A basic trial of gene therapy against peritoneal dissemination of ovarian cancer by calponin h1 gene transfer

Calponin H1(CNH1)是一种肌动蛋白结合蛋白，稳定α-平滑肌肌动蛋白(α-SMA)微丝，调节细胞生物学表型。我们已经报道，卵巢癌细胞可能通过下调形成血管壁的细胞中的CNH1和α-SMA而为血管内皮细胞的生长创造良好的环境。在本研究中，我们观察了(1)卵巢癌对腹膜间皮细胞和成纤维细胞的影响，(2)CNH1基因转染细胞的变化，(3)CNH1基因治疗对卵巢癌腹膜转移的影响。通过加入人卵巢癌细胞的条件培养液，CNH1和α-SMA在腹膜细胞和成纤维细胞中的表达减少，导致细胞回缩和抑制肌动蛋白应激纤维的形成。尤其是在培养的单层腹膜细胞中，可观察到细胞间的解离。血小板-…的浓度卵巢癌患者瘤内积液中较多的衍生生长因子(PDGF)水平明显高于良性卵巢肿瘤。此外，在卵巢癌细胞条件培养液中加入抗α中和抗体，可部分避免卵巢癌对成纤维细胞中PDGF一SMA表达的抑制作用。这些观察揭示了卵巢癌分泌因子包括PDGF通过下调腹膜细胞和成纤维细胞中CNH1和α-SMA的表达，为肿瘤的侵袭创造了有利的环境。腺病毒载体介导的CNH1基因导入腹膜细胞和卵巢癌细胞后，在每种细胞系中都形成了更长更厚的肌动蛋白无菌纤维，纤维的定位与外部转导的CNH1的表达相一致。同时，感染的单层腹膜细胞对卵巢癌细胞条件培养液诱导的细胞间解离具有稳定性，并抑制癌细胞穿透腹膜单层证实的癌细胞侵袭。另一方面，转导CNh1的卵巢癌细胞生长和侵袭能力减慢，后者伴随着细胞运动能力的下降。当同时转染腹膜细胞和卵巢癌细胞时，证实了通过腹膜细胞单层对癌细胞侵袭的相加抑制作用。通过裸鼠体内治疗实验，成功地避免了卵巢癌引起的腹膜细胞表面的改变，并显著延长了荷瘤小鼠的生存时间，因此，CNH1基因治疗有望通过抑制感染的腹膜细胞层对肿瘤的侵袭和直接抗肿瘤作用而有效地抑制卵巢癌的腹膜转移。目前的治疗方法可以被认为是一种新型的基因治疗，基于同一基因对癌症具有双功能治疗作用的概念：一是抑制癌细胞，二是加强宿主防御机制。较少

Calponin h1(CNh1), one of the actin-binding proteins, stabilizes the filaments of alpha-smooth muscle actin(α-SMA) and modulates cellular biological phenotypes. We have reported that the ovarian cancer cells probably make favorable environment for fheir intravasation through fhe down-regulation of CNh1 and α-SMA in the cells forming blood vessel walls. In this study, we investigated (1)the ovarian cancer-derived effects on peritoneal mesothelial cells and fibroblasfs, (2)the changes occurredin CNh1 gene-transfected cells, and (3)the efficacy of CNh1 gene therapy against the peritoneal dissemination of ovarian cancer.By adding the conditioned culture medium of human ovarian cancer cells, the reduction of both CNh1 andα-SMA expressions occurred in peritoneal cells and fibroblasts resulting in cellular retraction and suppression of actin stress fiber-formation. Especially in the monolayer of cultured peritoneal cells, intercellular dissociation was observed. The concentration of platelet- … More derived growth factor(PDGF) in an intratumoral fluid was significantly higher in ovarian cancers compared with that in benign ovarian tumors. Moreover, the ovarian cancer-derived inhibitory effect on α-SMA expression, which observed in cultured fibroblasts, was partially avoided by adding anti-PDGF neutralizing antibody into the conditioned medium of ovarian cancer cells. These observations opened up a possibility that ovarian cancer-derived secretor factors including PDGF made favorable environment for cancer invasion via down-regulated CNh1 and α-SMA in peritoneal cells and fibroblasts.Adenoviral vector-mediated CNh1 gene transfections into peritoneal cell lines and ovarian cancer cell lines induced the formation of longer and thicker actin steress fibers in each cell line, and the localization of fibers coincided with that of externally transducted CNh1 expression. Simultaneously, a monolayer of the infected peritoneal cells gained stability against the intercellular dissociation induced by the conditioned medium of ovarian cancer cells, and inhibited cancer cell invasion confirmed by cancer cell penetration through the peritoneal cell monolayer. On the other hand, CNh1-transfected ovarian cancer cells showed retarded growth property and invasiveness, the latter of which accompanied the impaired cell motility. When both the peritoneal cells and ovarian cancer cells were transfected at the same time, an additive inhibitory effect against the cancer cell invasion through the peritoneal cell monolayer was confirmed. By in vivo treatment experiments using nude mice, intraperitoneally injected CNh1-adenovirus successfully avoided ovarian cancer-induced change in peritoneal cell surface, and significantly prolonged the survival time of mice inoculated intraperitoneally with ovarian cancer cells.Consequently, CNh1 gene therapy against the peritoneal dissemination ot ovarian cancer is expected to be effective through the inhibitory effect in infected peritoneal cell layers against cancer invasion and the direct anti-tumor effect against cancer invasion and growth. The present therapy may be considered as a novel gene therapy based on the concept that the same gene plays bifunctional therapeutic roles against cancer : one is to repress cancer cells and the other is to reinforce host defense mechanism. Less