Abstract
We used computational and experimental biology approaches to identify candidate mechanisms of action of aTraditional Chinese Medicine, Compound Kushen Injection (CKI), in a breast cancer cell line (MDA-MB-231). Because CKI is a complex mixture of plant secondary metabolites, we used a high-performance liquid chromatography (HPLC) fractionation and reconstitution approach to define chemical fractions required for CKI to induce apoptosis. The initial fractionation separated major from minor compounds, and it showed that major compounds accounted for little of the activity of CKI. Furthermore, removal of no single major compound altered the effect of CKI on cell viability and apoptosis. However, simultaneous removal of two major compounds identified oxymatrine and oxysophocarpine as critical with respect to CKI activity. Transcriptome analysis was used to correlate compound removal with gene expression and phenotype data. Many compounds in CKI are required to trigger apoptosis but significant modulation of its activity is conferred by a small number of compounds. In conclusion, CKI may be typical of many plant based extracts that contain many compounds in that no single compound is responsible for all of the bioactivity of the mixture and that many compounds interact in a complex fashion to influence a network containing many targets.
Highlights
We introduce here a “subtractive fractionation approach” using high performance liquid chromatography (HPLC) that can pinpoint significant interacting compounds within a mixture when coupled with a suitable bioassay
As shown in this report, oxymatrine or oxysophocarpine or combined OmtOspc treatment caused no significant change in cell viability, the cell-cycle or apoptosis, in agreement with prior work that showed oxymatrine and oxysophocarpine exerting no significant effect on apoptosis, cell-cycle or cell proliferation in HCT116 human colon cancer cells[23]
The interactions between compounds in the mixture can be synergistic and antagonistic such that if two compounds are removed that have a synergistic effect that is antagonistic to the remainder of the mixture, the resulting depleted mixture will be dis-inhibited compared to Compound Kushen Injection (CKI)
Summary
We introduce here a “subtractive fractionation approach” using high performance liquid chromatography (HPLC) that can pinpoint significant interacting compounds within a mixture when coupled with a suitable bioassay We combined this approach with RNA sequencing (RNAseq) characterisation of our bioassay, correlating the removal of interacting compounds with concomitant alterations in gene expression. Gao and colleagues showed that treatment with each of four of the main compounds of CKI (oxymatrine, matrine, sophoridine and N-methylcytisine) at 4 mg/ml significantly decreased cell viability[6]. By removing one, two or three compounds, we have been able to map the effects of these compounds and their interactions to effects on specific pathways based on altered gene expression profiles in a cell-based assay This has illuminated the roles of several major compounds of CKI, which on their own have little or no activity in our bioassay. This approach can be used to dissect the roles and interactions of individual compounds from complex natural compound mixtures whose biological activity cannot be attributed to single purified compounds
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