Inhibited Tumor Cell Colony Formation Research Articles

Raltitrexed regulates proliferation and apoptosis of HGC-27 cells by upregulating RSK4

BackgroundRaltitrexed is a specific inhibitor of thymidylate synthase and a potential chemotherapeutic agent for the treatment of advanced gastric cancer. In this study, we investigated the effect of raltitrexed on the proliferation of HGC-27 human gastric cancer cells and its potential underlying molecular mechanism(s).MethodsRT-qPCR and western blotting were used to quantify RSK4 levels. Colony formation and flow cytometry assays were used to assess HGC-27 cell proliferation, cell cycle progression, mitochondrial membrane potential, and apoptosis. The expression of cell cycle and apoptosis markers were determined by western blotting.ResultsOur results demonstrate that raltitrexed upregulated RSK4 mRNA and protein levels in HGC-27 cells. Moreover, raltitrexed significantly inhibited tumor cell colony formation, arrested the cell cycle, decreased the mitochondrial membrane potential, and induced apoptosis. We observed that raltitrexed was capable of upregulating the expression of Bax, cyclin A1, and CDK3, and downregulating the expression of Bcl-2 and cleaved caspase-3. Importantly, siRNA-mediated RSK4 knockdown significantly reduced the inhibitory effect of raltitrexed on cell proliferation and its promotion of cell apoptosis. Moreover, silencing of RSK4 inhibited the raltitrexed-induced upregulation of cytochrome C. In addition, the changes in molecular markers related to the cell cycle and apoptosis induced by raltitrexed were reduced upon RSK4 depletion.ConclusionOur study shows that RSK4 is a key target of raltitrexed in the regulation of gastric cancer cell proliferation, cell cycle progression, and apoptosis.

Zinc-finger protein 382 antagonises CDC25A and ZEB1 signaling pathway in breast cancer

Our previous studies found that Zinc-finger protein 382 (ZNF382) played as a tumor suppressor gene in esophageal and gastric cancers, and a positive correlation between the high expression of ZNF382 and better outcome in breast cancer patients. However, the biological roles and mechanisms of ZNF382 in breast cancer remains unclear. We detected ZNF382 expression by reverse-transcription PCR (RT-PCR) and real-time quantitative PCR (qRT-PCR) in breast cancer cells and tissues, and explored the impacts and mechanisms of ectopic ZNF382 expression in breast cancer cells in vitro and in vivo, respectively. Our results revealed that ZNF382 was significantly down-regulated in breast cancer tissues compared with adjacent non-cancer tissues. Restoration of ZNF382 expression in silenced breast cancer cells not only inhibited tumor cell colony formation, viability, migration and invasion, and epithelial-mesenchymal-transition (EMT), but also induced apoptosis and G0/G1 arrest. In conclusion, ZNF382 could induce G0/G1 cell cycle arrest through inhibiting CDC25A signaling, and, inhibit cell migration, invasion and EMT by antagonizing ZEB1 signaling in breast cancer cells.

Synthesis and anticancer activity of ethyl 5-amino-1-N-substituted-imidazole-4-carboxylate building blocks.

A series of 5-amino-1-N-substituted-imidazole-4-carboxylate building blocks was synthesized and assayed for their antiproliferative potential against human cancer cell lines, including HeLa (cervical), HT-29, HCT-15 (colon), A549 (lung), and MDA-MB-231 (breast) cells. The preliminary screening results revealed that several derivatives containing alkyl chains at the N-1 position of the imidazole core demonstrate a certain inhibitory effect on growth and proliferation. A significant effect was observed following ethyl 5-amino-1-dodecyl-1H-imidazole-4-carboxylate (5e) treatment for 72 h. The IC50 value for HeLa cells was 0.737 ± 0.05 μM, whereas that for HT-29 cells was 1.194 ± 0.02 μM. Further investigations revealed that 5e significantly inhibited tumor cell colony formation and migration, and it exhibited antiadhesive effects on HeLa cells as well as antitubulin activity along with the induction of early apoptosis of HeLa and HT-29 cells. In addition, derivative 5e significantly reduced the cell mitochondrial membrane potential in a dose-dependent manner and induced early apoptosis of HeLa and HT-29 cells, indicating that 5e may serve as a lead compound for further drug discovery and development.

19q13 KRAB zinc-finger protein ZNF471 activates MAPK10/JNK3 signaling but is frequently silenced by promoter CpG methylation in esophageal cancer.

Zinc-finger proteins (ZFPs) are the largest transcription factor family in mammals, involved in the regulation of multiple physiologic processes including cell differentiation, proliferation, apoptosis and neoplastic transformation. Approximately one-third of ZFPs are Krüppel-associated box domain (KRAB)-ZFPs.Methods: ZNF471 expression and methylation were detected by reverse-transcription PCR and methylation-specific PCR. The impact and mechanism of ectopic ZNF471 expression in esophageal squamous cell carcinoma (ESCC) cells was evaluated in vitro and in vivo.Results: We identified a 19q13 KRAB-ZFP, ZNF471, as a methylated target in ESCC. We further found that ZNF471 is significantly downregulated in ESCC tissues compared with adjacent non-cancer tissues, due to its aberrant promoter CpG methylation, and further confirmed by methylation analysis and treatment with demethylation agent. Restoration of ZNF471 expression in silenced ESCC cells significantly altered cell morphology, induced apoptosis and G0/G1 arrest, and inhibited tumor cell colony formation, viability, migration and invasion. Importantly, ZNF471 was found to activate the expression of MAPK10/JNK3 and PCDH family genes, and further enhance MAPK10 signaling and downstream gene expression through binding to the MAPK10/JNK3 promoter.Conclusion: Our results demonstrate that ZNF471 is an important tumor suppressor and loss of ZNF471 functions hampers MAPK10/JNK3 signaling during esophageal carcinogenesis.

A novel uPAg-KPI fusion protein inhibits the growth and invasion of human ovarian cancer cells in vitro.

Urokinase-type plasminogen activator (uPA) acts by breaking down the basement membrane and is involved in cell proliferation, migration and invasion. These actions are mediated by binding to the uPA receptor (uPAR) via its growth factor domain (GFD). The present study evaluated the effects of uPAg-KPI, a fusion protein of uPA-GFD and a kunitz protease inhibitor (KPI) domain that is present in the amyloid β-protein precursor. Using SKOV-3 cells, an ovarian cancer cell line, we examined cell viability, migration, invasion and also protein expression. Furthermore, we examined wound healing, and migration and invasion using a Transwell assay. Our data showed that uPAg-KPI treatment reduced the viability of ovarian cancer SKOV-3 cells in both a concentration and time-dependent manner by arresting tumor cells at G1/G0 phase of the cell cycle. The IC50 of uPAg-KPI was 0.5 µg/µl after 48 h treatment. At this concentration, uPAg-KPI also inhibited tumor cell colony formation, wound closure, as well as cell migration and invasion capacity. At the protein level, western blot analysis demonstrated that uPAg-KPI exerted no significant effect on the expression of total extracellular signal-regulated kinase (ERK)1/ERK2 and AKT, whereas it suppressed levels of phosphorylated ERK1/ERK2 and AKT. Thus, we suggest that this novel uPAg-KPI fusion protein reduced cell viability, colony formation, wound healing and the invasive ability of human ovarian cancer SKOV-3 cells in vitro by regulating ERK and AKT signaling. Further studies using other cell lines will confirm these findings.

Pretreatment with the γ-secretase inhibitor DAPT sensitizes drug-resistant ovarian cancer cells to cisplatin by downregulation of Notch signaling

Notch signaling is implicated in ovarian cancer tumorigenesis and inhibition of Notch signaling with γ-secretase inhibitor DAPT resulted in reduction of tumor cell viability and induction of apoptosis in ovarian cancer cells. This study investigated whether DAPT has the same effect on ovarian cancer cells that are resistant to cisplatin and the underlying molecular events. Ovarian cancer cell lines resistant to cisplatin were treated with DAPT, cisplatin or combination for cell viability MTT, flow cytometric cell cycle, ELISA apoptosis and colony formation assays. qRT-PCR and western blotting were used to detect gene expressions. We found that pretreatment of ovarian cancer cisplatin-resistant cell lines with DAPT for 24 h and then with cisplatin for 72 h showed a synergistic antitumor activity in these cell lines, while cisplatin treatment and then addition of DAPT just showed an additive or antagonistic effects on these cisplatin-resistant ovarian cancer cells. Moreover, pretreatment of ovarian cancer cell lines with DAPT and then with cisplatin also inhibited tumor cell colony formation capacity, arrested tumor cells at G2 phase of the cell cycle and induced apoptosis. The cell cycle and apoptosis-related genes, such as cyclin B1, Bcl-2 and caspase-3, were also modulated by the treatment. Pretreatment of ovarian cancer cell lines with DAPT and then with cisplatin downregulated Notch1 and Hes1 expression dose- and time-dependently. The current data demonstrate that DAPT pretreatment was able to sensitize cisplatin-resistant human ovarian cancer cells to cisplatin by downregulation of Notch signaling.

NOR1 is an HSF1- and NRF1-regulated putative tumor suppressor inactivated by promoter hypermethylation in nasopharyngeal carcinoma

Promoter hypermethylation-mediated silencing of tumor suppressor genes (TSGs) is a hallmark of oncogenesis. Oxidored-nitro domain-containing protein 1 (NOR1) is a candidate TSG that is downregulated in nasopharyngeal carcinoma (NPC). In the present study, we identified a functional NOR1 promoter that is regulated by heat shock factor 1 and nuclear respiratory factor 1. The promoter is located within a CpG island. Hypermethylation of this CpG island was found in NPC tissue samples and cancer cell lines, whereas no aberrant promoter methylation was detected in non-cancerous nasopharyngeal tissue samples or normal nasopharyngeal epithelial cells. Treatment of NPC 6-10B cells and leukemia HL60 cells with 5'-aza-2'-deoxycytidine increased endogenous levels of NOR1 messenger RNA. Ectopic expression of NOR1 in NPC HNE1 cells inhibited tumor cell colony formation and viability. These findings suggest that promoter hypermethylation may participate in transcriptional inactivation of the NOR1 gene in NPC. Frequent epigenetic inactivation of the NOR1 gene in NPC suggests that it may be a critical tumor suppressor involved in the development of NPC.

Sorafenib Enhances the Antitumor Effects of Chemoradiation Treatment by Downregulating ERCC-1 and XRCC-1 DNA Repair Proteins

Head and neck squamous cell carcinoma remains a challenging clinical problem because of the persisting high rate of local and distant failure due to the acquisition of chemo- and radioresistance. In this study, we examined if treatment with sorafenib, a potent inhibitor of Raf kinase and VEGF receptor, could reverse the resistant phenotype in tumor and tumor-associated endothelial cells, thereby enhancing the therapeutic efficacy of currently used chemoradiation treatment. We used both in vitro and in vivo models to test the efficacy of sorafenib either as a single agent or in combination with chemoradiation. Sorafenib, as a single agent, showed antitumor and angiogenesis properties, but the effects were more pronounced when used in combination with chemoradiation treatment. Sorafenib significantly enhanced the antiproliferative effects of chemoradiation treatment by downregulating DNA repair proteins (ERCC-1 and XRCC-1) in a dose-dependent manner. In addition, combination treatment significantly inhibited tumor cell colony formation, tumor cell migration, and tumor cell invasion. Combination treatment was also very effective in inhibiting VEGF-mediated angiogenesis in vitro. In a severe combined immunodeficient mouse xenograft model, combination treatment was very well tolerated and significantly inhibited tumor growth and tumor angiogenesis. Interestingly, following combination treatment, low-dose sorafenib treatment alone was highly effective as a maintenance regimen. Taken together, our results suggest a potentially novel strategy to use sorafenib to overcome chemo- and radioresistance in tumor and tumor-associated endothelial to enhance the effectiveness of the chemoradiation therapy.

Abstract 2191: The Wnt/DVL signaling antagonist DACT2 is a pro-apoptotic tumor suppressor epigenetically silenced in multiple tumors and inhibits tumor cell proliferation and migration

Abstract Wnt/DVL/β-catenin signaling pathway is aberrantly activated in a wide range of human cancers which frequently involves the disruption of its negative regulators, hence we would like to identify tumor suppressor as the antagonist to this signaling pathway. We identified a DACT (Dpr/Frodo) family member DACT2 as a downregulated gene in human tumors. Further epigenetic study revealed that, in contrast to its broad expression in normal tissues and cell lines, DACT2 is silenced or downregulated in multiple tumor cell lines including nasopharyngeal (NPC), esophageal (ESCC), gastric, colorectal (CRC), breast, lung, renal (RCC), hepatocellular (HCC), prostate and cervix carcinomas, due to its promoter methylation which could be reversed by pharmacological demethylation. DACT2 promoter methylation was also frequently detected in multiple primary tumors, suggesting that DACT2 is a potential tumor marker. DACT2 encodes a cytoplasm protein localized in endosome. Ectopic expression of DACT2 dramatically inhibited tumor cell colony formation in silenced tumor cell lines, mainly through inducing apoptosis. DACT2 interacts and downregulates Dishevelled (DVL) protein, thus protecting glycogen synthase kinase 3β (GSK-3β) from inactivation by WNT/DVL and allowing GSK-3β to form a complex with Axin and APC to promote the phosphorylation and proteasomal degradation of β-catenin. Overexpression of DACT2 disrupted β-catenin activation and accumulation in nucleus, thus preventing its binding to transcription factors of the Lef/Tcf family. This caused the downregulation of target oncogenes such as c-Myc, CCND1 and fibronectin and the inhibition of tumor cell proliferation and migration. We also observed that DACT2 could upregulate epithelial cell marker E-cadherin, and modulate Akt/GSK-3β/β-catenin signaling pathway, another pathway involved in tumor cell epithelial mesenchymal transition. Our data demonstrate that DACT2 is a functional tumor suppressor epigenetically silenced in multiple tumors which acts as a negative regulator of the Wnt/DVL/β-Catenin pathway, and could be a promising biomarker for human tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2191. doi:10.1158/1538-7445.AM2011-2191

Abstract LB-354: Dysregulation of microRNA-34a expression in head and neck squamous cell carcinoma promotes tumor growth and tumor angiogenesis

Abstract Objectives of the study: Head and Neck squamous cell carcinoma (HNSCC) results from deregulation of multiple signaling pathways leading to tumor cells acquiring proliferative, invasive, angiogenic and metastatic properties. Interactions between neoplastic cells and the surrounding microenvironment also play a major role in tumor progression. Recent studies have demonstrated that microRNA's (miRs) play a critical role in cancer development where they can act as oncogenes or as onco-suppressors. A microRNA array analysis of a panel often HNSCC cell lines reveled that miR-34a is significantly downregulated in all these cell lines as compared to normal human oral keratinocyes (HOK). In addition, miR-34a expression was also downregulated in highly angiogenic endothelial cells (endothelial cells overexpressing Bcl-2; EC-Bcl-2) as compared to normal human endothelial cells. However, little is known about the role of miR-34a in HNSCC tumor growth and tumor angiogenesis. In this study, we examined if dysregulation of miR-34a in HNSCC promotes tumor growth and tumor angiogenesis. Methodology: The study was performed using a panel of established human head & neck cancer cell lines. miRs expression in tumor and endothelial cells was examined by miR-arrays and further validated by real time PCR. Lipofectamine-2000 was used to transfect miR-34a in 2 HNSCC cell lines (UM-SCC-74A and UM-SCC-47) and human endothelial cells. Cell-proliferation assay was performed by MTT and colony formation assay. Tumor cell and endothelial cell motility was assessed by scratch assay. Expression of miR-34a downstream mediators was measured by Western blot. Results: Ectopic expression of miR34a significantly inhibited cell proliferation of UM-SCC-74A cells (52% and 58% on days 2 and 4) and UM-SCC-47 cells (43% and 47% on days 2 and 4). In addition, enhanced expression of miR34a in UM-SCC-74A and UM-SCC-47 cells significantly inhibited tumor cell colony formation and tumor cell migration. Similar to tumor cells, miR-34a significantly inhibited EC-Bcl-2 proliferation and migration by down regulating c-met, Bcl-2 expression and upregulating phospho p38 MAPK expression. Conclusion: These findings indicate that dysregulation of miR-34a expression is common in HNSCC and modulation of miR34a activity might represent a novel therapeutic strategy for the treatment of head and neck cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-354.