Red Palm Oil Ameliorates Oxidative Challenge and Inflammatory Responses Associated with Lipopolysaccharide-Induced Hepatic Injury by Modulating NF-κß and Nrf2/GCL/HO-1 Signaling Pathways in Rats.

Lipopolysaccharide (LPS), a well-conserved cell wall component of Gram positive bacteria, exerts its toxic effects via inducing oxidative and pro-inflammatory responses. Red palm oil (RPO) is a unique natural product with a balanced ratio of saturated and unsaturated fatty acids, with reported antioxidant and anti-inflammatory effects. In this study, we assess the protective effect and mechanistic action of RPO using a lipopolysaccharide (LPS)-induced hepatic injury model. Male Wistar rats were assigned into four groups (10 animals/group): normal control (NC), RPO, LPS and RPO + LPS. Animals in the RPO and RPO + LPS groups were administered RPO (200 µL/day) for 28 days. On the 27th day of experiment, animals in LPS and RPO + LPS groups were injected with LPS (0.5 mg/kg body weight). Animals were sacrificed 24 h later, and blood and liver tissues harvested for biochemical and molecular analysis. RPO resolved hepatic histological dysfunction induced by LPS, and lowered alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and γ-glutamyl transferase activities in the serum. Hepatic malondialdehyde and conjugated dienes, as well as pro-inflammatory cytokines, including interleukin (IL)-1ß, IL-6 and TNFα were significantly diminished (p < 0.05) by RPO pre-treatment. Activity of hepatic antioxidant enzymes including superoxide dismutase, glutathione reductase, glutathione peroxidase, as well as glutathione redox status (GSH:GSSG), and markers of antioxidant capacity that decreased as a result of LPS injection were improved by RPO pre-treatment. Mechanistically, RPO up-regulated mRNA expression of redox sensitive transcription factor Nrf2 and its downstream targets GCL and HO-1, while also suppressing the expression of NFκß and associated inflammatory protein, Iκß kinase (IκKß). In conclusion, this study highlights the ameliorating effects of RPO against LPS-induced hepatic injury and revealed the Nrf2/GCL/HO-1 and NFκß signaling axis as potential contributing mechanisms.

A survey evaluating knowledge, perception, and use of skin lightening products among South African students.

BACKGROUND: In sub-Saharan Africa, the use of skin-lightening products (SLPs) for cosmetic purposes has become common practice among women with dark skin tones. Despite the associated risks, the practice is still significantly increasing in Africa. The objective of this study was to determine the knowledge, perceptions and practice toward skin lightening among young adults. METHODS: A cross-sectional survey among health science students at a tertiary institution in the Western Cape of South Africa was conducted. RESULTS: A total of 401 participants were included in the sample. There was a low prevalence (12%) of skin-lightening practice among students, which could possibly be a result of students being aware of the associated side effects. Participants believed that family and friends are most likely to influence this behavior (48%) and perceived that individuals who practice skin lightening do so because this provides a more fashionable look (76%). Men and women were found to be equally likely to use SLPs, and those residing in urban settings are 10 times more likely to engage in the practice compared with rural dwellers. CONCLUSION: This study contributes valuable information on the phenomenon of skin lightening among a diverse group of young adults. The results highlight the influential role social media platforms and family members play in motivating use of SLPs. Furthermore, the equal likelihood of use among both sexes suggests that the practice is growing among males.

Desensitization of metastatic melanoma cells to therapeutic treatment through repeated exposure to dacarbazine.

Aberrant anti-cancer drug efflux mediated by membrane protein ABC transporters (ABCB5 and ABCG2) is thought to characterize melanoma heterogeneous chemoresistant populations, presumed to have unlimited proliferative and self-renewal abilities. Therefore, this study primarily aimed to investigate whether continuous exposure of melanoma cells to dacarbazine (DTIC) chemotherapeutic drug enriches cultures with therapy resistant cells. Thereafter, we sought to determine whether combining the genotoxic activity of DTIC with the oxidative insults of hypericin activated photodynamic therapy (HYP-PDT) could synergized to kill heterogenous chemoresistant melanoma populations. This study revealed that DTIC resistant (UCT Mel-1DTICR2) melanoma cells were less sensitive to all therapies than parental melanoma cells (UCT Mel-1), yet combination therapy was the most efficient. At the exception of DTIC treatment, both HYP-PDT and the combination therapy were effective in significantly reducing the Hoechst non-effluxing dye melanoma main populations (MP) compared to their side population (SP) counterparts. Likewise, HYP-PDT and combination therapy significantly reduced self-renewal capacity, increased expression of ABCB5 and ABCG2 transporters and differentially induced cell cycle arrest and cell death (apoptosis or necrosis) depending on the melanoma MP cell type. Collectively, combination therapy could synergistically reduce melanoma proliferative and clonogenic potential. However, further research is needed to decipher the cellular mechanisms underlying this resistance which would enable combination therapy to reach therapeutic fruition.

Rooibos (Aspalathus linearis) and honeybush (Cyclopia species) modulate the oxidative stress associated injury of diesel exhaust particles in human umbilical vein endothelial cells.

BACKGROUND: Previous evidence show foods and beverages rich in polyphenolic compounds to have favourable effects on the cardiovascular system. HYPOTHESIS: The current study assessed the modulation of oxidative stress and associated inflammation induced by diesel exhaust particles (DEP - SRM 2975) by pre-treatment of human umbilical vein endothelial cells (HUVECs) with aqueous extracts of rooibos [fermented (FR) as well as green form (GR)] and honeybush [fermented form (FH)]. STUDY DESIGN: HUVEC are either exposed to DEP (10 µg/ml) for 4 h or pre-treated with 40 and 60 µg/ml of FR or GH or FR, or 50 µg/ml orientin (OR) for 6 h prior to DEP exposure. METHODS: In vitro antioxidant capacity of the extracts was assessed and the polyphenol contents were also assessed by HPLC. ROS, cell viability, lactate dehydrogenase leakage, lipid peroxidation, GSH:GSSG ratios, conjugated diene and protein carbonyl levels were determined as indices of oxidative stress and cytotoxicity. RT-qPCR and western blot were used to assess inflammatory cytokines and antioxidant genes expression. RESULTS: DEP caused a dose and time-dependent increase in ROS production, significant (p < 0.001) increase in protein carbonyl (PC) formation, thiobarbituric acid reactive substances and conjugated dienes levels (p < 0.01) and a significant reduction in glutathione (GSH) redox status. Pre-incubation with either the herbal extracts or orientin attenuated these effects. The significant increase in IL-1α, IL-6, IL-8, VCAM-1 and ATF4 gene expression caused by DEP (10 µg/ml) were also attenuated by the presence of the FR, GR and FH extracts, and OR . Pre-treatment with the rooibos extracts or flavone orientin enhanced cell viability, reduced LDH leakage, enhanced mRNA expression of NQO1 and Nrf2, but repressed CYP1B1 mRNA induced by DEP. Western blot showed both the herbal tea extracts and orientin to enhance NQO1 and γGSC protein induction by DEP. CONCLUSION: Taken together, the herbal extracts offer protection against DEP-induced oxidative stress and inflammatory response.

Skin cells for use in an alternate diagnostic method for Duchenne muscular dystrophy.

The importance of molecular diagnosis and identification of disease-associated variants for Duchenne muscular dystrophy (DMD) is evident in the age of gene-based therapies and personalised medicine. Detection of the causative DMD variant and determination of its effects on dystrophin expression is best achieved by analysis of RNA extracted from muscle biopsy material. However, this is not done routinely, as the procedure can be traumatic, especially to young children, and carries risk of complications related to the use of anaesthetic. As skin biopsies are safer and straightforward to perform than muscle biopsies, we investigated the utility of cultured human epidermal melanocytes and dermal fibroblasts as alternative tools for RNA-based diagnosis of DMD. Shallow skin biopsies from 5 boys with genetically confirmed diagnoses of DMD were used to culture fibroblasts and melanocytes. Biopsies were sampled, and tolerated without complications, using local anaesthetic cream. Dystrophin expression in the cultured cells was assessed using immunocytochemical staining, quantitative real-time PCR and cDNA sequencing methodologies. We observed differential expression of the full-length dystrophin muscle transcript, with significantly more robust expression in melanocytes, compared to that in fibroblasts. Our results suggest that cultured skin melanocytes may present an alternative tool for RNA-based genetic diagnosis of DMD.

Cancer stem cells: A product of clonal evolution?

The cancer stem cell (CSC) model has emerged as a prominent paradigm for explaining tumour heterogeneity. CSCs in tumour recurrence and drug resistance have also been implicated in a number of studies. In fact, CSCs are often identified by their expression of drug-efflux proteins which are also highly expressed in normal stem cells. Similarly, pro-survival or proliferation signalling often exhibited by stem cells is regularly reported as being upregulated by CSC. Here we review evidence suggesting that many aspects of CSCs are more readily described by clonal evolution. As an example, cancer cells often exhibit copy number gains of genes involved in drug-efflux proteins and pro-survival signalling. Consequently, clonal selection for stem cell traits may result in cancer cells developing "stemness" traits which impart a fitness advantage, without strictly following a CSC model. Finally, since symmetric cell division would give rise to more cells than asymmetric division, it is expected that more advanced tumours would depart from a CSC. Collectively, these observations suggest clonal evolution may explain many aspects of the CSC.

Intravenous glutathione for skin lightening: Inadequate safety data.

BACKGROUND: Glutathione (GSH) is the most abundant naturally occurring non-protein thiol that protects mammalian cells from oxidative stress. Intravenous (IV) GSH for skin lightening is advertised by clinics in South Africa and internationally online, yet to date no published review on the subject exists. Methods. We conducted a MEDLINE search (to 30 September 2015) of GSH use for skin lightening and of all indications in medicine, to evaluate its safety. Results. Two controlled clinical trials (GSH capsules: 60 patients; 2% glutathione disulphide lotion: 30 patients) and a case series (GSH lozenges: 30 patients) reported a significantly decreased melanin index. A case series (GSH soap: 15 patients) reported skin lightening based on photography. Two systematic reviews of IV GSH for preventing chemo-induced toxicity and a third review of adjuvant therapy for Parkinson's disease altogether included 10 trials. Most trials reported either no or minimal GSH adverse effects, but all had treatment durations of a few doses (IV) or 4 -12 weeks. No study reported long-term IV GSH use. Conclusion. In spite of widespread reported use, there are no studies of IV GSH use for skin lightening or of its safety for chronic use (for any indication). The switch from brown to red melanin production may increase the risk of sun-induced skin cancers in previously protected individuals. Regulatory assessment of systemic GSH administration for cosmetic use by the Medicines Control Council seems urgently warranted to protect consumers from potential side-effects and from complications of IV infusions. This is especially concerning because of reports of GSH bought online. Effective topical GSH may be useful for hyperpigmented skin disorders, but this requires scientific scrutiny. The debate on the merits of cosmetic skin lightening is best handled by multidisciplinary teams.

Melanocyte migration is influenced by E-cadherin-dependent adhesion of keratinocytes in both two- and three-dimensional in vitro wound models.

During wound healing, melanocytes are required to migrate into the wounded area that is still in the process of re-construction. The role and behaviour of melanocytes during this process is poorly understood, that is, whether melanocyte migration into the wound is keratinocyte-dependent or not. This paper attempts, through the use of both two- and three-dimensional in vitro models, to understand the role and behaviour of melanocytes during the process of wound healing. In addition, it sheds light on whether keratinocytes influence/contribute toward melanocyte migration and ultimately wound healing. Scratch assays were performed to analyse migration and Western blot analyses measured cellular E-cadherin expression. Immunohistochemistry was used to analyse the in vivo 3D wound healing effect. Scratch assays performed on co-cultures of melanocytes and keratinocytes demonstrated that melanocytes actively migrated, with the use of their dendrites, into the scratch ahead of the proliferating keratinocyte sheet. Migration of the melanocyte into the wound bed was accompanied by loss of attachment to keratinocytes at the wound front with concomitant downregulation of E-cadherin expression as observed through immunocytochemistry. This result suggests that, in vitro, melanocyte migration occurs independently of keratinocytes but that the migration is influenced by keratinocyte E-cadherin expression. We now demonstrate that melanocyte migration during re-pigmentation is an active process, and suggest that targeting of mechanisms involved in active melanocyte migration (e.g. the melanocyte dendrite) may enhance the re-pigmentation process.

St John's Wort (Hypericum perforatum L.) photomedicine: hypericin-photodynamic therapy induces metastatic melanoma cell death.

Hypericin, an extract from St John's Wort (Hypericum perforatum L.), is a promising photosensitizer in the context of clinical photodynamic therapy due to its excellent photosensitizing properties and tumoritropic characteristics. Hypericin-PDT induced cytotoxicity elicits tumor cell death by various mechanisms including apoptosis, necrosis and autophagy-related cell death. However, limited reports on the efficacy of this photomedicine for the treatment of melanoma have been published. Melanoma is a highly aggressive tumor due to its metastasizing potential and resistance to conventional cancer therapies. The aim of this study was to investigate the response mechanisms of melanoma cells to hypericin-PDT in an in vitro tissue culture model. Hypericin was taken up by all melanoma cells and partially co-localized to the endoplasmic reticulum, mitochondria, lysosomes and melanosomes, but not the nucleus. Light activation of hypericin induced a rapid, extensive modification of the tubular mitochondrial network into a beaded appearance, loss of structural details of the endoplasmic reticulum and concomitant loss of hypericin co-localization. Surprisingly the opposite was found for lysosomal-related organelles, suggesting that the melanoma cells may be using these intracellular organelles for hypericin-PDT resistance. In line with this speculation we found an increase in cellular granularity, suggesting an increase in pigmentation levels in response to hypericin-PDT. Pigmentation in melanoma is related to a melanocyte-specific organelle, the melanosome, which has recently been implicated in drug trapping, chemotherapy and hypericin-PDT resistance. However, hypericin-PDT was effective in killing both unpigmented (A375 and 501mel) and pigmented (UCT Mel-1) melanoma cells by specific mechanisms involving the externalization of phosphatidylserines, cell shrinkage and loss of cell membrane integrity. In addition, this treatment resulted in extrinsic (A375) and intrinsic (UCT Mel-1) caspase-dependent apoptotic modes of cell death, as well as a caspase-independent apoptotic mode that did not involve apoptosis-inducing factor (501 mel). Further research is needed to shed more light on these mechanisms.

Novel, biocatalytically produced hydroxytyrosol dimer protects against ultraviolet-induced cell death in human immortalized keratinocytes.

Compounds derived from botanicals, such as olive trees, have been shown to possess various qualities that make them function as ideal antioxidants and, in doing so, protect them against the damaging effect of ultraviolet (UV)-derived oxidative stress. The aim of this study was to biocatalytically synthesize a dimeric product (compound II) from a known botanical, 3-hydroxytyrosol, and test it for its antioxidant ability using a human immortalized keratinocyte cell line (HaCaT). 2,2-Diphenyl-picryhydrazyl (DPPH) antioxidant assays showed 33 and 86.7% radical scavenging activity for 3-hydroxytyrosol and its dimer, respectively. The ferric-reducing antioxidant power (FRAP) assay corroborated this by showing a 3-fold higher antioxidant activity for the dimer than 3-hydroxytyrosol. Western blot analyses, showing cells exposed to 500 µM of the dimeric product when ultraviolet A (UVA)-irradiated, increased the anti-apoptotic protein Bcl-2 expression by 16% and reduced the pro-apoptotic protein Bax by 87.5%. Collectively, the data show that the dimeric product of 3-hydroxytyrosol is a more effective antioxidant and could be considered for use in skin-care products, health, and nutraceuticals.

Hypericin-PDT-induced rapid necrotic death in human squamous cell carcinoma cultures after multiple treatment.

PDT (photodynamic therapy) has been used for the treatment of NMCC (non-melanoma cutaneous cancer) particularly, human SCC (squamous cell carcinoma). However, the nature of the photosensitizer, the activation light source and the mode of cell death induced post-PDT remains elusive. We tried to optimize PDT using the light-activated (320-400 nm) St John's Wort-derived compound, Hyp (hypericin). The work highlights the potential mode of cell death and the increased efficacy of the technique associated with multiple Hyp-PDT treatment. SCC cells were exposed to different concentrations of Hyp and activated with light at 1 J/cm2 for 1 or 2 days. Thereafter with the optimum dose of Hyp proliferation, ROS (reactive oxygen species), and apoptosis were analysed by XTT [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] assay, FACS analysis and Fluorescent/Phase contrast microscopy was carried out for morphological studies. Hyp-PDT produces more ROS after 1 day compared with 2 days and the mode of cell death is a necrotic caspase-independent mechanism. We propose a novel 'double-hit/2-day' strategy to reduce the viability in SCC using Hyp-based PDT as an adjunctive treatment modality.

Depigmentation in melanomas increases the efficacy of hypericin-mediated photodynamic-induced cell death.

Melanoma is the main cause of death in skin cancers. Despite combating with early detection, resection and post-operative therapy, melanoma treatment remains unsuccessful and investigations into other forms of adjuvant therapy such as photodynamic therapy (PDT) are prudent. This study proposes that depigmentation i.e. the removal of the free radical scavenging pigment, melanin, in melanotic melanoma cells increases their susceptibility to PDT-induced cell death. Two human melanoma cell lines: one pigmented (Mel-1) and one amelanotic (A(375)) cell lines were used. Kojic acid (KA), a tyrosinase-specific inhibitor, was optimised to 6 µg/ml and shown to quantifiably inhibit melanin synthesis after a 3-day exposure. PDT on these cells resulted in a 3.82 fold increase of intracellular ROS production which correlated to 11% increase in cell death susceptibility compared to untreated controls. Moreover, cells allowed to regain their pigment failed to return to normal even after 72 h thus proving the effectiveness of PDT. Using a DPPH* assay, the results confirmed the scavenging properties of melanin (IC(50) 18.30 µg/ml) proving that this pigment may be one of the reasons for melanoma chemoresistance. Overall this study shows that pigment plays an important role in the efficacy of adjunctive PDT treatment and its removal enhances cell death susceptibility in melanomas.

Simultaneous immunofluorescent labeling using anti-BrdU monoclonal antibody and a melanocyte-specific marker in formalin-fixed paraffin-embedded human skin samples.

Immunolabeling of tissue sections requires careful optimization of protocols in order to achieve accurate and consistent data. Multiple immunolabeling is desirable when determining the exact location and phenotype of cell populations in the same cellular compartment. 5-bromodeoxyuridine (BrdU)-immunolabeling is commonly used to assess cellular proliferation in vitro. However, the technical limitations of standard methods preclude multiple antigen immunolabeling. The aim was therefore to develop a robust protocol for simultaneous labeling using anti-BrdU and a melanocyte-specific marker in formalin-fixed paraffin-embedded (FFPE) skin samples. Human skin samples were obtained from patients undergoing elective plastic surgery. The tissue was incubated with BrdU, and a standard sample procedure for FFPE tissue was used. Heat-induced antigen retrieval was performed in a conventional pressure cooker, followed by immunolabeling with anti-BrdU and anti-Melan A/MART-1 antibodies. Fluorescent-conjugated secondary antibodies were used for signal detection. We have demonstrated both proliferating cells (BrdU-immunopositive) and melanocytes (Melan A/MART-1-immunopositive) in the basal compartment of the epidermis in our skin samples. Successful double labeling requires heat-induced epitope retrieval to replace the harsh pretreatment protocols of standard BrdU immunolabeling methods. We have optimized a robust protocol for the double labeling of proliferating cells and cells bearing melanocyte-specific antigens (melanocytes and/or melanoblasts) in FFPE human skin samples.

Photodynamic therapy-induced killing is enhanced in depigmented metastatic melanoma cells.

The resistance of pigmented human melanomas over their unpigmented counterparts to a number of therapies has suggested that the presence of intracellular melanin plays a role in rendering these cells less susceptible to cell death, probably through the ability of this pigment to act as an intracellular antioxidant, thus neutralizing chemotherapeutic-induced ROS (reactive oxygen species). PDT (photodynamic therapy) was recently suggested as an attractive, adjunctive therapy owing to its cellular specificity and limited side effects. In the present study, we propose that first depigmenting melanomas with a reversible TYR (tyrosinase) inhibitor such as PTU (phenylthiourea) increases their susceptibility to HYP-PDT (hypericin-mediated PDT). Pigmented [UCT Mel-1 (University of Cape Town melanoma cell line 1)] and unpigmented (A375) melanomas were first characterized with respect to their TYR activities and melanin quantities and then treated with a TYR inhibitor for 48 h. Cell viability assays after treatment with 3 µM HYP-PDT showed a significant increase in cell death in depigmented melanomas compared with untreated melanomas that returned to the level of untreated melanoma cells on removing the TYR inhibitor. The present study supports the hypothesis that combining the inhibition of melanogenesis with PDT should be explored as a valid therapeutic target for the management of advanced melanoma.

Photoprotection by honeybush extracts, hesperidin and mangiferin against UVB-induced skin damage in SKH-1 mice.

The possible mechanism of photoprotection by polyphenolic extracts of honeybush and the two most abundant polyphenols found in honeybush, hesperidin and mangiferin were determined using a mouse model. Ethanol: acetone soluble extracts and pure honeybush compounds were applied topically to the skin of SKH-1 mice before daily exposures to ultraviolet B (UVB) (180 mJ/cm²) for 10 days. The honeybush extracts reduced signs of sunburn, such as erythema, peeling and hardening of the skin and also significantly (P < 0.05) reduced edema, epidermal hyperplasia and the induction of cyclooxygenase-2 (COX-2), ornithine decarboxylase (ODC), GADD45 and OGG1/2 expression. The fermented honeybush extract significantly (P < 0.05) reduced lipid peroxidation and depletion of the antioxidant enzymes catalase and superoxide dismutase. Hesperidin and mangiferin were less effective. These results show that extracts of honeybush and to some extent, hesperidin and mangiferin, renders protection against UVB-induced skin damage. The mechanisms investigated suggest that honeybush extracts protected the skin via modulation of induced-oxidative damage, inflammation and cell proliferation. Other specific biological properties such as modulation of signaling pathways could also be involved.

Melanomas display increased cytoprotection to hypericin-mediated cytotoxicity through the induction of autophagy.

Photodynamic therapy (PDT) as a regime for melanoma is of limited success due to factors such as the efficacy of the photosensitizer used, penetration depth and the presence of pigment. We characterised a pigmented and an unpigmented melanoma cell line with respect to their phenotypes. Cell viability was assessed after exposure to hypericin, a UVA-activated photosensitizer. Exposure to 3 microM activated hypericin induced a cytoprotective (autophagic) response from both cell lines. However, the pigmented cells accumulated a large amount of glycogen in their cytoplasm. We hypothesise that the treatment induces an initial cytoprotective response through autophagy, but with increased stress results in a different mode of cell death in pigmented melanoma cells from unpigmented cells. These results indicate that hypericin-PDT could be an adjuvant therapy for melanoma.