High-content drug screening in zebrafish xenografts reveals high efficacy of dual MCL-1/BCL-XL inhibition against Ewing sarcoma.

Ewing sarcoma is a pediatric bone and soft tissue cancer with an urgent need for new therapies to improve disease outcome. To identify effective drugs, phenotypic drug screening has proven to be a powerful method, but achievable throughput in mouse xenografts, the preclinical Ewing sarcoma standard model, is limited. Here, we explored the use of xenografts in zebrafish for high-throughput drug screening to discover new combination therapies for Ewing sarcoma. We subjected xenografts in zebrafish larvae to high-content imaging and subsequent automated tumor size analysis to screen single agents and compound combinations. We identified three drug combinations effective against Ewing sarcoma cells: Irinotecan combined with either an MCL-1 or an BCL-XL inhibitor and in particular dual inhibition of the anti-apoptotic proteins MCL-1 and BCL-XL, which efficiently eradicated tumor cells in zebrafish xenografts. We confirmed enhanced efficacy of dual MCL-1/BCL-XL inhibition compared to single agents in a mouse PDX model. In conclusion, high-content screening of small compounds on Ewing sarcoma zebrafish xenografts identified dual MCL-1/BCL-XL targeting as a specific vulnerability and promising therapeutic strategy for Ewing sarcoma, which warrants further investigation towards clinical application.

Zinc Modulates the Response to Apoptosis in an In Vitro Model with High Glucose and Inflammatory Stimuli in C2C12 Cells.

Apoptosis is programmed cell death and its alteration is related to cancer, neurologic, autoimmune, and chronic diseases. A number of factors can affect this process. The aim of this paper is to study the effect of supplemental zinc on apoptosis-related genes in C2C12 myoblast cells after being challenged with a series of stimuli, such as high glucose, insulin, and an inflammatory agent. C2C12 myoblast cells were cultured for 24 h with zinc (Zn) (ZnSO4) 10 or 100 µM and/or glucose 10 or 30 mM. In addition to these stimuli, the cells were challenged with insulin 1 nM or interleukin-6 (IL-6) 5 nM. The mRNA expression of proapoptotic genes caspase 3 and Fas, the antiapoptotic genes, Xiap and Bcl-xL and the ratio of pro-/antiapoptotic genes Bax/Bcl-2, were determined by qRT-PCR. The expression of caspase-3 gene was significantly increased in the presence of the combination high Zn/high glucose with and without the presence of insulin and IL6 in the culture medium Fas expression instead, showed uneven responses. The expression of Bcl-xL and Xiap was increased in most conditions by having high Zn in the medium regardless of the presence of insulin or IL6. Bax/Bcl2 ratio was decreased in the presence of high Zn. Zn was able to stimulate the expression of antiapoptotic genes. This effect was specially noted in high-glucose conditions with and without the presence of insulin. This effect is partially overridden by the presence of an inflammatory agent such as IL-6.

Xiaoaiping injection enhances paclitaxel efficacy in ovarian cancer via pregnane X receptor and its downstream molecules.

ETHNOPHARMACOLOGICAL RELEVANCE: Xiaoaiping injection, a traditional Chinese medical injection extracted from root of Marsdenia tenacissima (Roxb.) Moon, has been exclusively used on curing malignant tumor in China and as adjuvant therapeutic agent for chemotherapeutics, including paclitaxel. AIM OF THE STUDY: The goal of this study was to investigate the synergistic inhibitory efficacy of Xiaoaiping injection and paclitaxel on ovarian cancer. The mechanism may be associated with nuclear receptor pregnane X receptor (PXR) regulating its downstream molecules. MATERIALS AND METHODS: In vitro, MTT assay, flow cytometry and Hoechst dyeing were used to evaluate the SK-OV-3 cell proliferation, apoptosis and cell cycle respectively. The mRNA and protein expression of PXR and its downstream CYP450 enzymes, transporters and Bcl-2 families were measured by qRT-PCR and Western blot. Rhodamine 123 efflux experiment was conducted to detect the P-gp efflux ability. PXR plasmid and PXR siRNA were transiently transfected into SK-OV-3 cells respectively to establish PXR-overexpressed or PXR-interfered cells. In vivo, xenograft tumor mice model was established by SK-OV-3 cells to estimate the antitumor effect of Xiaoaiping injection combined with paclitaxel. The expressions of PXR and its downstream molecules in tumor tissues were determined to further clarify the potential mechanism. RESULTS: Xiaoaiping injection significantly enhanced the anti-proliferation, pro-apoptosis effect of paclitaxel on SK-OV-3 cells. The synergetic effect was displayed by Xiaoaiping injection inhibiting paclitaxel-induced PXR and CAR expression, which subsequently inhibited CYP450 enzymes CYP2C8 and CYP3A4, transporter P-gp and anti-apoptotic proteins Bcl-2 and Bcl-xl in SK-OV-3 cells. In PXR-overexpressed cells, Xiaoaiping injection down-regulated the expression of PXR and its downstream molecules. The result of xenograft tumor model showed that Xiaoaiping injection combined with paclitaxel enhanced anti-tumor effect on ovarian cancer in vivo. CONCLUSIONS: Xiaoaiping injection enhances anti-tumor effect of paclitaxel by inhibiting cell proliferation, inducing apoptosis process. The mechanism may be associated with Xiaoaiping injection inhibiting PXR and its downstream metabolic enzymes CYP2C8, CYP3A4, transporter P-gp and anti-apoptosis protein Bcl-2.

Fridericia platyphylla (Cham.) L.G. Lohmann root extract exerts cytotoxic and antiproliferative effects on gastric tumor cells and downregulates BCL-XL, BIRC5, and MET genes.

Fridericia platyphylla (Cham.) L.G. Lohmann (FP) has cytotoxic, anti-inflammatory, and analgesic properties. We aimed to characterize the cytotoxic and antiproliferative effects of FP extract on normal (GAS) and tumor-derived (ACP02 and HepG2) cell lines. The effective concentrations (EC50s) by tetrazolium bromide assay (MTT) were 56.16, 43.68, and 42.57 µg mL-1 and 69.38, 41.73, and 52.39 µg mL-1 by neutral red assay for GAS, ACP02, and HepG2 cells, respectively. The extract decreased nuclear division indices, which was not reflected in cell proliferation curves. Flow cytometric analyses showed that even 30 µg mL-1 extract (shown to be noncytotoxic by MTT assay) increased the sub-G1 population, indicating cell death due to apoptosis and necrosis. A cytokinesis-block micronucleus cytome assay showed that 30 µg mL-1 of the extract increased the frequency of nuclear buds in tumor cells. Real-time quantitative polymerase chain reaction showed CCND1 upregulation in doxorubicin-treated GAS cells and BCL-XL, BIRC5, and MET downregulation in 5 or 30 µg mL-1 in FP extract-treated ACP02 cells. In conclusion, FP extract modulated apoptosis- and cell cycle-related genes and presented selective cytotoxicity toward tumor cells that deserves further investigation by testing other cell types. Our results demonstrated that even medicinal plants exert adverse effects depending on the extract concentrations used and tissues investigated.

5-FU preferably induces apoptosis in BRAF V600E colorectal cancer cells via downregulation of Bcl-xL.

Fluorouracil (5-FU) which has been widely used in postoperative adjuvant therapy in patients with colon cancer, remains the main backbone of combination treatment of patients with colon cancer. However, the efficacy of 5-FU alone in colorectal cancer patients with BRAFV600E is not clear. In this study, we demonstrated that BRAFV600E confers sensitivity to 5-FU in vitro and in vivo xenograft model, using the paired isogenic colorectal cancer cell lines RKO with either BRAF Wild Type (WT)(+/-) or mutant (Mut) (600E/-). Our results revealed 5-FU preferably induces marked apoptosis in BRAF-mutant colorectal cancer cells, through attenuating expression of Bcl-xL and activation caspase-3/9 pathway, eventually conferring the anti-tumor efficacy of 5-FU in vitro and in vivo. Meanwhile, expression of Bcl-xL remained unchanged in BRAF WT group after treatment of 5-FU, although low extent of anti-tumor activity of 5-FU still being observed. In conclusion, these results provided a better understanding of clinical outcome of 5-FU between BRAF WT and mutant colorectal cancer patients, and suggested the inhibition of Bcl-xL might present an alternative strategy to enhance the therapeutic efficacy of 5-FU in colorectal cancer patients with BRAF mutation.

Tooniliatone A sensitizes multidrug resistant cancer cells by decreasing Bcl-xL via activation of JNK MAPK signaling.

BACKGROUND: Multidrug resistance (MDR) refers to the phenotype of tumor cells that are resistant to various chemotherapeutic drugs with different structures and functions, which is clearly disadvantageous for patients. Finding a natural product that can effectively reverse the MDR of tumor cells is important for the treatment of patients. PURPOSE: To prove that tooniliatone A (TA), a novel typical limonoid, can effectively reverse the MDR of tumor cells and to explore its mechanism of action. METHODS: The MTT, CCK-8 and monoclonal formation assays, as well as flow cytometry, were used to evaluate the role of TA in reversing tumor multidrug resistance; then the mechanism of action for TA was explored by western blotting and real-time fluorescent quantitative PCR. RESULTS: TA significantly reversed the MDR of the K562/MDR and MCF-7/MDR cell lines. TA can inhibit the anti-apoptotic protein Bcl-xL to make cells sensitive to common chemotherapeutic drugs and activate the SAPK/JNK pathway to promote phosphorylation of JNK and its downstream cJun protein. Small interfering RNA-mediated knockdown of JNK and cJun could antagonize the MDR reversal effect of TA and the inhibition of Bcl-xL by TA. Therefore, we hypothesized that TA activates the JNK pathway to increase the transcription of the proapoptotic protein Bim, thereby inhibiting Bcl-xL and reversing MDR in tumor cells. CONCLUSION: Our study suggests that TA reverses tumor MDR by activating the SAPK/JNK pathway to inhibit the action of Bcl-xL. TA may be an effective tumor MDR reversal agent.

High-Dose Aspirin Reverses Tartrazine-Induced Cell Growth Dysregulation Independent of p53 Signaling and Antioxidant Mechanisms in Rat Brain.

Tartrazine, an azo dye used in food, cosmetics, and pharmaceuticals with the effects on cell cycle, is not well understood. Therefore, we investigated the toxicity of tartrazine in rat brain with high-dose aspirin. Male Wistar rats (n = 24) were divided into (C) control, (T) tartrazine (700 mg/kg body weight [BW] at weeks 1 and 2), (A) aspirin (150 mg/kg [BW] at weeks 1, 2, and 3), and (TA) aspirin + tartrazine (150 mg/kg [BW] aspirin at weeks 1, 2, and 3 and 700 mg/kg [BW] tartrazine at weeks 1 and 2) groups. The expression of p53, B cell lymphoma-2 extra-large (Bcl-xL), cyclin-dependent kinase 2 (CDK2), p27, and Ki67 was evaluated by quantitative reverse-transcription PCR. A histopathological analysis of brain tissue and oxidative stress level was assessed based on reduced glutathione (GSH), ascorbic acid (AA), and malondialdehyde levels. We found that Bcl-xL, Ki67, CDK2, and p27 were upregulated and p53 was downregulated in the tartrazine-treated group as compared to the control group. Aspirin administration reversed these changes except P53 expression. Tartrazine had no effect on lipid peroxidation but altered AA and GSH levels with no reversal by aspirin treatment. Histopathological analysis revealed that aspirin prevented tartrazine-induced damage including increased perivascular space and hemorrhage. These results indicate that aspirin protects the brain from tartrazine-induced toxicity independent of p53 signaling and antioxidant mechanisms.

Selenium Nanoparticle Protection of Fibroblast Stress: Activation of ATF4 and Bcl-xL Expression.

BACKGROUND: In recent years, selenium nanostructures have been researched due to their antibacterial properties, low toxicity to mammalian cells, and high biological efficacy. However, the clinical implementation of the use of selenium has received mixed results, and there is much work needed to improve the understanding of the biological mechanisms involved in the observed cellular responses. MATERIALS AND METHODS: In this work, an investigation into the mechanistic pathways of selenium nanoparticles (SeNPs) in biological systems was conducted by studying the changes in gene expression of ATF4, Bcl-xL, BAD2, HSP70, and SOD2 in non-cancerous human dermal fibroblasts (HDF) under oxidative stress, nutrient deprivation stress, and no treatment (control) conditions. RESULTS: This study revealed that SeNP incubation led to reduced internal reactive oxygen species (ROS) generation for all conditions tested, thus, providing a protective environment for HDF. At the stress conditions, the expression of ATF4 and Bcl-xL increased for cells treated with SeNP incubation, leading to attenuation of the cells under stress. These results also hint at reductive stress causing a detrimental impact to cell proliferation under routine cell passaging conditions. CONCLUSION: In summary, this study highlights some possible mechanistic pathways implicated in the action of SeNPs that warrant further investigation (specifically, reducing stress conditions for HDF) and continues to support the promise of SeNPs in a wide range of medical applications.

Ubiquinol promotes retinal ganglion cell survival and blocks the apoptotic pathway in ischemic retinal degeneration.

Coenzyme Q10 (CoQ10) protects retinal ganglion cells (RGCs) in experimental retinal ischemia and glaucoma by scavenging reactive oxygen species. We tested whether a diet supplemented with ubiquinol, the reduced form of CoQ10, promotes RGC survival and blocks the apoptotic pathway in ischemic mouse retina induced by acute high intraocular pressure (IOP) elevation. Ubiquinol (1%) treatment significantly promoted RGC survival at 2 weeks after ischemia/reperfusion. The ubiquinol treatment significantly blocked activation of astroglial and microglial cells in the ischemic retina at 2 weeks. While the ubiquinol treatment significantly decreased active Bax protein expression in the ischemic retina, phosphorylation of Bad at serine 112 and Bcl-xL protein expression were preserved in the ubiquinol-treated ischemic retina at 12 h. Consistently, the ubiquinol treatment prevented apoptotic cell death by blocking caspase-3 cleavage. These results suggest that the ubiquinol enhances RGC survival by modulating the Bax/Bad/Bcl-xL-mediated apoptotic pathway in the ischemic retina. Ubiquinol has therapeutic potential for ameliorating elevated IOP-induced ischemic retinal degeneration.

Resveratrol significantly improves the fertilisation capacity of bovine sex-sorted semen by inhibiting apoptosis and lipid peroxidation.

The aim of this study was to test the effects of five different concentrations (0, 10-3, 10-4, 10-5, and 10-6 M) of resveratrol (Res) supplementation in bull sperm washing and fertilisation medium on levels of reactive oxygen species (ROS), phosphatidylserine (PS) externalisation, mitochondrial membrane potential (Δψm), ATP and malondialdehyde (MDA), acrosomal integrity, blastocyst rate, and blastocyst quality after in vitro fertilisation (IVF). The results for sex-sorted sperm from three bulls showed: (1) ROS and MDA levels in 10-3 M and 10-4 M Res groups were significantly lower than those of controls (P < 0.05); (2) the percentage of viable sperm, percentage of sperm with high Δψm, and the ATP content in 10-3 M and 10-4 M Res groups were significantly higher than those of controls (P < 0.05); (3) the percentage of viable sperm with acrosomal integrity, and the blastocyst percentage and quality of the 10-4 M Res group were significantly higher than those of controls (P < 0.05). In conclusion, 10-4 M Res supplementation in washing and fertilisation medium of sex-sorted bull sperm significantly decreased ROS, PS externalisation, and MDA, and protected mitochondrial function and acrosomal integrity, thereby increasing blastocyst percentage and quality following IVF.

Anticancer activity of arborinine from Glycosmis parva leaf extract in human cervical cancer cells.

Glycosmis parva is a small shrub found in Thailand. Ethyl acetate (EtOAc) extract from its leaves has been shown to exert anticancer effects in vitro; however, the compound responsible for this activity has not been isolated and characterized. In this study, we demonstrate that arborinine, a major acridone alkaloid in the EtOAc fraction, decreased proliferation and was strongly cytotoxic to HeLa cervical cancer cells without significantly affecting normal cells. The compound also inhibited tumor spheroid growth much more potently than chemotherapeutic drugs bleomycin, gemcitabine, and cisplatin. In addition, unlike cisplatin, arborinine activated caspase-dependent apoptosis without inducing DNA damage response. We further show that arborinine strongly suppressed cancer cell migration by downregulating expression of key regulators of epithelial-mesenchymal transition. Taken together, our data provide important insights into the molecular mechanism of arborinine's anticancer activity, supporting its potential use for treating cervical cancer.

Licorice Pretreatment Protects Against Brain Damage Induced by Middle Cerebral Artery Occlusion in Mice.

Licorice is extracted from the roots of plants in the Glycyrrhiza genus, especially Glycyrrhiza uralensis in China and Korea. It has several pharmacological activities, including neuro-protective, anti-fungal, and anti-cariogenic effects. Ischemia/reperfusion-induced brain injury is a leading cause of adult disability and death; thus, the identification of anti-apoptotic, neuro-protective therapeutic agents is viewed as an attractive drug development strategy. Infarct volumes and the expression of several apoptosis-related proteins, including Bcl-xL, Bcl-2, caspase-8, and caspase-9, were evaluated by western blotting in the brains of mice subjected to middle cerebral artery occlusion (MCAO). Three consecutive days of oral pretreatment with the methanol extract of licorice (GRex) significantly reduced infarct volumes 24 h after MCAO. In addition, GRex effectively inhibited the activation of caspase-9 by upregulating protein expression of Bcl-xL and Bcl-2. The neuro-protective effect of licorice was due to its regulation of apoptosis-related proteins. These data suggest that licorice could be a potential candidate for the treatment of ischemia-induced brain damage.

Scorpion Venom Causes Upregulation of p53 and Downregulation of Bcl-xL and BID Protein Expression by Modulating Signaling Proteins Erk1/2 and STAT3, and DNA Damage in Breast and Colorectal Cancer Cell Lines.

Scorpion venoms efficiently block the normal neurotransmitter signaling pathway by prejudicing the ion channel operating mechanism in the body system. Besides its negative effect, venoms also possess some beneficial qualities for humans. They have also been shown to exhibit anticancer properties in various cancer types. This unique property of the venom as an anticancer agent is mainly a result of its role in initiating apoptosis and inhibiting several signaling cascade mechanisms that promote cancer cell proliferation and growth. In this study, we examine the effect of venom on phenotypic changes as well as changes at the molecular levels in colorectal and breast cancer cell lines. A dramatic decrease in cell invasion was observed in both cancer cell lines on venom treatment. Additionally, there was decrease in IL-6, RhoC, Erk1/2, and STAT3 in venom-treated cell lines, providing strong evidence of its anticancer properties. Furthermore, decrease in the expression of antiapoptotic proteins and also upregulation of proapoptotic ones by these lines were observed on venom treatment. Moreover, a vivid picture of DNA damage was also detected on venom treatment. In conclusion, scorpion venom possesses significant potential as an anticancer agent against colorectal and breast cancer cell lines.

Achyranthes bidentate saponins protect rat articular chondrocytes against interleukin-1ß-induced inflammation and apoptosis in vitro.

Achyranthes bidentate Blume (Niuxi) is often employed for treatment of arthritis in Traditional Chinese Medicine and possesses anti-inflammatory properties. Phytochemical and pharmacological studies proved the oleanane-type saponins to be the main bioactive principles. In the present study, protective effects of A. bidentata saponins (ABS) on inflammation and apoptosis in interleukine-1ß (IL-1ß)-induced chondrocytes were investigated. Rat chondrocytes were pretreated with ABS at 3 µg/mL, 10 µg/mL, and 30 µg/mL, and subsequently stimulated with IL-1ß (10 ng/mL). Methylthiazolyldiphenyl-tetrazolium bromide assay and annexin V/propidium iodide dual staining demonstrated that ABS could protect IL-1ß-induced chondrocyte injury. ABS suppressed IL-1ß-induced apoptosis by suppressing the activation of caspase-3, inhibiting levels of proapoptotic proteins Bax and Bad, decreasing p53 protein phosphorylation, and promoting the expression of antiapoptotic protein Bcl-xL and proliferating cell nuclear antigen. IL-1ß-induced inflammation and matrix degradation were also alleviated by ABS through the downregulation of the expressions of matrix metalloproteinases 3 and 9 and cyclooxygenase-2. Moreover, ABS inhibited IL-1ß-induced nuclear factor κB activation in rat chondrocytes. We demonstrated, for the first time, the protective effects of ABS on IL-1ß-stimulated chondrocytes and their molecular mechanisms. Thus, it is suggested that ABS might be a potential drug in the treatment of osteoarthritis.

Effect of melatonin supplementation on cryopreserved sperm quality in mouse.

BACKGROUND: Antioxidants protect spermatozoa against cell damage during cryopreservation. OBJECTIVE: To investigate whether melatonin supplement in the extender may improve the quality of cryopreserved mouse sperm. METHODS: Kunming mice sperm frozen in extender R18S3 (18% (w/v) raffinose and 3% (w/v) skim milk) supplemented with melatonin were thawed and evaluated. RESULTS: Mouse spermatozoa were cryopreserved in the freezing extender R18S3 that contained melatonin at 0, 0.125, 0.25 and 0.5 mg/mL melatonin. The extender without melatonin supplement was associated with increased formation of reactive oxygen species (ROS) and decreased sperm motility. Melatonin supplement at 0.125 mg/mL significantly increased the progressive motility of sperm in comparison to other melatonin concentration or control. The percentage of thawed viable sperm with ROS was lower in the melatonin-treated groups than in untreated group. Melatonin supplement also increased antiapoptotic gene Bcl-xl expression in the thawed sperm. CONCLUSION: Supplement of 0.125 mg/mL melatonin could reduce oxidative damage and apoptosis.

Soybean and Fish Oil Mixture With Different ω-6/ω-3 Polyunsaturated Fatty Acid Ratios Modulates Dextran Sulfate Sodium-Induced Changes in Small Intestinal Intraepithelial γδT-Lymphocyte Expression in Mice.

BACKGROUND: This study investigated the effect of different ω-6/ω-3 polyunsaturated fatty acid (PUFA) ratios on dextran sulfate sodium (DSS)-induced changes to small intestinal intraepithelial lymphocyte (IEL) γδT-cell expression. METHODS: Mice were assigned to 3 control and 3 DSS-treated groups and were maintained on a low-fat semipurified diet. One of the control (S) groups and a DSS (DS) group were provided with soybean oil; the other 2 control (Hω-3 and Lω-3) groups and 2 other DSS (DHω-3 and DLω-3) groups were fed either a soybean and fish oil mixture with a ω-6/ω-3 ratio of 2:1 or 4:1. After feeding the respective diets for 2 weeks, the DSS groups were given distilled water containing 2% DSS, and the control groups were given distilled water for 5 days. All groups were further provided distilled water 5 days for recovery, and the small intestinal IEL γδT-cell subset was isolated for analysis. RESULTS: DSS treatment resulted in a lower small intestinal IEL γδT-cell percentage and higher messenger RNA (mRNA) expressions of Reg IIIγ, keratinocyte growth factor (KGF), and complement 5a receptor (C5aR) by IEL γδT cells. Fish oil administration enhanced the proportion of small intestinal IEL γδT cells. Compared with the DLω-3 group, the DHω-3 group had lower Reg IIIγ, KGF, and C5aR mRNA expressions and higher expression of peroxisome proliferator-activated receptor (PPAR)-γ gene by small intestinal IEL γδT cells. CONCLUSIONS: Fish oil diets with a ω-6/ω-3 PUFA ratio of 2:1 were more effective than those with a ratio of 4:1 in improving DSS-induced small intestinal injury, and activation of PPAR-γ in IEL γδT cells may be associated with resolution of small intestinal inflammation.

Hyperoside induces apoptosis and inhibits growth in pancreatic cancer via Bcl-2 family and NF-κB signaling pathway both in vitro and in vivo.

Although advanced surgical operation and chemotherapy have been under taken, pancreatic cancer remains one of the most aggressive and fatal human malignancies with a low 5-year survival rate of less than 5 %. Therefore, novel therapeutic strategies for prevention and remedy are urgently needed in pancreatic cancer. This present research aimed to investigate the anti-cancer effects of hyperoside in human pancreatic cancer cells. Our in vitro results showed that hyperoside suppressed the proliferation and promoted apoptosis of two different human pancreatic cancer cell lines, which correlated with up-regulation of the ratios of Bax/Bcl-2 and Bcl-xL and down-regulation of levels of nuclear factor-κB (NF-κB) and NF-κB's downstream gene products. What's more, using an orthotopic model of human pancreatic cancer, we found that hyperoside also inhibited the tumor growth significantly. Mechanically, these outcomes could also be associated with the up-regulation of the ratios of Bax/Bcl-2 and Bcl-xL and down-regulation of levels of NF-κB and NF-κB's downstream gene products. Collectively, our experiments indicate that hyperoside may be a promising candidate agent for the treatment of pancreatic cancer.

High Dietary Folate in Mice Alters Immune Response and Reduces Survival after Malarial Infection.

Malaria is a significant global health issue, with nearly 200 million cases in 2013 alone. Parasites obtain folate from the host or synthesize it de novo. Folate consumption has increased in many populations, prompting concerns regarding potential deleterious consequences of higher intake. The impact of high dietary folate on the host's immune function and response to malaria has not been examined. Our goal was to determine whether high dietary folate would affect response to malarial infection in a murine model of cerebral malaria. Mice were fed control diets (CD, recommended folate level for rodents) or folic acid-supplemented diets (FASD, 10x recommended level) for 5 weeks before infection with Plasmodium berghei ANKA. Survival, parasitemia, numbers of immune cells and other infection parameters were assessed. FASD mice had reduced survival (p<0.01, Cox proportional hazards) and higher parasitemia (p< 0.01, joint model of parasitemia and survival) compared with CD mice. FASD mice had lower numbers of splenocytes, total T cells, and lower numbers of specific T and NK cell sub-populations, compared with CD mice (p<0.05, linear mixed effects). Increased brain TNFα immunoreactive protein (p<0.01, t-test) and increased liver Abca1 mRNA (p<0.01, t-test), a modulator of TNFα, were observed in FASD mice; these variables correlated positively (rs = 0.63, p = 0.01). Bcl-xl/Bak mRNA was increased in liver of FASD mice (p<0.01, t-test), suggesting reduced apoptotic potential. We conclude that high dietary folate increases parasite replication, disturbs the immune response and reduces resistance to malaria in mice. These findings have relevance for malaria-endemic regions, when considering anti-folate anti-malarials, food fortification or vitamin supplementation programs.

Omega-3 Polyunsaturated Fatty Acids Trigger Cell Cycle Arrest and Induce Apoptosis in Human Neuroblastoma LA-N-1 Cells.

Omega-3 (n-3) fatty acids are dietary long-chain fatty acids with an array of health benefits. Previous research has demonstrated the growth-inhibitory effect of n-3 fatty acids on different cancer cell lines in vitro, yet their anti-tumor effects and underlying action mechanisms on human neuroblastoma LA-N-1 cells have not yet been reported. In this study, we showed that docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) exhibited time- and concentration-dependent anti-proliferative effect on the human neuroblastoma LA-N-1 cells, but had minimal cytotoxicity on the normal or non-tumorigenic cells, as measured by MTT reduction assay. Mechanistic studies indicated that DHA and EPA triggered G0/G1 cell cycle arrest in LA-N-1 cells, as detected by flow cytometry, which was accompanied by a decrease in the expression of CDK2 and cyclin E proteins. Moreover, DHA and EPA could also induce apoptosis in LA-N-1 cells as revealed by an increase in DNA fragmentation, phosphatidylserine externalization and mitochondrial membrane depolarization. Up-regulation of Bax, activated caspase-3 and caspase-9 proteins, and down-regulation of Bcl-XL protein, might account for the occurrence of apoptotic events. Collectively, our results suggest that the growth-inhibitory effect of DHA and EPA on LA-N-1 cells might be mediated, at least in part, via triggering of cell cycle arrest and apoptosis. Therefore, DHA and EPA are potential anti-cancer agents which might be used for the adjuvant therapy or combination therapy with the conventional anti-cancer drugs for the treatment of some forms of human neuroblastoma with minimal toxicity.

Combined ginger extract & Gelam honey modulate Ras/ERK and PI3K/AKT pathway genes in colon cancer HT29 cells.

BACKGROUND: The interconnected Ras/ERK and PI3K/AKT pathways play a central role in colorectal tumorigenesis, and they are targets for elucidating mechanisms involved in attempts to induce colon cancer cell death. Both ginger (Zingiber officinale) and honey have been shown to exhibit anti-tumor and anti-inflammation properties against many types of cancer, including colorectal cancer. However, there are currently no reports showing the combined effect of these two dietary compounds in cancer growth inhibition. The aim of this study was to evaluate the synergistic effect of crude ginger extract and Gelam honey in combination as potential cancer chemopreventive agents against the colorectal cancer cell line HT29. METHODS: The cells were divided into 4 groups: the first group represents HT29 cells without treatment, the second and third groups were cells treated singly with either ginger or Gelam honey, respectively, and the last group represents cells treated with ginger and Gelam honey combined. RESULTS: The results of MTS assay showed that the IC50 of ginger and Gelam honey alone were 5.2 mg/ml and 80 mg/ml, respectively, whereas the IC50 of the combination treatment was 3 mg/ml of ginger plus 27 mg/ml of Gelam honey with a combination index of < 1, suggesting synergism. Cell death in response to the combined ginger and Gelam honey treatment was associated with the stimulation of early apoptosis (upregulation of caspase 9 and IκB genes) accompanied by downregulation of the KRAS, ERK, AKT, Bcl-xL, NFkB (p65) genes in a synergistic manner. CONCLUSIONS: In conclusion, the combination of ginger and Gelam honey may be an effective chemopreventive and therapeutic strategy for inducing the death of colon cancer cells.