Destruxin E, a Cyclodepsipeptide Antibiotic, Reduces Cyclin D1 Levels and Inhibits Anchorage-Independent Growth of v-Ki-ras-Expressed pMAM-ras-REF Cells

Abstract

Anchorage independence is a characteristic of malignant tumor cells. Tumor cells grow in the absence of substrate attachment, under conditions such as in soft agar cultures or suspension cultures. Their growth ability correlates well with their tumorigenicity. Inhibitors of anchorage-independent growth of tumor cells would be useful for the treatment of cancer. Fukazawa et al. developed a simple and rapid colorimetric assay method to quantify anchorage-independent growth using a microplate coated with an antiadhesive polymer poly(2-hydroxyethyl methacrylate) (polyHEMA).1) Normal cells and nontumorigenic cells do not grow on the microplate, but tumor cells and oncogenic-transformed cells grow easily on the polyHEMA-coated plates. Cell Culture pMAM-ras-REF is a cloned Fischer rat embryo fibroblast cell line transformed with pMAMneo-vKi-ras, a plasmid in which v-Ki-ras is linked to the dexamethasone-inducible MMTV-LTR promoter. MATERIALS AND METHODS Reagents and Antibodies1) The cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37°C with 5% CO 2 . The exponentially growing cells were trypsinized and resuspended in fresh medium. Then the cells were inoculated onto polyHEMA-coated (suspension culture) or -uncoated (substratum-attached culture) plates. Poly-HEMA-coated plates were prepared as described.1) v-Ki-ras expression in pMAM-ras-REF cells was induced by dexamethasone 1 mM. To assay the growth inhibition by DE, the cells were seeded at 5ϫ10 3 cells/well (0.32 cm 2 ) on IWAKI 96-well tissue culture plates. Each well contained 150 ml of DMEM and 10% FBS medium with or without dexamethasone 1 mM (day 0). Drugs were added to the wells on day 1, and incubation was continued until day 4. Cell growth was quantified by a colorimetric MTT assay as reported previously. 4) Preparation of Cell Lysates and Western BlottingCells (5ϫ10 5 ) were washed twice with ice-cold PBS containing Na 3 VO 4 100 mM and then lysed in a lysis buffer (20 mM Hepes [pH 7.5], 150 mM NaCl, 1% Triton X-100, 10% glycerol, 1 mM EDTA, 50 mM b-glycerophosphate, 1 mM Na 3 VO 4 , antipain 25 mg/ml, leupeptin 25 mg/ml). Cell extracts, each containing the same amount of protein, were separated by SDS-PAGE and transferred onto Immobilon PVDF membranes (Millipore). ECL Western blotting detection reagents (Amersham Pharmacia Biotech) were used to visualize the University; Machida, Japan: b Microbial Chemistry Research Center; Japan: and c

Cystathionine γ-Lyase Overexpression Inhibits Cell Proliferation via a H2S-dependent Modulation of ERK1/2 Phosphorylation and p21Cip/WAK-1

“…Cip/WAF-1 can inhibit cyclin D1 activation and thereby prevent cyclin D1/CRM1 association (24). However, our study demonstrated that cyclin D1 was not involved in CSE overexpression-induced cell growth inhibition.…”

Pro‐apoptotic effect of endogenous H2S on human aorta smooth muscle cells

“…Cyclin D1 is an activator of cdk4 and cdk6, which are rate‐limiting for cell cycle re‐entry (23). p21 Cip/WAK‐1 is one of the cdk inhibitory proteins, and it can prevent cell cycle progress by directly binding with phosphorylated cyclin D1 and cdk complexes (24, 25). The expression of cyclin D1 was decreased and that of p21 Cip/WAK‐1 significantly increased in Ad‐CSE infected cells compared with the cells infected with Ad‐lacZ or control cells (Fig.…”

“…Cyclin D1/cdk4/6 predominantly act sequentially during the G1/S transition and are regulated for cell cycle progression through this period (32). The kinase activity of this cyclin/cdk complex can be negatively regulated by cdk inhibitors, such as p21 Cip/WAK‐1 , which inhibit cyclin D1 activation and thereby prevent cyclin D1/CRM1 association (25). Both cyclin D1 and p21 Cip/WAK‐1 play important roles in regulating cellular differentiation, DNA repair, cell senescence and apoptosis.…”

Nonribosomal cyclic peptides: specific markers of fungal infections

“…167 Concentration of cyclic peptides generated by biocontrol should certainly be monitored, since recent studies indicate a distinct effect of cyclic peptides on mammalian cell lines as well. 168,169 Evolutionarily, fungi have developed various strategies for propagation and survival (S-strategy -stress tolerance; P-strategy -pathogenicity; R-strategy -ruderal, etc.). Some strains, including clinically important fungi, exert also a C-strategy, 170 where C stands for combative.…”