Nahee Park

Role of annexin a5 on mitochondria-dependent apoptosis induced by tetramethoxystilbene in human breast cancer cells.

We have previously shown that 2,4,3′,5′-tetramethoxystilbene (TMS), a trans-stilbene analogue, induces apoptosis in human cancer cells. However, the detailed mechanisms of mitochondria-dependent apoptosis induced by TMS are not fully understood. In the present study, the possible roles of annexin A5 in TMS-mediated apoptosis were investigated in MCF7 human breast cancer cells. Quantitative real-time PCR analysis and Western blot analysis showed that the expression of annexin A5 was strongly increased in TMS-treated cells. TMS caused a strong translocation of annexin A5 from cytosol into mitochondria. Confocal laser scanning microscopic analysis clearly showed that TMS induced translocation of annexin A5 into mitochondria. TMS increased the expression and oligomerization of voltage-dependent anion channel (VDAC) 1, which may promote mitochondria-dependent apoptosis through disruption of mitochondrial membrane potential. When cells were treated with TMS, the levels of Bax, and Bak as well as annexin A5 were strongly enhanced. Moreover, we found that the cytosolic release of apoptogenic factors such as cytochrome c, or apoptosis-inducing factor (AIF) in mitochondria was markedly increased. Annexin A5 depletion by siRNA led to decreased proapoptotic factors such as Bax, Bak, and annexin A5. Taken together, our results indicate that annexin A5 may play an important role in TMS-mediated mitochondrial apoptosis through the regulation of proapoptotic proteins and VDAC1 expression.

Embelin-Induced Apoptosis of Human Prostate Cancer Cells Is Mediated through Modulation of Akt and β-Catenin Signaling

There is increasing evidence that embelin, an active component of Embelia ribes, induces apoptosis in human cancer cells, but the detailed mechanisms are still unclear. Here, we have investigated the effect of embelin on the growth of human prostate cancer cells. Embelin strongly inhibited cell growth especially in human prostate cancer cell lines, including PC3, DU145, LNCaP-LN3 and normal prostate epithelial cell, RWPE-1 compared to breast cancer (MDA-MB-231, MCF-7, and T47D), hepatoma (HepG2, Hep3B, and HuH-7), or choriocarcinoma (JEG-3). We observed that embelin induced apoptosis of PC3 cells in a time-dependent manner correlated with decreased expression of Bcl-2, Bcl-xL, and Mcl-1, increased translocation of Bax into mitochondria, and a reduction in the mitochondrial membrane potential. Furthermore, embelin induced voltage-dependent anion channel (VDAC) 1 expression and oligomerization, which may promote cytochrome c and AIF release. Because embelin was able to inhibit Akt activation and cyclooxygenase-2 expression, the effects on Wnt/ β-catenin signaling were determined. Embelin activated glycogen synthase kinase (GSK)-3β by preventing phosphorylation and suppressed β-catenin expression. Attenuation of β-catenin-mediated TCF transcriptional activity and gene transcription, such as cyclin D1, c-myc, and matrix metalloproteinase (MMP)-7, were shown in embelin-treated cells. The changes in β-catenin levels in response to embelin were blocked by lithium chloride, a GSK-3 inhibitor, indicating that embelin may decrease β-catenin expression via GSK-3β activation. Furthermore, exposure of PC3 cells to embelin resulted in a significant decrease in cell migration and invasion. In conclusion, these findings suggest that inhibition of Akt signaling and activation of GSK-3β partially contributes to the pro-apoptotic effect of embelin in prostate cancer cells.

Auranofin promotes mitochondrial apoptosis by inducing annexin A5 expression and translocation in human prostate cancer cells.

Auranofin is a lipophilic gold compound with anti-inflammatory and immunosuppressive properties. This compound also exerts antiproliferative effects in several human cancer cell lines. Although auranofin induces apoptosis in human cancer cells, the underlying mechanisms remain unclear. This study investigated auranofin-mediated inhibition of cell growth and induction of mitochondrial apoptosis in PC3 human prostate cancer cells. Treatment with auranofin significantly inhibited cell viability with an IC50 value of 2.5 μM after 24 h. In particular, when cells were treated with 2.5 μM auranofin, there was a 2.2-fold increase in apoptotic cells compared to untreated cells. Auranofin activated caspase-3 and -8 in a concentration-dependent manner and decreased the levels of mitochondrial anti-apoptotic factors, such as Bcl-2 and Bcl-xL. In addition, auranofin enhanced oligomerization of the voltage-dependent anion channel (VDAC) in a concentration- and time-dependent manner. Interestingly, auranofin significantly enhanced annexin A5 mRNA and protein expression and promoted annexin A5 translocation into the mitochondria. In order to characterize the function of annexin A5 in auranofin-induced mitochondrial apoptosis, annexin A5 was depleted using siRNA. Annexin A5 siRNA suppressed auranofin-mediated annexin A5 expression and VDAC oligomerization. Accordingly, annexin A5 depletion rescued auranofin-induced apoptosis, which may be mediated by caspase-3 activation. In conclusion, the present findings suggest that auranofin induces mitochondrial apoptosis through induction of annexin A5 expression and translocation as well as VDAC oligomerization in human prostate cancer cells.

Role of Annexin A5 in Cisplatin-induced Toxicity in Renal Cells: MOLECULAR MECHANISM OF APOPTOSIS*

Background: There is increasing evidence that annexin A5 is related to cytotoxicity, but the precise function has yet to be elucidated. Results: Cisplatin induces mitochondrial translocation of annexin A5, and annexin A5 mediates VDAC oligomerization. Conclusion: Annexin A5 may play a role as a mediator of cisplatin-induced apoptosis in renal epithelial cells. Significance: Learning how annexin A5 is involved in the apoptotic pathway is crucial for understanding cisplatin-induced toxicity. Annexin A5 belongs to a large family of calcium-binding and phospholipid-binding proteins and may act as an endogenous regulator of various pathophysiological processes. There is increasing evidence that annexin A5 is related to cytotoxicity, but the precise function of this protein has yet to be elucidated. In this study, we aimed to verify the function of annexin A5 in the apoptosis of renal epithelial cells. Real-time PCR and Western blot analysis, together with immunofluorescence analysis, showed that the expression of annexin A5 significantly increased in the presence of cisplatin in both human and rat renal epithelial cells. With regard to the mechanism of cisplatin-induced apoptosis, apoptosis-inducing factor (AIF) release into the cytosol was observed, and the underlying mechanism was identified as voltage-dependent anion channel (VDAC) oligomerization. Mitochondrial membrane potential (Δψm) was found to be greatly disrupted in cisplatin-treated cells. Moreover, cisplatin strongly induced translocation of annexin A5 into mitochondria. To understand the functional significance of annexin A5 in renal cell death, we used a siRNA-mediated approach to knock down annexin A5. Annexin A5 depletion by siRNA led to decreased annexin A5 translocation into mitochondria and significantly reduced VDAC oligomerization and AIF release. Annexin A5 siRNA also increased cell viability compared with the control. Moreover, expression of annexin A5 was induced by other nephrotoxicants such as CdCl2 and bacitracin. Taken together, our data suggest that annexin A5 may play a crucial role in cisplatin-induced toxicity by mediating the mitochondrial apoptotic pathway via the induction and oligomerization of VDAC.

Bacterial Lipopolysaccharides Induce Steroid Sulfatase Expression and Cell Migration through IL-6 Pathway in Human Prostate Cancer Cells

Steroid sulfatase (STS) is responsiblefor the conversion of estrone sulfate to estrone that can stimulate growth in endocrine-dependent tumors such as prostate cancer. Although STS is considered as a therapeutic target for the estrogen-dependent diseases, cellular function of STS are still not clear. Previously, we found that tumor necrosis factor (TNF)-α significantly enhances steroid sulfatase expression in PC-3 human prostate cancer cells through PI3K/Akt-dependent pathways. Here, we studied whether bacterial lipopolysaccharides (LPS) which are known to induce TNF-α may increase STS expression. Treatment with LPS in PC-3 cells induced STS mRNA and protein in concentration- and time-dependent manners. Using luciferase reporter assay, we found that LPS enhanced STS promoter activity. Moreover, STS expression induced by LPS increased PC-3 tumor cell migration determined by wound healing assay. We investigated that LPS induced IL-6 expression and IL-6 increased STS expression. Taken together, these data strongly suggest that LPS induces STS expression through IL-6 pathway in human prostate cancer cells.

Induction of steroid sulfatase expression by tumor necrosis factor-α through phosphatidylinositol 3-kinase/Akt signaling pathway in PC-3 human prostate cancer cells

Steroid sulfatase (STS) is responsible for the hydrolysis of aryl and alkyl steroid sulfates and has a pivotal role in regulating the formation of biologically active estrogens. STS may be considered a new promising drug target for treating estrogen-mediated carcinogenesis. However, the molecular mechanism of STS expression is not well-known. To investigate whether tumor necrosis factor (TNF)-α is able to regulate gene transcription of STS, we studied the effect of TNF-α on STS expression in PC-3 human prostate cancer cells. RT-PCR and Western blot analysis showed that TNF-α significantly induced the expression of STS mRNA and protein in a concentration- and time-dependent manner. Treatment with TNF-α resulted in a strong increase in the phosphorylation of Akt on Ser-473 and when cells were treated with phosphatidylinositol (PI) 3-kinase inhibitors such as LY294002 or wortmannin, or Akt inhibitor (Akt inhibitor IV), induction of STS mRNA expression by TNF-α was significantly prevented. Moreover, activation of Akt1 by expressing the constitutively active form of Akt1 increased STS expression whereas dominant-negative Akt suppressed TNF-α-mediated STS induction. We also found that TNF-α is able to increase STS mRNA expression in other human cancer cells such as LNCaP, MDA-MB-231, and MCF-7 as well as PC-3 cells. Taken together, our results strongly suggest that PI 3-kinase/Akt activation mediates induction of human STS gene expression by TNF-α in human cancer cells.

Induction of steroid sulfatase expression in PC-3 human prostate cancer cells by insulin-like growth factor II.

Human steroid sulfatase (STS) plays an important role in regulating the formation of biologically active estrogens and may be a promising target for treating estrogen-mediated carcinogenesis. The molecular mechanism of STS gene expression, however, is still not clear. Growth factors are known to increase STS activity but the changes in STS expression have not been completely understood. To determine whether insulin-like growth factor (IGF)-II can induce STS gene expression, the effects of IGF-II on STS expression were studied in PC-3 human prostate cancer cells. RT-PCR and Western blot analysis showed that IGF-II treatment significantly increased the expression of STS mRNA and protein in concentration- and time-dependent manners. To understand the signaling pathway by which IGF-II induces STS gene expression, the effects of specific PI3-kinase/Akt and NF-κB inhibitors were determined. When the cells were treated with IGF-II and PI3-kinase/Akt inhibitors, such as LY294002, wortmannin, or Akt inhibitor IV, STS expression induced by IGF-II was significantly blocked. Moreover, we found that NF-κB inhibitors, such as MG-132, bortezomib, Bay 11-7082 or Nemo binding domain (NBD) binding peptide, also strongly prevented IGF-II from inducing STS gene expression. We assessed whether IGF-II activates STS promoter activity using transient transfection with a luciferase reporter. IGF-II significantly stimulated STS reporter activity. Furthermore, IGF-II induced expression of 17β-hydroxysteroid dehydrogenase (HSD) 1 and 3, whereas it reduced estrone sulfotransferase (EST) gene expression, causing enhanced estrone and β-estradiol production. Taken together, these results strongly suggest that IGF-II induces STS expression via a PI3-kinase/Akt-NF-κB signaling pathway in PC-3 cells and may induce estrogen production and estrogen-mediated carcinogenesis.

Annexin A5 suppresses cyclooxygenase-2 expression by downregulating the protein kinase C-ζ–nuclear factor-κB signaling pathway in prostate cancer cells

Annexin A5 (ANXA5) is a member of the annexin protein family. Previous studies have shown that ANXA5 is involved in anti-inflammation and cell death. However, the detailed mechanism of the role of ANXA5 in cancer cells is not well understood. In this study, we investigated the inhibitory effect of ANXA5 on cyclooxygenase-2 (COX-2) in prostate cancer cells. Expression of COX-2 induced by TNF-α was inhibited by overexpression of ANXA5 and inhibition of COX-2 expression by auranofin, which could induce ANXA5 expression, was restored by ANXA5 knockdown. In addition, ANXA5 knockdown induces phosphorylation of NF-κB p65 in prostate cancer cells, indicating that ANXA5 causes COX-2 downregulation through inhibition of p65 activation. We also found that protein kinase C (PKC)-ζ protein levels were upregulated by the inhibition of ANXA5, although the mRNA levels were unaffected. We have shown that upregulated COX-2 expression by inhibition of ANXA5 is attenuated by PKC-ζ siRNA. In summary, this study demonstrates that downregulation of PKC-ζ-NF-κB signaling by ANXA5 may inhibit COX-2 expression in prostate cancer.Annexin A5 (ANXA5) is a member of the annexin protein family. Previous studies have shown that ANXA5 is involved in anti-inflammation and cell death. However, the detailed mechanism of the role of ANXA5 in cancer cells is not well understood. In this study, we investigated the inhibitory effect of ANXA5 on cyclooxygenase-2 (COX-2) in prostate cancer cells. Expression of COX-2 induced by TNF-α was inhibited by overexpression of ANXA5 and inhibition of COX-2 expression by auranofin, which could induce ANXA5 expression, was restored by ANXA5 knockdown. In addition, ANXA5 knockdown induces phosphorylation of NF-κB p65 in prostate cancer cells, indicating that ANXA5 causes COX-2 downregulation through inhibition of p65 activation. We also found that protein kinase C (PKC)-ζ protein levels were upregulated by the inhibition of ANXA5, although the mRNA levels were unaffected. We have shown that upregulated COX-2 expression by inhibition of ANXA5 is attenuated by PKC-ζ siRNA. In summary, this study demonstrates that downregulation of PKC-ζ-NF-κB signaling by ANXA5 may inhibit COX-2 expression in prostate cancer.

Cloning and Expression in Pichia pastoris of a New Cytochrome P450 Gene from a Dandruff-causing Malassezia globosa.

The Malassezia fungi are responsible for various human skin disorders including dandruff and seborrheic dermatitis. Of the Malassezia fungi, Malassezia globosa (M. globosa) is one of the most common in human scalp. The completed genome sequence of M. globosa contains four putative cytochrome P450 genes. To determine the roles of Malassezia P450 enzymes in the biosynthesis of ergosterol, we isolated MGL3996 gene from M. globosa chromosomal DNA by PCR. The MGL3996 gene encodes an enzyme of 616 amino acids, which shows strong similarity with known CYP52s of other species. MGL3996 gene was cloned and expressed in Pichia pastoris (P. pastoris) heterologous yeast expression system. Using the yeast microsomes expressing MGL3996 protein, a typical P450 CO-difference spectrum was shown with absorption maximum at 448 nm. SDS-PAGE analysis revealed a protein band of apparent molecular weight 69 kDa and Western blot with anti-histidine tag antibody showed that MGL3996 was successfully expressed in P. pastoris. Cloning and expression of a new P450 gene is an important step to study the P450 monooxygenase system of M. globosa and to understand the role of P450 enzymes in pathophysiology of dandruff.

Abstract 2754: Induction of Sp1 factor by CYP1B1 inhibits TRAIL-mediated apoptotic pathway

Human cytochrome P450 1B1 (CYP1B1) belongs to the CYP1 family and is known as a major enzyme for estradiol 4-hydroxylation. It has been reported that CYP1B1 expression is higher in the tumor tissues than the normal ones, especially in hormone-related cancers such as breast, ovarian and prostate. Previously, we identified that CYP1B1 induces cell proliferation by activation of Wnt/β-catenin signal pathway. To explore the role of CYP1B1 on cancer cell proliferation further, we investigated whether CYP1B1 blocks apoptotic pathways. Using Western blot and RT-PCR, the expression of TRAIL was examined in MCF-7 cells, and we found that CYP1B1 overexpression or treatment with a CYP1B1 inducer, 7, 12-dimethylbenz[α]anthracene (DMBA) caused suppression of TRAIL and its receptors (DR4 and DR5) expression. However, when cells were treated with CYP1B1 siRNA or a selective CYP1B1 inhibitor, tetramethoxystilbene (TMS), expression of TRAIL and its receptors were significantly increased. Sp1, a zinc finger-containing transcription factor, has been reported to suppress TRAIL gene expression by binding TRAIL promoter. Interestingly, CYP1B1 overexpression and DMBA induced Sp1 expression and TMS was able to prevent DMBA-induced Sp1 expression. Sp1 knockdown with siRNA suppressed c-FLIP, an inhibitor protein of TRAIL-mediated apoptosis while it enhanced TRAIL and DR4 expression. These results were reversed by Sp1 overexpession. Moreover, attenuation of Sp1 DNA binding activity using mithramycin A, a Sp1 binding inhibitor, resulted in recovery of TRAIL expression suppressed by DMBA. Taken together, these data suggest that CYP1B1 prevents apoptosis via TRAIL suppression and this is mediated by Sp1 induction. Citation Format: Yeo-Jung Kwon, Nahee Park, Sangyun Shin, Dong-Jin Ye, Mihye Hong, Young-Jin Chun. Induction of Sp1 factor by CYP1B1 inhibits TRAIL-mediated apoptotic pathway. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2754. doi:10.1158/1538-7445.AM2014-2754