Purification of azurin from Pseudomonas aeruginosa and its cytotoxic effect on oral squamous carcinoma cells = Pseudomonas aeruginosa로 부터 azurin의 정제 및 구강편평상피암세포에 미치는 세포독성 효과

다국어 초록 (Multilingual Abstract)

The use of bacteria in the treatment of cancer has a long and interesting history. It has been reported that bacterial infections can sometimes elicit regression in many cancers. However, the use of live bacteria has produced the problems such as toxicity, so that purified low molecular weight bacterial proteins as anticancer agents were used to bypass the problems. A few years ago, azurin secreted by Psedomonas aeruginosa (P. aeruginosa) was isolated which can kill cancer cells but appears to have no harmful side effects. Tested in tumor-bearning mice, azurin shrank the malignancies.
The oral cancer is widely occurring cancer in the world. Oral cancer cells have a strong resistance to anticancer drug. Recently, the targeted elimination of oral squamous carcinoma cells by inducing apoptosis has emerged as a valued strategy to combat oral cancer. Thus, anticancer effect of purified azurin from P. aeruginosa were investigated in human oral squamous carcinoma (YD-9) cells.
Azurin DNA was cloned by PCR amplification and it was constructed into expression vector. Recombinant azurin clone was maintained in E. coli and purified by FPLC equiped with affinity column. In order to investigate the cytotoxicity of azurin, purified azurin was treated to YD-9 (p53 positive) cells and MG-63 (p53 negative) cells. The azurin showed cytotoxic effect in YD-9 cells dose dependently. The death of cells was further demonstrated to be due to apoptosis characterized by chromatin condensation and apoptotic bodies. Azurin treatment increased the expression level of p53, which was found to complex with azurin. This result suggests that azurin stabilize p53, leading to induce apoptosis in YD-9 cells. Moreover, combination treatment with azurin and 5-FU or etoposide in YD-9 cells and MG-63 cells showed much more cell death than single treatment of 5-FU or etoposide. And G2-M arrest-related proteins were involved in the synergistic effect induced by combination treatment.
These results indicate that azurin may be a potentially powerful anticancer agent to YD-9 cells. Therefore I suggest that more studies of application with azurin in various cancer cells could be needed for the advanced cancer therapy.

목차 (Table of Contents)

• Introduction 1
• Materials and Methods 8
• Results 24
• Discussion 45
• Summary 49
• References 51
• Korean abstract 58