Digitoxin inhibits proliferation of multidrug-resistant HepG2 cells through G2/M cell cycle arrest and apoptosis
Yuhe Lei1, Hua Gan2, Yuqing Huang2
1Department of Pharmacy, Shenzhen Hospital of Guangzhou University of Chinese Medicine, Shenzhen, Guangdong 518034, P.R. China.
Abstract:
Hepatocellular carcinoma (HCC) remains a challenge in the medical field due to its high malignancy and mortality rates particularly for HCC, which has developed multidrug resistance. Therefore, the identification of efficient chemotherapeutic drugs for multidrug resistant HCC has become an urgent issue. Natural products have always been of significance in drug discovery. In the present study, a cell-based method was used to screen a natural compound library, which consisted of 78 compounds, and the doxorubicin-resistant cancer cell line, HepG2/ADM, as screening tools. The findings of the present study led to the shortlisting of one of the compounds, digitoxin, which displayed an inhibitory effect on HepG2/ADM cells, with 50% inhibitory concentration values of 132.65±3.83, 52.29±6.26, and 9.13±3.67 nM for 24, 48, and 72 h, respectively. Immunofluorescence, western blotting and cell cycle analyses revealed that digitoxin induced G2/M cell cycle arrest via the serine/threonine-protein kinase ATR (ATR)-serine/threonine-protein kinase Chk2 (CHK2)-M-phase inducer phosphatase 3 (CDC25C) signaling pathway in HepG2/ADM cells, which may have resulted from a DNA double-stranded break. Digitoxin also induced mitochondrial apoptosis, which was characterized by changes in the interaction between Bcl-2 and Bax, the release of cytochrome c, as well as the activation of the caspase-3 and -9. To the best of our knowledge, the present study is the first report that digitoxin displays an anti-HCC effect on HepG2/ADM cells through G2/M cell cycle arrest, which was mediated by the ATR-CHK2-CDC25C signaling pathway and mitochondrial apoptosis. Therefore, digitoxin could be a promising chemotherapeutic agent for the treatment of patients with HCC.
Insights
Digitoxin shows promise as a treatment for multidrug-resistant hepatocellular carcinoma (HCC). This natural compound induces cell cycle arrest and apoptosis in resistant HCC cells, offering a potential new therapeutic strategy.
Area of Science:
- Oncology
- Pharmacology
- Biochemistry
Background:
- Hepatocellular carcinoma (HCC) presents significant challenges due to high mortality and the development of multidrug resistance.
- Natural products are a valuable source for novel drug discovery in oncology.
- Identifying effective treatments for multidrug-resistant HCC is an urgent medical need.
Purpose of the Study:
- To screen a natural compound library for agents effective against doxorubicin-resistant HCC cells (HepG2/ADM).
- To investigate the mechanism of action of promising compounds, specifically digitoxin, in resistant HCC.
- To evaluate digitoxin as a potential chemotherapeutic agent for HCC.
Main Methods:
- A cell-based screening assay using 78 natural compounds against the HepG2/ADM cell line.
- Determination of 50% inhibitory concentration (IC50) values for digitoxin.
- Analysis of cell cycle progression, apoptosis, and key signaling pathways (ATR-CHK2-CDC25C) using immunofluorescence and Western blotting.
Main Results:
- Digitoxin exhibited significant inhibitory effects on HepG2/ADM cells, with IC50 values in the nanomolar range.
- Digitoxin induced G2/M cell cycle arrest, likely due to DNA double-strand breaks, mediated by the ATR-CHK2-CDC25C pathway.
- Digitoxin triggered mitochondrial apoptosis, evidenced by altered Bcl-2/Bax interactions, cytochrome c release, and caspase activation.
Conclusions:
- Digitoxin demonstrates potent anti-HCC activity against a multidrug-resistant cell line.
- The anti-cancer effects are attributed to G2/M cell cycle arrest via the ATR-CHK2-CDC25C pathway and induction of mitochondrial apoptosis.
- Digitoxin represents a promising candidate for developing novel chemotherapeutic strategies against resistant HCC.
