Depletion of membrane cholesterol compromised caspase-8 imparts in autophagy induction and inhibition of cell migration in cancer cells

Abstract Background Cholesterol in lipid raft plays crucial role on cancer cell survival during metastasis of cancer cells. Cancer cells are reported to enrich cholesterol in lipid raft which make them more susceptible to cell death after cholesterol depletion than normal cells. Methyl-β-cyclodextrin (MβCD), an amphipathic polysaccharide known to deplete the membrane cholesterol, induces cell death selectively in cancer cells. Present work was designed to identify the major form of programmed cell death in membrane cholesterol depleted cancer cells (MDA-MB 231 and 4T1) and its impact on migration efficiency of cancer cells. Methods Membrane cholesterol alteration and morphological changes in 4T1 and MDA-MB 231 cancer cells by MβCD were measured by fluorescent microscopy. Cell death and cell proliferation were observed by PI, AO/EB and MTT assay respectively. Programme cell death was confirmed by flow cytometer. Caspase activation was assessed by MTT and PI after treatments with Z-VAD [OME]-FMK, mitomycin c and cycloheximide. Necroptosis, autophagy, pyroptosis and paraptosis were examined by cell proliferation assay and flow cytometry. Relative quantitation of mRNA of caspase-8, necroptosis and autophagy genes were performed. Migration efficiency of cancer cells were determined by wound healing assay. Results We found caspase independent cell death in cholesterol depleted MDA-MB 231 cells which was reduced by (3-MA) an autophagy inhibitor. Membrane cholesterol depletion neither induces necroptosis, paraptosis nor pyroptosis in MDA-MB 231 cells. Subsequent activation of caspase-8 after co-incubation of mitomycin c and cycloheximide separately, restored the cell viability in cholesterol depleted MDA-MB 231 cells. Down regulation of caspase-8 mRNA in cholesterol depleted cancer cells ensures that caspase-8 indirectly promotes the induction of autophagy. In another experiment we have demonstrated that membrane cholesterol depletion reduces the migration efficiency in cancer cells. Conclusion Together our experimental data suggests that membrane cholesterol is the crucial for the recruitment and activation of caspase-8 as well as its non-apoptotic functions in cancer cells. Enriched cholesterol in lipid raft of cancer cells may be regulating the cross talk between caspase-8 and autophagy machineries to promote their survival and migration. Therefore it can be explored to understand and address the issues of chemotherapeutic and drugs resistance

MOESM2 of Depletion of membrane cholesterol compromised caspase-8 imparts in autophagy induction and inhibition of cell migration in cancer cells

Additional file 2: Figure S2. Cholesterol depletion induced cell death in various cell lines. [a] cholesterol depletion induced cell death. MDA-MB 231, 4T1 and Balbc3T3 Cell lines were treated with different concentration of MβCD for 24 h. Cell viability was measured by MTT assay. 2[b] Vero, MDCK, 4T1, Balb/c3T3 and MDA-MB 231 cells were exposed to the 5mM MβCD for 16 h and cell death were measured by MTT assay. Statistical analysis: One way anova, post hock test Tukey. P*<0.05 P**<0.01, P**<0.001, N.S.-Not significant

MOESM3 of Depletion of membrane cholesterol compromised caspase-8 imparts in autophagy induction and inhibition of cell migration in cancer cells

Additional file 3: Figure S3. Assessment of role of caspase. 4T1 Cells for 4 hour [a] and Vero cells[b, c, d] for 2, 4 and 6 hour were incubated with 5 mM MβCD in the presence and absence of Z-VAD[OME]-FMK[60 ¾g/ml]. Cell viability was measured by Flow cytometer [a], MTT [b, c, d]. Statistical analysis: One way anova, post hock test Tukey. P*<0.05 P**<0.01, P**<0.001, N.S.-Not significant

MOESM4 of Depletion of membrane cholesterol compromised caspase-8 imparts in autophagy induction and inhibition of cell migration in cancer cells

Additional file 4: Figure S4. Role of Caspase-8 activation in cholesterol depleted cells. MDA-MB 231 cells were incubated with 5 mM MβCD and 3-Methyl adenine [3-MA] in presence and absence of mitomycin c for 6 Hours. Cell viability was measured by flow cytometer and MTT [a]-[b]. Statistical analysis: One way anova, post hock test Tukey. P*<0.05 P**<0.01, P**<0.001, N.S.-Not significant

MOESM1 of Depletion of membrane cholesterol compromised caspase-8 imparts in autophagy induction and inhibition of cell migration in cancer cells

Additional file 1: Figure S1. Cholesterol depletion induced cell death in cancer cells. MDA MB-231 Cells were incubated with 5mM MβCD at different concentration for 24 hours and cell viability was measured by MTT and PI respectively [a &b]. 4T1 Cells were treated with Methyl β Cyclodextrin at different concentration for 24 hour. Cytotoxicity was measured by MTT assay [c] and Acridine orange and ethidium bromide [AO/EB] [d]. Statistical analysis: One way anova, Post hock test Tukey. P*<0.05 P**<0.01, P**<0.001, N.S.-Not significant

Veterinary World, EISSN: 2231-0916 Available at www.veterinaryworld.org/Vol.7/July-2014/16.pdf RESEARCH ARTICLE Open Access

Evaluation of efficacy of saponin and freund&apos;s incomplete adjuvanted paratuberculosis vaccine in murine mode

Evaluation of efficacy of saponin and freund's incomplete adjuvanted paratuberculosis vaccine in murine model

Aim: To compare the efficacy between saponin adjuvanted paratuberculosis killed vaccine and Freund's incomplete (FIC) adjuvanted paratuberculosis vaccine in mice model. Materials and Methods: The study was conducted for a period of 11 months using 80 Swiss albino mice by dividing into four groups (Group I-mice vaccinated with killed Mycobacterium avium subspp. paratuberculosis (MAP) adjuvanted with saponin, Group II- for FIC, Group III- Saponin control and Gr IV- FIC control). Faecal shedding, changes in body weight, presence of local reactions and clinical symptoms, were evaluated. Serological assays for humoral and cell mediated immune response were determined by agar gel immunodiffusion (AGID) and nitric oxide test (NO) respectively. A total of 76 serum samples, 52 faecal samples and 20 tissue samples were collected at different intervals during the study period. ZN staining; AGID test and nitric oxide test were employed for analysis of samples. Results: A total of 38 samples (37 faecal and 1 tissue sample) and 18 serum samples were recorded as positive by ZN staining and AGID test respectively from all four groups of mice. Significant decrease in faecal shedding of organisms was observed in Group I mice vaccinated with saponin adjuvanted vaccine in comparison to Group II. Gain in body weight of Group I mice was recorded as higher than Group II. NO test revealed increase in the concentration of NO level in both vaccinated group but more significant in Group I. Conclusion: The tests results obtained revealed that killed MAP vaccine adjuvanted with saponin have higher protective efficacy in terms of reduced faecal shedding, gain in body weight, less side effects, absence of attributable specific symptoms of Johne's disease, etc. in comparison to killed MAP vaccine adjuvanted with FIC. The present study also reveals that AGID and NO estimation tests are specific tests which can be employed for diagnosis and comparison of efficacy of paratuberculosis vaccine

Lipid Storage and Interferon Response Determine the Phenotype of Ground Glass Hepatocytes in Mice and Humans

BACKGROUND AND AIMS: A histopathological hallmark of chronic hepatitis B virus (HBV) infection is the presence of ground glass hepatocytes (GGHs). GGHs are liver cells that exhibit eosinophilic, granular, glassy cytoplasm in light microscopy and are characterized by accumulation of HBV surface (HBs) proteins in the endoplasmic reticulum (ER). More important, GGHs have been accepted as a precursor of HCC and may represent preneoplastic lesions of the liver. METHODS: Here we show that the reason for ground glass phenotype of hepatocytes in patients with chronic hepatitis B (CHB) and in HBs transgenic mice is a complex formation between HBs proteins and lipid droplets (LDs) within the ER. RESULTS: As fat is a main component of LDs their presence reduces the protein density of HBs aggregates. Therefore, they adsorb less amount of eosin during hematoxylin-eosin staining and appear dull in light microscopy. However, after induction of interferon response in the liver LDs were not only co-localized with HBs but also distributed throughout the cytoplasm of hepatocytes. The uniform distribution of LDs weakens the contrast between HBs aggregates and the rest of the cytoplasm and complicates the identification of GGHs. Suppression of interferon response restored the ground glass phenotype of hepatocytes. CONCLUSIONS: Complex formation between HBs and LDs represents a very important feature of CHB that could affect LDs functions in hepatocytes. The strain specific activation of the interferon response in the liver of HBs/c mice prevented the development of GGHs. Thus, manipulation of LDs could provide a new treatment strategy in the prevention of liver cancer