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. 2019 Jan 24;19(1):103.
doi: 10.1186/s12885-018-5230-8.

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

Jian Cui  1   2 , Zhipeng Qu  1   2 , Yuka Harata-Lee  1   2 , Thazin Nwe Aung  1   2 , Hanyuan Shen  1   2 , Wei Wang  2   3 , David L Adelson  4   5
Affiliations

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

Jian Cui et al. BMC Cancer. .

Abstract

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.

Keywords: Alkaloid; Cell-cycle; Cyclin; Ku70; Ku80; Matrine.

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Conflict of interest statement

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests. While a generous donation was used to set up the Zhendong Centre by Shanxi Zhendong Pharmaceutical Co Ltd, they did not determine the research direction for this work or influence the analysis of the data. JC: no competing interests, ZQ: no competing interests, YHL: no competing interests, HS: no competing interests, TNA: no competing interests, WW: is an employee of Zhendong Pharma seconded to Zhendong Centre to learn bioinformatics methods and is a PI on The Special International Cooperation Project of Traditional Chinese Medicine (GZYYGJ2017035), DLA: Director of the Zhendong Centre which was set up with a generous donation from the Zhendong Pharmaceutical Co Ltd. Zhendong Pharmaceutical has had no control over these experiments, their design or analysis and have not exercised any editorial control over the manuscript.

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Figures

Fig. 1
Fig. 1
The energy metabolism determination assays in the two cell lines. a Growth curve (top panels) and comparison of glucose consumption analysis (lower panels) between the two cell lines at 3, 6, 12, 24 and 48 h. Overall glucose consumption is divided by cell number to calculate the consumption of glucose per million cells. b [ADP]/[ATP] ratio assay result for the two cell lines at 24 and 48 h. c Lactate content detection for the two cell lines at 24 and 48 h. Statistical analyses were performed using two-way ANOVA comparing treated with untreated (*p <0.05, **p <0.01, ***p <0.001, ****p <0.0001); bars show one standard deviation from the mean
Fig. 2
Fig. 2
Cell cycle shift by CKI and changing expression of key proteins. a Histogram and statistical results of cell cycle shift regulated by CKI over 48 h. In both cell lines, the earliest shifted cell cycle phase was S phase 6 h after treatment. Compared to Hep G2, MDA-MB-231 showed delayed responses. b Expression levels for five proteins as a result of CKI treatment at both 24 and 48 h. Statistical analyses were performed using two-way ANOVA comparing treated with untreated (*p <0.05, **p <0.01, ***p <0.001, ****p <0.0001); bars show one standard deviation from the mean
Fig. 3
Fig. 3
DSBs were increased by CKI treatment. a γ-H2AX expression from 3 to 48 h after treatment with 2 mg/ml CKI in two cell lines. b Localization of γ-H2AX in two cell lines after CKI treatment for 48 h. Blue is DAPI staining of nuclei, pink/red is staining of DSBs with antibody to γ-H2AX. The bar graph shows a quantification of the average number of γ-H2AX foci per cell detected in immunofluorescence images of 2 mg/ml CKI treated and untreated groups of 3 independent replicate experiments. c Expression of DSBs repair proteins, Ku70 and Ku80, as a result of treatment with 2 mg/ml CKI in two cell lines. Statistical analyses were performed using two-way ANOVA or t-test (for microscopy) comparing treated with untreated (*p <0.05, **p <0.01, ***p <0.001, ****p <0.0001); bars show one standard deviation from the mean
Fig. 4
Fig. 4
Effect of oxymatrine alone on validated pathways. Oxymatrine was tested at 0.5 mg/mL which is equivalent to its concentration in CKI. a Histogram and statistical results of cell cycle affected by oxymatrine over 48 h. b Effect of oxymatrine on γ-H2AX levels after 24 and 48 h. c Effect of oxymatrine on [ADP]/[ATP] ratio after 24 and 48 h. Statistical analyses were performed using two-way ANOVA comparing treated with untreated (*p <0.05, **p <0.01, ***p <0.001, ****p <0.0001); bars show one standard deviation from the mean
Fig. 5
Fig. 5
Integration of the three pathways altered by CKI. a General presentation of energy metabolism affected by CKI. Glucose utilisation is down-regulated by CKI. This is accompanied by increased lactate in the cytoplasm as CKI inhibits glucose metabolism downstream of glycolysis, leading to an increase in [ADP]/[ATP] and decrease in NADH/NADPH. b Effects on DNA repair in cancer cells by CKI. CKI may be able to directly induce DSBs, but may also indirectly induce DSBs by arresting checkpoint functions during the cell cycle. In addition, CKI may also inhibit NHEJ, the major repair mechanism for DSBs. c Reactome functional enrichment of cell cycle genes based on shared differentially expressed (DE) genes from previous studies. From M/G1 to S phase, the shared DE genes from both cell lines were significantly enriched. Most of these DE genes, were down-regulated
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