Cyclin D1 mRNA Levels Research Articles

Effects of basic fibroblast growth factor and cyclin D1 on cigarette smoke-induced pulmonary vascular remodeling in rats.

Cigarette smoking may contribute to pulmonary hypertension in chronic obstructive pulmonary disease by resulting in pulmonary vascular remodeling that involves pulmonary artery smooth muscle cell proliferation. This study investigated the effects of basic fibroblast growth factor (bFGF) and cyclin D1 on the pulmonary vascular remodeling in smoking-exposed rats. Twenty-four male Wistar rats were randomly divided into four groups. Three tobacco-exposed groups were exposed to the smoke produced by 20 cigarettes for 60 min, twice a day for two, four or eight weeks, and the control group were exposed to fresh air. The expression of bFGF and cyclin D1 in the pulmonary arterial smooth muscle cells were determined using immunohistochemistry. Quantitative polymerase chain reaction was conducted to determine the expression levels of bFGF and cyclin D1 mRNA. In addition, the expression of bFGF and cyclin D1 proteins was evaluated by western blotting. The expression of bFGF and cyclin D1 at the mRNA and protein levels was shown to increase with the duration of smoke exposure (P<0.05). The correlation analysis indicated the expression of bFGF and cyclin D1 was positively associated with the pulmonary vessel wall thickness. The expression of bFGF was positively associated with that of cyclin D1. Collectively, the data demonstrated that the upregulation of bFGF and cyclin D1 occurred in rats subjected to smoke exposure, which may be associated with the abnormal proliferation of the smooth muscle cells in the pulmonary arteries.

Chelidonium majus L. extract induces apoptosis through caspase activity via MAPK-independent NF-κB signaling in human epidermoid carcinoma A431 cells.

Chelidonium majus L. (C. majus L.) is known to possess certain biological properties such as anti-inflammatory, antimicrobial, antiviral and antitumor activities. We investigated the effects of C. majus L. extract on human epidermoid carcinoma A431 cells through multiple mechanisms, including induction of cell cycle arrest, activation of the caspase-dependent pathway, blocking of nuclear factor-κB (NF-κB) activation and involvement in the mitogen-activated protein kinase (MAPK) pathway. C. majus L. inhibited the proliferation of A431 cells in a dose- and time-dependent manner, increased the percentage of apoptotic cells, significantly decreased the mRNA levels of cyclin D1, Bcl-2, Mcl-1 and survivin and increased p21 and Bax expression. Exposure of A431 cells to C. majus L. extract enhanced the activities of caspase-3 and caspase-9, while co-treatment with C. majus L., the pan-caspase inhibitor Z-VAD-FMK and the caspase-3 inhibitor Z-DEVE-FMK increased the proliferation of A431 cells. C. majus L. extract not only inhibited NF-κB activation, but it also activated p38 MAPK and MEK/ERK signaling. Taken together, these results demonstrate that C. majus L. extract inhibits the proliferation of human epidermoid carcinoma A431 cells by inducing apoptosis through caspase activation and NF-κB inhibition via MAPK-independent pathway.

Ethyl acetate extract from Jiedu Xiaozheng Yin inhibits the proliferation of human hepatocellular carcinoma cells by suppressing polycomb gene product Bmi1 and Wnt/β-catenin signaling.

Jiedu Xiaozheng Yin (JXY) is a Chinese herbal decoction used to treat hepatocellular carcinoma (HCC). Previous studies have demonstrated that JXY can inhibit HCC cell proliferation via induction of G0/G1 phase arrest. In this study, we investigated whether the inhibitory effect of JXY on HCC cells is associated with the inhibition of the Wnt/β‑catenin pathway and the polycomb gene product Bmi1. Ethyl acetate extract from JXY (EE-JXY) was prepared. Methyl thiazolyl tetrazolium (MTT) and colony formation assays were used to measure cell proliferation. Immunofluorescence was used to analyze the expression and location of β-catenin and Bmi1. Immunohistochemistry was used to examine the expression of proliferating cell nuclear antigen (PCNA), c-myc and cyclin D1. β-catenin, Bmi1, c-myc, cyclin D1 and p16INK4A mRNA levels were detected by RT-PCR. The results demonstrated that EE-JXY inhibited the expression of PCNA, c-myc, cyclin D1 and Bmi1, and upregulated the expression of p16INK4A. We also found that EE-JXY could facilitate β-catenin translocation from the cytoplasm and nuclei to the cytomembrane. Finally, suppression of cell proliferation and expression of Bmi1 and Wnt/β-catenin by EE-JXY was confirmed in a mouse xenograft model of HCC. Thus, EE-JXY can inhibit the proliferation of HCC partially via suppression of the Bmi1 and Wnt/β-catenin signaling pathways.

Abstract 3186: Transplacental arsenic exposure modifies the number of hair follicle keratinocytes stem cells and alters their cell-cycle control

Abstract Drinking water contamination with arsenic is a worldwide problem and a concern in some areas of the United States, especially in the West (California, Nevada, Alaska and Utah) and small areas of New England. Inorganic arsenic is considered a human carcinogen with many target tissues such as the skin, urinary bladder and lung; and characterizing its mechanisms of action is key to designing methods of intervention. Arsenic has transplacental carcinogenic activity, and since their fetal abundance, stem cells (SCs) seem to be a main target of arsenic exposure during gestation. We have established that fetal arsenic exposure via the maternal drinking water, leads to a decrease number of keratinocyte's stem cells (KSC) in mouse epidermis of the offspring. Biochemical analysis of these KSCs shows increased mRNA levels of the cell-cycle regulators cyclin D1 and CDK4. Supporting this observation, overexpression of CDK4 in KSCs mimics arsenic exposure as demonstrated by the reduced number of KSCs, and the decreased number of benign skin tumors, and severe rise in the rate of malignant conversion to squamus cell carcinomas. We expect that these studies will enable to determine the consequence of in utero arsenic exposure on the proliferative potential of KSCs and the impact on the selection of KSCs that give origin to skin tumors with high risk of malignant conversion. Therefore, these studies will contribute to understanding how early alterations of somatic stem cells results in functional changes that impact a late stage of tumorigenesis such as malignant progression. Citation Format: Paula L. Miliani de Marval, Sun Hye Kim, Marcelo L. Rodriguez-Puebla. Transplacental arsenic exposure modifies the number of hair follicle keratinocytes stem cells and alters their cell-cycle control. [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 3186. doi:10.1158/1538-7445.AM2014-3186

Abstract 1405: Knockdown ARID5A suppresses proliferation of LNCaP prostate cancer cell through the inhibition of global protein synthesis

Abstract AT-Rich Interaction Domain-containing protein 5a (ARID5A; also known as Modulator Recognition Factor 1(MRF1)) was first cloned in our laboratory by screening a cDNA library of Tera-2 mRNA with an oligonucleotide probe from the major immediate early gene of human cytomegalovirus. It has been reported that ARID5A forms a complex with androgen receptor (AR) in yeast two hybrid assays and suppresses the transcriptional activity of AR in transient transfection assays. In this study, we report that the knockdown of Arid5a expression inhibits proliferation of LNCaP cells on the contrary to the expectation from the transient transfection assay. We found that proliferation of cells stimulated by the treatment with dihydro-testosterone (DHT) was suppressed when Arid5a expression was down-regulated by siRNA technology. Flow cytometer analysis showed that DHT-stimulated cell cycle was arrested at G1 phase after the knockdown of Arid5a expression. Oil Red O staining assays showed that the inhibition of Arid5a expression led to decreased DHT-induced lipid accumulation. Our data further proved that the knockdown of Arid5a expression inhibited the protein expression of DHT-induced sterol regulatory element binding transcription factor 1 (SREBP-1). In addition, Western blot analysis revealed that the down-regulation of Arid5a decreased the expression of cell survival/proliferation and cell cycle regulation proteins, such as hypoxia inducible factor 1 alpha (HIF1a), cyclin D1 and cyclin D3. Real time PCR data revealed that the knockdown of Arid5a expression did not affect the steady level of HIF1a, cyclin D1 and cyclin D3 mRNA. These suggested that knockdown of Arid5a expression affected protein expression at translational and/or post-translational level. Mammalian target of rapamycin complex 1 (mTORC1) is a well-known factor in regulating the cap-dependent protein synthesis. Western blot analysis indicated that the suppression of Arid5a expression had no effects on the activity of mTORC1. However, knockdown of Arid5a expression increased the phosphorylation of eukaryotic translation initiation factor 2a (eIF2a) which inhibited translation initiation. S35 labeling assay confirmed that the knockdown of Arid5a expression suppressed the global protein synthesis. In order to find out the mechanisms of Arid5a-dependent phosphorylation of eIF2a, we tested four known kinases which were responsible for the phosphorylation of eIF2a. Two of the four eIF2a kinases, general control non-derepressible 2 (GCN2) and RNA-dependent protein kinase (PKR)-like endoplasmic reticulum kinase (PERK), were activated by the suppression of Arid5a expression. We are currently studying on the mechanisms by which ARID5A regulates GCN2 and PERK kinase. Citation Format: Chao Sun, Viktor Chesnokov, Keiichi Itakura. Knockdown ARID5A suppresses proliferation of LNCaP prostate cancer cell through the inhibition of global protein synthesis. [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 1405. doi:10.1158/1538-7445.AM2014-1405

Placental estrogen suppresses cyclin D1 expression in the nonhuman primate fetal adrenal cortex.

We have previously shown that estrogen selectively suppresses growth of the fetal zone of the baboon fetal adrenal cortex, which produces the C19-steroid precursors, eg, dehydroepiandrosterone sulfate, which are aromatized to estrogen within the placenta. In the present study, we determined whether fetal adrenal expression of cell cycle regulators are altered by estrogen and thus provide a mechanism by which estrogen regulates fetal adrenocortical development. Cyclin D1 mRNA levels in the whole fetal adrenal were increased 50% (P < .05), and the number of cells in the fetal adrenal definitive zone expressing cyclin D1 protein was increased 2.5-fold (P < .05), whereas the total number of cells in the fetal zone and fetal serum dehydroepiandrosterone sulfate levels were elevated 2-fold (P < .05) near term in baboons in which fetal serum estradiol levels were decreased by 95% (P < .05) after maternal administration of the aromatase inhibitor letrozole and restored to normal by concomitant administration of letrozole plus estradiol throughout second half of gestation. However, fetal adrenocortical expression of cyclin D2, the cyclin-dependent kinase (Cdk)-2, Cdk4, and Cdk6, and Cdk regulatory proteins p27(Kip1) and p57(Kip2) were not changed by letrozole or letrozole plus estradiol administration. We suggest that estrogen controls the growth of the fetal zone of the fetal adrenal by down-regulating cyclin D1 expression and thus proliferation of progenitor cells within the definitive zone that migrate to the fetal zone. We propose that estrogen restrains growth and function of the fetal zone via cyclin D1 to maintain estrogen levels in a physiological range during primate pregnancy.

Thymoquinone inhibits proliferation and invasion of human nonsmall-cell lung cancer cells via ERK pathway.

Thymoquinone (TQ) is the primary bioactive component of Nigella sativa Linn seed oil and used as anti-inflammatory, anti-oxidant, and anti-neoplastic agent. Previous studies have shown that TQ exhibits inhibitory effects on multiple cancers. However, the detailed antineoplastic effects and its molecular mechanisms of TQ on lung cancer are not entirely elucidated yet. In the present study, we aimed to investigate the effects of TQ on cell proliferation, migration, and invasion as well as its underlying anti-metastatic mechanisms in A549 cells. Lung cancer cell line A549 cells were treated with different concentration of TQ for different period of time, and the growth-inhibitory effects of TQ was measured by MTT and cell count assays; cell cycle was determined by flow cytometry; wound healing and transwell assays were used to assess the cell migration and invasion activities; Western blot and real-time quantitative RT-PCR were used to determine the expression of proliferation and invasion associated genes as well as MAPKs pathway molecules; gelatinase activity was estimated using gelatin zymography assay. The results show that TQ played a role in inhibiting the proliferation, migration, and invasion of A549 lung cancer cells, it also inhibited the expression level of PCNA, cyclin D1, MMP2, and MMP9 mRNA and protein in a dose- and time-dependent manner especially at 10, 20, 40μmol/L concentrations. The cell cycle inhibitor P16 expression and the gelatinase activities of MMP2 and MMP9 were also inhibited by TQ dramatically. TQ reduced phosphorylation of ERK1/2; however, the proliferation and invasion inhibitory effects of TQ on A549 cells were neutralized by ERK1/2 inhibitor PD98059. In conclusion, our study confirmed that TQ could inhibit A549 cell proliferation, migration, and invasion through ERK1/2 pathway, as proposed the therapeutic potential of TQ as an anti-metastatic agent in human lung cancer treatment.

Duhuo Jisheng Decoction‑containing serum promotes proliferation of interleukin‑1β‑induced chondrocytes through the p16‑cyclin D1/CDK4‑Rb pathway.

Duhuo Jisheng Decoction (DHJSD) is a traditional Chinese herbal medicine that has multiple uses, including as a treatment for osteoarthritis (OA). However, the molecular mechanisms underlying the therapeutic effects of DHJSD on OA remain unknown. In the present study, a serum pharmacological method was applied to investigate the effects of DHJSD on the proliferation of chondrocytes treated with interleukin‑1β (IL‑1β) invitro. This is a cell model commonly used to reproduce the mechanisms involved in degenerative arthropathies, including OA. The most effective intervention conditions of DHJSD serum were examined by MTT assay. The degenerative chondrocyte model was established by IL‑1β‑culture for 24h, and was verified by optical microscopy and immunohistochemical analyses. Following the successful establishment of the degenerative chondrocyte model, the chondrocytes were subsequently randomly divided into two groups: The blank serum group and the DHJSD treatment group. Subsequent to treatment with the corresponding serum, cell proliferation was detected by MTT assay and DNA staining followed by FACS analysis, and the mRNA and protein expression levels of cyclinD1, cyclin‑dependent kinase4 (CDK4), retinoblastoma tumor suppressor protein (Rb) and p16 were measured by reverse transcription polymerase chain reaction and western blotting, respectively. The results indicated that the most effective condition for the promotion of chondrocyte proliferation was 10% concentration of DHJSD 2‑h serum, and the degenerative chondrocyte model was successfully reproduced by IL‑1β‑treatment for 24h. The mRNA and protein expression levels of cyclinD1, CDK4 and Rb in the DHJSD serum‑treated cells were significantly increased compared with those in the blank serum group, whereas p16 expression was significantly downregulated. These results indicate that treatment of cells with DHJSD‑containing serum is able to promote IL‑1β‑induced chondrocyte proliferation by promoting G1/Sphase transition via modulating the expressions of cyclinD1, CDK4, Rb and p16, which contribute to the clinical efficacy of DHJSD in OA.

The role of c2orf68 and PI3K/Akt/mTOR pathway in human colorectal cancer.

The aim of the research is to determine whether c2orf68 gene plays a role in the carcinogenesis of human colorectal cancer and to study the function of c2orf68 belonging to the UPF0561 family. The mRNA expression levels of c2orf68 were examined in 30 pairs of human colorectal adenocarcinoma tissues and adjacent normal colorectal tissues by qRT-PCR. The SW480 and SW620 cell lines were transfected with siRNA against the c2orf68 gene and set gene. The expressed mRNA levels of Akt, PI3K, Bax, Bcl-2, caspase3, c-Myc, cyclinD1, pp2a and set were determined by qRT-PCR, and the protein levels of C2ORF68, c-Myc, PP2A and SET were examined by Western blot. Cell proliferation was tested by MTT assay, and apoptosis and cell cycle were studied by flow cytometry. Cancer metastasis assay was performed by transwell chamber. The c2orf68 mRNA expression was down-regulated in 63.33 % of the cancer samples, and a positive correlation was found between the mRNA expression of c-Myc and pp2a that of c2orf68. Meanwhile, there was a negative correlation between the mRNA expression of c2orf68 and set. The c2orf68 mRNA was significantly down-regulated in SW480(-c2orf68) and SW620(-c2orf68) cells. The inhibitory rate in the two cell lines was, respectively, 65.2 and 71.6 % by qRT-PCR. A 22.7 % inhibition on cell proliferation in SW480(-c2orf68) cells and a 21.2 % inhibition in SW620(-c2orf68) cells were observed using the MTT assay. Flow cytometry analysis indicates that the cell apoptosis rate was 21.42 % in SW480(-c2orf68) cells and 17.78 % in SW620(-c2orf68) cells, whereas the percentage of G1 phase cells was 61.8 and 58.6 % in SW480(-c2orf68) and SW620(-c2orf68) cells, respectively. In addition, the mRNA expression of set and Bax was up-regulated after c2orf68 interfered in SW480(-c2orf68) and SW620(-c2orf68) cells, whereas that of Bcl-2, c-Myc, cyclinD1, caspase3 and pp2a was down-regulated. Consistent with the mRNA results, the protein expression of C2ORF68, PP2A and c-Myc was down-regulated, whereas that of SET was up-regulated. Our data thus suggest that c2orf68 promotes carcinogenesis through the regulation of mammalian target of rapamycin signaling pathway.

YB-1 promotes transcription of cyclin D1 in human non-small-cell lung cancers.

Cyclin D1, an oncogenic G1 cyclin, and YB-1, a transcription factor involved in cell growth, are both over-expressed in several human cancers. In human lung cancer, the functional association between YB-1 and cyclin D1 has never been elucidated. In this study, we show YB-1 is involved in the transcription of cyclin D1 in human lung cancer. Depletion of endogenous YB-1 by siRNA inhibited progression of G1 phase and down-regulated both the protein and mRNA levels of cyclin D1 in human lung cancer cells. Forced over-expression of YB-1 with a cyclin D1 reporter plasmid increased luciferase activity, and ChIP assay results showed YB-1 bound to the cyclin D1 promoter. Moreover, the amount of YB-1 mRNA positively correlated with cyclin D1 mRNA levels in clinical non-small-cell lung cancer (NSCLC) specimens. Immunohistochemical analysis also indicated YB-1 expression correlated with cyclin D1 expression in NSCLC specimens. In addition, most of the cases expressing both cyclin D1 and CDC6, another molecule controlled by YB-1, had co-existing YB-1 over-expression. Together, our results suggest that aberrant expression of both cyclin D1 and CDC6 by YB-1 over-expression may collaboratively participate in lung carcinogenesis.

The pterocarpanquinone LQB-118 inhibits tumor cell proliferation by downregulation of c-Myc and cyclins D1 and B1 mRNA and upregulation of p21 cell cycle inhibitor expression

The incidence of cancer grows annually worldwide and in Brazil it is the second cause of death. The search for anti-cancer drugs has then become urgent. It depends on the studies of natural and chemical synthesis products. The antitumor action of LQB-118, a pterocarpanquinone structurally related to lapachol, has been demonstrated to induce mechanisms linked to leukemia cell apoptosis. This work investigated some mechanisms of the in vitro antitumor action of LQB-118 on prostate cancer cells. LQB-118 reduced the expression of the c-Myc transcription factor, downregulated the cyclin D1 and cyclin B1 mRNA levels and upregulated the p21 cell cycle inhibitor. These effects resulted in cell cycle arrest in the S and G2/M phases and inhibition of tumor cell proliferation. LQB-118 also induced programmed cell death of the prostate cancer cells, as evidenced by internucleosomal DNA fragmentation and annexin-V positive cells. Except the cell cycle arrest in the S phase and enhanced c-Myc expression, all the mechanisms observed here for the in vitro antitumor action of LQB-118 were also found for Paclitaxel, a traditional antineoplastic drug. These findings suggest new molecular mechanisms for the LQB-118 in vitro antitumor action.

Substance P activates the Wnt signal transduction pathway and enhances the differentiation of mouse preosteoblastic MC3T3-E1 cells.

Recent experiments have explored the impact of Wnt/β-catenin signaling and Substance P (SP) on the regulation of osteogenesis. However, the molecular regulatory mechanisms of SP on the formation of osteoblasts is still unknown. In this study, we investigated the impact of SP on the differentiation of MC3T3-E1 cells. The osteogenic effect of SP was observed at different SP concentrations (ranging from 10−10 to 10−8 M). To unravel the underlying mechanism, the MC3T3-E1 cells were treated with SP after the pretreatment by neurokinin-1 (NK1) antagonists and Dickkopf-1 (DKK1) and gene expression levels of Wnt/β-catenin signaling pathway components, as well as osteoblast differentiation markers (collagen type I, alkaline phosphatase, osteocalcin, and Runx2), were measured using quantitative polymerase chain reaction (PCR). Furthermore, protein levels of Wnt/β-catenin signaling pathway were detected using Western blotting and the effects of SP, NK1 antagonist, and DKK1 on β-catenin activation were investigated by immunofluorescence staining. Our data indicated that SP (10−9 to 10−8 M) significantly up-regulated the expressions of osteoblastic genes. SP (10−8 M) also elevated the mRNA level of c-myc, cyclin D1, and lymphocyte enhancer factor-1 (Lef1), as well as c-myc and β-catenin protein levels, but decreased the expression of Tcf7 mRNA. Moreover, SP (10−8 M) promoted the transfer of β-catenin into nucleus. The effects of SP treatment were inhibited by the NK1 antagonist and DKK1. These findings suggest that SP may enhance differentiation of MC3T3-E1 cells via regulation of the Wnt/β-catenin signaling pathway.

Fangchinoline Inhibits Cell Proliferation Via Akt/GSK-3beta/cyclin D1 Signaling and Induces Apoptosis in MDA-MB-231 Breast Cancer Cells

Fangchinoline (Fan) inhibits cell proliferation and induces apoptosis in several cancer cell lines. The effects of Fan on cell growth and proliferation in breast cancer cells remain to be elucidated. Here, we show that Fan inhibited cell proliferation in the MDA-MB-231 breast cancer cell line through suppression of the AKT/Gsk- 3beta/cyclin D1 signaling pathway. Furthermore, Fan induced apoptosis by increasing the expression of Bax (relative to Bcl-2), active caspase 3 and cytochrome-c. Fan significantly inhibited cell proliferation of MDA- MB-231 cells in a concentration and time dependent manner as determined by MTT assay. Flow cytometry analysis demonstrated that Fan treatment of MDA-MB-231 cells resulted in cell cycle arrest at the G1 phase, which correlated with apparent downregulation of both mRNA and protein levels of both PCNA and cyclin D1. Further analysis demonstrated that Fan decreased the phosphorylation of AKT and GSK-3beta. In addition, Fan up-regulated active caspase3, cytochrome-c protein levels and the ratio of Bax/Bcl-2, accompanied by apoptosis. Taken together, these results suggest that Fan is a potential natural product for the treatment of breast cancer.

Expression of proliferation markers in colonic mucosa in normal volunteers with a high arginine-containing diet and effect of dietary grape ingestion.

431 Background: Resveratrol, which inhibits ODC and Wnt signaling, has colon cancer prevention activity in animal models. To assess the effects of a resveratrol-rich diet on the colon, 30 normal volunteers enrolled on a cancer prevention clinical trial (NCT00578396) involving the ingestion of 1/3, 2/3, or 1 pound of grapes per day for 14 days. Data analysis was stratified based on arginine intake because meat and dietary arginine have been linked to colon carcinogenesis. Methods: Patients were placed on a low-resveratrol diet 14 days prior to and during the grape ingestion period. Detailed dietary information and colonic biopsies were obtained before and after the grape intervention in a single-subject design. Tissue was processed for RNA for qRT- PCR analysis of genes implicated in colonic proliferation, Wnt signaling and colon carcinogenesis. The % of cells expressing Ki67 in the lower 1/3 of colonic crypts was determined by IHC. Results: Mean arginine consumption for the study population was 4.5 ± 2.27 gm/day. Participants with higher initial dietary arginine consumption (&gt; 4.5gm) had higher levels of cyclinD1 (p = 0.022), c-myc (p = 0.0002), axin2 (p = 0.0065) and CD133 (p = 0.041) mRNA in the colonic mucosa than participants with lower initial arginine consumption. No differences in the % of base of the colonic crypt cells staining for Ki67 were seen. Grape ingestion led to a significant reduction in cyclinD1 expression in participants with higher initial arginine consumption (p = 0.0039), with levels falling to those seen in low arginine consumption participants. Conclusions: Normal volunteers with a high amount of daily arginine ingestion had elevated levels of markers indicative of cell proliferation (cyclinD1, c-myc), Wnt signaling (axin2), and colonic stem cells (CD133). Expression of each of these markers was reduced following grape ingestion, though only the reduction in cyclinD1 reached statistical significance. Significance: Dietary arginine adversely affects colonic mucosa and may predispose individuals to the development of colon cancer. The effects are reversed in part by a resveratrol-rich grape-containing diet. Clinical trial information: NCT00578396.

Aberrant DNA Methylation and Epigenetic Inactivation of hMSH2 Decrease Overall Survival of Acute Lymphoblastic Leukemia Patients via Modulating Cell Cycle and Apoptosis

Altered regulation of many transcription factors has been shown to play important roles in the development of leukemia. hMSH2 can modulate the activity of some important transcription factors and is known to be a regulator of hematopoietic differentiation. Herein, we investigated epigenetic regulation of hMSH2 and its influence on cell growth and overall survival of acute lymphoblastic leukemia (ALL) patients. hMSH2 promoter methylation status was assessed by COBRA and pyrosequencing in 60 ALL patients and 30 healthy volunteers. mRNA and protein expression levels of hMSH2, PCNA, CyclinD1, Bcl-2 and Bax were determined by real time PCR and Western blotting, respectively. The influence of hMSH2 on cell proliferation and survival was assessed in transient and stable expression systems. mRNA and protein expression of hMSH2 and Bcl-2 was decreased, and that of PCNA, CyclinD1 and Bax was increased in ALL patients as compared to healthy volunteers (P<0.05). hMSH2 was inactivated in ALL patients through promoter hypermethylation. Furthermore, hMSH2 hypermethylation was found in relapsed ALL patients (85.7% of all cases). The median survival of patients with hMSH2 methylation was shorter than that of patients without hMSH2 methylation (log-rank test, P=0.0035). Over-expression of hMSH2 in cell lines resulted in a significant reduction in growth and induction of apoptosis. This study suggests that aberrant DNA methylation and epigenetic inactivation of hMSH2 play an important role in the development of ALL through altering cell growth and survival.

Esophageal Helicobacter pylori colonization aggravates esophageal injury caused by reflux.

To investigate esophageal Helicobacter pylori (H. pylori) colonization on esophageal injury caused by reflux and the related mechanisms. An esophagitis model, with acid and bile reflux, was surgically produced in male rats. The rats were randomly divided into either: (1) an esophagogastroduodenal anastomosis (EGDA) group; (2) an EGDA with H. pylori infection group; (3) a pseudo-operation with H. pylori infection group; or (4) a pseudo-operation group. All rats were kept for 36 wk. Based on the location of H. pylori colonization, the EGDA rats with H. pylori infection were subdivided into those with concomitant esophageal H. pylori colonization or those with only gastric H. pylori colonization. The esophageal injuries were evaluated grossly and microscopically. The expressions of CDX2 and MUC2 were determined by real-time polymerase chain reaction (RT-PCR) and immunohistochemistry. Ki-67 antigen expression was determined by immunohistochemistry. The mRNA levels of cyclin D1, c-Myc, Bax and Bcl-2 were determined by RT-PCR. Cell apoptosis was evaluated using the TdT-mediated dUTP nick-end labeling method. Esophagitis, Barrett's esophagus (BE), and esophageal adenocarcinoma (EAC) developed in rats that underwent EGDA. When comparing rats with EGDA and concomitant esophageal H. pylori colonization to EGDA-only rats, the severity of injury (87.9 ± 5.2 vs 77.2 ± 8.6, macroscopically, 92.5 ± 8.0 vs 83.8 ± 5.5, microscopically, both P < 0.05) and the incidences of BE (80.0% vs 33.3%, P = 0.055) and EAC (60.0% vs 11.1%, P < 0.05) were increased. These increases were associated with upregulation of CDX2 and MUC2 mRNA (10.1 ± 5.4 vs 3.0 ± 2.9, 8.4 ± 4.6 vs 2.0 ± 3.2, respectively, Ps < 0.01) and protein (8.1 ± 2.3 vs 3.3 ± 3.1, 7.3 ± 4.0 vs 1.8 ± 2.7, respectively, all P < 0.05). The expression of Ki-67 (8.9 ± 0.7 vs 6.0 ± 1.7, P < 0.01) and the presence of apoptotic cells (8.3 ± 1.1 vs 5.3 ± 1.7, P < 0.01) were also increased significantly in rats with EGDA and concomitant esophageal H. pylori colonization compared with rats with EGDA only. The mRNA levels of cyclin D1 (5.8 ± 1.9 vs 3.4 ± 1.3, P < 0.01), c-Myc (6.4 ± 1.7 vs 3.7 ± 1.2, P < 0.01), and Bax (8.6 ± 1.6 vs 5.1 ± 1.3, P < 0.01) were significantly increased, whereas the mRNA level of Bcl-2 (0.6 ± 0.3 vs 0.8 ± 0.3, P < 0.01) was significantly reduced in rats with EGDA and concomitant esophageal H. pylori colonization compared with rats with EGDA only. Esophageal H. pylori colonization increases esophagitis severity, and facilitates the development of BE and EAC with the augmentation of cell proliferation and apoptosis in esophageal mucosa.

Effects of Thapsigargin on the Proliferation and Survival of Human Rheumatoid Arthritis Synovial Cells

A series of experiments have been carried out to investigate the effects of different concentrations of thapsigargin (0, 0.001, 0.1, and 1 μM) on the proliferation and survival of human rheumatoid arthritis synovial cells (MH7A). The results showed that thapsigargin can block the cell proliferation in human rheumatoid arthritis synovial cells in a time- and dose-dependent manner. Results of Hoechst staining suggested that thapsigargin may induce cell apoptosis in MH7A cells in a time- and dose-dependent manner, and the percentages of cell death reached 44.6% at thapsigargin concentration of 1 μM treated for 4 days compared to the control. The protein and mRNA levels of cyclin D1 decreased gradually with the increasing of thapsigargin concentration and treatment times. Moreover, the protein levels of mTORC1 downstream indicators pS6K and p4EBP-1 were reduced by thapsigargin treatment at different concentrations and times, which should be responsible for the reduced cyclin D1 expressions. Our results revealed that thapsigargin may effectively impair the cell proliferation and survival of MH7A cells. The present findings will help to understand the molecular mechanism of fibroblast-like synoviocytes proliferations and suggest that thapsigargin is of potential for the clinical treatment of rheumatoid arthritis.

Tumor necrosis factor-α and NF-κB play a role in macrophage-like THP-1 cells promoting coal tar pitch extract-induced tumorigenic transformation of human bronchial epithelial cells

To characterize the role of tumor necrosis factor-α (TNF-α) and NF-κB play a role in macrophage-like THP-1 cells promoting coal tar pitch extract (CTPE)-induced tumorigenic transformation of human bronchial epithelial cells (BEAS-2B). From passage 10, CTPE-induced BEAS-2B cells cocultured with THP-1 cells were treated with NF-κB inhibitor-Pyrrolidine dithiocarbamate (PDTC) every 3 passages and TNF-α antibody every passage. Alterations of cell cycle, karyotype and colony formation in soft agar of BEAS-2B cells at passages 20, indicative of tumorigenicity, were determined, respectively. In addition, mRNA and protein levels of TNF receptor associated factor2 (TRAF2) and Cyclin D1 in BEAS-2B cells were measured with Real Time-PCR and Western blot, respectively. The percentages of S-phase BEAS-2B cells at passage 20 in PDTC group and TNF-α antibody group were (33.97±2.16)% and (34.29±2.04)% respectively, which were less than that in Co-culture+CTPE group of 20th passage [(44.46±0.83)%], P < 0.05; The number of cells with aneuploidy in 100 cells in 20th passage PDTC group and TNF-α antibody group were 40 and 37, and there were significantly different when comparing to that of 20th passage Co-culture+CTPE group (75); The number of colony formation and the rate of colony formation of BEAS-2B cells in soft agar at passage 20 in PDTC group were (15.17±2.48) and (1.51‰±0.25‰), (13.33±2.58)and (1.33‰±0.26‰) in TNF-α antibody group, which were less that those in 20th passage Co-culture+CTPE group [(172.33±12.09) and (17.23‰±1.20‰)], P < 0.05; at the same time, the mRNA and protein levels of TRAF2 and Cyclin D1 in BEAS-2B cells were decreased after PDTC and TNF-α antibody treatment. TNF-α and NF-κB could play an important role in THP-1 cells promoting coal tar pitch extract-induced tumorigenic transformation of BEAS-2B cells by influencing the expression of TRAF2 and Cyclin D1.

Diosgenin relieves goiter via the inhibition of thyrocyte proliferation in a mouse model of Graves' disease

To investigate the effects of diosgenin (Dio), a naturally occurring steroid saponin, on goiter formation in a mouse model of Graves' disease (GD) and the underlying mechanisms. Female BALB/c mice were injected with adenovirus expressing the A subunit of thyrotropin receptor to induce GD. The mice were treated with Dio (20, 100 mg·kg(-1)·d(-1), ip) for 12 or 24 d. The serum levels of TT4 and TRAb were examined using radioimmunoassay and electrochemiluminescence. The size and morphology of thyroid glands were examined. Thyrocyte proliferation was determined using BrdU incorporation assay. The expression of proliferation-associated proteins IGF-1, NF-κB, cyclin D1, and PCNA in thyroids was analyzed using immunohistochemistry and real-time PCR. The GD mice showed significantly high serum levels of TRAb and TT4 compared to the normal mice. Treatment of the GD mice with Dio for 24 d dose-dependently reduced the TT4 level and thyroid size, but did not affect the abnormal level of TRAb. Furthermore, Dio treatment dose-dependently reversed the morphological changes and reduced excessive thyrocyte proliferation in thyroids of the GD mice. Dio treatment also dose-dependently reduced the mRNA and protein levels of IGF-1, NF-κB, cyclin D1, and PCNA in thyroids of the GD mice. Dio relieves goiter in a mouse model of GD through the inhibition of thyrocyte proliferation. The mechanisms involve the suppression of IGF-1, NF-κB, cyclin D1, and PCNA expression.

Epstein-Barr Virus encoded LMP1 regulates cyclin D1 promoter activity by nuclear EGFR and STAT3 in CNE1 cells

The principal Epstein–Barr virus (EBV) oncoprotein, latent membrane protein 1 (LMP1) is strongly associated with nasopharyngeal carcinoma (NPC), a prevalent cancer in China. The epidermal growth factor receptor (EGFR) is important in carcinogenesis, as it is a ubiquitously expressed receptor tyrosine kinase. Signal transducer and activator of transcription 3 (STAT3) is a master transcriptional regulator in proliferation and apoptosis. Our previous study demonstrated that the nuclear EGFR could bind to the cyclin D1 promoter directly in the presence of LMP1, and the correlation between EGFR and STAT3 in NPC remains to be further explored. Here, we have shown that the interaction of EGFR and STAT3 increased in the nucleus in the presence of LMP1. LMP1 promoted both EGFR and STAT3 binding to the promoter region of cyclin D1, in turn, enhancing the promoter activity of cyclin D1. Furthermore, we demonstrated that both transcriptional activity and mRNA levels of cyclin D1 were decreased by small molecule interference of EGFR and STAT3 activity. These findings may provide a novel linkage between the EGFR and STAT3 signaling pathways and the activation of cyclin D1 by LMP1 in the carcinogenesis of NPC.