Andrographolide Induces Apoptosis and Cell Cycle Arrest through Inhibition of Aberrant Hedgehog Signaling Pathway in Colon Cancer Cells

Abstract

Background

Hedgehog signaling pathway (Hh) is abnormally stimulated in colon cancer. Evidence suggests the therapeutic effectiveness of andrographolide against several cancers. This study attempts to delineate the effect of andrographolide on Hh signaling pathway in colon cancer HCT-116 cells. Methods: Effects of andrographolide were studied on HCT-116 cells by evaluating cytotoxicity by MTT assay, morphology assessment, trypan blue exclusion, and colony formation assay; migratory potential by scratch assay; apoptosis by DAPI, Hoechst staining, FITC-Annexin V assay, and caspases activation; mitochondrial membrane potential (ΔΨm) by Mito Tracker and Rhodamine 123. Intracellular ROS by DCFH-DA staining. Cell cycle regulation by flow cytometry. Expression of BAX, BAD, BCL2, Cyclin B1, CDK1, Smo, and Gli1 by qRT-PCR. Interaction between andrographolide and Smo protein by in-silico molecular docking. Results: Andrographolide induced antiproliferative effect on HCT-116 cells in a dose-dependent and time-dependent manner. It also induced apoptosis and anti-migratory effect in HCT-116 cells. In combination with 5FU, andrographolide exhibited synergistic effect. It Induced G2/M phase arrest through downregulating CDK1 and Cyclin B1. Andrographolide also inhibited Hh signaling by downregulating Smo and Gli1 in HCT-116 cells. It showed high affinity toward Smo protein in-silico. Conclusion: Andrographolide repressed the colon cancer cell growth via inhibiting Hh signaling pathway.

Acknowledgments

The authors acknowledge Researchers Supporting Project Number (RSP-2020/86), King Saud University, Riyadh, Saudi Arabia.

Consent of Publication

Authors declare no conflict of interest and give full consent for publication.

Funding

This study was supported by Researchers Supporting Project Number (RSP-2020/86), King Saud University, Riyadh, Saudi Arabia.

Ethical Approval

N/A.

Authors Contribution

All the authors have contributed enough to be part of the present manuscript. The detailed authors contributions are as follows:

Imran Khan: Constructing an idea or hypothesis, performed all the experiment, analyzed all the data via planning methodology to reach the conclusion, wrote and revised the manuscript to its present form.

Sadaf Mahfooz: Analyzed all the data, wrote and revised the manuscript to its present form.

Mohammad Faisal: Assisted in manuscript revision, Evaluation of data and providing personnel, environmental, and financial support that are vital for the project.

Abdulrahman A. Alatar: Assisted in manuscript revision, Evaluation of data and providing personnel, environmental, and financial support that are vital for the project.

Irfan A. Ansari: Constructing an idea or hypothesis for research and manuscript, organizing, and supervising the course of the project and article writing.