## 4 Discussion  
The present study was based on the hypothesis that Khat is cytotoxic to NOFs and SSC4 cells. It was also hypothesized that Khat would stimulate positive expression of \(\alpha\)-SMA in fibroblasts. Interestingly, the study revealed the cytotoxicity of Khat for both NOFs and SSC4 cells, and increased cell death occurred as compared to the negative control. Moreover, fibroblasts also stained positive for \(\alpha\)-SMA after Khat exposure. Therefore, both postulated hypotheses were accepted. For cytotoxicity assessment, MTT assay was employed, as the technique was convenient, rapid, and reproducible [17]. Similarly, immunofluorescence was used to determine \(\alpha\)-SMA positive staining in fibroblast cells, as it showed high sensitivity and specificity over other techniques [18]. Khat was studied in relation to its damaging effects on oral mucosal keratinocytes, but its potential influence on the oral fibroblasts and cancer cell line was not studied. Multiple studies stated that Khat chewing is associated with oral hyperplasia, hyperkeratinization, and oral cancers [19]. The results of the present study showed that Khat caused cell death of both NOFs and SCC4 cells. The exact mechanism for Khat-induced cell death is not known. However, the available literature focused on the presence of alkaloids (cathine and other phytochemicals) as a cause of its cytotoxicity and suggests that Khat could induce cell death even in short tissue exposure [6]. In addition, Khat also triggers intracellular generation of free radical, i.e., reactive oxygen species (ROS) and Glutathione (GSH) depletion, which initiates programmed cell death (apoptosis) by causing injury to the DNA [20]. Furthermore, in the present study, it was also observed that higher concentrations (25 mg/mL) of Khat displayed variable outcomes of cytotoxicity, not showing significant cell death of NOF 316 cells, as compared to the other tested cells. Interestingly, Khat showed dose-dependent cell death of NOF 18, NOF 26, and SCC4 cells, indicating higher toxicity at higher doses. In contrast, NOF 316 and NOF 319 did not exhibit dose-dependent cytotoxicity due to Khat. A possible explanation for this might be derived from the fact that Khat (25 mg/mL) used in the present study was in the frozen from and might have lost some of its activity [21]. Moreover, the variations in backgrounds and oral habits of the patients from which NOFs were obtained, could have resulted in such outcomes [22]. Similar findings were also reported in studies by [PERSON] and [PERSON] [23] and [PERSON] et al. [24]. Furthermore, it was reported that Khat caused genetic material damage and its genotoxic effect on normal keratinocytes and fibroblasts increased with higher dose [25]. Their findings supported the outcomes of the present study. Therefore, clinically speaking, oral lesions including mucosal keratosis,  
Figure 11: SMA expression in NOF 316 after incubation with 40 mg/mL of TGF\(\beta\) (magnification \(\times\)100, Fluorescence at 1.6 s, DAPI at 0.100 s). pigmentation, and leukoplakia were not only induced by Khat, but could also progress and differentiate due to increased frequency and the extent of Khat chewing habit. Hence, patients should be educated that the Khat chewing increases the risk and incidence of oral lesions and a cessation of the habit should be encouraged. The present study reports a positive expression for \(\alpha\)-SMA staining in fibroblast, indicating a phenotypic change. SMA is a well appreciated marker for MFs differentiation in tumor stroma, particularly in relation to OSCC [26]. MFs display intermediate physical characteristics between fibroblasts and smooth muscle cells [27]. Various laboratory-based studies demonstrate the role of cancer cell line in acquisition of fibroblast transformation in MFs, by secreting various extracellular matrix proteins, matrix-degrading enzymes, and growth factors [28]. These MFs modulate the growth, adhesion, migration, invasion, and differentiation of cancer cells.