Mitigating transcriptional bottleneck using a constitutively active transcription factor, VP16-CREB, in mammalian cells.

High-level expression of recombinant proteins in mammalian cells has long been an area of interest. Inefficient transcription machinery is often an obstacle in achieving high-level expression of recombinant proteins in mammalian cells. Synthetic promoters have been developed to improve the transcription efficiency, but have achieved limited success due to the limited availability of transcription factors (TFs). Here, we present a TF-engineering approach to mitigate the transcriptional bottlenecks of recombinant proteins. This includes: (i) identification of cAMP response element binding protein (CREB) as a candidate TF by searching for TFs enriched in the cytomegalovirus (CMV) promoter-driven high-producing recombinant Chinese hamster ovary (rCHO) cell lines via transcriptome analysis, (ii) confirmation of transcriptional limitation of active CREB in rCHO cell lines, and (iii) direct activation of the transgene promoter by expressing constitutively active CREB at non-cytotoxic levels in rCHO cell lines. With the expression of constitutively active VP16-CREB, the production of therapeutic proteins, such as monoclonal antibody and etanercept, in CMV promoter-driven rCHO cell lines was increased up to 3.9-fold. VP16-CREB was also used successfully with synthetic promoters containing cAMP response elements. Taken together, this strategy to introduce constitutively active TFs into cells is a useful means of overcoming the transcriptional limitations in recombinant mammalian cells.

Collapsin response mediator protein 4 enhances the radiosensitivity of colon cancer cells through calcium­mediated cell signaling.

Radiation therapy is an effective treatment against various types of cancer, but some radiation­resistant cancer cells remain a major therapeutic obstacle; thus, understanding radiation resistance mechanisms is essential for cancer treatment. In this study, we established radiation­resistant colon cancer cell lines and examined the radiation­induced genetic changes associated with radiation resistance. Using RNA­sequencing analysis, collapsin response mediator protein 4 (CRMP4) was identified as the candidate gene associated with radiation sensitivity. When cells were exposed to radiation, intracellular Ca2+ influx, collapse of mitochondrial membrane potential, and cytochrome c release into the cytosol were increased, followed by apoptosis induction. Radiation treatment­ or Ca2+ ionophore A23187­induced apoptosis was significantly inhibited in CRMP4­deficient cells, including radiation­resistant or CRMP4­shRNA cell lines. Furthermore, treatment of CRMP4­deficient cells with low levels (<5 µM) of BAPTA­AM, a Ca2+ chelator, resulted in radiation resistance. Conversely, Ca2+ deficiency induced by a high BAPTA­AM concentration (>10 µM) resulted in higher cell death in the CRMP4­depleted cells compared to CRMP4­expressing control cells. Our results suggest that CRMP4 plays an important role in Ca2+­mediated cell death pathways under radiation exposure and that CRMP4 may be a therapeutical target for colon cancer treatment.

Cullin 3/KCTD5 Promotes the Ubiqutination of Rho Guanine Nucleotide Dissociation Inhibitor 1 and Regulates Its Stability.

Rho guanine nucleotide dissociation inhibitor 1 (RhoGDI1) plays important roles in numerous cellular processes, including cell motility, adhesion, and proliferation, by regulating the activity of Rho GTPases. Its expression is altered in various human cancers and is associated with malignant progression. Here, we show that RhoGDI1 interacts with Cullin 3 (CUL3), a scaffold protein for E3 ubiquitin ligase complexes. Ectopic expression of CUL3 increases the ubiquitination of RhoGDI1. Furthermore, potassium channel tetramerization domain containing 5 (KCTD5) also binds to RhoGDI1 and increases its interaction with CUL3. Ectopic expression of KCTD5 increases the ubiquitination of RhoGDI1, whereas its knockdown by RNA interference has the opposite effect. Depletion of KCTD5 or expression of dominant-negative CUL3 (DN-CUL3) enhances the stability of RhoGDI1. Our findings reveal a previously unknown mechanism for controlling RhoGDI1 degradation that involves a CUL3/KCTD5 ubiquitin ligase complex.

DNA Replication and Sister Chromatid Cohesion 1 (DSCC1) of the Replication Factor Complex CTF18-RFC is Critical for Colon Cancer Cell Growth.

DNA replication and sister chromatid cohesion 1 (DSCC1) combines with chromosome transmission-fidelity protein 18 (CTF18) to form a CTF18-DSCC1-CTF8 (CTF18-1-8) module, which in combination with CTF18-replication factor C (RFC) acts as a proliferating cell nuclear antigen (PCNA) loader during DNA replication-associated processes. It was found that DSCC1 was overexpressed in tumor tissues from patients with colon cancer and that the survival probability of patients with colon cancer was lower when the expression of cytosolic DSCC1 was higher in tumor regions (P=0.047). By using DSCC1- or CTF18-knockdown cell lines (HCT116-shDSCC1 or HCT116-shCTF18, respectively), it was confirmed that DSCC1-knockdown inhibits cell proliferation and invasion, but that CTF18-knockdown does not. Tumors in mice xenografted with shDSCC1 cells were significantly smaller compared with those in mice in the mock group or those xenografted with shCTF18 cells. The shDSCC1 cells were highly sensitive to γ-irradiation and other DNA replication inhibitory treatments, resulting in low cell viability. The present results suggested that DSCC1 is the most important component in the CTF18-1-8 module for CTF18-RFC and is highly relevant to the growth and metastasis of colon cancer cells, and, therefore, it may be a potential therapeutic target for colon cancer treatment.

Regulation of Rho GTPases by RhoGDIs in Human Cancers.

Rho GDP dissociation inhibitors (RhoGDIs) play important roles in various cellular processes, including cell migration, adhesion, and proliferation, by regulating the functions of the Rho GTPase family. Dissociation of Rho GTPases from RhoGDIs is necessary for their spatiotemporal activation and is dynamically regulated by several mechanisms, such as phosphorylation, sumoylation, and protein interaction. The expression of RhoGDIs has changed in many human cancers and become associated with the malignant phenotype, including migration, invasion, metastasis, and resistance to anticancer agents. Here, we review how RhoGDIs control the function of Rho GTPases by regulating their spatiotemporal activity and describe the regulatory mechanisms of the dissociation of Rho GTPases from RhoGDIs. We also discuss the role of RhoGDIs in cancer progression and their potential uses for therapeutic intervention.

Morin Hydrate Reverses Cisplatin Resistance by Impairing PARP1/HMGB1-Dependent Autophagy in Hepatocellular Carcinoma.

Chemoresistance is a major obstacle that limits the benefits of cisplatin-based chemotherapy in various cancers, including hepatocellular carcinoma. De-regulation of the poly(ADP-ribose) polymerase 1 (PARP1)/high-mobility group box 1 (HMGB1) signaling pathway has been proposed as an important mechanism involved in cisplatin-resistance. In this study, we investigated therapeutic potential of a natural flavonoid Morin hydrate against cisplatin-induced toxicity using the HepG2DR multi-drug resistant cell line, which is derived from the HepG2 human hepatocellular carcinoma cell line. HepG2DR cells were exposed to cisplatin and Morin hydrate alone or together after which autophagy and apoptotic signaling pathways were monitored by fluorometric assay and Western blot analysis. Xenograft mouse models were performed to confirm the in vitro effect of Morin hydrate. PARP1 was hyper activated in cisplatin-resistant HepG2DR cells. Cisplatin-induced PARP1 activation resulted in chemoresistance via increased autophagy. The cisplatin/Morin hydrate combination was effective in the reversal of the HepG2DR cell resistance via suppression of PARP1-mediated autophagy by regulating the HMGB1 and microtubule-associated protein 1A/1B light chain 3B (LC3) I/II. Moreover, PARP1 inhibition by 4-amino-1,8-naphthalimide or autophagy inhibition by a knockdown of the autophagy-related 5 (Atg5) gene resulted in sensitizing the HepG2DR cells to cisplatin (CP) through activation of the c-Jun N-terminal kinase (JNK) pathway. In a mouse xenograft model, the treatment of cisplatin with Morin hydrate reversed the increased expression of PARP and HMGB1 and significantly suppressed tumor growth. These findings indicate dysregulated expression of PARP1 confers cisplatin-resistance via autophagy activation in HepG2DR cells. Morin hydrate inhibits cisplatin-mediated autophagy induction, resulting in increased susceptibility of HepG2DR cells to cisplatin cytotoxicity. The combination of Morin hydrate with cisplatin may be a promising therapeutic strategy to enhance the efficacy of conventional chemotherapeutic drugs.

Efficient Interleukin-21 Production by Optimization of Codon and Signal Peptide in Chinese Hamster Ovarian Cells.

Interleukin-21 is a common γ-chain cytokine that controls the immune responses of B cells, T cells, and natural killer cells. Targeting IL-21 to strengthen the immune system is promising for the development of vaccines as well as anti-infection and anti-tumor therapies. However, the practical application of IL-21 is limited by the high production cost. In this study, we improved IL-21 production by codon optimization and selection of appropriate signal peptide in CHO-K1 cells. Codon-optimized or non-optimized human IL-21 was stably transfected into CHO-K1 cells. IL-21 expression was 10-fold higher for codon-optimized than non-optimized IL-21. We fused five different signal peptides to codon-optimized mature IL-21 and evaluated their effect on IL-21 production. The best result (a 3-fold increase) was obtained using a signal peptide derived from human azurocidin. Furthermore, codon-optimized IL-21 containing the azurocidin signal peptide promoted IFN-γ secretion and STAT3 phosphorylation in NK-92 cells similar to codon-optimized IL-21 containing original signal peptide. Collectively, these results indicate that codon optimization and azurocidin signal peptides provide an efficient approach for the high-level production of IL-21 as a biopharmaceutical.

RhoGDI2 promotes epithelial-mesenchymal transition via induction of Snail in gastric cancer cells.

Rho GDP dissociation inhibitor 2 (RhoGDI2) expression correlates with tumor growth, metastasis, and chemoresistance in gastric cancer. Here, we show that RhoGDI2 functions in the epithelial-mesenchymal transition (EMT), which is responsible for invasiveness during tumor progression. This tumorigenic activity is associated with repression of E-cadherin by RhoGDI2 via upregulation of Snail. Overexpression of RhoGDI2 induced phenotypic changes consistent with EMT in gastric cancer cells, including abnormal epithelial cell morphology, fibroblast-like properties, and reduced intercellular adhesion. RhoGDI2 overexpression also resulted in decreased expression of the epithelial markers E-cadherin and ß-catenin and increased expression of the mesenchymal markers vimentin and fibronectin. Importantly, RhoGDI2 overexpression also stimulated the expression of Snail, a repressor of E-cadherin and inducer of EMT, but not other family members such as Slug or Twist. RNA interference-mediated knockdown of Snail expression suppressed RhoGDI2-induced EMT and invasion, confirming that the effect was Snail-specific. These results indicate that RhoGDI2 plays a critical role in tumor progression in gastric cancer through induction of EMT. Targeting RhoGDI2 may thus be a useful strategy to inhibit gastric cancer cell invasion and metastasis.

VEGF-C mediates RhoGDI2-induced gastric cancer cell metastasis and cisplatin resistance.

Rho GDP dissociation inhibitor 2 (RhoGDI2) expression is correlated with tumor growth, metastasis and chemoresistance in gastric cancer. However, the mechanisms by which RhoGDI2 promotes tumor cell survival and metastasis remain unclear. In this study, we clearly demonstrate that RhoGDI2 upregulates VEGF-C expression and RhoGDI2 expression is positively correlated with VEGF-C expression in human gastric tumor tissues as well as parental gastric cancer cell lines. VEGF-C depletion suppressed RhoGDI2-induced gastric cancer metastasis and sensitized RhoGDI2-overexpressing cells to cisplatin-induced apoptosis in vitro and in vivo. Secreted VEGF-C enhanced gastric cancer cell invasion and conferred cisplatin resistance to RhoGDI2-overexpressing cells. We also show that RhoGDI2 positively regulates Rac1 activity in gastric cancer cells. Inhibition of Rac1 expression suppressed RhoGDI2-induced VEGF-C expression, and this inhibition was associated with decreased invasiveness and increased sensitivity to cisplatin in RhoGDI2-overexpressing cells. Our results indicate that RhoGDI2 might be a potential therapeutic target for simultaneously reducing metastasis risk and enhancing chemotherapy efficacy in gastric cancer.

14-3-3σ attenuates RhoGDI2-induced cisplatin resistance through activation of Erk and p38 in gastric cancer cells.

Rho GDP dissociation inhibitor 2 (RhoGDI2) promotes tumor growth and malignant progression and enhances chemoresistance of gastric cancer. Recently, we noted an inverse correlation between RhoGDI2 and 14-3-3σ expression, which suggests that 14-3-3σ is a target of gastric cancer metastasis and the chemoresistance-promoting effect of RhoGDI2. Herein, we evaluated whether 14-3-3σ is regulated by RhoGDI2 and is functionally important for the RhoGDI2-induced cisplatin resistance of gastric cancer cells. We used highly metastatic and cisplatin-resistant RhoGDI2-overexpressing SNU-484 cells and observed decreased 14-3-3σ mRNA and protein expression. Depletion of 14-3-3σ in SNU-484 control cells enhanced cisplatin resistance, whereas restoration of 14-3-3σ in RhoGDI2-overexpressing SNU-484 cells impaired cisplatin resistance in vitro and in vivo. We also found that the phosphorylation levels of Erk and p38 kinases significantly decreased in RhoGDI2-overexpressing SNU-484 cells and recovered after 14-3-3σ expression, and that decreased activities of these kinases were critical for RhoGDI2-induced cisplatin resistance. In conclusion, 14-3-3σ is a RhoGDI2-regulated gene that appears to be important for suppressing the chemoresistance of gastric cancer cells.

Proteomics-based strategy to delineate the molecular mechanisms of RhoGDI2-induced metastasis and drug resistance in gastric cancer.

Rho GDP dissociation inhibitor 2 (RhoGDI2) was initially identified as a regulator of the Rho family of GTPases. Our recent works suggest that RhoGDI2 promotes tumor growth and malignant progression, as well as enhances chemoresistance in gastric cancer. Here, we delineate the mechanism by which RhoGDI2 promotes gastric cancer cell invasion and chemoresistance using two-dimensional gel electrophoresis (2-DE) on proteins derived from a RhoGDI2-overexpressing SNU-484 human gastric cancer cell line and control cells. Differentially expressed proteins were identified using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF-MS). In total, 47 differential protein spots were identified; 33 were upregulated, and 14 were downregulated by RhoGDI2 overexpression. Upregulation of SAE1, Cathepsin D, Cofilin1, CIAPIN1, and PAK2 proteins was validated by Western blot analysis. Loss-of-function analysis using small interference RNA (siRNA) directed against candidate genes reveals the need for CIAPIN1 and PAK2 in RhoGDI2-induced cancer cell invasion and Cathepsin D and PAK2 in RhoGDI2-mediated chemoresistance in gastric cancer cells. These data extend our understanding of the genes that act downstream of RhoGDI2 during the progression of gastric cancer and the acquisition of chemoresistance.

PLCγ is required for RhoGDI2-mediated cisplatin resistance in gastric cancer.

Rho GDP dissociation inhibitor 2 (RhoGDI2) is a regulator of the Rho family GTPases. Recent work from our laboratory suggests that RhoGDI2 expression potentially enhances resistance to cisplatin as well as promotes tumor growth and malignant progression in gastric cancer. In this study, we demonstrate that phospholipase C-gamma (PLCγ) is required for RhoGDI2-mediated cisplatin resistance and cancer cell invasion in gastric cancer. The levels of phosphorylated PLCγ are markedly enhanced in RhoGDI2-overexpressing SNU-484 cells and, by contrast, repressed in RhoGDI2-depleted MKN-28 cells. Depletion of PLCγ expression or inhibition of its activity not only significantly increases cisplatin-induced apoptosis but also suppresses the invasive ability of RhoGDI2-overexpressing SNU-484 cells. Taken together, our results suggest that PLCγ plays a key role in RhoGDI2-mediated cisplatin resistance and cell invasion in gastric cancer cells.

RhoGDI2 confers gastric cancer cells resistance against cisplatin-induced apoptosis by upregulation of Bcl-2 expression.

Rho GDP dissociation inhibitor (RhoGDI)2 has been identified as a regulator of Rho family GTPase. Recently, we suggested that RhoGDI2 could promote tumor growth and malignant progression in gastric cancer. In this study, we demonstrate that RhoGDI2 contributes to another important feature of aggressive cancers, i.e., resistance to chemotherapeutic agents such as cisplatin. Forced expression of RhoGDI2 attenuated cisplatin-induced apoptosis, whereas RhoGDI2 depletion showed opposite effects in vitro. Moreover, the increased anti-apoptotic effect of RhoGDI2 on cisplatin was further validated in RhoGDI2-overexpressing SNU-484 xenograft model in nude mice. Furthermore, we identified Bcl-2 as a major determinant of RhoGDI2-mediated cisplatin resistance in gastric cancer cells. Depletion of Bcl-2 expression significantly increased cisplatin-induced apoptosis in RhoGDI2-overexpressing gastric cancer cells, whereas overexpression of Bcl-2 blocked cisplatin-induced apoptosis in RhoGDI2-depleted gastric cancer cells. Overall, these findings establish RhoGDI2 as an important therapeutic target for simultaneously enhancing chemotherapy efficacy and reducing metastasis risk in gastric cancer.

Gadd45b mediates Fas-induced apoptosis by enhancing the interaction between p38 and retinoblastoma tumor suppressor.

Gadd45b has been known as a positive mediator of apoptosis induced by certain cytokines and oncogenes. Here, we identified Gadd45b as an effector of Fas-induced apoptosis and found that p38-mediated Rb hyperphosphorylation is one of the mechanisms of Fas-induced apoptosis in murine hepatocyte AML12 cells. Gadd45b has been shown to activate p38 through its physical interaction with MTK1 and induce apoptosis. However, in this study, we have showed that the function of Gadd45b during Fas-induced apoptosis in AML12 cells is different from that reported in previous studies. Depletion of Gadd45b expression did not inhibit the phosphorylation of p38, but it suppressed p38-mediated Rb phosphorylation and apoptosis in response to Fas stimulation by reducing the interaction between p38 and Rb. Ectopic expression of Gadd45b was sufficient to enhance this interaction. These findings suggest that Gadd45b mediates p38-induced Rb phosphorylation by enhancing the interaction between p38 and Rb during Fas-induced apoptosis in murine hepatocytes.

RhoGDI2 as a therapeutic target in cancer.

IMPORTANCE OF THE FIELD: Rho GDP dissociation inhibitor 2 (RhoGDI2) has been identified as a regulator of Rho GTPases that play important roles in the development of numerous aspects of the malignant phenotype, including cell cycle progression, resistance to apoptotic stimuli, neovascularization, tumor cell motility, invasiveness, and metastasis. Although RhoGDI2 has been known to be expressed only in hematopoietic tissues, recent studies suggest that this protein is also aberrantly expressed in several human cancers and contributes to aggressive phenotypes, such as invasion and metastasis. Hence, RhoGDI2 appears to be a target of interest for therapeutic manipulation. AREAS COVERED IN THIS REVIEW: Here, we summarize the role of RhoGDI2 in human cancers, specifically metastasis-related processes, and discuss its potential as a therapeutic target. WHAT THE READER WILL GAIN: RhoGDI2 modulates the invasiveness and metastatic ability of cancer cells through regulation of Rac1 activity. TAKE HOME MESSAGE: RhoGDI2 may be a useful marker for tumor progression in human cancers, and interruption of the RhoGDI2-mediated cancer cell invasion and metastasis by an interfacial inhibitor may be a powerful therapeutic approach to cancer.

RhoGDI2 expression is associated with tumor growth and malignant progression of gastric cancer.

PURPOSE: Rho GDP dissociation inhibitor 2 (RhoGDI2) has been identified as a regulator of Rho family GTPase. However, there is currently no direct evidence suggesting whether RhoGDI2 activates or inhibits Rho family GTPase in vivo (and which type), and the role of RhoGDI2 in tumor remains controversial. Here, we assessed the effects of RhoGDI2 expression on gastric tumor growth and metastasis progression. EXPERIMENTAL DESIGN: Proteomic analysis was done to investigate the tumor-specific protein expression in gastric cancer and RhoGDI2 was selected for further study. Immunohistochemistry was used to detect RhoGDI2 expression in clinical samples of primary gastric tumor tissues which have different pathologic stages. Gain-of-function and loss-of-function approaches were done to examine the malignant phenotypes of the RhoGDI2-expressing or RhoGDI2-depleting cells. RESULTS: RhoGDI2 expression was correlated positively with tumor progression and metastasis potential in human gastric tumor tissues, as well as cell lines. The forced expression of RhoGDI2 caused a significant increase in gastric cancer cell invasion in vitro, and tumor growth, angiogenesis, and metastasis in vivo, whereas RhoGDI2 depletion evidenced opposite effects. CONCLUSION: Our findings indicate that RhoGDI2 is involved in gastric tumor growth and metastasis, and that RhoGDI2 may be a useful marker for tumor progression of human gastric cancer.

Upregulation and secretion of macrophage inhibitory cytokine-1 (MIC-1) in gastric cancers.

BACKGROUND: Macrophage inhibitory cytokine-1 (MIC-1), a distant member of the transforming growth factor (TGF)-beta superfamily, has been reported to be upregulated and secreted from several cancers. We examined MIC-1 expression and secretion in gastric cancers. METHODS: MIC-1 mRNA and protein levels in cancer tissues and cell lines were analyzed by RT-PCR and Western blot. MIC-1 expression in cancer tissues and its secretion in serum were analyzed using immunohistochemistry and ELISA. RESULTS: MIC-1 was significantly upregulated in gastric cancer tissues and cell lines. MIC-1 was secreted from gastric SNU620 cells and its levels in the serum of cancer patients were 10-fold higher than those of healthy controls. In addition, the staining of MIC-1 expression was strongly increased in metastatic gastric cancers. CONCLUSIONS: MIC-1 was obviously overexpressed in gastric cancers and MIC-1 secretion into blood may be useful for the prediction of gastric cancer progression.

A quantitative analysis of N-myc downstream regulated gene 2 (NDRG 2) in human tissues and cell lysates by reverse-phase protein microarray.

BACKGROUND: N-myc downstream regulated gene 2 (NDRG2) belongs to the NDRG family, which is comprised of 4 members, NDRG1-4. Recently, NDRG2 was reported as a new candidate for a tumor suppressor gene. We developed a reverse-phase protein microarray assay to access NDRG2 levels in human tissue specimens and cell lines. METHODS: We synthesized recombinant NDRG2 protein and produced monoclonal antibodies (mAb) to the NDRG2 protein. We selected 2 hybridomas producing mAb that specifically recognize the NDRG2 protein. To determine the NDRG2 concentration, the samples of serially-diluted NDRG2 protein, cell lysate, or tissue lysate were spotted onto a nitrocellulose membrane-coated slide glass and allowed to react with the mAb to the NDRG2 protein. The reaction was followed by additional incubation with biotin-linked anti-mouse IgG and horseradish peroxidase (HRP)-conjugated streptavidin, subsequently. The addition of dimethylaminobenzidine induced color development, which was measured using the GenePix program. We determined the NDRG2 concentration in various tissue specimens and cell lines using the new protein microarray technique. RESULTS: The dose-response relationship between NDRG2 and color intensity showed linearity in a range 0-10 ng/ml and a sensitivity of 50 pg/ml. The NDRG2 concentrations in the liver tissue lysates of patients with hepatocellular carcinoma (52.0+21.5 ng/mg) were significantly diminished as compared with those in the normal liver tissues (549.6+94.6 ng/mg). The results of the assay showed good agreement with those of Western blot analysis. CONCLUSIONS: The protein microarray is a highly sensitive and accurate method, and can adopted to assess specific proteins in human tissues or cell lines, particularly in the field of cancer and pathological research.

Snail is required for transforming growth factor-beta-induced epithelial-mesenchymal transition by activating PI3 kinase/Akt signal pathway.

Lens epithelial cells undergo epithelial-mesenchymal transition (EMT) after injury as in cataract extraction, leading to fibrosis of the lens capsule. We have previously shown that EMT of primary lens epithelial cells in vitro depends on TGF-beta expression and more specifically, on signaling via Smad3. In this report, we suggest phosphatidylinositol 3-OH kinase (PI3K)/Akt signaling is also necessary for TGF-beta-induced EMT in lens epithelial cells by showing that LY294002, an inhibitor of the p110 catalytic subunit of PI3K, blocked the expression of alpha-smooth muscle actin (alpha-SMA) and morphological changes. We also identify Snail as an effector of TGF-beta-induced EMT. Snail has been shown to be a mediator of EMT during metastasis of cancer. We show that Snail is an immediate-early response gene for TGF-beta and the proximal Snail promoter is activated by TGF-beta through the action of Smad2, 3, and 4. We show that antisense inhibition of Snail expression blocks TGF-beta-induced EMT and furthermore Akt activation. All of these findings suggest that Snail participates in TGF-beta-induced EMT by acting upstream of Akt activation.

Up-regulation of Bfl-1/A1 via NF-kappaB activation in cisplatin-resistant human bladder cancer cell line.

The potent anti-cancer agent cis-diamminedichloroplatinum (II) (cisplatin) is currently used for treating bladder cancer. However, clinical use of this drug for long periods is often limited because of the appearance of cisplatin-resistant bladder tumor cells. We employed the method of a differential display reverse transcriptase polymerase chain reaction to identify the differentially expressed genes in the parental human bladder cancer cell line, T24 and three cisplatin-resistant cell lines. We report here that cisplatin-resistant cell lines overexpress Bcl-2 family protein Bcl-2-related gene expressed in fetal liver (Bfl-1)/A1 as compared with their parental cell. Cisplatin and gamma-irradiation induced expression of Bfl-1/A1 in T24R2 cells but not in T24 cells. Among Bcl-2 family members, Bfl-1/A1 showed the most significant alteration of the expression level in resistant cells. The nuclear translocation of nuclear factor-kappaB (NF-kappaB) by cisplatin and gamma-irradiation selectively occurred in T24R2 cells. Mitochondrial depolarization and cell death by cisplatin were also prevented in T24R2 cells. Moreover, Bfl-1/A1 inhibited cisplatin- and TNF-alpha-induced apoptosis in BOSC23 cells. Our findings suggest that the induction of Bfl-1/A1 by NF-kappaB may be important in controlling resistance to cisplatin responses in bladder tumor cells.