Moringa oleifera and Autophagy: Evidence from In Vitro Studies on Chaperone-Mediated Autophagy in HepG2 Cancer Cells.

Hepatocellular carcinoma (HCC) is the most prevalent primary liver cancer in Sub-Saharan African countries, including South Africa (SA). Given the limitations in current HCC therapeutics, there is an increasing need for alternative adjuvant therapeutic options. As such, several cell survival mechanisms, such as autophagy, have been identified as potential adjuvant therapeutic targets in HCC treatment. Of the three most established autophagic pathways, the upregulation of chaperone-mediated autophagy (CMA) has been extensively described in various cancer cells, including HCC cells. CMA promotes tumor growth and chemotherapeutic drug resistance, thus contributing to HCC tumorigenesis. Therefore, the modulation of CMA serves as a promising adjuvant target for current HCC therapeutic strategies. Phytochemical extracts found in the medicinal plant, Moringa oleifera (MO), have been shown to induce apoptosis in numerous cancer cells, including HCC. MO leaves have the greatest abundance of phytochemicals displaying anticancer potential. However, the potential interaction between the pro-apoptotic effects of MO aqueous leaf extract and the survival-promoting role of CMA in an in vitro model of HCC remains unclear. This review aims to summarize the latest findings on the role of CMA, and MO in the progression of HCC.

The Hepatoprotective Effects of Moringa oleifera against Antiretroviral-Induced Cytotoxicity in HepG2 Cells: A Review.

The untreated human immunodeficiency virus (HIV), a lentivirus species that attacks immune cells (CD4+ T cells), causes acquired immunodeficiency syndrome (AIDS). HIV-positive people manage HIV/AIDS by using antiretroviral therapy (ART). The ART treatment regimen contains two nucleoside reverse transcriptase inhibitors (NRTIs) and one non-nucleoside reverse transcriptase inhibitor/integrase strand transfer inhibitor. Tenofovir, an NRTI approved for managing HIV infection, is associated with hepatic steatosis and lactic acidosis, which are linked to mitochondrial toxicity and oxidative stress. Due to side-effects associated with ART, people living with HIV often use medicinal plants or a combination of medicinal plants with ART to promote adherence and diminish the side-effects and cytotoxicity. The Moringa oleifera (MO) tree from the family of Moringaceae is among the medicinal trees studied in managing HIV/AIDS in sub-Saharan Africa. The MO tree extracts have been reported to have inhibitory activity primarily against HIV due to their bioactive compounds. However, there is a scarcity of knowledge about the use of the MO tree amongst HIV/AIDS patients receiving ART in South Africa and its effect on patient compliance and outcomes. Thus, this review aims to outline the impact of MO aqueous leaf extract on oxidative stress and antioxidant responses in human HepG2 liver cells after exposure to antiretrovirals such as tenofovir. The review will contribute to a comprehensive understanding of the potential protective effect of MO aqueous leaf extract on tenofovir-induced cytotoxicity.

Moringa oleifera: A Review on the Antiproliferative Potential in Breast Cancer Cells.

The global burden of female breast cancer and associated deaths has become a major concern. Many chemotherapeutic agents, such as doxorubicin, have been shown to have adverse side effects. The development of multi-drug resistance is a common occurrence, contributing to chemotherapeutic failure. The resistance of breast cancer cells to drug treatment leads to a decline in the treatment efficacy and an increase in cancer recurrence. Therefore, action is required to produce alternative drug therapies, such as herbal drugs. Herbal drugs have been proven to be beneficial in treating illnesses, including cancer. This review aims to highlight the antiproliferative potential of Moringa oleifera (MO), a medicinal tree native to India and indigenous to Africa, in breast cancer cells. Although MO is not yet considered a commercial chemopreventive drug, previous studies have indicated that it could become a chemotherapeutic agent. The possible antiproliferative potential of MO aqueous leaf extract has been previously proven through its antioxidant potential as well as its ability to induce apoptosis. This review will provide an increased understanding of the effect that MO aqueous leaf extract could potentially have against breast cancer.

miR-27b inhibition contributes to cytotoxicity in patulin-exposed HEK293 cells.

Patulin (PAT) is a mycotoxin produced by Penicillium and other fungi that contaminate fruit. PAT targets the kidney and is associated with nephrotoxicity. Micro-RNAs (miRNA) may offer new insights into PAT-induced nephrotoxicity. Cytochrome P450 family 1, subfamily B, polypeptide 1 (CYP1B1), involved in metabolism of dietary toxins is negatively regulated by miR-27b and linked with the nuclear factor kappa B (NF-κB) pathway and peroxisome proliferator activated receptor gamma (PPARÉ£) in renal fibrosis. This study investigated the effects of PAT on miR-27b, CYP1B1, PPARÉ£ and cytotoxicity in human kidney (HEK293) cells. HEK293 cells were exposed to PAT (2.5 µM, 24h). Protein expression of CYP1B1, PPARÉ£, NF-κB (p65), pNF-κB (p65) (phospho-Ser563) and cleaved PARP-1 was quantified using western blotting. QPCR evaluated mRNA levels of CYP1B1, IL-6, miR-27b, OGG1, mtDNA, TFAM and UCP2. Mitochondrial membrane potential and phosphatidylserine (PS) externalization was evaluated by flow cytometry while levels of ATP and caspase -9, -8, -3/7 activity was measured using luminometry. PAT significantly decreased miR-27b levels (p = 0.0014) and increased CYP1B1 mRNA (p = 0.0015) and protein (p = 0.0013) levels. PPARÉ£ protein expression was significantly increased (p = 0.0002) and associated with decreased NF-κB activation (p = 0.0273) and IL-6 mRNA levels (p = 0.0265). Finally, PAT significantly compromised mitochondrial repair mechanisms and increased apoptotic biomarkers. PAT altered miR-27b levels and PPARÉ£, with associated changes to NF-κB activation, downstream IL-6 and CYP1B1 expression. These results show that PAT impairs detoxification mechanisms leading to mitochondrial damage and apoptosis. In conclusion, PAT altered the epigenetic environment and impaired detoxification processes, supporting a mechanism for nephrotoxic outcomes.

Moringa oleifera Aqueous Leaf Extract Induces Cell-Cycle Arrest and Apoptosis in Human Liver Hepatocellular Carcinoma Cells.

Aim: Hepatocellular carcinoma is one of the leading global epidemics. A medicinal tree, Moringa oleifera (MO), has been part of traditional treatments including cancer therapies. We investigated the apoptosis inducing effects of MO crude aqueous leaf extract (MOE) in human liver hepatocellular carcinoma (HepG2) cells. Methods: HepG2, PBMCs and Hek293 cell viability was evaluated using MTT assay. Oxidative stress and DNA damage was determined using TBARS and comet assays, respectively. Apoptosis was assessed by caspase-9, -3/7 activities and ATP levels (luminometry). Cell cycle, γH2AX, and cleaved PARP-1 were determined (flow cytometry). Protein expression of c-myc, Bax, p-Bcl2, Smac/DIABLO, Hsp70, SRp30a and cleaved PARP-1 was assessed using western blotting. Results: MOE displayed minimal toxicity in PBMCs and Hek293 cells for 24 h. HepG2 cells were exposed to MOE (24 h) and an IC50 (4.479 mg/mL) was determined. MOE significantly increased lipid peroxidation, DNA damage and γH2AX levels. A significant decrease in G1, S and G2-M phase was seen. Significant increase in SRp30a protein expression activated caspase-9. Caspase-9 and -3/7 was significantly increased with significant decrease in ATP levels. Apoptosis was confirmed with significant decrease in c-myc, p-Bcl2 and Hsp70 protein expression and a significant increase in Bax, Smac/DIABLO and PARP-1 cleavage. Conclusion: MOE induces cell-cycle arrest and apoptosis in cancerous HepG2 cells.

Moringa oleifera and their phytonanoparticles: Potential antiproliferative agents against cancer.

Cancer is classified as one of the leading causes of global mortality. It has affected millions of people, often with poor prognosis. Having severe side-effects with conventional chemotherapy, alternate drugs and therapies are actively being investigated. There is a need for innovative drug discovery and design as existing cancer therapies are costly and not readily available. Ayurveda and traditional medicine have utilised natural resources such as plants and trees as part of their regime to treat various illness and diseases with positive outcomes. One such tree is Moringa oleifera (MO). Almost all parts have shown to be effective against several ailments including cancer which was attributed to the bioactive constituents. Targeted therapies had led to the development of nanoparticles which are extremely effective in various biomedical applications due to their small size. Green synthesis of gold nanoparticles have great potential as naturally occurring plants and trees such as MO can be used in the synthesis process. The resultant gold phytonanoparticles are useful in cancer therapies with improved survival rates and quality of life. The review highlights the importance of MO in natural medicine, synthesis of phytonanoparticles and the fundamental role as a potential antiproliferative agent against cancer.

HIV induced nitric oxide and lipid peroxidation, influences neonatal birthweight in a South African population.

HIV has been implicated in adverse birth outcomes, due to increased oxidative stress and inflammation. In addition, HIV has been reported to increase nitric oxide levels. Therefore the combined exposures to HIV and traffic-related air pollution, within South Durban, South Africa (SA), may lead to adverse birth outcomes. However, the exact mechanism is still unknown; this study aimed to identify a potential mechanism. First, the influence of HIV on oxidative and nitrosative stress markers in pregnant women was assessed. Secondly, the effect of these stress makers and exposure to oxides of nitrogen (NOx) on neonatal birthweight (BW) was evaluated. Finally, the effect HIV and traffic-related pollution exposure has on the oxidative and endoplasmic profile and epigenetic regulation of Nrf2-Keap1 pathway by miR-144 and miR-28 in pregnant women was determined. Women, in their third trimester with singleton pregnancies, who were HIV+ and HIV-, were recruited from Durban, SA. Biomarker levels of serum nitrites/nitrates (NO) and malondialdehyde (MDA) were analysed and mRNA expression levels of oxidative and endoplasmic stress response genes were assessed. Land regression modelling was performed to determine NOx exposure levels. HIV exposure during pregnancy was associated with increased NO levels. NO was shown to reduce neonatal BW. NO and MDA was found to reciprocally increase each other, with HIV differentially influencing MDA's effect on BW. HIV down-regulated miR-144 which was negatively associated with Nrf2, suggesting a potential mechanism for HIV associated chronic oxidative stress. This study proposes that NO plays a key role in neonatal BW reduction in response to HIV and traffic-related air pollution.

Toxicity assessment of mycotoxins extracted from contaminated commercial dog pelleted feed on canine blood mononuclear cells.

Raw ingredients of pet food are often contaminated with mycotoxins. This is a serious health problem to pets and causes emotional and economical stress to the pet owners. The aim of this study was to determine the immunotoxicity of the most common mycotoxins (aflatoxin, fumonisin, ochratoxin A and zearalenone) by examining 20 samples of extruded dry dog food found on the South African market [10 samples from standard grocery store lines (SB), 10 from premium veterinarian lines (PB)]. Pelleted dog food was subjected to extraction protocols optimized for the above mentioned mycotoxins. Dog lymphocytes were treated with the extracts (24 h incubation and final concentration 40 µg/ml) to determine cell viability, mitochondrial function, oxidative stress, and markers of cell death using spectrophotometry, luminometry and flow cytometry. Malondialdehyde, a marker of oxidative stress showed no significant difference between SB and PB, however, GSH was significantly depleted in SB extract treatments. Markers of apoptosis (phosphatidylserine externalization) and necrosis (propidium iodide incorporation) were elevated in both food lines when compared to untreated control cells, interestingly SB extracts were significantly higher than PB. We also observed decreased ATP levels and increased mitochondrial depolarization in cells treated with both lines of feed with SB showing the greatest differences when compared to the control. This study provides evidence that irrespective of price, quality or marketing channels, pet foods present a high risk of mycotoxin contamination. Though in this study PB fared better than SB in regards to cell toxicity, there is a multitude of other factors that need to be studied which may have an influence on other negative outcomes.

Fusaric Acid Induces DNA Damage and Post-Translational Modifications of p53 in Human Hepatocellular Carcinoma (HepG2 ) Cells.

Fusaric acid (FA), a common fungal contaminant of maize, is known to mediate toxicity in plants and animals; however, its mechanism of action is unclear. p53 is a tumor suppressor protein that is activated in response to cellular stress. The function of p53 is regulated by post-translational modifications-ubiquitination, phosphorylation, and acetylation. This study investigated a possible mechanism of FA induced toxicity in the human hepatocellular carcinoma (HepG2 ) cell line. The effect of FA on DNA integrity and post-translational modifications of p53 were investigated. Methods included: (a) culture and treatment of HepG2 cells with FA (IC50 : 580.32 µM, 24 h); (b) comet assay (DNA damage); (c) Western blots (protein expression of p53, MDM2, p-Ser-15-p53, a-K382-p53, a-CBP (K1535)/p300 (K1499), HDAC1 and p-Ser-47-Sirt1); and (d) Hoechst 33342 assay (apoptosis analysis). FA caused DNA damage in HepG2 cells relative to the control (P < 0.0001). FA decreased the protein expression of p53 (0.24-fold, P = 0.0004) and increased the expression of p-Ser-15-p53 (12.74-fold, P = 0.0126) and a-K382-p53 (2.24-fold, P = 0.0096). This occurred despite the significant decrease in the histone acetyltransferase, a-CBP (K1535)/p300 (K1499) (0.42-fold, P = 0.0023) and increase in the histone deacetylase, p-Ser-47-Sirt1 (1.22-fold, P = 0.0020). The expression of MDM2, a negative regulator of p53, was elevated in the FA treatment compared to the control (1.83-fold, P < 0.0001). FA also inhibited cell proliferation and induced apoptosis in HepG2 cells as evidenced by the Hoechst assay. Together, these results indicate that FA is genotoxic and post-translationally modified p53 leading to HepG2 cell death. J. Cell. Biochem. 118: 3866-3874, 2017. © 2017 Wiley Periodicals, Inc.

Pro-Inflammatory Cytokine Levels in HIV Infected and Uninfected Pregnant Women with and without Preeclampsia.

INTRODUCTION: Preeclampsia and HIV/AIDS are inflammatory conditions that contribute significantly to adverse maternal and foetal outcomes. The immune reconstitution effects of HAART on inflammatory mediators has not been adequately studied in pregnancy and may impact on the inflammatory cytokine network in women with co-morbid preeclampsia. Our study evaluated changes in pro-inflammatory cytokines IL-2, TNF-α, IFN-γ and IL-6 in HIV infected preeclamptic women on HAART. METHODS: A prospective experimental study was conducted at Prince Mshiyeni Memorial Hospital between July 2013 and September 2014. One hundred and ninety three pregnant women were recruited into 4 groups: uninfected normotensive (50; 26%), infected normotensive (45; 23%), uninfected preeclamptic (53; 28%) and infected preeclamptic women (45; 23%). Serum levels of cytokines TNF-α, IFN- γ, IL-2 and IL-6 were determined using commercially available kits and a Cytometric Bead Array (CBA). Comparative data was recorded and analysed descriptively. RESULTS: In the control groups (normotensive), significantly lower values were found in IL-2 (p = 0.010), TNF-α (p = 0.045), and IL-6 (p = 0.005); and a non-significant decrease was observed in IFN-γ (p = 0.345) in HIV infected women on HAART compared to uninfected controls. In the experimental group (preeclamptic) women, significantly reduced levels were observed in IL-2 and TNF-α (p = 0.001; p = 0.000) and non-significant decreases were observed in IFN-γ and IL-6 (p = 0.023; p = 0.086) in HIV infected women on HAART compared with uninfected preeclamptic women. Non-significant differences were observed between uninfected preeclamptic and normotensive women. CONCLUSION: In uncomplicated/normotensive pregnancies, HIV/HAART is associated with significant decreases in IL-2, TNF-α and IL-6, and in preeclamptic women significant decreases in IL-2 and TNF-α were observed. These findings suggest that HIV/HAART impacts on pro-inflammatory cytokines in women with co-morbid preeclampsia. This provides a platform for further research on immune reconstitution effects of HAART during pregnancy, and the development of potential immune modulation therapies for the management of preeclampsia.

Fusaric acid induces oxidative stress and apoptosis in human cancerous oesophageal SNO cells.

Oesophageal cancer (OC) is a global problem incrementally incident among black South African males. The high incidence of OC may be due to the consumption of corn as a staple, often contaminated with mycotoxins. Fusaric acid (FA), a neglected mycotoxin, is known to disrupt mitochondrial energy metabolism, chelates divalent metal cations and induces cell death in plants. This study investigated FA-induced cytotoxicity and apoptotic induction in the SNO OC cell line. Cells were treated with FA (IC50 = 78.81 µg/mL; 24 h; MTT assay) and assayed for oxidative stress and membrane damage (TBARS, LDH cytotoxicity and glutathione), apoptotic induction (ATP levels, caspase-8, -9, -3/7 activities) (Luminometry), single strand DNA and nuclear fragmentation (Comet and Hoechst assay). Additionally, relative expression of pro- and anti-apoptotic proteins were determined (Western Blotting). Significant antioxidant depletion was consistent with a concomitant increase in ROS-induced lipid peroxidation and extracellular LDH levels. FA induced apoptosis by significantly increasing Bax expression and caspase-8, -9 and -3/7 activities whilst decreasing ATP levels and Bcl-2 expression. Further, FA significantly increased comet tail lengths, PARP-1 expression and late stage apoptotic body formation in SNO cells. This study shows that FA is cytotoxic and induces increased apoptosis in SNO cells.

The Antiproliferative Effect of Moringa oleifera Crude Aqueous Leaf Extract on Human Esophageal Cancer Cells.

Esophageal cancer (EC) is commonly diagnosed in South Africa (SA), with high incidences occurring in SA's black population. Moringa oleifera (MO), a multipurpose tree, is used traditionally for its nutritional and medicinal properties. It has been used for the treatment of a variety of ailments, including cancer. We investigated the antiproliferative effect of MO crude aqueous leaf extract (MOE) on a cancerous esophageal cell line (SNO). SNO cells were exposed to a range of MOE dilutions to evaluate cytotoxicity (MTT assay). Oxidative stress was determined using the TBARS assay. The comet assay was used to assess DNA damage. We then determined cell death mechanisms by measuring phosphatidylserine (PS) externalization (flow cytometry), caspase-3/7 and caspase-9 activities, and adenosine triphosphate (ATP) levels (luminometry). Protein expression of Smac/DIABLO and PARP-1 was determined by western blotting. SNO cells were treated with a range of MOE dilutions to obtain an IC50 value of 389.2 µg/mL MOE (24 h), which was used in all subsequent assays. MOE significantly increased lipid peroxidation (P < .05) and DNA fragmentation (P < .0001) in SNO cells. The induction of apoptosis was confirmed by the increase in PS externalization (P < .0001), caspase-9 (P < .05) and caspase-3/7 (P = .22) activities, and decreased ATP levels (P < .0001). MOE significantly increased both the expression of Smac/DIABLO protein and cleavage of PARP-1, resulting in an increase in the 24-kDa fragment (P < .001). MOE possesses antiproliferative effects on SNO EC cells by increasing lipid peroxidation, DNA fragmentation, and induction of apoptosis.

Moringa oleifera Gold Nanoparticles Modulate Oncogenes, Tumor Suppressor Genes, and Caspase-9 Splice Variants in A549 Cells.

Gold 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. © 2016 Wiley Periodicals, Inc.

Cytotoxic Effect of a Novel Synthesized Carbazole Compound on A549 Lung Cancer Cell Line.

Increased death rates due to lung cancer have necessitated the search for potential novel anticancer compounds such as carbazole derivatives. Carbazoles are aromatic heterocyclic compounds with anticancer, antibacterial and anti-inflammatory activity. The study investigated the ability of the novel carbazole compound (Z)-4-[9-ethyl-9aH-carbazol-3-yl) amino] pent-3-en-2-one (ECAP) to induce cytotoxicity of lung cancer cells and its mechanism of action. ECAP was synthesized as a yellow powder with melting point of 240-247 °C. The 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), lipid peroxidation and comet assays were used to assess the cytotoxic effect of the compound on A549 lung cancer cells. Protein expression was determined using western blots, apoptosis was measured by luminometry (caspase-3/7, -8 and -9) assay and flow cytometry was used to measure phosphatidylserine (PS) externalisation. ECAP induced a p53 mediated apoptosis of lung cancer cells due to a significant reduction in the expression of antioxidant defence proteins (Nrf2 and SOD), Hsp70 (p < 0.02) and Bcl-2 (p < 0.0006), thereby up-regulating reactive oxygen species (ROS) production. This resulted in DNA damage (p < 0.0001), up-regulation of Bax expression and caspase activity and induction of apoptosis in lung cancer cells. The results show the anticancer potential of ECAP on lung cancer.

The antiproliferative effect of Moringa oleifera crude aqueous leaf extract on cancerous human alveolar epithelial cells.

BACKGROUND: The incidence of lung cancer is expected to increase due to increases in exposure to airborne pollutants and cigarette smoke. Moringa oleifera (MO), a medicinal plant found mainly in Asia and South Africa is used in the traditional treatment of various ailments including cancer. This study investigated the antiproliferative effect of MO leaf extract (MOE) in cancerous A549 lung cells. METHODS: A crude aqueous leaf extract was prepared and the cells were treated with 166.7 µg/ml MOE (IC50) for 24 h and assayed for oxidative stress (TBARS and Glutathione assays), DNA fragmentation (comet assay) and caspase (3/7 and 9) activity. In addition, the expression of Nrf2, p53, Smac/DIABLO and PARP-1 was determined by Western blotting. The mRNA expression of Nrf2 and p53 was assessed using qPCR. RESULTS: A significant increase in reactive oxygen species with a concomitant decrease in intracellular glutathione levels (p < 0.001) in MOE treated A549 cells was observed. MOE showed a significant reduction in Nrf2 protein expression (1.89-fold, p < 0.05) and mRNA expression (1.44-fold). A higher level of DNA fragmentation (p < 0.0001) was seen in the MOE treated cells. MOE's pro-apoptotic action was confirmed by the significant increase in p53 protein expression (1.02-fold, p < 0.05), p53 mRNA expression (1.59-fold), caspase-9 (1.28-fold, p < 0.05), caspase-3/7 (1.52-fold) activities and an enhanced expression of Smac/DIABLO. MOE also caused the cleavage and activation of PARP-1 into 89 KDa and 24 KDa fragments (p < 0.0001). CONCLUSION: MOE exerts antiproliferative effects in A549 lung cells by increasing oxidative stress, DNA fragmentation and inducing apoptosis.