A Benzimidazole-Based N-Heterocyclic Carbene Derivative Exhibits Potent Antiproliferative and Apoptotic Effects against Colorectal Cancer

Abstract

Background and Objectives: Colorectal cancer (CRC) remains a major global health issue. Although chemotherapy is the first-line treatment, its effectiveness is limited due to drug resistance developed in CRC. To overcome resistance and improve the prognosis of CRC patients, investigating new therapeutic approaches is necessary. Materials and Methods: Using human colorectal adenocarcinoma (HT29) and metastatic CRC (SW620) cell lines, the potential anticancer properties of a newly synthesized compound 1-(Isobutyl)-3-(4-methylbenzyl) benzimidazolium chloride (IMBZC) were evaluated by performing MTT cytotoxicity, cell migration, and colony formation assays, as well as by monitoring apoptosis-related protein and gene expression using Western blot and reverse transcription-quantitative polymerase chain reaction technologies. Results: Tested at various concentrations, the half-maximal inhibitory concentrations (IC₅₀) of IMBZC on HT29 and SW620 cell growth were determined to be 22.13 µM (6.97 μg/mL) and 15.53 µM (4.89 μg/mL), respectively. IMBZC did not alter the cell growth of normal HEK293 cell lines. In addition, IMBZC inhibited cell migration and significantly decreased colony formation, suggesting its promising role in suppressing cancer metastasis. Mechanistic analyses revealed that IMBZC treatment increased the expression of pro-apoptotic proteins p53 and Bax, while decreasing the expression of anti-apoptotic proteins Bcl-2 and Bcl-xL, thus indicating the induction of apoptosis in IMBZC-treated CRC cells, compared to untreated cells. Additionally, the addition of IMBZC to conventional chemotherapeutic drugs (i.e., 5-fluorouracil, irinotecan, and oxaliplatin) resulted in an increase in the cytotoxic potential of the drugs. Conclusions: This study suggests that IMBZC has substantial anticancer effects against CRC cells through its ability to induce apoptosis, inhibit cancer cell migration and colony formation, and enhance the cytotoxic effects of conventional chemotherapeutic drugs. These findings indicate that IMBZC could be a promising chemotherapeutic drug for the treatment of CRC. Further research should be conducted using in vivo models to confirm the anti-CRC activities of IMBZC.

Keywords: 1-isobutyl-benzimidazole chloride; antiproliferative; apoptosis; colorectal cancer; combination therapy; metastasis; migration.

Figure 1

Chemical structure of 1-(Isobutyl)-3-(4-methylbenzyl) benzimidazolium chloride (IMBZC) (MW = 314.85 g/mol).

Figure 2

Assessment of the cytotoxic potential of compound IMBZC on colon adenocarcinoma HT29 and mCRC SW620 cells, in comparison with normal HEK293 cells, using MTT assay. Determination of percent viability of (A) HT29, (B) SW620, and (C) HEK923 cells after 24 h of incubation in the absence (i.e., control) or presence of increasing concentrations of (3.97 to 31.76 µM) IMBZC. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. control. (ns: not significant).

Figure 3

Evaluation of the antiproliferative potential of IMBZC on CRC cell migration using wound healing assay. (A) HT29 and (B) SW620 cells were seeded in 6-well plates and incubated with complete medium until confluence, and then the cell monolayer was scratched with a sterile 200 µL tip and washed with PBS. The medium was replaced with or without IMBZC then incubated for 24 h. Microscopy was used to examine the cells, and digital images were taken.

Figure 4

The compound IMBZC inhibits HT29 and SW620 cell-based colony formation. Both HT29 (A) and SW620 (B) cells were incubated for 10–12 days at 37 °C for colony formation, along with untreated HT29 (A.A) and SW620 (B.A) cells (i.e., control). The number of colonies is represented by a bar graph and the data are presented as mean ± SD (N = 3). ** p < 0.01, *** p < 0.001, and **** p < 0.0001 vs. control.

Figure 5

Expression of mRNA levels of anti-apoptotic BCL-2 and BCL-xL (A,B) and pro-apoptotic BAX and TP53 (C,D) genes monitored in colorectal adenocarcinoma HT29 (A,C) and mCRC SW620 (B,D) (cell lines using RT-qPCR. Bar graphs show the relative gene expression levels calculated as a ratio to GAPDH, the internal control, and data are presented as mean ± SD (N = 3) * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. control.

Figure 6

The effect of IMBZC on the anti-apoptotic Bcl-2 and Bcl-xl and pro-apoptotic Bax and p53 protein expression levels. (A) HT29 and (B) SW620 cells were treated for 24 h at different concentrations (7.94, 15.88 and 31.76 µM) of IMBZC. Anti-Bcl-2, Bcl-xl, p53, and Bax antibodies were used to target these proteins in whole cell lysates. The strength of protein bands was semi-quantified relative to β-actin, used as a loading control, and was presented as relative to protein expression, compared to the untreated control. The bar graphs show the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 vs. control.

Figure 7

IMBZC potentiates the cytotoxicity of CRC conventional drugs (i.e., 5-FU, IRI and OXA) in (A) HT29 and (B) SW620 cells. 5-FU, IRI, and OXA drugs were tested at different concentrations (2.5, 5 and 10 µM) for 24 h of incubation. Combinations of conventional drugs with the IMBZC compound on HT29 (C) and SW620 (D) cell lines. The cell viability percentage was determined using MTT assay. The bar graphs show the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 vs. control.