Anticancer and Anti-Inflammatory Effects of Benzothiazole Derivatives Targeting NF-κB/COX-2/iNOS in a Hepatocellular Carcinoma Cell Line

Abstract

Objectives: Benzothiazole compounds, characterized by their diverse biological and pharmacological properties, have emerged as promising molecules for suppressing cancer cell proliferation and invasion due to their antiproliferative attributes. Prior research from our laboratory revealed that 2-substituted benzothiazole compounds inhibit the proliferation of glioma and cervical cancer cells and induce apoptosis in pancreatic cancer cells. However, there is limited research on the effectiveness of benzothiazoles against hepatocellular carcinoma cells (HCC). This study sought to elucidate the anticancer potential of 2-substituted benzothiazole derivatives through their modulation of oxidative stress and inflammation mediators.

Materials and methods: Antiproliferative effects of two-step synthesized 2-substituted benzothiazole derivatives were evaluated on HepG2 cells via MTT assay. Apoptosis induction was assessed using Annexin V/PI staining; cell cycle arrest effects were determined through cell cycle analysis; cell migration was examined via wound healing assay; and mitochondrial membrane damage was quantified using JC-1 staining. Spectrophotometric measurements of total antioxidant status (TAS), total oxidant status, superoxide dismutase (SOD), total thiol, and native thiol levels were used to assess cellular redox status. Expression of nuclear factor kappa B (NF-κB), an inflammatory marker, was assessed by western blot, while inflammation-related cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase levels were measured using ELISA.

Results: This investigation unveiled benzothiazole derivatives' antiproliferative and cytotoxic properties against HepG2 cells (IC₅₀ values of 56.98 μM and 59.17 μM at 24 h, and 38.54 μM 29.63 at 48 h). The synthesized compounds exhibited the ability to suppress cell migration and induce apoptosis, mediated by mitochondrial membrane potential loss (wound‑closure rates of 84.0 and 90.4% vs. 51.7% control at 48 h, apoptosis rates of 10.70% and 45.22% vs. 1.02% control). Furthermore, these derivatives reduced SOD activity (A and B at 100 μM p<0.001), TAS levels (A and B at 100 μM, p < 0.05, p < 0.001), and dynamic disulfide content. Notably, a decrease in NF-κB protein levels, closely associated with inflammation, was observed, along with a subsequent reduction in downstream effectors COX-2 (A and B at 100 μM, p<0.001) and iNOS (A and B at 100 μM, p < 0.001).

Conclusion: The findings of this study underscore the antiproliferative effects of benzothiazole derivatives in human HCCs, coupled with their anti-inflammatory potential by diminishing NF-κB levels.

Keywords: Benzothiazole; cyclooxygenase-2; inducible nitric oxide synthase; inflammation; nuclear factor kappa B; oxidative stress.

Figure 1

Synthesized 2-substituted benzothiazole compounds and inhibition effects of A and B on HepG2 and L929 cell lines (A, B) In the initial step, chalcone derivatives were synthesized by combining cis-bicyclo[3.2.0]hept-2-en-6-one with benzaldehyde (4-nitro or 4-fluoro) compounds in the presence of NaOH. Subsequently, the chalcone compounds obtained in the second step were subjected to a reaction with 2-amino-thiophenol in the presence of p-toluene sulfonic acid, forming benzothiazole compounds. The inhibition rates of A and B administrations on HepG2 and L929 cell lines were evaluated. The control groups, where no drug administration took place, showed an inhibition rate of 0%. (C) Cell viability of compound A in HepG2 cell line at 24 and 48 hours. (D) Cell viability of compound B in HepG2 cell line at 24 and 48 hours. (E) Cell viability of compounds A and B for 48 hours in the normal cell line (L929). (F) Cell viability of compound Sorafenib in HepG2 cell line at 24 and 48 hours

Figure 2

Annexin V flow cytometry analysis of the effects of A and B on HepG2 cell line. 100 µM concentration of A and B can cause apoptosis. FITC-Annexin V was utilized to evaluate apoptosis in HepG2 cells following a treatment period of 48 hours with 100 µM each of compounds A and B. The total number of Annexin V+/PI+ quadrants reflects the degree of apoptosis

Figure 3

Effects of A and B on cell cycle. The effects of A and B on the cell cycle of HepG2 cells were examined. Administration of 100 µM doses of A and B each caused the cells to shift to the sub-G1 phase

Figure 4

Effect of A and B on mitochondrial membrane potential. A red fluorescence image of HepG2 cells stained with JC-1 was taken after administering varying concentrations of compounds A and B, with each at concentrations of 50, 75, and 100 µM. The aggregation of JC-1 dye in cells decreased in a dose-dependent manner with the administration of A and B

Figure 5

Effect of A and B on wound healing in HepG2. Images of wound width in cells treated with A and B at 0, 24, and 48 hours. Treatment with benzothiazole derivatives decreased the closure of wound width in cells in a dose-dependent manner

Figure 6

Effects of A and B on NF-κB protein expression and COX-2 and iNOS activity. Western blot bands showing NF-κB protein expression levels after administration of A and B at 50 and 100 µM doses (A). Densitometric analysis of NF-κB protein levels (B). Different concentrations of compounds A and B affect COX-2 (C) and iNOS (D) levels in HepG2 cells NF-κB: Nuclear factor kappa B, COX-2: Cyclooxygenase-2

Figure 7

Effect of A and B on oxidative stress parameters in HepG2. HepG2 cells were treated with 50, 75, and 100 µM of A and B for 48 hours; TAS (A), TOS (B), OSI (C), SOD (D), total thiol (E), and native thiol (F) levels were measured TAS: Total antioxidant status, TOS: Total oxidant status, OSI: Oxidative stress index, SOD: Superoxide dismutase