Anticancer efficacy of selected South African phytomedicines in a three-dimensional colorectal cancer model

Abstract

Millions of patients die from cancer each year, with colorectal cancer being one of the leading causes. Chemotherapeutic drugs are routinely used in cancer treatment, but unfortunately these treatments have severe side effects. This contributes to the increasing popularity of phytomedicine use during cancer therapy. The use of phytomedicines is popular because they are seen as “safe and natural”, even though there is little concrete proof of their efficacy. Sutherlandia frutescens and Xysmalobium undulatum are South African medicinal plants that are widely used for a variety of diseases, and they have also been proposed to have anticancer potential. Both plants contain various bioactive compounds with potential activity in the treatment of cancer and other diseases. These compounds, however, could potentially interact with co-administered conventional drugs which can result in serious side effects or decreased pharmacological efficacy of the co-administered drug. The cytochrome P450 (CYP450) enzyme family is responsible for the metabolism of most commercially available drugs. Phytomedicines may change the expression or activity of these CYP450 enzymes, which may lead to phytomedicine-drug interactions. Three-dimensional (3D) cell culture models have been proposed to bridge the gap between in vitro anticancer and drug biotransformation studies, and the human in vivo system. The current gap is a result of the lack of physiological relevance of the highly used two-dimensional (2D) models. In this study, LS180 colorectal cancer cells were cultured as 3D sodium alginate encapsulated spheroids in clinostat based bioreactors. Their growth and viability were subsequently characterised for 20 days, and the ideal window in which to perform experiments was determined. The 3- (4,5- dimethylthiazol- 2- yl)- 2,5- diphenyltetrazolium bromide (MTT) assay was then used to establish half maximal inhibitory concentrations for S. frutescens and X. undulatum crude aqueous extracts, as wells as for the standard chemotherapeutic drug, paclitaxel. The determined MTT values were then used to validate and implement the established 3D model. During model characterization, validation and implementation, the following parameters were measured: soluble protein content, intracellular adenosine triphosphate levels (ATP), extracellular adenylate kinase (AK), glucose consumption and CYP3A4 and CYP2D6 gene expression. Use of the model for anticancer treatment screening was validated using two concentrations of paclitaxel, and treatment continued for 96 h. It was established that the LS180 3D cell model could be used for future anticancer activity screening, as paclitaxel caused a decrease in cell growth, viability and glucose consumption in the model. Furthermore, relative expression of the CYP3A4, CYP2D6 and P-glycoprotein genes all increased relative to the untreated control group. These are typical resistance-producing changes known to be a result of paclitaxel treatment. The model was then used to evaluate the anticancer potential of the two selected South African phytomedicines. The LS180 cell spheroids were treated with two concentrations of each of the phytomedicines for 96 h. Crude aqueous S. frutescens extract caused a marked decrease in the soluble protein content, and caused the ATP per protein content and AK per protein content to decrease below detectable limits after only 4 h exposure. S. frutescens also resulted in a decrease in glucose consumption. Treatment with the X. undulatum aqueous extract also resulted in decreased soluble protein content, as well as decreased cell viability and glucose consumption. The results suggested that S. frutescens and X. undulatum could have treatment potential against colorectal cancer. It was concluded that the LS180 sodium alginate encapsulated spheroid model could be used for future anticancer treatment and drug biotransformation screening. Furthermore, the two phytomedicines have colorectal anticancer potential as determined by in vitro tests, and this needs to be studied further to determine the clinical significance of their activities.