The bioactive carbazole alkaloid, i.e., isomahanine, was isolated and purified from the CH₂Cl₂ extract of M. koenigii leaves by repeated silica gel and Sephadex LH-20 column chromatography. The spectroscopic data of isomahanine were consistent with the reported values (28). The purified carbazole alkaloid was dissolved in dimethyl sulfoxide (DMSO) prior to conducting experiments.

cis-diamminedichloroplatinum(II) (cisplatin), camptothecin, DMSO, 3-MA, 4-phenylbutyric acid (4-PBA), chloroquine (CQ) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). Benzyloxycarbonyl-ValAla-Asp (OMe) and fluoromethylketone (z-VAD-fmk) were purchased from InvivoGen (San Diego, CA, USA). Luminita™ Chemiluminescent HRP substrate was purchased from EMD Millipore (Billerica, MA, USA). All primary antibodies (rabbit), HRP-conjugated secondary antibodies (anti-rabbit) and MAPK inhibitors (U0126, SB203580 and SP600125) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Human OSCC CLS-354 cells (CLS Cell Lines Service GmbH, Eppelheim, Germany) at passage no. 30–40 were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (Biochrom GmbH, Berlin, Germany), 1% penicillin/streptomycin and 2 mM stable L-glutamine (PAA Laboratories GmbH, Pasching, Austria). The cancer cell line was maintained in an atmosphere of 95% humidity and 5% CO₂ at 37̊C. CLS-354 cells, as parental cells, were oral epithelial cancer cells exhibiting epithelial-like shape, namely CLS-354/WT (Fig. 1A, left). The mesenchymal phenotype with the elongated shape was isolated by differential trypsinization as previously described (29). The cell line was named as CLS-354/DX (Fig. 1A, right).

Cancer cells (8×10⁵) were seeded onto a 60-mm tissue culture dish and allowed to grow for 36 h. Cells (2×10⁵ cells/sample) were collected, washed with phosphate-buffered saline (PBS), and subjected to MDR assay using eFluxx-ID^® Green MDR assay kit (Enzo Life Sciences, Inc., Farmingdale, NY, USA) according to the manufacturer's instructions. The cells were analyzed immediately by flow cytometry. The mean fluorescence intensity (MFI) values were analyzed using CellQuest™ Pro (BD Biosciences, San Jose, CA, USA). The MDR activity factor (MAF) for each transporter was calculated according to the formula: MAF = 100 × (MFI_{with inhibitor} - MFI control)/MFI_{with inhibitor}.

Cells (1.5×10⁴ cells/well) were seeded into 96-well plates and grown for 48 h. Cells were treated with various concentrations of cisplatin (10–25 µM) and camptothecin (0.3–15 µM) or isomahanine (10–25 µM) for 24 h or for various time-points. After treatment, cell viability was assessed by MTT assay. The half-maximal inhibitory concentration (IC₅₀) was calculated from concentration-response curves following a 24-h treatment. The resistance index (RI) was defined as the ratio of the IC₅₀ value of the resistant cell line to the IC₅₀ value of the parental cell line of each drug.

Cells (4×10⁵ cells/well seeded on a 6-well plate) were incubated with isomahanine for 0, 6, 12 and 24 h. The cells were harvested, washed with PBS, and stained with Annexin V-FITC and Annexin V-FITC/PI according to the manufacturer's instructions (Roche Diagnostics Deutschland GmbH, Mannheim, Germany). Fluorescence intensity (FL-1 and FL-2) was immediately determined by flow cytometry. For each measurement, at least 15,000 cells were counted. The percentages of Annexin V-FITC- and Annexin V-FITC/PI-positive cells were analyzed as apoptotic cells using CellQuest™ Pro (BD Biosciences).

CLS-354/DX cells (2×10⁴ cells/well seeded on a 24-well plate) were transduced with BacMam LC3B-GFP viral particles (MIO=30 plaque-forming units/cell), according to the manufacturer's instructions of the Premo™ Autophagy Sensor kit (Invitrogen, Carlsbad, CA, USA). After 24 h, the cells were treated with 20 µM isomahanine or 50 µM CQ. After a 16-h incubation, green fluorescent signals were monitored and imaged using a Zeiss Axio Vert.A1 inverted microscope (magnification, ×40). Cells with >5 GFP-LC3B puncta were considered autophagy-positive. The percentage of cells with punctate GFP-LC3B/total GFP-LC3B-positive cells was determined.

After isomahanine treatment, the cells were harvested and lysed in lysis buffer containing protease inhibitors. Protein samples (50 µg) were then prepared in Laemmli sample buffer. The proteins were separated on 8–12.5% SDS-PAGE gels and transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with 5% non-fat dry milk, washed with a mixture of Tris-buffered saline and Tween-20, and probed with primary antibodies (1:250-1:1,000) at 4̊C overnight. The blot was then probed with the HRP-conjugated secondary antibody (1:5,000) at room temperature for 1 h. The proteins were visualized using the chemiluminescent detection system.

All results are expressed as the mean ± SEM. Data were obtained from at least 3 independent experiments. Significant differences among the different groups were considered at a p-value <0.05, using Student's t-test or one-way ANOVA. The statistical analyses were performed using GraphPad Prism 6 (GraphPad Software, Inc., La Jolla, CA, USA).

The multidrug-resistant OSCC cell line was spontaneously generated from the parental human OSCC cell line CLS-354 (passage no. 27), which is epithelial-like in shape (CLS-354/WT) (Fig. 1A, left). After being cultured for >10 passages, some cells became elongated and mesenchymal-like. These mesenchymal-like cells were isolated by differential adhesion (29) and named CLS-354/DX (Fig. 1A, right). These two cell lines had similar growth characteristics. The population doubling times of CLS-354/WT and CLS-354/DX were 31.25±0.64 and 29.17±1.20 h, respectively, which were not significantly different.

To investigate the drug-resistance profiling, the activity of a particular multidrug-resistant transporter [P-gp, MRP1/2 and breast cancer resistance protein (BCRP)] was assessed using eFluxx-ID^® Green probe. The probe is a hydrophilic fluorescent dye, which is trapped within the cells unless actively pumped out by these transporters and is detectable by flow cytometry. As shown in Fig. 1B, CLS-354/WT cells exhibited an equal increase of fluorescent signals in the presence of a P-gp inhibitor (verapamil) and an MRP1/2 inhibitor (MK-571) in comparison to the control (Fig. 1B, left). We clearly found a higher fluorescent signal from the CLS-354/DX cells in the presence of verapamil and MK-571 when compared with the control (Fig. 1B, right). This result indicated the essential roles of P-gp and MRP1/2 in these cells. The comparative analysis of MDR between the CLS-354/WT and CLS-354/DX cell lines was estalished by calculating the MAF values. The MAF values of P-gp and MRP1/2 in CLS-354/DX cells were 66.48±0.73 and 86.10±0.58, respectively, which were significantly greater than those of the CLS-354/WT cells (Fig. 1C). As a result, the highest MAF value of MRP1/2 was observed in the CLS-354/DX cells (Fig. 1C). Hence, the expression levels of the MRP1 and MRP2 proteins in these cell lines were examined by western blotting. We found that the MRP1 expression level in the CLS-354/DX cells was significantly greater than that in the CLS-354/WT cell line (Fig. 1D), while MRP2 was undetectable in both cell lines. These results indicated that the CLS-354/DX cells developed the multidrug-resistant phenotype via MRP1 overexpression.

The responsiveness to cisplatin, a platinum-based anticancer drug and camptothecin, an anticancer alkaloid (Fig. 2A, left and middle), was investigated in the CLS-354/WT and CLS-354/DX cells following 24-h treatments using the cell viability assay. The results showed that the response of CLS-354/DX cells to cisplatin and camptothecin was significantly lower than that of CLS-354/WT cells (Fig. 2B, left and middle). The IC₅₀ values for CLS-354/DX cell line to cisplatin and camptothecin were 33.23±0.96 and >15 µM, respectively, which were 2 times and >65 times, respectively, greater than those of the CLS-354/WT cells as indicated by the RI (Fig. 2C). These data strongly support the contribution of MDR in CLS-354/DX cells. Notably, isomahanine (Fig. 2A, right) at a concentration starting from 20 µM effectively suppressed the viability of CLS-354/DX cells without relevance of resistance (Fig. 2B, right). The IC₅₀ values observed in both cell lines were not significantly different, with an RI value of 1.1 (Fig. 2C).

The mechanism of cell death was further examined in the CLS-354/DX cells. Cell morphology was preliminarily observed in a time-course study following isomahanine treatment (20 µM) for 0, 6, 12 and 24 h. As shown in Fig. 3A, the cells after exposure to isomahanine for 6 h presented small cytoplasmic vacuoles. After 12 h, the cells detached and shrank in size. Eventually, several cells became round in shape and cell death followed after 24-h treatment. Hence, apoptosis was further confirmed by flow cytometric analysis as well as western blot analysis of caspase-3 and poly(ADP-ribose) polymerase (PARP). Isomahanine time-dependently induced early apoptotic and late apoptotic cells to 5.09 and 15.55%, respectively (Fig. 3B, upper panel). Total apoptosis significantly increased in a time-dependent manner, while the maximal induction was increased to 20% following the 24-h treatment (Fig. 3B, lower panel). Isomahanine activated cleavage of caspase-3 and PARP in a time-dependent manner, and the greatest level of increase was observed upon 24-h treatment (Fig. 3C). To confirm caspase-dependent apoptotic death, the cells were treated with isomahanine along with z-VAD-fmk, a pan-caspase inhibitor. The inhibition of caspase activity significantly rescued isomahanine-induced cell death (Fig. 3D). Thus, isomahanine induced apoptotic death via a caspase-dependent pathway.

We hypothesized that autophagy may be involved in cell death upon the morphological observations. We determined autophagic induction by analysis of GFP-LC3B puncta. Cells treated with isomahanine displayed a punctate pattern of GFP-LC3B localized in the perinuclear region, indicating autophagic induction. Isomahanine-induced GFP-LC3B puncta formation by 50%, which was comparable to that of CQ (~60%), a positive control (Fig. 4A). We further performed western blotting of microtubule-associated protein 1B-light chain 3 (LC3B-II), which is an autophagosomal marker. Isomahanine induced the expression of LC3B-II in a time-dependent manner, and the expression of LC3B-II/LC3B-I ratio significantly increased after 12-h treatment (Fig. 4B). Thus, isomahanine induced autophagy in CLS-354/DX cells.

An increase in the number of autophagosomes may be due to activation or inhibition of autophagic flux. During autophagy, the cellular level of the p62 protein is typically degraded, and its decreased level serves as an indicator of autophagic flux (14). We further examined the expression of p62 with isomahanine treatment. Isomahanine markedly decreased the expression level of p62 in contrast with LC3B-II after 24 h, indicating autophagic flux (Fig. 4C). We also excluded the effect of proteasomal degradation activity on the p62 protein using MG-132, a proteasome inhibitor. The result revealed that isomahanine decreased the level of p62 as usual, while MG-132 did not inhibit its decrease (Fig. 4D). To assess the contribution of autophagy to cell death, the effect of autophagy inhibitors on cell viability was determined. Results revealed that the autophagy inhibitors 3-MA and CQ significantly inhibited isomahanine-induced cell death in the CLS-354/DX cells (Fig. 4E-F). Collectively, these findings suggest that autophagic flux induced by isomahanine was involved in the cytotoxic effects of the CLS-354/DX cells.

To further elucidate the cell-death mechanisms induced by isomahanine, we examined the effect of this compound on the activation of ER stress and MAPK phosphorylation. As shown in Fig. 5A, isomahanine activated the phosphorylation of protein kinase PNA (PKR)-like ER kinase (PERK), an ER stress sensor, in a time-dependent manner. Concomitantly, the compound induced the expression of the transcription factor CHOP, which is a marker of UPR (Fig. 5A). Activation of the MAPK family members, p38, ERK1/2 and JNK1/2 was also determined. The phosphorylation of p38, ERK1/2 and JNK1/2 was markedly upregulated after exposure to isomahanine for 6 h. At 12 h of treatment, the phosphorylation of p38 and ERK1/2 was strongly activated, while their activation slightly decreased after 24 h (Fig. 5A). JNK1/2 was activated to a maximal level at 6 h and decreased after 12 h (Fig. 5A). Next, we applied 4-PBA, an inhibitor of ER stress, to demonstrate the relation between ER stress and MAPK pathway. As shown in Fig. 5B, the activated PERK and CHOP proteins were significantly decreased in the presence of 4-PBA. ER stress inhibition protected against isomahanine-activated p38 MAPK phosphorylation. Meanwhile, the activation of ERK1/2 and JNK1/2 were concomitantly increased by 4-PBA (Fig. 5B). Collectively, these results suggest that ER stress mediated the activation of p38 MAPK, but not that of ERK1/2 and JNK1/2.

Activation of MAPK in response to ER stress can promote cell death by apoptosis and autophagy. To study the role of MAPK in apoptosis- and autophagy-induced cell death, we assessed the cell viability with isomahanine treatment in the presence of inhibitors of p38 MAPK (SB203580), ERK (U0126) and JNK (SP600125). As shown in Fig. 6A, SB203580 and U0126 significantly suppressed isomahanine-induced cell death, implying that p38 MAPK and ERK participate in cell death. The function of p38 MAPK and ERK in the induction of apoptosis and autophagy was further examined using inhibitors SB203580 and U0126. Isomahanine activated caspase-3, LC3B-II and CHOP and concomitantly decreased the expression level of p62 as usual. SB203580 inhibited MAPKAPK-2, a downstream effector of p38 MAPK (Fig. 6B). The blockade of p38 MAPK completely inhibited isomahanine-activated caspase-3, LC3B-II and p62, but SB203580 did not affect CHOP expression (Fig. 6C). U0126 markedly inhibited ERK phosphorylation (Fig. 6B), and this attenuated only isomahanine-activated caspase-3 (Fig. 6C). These data indicated that p38 MAPK was an important cytotoxic mediator mediating both apoptosis and autophagic flux in response to isomahanine. Meanwhile, the activation of ERK, in part, regulated apoptosis under the tested conditions. As CHOP was not affected by MAPK inhibition, ER stress may be an upstream regulator involved in this activation. The overall cytotoxic mechanism of isomahanine against CLS-354/DX cells is illustrated in Fig. 6D.