Reversal effect of bufalin on multidrug resistance in human hepatocellular carcinoma BEL-7402/5-FU cells

Gu,Wei; Liu,Long; Fang,Fan-Fu; Huang,Feng; Cheng,Bin-Bin; Li,Bai

doi:10.3892/or.2013.2817

Reversal effect of bufalin on multidrug resistance in human hepatocellular carcinoma BEL-7402/5-FU cells

Authors:
- Wei Gu
- Long Liu
- Fan-Fu Fang
- Feng Huang
- Bin-Bin Cheng
- Bai Li
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Affiliations: Department of Traditional Chinese Medicine, Changhai Hospital, Second Military Medical University, Shanghai 200433, P.R. China
Published online on: October 24, 2013 https://doi.org/10.3892/or.2013.2817
Pages: 216-222

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Abstract

Multidrug resistance (MDR) is a major obstacle to chemotherapy in patients with hepatocellular carcinoma (HCC). To overcome MDR and improve chemotherapeutic efficacy, novel reversal agents with higher efficacy and lower toxicity are urgently needed for HCC. The present study was designed to examine the potential reversal activity of bufalin, a toxic ligand isolated from the traditional Chinese medicine ‘Chansu’ and to elucidate the possible related mechanisms. A multidrug-resistant HCC cell line, BEL-7402/5-FU, was used as the cell model. The working concentration of bufalin as an effective reversal agent, and the cell viability in the reversal experiments were determined by MTT assay. The effects of bufalin at a non-cytotoxic dose on cell cycle distribution, apoptosis and drug efflux pump activity were measured by flow cytometry. Qualitative observation of apoptosis was also carried out by confocal microscopy. Furthermore, the effects of bufalin on the expression of potential genes involved in MDR of BEL-7402/5-FU cells, including thymidylate synthase (TS), P-glycoprotein (P-gp), multidrug resistance protein 1 (MRP1), B-cell lymphoma-extra large (Bcl-xL) and Bcl-2-associated X protein (Bax), were determined using real-time PCR and western blot analysis. The results showed that bufalin at a concentration of 1 nM enhanced the chemosensitivity of BEL-7402/5-FU cells to 5-FU with a reversal fold of 3.8 which was similar to that of 1 µM verapamil. Bufalin significantly arrested the cell cycle at the G0/G1 phase, induced apoptosis through an increase in the Bax/Bcl-xL ratio, inhibited drug efflux pump activity via downregulation of MRP1, and reduced the expression of TS in BEL-7402/5-FU cells. The present study revealed that bufalin effectively reversed MDR in BEL-7402/5-FU cells through multiple pathways. The combination of bufalin with cytotoxic drugs may serve as a promising strategy for the chemotherapy of HCC.

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