Resveratrol, Acetyl-Resveratrol, and Polydatin Exhibit Antigrowth Activity against 3D Cell Aggregates of the SKOV-3 and OVCAR-8 Ovarian Cancer Cell Lines

Resveratrol has aroused significant scientific interest as it has been claimed that it exhibits a spectrum of health benefits. These include effects as an anti-inflammatory and an antitumour compound. The purpose of this study was to investigate and compare any potential antigrowth effects of resveratrol and two of its derivatives, acetyl-resveratrol and polydatin, on 3D cell aggregates of the EGFR/Her-2 positive and negative ovarian cancer cell lines SKOV-3 and OVCAR-8, respectively. Results showed that resveratrol and acetyl-resveratrol reduced cell growth in the SKOV-3 and OVCAR-8 in a dose-dependant manner. The growth reduction was mediated by the induction of apoptosis via the cleavage of poly(ADP-ribose) polymerase (PARP-1). At lower concentrations, 5 and 10 µM, resveratrol, acetyl-resveratrol, and polydatin were less effective than higher concentrations, 50 and 100 µM. In SKOV-3 line, at higher concentrations, resveratrol and polydatin significantly reduced the phosphorylation of Her-2 and EGFR and the expression of Erk. Acetyl-resveratrol, on the other hand, did not change the activation of Her-2 and EGFR. Resveratrol, acetyl-resveratrol, and polydatin suppressed the secretion of VEGF in a dose-dependant fashion. In the OVCAR-8 cell line, resveratrol and acetyl-resveratrol at 5 and 10 µM increased the activation of Erk. Above these concentrations they decreased activation. Polydatin did not produce this effect. This study demonstrates that resveratrol and its derivatives may inhibit growth of 3D cell aggregates of ovarian cancer cell lines via different signalling molecules. Resveratrol and its derivatives, therefore, warrant further in vivo evaluation to assess their potential clinical utility

The resistance of intracellular mediators to doxorubicin and cisplatin are distinct in 3D and 2D endometrial cancer

Abstract Background Advanced endometrial cancer often shows resistance to clinical chemotherapy although potencies of anticancer drugs in vitro are promising. The disparity suggests that in vivo microenvironments are not recapitulated by in vitro models used for preclinical testing. However, spheroids replicate some important properties of tumours in vivo. Therefore, for the first time, we compared effects of doxorubicin and cisplatin on 3D multicellular structures and 2D cell monolayers of endometrial cancer cells. Methods 3D multicellular structures were generated by culturing cancer cells on non-adherent surfaces; and for comparison cell monolayers were cultured on adherent culture plates. Ishikawa, RL95-2, and KLE cell lines were studied. Morphologies of 3D multicellular structures were examined. After 48 hours treatment with anticancer drugs, apoptosis, proliferation, glucose metabolism and vascular endothelial growth factor (VEGF) were analysed. Immunostaining of PCNA, Glut-1, p-Erk1/2, SOD-1 and p-Akt1/2/3 was also performed. Results Distinct 3D multicellular morphologies were formed by three different endometrial cancer cell lines. Doxorubicin induced less apoptosis in 3D multicellular structures of high grade cancer cells (RL95-2 and KLE cell lines) than in cell monolayers. Parallel alterations in Erk1/2 phosphorylation and cell proliferation might suggest they were linked and again doxorubicin had less effect on 3D multicellular structures than cell monolayers. On the other hand, there was no correlation between altered glucose metabolism and proliferation. The responses depended on cancer cell lines and were apparently not mediated by altered Glut-1 levels. The level of SOD-1 was high in 3D cell cultures. The effects on VEGF secretion were various and cancer cell line dependent. Importantly, both doxorubicin and cisplatin had selective paradoxical stimulatory effects on VEGF secretion. The microenvironment within 3D multicellular structures sustained Akt phosphorylation, consistent with it having a role in anchorage-independent pathways. Conclusions The cancer cells responded to microenvironments in a distinctive manner. 3D multicellular structures exhibited greater resistance to the agents than 2D monolayers, and the differences between the culture formats were dependent on cancer cell lines. The effects of anticancer drugs on the intracellular mediators were not similar in 3D and 2D cultures. Therefore, using 3D cell models may have a significant impact on conclusions derived from screening drugs for endometrial carcinomas.</p

The Anti-Proliferative Effect of PI3K/mTOR and ERK Inhibition in Monolayer and Three-Dimensional Ovarian Cancer Cell Models

Most ovarian cancer patients are diagnosed with advanced stage disease, which becomes unresponsive to chemotherapeutic treatments. The PI3K/AKT/mTOR and the RAS/RAF/MEK/ERK kinase signaling pathways are attractive targets for potential therapeutic inhibitors, due to the high frequency of mutations to PTEN, PIK3CA, KRAS and BRAF in several ovarian cancer subtypes. However, monotherapies targeting one of these pathways have shown modest effects in clinical trials. This limited efficacy of the agents could be due to upregulation and increased signaling via the adjacent alternative pathway. In this study, the efficacy of combined PI3K/mTOR (BEZ235) and ERK inhibition (SCH772984) was investigated in four human ovarian cancer cell lines, grown as monolayer and three-dimensional cell aggregates. The inhibitor combination reduced cellular proliferation in a synergistic manner in OV-90 and OVCAR8 monolayers and in OV-90, OVCAR5 and SKOV3 aggregates. Sensitivity to the inhibitors was reduced in three-dimensional cell aggregates in comparison to monolayers. OV-90 cells cultured in large spheroids were sensitive to the inhibitors and displayed a robust synergistic antiproliferative response to the inhibitor combination. In contrast, OVCAR8 spheroids were resistant to the inhibitors. These findings suggest that combined PI3K/mTOR and ERK inhibition could be a useful strategy for overcoming treatment resistance in ovarian cancer and warrants further preclinical investigation. Additionally, in some cell lines the use of different three-dimensional models can influence cell line sensitivity to PI3K/mTOR and RAS/RAF/MEK/ERK pathway inhibitors

Uptake of Helicobacter pylori Outer Membrane Vesicles by Gastric Epithelial Cells▿

Helicobacter pylori bacteria colonize the human stomach where they stimulate a persistent inflammatory response. H. pylori is considered noninvasive; however, lipopolysaccharide (LPS)-enriched outer membrane vesicles (OMV), continuously shed from the surface of this bacterium, are observed within gastric epithelial cells. The mechanism of vesicle uptake is poorly understood, and this study was undertaken to examine the roles of bacterial VacA cytotoxin and LPS in OMV binding and cholesterol and clathrin-mediated endocytosis in vesicle uptake by gastric epithelial cells. OMV association was examined using a fluorescent membrane dye to label OMV, and a comparison was made between the associations of vesicles from a VacA+ strain and OMV from a VacA− isogenic mutant strain. Within 20 min, essentially all associated OMV were intracellular, and vesicle binding appeared to be facilitated by the presence of VacA cytotoxin. Uptake of vesicles from the VacA+ strain was inhibited by H. pylori LPS (58% inhibition with 50 μg/ml LPS), while uptake of OMV from the VacA− mutant strain was less affected (25% inhibition with 50 μg/ml LPS). Vesicle uptake did not require cholesterol. However, uptake of OMV from the VacA− mutant strain was inhibited by a reduction in clathrin-mediated endocytosis (42% with 15 μg/ml chlorpromazine), while uptake of OMV from the VacA+ strain was less affected (25% inhibition with 15 μg/ml chlorpromazine). We conclude that VacA toxin enhances the association of H. pylori OMV with cells and that the presence of the toxin may allow vesicles to exploit more than one pathway of internalization

A Combination of Two Receptor Tyrosine Kinase Inhibitors, Canertinib and PHA665752 Compromises Ovarian Cancer Cell Growth in 3D Cell Models

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