Anti-tumor Effects of Epigallocatechin-3-gallate Extracted from Green Tea on Ovarian Cancer Cell Lines / 대한산부인과학회잡지

OBJECTIVE: A constituent of green tea, (-)-epigallocatechin-3-gallate (EGCG), has been known to possess anti-diabetes, anti-hypertension and anti-cancer properties. In this study, we investigated the anticancer effects of EGCG on human ovarian cancer cell lines. The growth inhibitory mechanism(s) and regulation of cell cycle-related proteins by EGCG were also evaluated. METHODS: To carry out cell counting assay to observe the anti-proliferative effects, we treated 25, 50, and 100 uM EGCG to both ovarian cancer cell lines SKOV-3 and OVCAR-3, respectively. Also, we treated EGCG to PA-1 cells with 6.25, 12.5 and 25 uM, respectively. Six days later, we examined the characteristics of apoptosis and changes in cell cycle regulation by cell counting assay, Annexin V-FITC staining and DNA fragmentation assay, and FACS analysis. In addition, protein and gene expression patterns in SKOV-3 cell were investigated by using cell cycle cDNA chip, RT-PCR, and Western blot analyses. RESULTS: Inhibition of cell growth by cell counts showed in SKOV-3 cells with 48.8%, 82.5%, 99.2% after six days of the treatment with 25, 50, 100 uM of EGCG, respectively. OVCAR-3 cells showed 53.9%, 84.8%, and 97.7% growth inhibition patterns. And PA-1 cells showed 17.1%, 48.4%, and 74.1%, as compared to control. When SKOV-3 cells were tested for EGCG-induced apoptosis, apoptotic cells were observed with 8.6, 11.4, and 23.3-fold at 25, 50, 100 uM EGCG, respectively. And PA-1 cells showed 1.7, 2.4, and 4.2-fold, as compared to control. In contrast, OVCAR-3 did not show EGCG-induced apoptosis. When SKOV-3 cells were tested for their gene expression using cell cycle cDNA chip after treatment with 24.5 uM of EGCG, up-regulations of p21, Bax and cyclin G were shown, while down-regulations of CDK6, E2F-4, and cyclin A were shown. In Western blot assay, up-regulations of Bax and p21 proteins were shown, while down- regulations of cyclin D1, Bcl-XL, Rb, CDK2, E2F-1, E2F-4, PCNA proteins were shown. CONCLUSION: These data support that EGCG can inhibit ovarian cancer cell growth through induction of apoptosis and cell cycle arrest as well as regulation of gene and protein expressions. Thus, EGCG likely provides an additional option for a new and potential drug approach for ovarian cancer.

Induction of Anti-tumor Immunity by Vaccination with E7 of HPV16 and IL-12 / 대한산부인과학회잡지

OBJECTIVE: Interleukin-12 is well known to induce cellular immune response materials and suppress the tumor growth. HPV infection has significant roles in cervical carcinogenesis, and HPV oncoprotein E6 and E7 are important roles in formation and maintenance of cervical cancer. E7 specific immune response was detected in cervical cancer patients, and this shows that E7 protein would be important in potential immunetherapy in cervical cancer. This study is aimed to investigate antitumor effect and E7 immune response by injection of adenovirus IL-12 and E7 in cervical cancer animal model. METHODS: In the cervical cancer animal model using C57BL/6 mice and HPV16 E7 immortalized hosts, 5 X 10(8) pfu/100 ul of PBS, AdLacZ, AdE7 and AdIL-12 were injected into the tumor mass when the tumor sized is increased to 7-8 mm. After the injection, the tumor size was caliperated every 2-3 days, and pathologic and blood studies were done on day 1, 3, 5, 7, 11, 12, and 21 days. The expression level of IL-12 and INF- and E7 specific immune response were measured by ELISA. RESULTS: After the injection of AdIL-12 into the tumor mass, 45% of tumor growth suppression was noted in comparison with control group. In the cases of combination injections of AdIL-12 and AdE7, 80% growth suppression was observed, and complete regression was shown in 40% of the study group. After injection of AdIL-12, the expression of IL-12 in the tumor mass was 9 time higher than that of control group, and 6 times higher in blood sample in comparison with control group. In the group with combined AdIL-12 and AdE7, the highest expression of INF- was noted in comparison with single injection of AdIL-12 or control group. IgGI and IgG2b isotype expression level increased 2.5 times and 2.2 times respectively 3 weeks after adenovirus injection. CONCLUSION: In cervical cancer animal model, IL-12 and E7 application using Adenovirus vector is significant antitumor effect and this demonstrates the potential immunotherapy in near future.

The Effects of Green Tea Extracts (Polyphenon E, EGCG) in Human Papilloma Virus Positive Cervical Lesion / 대한산부인과학회잡지

OBJECTIVE: We tried to confirm the effects of green tea extracts (polyphenon E, EGCG) in patients with human papilloma virus (HPV) positive cervical lesion. METHODS: We divided 51 HPV positive cervical lesion patients (chronic cervicitis, mild dysplasia, moderate dysplasia and severe dysplasia) into 4 group and 37 patient as placebo control. We applied poly E ointment two times per week (27 patients), poly E ointment plus poly E capsule (8 patients), poly E capsule (6 patients), EGCG capsule (10 patients) 200 mg each for 8 to 12 weeks. RESULTS: Among 27 patients with poly E ointment group, 20 patients responded (74%), such as chronic cervicitis (12/18), mild dysplasia (4/5), moderate dysplasia (2/2) and severe dysplasia (1/2). Among 8 patients with poly E ointment and poly E capsule group, 6 patients responded (75%), 6 patients poly E capsule group responded 3 patients (50%). 10 EGCG capsule patients group responded 6 patients (60%). Overall responsive rate is 69% (35/51) in case of green tea extracted treated group and 10% (4/39) in placebo controlled group (P<0.05). CONCLUSION: The effects of green tea extract in HPV positive cervical lesion were statistically significant (P<0.05). This result suggests that green tea extract has highly potential of new treatment agent for HPV infected cervical lesion.

The Changes of Cell Cycle Regulatory Proteins Expression by AdCMVp53 in Cervical Cancer Cell Lines / 대한산부인과학회잡지

OBJECTIVE: In this study, we investigated the cell growth inhibition, regulation of cell cycle and induction of apoptosis through recombinant p53 adenoviral vector delivery into cervical cancer cell line SiHa, to explore the possibility of p53 gene therapy. METHODS: We infected SiHa with AdCMVp53 at 50 MOI. After 48 hours, the regulation of cell cycle and apoptosis were investigated with FACS. The gene expression profiling associated with cell cycle was also investigated with cell cycle DNA membrane chip. RESULTS: SiHa cells were arrested in the G1 phase by AdCMVp53 and showed cell growth inhibition via apoptosis. The gene expression profiles involved in cell cycle including cyclin-dependent kinase inhibitor 1C (p57, Kip2), RAD9 (S.pombe) homolog, and MAD2 (mitoticarrest deficient, yeast, homolog)-like 2 were up-regulated by more than three-fold, as compared to control group. In contrast, 6 genes such as retinoblastoma-like 2 (p130), and cyclin H were down-regulated by more than three-fold. Several genes known as being differentially up- or down-regulated compared to control were confirmed by RT-PCR and Western blotting assays. CONCLUSION: The adenoviral p53 gene delivery into cervical cancer cell line, suggesting the possibility of p53 gene therapy in cervical neoplasia make the cell growth inhibition and changes of cell cycle-associated gene expression.

Photogem Induces Necrosis in Various Uterine Cervical Cancer Cell Lines by PDT / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: In order to elucidate the antitumor effect of photodynamic therapy (PDT), using a derivative of the photosensitizing agent hematoporphyrin (Photogem) and a diode laser, the cell death of uterine cancer cell lines (CaSki, HT3, HeLa, and SKOV-3), and mice transplanted with TC-1 lung cancer cells, were evaluated. MATERIALS AND METHODS: The morphological changes, MTT assay, flow cytometry, cytotoxicity and tumor growth inhibition study were evaluated at various time intervals after the PDT. RESULTS: The results showed that the survival rates of each cell line decreased with time and dose response after performing the PDT. Also, the PDT-induced damage of cancer cells was almost entirely confined to necrosis of the tumor cells in the early time courses. The irradiation of CaSki cells in the presence of Photogem induced plasma membrane disruption and cell shrinkage, indicating the plasma membrane as the main target for Photogem. In the in vivo experiment, significantly longer survival and a significantly smaller tumor size were seen over the time courses of the Photogem with irradiation compared to the untreated control groups; resorption of the tumor was also observed after the PDT treatment. CONCLUSION: Collectively, our results indicated that Photogem possesses anti-tumor effects, and necrosis-like death, with plasma membrane damage, was postulated to be the principal mechanism of the antitumor effect of the PDT using Photogem.