Protein kinase CK2 modulation of pyruvate kinase M isoforms augments the Warburg effect in cancer cells.

Protein kinase CK2 is active in cancer cells. Previously, we reported that increased CK2 activity could induce epithelial mesenchymal transition of cancer cells. CK2 also induced epithelial mesenchymal transition in colon cancer cell lines such as HT29 and SW620, and the transitioned cells (CK2α cells) became more proliferative than the controls. We assumed that CK2 could affect cancer cell growth by modulating their energy metabolism. Here, we examined the molecular effects of CK2 on the glucose metabolism of cancer cells. We found that CK2α cells consumed more glucose and produced more lactate than control cells did. An XF glycolysis stress test showed that aerobic glycolysis was augmented up to the cancer cell's maximal glycolytic capacity in CK2α cells. Molecular analysis revealed that pyruvate kinase M1 was downregulated and pyruvate kinase M2 was nuclear localized in CK2α cells. Consequently, the expression and activity of lactate dehydrogenase A (LDHA) were upregulated. Treatment with FX11-a specific LDHA inhibitor-or clustered regularly interspaced short palindromic repeats (CRISPR)-mediated knockout of LDHA inhibited the CK2-driven proliferation of cancer cells. We conclude that CK2 augments the Warburg effect, resulting in increased proliferation of cancer cells.

Zbtb7c is a molecular 'off' and 'on' switch of Mmp gene transcription.

Matrix metalloproteinases (MMPs) are zinc-containing endopeptidases that play roles in cell proliferation, migration, differentiation, angiogenesis, and apoptosis. The expression of MMP gene is tightly regulated and shows cell- and tissue-specific expression patterns. Despite their differential expression, MMP genes have AP-1 (activator protein-1) binding elements within their promoters. Interestingly, c-JUN phosphorylation by cytokine signaling decreased its interaction with NCoR, but increased its interaction with p300, resulting in activation of MMP gene transcription. Here, we found that Zbtb7c (Kr-pok) is a critical component of a transcriptional repressor complex containing c-Jun and NCoR. c-Jun, bound at AP-1, interacts with Zbtb7c, which in turn recruits an NCoR/Hdac3 complex to repress several Mmp (-8, -10, -13, and -16) genes. The molecular interaction between c-Jun and Zbtb7c also prevents phosphorylation of c-Jun by p-Jnk, However, Zbtb7c phosphorylation by p-Jnk (induced by TNFα), and its (Zbtb7c) subsequent degradation by the ubiquitin-mediated proteasomal pathway, leads to c-Jun phosphorylation by p-Jnk. Promoter-bound p-c-Jun then recruits the coactivator p300 to upregulate Mmp gene. Overall, these findings show that Zbtb7c is a key molecule that recruits an NCoR/Hdac3 complex to inhibit phosphorylation of c-Jun, and thereby repress Mmp gene expression.

ZBTB2 increases PDK4 expression by transcriptional repression of RelA/p65.

The NF-κB is found in almost all animal cell types and is involved in a myriad of cellular responses. Aberrant expression of NF-κB has been linked to cancer, inflammatory diseases and improper development. Little is known about transcriptional regulation of the NF-κB family member gene RelA/p65. Sp1 plays a key role in the expression of the RelA/p65 gene. ZBTB2 represses transcription of the gene by inhibiting Sp1 binding to a Sp1-binding GC-box in the RelA/p65 proximal promoter (bp, -31 to -21). Moreover, recent studies revealed that RelA/p65 directly binds to the peroxisome proliferator-activated receptor-γ coactivator1α (PGC1α) to decrease transcriptional activation of the PGC1α target gene PDK4, whose gene product inhibits pyruvate dehydrogenase (PDH), a key regulator of TCA cycle flux. Accordingly, we observed that RelA/p65 repression by ZBTB2 indirectly results in increased PDK4 expression, which inhibits PDH. Consequently, in cells with ectopic ZBTB2, the concentrations of pyruvate and lactate were higher than those in normal cells, indicating changes in glucose metabolism flux favoring glycolysis over the TCA cycle. Knockdown of ZBTB2 in mouse xenografts decreased tumor growth. ZBTB2 may increase cell proliferation by reprogramming glucose metabolic pathways to favor glycolysis by upregulating PDK4 expression via repression of RelA/p65 expression.

Nanoparticulation improves bioavailability of Erlotinib.

OBJECTIVES: Nanoparticulation using fat and supercritical fluid (NUFSTM) is a drug delivery platform technology enabling efficient and effective formulation of poorly soluble drugs. We performed experiments to examine whether NUFS™ could improve poor bioavailability and reduce fed-fasted bioavailability variances of erlotinib (Ert). METHODS: NUFS-Ert was prepared using NUFS™ technology; its physical properties were characterized, and drug release was measured. Furthermore, in vitro and in vivo efficacy tests and pharmacokinetic analysis were performed. RESULTS: NUFS-Ert nanoparticles had an average size of 250 nm and were stable for 2 months at 40 °C, 4 °C, and room temperature. The dissolution rate of NUFS-Ert increased in bio-relevant dissolution media. NUFS-Ert was more potent in inhibiting EGF signaling and in suppressing the proliferation of A549, a human non-small cell lung cancer cell line. Furthermore, A549 xenografts in BALB/c nude mice treated with NUFS-Ert regressed more efficiently than those in the mice treated with vehicle or Tarceva®. In addition, experimental lung metastasis was more efficiently inhibited by NUFS-Ert than by Tarceva®. The relative bioavailability of NUFS-Ert compared with that of Tarceva® was 550% and the ratio of the area under the concentration-time curve (AUC) of fed state to the AUC of fasted state was 1.8 for NUFS-Ert and 5.8 for Tarceva®. CONCLUSIONS: NUFS-Ert could improve poor bioavailability and reduce fed-fasted bioavailability variances of Ert. NUFS-Ert was more efficacious than Tarceva®.

Promyelocytic leukemia zinc finger-retinoic acid receptor α (PLZF-RARα), an oncogenic transcriptional repressor of cyclin-dependent kinase inhibitor 1A (p21WAF/CDKN1A) and tumor protein p53 (TP53) genes.

Promyelocytic leukemia zinc finger-retinoic acid receptor α (PLZF-RARα) is an oncogene transcriptional repressor that is generated by a chromosomal translocation between the PLZF and RARα genes in acute promyelocytic leukemia (APL-type) patients. The molecular interaction between PLZF-RARα and the histone deacetylase corepressor was proposed to be important in leukemogenesis. We found that PLZF-RARα can repress transcription of the p21WAF/CDKN1A gene, which encodes the negative cell cycle regulator p21 by binding to its proximal promoter Sp1-binding GC-boxes 3, 4, 5/6, a retinoic acid response element (RARE), and distal p53-responsive elements (p53REs). PLZF-RARα also acts as a competitive transcriptional repressor of p53, RARα, and Sp1. PLZF-RARα interacts with co-repressors such as mSin3A, NCoR, and SMRT, thereby deacetylating histones Ac-H3 and Ac-H4 at the CDKN1A promoter. PLZF-RARα also interacts with the MBD3-NuRD complex, leading to epigenetic silencing of CDKN1A through DNA methylation. Furthermore, PLZF-RARα represses TP53 and increases p53 protein degradation by ubiquitination, further repressing p21 expression. Resultantly, PLZF-RARα promotes cell proliferation and significantly increases the number of cells in S-phase.

Role of promyelocytic leukemia zinc finger (PLZF) in cell proliferation and cyclin-dependent kinase inhibitor 1A (p21WAF/CDKN1A) gene repression.

Promyelocytic leukemia zinc finger (PLZF) is a transcription repressor that was initially isolated as a fusion protein with retinoic acid receptor α. PLZF is aberrantly overexpressed in various human solid tumors, such as clear cell renal carcinoma, glioblastoma, and seminoma. PLZF causes cellular transformation of NIH3T3 cells and increases cell proliferation in several cell types. PLZF also increases tumor growth in the mouse xenograft tumor model. PLZF may stimulate cell proliferation by controlling expression of the genes of the p53 pathway (ARF, TP53, and CDKN1A). We found that PLZF can directly repress transcription of CDKN1A encoding p21, a negative regulator of cell cycle progression. PLZF binds to the proximal Sp1-binding GC-box 5/6 and the distal p53-responsive elements of the CDKN1A promoter to repress transcription. Interestingly, PLZF interacts with Sp1 or p53 and competes with Sp1 or p53. PLZF interacts with corepressors, such as mSin3A, NCoR, and SMRT, thereby deacetylates Ac-H3 and Ac-H4 histones at the CDKN1A promoter, which indicated the involvement of the corepressor·HDACs complex in transcription repression by PLZF. Also, PLZF represses transcription of TP53 and also decreases p53 protein stability by ubiquitination. PLZF may act as a potential proto-oncoprotein in various cell types.

Ethyl 2-(benzylidene)-7-methyl-3-oxo-2,3-dihydro-5H-thiazolo[3,2-a]pyrimidine-6-carboxylate analogues as a new scaffold for protein kinase casein kinase 2 inhibitor.

Protein kinase casein kinase 2 (PKCK2) is a constitutively active, growth factor-independent serine/threonine kinase, and changes in PKCK2 expression or its activity are reported in many cancer cells. To develop a novel PKCK2 inhibitor(s), we first performed cell-based phenotypic screening using 4000 chemicals purchased from ChemDiv chemical libraries (2000: randomly selected; 2000: kinase-biased) and performed in vitro kinase assay-based screening using hits found from the first screening. We identified compound 24 (C24)[(Z)-ethyl 5-(4-chlorophenyl)-2-(3,4-dihydroxybenzylidene)-7-methyl-3-oxo-3,5-dihydro-2H-thiazolo[3,2-a] pyrimidine-6-carboxylate] as a novel inhibitor of PKCK2 that is more potent and selective than 4,5,6,7-tetrabromobenzotriazole (TBB). In particular, compound 24 [half maximal inhibitory concentration (IC50)=0.56µM] inhibited PKCK2 2.2-fold more efficiently than did TBB (IC50=1.24µM), which is quite specific toward PKCK2 with respect to ATP binding, in a panel of 31 human protein kinases. The Ki values of compound 24 and TBB for PKCK2 were 0.78µM and 2.70µM, respectively. Treatment of cells with compound 24 inhibited endogenous PKCK2 activity and showed anti-proliferative and pro-apoptotic effects against stomach and hepatocellular cancer cell lines more efficiently than did TBB. As expected, compound 24 also enabled tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-resistant cancer cells to be sensitive toward TRAIL. In comparing the molecular docking of compound 24 bound to PKCK2α versus previously reported complexes of PKCK2 with other inhibitors, our findings suggest a new scaffold for specific PKCK2α inhibitors. Thus, compound 24 appears to be a selective, cell-permeable, potent, and novel PKCK2 inhibitor worthy of further characterization.

Lactate Dehydrogenase A as a Potential New Biomarker for Thyroid Cancer.

BACKGROUND: Several cancers show increased levels of lactate dehydrogenase A (LDHA), which are associated with cancer progression. However, it remains unclear whether LDHA levels are associated with papillary thyroid cancer (PTC) aggressiveness or with the presence of the PTC prognostic marker, the BRAFV600E mutation. This study aimed to evaluate the potential of LDHA as a PTC prognostic marker. METHODS: LDHA expression was examined in 83 PTC tissue specimens by immunohistochemistry. Human thyroid cell lines were genetically manipulated to overexpress BRAFV600E or were treated with a BRAF-specific short hairpin RNA (shBRAF), whose effects on LDHA expression were evaluated by Western blotting. Data from 465 PTC patients were obtained from The Cancer Genome Atlas (TCGA) database and analyzed to validate the in vitro results. RESULTS: LDHA was aberrantly overexpressed in PTC. Intense immunostaining for LDHA was observed in PTC specimens carrying mutated BRAF, whereas the intensity was less in wild-type BRAF samples. Overexpression of BRAFV600E resulted in LDHA upregulation, whereas treatment with shBRAF downregulated LDHA in human thyroid cell lines. Furthermore, LDHA mRNA expression was significantly elevated and associated with BRAFV600E expression in thyroid cancer tissues from TCGA database. Additionally, LDHA overexpression was found to be correlated with aggressive clinical features of PTC, such as lymph node metastases and advanced tumor stages. CONCLUSION: LDHA overexpression is associated with the BRAFV600E mutation and an aggressive PTC behavior. Therefore, LDHA may serve as a biomarker and therapeutic target in PTC.

The functional relationship between co-repressor N-CoR and SMRT in mediating transcriptional repression by thyroid hormone receptor alpha.

A central issue in mediating repression by nuclear hormone receptors is the distinct or redundant function between co-repressors N-CoR (nuclear receptor co-repressor) and SMRT (silencing mediator of retinoid and thyroid hormone receptor). To address the functional relationship between SMRT and N-CoR in TR (thyroid hormone receptor)-mediated repression, we have identified multiple TR target genes, including BCL3 (B-cell lymphoma 3-encoded protein), Spot14 (thyroid hormone-inducible hepatic protein), FAS (fatty acid synthase), and ADRB2 (beta-adrenergic receptor 2). We demonstrated that siRNA (small interfering RNA) treatment against either N-CoR or SMRT is sufficient for the de-repression of multiple TR target genes. By the combination of sequence mining and physical association as determined by ChIP (chromatin immunoprecipitation) assays, we mapped the putative TREs (thyroid hormone response elements) in BCL3, Spot14, FAS and ADRB2 genes. Our data clearly show that SMRT and N-CoR are independently recruited to various TR target genes. We also present evidence that overexpression of N-CoR can restore repression of endogenous genes after knocking down SMRT. Finally, unliganded, co-repressor-free TR is defective in repression and interacts with a co-activator, p300. Collectively, these results suggest that both SMRT and N-CoR are limited in cells and that knocking down either of them results in co-repressor-free TR and consequently de-repression of TR target genes.

Impact of grade, hormone receptor, and HER-2 status in women with breast cancer on response to specific chemotherapeutic agents by in vitro adenosine triphosphate-based chemotherapy response assay.

This study was designed to assess whether histological and biological factors of breast cancer can predict chemoresponse to specific agents. Adenosine triphosphate-based chemotherapy response assay (ATP-CRA) was employed to retrieve chemoresponse to 5-fluorouracil (5-FU), doxetaxel, doxorubicin, epirubicin, and paclitaxel in 49 patients. Tumors with high histologic and nuclear grade have higher response rate to doxorubicin (P<0.05) and palitaxel (P<0.05). Estrogen receptor (ER)-negative tumors respond well to doxorubicin (P=0.038), and progesterone receptor (PR)-negative tumors to 5-FU (P=0.039), doxetaxel (P=0.038), doxorubicin (P=0.000), epirubicin (P=0.010), and paclitaxel (P=0.003). Among the breast cancer subtypes determined by ER, PR, and HER-2 immunohistochemical stains, the HER-2+/ER- subtype has a higher response rate to doxorubicin (P=0.008). This in vitro result suggests that the combination of histologic and nuclear grade, hormone receptor, and HER-2 status can be a predictive factor of response to specific chemotherapy agents. Further in vivo study should be followed for clinical trials.

Protein kinase CK2 activation is required for transforming growth factor ß-induced epithelial-mesenchymal transition.

Transforming growth factor ß (TGFß) is overexpressed in advanced cancers and promotes tumorigenesis by inducing epithelial-mesenchymal transition (EMT), which enhances invasiveness and metastasis. Although we previously reported that EMT could be induced by increasing CK2 activity alone, it is not known whether CK2 also plays an essential role in TGFß-induced EMT. Therefore, in the present study, we investigated whether TGFß signaling could activate CK2 and, if so, whether such activation is required for TGFß-induced EMT. We found that CK2 is activated by TGFß treatment, and that activity peaks at 48 h after treatment. CK2 activation is dependent on TGFß receptor (TGFBR) I kinase activity, but independent of SMAD4. Inhibition of CK2 activation through the use of either a CK2 inhibitor or shRNA against CSNK2A1 inhibited TGFß-induced EMT. TGFß signaling decreased CK2ß but did not affect CK2α protein levels, resulting in a quantitative imbalance between the catalytic α and regulatory ß subunits, thereby increasing CK2 activity. The decrease in CK2ß expression was dependent on TGFBRI kinase activity and the ubiquitin-proteasome pathway. The E3 ubiquitin ligases responsible for TGFß-induced CK2ß degradation were found to be CHIP and WWP1. Okadaic acid (OA) pretreatment protected CK2ß from TGFß-induced degradation, suggesting that dephosphorylation of CK2ß by an OA-sensitive phosphatase might be required for CK2 activation in TGFß-induced EMT. Collectively, our results suggest CK2 as a therapeutic target for the prevention of EMT and metastasis of cancers.

Phosphorylation-dependent stabilization of MZF1 upregulates N-cadherin expression during protein kinase CK2-mediated epithelial-mesenchymal transition.

Epithelial-mesenchymal transition (EMT) is a critical process in invasion and metastasis of cancer cells. E-cadherin to N-cadherin switching is considered a molecular hallmark of EMT. Recently, we reported that increased CK2 activity fully induces E-cadherin to N-cadherin switching, but the molecular mechanisms of N-cadherin upregulation are unknown. In this study, we examined how N-cadherin is upregulated by CK2. N-cadherin promoter analysis and ChIP analysis identified and confirmed myeloid zinc finger 1 (MZF1) as an N-cadherin transcription factor. Molecular analysis showed that MZF1 directly interacts with CK2 and is phosphorylated at serine 27. Phosphorylation stabilizes MZF1 and induces transcription of N-cadherin. MZF1 knockdown (MKD) in N-cadherin-expressing cancer cells downregulates N-cadherin expression and reverts the morphology from spindle and fibroblast-like to a rounded, epithelial shape. In addition, we showed that that MKD reduced the motility and invasiveness of N-cadherin-expressing cancer cells. Collectively, these data indicate that N-cadherin upregulation in CK2-mediated E-cadherin to N-cadherin switching is dependent on phosphorylation-mediated MZF1 stabilization. CK2 could be a good therapeutic target for the prevention of metastasis.

The functional role of the CARM1-SNF5 complex and its associated HMT activity in transcriptional activation by thyroid hormone receptor.

We have investigated the function and mechanisms of the CARM1-SNF5 complex in T3-dependent transcriptional activation. Using specific small interfering RNAs (siRNA) to knock down coactivators in HeLa alpha2 cells, we found that coactivator associated arginine methyltransferase 1 (CARM1) and SWI/SNF complex component 5 (SNF5) are important for T3-dependent transcriptional activation. The CARM1- SWI/SNF chromatin remodeling complex serves as a mechanism for the rapid reversal of H3-K9 methylation. Importantly, siRNA treatment against CARM1 and/or SNF5 increased the recruitment of HMTase G9a to the type 1 deiodinase (D1) promoter even with T3. Knocking-down either CARM1 or SNF5 also inhibited the down-regulation of histone macroH2A, which is correlated with transcriptional activation. Finally, knocking down CARM1 and SNF5 by siRNA impaired the association of these coactivators to the D1 promoter, suggesting functional importance of CARM1- SNF5 complex in T3-dependent transcriptional activation.

ZNF509S1 downregulates PUMA by inhibiting p53K382 acetylation and p53-DNA binding.

Expression of the POK family protein ZNF509L, and -its S1 isoform, is induced by p53 upon exposure to genotoxic stress. Due to alternative splicing of the ZNF509 primary transcript, ZNF509S1 lacks the 6 zinc-fingers and C-terminus of ZNF509L, resulting in only one zinc-finger. ZNF509L and -S1 inhibit cell proliferation by activating p21/CDKN1A and RB transcription, respectively. When cells are exposed to severe DNA damage, p53 activates PUMA (p53-upregulated modulator of apoptosis) transcription. Interestingly, apoptosis due to transcriptional activation of PUMA by p53 is attenuated by ZNF509S1. Thus we investigated the molecular mechanism(s) underlying the transcriptional attenuation and anti-apoptotic effects of ZNF509S1. We show that ZNF509S1 modulation of p53 activity is important in PUMA gene transcription by modulating post-translational modification of p53 by p300. ZNF509S1 directly interacts with p53 and inhibits p300-mediated acetylation of p53 lysine K382, with deacetylation of p53 K382 leading to decreased DNA binding at the p53 response element 1 of the PUMA promoter. ZNF509S1 may play a role not only in cell cycle arrest, by activating RB expression, but also in rescuing cells from apoptotic death by repressing PUMA expression in cells exposed to severe DNA damage.

The combination of TRAIL treatment and cancer cell selective expression of TRAIL-death receptor DR4 induces cell death in TRAIL-resistant cancer cells.

The human telomerase reverse transcriptase (hTERT) promoter can be used for the tumor-specific expression of transgenes in order to induce selective cancer cell death. The hTERT core promoter is active in cancer cells but not in normal cells. To examine whether the combination of TNF-related apoptosis inducing ligand (TRAIL) treatment and cancer cell-selective expression of the TRAIL-death receptor could induce cell death in TRAIL-resistant cancer cells, we generated a death receptor-4 (DR4)-expressing adenovirus (Ad-hTERT-DR4), in which the expression of DR4 is driven by the hTERT promoter. Upon infection, DR4 expression was slightly increased in cancer cell lines, and cell death was observed in TRAIL-resistant cancer cell lines but not in normal human cells when DR4 infection was combined with TRAIL treatment. We also generated an adenovirus that expresses a secretable isoleucine zipper (ILZ)-fused, extracellular portion of TRAIL (Ad-ILZ- TRAIL). In cells infected with Ad-ILZ-TRAIL, TRAIL was expressed, secreted, oligomerized and biologically active in the induction of apoptosis in TRAIL-sensitive cancer cells. When Ad-hTERT-DR4 infected TRAIL-resistant HCE4 cells and Ad-ILZ-TRAIL infected TRAIL-resistant HCE7 cells were co-cultured, cell deaths were evident 24 h after co-culture. Taken together, our results reveal that the combination of TRAIL and cancer cell-specific expression of DR4 has the potential to overcome the resistance of cancer cells to TRAIL without inducing significant cell death in normal cells.

Antiobesity and Antidiabetes Effects of a Cudrania tricuspidata Hydrophilic Extract Presenting PTP1B Inhibitory Potential.

Diabetes and obesity represent the major health problems and the most age-related metabolic diseases. Protein-tyrosine phosphatase 1B (PTP1B) has emerged as an important regulator of insulin signal transduction and is regarded as a pharmaceutical target for metabolic disorders. To find novel natural materials presenting therapeutic activities against diabetes and obesity, we screened various herb extracts using a chip screening allowing the determination of PTP1B inhibitory effects of the tested compounds using insulin receptor (IR) as the substrate. Cudrania tricuspidata leaves (CTe) had a strong inhibitory effect on PTP1B activity and substantially inhibited fat accumulation in 3T3-L1 cells. CTe was orally administrated to diet-induced obesity (DIO) mice once daily for 3 weeks after which changes in glucose, insulin metabolism, and fat accumulation were examined. Hepatic enzyme markers (aspartate aminotransferase, AST, and alanine aminotransferase, ALT) and total fat mass and triglyceride levels decreased in CTe-treated mice, whereas body weight and total cholesterol concentration slightly decreased. CTe increased the phosphorylation of IRS-1 and Akt in liver tissue. Furthermore, CTe treatment significantly lowered blood glucose levels and improved insulin secretion in DIO mice. Our results strongly suggest that CTe may represent a promising therapeutic substance against diabetes and obesity.

The long noncoding RNA HOXA11 antisense induces tumor progression and stemness maintenance in cervical cancer.

Recent research has focused on the impact of long noncoding RNA (lncRNA) in cervical carcinogenesis. However, whether HOXA11 antisense (HOXA11-AS) is involved in cervical cancer remains to be elucidated. In the present study, we examined HOXA11-AS expression levels in cervical cancer patients and determined the relationships between HOXA11-AS expression and clinicopathological factors. We also investigated the bio-functional consequences of HOXA11-AS overexpression both in vitro and in vivo. HOXA11-AS expression was significantly greater in tissues from patients with cervical cancer than in control patients (P<0.001). Multivariate analysis showed that high HOXA11-AS was an independent prognosticator of overall survival (Hazard ratio=2.450, P=0.032). HOXA11-AS overexpression enhanced cell proliferation, migration, and tumor invasion in vitro, whereas HOXA11-AS knockdown inhibited these biologic aggressive features. These adverse changes were accompanied by characteristics of epithelial-mesenchymal transition (EMT). In vivo xenograft experiments using the siHOXA11-AS-transfected HeLa cells revealed that HOXA11-AS strongly induced tumor growth. Furthermore, we found that HOXA11-AS knockdown decreased cancer stemness and triggered the EMT program. In conclusion, HOXA11-AS overexpression correlated with poor survival in patients with cervical cancer. Thus, HOXA11-AS may be a pivotal target for exploring novel cervical cancer therapeutics.

Cisplatin-induced apoptosis in Hep3B cells: mitochondria-dependent and -independent pathways.

Human hepatoma cell lines undergo apoptosis after treatment with cisplatin (CP), by mechanisms that are not fully understood, although our previous study demonstrated that Fas-dependent or -independent pathways are involved. To elucidate the mechanisms of CP-induced apoptosis in Hep3B cells, which are Fas- and p53-negative, we investigated mitochondria associated pathways, the involvement of NF-kappaB, and p73 activation. Results of Western blot and flow cytometry assay revealed that the translocation of Bax, resulted in the loss of mitochondrial membrane potential (Deltaphi(m)) and the efflux of cytochrome c and of second mitochondria-derived activator of caspase/DIABLO from mitochondria into the cytosol. Caspase-3, -8 and -9 were activated by CP treatment, however, CP-induced apoptosis was not completely blocked by pretreating with the pan-caspase inhibitor, benzyloxycarbonyl-valinyl-alaninyl-aspartyl-(O-methyl)-fluoromethylketone, indicating that caspase-independent apoptotic pathways might also be involved. RNase protection assay confirmed that NF-kappaB downregulation leading to the suppression of its target genes, such as XIAP and TRAF2, and p73 accumulation were also observed in Hep3B cells treated with CP. CP-induced apoptosis was inhibited to some extent by transiently overexpressed p73 dominant negative and XIAP, but not by p73DN or XIAP alone. In conclusion, this study demonstrates that CP-induced apoptosis in Hep3B cells is associated with mitochondrial dysregulation, NF-kappaB downregulation and p73 accumulation.

Human hepatocellular carcinoma cells resist to TRAIL-induced apoptosis, and the resistance is abolished by cisplatin.

TNF-related apoptosis-inducing ligand (TRAIL), a member of the TNF family, selectively induce apoptosis in various transformed cell lines but not in almost-normal tissues. It is regulated by 2 death receptors, TRAIL receptor 1 (TRAIL-R1) and TRAIL-R2 and 2 decoy receptors, TRAIL-R3 and TRAIL-R4. However, the determining factors of the sensitivity to TRAIL-induced apoptosis are not clearly understood. Herein, we investigated the expression of TRAIL-R, c-FLIP, FADD-like interleukin-1beta-converting enzyme inhibitory protein, and TRAIL-induced apoptosis in human hepatocellular carcinoma (HCC) cell lines. Seven of ten HCC cell lines showed resistance to TRAIL-induced apoptosis and five of seven TRAIL-resistant cell lines became sensitive to TRAIL by co-treatment with cycloheximide. In HCC cell lines, their TRAIL resistance did not correlate with the basal expression level of TRAIL receptors or c-FLIP, however, in human tissues, TRAIL-R1 and TRAIL-R2 expressions were notably decreased compared to normal counterpart. Cisplatin showed synergistic effect on TRAIL-induced apoptosis in most HCC cell lines regardless of their p53 status and TRAIL-R1 was induced by cisplatin treatment in certain cell lines. Inhibition of nuclear factor K B (NF-kappaB) by SN50, a peptide inhibitor of NF-KB activity, had no effect on TRAIL-induced apoptosis in HCC cells. These results suggest that (a) the majority of human HCC cell lines are resistant to TRAIL-induced apoptosis and cycloheximide-sensitive short-lived antiapoptotic molecule(s) is responsible for this resistance, (b) the expression of TRAIL-R1 and TRAIL-R2 is reduced in HCC tissues, and the increased expression of TRAIL-R1 may be a mechanism of cisplatininduced sensitization to TRAIL-induced apoptosis in some HCC cells, and (c) the activation of NF-kappaB may not be involved in the TRAIL resistance of HCC cells

Neobavaisoflavone sensitizes apoptosis via the inhibition of metastasis in TRAIL-resistant human glioma U373MG cells.

AIMS: Neobavaisoflavone (NBIF), an isoflavone isolated from Psoralea corylifolia (Leguminosae), has striking anti-inflammatory and anti-cancer effects. NBIF inhibits the proliferation of prostate cancer in vitro and in vivo. MAIN METHODS: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a key endogenous molecule that selectively induces apoptosis in cancer cells with little or no toxicity in normal cells. However, some cancer cells, including U373MG cells, are resistant to TRAIL-mediated apoptosis. We demonstrated that the cell viability, migration and invasion assay were used in U373MG glioma cells. KEY FINDINGS: In this study, we found that NBIF sensitizes human U373MG glioma cells to TRAIL-mediated apoptosis. Co-treatment of TRAIL and NBIF effectively induced Bid cleavage and activated caspases 3, 8, and 9. Importantly, DR5 expression was upregulated by NBIF. We also observed that the combination NBIF and TRAIL increased expression of BAX. We further demonstrate that NBIF induced TRAIL-mediated apoptosis in human glioma cells by suppressing migration and invasion, and by inhibiting anoikis resistance. SIGNIFICANCE: Taken together, our results suggest that NBIF reduces the resistance of cancer cells to TRAIL and that the combination of NBIF and TRAIL may be a new therapeutic strategy for treating TRAIL-resistant glioma cells.