Antiproliferation and colony-forming inhibition activities of ã- tocotrienol in human colon carcinoma HT-29 cells

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Author: Weili Xu , Bingqing Chen

Introduction

Tocotrienol rich fraction (TRF), a mixture of á-, ã-, ä-tocotrienol isomers and some á-tocopherols, has anticarcinogenic effects on various cancer cell lines such as human colon carcinoma cells (Agarwal et al., 2004) and human prostate cancer cells (Srivastava et al., 2006).

Tocotrienols, a subgroup compound of the vitamin E family, has been reported to have various activities such as anti-oxidant, anti-inflammatory, anti-angiogenic, anti-proliferative, hypochol esterolemic and immunoregulatory activities(Asaf et al., 2000; Yamada et al., 2002; Packer et al., 2001; Mizushina et al., 2006). It has been shown to display a potent anticancer activity. The data have been shown that TRF activates p53, modulates Bax/Bcl-2 ratio and induces apoptosis in HT-29 cells (Agarwal et al., 2004). The others in vitro indicated that tocotrienols inhibited the proliferation of rat hepatoma dRLh-84 cells (Sakai et al., 2004), normal mouse mammary epithelial cells (Barry et al., 2000), MCF-7, MDA-MB-231 and MDA-MB-435 breast carcinoma cells (Guthrie et al., 1997; Nesaretnam et al., 1998) and murine B16 melanoma cells(He et al., 1997). More recent studies have shown that ã- tocotrienols inhibit neoplastic +SA mammary epithelial cell(Shah et al., 2005), human hepatoma Hep3B (Sakai et al., 2006) and prostate cancer cell proliferation (Carmela Conte et al., 2004) at treatment doses that have little or no effect on normal cell growth and function.

Colon carcinoma is a serious health problem in most developed countries and is one of the leading causes of cancer mortality throughout the world (Labianca et al., 2004). Up to date, chemoprevention is a major strategy since other therapies have not been effective in controlling either the high incidence or low survival rate of human colon carcinoma (Gustin et al., 2002).

The prevention and treatment of cancer by dietary factors that inhibit cell proliferation is an exciting prospect. Several compounds of the flavone subgroup displayed strong antiproliferative activity in HT-29 cells (Wenzel et al., 2000). Song (Song et al., 2005) reported the inhibitory effects of curcumin on the proliferation of HT-29 cells by MTT method. A 4-year study provided convincing data that a high intake of vitamin E was associated with a reduced risk of colon carcinoma, particularly for women under 65 years of age (William et al., 1997). It has recently been shown that tocotrienol rich fraction (TRF) exert anti-proliferative effects in human colon carcinoma RKO cells (Agarwal et al., 2004). Although there are a great of studies about anticancer of ã- tocotrienol, the effects of TRF monomer in human colon carcinoma cell proliferation and the possible mechanisms by which their influence in cancer cell proliferation are still unclear. ã- tocotrienol is an agent with potent biological and pharmacological action. Therefore, it is of great meaning to elucidate the cancer-preventing mechanism of ã- tocotrienol. Up to date, the effects of ã- tocotrienol in HT-29 cells have not yet been reported. The objective of this study, the growth inhibitory effect of ã- tocotrienol was investigated in HT-29 cells.

Material and methods

Material Human colon carcinoma HT-29 cells line was obtained from the laboratory of Health Toxicology, Public Health School (Harbin Medical University). ã- tocotrienol (Davos Life Science Pte Ltd, Singapore) was dissolved in alcohol（100% purified）and stored at -20℃. Cell culture Human colon carcinoma HT-29 cells were cultured in RPMI-1640 containing 10% fetal bovine serum (FBS; GIBCO), 2 ìM glutamine, 100unit/ml penicillin and 100 ìg/ml streptomycin. The cultures conditions were maintained at 37℃ in a humidified atmosphere of 5% CO2. The cells were passaged at pre confluent densities by the use of a mixture solution containing 0.25% trypsin/0.02% EDTA.

Cell Viability The effect of ã- tocotrienol on the cell viability was determined by MTT assay. Briefly, the cells were seeded at 1.0×104 cells per well after 24 h, The cells were changed 200ìl of complete culture medium containing 15, 30,45 and 60 ìM concentrations of ã-tocotrienol in 96-well microtiter plates. Each concentration of ã- tocotrienol was repeated in five wells. After incubation for different time at 37℃, cell viability was determined. 20 ìl MTT (5mg/ml in phosphate-buffered saline) was added to each well and incubated for 4h. The MTT solution was removed from the wells by aspiration. After careful removal of the medium, 150 ìl DMSO was added to each well and plates were shaken.

The absorbance was recorded on a microplate reader at the wavelength of 570 nm. The growth inhibition of ã- tocotrienol was assessed as percent cell viability where vehicle-treated cells were taken as 100% viable. Mitotic index HT-29 cells were seeded in 24 well plates in 1.0×105 cells/well and cultured for 24h to cause the cell to attach. Triplicate samples of cultured cells were treated with ã- tocotrienol at dose of 15, 30, 45 and 60ìM for 24 h and 48 h, the cells were fixed with methanol and stained with Giemsa solution. After that, the cells were washed with distilled water for 3 times. The mitotic index was counted in 2000 cells. Mitotic index (MI) =the number of mitotic cells/total number of cells×100% Colony Formation Assay Exponentially growing cells were seeded at 400 cells per well in 6-well plates and allowed to attach for 36 h. Different concentrations of ã- tocotrienol were added directly to dishes. Control plates received the vehicle treatment. After 24h and 48h treatment, cells were rinsed by PBS, and fresh medium was added continued to culture 12 days. The cells were fixed with methanol, and then stained with Giemsa. Cell colonies which originated from one cell and contained more than 50 cells were counted under the invert microscope.

Statistical Analysis

Statistical analysis was used by SPSS 13.0 software. The data were expressed as mean ± SD. Differences between the control and treated groups were performed by Student's t test and P value less than 0.05 was considered statistically significant. Results Cell Viability We examined the effects of different concentration of ã- tocotrienol on the viability of HT-29 cells for 48 h by the MTT method (Figure 1).

The results showed that ã- tocotrienol could inhibit the survival of HT-29 cells, The IC50 value was 31.7±1.6ìM. ã- tocotrienol inhibited the growth of HT-29 cells in a dose- and time dependent manners. As shown in Figure1, treatment of HT-29 with 15, 30, 45 and 60ìM concentrations of ã- tocotrienol for 48 h, resulted in 26.8%, 52.3%, 64.2% and 68.4% inhibition of cell growth, compared to the vehicle treated control group. At the concentration of ã- tocotrienol at 45 and 60 ìM, a significant loss of viability was detectable during the 48 h of treatment. In addition, treatment of HT-29 cells to 30ìM concentration of ã- tocotrienol resulted in time-dependent inhibition of cell growth and the effect was more pronounced at 48 h.

Mitotic index

The effect of ã- tocotrienol on mitotic index of HT-29 cells was investigated. In this study, ã- tocotrienol could reduce the mitotic index of HT-29 cells with increasing both treatment time and concentration increase compared to the control group. The results as shown in Table 1, the lowest of mitotic index was observed at dose of 60ìM ã- tocotrienol in HT-29 cells. The inhibitory frequencies (IF, %) of cell mitosis were 3.8 %~26.3% at 24 h and 17.1 %~68.4 % at 48 h, respectively. The mitotic index of the cells treated with ã- tocotrienol for 48 h showed that mitotic index was decreased as concentrations increasing.

Colony Formation

Assay HT-29 cells were investigated to determine whether ã-tocotrienol could inhibit colony formation. The results as shown in Figure 2, ã-tocotrienol could decrease the number of colony compared to the control group, inhibitory rate were from 22.8 % to 85.3% at 24 h and from 38.9% to 93.2% at 48h in HT-29 cells. The results showed that ã- tocotrienol significantly inhibited colony formation (P < 0.05) of HT-29 cells and this inhibitory action showed a dose -dependent manner.

Discussion

Chemoprevention by naturally occurring compounds appears to be a practical approach to fight with colon carcinoma. Previous studies have been shown that ã- tocotrienol at very low doses (ìM) can possess activity of antiproliferation in various cancer cell types (Shah et al., 2003; McIntyre et al., 2000; Sylvester et al., 2005; Carmela Conte et al., 2004). These cytostatic effects could be attained by the ingestion of ã- tocotrienol in the diet of healthy individuals who are at increased risk of developing colon carcinoma. Cytostatic effects may also be beneficial by limiting the possibility of developing drug resistance and drug-induced toxicity. Therefore, we studied whether ã- tocotrienol has potential to slow the proliferation of HT-29 cells. Treatment with ã- tocotrienol has been reported to exert antiproliferative and cytotoxic effects on other cell types. Sylvester's studies(Sylvester et al., 2005) have shown that treatment with lower doses (2-6 ìM)of ã- tocotrienol was found to significantly inhibit +SA cell growth while having no effect on cell viability. In contrast, treatment with higher doses (10-20ìM) of ã- tocotrienol resulted in a significant reduction in viable +SA cell number within 24 h. Previously, Hep3B cells were treated with 25, 50 or 75ìM of ã- tocotrienol, the results showed that ã- tocotrienol derivatives exert a strong cytotoxic effect against Hep3B cells (Sakai at al., 2006).

In vitro studies showed that ã- tocotrienol possesses cytotoxicity at a variety of human tumor cell lines. The IC50 value of ã- tocotrienol has been reported in various cells; the IC50 for ã- tocotrienol were found to be approximately 4-5 ìM (5-day) in mammary malignant epithelial cells (McIntyre et al., 2000). The IC50 for ã- tocotrienol in murine melanoma cells and human hepatocellular carcinoma cells were 20 ìM (1-day) (He et al., 1997) and 27.4 ìM (3-day) (Wada et al., 2005). In this study, we evaluated the growth-inhibitory effects of ã- tocotrienol in HT-29 cells. At first, we assessed the effect of ã- tocotrienol on the viability of HT-29 cells. HT-29 cells were very sensitive to ã- tocotrienol-mediated loss of viability, which occurred at lower doses. As shown in Figure1, HT-29 cells treated with 15, 30, 45 and 60 ìM of ã- tocotrienol for 48 h, resulted in 26.8%, 52.3%, 64.2% and 68.4% inhibition of cell growth, compared to the control group. The IC50 value for ã- tocotrienol was 31.7±1.6 ìM (2-day) in HT-29 cells. These results partly support earlier similar observations regarding inhibition of cell proliferation (McIntyre et al., 2000; He et al., 1997; Wada et al., 2005).

Traditionally, mitotic index is defined as the ratio between the number of cells in mitosis and the total number of cells. It is a simple and useful method to evaluate the proliferative activity (Cui et al., 1977; Suehiro et al., 1995). In the present study, mitotic index of HT-29 cells was decreased after treatment with various concentrations of ã- tocotrienol. The inhibitory frequencies (IF, %) of cellular mitosis were 3.8 %~26.3% at 24 h and 17.1 %~68.4 % at 48 h, respectively (Table 1). When compared to the control group, this decrease was found statistically significant in each group (P<0.05). Briefly, the ã- tocotrienol at dose of 15-60ìM showed to possess the inhibitory effect on proliferation of HT-29 cells. ã- tocotrienol could also inhibit colony formation in HT-29 cells (Figure 2). The inhibitory rate was from 22.8 % to 85.3% at 24 h and from 38.9% to 93.2% at 48h in HT-29 cells treated with ã- tocotrienol. In this study, our data showed a significant decrease in the ability of colonies formation by ã- tocotrienol and observed in a dose-dependent manner.

In summary, the results in this study showed that not only the rate of cell proliferation was inhibited, but also nucleus mitosis and colony formation were decreased in HT-29 cells induced by ã-tocotrienol. The mechanism for this impressive growth inhibition of ã- tocotrienol in HT-29 cells is not yet elucidated and need to be further studied. Acknowledgements We would like thank Prof. Dr. Ma Ying of School of Food Science and Engineering, Harbin Institute of Harbin Medical University, for her helpful suggestions and assistance.

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