Metformin improves polycystic ovary syndrome and activates female germline stem cells in mice / 生理学报

Polycystic ovary syndrome (PCOS) is a common disease caused by complex endocrine and metabolic abnormalities in women of childbearing age. Metformin is the most widely used oral hypoglycemic drug in clinic. In recent years, metformin has been used in the treatment of PCOS, but its mechanism is not clear. In this study, we aimed to investigate the effect of metformin on PCOS and its mechanism through PCOS mouse model. Female C57BL/6J mice aged 4-5 weeks were intragastrically given letrozole (1 mg/kg daily) combined with a high-fat diet (HFD) for 21 days to establish the PCOS model. After modeling, metformin (200 mg/kg daily) was intragastrically administered. One month later, the body weight and oral glucose tolerance test (OGTT) were measured. Hematoxylin eosin (H&E) staining was used to detect the pathological changes of ovary. The serum levels of anti-Mullerian hormone (AMH), follicle-stimulating hormone (FSH), luteinizing hormone (LH), E2 and testosterone (T) were measured by ELISA. The expression of DDX4/MVH was detected by immunohistochemistry. DDX4/MVH and PCNA were co-labeled by immunofluorescence. The protein levels of DDX4/MVH, PCNA, cyclin D2, AMPK and mTOR were detected by Western blot. The results showed that after metformin treatment, the body weights of PCOS mice were gradually returned to normal, glucose tolerance was significantly improved, serum E2 levels were increased, while AMH, LH, T levels and LH/FSH ratio were decreased. Ovarian polycystic lesions were reduced with reduced atresia follicles. Furthermore, the number of proliferative female germline stem cells (FGSCs) and levels of proliferation related proteins (PCNA, cyclin D2) were significantly increased, and the p-mTOR and p-AMPK levels were markedly up-regulated. These results suggest that metformin treatment not only improves hyperandrogenemia, glucose intolerance and polycystic ovarian lesions in PCOS, but also activates the function of FGSCs. The underlying mechanism may be related to the phosphorylation of AMPK and mTOR. These findings provide new evidence to use metformin in the treatment of PCOS and follicular development disorder.

Notoginsenoside R1 attenuates breast cancer progression by targeting CCND2 and YBX3 / 中华医学杂志(英文版)

BACKGROUND@#Breast cancer (BC) is a common malignancy with highly female incidence. So far the function of notoginsenoside R1 (NGR1), the extract from Panax notoginseng, has not been clearly elucidated in BC.@*METHODS@#Optimal culture concentration and time of NGR1 were investigated by cell counting kit-8 assay. Cell proliferation ability was measured by colony formation assays. Transwell assay was used to detect the effect of NGR1 on cell migration and invasion. The apoptosis rate of cells between each group was measured by TUNEL assay.@*RESULTS@#NGR1 treatment has an inhibitory effect on proliferation, migration, invasion, and angiogenesis and a stimulating effect on cell cycle arrest and apoptosis of Michigan Cancer Foundation-7 (MCF-7) cells. The 50% growth inhibitory concentration for MCF-7 cells at 24 h was 148.9 mmol/L. The proportions of MCF-7 cells arrested in the G0/G1 phase were 36.94±6.78%, 45.06±5.60%, and 59.46±5.60% in the control group, 75, and 150 mmol/L groups, respectively. Furthermore, we revealed that NGR1 treatment attenuates BC progression by targeted downregulating CCND2 and YBX3 genes. Additionally, YBX3 activates phosphatidylinositol 3-phosphate kinase (PI3K)/protein kinase B (Akt) signaling pathway by activating kirsten rat sarcoma viral oncogene, which is an activator of the PI3K/Akt signaling pathway.@*CONCLUSION@#These results suggest that NGR1 can act as an efficacious drug candidate that targets the YBX3/PI3K/Akt axis in patients with BC.

CyPA-CD147-ERK1/2-cyclin D2 signaling pathway is upregulated during rat left ventricular hypertrophy / 生理学报

The changes of serum cyclophilin A (CyPA), its receptor CD147 and the downstream signaling pathway during the process of cardiac hypertrophy remain unknown. The present study aims to investigate the relationships between CyPA-CD147-ERK1/2-cyclin D2 signaling pathway and the development of cardiac hypertrophy. Left ventricular hypertrophy was prepared by 2-kidney, 2-clip in Sprague-Dawley rats and observed for 1 week, 4 and 8 weeks. Left ventricular hypertrophy was evaluated by ratio of left ventricular heart weight to body weight (LVW/BW) and cardiomyocyte cross sectional area (CSA). CyPA levels in serum were determined with a rat CyPA ELISA kit. Expressions of CyPA, CD147, phospho-ERK1/2 and cyclin D2 in left ventricular myocytes were determined by Western blot and immunostaining. Compared with sham groups, systolic blood pressure reached hypertensive levels at 4 weeks in 2K2C groups. LVW/BW and CSA in 2K2C groups were significantly increased at 4 and 8 weeks after clipping. ELISA results indicated a prominent increase in serum CyPA level associated with the degree of left ventricular hypertrophy. Western blot revealed that the expressions of CyPA, CD147, phospho-ERK1/2 and cyclin D2 in left ventricular tissues were also remarkably increased as the cardiac hypertrophy developed. The results of the present study demonstrates that serum CyPA and CyPA-CD147-ERK1/2-cyclin D2 signaling pathway in ventricular tissues are time-dependently upregulated and activated with the process of left ventricular hypertrophy. These data suggest that CyPA-CD147 signaling cascade might play a role in the pathogenesis of left ventricular hypertrophy, and CyPA might be a prognosticator of the degree of left ventricular hypertrophy.

Study on reversion of malignant phenotype of glioma by siRNA targeting p75 neurotrophin receptor / 中华病理学杂志

<p><b>OBJECTIVE</b>To study the therapeutic efficacy of siRNA fragments silencing p75 neurotrophin receptor (p75(NTR)), which may be a key regulator of glioma cell apoptosis and invasion.</p><p><b>METHODS</b>The siRNA sequence fragments targeting p75(NTR) were designed and transferred into human glioma cell line U251. RT-PCR and immunocytochemistry method were used to explore the expression of p75(NTR) mRNA and protein. Cell adhesion assay was employed to detect cellular adhesion ability, and soft agar clone formation assay was adopted to identify oncogenicity, and a U251 glioma model was established in nude mice. The intracranial tumor volume was detected by MRI. The expression of p75(NTR), NGF and cyclin D2 were identified using immunohistochemistry. Cell apoptosis was detected by apoptosis kit in situ.</p><p><b>RESULTS</b>The siRNA fragments targeting p75(NTR) were capable of decreasing mRNA and protein expression of p75(NTR) in U251 glioma cell line. Both the cellular adhesion ability and oncogenicity were weakly relevant. The p75(NTR) expression level was negatively correlated with cyclin D2 and apoptosis, and positively correlated with NGF expression. The siRNA sequence fragments targeting p75(NTR) were effective in decreasing the gross volume of tumor; prolonged the survival time of mice, and the edge of tumor was much sharper than that of the control group.</p><p><b>CONCLUSIONS</b>The gene silencing technique by siRNA targeting p75(NTR) is capable of decreasing tumor invasion and cell proliferation as well as inducing cell apoptosis. It is expected to be a new choice for glioma gene therapy.</p>

Time-course Transcriptional Profiling of Human Amniotic Fluid-derived Stem Cells Using Microarray / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: To maintain the homeostasis of stem cells and prevent their ability to initiate tumorigenesis, it is important to identify and modify factors that prevent or accelerate stem cell senescence. We used microarrays to attempt to identify such factors in human amniotic fluid (HAF)-derived stem cells. MATERIALS AND METHODS: To identify gene expression changes over a time course, we compared gene expression profiles of HAF-derived stem cells in different passages (1st, 2nd, 4th, 6th, 8th, and 10th) using a Sentrix Human illumina microarray. RESULTS: Of the 25,804 genes in the microarray chip, 1,970 showed an over 2-fold change relative to the control (the 1st passage)-either upregulated or downregulated. Quantitative real-time PCR validated the microarray data for selected genes: markedly increased genes were CXCL12, cadherin 6 (CDH6), and folate receptor 3 (FOLR3). Downregulated genes included cyclin D2, keratin 8, insulin-like growth factor 2 (IGF2), natriuretic peptide precursor B (NPPB) and cellular retinoic acid binding protein 2 (CRABP2). The expression pattern of the selected genes was consistent with the microarray data except for CXCL12 and IGF2. Interestingly, the expression of NPPB was dramatically downregulated along the time course; it was almost completely shut-down by the 10th passage. In contrast, FOLR3 mRNA expression was dramatically increased. CONCLUSION: Taken together, although a function for NPPB and FOLR3 in stem cell senescence has not been reported, our results strongly suggest that NPPB and/or FOLR3 play a significant role in the regulation of stem cell senescence.

Reversed effect of valproic acid on transcription inhibition of AML1-ETO fusion protein of kasumi-1 leukemic cell line / 中国实验血液学杂志

This study was aimed to investigate the mechanism of histone deacetylase (HDAC) inhibitor, valproic acid (VPA), reversing transcription inhibition of AML1-ETO fusion protein in Kasumi-1 cell line. The mRNA expressions of AML1-ETO, AML1 and cyclin D2 were detected by semi-quantitation RT-PCR after treating kasumi-1 cells with VPA at different doses/and different time points. The results indicated that the mRNA expression of AML1-ETO showed no obvious change, when kasumi-1 cells were treated with VPA. Compared with control group, the expression level of AML1 mRNA significantly increased in a dose-dependent manner. Compared with control group, the expression level of cyclin D2 mRNA significantly decreased when kasumi-1 cells had been treated with 3 mmol/L VPA as well as kasumi-1 cells were treated with different concentrations of VPA for 3 days. In conclusion, VPA could remove transcription inhibition of AML1-ETO fusion protein, increase transcription of AML1 and down-regulate mRNA expression of AML1 target gene cyclin D2 through HDAC inhibiting activity.

Adenomatous Polyposis Coli and Ras Association Domain Family 1A Gene Methylation in Breast Cancer Patients / 한국유방암학회지

PURPOSE: Of the many carcinogenic mechanisms, DNA methylation is a strong factor in various cancers, including cancer of the breast. The genes related to breast cancer include 14.3.3 sigma, Cyclin D2, RARbeta, Twist, Ras association domain family 1A gene (BASSF1A), HIN-1, p16, and Adenomatous polyposis coli (APC). Of these, hypermethylation of the APC and RASSF1A genes is, found in breast cancer patients, and especially in those with a poor prognosis. This study investigated whether hypermethylation of the APC and RASSF1A genes is related with breast cancer metastasis. METHODS: Of the 110 patients who received surgical operation at our hospital's department of surgery from January 2001 to December 2003, 16 patients with metastatic lesion found during the follow-up period were selected. Seventeen patients without metastasis selected as the tissue group after considering their age, cancer stage, and physical state. Forty seven patients were selected as the serum group, including 6 patients with metastasis, and they were evaluated for metastasis and methylation. Serum and tissue were collected and after being processed by the methylation specific PCR (MSP), and the methylation of the APC and RASSF1A genes was observed. RESULTS: In the tissue study group, the APC gene methylation ratio of the patients whose stages are between the stage 2 and 3 was 50:94%, and that of RASSF1A gene methylation was 68.7:65% respectively. Methylation rates of both genes was found in 42.9% of the stage 2 recurrent patients (non-recurrent patients: 22%) and in 77.8% (non-recurrent patients: 50%) in stage 3 recurrent patients. In the serum study group, a statistical correlation was shown (p=0.013) between methylation of RASSF1A and recurrence, where 5 of the 16 patients with methylation showed recurrences and only 1 patient of the 31 nonmethylated patients showed recurrence. CONCLUSION: We determined the correlation between APC and RASSF1A methylation and recurrence of breast cancer. Further studies with large sample populations and more advanced method are needed to confirm our findings.

The effect of cyclin D2 shRNA on the proliferation and apoptosis of LP-1 cell / 中华血液学杂志

<p><b>OBJECTIVE</b>To construct cyclin D2 (CCND2) short hairpin RNA ( shRNA) plasmid for repressing the expression of CCND2 in human myeloma cell line LP-1,and to detect its effect on the proliferation and apoptosis of LP-1 cell.</p><p><b>METHODS</b>A CCND2 shRNA model was constructed and cloned into plasmid pGensil-2, then the plasmid was transfected into LP-1 cell in vitro. The CCND2 expression cell proliferation, cell cycle and cell apoptosis of the transfected LP-1 cells were studied by RT-PCR, trypanosome staining, flow cytometry and annexin V assay.</p><p><b>RESULTS</b>The transfection efficiency of LP-1 cell was 34. 2%. In the transfected LP-1 cell CCND2 mRNA expression was reduced significantly, the cell growth was inhibited significantly and the cell cycle was partly arrested in G, phase. The apoptosis rate of the transfected LP-1 cell after 72 h was (25.7+/-4.8)%.</p><p><b>CONCLUSION</b>The inhibition of CCND2 in LP-1 cells could inhibit the cell growth and induce cell apoptosis. CCND2 maybe a new therapeutic target.</p>

Peroxisome proliferator-activated receptor gamma activator inhibits cell growth of MDA-MB-231 breast cancer cells through induction of apoptosis / 한국유방암학회지

PURPOSE: Peroxisome proliferator-activated receptor gamma (PPARgamma) has become a potential target for the prevention and treatment of human cancers. PPARgamma ligands inhibit cell proliferation of estrogen receptoralpha(ERalpha)-positive breast cancer cells. However, it has recently been shown that ERalpha-negatively inhibits PPARgamma signaling in breast cancer cells, indicating that PPARgamma ligand may be more useful for treating ERalpha-negative breast cancer cells compared to ERalpha-positive breast cancer cells. In this study, we attempted to elucidate the role of PPARg in ERalpha-negative breast cancer cells. METHODS: The effect of PPARgamma ligand on the growth of MDA-MB-231 cells was measured by MTT assay and flow cytometric analysis. TUNEL staining and Hoechst 33342 fluorescent staining were used to observe the effects of PPARgamma ligand on cell apoptosis. The regulatory proteins of the cell cycle were measured by Western blot. RESULTS: The treatment of MDA-MB-231 human breast cancer cells with the PPARgamma ligand, trgoglitazone, was shown to induce inhibition of cell growth in a dose-dependent manner. Cell cycle analysis showed a G1 arrest in MDA-MB-231 cells exposed to troglitazone. The apoptotic effect by troglitazone demonstrated that apoptotic cells were elevated from 2.5-fold of the control level at 10 mM, to 3.1-fold at 50micrometer and to 3.5-fold at 75 mM of troglitazone. Moreover, troglitazone treatment dose-dependently caused a marked decrease in the pRb, cyclin D1, cyclin D2, cyclin D3, cdk2, Cdk4 and Cdk6 expressions and there was a significant increase in the p21 and p27 expressions. CONCLUSION: These results indicate that trgoglitazone induces cell-cycle G1 arrest and apoptosis in ERalpha-negative MDA-MB-231 breast cancer cells. Collectively, this paper shows that PPARgamma ligand is an important player as a member of the chemotherapeutic candidates for treating ERalpha-negative breast cancer.

Gene Promoter Hypermethylation in Tumors and Plasma of Breast Cancer Patients / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: To measure the hypermethylation of four genes in primary tumors and paired plasma samples to determine the feasibility of gene promoter hypermethylation markers for detecting breast cancer in the plasma. MATERIALS AND METHODS: DNA was extracted from the tumor tissues and peripheral blood plasma of 34 patients with invasive breast cancer, and the samples examined for aberrant hypermethylation in cyclin D2, retinoic acid receptor beta (RARbeta), twist and high in normal-1 (HIN-1) genes using methylation-specific PCR (MSP), and the results correlated with the clinicopathological parameters. RESULTS: Promoter hypermethylation was detected at high frequency in the primary tumors for cyclin D2 (53%), RARbeta (56%), twist (41%) and HIN-1 (77%). Thirty-three of the 34 (97%) primary tumors displayed promoter hypermethylation in at least one of the genes examined. The corresponding plasma samples showed hyperme thylation of the same genes, although at lower frequencies (6% for cyclin D2, 16% for RARbeta, 36% for twist, and 54% for HIN-1). Overall, 22 of the 33 (67%) primary tumors with hypermethylation of at least one of the four genes also had abnormally hypermethylated DNA in their matched plasma samples. No significant relationship was recognized between any of the clinical or pathological parameters (tumor size, axillary lymph node metastasis, stage, or Ki-67 labeling index) with the frequency of hypermethylated DNA in the primary tumor or plasma. CONCLUSION: The detection of aberrant promoter hypermethylation of cancer-related genes in the plasma may be a useful tool for the detection of breast cancer.

Cyclin D2 expression in chronic myelogenous leukemia / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the relationship between cyclin D2 and P210(BCR/ABL) tyrosine kinase in chronic myelogenous leukemia (CML).</p><p><b>METHODS</b>RT-PCR, Western blot and flow cytometry were performed to detect the expression of cyclin D2 in K562 cells and in K562-ib-eGFP cells which express intracellular single-chain antibody (sFv, intrabody) against ABL tyrosine kinase domain.</p><p><b>RESULTS</b>Cyclin D2 expression in K562-ib-eGFP cells was 18.90% which was lower than that of control K562 cells (48.10%), and the number of S-phase cells in K562-ib-eGFP was 40.40% which was much lower than that in K562 cells (64.34%).</p><p><b>CONCLUSION</b>Cyclin D2 is a potential down-stream signal molecule of the p210(BCR/ABL) tyrosine kinase in CML. The altered expression of cyclin D2 may contribute to the over proliferation of CML cells.</p>

Expression of cyclin genes in human gastric cancer and in first degree relatives / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>To clarify the role of these cyclins in human gastric cancer.</p><p><b>METHODS</b>38 gastric cancer patients, 29 first degree relatives of gastric cancer patients, as well as 18 healthy subjects were included. The mRNA expression of cyclins D1, D2, D3 and E in gastric biopsies was evaluated by RT-PCR analysis using specific primers. Histomorphological features such as intestinal metaplasia, atrophy, H. pylori infection and severity of gastritis were determined by the updated Sydney System.</p><p><b>RESULTS</b>Significant mRNA overexpression was found for cyclins D2, D3 and E compared with healthy normal specimen, but cyclin D1 expression was not different between tumor and normal tissues. In addition, cyclin D2 and D3 overexpression was significantly more frequent in first degree relatives than in healthy controls (P < 0.05). Among the various pathological findings, the overexpression of cyclins D2 and E was associated with intestinal metaplasia, and the overexpression of cyclin D3 was associated with intestinal metaplasia as well as atrophy. The overexpression of cyclins D2 and D3 was significantly correlated with H. pylori infection. No correlation was observed between the overexpression of cyclin D1 and any pathological variables.</p><p><b>CONCLUSION</b>The overexpression of cyclins D2, D3 and E is a frequent event in patients with gastric cancer and their first degree relatives and may be an early event in gastric carcinogenesis.</p>

A Comparative Study of Gene Array of Cell Cycle Modulators in Human Corneal Epithelial Cells Stimulated with Epidermal Growth Factor(EGF), Hepatocyte Growth Factor(HGF), or Keratinocyte Growth Factor(KGF)

PURPOSE: To identify and differentiate genes that are up-regulated or down-regulated in human corneal epithelial cells in response to epidermal growth factor(EGF), hepatocyte growth factor(HGF) or keratinocyte growth factor(KGF). METHODS: Primary cultures of human corneal epithelial cell(HCE) were treated with 25 ng/ml of EGF, 25 ng/ml HGF, 25 ng/ml KGF, or vehicle in serum-free medium for 8 hours. Total RNA was isolated with TRIZOL(GIBCO, NY), and treated with DNAse I.P 32-labeled complementary DNA(cDNA) probes were synthesized using 6 ug of total RNA made from HCE cells. Equivalent counts of P 32-labeled cDNA probes were hybridized with the membrane of Atlas human cell cycle array at 68degreesC overnight. After sequential washing, the membranes were exposed to X-ray film for three days. These results were analyzed using Atlas Image TM 1.1 Software. RNAse protection assay was used to confirm one of known genes on the array, which was up-regulated by EGF, KGF, and HGF in the human corneal epithelial cells. RESULTS: Autoradiographic analysis showed that out of 111 genes analyzed, 22 were up- or down-regulated in EGF, 26 in HGF and 7 in KGF compared to untreated corneal epithelial cell. After different signal intensity was normalized more than 2000 by Atlas Image TM 1.1 Software, 12 genes were up-regulated and 10 genes down-regulated in EGF. HGF have 6 up-regulated genes and 1 down-regulated gene and KGF had all up-regulated 7 genes. EGF, HGF and KGF all up-regulated the expression of cyclin D1(BCL-1 oncogene) and serine/threonine-protein kinase PITALRE in the primary cultured human corneal epithelial cells. EGF and KGF both up-regulated E2F-1 pRB-binding protein gene. HGF and KGF up-regulated cyclin D2 gene. Proto-oncogene raf was down-regulated by EGF and HGF. CONCLUSIONS: The three growth factors seemed to have similar effects on the genes that contribute to cell cycle control. Studies to analyze the significance of the differences among these growth factors are ongoing.