Ashwagandha (Withania somnifera) targets liver cancer stem cells via inhibiting Hedgehog signaling pathway in hepatocellular carcinoma

Samah E Ismail; Amany I Youssef; Esraa E Fawzy; Eman A Khalifa; Amani H Kazem; Marwa A Kholief; Zainab A Saleh

doi:10.1007/s12032-025-03215-0

Ashwagandha (Withania somnifera) targets liver cancer stem cells via inhibiting Hedgehog signaling pathway in hepatocellular carcinoma

Med Oncol. 2026 Feb 5;43(3):137. doi: 10.1007/s12032-025-03215-0.

Authors

Samah E Ismail¹, Amany I Youssef², Esraa E Fawzy³, Eman A Khalifa⁴, Amani H Kazem⁵, Marwa A Kholief^{6

7}, Zainab A Saleh²

Affiliations

¹ Applied Medical Chemistry Department, Medical Research Institute, Alexandria University, Alexandria, Egypt. Samah.diab@alexu.edu.eg.
² Applied Medical Chemistry Department, Medical Research Institute, Alexandria University, Alexandria, Egypt.
³ Botany and Microbiology Department, Faculty of Science, Alexandria University, Alexandria, Egypt.
⁴ Histochemistry and Cell Biology Department, Medical Research Institute, Alexandria University, Alexandria, Egypt.
⁵ Pathology Department, Medical Research Institute, Alexandria University, Alexandria, Egypt.
⁶ Forensic Medicine and Clinical Toxicology Department, Faculty of Medicine, Alexandria University, Alexandria, Egypt.
⁷ Center of Excellence for Research in Regenerative Medicine and Applications (CERRMA), Faculty of Medicine, Alexandria University, Alexandria, Egypt.

Abstract

Ashwagandha (W. somnifera), known for its broad health benefits, has shown potential in cancer prevention and treatment. The aim of this study was to investigate the potential effect of ashwagandha aqueous extract (ASH-AE) on cell proliferation and therapy resistance markers in hepatocellular carcinoma (HCC). An in vitro study was conducted using the HepG2 cell line. The HepG2 cells were divided into 4 groups according to the treatment regimen received. ASH-AE was extracted from the whole plant and characterized by GC-MS and HPLC. HepG2 cell viability was determined for all groups. The protein expression of cluster of differentiation 90 (CD90) was determined by flow cytometry technique. Also, the gene expression of Sonic Hedgehog (SHH), Patched 1(PTCH1) and ATP-binding cassette subfamily C1 (ABCC1) was assayed by qRT-PCR. The protein expression and localization of glioma-associated oncogene 1 (Gli1) in HepG2 cells were determined by immunocytochemistry (ICC) assay. The results indicated that ASH-AE either alone or in combination with sorafenib (SOR) significantly reduced HepG2 cell viability in a concentration dependent manner (P ˂0.001) with IC50 was 6.65 mg/ml for ASH-AE, 11.3 µM for SOR, and 5.6 mg/ml + 18.6 µM for ASH-AE in combination with SOR. Moreover, SOR significantly increased the percentage of CD90⁺ cells and Gli1 protein expression and nuclear translocation as well as ABCC1 gene expression compared to untreated cells. On the other hand, ASH-AE either alone or in combination with SOR significantly decreased the percentage of CD90⁺ cells and Gli1 expression and nuclear translocation as well as SHH, PTCH1 and ABCC1 gene expression compared to untreated cells and that treated with SOR. We concluded for the first time that the combination of SOR and ASH-AE generates antagonistic antitumor effect in HepG2 cells. Moreover, ASH-AE can inhibit proliferation of HepG2 cells and mitigate sorafenib-induced resistance-associated markers in HepG2 cells by targeting CD90⁺ cells via Hedgehog pathway modulation.

Keywords: Ashwagandha; Hedgehog signaling pathway; Liver cancer stem cells; Sorafenib.

MeSH terms

ATP-Binding Cassette, Sub-Family C Proteins
Carcinoma, Hepatocellular* / drug therapy
Carcinoma, Hepatocellular* / metabolism
Carcinoma, Hepatocellular* / pathology
Cell Proliferation / drug effects
Cell Survival / drug effects
Hedgehog Proteins* / antagonists & inhibitors
Hedgehog Proteins* / metabolism
Hep G2 Cells
Humans
Liver Neoplasms* / drug therapy
Liver Neoplasms* / metabolism
Liver Neoplasms* / pathology
Neoplastic Stem Cells* / drug effects
Neoplastic Stem Cells* / metabolism
Neoplastic Stem Cells* / pathology
Plant Extracts* / pharmacology
Signal Transduction / drug effects
Sorafenib / pharmacology
Withania* / chemistry
Zinc Finger Protein GLI1 / metabolism

Substances

Hedgehog Proteins
Plant Extracts
Ashwagandha
GLI1 protein, human
Zinc Finger Protein GLI1
multidrug resistance-associated protein 1
Sorafenib
ATP-Binding Cassette, Sub-Family C Proteins