Dulbecco’s modified Eagle’s medium (DMEM) was obtained from HyClone Laboratories (Logan, UT, USA). Fetal bovine serum (FBS) was provided by Hangzhou Sijiqing Biological Engineering Materials Co., Ltd. (Hangzhou, China). Antibodies against caspase-8 (13423-1-AP) and caspase-9 (10380-1-AP) were supplied from the Proteintech Group, Inc. (Chicago, IL, USA). Antibodies specific for Fas (sc-1023), Bcl-2 (sc-7382), Bax (sc-7480), extracellular signal-regulated kinase (ERK; sc-271270), phospho-ERK (p-ERK; sc-81492), p38 (sc-7149), phospho-p38 (p-p38; sc-101759), c-Jun NH2-terminal kinase (JNK; sc-1648), phospho-JNK (p-JNK; sc-135642) and β-actin (Sc-1616r) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). All other chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise specified.

Catha edulis Forsk (khat) purchased from Sana’a, Yemen was used in this study. Fresh khat leaves along with soft stems were collected during the summer period, weighed, washed with distilled water three times and allowed to dry for three days in a clean, dry room protected from sunlight. After drying, the plant was weighed, packed in a closed foil packet and stored at 4°C until use. Khat was extracted from the leaves as described previously (14,21,22). Briefly, dried khat leaves (100 g) were swiftly chopped into small (5 mm) pieces and dissolved in 100 ml of 95% ethanol, centrifuged at 5,000 rpm for 5 min and the supernatant then filtered with Whatman filter paper. Ethanol (100 ml) was added to the remaining leaves and the procedure was repeated. The ethanol khat extract was concentrated using a rotary evaporator (Labtech, Inc., Hopkinton, MA, USA) at 30°C with a rotation speed of 70 rpm until 70% of the ethanol solvent had evaporated. The resulting viscous solution was diluted with 100 ml of distilled water and then stirred at 1,000 rpm with for 1 h at room temperature. The filtrate was kept frozen at -70°C for 24 h and then dried by lyophilization (Lyophilization Technology, Inc., Ivyland, PA, USA). Typically, 100 g of dried leaves yielded 8 g of khat extract powder. As previously described, high performance liquid chromatography analysis confirmed that the alkaloids in the khat extract consisted of 80% cathine and 20% norephedrine; no cathinone was detected (14,21,22). Lyophilized khat extract was dissolved in Hank’s buffered salt solution (HBSS) without Ca²⁺, Mg²⁺ to a final concentration of 200 μg/ml and filter (0.2 μm) sterilized before being added to the cells.

L02 cells were cultured in DMEM containing 10% FBS and maintained at 37°C in a 5% CO₂ humidified incubator. The cells were grown to 60–70% confluence before the experiments were conducted. L02 cells were exposed to various concentrations of khat (10, 50 and 100 μg/ml) and incubated at 37°C, 5% CO₂ for up to 24 h. The cells not treated with khat served as the control group.

The number of viable cells was determined using the trypan blue exclusion assay as previously described by Ahmed et al (23). Briefly, the cells were seeded in 6-well plates to 60–70% confluence, incubated overnight, and then exposed to the indicated concentrations of khat for 4, 8, 16 and 24 h. Floating and adhering cells were collected and stained with 0.2% trypan blue for 5 min at room temperature before they were examined under a fluorescence microscope (Olympus, Tokyo, Japan). Following the internalization of the dye into the cells, the cells whose nuclei were stained blue were considered dead. The results are expressed as a percentage of the control.

Following treatment with khat, the cells were washed with 0.1 mol/l PBS (pH 7.2) and resuspended in the same buffer. The cells contained in 100 μl of cell suspension (1×10⁶ cells/ml) were incubated with 1 ml Hoechst 33258 (1 mg/ml in distilled water) for 10 min. Apoptotic cells were evaluated under a fluorescence microscope.

Apoptosis was assessed by measuring the membrane redistribution of phosphatidylserine using an Annexin V-FITC apoptosis detection kit according to the manufacturer’s instructions. After drug treatment, the cells were collected, washed twice with PBS and resuspended in 500 ml of staining solution containing FITC-conjugated Annexin V antibody (5 ml) and PI (5 ml of 250 mg/ml stock). After incubating the cells in ice for 30 min, the cells were analyzed by flow cytometry (FACSCalibur; Becton-Dickinson, San Jose, CA, USA). Basal apoptosis and necrosis were also determined in the untreated cells. The percentage of cells undergoing apoptosis was determined by three independent experiments.

The cells were fixed in 0.1 M Na-cacodylate buffer, pH 7.4 containing 2% glutaraldehyde. The samples were rinsed with buffer and post-fixed in 1% osmium tetroxide. The specimens were dehydrated using graded ethanol and embedded in epoxy resin. Ultra-thin sections were double-stained with uranyl acetate and lead citrate. Specimens were examined under an electron microscope (Jeol 1230; Jeol Ltd., Tokyo, Japan), and the micrographs were processed using an Agfa Arcus II scanner and Adobe Photoshop 7.0.1 software.

The cells were harvested and lysed on ice for 30 min in modified radioimmunoprecipitation assay buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, 50 mM NaF, 1 mM Na₃VO₄, 5 mM sodium pyrophosphate and a protease inhibitor tablet). The cell lysates were centrifuged at 14,000 × g for 15 min, and the supernatant was recovered. The total protein concentration was determined using the BCA Protein Assay Reagent (Pierce, Rockford, IL, USA). The lysates were denatured by boiling in SDS sample buffer. The proteins were separated on SDS/PAGE 4–20% SDS-polyacrylamide gels and then transferred onto polyvinylidene difluoride membranes (Amersham Pharmacia Biotech, Inc., Piscataway, NJ, USA) using a semi-dry transfer cell (Bio-Rad). After blocking the membranes, the membranes were probed with the appropriate primary antibodies. Membrane-bound primary antibodies were detected using secondary antibodies that were conjugated to horseradish peroxidase. Western blots were visualized using enhanced chemiluminescence detection reagents (Sigma) according to the manufacturer’s instructions. The quantification of protein bands was performed via scanning using the Bio-Rad Gel Doc™ XR and ChemiDoc™ XRS systems and analyzed using Quantity One 1-D analysis software version 4.6.3.

The generation of ROS was measured by flow cytometric analysis using dichlorofluorescein-diacetate (DCFH-DA) as a substrate (24). Briefly, following treatment with khat at a concentration of 100 μg/ml for 4, 8, 16 and 24 h, the cells were harvested, washed twice with cold PBS, and suspended in PBS (1×10⁶ cells/ml). The cell suspension (500 μl) was placed in a tube, loaded with DCFH-DA to a final concentration of 5 μM, and incubated for 30 min at 37°C. ROS production was assessed based on the DCF fluorescence intensity from 10,000 cells that was obtained by flow cytometry.

Data are expressed as the means ± SD and analyzed using SPSS 10.0 statistical software (SPSS Inc., Chicago, IL, USA). The one-way ANOVA procedure followed by LSD post hoc tests was used to determine the significance of differences among groups (P<0.01 and P<0.05).

The inhibitory effect of khat was evaluated by measuring the viability of L02 cells. At the indicated concentrations of 10, 50 and 100 μg/ml, treatment with khat inhibited the growth of the L02 cells in a time-dependent manner, compared to the control group (P<0.05, P<0.01). In the current study, we also found that khat significantly decreased the viability of the L02 cells at the concentration of 100 μg/ml following treatment for 8 h (Fig. 1). Therefore, the concentration of 100 μg/ml of khat was selected for the remaining experiments.

To investigate whether the reduction in cell viability was due to apoptosis, cytometric analysis and morphological observation of the cells were performed. We double-stained the cells with Annexin V and PI and analyzed the results using flow cytometry. Following serum starvation for 24 h, the L02 cells were exposed to 100 μg/ml of khat for 4, 8, 16 and 24 h, yielding apoptotic rates of 4.7±1.4, 21.7±3.1, 33.4±4.4 and 39.5±4.7%, respectively (Fig. 2). For a further assessment of apoptosis, we analyzed chromatin condensation and apoptotic bodies. the L02 cells were treated with the indicated concentrations of khat for 8 h. The vehicle-treated cells exhibited regular and round-shaped nuclei. Bu contrast, the majority of cells treated with khat (100 μg/ml) were stained with Hoechst 33258, which is bound to chromatin. The treated cells exhibited primary characteristics of apoptotic cells, namely the condensation and fragmentation of nuclei. When observed under an electron microscope, the khat-treated cells showed chromatin condensation and nuclear shrinkage consistent with apoptosis (Fig. 3).

We assessed the activated caspase-8 and -9 levels in the L02 cells before and after treatment with khat by western blot analysis. Khat exposure significantly activated caspase-8 and -9 in a time-dependent manner (Fig. 4). After incubation with khat at the concentration of 100 μg/ml for different periods of time (0, 4, 8, 16 and 24 h), the intensity of the bands corresponding to procaspase-8 and -9 decreased. However, under the same experimental conditions, the expression level of Fas remained stable following treatment with khat, which suggested that caspase activation not stimulated by Fas but by other death signaling pathways.

In addition, Bax protein expression levels in L02 cells treated with khat demonstrated a marked increase compared to the control protein expression levels. Conversely, the L02 cells treated with khat showed a marked decrease in Bcl-2 protein levels.

In order to clarify the involvement of MAPKs in the khat-induced cell death of L02 cells, the levels of phosphorylated MAPKs (p38, ERK and JNK) were investigated by western blot analysis (Fig. 5). The phosphorylation of JNK and ERK was significantly increased following treatment with khat at the concentration of 100 μg/ml. However, the phosphorylation levels of p38 were not altered under the same conditions. The total levels of each MAPK were not altered during the incubation period. Therefore, the ERK and JNK signaling pathways may be involved in the response of L02 cells to khat.

To further evaluate the possible roles of MAPKs in khat-induced apoptosis, we examined cell viability and apoptotic rates in the presence or absence of specific inhibitors of JNK (SP600125) and ERK1/2 (PD98059). SP600125 and PD98059 prominently reversed the khat (100 μg/ml)-induced cell death observed in the typan blue exclusion assay and flow cytometric analysis (Fig. 6), which was concomitant with the block of Bcl-2 reduction as well as caspase-8 and -9 activation (data not shown). These results suggest that both JNK and ERK are involved in the apoptotic progression caused by khat in L02 cells.

ROS have been implicated as potential modulators of apoptosis. In the L02 cells, treatment with khat caused a dose-dependent accumulation of intracellular ROS (Fig. 7a).

To further determine the role of khat-induced ROS generation in the activation of JNK and ERK, the L02 cells were pre-treated with 5 mM N-acetyl-L-cysteine (NAC), a specific inhibitor of ROS, for 2 h and then incubated with 100 μg/ml of khat for 8 h.

NAC prominently reversed khat (100 μg/ml)-induced cell death, which was observed using the trypan blue exclusion assay (Fig. 7b). NAC markedly decreased the activity of ERK and JNK induced by khat (Fig. 7c). However, NAC had no effect on total ERK1/2 and JNK expression. These results suggest that khat-induced ROS accumulation may contribute to the activation of ERK and JNK in L02 cells.