Studies on Combination Treatment Using Cytostatic Agents and Chemotherapy Drugs for Cancer Therapy

• Main Authors: Chia-Ying Lin, 林佳穎
• Other Authors: Chieh-Hsien Tu
• Format: Others
• Language: en_US
• Published: 2002
• Online Access: http://ndltd.ncl.edu.tw/handle/18140632554094882009

碩士 === 國立屏東科技大學 === 獸醫學系 === 90 === Abstract The major procedures for cancer therapy include surgery, chemotherapy and ionizing radiation therapy. However, none of these approaches were completely effective. Besides, they frequently caused severe side effects and serious complication during clinical application. Therefore, the development of combinatory therapy using chemotherapy drugs and novel anti-cancer agents is highly demanded, which would improve the therapeutic efficacy while minimizing undesired effects. In present study, we produced recombinant cytotoxic A chain of abrin (ABR-A) as well as its fusion form with cell-penetrating peptide TAT (TAT-ABR-A). After expression and purification, the molecular weight of recombinant ABR-A and TAT-ABR-A were determined at 32 and 34 kDa, respectively. ABR-A and TAT-ABR-A exhibited differential abilities in inhibiting the proliferation of Lewis lung carcinoma cells (LLC) and bovine aortic endothelial cell (BAEC). Nucleus staining indicated treatment of cells with TAT-ABR-A resulted in chromosome condensation, DNA fragmentation, and attenuated mitosis. Flow cytometry analysis revealed the TAT-ABR-A increased the number of cells in sub-G0/G1. TAT-ABR-A decreased the levels of bcl-2, cdk-1, and cdk-2 as revealed by western blot analysis. Together, these results indicated the potency of TAT-ABR-A in inhibiting cell proliferation is 5-10 folds higher than ABR-A. In rabbit reticulocyte translation system, the efficacy of TAT-ABR-A in inhibition of protein synthesis is similar to that of ABR-A. Furthermore, pretreatment with antioxidants such as superoxide dismutase- (SOD) or N-acetyl-L-cysteine (NAC) attenuated the cytotoxicity of TAT-ABR-A and ABR-A, indicating generation of reactive oxygen species participated in the cytotoxic mechanism of both proteins. By chemical conjugation with FITC (fluorescein 5-isothiocyanate), cellular uptake of TAT-ABR-A was detected within less than 30 min, whereas there was no significant uptake of ABR-A even after 16 h. These data indicated that fusion with TAT promoted the entry and increased the cytotoxicity of ABR-A. Subsequently, we analyzed the efficacy of cisplatin on LLC proliferation in the presence of TAT-ABR-A or ABR-A, and found that TAT-ABR-A significantly enhanced the cytotoxicity of cisplatin. The combinatory effect of TAT-ABR-A or ABR-A with cisplatin was evaluated in Lewis lung carcinoma grown in C57BL/6 mice, in which the tumors were grown to ~100 mm3 before treatment. Administration of TAT-ABR-A or ABR-A alone had no significant inhibitory effect on tumor growth. However, the combination therapy using TAT-ABR-A and cisplatin significantly reduced the tumor growth. Examination of body weight of tumor-bearing mice indicated that the injection of TAT-ABR-A or ABR-A did not aggravate the side effects of cisplatin. In summary, we demonstrated the feasibility of using genetic engineering techniques to produce biologically functional toxin, ABR-A. Furthermore, the efficacy of this recombinant toxin could be improved through fusion with cell-penetrating peptide. Above all, this improved toxin might be use TAT-ABR-A as adjuvant agents in conjunction with chemotherapy drugs to improve the therapeutic efficacy for treatment of cancer.