Cytotoxic effects of Phyllanthus watsonii airy shaw extract in combination with 5-fluorouracil on human colon cancer cells / Amira Nadirah Roslan

Amira Nadirah, Roslan (2018) Cytotoxic effects of Phyllanthus watsonii airy shaw extract in combination with 5-fluorouracil on human colon cancer cells / Amira Nadirah Roslan. Masters thesis, University of Malaya.

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Abstract

Colorectal cancer (CRC) is one of the most common cancers that affects both men and women in Malaysia and is considered as one of the leading causes of death in the world. The chemotherapeutic agent that is used to treat CRC is usually 5-Fluorouracil (5-FU). However, the resistance of CRC cells to this drug and the side effects caused by this drug have prompted new strategies to overcome the shortcomings. The aims of this study are (i) to investigate the cytotoxic effects of Phyllanthus watsonii ethyl acetate extract (PW-E) in combination with 5-FU on human colon cancer cell lines HT-29 and HCT-116, (ii) to evaluate the possible interaction (synergistic, antagonistic or additive) occurs during combination between PW-E and 5-FU in their cytotoxicity based on the median-effect principle, and (iii) to determine the potential cell death mechanisms via apoptosis that is triggered by the combination of PW-E and 5-FU. The cytotoxic effects of PW-E, 5-FU and PW-E/5-FU combination on HCT-116 and HT-29 after 72 hours of were determined by Neutral Red Uptake (NRU) assay. The IC50 of PW-E, 5-FU and PW-E/5-FU combination (at a ratio of 3:1) on HCT-116 cell were 1.522 ± 0.16 μg/ml, 1.588 ± 0.46 μg/ml and 1.482 ± 0.25 μg/ml respectively while the IC50 on HT-29 were 0.030 ± 0.003 μg/ml, 19.70 ± 1.72 μg/ml and a combination of 0.03 μg/ml of PW-E and 10.0 μg/ml of 5-FU respectively. By combining PW-E with 5-FU, lower dose of drug was needed, indicating that the cytotoxicity effect of 5-FU towards the cancer cells was enhanced in the presence of PW-E. Our result also shows that the Combination Index (CI) of PW-E/5-FU combination analysed by Compusyn 1.0 software was < 1 on both HCT-116 and HT-29 cells. This indicates that PW-E/5-FU combination exerts a synergistic effect on both CRC cells. In addition, PW-E/5-FU combination is selectively cytotoxic towards the CRC cells in comparison with the normal lung fibroblast MRC-5 cells. Drug Reduction Index (DRI) for PW-E/5-FU combination was measured on how much (-fold) the dose of a drug or agent in synergistic combination may be reduced at a given effect level compared with the dose of each drug alone. The DRI value for PW-E/5-FU combination on both HCT-116 and HT-29 is > 1 and shows that the combination is favourable in terms of clinical therapy. Morphological assessment by Acridine Orange / Ethidium Bromide (AO/EB) double staining showed that cell death mainly occurs by apoptosis instead of necrosis. Increase in caspase-3 activity was observed in CRC cells treated with PW-E/5-FU combination. PW-E was subjected to Liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis and six main compounds were identified. The compounds detected were quercetin, kaempferol rhamnoside, kaempferol glucoside, ellagic acid, hyperin and strictinin isomer. In conclusion, herb-drug combination enhances the cytotoxic activity of 5-FU towards colorectal cancer cells and the cell death is mediated by apoptosis. Hence, further studies should be done to understand further the interactions between the herb and drug.

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