Su-Hyeong Kim

Celecoxib induces apoptosis in cervical cancer cells independent of cyclooxygenase using NF-κB as a possible target

Purpose Recently, many studies have shown that celecoxib induces apoptosis in various cancer cells by different mechanisms depending on the cell type. This study examined the apoptotic effect of celecoxib in cervical cancer cells and its mechanism.Methods Cell viability was measured by MTT assay and apoptosis was examined by DNA fragmentation and flow cytometry. Western blotting and immunoprecipitation were used to explore various mechanisms of celecoxib-induced apoptosis. The activation of NF-κB was confirmed by EMSA.Results Celecoxib induced apoptosis independent of COX-2 activity. This event accompanied the activation of caspase-8 and -9 with Bid cleavage and the loss of mitochondrial membrane potential. The protective effect of caspase-8 and -9 inhibitors on celecoxib-induced apoptosis suggests the importance of caspase-8 and -9 activation in this apoptotic pathway. Fas/FADD-mediated apoptotic pathway was detected only in C33A cells, demonstrated by the immunoprecipitation of Fas-FADD in celecoxib-treated cells and the protective effect of FADD dominant negative mutant. Finally, NF-κB appeared to be involved in celecoxib-induced apoptosis, as revealed by increased NF-kB DNA binding activity in a time-dependent manner and attenuation of its proapoptotic effect by N-tosyl-L-phenylalanyl-chloromethyl ketone, an NF-kB blocker.Conclusions These data show that caspase-8 and -9 are involved in the apoptotic effect of celecoxib in cervical cancer cells. This requires the FADD-dependent pathway in a cell type-specific manner. In addition, NF-κB may play a key role in celecoxib-induced apoptosis.

Akt involvement in paclitaxel chemoresistance of human ovarian cancer cells

Abstract:  Paclitaxel (taxol) is extensively used for chemotherapy of various cancers including ovarian cancer. Although paclitaxel induces apoptosis in cancer cells, its exact mechanism of action still remains to be determined. Akt mediates survival signals which preserve various cancer cells from apoptosis pathway. Thus, Akt is considered an exciting target for therapeutics. Here, we demonstrated that inhibition of Akt increases the efficacy of the paclitaxel‐induced apoptosis in SKOV3 and PA‐1 human ovarian cancer cells. The sensitivity to paclitaxel of SKOV3 and PA‐1 cells was examined using the MTT assay. At a concentration of 30 nM, PA‐1 cells were more sensitive to paclitaxel than SKOV3 cells. Apoptosis was accompanied by the release of cytochrome c into the cytoplasm and cleavage of poly (ADP‐ribose) polymerase (PARP). To further elucidate the mechanism of apoptosis by paclitaxel, we compared the levels of phosphorylation of Akt between paclitaxel‐resistant SKOV3 cells and paclitaxel‐sensitive PA‐1 cells. The higher level of phosphorylated Akt was shown in SKOV3 cells than in PA‐1 cells. Interestingly, the treatment of paclitaxel decreased the amount of phosphorylated Akt in a time‐dependent manner in both cell lines. Furthermore, inhibition of Akt by specific phosphatidyinositol‐3‐kinase (PI3K)‐Akt inhibitors (Wortmannin, and LY294002) synergistically increased the efficacy of the paclitaxel‐induced apoptosis in both cell lines. These results suggest that the addition of the Akt inhibitor may increase the therapeutic efficacy of paclitaxel for patients with ovarian cancer.

Genistein Inhibits Cell Growth by Modulating Various Mitogen-Activated Protein Kinases and AKT in Cervical Cancer Cells

Genistein, a soy‐derived isoflavone, inhibits growth of tumor cells from various malignancies. Here we investigated the effect of genistein on the growth of cervical cancer cells (HeLa and CaSki) and its possible mechanism. Genistein significantly suppressed cell growth of HeLa and CaSki cells at concentrations of 20 and 60 μmol/L, respectively, for 24 h. Western blotting analysis showed that genistein reduced phosphorylation of AKT and extracellular signal–regulated kinase (ERK)‐1/2 and induced phosphorylation of p38 mitogen‐activated protein kinase (MAPK) and c‐Jun N‐terminal kinase (JNK). Moreover, inhibition of ERK1/2 activity enhanced cell growth inhibition by genistein, whereas inhibition of p38 MAPK activity rescued from genistein‐mediated growth inhibition. Interestingly, inhibition of AKT activity recovered genistein‐induced growth inhibition in CaSki cells but did not in HeLa cells. However, inhibition of JNK activity seemed to have little effect on cell growth inhibition by genistein. Taken together, these results suggest that genistein could inhibit cell growth by inhibiting ERK1/2 activity and activating p38 MAPK.

Human papillomavirus type 16 E5 protein inhibits hydrogen-peroxide-induced apoptosis by stimulating ubiquitin-proteasome-mediated degradation of Bax in human cervical cancer cells.

To investigate the mechanism by which the human papillomavirus (HPV) E5 protein contributes to the carcinogenesis of uterine cervical cancer, we studied the effect of HPV E5 on apoptosis of cervical cancer cells and its underlying mechanism. Expression of HPV16 E5 protein inhibited hydrogen peroxide-induced apoptosis in C-33A cervical cancer cells. E5 decreased the expression of Bax protein, and exogenous expression of Bax abolished the anti-apoptotic effect of E5. Knockdown of E5 by small interfering RNA sensitized CaSki cervical cancer cells to hydrogen peroxide-induced apoptosis with concurrent increase in Bax expression. Transient expression of E5 significantly increased the degradation rate of Bax protein by inducing the ubiquitination. The E5-induced decrease in Bax expression was inhibited by a cyclooxygenase-2 (COX-2) inhibitor, prostaglandin E2 (PGE(2)) receptor antagonists and cyclic adenosine monophosphate-dependent protein kinase (PKA) inhibitor. Treatment with PGE(2) decreased the expression of Bax and inhibited hydrogen peroxide-induced apoptosis of C-33A cells. We concluded that HPV16 E5 protein inhibits hydrogen peroxide-induced apoptosis of cervical cancer cells by stimulating the ubiquitin-proteasome-mediated degradation of Bax protein, and the pathway involves COX-2, PGE(2) and PKA. This finding suggests the possibility that HPV 16 E5 protein contributes to cervical carcinogenesis by inhibiting apoptosis of transformed cervical epithelial cells.

Human papillomavirus E5 protein induces expression of the EP4 subtype of prostaglandin E2 receptor in cyclic AMP response element-dependent pathways in cervical cancer cells

Human papillomavirus (HPV) is the major cause of uterine cervical cancer, but the role of the HPV E5 in carcinogenesis is not clearly understood. Prostaglandins are known to contribute to carcinogenesis of cervical cancer, and we therefore investigated the effect of HPV16 E5 on the expression of prostaglandin E2 (PGE2) receptors and underlying mechanisms. Stable expression of the E5 induced expression of the EP4 subtype of PGE2 receptors in C33A cervical cancer cells, and transfection of E5 small interfering RNA (siRNA) decreased it. EP4 protein expression was increased in human cervical cancer tissues, and EP4 mediated E5-induced increase in anchorage-independent colony formation and vascular endothelial growth factor expression. E5 induced cyclooxygenase-2 (COX-2) expression, and COX-2 increased PGE2 secretion and EP4 expression. The induction of EP4 by PGE2 and E5 was inhibited by an EP4 antagonist, inhibitors of cyclic adenosine monophosphate-dependent protein kinase or phosphatidylinositol 3-kinase, and a cyclic adenosine monophosphate response element (CRE) decoy. E5 increased the luciferase expression controlled by a variant CRE of the EP4 promoter, and it also increased the binding of cyclic adenosine monophosphate response element binding protein (CREB) to oligonucleotides containing this CRE. We conclude that the HPV16 E5 protein induces EP4 receptor protein in cervical cancer cells and that this induction involves epidermal growth factor receptor, COX-2, PGE2, EP2 and EP4, protein kinase A, CREB and CRE.

Involvement of Both Extrinsic and Intrinsic Apoptotic Pathways in Apoptosis Induced by Genistein in Human Cervical Cancer Cells

Genistein, a naturally occurring isoflavonoid abundant in soy products, has anticancer activity in multiple tumor cells. In this study, we evaluated the apoptotic effect of genistein on cervical cancer cells and its mechanism of apoptosis. Genistein inhibited the proliferation of cervical cancer cells (HeLa, CaSki, and C33A). HeLa cells were the most sensitive to genistein, whereas CaSki and C33A cells were less sensitive. Sub‐G1 analysis showed that genistein increased apoptotic cells up to 45% at a concentration of 60 μmol/L in HeLa cells, whereas it produced 21% and 17% apoptotic cells in CaSki and C33A cells, respectively, at the same concentration. To determine the apoptotic pathway induced by genistein in the cervical cancer cells, we assessed activation of caspase‐3, ‐8, and ‐9 by immunoblotting. Procaspase‐3, ‐8, and ‐9 were decreased and PARP cleavage increased in a time‐dependent manner after the treatment of genistein in HeLa cells. Also, inhibition of caspase‐3, ‐8, and ‐9 with pharmacological inhibitors reduced genistein‐mediated apoptosis. Interestingly, inhibition of caspase‐8 resulted in remarkable reduction of genistein‐induced apoptosis. Bax expression was increased and total bid decreased, whereas bcl‐2 level was not changed by genistein. Taken together, these results suggest that genistein could induce apoptosis through both extrinsic and intrinsic pathways in human cervical cancer cells.

Expression of MMP‐2, MMP‐9, and Urokinase‐Type Plasminogen Activator in Cervical Intraepithelial Neoplasia

Matrix metalloproteinase‐2 (MMP‐2), MMP‐9, and urokinase‐type plasminogen activator (uPA) are important factors for cancer invasion and metastasis, degrading the extracellular matrix. They are also associated with angiogenesis. Angiogenic phenotype is another feature of high‐grade cervical intraepithelial neoplasia (CIN). However, their associations with the progression of low‐grade CIN to high‐grade CIN are unexplored. We investigated whether these proteolytic enzyme expressions correlate with the progression of CIN. A total of 39 paraffin‐embedded specimens from 10 patients with CIN grade 1, nine with CIN grade 2, and 20 with CIN grade 3 were assessed immunohistochemically by specific antibodies against MMP‐2, MMP‐9, and uPA. MMP‐9 expression was higher in CIN 3 lesions (47.4%) than in CIN 1 (22.2%) and CIN 2 (20.2%) lesions, although the difference failed to reach statistical significance. The expression level of uPA and MMP‐2 was not associated with the grade of CIN lesions. Interestingly, we found a significant association between expressions of uPA and MMP‐2 (P= 0.028). Our results suggest that MMP‐9 might play a role in the progression of CIN.

Apoptotic effect of celecoxib dependent upon p53 status in human ovarian cancer cells

Abstract:  Celecoxib, a selective cyclooxygenase‐2 (COX‐2) inhibitor, induces the apoptosis in various cancers in COX‐2 dependent and/or independent manners. The p53 protein is mutated in 50% of all human tumors and plays a key role in apoptosis, cell cycle, and the expression of several proteins. In ovarian cancer, the rate of p53 mutation has been shown to be very high and associated with poor prognosis. To explore the importance of functional status of p53 in apoptosis by celecoxib in ovarian cancer cells, the cellular response to celecoxib was determined in SK‐OV3 ovarian cancer cells with null type p53 and PA‐1 with wild‐type p53. Our results showed that celecoxib inhibited cell growth more in PA‐1 than in SK‐OV3. The underlying antiproliferative mechanism may differ between these two cell types dependent upon the functional status of p53, which plays integral roles in regulating cell cycle and survival. Higher sub‐G1 was shown in PA‐1 than in SK‐OV3 in response to celecoxib (PA‐1 versus SK‐OV3; 60.28% versus 6.69%). Caspase ‐8, ‐9, and ‐3 were activated in PA‐1 cells, but not in SK‐OV3 cells. These results suggest that death receptor and mitochondria‐mediated apoptotic pathways may be involved in celecoxib‐induced apoptosis dependent upon the functional status of p53. Our article demonstrated that the celecoxib effectively inhibited cell growth and induced apoptosis in human ovarian cancer cells with wild‐type p53. Thus, apoptotic effect by celecoxib seemed to be different dependent upon the functional status of p53.

Electron-beam lithography resist profile simulation for highly sensitive resist

Abstract A very highly sensitive resist is difficult to simulate its resist profile because of its extreme difference of development rate which can be determined from absorbed energy when electron beam is exposed. We developed resist profile simulator named ELIS (Electron-beam LIthography Simulator) that can predict the development profile of a very highly sensitive resist: Hybrid scattering model, Gaussian beam convolution and its superposition were used for Monte Carlo simulation and the ray tracing model for resist development.