Abstract
The specialized cholesterol/sphingolipid-rich membrane domains termed lipid rafts are highly dynamic in the cancer cells, which rapidly assemble effector molecules to form a sorting platform essential for oncogenic signaling transduction in response to extra- or intracellular stimuli. Density-based membrane flotation, subcellular fractionation, cell surface biotinylation, and co-immunoprecipitation analyses of bichalcone analog ((E)-1-(4-Hydroxy-3-((4-(4-((E)-3-(pyridin-3-yl)acryloyl)phenyl)piperazin-1-yl)methyl)phenyl)-3-(pyridin-3-yl)prop-2-en-1-one (TSWU-BR4)-treated cancer cells showed dissociation between GRP78 and p85α conferring the recruitment of PTEN to lipid raft membranes associated with p85α. Ectopic expression of GRP78 could overcome induction of lipid raft membrane-associated p85α–unphosphorylated PTEN complex formation and suppression of GRP78−PI3K−Akt−GTP-Rac1-mediated and GRP78-regulated PERK−Nrf2 antioxidant pathway and cancer cell invasion by TSWU-BR4. Using specific inducer, inhibitor, or short hairpin RNA for ASM demonstrated that induction of the lipid raft membrane localization and activation of ASM by TSWU-BR4 is responsible for perturbing homeostasis of cholesterol and ceramide levels in the lipid raft and ER membranes, leading to alteration of GRP78 membrane trafficking and subsequently inducing p85α–unphosphorylated PTEN complex formation, causing disruption of GRP78−PI3K−Akt−GTP-Rac1-mediated signal and ER membrane-associated GRP78-regulated oxidative stress balance, thus inhibiting cancer cell invasion. The involvement of the enrichment of ceramide to lipid raft membranes in inhibition of NF-κB-mediated MMP-2 expression was confirmed through attenuation of NF-κB activation using C2-ceramide, NF-κB specific inhibitors, ectopic expression of NF-κB p65, MMP-2 promoter-driven luciferase, and NF-κB-dependent reporter genes. In conclusion, localization of ASM in the lipid raft membranes by TSWU-BR4 is a key event for initiating formation of ceramide-enriched lipid raft membrane platforms, which causes delocalization of GRP78 from the lipid raft and ER membranes to the cytosol and formation of p85α–unphosphorylated PTEN complexes to attenuate the GRP78-regulated oxidative stress balance and GRP78−p85α−Akt−GTP-Rac1−NF-κB−MMP-2-mediated cancer cell invasion.
Highlights
Increased level of cholesterol/sphingolipid-rich lipid rafts in cancer cells is related to high synthesis of lipid and cholesterol by changing in lipid- and cholesterol-associated pathways [1]
While investigating the cytotoxic action of the bichalcone analog TSWU-BR4 (Figure 1A) on human cancer cell lines, we found that TSWU-BR4 exhibits a strong inhibitory effect on the growth of both nasopharyngeal carcinoma NPC-TW039 and pharyngeal squamous carcinoma FaDu cells
reactive oxygen species (ROS) scavenger N-acetyl-L-cysteine (NAC) co-treatment suppressed the increase of ROS; NAC can only partially overcome from TSWU-BR4-induced decrease of cholesterol and invaded cell levels. These results indicate that TSWU-BR4 attenuates the association of glucose regulated protein 78 (GRP78) with p85α at the lipid raft membranes to induce the formation of lipid raft membrane-associated p85α–unphosphorylated PTEN complexes, thereby suppressing the regulatory effects of GRP78 on the endoplasmic reticulum (ER) stress-mediated cholesterol, ROS generation, and cell invasion
Summary
Increased level of cholesterol/sphingolipid-rich lipid rafts in cancer cells is related to high synthesis of lipid and cholesterol by changing in lipid- and cholesterol-associated pathways [1]. The dynamic compartmentalization of the lipid rafts modulates the lateral compartmentalization of receptors at the cell surface to facilitate the interaction of receptor and signaling molecules during advanced and metastatic stages of carcinogenesis [1,2]. 3-kinase (PI3K)−protein kinase B (Akt) pathway is compartmentalized within the lipid rafts to play functional roles in the regulation of cancer cell metabolism, survival, and invasion [4]. Akt activation is triggered by PI3K induction of generation of phosphatidylinositol-3,4,5-trisphosphate (PIP3 ). PIP3 accumulation in the plasma membrane formed by PI3K can facilitate guanine nucleotide factor-mediated guanosine 50 -triphosphate (GTP) binding to Ras-related C3 botulinum toxin substrate
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