Involvement of ER stress and activation of apoptotic pathways in fisetin induced cytotoxicity in human melanoma

Abstract

The prognosis of malignant melanoma remains poor in spite of recent advances in therapeutic strategies for the deadly disease. Fisetin, a dietary flavonoid is currently being investigated for its growth inhibitory properties in various cancer models. We previously showed that fisetin inhibited melanoma growth in vitro and in vivo. Here, we evaluated the molecular basis of fisetin induced cytoxicity in metastatic human melanoma cells. Fisetin treatment induced endoplasmic reticulum (ER) stress in highly aggressive A375 and 451Lu human melanoma cells, as revealed by up- regulation of ER stress markers including IRE1α, XBP1s, ATF4 and GRP78. Time course analysis indicated that the ER stress was associated with activation of the extrinsic and intrinsic apoptotic pathways. Fisetin treated 2-D melanoma cultures displayed autophagic response concomitant with induction of apoptosis. Prolonged treatment (16 days) with fisetin in a 3-D reconstituted melanoma model resulted in inhibition of melanoma progression with significant apoptosis, as evidenced by increased staining of cleaved Caspase-3 in the treated constructs. However, no difference in the expression of autophagic marker LC-3 was noted between treated and control groups. Fisetin treatment to 2-D melanoma cultures resulted in phosphorylation and activation of the multifunctional AMPK-activated protein kinase (AMPK) involved in the regulation of diverse cellular processes, including autophagy and apoptosis. Silencing of AMPK failed to prevent cell death indicating that fisetin induced cytotoxicity is mediated through both AMPK-dependent and -independent mechanisms. Taken together, our studies confirm apoptosis as the primary mechanism through which fisetin inhibits melanoma cell growth and that activation of both extrinsic and intrinsic pathways contributes to fisetin induced cytotoxicity.

Fisetin Induces Apoptosis of HSC3 Human Oral Cancer Cells Through Endoplasmic Reticulum Stress and Dysfunction of Mitochondria-mediated Signaling Pathways

“…It was reported that chemotherapeutic agents induced necrotic or apoptotic cell death in cancer cells via the generation of ROS (41). But another report showed that fisetin treatment of A375.S2 melanoma cells resulted in a significant decrease in ROS level (28), while another reported that fisetin stimulated generation of ROS in U266 multiple myeloma cells and induced apoptosis through activation of AMP-activated protein kinase pathways in a ROS-dependent manner (42). Thus, based on these observations, fisetininduced modulation of ROS may be cell type-dependent.…”

“…Numerous reports have shown that fisetin induced cytotoxic effects on many human cancer cell lines through cell-cycle arrest and apoptosis (20,(22)(23)(24)(25)(26)(27)(28)(29), however, there is no report demonstrating that fisetin induces apoptosis of human oral cancer cells, therefore, we investigated the cytotoxic effects of fisetin on HSC3 human oral cancer cells in vitro. It is well documented that the best strategy for anticancer drugs is to induce cancer cell apoptosis.…”

“…Fisetin (3,7,3',4'-tetrahydroxyflavone), a naturally-occurring flavonoid, is widely distributed among edible vegetables and fruits such as apple, cucumber, grapes, kiwis, onion, persimmon and strawberry, the highest level of fisetin (160 μg/g wet food) being found in strawberries (14). Numerous studies have shown that fisetin has pharmacological activities such as antioxidant (15), anti-inflammatory (16), anti-invasive (17,18), anti-angiogenesis (19,20), anti-proliferation (21,22) and anticancer effects in several cancer types, such as prostate cancer (22,23), lung adenocarcinoma (20,24), pancreatic cancer (25), colon cancer (26), cervical cancer (27), melanoma (28) and acute monocytic leukemia cells (29). Recently, it was reported that combination treatment of fisetin and sorafenib effectively inhibited B-Raf proto-oncogene serine/threonine-protein kinase (BRAF)-mutated melanoma cell growth, induced apoptosis, and down-regulated mitogenactivated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) signaling pathways in vitro and in vivo.…”

Exploring the molecular targets of dietary flavonoid fisetin in cancer

“…In addition to its anti-oxidant activity, we [3,5,26–31] and others [4,7] have extensively shown the potential of fisetin in affecting signaling pathways that control cell survival, growth and proliferation both in vitro and in vivo . Haddad et al [7] observed a decrease in proliferation with concomitant induction of apoptosis in prostate cancer cells upon fisetin treatment.…”

“…In addition, the sensitivity of cancer cells to apoptosis in vitro may be a predictor of their sensitivity to these drugs in vivo . We [3,5,28] and others [4,6,34,42,66] have shown that fisetin has significant pro-apoptotic activities against cancer cells. A structure-activity relationship study of 22 flavonoids showed that at least two hydroxylations in positions 3, 5, and 7 of the A ring were needed to induce apoptosis, whereas hydroxylation in 3' and/or 4' of the B ring enhanced proapoptotic activity [70].…”