Protein Expression Levels Of Cyclin Research Articles

Identification of Hair Growth Promoting Components in the Kernels of Prunus mira Koehne and Their Mechanism of Action.

The application of the seed oil of Prunus mira Koehne (Tibetan name ཁམབུ།), a plant belonging to the Rosaceae family, for the treatment of alopecia has been recorded in Jingzhu Materia Medica (ཤེལ་གོང་ཤེལ་ཕྲེང་།) (the classic of Tibetan medicine) and Dictionary of Chinese Ethnic Medicine. This study aims to reveal the effective components and mechanism of hair growth promotion in the kernel of Prunus mira Koehne. Network pharmacology was used to predict the mechanism of action and effective components in the treatment of the kernel of Prunus mira Koehne. The contents of amygdalin in 12 batches of the kernel of Prunus mira Koehne were determined by HPLC. An animal model of the depilation of KM mice induced by sodium sulfide was created, and five effective components that promoted hair growth were initially screened. In the study of the effectiveness and mechanism of action, KM and C57BL/6 mice are selected as experimental objects, three screening tests for active components of the kernel of P. mira are performed, and three effective components are screened out from the eight components. HE staining was used to detect the number of hair follicles and the thickness of the dermis. RT-PCR and immunohistochemistry were used to evaluate the influence of the expression of indicators in the Wnt/β-catenin signaling pathway in skin, including β-catenin, GSK-3β, and mRNA and protein expression levels of Cyclin D 1 and LEF 1. The network pharmacology study showed 12 signaling pathways involving 25 targets in the treatment of alopecia by the kernel of Prunus mira Koehne. vitamin E (3.125 mg/cm2/d), β-sitosterol (0.061 mg/cm2/d), and linoleic acid (0.156 mg/cm2/d) in the kernel of Prunus mira Koehne can promote hair growth in mice, and the mechanism of action may be related to the Wnt/β-catenin pathway.

Bmi1 regulate tooth and mandible development by inhibiting p16 signal pathway.

To determine whether the deletion of p16 can correct tooth and mandible growth retardation caused by Bmi1 deficiency, we compared the tooth and mandible phenotypes of homozygous p16‐deficient (p16−/−) mice, homozygous Bmi1‐deficient (Bmi1−/−) mice, double homozygous Bmi1 and p16‐deficient (Bmi1−/−p16−/−) mice to those of their wild‐type littermates at 4 weeks of age by radiograph, histochemistry and immunohistochemistry. Results showed that compared to Bmi1−/− mice, the dental mineral density, dental volume and dentin sialoprotein immunopositive areas were increased, whereas the ratio of the predentin area to total dentin area and that of biglycan immunopositive area to dentin area were decreased in Bmi1−/−p16−/− mice. These results indicate that the deletion of p16 can improve tooth development in Bmi1 knockout mice. Compared to Bmi1−/− mice, the mandible mineral density, cortical thickness, alveolar bone volume, osteoblast number and activity, alkaline phosphatase positive area were all increased significantly in Bmi1−/−p16−/− mice. These results indicate that the deletion of p16 can improve mandible growth in Bmi1 knockout mice. Furthermore, the protein expression levels of cyclin D, CDK4 and p53 were increased significantly in p16−/− mice compared with those from wild‐type mice; the protein expression levels of cyclin D and CDK4 were decreased significantly, whereas those of p27 and p53 were increased significantly in Bmi1−/− mice; these parameters were partly rescued in Bmi1−/−p16−/− mice compared with those from Bmi1−/− mice. Therefore, our results indicate that Bmi1 plays roles in regulating tooth and mandible development by inhibiting p16 signal pathway which initiated entry into cell cycle.

Kang-Ai Injection Inhibits Gastric Cancer Cells Proliferation through IL-6/STAT3 Pathway.

To explore the mechanisms underlying the proliferative inhibition of Chinese herbal medicine Kang-Ai injection (KAI) in gastric cancer cells. Gastric cancer cell lines MGC803 and BGC823 were treated by 0, 0.3%, 1%, 3% and 10% KAI for 24, 48 and 72 h, respectively. The cell proliferation was evaluated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. The apoptosis and cell cycle were evaluated by flow cytometry. Interleukin (IL)-6 mRNA and protein expression levels were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immune sorbent assay (ELISA), respectively. The protein expression levels of cyclin A, cyclin E, cyclin B1, cyclin D1, p21, retinoblastoma (RB), protein kinase B (AKT), extracellular regulated protein kinases (ERK), signal transducer and activator of transcription (STAT) 1 and STAT3 were detected by Western blot. KAI inhibited the proliferation of MGC803 and BGC823 gastric cancer cells in dose- and time-dependent manner. After treated with KAI for 48 h, the proportion of G1 phase was increased, expression level of cyclin D1 and phosphorylation-RB were down-regulated, whereas the expression of p21 was up-regulated (all P<0.01). Furthermore, 48-h treatment with KAI decreased the phosphorylation level of STAT3, inhibited the mRNA and protein expressions of IL-6 (all P<0.01). IL-6 at dose of 10 ng/mL significantly attenuated the proliferative effect of both 3% and 10% KAI, and recovered KAI-inhibited STAT3 phosphorylation and cyclin D1 expression level (all P<0.01). KAI exerted an anti-proliferative function by inhibiting IL-6/STAT3 signaling pathway followed by the induction of G1 phase arrest in gastric cancer cells.

4'-O-?-d-Glucopyranosyl-4-hydroxy-3,3',5-trimethoxychalcone derived from Brassica rapa L. induces cell cycle arrest and apoptosis in neuroblastoma cells.

Introduction: Neuroblastoma is one of the most commonly encountered malignant solid tumors in the pediatric age group. We examined the antitumor effects of chalcone glycosides derived from Brassica rapa L against neuroblastoma cell lines. Materials and Methods: We used the WST-8 assay to evaluate the cytotoxicity of the glycosides against human neuroblastoma cell lines and normal cell lines. We also performed analysis of cell cycle arrest by flow cytometry, and examined the expression levels of cell cycle-and apoptosis-related proteins by western blot analysis. Results: One of the derivative compounds (compound 6,4'-O-β-d-glucopyranosyl-4-hydroxy-3,3',5- trimethoxychalcone) was found to exert cell cytotoxicity against neuroblastoma cells. Examination of the effects on the cell cycle revealed cell cycle arrest in the G0/G1 phase. Furthermore, exposure of the cells to compound 6 was associated with decreased protein expression levels of cyclin D, phospho-retinoblastoma protein (Rb) and E2 factor (E2F), as evaluatedby western blot analysis. In addition, a Hoechst 33342 staining experiment revealed apoptosis, characterized by cell shrinkage, nuclear chromatin condensation and fragmentation. Annexin V-propidium iodide (PI) double staining also showed an increase in the number of early apoptotic cells. Compound 6 induced activation of executioner caspases (caspase-3 and caspase-7) and Poly (ADP-ribose) polymerase (PARP) cleavage. Conclusion: Compound 6 exerted inhibition of cell growth of neuroblastoma cells by inducing G0/G1 arrest and caspase-dependent apoptosis. This compound may offer promise for development as a useful drug for the treatment of advanced neuroblastoma.

PPeOp from Omphalia lapidescens Schroet induces cell cycle arrest and inhibits the migration of MC-4 gastric tumor cells.

The aim of the present study was to investigate the effect of purified Omphalia lapidescens protein (pPeOp) extracted by polyvinylpyrrolidone from the fungus Omphalia lapidescens Schroet on the proliferation and cell cycle progression of MC-4 human gastric tumor cells. Using polyvinylpyrrolidone, pPeOp was extracted from O. lapidescens Schroet. MC-4 cells were cultured with 30, 60 or 90 µg/ml pPeOp, with 5-fluorouracil used as a positive control. Survival rates of treated cells were significantly decreased compared with those of the untreated control group in a dose-dependent manner. Using flow cytometric analysis, cells treated with pPeOp were demonstrated to arrest in S phase and exhibit abnormal G0/G1 and G2/M phase cell cycle distribution. In addition, a wound healing assay demonstrated that pPeOp significantly inhibited the migration of MC-4 cells. The mRNA and protein expression levels of cyclin D1/cyclin-dependent kinase (CDK) 4, cyclin B/CDK1, cyclin A/CDK2, matrix metalloproteinase (MMP)-2 and MMP-9 were determined using reverse transcription-quantitative polymerase chain reaction analysis and western blotting. The mRNA expression level of CDK4 and cyclin A was significantly increased compared with the untreated control; however, cyclin D1, CDK1, CDK2, cyclin B, MMP-2, and MMP-9 exhibited a significantly decreased mRNA expression level, indicating that there is a negative association between concentration and cyclin D1 expression levels. The expression of the cycle arrest-associated proteins and migration-associated proteins examined were similar to the observed mRNA expression levels. In conclusion, pPeOp was identified to inhibit migration of and cause S phase cell cycle arrest in MC-4 cells.

Ursolic Acid Mediated Inhibition of Human Prostate Cancer Cell Growth in vitro Involves Alterations in Cell Cycle Related Protein Expression

Ursolic acid is a constituent of the American cranberry (Vaccinium macrocarpon) and other Vaccinium fruit found primarily in the peel. In this study, the effects of ursolic acid (UA) on the cell cycle behaviour of DU145 human prostate cancer cells in vitro was examined. Following treatment of cells with UA (50 μM) for 6 hours, the cellular viability of the cells decreased. UA treatment increased the proportion of cells in the G1 phase of the cell cycle. The relative proportion of cells in G2-M phase was similar in both control and UA-treated cells. UA treatment also caused a decrease in the proportion of cells in S-phase relative to untreated cells. These cell cycle alterations were associated with changes in expression of cell cycle associated regulatory proteins. UA decreased the protein expression levels of cyclin A, cyclin D, p16 , pRBp107 and increased the expression of CDK2, CDK4, cyclin E, p21, p27 and pRBp130. CyclinB1 protein expression levels were apparently unaffected by UA. These findings demonstrate that ursolic acid can affect the cell growth behaviour of human prostate cancer cells in vitro. [Cranberry Institute (Wisconsin Board) & Telus MRFD Prostate Cancer Research Fund (PEI Division) funded]

Lipoxin A4 Inhibits NF-κB Activation and Cell Cycle Progression in RAW264.7 Cells

Lipoxins (LXs), including lipoxin A4 (LXA4), etc., have been approved for potent anti-inflammatory and immunomodulatory properties. Based on the important roles of macrophages in inflammation and immunomodulation, we investigate the effects of LXA4 on lipopolysaccharide (LPS)-induced proliferation and the possible signal transduction pathways in RAW264.7 macrophages. RAW264.7 cells were treated in vitro with or without LPS in the absence or presence of LXA4. [(3)H]-TdR incorporation assay and flow cytometry were used for detecting cell proliferation and cycle, respectively. Moreover, Western blot was applied to evaluate the protein expression levels of Cyclin E, IκBα, nuclear factor-κB (NF-κB), and IκB kinase (IKK). Our research showed that LXA4 suppressed LPS-induced proliferation, increased the proportion of the G0/G1 phase, decreased the proportion of the S phase, and downregulated the expression of Cyclin E. Besides these, LXA4 suppressed LPS-induced IκBα degradation, NF-κB translocation, and the expression of IKK. The data suggested that LXA4 inhibited LPS-induced proliferation through the G0/G1 phase arrest in RAW264.7 macrophages, and the inhibitory effect might depend on NF-κB signaling transduction pathway.

Effect of long-term power frequency electromagnetic field exposure on proliferation and apoptosis of SRA01/04 cells

To investigate the effect of long-term power frequency electromagnetic field (50 Hz) exposure on the proliferation and apoptosis of human lens epithelial cells (SRA01/04 cells). SRA01/04 cells in the exponential growth phase were exposed or sham-exposed to power frequency electromagnetic field (50 Hz, 2.3 mT) for 2 hours per day, 5 days every week. After 11 weeks of exposure, the cells were collected; the cell morphology was observed under a microscope, the cell viability was measured by MTT assay, the cell cycle and apoptosis were examined by flow cytometry, and the protein expression levels of cyclin D and proliferating cell nuclear antigen (PCNA) were determined by western blot. Compared with the sham-exposed SRA01/04 cells, most exposed cells became rounded and more stereoscopic, and heterochromatin gathered near the nuclear membrane in some exposed cells. The MTT assay showed that the viability of exposed cells was significantly increased compared with that of the sham-exposed cells (P < 0.05). Long-term power frequency electromagnetic field exposure led to significantly increased number of cells in S phase (P < 0.05), and the proliferation index was significantly higher in the exposed cells than in the sham-exposed cells (P < 0.05). There was no significant difference in apoptotic rate between the exposed cells and sham-exposed cells (P > 0.05). The exposed cells had significantly higher protein expression levels of cyclin D and PCNA than the sham-exposed cells (P < 0.05). Long-term power frequency electromagnetic field exposure can promote cellular proliferation and change cell cycle in SRA01/04 cells, but it has no marked effect on the apoptosis of SRA01/04 cells.

Cranberry proanthocyanidins affect human prostate cancer cell growth via cell cycle arrest by modulating expression of cell cycle regulators

The effects of a proanthocyanidin‐enriched fraction (PACs) from American cranberry(V. macrocarpon) on DU145 cancer cells’ behavior in vitro was studied. PACs was characterized by MALDI‐TOF MS to contain PAC‐oligomers ranging in size from 2–12 epicatechin units with at least one A type linkage. PACs (25 ug/mL) decreased cellular viability by ~30 % post 6h.of treatment. PACs (post 6h. exposure) increased the proportion of cells in G2‐M and decreased the proportion of cells in G1. These alterations in cell cycle were associated with changes in cell cycle regulatory proteins and other cell cycle associated activities. PACs decreased the protein expression levels of cyclin A &amp; cyclin B1 and increased the expression of cyclin D1 &amp; cyclin E. PACs treatment resulted in an increase in the protein expression levels of CDK2 &amp; CDK4. Decreased p21, p16, &amp; pRBp107 protein expression levels and increased p27 protein expression levels were evident in response to PACs treatment with no apparent alteration in the levels of pRbp130 protein. These findings demonstrate that PACs contribute to the cranberry mediated alterations in cell cycle proteins. Cranberry extracts can affect the behavior of DU145 prostate cancer cells in vitro further supporting the potential health benefits associated with cranberries. [N.C.I.C.‐ CCS, P.E‐HRP, The Cranberry Institute (Wisconsin Cranberry Board), Telus MRFD Prostate Cancer Fund (PEI Division)]

Bufalin increases sensitivity to AKT/mTOR-induced autophagic cell death in SK-HEP-1 human hepatocellular carcinoma cells

Bufalin is the major component of Chan-Su (a traditional Chinese medicine, TCM) extracts from the venom of Bufo bufo gargarizan. In the present study, we investigated the pharmacological mechanisms of cell cycle arrest and autophagic cell death induced by bufalin in SK-HEP-1 human hepatocellular carcinoma cells in vitro. Bufalin inhibited cell survival by MTT assay and increased cell death by trypan blue exclusion assay in a concentration-dependent manner. In addition, bufalin induced G2/M phase arrest by reducing CDK1 activity. Bufalin triggered DNA fragmentation and apoptotic cell death in SK-HEP-1 cells by DNA gel electrophoresis, TUNEL and caspase-3 activity assay, while bufalin induced autophagic cell death by double-membrane vacuoles (transmission electron microscopy, TEM), acidic vesicular organelles (acridine orange staining) and cleavage of microtubule-associated protein 1 light chain 3 (LC3). Protein expression levels of cyclin A and B, CDK1, phospho-CDK1 (Thr161), Cdc25c, phospho-Cdc25c (Ser198), phospho-AKT (Thr308), phospho-AKT (Ser473), phospho‑mTOR (Ser2481) were downregulated. In contrast, protein expression levels of the Chk1, Wee1, LC3-II, Beclin-1, Atg 5, Atg 7 and Atg 12 were upregulated in SK-HEP-1 cells after bufalin treatment. Inhibition of autophagy by 3-methyladenine (an inhibitor of class III phosphatidylinositol-3 kinase; 3-MA) or bafilomycin A1 (an inhibitor of the vacuolar proton pump of lysosomes and endosomes) reduced the effect of bufalin on cell viability and enhanced the effect of bufalin on apoptosis. In conclusion, bufalin triggered autophagic cell death and G2/M phase arrest through the AKT/mTOR signaling pathway in SK-HEP-1 cells. Our findings showed that bufalin may be potentially efficacious in the treatment of human hepatocellular carcinoma.

Limited prognostic value of tissue protein expression levels of cyclin E in Danish ovarian cancer patients: from the Danish ‘MALOVA’ ovarian cancer study

The primary objective of this study was to assess the expression of cyclin E in tumour tissues from 661 patients with epithelial ovarian tumours. The second was to evaluate whether cyclin E tissue expression levels correlate with clinico-pathological parameters and prognosis of the disease. Using tissue arrays (TA), we analysed the cyclin E expression levels in tissues from 168 women with borderline ovarian tumours (BOT) (147 stage I, 4 stage II, 17 stage III) and 493 Ovarian cancer (OC) patients (127 stage I, 45 stage II, 276 stage III, 45 stage IV). Using a 10% cut-off level for cyclin E overexpression, 20% of the BOTs were positive with a higher proportion of serous than mucinous tumours. Sixty-two per cent of the OCs were positive for cyclin E expression with the highest percentage found in clear cell carcinomas. Results based on univariate and multivariate survival analyses with a 10% cut-off value showed that cyclin E had no independent prognostic value. In conclusion, we found cyclin E expression in tumour tissue to be of limited prognostic value to Danish OC patients.

Lewis y Regulate Cell Cycle Related Factors in Ovarian Carcinoma Cell RMG-I in Vitro via ERK and Akt Signaling Pathways

ObjectiveTo investigate the effect of Lewis y overexpression on the expression of proliferation-related factors in ovarian cancer cells.MethodsmRNA levels of cyclins, CDKs, and CKIs were measured in cells before and after transfection with the α1,2-fucosyltransferase gene by real-time PCR, and protein levels of cyclins, CDKs and CKIs were determined in cells before and after gene transfection by Western blot.ResultsLewis y overexpression led to an increase in both mRNA and protein expression levels of cyclin A, cyclin D1 and cyclin E in ovarian cancer cells, decrease in both mRNA and protein expression levels of p16 and p21, and decrease of p27 at only the protein expression level without change in its mRNA level. There were no differences in proteins and the mRNA levels of CDK2, CDK4 and CDK6 before and after gene transfection. Anti-Lewis y antibody, ERK and PI3K pathway inhibitors PD98059 and LY294002 reduced the difference in cyclin and CKI expression caused by Lewis y overexpression.ConclusionLewis y regulates the expression of cell cycle-related factors through ERK/MAPK and PI3K/Akt signaling pathways to promote cell proliferation.

Inhibition of DNA synthesis by downregulation of cyclin A but not Skp 2 overexpression in human hepatocellular carcinoma cells

Cyclin A is an S and G2/M phase regulatory protein and associates with Skp 2 in many transformed cells. Our previous results showed that 12 (39%) and 17 (55%) out of 31 hepatocellular carcinoma (HCC) patients exhibited higher protein expression levels of cyclin A and Skp 2, respectively, in their tumorous compared to non-tumorous tissues. In the present study, we used Western blot analysis to show that 3 out of 6 HCC cell lines, HA59T, HA22T and HCC36, exhibited overexpression of cyclin A, among which the HCC36 cell line also expressed a higher Skp 2 protein level. Moreover, we used the antisense oligonucleotide phosphorothioates to down regulate the overexpression of cyclin A and Skp 2 proteins to determine whether or not these two proteins are involved in the mitogenesis of HCC36 cells. After treatment with antisense oligonucleotide phosphorothioates, the gene product of cyclin A or Skp 2 was suppressed dose-dependently as revealed by Western blot analyses. By [3H]thymidine incorporation assay, we found that downregulation of cyclin A but not Skp 2 overexpression could inhibit the DNA synthesis ability of HCC36 cells, suggesting that abnormal Skp 2 expression is not directly correlated with the HCC proliferation.