Cycle Regulatory Proteins Cyclin D1 Research Articles

Selenium modulates AR/IGF-1R/EGFR and TROP2 signaling pathways and improves anticancer efficacy in murine mammary carcinoma 4T1

The micronutrient selenium (Se) has been shown to exert potential anticancer properties. This study aimed to evaluate the effects of Se (in Se yeast form) on the selenoproteins (SELENO), AR/IGF-1R/EGFR, PI3K/Akt/mTOR and Ras/Raf/ERK cascades, and immune checkpoint blockade in TNBC murine 4T1 cells. We also assessed the effects of combination treatment with chemotherapeutic doxorubicin and Se on trophoblast cell surface antigen 2 (TROP2) levels. Compared with the control groups, cells incubated with Se (0.25, 0.5, 0.75, 1.0, 1.5 µg Se/mL) have lower viability, raised intracellular Se concentrations and SELENO expression, and higher malondialdehyde products in a dose-dependent manner. Se induced the inactivation of AR/IGF-1R/EGFR and downregulation of the PI3K/Akt/mTOR and Ras/Raf/ERK signaling molecules. Se-treated cells also exhibited decreased mitochondrial membrane potential, reduced levels of the cell cycle regulatory protein cyclin D1, cancer stemness, metastatic and EMT-related markers, and increased apoptosis. Subsequently, Se treatment significantly suppressed PD-1/PD-L1 and CTLA-4 mRNA levels and proteins. Doxorubicin decreased 4T1 cell viability and TROP2 expression levels, but the addition of Se to doxorubicin contributed to further reductions. Similar responses to Se treatment were also observed in the human MDA-MB-231 and MCF-7 breast cancer cells. These results show that Se upregulates SELENO and anti-AR/IGF-1R/EGFR signaling in TNBC cells, thus inducing oxidative stress-dependent apoptosis and cell cycle arrest, stemness, EMT, and metastasis, as well as blocking the immune checkpoint molecules. TROP2 down-regulation with Se is also a potential anti-TNBC therapeutic target.

Expression of Cell-Cycle Regulatory Proteins pRb, Cyclin D1, and p53 Is Not Associated with Recurrence Rates of Equine Sarcoids.

Simple SummaryExpression of cell-cycle regulatory proteins pRB, Cyclin D1, and p53 is not associated with recurrence rates of equine sarcoids. Sarcoids are common tumors of equids, and since the pathogenesis and Bovine Papillomavirus (BPV) mechanism of tumorigenesis is not fully understood yet, we investigated the expression of cell cycle regulatory proteins pRB, Cyclin D1, and p53 to understand whether BPV and Human Papillomavirus (HPV) share similar mechanisms of action. The expression pattern of these proteins in a series of BPV-positive equine sarcoids is different from HPV-induced tumors. Moreover, their expression is not correlated with the recurrence or occurrence of sarcoids in new body sites.Sarcoids are among the most common tumors diagnosed in equids; their association with bovine papillomaviruses (BPV) infection has been widely reported, but the mechanism of carcinogenesis has not been fully elucidated. To verify whether BPV infection causes dysregulation of the pRb-Cyclin D1-p16CDKN2A-p53 pathway as reported for human papillomavirus (HPV), the study employed immunohistochemistry to test 55 equine sarcoid biopsies for the expression of pRb, Cyclin D1, and p53 cell cycle regulatory proteins and to evaluate the proliferative rate through Ki67. High Cyclin D1 and pRb expression were observed in 51% and 80% of cases, respectively, while low expression was observed in 49% and 20% of cases, respectively. Significantly higher Ki67 proliferation indexes were observed in fibroblastic, nodular, and mixed sarcoids compared to the occult and verrucous. High proliferation was significantly associated with high Cyclin D1 expression. In contrast with previous studies, p53 positivity was not observed in the cases examined in this study. Moreover, follow-up analysis revealed that fibroblastic, mixed sarcoids were associated with significantly higher local recurrence rates while the verrucous subtype was associated with higher rates of new sarcoid development at distant sites.

Phycocyanin inhibits Helicobacter pylori-induced hyper-proliferation in AGS cells via activation of the ROS/MAPK signaling pathway.

BackgroundReactive oxygen species (ROS)-induced oxidative stress (OS) and hyper-proliferation of gastric epithelial cells (GECs) due to Helicobacter pylori (Hp) infection are important mechanisms that lead to gastric carcinoma. Phycocyanin is a marine functional food additive with antioxidant and anti-inflammatory properties.MethodsThe flow cytometry was used to detect the effect of 150 µM phycocyanin intervention on the cell cycle of human gastric adenocarcinoma cell line (AGS) infected with Hp. The transcriptomics of AGS cells intervened by 150 µM phycocyanin for 24 h and infected by Hp were detected. Differential gene expression analysis was performed using a cutoff at the normalized gene expression (log2) of 2 and a P-value of <0.05. Comparisons of the transcriptomes were made between the following groups: (I) AGS cells not infected with Hp and not using phycocyanin action and AGS cells infected with Hp only; (II) AGS cells not infected with Hp and not using phycocyanin action and AGS cells infected with phycocyanin action only; and (III) AGS cells infected with Hp only and phycocyanin treated and infected with Hp cells. c-myc and CyclinD1 was detected by quantitative real-time polymerase chain reaction (qRT-PCR) and immunoblotting. Phosphorylation and non-phosphorylation of c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38 Mitogen-activated protein kinase (p38MAPK) were detected by immunoblotting. Intracellular ROS was detected by immunofluorescence.ResultsPhycocyanin alleviates the Hp infection-induced increased cell viability and expression of cell cycle regulatory proteins c-myc and CyclinD1. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that the differentially expressed genes in phycocyanin-treated Hp-infected AGS cells were most significantly enriched in the mitogen-activated protein kinase (MAPK) signaling pathway. Phycocyanin could inhibit the Hp infection-induced phosphorylation of JNK, ERK, and p38MAPK and reduce the level of cellular ROS.ConclusionsThis study suggests that phycocyanin can regulate the ROS/MAPK signaling pathway and reduce c-myc and CyclinD1 expression to inhibit the hyper-proliferation of AGS cells. Phycocyanin may serve as an inhibitor of malignant progression of Hp infection-induced gastric disease.

3200 – USING QUANTITATIVE PROTEOMICS TO IDENTIFY NEW THERAPEUTIC TARGETS IN MANTLE CELL LYMPHOMA

Mantle cell lymphoma (MCL) is a B cell non-Hodgkin's lymphoma that affects 4-6% of the population. Despite the relatively low incidence of MCL compared to other lymphomas, MCL only has a 5-year survival rate of 50%, making it one of the most deadly. While 90% of patients exhibit the t(11;14) translocation resulting in overexpression of the cell cycle regulatory protein CyclinD1 (CCND1), this alone is not enough for disease initiation. Patients must also have secondary mutations for disease manifestation; however, the number and variety of these mutations differ between every patient. This genetic complexity lends to the difficulty in disease treatment, and there are no standard therapies currently available. Although some therapies have shown promise for treatment in the early stages of the disease, all cases eventually stop responding, resulting in relapse. As such, it is imperative that new therapies be identified to treat this deadly disease. To this end, our lab performed quantitative proteomics on B cells from five healthy donors and tumors from different lymphoma patients including sixteen MCL, seven follicular lymphoma, and seven chronic lymphocytic lymphoma in order to identify changes at the proteomic level that are specific to MCL. This analysis has identified eight proteins that are specifically upregulated in MCL but are unchanged transcriptionally and would have gone unidentified by typical patient screening methods. Dysregulation of one of these proteins, HMGB3, has been shown to lend drug resistance in ovarian cancer and poor prognosis in neuroblastoma; however, has never been associated with MCL. Knockdown of HMGB3 in MCL cell lines results in reduced proliferation, increased apoptosis, and alterations to cell cycle, indicating it may prove to be a potential therapeutic target for MCL treatment. Mantle cell lymphoma (MCL) is a B cell non-Hodgkin's lymphoma that affects 4-6% of the population. Despite the relatively low incidence of MCL compared to other lymphomas, MCL only has a 5-year survival rate of 50%, making it one of the most deadly. While 90% of patients exhibit the t(11;14) translocation resulting in overexpression of the cell cycle regulatory protein CyclinD1 (CCND1), this alone is not enough for disease initiation. Patients must also have secondary mutations for disease manifestation; however, the number and variety of these mutations differ between every patient. This genetic complexity lends to the difficulty in disease treatment, and there are no standard therapies currently available. Although some therapies have shown promise for treatment in the early stages of the disease, all cases eventually stop responding, resulting in relapse. As such, it is imperative that new therapies be identified to treat this deadly disease. To this end, our lab performed quantitative proteomics on B cells from five healthy donors and tumors from different lymphoma patients including sixteen MCL, seven follicular lymphoma, and seven chronic lymphocytic lymphoma in order to identify changes at the proteomic level that are specific to MCL. This analysis has identified eight proteins that are specifically upregulated in MCL but are unchanged transcriptionally and would have gone unidentified by typical patient screening methods. Dysregulation of one of these proteins, HMGB3, has been shown to lend drug resistance in ovarian cancer and poor prognosis in neuroblastoma; however, has never been associated with MCL. Knockdown of HMGB3 in MCL cell lines results in reduced proliferation, increased apoptosis, and alterations to cell cycle, indicating it may prove to be a potential therapeutic target for MCL treatment.

Anti-prostate cancer activity of a nanoformulation of the spleen tyrosine kinase (SYK) inhibitor C61.

Patients with advanced or metastatic castration-resistant prostate cancer have a dismal prognosis and are therefore in urgent need for therapeutic innovations. Spleen tyrosine kinase has emerged as a new molecular target for castration-resistant prostate cancer. This study was done to test the cytotoxicity of the lead nanoformulation of a potent spleen tyrosine kinase inhibitor, C61-LNP, against the human prostatic carcinoma cell line, PC-3. PC-3 cells were treated with various concentrations of C61-LNP either alone or in combination with cisplatin (CDDP) for 24, 48 and 72 hours. The cell viability was evaluated by MTS assay. Cellular expression levels of various regulatory proteins in treated PC-3 cells were evaluated by Western blot analyses. C61-LNP exhibited dose-dependent cytotoxicity against PC-3 cells. C61-LNP, as well as C61-LNP + CDDP treatments, caused pro-apoptotic proteomic changes including an increase in cleaved fragments of caspases-3 and -9 consistent with caspase activation as well as an improvement in the anti-apoptotic Bcl2 and Bax levels. The combination of C61-LNP and CDDP changed in alterations of the cell cycle regulatory proteins p53, p21, p27, cyclin D1 and cyclin E levels. C61-LNP exhibited cytotoxicity against the castration-resistant prostate cancer cell line PC3. It also caused alterations in expression levels of regulatory proteins involved in apoptosis and cell cycle regulation and these effects were not abrogated by the standard chemotherapy drug CDDP. We are planning to further develop C61-LNP as a selective spleen tyrosine kinase inhibitor as part of a multi-modality treatment strategy for advanced/metastatic castration-resistant prostate cancer.

Management of Drug Resistance in Mantle Cell Lymphoma.

Mantle cell lymphoma (MCL) is a rare but aggressive B-cell hemopathy characterized by the translocation t(11;14)(q13;q32) that leads to the overexpression of the cell cycle regulatory protein cyclin D1. This translocation is the initial event of the lymphomagenesis, but tumor cells can acquire additional alterations allowing the progression of the disease with a more aggressive phenotype and a tight dependency on microenvironment signaling. To date, the chemotherapeutic-based standard care is largely inefficient and despite the recent advent of different targeted therapies including proteasome inhibitors, immunomodulatory drugs, tyrosine kinase inhibitors, relapses are frequent and are generally related to a dismal prognosis. As a result, MCL remains an incurable disease. In this review, we will present the molecular mechanisms of drug resistance learned from both preclinical and clinical experiences in MCL, detailing the main tumor intrinsic processes and signaling pathways associated to therapeutic drug escape. We will also discuss the possibility to counteract the acquisition of drug refractoriness through the design of more efficient strategies, with an emphasis on the most recent combination approaches.

Alisol A Suppresses Proliferation, Migration, and Invasion in Human Breast Cancer MDA-MB-231 Cells.

Natural products are a precious source of promising leads for the development of novel cancer therapeutics. Recently, triterpenoids in Alismatis rhizoma has been widely demonstrated for their anti-cancer activities in cancer cells. In this study, we examined the inhibitory effects of alisol A in human breast cancer cells. We demonstrated that alisol A exhibited significant anti-proliferative effects in MDA-MB-231 cells and this response was related to autophagy induction. Alisol A-induced autophagy was supported by the triggered autophagosome formation and increased LC3-II levels. Interestingly, autophagy inhibitor 3-MA significantly reversed the cytotoxic effects induced by alisol A. Meanwhile, alisol A-induced autophagy was significantly inhibited by 3-MA in MDA-MB-231 cells. Cell cycle analysis revealed that alisol A arrested the cell cycle at G0/G1 phase. The expression level of cell cycle regulatory proteins cyclin D1 was significantly down regulated. In addition, the suppression of NF-κB and PI3K/Akt/mTOR pathways in MDA-MB-231 cells was observed. Furthermore, alisol A significantly suppressed the migration and invasion of MDA-MB-231 cells by inhibiting the expression levels of MMP-2 and MMP-9. Taken together, our results demonstrated that alisol A could inhibit the proliferation and metastasis of MDA-MB-231 cells. It could be a promising agent for breast cancer therapy.

The prognostic value of C-X-C motif chemokine receptor 4 in patients with sporadic malignant peripheral nerve sheath tumors

BackgroundRecent studies indicate that C-X-C motif chemokine receptor 4 (CXCR4) and its ligand, C-X-C motif chemokine ligand 12 (CXCL12), stimulate expression of the cell cycle regulatory protein Cyclin D1 in neurofibromatosis 1-associated malignant peripheral nerve sheath tumor (MPNST) cells and promote their proliferation. In this study, we measured the expression of CXCR4, CXCL12, and Cyclin D1 proteins in sporadic MPNST tissues from Chinese patients and investigated their prognostic values.MethodsCXCR4, CXCL12, and Cyclin D1 protein expression in samples from 58 Chinese patients with sporadic MPNST was assessed with immunohistochemical staining. Their prognostic values were evaluated with Kaplan–Meier analysis and a log-rank test. Multivariate Cox regression analysis was used to identify independent prognostic factors.ResultsHigh expression of CXCR4, CXCL12, and Cyclin D1 was observed in 19 (32.8%), 32 (55.2%), and 16 (27.6%) samples, respectively. CXCR4 expression was positively correlated with CXCL12 expression (r = 0.334, P = 0.010) and Cyclin D1 expression (r = 0.309, P = 0.018). Patients with high CXCR4 expression showed longer overall survival than those with low CXCR4 expression (χ2 = 4.642, P = 0.031).ConclusionHigh CXCR4 expression may define a specific subtype of sporadic MPNST with favorable prognosis.

Pro-oxidant and pro-apoptotic activity of polyphenol extract from Annurca apple and its underlying mechanisms in human breast cancer cells.

Among nutraceuticals, polyphenols represent the most intriguing and studied class of compounds that can be therapeutics for a large spectrum of the most common diseases, including cancer. Although polyphenols are well known as potent antioxidants, a pro-oxidant effect has been associated with a pro-apoptotic function of these compounds in various types of tumor cells. Annurca apple, a southern Italian variety, is characterized by an extremely high content of polyphenols and displays a stronger antioxidant activity compared with other varieties. In the present study we explored the antiproliferative effect of Annurca apple polyphenol extract (APE) in human breast cancer MCF-7 cells and we investigated the underlying mechanisms. Results showed that at 500µM catechin equivalent (EqC) APE acts as a pro-oxidant increasing thiobarbituric acid-reactive species cell content of approximately 6-fold more than the untreated cells. We found that APE strongly inhibits the proliferation of MCF-7 cells by inducing G2/M cell cycle arrest and apoptosis. Immunoblot analysis demonstrated that APE treatment increases the levels of p53 and p21, downregulates the expression of the cell cycle regulatory protein cyclinD1, and inhibits ERK1/2 phosphorylation. Moreover, APE treatment caused a marked increase of pro-apoptotic Bax/Bcl-2 ratio paralleled by caspase-9, -6, -7, and poly(ADP ribose) polymerase cleavage. Altogether our data indicate that APE, at elevated concentrations, acts as a potent pro-oxidant and antiproliferative agent able to downregulate ERK1/2 pathway leading to cell cycle inhibition and apoptosis and provides a rationale for its potential use in the development of novel therapeutics towards breast cancer.

Abstract LB-325: Tumor suppressor effects of miR-17-92a cluster miRNAs on prostate cancer

Abstract MicroRNAs in miR-17-92a cluster are transcribed from a polycistronic transcription unit C13orf25 that generates six mature miRNAs, miR-17, miR-18a, miR-19a, miR- 19b, miR-20a. These miRNAs are reported to be overexpressed in colon and lung cancers, however, we noted a loss of expression of all members of this miR cluster in prostate cancer tissues compared to uninvolved tissues and in aggressive prostate cancer cells. In this study, we show that replenishment of miR-17-92a cluster as a whole showed antiproliferative effects, reduced activation of AKT and MAP kinases, delayed tumorigenicity and reduced tumor growth. Analysis of the expression of mRNA targets showed reduced expression of cell cycle regulatory proteins cyclin D1 and SSH1; and LIMK1 and FGD4 of RhoGTPase signaling pathway through direct targeting. miR-17-92 cluster expression also inhibited EMT via reduced cell migration and expression of mesenchymal markers while elevating expression and surface localization of the epithelial marker E-Cadherin. miR-17-92a miRNAs improved sensitivity of androgen dependent LNCaP 104-S prostate cancer cells to anti-androgen drug Casodex, AKT inhibitor MK-2206 2HCl, and docetaxel. The androgen refractory PC-3 cells also showed increased sensitivity to docetaxel, MK-2206 2HCl and Aurora kinase inhibitor VX680 upon ectopic expression of miR-17-92a cluster miRNAs. RNA seq analysis of prostate cancer cells expressing miR-17-92a cluster revealed altered expression of a number of oncogenic and tumor suppressor mRNAs, and noncoding RNAs, which may have implications in the altered phenotype of these cells. Our data demonstrate a tumor suppressor effect of miR-17-92a cluster miRNAs in prostate cancer cells and restoration of expression of these miRNAs has a therapeutic benefit for both androgen-dependent and -independent prostate cancer cells. Note: This abstract was not presented at the meeting. Citation Format: Ratna Chakrabarti, Richard Ottman, Md Faqrul Hasan. Tumor suppressor effects of miR-17-92a cluster miRNAs on prostate cancer [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 LB-325. doi:10.1158/1538-7445.AM2017-LB-325

MCM7 amplification and overexpression promote cell proliferation, colony formation and migration in esophageal squamous cell carcinoma by activating the AKT1/mTOR signaling pathway.

The roles and mechanisms of mini-chromosome maintenance complex component7 (MCM7) amplification and overexpression in esophageal carcinogenesis were investigated. By analyzing the TCGA datasets, we found that MCM7 was amplified in approximately 12% of esophageal squamous cell carcinomas (ESCCs), and in more than 4% of head and neck squamous cell carcinomas and stomach carcinomas. Overexpression of MCM7 was further verified in three independent GEO datasets of esophageal cancer. Knockdown of MCM7 using two siRNAs significantly inhibited cell proliferation, colony formation and migration of KYSE510 and EC9706 cells invitro. Noteworthy, we further found that silencing of MCM7 suppressed the phosphorylation of AKT1 and mTOR both in KYSE510 and EC9706 cells, and reduced the cell cycle regulatory proteins cyclinD1, cyclinE2 and CDK2. Taken together, our findings suggested that MCM7 promoted tumor cell proliferation, colony formation and migration of ESCC cells via activating AKT1/mTOR signaling pathway.

Inactivation of EGFR/AKT signaling enhances TSA-induced ovarian cancer cell differentiation.

Ovarian tumor is one of the most lethal gynecologic cancers, but differentiation therapy for this cancer is poorly characterized. Here, we show that thrichostatin A (TSA), the well known inhibitor of histone deacetylases (HDACs), can induce cell differentiation in HO8910 ovarian cancer cells. TSA-induced cell differentiation is characterized by typical morphological change, increased expression of the differentiation marker FOXA2, decreased expression of the pluripotency markers SOX2 and OCT4, suppressing cell proliferation, and cell cycle arrest in the G1 phase. TSA also induces an elevated expression of cell cycle inhibitory protein p21Cip1 along with a decrease in cell cycle regulatory protein cyclinD1. Significantly, blockage of epidermal growth factor receptor(EGFR) signaling pathway with specific inhibitors of this signaling cascade promotes the TSA-induced differentiation of HO8910 cells. These results imply that the EGFR cascade inhibitors in combination with TSA may represent a promising differentiation therapy strategy for ovarian cancer.

Alpinia oxyphylla Miq. fruit extract activates IGFR-PI3K/Akt signaling to induce Schwann cell proliferation and sciatic nerve regeneration

BackgroundIt is known that the medicinal herb Alpinia oxyphylla Miq. is widely used as a remedy for diarrhea as well as the symptoms accompanying hypertension and cerebrovascular disorders. Moreover, it has also been reported that Alpinia oxyphylla Miq. has beneficial effects on anti-senescence and neuro-protection. This study focuses on the molecular mechanisms by which the Alpinia oxyphylla Miq. fruits promote neuron regeneration.MethodsA piece of silicone rubber was guided across a 15 mm gap in the sciatic nerve of a rat. This nerve gap was then filled with various doses of Alpinia oxyphylla Miq. fruits to assess their regenerative effect on damaged nerves. Further, we investigated the role of Alpinia oxyphylla Miq. fruits in RSC96 Schwann cell proliferation.ResultsOur current results showed that treatment with the extract of Alpinia oxyphylla Miq. fruits triggers the phosphorylated insulin-like growth factor-1 receptor- phosphatidylinositol 3-kinase/serine-threonine kinase pathway, and up-regulated the proliferating cell nuclear antigen in a dose-dependent manner. Cell cycle analysis on RSC96 Schwann cells showed that, after exposure to Alpinia oxyphylla Miq. fruit extract, the transition from the first gap phase to the synthesis phase occurs in 12–18 h. The expression of the cell cycle regulatory proteins cyclin D1, cyclin E and cyclin A increased in a dose-dependent manner. Transfection with a small interfering RNA blocked the expression of phosphatidylinositol 3-kinase and induced down-regulation both on the mRNA and protein levels, which resulted in a reduction of the expression of the survival factor B-cell lymphoma 2.ConclusionWe provide positive results that demonstrate that Alpinia oxyphylla Miq. fruits facilitate the survival and proliferation of RSC96 cells via insulin-like growth factor-1 signaling.

AG36 Inhibits Human Breast Cancer Cells Proliferation by Promotion of Apoptosis In vitro and In vivo.

AG36 is the biotransformation product of triterpenoid saponin from Ardisia gigantifolia stapf. In this study, the antitumor activity and underlying molecular mechanisms of AG36 against human breast MCF-7, MDA-MB-231, and SK-BR-3 cancer cells were investigated. AG36 inhibited the viability of MCF-7, MDA-MB-231, and SK-BR-3 cells in a dose and time-dependent manner, with an IC50 of approximately 0.73, 18.1, and 23.4 μM at 48 h, respectively. AG36 obviously induced apoptosis and G2/M arrest of all the three breast cancer cells. Moreover, AG36 decreased the protein expression of cycle regulatory proteins cyclin B1 or cyclin D1. In MCF-7 and MDA-MB-231 cells, AG36 strongly increased the cleaved caspase-3 and -8 protein expressions, while in SK-BR-3 cells, AG36 only increased the protein expression of cleaved caspase-3. In all the three breast cancer cells, the ratio of Bax/Bcl-2 and cytosolic cytochrome c content increased significantly compared with control group. The death receptor-related proteins Fas/FasL, TNFR1, and DR5 were detected by Western blot, it showed that different breast cancer cells activated the death receptor-mediated extrinsic caspase-8 pathway through different receptors. In addition, the caspase-8 inhibitor z-IETD-fmk could significantly block AG36-triggered MCF-7 cells apoptosis. The in vivo studies showed that AG36 significantly inhibited the growth of MCF-7 xenograft tumors in BALB/c nude mice comparing with control. In conclusion, AG36 inhibited MCF-7, MDA-MB-231, and SK-BR-3 cells proliferation by the intrinsic mitochondrial and the extrinsic death receptor pathways and AG36 might be a potential breast cancer therapeutic agent.

A3 adenosine receptor agonist induce G1 cell cycle arrest via Cyclin D and cyclin-dependent kinase 4 pathways in OVCAR-3 and Caov-4 cell lines.

The cell cycle, a vital process that involves in cells' growth and division, lies at the heart of cancer. It has been shown that IB-MECA, an A3 adenosine receptor agonist inhibits the proliferation of cancer cells by inducing cell cycle arrest in several tumors. In this study, we evaluated the role of IB-MECA inhibition in cell cycle progression in ovarian cancer cells. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay in Caov-4 and OVCAR-3. Analysis of cell cycle distribution was carried out by flow cytometry. To determine the mechanisms of IB-MECA-mediated induction of cell cycle arrest, the expression of cell cycle regulatory proteins Cyclin D1 and cyclin-dependent kinase 4 (CDK4) was evaluated. Our results showed that IB-MECA significantly reduced cell viability in a dose-dependent manner. Moreover, our results indicated that a low concentration of IB-MECA induced G1 cell cycle arrest. Reduction of Cyclin D1 and CDK4 protein levels was also observed after treating cancer cells with IB-MECA. This study demonstrated that IB-MECA induces G1 phase cell cycle arrest through Cyclin D1/CDK4-mediated pathway in ovarian cancer cells.

The EMSY Gene Collaborates with CCND1 in Non-Small Cell Lung Carcinogenesis.

Background: Lung cancer is the leading cause of cancer deaths. The main risk factor is smoking but the risk is also associated with various genetic and epigenetic components in addition to environmental factors. Increases in the gene copy numbers due to chromosomal amplifications constitute a common mechanism for oncogene activation. A gene-dense region on chromosome 11q13 which harbors four core regions that are frequently amplified, has been associated with various types of cancer. The important cell cycle regulatory protein cyclin D1 (CCND1) is an essential driver of the first core region of the Chr11q13 amplicon. Deregulation of CCND1 has been associated with different kinds of human malignancies including lung cancer. The EMSY (c11orf30) gene has been proposed as the possible driver of the fourth core of the 11q13 amplicon and its amplification has been associated with breast and ovarian cancers. There is no report in the literature investigating the EMSY gene in lung cancer.Methods: In this study, expression levels of the EMSY and CCND1 genes were investigated in 85 patients with non small cell lung cancer by Real Time PCR.Results: Expression of the EMSY and CCND1 genes were increased in 56 (65.8%) and 50 (58.8%) of the patients, respectively. Both genes showed a higher expression in the tumors when compared to normal tissues. A strong correlation was present between the expression rates of both genes (p<0.001). Patients with adenocarcinoma had higher expression levels of both genes (p=0.02).Conclusion: We conclude that EMSY and CCND1 work in collaboration and contribute to the pathogenesis of lung cancer.

The other face of miR-17-92a cluster, exhibiting tumor suppressor effects in prostate cancer.

miR-17-92a cluster miRNAs are transcribed from a polycistronic transcription unit C13orf25 that generates six mature miRNAs, miR-17, miR-18a, miR-19a, miR-19b, miR-20a and miR-92a that are overexpressed in lung and colon cancers. Here we show that the expression of miR-17-92a miRNAs are reduced in cancerous prostate tissues compared to uninvolved areas and also in aggressive prostate cancer cells. Restoration of expression of all members of miR-17-92a cluster showed, decreased expression of cell cycle regulatory proteins cyclin D1 and SSH1; and LIMK1 and FGD4 of RhoGTPase signaling pathway. Expression of miR-17-92a miRNAs caused decreased cell proliferation, reduced activation of AKT and MAP kinases, delayed tumorigenicity and reduced tumor growth in animals. Expression of miR-17-92a miRNAs inhibited EMT via reduced cell migration and expression of mesenchymal markers while elevating expression and surface localization of the epithelial marker E-Cadherin. Expression of miR-17-92a miRNAs improved sensitivity of androgen dependent LNCaP 104-S prostate cancer cells to anti-androgen drug Casodex, AKT inhibitor MK-2206 2HCl, and docetaxel. The androgen refractory PC-3 cells also showed increased sensitivity to docetaxel, MK-2206 2HCl and Aurora kinase inhibitor VX680 upon ectopic expression of miR-17-92a cluster miRNAs. Our data demonstrate a tumor suppressor effect of miR-17-92a cluster miRNAs in prostate cancer cells and restoration of expression of these miRNAs has a therapeutic benefit for both androgen-dependent and -independent prostate cancer cells.

Antiangiogenic and antihepatocellular carcinoma activities of the Juniperus chinensis extract

BackgroundTo identify a novel therapeutic agent for hepatocellular carcinoma (HCC), for which no promising therapeutic agent exists, we screened a panel of plants and found that Juniperus chinensis exhibited potential antiangiogenic and anti-HCC activities. We further investigated the antiangiogenic and anti-HCC effects of the active ingredient of J. chinensis extract, CBT-143-S-F6F7, both in vitro and in vivo.MethodsA tube formation assay conducted using human umbilical vein endothelial cells (HUVECs) was first performed to identify the active ingredient of CBT-143-S-F6F7. A series of angiogenesis studies, including HUVEC migration, Matrigel plug, and chorioallantoic membrane (CAM) assays, were then performed to confirm the effects of CBT-143-S-F6F7 on angiogenesis. The effects of CBT-143-S-F6F7 on tumor growth were investigated using a subcutaneous and orthotopic mouse model of HCC. In vitro studies were performed to investigate the effects of CBT-143-S-F6F7 on the cell cycle and apoptosis in HCC cells. Moreover, protein arrays for angiogenesis and apoptosis were used to discover biomarkers that may be influenced by CBT-143-S-F6F7. Finally, nuclear magnetic resonance analysis was conducted to identify the compounds of CBT-143-S-F6F7.ResultsCBT-143-S-F6F7 showed significantly antiangiogenic activity in various assays, including HUVEC tube formation and migration, CAM, and Matrigel plug assays. In in vivo studies, gavage with CBT-143-S-F6F7 significantly repressed subcutaneous Huh7 tumor growth in severe combined immunodeficient (SCID) mice, and prolonged the survival of orthotopic Huh7 tumor-bearing SCID mice (a 40 % increase in median survival duration compared with the vehicle-treated mice). Immunohistochemical staining of subcutaneous Huh7 tumors in CBT-143-S-F6F7-treated mice showed a significantly decrease in the cell cycle regulatory protein cyclin D1, cellular proliferation marker Ki-67, and endothelial marker CD31. CBT-143-S-F6F7 caused arrest of the G2/M phase and induced Huh7 cell apoptosis, possibly contributing to the inhibition of HCC tumors. Protein array analysis revealed that several angiogenic and antiapoptotic factors were suppressed in CBT-143-S-F6F7-treated Huh7 cells. Finally, five compounds from CBT-143-S-F6F7 were identified.ConclusionsAccording to these results, we report for the first time the antiangiogenic and anti-HCC activities of CBT-143-S-F6F7, the active fractional extract of J. chinensis. We believe that CBT-143-S-F6F7 warrants further evaluation as a new anti-HCC drug.Electronic supplementary materialThe online version of this article (doi:10.1186/s12906-016-1250-6) contains supplementary material, which is available to authorized users.

Gartanin induces cell cycle arrest and autophagy and suppresses migration involving PI3K/Akt/mTOR and MAPK signalling pathway in human glioma cells.

In central nervous system, glioma is the most common primary brain tumour. The diffuse migration and rapid proliferation are main obstacles for successful treatment. Gartanin, a natural xanthone of mangosteen, suppressed proliferation, migration and colony formation in a time‐ and concentration‐dependent manner in T98G glioma cells but not in mouse normal neuronal HT22 cells. Gartanin, at low micromole, led to cell cycle arrest in G1 phase accompanied by inhibited expression level of G1 cell cycle regulatory proteins cyclin D1, while increased expression level of cyclin‐dependent kinase inhibitor p27Kip1. In addition, the secretion and activity of matrix metalloproteinases 2/9 (MMP‐2/‐9) were significantly suppressed in T98G cells treated with gartanin, and it might result from modulating mitogen‐activated protein kinases (MAPK) signalling pathway in T98G glioma cells. Moreover, gartanin significantly induced autophagy in T98G cells and increased GFP‐LC3 punctate fluorescence accompanied by the increased expression level of Beclin 1 and LC3‐II, while suppressed expression level of p62. Gartanin treatment resulted in obvious inhibition of PI3K/Akt/mTOR signalling pathway, which is important in modulating autophagy. Notably, gartanin‐mediated anti‐viability was significantly abrogated by autophagy inhibitors including 3‐methyladenine (3‐MA) and chloroquine (CQ). These results indicate that anti‐proliferation effect of gartanin in T98G cells is most likely via cell cycle arrest modulated by autophagy, which is regulated by PI3K/Akt/mTOR signalling pathway, while anti‐migration effect is most likely via suppression of MMP‐2/‐9 activity which is involved in MAPK signalling pathway.

FoxF1 and FoxF2 transcription factors synergistically promote rhabdomyosarcoma carcinogenesis by repressing transcription of p21Cip1 CDK inhibitor.

The role of Forkhead Box F1 (FoxF1) transcription factor in carcinogenesis is not well characterized. Depending on tissue and histological type of cancer, FoxF1 was shown to be either oncogene or tumor suppressor. Alveolar rhabdomyosarcoma (RMS) is the most aggressive pediatric soft tissue sarcoma. While FoxF1 is highly expressed in alveolar RMS, the functional role of FoxF1 in RMS is unknown. The present study demonstrates that expression of FoxF1 and its closely related transcription factor FoxF2 are essential for rhabdomyosarcoma tumor growth. Depletion of FoxF1 or FoxF2 in rhabdomyosarcoma cells decreased tumor growth in orthotopic mouse models of RMS. The decreased tumorigenesis was associated with the reduced tumor cell proliferation. Cell cycle regulatory proteins Cdk2, Cdk4/6, Cyclin D1 and Cyclin E2 were decreased in FoxF1- and FoxF2-deficient RMS tumors. Depletion of either FoxF1 or FoxF2 delayed G1-S cell cycle progression, decreased levels of phosphorylated Rb and increased protein levels of the CDK inhibitors, p21Cip1 and p27Kip1. Depletion of both FoxF1 and FoxF2 in tumor cells completely abrogated RMS tumor growth in mice. Overexpression of either FoxF1 or FoxF2 in tumor cells was sufficient to increase carcinogenesis in orthotopic RMS mouse model. FoxF1 and FoxF2 directly bound to and repressed transcriptional activity of p21Cip1 promoter through −556/−545 bp region, but did not affect p27Kip1 transcription. Knockdown of p21Cip1 restored cell cycle progression in the FoxF1- or FoxF2-deficient tumor cells. Altogether, FoxF1 and FoxF2 promoted RMS tumorigenesis by inducing tumor cell proliferation via transcriptional repression of p21Cip1 gene promoter. Due to robust oncogenic activity in RMS tumors, FoxF1 and FoxF2 may represent promising targets for anti-tumor therapy.