Inik Chang

Resistance of Mitochondrial DNA-depleted Cells against Cell Death ROLE OF MITOCHONDRIAL SUPEROXIDE DISMUTASE

We have shown that mitochondrial DNA-depleted (ρ0) SK-Hep1 hepatoma cells are resistant to apoptosis, contrary to previous papers reporting normal apoptotic susceptibility of ρ0 cells. We studied the changes of gene expression in SK-Hep1 ρ0 cells. DNA chip analysis showed that MnSOD expression was profoundly increased in ρ0 cells. \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{{\bar{{\cdot}}}}\) \end{document} contents increased during ρ0 cell derivation but became normalized after establishment of ρ0 phenotypes, suggesting that MnSOD induction is an adaptive process to increased \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{{\bar{{\cdot}}}}\) \end{document}. ρ0 cells were resistant to menadione, paraquat, or doxorubicin, and \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{{\bar{{\cdot}}}}\) \end{document} contents after treatment with them were lower in ρ0 cells compared with parental cells because of MnSOD overexpression. Expression levels and activity of glutathione peroxidases were also increased in ρ0 cells, rendering them resistant to exogenous H2O2. ρ0 cells were resistant to p53, and intracellular ROS contents after p53 expression were lower compared with parental cells. Other types of ρ0 cells also showed increased MnSOD expression and resistance against ROS. Heme oxygenase-1 expression was increased in ρ0 cells, and a heme oxygenase-1 inhibitor decreased the induction of MnSOD in ρ0 cells and their resistance against ROS donors. These results indicate that ρ0 cells are resistant to cell death contrary to previous reports and suggest that an adaptive increase in the expression of antioxidant enzymes renders cancer cells or aged cells with frequent mitochondrial DNA mutations to resist against oxidative stress, host anti-cancer surveillance, or chemotherapeutic agents, conferring survival advantage on them.

Genistein Inhibits Prostate Cancer Cell Growth by Targeting miR-34a and Oncogenic HOTAIR

Objective Genistein is a soy isoflavone that has antitumor activity both in vitro and in vivo. It has been shown that genistein inhibits many type of cancers including prostate cancer (PCa) by regulating several cell signaling pathways and microRNAs (miRNAs). Recent studies suggest that the long non-coding RNAs (lncRNAs) are also involved in many cellular processes. At present there are no reports about the relationship between gensitein, miRNAs and lncRNAs. In this study, we focused on miRNAs, lncRNA that are regulated by genistein and investigated their functional role in PCa. Method Microarray (SurePrint G3 Human GE 8×60K) was used for expression profiling of genistein treated and control PCa cells (PC3 and DU145). Functional assay (cell proliferation, migration, invasion, apoptosis and cell cycle assays) were performed with the PCa cell lines, PC3 and DU145. Both in vitro and in vivo (nude mouse) models were used for growth assays. Luciferase reporter assays were used for binding of miR-34a to HOTAIR. Results LncRNA profiling showed that HOTAIR was highly regulated by genistein and its expression was higher in castration-resistant PCa cell lines than in normal prostate cells. Knockdown (siRNA) of HOTAIR decreased PCa cell proliferation, migration and invasion and induced apoptosis and cell cycle arrest. miR-34a was also up-regulated by genistein and may directly target HOTAIR in both PC3 and DU145 PCa cells. Conclusions Our results indicated that genistein inhibited PCa cell growth through down-regulation of oncogenic HOTAIR that is also targeted by tumor suppressor miR-34a. These findings enhance understanding of how genistein regulates lncRNA HOTAIR and miR-34a in PCa.

Up-Regulation of MicroRNA-21 Correlates with Lower Kidney Cancer Survival

Background MicroRNA-21 is up-regulated in a variety of cancers like, breast, colorectal, lung, head and neck etc. However, the regulation of miR-21 in renal cell carcinoma (RCC) has not yet been studied systematically. Methods and Results We measured miR-21 levels in 54 pairs of kidney cancers and their normal matched tissues by real-time PCR. The expression level of miR-21 was correlated with 5 year survival and the pathological stage. Functional studies were done after inhibiting miR-21 in RCC cell lines. We studied in vitro and in vivo effects of the chemo preventive agent genistein on miR-21 expression. In 48 cases (90%), miR-21 was increased. All patients with low miR-21 expression survived 5 years, while with high miR-21 expression, only 50% survived. Higher expression of miR-21 is associated with an increase in the stage of renal cancer. Functional studies after inhibiting miRNA-21 in RCC cell lines show cell cycle arrest, induction of apoptosis and reduced invasive and migratory capabilities. Western blot analysis showed an increase in the expression of p21 and p38 MAP kinase genes and a reduction in cyclin E2. Genistein inhibited the expression of miR-21 in A-498 cells and in the tumors formed after injecting genistein treated A-498 cells in nude mice besides inhibiting tumor formation. Conclusions The current study shows a clear correlation between miR-21 expression and clinical characteristics of renal cancer. Thus we believe that miR-21 can be used as a tumor marker and its inhibition may prove to be useful in controlling cancers with up-regulated miR-21.

miR-23b Represses Proto-oncogene Src Kinase and Functions as Methylation-Silenced Tumor Suppressor with Diagnostic and Prognostic Significance in Prostate Cancer

The miRNAs have great potential as biomarkers and therapeutic agents owing to their ability to control multiple genes and potential to influence cellular behavior. Here, we identified that miR-23b is a methylation-silenced tumor suppressor in prostate cancer. We showed that miR-23b expression is controlled by promoter methylation and has great promise as a diagnostic and prognostic biomarker in prostate cancer. High levels of miR-23b expression are positively correlated with higher overall and recurrence-free survival in patients with prostate cancer. Furthermore, we elucidated the tumor suppressor role of miR-23b using in vitro and in vivo models. We showed that proto-oncogene Src kinase and Akt are direct targets of miR-23b. Increased expression of miR-23b inhibited proliferation, colony formation, migration/invasion, and triggered G(0)-G(1) cell-cycle arrest and apoptosis in prostate cancer. Overexpression of miR-23b inhibited epithelial-to-mesenchymal transition (EMT) causing a decline in mesenchymal markers Vimentin and Snail and increasing the epithelial marker, E-cadherin. Depletion of Src by RNA interference conferred similar functional effects as that of miR-23b reconstitution. miR-23b expression caused a dramatic decrease in tumor growth in nude mice and attenuated Src expression in excised tumors compared with a control miR. These findings suggest that miR-23b is a methylation-silenced tumor suppressor that may be a useful biomarker in prostate cancer. Loss of miR-23b may confer proliferative advantage and promote prostate cancer migration and invasion, and reexpression of miR-23b may contribute to the epigenetic therapy for prostate cancer.

Long Non-coding RNA HOTAIR Is Targeted and Regulated by miR-141 in Human Cancer Cells

Background: Silencing of long non-coding RNA (lncRNA) by microRNA (miRNA) has only been recently observed. Results: miR-141 binds to HOTAIR and suppresses its oncogenic function in Ago2 (Argounaute2). Conclusion: miR-141 targets and silences HOTAIR in an Ago2-dependent manner in cancer cells. Significance: Our results suggest that regulation of lncRNA expression by miRNA plays essential roles in gene expression and cellular functions. HOTAIR is a long non-coding RNA that interacts with the polycomb repressive complex and suppresses its target genes. HOTAIR has also been demonstrated to promote malignancy. MicroRNA-141 (miR-141) has been reported to play a role in the epithelial to mesenchymal transition process, and the expression of miR-141 is inversely correlated with tumorigenicity and invasiveness in several human cancers. We found that HOTAIR expression is inversely correlated to miR-141 expression in renal carcinoma cells. HOTAIR promotes malignancy, including proliferation and invasion, whereas miR-141 suppresses malignancy in human cancer cells. miR-141 binds to HOTAIR in a sequence-specific manner and suppresses HOTAIR expression and functions, including proliferation and invasion. Both HOTAIR and miR-141 were associated with the immunoprecipitated Ago2 (Argonaute2) complex, and the Ago2 complex cleaved HOTAIR in the presence of miR-141. These results demonstrate that HOTAIR is suppressed by miR-141 in an Ago2-dependent manner.

Genistein Up-Regulates Tumor Suppressor MicroRNA-574-3p in Prostate Cancer

Genistein has been shown to inhibit cancers both in vitro and in vivo, by altering the expression of several microRNAs (miRNAs). In this study, we focused on tumor suppressor miRNAs regulated by genistein and investigated their function in prostate cancer (PCa) and target pathways. Using miRNA microarray analysis and real-time RT-PCR we observed that miR-574-3p was significantly up-regulated in PCa cells treated with genistein compared with vehicle control. The expression of miR-574-3p was significantly lower in PCa cell lines and clinical PCa tissues compared with normal prostate cells (RWPE-1) and adjacent normal tissues. Low expression level of miR-574-3p was correlated with advanced tumor stage and higher Gleason score in PCa specimens. Re-expression of miR-574-3p in PCa cells significantly inhibited cell proliferation, migration and invasion in vitro and in vivo. miR-574-3p restoration induced apoptosis through reducing Bcl-xL and activating caspase-9 and caspase-3. Using GeneCodis software analysis, several pathways affected by miR-574-3p were identified, such as ‘Pathways in cancer’, ‘Jak-STAT signaling pathway’, and ‘Wnt signaling pathway’. Luciferase reporter assays demonstrated that miR-574-3p directly binds to the 3′ UTR of several target genes (such as RAC1, EGFR and EP300) that are components of ‘Pathways in cancer’. Quantitative real-time PCR and Western analysis showed that the mRNA and protein expression levels of the three target genes in PCa cells were markedly down-regulated with miR-574-3p. Loss-of-function studies demonstrated that the three target genes significantly affect cell proliferation, migration and invasion in PCa cell lines. Our results show that genistein up-regulates tumor suppressor miR-574-3p expression targeting several cell signaling pathways. These findings enhance understanding of how genistein regulates with miRNA in PCa.

miRNA-708 Control of CD44 + Prostate Cancer–Initiating Cells

Tumor recurrence in prostate cancer has been attributed to the presence of CD44-expressing tumor-initiating cells. In this study, we report that miR-708 is a key negative regulator of this CD44(+) subpopulation of prostate cancer cells, with important implications for diagnosis and prognosis of this disease. miR-708 was underexpressed in CD44(+) cells from prostate cancer xenografts. Reconstitution of miR-708 in prostate cancer cell lines or CD44(+) prostate cancer cells led to decreased tumorigenicity in vitro. Intratumoral delivery of synthetic miR-708 oligonucleotides triggered regression of established tumors in a murine xenograft model of human prostate cancer. Conversely, miR-708 silencing in a purified CD44(-) population of prostate cancer cells promoted tumor growth. Functional studies validated CD44 to be a direct target of miR-708 and also identified the serine/threonine kinase AKT2 as an additional target. Clinically, low miR-708 expression was associated significantly with poor survival outcome, tumor progression, and recurrence in patients with prostate cancer. Together, our findings suggest that reduced miR-708 expression leads to prostate cancer initiation, progression, and development by regulating the expression of CD44 as well as AKT2. miR-708 therefore may represent a novel therapeutic target or diagnostic and prognostic biomarker in prostate cancer.

Role of Calcium in Pancreatic Islet Cell Death by IFN-γ/TNF-α

We studied the intracellular events associated with pancreatic β cell apoptosis by IFN-γ/TNF-α synergism. IFN-γ/TNF-α treatment of MIN6N8 insulinoma cells increased the amplitude of high voltage-activated Ca2+ currents, while treatment with IFN-γ or TNF-α alone did not. Cytosolic Ca2+ concentration ([Ca2+]c) was also increased by IFN-γ/TNF-α treatment. Blockade of L-type Ca2+ channel by nifedipine abrogated death of insulinoma cells by IFN-γ/TNF-α. Diazoxide that attenuates voltage-activated Ca2+ currents inhibited MIN6N8 cell death by IFN-γ/TNF-α, while glibenclamide that accentuates voltage-activated Ca2+ currents augmented insulinoma cell death. A protein kinase C inhibitor attenuated MIN6N8 cell death and the increase in [Ca2+]c by IFN-γ/TNF-α. Following the increase in [Ca2+]c, calpain was activated, and calpain inhibitors decreased insulinoma cell death by IFN-γ/TNF-α. As a downstream of calpain, calcineurin was activated and the inhibition of calcineurin activation by FK506 diminished insulinoma cell death by IFN-γ/TNF-α. BAD phosphorylation was decreased by IFN-γ/TNF-α because of the increased calcineurin activity, which was reversed by FK506. IFN-γ/TNF-α induced cytochrome c translocation from mitochondria to cytoplasm and activation of caspase-9. Effector caspases such as caspase-3 or -7 were also activated by IFN-γ/TNF-α treatment. These results indicate that IFN-γ/TNF-α synergism induces pancreatic β cell apoptosis by Ca2+ channel activation followed by downstream intracellular events such as mitochondrial events and caspase activation and also suggest the therapeutic potential of Ca2+ modulation in type 1 diabetes.

Resistance of mitochondrial DNA-deficient cells to TRAIL: role of Bax in TRAIL-induced apoptosis

Mitochondrion is one of the master players in both apoptosis and necrosis. We studied the role of mitochondrial function in TRAIL-induced apoptosis. TRAIL killed SK-Hep1 cells with characteristic features of apoptosis such as DNA fragmentation, sub-G1 ploidy peak and cytochrome c translocation. In contrast, mitochondrial DNA-deficient SK-Hep1 ρ0 cells were resistant to TRAIL. Dissipation of mitochondrial potential or cytochrome c translocation did not occur in ρ0 cells after TRAIL treatment. TRAIL induced translocation of Bax subsequent to the cleavage of Bid in parental cells. However, Bax translocation was absent in ρ0 cells, accounting for the failure of cytochrome c release in ρ0 cells. Forced expression of Bax induced caspase-3 activity in ρ0 cells. Incubation of ρ0 cells with ADP+Pi to increase intracellular ATP restored sensitivity to TRAIL. Despite different sensitivity to TRAIL, parental cells and ρ0 cells did not show significant difference in susceptibility to agonistic anti-Fas antibody, TNF-α or staurosporine. Our results indicate that TRAIL-induced apoptosis is dependent on intact mitochondrial function and susceptibility of mitochondrial DNA-deficient cells to apoptosis depends on the type of apoptotic stimuli. Tumor cells with mitochondrial mutations or dysfunction might have the ability to evade tumor surveillance imposed by TRAIL in vivo.

Inhibition of PTEN Gene Expression by Oncogenic miR-23b-3p in Renal Cancer

Background miR-23b is located on chromosome number 9 and plays different roles in different organs especially with regards to cancer development. However, the functional significance of miR-23b-3p in renal cell carcinoma (RCC) has not been reported. Methods and Results We measured miR-23b-3p levels in 29 pairs of renal cell carcinoma and their normal matched tissues using real-time PCR. The expression level of miR-23b-3p was correlated with the 5 year survival rate of renal cancer patients. In 15 cases (52%), miR-23b-3p expression was found to be high. All patients with moderate to low miR-23b-3p expression survived 5 years, while those with high miR-23b-3p expression, only 50% survived. After knocking down miRNA-23b-3p expression in RCC cell lines, there was an induction of apoptosis and reduced invasive capabilities. MiR-23b-3p was shown to directly target PTEN gene through 3′UTR reporter assays. Inhibition of miR-23b-3p induces PTEN gene expression with a concomitant reduction in PI3-kinase, total Akt and IL-32. Immunohistochemistry showed the lack of PTEN protein expression in cancerous regions of tissue samples where the expression of miR-23b-3p was high. We studied the in vitro effects of the dietary chemo preventive agent genistein on miR-23b-3p expression and found that it inhibited expression of miR-23b-3p in RCC cell lines. Conclusions The current study shows that miR-23b-3p is an oncogenic miRNA and inhibits PTEN tumor suppressor gene in RCC. Therefore, inhibition of miR-23b-3p may be a useful therapeutic target for the treatment of renal cell carcinoma.