Corydalis yanhusuo Suppresses Prostate Cancer Progression by Targeting PI3K Pathway and Upregulating miR-192-5p : Insights from Network Pharmacology, In Silico, and In Vitro Studies

다국어 초록 (Multilingual Abstract)

Corydalis yanhusuo Suppresses Prostate Cancer Progression by Targeting PI3K Pathway and Upregulating miR-192-5p: Insights from Network Pharmacology, In Silico, and In Vitro Studies By Md. Maharub Hossain Fahim Master of Science Graduate School of Kyung Hee University Advised by Prof. Bonglee Kim Prostate cancer (PCa) is a common and mortal malignancy often driven by abnormalities of the signaling pathway. Traditional Chinese medicine Corydalis yanhusuo (CDY) has shown great promise for treating cancers due to its bioactive compounds. This study aims to discuss the anticancer potential of CDY, with an emphasis on how modulation of the PI3K pathway and miR-192-5p in PCa cells occurs. In this study, Network pharmacology assessment identified potential bioactive components of CDY and their targets associated with PCa, apoptosis, and ROS pathway. In silico analyses predicted interactions and confirmed binding affinities with molecular docking. In vitro experiments using DU145 and PC-3 cell lines assessed the effects of CDY on cell migration, protein expression, and miRNA regulation. To validate the findings, Western blot, qRT-PCR, Terminal deoxynucleotidyl transferase dUTP nick endlabeling (TUNEL), reactive oxygen species ROS, wound healing, etc. assays were performed. Here we found CDY treatment downregulated PI3K/AKT/mTOR pathway components, including EGFR, p- EGFR, AKT, p-AKT, and mTOR, and modulated epithelial-mesenchymal transition markers, reducing N-cadherin and Snail while increasing E-cadherin. miR-192-5p was significantly upregulated in CDY- treated cells, contributing to pathway suppression. Functional assays demonstrated reduced migration and wound closure in CDY-treated groups. Finally, through an in silico study, we identified the most potent bioactive compound quercetin from CDY and showed top targeted proteins HSP90AA1, AKT1, STAT3, SRC, EGFR, and PIK3R1 linked molecular docking analysis. However, CDY suppresses PCa by targeting the PI3K/AKT pathway and upregulating miR-192-5p. This integrative study used network pharmacology, in silico, and in vitro approaches that provide mechanistic insights into the anticancer effects of CDY and support its potential use as a therapeutic agent for PCa management. Keywords; Prostate cancer, CDY, PI3K/AKT pathway, miR-192-5p.

국문 초록 (Abstract)

전립선암(PCa)은 흔하면서도 치명적인 악성 질환으로, 종종 신호 전달 경로의 이상에 의해 유발된다. 전통 중국 의약품인 현호색(Corydalis yanhusuo, CDY)은 생리활성 화합물로 인해 암 치료에 있어 큰 잠재력을 보여주고 있다. 본 연구는 CDY의 항암 가능성을 논의하며, 특히 전립선암 세포에서 PI3K 경로와 miR-192-5p의 조절 메커니즘에 중점을 두었다. 본 연구에서는 네트워크 약리학 분석을 통해 CDY의 잠재적 생리활성 성분과 전립선암, 세포자멸사 및 ROS(활성산소) 경로와 관련된 표적들을 확인하였다. In silico 분석은 상호작용을 예측하고, 분자 도킹을 통해 결합 친화성을 확인하였다. DU145 및 PC-3 세포주를 이용한 In vitro 실험에서는 CDY가 세포 이동, 단백질 발현 및 miRNA 조절에 미치는 영향을 평가하였다. 결과를 검증하기 위해 웨스턴 블랏(Western blot), qRT-PCR, TUNEL(Terminal deoxynucleotidyl transferase dUTP nick end-labeling), ROS 분석, 상처 치유(wound healing) 실험 등을 수행하였다. 연구 결과, CDY 처리는 PI3K/AKT/mTOR 경로의 주요 구성 요소인 EGFR, p-EGFR, AKT, p-AKT, mTOR을 하향 조절하였으며, 상피-중간엽 전이(EMT) 마커를 조절하여 N-cadherin과 Snail의 발현을 감소시키고, E-cadherin의 발현을 증가시켰다. 또한 CDY 를 처리한 세포에서 miR-192-5p가 유의미하게 상향 조절되어 경로 억제에 기여하였다. 기능적 실험에서는 CDY 처리군에서 세포 이동과 상처 치유 속도가 감소하는 것이 관찰되었다. 마지막으로 In silico 연구를 통해 CDY의 가장 강력한 생리활성 화합물인 퀘르세틴(quercetin)을 확인하였으며, HSP90AA1, AKT1, STAT3, SRC, EGFR, PIK3R1과 같은 주요 표적 단백질들과의 상호작용을 분자 도킹 분석으로 밝혔다. 결론적으로, CDY는 PI3K/AKT 경로를 표적화하고 miR-192-5p를 상향 조절하여 전립선암을 억제하였다. 본 통합 연구는 네트워크 약리학, In silico 및 In vitro 접근법을 통해 CDY의 항암 효과에 대한 기전을 제시하며, 전립선암 치료제로서 CDY의 잠재적 활용 가능성을 뒷받침한다.
키워드: 전립선암, 현호색(CDY), PI3K/AKT 경로, miR-192-5p.

목차 (Table of Contents)

• Abstract vi
• 1. Introduction 1
• 2. Materials and Methods 3
• 2.1 Compound identification 3
• 2.2 Estimating the activities of bioactive compounds, analyzing toxicity, and drug likeness 3
• 2.3 Identification and analysis of protein targets 4
• 2.4 Network pharmacology analysis 4
• 2.5 Molecular docking simulation 4
• 2.6 CDY sample preparation 5
• 2.7 Chemicals and reagents 5
• 2.8 Cell culture 5
• 2.9 Cytotoxicity assay 6
• 2.10 Colony formation assay 6
• 2.11 JC-1 assay 6
• 2.12 ROS generation assay 7
• 2.13 Wound healing assay 7
• 2.14 TUNEL assay 7
• 2.15 Western blot analysis 8
• 2.16 Time dependent western blot 8
• 2.17 Quantitative real-time Polymerase Chain Reaction 9
• 2.18 Statistical analysis 9
• 3. Results 10
• 3.1 Compound identification 10
• 3.2 Pa value activities of bioactive compounds, analyzing toxicity, drug likeness and analysis
• of network pharmacology 12
• 3.3 Gene ontology analysis 15
• 3.4 Effective compounds of CDY and Molecular docking analysis 20
• 3.5 Cytotoxic impact of CDY in DU145 and PC-3 21
• 3.6 CDY prevented PCa colony formation 23
• 3.7 CDY reduced potential of mitochondrial membrane in PCa cell lines 25
• 3.8 CDY promoted reactive oxygen species in PCa cell lines 27
• 3.9 CDY inhibited the migration of PCa cell lines 29
• 3.10 CDY increased the TUNEL-positive cells in PCa 31
• 3.11 CDY inhibited the PI3K/AKT and EMT pathway in PCa 33
• 3.12 CDY inhibited apoptosis, ROS, and PCa-related key signaling protein expression identified
• from network pharmacology 36
• 3.13 CDY decreased PI3K and AKT expression over time 38
• 3.14 CDY inhibited PI3K by regulating miR-192-5p 40
• 4. Discussion 42
• 5. Conclusion 45
• 6. References 46