Rebecca H. Roubin

Ethnopharmacological and bioactivity guided investigation of five TCM anticancer herbs

ETHNOPHARMACOLOGICAL RELEVANCE Five herbs, Curcuma longa L. (CL), Scutellaria baicalensis Georgi (SBC), Scutellaria barbata D. Don (SBB), Hedyotis diffusa Willd. (HD) and Solanum nigrum L. (SN), are often prescribed in the polyherbal formulas for cancer treatment by traditional Chinese medicine (TCM) practitioners. The purpose of the present study was to identify important anticancer herbs used in TCM and carry out bioactivity-directed fractionation and isolation (BDFI) using six cancer cell lines as well as peripheral blood mononuclear cells (PBMCs), to identify constituents with anticancer activity but devoid of toxic effects against healthy immune cells. MATERIALS AND METHODS Of 243 document anticancer TCM treatments, 199 anticancer TCM herbs were ranked by the number of literature reports for each herb. Five herbs were identified from the top 50 ranked herbs by at least two out of three TCM practitioners as frequently used in the TCM treatment of cancer. BDFI using MTS assay was applied to determine the active anticancer extracts, fractions, and finally discrete compounds. RESULTS Five herbs were selected for study of their anticancer activities. The extracts of Curcuma longa L., Scutellaria barbata D. Don, and Hedyotis diffusa showed antiproliferative activity to various extents, extracts of Scutellaria baicalensis Georgi and Solanum nigrum L. showed little anticancer activity. Seven out of the 21 fractions obtained from Hedyotis diffusa Willd. showed anticancer activity. One new compound, ethyl 13(2) (S)-hydroxy-chlorophyllide a(1), along with 10 known compounds, i.e. 2-methyl-3-methoxyanthraquinone (2), 2-hydroxymethylanthraquinone(3), 2-hydroxy-3-methylanthraquinone(4), 2-hydroxymethy-1-hydroxyanthraquinone(5), 1-methoxy-2-hydroxyanthraquinone(6), 2-hydroxy-3-methyl-1-methoxyanthraquinone (7), oleanolic acid (8), ursolic acid (9), stigmasterol (10) and docosanoic acid (11), were isolated and identified. Compounds 2-6, 8 and 9 dose-dependently inhibited the cell viability of cancer cells within a concentration range of 1-200µM. Furthermore, compounds 2, 3, 5 and 9 showed significantly stronger inhibition of tested cancer cell lines than on that of PBMCs. CONCLUSION This study identified anticancer herbs, extracts, fractions and eventually compounds from the documented anticancer TCM herbs by using BDFI. It also determined the antiproliferative activity in cancer and healthy immune cells of the isolated compounds from Hedyotis diffusa. The results will be useful in the validation of the clinical application of these herbs and the development of novel anticancer therapeutics.

High-Throughput Screening Platform for Anticancer Therapeutic Drug Cytotoxicity

There are substances that kill cancer cells, but induce T cell proliferation, like thalidomide. To find more of these, a new anticancer drug screening strategy is vital. In this study we report the development of a differential cytotoxicity screening or evaluation platform using the CellTiter-Glo (Promega, Annandale, NSW, Australia) luminescent cell viability assay (ATP assay) and also the CellTiter 96 AQueous (Promega) one solution cell proliferation assay [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay]. The results suggested the platform consisting of the combination of the ATP assay be used for quantifying peripheral blood mononuclear cells, while the more economic MTS colorimetric assay is well suited to be used detecting cell viability of cancer cells. In addition, we found paclitaxel (Taxol, MP Biomedicals Australasia Pty Ltd., Seven Hills, NSW, Australia) to be a useful control for this routine screening methodology. Taxol exhibits the desirable in vitro feature of differential cytotoxicity that spares the immunological cells, when used at a concentration that will kill the majority of the cancer cell population.

Characterization and purification of glycosaminoglycans from crude biological samples.

Chondroitin sulfate (CS) is a glycosaminoglycan derived from cartilage and commonly used to treat osteoarthritis, psoriasis, and other conditions. The dimethylmethylene blue (DMMB) assay has been used often to measure glycosaminoglycan levels in relatively pure samples. In this study, we verified the accuracy of the DMMB assay in measuring CS levels in unpurified extract from bovine trachea and shark cartilage, despite potential interference from salts, proteins, and DNA. We found that the glycosaminoglycan signal obtained was due to CS and not to other glycosaminoglycan species. This was confirmed using fluorophore-assisted carbohydrate electrophoresis, which also revealed that the majority of the CS was monosulfated at the C4 or C6 position. Finally, we used anion-exchange chromatography to purify the bovine extract and obtained complete recovery of the glycosaminoglycans, with no contaminating protein. The results of this study should be very useful for future purification and analysis of this common supplement.

PSP activates monocytes in resting human peripheral blood mononuclear cells: Immunomodulatory implications for cancer treatment

Polysaccharopeptide (PSP), from Coriolus versicolor, has been used as an adjuvant to chemotherapy, and has demonstrated anti-tumor and immunomodulating effects. However its mechanism remains unknown. To elucidate how PSP affects immune populations, we compared PSP treatments both with and without prior incubation in phytohaemagglutinin (PHA) - a process commonly used in immune population experimentation. We first standardised a capillary electrophoresis fingerprinting technique for PSP identification and characterisation. We then established the proliferative capability of PSP on various immune populations in peripheral blood mononuclear cells, using flow cytometry, without prior PHA treatment. It was found that PSP significantly increased the number of monocytes (CD14(+)/CD16(-)) compared to controls without PHA. This increase in monocytes was confirmed using another antibody panel of CD14 and MHCII. In contrast, proliferations of T-cells, NK, and B-cells were not significantly changed by PSP. Thus, stimulating monocyte/macrophage function with PSP could be an effective therapeutic intervention in targeting tumors.

Small-Scale Enzymatic Digestion of Glycoproteins and Proteoglycans for Analysis of Oligosaccharides by LC-MS and FACE Gel Electrophoresis

Structural characterization of oligosaccharides from proteoglycans and other glycoproteins is greatly enhanced through the use of mass spectrometry and gel electrophoresis. Sample preparation for these sensitive techniques often requires enzymatic treatments to produce oligosaccharide sequences for subsequent analysis. This chapter describes several small-scale methods for in-gel, on-blot, and in-solution enzymatic digestions in preparation for graphitized carbon liquid chromatography-mass spectrometry (LC-MS) analysis, with specific applications indicated for glycosaminoglycans (GAGs) and N-linked oligosaccharides. In addition, accompanying procedures for oligosaccharide reduction by sodium borohydride, sample desalting via carbon microcolumn, desialylation by sialidase enzyme treatment, and small-scale oligosaccharide species fractionation are included. Fluorophore-assisted carbohydrate electrophoresis (FACE) is another useful method to isolate derivatized oligosaccharides. Overall, the modularity of these techniques provides ease and flexibility for use in conjunction with mass spectrometric and electrophoretic tools for glycomic research studies.

Evaluation of Selected Immunomodulatory Glycoproteins as an Adjunct to Cancer Immunotherapy

Polysaccharopeptide (PSP), from Coriolus versicolor, has been used widely as an adjuvant to chemotherapy with demonstrated anti-tumor and broad immunomodulating effects. While PSP’s mechanism of action still remains unknown, its enhanced immunomodulatory potential with acacia gum is of great interest. Acacia gum, which also contains polysaccharides and glycoproteins, has been demonstrated to be immunopotentiating. To elucidate whether PSP directly activates T-cell-dependent B-cell responses in vivo, we used a well-established hapten carrier system (Nitrophenyl-chicken gamma globulin (NP-CGG)). 6-week C57BL/6 male mice were immunised with 50 μg of NP25-CGG alum precipitate intraperitoneally. Mice were gavaged daily with 50mg/kg PSP in a vehicle containing acacia gum and sacrificed at days 0, 4, 7, 10, 14 and 21. ELISA was used to measure the total and relative hapten-specific anti-NP IgA, IgM and IgG titre levels compared to the controls. It was found that PSP, combined with acacia gum, significantly increased total IgG titre levels at day 4 (P< 0.05), decreased IgM titre levels at days 4 and 21 (P< 0.05) with no alterations observed in the IgA or IgE titre levels at any of the time points measured. Our results suggest that while PSP combined with acacia gum appears to exert weak immunological effects through specific T-cell dependent B-cell responses, they are likely to be broad and non-specific which supports the current literature on PSP. We report for the first time the application of a well-established hapten-carrier system that can be used to characterise and delineate specific T-cell dependent B-cell responses of potential immunomodulatory glycoprotein-based herbal medicines combinations in vivo.