Compound Kushen injection reduces severity of radiation-induced gastrointestinal mucositis in rats.

Mucositis, or damage/injury to mucous membranes of the alimentary, respiratory, or genitourinary tract, is the major side effect associated with anticancer radiotherapies. Because there is no effective treatment for mucositis at present, this is a particular issue as it limits the dose of therapy in cancer patients and significantly affects their quality of life. Gastrointestinal mucositis (GIM) occurs in patients receiving radiotherapies to treat cancers of the stomach, abdomen, and pelvis. It involves inflammation and ulceration of the gastrointestinal (GI) tract causing diarrhea, nausea and vomiting, abdominal pain, and bloating. However, there is currently no effective treatment for this debilitating condition. In this study, we investigated the potential of a type of traditional Chinese medicine (TCM), compound Kushen injection (CKI), as a treatment for GIM. It has previously been shown that major groups of chemical compounds found in CKI have anti-inflammatory effects and are capable of inhibiting the expression of pro-inflammatory cytokines. Intraperitoneal administration of CKI to Sprague Dawley (SD) rats that concurrently received abdominal irradiation over five fractions resulted in reduced severity of GIM symptoms compared to rats administered a vehicle control. Histological examination of the intestinal tissues revealed significantly less damaged villus epithelium in CKI-administered rats that had reduced numbers of apoptotic cells in the crypts. Furthermore, it was also found that CKI treatment led to decreased levels of inflammatory factors including lower levels of interleukin (IL)-1ß and IL-6 as well as myeloperoxidase (MPO)-producing cells in the intestinal mucosa. Together, our data indicate a novel effect of CKI to reduce the symptoms of radiation-induced GIM by inhibiting inflammation in the mucosa and apoptosis of epithelial cells.

The effect of compound kushen injection on cancer cells: Integrated identification of candidate molecular mechanisms.

Traditional Chinese Medicine (TCM) preparations are often extracts of single or multiple herbs containing hundreds of compounds, and hence it has been difficult to study their mechanisms of action. Compound Kushen Injection (CKI) is a complex mixture of compounds extracted from two medicinal plants and has been used in Chinese hospitals to treat cancer for over twenty years. To demonstrate that a systematic analysis of molecular changes resulting from complex mixtures of bioactives from TCM can identify a core set of differentially expressed (DE) genes and a reproducible set of candidate pathways. We used in vitro cancer models to measure the effect of CKI on cell cycle phases and apoptosis, and correlated those phenotypes with CKI induced changes in gene expression. We treated two cancer cell lines with or without CKI and assessed the resulting phenotypes by employing cell viability and proliferation assays. Based on these results, we carried out high-throughput transcriptome data analysis to identify genes and candidate pathways perturbed by CKI. We integrated these differential gene expression results with previously reported results and carried out validation of selected differentially expressed genes. CKI induced cell-cycle arrest and apoptosis in the cancer cell lines tested. In these cells CKI also altered the expression of 363 core candidate genes associated with cell cycle, apoptosis, DNA replication/repair, and various cancer pathways. Of these, 7 are clinically relevant to cancer diagnosis or therapy, 14 are cell cycle regulators, and most of these 21 candidates are downregulated by CKI. Comparison of our core candidate genes to a database of plant medicinal compounds and their effects on gene expression identified one-to-one, one-to-many and many-to-many regulatory relationships between compounds in CKI and DE genes. By identifying genes and promising candidate pathways associated with CKI treatment based on our transcriptome-based analysis, we have shown that this approach is useful for the systematic analysis of molecular changes resulting from complex mixtures of bioactives.

A New Strategy for Identifying Mechanisms of Drug-drug Interaction Using Transcriptome Analysis: Compound Kushen Injection as a Proof of Principle.

Drug-drug interactions (DDIs), especially with herbal medicines, are complex, making it difficult to identify potential molecular mechanisms and targets. We introduce a workflow to carry out DDI research using transcriptome analysis and interactions of a complex herbal mixture, Compound Kushen Injection (CKI), with cancer chemotherapy drugs, as a proof of principle. Using CKI combined with doxorubicin or 5-Fu on cancer cells as a model, we found that CKI enhanced the cytotoxic effects of doxorubicin on A431 cells while protecting MDA-MB-231 cells treated with 5-Fu. We generated and analysed transcriptome data from cells treated with single treatments or combined treatments and our analysis showed that opposite directions of regulation for pathways related to DNA synthesis and metabolism which appeared to be the main reason for different effects of CKI when used in combination with chemotherapy drugs. We also found that pathways related to organic biosynthetic and metabolic processes might be potential targets for CKI when interacting with doxorubicin and 5-Fu. Through co-expression analysis correlated with phenotype results, we selected the MYD88 gene as a candidate major regulator for validation as a proof of concept for our approach. Inhibition of MYD88 reduced antagonistic cytotoxic effects between CKI and 5-Fu, indicating that MYD88 is an important gene in the DDI mechanism between CKI and chemotherapy drugs. These findings demonstrate that our pipeline is effective for the application of transcriptome analysis to the study of DDIs in order to identify candidate mechanisms and potential targets.

Understanding the Mechanistic Contribution of Herbal Extracts in Compound Kushen Injection With Transcriptome Analysis.

Herbal compatibility is the knowledge of which herbs to combine in traditional Chinese medicine (TCM) formulations. The lack of understanding of herbal compatibility is one of the key problems for the application and popularization of TCM in western society. Because of the chemical complexity of herbal medicines, it is simpler to begin to conduct compatibility research based on herbs rather than component plant secondary metabolites. We have used transcriptome analysis to explore the effects and interactions of two plant extracts (Kushen and Baituling) combined in Compound Kushen Injection (CKI). Based on shared chemical compounds and in vitro cytotoxicity comparisons, we found that both the major compounds in CKI, and the cytotoxicity effects of CKI were mainly derived from the extract of Kushen (Sophorae flavescentis). We generated and analyzed transcriptome data from MDA-MB-231 cells treated with single-herb extracts or CKI and results showed that Kushen contributed to the perturbation of the majority of cytotoxicity/cancer related pathways in CKI such as cell cycle and DNA replication. We also found that Baituling (Heterosmilax yunnanensis Gagnep) could not only enhance the cytotoxic effects of Kushen in CKI, but also activate immune-related pathways. Our analyses predicted that IL-1ß gene expression was upregulated by Baituling in CKI and we confirmed that IL-1ß protein expression was increased using an ELISA assay. Altogether, these findings help to explain the rationale for combining Kushen and Baituling in CKI, and show that transcriptome analysis using single herb extracts is an effective method for understanding herbal compatibility in TCM.

An effective drug sensitizing agent increases gefitinib treatment by down regulating PI3K/Akt/mTOR pathway and up regulating autophagy in non-small cell lung cancer.

Gefitinib is one of commonly used first-line treatment options for patients with positive EGFR mutation in non-small cell lung cancer (NSCLC). However, most patients with gefitinib treatment relapse over time due to the loss of drug sensitivity. Compound Kushen injection (CKI) has been used to treat lung cancer, including EGFR-mutated NSCLC. In this report, we examined the anti-cancer and drug sensitivity increased activities of CKI in gefitinib less sensitive NSCLC cell lines H1650 and H1975. Bioinformatics analysis was applied to uncover gene regulation and molecular mechanisms of CKI. Our results indicated that when associating with gefitinib in a dose-dependent fashion, CKI demonstrated the ability to inhibit the proliferation and to increase the sensitivity to gefitinib treatment in gefitinib less sensitive cell lines. This could be the results of down regulation of the PI3K/Akt/mTOR pathway and up regulation of autophagy, which were identified as the potential primary targets of CKI to increase gefitinib treatment effect.

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.

Traditional Chinese Medicines are promising sources of new agents for controlling cancer metastasis. Compound Kushen Injection (CKI), prepared from medicinal plants Sophora flavescens and Heterosmilax chinensis, disrupts cell cycle and induces apoptosis in breast cancer; however, effects on migration and invasion remained unknown. CKI, fractionated mixtures, and isolated components were tested in migration assays with colon (HT-29, SW-480, DLD-1), brain (U87-MG, U251-MG), and breast (MDA-MB-231) cancer cell lines. Human embryonic kidney (HEK-293) and human foreskin fibroblast (HFF) served as non-cancerous controls. Wound closure, transwell invasion, and live cell imaging showed CKI reduced motility in all eight lines. Fractionation and reconstitution of CKI demonstrated combinations of compounds were required for activity. Live cell imaging confirmed CKI strongly reduced migration of HT-29 and MDA-MB-231 cells, moderately slowed brain cancer cells, and had a small effect on HEK-293. CKI uniformly blocked invasiveness through extracellular matrix. Apoptosis was increased by CKI in breast cancer but not in non-cancerous lines. Cell viability was unaffected by CKI in all cell lines. Transcriptomic analyses of MDA-MB-231indicated down-regulation of actin cytoskeletal and focal adhesion genes with CKI treatment, consistent with observed impairment of cell migration. The pharmacological complexity of CKI is important for effective blockade of cancer migration and invasion.

Cell cycle, energy metabolism and DNA repair pathways in cancer cells are suppressed by Compound Kushen Injection.

BACKGROUND: In this report we examine candidate pathways perturbed by Compound Kushen Injection (CKI), a Traditional Chinese Medicine (TCM) that we have previously shown to alter the gene expression patterns of multiple pathways and induce apoptosis in cancer cells. METHODS: We have measured protein levels in Hep G2 and MDA-MB-231 cells for genes in the cell cycle pathway, DNA repair pathway and DNA double strand breaks (DSBs) previously shown to have altered expression by CKI. We have also examined energy metabolism by measuring [ADP]/[ATP] ratio (cell energy charge), lactate production and glucose consumption. Our results demonstrate that CKI can suppress protein levels for cell cycle regulatory proteins and DNA repair while increasing the level of DSBs. We also show that energy metabolism is reduced based on reduced glucose consumption and reduced cellular energy charge. RESULTS: Our results validate these pathways as important targets for CKI. We also examined the effect of the major alkaloid component of CKI, oxymatrine and determined that it had no effect on DSBs, a small effect on the cell cycle and increased the cell energy charge. CONCLUSIONS: Our results indicate that CKI likely acts through the effect of multiple compounds on multiple targets where the observed phenotype is the integration of these effects and synergistic interactions.

Long read reference genome-free reconstruction of a full-length transcriptome from Astragalus membranaceus reveals transcript variants involved in bioactive compound biosynthesis.

Astragalus membranaceus, also known as Huangqi in China, is one of the most widely used medicinal herbs in Traditional Chinese Medicine. Traditional Chinese Medicine formulations from Astragalus membranaceus have been used to treat a wide range of illnesses, such as cardiovascular disease, type 2 diabetes, nephritis and cancers. Pharmacological studies have shown that immunomodulating, anti-hyperglycemic, anti-inflammatory, antioxidant and antiviral activities exist in the extract of Astragalus membranaceus. Therefore, characterising the biosynthesis of bioactive compounds in Astragalus membranaceus, such as Astragalosides, Calycosin and Calycosin-7-O-ß-d-glucoside, is of particular importance for further genetic studies of Astragalus membranaceus. In this study, we reconstructed the Astragalus membranaceus full-length transcriptomes from leaf and root tissues using PacBio Iso-Seq long reads. We identified 27 975 and 22 343 full-length unique transcript models in each tissue respectively. Compared with previous studies that used short read sequencing, our reconstructed transcripts are longer, and are more likely to be full-length and include numerous transcript variants. Moreover, we also re-characterised and identified potential transcript variants of genes involved in Astragalosides, Calycosin and Calycosin-7-O-ß-d-glucoside biosynthesis. In conclusion, our study provides a practical pipeline to characterise the full-length transcriptome for species without a reference genome and a useful genomic resource for exploring the biosynthesis of active compounds in Astragalus membranaceus.

Identification of candidate anti-cancer molecular mechanisms of Compound Kushen Injection using functional genomics.

Compound Kushen Injection (CKI) has been clinically used in China for over 15 years to treat various types of solid tumours. However, because such Traditional Chinese Medicine (TCM) preparations are complex mixtures of plant secondary metabolites, it is essential to explore their underlying molecular mechanisms in a systematic fashion. We have used the MCF-7 human breast cancer cell line as an initial in vitro model to identify CKI induced changes in gene expression. Cells were treated with CKI for 24 and 48 hours at two concentrations (1 and 2 mg/mL total alkaloids), and the effect of CKI on cell proliferation and apoptosis were measured using XTT and Annexin V/Propidium Iodide staining assays respectively. Transcriptome data of cells treated with CKI or 5-Fluorouracil (5-FU) for 24 and 48 hours were subsequently acquired using high-throughput Illumina RNA-seq technology. In this report we show that CKI inhibited MCF-7 cell proliferation and induced apoptosis in a dose-dependent fashion. We integrated and applied a series of transcriptome analysis methods, including gene differential expression analysis, pathway over-representation analysis, de novo identification of long non-coding RNAs (lncRNA) as well as co-expression network reconstruction, to identify candidate anti-cancer molecular mechanisms of CKI. Multiple pathways were perturbed and the cell cycle was identified as the potential primary target pathway of CKI in MCF-7 cells. CKI may also induce apoptosis in MCF-7 cells via a p53 independent mechanism. In addition, we identified novel lncRNAs and showed that many of them might be expressed as a response to CKI treatment.