Sinulariolide Induced Hepatocellular Carcinoma Apoptosis through Activation of Mitochondrial-Related Apoptotic and PERK/eIF2α/ATF4/CHOP Pathway

Abstract

Sinulariolide, an active compound isolated from the cultured soft coral Sinularia flexibilis, has potent anti-microbial and anti-tumorigenesis effects towards melanoma and bladder cancer cells. In this study, we investigated the effects of sinulariolide on hepatocellular carcinoma (HCC) cell growth and protein expression. Sinulariolide suppressed the proliferation and colony formation of HCC HA22T cells in a dose-dependent manner and induced both early and late apoptosis according to flow cytometry, Annexin V/PI stain and TUNEL/DAPI stain analyses. A mechanistic analysis demonstrated that sinulariolide-induced apoptosis was activated through a mitochondria-related pathway, showing up-regulation of Bax, Bad and AIF, and downregulation of Bcl-2, Bcl-xL, MCl-1 and p-Bad. Sinulariolide treatment led to loss of the mitochondrial membrane potential, release of mitochondrial cytochrome c to the cytosol, and activation of both caspase-9 and caspase-3. Sinulariolide-induced apoptosis was significantly blocked by the caspase inhibitors Z-VAD-FMK and Z-DEVD-FMK. The increased expression of cleaved PARP also suggested that caspase-independent apoptotic pathway was involved. In the western blotting; the elevation of ER chaperones GRP78; GRP94; and CALR; as well as up-regulations of PERK/eIF2α/ATF4/CHOP; and diminished cell death with pre-treatment of eIF2α phosphatase inhibitor; salubrinal; implicated the involvement of ER stress-mediated PERK/eIF2α/ATF4/CHOP apoptotic pathway following sinulariolide treatment in hepatoma cells. The current study suggested sinulariolide-induced hepatoma cell cytotoxicity involved multiple apoptotic signal pathways. This may implicate that sinulariolide is a potential compound for the treatment of hepatocellular carcinoma.

The effect of oleuropein from olive leaf (Olea europaea) extract on Ca2+ homeostasis, cytotoxicity, cell cycle distribution and ROS signaling in HepG2 human hepatoma cells

“…CHOP and ATF4 protein expressions are associated with ER stress (Cao et al, 2012;Chen et al, 2013). During ER stress, the levels of CHOP and ATF4 are elevated leading to apoptosis (Cao et al, 2012;Chen et al, 2013). However, the results show that oleuropein did not induce caspase-12 activation or increased CHOP and ATF4 protein levels in HepG2 cells.…”

“…Data are mean ± SEM of three separate experiments. CHOP and ATF4 protein expressions are associated with ER stress (Cao et al, 2012;Chen et al, 2013). During ER stress, the levels of CHOP and ATF4 are elevated leading to apoptosis (Cao et al, 2012;Chen et al, 2013).…”

Improvement and enhancement of antibladder carcinoma cell effects of heteronemin by the nanosized hyaluronan aggregation

“…9,29 Several crucial ER stress-related pathways, including the PERK/eIF2α/ATF4/CHOP, ATF6/ CHOP, and IRE1α/CHOP, have been revealed to be able to induce apoptosis in cancer cells. 9,10,29 Our Western blot results showed that treatment with pure HET results in increases in p-PERK, p-eIF2α, ATF4, IRE1α, and CHOP and a decrease in eIF2α, suggesting that HET is capable of eliciting apoptosis via several ER stress-associated pathways in bladder carcinoma cells. HA nanoparticles-aggregated HET at 0.1 and 0.2 µg/mL further enhanced these crucial ER stress apoptotic proteins compared with pure HET, implicating that HA nanoparticle aggregation may strengthen ER stress-related apoptosis induced by pure HET against bladder carcinoma cells.…”

“…It has been found that several natural compounds from soft corals have cytotoxic and apoptosis-inducing effects against melanoma cells, hepatocellular carcinoma cells, and bladder cancer cells in our previous studies. 2,9,10 Heteronemin (HET) is a known sesterterpenoid extracted from the sponge Hippospongia sp. This natural marine compound was found to inhibit the enzyme protein farnesyltransferase and reduce the viability of leukemia cells, colon cancer cells, and breast cancer cells.…”

“…[28][29][30] Natural compounds extracted from marine soft corals have been shown to display apoptotic effects against tumor cells via the ER stress-related pathways. 9,10 The start of ER stress is usually a result from affecting ER homeostasis and interfering with proper protein folding, followed by imbalance between protein folding load and capacity, leading to eventual accumulation of unfolded/misfolded proteins in ER lumens that elicit unfolded protein response and ER-associated protein degradation. 9,29 Several crucial ER stress-related pathways, including the PERK/eIF2α/ATF4/CHOP, ATF6/ CHOP, and IRE1α/CHOP, have been revealed to be able to induce apoptosis in cancer cells.…”