Kazi Rejvee Ahmed ¹ Ji Soo Myong ^1* Md Ataur Rahman ² Md. Maharub Hossain Fahim ^1* Min Choi ^1* Muntajin Rahman ¹ JinWon Choi ¹ Kiryang Kim ^1* Seungjoon Moon ^1* Mohammed DALLI ³ Rony Abdi Syahputra ⁴ Moon Nyeo Park ¹ Sang-Won Shin ^5* Abdel Halim Harrath ⁶ Bonglee Kim ^1* Hwa-Seung Yoo ^7*

• ¹ Kyung Hee University, Seoul, Republic of Korea
• ² Wayne State University, Detroit, Michigan, United States
• ³ Higher Institute of Nursing Professions and Health Techniques, Agadir, Souss-Massa, Morocco
• ⁴ University of North Sumatra, Medan, North Sumatra, Indonesia
• ⁵ Pusan National University, Busan, Busan, Republic of Korea
• ⁶ King Saud University, Riyadh, Riyadh, Saudi Arabia
• ⁷ Daejeon University, Daejeon, Daejeon, Republic of Korea

Prostate cancer (PCa) is a malignancy arising from abnormal cell proliferation within the prostate gland, situated behind the bladder in the male reproductive system. Atractylodes lancea DC. (ALD), a medicinal herb widely used in traditional Asian medicine, is highly valued in China and other regions for its therapeutic properties, including antioxidant, antidiabetic, and anticancer activities. However, its specific anticancer and anti-metastatic effects on prostate cancer have not been fully elucidated. Virtual docking studies indicate that certain components of Traditional Chinese Medicine (TCM), such asAtractylenolide II and III demonstrated binding potential to inhibit androgen receptor (AR) activity, with docking scores of -8.9 and -9.3, respectively. The combined interaction strength of ALD's active compounds may rival or even exceed that of enzalutamide (ENZ), suggesting a synergistic effect that enhances androgen receptor (AR) inhibition and stability in ALD's therapeutic action. In this study, we investigated the cytotoxic effects of ALD on PC3 and DU145 prostate cancer cells, as well as on the normal prostate cell line BPH-1.To assess cell viability, we utilized the EZ-Cytotoxic kit. We also performed colony formation assays and analyzed apoptosis through TUNEL staining and annexin V-FITC/PI staining.Western blotting was conducted to explore the underlying mechanisms of ALD's effects.Our findings indicate that ALD selectively reduces the viability of prostate cancer cells while having minimal impact on BPH-1 normal cells, demonstrating a degree of cancer selectivity. Furthermore, ALD disrupts mitochondrial function by decreasing mitochondrial membrane potential (ΔΨm) and increasing intracellular calcium levels. We observed a concentration-dependent increase in reactive oxygen species (ROS) generation in both PC3 and DU145 cells, which was completely inhibited by pretreatment with N-acetylcysteine (NAC), confirming a ROS-mediated mechanism. Overall, our results suggest that ALD induces ROS-mediated apoptosis and disrupts critical survival pathways in prostate cancer cells, presenting a promising therapeutic approach with selective cytotoxicity against metastatic prostate cancer This findings highlight the potential of ALD, containing atractylenolide II and III, to synergistically engage multiple pathways related to anti-４ proliferation and metastasis, promoting apoptosis. This multifaceted interaction enhances ALD's therapeutic efficacy, offering new hope in the fight against this aggressive cancer.