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Item Moringa oleifera gold nanoparticles modulate oncogenes, tumor suppressor genes, and Caspase-9 splice variants in A549 cells(Wiley Online Library, 2016) Tiloke, Charlette; Phulukdaree, Alisa; Anand, Krishnan; Gengan, Robert MoonsamyGold nanoparticles (AuNP's) facilitate cancer cell recognition and can be manufactured by green synthesis using nutrient rich medicinal plants such as Moringa oleifera (MO). Targeting dysregulated oncogenes and tumor suppressor genes is crucial for cancer therapeutics. We investigated the antiproliferative effects of AuNP synthesized from MO aqueous leaf extracts (MLAuNP) in A549 lung and SNO oesophageal cancer cells. A one‐pot green synthesis technique was used to synthesise MLAuNP. A549, SNO cancer cells and normal peripheral blood mononuclear cells (PBMCs) were exposed to MLAuNP and CAuNP to evaluate cytotoxicity (MTT assay); apoptosis was measured by phosphatidylserine (PS) externalization, mitochondrial depolarization (ΔΨm) (flow cytometry), caspase‐3/7, −9 activity, and ATP levels (luminometry). The mRNA expression of c‐myc, p53, Skp2, Fbw7α, and caspase‐9 splice variants was determined using qPCR, while relative protein expression of c‐myc, p53, SRp30a, Bax, Bcl‐2, Smac/DIABLO, Hsp70, and PARP‐1 were determined by Western blotting. MLAuNP and CAuNP were not cytotoxic to PBMCs, whilst its pro‐apoptotic properties were confirmed in A549 and SNO cells. MLAuNP significantly increased caspase activity in SNO cells while MLAuNP significantly increased PS externalization, ΔΨm, caspase‐9, caspase‐3/7 activities, and decreased ATP levels in A549 cells. Also, p53 mRNA and protein levels, SRp30a (P = 0.428), Bax, Smac/DIABLO and PARP‐1 24 kDa fragment levels were significantly increased. Conversely, MLAuNP significantly decreased Bcl‐2, Hsp70, Skp2, Fbw7α, c‐myc mRNA, and protein levels and activated alternate splicing with caspase‐9a splice variant being significantly increased. MLAuNP possesses antiproliferative properties and induced apoptosis in A549 cells by activating alternate splicing of caspase‐9. J. Cell. Biochem. 117: 2302–2314, 2016.Item Anticancer activity of ceratotheca triloba(2016) Naicker, Leeann; Odhav, Bharti; Matsabisa, M.G.; Mohanlall, VireshPlants have provided a source of medicine from the beginning of human history and are the core of modern medicine. Moreover, plant based drug discovery has led to the development of various anticancer drugs (such as vincristine, vinblastine, etoposide, paclitaxel, camptothecin, topotecan and irinotecan). The use of botanical, phytochemical, biological and molecular techniques have facilitated the discovery of anthraquinones from Ceratotheca triloba that can inhibit the human topoisomerase II enzyme (target for anticancer drugs) and kill cancer cells. However, the C. triloba plant has not been extensively studied for its anticancer activity. Therefore, the aim of this study was to further investigate the anticancer activity of C. triloba and determine the classes of compounds that contributed towards its activity. In this study the leaf and root extracts were prepared by using hexane, DCM, hexane: DCM (1:1), methanol and/or water. These extracts were examined for their growth inhibitory potential on three cancer cell lines (A375 [melanoma], MDA-MB-231[breast] and WHCO1 [esophageal]) by using the MTT assay. Then, different mobile phases were prepared for optimizing the separation of the compounds of the active extract by TLC. Column chromatography was performed with the active extract by using five mobile phases (hexane : DCM [60 : 40, 40 : 60], DCM, DCM : ethyl acetate [90 : 10, 70 : 30, 60 : 40, 50 : 50, 50 : 60, 30: 60, 20 : 80], ethyl acetate and ethyl acetate: methanol [80 : 20, 70 : 30, 50 : 50]). The fractions collected from the column were examined for their growth inhibitory potential on two melanoma cell lines (A375 and UACC-62). The IC50 and TGI (total growth inhibition) values of the active fractions were determined. Also, the apoptosis inducing effects of the active fractions and standards (camptothecin and doxorubicin) were determined by using flow cytometer based assays (FITC annexin assay, PE active caspase 3 assay and BD MitoScreen assay). Subsequently, the chemical structures of the compounds that contributed towards the activity of these fractions were obtained by EI-LC-MS analysis. The results demonstrated that the hexane root extract exhibited the best percentage of growth inhibition (%GI) on all three cancer cell lines. The separation of the compounds of the hexane root extract was optimized on TLC plates by using different ratios of hexane and DCM. Column chromatography allowed for fractionation of this extract. Purified compounds were not obtained due to co-elution. Further research would have to be conducted to obtain purified compounds. This may involve the use of mini-column chromatography and PTLC. Overall a total of ten combined fractions were collected from the column. Four of these fractions (F2, F4, F5 and F8) displayed a high %GI on the A375 and UACC-62 cell lines. Moreover, fraction F4 was the most active fraction as it had the lowest IC50 (0.70 µg.ml-1 [A375] and 0.39 µg.ml-1 [UACC-62]) and TGI (12.50 µg.ml-1[A375] and 25 µg.ml-1 [UACC-62]) values in comparison to the other fractions. All four fractions induced depolarization of the mitochondria membrane potential (ΔΨ), caspase 3 activation, early apoptosis (phospholipid phosphatidylserine exposure) and/or late apoptosis in the melanoma cells. The results also revealed that fraction F4 (25 µg.ml-1) induced depolarization of the ΔΨ in a higher percentage of A375 (78.11%) and UACC-62 (87.4%) cells than the other fractions and standards. This fraction also induced caspase 3 activation in a high percentage of A375 (90.56%) and UACC-62 (96.78%) cells. Therefore fraction F4 was also the most active fraction in terms of apoptosis activity. Based on our results and literature findings we can deduce that the active fractions induced the intrinsic or extrinsic (type II) apoptosis pathway in the melanoma cells. Six classes of compounds were identified from the four active fractions. These were: benzothiophenones, benzopyranones, naphthoquinones, anthraquinones, androstanes and quinazolines. In conclusion, this is the first study that evaluated the growth inhibition potential of the leaf and root extracts of C. triloba on a panel of cancer cells. This research indicated that the hexane root extract displayed the best levels of cell growth inhibition. The active constituents of this extract were isolated into four fractions which elicited apoptosis inducing effects that promoted the extrinsic (type II) or intrinsic apoptosis pathway in the melanoma cells. Furthermore, fraction F4 contained the most active compounds from C. triloba as it had the lowest IC50 and TGI values (in comparison to the other fractions) and induced depolarization of the ΔΨ in the highest percentage of melanoma cells. It was confirmed that six classes of compounds were accountable for the anticancer activity of these fractions. Thus, the C. triloba plant is a rich source of anticancer compounds.Item Molecular immunogenetics of apoptosis : experimental dilemas(International Journal of Biological & Pharmaceutical Research, 2012) Singh, Rishan; Reddy, LaliniThere have been several research articles published on the biological and biochemical nature of apoptosis. These have included studies on the molecular genetics of apoptosis. Apart from the genes that are involved in the apoptotic cascade, there are several other genes that are either activated or inhibited when cell lines are exposed to apoptotic stimuli. This article addresses the simplicity and complexity of the genetic nature of apoptosis in a variety of cell lines.Item Apoptosis in the human laryngeal carcinoma (HEp-2) cell line by Bulbine natalensis and B. Frutescens fractions(IJBPR, 2012) Singh, Rishan; Reddy, LaliniMany plants that belong to the genus Bulbine have compounds in their roots and leaves which are considered important for traditional treatments. The stems and roots of Bulbine species are believed to contain anticancer compounds such as anthraquinones, including chrysophanol and knipholone. However, in general, people utilise plants of the Bulbine genus for the treatment of rashes, itches, wounds, burns, cracked lips and cracked skin. This study assessed the effect of aqueous and organic fractions of Bulbine natalensis and Bulbine frutescens on the human laryngeal carcinoma cell line (HEp-2) for apoptosis. The MTT assay was used to determine the cytotoxicity of the fractions administered and to select fractions for analysis of bax and caspase-3 gene expression, which are induced during programmed cell death type 1, known as apoptosis. All of the B. natalensis fractions induced expression of caspase-3, while the tested B. frutescens aqueous root fractions failed to induce expression of caspase-3. The variation in bax gene expression indicated that HEp-2 cell death was due to apoptosis and other unknown forms of cell death that may or may not activate caspase-3 gene expression.Item Silver nanoparticles of Albizia adianthifolia : the induction of apoptosis in human lung carcinoma cell line(BioMed Central, 2013) Govender, Rishalan; Phulukdaree, Alisa; Gengan, Robert Moonsamy; Anand, Krishnan; Chuturgoon, Anil A.Background Silver nanoparticles (AgNP), the most popular nano-compounds, possess unique properties. Albizia adianthifolia (AA) is a plant of the Fabaceae family that is rich in saponins. The biological properties of a novel AgNP, synthesized from an aqueous leaf extract of AA (AAAgNP), were investigated on A549 lung cells. Cell viability was determined by the MTT assay. Cellular oxidative status (lipid peroxidation and glutathione (GSH) levels), ATP concentration, caspase-3/-7, -8 and −9 activities were determined. Apoptosis, mitochondrial (mt) membrane depolarization (flow cytometry) and DNA fragmentation (comet assay) were assessed. The expression of CD95 receptors, p53, bax, PARP-1 and smac/DIABLO was evaluated by flow cytometry and/or western blotting. Results Silver nanoparticles of AA caused a dose-dependent decrease in cell viability with a significant increase in lipid peroxidation (5-fold vs. control; p = 0.0098) and decreased intracellular GSH (p = 0.1184). A significant 2.5-fold decrease in cellular ATP was observed upon AAAgNP exposure (p = 0.0040) with a highly significant elevation in mt depolarization (3.3-fold vs. control; p < 0.0001). Apoptosis was also significantly higher (1.5-fold) in AAAgNP treated cells (p < 0.0001) with a significant decline in expression of CD95 receptors (p = 0.0416). Silver nanoparticles of AA caused a significant 2.5-fold reduction in caspase-8 activity (p = 0.0024) with contrasting increases in caspase-3/-7 (1.7-fold vs. control; p = 0.0180) and −9 activity (1.4-fold vs. control; p = 0.0117). Western blots showed increased expression of smac/DIABLO (4.1-fold) in treated cells (p = 0.0033). Furthermore, AAAgNP significantly increased the expression of p53, bax and PARP-1 (1.2-fold; p = 0.0498, 1.6-fold; p = 0.0083 and 1.1-fold; p = 0.0359 respectively). Conclusion Data suggests that AAAgNP induces cell death in the A549 lung cells via the mt mediated intrinsic apoptotic program. Further investigation is required to potentiate the use of this novel compound in cancer therapy trials.Item Cytotoxicity and gene expression of selected apoptotic markers in the human laryngeal carcinoma cell line (HEp-2) by Bulbine spp. fractions(2013-07-30) Singh, Rishan; Reddy, Lalini; Permaul, KugenApoptosis, or programmed cell death, is a process which is pivotal in eliminating damaged, infected, or unwanted cells from the body. It has been studied in numerous types of cell lines ranging from normal to infected cell lines, and there have been a wide range of studies on apoptosis in laryngeal cancer because this type of cancer has become one of the most common types of head or neck cancer due to the high incidence of alcohol consumption, tobacco smoking and chewing of betel quid amongst populations. Laryngeal cancer is usually treated with radiotherapy or is surgically removed, but due to the loss of the function of the larynx after surgery, it has been suggested that alternative strategies or ways of treating laryngeal cancer are required. This has prompted the use of, and research in the field of, plant medicine to combat laryngeal cancer. Plant medicine has been used for centuries by the Chinese, Indian and Arabian population in Uhani, Ayurveda and Siddha as a form of replacing conventional medicine with complementary and alternative medicine, these include many plants from the family Asphodelaceae, which have become marketable commodities owing to their medicinal values and traditional uses. Amongst these plants, the genus Bulbine has been used as a form of natural medicine in rural Africa and they are also exploited for their aloe vera properties as well as their possession of phytochemical compounds such as isoflavanoids, nor-lignans, naphthalene derivatives, anthracene and poly prenylated flavonoids. There has been a compelling amount of literature on the traditional uses of the Bulbine spp. because these are linked to the Bulbine spp. having secondary metabolites such as pyroles, chromones, coumarins, bianthraceane, benzene as well as alkaloids. However, for Bulbine natalensis and B. frutescens, the plants of interest in this study, the location of anticancer compounds in them are the only amounts of information available. It has been reported, traditionally, that B. natalensis possesses the anticancer potential in the roots, while the anticancer potential for B. frutescens is in the leaves. However, this requires scientific clarification. Therefore, this study was conducted to assess programmed cell death or apoptosis by analysing the responses of the human laryngeal carcinoma cell line (HEp-2) to crude aqueous and organic (50% and 100% ethanol) fractions of B. natalensis and B. frutescens. In order to have achieved this, the HEp-2 cell line was exposed to the above mentioned fractions at three different final concentrations (20, 2 and 1μg/ml) and assessed for cytotoxicity using the 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assay as an indicator of cell death after fraction utilisation (3 days) for 5 and 8 days. The differences in the potency of the Bubline spp. fractions were confirmed using the non-parametric ANOVA test. Thereafter, selected fractions were screened for apoptotic potential using reverse transcriptase-polymerase chain reaction (RT-PCR) to determine the expression of bax and caspase-3 biomarkers, which are the biomarkers that participate in mitochondrial, endoplasmic reticulum and receptor-ligand mechanism of apoptosis. The fractions of B. frutescens were selected relative to those of B. natalensis for the RT-PCR procedure (read section 3.4.1. for the selection procedure) and links between the cytotoxicity and gene expression results were analysed. It was found that the B. natalensis fractions had a much greater cytotoxic effect on the HEp-2 cell line compared to fractions of B. frutescens by the fifth day of the MTT assay. On the eight day of incubation, there was an increase in HEp-2 cell line proliferation by the fractions of both plant species administered. The fractions selected for bax and caspase-3 gene expression analysis for B. natalensis were the: 20 μg/ml root and corm aqueous fractions, 20 μg/ml leaf and corm 100% ethanol fractions, 20 μ g/ml corm 50% ethanol fraction, 2 μg/ml root aqueous fraction, 2 μg/ml leaf 100% ethanol fraction and the corm 1 μg/ml aqueous and 50% ethanol fractions. The fractions that were compared to B. natalensis were the 20 μg/ml root and leaf aqueous and 100% ethanol fractions respectively, the 2 μg/ml root aqueous fraction and the 2 μg/ml leaf 100% ethanol fraction. It was found from RT-PCR analysis that all of the B. natalensis fractions tested induced expression of caspase-3, which indicated that those fractions were capable of inducing apoptosis in laryngeal carcinoma in vitro, since caspase-3 is the molecular indicator of apoptosis. The aqueous B. frutescens root fraction, did not induce expression of caspase-3 gene, although it caused expression of bax. This implied that the root aqueous B. frutescens fraction, may be involved in some other form of cell death, other than apoptosis. It was also found that there was variability in the response of the HEp-2 cell line to the Bulbine spp. fractions because of the variation in bax expression among fractions of different concentration. It was difficult, from this study, to classify fractions into categories for their mechanism of action, because not all of the fractions that caused the expression of capase-3, induced bax gene expression. Hence, proper conclusions were unable to be made, more so, because all the mechanisms of apoptosis mentioned, involve bax gene activation in order to proceed to completion. Therefore for those Bulbine spp. fractions to which the HEp-2 cell line exhibited a variable response to, it was postulated that cell death or apoptosis occurred through some other unknown mechanism. Overall, the cytotoxicity result didn’t correlate to the gene expression results because fractions that promoted HEp-2 cell line growth by day five, expressed apoptotic markers, which highlighted the sensitivity and accuracy of the cells-to-cDNATM II kit for detecting a few possibly apoptosed cells. This was confirmed by the fact that the HEp-2 cell line used in the MTT cytotoxicity assay and gene expression study had the same passage number and were viable, the latter being achieved because the MTT assay only measures the cytotoxicity of compounds once they have been taken up by viable cells – measuring mitochondrial activities expressed as absorbances. Therefore, the deduction that HEp-2 cell death may be due to bax/caspase-3 expression was valid because the mRNA was isolated from viable HEp-2 cells that had been killed by Bulbine spp. fractions of different polarity. Furthermore, the lack of correlation between the cytotoxicity and gene expression results indicated the amount of HEp-2 cell line proliferation by the fraction out-competes those that died, thereby producing a negative cytotoxicity result. There was a relationship between the traditional information about the anticancer potential for B. natalensis and B. frutescens. For example, the aqueous root fractions of B. natalensis were found to be non-toxic to the HEp-2 cell line, but did express caspase-3, which indicated the possibility of apoptosis. Similarly, the 100% ethanol leaf B. frutescens fractions were non-toxic to the HEp-2 cell line, but were able to induce apoptosis as well. This emphasised that the MTT cytotoxicity assay should be compared with other methods of measuring cytotoxicity when performing studies like this, because although literature has emphasised many advantages of using the MTT cytotoxicity assay in apoptotic studies, this study proved otherwise. When identical HEp-2 cells were treated with the same extract, only some cells were killed (apoptosis) whereas others proliferated. This was because although the cells were identical phenotypically, they were all probably at different phases of the cell cycle resulting in the HEp-2 cells responding variably to the same fraction at different concentrations. It was also found that the responses were concentration independent. For example, the 1 μg/ml B. natalensis corm fraction exhibited the highest toxicity of the three concentrations administered. The lowest cytotoxicity was achieved for the 20 μg/ml fraction – showing a proliferative effect on the HEp-2 cell line. Similarly, the 2 μg/ml aqueous B. natalensis leaf fraction induced the highest cytotoxicity level in the HEp-2 cell line followed by the 1 μg/ml and then the 20 μg/ml fractions. Apart from the genetic variation in identical HEp-2 cells; this indicated that the HEp-2 cell line was selective to particular fractions of the Bulbine spp. for utilisation. Concentration independence and HEp-2 cell preferential selection has been reported in many other studies involving plant fractions/extracts and natural products. This study demonstrated that although all the tested B. natalensis fractions were capable of inducing HEp-2 cell death possibly via. apoptosis (caspase-3 induction), a lack of any link between apoptosis and the cytotoxicity results (hence the 20 μg/ml corm fraction had a negative cytotoxicity but expressed both apoptotic markers), indicated the need for phytochemical screening of both Bulbine spp. in future, to determine the compounds that are responsible for the cytotoxicity and gene expression result outcomes of both Bulbine spp. fractions. Furthermore, procaspase genes also have to be analysed since genes are expressed to form procaspases, which then form active caspases. Although normal cells also express caspase-3 genes during apoptosis, this study focused exclusively on the effect of Bulbine natalensis and B. frutescens fractions (selected relative to the cytotoxicity results of B. natalensis) on the HEp-2 cell line (read cell culture and cytotoxicity discussion for selection of HEp-2 cell line). The validity of this study is confirmed by similar experimental designs that assayed the cytotoxicity of plant-derived or natural compounds on cancer cell lines only, and the detection of apoptosis through caspase- 3 induction and other unrelated methods. This is the first study to report the induction of apoptosis in cancer cell lines by Bulbine spp. fractions using cytotoxicity and the expression of bax and caspase-3 apoptotic markers. It provides insight into the interaction between the HEp-2 cell line and the aqueous and organic fractions of B. natalensis and B. frutescens by analyzing links between cytotoxicity and bax and caspase-3 gene expression; which could probably contribute to drug design with selected Bulbine spp. fractions. Further investigations are required in future, to confirm the possible drug targets of the studied Bulbine spp. fractions in an attempt of assaying their therapeutic importance.Item Anti-carcinogenic activity of Centella asiatica and Elytropappus rhinocerotis on a human colon cancer cell line(2012) Dwarka, Depika; Odhav, Bharti; Sewram, VikashRecently our understanding of cancer has advanced in the realization that apoptosis and the genes that control it have a profound effect on the malignant phenotype. It is now clear that some oncogenic mutations disrupt apoptosis, leading to tumor initiation, progression or metastasis. Conversely, compelling evidence indicates that other oncogenic changes promote apoptosis, thereby producing selective pressure to override apoptosis during multistage carcinogenesis. Finally, it is now well documented that most cytotoxic anti-cancer agents induce apoptosis, raising the intriguing possibility that defects in apoptotic programs contribute to treatment failure. Because the same mutations that suppress apoptosis during tumor development also reduce treatment sensitivity, apoptosis provides a conceptual framework to link cancer genetics with cancer therapy. An intense research effort is uncovering the underlying mechanisms of apoptosis, such that, in the next decade, one envisions that this information will produce new strategies to exploit apoptosis for therapeutic benefit. Plants have a long history in cancer treatment. More than 3000 species have been known for their anti-cancer potential. Over 60% of currently used anti-cancer agents are derived in one way or another from higher plants. Indeed, compounds derived from natural sources, including plants, have played, and continue to play, a dominant role in the discovery of leads for the development of conventional drugs for the treatment of most human diseases especially cancer. Thus the aim of this study was to investigate if Centella asiatica and Elytropappus rhinocerotis possess anti-cancer potential and determine the effect on the modulation of apoptosis. In South Africa C. asiatica is known anecdotally to treat various forms of cancers and E. rhinocerotis is known to treat colic and diarrhoea. The anti-cancer activity of C. asiatica has been studied in some parts India but E. rhinocerotis has not been investigated. This study was conducted using polarity guided fractionation (aqueous, ethanolic, methanolic and hexane), thereafter these extracts were tested for their toxicity on a colon cancer cell line (CaCO-2) and on normal cells vi (PBMC). Subsequently, the most active extract was used to isolate the active fraction. The fraction that displayed toxicity on the CaCO-2 cells were further investigated for their ability to induce apoptosis by observing the morphological effects and DNA changes using acridine orange-ethidium bromide staining. Apoptosis was confirmed using Annexin V- PI staining. Nuclear effects were studied by DNA fragmentation and by agarose gel electrophoresis. Nuclear fragmentation was studied by flow cytometry using bromodeoxyuridine (BrDU). Pro-apoptotic changes were determined with Caspase III enzyme levels using flow cytometry. The results were compared to the effect of a known anti-carinogen - Taxol. The anti-oxidant activity was also evaluated for the different extracts. The ethanolic extracts of both C. asiatica and E. rhinocerotis showed more than 100% radical scavenging activity. The methanolic extract (125 μg/ml -500 μg/ml) showed cytotoxicity on the CaCO-2 cells and a proliferative effect on the PBMC. Apoptosis was confirmed in the methanolic extract for both plants and was therefore used to carry forth this study. This included early apoptotic changes observed by the morphological study i.e., membrane blebbing, nuclear condensation and the presence of apoptotic bodies, in both C. asiatica and E. rhinocerotis fractions demonstrated more non-viable apoptotic cells than the methanolic extracts. Late changes of apoptosis were also found as indicated by DNA laddering and a positive outcome with BrDU. Both the active fractions from C. asiatica and E. rhinocerotis showed more DNA laddering and active caspase III than the methanolic extract. These features indicate that C. asiatica and E. rhinocerotis cause apoptotic death of colon cancer cells CaC0-2. In conclusion, there was a significant increase in apoptosis of CaCO-2 cells with little alteration of PBMC in the presence of the methanolic extract of C. asiatica and E. rhinocerotis. The semipure fractions resulted in changes related to late apoptosis. The results suggest that C. asiatica and E. rhinocerotis induces apoptosis in CaCO-2 cells which is an important step in elucidating the underlying molecular mechanism for anti-tumour activity.