http://hdl.handle.net/10386/70 Sun, 05 Apr 2026 15:25:50 GMT 2026-04-05T15:25:50Z http://hdl.handle.net/10386/5198 Examination of the effects of HIV protease inhibitors (HIV-PIs) on the function of the novel s-DAPK-1 in Human Papilloma Virus (HPV)-related cervical cancers Makgoo, Lillian Cervical cancer treatment continues to be every country’s nightmare due to ineffectiveness and non-specificity of the current therapeutic options, late diagnosis and chemo drug resistance. An escalating resistance of cervical cancer cells to chemotherapy coupled with severe side effects of commonly used cytotoxic drugs has intensified the need to search for new anti-cancer agents. Several drugs initially approved for non-cancerous conditions have recently been found to possess cytostatic effects on cancer cells. Thus, these drugs could be expediently repurposed for use as anti-cancer agents because they have already been tested for safety in animals and humans. In light of this, this study sought to investigate the possibility of adapting pure HIV protease inhibitors (HIV-PIs) and their over-the counter tablets for anti-cervical cancer therapeutic purposes. Additionally, since cervical cancer is viewed as a pathology that is partly driven by genes, it was of interest to understand the expression of short-DAPK-1 known as s-DAPK-1, which remains unexplored in cervical cancer, and the HIV-PI s' mechanisms of action. Therefore, this study was aimed at investigating the effect of HIV-PIs on s-DAPK-1 and other cancer-related genes in HPV-induced cervical cancer cells. To address the aim of this study, the MTT viability and Muse™ Count & Viability assays were used to evaluate the effect of the pure HIV protease inhibitors and their tablet forms on the viability of CaSki and HeLa cervical cancer cell lines, as well as on the non-cancerous cells (HEK-293). To detect the mode of death induced by pure HIV-PIs (lopinavir and atazanavir) and their tablet forms (Aluvia and Ritoataz) in HPV-associated cervical cancer cells, apoptosis was assessed using the Annexin V Assay. Apoptosis-related proteins regulated by HIV-PIs and their tablet forms were detected using the Human Apoptosis Array profiler. In addition, the Muse™ Cell Cycle assay was used to assess the effect of HIV-PIs and their tablet forms on cell cycle progression of the cervical cancer cells. The Polymerase Chain Reaction (PCR) was used to determine the expression of s-DAPK-1 in HIV-PIs-treated and untreated cervical cancer cells, and to elucidate the effect of pure HIV protease inhibitors and HIV protease inhibitor tablets on its expression. In addition to the use of PCR, the proteome profiler human apoptosis array kit was used to detect other cellular targets of pure HIV-PIs and their over-the counter tablets in HPV-associated cervical cancer cells. Furthermore, various bioinformatics tools such as ProtScale, ProteinPrompt, I-TASSER, PSIPRED, ProtParam, ScooP, Hawkdock, Phyre2, SAVES and PROCHECK along with user-friendly databases such as NCBI, TarBase and Protein Data Bank (PDB) were used to understand s-DAPK-1 regulation, 3D structure, physicochemical and thermodynamic properties. This study demonstrated that lopinavir and atazanavir pure HIV-PIs, as well as Aluvia and Ritoataz tablets did not affect the viability of non-cancerous cells (HEK-293), confirming the safety of the HIV-PIs. However, they have significantly decreased the viability of the CaSki and HeLa cervical cancer cells in a dose- and time-dependent manner. It is important to note that, relative to HeLa cells, a higher concentration of lopinavir (IC50=150 μM) and Ritoataz (IC50=180 μM) was required to reduce the viability of CaSki cells by 50%. All the HIV-PIs triggered apoptosis in CaSki and HeLa cervical cancer cells. However, lopinavir (39.925±1.483 in CaSki and 41.583±1.001 in HeLa) and atazanavir (49.092±0.9376 in CaSki and 36.717±1.729 in HeLa) significantly (p ≤ 0.001) exhibited the highest induction of apoptosis. In contrast, the corresponding tablets of Aluvia (24.418± 2.346 in CaSki and 26.795±0.6805 in HeLa) and Ritoataz (25.310±1.323 in CaSki and 28.432±2.374 in HeLa) induced a significantly (p ≤ 0.001) lower levels of apoptosis. In addition, pure HIV protease inhibitors along with their tablet forms significantly (p ≤ 0.05) regulated the activity of various apoptosis-related proteins, including phosphorylation p53 (S392) and Rad17 (S635). The HIV-PIs upregulated SMAC/Diablo, and Bcl-2, suggesting induction of an intrinsic apoptosis pathway, with cervical cells resisting cell death by upregulating Bcl-2. The s-DAPK-1 variant was significantly downregulated in HeLa cells relative to non-cancerous HEK-293 cells, suggesting that it may be a tumour suppressor. In addition, pure HIV protease inhibitors and the HIV protease inhibitor tablets did not influence the expression of s-DAPK-1 in cervical cancer cells. Furthermore, the in-silico approach, to determine s-DAPK-1 regulation, successfully identified several s-DAPK-1-specific microRNAs. In addition, phyre2 database demonstrated that the s-DAPK-1 isoform possesses 40% alpha helices and 4% beta strands, forming a stable 3D structure. Moreover, s-DAPK-1 was discovered to withstand high temperatures and to interact with a variety of proteins involved in tumour progression and gene regulation, including Prion protein and Histone H2B type 2-E (H2B2E). The findings of this study highlight the HIV protease inhibitors as promising anticancer agents, demonstrating significant effects on inducing cell death and suppressing proliferation. Furthermore, this work has discovered more anticancer drug targets that should be exploited for drug development. Moreover, since this is the first study to explore the expression and regulation of s-DAPK-1 by therapeutic agents, there is a pressing need to identify novel compounds that can modulates s-DAPK-1 and to explore its potential tumour suppressor function in cancer cells. Thesis (Ph. D. (Biochemistry)) -- University of Limpopo, 2025 Wed, 01 Jan 2025 00:00:00 GMT http://hdl.handle.net/10386/5198 2025-01-01T00:00:00Z http://hdl.handle.net/10386/5150 Exploration of the regulation of immunomodulatory genes by commelina benghalensis aqueous extra in breast cancer cells Mnisi, Hellen Laura Cancer is a noteworthy global health issue, and according to predictions by the World Health Organization, the number of new cases was projected to increase to approximately 29-37 million by 2040, worldwide (WHO, 2020, Sung et al., 2021). Female breast cancer has been shown to be the leading cause of global cancer incidence in 2020, with reported 2.3 million new cases, representing 11.7% of all cancer cases (Reyes-Monasterio et al., 2022). Treatments such as surgery, radiotherapy, and chemotherapy are used to counteract breast cancer; however, these treatment approaches are now ineffective, which has led to the shift of interest towards the use of medicinal plants such as Commelina benghalensis (Cb), which has been traditionally used in folk medicine to treat various ailments, including inflammation-related conditions. Studies reveal that the plant contains bioactive compounds, such as flavonoids, tannins and phenolics, which possess anti-inflammatory, antioxidant, and antitumor properties (Islam et al., 2018; Islam et al., 2017; Islam et al., 2016). Thus, the aim of the study was to determine the antioxidant and anticancer properties of Cb aqueous extract in breast cancer cells. Breast cancer cell lines, MDA-MB 231 and MCF-7, were cultured and maintained in appropriate growth media. The Cb was extracted using only water, and this was done to mimic what the traditional healers do, when they prepare medicinal plants concoctions for medicinal purposes. The profiling of the bioactive compounds was done using Thin Layer Chromatography (TLC), phytochemical screening, and the Liquid Chromatography-Mass Spectrometry (LC-MS). The total amount of phenols, flavonoids and tannin in the Cb aq-extract was quantified using the Folin-Ciocalteu Colorimetric Assay. Additionally, the antioxidant activity was done using 2, 2-Diphenyl-1-picrylhydrazyl (DPPH) and free radical antioxidant power (FRAP) assays. Furthermore, the cytotoxicity effect of Cb aqueous extract was determined against the breast cancer using 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) assay. The Muse® Cell Count and Viability assay was done to confirm the MTT results. Morphological changes were observed using light microscopy and the images were captured using computer connected to microscope. Moreover, Annexin V assay was conducted to investigate the potential cell death that may be induced by the Cb leaf-aq extract. The TLC plates demonstrated that the Cb root, leaf and stem-aq extract have bioactive molecules. The quantitative phytochemical analysis using Folin-Ciocalteu Colorimetric Assays, showed that the Cb aq extracts had high phenolic, flavonoid and low tannin contents Folin-Ciocalteu Colorimetric Assays. Moreover, the Cb aq-extracts demonstrated antioxidant properties, and this was mostly observed in the leaf and the root parts of the plant. The LC-MS results of Cb leaf-aq extract demonstrated the presence of bioactive compounds that have been previously shown to exhibit anticancer cancer immunomodulatory activities. In this study, three compounds with immunomodulatory effects; namely, Abietic acid, Vorinostat and Lauramide, were identified. These compounds regulate a number of cytokine genes, and these include TGF-b1, IGF1R, IFN, IL-2, IL-4, IL-10, IL-13. Additionally, the Cb leaf-aq significantly (**P ˂ 0.01, ***P ˂0.001 and ****P ˂0.0001) reduced the viability of MDA-MB 231 cells after 24- and 48-hours treatment with 500 and 1000μg/ml compared to other parts of the plant. Furthermore, the Cb leaf aqueous extract significantly (****P ˂0.0001) induced apoptosis of MDA-MB-231 cells after their treatment with the IC50 concentration (750μg/mL). These findings collectively support the conclusion that the phytocompounds identified in Cb have anticancer properties and a potential to modulate immunological responses in breast cancer cells. Thesis (M.Sc. (Biochemistry)) -- University of Limpopo, 2025 Wed, 01 Jan 2025 00:00:00 GMT http://hdl.handle.net/10386/5150 2025-01-01T00:00:00Z http://hdl.handle.net/10386/5043 Screening of carpobrotus edulis extracts for anticancer activity against cervical cancer cells Phoogole, Walter Introduction: Cancer is a primary cause of death and a significant impediment towards extending the life expectancy of human-kind. In terms of both incidence and fatality rates, cervical cancer is the fourth most common malignancy after lung cancer, breast cancer and colorectal cancer, worldwide. It is the second most common cancer and the primary cause of cancer fatalities in South African women. This cancer is primarily attributed to the cancer-causing Human papilloma virus (HPV). However, not all cases of cervical cancer are credited to this oncovirus. The soaring numbers can be ascribed to both inefficiencies associated with the current therapeutic strategies that are also related with adverse effects, and inadequacies linked to cancer diagnostic tools. Therefore, the development of novel anticancer medicines with improved efficacy and fewer side effects is critical. Thus, this study was aimed at evaluating the potential anticancer activity of Carpobrotus edulis extracts (C. edulis) against different cervical cancer cell lines. Methodology: C. edulis extracts were screened for the presence of various phytochemicals using the standard conventional phytochemical tests. The potential antioxidant activities of the plant extracts were quantified using both the 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) and ferric reducing antioxidant power (FRAP) assays. The probable identities of the compounds found in C. edulis extracts was determined utilizing the Liquid Chromatography-Mass Spectrophotometry (LC-MS) for identification of compounds in C. edulis. The 3-(4,5-dimethyl-thiozol-2yl)-2,5-diphenyltetrazolim bromide (MTT) assay was utilized to assess the effects of the C. edulis extracts on HeLa, CaSki and non-cancerous (Hek-293) cell lines. To further validate the cytotoxic effects of the extracts (IC50s) against the mentioned cells, the Muse™ Count & Viability assay was utilized. The Annexin V Apoptosis analysis was employed to investigate the potential cell death-inducing capacity of the C. edulis water extract against the cervical cancer cells. Furthermore, the proteome profiler human angiogenesis array was used to examine the potential effect of the water extract of C. edulis on angiogenesis in cervical cancer cell lines. xviii Results and Discussion: All the extracts were proven to possess various phytochemicals. C. edulis root extracts (acetone and ethanol) promoted the viability of cervical cancer cells while the aqueous extract had reduced the viability of both HeLa (IC50 = 1000μg/mL) and CaSki (IC50 = 1000μg/mL) cells but showed no effect against the non-cancer cells (Hek293).. The C. edulis aqueous extract induced minimal apoptosis in both CaSki (7.950 ± 1.422) and HeLa (8.933 ± 0.361) cells studied. C. edulis root water extract (1000 μg/ml) treated cells had higher levels of urokinase type-plasminogen activator (uPA), and vascular endothelial growth factor (VEGF) was somewhat higher in C. edulis root water extract treated cells than in curcumin-treated and untreated cells. Conclusion: The C. edulis root water extract was found to have potential anticancer effects on HeLa and CaSki cells, but safe against non-cancerous Hek-293 cells. Extracts from the roots of C. edulis in acetone or ethanol did not have any cytotoxic effects on HeLa or CaSki cells. HeLa or CaSki cell death were unaffected by the C. edulis root water extract, while VEGF and uPA protein levels, which promote angiogenesis, were modestly elevated. Thesis (M. Sc. (Biochemistry)) -- University of Limpopo, 2024 Mon, 01 Jan 2024 00:00:00 GMT http://hdl.handle.net/10386/5043 2024-01-01T00:00:00Z http://hdl.handle.net/10386/5040 The effects of curcumin derivatives on SARS-CoV-2 spike S1 protein induced oxidative stress on macrophage cells Nkwana, Maureen Ramadimetsa The corona virus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has emerged as a global health crisis that claimed almost 7 million lives and a total of more than 700 million cases to date with new variants still being reported in various countries. Currently, there is no specific treatment for COVID-19 patients and the sporadic emergence of new variants make vaccine development a delayed response towards management of future outbreaks. Infection of host cells via interaction with SARS-CoV-2 spike proteins is characterised by hyper-inflammation of the innate immune response leading to overproduction of reactive oxygen species (ROS) by macrophage. The increased ROS production ultimately overwhelms the antioxidant pool and promote elevated oxidative stress levels, converting mild symptoms to severe COVID-19 outcomes. Curcumin has shown potent antioxidant and anti-inflammatory properties in various pathological conditions. Thus, this study investigated the antioxidant potential of curcumin derivatives in ameliorating SARS-CoV-2 spike protein-induced oxidative stress using macrophage cells as the innate immune model system. The cytotoxicity effect of curcumin derivatives was assessed by using two viability assays, namely the colorimetric MTT and propidium iodide (PI) fluorescence assays on Raw 264.7 macrophage cells. The curcumin derivatives were shown to exert no cytotoxic effect on Raw 264.7 cells at low doses (1.25-0.625 mM). The Annexin-V/ PI assay was employed to evaluate the mode of cell death induced by these curcumin derivatives. The results obtained showed that the derivatives at 5 mM induce apoptosis as a mode cell death. The production of IL-6 pro-inflammatory cytokines was examined using ELISA from supernatant after stimulation with 100 ng/ml SARS-CoV-2 spike S1 and treated with 1 mM curcumin derivatives. Treatment of SARS-CoV-2 spike S1-stimulated Raw 264.7 macrophages with curcumin resulted in lower production of IL-6 compared to untreated SARS-CoV-2 spike S1 stimulated control cells. The oxidative stress muse kit and DAF-2 DA were used to determine the levels of reactive oxygen species (ROS) and nitric oxide (NO) produced by Raw 264.7 cells post SARS-CoV-2 spike S1 stimulation and curcumin 13 derivative treatment. Treatment with 1 mM curcumin derivatives reduced the production of ROS and NO. The Western blot assay was used to measure the expression levels of Nrf2, IκΒ-α and NF-κB inflammatory proteins. Western blot protein expression assay demonstrated that these curcumin derivatives upregulate the expression of Nrf2 and IκΒ-α while downregulating that of NF-κB. Collectively, this study suggests that curcumin derivatives possess remarkable anti-inflammatory and antioxidant properties, making them a potential therapeutic treatment option to explore for the management of the SARS-Cov-2 induced oxidative stress and hyper-inflammation associated with COVID-19 infection. Thesis (M. Sc. (Biochemistry)) -- University of Limpopo, 2025 Wed, 01 Jan 2025 00:00:00 GMT http://hdl.handle.net/10386/5040 2025-01-01T00:00:00Z