An in vitro assessment of pharmacodynamic interactions between selected herbal extracts and anticancer chemotherapeutic agents
- Authors: Hwehwe, Nyashadzashe , Swanepoel, Bresler
- Date: 2024-04
- Subjects: Chemotherapy , Herbs -- Therapeutic use , Antineoplastic agents
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10948/63506 , vital:73383
- Description: Cancer is a disease caused by uncontrolled growth and spreading of abnormal cells to distant body parts. Approximately 19.3 million and 10 million new cancer cases and cancer deaths, respectively were recorded in 2020. Despite advancements in prevention and therapeutic strategies, cancer remains a global health problem. Cancer patients are increasingly seeking complementary and alternative medicines, of which herbal medicines are the most common form of CAM used by patients. Numerous in vitro and in vivo studies of herbal medicines in cancer have shown that they have antioxidant and anticancer (antitumor and anti-proliferative) properties. While concurrent use with conventional cancer treatments may improve treatment efficacy, alleviate chemotherapy-related side effects, boost the immune system, or impede drug resistance, it can also prompt drug-herbal interactions, and this may affect the pharmacodynamics and pharmacokinetics of the chemotherapeutic drug. This study aimed to determine relevant pharmacodynamic interactions with chemotherapeutic drugs and investigate such interactions' mechanisms. The objectives of the study were to screen the cytotoxicity effects of drug compounds, herbal extracts, and drug-herb combinations, to determine the ability of treatments to induce apoptosis, and to determine the most beneficial treatment. The cytotoxic effects of cisplatin in HeLa cells, tamoxifen in MCF7 cells, 5-fluorouracil in Caco-2 cells, and grapeseed, green tea, fermented rooibos, and green rooibos in all the listed cell lines were evaluated individually and in combination using the bis-Benzamide H 33342 trihydrochloride/propidium iodide (Hoechst 33342/PI) dual staining method. CompuSyn 1.0 Software was used to quantify synergism and antagonism. The mechanism of apoptosis induction of the different synergetic combinations, drug compounds, and herbal extracts was illustrated by quantitative fluorescence image analysis, specifically cell cycle analysis, phosphatidylserine translocation, mitochondrial membrane potential analysis, caspase 3 activation, and reactive oxygen species production using the relevant contrast dyes. Grapeseed displayed cytotoxicity towards MCF7 and HeLa cells (IC50 57.98 and 83.28 μg/mL, respectively). Green tea was only cytotoxic against HeLa cells at an IC50 of 91,92 μg/mL. None of the extracts displayed cytotoxicity against Caco-2 cells (IC50 values > 200μg/mL). If the herbal extracts had inconclusive IC50 values in the three cell lines subsequent experiments were conducted using 100 μg/mL. The results showed that most of the combinations were antagonistic but, some combinations had synergistic or enhancement effects(1:3 for cisplatin with grape seed or green tea and tamoxifen with grapeseed or green tea, and 3:1 for cisplatin with green tea as well as for tamoxifen with fermented rooibos or green rooibos, and all the 1:1 combinations of 5- fluorouracil with all herbal extracts) with a combination index (CI) < 1. Grape seed and green tea were found to induce apoptosis in the three cell lines. Assays that were conducted to detect apoptosis induction showed positive staining for phosphatidylserine (PS), activated caspase 3, and reactive oxygen species (ROS), mitochondrial membrane depolarization. Analysis of the cell cycle showed two things; 1) that grape seed and green tea were apoptotic in HeLa and MCF7 cells only, and 2) the extracts of green tea and green rooibos, and the combinations of grape seed with all drug compounds arrested the cell in more than one phase of the cell cycle. The combinations of grapeseed and green tea potentially induced apoptosis in various manners but those with both rooibos extracts were unclear in all the cell lines. The results for combinations with grapeseed and green tea are promising and provide a basis for further research as combinations of chemotherapeutic drugs and herbal extracts may be effective therapeutic strategies. , Thesis (MPharm) -- Faculty of Health Sciences, School of Clinical Care & Medicinal Sciences, 2024
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- Date Issued: 2024-04
Establishment of a high-content neurodegenerative disease model screening platform
- Authors: Swanepoel, Bresler
- Date: 2023-12
- Subjects: Molecular neurobiology , Nervous system -- Diseases , Nervous system -- Degeneration
- Language: English
- Type: Doctorial theses , text
- Identifier: http://hdl.handle.net/10948/62644 , vital:72906
- Description: The identification of viable therapeutic targets and new treatments for central nervous system disorders, especially neurodegenerative diseases, remain major challenges in the field of drug discovery. Over the past few years there has been a steady decline in the turnaround time of current screening processes to yield viable drugs. Therefore, an increasing need exists for better technologies, protocols, and the screening of larger libraries. High-throughput screening provides the best solution to this problem and has become a key part in the drug discovery and development process. Likewise, high-content analysis has gained popularity over the past few years and is suitable for high-throughput screening. The aim of this study was to establish a comprehensive in vitro neuroprotective screening platform incorporating high throughput screening, using Parkinson’s disease as the neurodegenerative disease of interest. To evaluate the success of this platform, the neuroprotective potential of two mushrooms (Hericium erinaceus and Phlebopus sudanicus), two plants (Lippia javanica and Myrothamnus flabellifolia) and two seaweeds (Eucheuma denticulatum and Kappaphycus alvarezii) were investigated. Aqueous and ethanolic extracts of the selected natural products were evaluated across 21 parameters associated with four hallmarks of neurodegeneration: acquiring senescence, acquiring cell death, neuroinflammation and altered metabolism/cell survival. Based on the effects of these selected natural products on the 21 parameters, their potential mechanisms of action were elucidated. In addition to this, the natural products were scored under each of these therapeutic targets in an attempt to identify the most suitable animal models for future studies. The scoring system indicated that animal models investigating anti-senescence ability would be more suited for extracts of H. erinaceus, P. sudanicus and E. denticulatum whereas studies investigating the prevention of cell death would be more suited for extracts of E. denticulatum, L. javanica and K. alvarezii. Likewise, models based on neuroinflammation would be best suited for extracts of H. erinaceus, E. denticulatum and L. javanica while studies examining altered metabolism/cell survival would be best suited to extracts of H. erinaceus, E. denticulatum, K. alvarezii and M. flabellifolia. Considering the pleiotropic nature of the selected natural products, models that can combine these therapeutic targets could be of great interest. 6-OHDA proved to be capable of inducing favourable effects, in all the parameters investigated, with regard to a neurodegenerative state. However, it is known to have some disadvantages when it comes to pathological features such as the lack of the ability to induce Lewy body formation. Choosing the correct inducer remains a daunting task and no model, whether cell-based or animal-based, exists yet in which all the features of neurodegenerative diseases have been successfully replicated. The limitations of the current models, however, does not mean that they do not produce valuable insights. This is especially true if the mechanism of action for a specific compound or natural product is unknown. Animal models are still indispensable for the validation and interpretation of the results obtained from cell models with particular importance to toxicity. Therefore, this study assessed the best studied extract with the highest overall score for its toxicity using a zebrafish larvae-based model. Assessment of the toxicity of H. erinaceus revealed that both aqueous and ethanolic extracts resulted in death at the highest concentrations. This was supported by the results obtained in the in vitro cytotoxicity screening. In conclusion, this highlighted the importance of using physiologically relevant concentrations and supported the translational value of the current cell-based screening model to animal models and possibly humans. The findings of the present study suggest that a scoring system, which categorizes the different activities of selected natural products into distinct groups, can be a useful tool to improve the translatability of in vitro results to animal models. Furthermore, the current study arguably provides the most comprehensive neuroprotective screening platform in existence. Future research can look at expanding the platform through incorporation of additional parameters based on other hallmarks of neurodegeneration, not covered in this study, including protein folding and aggregation, altered epigenetics and the examination of other neuronal markers such as the involvement of astrocytes, oligodendrocytes, and microglia. In addition to this, future research can make use of more sophisticated cell models such as differentiated, human induced pluripotent stem cells and three-dimensional cultures. , Thesis (PhD) -- Faculty of Science, School of Biomolecular and Chemical Sciences, 2023
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- Date Issued: 2023-12
New synergic biomaterials for anti-cancer therapy
- Authors: Swanepoel, Bresler
- Date: 2019
- Subjects: Pharmaceutical chemistry , Cancer -- Research , Biomedical materials
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10948/43957 , vital:37087
- Description: In the last two decades, anti-cancer therapy has grown considerably with the help of both natural and synthetic anti-cancer compounds but, the search for new and improved cancer treatment remains an ongoing and important issue. Some anti-cancer compounds such as cisplatin are limited by their toxicity in normal tissues and the development of drug resistance. Therefore, in order to address drug resistance and side-effects of anti-cancer agents, recent research has been focusing on finding novel combinations of anti-cancer agents that have non-overlapping mechanisms of action. The first objective of this study was to determine the mechanism of action of Anemone nemorosa, Artemisia afra, N-[[3-(4-bromophenyl)-1H-pyrazol-5-yl]-carbamothioyl]-4-chloro-benzamide (BC-7) and N-benzoyl-N’-(3-(4-bromophenyl)-1H-pyrazol-5-yl)-thiourea (BT-7) through cell cycle arrest, phosphatidylserine translocation (PS), caspase activation and mitochondrial membrane depolarization. This study has shown that A. nemorosa, BC-7 and A. afra are capable of inducing cell death within three cancer cell lines namely HeLa, MeWo and HepG2, at varying degrees. HeLa cells were the most susceptible to treatment with A. nemorosa and BC-7 with IC50 values of 20.33 ± 2.480 μg/ml and 65.58 ± 8.400 μM (28.58 ± 3.660 μg/ml), respectively. A. afra was the most active against HepG2 cells with an IC50 value of 37.55 μg/ml. BT-7 was not cytotoxic against any of the cancer cell lines. The effects on HeLa cells and their progression through the cell cycle indicated that cells were arrested in the early M phase for all treatments. The induction of apoptosis was confirmed by an increase in PS translocation and activation of caspase 3 and 8 as well as a decrease in the mitochondrial membrane potential. It was deduced that A. nemorosa, A. afra and BC-7 induce caspase-dependent apoptosis in a mitochondrial dependent manner. The second objective of this study was to investigate the potential of A. nemorosa, A. afra and BC-7 to target various mediators involved in the inflammatory response as an alternative method in which cell death may be induced. Most treatments indicated that a tumour-elicited inflammatory response is indeed induced in HeLa cells and that the significant activation of nuclear factor kappa B (NF-κB) favoured the production of nitric oxide (NO) over cyclo-oxygenase 2 (COX-2). However, treatments with A. nemorosa, BC-7 and A. afra at their IC10 showed the potential of inhibiting this response. ROS levels were increased by most treatments and support the idea of ROS-mediated apoptosis. The third objective was to investigate combination treatments of these extracts and compounds for their potential synergistic cytotoxic activity and thus formulating the combinations as potential anti-cancer agents. Thirty combination mixtures were prepared using the IC50 values of each extract or compound at ratios of 1:3, 1:2, 1:1, 2:1 and 3:1, respectively. The cytotoxic/anti-proliferative activity of each mixture was determined by the bisBenzamide H 33342 trihydrochloride/propidium iodide (Hoechst 33342/PI) dual staining method on HeLa cervical cancer cells. The combination index (CI) values, at inhibition of 50% of HeLa cell growth, for each combination mixture, were determined by means of the Chou and Talalay method. The combined effect can then be indicated as CI < 1, synergism; CI = 1, additive effect or CI > 1, antagonism, respectively. Most combination treatments showed to have an antagonistic effect except for cisplatin:BC-7 (1:3, 1:1, 2:1, 3:1) and cisplatin:A. afra (1:3, 1:2, 1:1, 3:1) combinations that showed synergism. The 1:2 ratio of cisplatin:BC-7 and the 2:1 ratio of cisplatin:A. afra were additive. CI values were also calculated at inhibition of 10, 25 and 75% of HeLa cell growth, for each combination mixture. Antagonistic effects were frequently observed at lower effect levels such as at 10 and 25% inhibition of growth. However, this was not seen for the cisplatin:BC-7 combinations as all the ratios indicated synergism. Some of these ratios, such as the 1:3 and 1:2, even led to a greater degree of synergism being obtained, with noticeable antagonistic effects seen at 50 and 75% inhibition of growth. The current finding is that BC-7 and A. afra could lower the dose of cisplatin in combination to achieve a similar anti-cancer efficacy compared to the higher cisplatin dose when used alone. The lower dosage in combination could result in reduced drug resistance as well as limit the toxicity on normal cells associated with cisplatin treatment. In conclusion, this study shows, for the first time, that A. nemorosa has the potential to induce apoptosis and also has some anti- and pro-inflammatory activity in HeLa cancer cells. This study also enhanced the knowledge of the mechanism of apoptosis induction of BC-7, in a more detailed manner, as well as investigated its inflammatory effects for the first time. Results obtained for A. afra correlated nicely to previously reported studies and confirmed that the methods used in this study, although different, leads to the same conclusions. Combination treatments also indicated, for the first time, that BC-7 and A. afra have the ability to function in a synergic manner with cisplatin and proves that, although extensive research may have been done on a plant or compound, more can be discovered. This new information can lead to identification of new compounds in the plants and the integration of signalling pathways that can be targeted for treatment of cancer.
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- Date Issued: 2019