Synergistic actions of atorvastatin with gamma-tocotrienol and celecoxib against human colon cancer HT29 and HCT116 cells - PubMed (original) (raw)

Synergistic actions of atorvastatin with gamma-tocotrienol and celecoxib against human colon cancer HT29 and HCT116 cells

Zhihong Yang et al. Int J Cancer. 2010.

Abstract

The synergistic actions of atorvastatin (ATST) with gamma-tocotrienol (gamma-TT) and celecoxib (CXIB) were studied in human colon cancer cell lines HT29 and HCT116. The synergistic inhibition of cell growth by ATST and gamma-TT was demonstrated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and isobologram analysis. delta-TT exhibited a similar inhibitory action when combined with ATST. Mevalonate and geranylgeranyl pyrophosphate eliminated most of the growth inhibitory effect of ATST, but only marginally decreased that of gamma-TT; whereas farnesyl pyrophosphate and squalene exhibited little effect on the inhibitory action of ATST and gamma-TT, indicating protein geranylgeranylation, but not farnesylation are involved in the inhibition of colon cancer cell growth. Both mevalonate and squalene restored the cellular cholesterol level that was reduced by ATST treatment, but only mevalonate eliminated the cell growth inhibitory effect, suggesting that the cholesterol level in cells does not play an essential role in inhibiting cancer cell growth. Protein level of HMG-CoA reductase increased after ATST treatment, and the presence of gamma-TT attenuated the elevated level of HMG-CoA reductase. ATST also decreased membrane-bound RhoA, possibly due to a reduced level of protein geranylgeranylation; addition of gamma-TT enhanced this effect. The mediation of HMG-CoA reductase and RhoA provides a possible mechanism for the synergistic action of ATST and gamma-TT. The triple combination of ATST, gamma-TT and CXIB showed a synergistic inhibition of cancer cell growth in MTT assays. The synergistic action of these three compounds was also illustrated by their induction of G(0)/G(1) phase cell cycle arrest and apoptosis.

PubMed Disclaimer

Figures

Fig. 1

A diagram of the mevalonate cascade and the structure of the HMG-CoA reductase inhibitor ATST used in this study (modified from Holstein et al.1).

Fig. 2

Growth inhibitory effects of ATST, TTs, and their combination. Human colon cancer cells HT29 (A) and HCT116 (B) were treated with different concentrations of ATST, γ-TT (or δ-TT), and their combination for 48 h. Viable cells were measured by MTT assay, and are shown as a percentage of the value of the respective control. The synergistic effect between ATST and TTs was determined by the interaction index plot constructed using the method of Chou and Talalay. Synergy was indicated by interaction index lower than 1.0. Error bars in interaction index plots represent 95% confidence intervals for the interaction indices and were calculated using the delta method (n=6).

Fig. 3

Effects of mevalonate (MVL), farnesyl pyrophosphate (Fpp), geranylgeranyl pyrophosphate (GGpp), and squalene (SQL) on cell growth inhibition induced by ATST, γ-TT, and their combination. (A) HT29 cells were treated with different concentrations of ATST, γ-TT, and their combination in the absence or presence of mevalonate (30 and 500 μM), and other intermediates in the cholesterol synthesis pathway including Fpp (10 μM), GGpp (10 μM), and SQL (30 μM) for 48 h. Viable cells were measured by MTT assay. (B) HT29 cells were treated with 20 μM ATST alone, or ATST plus MVL (250 μM) or SQL (250 μM). The total cellular cholesterol level of each treatment was determined by Wako cholesterol E kit.

Fig. 4

Effects of ATST, γ-TT, and their combination on the level of HMG-CoA reductase and the cellular distribution of RhoA. (A) HT29 cells were treated with ATST, γ-TT, and their combination for 24 h at the concentrations indicated. The protein level of HMG-CoA reductase was determined by Western-blot with an anti-HMG-CoA reductase antibody. β-actin served as an equal loading control. (B) HT29 cells were treated with ATST, γ-TT, and their combination for 24 h at the concentrations indicated, with the supplement of different intermediate products in the cholesterol biosynthesis pathway including mevalonate (MVL, 100 μM), Fpp (10 μM), GGpp (10 μM) and squalene (SQL, 100 μM). The cytosolic and membrane fractions of HT29 cells were prepared as described previously and the levels of RhoA and k-Ras proteins were determined by Western-blots. β-actin and Na+/K+ ATPase served as equal loading controls for the cytosolic and membrane fractions, respectively.

Fig. 5

Synergistic cell growth inhibitory effect of the triple combination of ATST, γ-TT, and CXIB. (A) HT29 cells were treated with different concentrations of γ-TT, ATST, CXIB, and with their double and triple combinations for 48 h. Viable cells were measured by MTT assay. The synergy between any double combination and the third compound was determined by the interaction index plot constructed using the method of Chou and Talalay. AT, AC, and TC represent the double combinations of ATST plus γ-TT, ATST plus CXIB, and γ-TT plus CXIB, respectively; ATC represents the triple combination of ATST, γ-TT, and CXIB. (B) G0/G1 phase cell cycle arrest effect of ATST, γ-TT, CXIB, and their combinations. HT29 and HCT116 cells were treated with different concentrations of ATST, γ-TT, CXIB, and with their double and triple combinations for 24 h. Cells were stained with propidium iodide and cell cycle was analyzed by flow cytometry. The mean difference among groups was analyzed by one-way ANOVA followed by Tukey’s B test. The level of p21Cip1/Waf1, determined by Western-blot, is shown below the cell cycle analysis data. The numbers below the p21Cip1/Waf1 band are the folds of increase of p21Cip1/Waf1 as compared to control, after normalization by β-actin.

Fig. 6

Effect of ATST, γ-TT, CXIB, and their combinations on cancer cell apoptosis. (A) HCT116 cells were treated with ATST, γ-TT, CXIB, and their double and triple combinations with the concentrations indicated for 48 h. The cells were then co-stained with Annexin-V and propidium iodide (PI) and analyzed by flow cytometry. Dots located in the B4 region of the intensity dot plot are considered as early apoptosis. The percentage of early apoptotic cells among total gated cells is shown in the figure. (B). HT29 and HCT116 cells were treated with different concentrations of ATST, γ-TT, CXIB, and their double and triple combinations as indicated in the absence or presence of mevalonate (MVL, 250 μM) for 48 h. Cell apoptosis was determined by Western blot with antibodies against cleaved caspase-3 and cleaved PARP, and by DNA fragmentation assay.

References

1. Holstein SA, Wohlford-Lenane CL, Wiemer DF, Hohl RJ. Isoprenoid pyrophosphate analogues regulate expression of Ras-related proteins. Biochemistry. 2003;42:4384–91. - PubMed
1. Aznar S, Lacal JC. Rho signals to cell growth and apoptosis. Cancer Lett. 2001;165:1–10. - PubMed
1. Fromigué O, Haÿ E, Modrowski D, Bouvet S, Jacquel A, Auberger P, Marie PJ. RhoA GTPase inactivation by statins induces osteosarcoma cell apoptosis by inhibiting p42/p44-MAPKs-Bcl-2 signaling independently of BMP-2 and cell differentiation. Cell Death Differ. 2006;13:1845–56. - PubMed
1. Fromigué O, Hamidouche Z, Marie PJ. Blockade of the RhoA-JNK-c-Jun-MMP2 cascade by atorvastatin reduces osteosarcoma cell invasion. J Biol Chem. 2008;283:30549–56. - PMC - PubMed
1. Ahn KS, Sethi G, Krishnan K, Aggarwal BB. Gamma-tocotrienol inhibits nuclear factor-kappaB signaling pathway through inhibition of receptor-interacting protein and TAK1 leading to suppression of antiapoptotic gene products and potentiation of apoptosis. J Biol Chem. 2007;282:809–20. - PubMed

Synergistic actions of atorvastatin with gamma-tocotrienol and celecoxib against human colon cancer HT29 and HCT116 cells - PubMed (original) (raw)