Cytoprotective effect of neuropeptides on cancer stem cells: vasoactive intestinal peptide-induced antiapoptotic signaling.

Cancer stem cells (CSCs) are increasingly considered to be responsible for tumor initiation, metastasis and drug resistance. The drug resistance mechanisms activated in CSCs have not been thoroughly investigated. Although neuropeptides such as vasoactive intestinal peptide (VIP) can promote tumor growth and activate antiapoptotic signaling in differentiated cancer cells, it is not known whether they can activate antiapoptotic mechanisms in CSCs. The objectives of this study are to unravel the cytoprotective effects of neuropeptides and identify antiapoptotic mechanisms activated by neuropeptides in response to anticancer drug treatment in CSCs. We enriched and purified CSCs (CD44+/high/CD24-/low or CD133+ population) from breast and prostate cancer cell lines, and demonstrated their stemness phenotype. Of the several neuropeptides tested, only VIP could protect CSCs from drug-induced apoptosis. A functional correlation was found between drug-induced apoptosis and dephosphorylation of proapoptotic Bcl2 family protein BAD. Similarly, VIP-induced cytoprotection correlated with BAD phosphorylation at Ser112 in CSCs. Using pharmacological inhibitors and dominant-negative proteins, we showed that VIP-induced cytoprotection and BAD phosphorylation are mediated via both Ras/MAPK and PKA pathways in CSCs of prostate cancer LNCaP and C4-2 cells, but only PKA signaling was involved in CSCs of DUVIPR (DU145 prostate cancer cells ectopically expressing VIP receptor) and breast cancer MCF7 cells. As each of these pathways partially control BAD phosphorylation at Ser112, both have to be inhibited to block the cytoprotective effects of VIP. Furthermore, VIP is unable to protect CSCs that express phosphorylation-deficient mutant-BAD, suggesting that BAD phosphorylation is essential. Thus, antiapoptotic signaling by VIP could be one of the drug resistance mechanisms by which CSCs escape from anticancer therapies. Our findings suggest the potential usefulness of VIP receptor inhibition to eliminate CSCs, and that targeting BAD might be an attractive strategy for development of novel therapeutics.

Identification of a novel oxidative stress induced cell death by Sorafenib and oleanolic acid in human hepatocellular carcinoma cells.

The lack of effective chemotherapies in hepatocellular carcinoma (HCC) is still an unsolved problem and underlines the need for new strategies in liver cancer treatment. In this study, we present a novel approach to improve the efficacy of Sorafenib, today's only routinely used chemotherapeutic drug for HCC, in combination with triterpenoid oleanolic acid (OA). Our data show that cotreatment with subtoxic concentrations of Sorafenib and OA leads to highly synergistic induction of cell death. Importantly, Sorafenib/OA cotreatment triggers cell damage in a sustained manner and suppresses long-term clonogenic survival. Sorafenib/OA cotreatment induces DNA fragmentation and caspase-3/7 cleavage and the addition of the pan-caspase inhibitor zVAD.fmk shows the requirement of caspase activation for Sorafenib/OA-triggered cell death. Furthermore, Sorafenib/OA co-treatment stimulates a significant increase in reactive oxygen species (ROS) levels. Most importantly, the accumulation of intracellular ROS is required for cell death induction, since the addition of ROS scavengers (i.e. α-tocopherol, MnTBAP) that prevent the increase of intracellular ROS levels completely rescues cells from Sorafenib/OA-triggered cell death. In conclusion, OA represents a novel approach to increase the sensitivity of HCC cells to Sorafenib via oxidative stress.

Alleviation of isoproturon toxicity to wheat by exogenous application of glutathione.

Treatment with the recommended field dose of isoproturon to 7-d-old wheat seedlings significantly decreased shoot height, fresh and dry weights during the subsequent 15days. Meanwhile contents of carotenoids, chlorophylls and anthocyanin as well as activities of δ-aminolevulinate dehydratase (ALA-D), phenylalanine ammonia lyase (PAL) and tyrosine ammonia lyase (TAL) were significantly inhibited. On the other hand, the herbicide significantly increased malondialdehyde (MDA), a naturally occurring product of lipid peroxidation and H2O2, while it significantly decreased the contents of glutathione (GSH) and ascorbic acid (AsA) and reduced the activities of superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX). These findings indicate an induction of a stress status in wheat seedlings following isoproturon treatment. However, exogenous GSH appeared to limit the toxic effects of isoproturon and seemed to overcome this stress status. Most likely, contents of pigment and activities of enzymes were raised to approximate control levels. Moreover, antioxidants were elevated and the oxidative stress indices seemed to be alleviated by GSH application. These results indicate that exogenous GSH enhances enzymatic and nonenzymatic antioxidants to alleviate the effects of isoproturon.

PPARalpha agonists inhibit nitric oxide production by enhancing iNOS degradation in LPS-treated macrophages.

BACKGROUND AND PURPOSE: Nitric oxide (NO) production through the inducible nitric oxide synthase (iNOS) pathway is increased in response to pro-inflammatory cytokines and bacterial products. In inflammation, NO has pro-inflammatory and regulatory effects. Peroxisome proliferator-activated receptors (PPARs), members of the nuclear steroid receptor superfamily, regulate not only metabolic but also inflammatory processes. The aim of the present study was to investigate the role of PPARalpha in the regulation of NO production and iNOS expression in activated macrophages. EXPERIMENTAL APPROACH: The effects of PPARalpha agonists were investigated on iNOS mRNA and protein expression, on NO production and on the activation of transcription factors NF-kappaB and STAT1 in J774 murine macrophages exposed to bacterial lipopolysaccharide (LPS). KEY RESULTS: PPARalpha agonists GW7647 and WY14643 reduced LPS-induced NO production in a dose-dependent manner as measured by the accumulation of nitrite into the culture medium. However, PPARalpha agonists did not alter LPS-induced iNOS mRNA expression or activation of NF-kappaB or STAT1 which are important transcription factors for iNOS. Nevertheless, iNOS protein levels were reduced by PPARalpha agonists in a time-dependent manner. The reduction was markedly greater after 24 h incubation than after 8 h incubation. Treatment with the proteasome inhibitors, lactacystin or MG132, reversed the decrease in iNOS protein levels caused by PPARalpha agonists. CONCLUSIONS AND IMPLICATIONS: The results suggest that PPARalpha agonists reduce LPS-induced iNOS expression and NO production in macrophages by enhancing iNOS protein degradation through the proteasome pathway. The results offer an additional mechanism underlying the anti-inflammatory effects of PPARalpha agonists.