Author: Babak Esmaeelian
Esmaeelian, Babak, 2014 Preclinical in vitro and in vivo effects of purified and synthetic bioactive compounds from marine mollusc Dicathais orbita on colorectal cancer: Cancer prevention and toxicity study, Flinders University, School of Biological Sciences
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Marine indole-based compounds, including precursors and isomers of the ancient purple dye, Tyrian purple, are known for their biological activity. In particular, the precursors 6-bromoisatin and tyrindoleninone from the Australian whelk, Dicathais orbita are compounds that have gained specific interest over the past few years for their anticancer effects in several cancer cell lines. Previous in vivo studies in mice by administration of D. orbita extract has indicated the potential for these bioactive compounds to prevent colon cancer, but with possible idiosyncratic liver toxicity. Therefore, purification of the most likely bioactive compounds (tyrindoleninone and 6-bromoisatin) from D. orbita could be helpful to enhance the anticancer properties and potentially reduce the toxicity associated with the crude extract. Synthetic 6-bromoisatin is commercially available, so testing the pure synthetic compound will also help confirm any activity associated with this compound. Tyrindoleninone is a compound which can be easily oxidized to other components. Therefore, stabilizing it by using antioxidants might be beneficial to increase its bioactive effects. The objective of this project was to optimize the purification of tyrindoleninone and 6-bromoisatin and examine the effects of these compounds, along with crude extract from D. orbita, on colorectal cancer in vitro and in vivo. The toxicity of these compounds and extracts was also assessed in vivo to establish the safety of these compounds in the body system. To optimize the purification of tyrindoleninone and 6-bromoisatin, initially the compounds were separated from the extract using thin layer chromatography (TLC) using a gradient of hexane, dichloromethane and methanol. Then flash chromatography was used to purify these compounds using the most effective solvent system from TLC. The purified compounds were analysed using liquid chromatography/mass spectrometry (LC/MS) to confirm their identity and purity. The chemical composition of crude extracts from egg masses and hypobranchial glands were also compared by LC/MS and found to contain a very similar percent composition of the main brominated compounds. In order to inhibit the degradation of tyrindoleninone, a fraction containing tyrindoleninone and tyrindolenine was exposed to oxygen overnight in the presence of two antioxidants, Vitamin A and Vitamin E, and then reanalysed by LC/MS. The synergic anti-proliferative effect of tyrindoleninone with the most effective antioxidant was then tested on HT29 cells using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl) tetrazolium bromide (MTT) assay. The antioxidant experiment showed that 0.1 % Vitamin E was the most effective antioxidant for inhibiting tyrindoleninone degradation, but it failed to increase the cytotoxic effect of tyrindoleninone on HT29 cells and in fact appeared to provide some protection against the cytotoxic properties of tyrindoleninone. In the next in vitro experiments, an egg mass extract was used for purification of the bioactive compounds with the optimised flash silica chromatography method. Bioassay guided fractionation was performed to identify the compounds with the greatest antiproliferative effects against colon cancer cells. The identity of the main bioactive compounds was confirmed by LC/MS, GC/MS and NMR as tyrindoleninone (more than 99% purity) and 6-bromoisatin (90% purity). These compounds were then tested for cytotoxic, apoptotic or necrotic effects using MTT, caspase 3/7 and membrane integrity assays respectively, on HT29 and Caco2 cells. The apoptotic effects of the bioactive compounds were confirmed by flow cytometry using Annexin-V-FITC and PI staining. Cell cycle analysis was also performed on HT29 cells treated with the most bioactive compound. The 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay showed that semi-purified 6-bromoisatin inhibited the viability of both cell lines (IC50= 100 μM). The fraction containing 6-bromoisatin activated caspase-3 and -7 enzymes in Caco2 and HT29 cells at approximately 100 μM (0.025 mg/mL) and 200 μM (0.05 mg/mL) respectively, much lower concentrations than those required to cause LDH release and necrosis (~1000 to ~2000 μM). Flow cytometry showed that semi-purified 6-bromoisatin (~200 μM) induced 77.6% apoptosis in HT29 cells. Cell cycle analysis showed the accumulation of 25.7% of HT29 cells treated with semi-purified 6-bromoisatin (~100 μM) in G2/M phase. The other compound, tyrindoleninone, was also found to inhibit the proliferation of Caco2 cells (IC50= 98 μM) and HT29 (IC50= 390 μM). Caspase-3 and -7 activity significantly increased only in HT29 cells treated with 195 μM (0.05 mg/mL) tyrindoleninone. LDH was released in both cell lines treated with high concentrations of tyrindoleninone. In an in vivo trial, the effects of the purified tyrindoleninone and semi-purified 6-bromoisatin, along with the crude extract were tested for prevention of colorectal cancer in a two week mouse trial to determine whether these compounds can enhance the acute apoptotic response to genotoxic carcinogens (AARGC). The anti-proliferative effects of the extract and purified/semi-purified compounds were also tested by immunohistological examination using Ki-67 antibody. To evaluate any possible toxicity of the compounds, mouse general health, behavior, body weight and liver weight were assessed. Liver damage was also tested using histopathology and also biochemistry by measuring liver enzymes (ALT, AST and ALP) in the serum. Some other biochemical and also hematological blood tests were performed to evaluate any other toxicity or side effects in blood and kidney. Semi-purified 6-bromoisatin (0.05 mg/g) was found to be the most bioactive compound in the crude extract capable of enhancing the apoptotic index in distal colon of mice. Tyrindoleninone did not increase the apoptotic index significantly. Semi-purified 6-bromoisatin did not show any toxic effect on liver, as indicated by no significant difference in the liver enzymes in comparison to the controls. In contrast, tyrindoleninone caused an increase in AST level compared to the saline control and also caused a reduction in red blood cell counts. In my last experiment, pure synthetic 6-bromoisatin was tested for in vitro anticancer activity and prevention of the colorectal cancer using the same in vivo model. Administration of pure synthetic 6-bromoisatin to the mice, confirmed the results from the semi-purified 6-bromoisatin, with a significant increase in apoptosis at 0.05 mg/g, without any sign of toxicity in the liver or blood cells. However, a decrease in the potassium levels in the blood indicated the possibility of a diuretic effect associated with synthetic 6-bromoisatin. This research confirmed the anticancer effects of 6-bromoisatin against two colorectal cancer cell lines in vitro, as well as the potential cancer preventative effects in vivo based on the ability to induce apoptosis in DNA damaged cells. This supports the potential development of this molluscan extract or natural 6-bromoisatin as a nutraceutical for chemoprevention of colorectal cancer. In addition, synthetic 6-bromoisatin is a promising lead for further pharmaceutical development for prevention of this disease. However, future studies in longer term animal models are required to confirm that the early stage prevention of tumors by apoptosis in DNA damaged cells by 6-bromoistain does prevent the formation of actual tumors at the later developmental stages.
Keywords: marine mollusc,colorectal cancer,cancer prevention
Subject: Biological Sciences thesis
Thesis type: Doctor of Philosophy
School: School of Biological Sciences
Supervisor: Associate Professor Catherine Abbott