Studies of the Enhancement of Cisplatin on Ionizing Radiation-induced Cytotoxicity in Human Cervical Cancer Cells

• Main Authors: HsiangYi Huang, 黃湘怡
• Other Authors: Huang, Haimei
• Format: Others
• Language: en_US
• Published: 2003
• Online Access: http://ndltd.ncl.edu.tw/handle/02375996230295904683

碩士 === 國立清華大學 === 生命科學系 === 91 === Cocurrent cisplatin-based chemotherapy and ioinizing radiation (IR) has become the stand therapy since 1999, because single cisplatin treatment may induce cisplatin resistance for cancer cells. In this study, cisplatin and IR were two major agents for cell killing, the possible mechanisms for additive or synergistic killing effects in SiHa cells were investigated. The results were summaried as followed. To test whether cisplatin increase cisplatin-DNA adducts in SiHa cells, MAb 62-5 antibodies were used to probe cisplatin-DNA adducts in cisplatin-treated cells. The results indicated that the amount of cisplatin-DNA adducts increased with increasing concentration of cisplatin in a dose dependent manner. At 25 μM treatment dose, about twice of cisplatin-DNA adducts was shown (Fig. 1A). Similarly, results from immunostaining have shown that the fluorescence intensity of cisplatin-treated cells increased in a dose-dependent manner (Fig. 1B). In this study, the effect of cisplatin—based therapy was also explored on HPV-16 E6 inserted SiHa cervical carcinoma cells. After treatment with cisplatin 10μM for 4~24 hr and 2.5~20 μM for 24 hr, it was found that expression of p53 proteins were restored in time and dose dependent manners and apoptotic cells were also increased during drug treatment(Fig. 2A, 2B; Fig. 4). In coincidence with the appearance of p53 protein, the level of HPV-16 E6 mRNA reduced to a minimum at 20 μM cisplatin for 24 hr (Fig. 2 and Fig. 3). Furthermore, cisplatin was treated concurrently with X-irradiation or with sodium arsenite (SA) on SiHa cells. Since previous data have shown that pretreatment with SA increased cisplatin-induced cisplatin-DNA adducts in HeLa cells, pretreatment with SA might improve the effects of cisplatin in clinical therapies. Results from SRB cell survival assay and Western blot analysis have shown that cisplatin combined with X-irradiation or SA treatment in SiHa cells not only enhanced cell cytotoxicity(Fig. 6,Fig. 9A) but also stabilized and accumulated more p53 protein expression in this study(Fig. 7A,C,Fig. 9B). In addition, more apoptotic cells appeared in SiHa cells through flow cytometric analysis after combined treatment (Fig. 8).