Fractional Deletion Of Compound Kushen Injection, A Natural Compound Mixture, Indicates Cytokine Signaling Pathways Are Critical For Its Perturbation Of The Cell Cycle

Abstract

We have used computational and experimental biology approaches to identify candidate mechanisms of action of a traditional Chinese medicine. Compound Kushen Injection (CKI), in a breast cancer cell line in which CKI causes apoptosis. 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 in MDA-MB-231 cells. Our initial fractionation separated major from minor compounds, and showed that the major compounds accounted for little of the activity of CKI.By systematically perturbing the major compounds in CKI we found that removal of no single major compound could alter the effect of CKI on cell viability and apoptosis. However, simultaneous removal of two major compounds identified oxymatrine and oxysophocarpine as critical compounds with respect to CKI activity. We then used RNA sequencing and transcriptome analysis to correlate compound removal with gene expression and phenotype data. We determined that many compounds in CKI are required for its effectiveness in triggering apoptosis but that 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.

Effect of Compound Kushen Injection, a Natural Compound Mixture, and Its Identified Chemical Components on Migration and Invasion of Colon, Brain, and Breast Cancer Cell Lines

“…Transcriptome (23) analyses were performed to identify over-represented Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) for all differentially expressed (DE) genes by comparing MDA-MB-231 gene expression profiles with and without CKI treatment (Figure 1 and Supplementary Image 1). Differences in gene expression levels were used to identify migration related GO terms and pathways of interest, which were classified by functional roles via GO and KEGG over-representation analyses.…”

“…Concentrations of the nine major compounds in CKI were measured using LC-MS/MS (see Materials and Methods). The concentrations of MJ components were determined (Supplementary Images 3, 4) from calibration curves of nine standard compounds as previously described (23) and summarized in Supplementary Table 1. The total concentration of the nine major compounds in CKI was 9.99 mg/ml which accounted for approximately 40 % of the dry mass of CKI, and the total concentration of the nine compounds in MJ was 8.62 mg/ml, indicating that the major compounds were clearly fractionated from CKI, and that MJ and MN components were well-separated.…”

“…Work here showed that the major components trifolirhizin and adenine, not previously characterized, when added to CKI minor fraction further slowed cell migration, suggesting these agents merit further study as potential therapeutics, singly and in combination. Not all components of CKI enhance its anti-cancer activities; for example, depletion of three compounds (oxymatrine, oxysophocarpine and macrozamin) increased the anti-proliferative activity of CKI (23).…”

“…High performance liquid chromatography (HPLC) fractionation and liquid chromatography-mass spectrometry (LC-MS/MS) were used to analyse single compounds and confirm their concentrations in CKI. Fractionation of CKI was done by using Shimadzu HPLC SPC-M20A photodiode-array UV-Vis detector (Japan) equipped with a C 18 column (5 μm, 250 × 10 mm; Phenomenex, CA, USA), with methods for fractionation and concentration determinations as described previously (23) (Supplementary Images 2, 3). The nine compounds comprising the major fraction (MJ): oxymatrine, oxysophocarpine, n-methylcytisine, matrine, sophocarpine, trifolirhizin, adenine, and sophoridine were purchased as isolated compounds from Beina Biotechnology Institute Co., Ltd (Shanxi, China), and macrozamin was obtained from Zhendong Pharmaceutical Co. Ltd (Beijing, China).…”

“…RNA-seq (RNA-sequencing) data (23) of MDA-MB-231 cells treated with CKI were used to identify cell migration genes affected by CKI. Gene Ontology (GO) enrichment analyses were carried out with R package clusterProfiler 3.8.0 (10).…”