α-TEA inhibits the growth and motility of human colon cancer cells via targeting RhoA/ROCK signaling

Abstract

Colon or colorectal cancer is a common type of human cancer, which originates in the intestine crassum or the rectum. In the United States, colorectal cancer has one of the highest rates of cancer‑related mortality. Investigating novel chemotherapeutic approaches is significant in the treatment of cancers, such as colorectal cancer. α-tocopherol ether-linked acetic acid (α-TEA) is a potent anticancer agent in multiple types of human cancer. However, its effect remains to be determined in colon cancer. In this study, HCT116 and SW480 human colon cancer cells were used to investigate the anticancer role of α-TEA. It was demonstrated that α-TEA inhibited cell proliferation, migration and invasion in colon cancer cells. Furthermore, it was shown that α-TEA downregulated the activity of RhoA and phosphorylated Rho-associated protein kinase (ROCK) substrate myosin light chain (MLC) using a pull-down assay and western blotting, respectively, implying that the RhoA/ROCK pathway is involved in α-TEA-mediated cell growth and motility inhibition. In order to confirm this hypothesis a RhoA inhibitor (clostridium botulinum C3 exoenzyme), a ROCK inhibitor (Y27632) and RhoA small interfering (si)RNA were applied to block RhoA/ROCK signaling. This resulted in the attenuation of MLC phosphorylation, and augmentation of α-TEA-mediated growth and motility inhibition in colon cancer cells. In conclusion, these results indicate that α-TEA inhibits growth and motility in colon cancer cells possibly by targeting RhoA/ROCK signaling. Moreover, combined with RhoA or ROCK inhibitors, α-TEA may exhibit a more effective inhibitory role in colon cancer.

Figure 1

α-TEA attenuated migration and invasion of colon cancer cells. (A) Migration and (B) invasion of HCT116 and SW480 cells measured by a Transwell assay. Cells were seeded into the upper chamber in the absence or presence of α-TEA. Following incubation, the migrating and invading cells were stained and counted. (C) Proliferation of cells was assessed by an MTT assay. Migration, invasion or proliferation ability was expressed as a ratio relative to non-treated control. Data are presented as the mean ± standard error of the mean of three independent experiments. ^*P<0.05. N.S., not significant; α-TEA, α-tocopherol ether-linked acetic acid.

Figure 2

RhoA activation was inhibited by α-TEA. (A) HCT116 cells were cultured in serum-free Dulbecco's modified Eagle's medium for 24 h, and then exposed to α-TEA. Following treatment with α-TEA, the cells were harvested. RhoA activity was assessed by a rhotekin-based pull-down assay over time following treatment with 10 µM α-TEA. Active RhoA (in pull-down samples) and total RhoA (in total lysates) were detected by western blotting using an anti-RhoA antibody. The blots were quantified by densitometry, and the results were expressed as ratio relative to the values obtained in non-treated control cells (0 min). ^**P<0.01 vs. control. α-TEA, α-tocopherol ether-linked acetic acid.

Figure 3

α-TEA reduced MLC phosphorylation. HCT116 cells were starved in serum-free Dulbecco's modified Eagle's medium for 24 h, and then treated with 10 µM α-TEA and monitored over time. MLC phosphorylation was evaluated by western blotting. The blots were quantified by densitometry, and the results were expressed as a ratio relative to the values obtained in non-treated control cells (0 min). ^**P<0.01 vs. control. α-TEA, α-tocopherol ether-linked acetic acid; MLC, myosin light chain; pMLC, phosphorylated MLC.

Figure 4

Effects of RhoA and ROCK inhibitors and RhoA siRNA combined with α-TEA on the migration and invasion of colon cancer cells. Untreated control and 10 µM α-TEA-treated HCT116 and SW480 cells were induced with or without 50 µg/ml RhoA inhibitor C3 exoenzyme or 50 µM ROCK inhibitor Y27632. (A) Migration and (B) invasion were detected using a Transwell assay. (C) Impact of inhibitors of RhoA and ROCK combined with α-TEA on MLC phosphorylation in HCT116 cells. Untreated control and α-TEA-treated cells were induced with or without inhibitors of RhoA and ROCK. MLC phosphorylation was evaluated by western blotting using anti-MLC and anti-pMLC antibodies. The blots were quantified by densitometry, and the results are expressed as a ratio relative to the values obtained in untreated control cells. Data are presented as the mean ± standard error of the mean of three independent experiments. ^*P<0.05 and ^**P<0.01. (D) Impact of RhoA siRNA on RhoA mRNA and protein expression. HCT116 cells were transfected with RhoA siRNA for 48 h. mRNA and protein were extracted, and then reverse transcription-polymerase chain reaction and western blotting were used to detect RhoA mRNA and protein expression, respectively. (E) HCT116 cells were transfected with or without RhoA siRNA for 48 h, and then treated with 10 µM α-TEA and cell migration was evaluated by Transwell assay. ^*P<0.05 and ^**P<0.01 vs. control. (F) Activity of RhoA and MLC phosphorylation were assessed by western blotting using anti-RhoA, anti-pMLC and anti-MLC antibodies. The blots were quantified by densitometry. ^**P<0.01 vs. control. MLC, myosin light chain; pMLC, phosphorylated MLC; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; siRNA, small interfering RNA; α-TEA, α-tocopherol ether-linked acetic acid; ROCK, Rho-associated protein kinase.

Figure 5

Effect of treatment with RhoA and ROCK inhibitors in combination with α-TEA on proliferation. HCT116 cells was treated with 20 µM α-TEA, (A) plus RhoA inhibitor C3 exoenzyme, or (B) plus ROCK inhibitor Y27632 for 24 h. Cell proliferation was assessed by an MTT assay. MLC phosphorylation was evaluated by western blotting. The blots were quantified by densitometry. ^*P<0.05 and ^**P<0.01 vs. control. ROCK, Rho-associated protein kinase; α-TEA, α-tocopherol ether-linked acetic acid; Ctrl, control; MLC, myosin light chain; pMLC, phosphorylated MLC.