Effect And Mechanism Of Cucurbitacin E On Proliferation And Migration In Human Colonic Cancer Cells Caco-2

ObjectiveCucurbitacin E (CuE), extracted from Muskmelon predicel of traditional Chinese medicine, posses various biological activities, such as antitumor effect, liver-protection, anti-chemical carcinogenesis and enhancement of immunity etc. Previous studies have shown that CuE is able to inhibit the proliferation and migration of some tumor cells both in vitro and in vivo. However, it is still not clear whether CuE can also inhibit the proliferation and migration in human colonic cancer cell lines Caco-2. It is well known that both proliferation and migration of tumor cells are the important causes of cancer progression. Thus, the purpose of this study was to investigate the effect of different concentrations of CuE on the proliferation and migration of human colonic cancer cell lines Caco-2, and to elucidate the underlying molecular mechanism on the action of CuE, so as to provide new experimental evidence for the treatment of colon cancer.MethodsHuman colonic cancer cell lines Caco-2, cultured with DMEM medium, were used for this study at the cellular and molecular levels in combination in vitro.1. The cultured Caco-2 cells were treated with CuE at 0.001,0.01,0.1,1, 1O?mol/L respectively, and CCK-8 method was used to detect the proliferation of Caco-2 cells. The migration of Caco-2 cells was analyzed by both the scratch assay and transwell chamber assay;2. The cultured Caco-2 cells were treated with CuE at 0.1?mol/L for 24h. The fluorescence assay with DNasel and phalloidin as the probes was empolyed to determine the relative content of G-actin and F-actin, respectively;3. The cultured Caco-2 cells were treated with CuE at 0.1?mol/L for 24h. The fluorescence assay with phalloidin as the probe was adopted to mark F-actin of the actin cytoskeleton, and the laser confocal microscopy was used to observe the cytoskeleton F-actin;4. The cultured Caco-2 cells were respectively treated with CuE at 0.001,0.01,0.1, 1?mol/L and 10?mol/L for 24h. The Western blot assay was used to detect the protein expression of cofilin, phosphorylated cofilin (P-cofilin), LIMK2 and phosphorylated LIMK2 (P-LIMK2).Results1. The results from CCK-8 assay, scratch assay and Transwell chamber assay demonstrated that the inhibitory effects of CuE on the proliferation and migration of Caco-2 cells were not significantly at the concentration of 0.001 and 0.01 ?mol/L. However, CuE significantly inhibited both the proliferation and migration of Caco-2 cells at the concentration of 0.1,1 ?mol/L and 10?mol/L respectively, in a dose-dependent and time-dependent manner (P<0.05);2. The fluorescence assay showed a significant increase of G-actin content (P<0.05), and the disorder of F-actin/G-actin dynamics in Caco-2 cells treated with 0.1 ?mol/L CuE for 24h;3. The results from laser confocal microscopy revealed an obvious disruption of cytoskeleton F-actin in Caco-2 cells treated with 0.1 ?mol/L CuE for 1h,2h,6h,12h and 24h, and the cytoskeleton F-actin was changed into breakage, aggregation, and salvation etc;4. Western blot analysis demonstrated that the protein expression of cofilin, phosphorylated cofilin and phosphorylated LIMK2 were decreased significantly in Caco-2 cells treated with different concentrations of CuE for 24h (P<0.05), while the expression of LIMK2 protein had no significant difference as compared with control.Conclusions1. Both the proliferation and migration of human colonic cancer Caco-2 cells were significantly inhibited by CuE in a dose-dependent and time-dependent manner;2. The G-actin content of human colonic cancer Caco-2 cells was increased by CuE, leading to the disorder of F-actin/G-actin dynamics;3. The cytoskeleton F-actin in human colonic cancer Caco-2 cells was disrupted seriously by CuE;4. The protein expression of cofilin, phosphorylated cofilin and phosphorylated LIMK2 were decreased in Caco-2 cells treated with different concentrations of CuE, while LIMK2 had no significant change as compared with control. So, CuE may disrupt the cytoskeleton actin by inhibiting LIMK/cofilin signaling pathway, which contributes to the suppression of proliferation and migration in human colonic cancer Caco-2 cells.