Levels Of Cell Cycle-related Proteins Research Articles

RNA m6 A methylation regulates uveal melanoma cell proliferation, migration, and invasion by targeting c-Met.

N6 -methyladenosine (m6 A) is a novel epitranscriptomic marker that contributes to regulating diverse biological processes through controlling messenger RNA metabolism. However, it is unknown if m6 A RNA methylation affects uveal melanoma (UM) development. To address this question, we probed its function and molecular mechanism in UM. Initially, we demonstrated that global RNA m6 A methylation levels were dramatically elevated in both UM cell lines and clinical specimens. Meanwhile, we found that METTL3, a main m6 A regulatory enzyme, was significantly increased in UM cells and specimens. Subsequently, cycloleucine (Cyc) or METTL3 targeted small interfering RNA was used to block m6 A methylation in UM cells. We found that Cyc or silencing METTL3 significantly suppressed UM cell proliferation and colony formation through cell cycle G1 arrest, as well as migration and invasion by functional analysis. On the other hand, overexpression of METTL3 had the opposite effects. Furthermore, bioinformatics and methylated RNA immunoprecipitation-quantitative polymerase chain reaction identified c-Met as a direct target of m6 A methylation in UM cells. In addition, western blot analysis showed that Cyc or knockdown of METTL3 downregulated c-Met, p-Akt, and cell cycle-related protein levels in UM cells. Taken together, our results demonstrate that METTL3-mediated m6 A RNA methylation modulates UM cell proliferation, migration, and invasion by targeting c-Met. Such a modification acts as a critical oncogenic regulator in UM development.

Alpinumisoflavone suppresses human Glioblastoma cell growth and induces cell cycle arrest through activating peroxisome proliferator-activated receptor-γ.

As a common subtype of malignant gliomas, glioblastoma multiforme (GBM) is associated with poor prognosis. This study is aimed to examine the anticancer activities of alpinumisoflavone (AIF) and its underlying mechanisms. Our results demonstrated that AIF inhibited the proliferation of GBM cells (U373 and T98G) in a time and dose-dependent manner. In addition, flow cytometry analysis not only confirmed AIF arrested cell cycle at the G0/G1 phase but also the induced apoptosis of U373 and T98G cells. Western blotting also confirmed that AIF altered the expression levels of cell cycle-related proteins. Further mechanism studies revealed that AIF inhibited cell proliferation, induced G0/G1 phase arrest and induced apoptosis of U373 and T98G cells through activating PPARγ, as evidenced by the fact that GW9662 (PPARγ inhibitor) could effectively reverse the effects of AIF on U373 and T98G cells. Furthermore, the in vivo study also revealed that AIF suppressed tumor growth and caused cell cycle arrest. Collectively, these results highlighted the potential use of AIF in the treatment of GBM.

Cytoprotective effects of taxifolin against cadmium-induced apoptosis in human keratinocytes.

Cadmium (Cd) is a heavy metal widely used in industry, and the skin is an important target of this metal. Taxifolin (Tax), a natural source of bioflavonoids found in various conifers, exerts multiple biologic effects on skin cells. However, the mechanisms by which Tax protects keratinocytes against Cd are currently unclear. We investigated the cytoprotective effects of Tax against Cd-induced apoptosis in the human HaCaT keratinocyte. The water-soluble tetrazolium salt (WST-1) assay and Annexin V/propidium iodide double-staining assay results showed that Cd-induced cell death was lower in cells treated with Tax (0-100 μM) than in cells treated with Cd alone. Additionally, a reduction of Cd-induced DNA fragmentation by Tax was shown by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling assay. The levels of reactive oxygen species were also lower in Cd/Tax-treated cells than in Cd-treated cells. We employed a two-dimensional electrophoresis-based proteomic analysis to identify treatment-related alterations in protein expression. Tax downregulated cathepsin B and D and upregulated hsp27, cyclophilin A, and peroxiredowin-1. Western blotting confirmed the downregulation of cathepsin B and D and the upregulation of hsp27. The cytoprotective effects of Tax against Cd-induced apoptosis were also characterized by the changes in the activity of caspase 3, -7, poly ADP-ribose polymerase, the cellular proliferation-related ERK1/2, and AKT. Furthermore, the levels of cell cycle-related proteins, such as SP1 and p21, decreased, whereas p53 level increased. We concluded that Tax reduced Cd cytotoxicity and Cd-induced apoptosis by inhibiting the apoptotic pathway.

The protective effects of the antioxidant N-acetylcysteine (NAC) against oxidative stress-associated apoptosis evoked by the organophosphorus insecticide malathion in normal human astrocytes

Malathion is one of the most widely used organophosphorus insecticides in agriculture. However, malathion may be involved in the etiology of human brain dysfunction. Induction of ROS has been proposed as a mechanism of malathion-induced poisoning cases, but there are few data regarding the effects of malathion on oxidative stress-associated neurotoxicity in human glial cells. The aim was to explore the mechanism underlying effects of malathion on neurotoxicity in Gibco® Human Astrocytes (GHA cells) and evaluate the protective effects of the antioxidant (N-acetylcysteine, NAC). Cell viability was measured by the cell proliferation reagent (WST-1). Antioxidant enzymes (glutathione peroxidase and catalase) were measured by an ELISA reader. Cell cycle distribution and ROS productions were detected by flow cytometry. Cell cycle-related protein levels (cyclin E1, CDK2, cyclin A2, CDK1/CDC2, or cyclin B1) and apoptotic protein levels (Bcl-2, Bax, and cleaved caspase-9/caspase-3) were analyzed by Western blotting. In GHA cells, treatment with malathion (10–25 μM) for 24 h concentration-dependently induced cytotoxicity and cell cycle arrest. In terms of oxidative stresses, malathion elevated intracellular ROS levels, but reduced glutathion and antioxidant enzyme levels. Treatment with NAC (5 μM) reversed malathion-induced oxidative stress responses, and prevented malathion-evoked apoptosis by regulating apoptotic protein expressions. Together, in GHA cells, NAC mediated inhibition of malathion-activated mitochondrial apoptotic pathways that involved cell cycle arrest and ROS responses. These data provide further insights into the molecular mechanisms behind malathion poisoning, and might suggest that NAC with its protective effects may be a potential compound for prevention of malathion-induced brain injury.

SiRNA against TSG101 reduces proliferation and induces G0/G1 arrest in renal cell carcinoma \u2013 involvement of c-myc, cyclin E1, and CDK2

ObjectiveThe tumor susceptibility gene 101 (TSG101) is closely associated with various tumor types, but its role in the pathogenesis of renal cell carcinoma (RCC) is still unknown. This study used RNA interference to silence the expression of TSG101 in RCC cell lines and explore the role of TSG101 in RCC.MethodsImmunohistochemistry and western blot were performed to detect the expression of TSG101 in 15 paired renal tumor samples. A small interfering RNA (siRNA) targeting TSG101 was transfected into A498 and 786-O cell lines. The Cell Counting Kit-8 (CCK-8) assay and colony formation assay were used to observe the changes in cell proliferation after transfection. Flow cytometry was used to detect the effect on the cell cycle. Western blot was conducted to study the changes of related functional proteins.ResultsThe expression of TSG101 was higher in RCC tissues than in adjacent normal tissues. The CCK-8 assay showed that the proliferation and colony formation of the A498 and 786-O cell lines were attenuated after suppression of TSG101. Flow cytometry showed that silencing of TSG101 induced G0/G1 arrest. The western blot results revealed that the levels of cell cycle-related proteins (c-myc, cyclin E1 and cyclin-dependent kinase 2 (CDK2)) were markedly decreased in the siRNA groups.ConclusionsTSG101 promotes proliferation of RCC cells. This positive effect on tumor growth involves activation of c-myc and cyclin E1/CDK2 and their effect on cell cycle distribution.

MicroRNA-181b suppresses the metastasis of lung cancer cells by targeting sex determining region Y-related high mobility group-box 6 (Sox6)

BackgroundThe aim of the study was to measure the expression of microRNA (miR)-181b in patients with lung cancer, investigate its biological function and elucidate the underlying mechanisms associated with the development of lung cancer. MethodsmiR-181b expression in tissues was measured via RT-qPCR. After A549 cells were transfected with miR-181b mimic or si-Sox6, the proliferation, migration and cell cycle distribution of A549 were evaluated using cell counting kit-8 assay, transwell assay and flow cytometry. The levels of cell cycle-related proteins and Sox6 were analyzed by western blotting. Gene targets of miR-181b were predicted via bioinformatics analysis and verified using a dual-luciferase reporter gene assay. ResultsExpression of miR-181b was significantly downregulated in lung cancer tissues (P < 0.05), and was inversely correlated with the degree of cell differentiation and clinical stages of lung cancer (both P < 0.05). Additionally, the expression of miR-181b was significantly lower in adenocarcinoma compared with squamous cell carcinoma in the lungs (P < 0.05). Overexpression of miR-181b significantly decreased the protein level of Sox6 and significantly suppressed the cell proliferation and metastasis (both P < 0.05); this effect was also observed in A549 cells transfected with si-Sox6. The luciferase activity of a Sox6 3′-untranslated region-based reporter construct was significantly lower when transfected with miR-181b (P < 0.05), which suggests that Sox6 is a direct target of miR-181b. ConclusionThe results of the present study suggest that miR-181b may function as a tumor inhibitor in the development of lung cancer via targeting Sox6 to decrease the proliferation and metastasis of lung cancer cells.

Natural product pectolinarigenin inhibits proliferation, induces apoptosis, and causes G2/M phase arrest of HCC via PI3K/AKT/mTOR/ERK signaling pathway.

BackgroundHepatocellular carcinoma (HCC) is characterized by considerable phenotypic and molecular heterogeneity, but the overall survival of HCC patients remains extremely poor. Thus, novel and efficient alternatives to antitumor agents are urgently needed. Pectolinarigenin, a flavonoid compound extract, has been previously reported for the treatment of nasopharyngeal cancer. However, the potential antitumor roles of pectolinarigenin in HCC have not been clearly elaborated. In the present study, we investigated its role in HCC treatment and explored the potential molecular mechanism(s).Materials and methodsHCC cell lines SMMC7721 and PLC5 were cultured and treated with indicated concentrations of pectolinarigenin. For the HCC cell proliferation, after HCC cells were stimulated with indicated concentrations of pectolinarigenin, the cell viability was detected in CCK-8 and colony-forming assays. HCC cell invasion/migration assay was performed by Transwell and wound scratch methods. Additionally, cellular apoptosis and cell cycle arrest analysis was performed with flow cytometric analysis. Finally, the involved underlying signaling pathway, the PI3K/AKT/mTOR/ERK signaling-related molecular markers were detected through Western blot methods with indicated antibodies. Meanwhile, antitumor activity of pectolinarigenin was also assessed in tumor-bearing mice.ResultsThe results indicated that the treatment with pectolinarigenin significantly inhibited cell proliferation and migratory and invasive abilities of SMMC7721 and PLC5 cells in concentration- and time-dependent manner. Meanwhile, pectolinarigenin markedly induced cell apoptosis and G2/M phase arrest in SMMC7721 and PLC5 cells, which was associated with apoptosis- and cell cycle-related protein levels, respectively. Furthermore, pectolinarigenin inhibited PI3K/AKT/mTOR/ERK signaling pathway. It also significantly suppressed HCC tumor growth in vivo.ConclusionPectolinarigenin could suppress the viability and motility and cause apoptosis and G2/M phase arrest in HCC cell lines by inhibiting the PI3K/AKT/mTOR/ERK signaling pathway. This might be an appealing potential therapeutic agent for HCC treatment.

Ubiquitin-like modifier activating enzyme 2 promotes cell migration and invasion through Wnt/β-catenin signaling in gastric cancer.

AIMTo investigate the function and mechanism of ubiquitin-like modifier activating enzyme 2 (Uba2) in progression of gastric cancer (GC) cells.METHODSUba2 level in patients with GC was analyzed by Western blotting and immunohistochemistry. MTT and colony formation assays were performed to examine cell proliferation. Flow cytometry was used for cell cycle analysis. Wound healing and Transwell assays were conducted to examine the effects of Uba2 on migration and invasion. Expression levels of cell cycle-related proteins, epithelial-mesenchymal transition (EMT) biomarkers, and involvement of the Wnt/β-catenin pathway was assessed by Western blotting. Activation of the Wnt/β-catenin pathway was confirmed by luciferase assay.RESULTSUba2 expression was higher in GC than in normal tissues. Increased Uba2 expression was correlated with tissue differentiation, Lauren’s classification, vascular invasion, and TNM stage, as determined by the analysis of 100 GC cases (P < 0.05). Knock-down of Uba2 inhibited GC cell proliferation, induced cell cycle arrest, and altered expression of cyclin D1, P21, P27, and Bcl-2, while up-regulation of Uba2 showed the opposite effects. The wound healing and Transwell assays showed that Uba2 promoted GC cell migration and invasion. Western blotting revealed alterations in EMT biomarkers, suggesting the role of Uba2 in EMT. Furthermore, the luciferase reporter assay indicated the involvement of the Wnt/β-catenin signaling pathway as a possible modulator of Uba2 oncogenic functions.CONCLUSIONUba2 plays a vital role in GC cell migration and invasion, possibly by regulating the Wnt/β-catenin signaling pathway and EMT.

Knockdown of IARS2 suppressed growth of gastric cancer cells by regulating the phosphorylation of cell cycle-related proteins

The purpose of the article is to investigate the role of IARS2 in proliferation, apoptosis, and cell cycle of gastric cancer (GC) cells in vitro. The IARS2-shRNA lentiviral vector was established and used to infect the GC cell line AGS. qRT-PCR and Western blot were employed to determine the efficiency of IARS2 knockdown. The effects of IARS2 knockdown on cell proliferation, cell clone formation, and cell cycle were assessed by MTT assay, colony formation assay, and flow cytometer analysis, respectively. Finally, a PathScan Antibody Array Kit was used to detect the expression levels of cell cycle-related proteins after IARS2 knockdown in AGS cells to elucidate the underlying mechanisms. Compared with negative control group, IARS2 was significantly knocked down by transfection with lentivirus encoding shRNA of IARS2 in AGS cells. IARS2 knockdown significantly inhibited the proliferation and colony formation ability and induced cycle arrest at G2/M phase of AGS cells. IARS2 knockdown significantly decreased the expression levels of phosphorylation of (p-Smad2), p-SAPK/JUK, cleavage-Caspase-7, and p-TAK1, but increased the expression levels of p-53 and cleavage-PARP in AGS cells compared to shCtrl group. We demonstrated that IARS2 knockdown inhibits proliferation, suppresses colony formation, and causes cell cycle arrest in AGS cells. We also found that IARS2 regulates key molecules of cell apoptosis-related signaling pathway.

Effects of microRNA-200c on the proliferation and cell cycle of bladder cancer T24 cells

Objective To investigate the influence of microRNA (miRNA, miR)-200c on cell proliferation and cell cycle in bladder cancer T24 cells and the preliminary mechanism. Methods The expression of miR-200c in bladder cancer cell lines was detected using real-time quantitative polymerase chain reaction (Real-time PCR). T24 cells were transfected with miR-200c mimics (MU group) or nonsense control (NC group) individually to generate negative control group and over-expression group. Then cell proliferation was assessed using cell counting kit-8 (CCK-8) kits at 24, 48 and 72 h while cell cycle distribution was detected by flow cytometry. Finally, Western blotting was applied to detect the levels of cell cycle-related proteins. Results MiR-200c was up-regulated in bladder cancer cell lines EJ (13.204±0.946), T24 (18.259±0.776), 253J (8.333±0.543), 5637 (6.594±0.326), RT4 (11.348±0.513) as compared with normal urothelial cell line SV-Huc (1.053±0.078, P=0.000). MiR-200c mimics promoted cell proliferation at 48 and 72 h after transfection [(1.485±0.131 and 2.396±0.110) vs. (1.158±0.182 and 1.893±0.216), P=0.050, P=0.023] and induced G1/S checkpoint release (P=0.013). Meanwhile, the protein levels of cyclin-dependent kinase (CDK)2 (1.44±0.10 vs. 0.86±0.15, P=0.005), CDK4 (3.05±0.17 vs. 1.24±0.07, P=0.001), E2F3 (1.81±0.22 vs. 1.18±0.20, P=0.021) were upregulated after transfection. Conclusion MiR-200c promotes cell proliferation and induces G1/S transition in bladder cancer T24 cell lines via regulating cell cycle-related proteins. Key words: Bladder cancer; MicroRNA-200c; Cell cycle; Proliferation

Abstract 3475: Inhibitory effects of ZFP36L1 and ZFP36L2 on the cell proliferation in human colorectal cancer cells

Abstract ZFP36 family members include ZFP36 (also named TTP and TIS11), ZFP36L1 (also called BRF1, ERF1 or TIS11B) and ZFP36L2 (also called BRF2, ERF2 and TIS11D), which belong to the CCCH-type zinc finger protein with 2 tandem zinc finger region (TZF). These ZFP36 family proteins can function as RNA-binding protein through binding to AU-rich elements (AREs) in the 3’ untranslated region (3’ UTR) of mRNA, which promote the mRNA degradation or translation repression. Previously, ZFP36 has been found to inhibit cell proliferation through p53-dependent manner. In this study, we’d like to investigate whether ZFP36L1and ZFP36L2 have anti-proliferative activity as same as ZFP36 work. Tetracycline-inducible (Tet-On) system was used to induce the overexpression of ZFP36L1 or ZFP36L2 proteins in T-REx-293 cells by doxycycline (Dox) treatment. While ZFP36L1 or ZFP36L2 was overexpressed, we found that cell proliferation was dramatically inhibited, but didn’t cause cell death significantly. The importance of TZF was confirmed by using TZF mutants, and found that cell proliferation was deprived in HEK-293 cells with overexpression of ZFP36L1 mutant (C135/173R). Using western blot analysis, it was revealed that expression level of p53 protein was increased after the overexpression of ZFP36L1 or ZFP36L2 protein. Whereas, the levels of cell cycle-related proteins including cyclin B1, cyclin D1, cyclin A2 and p21 were decrease. Next, three human colorectal cancer cells HCT116 p53+/+, HCT116 p53-/- and SW620 (mutated p53) cells were used and ZFP36L1 or ZFP36L2 gene was transduced into these three cells by lentivirus. Overexpression of ZFP36L1 or ZFP36L2 also inhibited the cyclin D1 protein expression and cell proliferation in these three cells, however, increased the p53 and p21 protein expression in HCT116 p53+/+ cells, and decreased the c-Myc expression only in SW620 cells. On the other hand, knockdown of ZFP36L1 or ZFP36L2 increased cell proliferation, and mutation in TZF of ZFP36L1 (C135/173R) or ZFP36L2 (C174/212R) lost their anti-proliferative abilities in these three cells. Taken together, the results suggest that ZFP36L1 and ZFP36L2 play a negative role in cell proliferation of human colorectal cancer cells, and the underlying mechanisms might be mediated through downregulation of cyclin D1 and p53-independent pathway. Citation Format: Fat-Moon Suk, Ya-Ting Chen, Yu-Chih Liang. Inhibitory effects of ZFP36L1 and ZFP36L2 on the cell proliferation in human colorectal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3475. doi:10.1158/1538-7445.AM2017-3475

MiR-143 inhibits cell proliferation and invasion by targeting DNMT3A in gastric cancer.

Increasing evidence has suggested that MircroRNAs (miRNAs) dysregulated in pathogenesis and tumorigenicity in human cancers including gastric cancer (GC). MiR-143 had been reported to function as tumor suppressor in GC progression, however, the underlying function of miR-143 in GC still need to be well known. In the study, we revealed that miR-143 was significantly down-regulated in GC cell lines. Upregulation of miR-143 inhibited cell proliferation, invasion, S phase cell proportion and cell cycle related protein levels of Cyclin D1, CDK4 and CDK6 in GC. Furthermore, luciferase reporter assays demonstrated that DNMT3A was a direct target of miR-143 and Upregulation of miR-143 inhibited the DNMT3A mRNA and protein expression levels in GC cells. Moreover, we demonstrated that DNMT3A knockdown rescued the promoting effect of miR-143 inhibitor on cell proliferation in GC. Thus, these results demonstrated that miR-143 targeted DNMT3A in GC cells and inhibit GC tumorigenesis and progression, which may provide a novel therapeutic target of GC.

Down-regulation of long non-coding RNA RP11-708H21.4 is associated with poor prognosis for colorectal cancer and promotes tumorigenesis through regulating AKT/mTOR pathway.

Long non-coding RNAs (lncRNAs) serve critical roles in cancer development and progression. Herein, through next generation RNA sequencing and experimental validations, we determined the expression status of RP11-708H21.4 in colorectal cancer (CRC) and explored its clinical significance and biological functions in CRC. Differentially expressed lncRNAs from CRC samples and corresponding normal mucosa tissues was screened through RNA sequencing, and RP11-708H21.4 was selected for further experimental validation. The expression levels of RP11-708H21.4 in CRC tissues and cell lines were determined using qRT-PCR. Also, the relationship between the clinicopathological features and RP11-708H21.4 expression was analyzed. Cell viability was examined by CCK-8 and colony assays; cell migration and invasion were detected by transwell assays; cell cycle and cell apoptosis were analyzed by flow cytometry. The chemosensitivity of CRC cells to 5-Fluorouracil (5-FU) was also determined using CCK-8 assay. CRC xenograft tumor models were established to determine the biological functions of RP11-708H21.4 in vivo. Levels of cell cycle-related proteins and AKT/mTOR pathway-related proteins were detected by western blot assay. RP11-708H21.4 expression was aberrantly decreased in CRC, and its expression was closely associated with aggressive clinicopathologic features and unfavorable prognosis of CRC patients. Overexpressed RP11-708H21.4 suppresses CRC cell proliferation through inducing G1 arrest. Moreover, up-regulation of RP11-708H21.4 inhibits cell migration and invasion, causes cell apoptosis, and enhances 5-FU sensitivity of CRC cells. Finally, increased RP11-708H21.4 expression blocked AKT/mTOR pathway, and repressed in vivo CRC xenograft tumor growth. The results indicated that RP11-708H21.4 might have potential roles as a biomarker and a therapeutic target for CRC.

Effects of galectin-9 on apoptosis, cell cycle and autophagy in human esophageal adenocarcinoma cells

The incidence of esophageal adenocarcinoma (EAC) is rapidly increasing in western countries. The overall mortality of this disease remains high with a 5-year survival rate of less than 20%, despite remarkable advances in the care of patients with EAC. Galectin-9(Gal-9) is a tandem-repeat type galectin that exerts anti-proliferative effects on various cancer cell types. The aim of the present study was to evaluate the effects of Gal-9 on human EAC cells and to assess the expression of microRNAs (miRNAs) associated with the antitumor effects of Gal-9 invitro. Gal-9 suppressed the proliferation of the EAC cell lines OE19, OE33, SK-GT4, and OACM 5.1C. Additionally, Gal-9 treatment induced apoptosis and increased the expression levels of caspase-cleaved cytokeratin 18, activated caspase-3 and activated caspase-9. However, it did not promote cell cycle arrest by reducing cell cycle-related protein levels. Furthermore, Gal-9 increased the level of the angiogenesis-related protein interleukin-8(IL-8) and markedly altered miRNA expression. Based on these findings, Gal-9 may be of clinical use for the treatment of EAC.

Licochalcone B Arrests Cell Cycle Progression and Induces Apoptosis in Human Breast Cancer MCF-7 Cells

Recent patent of licochalcone B (LCB) as an antiinflammatory agent has been developed. Emerging evidence shows that LCB may be a promising alternative compound with anti-cancer activities. However, the anticancer mechanism of LCB in MCF-7 cells has not been fully investigated. We aimed to unearth the anti-cancer effect and mechanism of LCB in MCF-7 cells. Cell proliferation activity and cell-cycle progression were determined by sulforhodamine B assay and flow cytometry, respectively. The mRNA and protein levels of cell cycle-related proteins and apoptosis-associated proteins were examined by RT-qPCR and western blot, respectively. Mitochondrial membrane potential (MMP) was measured by flow cytometry after JC-1 staining. We found that LCB inhibited MCF-7 cells proliferation in a concentration- and time-dependent manner. Moreover, LCB-treatment led to S phase arrest in MCF-7 cells, which could be elucidated by the decreased mRNA and protein levels of Cyclin A, Cdk2 and Cdc25 A, and the increased protein level of p21. LCB also induced such apoptosis morphology as phosphatidylserine externalization, chromatin condensation and DNA fragmentation. Moreover, LCB led to the loss of MMP, resulting in the release of cytochrome C. The above apoptotic events were supported by the fact that LCB upregulated the mRNA and protein levels of Caspase 3, Caspase 9 and Bax, and downregulated the mRNA and protein level of Bcl-2, which was triggered by the increased p53 protein level in LCB-treated MCF-7 cells. These findings suggested that LCB could be a promising agent for treatment of human breast cancer.

Abstract 3194: Platycodin D induces growth arrest and cell cycle arrest by activating MDM2 and interacting with FOXO3a in prostate cancer

Abstract Purpose: The present study was designed to evaluate the anti-tumor activity and possible mechanism of action of Platycodin D (PD), a major saponin derived from Platycodin grandiflorum, in human prostate cancer. Experimental Design: Human prostate cancer cells with different p53 status (p53 wildtype LNCaP cells, p53 null PC3 cells and p53 mutant DU145 cells), and the normal human prostate cell line, RWPE-1, were exposed to various concentrations of PD to compare its cytotoxicity in vitro (via the MTT assay). Detailed invitro and invivo studies on the anti-tumor effects of PD were then performed. Results: PD exerted cytotoxicity against all three prostate cancer cell lines, with half-maximal inhibitory concentrations in the range of 11.16 to 26.13μmol/L. The PC3 cells were the most sensitive to PD, whereas RWPE-1 cells were not sensitive to PD.A further study in these cell lines showed that PD could potently affect the cell proliferation (indicated by the bromodeoxyuridine assay), induce cell apoptosis (by Annexin V-FITC flow cytometry) and cause cell cycle arrest (PI staining). After being treated with PD for 48 hours,the DU145 and LNCaP cells were arrested in the G0 /G1 phase, and the PC3 cells were arrested in the G2/M phase. A Western blot analysis indicated that PD decreased the expression of MDM2 and increased the expression levels of FOXO3a, p21 and p27 in the three cell lines. In PC3 cells, PD exposure decreased the levels of cell cycle-related proteins, including E2F1, cdk2, cdk4, cdk6, cyclin D1 and cdc2. PD exposure increased apoptosis-related proteins, Bax, caspase9 and caspase3, and downregulated Bcl-2, PARP, and caspase8. Real-time PCR indicated that the same changes had occurred at the transcriptional level for all of these targets. Moreover, PD dose-dependently inhibited the growth of PC3 xenograft tumors in BALB/c nude mice. A Western blot analysis of the xenograft tumors indicated that similar changes in protein expression also occurred in vivo. Conclusion: PD exhibits significant activity against prostate cancer cells in a p53-independent manner. MDM2 and the FOXO3a transcription factor appear to be involved in the mechanism of action of PD, which provides a basis for future development of this agent for human prostate cancer therapy. Citation Format: Hongxia Xu, Rui Zhou, Zongliang Lu, Kai Liu. Platycodin D induces growth arrest and cell cycle arrest by activating MDM2 and interacting with FOXO3a in prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3194. doi:10.1158/1538-7445.AM2014-3194

Abstract 2224: Identification of molecular drivers of human hemangioblastoma

Abstract Hemangioblastoma of the central nervous system is the most common manifestation of Von Hippel-Lindau (VHL) disease, an autosomal dominant inherited cancer syndrome. The underlying molecular mechanism of hemangioblastoma is not known. To discover the molecular drivers of hemangioblastomas, 17 hemangioblastoma human tissue cases were submitted to MIP (molecular inversion probe) analysis to assess copy number alteration. Surprisingly, a focal loss of chromosome 17q was observed in 15 of 17 cases. Further analysis discovered a specific deletion involving HNF1B (Hepatocyte Nuclear Factor 1 homeobox B), a nuclear transcription factor. To elucidate the molecular effects of HNF1B loss, we used three different sequences of shRNA to knock down Hnf1b in mouse embryonic fibroblast (MEF) cells. Loss of Hnf1b resulted in significantly fewer cells arrested in G2/M in response to nocodazole treatment. Cell cycle related protein levels such as cyclin B1, cyclin D1 and phospho-Tyr15-Cdk1 were correspondingly changed. Of note, the cyclin dependent kinase inhibitor Cdkn1b (p27) was remarkably suppressed while another cyclin dependent kinase inhibitor, p21, was not affected at all. To elucidate the mechanism of Hnf1b dependent p27 regulation, the proteasome inhibitor MG132 was used to treat Hnf1b knockdown cells, which failed to increase p27 levels. An in vitro ubiquitination assay demonstrated that Hnf1b did not affect p27 ubiquitination. Both results indicated that Hnf1b regulation of p27 is not via posttranslational ubiquitin-proteasome system. Real time-polymerase chain reaction (PCR) and chromatin immunoprecipitation (ChIP) experiments demonstrated that Hnf1b regulated p27 at the mRNA level and specific consensus binding regions were identified. We then showed that p27 overexpression by lenti-virus infection in Hnf1b knockdown cells normalized the percentage of G2/M arrested cells. Hemangioblastoma are highly vascularized tumors. We discovered a dramatic up-regulation of vascular endothelial growth factor (Vegf) by enzyme-linked immunosorbent assay in cells with Hnf1b knockdown. These findings provided further evidence that loss of Hnf1b is one of the major drivers of the hemangioblastoma disease phenotype. Taken together, our findings for the first time identified HNF1B as a major gene loss in hemangioblastoma patients, and provide molecular evidence that Hnf1b transcriptionally regulates cell cycle protein p27, and drives the proangiogenic phenotype. This novel study sheds insights into molecular mechanisms of hemangioblastoma and provides tools to develop prevention and treatment strategies for the disease. Citation Format: Mianen Sun, Federico Monzon, Lijun Zhou, Xiande Liu, Zhiyong Ding, Xuesong Zhang, Shanshan Bai, Peter German, Sarathi Kalra, In Young ‎ Park, Ruhee Dere, Tia Berry, Xuefei Tong, Cheryl Walker, Nizar Tannir, Surena Matin, Gregory Fuller, Ian McCutcheon, Eric Jonasch. Identification of molecular drivers of human hemangioblastoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2224. doi:10.1158/1538-7445.AM2014-2224

Curcumin cytotoxicity is enhanced by PTEN disruption in colorectal cancer cells

To investigate the effects of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) deficiency on the cytotoxicity of chemotherapeutic agents toward colorectal cancer cells. PTEN-deficient colorectal cancer (CRC) cells were generated by human somatic cell gene targeting using the adeno-associated virus system. The cytotoxic effects of compounds including curcumin, 5-fluorouracil (5-FU), dihydroartemisinin (DHA), irinotecan (CPT-11) and oxaliplatin (OXA) on cancer cells were determined using the MTT assay. Enhanced cytotoxicity of curcumin in PTEN-deficient CRC cells was observed, and this was confirmed using clonogenic assays. Apoptosis and cell cycle progression were analyzed by flow cytometry. Levels of apoptosis and cell cycle-related proteins were examined by Western blotting. We developed an isogenic set of CRC cell lines that differed only in their PTEN status. Using this set of cell lines, we found that disruption of the PTEN gene had no effect on the sensitivity of CRC cells to 5-FU, CPT-11, DHA, or OXA, whereas PTEN disruption increased the sensitivity of CRC cells to curcumin. Loss of PTEN did not alter the curcumin-induced apoptosis in CRC cells. However, PTEN deficiency led to an altered pattern of curcumin-mediated cell cycle arrest. In HCT116 PTEN (+/+) cells, curcumin caused a G2/M phase arrest, whereas it caused a G0/G1 phase arrest in HCT116 PTEN (-/-) cells. Levels of cell cycle-related proteins were consistent with these respective patterns of cell cycle arrest. Curcumin shows enhanced cytotoxicity toward PTEN-deficient cancer cells, suggesting that it might be a potential chemotherapeutic agent for cancers harboring PTEN mutations.

Ligand-free estrogen receptor activity complements IGF1R to induce the proliferation of the MCF-7 breast cancer cells

BackgroundLigand-dependent activation of the estrogen receptor (ER) as well as of the insulin-like growth factor type 1 (IGF1R) induces the proliferation of luminal breast cancer cells. These two pathways cooperate and are interdependent. We addressed the question of the mechanisms of crosstalk between the ER and IGF1R.MethodsWe evaluated the mitogenic effects of estradiol (E2; agonist ligand of ER) and of insulin (a ligand of IGF1R) in the MCF-7 cells by flow cytometry and by analyzing the cell levels of cell cycle-related proteins (immunoblotting) and mRNA (RT-QPCR). To verify the requirement for the kinase activity of Akt (a downstream target of IGF1R) in the mitogenic action of estradiol, we used shRNA strategy and shRNA-resistant expression vectors.ResultsThe activation of the ER by E2 is unable to induce the cell cycle progression when the phosphatidyl inositol-3 kinase (PI3K)/Akt signaling is blocked by a chemical inhibitor (LY 294002) or by shRNA targeting Akt1 and Akt2. shRNA-resistant Akt wild-type constructs efficiently complemented the mitogenic signaling activity of E2 whereas constructs with inactivated kinase function did not. In growth factor-starved cells, the residual PI3K/Akt activity is sufficient to complement the mitogenic action of E2. Conversely, when ER function is blocked by the antiestrogen ICI 182780, IGF1R signaling is intact but does not lead to efficient reinitiation of the cell cycle in quiescent, growth factor-starved MCF-7 cells. The basal transcription-promoting activity of ligand-free ER in growth factor-starved cells is sufficient to complement the mitogenic action of the IGF1R-dependent signaling.ConclusionsThe basal ER activity in the absence of ligand is sufficient to allow efficient mitogenic action of IGF1R agonists and needs to be blocked to prevent the cell cycle progression.

CD44 is a biomarker associated with human prostate cancer radiation sensitivity

CD44 plays an important role in cancer metastasis, chemotherapy, and radiation resistance. The present study investigated the relationship of CD44 expression and radioresistance, and the potential mechanisms of CD44 in radiosensitivity using prostate cancer (CaP) cell lines. CD44 was knocked down in three CaP cell lines (PC-3, PC-3M-luc, and LNCaP) using small interfering RNA (siRNA) and clonogenic survival fractions after single dose irradiation were compared before and after CD44 knocking down (KD). The effect of radiation on cell cycle distribution was examined by flow cytometry and the cell cycle-related protein levels of phospho-Chk1 and phospho-Chk2 were ascertained by Western blotting. The expression of the DNA double strand break (DSB) marker-γH2AX was also quantified by immunofluorescence staining. Our results indicate that the down-regulation of CD44 enhanced radiosensitivity in PC-3, PC-3M-luc, and LNCaP CaP cells, the sensitizing enhancement ratio for these cell lines was 2.3, 1.3, and 1.5, respectively and that the delay of DNA DSB repair in low CD44-expressing KD CaP cells correlated with ineffective cell cycle arrest and the delayed phosphorylation of Chk1 and Chk2. These findings suggest that CD44 may be a valuable biomarker and a predictor of radiosensitivity in CaP treatment.