Sang Hoon Kwon

Detoxification: A Novel Function of BRCA1 in Tumor Suppression?

Our studies found that BRCA1 levels negatively correlate with DNA adducts induced by Benzo(a)pyrene (BaP). Pulse-chase experiments showed that the increase in BaP-induced DNA adducts in BRCA1 knockdown cells may not be associated with BRCA1’s function in nucleotide excision repair activity; rather, it may be associated with its function in modulating transcriptional regulation. BRCA1 knockdown in MCF-10A cells significantly attenuated the induction of CYP1A1 following BaP treatment indicating that the increase in BaP-induced adducts in BRCA1 knockdown cells is not CYP1A1 dependent. However, our study shows that BRCA1 defective cells may still be able to biotransform BaP by regulating other CYP enzymes, including CYP1B1. Knockdown of BRCA1 also severely affected the expression levels of two types of uridine diphosphate glucorunyltransferase (UGT1A1 and UGT1A9) and NRF2. Both UGTs are known as BaP-specific detoxification enzymes, and NRF2 is a master regulator of antioxidant and detoxification genes. Thus, we concluded that the increased amount of BaP-induced DNA adducts in BRCA1 knockdown cells is strongly associated with its loss of functional detoxification. Chromatin immunoprecipitation assay revealed that BRCA1 is recruited to the promoter/enhancer sequences of UGT1A1, UGT1A9, and NRF2. Regulation of UGT1A1 and UGT1A9 expression showed that the induction of DNA adducts by BaP is directly affected by their expression levels. Finally, overexpression of UGTs, NRF2, or ARNT significantly decreased the amount of BaP-induced adducts in BRCA1-deficient cells. Overall, our results suggest that BRCA1 protects cells by reducing the amount of BaP-induced DNA adducts possibly via transcriptional activation of detoxification gene expression.

Augmentation of Sodium Butyrate-induced Apoptosis by Phosphatidylinositol 3-kinase Inhibition in the Human Cervical Cancer Cell-line

PURPOSE Sodium butyrate (NaBT) is principally a histone deacetylase (HDAC) inhibitor, and it has the potential to arrest HPV-positive carcinoma cells at the G1 to S phase transition of the cell cycle. The aim of study was to determine whether phosphatidylinositol 3-kinase (PI3K) inhibition can enhance the inhibitory effect of NaBT on a human cervical cancer cell line (HeLa). MATERIALS AND METHODS Cervical cancer cells (HeLa) were treated with NaBT alone or in combination with the PI3K inhibitors wortmannin or LY294002. Cell viability analysis and FACS analysis were carried out. The expressions of the cell cycle related proteins were evaluated by Western-blot analysis. RESULTS Inhibition of PI3K enhanced NaBT-mediated apoptosis and this decreased the HeLa cell viability. Either wortmannin or LY294002, combined with NaBT, enhanced the activation of caspase 3 and caspase 9, and this enhanced the subsequent cleavage of poly (ADP-ribose) polymerase (PARP). Cervical cancer cells were arrested in the subG1 and G2/M phase, as was detected by FACS analysis. NaBT treatment in combination with PI3K inhibitors showed the increased expression of the CDK inhibitors p21(Cip1/Waf1) and p27(Kip1), in a p53 dependent manner, and also the increased dephosphorylation of Rb whereas there was a reduction in the expression levels of cyclin A, cyclin D1 and cyclin B1. CONCLUSION The results demonstrate that inhibition of PI3K enhances NaBT-mediated cervical cancer cell apoptosis through the activation of the caspase pathway. Moreover, these findings will support future investigation using the PI3K inhibitors in combination with adjuvant treatment for treating carcinoma of the cervix.

Salinomycin inhibited cell proliferation and induced apoptosis in human uterine leiomyoma cells

Objective The aim of this study was to investigate the anti-proliferative effect of the salinomycin in cell proliferation and apoptosis in primary cultured human uterine leiomyoma cells. Methods Cell viability was measured by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Caspase-3 activity assay and DNA fragmentation assay were performed to determine the effect of apoptosis. The expression of apoptosis regulatory-related proteins was evaluated by western blot. Results The cell viability and proliferation of uterine leiomyoma cells were significantly reduced by salinomycin treatment in a dose-dependent manner. DNA fragmentation assay results showed apoptotic cell death after salinomycin incubation. Salinomycin activated caspase-3, -8, and -9, causing apoptosis in uterine leiomyoma cells. Down-regulation of Bcl-2, XIAP, and FLIP with a concomitant increase in Bax, Fas, and DR5 were observed. Conclusion These results provided the first evidence that salinomycin induce both intrinsic and extrinsic apoptosis. Therefore, salinomycin may be a promising chemopreventive and therapeutic agent against human uterine leiomyoma.

Cyclin-Dependent Kinase Inhibitor p27Kip1 Controls Growth and Cell Cycle Progression in Human Uterine Leiomyoma

The molecular mechanism of the cell-cycle machinery in uterine leiomyoma has not yet been fully elucidated. Among the various types of cell-cycle regulators, p27Kip1 (p27) is considered to be a potent tumor suppressor. To provide further molecular basis for understanding the progression of uterine leiomyoma, our objective was to evaluate the expression level of p27 in normal myometrium and uterine leiomyoma tissue and its effect on cytogenic growth. Western blot analysis, real-time polymerase chain reaction (PCR) and immunohistochemical staining revealed that p27 protein and messenger RNA were down-regulated in uterine leiomyoma tissue and cultured cells compared to normal myometerium. Full-length human p27 cDNA was transferred using a replication-deficient recombinant adenoviral vector (Ad.p27) into uterine leiomyoma cells and evaluated the effect on cell proliferation. Transfection of Ad.p27 into uterine leiomyoma cells resulted in the induction of apoptosis, reduction in viability and proliferation of uterine leiomyoma cells. Our results suggest a new paradigm that down-regulated p27 protein expression is the possible underlying mechanism for the growth of uterine leiomyoma and over-expression of p27 induces cell death. This study provides better understanding of the control exerted by p27 in regulating growth and disease progression of uterine leiomyoma.

The effect of salinomycin on ovarian cancer stem-like cells

Objective The identification of cancer stem-like cells is a recent development in ovarian cancer. Compared to other cancer cells, cancer stem-like cells present more chemo-resistance and more aggressive characteristics. They play an important role in the recurrence and drug resistance of cancer. Therefore, the target therapy of cancer stem-like cell may become a promising and effective approach for ovarian cancer treatment. It may also help to provide novel diagnostic and therapeutic strategies. Methods The OVCAR3 cell line was cultured under serum-free conditions to produce floating spheres. The CD44+CD117+ cell line was isolated from the human ovarian cancer cell line OVCAR3 by using immune magnetic-activated cell sorting system. The expression of stemness genes such as OCT3/4, NANOG and SOX2 mRNA were determined by reverse transcription polymerase chain reaction. OVCAR3 parental and OVCAR3 CD44+CD117+ cells were grown in different doses of paclitaxel and salinomycin to evaluate the effect of salinomycin. And growth inhibition of OVCAR3 CD44+CD117+ cells by paclitaxel combined with salinomycin was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Results Tumor spheroids generated from the OVCAR3 cell line are shown to have highly enriched CD44 and CD117 expression. Treatment with a combination of paclitaxel and salinomycin demonstrated growth inhibition of OVCAR3 CD44+CD117+ cells. Conclusion The present study is a detailed investigation on the expression of CD44 and CD117 in cancer stem cells and evaluates their specific tumorigenic characteristics in ovarian cancer. This study also demonstrates significant growth inhibition of cancer stem-like cells by paclitaxel combined with salinomycin. Identification of these cancer stem-like cell markers and growth inhibition effect of salinomycin may be the next step to the development of novel target therapy in ovarian cancer.

Hsp90 inhibitor SY‑016 induces G2/M arrest and apoptosis in paclitaxel-resistant human ovarian cancer cells

The aim of the present study was to evaluate the in vitro effect of a heat shock protein (Hsp)90 inhibitor, SY-016, on the paclitaxel (PTX)-resistant human ovarian cancer cell line OVCAR-3PTX, and explore its mechanism of apoptosis. In the present study, SY-016 was used in combination with PTX to determine its effect on the cell proliferation and apoptosis of OVCAR-3PTX cells. The drug-resistant tumor cells were established in vitro by stepwise sequential exposure to increasing concentrations of PTX. The cell viability and cell cycle distribution were measured by MTT assay and flow cytometric analysis, respectively. The induction of apoptosis was measured by caspase-3 activity, DNA fragmentation and western blot analyses. The cell viability significantly decreased following treatment with PTX and SY-016 as compared with either drug alone. The DNA fragmentation assay revealed an induction of apoptosis. The results from the flow cytometric analysis revealed an increase in the percentage of cells in the G2/M phase. Downregulation of B-cell lymphoma (Bcl)-2, X-linked inhibitor of apoptosis protein, survivin, Akt, nuclear factor-κB and cyclin-dependent kinase 4, as well as upregulation of Bcl-2-associated X protein, were observed. SY-016 may contribute to the induction of apoptosis in OVCAR-3PTX cells. These results suggest that SY-016 in combination with PTX may be a beneficial chemotherapeutic strategy, particularly in patients with tumors refractory to PTX.

A case of primary fallopian tube carcinoma diagnosed radiologically before operation

Primary fallopian tube carcinoma is one of the rarest gynecological malignancies, accounting for 0.18% to 1.6% of all malignant neoplasms of the female reproductive tract. Preoperative diagnosis was difficult due to nonspecific symptoms and signs. This case of primary tubal cancer was diagnosed preoperatively on the basis of ultrasonography, computed tomography and magnetic resonance imaging. We have experienced a case of primary fallopian tube carcinoma before operation and so report with brief review of the literature.

Metastatic gestational trophoblastic tumor presenting as spontaneous kidney rupture: Treatment with embolization

Choriocarcinoma is a rapidly growing tumor that characteristically outgrows its blood supply. We report a rare case of metastatic choriocarcinoma presenting with acute right flank pain due to kidney rupture secondary to renal metastasis. The renal metastasis was embolised to stanch blood for control of hemorrhage. A brief review of the imaging features and therapeutic options for the ruptured renal metastases is discussed along with the case.