Anti-Proliferation Effects of Benzimidazole Derivatives on HCT-116 Colon Cancer and MCF-7 Breast Cancer Cell Lines

Many benzimidazoles are pharmaceutical agents and are used widely in biological system applications (Townsend and Revankav, 1970; Trivedi et al., 2006). Some derivatives of benzimidazoles were reported and used as antiviral agents (Gudmundsson et al., 2000; Cheng et al., 2005), topoisomerase I inhibitors (Kim et al., 1996: 1997; Rangarajan et al., 2000; Mekapati and Hansch, 2001) and as antiproliferative agent (Hong et al., 2004). Some of 4,5,6,7-tetrahalo-1H-benzimidazoles were synthesised and showed antiprotozoal activity of Acanthamoeba castellanii (Kopanska et al., 2004), antimycobacterial activity against Mycobacterioum strains (Kazimierczuk et al., 2005). In other studies, they also can act as antibacterial agent (Nezhad et al., 2005; Ozden et al., 2005), and showed anthelmintics activity of Trichinella spiralis (Mavrova et al., 2005), anti-inflammatory and analgesic activities (Sondhi et al., 2006) and as inhibitors for the hepatitis B (Li et al., 2006) and C viruses (Beaulieu et al., 2004). Some benzimidazoles were also tested as anti-HIV (Roth et al., 1997; Smith et al., 2003), anticancer agents (Craigo et al., 1999; Rida et al., 2006) and antioxidants (Kus et al., 2008). Recent publication also reported the use of phenolic and anisolic benzimidazole derivatives in vasodilator and antihypertensive studies (Soto et al., 2006), while other alkyloxyaryl benzimidazole derivatives have been tested


Anti-Proliferation Effects of Benzimidazole
Most benzimidazoles have functional groups at the positions 4, 5, 6 or 7 of the benzimidazole ring, which increase the activity of the compounds against the tumor cells (Kim et al., 1996(Kim et al., : 1997;;Garuti et al., 2000).Garuti et al. (2000) reported that benzimidazole 5 is a cytotoxic compound against MCF-7 and the IC 50 was 26.4 μM (Figure 1).
On the other hand, Kumar et al. (2002) evaluated both benzimidazoles 6 and 7 against MCF-7 and HT-29 (colon cancer cell line) with IC 50 >100 μM, while the IC 50 of benzimidazole 6 with leukemic cell line (leukemia) HL-60 was 70 μM.Benzimidazoles 6 and 7 have two substituted functional groups at positions 2 and 4, while benzimidazole 8 has three substituted functional groups at positions 1, 2 and 5 in benzimidazole ring (see Figure 2).It has been shown that benzimidazole 8 inhibited the proliferation of MDA-MB-231 (human breast cancer cells) by 26.1% (Timmegowda et al., 2008).Starcevic et al. (2007) have tested several benzimidazoles which inhibit the growth of MCF-7 cells, the substitution functional groups of those benzimidazoles are at the positions 2 and 5 of the benzimidazole ring.
In view of its importance, we report here the study of the ability of the synthesized benzimidazoles 1-4 (see Figure 3) to inhibit cell proliferation for breast cancer cell MCF-7 and colon cancer cell HCT-116 using MTT assay.

Routine cell culture
The cell used was adherent and of human origin.Cell was grown on tissue culture flasks (Nunc, Denmark) at 37 o C in a humidified incubator with the CO 2 level maintained at 5% (Freshney, 2000).The MCF-7 breast cancer cell line was cultured in DMEM growth medium (Gibco, UK), while the HCT-116 colon cancer cell line was cultured in McCoy's growth medium (Gibco, UK).The media was supplemented with 10% heat inactivated fetal calf serum (HIFCS, Gibco, UK) and 1% penicillin/ streptomycin (Sigma-Aldrich, Germany).The cells used for the experiments were between passage 4 and 7.The cell line was obtained from American Type Culture Collection (ATCC, Rockville, MD, USA).

Cytotoxicity evaluation
Tetrazolium dye, 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay was used as a measurement of cell proliferation.The MTT assay requires cellular metabolic activity to convert the colourless tetrazolium to the purple-coloured formazon product.Therefore, this assay measure only viable cells.MTT viability assay was performed with slight modification as described by Mosmann (Mosmann, 1983).

MTT assay
Cell was grown overnight in 96-well microtiter plate at 50,000 cells/well.A dilution of 1-4 were prepared by dissolving the samples in DMSO and were diluted further with cell culture medium to the required concentrations.
Cell was treated with different concentrations of the benzimidazoles 1-4 (1-50 μg/mL) for 48 hr.The stock solution was used to make the final concentration of 50, 30, 20, 10, 5 and 1 μg/mL.The final DMSO concentration in the medium was 1%.The untreated cells received only DMSO as a negative control, while  MTT was first prepared as a stock solution in 5 mg/mL of phosphate buffer saline PBS.At the end of the treatment period (48 hr), 20 μL of MTT solution was added to each well.The plates were incubated at 37 o C, 5% CO 2 for 5 hr.The medium was removed and 200 μL of DMSO was added to each well to dissolve the formazon crystals.The plates were shaken vigorously for one minute at room temperature.
The optical density reading (OD) was recorded using a micro plate reader (Hitachi U-2000, Japan) at 570 nm for absorbance and 650 nm as reference filter.Percentage of cell growth inhibition is determined by mean±SD, using the following equation: Percentage of cell growth inhibition=1-[(OD Sample -OD Blank )/ (OD Control -OD Blank )] IC 50 is the concentration that inhibits 50% from the cell growth.Cells proliferation (IC 50 ) was analysed and calculated for the benzimidazoles 1-4 by linear regression equation: y=mx+b Where y is the percentage of inhibition and it set to be 50%, m is the slope of the standard curve, x is the concentration of compound tested in μg/mL, and b is the y-intercept of the line of standard curve.

Cytotoxicity effect of benzimidazoles 1-4 on MCF-7 and HCT-116
As seen in Figure 3, the chemical structures of the benzimidazoles studied show the functional groups as a hydroxyl and benzyloxy groups.Benzimidazole 1 has hydroxyl group at the position 2 of the substituted aromatic ring in the position C2 of the benzimidazole ring, while in 3; the hydroxyl group was replaced with benzyloxy group in the position 2. Both benzimidazoles 1 and 3 have no substituted group in the position N1 of the benzimidazole ring.Benzimidazole 2 has two hydroxyl groups in the positions 2 and 2` of the substituted aromatic rings in the positions N1 and C2 of the benzimidazole ring.For benzimidazole 4, the hydroxyl groups were replaced with two benzyloxy groups in the positions 2 and 2` of the substituted aromatic rings.
The benzimidazoles 1-4 have been evaluated against MCF-7 and HCT-116 cells using Mosmann's method (1983) to show the cytotoxic effect of both hydroxyl and benzyloxy functional groups in the positions N1 and C2 of the benzimidazole ring.Our preliminary results on MCF-7 showed that benzimidazole 4 exhibits the lowest IC 50 value at 8.86±1.10μg/mL.Benzimidazoles 1 and 2 showed moderate toxicity, where the IC 50 for both compounds were 31.21±4.49μg/mL and 29.29±6.39μg/mL (Figure 4), respectively.Benzimidazole 3 was considered not toxic to MCF-7 cells with the IC 50 >50 μg/mL.The HCT-116 cells showed that benzimidazole 2 exhibited the lowest IC 50 value at 16.18±3.85μg/mL.Both 1 and 2 benzimidazoles showed moderate toxicity towards HCT-116 with the IC 50 values of 28.54±2.91μg/mL and 24.08±0.31μg/mL, respectively.Compound 3 was considered not cytotoxic with the IC 50 value >50 μg/mL.Table 1 summarised all the IC 50 values for all the compounds tested.
The substitution of hydroxyl group (-OH) with the benzyloxy ring at the position 2 of benzimidazole 1, appeared to decrease the cytotoxic effect of 3 on both MCF-7 and HCT-116 cell lines.In benzimidazole 1, position N1 is hydrogen atom, but in 2, position N1 has trisubstituted aromatic ring, and these differences does not seem to affect the IC 50 values of both benzimidazoles in MCF-7.However, hydrogen atom in position N1 appeared to contribute to the slight improvement of IC 50 value of HCT-116.
Compounds 2 and 4 have the same benzimidazole nucleus.Compound 4 has two benzyloxy rings in the positions 2 and 2` of the substituted aromatic ring in the positions N1 and C2 of the benzimidazole ring, making it bulkier than 2. Benzimidazole 2 has two hydroxyl groups in the positions 2 and 2` of the substituted aromatic ring in the positions N1 and C2 of the benzimidazole ring, making it more stable in aqueous solution.This may contribute to the stronger cytotoxic effect of benzimidazole 2 in HCT-116.In contrast, benzimidazole 4 showed a stronger cytotoxic effect in MCF-7 compared to benzimidazole
From this preliminary study, benzimidazoles 2 and 4 showed more promising results compared to other benzimidazoles.However, it is imperative to expand the study to include other types of cancer cell lines as well as normal cells in order to determine whether these two benzimidazoles 2 and 4 are suitable candidates for the development of new anti-cancer drugs.