Synergistic Effect of Forbesione From Garcinia hanburyi in Combination with 5-Fluorouracil on Cholangiocarcinoma

Document Type: Research Articles

Authors

1 Department of Microbiology, Khon Kaen University, Khon Kaen 40002, Thailand.

2 Liver Fluke and Cholangiocarcinoma Research Center, Cholangiocarcinoma Screening and Care Program (CASCAP), Khon Kaen 40002, Thailand.

3 Center of Excellence for Innovation in Chemistry, Khon Kaen University, Khon Kaen 40002, Thailand.

4 Department of Parasitology, Khon Kaen University, Khon Kaen 40002,Thailand.

5 Department of Pharmacognosy, Faculty of Pharmacy, Mahidol University, Bangkok, 10400, Thailand.

6 Department of Chemistry, Faculty of Science, Mahidol University, Bangkok, 10400, Thailand.

Abstract

 
Background: Chemotherapy for advanced cholangiocarcinoma (CCA) is largely ineffective; thus innovative combinations of chemotherapeutic agents and natural compounds represent a promising strategy. This study aimed to investigate the synergistic effects of forbesione combined with 5-fluorouracil (5-FU) in hamster cholangiocarcinoma (Ham-1) cells both in vitro and in vivo. The anti-tumor effects of 5-FU combined with forbesione in vitro were determined using the Sulforhodamine B (SRB) assay and the effects in vivo were assessed in transplanted Ham-1 allograph models. Using ethidium bromide/acridine orange (EB/AO) staining, the morphological changes of apoptotic cells was investigated. The expressions of apoptosis-related molecules after combined treatment with forbesione and 5-FU were determined using real-time RT-PCR and western blot analysis. Forbesione or 5-FU alone inhibited proliferation of Ham-1 cells in a dose-dependent manner and their combination showed a synergistic proliferation inhibitory effect in vitro. In vivo studies, forbesione in combination with 5-FU exhibited greater inhibition of the tumor in the hamster model compared with treatment using either drug alone. Forbesione combined with 5-FU exerted stronger apoptotic induction in Ham-1 cells than did single drug treatment. The combination of drugs strongly suppressed the expression of B-cell lymphoma 2 (Bcl-2) and procaspase-3 while enhancing the expression of p53, Bcl-2-associated X protein (Bax), apoptotic protease activating factor-1 (Apaf-1), caspase-9 and caspase-3, compared with single drug treatments. These results explained the decreased expression of cytokeratin 19 (CK19) positive cells and proliferation cell nuclear antigen (PCNA) positive cells in Ham-1 cell tumor tissues of the treated hamsters. There was no apparent systemic toxicity observed in the treated animals compared with the control groups. Forbesione combined with 5-FU strongly induced apoptosis in Ham-1 cells. The growth inhibitory effect of combined treatment using these two drugs was much greater than treatment with either drug alone, both in vitro and in vivo.

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