Magnolol induces apoptosis in osteosarcoma cells via G0/G1 phase arrest and p53-mediated mitochondrial pathway.

Osteosarcoma is a highly invasive primary malignancy of bone. Magnolol is biologically active, which shows antitumor effects in a variety of cancer cell lines. However, it has not been elucidated magnolol's effects on human osteosarcoma cells (HOC). This study aimed to determine antitumor activity of magnolol and illustrate the molecular mechanism in HOC. Magnolol showed significant inhibition effect of growth on MG-63 and 143B cells and induced apoptosis and cell cycle arrest at G0/G1. In osteosarcoma cells, magnolol upregulated expressions of proapoptosis proteins and suppressed expressions of antiapoptosis proteins. Additionally, under the pretreatment of pifithrin-a (PFT-a, a p53 inhibitor), the magnolol-induced apoptosis was significantly reversed. The results above indicated that magnolol induces apoptosis in osteosarcoma cells may via G0/G1 phase arrest and p53-mediated mitochondrial pathway.

Quinacrine upregulates p21/p27 independent of p53 through autophagy-mediated downregulation of p62-Skp2 axis in ovarian cancer.

We have previously shown that the anti-malarial compound Quinacrine (QC) inhibits ovarian cancer (OC) growth by modulating autophagy. In the present study we extended these studies to identify the molecular pathways regulated by QC to promote apoptosis independent of p53 status in OC. QC exhibited strong anti-cancer properties in OC cell lines in contrast to other anti-malarial autophagy inhibiting drugs. QC treatment selectively upregulated cell cycle inhibitor p21, and downregulated F box protein Skp2 and p62/SQSTM1 expression independent of p53 status. Genetic downregulation of key autophagy protein ATG5 abolished QC-mediated effects on both cell cycle protein p21/Skp2 as well as autophagic cargo protein p62. Furthermore, genetic silencing of p62/SQSTM1 resulted in increased sensitivity to QC-mediated apoptosis, downregulated Skp2 mRNA and increased accumulation of p21 expression. Likewise, genetic knockdown of Skp2 resulted in the upregulation of p21 and p27 and increased sensitivity of OC cells to QC treatment. In contrast, transient overexpression of exogenous p62-HA plasmid rescued the QC-mediated Skp2 downregulation indicating the positive regulation of Skp2 by p62. Collectively, these data indicate that QC-mediated effects on cell cycle proteins p21/Skp2is autophagy-dependent and p53-independent in high grade serious OC cells.

Roscovitine Protects From Arterial Injury by Regulating the Expressions of c-Jun and p27 and Inhibiting Vascular Smooth Muscle Cell Proliferation.

PURPOSE: Roscovitine (Rosc) is a selective inhibitor of cyclin-dependent kinases (CDKs) and a promising therapy for various cancers. However, limited information is available on the biological significance of Rosc in vascular smooth muscle cells (VSMCs), the cell type critical for the development of proliferative vascular diseases. In this study, we address the effects of Rosc in regulating VSMC proliferation, both in vitro and in vivo, exploring the underlying molecular mechanisms. METHODS: The proliferations and cell-cycle distributions of in vitro cultured VSMCs, as well as several other cancer cell lines, were examined by cell-counting assay and flow cytometry, respectively. Molecular changes in various CDKs, cyclins, and other regulatory molecules were examined by reverse transcription polymerase chain reaction, Western blot, or immunocytochemistry. The in vivo effects of Rosc were examined on a carotid arterial balloon-injury model. RESULTS: Rosc significantly inhibited VSMC proliferation in response to serum or angiotensin II and arrested these cells at the G0/G1 phase. These changes were associated with a specific and robust decrease in CDK4, cyclin E, c-Jun, and a dramatic increase in p27kip1 in VSMCs, which was also translated in vivo and correlated with the protection of Rosc on injury-induced neointimal hyperplasia. CONCLUSIONS: Acting on distinct molecular targets in VSMCs versus cancer cells, Rosc inhibits VSMC proliferation and protects from proliferative vascular diseases.

Stellettin B Induces G1 Arrest, Apoptosis and Autophagy in Human Non-small Cell Lung Cancer A549 Cells via Blocking PI3K/Akt/mTOR Pathway.

Until now, there is not yet antitumor drug with dramatically improved efficacy on non-small cell lung cancer (NSCLC). Marine organisms are rich source of novel compounds with various activities. We isolated stellettin B (Stel B) from marine sponge Jaspis stellifera, and demonstrated that it induced G1 arrest, apoptosis and autophagy at low concentrations in human NSCLC A549 cells. G1 arrest by Stel B might be attributed to the reduction of cyclin D1 and enhancement of p27 expression. The apoptosis induction might be related to the cleavage of PARP and increase of ROS generation. Moreover, we demonstrated that Stel B induced autophagy in A549 cells by use of various assays including monodansylcadaverine (MDC) staining, transmission electron microscopy (TEM), tandem mRFP-GFP-LC3 fluorescence microscopy, and western blot detection of the autophagy markers of LC3B, p62 and Atg5. Meanwhile, Stel B inhibited the expression of PI3K-p110, and the phosphorylation of PDK1, Akt, mTOR, p70S6K as well as GSK-3ß, suggesting the correlation of blocking PI3K/Akt/mTOR pathway with the above antitumor activities. Together, our findings indicate the antitumor potential of Stel B for NSCLC by targeting PI3K/Akt/mTOR pathway.

The Chemopreventive Effect of Tanacetum Polycephalum Against LA7-Induced Breast Cancer in Rats and the Apoptotic Effect of a Cytotoxic Sesquiterpene Lactone in MCF7 Cells: A Bioassay-Guided Approach.

BACKGROUND: Tanacetum polycephalum L. Schultz-Bip is a member of the Asteraceae family. This study evaluated the chemopreventive effect of a T. polycephalum hexane extract (TPHE) using in in vivo and in vitro models. METHODS AND RESULTS: Five groups of rats: normal control, cancer control, TPHE low dose, TPHE high dose and positive control (tamoxifen) were used for the in vivo study. Histopathological examination showed that TPHE significantly suppressed the carcinogenic effect of LA7 tumour cells. The tumour sections from TPHE-treated rats demonstrated significantly reduced expression of Ki67 and PCNA compared to the cancer control group. Using a bioassay-guided approach, the cytotoxic compound of TPHE was identified as a tricyclic sesquiterpene lactone, namely, 8ß- hydroxyl- 4ß, 15- dihydrozaluzanin C (HDZC). Signs of early and late apoptosis were observed in MCF7 cells treated with HDZC and were attributed to the mitochondrial intrinsic pathway based on the up-regulation of Bax and the down-regulation of Bcl-2. HDZC induced cell cycle arrest in MCF7 cells and increased the expression of p21 and p27 at the mRNA and protein levels. CONCLUSION: This results of this study substantiate the anticancer effect of TPHE and highlight the involvement of HDZC as one of the contributing compounds that act by initiating mitochondrial-mediated apoptosis.

The cytotoxic activity of Ziziphus Jujube on cervical cancer cells: In Vitro study.

Recently, there are tendency to use natural products such as Ziziphus Jujube (Jujube) as therapeutic agents for cancer. Understanding the molecular mechanisms of anti-cancer effects of Jujube may improve the current therapeutic strategies against cervical cancer. Our MTT data showed a significant dose- and time-dependent inhibition of OV-2008 cell proliferation following Jujube administration. Moreover, qRT-PCR analyses significantly revealed the suppression of cyclin D1 and the enhancement of P53, P21 and P27 expression in treated cells. These results suggest that the herb exerts a cytotoxic effect on cervical cancer cells through alternation of the expression of the genes that are involved in regulation of cell cycle.

The cytotoxic role of intermittent high glucose on apoptosis and cell viability in pancreatic beta cells.

OBJECTIVES: Glucose fluctuations are both strong predictor of diabetic complications and crucial factor for beta cell damages. Here we investigated the effect of intermittent high glucose (IHG) on both cell apoptosis and proliferation activity in INS-1 cells and the potential mechanisms. METHODS: Cells were treated with normal glucose (5.5 mmol/L), constant high glucose (CHG) (25 mmol/L), and IHG (rotation per 24 h in 11.1 or 25 mmol/L) for 7 days. Reactive oxygen species (ROS), xanthine oxidase (XOD) level, apoptosis, cell viability, cell cycle, and expression of cyclinD1, p21, p27, and Skp2 were determined. RESULTS: We found that IHG induced more significant apoptosis than CHG and normal glucose; intracellular ROS and XOD levels were more markedly increased in cells exposed to IHG. Cells treated with IHG showed significant decreased cell viability and increased cell proportion in G0/G1 phase. Cell cycle related proteins such as cyclinD1 and Skp2 were decreased significantly, but expressions of p27 and p21 were increased markedly. CONCLUSIONS: This study suggested that IHG plays a more toxic effect including both apoptosis-inducing and antiproliferative effects on INS-1 cells. Excessive activation of cellular stress and regulation of cyclins might be potential mechanism of impairment in INS-1 cells induced by IHG.

Bmi-1-shRNA inhibits the proliferation of lung adenocarcinoma cells by blocking the G1/S phase through decreasing cyclin D1 and increasing p21/p27 levels.

B lymphoma Mo-MLV insertion region 1 (Bmi-1) is highly expressed in a variety of cancers and has been shown to regulate cell proliferation. The INK4a/ARF tumor suppressor gene locus is one of the major targets of Bmi-1. In the present study, we chose two lung adenocarcinoma cell lines, A549 cells (without INK4a locus) and SPC-A1 cells (with INK4a locus), to investigate if the small hairpin RNA-mediated knockdown of Bmi-1 could inhibit the proliferation of lung adenocarcinoma cells, and to delineate the possible mechanism underlying Bmi-1 modulation of cell proliferation. We also investigated the potential pathway underlying Bmi-1 regulation of lung adenocarcinoma cell proliferation in different genetic backgrounds. To this end, we used shRNA to knockdown Bmi-1 expression in lung adenocarcinoma cells, which led to inhibition of cell growth, colony formation in vitro, and tumorigenesis in vivo. In addition, phosphorylated Akt and cyclin D1 expression were downregulated, p21 and p27 levels were upregulated, and p16 expression remained unchanged in SPC-A1 cells. These data indicate that Bmi-1 might modulate the growth of lung adenocarcinoma cells in an INK4a-p16 independent pathway.

Berberine attenuates high glucose-induced proliferation and extracellular matrix accumulation in mesangial cells: involvement of suppression of cell cycle progression and NF-κB/AP-1 pathways.

Berberine has been shown to have renoprotective effects on diabetes through attenuating TGF-ß1 and fibronectin (FN) expression. However, how berberine regulates TGF-ß1 and FN is not fully clear. Here we investigated whether berberine inhibited TGF-ß1 and FN expression in high glucose-cultured mesangial cells. Berberine significantly inhibited mesangial cell proliferation and hypertrophy by increasing the cell population in G1-phase and reducing that in S-phase. In addition, berberine reversed high glucose-induced down-regulation of cyclin-dependent kinase inhibitor p21(Waf1)/(Cip1) and p27(Kip1). Berberine inhibited p65 translocation to the nucleus and c-jun phosphorylation induced by high glucose. Furthermore, berberine attenuated high glucose-induced expression of TGF-ß1 and FN. Using a luciferase reporter assay, we found that high glucose-induced transcription activity of NF-κB and AP-1 was blocked by berberine. Electrophoretic mobility shift assay showed that high glucose increased that NF-κB and AP-1 DNA binding activity. These data indicate that berberine inhibited mesangial cell proliferation and hypertrophy by modulating cell cycle progress. In addition, berberine suppressed high glucose-induced TGF-ß1 and FN expression by blocking NF-κB/AP-1 pathways.

Amplified inhibition of stellate cell activation pathways by PPAR-γ, RAR and RXR agonists.

Peroxisome proliferator activator receptors (PPAR) ligands such as 15-Δ12,13-prostaglandin L(2) [PJ] and all trans retinoic acid (ATRA) have been shown to inhibit the development of liver fibrosis. The role of ligands of retinoic X receptor (RXR) and its ligand, 9-cis, is less clear. The purpose of this study was to investigate the effects of combined treatment of the three ligends, PJ, ATRA and 9-cis, on key events during liver fibrosis in rat primary hepatic stellate cells (HSCs). We found that the anti-proliferative effect of the combined treatment of PJ, ATRA and 9-cis on HSCs was additive. Further experiments revealed that this inhibition was due to cell cycle arrest at the G0/G1 phase as demonstrated by FACS analysis. In addition, the combined treatment reduced cyclin D1 expression and increased p21 and p27 protein levels. Furthermore, we found that the three ligands down regulated the phosphorylation of mTOR and p70(S6K). The activation of HSCs was also inhibited by the three ligands as shown by inhibition of vitamin A lipid droplets depletion from HSCs. Studies using real time PCR and western blot analysis showed marked inhibition of collagen Iα1 and αSMA by the combination of the three ligands. These findings suggest that the combined use of PJ, ATRA and 9-cis causes inhibition of cell proliferation by cell cycle arrest and down-regulation of fibrotic markers to a greater extent compared to each of the ligands alone.

ErbB receptor tyrosine kinases contribute to proliferation of malignant melanoma cells: inhibition by gefitinib (ZD1839).

Members of the epidermal growth factor (EGF) family of structurally related tyrosine kinase receptors, known as the ErbB receptors (EGFR/ErbB1/HER1, ErbB2/HER2/neu, ErbB3/HER3 and ErbB4/HER4) and their respective ligands, have been suggested to be involved in the development and progression of malignant melanoma. Here we investigate the effects of the ErbB1 tyrosine kinase inhibitor gefitinib (ZD1839, Iressa) on human malignant melanoma cells (RaH3 and RaH5) in vitro. ZD1839 inhibited proliferation of exponentially growing RaH3 and RaH5 cells in a dose-dependent manner with a half-maximally effective dose of 3.5 and 2.0 micromol/l, respectively. Cell growth was inhibited at 0.1 micromol/l ZD1839 in both cell lines. Maximal inhibition was accomplished at 10 micromol/l ZD1839; however, the effect was not complete as both cell lines showed a continuous slow growth during the treatment period. Flow cytometry analysis of cell-cycle distribution showed that ZD1839 treatment caused accumulation of RaH3 and RaH5 cells in the G1 phase. The growth arrest induced by ZD1839 coincided with upregulation of the cyclin-dependent kinase inhibitor p27 KIP 1. There was no increase in apoptosis as determined by analysis of plasma phosphatidyl serine redistribution. Western blot analysis revealed that ZD1839 substantially reduced tyrosine phosphorylation of ErbB1 as well as ErbB2 and ErbB3. This was accompanied by a concomitant decrease in Akt-phosphorylation, Erk1/2-phosphorylation, and Stat3-phosphorylation. Our results show that ZD1839 interferes with the growth of human malignant melanoma cells by cytostatic effects. These findings indicate the possible use of ErbB receptor kinase inhibitors as a novel treatment strategy in malignant melanoma.