Abstract
Malignant melanoma is the leading cause of death from skin cancer. Drug toxicity and resistance represent a serious challange for melanoma treatments. Evidence demonstrates that natural compounds may play a crucial role in cancer prevention, growth and progression. Vitamin E tocotrienols (TT) were shown to possess antitumor activity. Here, we analyzed the effects of δ-TT on melanoma cell growth and the involvement of the endoplasmic reticulum (ER) stress in this activity. The experiments were performed on human melanoma cell lines, BLM and A375. δ-TT exerted a significant proapoptotic effect on both cell lines, involving the intrinsic apoptosis pathway; importantly, this compound did not affect the viability of normal human melanocytes. In melanoma cells, δ-TT exerted its antitumor effect through activation of the PERK/p-eIF2α/ATF4/CHOP, IRE1α and caspase-4 ER stress-related branches. Salubrinal, an inhibitor of the ER stress, counteracted the cytotoxic activity of δ-TT. In vivo experiments performed in nude mice bearing A375 xenografts evidenced that δ-TT reduces tumor volume and tumor mass; importantly, tumor progression was significantly delayed by δ-TT treatment. In conclusion, δ-TT exerts a proapoptotic activity on melanoma cells, through activation of the ER stress-related pathways. δ-TT might represent an effective option for novel chemopreventive/therapeutic strategies for melanoma.
Highlights
Cells react to endoplasmic reticulum (ER) stress with an initial defensive process, the so called unfolded protein response (UPR), aimed at restoring homeostasis by enhancing protein folding capacity[23]; in conditions of severe stress, misfolded proteins accumulate in the ER triggering a set of prodeath programs[21]
To investigate whether δ-TT might induce melanoma cell death, BLM and A375 cells were treated with different doses of δ-TT (5–20 μg/ml) for 24 h, floating and adherent cells were harvested, stained with 0.4% of Trypan blue (1:1) and counted with an automated cell counter, differentiating from total, living and dead cells
By Western blotting we evaluated the expression levels of ER stress markers (BiP, p-eIF2α, eIF2α, PKR-like ER kinase (PERK), IRE1αand PDI, protein disulfide isomerase, a Ca2+-dependent chaperon protein located in the ER lumen) and ER stress-related apoptosis markers (ATF4, CHOP and ERO1α, a target of CHOP, involved in the hyperoxidation of the ER environment, through generation of ROS)
Summary
These compounds did not provide the expected improvement on overall survival, being accompanied by severe toxicity[5] Based on these disappointing observations, combination treatments targeting different intracellular pathways are currently investigated as potential effective therapeutic strategies for aggressive melanomas[6]. The role of dietary factors in preventing cancers was investigated in a large body of epidemiological studies Natural compounds, such as epigallocatechin-3-gallate (EGCG), resveratrol, lycopene, polyunsaturated omega-3 fatty acids (PUFA) and genistein, were reported to exert antitumor effects on several cancer cell lines[8,9]. Three major proteins are known to act as stress sensors in the ER: double-stranded RNA-dependent protein kinase PKR-like ER kinase (PERK), inositol-requiring enzyme 1α(IRE1α), and activating transcription factor 6 (ATF6)[24] In physiological conditions, these sensors are inactivated by the association with the chaperon protein immunoglobulin-heavy-chain-binding protein (BiP, known as GRP78). Different natural compounds have been shown to induce ER stress-mediated death in cancer cells[32]
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