Shih-Ming Huang

Dual Degradation of Aurora A and B Kinases by the Histone Deacetylase Inhibitor LBH589 Induces G2-M Arrest and Apoptosis of Renal Cancer Cells

Purpose: This study is aimed at investigating antineoplastic efficacy of histone deacetylase inhibitor (HDACI) LBH589 on renal cell carcinoma (RCC) and elucidating the novel molecular mechanisms involved in growth arrest and apoptosis by targeting the important nonhistone molecules. Experimental Design: We analyzed the growth-inhibitory effect of LBH589 on RCC by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay in vitro and antitumor efficacy by xenograft experiments in vivo. To verify the associated molecular mechanisms involved in LBH589-mediated cell death and cell cycle progression by Western blotting and fluorescence-activated cell sorting analysis. Results: HDACI LBH589 induced degradation of both Aurora A and B kinases through a proteasome-mediated pathway by targeting HDAC3 and HDAC6. The dual degradation of Aurora A and B kinases mediated by LBH589 resulted in inducing G2-M arrest and apoptosis of renal cancer cell lines and our results also showed that LBH589 potently inhibited renal cancer cell growth in vitro and suppressed tumor formation in vivo. The Aurora A and B kinases and HDAC3 are overexpressed in the human RCC tumor tissues examined, which make them perfect targets for HDACI LBH589 treatment. Conclusions: Our in vitro and in vivo data showed that LBH589 has potent anticancer effect of renal cancer cells. LBH589 and other HDACI treatment resulted in inducing G2-M arrest and apoptosis of renal cancer cells through degradation of Aurora A and B kinases by inhibition of HDAC3 and HDAC6. The clinical efficacy of LBH589 in the treatment of patients with metastatic RCC, especially those with high Aurora kinase and HDAC expression, is worthy of further investigation.

Adipose-derived stem cells seeded on acellular dermal matrix grafts enhance wound healing in a murine model of a full-thickness defect.

IntroductionThe promotion of wound healing using dermal substitutes has become increasingly widespread, but the outcomes of substitute-assisted healing remain functionally deficient. Adipose-derived stem cells (ASCs) have been investigated widely in regenerative medicine and tissue engineering, and they have the potential to enhance wound healing. In this study, we focused on investigating the effects and mechanism of ASCs combined with an acellular dermal matrix (ADM) to treat full-thickness cutaneous wounds in a murine model. MethodsThe ADM was prepared from the dorsal skin of nude mice by decellularization by treatment with trypsin followed by Triton X-100. The human ASCs were isolated and cultured from abdominal lipoaspirate. We created a rounded, 8-mm, full-thickness cutaneous wound in nude mice and divided the mice into the following 4 groups: silicon sheet cover only, silicon sheet with spreading ASCs, ADM only, and ASCs seeded on ADM. The granulation thickness was evaluated by histology after 7 days. Further comparisons between the ADM only and ASC-seeded ADM groups were undertaken by assessing the reepithelialization ratio and blood vessel density at postoperative days 9 and 14. Statistical analyses were conducted using Student 2-tailed t test. Immunofluorescent histology and ASC labeling were also performed to identify possible mechanisms. ResultsThe ADM was successfully prepared, and the cytometry analysis and differentiation assay provided the characterization of the human ASCs. A marked improvement in granulation thickness was detected in the ADM-ASC group in comparison with other 3 groups. A significantly increased rate of reepithelialization in the ADM-ASC group (80 ± 6%) compared to the ADM only group (60 ± 7%) was noted on postoperative day 9. The blood vessel density was evidently increased in the ADM-ASC group (7.79 ± 0.40 vessels per field) compared to the ADM only group (5.66 ± 0.23 vessels) on day 14. Cell tracking experiments demonstrated that labeled ASCs were colocalized with staining for VEGF or endothelial cell maker vWF after the transplantation of ADM-ASCs on postoperative day 14. ConclusionsAdipose-derived stem cells seeded on an ADM can enhance wound healing, promote angiogenesis, and contribute to newly formed vasculature, and VEGF-expressing ASCs can be detected after transplantation. This model could be used to improve the other clinical applications of ASCs and to decipher the detailed mechanism by which ASCs interact with wound tissue.

Molecular pathogenesis of Gilbert's syndrome: decreased Tata-binding protein binding affinity of ugt1a1 gene promoter

Objectives Gilberts syndrome is a congenital, nonhemolytic, unconjugated hyperbilirubinemia. The most common genotype of Gilberts syndrome is the homozygous polymorphism, A(TA)7TAA, in the promoter of the gene for UDP-glucuronosyltransferase 1A1 (UGT1A1), with a thymine adenine insertion in the TATA-box-like sequence, which results in a decrease in UGT1A1 activity. The mechanism responsible for this decrease in UGT1A1 activity, however, has not been elucidated. To clarify the mechanism underlying this deficiency in UGT1A1 activity in patients with Gilberts syndrome. Methods The promoter activity assay using the wild-type A(TA)6TAA or the mutant A(TA)7TAA promoter and a luciferase reporter was performed in two different hepatoma cell lines. The binding affinity for a nuclear protein complex or for TATA-binding protein was evaluated by a competitive electophoretic mobility shift assay using wild-type or mutant TATA-box-like oligonucleotide probes and nuclear extract or TATA-binding protein. The formation of complexes between TATA-binding protein and wild-type or mutant oligonucleotide probes was also studied by a quantitive electophoretic mobility shift assay. Results A TA insertion in the TATA-box-like sequence of the promoter activity of UGT1A1 gene. A competitive electrophoretic mobility shift assay showed a decrease in nuclear proteincomplex binding affinity and TATA-binding protein binding affinity of the mutant TATA-box-like sequence A(TA)7TAA. When the mutants A(TA)5TAA and A(TA)8TAA were also compared, quantitative electrophoretic mobility shift assay demonstrated that the TATA-binding protein binding affinity progressively decreased as the number of TA repeats in the TATA-box-like sequence increased. Conclusion TA insertion in the TATA-box-like sequence of the UGT1A1 promoter affected its binding affinity for TATA-binding protein, causing a decrease in its activity. This explains the pathogenesis of Gilberts syndrome.

Inhibition of Nodal suppresses angiogenesis and growth of human gliomas

Angiogenesis is the hallmark of malignant gliomas positively correlated with the vascular endothelial growth factor (VEGF) expression. We previously reported that expression levels of Nodal, a member of transforming growth factor-β super family, correlate with the malignant invasive behavior of human glioma cells. In this study, we show that knockdown of Nodal suppresses glioma angiogenesis by inhibition of VEGF. In human primary glioma specimens, expression of Nodal positively correlates with WHO glioma tumor grades and expression of VEGF in the corresponding glioma specimens. In human U87MG glioma cells, knockdown of endogenous Nodal by RNA interference (RNAi) significantly decreases colony formation and secretion of VEGF. In vivo, cellular depletion of Nodal in U87MG inhibited brain glioma growth and prolonged the survival of mice with U87MG/shNodal glioma compared with controls. Inhibition of Nodal suppressed tumor vessel growth in U87MG gliomas. Using Nodal inhibitor (SB431542), silencing Nodal, or overexpressing Nodal in the U87MG, GBM8401, and GBM glioma cells, our further experiments revealed that Nodal-induced VEGF expression might, at least in part, mediate through the ERK1/2-HIF-1α-mediated signaling pathway. Taken together, our data revealed that alteration of Nodal expression in glioma cells resulted in changes to VEGF secretion, and subsequent colony formation, in vivo tumor growth, and angiogenesis, all of which are consistent with the regulation of VEGF through the ERK1/2-HIF-1α-mediated signaling, suggesting that Nodal may serve as a potential therapeutic target for the treatment of human gliomas.

Isolation and characterization of tumor stem-like cells from human meningiomas

Recent advances in research have found that tumor stem-like cells are resistant to surgery, radiotherapy, and chemotherapy. Tumor stem-like cells play critical roles in tumor recurrence, angiogenesis, and invasion in malignant brain tumors. However, the identification of tumor stem-like cells in meningiomas has not been clarified. In this study, we identified the stem-like features of sphere-forming cells in human meningiomas. The results showed that meningioma stem-like cells possess the ability to form spheres in identical stem cell culture condition. These meningioma sphere cells (MgSCs) expressed progenitor cell marker, CD133, but not differentiated cell marker, epithelial membrane antigen (EMA) on immunofluorescence staining. Importantly, the mRNA expression of ABCG, and CD133 was higher in MgSCs than daughter meningioma adherent cells (MgACs). In addition, cells displayed chemotherapeutic resistance to vincristine more in MgSCs than MgACs. This phenomenon was also found in single cell form from dissociated spheres than MgACs. Moreover, MgSCs are more resistant to irradiation treatment than MgACs. Furthermore, MgSCs revealed high tumorigenicity in vivo following orthotopic inoculation into the brains of immunodeficient mice. The corresponding immunohistochemical (IHC) staining found that MgSCs are positive for EMA, vimentin, and CD133, consistent with IHC of primary human meningiomas. These findings have provided better understanding of meningioma cell biology and suggested that meningioma stem-like cells may serve as a novel target in therapeutic resistant meningiomas.

Antiangiogenesis Targeting Tumor Microenvironment Synergizes Glucuronide Prodrug Antitumor Activity

Purpose: This study is aimed at investigating the in vivo antitumor activity of a novel cell-impermeable glucuronide prodrug, 9-aminocamptothecin glucuronide (9ACG), and elucidating the synergistically antitumor effects of antiangiogenesis therapy by targeting the tumor microenvironment. Experimental Design: We analyzed the antitumor effects of 9ACG alone or combined with antiangiogenic monoclonal antibody DC101 on human tumor xenografts by measuring tumor growth and mouse survival in BALB/c nu/nu nude and NOD/SCID mice. The drug delivery, immune response, and angiogenesis status in treated tumors were assessed by high performance liquid chromatography, immunohistochemistry, and immunofluorescence assays. Results: We developed a nontoxic and cell-impermeable glucuronide prodrug, 9ACG, which can only be activated by extracellular β-glucuronidase to become severely toxic. 9ACG possesses potent antitumor activity against human tumor xenografts in BALB/c nu/nu nude mice but not for tumors implanted in NOD/SCID mice deficient in macrophages and neutrophils, suggesting that these cells play an important role in activating 9ACG in the tumor microenvironment. Most importantly, antiangiogenic monoclonal antibody DC101 potentiated single-dose 9ACG antitumor activity and prolonged survival of mice bearing resistant human colon tumor xenografts by providing strong β-glucuronidase activity and prodrug delivery through enhancing inflammatory cell infiltration and normalizing tumor vessels in the tumor microenvironment. We also show that inflammatory cells (neutrophils) were highly infiltrated in advanced human colon cancer tissues compared with normal counterparts. Conclusions: Our study provides in vivo evidence that 9ACG has potential for prodrug monotherapy or in combination with antiangiognesis treatment for tumors with infiltration of macrophage or neutrophil inflammatory cells.

Resveratrol increases anti-aging Klotho gene expression via the activating transcription factor 3/c-Jun complex-mediated signaling pathway.

The Klotho gene functions as an aging suppressor gene. Evidence from animal models suggests that induction of Klotho expression may be a potential treatment for age-associated diseases. However, the molecular mechanism involved in regulating renal Klotho gene expression remains unclear. In this study, we determined that resveratrol, a natural polyphenol, induced renal Klotho expression both in vivo and in vitro. In the mouse kidney, resveratrol administration markedly increased both Klotho mRNA and protein expression. In resveratrol-treated NRK-52E cells, increased Klotho expression was accompanied by the upregulation and nuclear translocation of activating transcription factor 3 (ATF3) and c-Jun. ATF3 or c-Jun overexpression enhanced the transcriptional activation of Klotho. Conversely, resveratrol-induced Klotho expression was attenuated in the presence of dominant-negative ATF3 or c-Jun. Coimmunoprecipitation and a chromatin immunoprecipitation assay revealed that ATF3 physically interacted with c-Jun and that the ATF3/c-Jun complex directly bound to the Klotho promoter through ATF3- and AP-1-binding elements. c-Jun cotransfection augmented the effects of ATF3 on Klotho transcription in vitro. Although Sirtuin 1 mRNA expression was induced by resveratrol and involved in regulating Klotho mRNA expression, it was not the primary cause for the aforementioned ATF3/c-Jun pathway. In summary, resveratrol enhances the renal expression of the anti-aging Klotho gene, and the transcriptional factors ATF3 and c-Jun functionally interact and coordinately regulate the resveratrol-mediated transcriptional activation of Klotho.

Modulation of the Cyclin-Dependent Kinase Inhibitor p21WAF1/Cip1 Gene by Zac1 through the Antagonistic Regulators p53 and Histone Deacetylase 1 in HeLa Cells

Zac1 is a novel seven–zinc finger protein which possesses the ability to bind specifically to GC-rich DNA elements. Zac1 not only promotes apoptosis and cell cycle arrest but also acts as a transcriptional cofactor for p53 and a number of nuclear receptors. Our previous study indicated that the enhancement of p53 activity by Zac1 is much more pronounced in HeLa cells compared with other cell lines tested. This phenomenon might be due to the coactivator effect of Zac1 on p53 and the ability of Zac1 to reverse E6 inhibition of p53. In the present study, we showed that Zac1 acted synergistically with either p53 or a histone deacetylase inhibitor, trichostatin A, to enhance p21WAF1/Cip1 promoter activity. We showed that Zac1 physically interacted with some nuclear receptor corepressors such as histone deacetylase 1 (HDAC1) and mSin3a, and the induction of p21WAF1/Cip1 gene and protein by Zac1 was suppressed by either overexpressing HDAC1 or its deacetylase-dead mutant. In addition, our data suggest that trichostatin A–induced p21WAF1/Cip1 protein expression might be mediated through a p53-independent and HDAC deacetylase–independent pathway. Taken together, our data suggest that Zac1 might be involved in regulating the p21WAF1/Cip1 gene and protein expression through its protein-protein interaction with p53 and HDAC1 in HeLa cells. (Mol Cancer Res 2008;6(7):1204–14)

Importin α1 is involved in the nuclear localization of Zac1 and the induction of p21WAF1/CIP1 by Zac1

Zac1, a novel seven-zinc-finger transcription factor, preferentially binds GC-rich DNA elements and has intrinsic transactivation activity. To date, the NLS (nuclear localization signal) of Zac1 has not been empirically determined. We generated a series of EGFP (enhanced green fluorescence protein)-tagged deletion mutants of Zac1 and examined their subcellular localization, from which we defined two NLSs within the DNA-binding (or zinc-finger) domain. Fusion proteins consisting of the two EGFP-tagged zinc-finger clusters (zinc finger motifs 1-3 and 4-7) were located exclusively in the nucleus, demonstrating that each of the zinc-finger clusters is sufficient for nuclear localization. Physical interactions between these two zinc-finger clusters and importin alpha1 were demonstrated using an in vitro glutathione S-transferase pull-down assay. Finally, our results indicate that the association of Zac1 with importin alpha1 is also involved in regulating the transactivation activity of Zac1 on the p21WAF1/CIP1 gene and protein expression.

Hepatitis C Virus Core Protein Down-Regulates p21Waf1/Cip1 and Inhibits Curcumin-Induced Apoptosis through MicroRNA-345 Targeting in Human Hepatoma Cells

Background Hepatitis C virus (HCV) has been reported to regulate cellular microRNAs. The HCV core protein is considered to be a potential oncoprotein in HCV-related hepatocellular carcinoma, but HCV core-modulated cellular microRNAs are unknown. The HCV core protein regulates p21Waf1/Cip1 expression. However, the mechanism of HCV core-associated p21Waf1/Cip1 regulation remains to be further clarified. Therefore, we attempted to determine whether HCV core-modulated cellular microRNAs play an important role in regulating p21Waf1/Cip1 expression in human hepatoma cells. Methods Cellular microRNA profiling was investigated in core-overexpressing hepatoma cells using TaqMan low density array. Array data were further confirmed by TaqMan real-time qPCR for single microRNA in core-overexpressing and full-length HCV replicon-expressing cells. The target gene of microRNA was examined by reporter assay. The gene expression was determined by real-time qPCR and Western blotting. Apoptosis was examined by annexin V-FITC apoptosis assay. Cell cycle analysis was performed by propidium iodide staining. Cell proliferation was analyzed by MTT assay. Results HCV core protein up- or down-regulated some cellular microRNAs in Huh7 cells. HCV core-induced microRNA-345 suppressed p21Waf1/Cip1 gene expression through targeting its 3′ untranslated region in human hepatoma cells. Moreover, the core protein inhibited curcumin-induced apoptosis through p21Waf1/Cip1-targeting microRNA-345 in Huh7 cells. Conclusion and Significance HCV core protein enhances the expression of microRNA-345 which then down-regulates p21Waf1/Cip1 expression. It is the first time that HCV core protein has ever been shown to suppress p21Waf1/Cip1 gene expression through miR-345 targeting.