Isomahanine induces endoplasmic reticulum stress and simultaneously triggers p38 MAPK-mediated apoptosis and autophagy in multidrug-resistant human oral squamous cell carcinoma cells

Abstract

Advanced oral squamous cell carcinoma (OSCC) is typically aggressive and closely correlated with disease recurrence and poor survival. Multidrug resistance (MDR) is the most critical problem leading to therapeutic failure. Investigation of novel anticancer candidates targeting multidrug-resistant OSCC cells may provide a basis for developing effective strategies for OSCC treatment. In the present study, we investigated the cytotoxic mechanism of a carbazole alkaloid, namely isomahanine, in a multidrug‑resistant OSCC cell line CLS-354/DX. We demonstrated that CLS-354/DX cells overexpressing multidrug resistance-associated protein 1 (MRP1) were resistant to anticancer drugs cisplatin and camptothecin. Isomahanine effectively induced cytotoxicity against CLS-354/DX cells regardless of resistance. Apoptosis as determined by FITC‑Annexin V/PI staining and western blot analysis of cleaved caspase-3 and cleaved poly(ADP‑ribose) polymerase (PARP) was significantly induced in a time-dependent manner upon isomahanine treatment. Isomahanine-induced caspase‑dependent apoptosis was determined using z-VAD‑fmk. The effects on autophagy in isomahanine-treated cells were investigated via conversion of LC3B and degradation of p62/SQSTM1 (p62). Isomahanine obviously induced autophagic flux as shown by an increase in punctate GFP-LC3B and the LC3B-II/LC3B-I ratio with a concomitant decrease in p62 levels. Autophagy inhibitors 3-methyladenine (3-MA) and chloroquine (CQ) protected isomahanine-induced cell death, indicating the activation of autophagic cell death. Endoplasmic reticulum (ER) stress and MAPK activation were examined to elucidate the mechanism underlying cell death. The expression levels of PERK, CHOP and phosphorylated MAPK (p38, ERK1/2 and JNK1/2) were upregulated following isomahanine treatment. We found that p38 MAPK inhibitor (SB203580) significantly attenuated isomahanine-induced apoptosis and autophagic flux and this prevented cell death. Collectively, the present study demonstrated that isomahanine was able to induce ER stress and trigger p38 MAPK-mediated apoptosis and autophagic cell death in multidrug-resistant OSCC cells. The potential cytotoxic action of isomahanine may provide the development of anticancer candidates for treating multidrug-resistant cancer.

Autophagy induction enhances tetrandrine-induced apoptosis via the AMPK/mTOR pathway in human bladder cancer cells

“…Research has shown that cobalt chloride induced autophagy and apoptosis in glioma cells by the p53 pathway (26). Another study revealed that isomahanineinduced autophagy was mediated by p38 mitogen-activated protein kinase (MAPK) signaling pathway in oral squamous cell carcinoma cells (27). Among the various pathways, the AMPK pathway has attracted great attention.…”

AMPK‐mTOR‐ULK1 axis activation‐dependent autophagy promotes hydroxycamptothecin‐induced apoptosis in human bladder cancer cells

“…In glioma cells, autophagy is induced by cobalt chloride mainly through the p53 pathway (B.-C. Cheng et al, 2017). Another study found that drug-induced MAPK signaling pathway activation might initiate protective autophagy (Utaipan, Athipornchai, Suksamrarn, Chunsrivirot, & Chunglok, 2017). Among various autophagy-related signaling pathways, AMPK, a kinase sensing the energy or nutrient state of cells, has always been a hotspot (Hardie, 2007).…”

A trienone analog of curcumin, 1,7-bis(3-hydroxyphenyl)-1,4,6-heptatrien-3-one, possesses ROS- and caspase-mediated apoptosis in human oral squamous cell carcinoma cells in vitro

“…CLS-354/WT and CLS-354/DX cell lines were established from human OSCC cell line CLS-354 (CLS Cell line service, GmbH, Eppellheim, Germany) as has been described earlier [23,24]. A monkey kidney epithelial cell line (Vero) was purchased from CLS Cell line service (GmbH, Eppellheim, Germany).…”

“…After the 24 h-treatment, the spent media were removed, 200 μl of 0.5 mg/ml 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Invitrogen, Life Technologies, USA) solution was added to each well and incubated for 4 h. The formazan crystals were dissolved in DMSO. The absorbance (Abs) was detected at 560 nm and background at 670 nm was subtracted, using a microplate reader [23]. Cell viability was calculated as follows:…”

“…After staining, the stained cells were subjected to a flow cytometer (BD Biosciences 2 Laser FacsCalibur, USA) using FL-1 and FL-2. Percentages of Annexin V-FITC-and Annexin V-FITC/PI-positive cells were analyzed as apoptotic cells by CellQuest ™ Pro (BD Biosciences) [23].…”