Cordier, Werner2024-12-122024-12-122025-042024-08-01*A2025http://hdl.handle.net/2263/99954Dissertation (MSc (Pharmacology))--University of Pretoria, 2024.Tamoxifen remains the preferred standard choice of treatment for oestrogen receptor- and/or progesterone-positive breast cancer among females. Unfortunately, approximately 30% of these patients develop resistance to tamoxifen (de novo or acquired), despite an initial positive response to the treatment. Tamoxifen-resistance presents an obstacle to hormone treatment since it has often been associated with poor survival rates. Due to such resistance, alternatives have been investigated either to replace treatment, or reverse resistance mechanisms. The use of complementary and alternative medicine has drastically increased among breast cancer patients. It is used either exclusively, or in addition to prescribed treatment (termed integrative medicine). Garlic (Allium sativum L.) is known to contain a variety of phytochemicals potentially beneficial in disease treatment due to anti-proliferative, anti-oxidative, and anti-inflammatory effects. However, sparse data is available on the combinational use of crude garlic extracts with tamoxifen in breast cancer treatment. The aim of the project was to determine the effects of crude garlic extracts in combination with tamoxifen in tamoxifen-sensitive breast adenocarcinoma cell lines. Ethnomedicinal (hot-water) and pharmaceutical-representative (methanol) extracts of the garlic bulb were prepared by brewing and ultrasonic maceration, respectively. Preliminary cytotoxicity evaluation of the crude garlic extracts and 4-hydroxytamoxifen on MCF-7 breast adenocarcinoma cells were performed using the sulforhodamine B (SRB) assay. Thereafter, the synergistic cytotoxic evaluation of the combination of the crude garlic extracts and 4-hydroxytamoxifen were performed in a checkerboard manner using the SRB assay. The mechanism of action of the crude garlic extract, 4-hydroxytamoxifen, and combination thereof, was assessed by determining the alterations to the cell cycle, levels of nitric oxide and lipid peroxidation, and the activity of caspase-3/7. Crude hot-water and methanol extracts were prepared successfully; however, the extracts showed minimal inherent cytotoxicity as a half-maximal inhibitory (IC50) concentration could not be calculated for either extract at the highest concentration tested (100 µg/mL). The cytotoxicity of 4-hydroxytamoxifen was determined with an IC50 of 10.99 µM. Combining the extracts (7.5, 15 and 30 µg/mL) with 4-hydroxytamoxifen (¼IC50, ½IC50 and IC50) yielded a range of compound interactions including synergistic, additive, and antagonistic effects. The combination with the highest synergistic activity was 7.5 µg/mL of the hot-water extract and 10.99 µM of 4-hydroxytamoxifen (combination index = 0.624). The hot-water extract (7.5 µg/mL) potentially induced cell cycle arrest in MCF-7 cells at G0/G1 phase. The cells treated with 4-hydroxytamoxifen (10.99 µM) indicated cell death as most cells were in the sub-G1 phase. The combination of the hot-water extract and 4-hydroxytamoxifen arrested more MCF-7 cells in the G0/G1-phase similarly to the solo treatment with the hot-water extract. The hot-water extract (7.5 µg/mL; p < 0.05) and 4-hydroxytamoxifen (10.99 µM; p < 0.001) alone significantly increased nitric oxide levels. Furthermore, the combination of the of hot-water extract and 4-hydroxytamoxifen increased nitric oxide levels significantly (p < 0.001). Treatment with the hot-water extract (7.5 µg/mL) significantly (p < 0.001) decreased lipid peroxidation suggestive thereof that the hot-water extracts possess intrinsic anti-oxidant properties, while 4-hydroxytamoxifen (10.99 µM) significantly (p < 0.001) increased lipid peroxidation. Though, the combination of hot-water extract and 4-hydroxytamoxifen increased lipid peroxidation significantly (p < 0.001), the amplified nitric oxide levels is also associated with nitrosative stress resulting in lipid peroxidation. The hot water extract (7.5 µg/mL) did not significantly decrease nor increase caspase-3/7 activity after 24 or 48 h, respectively. However, 4-hydroxytamoxifen did increase caspase-3/7 activity significantly after 24 (p < 0.01) and 48 h (p < 0.001). The combination of the hot-water extract and 4-hydroxytamoxifen increased caspase-3/7 activity significantly (p < 0.001; for both) after 24 and 48 h incubation. The mechanism of cell death induced by the combination of the hot-water extract and 4-hydroxytamoxifen appears to be mediated by the synergism of the individual treatments. The use of a synergistic combination in tamoxifen-resistant breast cancer cells have the potential to re-sensitise resistant cells to tamoxifen. This could be a step towards overcoming resistance in breast cancer patients, thereby improving prognosis.en© 2023 University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria.UCTDSustainable Development Goals (SDGs)4-hydroxytamoxifenBreast cancerCombinational treatmentGarlicMCF-7 cellsDissertationu1820748210.25403/UPresearchdata.27765561