Abstract
Objective To investigate the effect of chaetocin on the intrahepatic cholangiocarcinoma CCLP-1 and explore the potential mechanism. Methods The concentration of chaetocin were divided into 4 groups: Group 1: 0 nmol/L, Group 2: 50 nmol/L, Group 3: 100 nmol/L and Group 4: 200 nmol/L, and all were co-cultured with CCLP-1 cell, respectively. Cell viability was detected by Cell Counting Kit-8 assay; Giemsa staining was used to observe the cell morphological and flow cytometry technique were used to detect cell apoptosis; The intracellular reactive oxygen species (ROS) level was detected using fluorescent probe under fluorescence microscope; Western blotting were used to measure the expression of Apoptosis signal-regulating kinase 1 (ASK-1) and c-Jun N-terminal kinase (JNKs). Results The proliferation of CCLP-1 cell was inhibited by chaetocin in dose dependent (F=102.369, P=0.000) or in time dependent (F=141.377, P=0.000). Furthermore, both have interaction (F=15.418, P=0.000). Giemsa staining showed chaetocin brought about CCLP-1 cell to present morphological characteristics of apoptosis, and flow cytometry indicated title rate of apoptosis of CCLP-1 cell were (6.42±3.10)%, (10.82±2.84)%, (13.67±1.71)% and (17.91±6.16)%, and apoptosis was significantly increased (F=4.788, P=0.034). The intracellular ROS level of treatment group (24.126±9.587) was much higher than that of control group (5.114±1.937) and had significantly difference (P=0.006). Western blotting showed the expressions of p-ASK-1 were 0.255±0.150, 0.680±0.039, 1.356±0.125 and 1.916±0.367. The expressions of JNKs were 0.493±0.233, 1.542±0.573, 1.639±0.342 and 2.480±1.001.Both proteins expressed increasingly as dose of chaetocin increased. (ASK-1: F=36.946, P=0.000, JNKs: F=5.291, P=0.027). Conclusion Chaetocin could inhibit CCLP-1 cell and induce it apoptosis via promote ROS mediate ASK-1/JNK signal. Key words: Chaetocin; Human intrahepatic cholangiocarcinoma cell; Apoptosis; Oxygen species
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