Low inducible expression of p21Cip1 confers resistance to paclitaxel in BRAF mutant melanoma cells with acquired resistance to BRAF inhibitor.

Jang, Gun-Hee,Kim, Na-Yeon,Lee, Michael Dr. W. Junk B. V. Publishers ; Kluwer Academic Pub 2015 MOLECULAR AND CELLULAR BIOCHEMISTRY - Vol.406 No.1

<P>The therapeutic efficacy of oncogenic BRAF inhibitor is limited by the onset of acquired resistance. In this study, we investigated the potential therapeutic effects of the mitotic inhibitor paclitaxel on three melanoma cell lines with differing sensitivity to the BRAF inhibitor. Of the two BRAF inhibitor-resistant cell lines, A375P/Mdr cells harboring the BRAF V600E mutant were resistant and the wild-type BRAF SK-MEL-2 cells were sensitive to paclitaxel. In particular, paclitaxel caused the growth inhibition of SK-MEL-2 cells to a much greater extent than it caused growth inhibition of A375P cells. Paclitaxel exhibited no significant effect on the phosphorylation of MEK-ERK in any cell lines tested, regardless of both the BRAF mutation and the drug resistance, implying that paclitaxel activity is independent of MEK-ERK inhibition. In A375P cells, paclitaxel treatment resulted in a marked emergence of apoptotic cells after mitotic arrest, concomitant with a remarkable induction of p21(Cip1). However, paclitaxel only moderately increased the levels of p21(Cip1) in A375P/Mdr cells, which exhibited a strong resistance to paclitaxel. The p21(Cip1) overexpression partially conferred paclitaxel sensitivity to A375P/Mdr cells. Interestingly, we found an extremely low background expression level of p21(Cip1) in SK-MEL-2 cells lacking normal p53 function, which caused much greater G2/M arrest than that seen in A375P cells. Taken together, these results suggest that paclitaxel may be an effective anticancer agent through regulating the expression of p21(Cip1) for the treatment of BRAF mutant melanoma cells resistant to BRAF inhibitors.</P>

Radicicol, an inhibitor of Hsp90, enhances TRAIL-induced apoptosis in human epithelial ovarian carcinoma cells by promoting activation of apoptosis-related proteins.

Kim, Yun Jeong,Lee, Seon Ae,Myung, Soon Chul,Kim, Wonyong,Lee, Chung Soo Dr. W. Junk B. V. Publishers ; Kluwer Academic Pub 2012 MOLECULAR AND CELLULAR BIOCHEMISTRY - Vol.359 No.1

<P>Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in various cancer cells. Hsp90 is known to be involved in cell survival and growth in tumor cells. Nevertheless, Hsp90 inhibitors exhibit a variable effect on the cytotoxicity of anticancer drugs. Furthermore, the combined effect of Hsp90 inhibitors on TRAIL-induced apoptosis in epithelial ovarian cancer cells has not been determined. To assess the ability of an inhibitor of Hsp90 inhibitor radicicol to promote apoptosis, we investigated the effect of radicicol on TRAIL-induced apoptosis in the human epithelial ovarian carcinoma cell lines OVCAR-3 and SK-OV-3. TRAIL induced a decrease in Bid, Bcl-2, Bcl-xL, and survivin protein levels, increase in Bax levels, loss of the mitochondrial transmembrane potential, cytochrome c release, activation of caspases (-8, -9, and -3), cleavage of PARP-1 and an increase in the tumor suppressor p53 levels. Radicicol enhanced TRAIL-induced apoptosis-related protein activation, nuclear damage and cell death. These results suggest that radicicol may potentiate the apoptotic effect of TRAIL on ovarian carcinoma cell lines by increasing the activation of the caspase-8- and Bid-dependent pathway and the mitochondria-mediated apoptotic pathway, leading to caspase activation. Radicicol may confer a benefit in the TRAIL treatment of epithelial ovarian adenocarcinoma.</P>

Involvement of the TLR4 (Toll-like receptor4) signaling pathway in palmitate-induced INS-1 beta cell death.

Lee, Sung-Mi,Choi, Sung-E,Lee, Ji-Hyun,Lee, Jung-Jin,Jung, Ik-Rak,Lee, Soo-Jin,Lee, Kwan-Woo,Kang, Yup Dr. W. Junk B. V. Publishers ; Kluwer Academic Pub 2011 MOLECULAR AND CELLULAR BIOCHEMISTRY - Vol.354 No.1

<P>Fatty acid-induced cytotoxicity is believed to recapitulate lipotoxicity seen in obese type-2 diabetes, and, thus, contribute to beta cell loss in the disease. These studies were initiated to determine whether the Toll-like receptor (TLR) signaling pathway was involved in palmitate-induced beta cell death. Treatment of INS-1 beta cells with palmitate enhanced interaction between TLR and myeloid differentiation factor88 (MyD88). Concomitant with TLR/MyD88 interaction, the level of phospho-C-Jun N-terminal kinase (phospho-JNK) showed an increase; however, the level of inhibitory factor kappa B alpha (IκBα) showed a decrease. Gene knockdown of TLR4 prevented palmitate-induced INS-1 cell death, while knockdown of TLR2 did not. In addition, gene knockdown of TLR4 prevented palmitate-induced increase of phospho-JNK and decrease of IκBα. JNK inhibitor SP60125 significantly protected against palmitate-induced INS-1 cell death, while IκB kinase (IKK) inhibitor acetylsalicylate did not. These data suggest involvement of JNK activation through the TLR4 signaling pathway in palmitate-induced INS-1 beta cell death.</P>

RC3/neurogranin negatively regulates extracellular signal-regulated kinase pathway through its interaction with Ras.

Ryoo, Kanghyun,Hwang, Sang-Gil,Kim, Kwang Je,Choi, Eui-Ju Dr. W. Junk B. V. Publishers ; Kluwer Academic Pub 2015 MOLECULAR AND CELLULAR BIOCHEMISTRY - Vol.402 No.1

<P>RC3/neurogranin is a postsynaptic protein and plays pivotal roles in spatial learning and emotional anxiety as well as synaptic plasticity. The expression level of RC3 is dynamically changed during developmental stages, but the function of RC3 in brain development is not well understood yet. Neurotrophins interact with tropomyosin-related kinase receptors to activate Ras-extracellular signal-regulated kinase (ERK) pathway and can also induce neuronal differentiation. In this study, we demonstrate that RC3 inhibits Ras-ERK pathway by interaction with Ras and controls neurite outgrowth induced by neurotrophins. In PC12 cells, RC3 inhibits nerve growth factor (NGF)-induced activation of Ras and thereby ERK1/2 signaling cascade as well as neurite outgrowth induced by NGF. We found Ras is the target of the inhibitory function of RC3, because RC3 interacts with Ras and suppresses the elevated affinity of Ras to Ras-binding domain of Raf-1. Meanwhile, already activated Raf-1 by Ras activity is not affected by RC3. Furthermore, depletion of RC3 by RNA interference drastically enhances the stimulation of ERK1/2 and neurite outgrowth induced by brain-derived neurotrophic factor in hippocampal neurons. These findings suggest that RC3 is a novel natural inhibitor of Ras-ERK1/2 signaling axis, leading to negatively regulate neuronal differentiation induced by neurotrophins.</P>

Biogenesis of Epstein-Barr virus microRNAs.

Kim, Do Nyun,Lee, Suk Kyeong Dr. W. Junk B. V. Publishers ; Kluwer Academic Pub 2012 MOLECULAR AND CELLULAR BIOCHEMISTRY - Vol.365 No.1

<P>Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus implicated in lymphomas, such as Burkitt's lymphoma, Hodgkin's lymphoma, and NK/T cell lymphoma. MicroRNAs (miRNAs) are 19-25 nucleotide long single-stranded RNAs involved in post-transcriptional gene regulation. miRNAs are mainly transcribed by RNA polymerase II (pol II) to have stem-loop structures and subsequently processed by Drosha and Dicer. EBV miRNAs are expressed in B cells, nasopharyngeal carcinoma cells, and gastric carcinoma cells infected with EBV. EBV miRNAs can be divided into two groups: BHRF1 miRNAs and BART miRNAs. In this study, we investigated the biogenesis of EBV miRNAs. Treatment of the SNU-719 EBV-positive gastric cancer cell line with 관-amanitin at a concentration that selectively inhibits RNA polymerase II activity decreased the expression levels of BART miRNAs. The expression levels of BART miRNAs were also reduced by RNA interference targeting Drosha and Dicer. Two of each C/EBP관 and c-Myc binding sites are located upstream of the proposed initiation sites for primary BART miRNA transcripts. Knockdown of C/EBP관 but not c-Myc using siRNAs reduced BART miRNA expression by 25-55% compared with the control. These results suggest that BART miRNAs are transcribed by pol II and undergo a similar biogenesis process with cellular miRNAs.</P>

Cytotoxic effect of gambogic acid on SH-SY5Y neuroblastoma cells is mediated by intrinsic caspase-dependent signaling pathway.

Rahman, Md Ataur,Kim, Nam-Ho,Huh, Sung-Oh Dr. W. Junk B. V. Publishers ; Kluwer Academic Pub 2013 MOLECULAR AND CELLULAR BIOCHEMISTRY - Vol.377 No.1

<P>Gambogic acid (GA) is the dry resin of Garcinia hanburyi (Guttiferae) with potent anti-tumor activity, various bioactivities, including detoxification, homeostasis, anti-inflammatory, and parasiticide, whereas the effect of this natural compound on cancer cells has not been clearly clarified. Here, we examined cellular cytotoxicity by cell viability assay and DNA fragmentation by DNA-ladder assay. Activation of different protein expressions were detected by western blot analyses. We first demonstrated that GA reduces the human SH-SY5Y neuroblastoma cell viability with IC50 of 1.28 μM at 6 h which has less toxicity in fibroblast cells. However, lower concentration GA significantly downregulated the expression of anti-apoptotic protein including Bcl-2, Bcl-xL, and Mcl-1, which also dramatically activated cleaved caspase-9 and -3 in a dose- and time-dependent manner. Consequently, GA-induced cytotoxicity was not mediated by the Fas/FasL and PI3 K/AKT/GSK-3β signaling pathway. In addition, GA-induced cells showed damage morphology which had become cell rounding, neurite retraction, membrane blebbing and shrunken in a dose- and time-dependent manner that clearly indicates this morphological change might be due to the process of apoptosis which shows fragmented DNA. Therefore, the findings presented in this study demonstrate that apoptotic effects of GA on SH-SY5Y cells are mediated by intrinsic mitochondrion-dependent caspase pathway which suggests this natural compound might be effective as an anti-cancer agent for neuroblastoma malignancies.</P>

Benzyl isothiocyanate inhibits basal and hepatocyte growth factor-stimulated migration of breast cancer cells.

Kim, Eun Ji,Eom, Soon Ju,Hong, Ji Eun,Lee, Jae-Yong,Choi, Myung-Sook,Park, Jung Han Yoon Dr. W. Junk B. V. Publishers ; Kluwer Academic Pub 2012 MOLECULAR AND CELLULAR BIOCHEMISTRY - Vol.359 No.1

<P>Benzyl isothiocyanate (BITC), which is found in cruciferous vegetables, has been shown to have anti-carcinogenic properties. Hepatocyte growth factor (HGF) has the ability to stimulate dissociation, migration, and invasion in various tumor cells, and abnormally increased expressions of HGF and its transmembrane tyrosine kinase receptor, c-Met, have previously been detected in human breast cancer, and are associated with high tumor grade and poor prognosis. In this study, in order to assess the mechanisms relevant to the BITC-induced regulation of breast cancer cell migration and invasion, MDA-MB-231 human breast cancer cells and 4T1 murine mammary carcinoma cells were cultured in the presence of 0-4?μmol/l BITC with or without 10?μg/l of HGF. BITC inhibited both the basal and HGF-induced migration of MDA-MB-231 and 4T1 cells in a dose-dependent manner. In MDA-MB-231 cells, BITC reduced both basal and HGF-induced secretion and activity of urokinase-type plasminogen activator (uPA). In addition, BITC increased the protein levels of plasminogen activator inhibitor-1. HGF stimulated c-Met and Akt phosphorylation, but did not affect the phosphorylation of extracellular signal-regulated kinase-1/2 or stress-activated protein/c-jun N-terminal kinase. BITC suppressed NF-κB activity and reduced the HGF-induced phosphorylation of c-Met and Akt in a dose-dependent manner. LY294002, a specific Akt inhibitor, reduced both basal and HGF-induced uPA secretion and migration of MDA-MB-231 cells. In this study, we demonstrated that BITC profoundly inhibits the migration and invasion of MDA-MB-231 cells, which is associated with reduced uPA activity, and also that these phenomena are accompanied by the suppression of Akt signaling.</P>

Effect of the modulation of leucine zipper tumor suppressor 2 expression on proliferation of various cancer cells functions as a tumor suppressor.

Kim, Jong Myung,Song, Ji Sun,Cho, Hyun Hwa,Shin, Keun Koo,Bae, Yong Chan,Lee, Byung Ju,Jung, Jin Sup Dr. W. Junk B. V. Publishers ; Kluwer Academic Pub 2011 MOLECULAR AND CELLULAR BIOCHEMISTRY - Vol.346 No.1

<P>β-catenin is a component of the adhesion complex linking cadherin and actin cytoskeleton, as well as a major mediator of the Wnt pathway, which is a critical signal cascade regulating embryonic development, cell polarity, carcinogenesis, and stem cell function. NF-κB functions as a key regulator of immune responses and apoptosis, and mutations in NF-κB signaling can lead to immune diseases and cancers. We previously showed that NF-κB-mediated modulation of β-catenin/Tcf signaling is mediated by leucine zipper tumor suppressor 2 (Lzts2) and that lzts2 expression is differentially regulated in various cancer cells. Its functional significances, however, are poorly understood. We showed that NF-κB-induced modulation of β-catenin/Tcf pathway is regulated by lzts2 expression in mesenchymal stem cells (MSCs) and several cancer cells, and that NF-κB-induced lzts2 expression is differentially regulated among cancer cell types. Here, using a promoter-reporter assay and EMSA, we demonstrate that NF-κB regulates lzts2 transcription by directly binding to the lzts2 promoter, and that NF-κB-induced lzts2 transcription differs by cell types. Modulation of lzts2 expression by lentiviral techniques affected proliferation and tumorigenicity of several cancer cell lines such as breast, colon, prostate cancer, and glioma, but did not affect cisplatin sensitivity or cell migration. Our data indicate that lzts2 expression is transcriptionally regulated by NF-κB activities, and the modulation of lzts2 expression affects cell proliferation and tumor growth through the Wnt/β-catenin pathway in various cancer cell lines.</P>

Molecular characterization and transcriptional analysis of the olive flounder (Paralichthys olivaceus) YGHL1 gene in response to hypoxia and infection.

Kim, Young-Ok,Park, Eun-Mi,Moon, Ji Young,Kong, Hee Jeong,Nam, Bo-Hye,Kim, Woo-Jin,Lee, Jeong-Ho,Kim, Kyung-Kil,Lee, Sang-Jun Dr. W. Junk B. V. Publishers ; Kluwer Academic Pub 2011 MOLECULAR AND CELLULAR BIOCHEMISTRY - Vol.357 No.1

<P>The hypoxia-inducible gene 1, YGHL1 from olive flounder (Paralichthys olivaceus) (fYGHL1) was cloned, and its structural organization and expression profiles were determined. A 1,400?kb full-length cDNA encoding a predicted polypeptide of 91 amino acids was sequenced. The fYGHL1 gene comprises three introns, four exons, and several transcriptional elements upstream of the transcriptional start site. The mRNA transcript is expressed in almost all tissues, with high expression in the intestine and brain of normal-conditioned fish, and is expressed constitutively in early developmental stages after hatching. The mRNA expression of fYGHL1 is highly regulated by hypoxia and E. tarda infection. The expression of fYGHL1 mRNA was down regulated in the gill, spleen, intestine, and stomach of flounder under hypoxic conditions, whereas the expression level was increased in flounder embryonic cells treated with the hypoxia-mimic CoCl(2) (a HIF-1 inducer). Pathogen challenge induced fYGHL1 expression in the spleen of juvenile fish. Taken together, these results suggest that fYGHL1 is a hypoxia-related gene with potential roles in the hypoxia response mechanism as well as in defense, immune responses, growth, and regulation of reproduction.</P>

PHB2 interacts with RNF2 and represses CP2c-stimulated transcription.

Lee, Sun-Joo,Choi, Dongwon,Rhim, Hyangshuk,Choo, Hyo-Jung,Ko, Young-Gyu,Kim, Chul Guen,Kang, Seongman Dr. W. Junk B. V. Publishers ; Kluwer Academic Pub 2008 MOLECULAR AND CELLULAR BIOCHEMISTRY - Vol.319 No.1

<P>RNF2, a polycomb group protein, is an important component of PRC complex regulating transcriptional activity. Recently, several RNF2 interacting proteins have been identified. Thus, RNF2 might have multiple activities, depending on its interacting partner proteins. In the present study, using the yeast two-hybrid system, we have found that RNF2 interacts with the PHB2 protein. Luciferase reporter assays showed that RNF2 represses the CP2c-stimulated luciferase activity in a PHB2 dose-dependent manner. Further experiments with RNF2 deletion mutants indicated that RNF2(1-158) is sufficient for both the physical association and functional co-operation with the PHB2 protein. Co-immunoprecipitation experiments revealed that PHB2 and CP2c bind to the N- and C-terminals of RNF2, respectively. Luciferase reporter assays using alpha-globin promoter with CP2-binding elements hinted that RNF2 and PHB2 are involved in the CP2-stimulated expression of the alpha-globin gene. Our study suggests a novel mechanism by which RNF2 and PHB2 modulate the CP2-mediated transcriptional pathway.</P>