Introduction: Pancreatic cancer is the fourth leading cause of cancer-related deaths worldwide. 5-Fluorouracil is one of the commonly used chemotherapeutic drugs. The plant Artemisia absinthium has attracted attention as a potential herbal anticancer agent. This study investigated the effect of the methanolic extract of this plant on the expression of miR-34a and the apoptotic genes BAX, Caspase-3, and Caspase-9 in AsPC-1 pancreatic cancer cells.

Aims: This study aimed to evaluate the cytotoxic and pro-apoptotic effects of Artemisia absinthium methanol extract on pancreatic cancer cells. The research specifically investigated the molecular mechanism by analyzing expression changes in the tumor suppressor miR-34a and key apoptotic genes BAX, Caspase-3, and Caspase-9 to elucidate the extract's anti-cancer mode of action.

Materials and methods: To evaluate the cytotoxic effects of the plant extract on cancer cells, an MTT assay was performed to determine the viability and survival rate of the cells following treatment with various concentrations of the extract, the chemotherapeutic drug fluorouracil (5-FU), and the combined treatment of the extract and the drug. This assay measures cellular metabolic activity and allows quantification of live and dead cells after exposure to different treatments. Based on the obtained results, the IC₅₀ value for each treatment was calculated, representing the concentration at which 50% of the cells were inhibited or killed.

After determining the IC₅₀ value, cells were treated with concentrations equal to, lower, and higher than the IC₅₀ to further investigate the cytotoxic effects and the mode of cell death induced by the treatments. To distinguish between apoptotic and necrotic cell death, the Annexin V-FITC/PI assay was employed. This assay detects phosphatidylserine externalization on the cell membrane and enables differentiation between live, early apoptotic, late apoptotic, and necrotic cells.



In addition to the morphological and physiological assessments, molecular analyses were conducted to examine the expression levels of key apoptosis-related genes, including Caspase-3, Caspase-9, and BAX, as well as the regulatory microRNA miR-34a. Gene expression analysis was performed using Real-time PCR (qPCR).



Results: The results of the MTT assay demonstrated that the proliferation of AsPC-1 pancreatic cancer cells was inhibited by treatment with the extract of Artemisia absinthium and the chemotherapeutic drug fluorouracil (5-FU) in a concentration-dependent manner. As the concentration of each treatment increased, cell viability significantly decreased, indicating a marked cytotoxic effect of both the plant extract and the drug. Moreover, a possible synergistic effect between the extract and fluorouracil in suppressing. To determine the mode of cell death induced by these treatments, the Annexin V-FITC/PI assay was performed. The results revealed that a considerable proportion of treated cells underwent programmed cell death (apoptosis), while the percentage of necrotic cells remained relatively low. These findings suggest that the observed reduction in cell viability is mainly mediated through the activation of apoptotic pathways rather than necrosis. Furthermore, Real-time PCR analysis showed a significant upregulation in the expression of the regulatory microRNA miR-34a and the apoptosis-related genes Caspase-3, Caspase-9, and BAX in the treated groups compared to the control group.

Discussion: The obtained results suggest that Artemisia absinthium extract exerts its cytotoxic effect primarily through the induction of apoptosis rather than necrosis in AsPC-1 pancreatic cancer cells. The observed upregulation of miR-34a, Caspase-3, Caspase-9, and BAX implies activation of intrinsic apoptotic pathways. These findings are consistent with previous studies reporting pro-apoptotic properties of A. absinthium and other Artemisia species. Therefore, the extract may enhance the therapeutic response of pancreatic cancer cells when combined with conventional chemotherapeutic agents such as fluorouracil.

Conclusion: In conclusion, Artemisia absinthium extract demonstrated strong antiproliferative and apoptosis-inducing effects on AsPC-1 cancer cells in a dose-dependent manner. Its combination with fluorouracil produced a synergistic cytotoxic impact, significantly enhancing cell death through apoptotic signaling. The molecular findings support the potential of this extract as a complementary therapeutic agent. Further studies are recommended to explore its mechanisms and evaluate its efficacy in in vivo models of pancreatic cancer.