The Anticancer Effects of the Pro-Apoptotic Benzofuran-Isatin Conjugate (5a) Are Associated With p53 Upregulation and Enhancement of Conventional Chemotherapeutic Drug Efficiency in Colorectal Cancer Cell Lines

Abstract

The present study aimed to investigate in-depth a cytotoxic novel benzofuran-isatin conjugate (5a, 3-methyl-N'-(2-oxoindolin-3-ylidene)benzofuran-2-carbohydrazide) with promising potential anticancer activities in colorectal adenocarcinoma HT29 and metastatic colorectal cancer (CRC) SW620 cell lines. Thus, the primary cell events involved in tumorigenicity, tumor development, metastasis, and chemotherapy response were explored. Both CRC cell lines were exposed to different concentrations of Compound 5a and then subjected to real-time cell viability, migration, and invasion assays, colony formation and cytotoxicity assays, and flow cytometry for cell cycle analysis and apoptosis determination. Western blot and RT-qPCR were performed to assess the protein and transcript expression levels of epithelial-mesenchymal transition (EMT), cell cycle, and apoptosis markers. We showed that the Compound 5a treatment exhibited anticancer effects through inhibition of HT29 and SW620 cell viability, migration, and invasion, in a dose-dependent manner, which were associated with the upregulation of the tumor suppressor p53. Compound 5a also inhibited the colony formation ability of HT29 and SW620 cells and reversed EMT markers E-cadherin and N-cadherin expression. CRC cell exposure to Compound 5a resulted in a cell cycle arrest at the G1/G0 phase in HT29 cells and at the G2/M phase in SW620 cells, along with the downregulation of cyclin A1 expression, described to be involved in the S phase entry. Furthermore, Compound 5a-induced apoptosis was associated with the downregulation of the anti-apoptotic Bcl-xl marker, upregulation of pro-apoptotic Bax and cytochrome c markers, and increased mitochondrial outer membrane permeability, suggesting the involvement of mitochondria-dependent apoptosis pathway. In addition, the combination studies of Compound 5a with the main conventional chemotherapeutic drugs 5-fluorouracil, irinotecan, and oxaliplatin showed a more potent cytotoxic effect in both CRC cells than a single treatment. In conclusion, our findings described the interesting in vitro anticancer properties of Compound 5a, shown to have possible antitumor, antimetastatic, and pro-apoptotic activities, with the enhancement of the cytotoxic efficiency of conventional chemotherapeutic drugs. In vivo studies are requested to confirm the promising anticancer potential of Compound 5a for CRC therapy.

Synthesis and Biological Studies of Benzo[b]furan Derivatives: A Review from 2011 to 2022

“…Isatin, also known as 1 H -indole-2,3-dione, is an important N -heterocycle in medicinal chemistry and drug discovery [ 55 ]. In their study, Mohammed et al synthesized an isatin–benzofuran hybrid 126 and investigated its antiproliferative activity against HT29 and SW620 cancer cell lines, along with its impact on tumor and metastatic development involved in primary cellular processes [ 56 ]. Scheme 20 shows the three-step synthesis of the isatin–benzofuran hybrid 126 .…”

“…Apoptotic studies were performed to evaluate the pro-apoptotic effects of hybrid 126 , involving its role in suppressing the expression levels of mitochondrial proteins Bcl-x, Bax, and cytochrome C utilizing flow cytometry [ 56 ]. The results showed a significant suppression of the Bcl-x protein expression by 50% in the HT29 cells, and by 75% and 90% in the SW620 cells at concentrations of 5 and 10 μM, respectively, in comparison to untreated cells ( Figure 46 a,b).…”

“…In this particular study, they assessed the cytotoxic effect of hybrid 126 both alone and in combination with three anticancer drugs: irinotecan (IRI), 5-fluorouracil (5-FU), and oxaliplatin (OXA) in the HT29 and SW620 cancer cell lines at different concentrations ( Figure 47 a,b) [ 56 ]. Notably, when combining hybrid 126 with IRI in the HT29 cell line ( Figure 47 a), there was a significant inhibition of cell proliferation at 10 μM (−75% vs. −50%) and 20 μM (−90% vs. −65%) compared to the single treatment with IRI alone.…”

“…The data were considered significant when reporting * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 vs. control. This is an open-access article distributed under the terms of the Creative Commons CC BY license [ 56 ].…”

“…** p < 0.01 and **** p < 0.0001 vs. control. This is an open-access article distributed under the terms of the Creative Commons CC BY license [ 56 ].…”