Molecular Mechanisms of Natural Compounds : Compound Kushen Injection (CKI) in Cancer

Abstract

Chemotherapy is a treatment that uses cytotoxic drugs to kill rapidly dividing cancer cells. There are many anti-cancer chemotherapeutic drugs used alone or in combination with others to kill cancerous cells, and some of these, are of plant origin. Naturally occurring compounds, such as Taxol, are used in chemotherapy and have very specific, unique, molecular targets. However, according to the World Health Organization (Ekor, 2014), approximately eighty percent of the world’s population depends on natural compounds from traditional medicine and these compounds are widely used in complementary medicine as anti-cancer drugs (Foster et al., 2000). Traditional Chinese medicine (TCM) uses treatments that contain multiple natural compounds, a number of which have been claimed to be of therapeutic benefit to cancer sufferers (Chung et al., 2015). Some TCM preparations have shown anti-cancer, anti-migratory and anti-metastatic properties in laboratory settings (Wang et al., 2009;Pan et al., 2011;Qu et al., 2016). Research suggests that TCM natural compound mixtures might synergistically trigger therapeutic benefits through the action of multiple components affecting multiple regulatory signaling targets (Wang et al., 2008). Compound Kushen injection (CKI) is a TCM anticancer agent which has been approved by the Chinese State Food and Drug Administration to treat solid tumors in combination with chemotherapy drugs in clinics for pain relief, cancer metastasis and enhancement of the immune system since 1995, and is used to treat approximately 30,000 patients daily. Although a large body of evidence has suggested CKI has anti-cancer properties (Xu et al., 2011;Gao et al., 2018) the anti-cancer mechanisms attributable to specific compounds within the mixture remain unknown. CKI contains multiple alkaloid and flavonoid compounds and the main bioactive compounds such as matrine and oxymatrine have shown to affect cancer cells in the lab. However, other medicinal herbs containing these two compounds as main components have demonstrated patient toxicity. It is therefore important to better understand the effects of CKI, particularly with respect the contributions of individual compounds within the mixture. In this thesis, I describe a multi-disciplinary approach including analytical chemistry, cellular assays and transcriptome analysis to explore the effects of several major compounds present in CKI. Through the application of a subtractive fractionation method that removed individual compounds one, two or three at a time, I have been able to map these compounds and their interactions to specific pathways based on altered gene expression profiles. This has illuminated the roles of several major compounds of CKI, that on their own, have no, or minimal, activity in our bioassay. This approach has enabled us to identify the interactions between compounds in a mixture as shown by the response of cancer cell cultures. Using a systems biology approach along with cellular migration and invasion assays, I have mapped the activity of related proteins and pathways which may contribute to the migrastatic activity of CKI. Altogether, this thesis presents an initial characterization of the underlying mechanistic changes induced by CKI. First, by comparing differentially expressed genes across treatment combinations generated using our subtractive fractionation approach, I identified specific candidate pathways that were altered by the removal of compounds from the mixture. Second, by using transcriptome data of a breast cancer cell line, the effects of CKI on candidate anti-migratory pathways for six different cancer cell lines were assessed. These experiments identified specific candidate target pathways through which CKI might act. These approaches can be used to understand the roles and interactions of individual compounds from any complex natural compound mixture whose biological activity cannot be associated with purified compounds.