Abstract
Objective:The study aimed to investigate the inhibitory effects of AL on the ERK signaling molecules (ERK, p-ERK, cyclin D, and eIF4B) and the growth and proliferation of CCA cells. Materials and methods:The viability of the three CCA cell lines CL-6, HuCCT1, and HuH28 was determined using MTT assay. The effect of Ras/ERK inhibitors on protein expression in the presence of AL extract was investigated. The protein extracted from each CCA cell following exposure to AL and/or Ras/ERK inhibitors were separated on 12.5% SDS-PAGE. The analysis of mRNA expression following 48 and 72 hours of AL exposure in comparison with 0 hours (non-exposed cells) was performed by using RT-PCR. Results:The potency of cytotoxic activity of AL (by MTT assay) was about three times higher than the standard drug 5-fluorouracil. The IC50 (concentration that inhibits cell growth by 50%) of AL for the CL-6, HuCCT-1 and HuH28 cell lines were 29.77±6.64, 35.45±4.96, and 35.32±6.69 µg/mL (mean+SD), respectively. The cells were exposed to AL extract at the IC50 for 0, 12, 24, 48, and 72 hours in the absence and presence of Ras/ERK inhibitors (salirasib and XMD8-92). Protein expression was determined by Western blot analysis. The results suggested the lack of significant inhibitory effect of AL on ERK at 48 and 72 hours of exposure in all CCA cell types. On the other hand, a significant inhibitory effect was observed with p-ERK expression in all CCA cell types. Cyclin D was significantly down-regulated at 72 hours of exposure in all cell types with different potencies. The expression of eIF4B was markedly inhibited in HuCCT-1 but slightly inhibited in CL-6 and HuH28 cells. Real-time PCR analysis revealed significant down-regulation of ERK following 72 hours of AL exposure in the HuCCT1 and HuH28, but not CL-6 cell. Conclusion:The ERK signaling cascade and downstream molecules are potential targets of action of AL in CCA.
Highlights
Cholangiocarcinoma (CCA), the biliary ductal cancer, is recognized as a highly progressive cancer that originates from the transformed intra- and extrahepatic cholangiocytes or sometimes metastasis from other adjacent cancers (Alsaleh et al, 2019)
The results suggested the lack of significant inhibitory effect of Atractyloides lancea (Thunb.) DC. (AL) on extracellular signal-regulated kinase (ERK) at 48 and 72 hours of exposure in all CCA cell types
Inhibitory effect of AL extract on CCA cells growth The growth of CL-6 and HuCCT1 cells showed similar patterns during the 72 hours, characterized by the rapid growth phase during the first 48 hours, followed thereafter, by the relatively slow growth phase
Summary
Cholangiocarcinoma (CCA), the biliary ductal cancer, is recognized as a highly progressive cancer that originates from the transformed intra- and extrahepatic cholangiocytes or sometimes metastasis from other adjacent cancers (Alsaleh et al, 2019). High prevalence rates have been reported from several countries, especially in the Mekong subregion, where liver fluke infections (Opisthorchis viverrini, Opisthorchis felineus, and Clonorchis sinensis) are endemic (Kirstein and Vogel, 2016). Long-term parasitic infection, coupled with the consumption of the precarcinogen dimethyl-nitrosamine (DMN) in the fermented fish and meats, is the major risk factor of CCA in this area (Sripa et al, 2007). The prognosis of CCA patients is generally poor due to the rapid and aggressive cell proliferation. The most effective treatment is surgery, but the prognosis is markedly variable (Charonpongsuntorn et al, 2019). Effective control of CCA has been limited by the lack of specific and sensitive diagnostic tools for early detection of CCA as well as effective chemotherapeutic
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