Evaluation of BK002 and Ojeoksan Combination Therapy in Prostate Cancer Cell Lines DU145 and PC3

다국어 초록 (Multilingual Abstract)

Evaluation of BK002 and Ojeoksan Combination Therapy in Prostate Cancer Cell Lines DU145 and PC3

By Kiryang Kim
Master of Korean Medicine
Department of Cancer Preventive Material Development
Advised by Prof. Bum sang Shim and Bonglee Kim

Prostate cancer is one of the most commonly diagnosed malignancies in men worldwide, yet cur- rent treatments are often limited by resistance and toxicity. In this study, we evaluated the syner- gistic anticancer effects of BK002—combined herbal mixture of Achyranthes japonica (AJN) and Melandrium firmum (MFR)—combined with Ojeoksan (OJS), a traditional multi-herb formula- tion, in DU145 and PC3 prostate cancer cells. Using combination index (CI) analysis, we identi- fied optimal synergistic concentrations. Cell viability assays and western blotting revealed signif- icant inhibition of DNA methyltransferase 1 (DNMT1) and phosphoinositide 3-kinase (PI3K), alongside reduced expression of immune-related proteins Signal Transducer and Activator of Transcription 3 (STAT3) and Programmed Death-ligand 1(PD-L1). Phytochemical profiling con- firmed the presence of β-ecdysterone (20-hydroxyecdysone) in the combined formulation using High-Performance Liquid Chromatography (HPLC) and Liquid Chromatography-Mass Spec- trometry (LC-MS)/MS, supporting its potential role as a bioactive marker compound. Molecular docking analysis showed strong binding affinities of β-ecdysterone to prostate cancer-associated targets, including DNMT1 and PI3K. Given that both targets are actively being explored in clin- ical trials due to their roles in tumor progression and resistance, our findings highlight the trans- lational value of dual-targeted herbal strategies. Overall, this study demonstrates that the BK002 + OJS combination exerts potent anticancer effects via biochemical and immunoregulatory mech- anisms, supporting its potential for further development in prostate cancer therapy.

Keywords; BK002, Ojeoksan, Prostate cancer, DNMT1, PI3K, miR-148a-3p, miR192-3p

목차 (Table of Contents)

• Abstract vi
• 1. Introduction 1
• 2. Materials and Methods 3
• 1) Chemicals and reagents 3
• 2) LC/MS-based analysis of β-ecdysterone in the BK002 and OJS combination 3
• 3) Network pharmacology and multi-omics analysis of BK002 and OJS 4
• 4) Molecular docking analysis 5
• 5) Cell culture 6
• 6) Cytotoxicity assay. 6
• 7) TNUNEL assay 6
• 8) Colony formation assay 7
• 9) Reactive oxygen species assay 7
• 10) Mitochondrial membrane potential (ΔΨm) analysis. 8
• 11) Western blot analysis. 8
• 12) Wound healing assay. 9
• 13) miRNA silencing and cytotoxicity assessment 9
• 13) Statistical analysis 10
• 3. Results 11
• 1) Transcriptomic expression and prognostic relevance of DNMT1, DICER1, PDCD1,
• and CD274 in prostate cancer and their association with Gleason score.. . 11
• 2) Bioinformatics identification of prostate cancer-relevant targets of BK002 + OJS 15
• 3) Target specific binding of β-ecdysterone to DNMT1 and PI3K: A molecular docking
• study...18
• 4) HPLC and LC-MS/MS analysis identifies β-ecdysterone in BK002 and OJS combina-
• tion 22
• 5) BK002 exhibits synergistic cytotoxic effects against prostate cancer cells 24
• 6) BK002 combined with OJS enhances cytotoxicity in prostate cancer cells 27
• 7) BK002 combined with OJS Suppresses migration in prostate cancer cells.. 29
• 8) BK002 combined with OJS downregulated STAT3/PD-L1-IL-6 axis in prostate can-
• cer Cells.. 31
• 9) BK002 and OJS combination suppresses DNMT-1 and PI3K expression in prostate
• cancer cells 33
• 10) BK002 and OJS combination enhances intracellular ROS generation to promote cell
• death in prostate cancer cells 35
• 11) BK002 and OJS combination induces mitochondrial dysfunction in prostate cancer
• cells via loss of membrane potential 37
• 12) BK002 and OJS promote apoptotic cell death in prostate cancer cells, confirmed by
• TUNEL assay. 39
• 13) Inhibition of miR-148-3p and miR-192-5p attenuates the cytotoxicity of BK002 and
• OJS in prostate cancer cells... .41
• 4. Discussion 43
• 5. References 47
• 6. ABSTRACT IN KOREAN. 51