In vitro effect of a sirtuin inhibitor and its possible synergistic effect with a potential tubulin inhibitor on breast cancer cell lines

Abstract

Introduction Breast cancer is among the most prevalent cancers, constituting 11.7% of global incidences and 14.32% of new diagnoses in South Africa in 2020. Owing to the high prevalence of breast cancer, there is an increasing need for anti-cancer drugs with high efficacy and minimal or no side effects. The process of drug design is integral for producing drugs that meet these needs. This is achieved by developing drugs targeting specific pathways known to be involved in the development of cancers. One of the most promising avenues in cancer drug development—currently used in clinical treatment of cancer—is combination therapy, that allows for improved efficacy through simultaneous targeting of different pathways such that the net effect exceeds that of the individual drugs. In this study the in vitro effects of a newly synthesised sirtuin inhibitor 6-methoxy-4',6'-dimethylisoflavone-2',5'-quinone (PK108-C3) and a potential tubulin inhibitor 5-hydroxy-3-(2',3',4'-trimethoxybenzoyl)-4,7-dimethylbenzo[b]furan (PK-92) were investigated individually and in combination. The benzofuran and isoflavone derivatives were assessed for the ability to reduce proliferation through cell death induction. Methods The effects of PK-92 and PK108-C3 on cell proliferation and metabolic activity were evaluated spectrophotometrically using crystal violet staining and dimethylthiazolyl-diphenyl-tetrazolium bromide assays in both the Monroe Dunaway Anderson Foundation Metastatic Breast Cancer 231 (MDA-MB-231) and Michigan Cancer Foundation-7 (MCF-7) cell lines. Morphological changes were investigated using light microscopy (haematoxylin and eosin staining) and polarisation-optical transmitted light differential interference contrast (PlasDIC) microscopy. Cell cycle progression, possible induction of apoptosis (annexin V- fluorescein isothiocyanate (FITC)) and mitochondrial membrane potential (MMP) disruption were investigated using flow cytometry. Spectrophotometry was conducted to assess the influence of individual and combination therapy on initiator and executioner caspase 8 and -6 activation. Results The half maximal growth inhibitory concentration (GI50) after 48 hours exposure was determined to be 0.125 μM for PK-92 and 7.0 μM for PK108-C3 for both cell lines. Data from microscopy techniques showed compromised cell density and characteristics of apoptosis, including cell shrinkage, hypercondensed deoxyribonucleic acid (DNA), membrane blebbing, as well as the presence of apoptotic bodies in PK-92, PK108-C3 and combination-treated cells. Cell cycle progression data revealed an increase in the number of cells in sub-G1 and G2/M in the PK-92 and combination–treated cells. MMP disruption was identified in PK-92, PK108-C3 and combination-treated cells. Annexin V-FITC analysis revealed that all treatments increased the number of apoptotic cell populations, while spectrophotometry indicated the increase of caspase 6 (for all treatments) and caspase 8 (in PK108-C3) activity for both cell lines. Conclusions This in vitro study provides evidence that PK-92 and PK108-C3 have potential anti-proliferative effects and induce apoptosis, that may be of mitochondrial origin, in the MDA-MB-231 and MCF-7 breast cell lines at concentrations lower than some commercially available drugs. These findings provide information on newly synthesised, non-commercially available, derivatives of naturally occurring benzofuran and isoflavones contributing to the knowledge base with specific focus on alternative cancer treatment regimens.