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
The anti-proliferative activity of drimia altissima and a novel isolated flavonoid glycoside against hela cervical cancer cells
- Authors: Nyambe, Mutenta Nsokolo
- Date: 2019
- Subjects: Cancer -- Research , Cervix uteri -- Cancer , Cervix uteri -- Cancer -- Diagnosis -- Developing countries
- Language: English
- Type: Thesis , Doctoral , DPhil
- Identifier: http://hdl.handle.net/10948/42770 , vital:36690
- Description: Cancer is one of the leading causes of mortality worldwide. About 44% of all cancer morbidity and 53% of all cancer mortality occur in countries with a low to medium Human Development Index (HDI). Thus, cancer is rapidly emerging as a serious threat to public health in Africa and most especially, sub-Saharan Africa. The International Agency for Research on Cancer (IARC) projects that there will be 1.28 million new cancer cases and 970 000 cancer deaths in Africa by the year 2030 owing to the increase in economic development associated lifestyles. The dominant types of cancer in Africa are those related to infectious diseases such as Kaposi’s sarcoma and cervical, hepatic and urinary bladder carcinomas. The main challenge to cancer treatment in Africa is the unavailability of efficacious anticancer drugs. This is because most developing countries can only afford to procure the most basic anticancer drugs, which are also frequently unavailable due to intermittent supplies. This results in patients progressing to more advanced cancer states. One way of combating this African problem is to focus on research that aims at discovering efficacious and cost effective cancer therapies from available natural resources within the African continent. This study investigated the potential anti-proliferative activity (against HeLa cervical cancer cells) of four plants (Adansonia digitata, Ceiba pentandra, Maytenus senegalensis and Drimia altissima) commonly used in the African traditional treatment of malignancies. After in vitro bio-assay screening using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, M. senegalensis root extract (MS-R) and D. altissima bulb extract (DA-B) showed anti-proliferative activity against HeLa cervical cancer cells with IC50 values of 25 μg/mL and 1.1 μg/mL respectively. By possessing the strongest anti-proliferative activity among the tested extracts, D. altissima was selected for further studies. Liquid-liquid partitioning of the Drimia altissima bulb extract with n-hexane, ethyl acetate, and n-butanol, yielded partitions 79a – d, with the n-butanol fraction, 79d, exhibiting the strongest cytotoxic activity (IC50 = 0.497 μg/mL). Through High Content Analysis (HCA) screening, fraction 79d was found to induce marked early mitotic cell cycle arrest. Fractionation of 79d using Diaion® HP-20 open column chromatography and a stepwise gradient of reducing polarity (water-methanol-ethanol-ethyl acetate) yielded cytotoxic fractions 82b, 82c, 82d and 82e, all with significant anti-proliferative activities at the tested concentrations of 0.1, 1.0 and 10 μg/mL. Bio-assay guided fractionation of 82c (the most effective fraction at the lowest tested concentration of 0.1 μg/mL) using Sephadex® LH-20 open column chromatography and 50% MeOH led to the isolation of compound 3.17. After structural elucidation using 1D and 2D Nuclear Magnetic Resonance spectroscopy (NMR), High resolution Mass spectrometry (HRMS), Fourier-Transform Infrared spectroscopy (FT-IR), ultraviolet spectroscopy (UV) and Circular Dichroism (CD), compound 3.17 was identified as a novel C-glucosylflavonoid-O-glucoside, 6-C-[-apio-α-D-furanosyl-(1→6)-β-glucopyranosyl]-4′, 5, 7-trihydroxyflavone (Altissimin, 3.17). Compound 3.17 exhibited a dose dependant anti-proliferative activity with an IC50 of 2.44 μM. The mechanism of action for compound 3.17 was investigated through cell cycle arrest, phosphatidylserine translocation (PS), caspase activation and mitochondrial membrane depolarization. The mechanism of cell death elicited by compound 3.17 in HeLa cells was found to involve the induction of M phase cell cycle arrest with consequent activation of apoptotic cell death which was evident from annexin V staining, mitochondrial membrane potential (ΔΨm) collapse and the activation of caspases -8 and -3. In silico computational techniques were employed to virtually determine potential biological targets of compound 3.17. Target fishing using the Similarity Ensemble Approach (SEA) target prediction gave human aldose reductase (hAR, AKR1B1) the highest ranking with a p value of 2.85 x 10-24, a max Tc of 0.35 and a Z-score of 41.8217. Using AutoDock4 and the AutoDock tools suite (ADT), molecular docking of compound 3.17 in the hAR binding pocket was successfully achieved with a lower ΔG free energy binding (-9.4 kcal/mol) than that of positive control ligand 393 (-8.7 kcal/mol). In conclusion, this study identified the genus Drimia and particularly D. altissima as a potential source for novel cytotoxic compounds. The discovery of altissimin (3.17), the first flavonoid glycoside to be isolate from D. altissima, enquires into the possible existence of similar compounds within the species. In addition to the observed in vitro cytotoxic activity against HeLa cells, the potential of altissimin (3.17) as a hAR enzyme inhibitor opens up the possibility of its use as an adjunct to increase cancer cell sensitivity to chemotherapy. Thus, altissimin (3.17) shows promise as a potential anticancer agent.
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- Date Issued: 2019