A combination of Chinese herb Hedyotis diffusa and Scutellaria barbata impairs mitotic entry of prostate cancer cells without eliciting DNA damage

Abstract

Prostate cancer (PCa) is one of the most frequently diagnosed malignancies and second leading causes of cancer related death in men worldwide. PCa is remarkably heterogeneous, with a range of manifestations from asymptomatic to widespread metastatic disease. Active surveillance (AS) is one of the options for men with low grade and low volume early PCa. AS involves regular PSA testing, digital examination, and biopsy without providing any medical intervention. Although AS decreases unnecessary overtreatment for some men, it may cause anxiety over disease progression in others. The number of men under AS is increasing as concern about over-treatment has developed in recent decades. Hence, in those men undertaking AS, who are otherwise on no specific treatment, there is an urgent need to develop an approach to prevent or impede PCa progression with no adverse effects that is cost effective for long-term use. Traditional Chinese Medicine has been used for centuries in treating and improving the symptoms experienced by cancer patients in China. Detoxification recipe (DR) is an anticancer Chinese Medicine formula and consists of two herbs, namely Hedyotis diffusa (HD) and Scutellaria barbata (SB). In a recent survey in Taiwan, the combination of HD and SB was shown to be the most commonly described herbal medicine in the treatment of cancer. However, how HD and SB in combination exerts its anti-cancer action is elusive. The aims of this project were to determine the anti-proliferative and its modes of action of DR. Specifically, this study aimed to firstly establish the cytostatic action of DR and its impact on the cell cycle phase distribution and secondly to determine the underlying molecular mechanism contributing to the DR induced cell cycle alteration. Our study revealed that DR has anti-proliferative activity in PCa cells, evidenced by a reduction in DNA synthesis by SYBR Green DNA analysis assay and BrdU incorporation. This is also supported by the reduction in proliferative cells by Ki-67 immunocytochemical staining. The colony formation assay also revealed the long-term inhibitory antiproliferative effect of DR. The pH and osmolality test of DR-containing culture medium showed that the anti-proliferative activity of DR is intrinsic. Studies in normal mice demonstrated that DR did not have any apparent adverse effects at least in the short-term. In order to verify the effect of DR on cell cycle progression, flow cytometric cell cycle analysis was performed using propidium iodide (PI) staining. PI-stained PCa cells indicated that DR accumulates cells in the G2/M phase. Subsequent flow cytometric analysis of PI and phospho-Histone H3 co-staining revealed that treatment with DR resulted in no increase in M phase cells compared to control cells, suggesting that DR arrests the PCa cells at G2 phase. To examine the mechanism causing the G2 phase arrest by DR, a DNA damage scenario leading to G2 arrest was examined. With irradiated cells as a positive control, we found no equivalent foci of phospho-H2AX and 53BP1 in DR-treated cells by immunofluorescence. For further verification, immunoblotting of DNA damage response proteins, including total and phosphorylated form of ATM, ATR, CHK1 and CHK2, was performed. No activation of DNA damage signalling pathways was observed in the presence of DR treatment comparing to irradiated cells. Cell cycle transition from G2 to M phase is controlled by M phase promoting complex Cyclin B1-CDK1 and their positive regulators PLK1 and Aurora A kinases. DR reduced all four at both protein and mRNA levels. Since Cyclin B1 and CDK1 confer the G2-M transition, the reduction indicates that DR-induced G2 phase cell cycle arrest is associated with the impairment in M phase promoting complex and regulators. Lastly, to determine the clinical significance of M phase promoting complex and regulators in PCa progression, an online database for genomic analysis and mRNA expression was analysed. mRNA expression levels of all four were highly correlated with PCa disease progression. We found a deletion of Cyclin B1 and amplification of CDK1, PLK1 and Aurora A genes in PCa tissue. In summary, the commonly prescribed Chinese herb medicine combination DR has significant anti-proliferative activity. DR can diminish the transition from G2 to M phase in PCa cells. DR-induced reduction of key proteins essential for mitotic entry is likely responsible for the observed impact of DR on the G2-M transition. The potential to use DR as a method to suppress PCa progression for men under AS warrants further investigation.

Date of Award	2017
Original language	English