change after radiation. Expression of bcl-2 was decreased by celecoxib; radiation induced the expression of bcl-2 ; combination of celecoxib and radiation significantly suppressed the expression of bcl-2. In vitro, angiogenesis and cell invasion potential of pancreatic cancer cells were inhibited by celecoxib, and celecoxib combined with radiation, but without significant change in radiation group compared with the control group. Expression and secretion of MMP-2 and MMP-9 were closely related to the changes in angiogenesis and cell invasion potential, while the expressions of TIMP-1 and TIMP-2 did not alter significantly in all groups. Conclusions The selective cyclooxygenase 2 inhibitor celecoxib potently enhances the effect of radiation on the treatment of pancreatic cancer. Induction of apoptosis, inhibition of angiogenesis and invasion are involved in the mechanism of celecoxib treatment.