Molecular mechanisms of cholangiocarcinoma cell inhibition by medicinal plants

Abstract

Cholangiocarcinoma (CCA) is one of the most common causes of cancer-associated mortality in Thailand. Certain phytochemicals have been demonstrated to modulate apoptotic signaling pathways, which may be targeted for the prevention and treatment of cancer. Therefore, the aim of the present study was to investigate the effect of specific medicinal plants on the inhibition of CCA cell proliferation, and to identify the molecular mechanisms underlying this. A WST-1 cell proliferation assay was performed using an RMCCA1 cell line, and apoptotic signaling pathways were also investigated using a PathScan Stress and Apoptosis Signaling Antibody Array Kit. The cell proliferation assay indicated that extracts from the Phyllanthus emblica fruit pulp (PEf), Phyllanthus emblica seed (PEs), Terminalia chebula fruit pulp (TCf), Terminalia chebula seed (TCs), Areca catechu seed (ACs), Curcuma longa (CL) and Moringa oleifera seed (MOs) exerted anti-proliferative activity in RMCCA1 cells. In addition, the PathScan assay revealed that certain pro-apoptotic molecules, including caspase-3, poly (ADP-ribose) polymerase, checkpoint kinase 2 and tumor protein 53, exhibited increased activity in RMCCA1 cells treated with the aforementioned selected plant extracts, with the exception of PEf. The mitogen-activated protein kinase (MAPK) pathways (including ERK1/2 and p38 MAPK) expression level was significantly increased in RMCCA1 cells pre-treated with extracts of PEs, TCf, CL and MOs. The activation of protein kinase B (Akt) was significantly demonstrated in RMCCA1 cells pre-treated with extracts of TCf, ACs and MOs. In summary, the present study demonstrated that extracts of PEs, TCf, TCs, ACs, CL and MOs exhibited anti-proliferative effects in CCA cells by inducing pro-apoptotic signals and modulating signal transduction molecules. Further studies in vivo are required to demonstrate the potential applications of specific plant extracts for the treatment of human cancer.

Keywords: anticancer; apoptosis; cholangiocarcinoma; medicinal plants; signal transduction.

Figure 1.

Cell proliferation assay of RMCCA1 cells treated with selected medicinal plants. The cells were treated with extracts of specific medicinal plants at various concentrations for 48 h. Cell proliferation was measured by WST-1 and analyzed by spectrophotometry (absorbance at 450 nm). The data are presented as a percentage of cell proliferation, for which the optical density value obtained from control cells, treated with HAM's F12 medium containing 0.01% Dimethyl sulfoxide, was set at 100%. The results are presented as the mean ± standard deviation of three independent experiments. *P<0.05 vs. control.

Figure 2.

The effects of medicinal plants on the phosphorylation or cleavage of certain signaling proteins in RMCCA1 cells. Chemiluminescent array images obtained using the PathScan Stress and Apoptosis Signaling Antibody Array Kit allowed for the simultaneous detection of 19 signaling molecules that involved in the regulation of the stress response and apoptosis. Images were captured following brief exposure of the slide to a standard chemiluminescent film. Numbers on the target map correspond to the numbered targets shown on the right. The expression levels of α-tubulin were used to normalize the signals between various samples.

Figure 3.

Array image pixel intensity ratio of phosphorylated/cleaved pro-apoptotic signaling molecules in RMCCA1 cells treated with medicinal plants. RMCCA1 cells were treated with seven selected plants that had exhibited apoptotic signals. The phosphorylation levels of Bad at Ser136, HSP27 at Ser82, Smad2 at Ser465/467, p53 at Ser15, IκBα at Ser32/36, Chk1 at Ser345 and Chk2 at Thr68, and the cleavage of PARP at Asp214 and caspase-3 at Asp175, were significantly altered. The expression of survivin decreased only in RMCCA1 cells treated with the extract of TCf. The expression levels of α-tubulin were used to normalize the signals between various samples, and these were observed to be consistent. *P<0.05 vs. control. Bad, BCL-2-associated death promoter; HSP27, heat shock protein 27; PARP, poly (ADP-ribose) polymerase; IκBα, NF-κB inhibitor α; Chk, checkpoint kinase.

Figure 4.

Detection of the phosphorylation of intracellular signal molecules in RMCCA1 cells treated with medicinal plant extracts. The phosphorylation levels of p44/42 MAPK (ERK1/2) at Thr202/Tyr204, Akt at Ser473, p38 MAPK at Thr180/Tyr182 and SAPK/JNK at Thr183/Tyr185 were significantly altered. The expression levels of α-tubulin were used to normalize the signals between various samples. *P<0.05 vs. control. MAPK, mitogen-activated protein kinase; Akt, protein kinase B; SAPK/JNK, stress-activated protein kinases/Jun amino-terminal kinases.