Translocation of insulin receptors into plasma membrane microdomains in response to insulin and to insulin-enhancing vanadium and chromium compounds

2010 Summer.Includes bibliographic references (pages 135-156).Covers not scanned.Print version deaccessioned 2022.We have examined the translocation of insulin receptors into specialized, cholesterol-enriched membrane microdomains called lipid rafts following treatment of RBL-2H3 cells with insulin, bis-maltolatooxovanadium (BMOV) and tris(pyridinecarbxylato) chromium(III) (Cr(pic)3). Isopycnic sucrose gradient ultracentrifugation was used to subfractionate membrane fragments and insulin receptors were identified within low or high buoyant density membrane fractions using insulin receptor-specific antibodies and western blotting. Single particle tracking methods were used to confirm the confinement of individual insulin receptors within small membrane compartments on intact, viable RBL-2H3 cells. We demonstrated that insulin receptors translocate into lipid rafts upon binding insulin or following exposure to BMOV or Cr(pic)3 Phosphorylated insulin receptors also appeared in membrane raft fragments in response to insulin and/or insulin-mimicking compounds. Extraction of cholesterol from lipid rafts disrupted these microdomains and caused a decrease in the number of unphosphorylated and phosphorylated insulin receptors within these compartments. In addition to their ability to induce translocation of insulin receptors into lipid rafts, BMOV and Cr(pic)3 caused an increase in the number of phosphorylated IRS-1 molecules within these membrane fragments. To determine why Cr(pic)3 and BMOV might affect the distribution of insulin receptors in non-raft and raft compartments, membrane fluidity was evaluated in Cr(pic)3 and BMOV treated cells. Fluidity, as suggested by a decrease in lipid packing, was increased following treating 2H3 cells with either BMOV or Cr(pic)3 These results suggest that changes in lipid packing resulting from exposure of cells to either Cr(pic)3 and BMOV may affect the distribution of receptors in non-raft and raft compartments. Increased receptor localization in rafts or small membrane compartments evaluated by single particle tracking studies, would result in increased likelihood of insulin receptor phosphorylation within these signaling platforms. Thus rafts may be an important membrane structures involved in cell signaling events mediated by insulin receptors

Combined pitavastatin and dacarbazine treatment activates apoptosis and autophagy resulting in synergistic cytotoxicity in melanoma cells

Melanoma is an aggressive skin cancer and its incidence is increasing faster than any other type of cancer. Whilst dacarbazine (DTIC) is the standard chemotherapy for metastatic melanoma, it has limited success. Statins, including pitavastatin, have been demonstrated to have a range of anti-cancer effects in a number of human cancer cell lines. The present study therefore explored the anti-cancer activity of combined DTIC and pitavastatin in A375 and WM115 human melanoma cells. Cell survival assays demonstrated that combined DTIC and pitavastatin treatment resulted in synergistic cell death. Cell cycle analyses further revealed that this combined treatment resulted in a G1 cell cycle arrest, as well as a sub-G1 population, indicative of apoptosis. Activation of apoptosis was confirmed by Annexin V-fluorescein isothiocyanate/propidium iodide double-staining and an increase in the levels of active caspase 3 and cleaved poly (ADP-ribose) polymerase. Furthermore, it was demonstrated that apoptosis occurs through the intrinsic pathway, evident from the release of cytochrome c. Finally, combined DTIC and pitavastatin treatment was demonstrated to also activate autophagy as part of a cell death mechanism. The present study provides novel evidence to suggest that the combined treatment of DTIC and pitavastatin may be effective in the treatment of melanoma.Melanoma is an aggressive skin cancer and its incidence is increasing faster than any other type of cancer. Whilst dacarbazine (DTIC) is the standard chemotherapy for metastatic melanoma, it has limited success. Statins, including pitavastatin, have been demonstrated to have a range of anti-cancer effects in a number of human cancer cell lines. The present study therefore explored the anti-cancer activity of combined DTIC and pitavastatin in A375 and WM115 human melanoma cells. Cell survival assays demonstrated that combined DTIC and pitavastatin treatment resulted in synergistic cell death. Cell cycle analyses further revealed that this combined treatment resulted in a G1 cell cycle arrest, as well as a sub-G1 population, indicative of apoptosis. Activation of apoptosis was confirmed by Annexin V-fluorescein isothiocyanate/propidium iodide double-staining and an increase in the levels of active caspase 3 and cleaved poly (ADP-ribose) polymerase. Furthermore, it was demonstrated that apoptosis occurs through the intrinsic pathway, evident from the release of cytochrome c. Finally, combined DTIC and pitavastatin treatment was demonstrated to also activate autophagy as part of a cell death mechanism. The present study provides novel evidence to suggest that the combined treatment of DTIC and pitavastatin may be effective in the treatment of melanoma

Investigation of gene variation among non-alcoholic fatty liver disease patients in Gaza strip: a preliminary study

The prevalent hepatic presentation of the metabolic syndrome is a non-alcoholic fatty liver disease (NAFLD), one of the most common types of chronic liver illnesses. Patients with NAFLD may develop liver damage depending on their genetic heritage. In this preliminary study, our main aim was to detect the genetic association of p85α (Met326Ile), PNPLA3 (C>G), IL28B275 (A>G), and IL28B860 (C>T) single nucleotide polymorphisms (SNPs) with steatosis and NASH in patients from Gaza Strip. We performed an SNP analysis by RFLP-PCR in 33 cases of steatosis and 28 cases of non-alcoholic steatohepatitis (NASH), in addition to 29 age- and sex-matched controls. We found that only the mutant T allele of IL28B860 was significantly associated with an increased risk of steatosis (P = 0.04). The other studied alleles and genotypes were not significantly associated with increased or decreased risk of steatosis, NASH, or combined steatosis or NASH groups. Among all of the studied variables (age, sex, diabetes, and BMI), only BMI was significantly associated with an increased risk of steatosis as well as NASH. A linkage disequilibrium analysis showed that the association between the two SNPs of IL28B860 and IL28B275 was significant. Having the TG haplotype increased the risk of steatosis by 2.97 fold and the risk of combined steatosis or NASH by 2.44 fold. This haplotype increased the risk of NASH, but the effect was not significant

Statins: Could an old friend help the fight against COVID-19?

This is the peer reviewed version of the following article: "Statins: Could an old friend help the fight against COVID-19?" . British Journal of Pharmacology (2020): 19 June, which has been published in final form at https://doi.org/10.1111/bph.15166. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versionshe COVID-19 pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has overwhelmed healthcare systems requiring the rapid development of treatments, at least, to reduce COVID-19 severity. Drug repurposing offers a fast track. Here, we discuss the potential beneficial effects of statins in COVID-19 patients based on evidence that they may target virus receptors, replication, degradation, and downstream responses in infected cells, addressing both basic research and epidemiological information. Briefly, statins could modulate virus entry, acting on the SARS-CoV-2 receptors, ACE2 and CD147, and/or lipid rafts engagement. Statins, by inducing autophagy activation, could regulate virus replication or degradation, exerting protective effects. The well-known anti-inflammatory properties of statins, by blocking several molecular mechanisms, including NF-κB and NLRP3 inflammasomes, could limit the "cytokine storm" in severe COVID-19 patients which is linked to fatal outcome. Finally, statin moderation of coagulation response activation may also contribute to improving COVID-19 outcomesThis work and data discussed here were supported by grants from the Instituto de Salud Carlos III (ISCIII) and Fondos FEDER European Union (PI17/00119 and Red de Investigación Renal (REDINREN): RD16/0009, to M.R-O, PI17/01495 to J.E, PI18/01133 to AMR, PI19/00815 to A.O); Comunidad de Madrid (“NOVELREN” B2017/BMD3751 to M.R-O, B2017/BMD-3686 CIFRA2-CM to A.O); Spanish Ministry of Economy and Competitiveness MINECO (DTS17/00203, DTS19/00093) to J,E; “Convocatoria Dinamización Europa Investigación 2019” MINECO (EIN2019-103294 to M.R-O and SR-M); ERA-PerMed-JTC2018 (KIDNEY ATTACK AC18/00064 and PERSTIGAN AC18/00071) and DTS18/00032 to A.O; The “Sara Borrell” postdoctoral training program of the ISCIII supported the salary of SR-M (CD19/00021), IMPROVE-PD project (“Identification and Management of Patients at Risk–Outcome and Vascular Events in Peritoneal Dialysis”) funding from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie Grant Agreement No. 812699 to M.R.O

Combined Caffeine and Cisplatin Treatment Induces Synergistic Cytotoxicity in Hela Cell Line

Cisplatin is a common alkylating anticancer agent that has been used to treat several cancers. However, the efficiency of cisplatin treatment is limited due to the severe side effects and the resistance to the drug, which eventually results in treatment failure. Caffeine is a natural ingredient contained in many food sources. Caffeine has been shown to induce cell cycle arrest and apoptosis in different cancer cell types. The effect of caffeine on cisplatin treatment on cervical cancer cells is not well known. Here we examined the combined effect of caffeine and cisplatin in human Hela cells. The cancer cells were exposed to different concentrations of caffeine and cisplatin and IC50’s were determined by MTT assay. Cell number and viability were measured by cell counting and trypan blue assays. Data obtained show that, caffeine treatment enhances the anti-proliferation effect of cisplatin and lowered the IC50 of cisplatin from 8.93 µM to be 2.75 µM. These results suggest that caffeine-assisted chemotherapy is useful for cervical cancer treatment