Abstract
BackgroundCholesterol 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.MethodsMembrane 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.ResultsWe 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.ConclusionTogether 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.
Highlights
Cholesterol in lipid raft plays crucial role on cancer cell survival during metastasis of cancer cells
Conclusively our data suggests that lipid raft disruption induces caspase-independent cell death where compromised caspase-8 promotes autophagy
Pyroptosis nor paraptosis are associated with cell death after cholesterol depletion in MDA-MB 231 cells
Summary
Cholesterol in lipid raft plays crucial role on cancer cell survival during metastasis of cancer cells. Enrichment of cholesterol in the lipid raft in cancer cells make them more sensitive to cholesterol depletion and induces anoikis like cell death [6]. It shows the cholesterol in lipid raft having immense role in cancer cells survival and its progression [7]. Cholesterol enrichment in lipid raft requires for the recruitment and activation of caspase-8 and FADD in death inducing signaling complex (DISC) which execute apoptotic and non-apoptotic functions [8, 9]. In recent years there are some reports suggesting induction of apoptosis and autophagy as well as anoikis like cell death mechanism in cholesterol depleted cells [10, 11]. Disruption of lipid raft inhibits the migration and activation of caspase-8 [15] as well as FADD which can create opportunity for caspase independent cell death [16]
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