Suppressed expression of NDRG2 correlates with poor prognosis in pancreatic cancer

Pancreatic cancer is a highly lethal disease with a poor prognosis; the molecular mechanisms of the development of this disease have not yet been fully elucidated. N-myc downstream regulated gene 2 (NDRG2), one of the candidate tumor suppressor genes, is frequently downregulated in pancreatic cancer, but there has been little information regarding its expression in surgically resected pancreatic cancer specimens. We investigated an association between NDRG2 expression and prognosis in 69 primary resected pancreatic cancer specimens by immunohistochemistry and observed a significant association between poor prognosis and NDRG2-negative staining (P = 0.038). Treatment with trichostatin A, a histone deacetylase inhibitor, predominantly up-regulated NDRG2 expression in the NDRG2 low-expressing cell lines (PANC-1, PCI-35, PK-45P, and AsPC-1). In contrast, no increased NDRG2 expression was observed after treatment with 5-aza-2′ deoxycytidine, a DNA demethylating agent, and no hypermethylation was detected in either pancreatic cancer cell lines or surgically resected specimens by methylation specific PCR. Our present results suggest that (1) NDRG2 is functioning as one of the candidate tumor-suppressor genes in pancreatic carcinogenesis, (2) epigenetic mechanisms such as histone modifications play an essential role in NDRG2 silencing, and (3) the expression of NDRG2 is an independent prognostic factor in pancreatic cancer.     

Clinical and pathological significance of N-Myc downstream-regulated gene 2 (NDRG2) in diverse human cancers

Human N-Myc downstream-regulated gene 2 (NDRG2), located at chromosome 14q11.2, has been reported to be down-regulated and associated with the progression and prognosis of diverse cancers. Collectively, previous studies suggest that NDRG2 functions as a candidate tumor-suppressor gene; thus, up-regulation of NDRG2 protein might act as a promising therapeutic strategy for malignant tumors. The aim of this review was to comprehensively present the clinical and pathological significance of NDRG2 in human cancers.     

Podocalyxin is crucial for the growth of oral squamous cell carcinoma cell line HSC-2

Oral cancers constitute approximately 2% of all cancers, with the most common histological type being oral squamous cell carcinoma (OSCC), representing 90% of oral cancers. Although diagnostic technologies and therapeutic techniques have progressed, the survival rate of patients with OSCC is still 60%, whereas the incidence rate has increased. Podocalyxin (PODXL) is a highly glycosylated type I transmembrane protein that is detected in normal tissues such as heart, breast, and pancreas as well as in many cancers, including lung, renal, breast, colorectal, and oral cancers. This glycoprotein is associated with the progression, metastasis, and poor outcomes of oral cancers. PODXL overexpression was strongly detected using our previously established anti-PODXL monoclonal antibody (mAb), PcMab-47, and its mouse IgG_2a-type, 47-mG_2a. In previous studies, we also generated PODXL-knock out (PODXL-KO) cell lines using SAS OSCC cell lines, in order to investigate the function of PODXL in the proliferation of oral cancer cells. The growth of SAS/PODXL-KO cell lines was observed to be lower than that of parental SAS cells. For this study, PODXL-KO OSCC cell lines were generated using HSC-2 cells, and the role of PODXL in the growth of OSCC cell lines in vitro was assessed. Decreased growth was observed for HSC-2/PODXL-KO cells compared with HSC-2 parental cells. The influence of PODXL on tumor growth of OSCC was also investigated in vivo, and both the tumor volume and the tumor weight were observed to be significantly lower for HSC-2/PODXL-KO than that for HSC-2 parental cells. These results, taken together, indicate that PODXL plays an important role in tumor growth, both in vitro and in vivo.     

Chemoresistance to Concanamycin A1 in Human Oral Squamous Cell Carcinoma Is Attenuated by an HDAC Inhibitor Partly via Suppression of Bcl-2 Expression

V-ATPase is involved in the acidification of the microenvironment around/in solid tumors, such as oral squamous cell carcinoma (OSCC). V-ATPase is thought to induce tumor invasion and multi-drug resistance in several malignant tumors, and it also contributes to maintaining the intracellular pH under an acidic microenvironment by inducing proton extrusion into the extracellular medium. However, there is little information regarding the effects of V-ATPase inhibitors on OSCCs. In this study, the effects of a V-ATPase inhibitor, concanamycin A1 (CMA), on the proliferation and apoptosis of OSCC were investigated in vitro. We used four OSCC cell lines, MISK81-5, SAS, HSC-4 and SQUU-B. Acridine orange staining revealed that the red fluorescence was reduced in all of the low concentration CMA-treated OSCC cells, indicating that the acidification of vesicular organelles in the OSCCs was prevented by the treatment with low-concentration of CMA. CMA treatment induced apoptosis in MISK81-5, SAS and HSC-4 cells, but not in SQUU-B cells. The p-p38 expression was not altered in CMA-treated SQUU-B cells, but their levels were increased in the other cells. The Bax/Bcl-2 ratio in CMA-treated SQUU-B cells was dramatically decreased in comparison with that in the other cell lines treated with CMA. However, when the SQUU-B cells were treated with CMA and a histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA), the SQUU-B cells became more susceptible to the CMA-induced apoptosis. SAHA treatment led to a significantly decrease in the Bcl-2 expression in CMA-treated SQUU-B cells, resulting in a dramatically increased Bax/Bcl-2 ratio in comparison with that observed in the SQUU-B cells treated with CMA alone. These findings suggest that CMA could have an anti-tumor effect on OSCCs. In addition, combination of CMA with other agents, such as SAHA, could help improve the pro-apoptotic effects of CMA even in CMA-resistant OSCC cells.     

Over-expression of PTEN on proliferation and apoptosis in canine mammary tumors cells

Phosphatase and tensin homolog (PTEN) is an important tumor-suppressor gene which constitutes an important PI3K/Akt pathway by regulating the signaling of multiple biological processes, including apoptosis, metabolism, cell proliferation, and cell growth has been gaining increasing attention. However, the role of PTEN in regulating apoptosis of canine mammary tumors cells still needs further investigation. In this experiment, the effect of PTEN on proliferation and apoptosis in canine mammary tumors (CMT) cells was analyzed. As a result, gene and protein expression levels of apoptosis-related genes were detected. Eukaryotic expression vector pcDNA3.1+-PTEN were successfully constructed and stably transferred into canine CMT cells after geneticin (G418) selection. After pcDNA3.1+-PTEN transfection, compared with control group, the cells proliferation was inhibited and the cell apoptosis was increased in CMT cells. The expression of p-Akt was decreased and the apoptosis-related genes, such as caspase-3, caspase-9, and Bax, were increased. These data serve to demonstrate the function of PTEN on apoptosis and gene regulatory in PI3K/Akt pathway in CMT cells. Collectively, our data link the tumor-suppressor activities of PTEN to the machinery controlling cell cycle through the modulation of signaling molecules whose signal target is the functional inactivation of the apoptosis gene product.     

G15, a GPR30 antagonist,induces apoptosis and autophagy in human oral squamous carcinoma cells

As GPR30 has been implicated in mediating cancer cell proliferation, this study aimed to examine the antitumor effect of the GPR30 antagonist G15 in human oral squamous cell carcinoma (OSCC). G15 induced dose-dependent cytotoxicity, apoptosis and G2/M cell cycle arrest in a panel of OSCC cells. The results showed that G15 could inhibit the growth of the oral cancer cells with IC₅₀ value 11.2 μM for SCC4, 15.6 μM for SCC9, and 7.8 μM for HSC-3, respectively. Flow cytometric analysis and Comet assay indicated that G15 suppressed the viability of SCC4 and HSC-3 cells by inducing apoptosis and G2/M arrest. In addition, G15 down regulated the expression of Akt, cell cycle-related proteins, and mitogen-activated protein kinases, but increased the levels of LC3B-II and the accumulation of autophagosomes. Inhibition of autophagy by chloroquine does not affect the G15-induced apoptosis in SCC4 cells. Mechanistic evidence indicated that the antiproliferative effect was mediated through the downregulation of cdc2, cdc25c and NF-κB expression. Taken together, our findings suggest the potential of G15 in treating OSCC.     

Role of EZH2 in oral squamous cell carcinoma carcinogenesis

Oral squamous cell carcinoma (OSCC) is a common human malignancy with high incidence rate and poor prognosis. Although the polycomb group protein enhancer of zeste homolog 2 (EZH2) plays a crucial role in cell proliferation and differentiation during the occurrence and development progress of several kinds of malignant tumors, the impact of EZH2 on the development and progression of OSCC is unclear. In this study, we demonstrate that EZH2 is overexpressed in OSCC cells and clinical tissue. With in vitro RNAi analysis, we generated stable EZH2 knocking down cell lines from two OSCC cell lines, with two sh-RNAs targeting to EZH2, respectively. We found that knocking down of EZH2 could decrease the proliferation ability and induce apoptosis of OSCC cells. Moreover, we demonstrated that of EZH2 inhibition decreased the migration and metastasis of OSCC cells. In conclusion, the results of the current study demonstrated an association between EZH2 expression and OSCC cell development. We recommend that EZH2 acts as an oncogene and plays an important role in OSCC carcinogenesis.     

NDRG2 is one of novel intrinsic factors for regulation of IL-10 production in human myeloid cell

N-myc downstream-regulated gene 2 (NDRG2) implicated in cellular growth and differentiation was previously reported as it is specifically expressed in primary and in vitro-differentiated dendritic cells (DCs) from monocytes and CD34⁺ progenitor cells. However, its function has yet to be investigated in DCs. Here, the novel NDRG2 function about modulation of cytokines in DC was observed in this study. The secretion of IL-10 was not found in the monocyte-derived DC cells with high level of NDRG2 expression, but IL-10 was abundantly secreted up to 1 ng/ml in the monocyte-derived macrophages with low level of NDRG2 expression, and further confirmed that the expression of IL-10 was dramatically increased in NDRG2-silenced DCs under presence of LPS, and significantly reduced in the NDRG2-overexpressed U937 cells under stimulation of PMA. The secretion of IL-12p70 was significantly reduced in the siNDRG2 introduced DC cells. The intracellular signaling of IL-10 secretion was markedly inhibited by SB203580, inhibitor of p38 MAPK, in the LPS-activated DCs and phosphorylation of p38 MAPK was decreased in the NDRG2 introduced U937 cells under PMA-stimulation. Taken together, NDRG2 might have a pivotal role as one of intrinsic factors for the modulation of p38 MAPK phosphorylation, and subsequently involve in controlling of IL-10 production.     

Akt基因转染对骨髓间充质干细胞缺氧时凋亡和增殖的影响

目的采用Akt基因转染鼠骨髓MSCs探讨Akt基因是否减轻MSCs缺氧时的凋亡和提高缺氧时的增殖能力,即耐缺氧能力。方法将转染和未转染Akt基因的MSCs置于94%N2、1%O2和5%CO2缺氧箱中37℃孵育不同时间（常氧、缺氧0.5h、1h、2h、4h和8h）后,Annexin V/PI双染法行流式细胞仪分析凋亡率（apoptoticrate,AR）和死亡率（deadrate,DR）、MTT法分析细胞增殖状态、Rt-PCR和Western blot等检测Akt和p-Akt表达以及放射同位素法检测MSCs对氚标-葡萄糖（^3H-G）的摄取等。结果1.Akt基因显著降低MSCs缺氧时AR和DR（P〈0.01）,而各缺氧时间点没有统计学意义（P〉0.05）;2.Akt基因显著增高MSCs常氧和缺氧（与未转染Akt基因MSCs同等条件下比较）时增殖能力（P〈0.01）,缺氧时增殖能力显著低于常氧时（P〈0.01）;3.Akt基因显著增高常氧时MSCsAkt mRNA（P〈0.01）和蛋白（P〈0.01）表达,而不增高p-Akt蛋白（P〉0.05）表达;Akt基因显著提高缺氧时p-Akt蛋白（P〈0.01）表达,而不提高常氧时p-Akt蛋白（P〉0.05）表达;4.Akt基因显著增高MSCs常氧和缺氧（与未转染Akt基因MSCs同等条件下比较）时^3H-G的摄取（P〈0.01）,缺氧时^3H-G的摄取显著性低于常氧培养时（P〈0.01）;^3H-G的摄取与细胞增殖显著正相关（r=0.79,P=0.015）而与细胞凋亡显著负相关（r=-1.47,P=0.023）。结论Akt基因转染可显著提高MSCs耐缺氧能力,此可能与缺氧时改善MSCs葡萄糖摄取等有关。     

Knockdown of Akt isoforms by RNA silencing suppresses the growth of human prostate cancer cells in vitro and in vivo

The serine/threonine kinase Akt has three highly homologous isoforms in mammals: Akt1, Akt2, and Akt3. Recent studies indicate that Akt is often constitutively active in many types of human malignancy. Here we investigated the expression and function of Akt isoforms in human prostatic carcinoma cells. Initially, we used Western blotting to examine Akt expression in four human prostate cancer cell lines. Next, small-interfering RNAs (siRNAs) specific for Akt isoforms were used to elucidate their role on the in vitro and in vivo growth of prostate cancer cells. Expression of Akt1 and Akt2 was detected in all cells tested, but Akt3 was expressed only in cancer cells that did not express androgen receptors. All synthetic siRNAs against Akt isoforms suppressed their expression and inhibited the growth of cancer cells in vitro. Furthermore, atelocollagen-mediated systemic administration of siRNAs significantly reduced the growth of tumors that had been subcutaneously xenografted. These results suggest that targeting Akt isoforms could be an effective treatment for prostate cancers.     

Overexpression of N-Myc Downstream-Regulated Gene 2 (NDRG2) Regulates the Proliferation and Invasion of Bladder Cancer Cells In Vitro and In Vivo

N-Myc downstream-regulated gene 2 (NDRG2) is a candidate tumor suppressor gene, which plays an important role in controlling tumor growth. The aim of this study was to investigate the expression of NDRG2 gene in bladder cancer (BC) tissues and several bladder cancer cell lines, and to seek its clinical and pathological significance. Ninety-seven bladder carcinoma and 15 normal bladder tissue sections were analyzed retrospectively with immunohistochemistry. The human bladder cancer cell line T24 was infected with LEN-NDRG2 or LEN-LacZ. The effects of NDRG2 overexpression on T24 cells and T24 nude mouse xenografts were measured via cell growth curves, tumor growth curves, flow cytometric analysis, western blot and Transwell assay. NDRG2 was highly expressed in normal bladder tissue, but absent or rarely expressed in cacinomatous tissues (χ²=8.761, p < 0.01). The NDRG2 level was negatively correlated with tumor grade and pathologic stage(r=-0.248, p < 0.05), as well as increased c-myc level (r=-0.454, p< 0.001). The expression of NDRG2 was low in the three BC cell lines. T24 cells infected with LEN-NDRG2 showed inhibition of proliferation both in vitro and in vivo, and NDRG2 overexpression can inhibit tumor growth and invasion in vitro.     

Characterization of arsenic trioxide resistant clones derived from Jurkat leukemia T cell line: Focus on PI3K/Akt signaling pathway

In this study the role of PI3K/Akt signaling pathway in arsenic trioxide (ATO)-treated parental Jurkat cells and also in derived ATO-resistant clones grown in the presence of given ATO concentration was investigated. ATO-resistant clones (cultured for 8–12 weeks in the presence of 1, 2.5 and 5 μM ATO) were characterized by high viability in the presence of ATO but slower growth rate compared to the parental cells. Morphological and functional characterization of derived ATO-resistant clones revealed that they did not differ fundamentally from parental Jurkat cells in terms of cell size, level of GSH, the lysosomal fluorescence or CD95/Fas surface antigen expression. However, a slight increase in the mitochondrial potential (JC-1 staining) was detected in the clones compared to parental Jurkat cells. Side population analysis (Vybrant DyeCycle Violet™ staining) in ATO resistant clones did not indicate any enrichment withcancer stem cells. Akt1/2, AktV or wortmannin inhibitors decreased viability of ATO-resistant clones grown in the presence of ATO, with no effect on ATO-treated parental cells. Flow cytometry analysis showed that ATO decreased the level of p-Akt in ATO-treated parental cells, while the resistant clones exhibited higher levels of p-Akt immunostaining than parental Jurkat cells. Expression analysis of 84 genes involved in the PI3K/Akt pathway revealed that this pathway was predominantly active in ATO-resistant clones. c-JUN seems to play a key role in the induction of cell death in ATO-treated parental Jurkat cells, as dose-dependent strong up-regulation of JUN was specific for the ATO-treated parental Jurkat cells. On the other hand, changes in expression of cyclin D1 (CCND1), insulin receptor substrate 1 (IRS1) and protein kinase C isoforms (PRKCZ,PRKCB and PRKCA) may be responsible for the induction of resistance to ATO. The changes in expression of growth factor receptor-bound protein 10 (GRB10) observed in ATO-resistant clones suggest a possibility of induction of different mechanisms in development of resistance to ATO depending on the drug concentration and thus involvement of different signaling mediators.     

Frequent mutations in NOTCH1 ligand-binding regions in Japanese oral squamous cell carcinoma

Recent studies showed that head and neck squamous cell carcinoma (HNSCC) including oral squamous cell carcinoma (OSCC) of Caucasian, Chinese and Indian patients frequently have NOTCH1 mutations. We found eight of 84 OSCC in Japanese patients have point mutations (9.5%) correspond to the ligand binding region of NOTCH1 protein. Two set of them are the same mutations and all mutations are non-synonymous G > A transitions. In addition, median disease-free survival is significantly longer in patients with NOTCH1-mutated tumors as compared to those without the mutation (P < 0.05). The protein structure simulation based on X-ray crystallography indicated that new p.A465T mutation leads to a conformational change of NOTCH1 ligand binding domain as well as the p.G481S mutant NOTCH1 with a loss of flexibility around this residue. These results suggest that NOTCH1 mutation occurs frequently in Japanese OSCC in the vicinity of the ligand binding region and, these mutations cause downregulation of the NOTCH1 function.     

Prognostic significance of NDRG1 expression in oral and oropharyngeal squamous cell carcinoma

Human N-myc downstream-regulated gene 1 (NDRG1) is a metastasis suppressor gene with several potential functions, including cell differentiation, cell cycle regulation and response to hormones, nickel and stress. The purpose of this study was to investigate the immunoexpression of NDRG1 in oral and oropharyngeal squamous cell carcinomas searching for its role in the clinical course of these tumors. We investigated immunohistochemical expression of NDRG1 protein in 412 tissue microarray cores of tumor samples from 103 patients with oral and oropharyngeal squamous cell carcinomas and in 110 paraffin-embedded surgical margin sections. The results showed NDRG1 up-regulation in 101/103 (98.1?%) tumor samples, but no expression in any normal tissue sample. Western blot assays confirmed the immunohistochemical findings, suggesting that lower levels of NDRG1 are associated with a high mortality rate. NDRG1 overexpression was related to long-term specific survival (HR?=?0.38; p?=?0.009), whereas the presence of lymph-node metastasis showed the opposite association with survival (HR?=?2.45; p?=?0.013). Our findings reinforce the idea that NDRG1 plays a metastasis suppressor role in oral and oropharyngeal squamous cell carcinomas and may be a useful marker for these tumors.