In vitro P-glycoprotein activity of alkaloid-enriched fractions from Solanum aculeastrum and its synergistic potential with doxorubicin

Abstract

Cancer is one of the most life-threatening groups of diseases that affect humanity. Current treatment options include surgery, radiation treatment as well as chemotherapeutic agents such as alkylating agents, antimetabolites and antimitotic antibiotics. Resistance is often experienced due to overexpression of P-glycoprotein (P-gp) which leads to treatment failure. Due to prevalence of resistance and lack of effective combination treatment, novel treatment modalities are sought. Solanum aculeastrum Dunal, also known as the goat apple, is used to treat cancer, in the Eastern Cape. Literature is scarce concerning its anticancer activity, but steroidal alkaloids, such as solasodine and solamargine, are the proposed bioactive components. The aim of this project was therefore to assess the anticancer and P-glycoprotein inhibitory potential of alkaloid-enriched fractions from S. aculeastrum fruits as well as its synergistic potential with doxorubicin in vitro. A crude extract was prepared by exhaustive ultrasonic maceration of S. aculeastrum fruits. Sequential liquid-liquid extraction was done to prepare one aqueous and two organic fractions from an acidified aliquot of the crude extract. Phytochemical screening for steroidal alkaloid employed thin layer chromatography (TLC), with ultra violet (UV) and spraying with Dragendorff s reagent. The extract and fractions were assessed for cytotoxicity in cancerous (A2780, Caco-2, DU145, HepG2, MCF-7, MDA-MB-231, SH-SY5Y, SK-Br3) and non-cancerous (3T3-L1, C2C12, EA.hy926, SC-1) cell lines using the sulphorhodamine B (SRB) assay, whereas P-glycoprotein inhibitory activity was determined utilising the rhodamine-123 assay. The aqueous fraction was further fractionated using High Performance Liquid Chromatography, followed by bioactivity screening (cytotoxicity and P-gp activity) of sub-fractions. Isolation of active components was by means of column chromatography, solid phase extraction and preparative TLC. Identities of isolated compounds were confirmed using Nuclear Magnetic Resonance (NMR) and Ultra-Performance Liquid Chromatography Tandem-Mass Spectrometry (UPLC-MS). The isolated compounds were also subjected to cytotoxicity and P-gp activity determination. Synergism was evaluated by combining doxorubicin with either the active isolated compound or the aqueous fraction using the checkerboard assay. Phytochemical screening indicated the presence of steroidal alkaloids. The crude extract and aqueous fraction were the most cytotoxic in all cell lines, whereas the ether and chloroform fractions showed no cytotoxicity. The crude extract and aqueous fraction were most cytotoxic towards the SH-SY5Y neuroblastoma cell line, with half-maximal inhibitory concentrations (IC50) of 10.72 ?g/ml (crude) and 17.21 ?g/ml (aqueous) respectively. Dose-dependent P-gp inhibition was observed for the crude extract (5.9 to 18.9-fold at 100 ?g/ml) and the aqueous fraction (2.9 to 21.2 at 100 ?g/ml). Sub-fractions 10 and 11 were the most active (cell density reduction: 91.06 and 91.72%, P-gp inhibition: 7.13 and 12.69-fold). Isolation and structural elucidation yielded the steroidal alkaloid solamargine as the bioactive component with an IC50 of 15.62 ?g/ml and 9.1-fold P-gp inhibitory activity at 100 ?g/ml against the SH-SY5Y cell line. The crude extract, aqueous fraction and solamargine also showed dose-dependent cytotoxicity and P-gp inhibition against primary cell lines. The steroidal alkaloid solasonine was also isolated but was found to be inactive. No synergistic combinations with doxorubicin were found in any of the cell lines tested. Additive effects were noted for combinations of doxorubicin with the aqueous fraction as well as solamargine against the SH-SY5Y cell line with FIX values of 0.71 and 0.51 respectively. In the C2C12 cell line, antagonistic interactions were noted with FIX values > 2. The crude extract and aqueous fraction displayed potent non-selective cytotoxicity, and noteworthy P-gp inhibition against various cancer types, which was attributed to the steroidal alkaloid solamargine. Solamargine and the aqueous fraction were also shown to enhance doxorubicin through additive effects in select cell lines as well as indifferent and antagonistic responses in others. Although P-gp inhibition was present concurrently with cytotoxicity, it is not the proposed mechanism of the enhanced effect. Other processes need to be investigated.