The present study investigated the cellular toxicity of Adenium obesum plant extract on EJ138 bladder cancer cells under in vitro conditions. The cytotoxic effect of Adenium obesum on EJ138 cell viability was determined by MTT, NRU, and NO assays. The oxidative stress induction by Adenium obesum in EJ138 cells was measured by GSH and catalase tests. The induction of apoptosis by Adenium obesum in EJ138 cells was detected by the COMET assay. The biochemical analysis of Adenium obesum led to the isolation and identification of 53 bioactive compounds, some of which exhibit toxic properties. The biochemical analysis of Adenium obesum led to the isolation and identification of 53 bioactive compounds, some of which exhibit toxic properties. The plant contains various chemical compounds such as carbohydrates, cardiac glycosides, flavonoids, prenylated flavonoids, terpenoids, and more (9). Previous studies have demonstrated that Adenium obesum possesses antimicrobial, antioxidant, anticancer, antiviral, and immunomodulatory activities (9–11). Other studies have revealed its anti-malarial and anti-trypanosomal properties (12). The biochemical properties of Adenium obesum may be utilized as a novel drug for cancer treatment and prevention.
The MTT test, a cellular toxicity indicator, was used in the study to assess the impact of Adenium obesum plant extract on malignant cell survival, and the neutral red uptake assay was used to confirm the findings (26). Following treatment with Adenium obesum extract, the EJ138 cell viability was shown to decline in a concentration-dependent manner according to the MTT and neutral red tests. Cells of bladder cancer were destroyed by the extract, which was extremely poisonous. These experiments showed that Adenium obesum extracts at different doses (0.25, 0.5, and 1 microgram per milliliter) significantly decreased the survival rate of EJ138 cellsThe results align with the findings of Al-Mudhaffer et al., who observed a decrease in cell viability linked to the concentration of Adenium obesum extract when utilizing the MTT assay on MCF-7, HFB4, HEPG2, and HEK293 cell lines indicating a potential cytotoxic effect of the extract (27). However, differences in concentrations and results compared to our study may be attributed to factors such as the composition of the plant, extraction methods, and the specific cell lines used. The findings from the research conducted by Qasim Ali et al., examining the cytotoxic impact of the ethanol extract from various components of Adenium obesum on the MCF-7 cell line, align with our results, demonstrating a reduction in cancer cell viability dependent on concentration (28). Nevertheless, the concentrations employed in their study differed from ours, which could be due to factors akin to those mentioned in the preceding paragraph.
Based on the results of a study by Al-Shahri, which parallels our own, the cytotoxic impact of the ethanol extract from Adenium obesum leaves on the A549 lung cancer cell line was assessed. The findings indicated a decrease in cell survival rate with escalating concentration, aligning with our own results (29). Nonetheless, disparities in the concentrations utilized in Al-Shahri's study compared to ours have been previously noted.
The results of the study conducted by Abuelfarh et al., who investigated the cytotoxic effects of silver nanoparticles synthesized from Adenium obesum leaf extract on the MCF-7 cell line, are also consistent with our findings. The survival rate of cancer cells decreased in a concentration-dependent manner (30). However, the concentrations and IC50 values used in their study were different from ours, which may be due to the use of nanoparticles, the method of extract preparation, and the cell line used in the experiment.
Nitric oxide regulates molecular signaling and physiological and pathophysiological processes such as vascular functions, neural functions, and cytotoxic functions at high concentrations (31). Nitric oxide can induce mitochondrial apoptosis, leading to chromatin condensation, DNA fragmentation, and caspase activation (32). NO regulates apoptosis in various cells, but its effects depend on the NO amount. It has both inhibitory and inducible effects on apoptosis. Nitric oxide inhibits apoptosis in some cells, such as leukocytes, hepatocytes, trophoblasts, and endothelial cells (33). In the present study, nitrite, the main product of NO metabolism, was determined in EJ138 cells after treatment with Adenium obesum extract. The findings revealed that the extract led to a reduction in nitrite production, which was notably lower than the control. However, we noted a substantial decrease in NO levels in cells in a concentration-dependent fashion. Thus, our study exhibited a noteworthy decline in NO levels in cells in a concentration-dependent pattern.
In this study, methods such as GSH assay and catalase measurement were used to investigate the ability of Adenium obesum crude extract to induce oxidative stress in MCF-7 cells (22). Glutathione (GSH) participates in various cellular processes, such as differentiation, proliferation, and apoptosis, Its disruption is associated with numerous human conditions, including cancer. GSH deficiency, or a low ratio of glutathione disulfide (GSSG)/GSH contributes to cancer progression via oxidative stress, whereas elevated GSH levels enhance antioxidant capacity. GSH in cancer cells modulates carcinogenic mechanisms, responsiveness to cytotoxic agents, radiation, and cytokines, DNA synthesis and replication, and cell death. The main function of GSH is to detoxify antibiotics and some intracellular compounds (34, 35).
In the present study, EJ138 cells exhibited a significant increase in GSH levels at higher concentrations following treatment with Adenium obesum extract. To the best of our knowledge, this is the first report of GSH levels in EJ138 cells treated with Adenium obesum extract. Gibelli et al. reported that modulation of GSH levels did not affect the apoptosis rate. Therefore, it can be inferred that GSH depletion in apoptosis is not solely due to oxidative stress and that GSH reduction is not sufficient to induce apoptosis (36), which is consistent with our results.
Catalase converts hydrogen peroxide into water and oxygen (37). Catalase, a hemoprotein, protects cells from hydrogen peroxide damage. Nitric oxide (NO) inhibits hydrogen peroxide consumption, and catalase and hydrogen peroxide consume NO, leading to cellular imbalance (38). The findings of this research indicate that the crude extract of the Adenium obesum plant considerably enhances the catalase enzyme production in EJ138 cancer cells. This outcome aligns with the results obtained from the original investigations examining nitic oxide and glutathione levels, which are herein reported for the first time.
The impact of Adenium obesum crude extract on the induction of apoptosis in EJ138 cells was investigated using the comet assay. Apoptosis is started by caspases, which are particular cysteine proteases. Caspase-3 activation is a crucial stage in apoptosis and is important for cellular functions such as DNA fragmentation, chromatin compaction, cell bursting, and shrinkage (39, 40). According to the study's findings, Adenium obesum crude extract causes EJ138 cells to undergo apoptosis in a concentration-dependent manner. This is in contrast to Ghasem Ali et al.'s findings, which showed that in MCF-7 cells, apoptosis increased after 12 hours but decreased after 24 hours in a concentration-dependent manner (28). Our findings, however, are consistent with those of Abolfarh et al., who assessed the cytotoxic impact of silver nanoparticles made from Adenium obesum leaf extract on MCF-7 cells and discovered a concentration-dependent rise in apoptosis (30). The use of nanoparticles, variations in the processing of the crude extract, and the cell line employed are only a few examples of the elements that might be responsible for the study's varying amounts. In his study, Mr. Alshahri examined the cytotoxic effects of Adenium obesum ethanol leaf extract on A549 lung cancer cells. His results showed a concentration-dependent increase in apoptosis, which is consistent with our findings (29).