Plants against cancer: the immune-boosting herbal microbiome: not of the plant, but in the plant. Basic concepts, introduction, and future resource for vaccine adjuvant discovery.

The presence of microorganism communities (MOCs) comprised of bacteria, fungi, archaea, algae, protozoa, viruses, and the like, are ubiquitous in all living tissue, including plant and animal. MOCs play a significant role in establishing innate and acquired immunity, thereby influencing susceptibility and resistance to disease. This understanding has fostered substantial advancements in several fields such as agriculture, food science/safety, and the development of vaccines/adjuvants, which rely on administering inactivated-attenuated MOC pathogens. Historical evidence dating back to the 1800s, including reports by Drs Busch, Coley, and Fehleisen, suggested that acute febrile infection in response to "specific microbes" could trigger spontaneous tumor remission in humans. This discovery led to the purposeful administration of the same attenuated strains, known as "Coley's toxin," marking the onset of the first microbial (pathogen) associated molecular pattern (MAMPs or PAMPs)-based tumor immunotherapy, used clinically for over four decades. Today, these same MAMPS are consumed orally by billions of consumers around the globe, through "specific" mediums (immune boosting "herbal supplements") as carriers of highly concentrated MOCs accrued in roots, barks, hulls, sea algae, and seeds. The American Herbal Products Association (AHPA) mandates microbial reduction in botanical product processing but does not necessitate the removal of dead MAMP laden microbial debris, which we ingest. Moreover, while existing research has focused on the immune-modulating role of plant phytochemicals, the actual immune-boosting properties might instead reside solely in the plant's MOC MAMP laden biomass. This assertion is logical, considering that antigenic immune-provoking epitopes, not phytochemicals, are known to stimulate immune response. This review explores a neglected area of research regarding the immune-boosting effects of the herbal microbiome - a presence which is indirectly corroborated by various peripheral fields of study and poses a fundamental question: Given that food safety focuses on the elimination of harmful pathogens and crop science acknowledges the existence of plant microbiomes, what precisely are the immune effects of ingesting MAMPs of diverse structural composition and concentration, and where are these distributed in our botanicals? We will discuss the topic of concentrated edible MAMPs as acid and thermally stable motifs found in specific herbs and how these would activate cognate pattern recognition receptors (PPRs) in the upper gut-associated lymphoid tissue (GALT), including Peyer's patches and the lamina propria, to boost antibody titers, CD8+ and CD4+ T cells, NK activity, hematopoiesis, and facilitating M2 to M1 macrophage phenotype transition in a similar manner as vaccines. This new knowledge could pave the way for developing bioreactor-grown/heat-inactivated MOC therapies to boost human immunity against infections and improve tumor surveillance.

Disparities in Infant Nutrition: WIC Participation and Rates of Breastfeeding in Florida.

Being cognizant of the pronounced health advantages of breastfeeding for both the nursing mother and her infant, the breastfeeding dyad, we examined breastfeeding rates among Floridian women who gave birth from 2012 to 2014 (N = 639,052). We investigated the associations between breastfeeding initiation and WIC-based breastfeeding support (the Special Supplemental Nutrition Program for Women, Infants, and Children), education level, and race and ethnicity. We compared the percentage of breastfeeding mothers between those in the WIC program and those who were not, and we compared breastfeeding rates across racial and ethnic groups. Consistent with previous reports, black newborns in this study were breastfed at lower rates than other racial groups, and WIC program participants were less likely to breastfeed than non-WIC program participants. However, by breaking down the data by education level and race, and ethnicity, we see a significantly increased rate of breastfeeding due to WIC participation for both Hispanic and black women with less than a high school education. Further, we assessed differences by insurance type, race, and WIC participation. In multivariable logistic regression, we showed that the WIC program has a significant positive impact on breastfeeding rates for all but white non-Hispanic mothers, independent of sociodemographic and geographic variables. We also note a trend of increasing breastfeeding rates over the study period (p-value < 0.0001), which has positive public health implications.

Triple Isozyme Lactic Acid Dehydrogenase Inhibition in Fully Viable MDA-MB-231 Cells Induces Cytostatic Effects That Are Not Reversed by Exogenous Lactic Acid.

A number of aggressive human malignant tumors are characterized by an intensified glycolytic rate, over-expression of lactic acid dehydrogenase A (LDHA), and subsequent lactate accumulation, all of which contribute toward an acidic peri-cellular immunosuppressive tumor microenvironment (TME). While recent focus has been directed at how to inhibit LDHA, it is now becoming clear that multiple isozymes of LDH must be simultaneously inhibited in order to fully suppress lactic acid and halt glycolysis. In this work we explore the biochemical and genomic consequences of an applied triple LDH isozyme inhibitor (A, B, and C) (GNE-140) in MDA-MB-231 triple-negative breast cancer cells (TNBC) cells. The findings confirm that GNE-140 does in fact, fully block the production of lactic acid, which also results in a block of glucose utilization and severe impedance of the glycolytic pathway. Without a fully functional glycolytic pathway, breast cancer cells continue to thrive, sustain viability, produce ample energy, and maintain mitochondrial potential (ΔΨM). The only observable negative consequence of GNE-140 in this work, was the attenuation of cell division, evident in both 2D and 3D cultures and occurring in fully viable cells. Of important note, the cytostatic effects were not reversed by the addition of exogenous (+) lactic acid. While the effects of GNE-140 on the whole transcriptome were mild (12 up-regulated differential expressed genes (DEGs); 77 down-regulated DEGs) out of the 48,226 evaluated, the down-regulated DEGS collectively centered around a loss of genes related to mitosis, cell cycle, GO/G1-G1/S transition, and DNA replication. These data were also observed with digital florescence cytometry and flow cytometry, both corroborating a G0/G1 phase blockage. In conclusion, the findings in this work suggest there is an unknown element linking LDH enzyme activity to cell cycle progression, and this factor is completely independent of lactic acid. The data also establish that complete inhibition of LDH in cancer cells is not a detriment to cell viability or basic production of energy.

Effects of Wild Yam Root (Dioscorea villosa) Extract on the Gene Expression Profile of Triple-negative Breast Cancer Cells.

BACKGROUND/AIM: Wild yam extract [Dioscorea villosa, (WYE)] is consistently lethal at low IC50s across diverse cancer-lines in vitro. Unlike traditional anti-cancer botanicals, WYE contains detergent saponins which reduce oil-water interfacial tensions causing disintegration of lipid membranes and causing cell lysis, creating an interfering variable. Here, we evaluate WYE at sub-lethal concentrations in MDA-MB-231 triple-negative breast cancer (TNBC) cells. MATERIALS AND METHODS: Quantification of saponins, membrane potential, lytic death and sub-lethal WYE changes in whole transcriptomic (WT) mRNA, miRNAs and biological parameters were evaluated. RESULTS: WYE caused 346 differentially expressed genes (DEGs) out of 48,226 transcripts tested; where up-regulated DEGS reflect immune stimulation, TNF signaling, COX2, cytokine release and cholesterol/steroid biosynthesis. Down-regulated DEGs reflect losses in cell division cycle (CDC), cyclins (CCN), cyclin-dependent kinases (CDKs), centromere proteins (CENP), kinesin family members (KIFs) and polo-like kinases (PLKs), which were in alignment with biological studies. CONCLUSION: Sub-lethal concentrations of WYE appear to evoke pro-inflammatory, steroid biosynthetic and cytostatic effects in TNBC cells.

Transcriptome Profile Analysis of Triple-Negative Breast Cancer Cells in Response to a Novel Cytostatic Tetrahydroisoquinoline Compared to Paclitaxel.

The absence of chemotherapeutic target hormone receptors in breast cancer is descriptive of the commonly known triple-negative breast cancer (TNBC) subtype. TNBC remains one of the most aggressive invasive breast cancers, with the highest mortality rates in African American women. Therefore, new drug therapies are continually being explored. Microtubule-targeting agents such as paclitaxel (Taxol) interfere with microtubules dynamics, induce mitotic arrest, and remain a first-in-class adjunct drug to treat TNBC. Recently, we synthesized a series of small molecules of substituted tetrahydroisoquinolines (THIQs). The lead compound of this series, with the most potent cytostatic effect, was identified as 4-Ethyl-N-(7-hydroxy-3,4-dihydroisoquinolin-2(1H)-yl) benzamide (GM-4-53). In our previous work, GM-4-53 was similar to paclitaxel in its capacity to completely abrogate cell cycle in MDA-MB-231 TNBC cells, with the former not impairing tubulin depolymerization. Given that GM-4-53 is a cytostatic agent, and little is known about its mechanism of action, here, we elucidate differences and similarities to paclitaxel by evaluating whole-transcriptome microarray data in MDA-MB-231 cells. The data obtained show that both drugs were cytostatic at non-toxic concentrations and caused deformed morphological cytoskeletal enlargement in 2D cultures. In 3D cultures, the data show greater core penetration, observed by GM-4-53, than paclitaxel. In concentrations where the drugs entirely blocked the cell cycle, the transcriptome profile of the 48,226 genes analyzed (selection criteria: (p-value, FDR p-value < 0.05, fold change -2< and >2)), paclitaxel evoked 153 differentially expressed genes (DEGs), GM-4-53 evoked 243 DEGs, and, of these changes, 52/153 paclitaxel DEGs were also observed by GM-4-53, constituting a 34% overlap. The 52 DEGS analysis by String database indicates that these changes involve transcripts that influence microtubule spindle formation, chromosome segregation, mitosis/cell cycle, and transforming growth factor-ß (TGF-ß) signaling. Of interest, both drugs effectively downregulated "inhibitor of DNA binding, dominant negative helix-loop-helix" (ID) transcripts; ID1, ID3 and ID4, and amphiregulin (AREG) and epiregulin (EREG) transcripts, which play a formidable role in cell division. Given the efficient solubility of GM-4-53, its low molecular weight (MW; 296), and capacity to penetrate a small solid tumor mass and effectively block the cell cycle, this drug may have future therapeutic value in treating TNBC or other cancers. Future studies will be required to evaluate this drug in preclinical models.

Metabolic Response to the Mitochondrial Toxin 1-Methyl-4-phenylpyridinium (MPP+) in LDH-A/B Double-knockout LS174T Colon Cancer Cells.

BACKGROUND/AIM: Rapid glycolytic substrate-level phosphorylation (SLP) and accumulation of lactic acid are characteristics of diverse cancers. Recent advances in drug discovery have included the use of glycolytic inhibitors with mitochondrial targeting drugs to attempt to invoke an energy crisis in aggressive metabolically active chemo-resistant cancers. In this work, we examine the consequences of inhibiting mitochondrial oxidative phosphorylation (OXPHOS) with 1-methyl-4-phenylpyridinium (MPP+) in LS14T colon cancer cells containing a genetic double knock out (DKO) of lactic acid dehydrogenase (LDHA and LDHB). MATERIALS AND METHODS: Several metabolic parameters were evaluated concomitant to whole transcriptomic (WT) mRNA, microRNA, and long intergenic non-coding RNAs using Affymetrix 2.1 human ST arrays. RESULTS: MPP+ effectively blocked OXPHOS where a compensatory shift toward anaerobic SLP was only observed in the control vector (CV), and not observed in the LDH-A/B DKOs (lacking the ability to produce lactic acid). Despite this, there was an unexpected resilience to MPP+ in the latter in terms of energy, which displayed significantly higher resting baseline respiratory OXPHOS capacity relative to controls. At the transcriptome level, MPP+ invoked 1738 differential expressed genes (DEGs) out of 48,226; LDH-A/B DKO resulted in 855 DEGs while 349 DEGs were found to be overlapping in both groups versus respective controls, including loss of mitochondrial complex I (subunits 3 and 6), cell cycle transcripts and fluctuations in epigenetic chromatin remodeling systems. In terms of energy, the effects of MPP+ in the CV transcripts reflect the funneling of carbon intermediates toward glycolysis. The LDH-A/B DKO transcripts reflect a flow of carbons away from glycolysis toward the production of acetyl-CoA. CONCLUSION: The findings from this study suggest a metabolic resilience to MPP+ in cancer cells devoid of LDH-A/B, explainable in-part by higher baseline OXPHOS respiratory ATP production, necessitating more toxin to suppress the electron transport chain.

Cocaine potentiates an inflammatory response in C6 astroglia-like cells.

Cocaine is a highly addictive drug that mediates its effect through altering dopamine metabolism in the central nervous system (CNS), resulting in a feeling of euphoria. Owing to its high lipophilicity, cocaine easily crosses the blood brain barrier of the CNS and reaches various domains of the brain, where it can trigger cellular damage. Cocaine-induced CNS damage may arise due to increased levels of free radicals and nitric oxide (NO) in immunecompetent astroglial cells. In the present study, the potential ability of cocaine to exacerbate the production of inflammatory products, primarily superoxide free radicals (O2 -), hydrogen peroxide (H2O2) and NO/nitrite (NO2 -) was examined in rat C6 astroglia-like cells challenged with lipopolysaccharide (LPS), a bacterial endotoxin, and interferon gamma (IFNγ), a pro-inflammatory cytokine. Furthermore, the role of cocaine in increasing the expression of hypoxia inducible factor-1 (HIF-1α) and vascular endothelial growth factor (VEGF) in cells was also determined. First, the viability of the cells was assessed when treated with cocaine (0.5-7 mM) for 24 and 48 h. The results showed that cocaine toxicity was both time and dose-dependent. In subsequent studies, cells were challenged with or without LPS and IFNγ, followed by co-treatment with cocaine (1-4 mM) for 24 h. Cocaine treatment did not increase O2 - or H2O2 production in the challenged or unchallenged cells. Similarly, cocaine treatment did not increase NO/NO2 - production in the unchallenged cells; however, NO/NO2 - levels in the challenged cells was increased 40-50-fold upon cocaine treatment compared with the corresponding unchallenged group. The HIF-1α and VEGF levels were significantly increased in the challenged cells at higher cocaine doses compared with the unchallenged cells. Since high concentrations of NO are associated with inflammation, the high levels of NO production observed in the present study suggested that cocaine may have potentiated the inflammatory response in the challenged C6 astroglia-like cells.

Therapeutic Potential of Thymoquinone in Triple-Negative Breast Cancer Prevention and Progression through the Modulation of the Tumor Microenvironment.

To date, the tumor microenvironment (TME) has gained considerable attention in various areas of cancer research due to its role in driving a loss of immune surveillance and enabling rapid advanced tumor development and progression. The TME plays an integral role in driving advanced aggressive breast cancers, including triple-negative breast cancer (TNBC), a pivotal mediator for tumor cells to communicate with the surrounding cells via lymphatic and circulatory systems. Furthermore, the TME plays a significant role in all steps and stages of carcinogenesis by promoting and stimulating uncontrolled cell proliferation and protecting tumor cells from the immune system. Various cellular components of the TME work together to drive cancer processes, some of which include tumor-associated adipocytes, fibroblasts, macrophages, and neutrophils which sustain perpetual amplification and release of pro-inflammatory molecules such as cytokines. Thymoquinone (TQ), a natural chemical component from black cumin seed, is widely used traditionally and now in clinical trials for the treatment/prevention of multiple types of cancer, showing a potential to mitigate components of TME at various stages by various pathways. In this review, we focus on the role of TME in TNBC cancer progression and the effect of TQ on the TME, emphasizing their anticipated role in the prevention and treatment of TNBC. It was concluded from this review that the multiple components of the TME serve as a critical part of TNBC tumor promotion and stimulation of uncontrolled cell proliferation. Meanwhile, TQ could be a crucial compound in the prevention and progression of TNBC therapy through the modulation of the TME.

Synergistic effects of methyl 2-cyano-3,11-dioxo-18beta-olean-1,-12-dien-30-oate and erlotinib on erlotinib-resistant non-small cell lung cancer cells.

Non-small cell lung cancer (NSCLC) is often characterized by an underlying mutation in the epidermal growth factor receptor (EGFR), contributing to aggressive metastatic disease. Methyl 2-cyano-3,11-dioxo-18beta-olean-1,12-dien-30-oate (CDODA-Me), a glycyrrhetinic acid derivative, reportedly improves the therapeutic response to erlotinib (ERL), an EGFR tyrosine kinase inhibitor. In the present study, we performed a series of studies to demonstrate the efficacy of CDODA-Me (2 µM) in sensitizing HCC827R (ERL-resistant) cells to ERL. Herein, we first established the selectivity of ERL-induced drug resistance in the HCC827R cells, which was sensitized when ERL was combined with CDODA-Me (2 µM), shifting the IC50 from 23.48 µM to 5.46 µM. Subsequently, whole transcriptomic microarray expression data demonstrated that the combination of ERL + CDODA-Me elicited 210 downregulated genes (0.44% of the whole transcriptome (WT)) and 174 upregulated genes (0.36% of the WT), of which approximately 80% were unique to the ERL + CDODA-Me group. Synergistic effects centered on losses to cell cycle progression transcripts, a reduction of minichromosome maintenance complex components (MCM2-7), all key components of the Cdc45·MCM2-7GINS (CMG) complex, and replicative helicases; these effects were tantamount to the upregulation of processes associated with the nuclear factor erythroid 2 like 2 translational response to oxidative stress, including sulfiredoxin 1, heme oxygenase 1, and stress-induced growth inhibitor 1. Collectively, these findings indicate that the synergistic therapeutic effects of ERL + CDODA-Me on resistant NSCLC cells are mediated via the inhibition of mitosis and induction of oxidative stress.

Whole-transcriptome Analysis of Fully Viable Energy Efficient Glycolytic-null Cancer Cells Established by Double Genetic Knockout of Lactate Dehydrogenase A/B or Glucose-6-Phosphate Isomerase.

BACKGROUND/AIM: Nearly all mammalian tumors of diverse tissues are believed to be dependent on fermentative glycolysis, marked by elevated production of lactic acid and expression of glycolytic enzymes, most notably lactic acid dehydrogenase (LDH). Therefore, there has been significant interest in developing chemotherapy drugs that selectively target various isoforms of the LDH enzyme. However, considerable questions remain as to the consequences of biological ablation of LDH or upstream targeting of the glycolytic pathway. MATERIALS AND METHODS: In this study, we explore the biochemical and whole transcriptomic effects of CRISPR-Cas9 gene knockout (KO) of lactate dehydrogenases A and B [LDHA/B double KO (DKO)] and glucose-6-phosphate isomerase (GPI KO) in the human colon cancer cell line LS174T, using Affymetrix 2.1 ST arrays. RESULTS: The metabolic biochemical profiles corroborate that relative to wild type (WT), LDHA/B DKO produced no lactic acid, (GPI KO) produced minimal lactic acid and both KOs displayed higher mitochondrial respiration, and minimal use of glucose with no loss of cell viability. These findings show a high biochemical energy efficiency as measured by ATP in glycolysis-null cells. Next, transcriptomic analysis conducted on 48,226 mRNA transcripts reflect 273 differentially expressed genes (DEGS) in the GPI KO clone set, 193 DEGS in the LDHA/B DKO clone set with 47 DEGs common to both KO clones. Glycolytic-null cells reflect up-regulation in gene transcripts typically associated with nutrient deprivation / fasting and possible use of fats for energy: thioredoxin interacting protein (TXNIP), mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), PPARγ coactivator 1α (PGC-1α), and acetyl-CoA acyltransferase 2 (ACAA2). Other changes in non-ergometric transcripts in both KOs show losses in "stemness", WNT signaling pathway, chemo/radiation resistance, retinoic acid synthesis, drug detoxification, androgen/estrogen activation, and extracellular matrix reprogramming genes. CONCLUSION: These findings demonstrate that: 1) The "Warburg effect" is dispensable, 2) loss of the LDHAB gene is not only inconsequential to viability but fosters greater mitochondrial energy, and 3) drugs that target LDHA/B are likely to be ineffective without a plausible combination second drug target.

Effect of apigenin on whole transcriptome profile of TNFα-activated MDA-MB-468 triple negative breast cancer cells.

The lack of hormone receptors in triple negative breast cancer (TNBC) is associated with the inefficacy of anti-estrogen chemotherapies, leaving fewer options for patient treatment and higher mortality rates. Additionally, as with numerous types of inflammatory breast cancer, infiltration of tumor associated macrophages and other leukocyte sub-populations within the tumor inevitably lead to aggressive, chemo-resistant, metastatic and invasive types of cancer which escape immune surveillance. These processes are orchestrated by the release of potent cytokines, including TNFα, IL-6 and CCL2 from the stroma, tumor and immune cells within the tumor microenvironment. The present study evaluated apigenin modulating effects on the pro-inflammatory activating action of TNFα in TNBC MDA-MB-468 cells, derived from an African American woman. Initially, cell viability was determined to establish an optimal sub-lethal dose of TNFα and apigenin in MDA-MB-468 cells. Subsequently, various treatments effects were evaluated using whole transcriptomic analysis of mRNA and long intergenic non-coding RNA with Affymetrix HuGene-2.1-st human microarrays. Gene level differential expression analysis was conducted on 48,226 genes where TNFα caused significant upregulation of 53 transcripts and downregulation of 11 transcripts. The largest upward differential shift was for CCL2 [+61.86 fold change (FC); false discovery rate (FDR), P<0.0001]; which was down regulated by apigenin (to +10.71 FC vs. Control; FDR P-value <0.001), equivalent to an 83% reduction. Several TNFα deferentially upregulated transcripts were reduced by apigenin, including CXCL10, C3, PGLYRP4, IL22RA2, KMO, IL7R, ROS1, CFB, IKBKe, SLITRK6 (a checkpoint target) and MMP13. Confirmation of CCL2 experimentally induced transcript alterations was corroborated at the protein level by ELISA assays. The high level of CCL2 transcript in the cell line was comparable to that in our previous studies in MDA-MB-231 cells. The differential effects of TNFα were corroborated by ELISA, where the data revealed a >10-fold higher releasing rate of CCL2 in MDA-MB-468 cells compared with in MDA-MB-231 cells, both of which were attenuated by apigenin. The data obtained in the present study demonstrated a high level of CCL2 in MDA-MB-468 cells and a possible therapeutic role for apigenin in downregulating TNFα-mediated processes in these TNBC cells.

Whole Transcriptomic Analysis of Apigenin on TNFα Immuno-activated MDA-MB-231 Breast Cancer Cells.

BACKGROUND: Triple-negative breast cancer is categorized by a lack of hormone receptors, inefficacy of anti-estrogen or aromatase inhibitor chemotherapies and greater mortality rates in African American populations. Advanced-stage breast tumors have a high concentration of tumor necrosis factor-α (TNFα) throughout the tumor/stroma milieu, prompting sustained release of diverse chemokines (i.e. C-C motif chemokine ligand 2 (CCL2)/CCL5). These potent chemokines can subsequently direct mass infiltration of leukocyte sub-populations to lodge within the tumor, triggering a loss of tumor immune surveillance and subsequent rapid tumor growth. Previously, we demonstrated that in the MDA-MB-231 TNBC cell line, TNFα evoked a rise in immune signaling proteins: CCL2, granulocyte macrophage colony-stimulating factor, interleukin (IL)1α, IL6 and inhibitor of nuclear factor kappa-B kinase subunit epsilon (IKBKε) all of which were attenuated by apigenin, a dietary flavonoid found in chamomile and parsley. MATERIALS AND METHODS: The present work elucidates changes evoked by TNFα in the presence or absence of apigenin by examining the entire transcriptome for mRNA and long intergenic non-coding RNA with Affymetrix Hugene-2.1_ST human microarrays. Differential gene-expression analysis was conducted on 48,226 genes. RESULTS: TNFα caused up-regulation of 75 genes and down-regulation of 10. Of these, apigenin effectively down-regulated 35 of the 75 genes which were up-regulated by TNFα. These findings confirm our previous work, specifically for the TNFα-evoked spike in IL1A vs. untreated controls [+21-fold change (FC), p<0.0001] being attenuated by apigenin in the presence of TNFa (-15 FC vs. TNFα, p<0.0001). Similar trends were seen for apigenin-mediated down-regulation of TNFα-up-regulated transcripts: IKBKE (TNFα: 4.55 FC vs. control, p<0.001; and TNFα plus apigenin: -4.92 FC, p<0.001), CCL2 (2.19 FC, p<0.002; and -2.12 FC, p<0.003), IL6 (3.25 FC, p<0.020; and -2.85 FC, p<0.043) and CSF2 (TNFα +6.04 FC, p<0.001; and -2.36 FC, p<0.007). In addition, these data further establish more than a 65% reduction by apigenin for the following transcripts which were also up-regulated by TNFα: cathepsin S (CTSS), complement C3 (C3), laminin subunit gamma 2 (LAMC2), (TLR2), toll-like receptor 2 G protein-coupled receptor class C group 5 member B (GPRC5B), contactin-associated protein 1 (CNTNAP1), claudin 1 (CLDN1), nuclear factor of activated T-cells 2 (NFATC2), C-X-C motif chemokine ligand 10 (CXCL10), CXCL11, interleukin 1 receptor-associated kinase 3 (IRAK3), nuclear receptor subfamily 3 group C member 2 (NR3C2), interleukin 32 (IL32), IL24, slit guidance ligand 2 (SLIT2), transmembrane protein 132A (TMEM132A), TMEM171, signal transducing adaptor family member 2 (STAP2), mixed lineage kinase domain-like pseudokinase (MLKL), kinase insert domain receptor (KDR), BMP-binding endothelial regulator (BMPER), and kelch-like family member 36 (KLHL36). CONCLUSION: There is a possible therapeutic role for apigenin in down-regulating diverse genes associated with tumorigenic leukocyte sub-population infiltration by triple-negative breast cancer. The data have been deposited into the Gene Expression Omnibus for public analysis at https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE120550.

In Silico Investigation of the Binding of MCoTI-II Plant Defense Knottin to the γ-NGF Serine Protease of the 7S Nerve Growth Factor Complex and Biological Activity of Its NGF Mimetic Properties.

Nerve growth factor (NGF) is an endogenously produced polypeptide that promotes the differentiation, survival, and repair of neurons in the central and peripheral nervous systems. While trophic proteins hold promise for the treatment of neuronal injury and disease, use of NGF is limited by its large molecular weight, lack of permeability through the blood-brain barrier, and peripheral side effects. Previously, we found that an extract of the Momordica cochinchinensis seed stimulated PC-12 neurite outgrowth. Bioactivity-guided fractioning of the seed extract suggested that the NGF mimetic agent was one of few defined proteins from this plant: one group being the defense Knottins and the other group of the lowest mass is the potent trypsin inhibitor MCoTI-II. Here, the NGF mimetic potential of this latter protein was investigated using two concurrent but different approaches. A biological study used recombinant purified MCoTI-II, which when tested in rat PC-12 cells grown on collagen, failed to initiate outgrowth relative to the positive control 7S NGF. In a separate computational study, the possibility was investigated such that MCoTI-II could exert an effect through binding to the serine protease γ-NGF subunit of the 7S NGF complex, analogous to its binding to its native receptor trypsin. Molecular dynamics simulations showed that MCoTI-II can bind stably to γ-NGF for >350 ns. Modeling indicated that this interaction could sterically inhibit 7S NGF complex formation, potentially altering the equilibrium between inactive complexed and free active NFG protein. In conclusion, the biological study now excludes the MCoTI-II protein as the NGF mimetic factor in the Momordica extract, an important and required step to identify the active component in this seed. On the other hand, the theoretical study has revealed a novel observation that may be of use in the development of strategies to affect NGF activity.

Cellular and molecular responses to acute cocaine treatment in neuronal-like N2a cells: potential mechanism for its resistance in cell death.

Cocaine is a highly abused drug that causes psychiatric and neurological problems. Its entry into neurons could alter cell-biochemistry and contribute in the manifestation of early pathological symptoms. We have previously shown the acute cocaine effects in rat C6 astroglia-like cells and found that these cells were highly sensitive to cocaine in terms of manifesting certain pathologies known to underlie psychological disorders. The present study was aimed to discern acute cocaine effects on the early onset of various changes in Neuro-2a (N2a) cells. Whole-cell patch-clamp recording of differentiated cells displayed the functional voltage-gated Na+ and K+ channels, which demonstrated the neuronal characteristics of the cells. Treatment of these cells with acute cocaine (1 h) at in vivo (nM to µM) and in vitro (mM) concentrations revealed that the cells remained almost 100% viable. Cocaine administration at 6.25 µM or 4 mM doses significantly reduced the inward currents but had no significant effect on outward currents, indicating the Na+ channel-blocking activity of cocaine. While no morphological change was observed at in vivo doses, treatment at in vitro doses altered the morphology, damaged the neurites, and induced cytoplasmic vacuoles; furthermore, general mitochondrial activity and membrane potential were significantly decreased. Mitochondrial dysfunction enabled the cells switch to anaerobic glycolysis, evidenced by dose-dependent increases in lactate and H2S, resulting unaltered ATP level in the cells. Further investigation on the mechanism of action unfolded that the cell's resistance to cocaine was through the activation of nuclear factor E2-related factor-2 (Nrf-2) gene and subsequent increase of antioxidants (glutathione [GSH], catalase and GSH peroxidase [GPx]). The data clearly indicate that the cells employed a detoxifying strategy against cocaine. On a broader perspective, we envision that extrapolating the knowledge of neuronal resistance to central nervous system (CNS) diseases could delay their onset or progression.

Whole-transcriptomic Profile of SK-MEL-3 Melanoma Cells Treated with the Histone Deacetylase Inhibitor: Trichostatin A.

BACKGROUND: Malignant melanoma cells can rapidly acquire phenotypic properties making them resistant to radiation and mainline chemotherapies such as decarbonize or kinase inhibitors that target RAS-proto-oncogene independent auto-activated mitogen-activated protein kinases (MAPK)/through dual specificity mitogen-activated protein kinase (MEK). Both drug resistance and inherent transition from melanocytic nevi to malignant melanoma involve the overexpression of histone deacetylases (HDACs) and a B-Raf proto-oncogene (BRAF) mutation. MATERIALS AND METHODS: In this work, the effects of an HDAC class I and II inhibitor trichostatin A (TSA) on the whole transcriptome of SK-MEL-3 cells carrying a BRAF mutation was examined. RESULTS: The data obtained show that TSA was an extremely potent HDAC inhibitor within SK-MEL-3 nuclear lysates, where TSA was then optimized for appropriate sub-lethal concentrations for in vitro testing. The whole-transcriptome profile shows a basic phenotype dominance in the SK-MEL-3 cell line for i) synthesis of melanin, ii) phagosome acidification, iii) ATP hydrolysis-coupled proton pumps and iv) iron transport systems. While TSA did not affect the aforementioned major systems, it evoked a dramatic change to the transcriptome: reflected by a down-regulation of 810 transcripts and up-regulation of 833, with fold-change from -15.27 to +31.1 FC (p<0.00001). Largest differentials were found for the following transcripts: Up-regulated: Tetraspanin 13 (TSPAN13), serpin family i member 1 (SERPINI1), ATPase Na+/K+ transporting subunit beta 2 (ATP1B2), nicotinamide nucleotide adenylyl transferase 2 (NMNAT2), platelet-derived growth factor receptor-like (PDGFRL), cytochrome P450 family 1 subfamily A member 1 (CYP1A1), prostate androgen-regulated mucin-like protein 1 (PARM1), secretogranin II (SCG2), SYT11 (synaptotagmin 11), rhophilin associated tail protein 1 like (ROPN1L); down-regulated: polypeptide N-acetylgalactosaminyltransferase 3 (GALNT3), carbonic anhydrase 14 (CAXIV), BCL2-related protein A1 (BCL2A1), protein kinase C delta (PRKCD), transient receptor potential cation channel subfamily M member 1 (TRPM1), ubiquitin associated protein 1 like (UBAP1L), glutathione peroxidase 8 (GPX8), interleukin 16 (IL16), tumor protein p53 (TP53), and serpin family H member 1 (SERPINH1). There was no change to any of the HDAC transcripts (class I, II and IV), the sirtuin HDAC family (1-6) or the BRAF proto-oncogene v 599 transcripts. However, the data showed that TSA down-regulated influential transcripts that drive the BRAF-extracellular signal-regulated kinase (ERK)1/2 oncogenic pathway (namely PRKCD and MYC proto-oncogene which negatively affected the cell-cycle distribution. Mitotic inhibition was corroborated by functional pathway analysis and flow cytometry confirming halt at the G2 phase, occurring in the absence of toxicity. CONCLUSION: TSA does not alter HDAC transcripts nor BRAF itself, but down-regulates critical components of the MAPK/MEK/BRAF oncogenic pathway, initiating a mitotic arrest.

Effects of Sepantronium Bromide (YM-155) on the Whole Transcriptome of MDA-MB-231 Cells: Highlight on Impaired ATR/ATM Fanconi Anemia DNA Damage Response.

Sepantronium bromide (YM-155) is believed to elicit apoptosis and mitotic arrest in tumor cells by reducing (BIRC5, survivin) mRNA. In this study, we monitored changes in survivin mRNA and protein after treating MDA-MB-231 cells with YM-155 concurrent with evaluation of whole transcriptomic (WT) mRNA and long intergenic non-coding RNA at 2 time points: 8 h sub-lethal (83 ng/mL) and 20 h at the LC50 (14.6 ng/mL). The data show a tight association between cell death and the precipitating loss of survivin protein and mRNA (-2.67 fold-change (FC), p<0.001) at 20 h, questioning if the decline in survivin is attributed to cell death or drug impact. The meager loss of survivin mRNA was overshadowed by enormous differential change to the WT in both magnitude and significance for over 2000 differentially up/down-regulated transcripts: (+22 FC to -12 FC, p<0.001). The data show YM-155 to up-regulate transcripts in control of circadian rhythm (NOCT, PER, BHLHe40, NFIL3), tumor suppression (SIK1, FOSB), histone methylation (KDM6B) and negative feedback of NF-kappa B signaling (TNFAIP3). Down-regulated transcripts by YM-155 include glucuronidase (GUSBP3), numerous micro-RNAs, DNA damage repair elements (CENPI, POLQ, RAD54B) and the most affected system was the ataxia-telangiectasia mutated (ATM)/Fanconi anemia E3 monoubiquitin ligase core complexes (FANC transcripts - A/B/E/F/G/M), FANC2, FANCI, BRCA1, BRCA2, RAD51, PALB2 gene and ATR (ATM- and Rad3-Related) pathway. In conclusion, these findings suggest that a primary target of YM-155 is the loss of replicative DNA repair systems.

Identification of biochemical and cytotoxic markers in cocaine treated PC12 cells.

Cocaine is one of the powerful addictive drugs, widely abused in most Western countries. Because of high lipophilic nature, cocaine easily reaches various domains of the central nervous system (CNS) and triggers different levels of cellular toxicity. The aim of this investigation was to reproduce cocaine toxicity in differentiated PC12 cells through quantitative knowledge on biochemical and cytotoxicity markers. We differentiated the cells with 0.1 µg/ml nerve growth factor (NGF) for 5 days, followed by treatment with cocaine for 48 h at in vivo and in vitro concentrations. Results indicated that cocaine at in vivo concentrations neither killed the cells nor altered the morphology, but decreased the mitochondrial membrane potential that paralleled with increased lactate and glutathione (GSH) levels. On the other hand, cocaine at in vitro concentrations damaged the neurites and caused cell death, which corresponded with increased reactive oxygen species (ROS) generation, plasma membrane damage, and GSH depletion with no detectable nitric oxide (NO) level. While direct understanding of cocaine and cell interaction under in vivo animal models is impeded due to high complexity, our present in vitro results assisted in understanding the onset of some key events of neurodegenerative diseases in cocaine treated neuronal cells.

Transcriptomic Profiling of MDA-MB-231 Cells Exposed to Boswellia Serrata and 3-O-Acetyl-B-Boswellic Acid; ER/UPR Mediated Programmed Cell Death.

BACKGROUND/AIM: Triple-negative breast cancer (TNBC) is characterized by the absence of hormone receptors (estrogen, progesterone and human epidermal growth factor receptor-2) and a relatively poor prognosis due to inefficacy of hormone receptor-based chemotherapies. It is imperative that we continue to explore natural products with potential to impede growth and metastasis of TNBC. In this study, we screened over 1,000 natural products for capacity to induce cell death in TNBC (MDA-MB -231) cells. MATERIALS AND METHODS: Frankincense (Boswellia serrata extract (BSE)) and 3-O-Acetyl-ß-boswellic acid (3-OAßBA) were relatively potent, findings that corroborate the body of existing literature. The effects of BSE and 3-OAßBA on genetic parameters in MDA-MB-231 cells were evaluated by examining whole-transcriptomic influence on mRNAs, long intergenic non-coding RNA transcripts (lincRNA) and non-coding miRNAs. RESULTS: Bio-statistical analysis demarcates the primary effect of both BSE/3-OAßBA on the up-regulation of PERK (protein kinase RNA-like endoplasmic reticulum kinase)- endoplasmic reticulum (ER)/unfolded protein response (UPR) pathways that are closely tied to activated programmed cell death (APCD). Global profiling confirms concomitant effects of BSE/3-OAßBA on upwardly expressed ER/URP APCD key components PERK (EIF2AK3), XBP1, C/EBP homologous protein transcription factor (CHOP), ATF3 and DDIT3,4/DNA-damage-inducible transcript 3,4 (GADD34). Further, BSE and/or 3-OAßBA significantly down-regulated oncogenes (OG) which, heretofore, lack functional pathway mapping, but are capable of driving epithelial-mesenchymal transition (EMT), cell survival, proliferation, metastasis and drug resistance. Among these are cell migration-inducing protein hyaluronan binding (CEMIP) [-7.22]; transglutaminase 2 [-4.96], SRY box 9 (SOX9) [-4.09], inhibitor of DNA binding 1, dominant negative helix-loop-helix protein (ID1) [-6.56]; and endothelin 1 (EDN1, [-5.06]). Likewise, in the opposite manner, BSE and/or 3-OAßBA induced the robust overexpression of tumor suppressor genes (TSGs), including: glutathione-depleting ChaC glutathione-specific gamma-glutamylcyclotransferase 1 (CHAC1) [+21.67]; the mTOR inhibitors - sestrin 2 (SESN2) [+16.4] Tribbles homolog 3 (TRIB3) [+6.2], homocysteine-inducible, endoplasmic reticulum stress-inducible, ubiquitin-like domain member 1 (HERPUD1) [+12.01]; and cystathionine gamma-lyase (CTH) [+11.12]. CONCLUSION: The anti-cancer effects of the historically used frankincense sap (BSE) appear to involve major impact on the ER/UPR response, concomitant to effecting multiple targets counter to the growth, proliferation and metastasis of TNBC cancer cells. The microarray data are available at Expression Omnibus GEO Series accession number GSE102891.

Apigenin inhibits TNFα/IL-1α-induced CCL2 release through IKBK-epsilon signaling in MDA-MB-231 human breast cancer cells.

Mortality associated with breast cancer is attributable to aggressive metastasis, to which TNFα plays a central orchestrating role. TNFα acts on breast tumor TNF receptors evoking the release of chemotactic proteins (e.g. MCP-1/CCL2). These proteins direct inward infiltration/migration of tumor-associated macrophages (TAMs), tumor-associated neutrophils (TANs), myeloid-derived suppressor cells (MDSCs), T-regulatory cells (Tregs), T helper IL-17-producing cells (Th17s), metastasis-associated macrophages (MAMs) and cancer-associated fibroblasts (CAFs). Tumor embedded infiltrates collectively enable immune evasion, tumor growth, angiogenesis, and metastasis. In the current study, we investigate the potential of apigenin, a known anti-inflammatory constituent of parsley, to downregulate TNFα mediated release of chemokines from human triple-negative cells (MDA-MB-231 cells). The results show that TNFα stimulation leads to large rise of CCL2, granulocyte macrophage colony-stimulating factor (GMCSF), IL-1α and IL-6, all suppressed by apigenin. While many aspects of the transcriptome for NFkB signaling were evaluated, the data show signaling patterns associated with CCL2 were blocked by apigenin and mediated through suppressed mRNA and protein synthesis of IKBKe. Moreover, the data show that the attenuation of CCL2 by apigenin in the presence TNFα paralleled the suppression of phosphorylated extracellular signal-regulated kinase 1 (ERK 1/ 2). In summary, the obtained findings suggest that there exists a TNFα evoked release of CCL2 and other LSP recruiting cytokines from human breast cancer cells, which can be attenuated by apigenin.

Stable shRNA Silencing of Lactate Dehydrogenase A (LDHA) in Human MDA-MB-231 Breast Cancer Cells Fails to Alter Lactic Acid Production, Glycolytic Activity, ATP or Survival.

BACKGROUND: In the US, African Americans have a high death rate from triple-negative breast cancer (TNBC), characterized by lack of hormone receptors (ER, PR, HER2/ERRB2) which are otherwise valuable targets of chemotherapy. There is a need to identify novel targets that negatively impact TNBC tumorigenesis. TNBCs release an abundance of lactic acid, under normoxic, hypoxic and hyperoxic conditions; this referred to as the Warburg effect. Accumulated lactic acid sustains peri-cellular acidity which propels metastatic invasion and malignant aggressive transformation. The source of lactic acid is believed to be via conversion of pyruvate by lactate dehydrogenase (LDH) in the last step of glycolysis, with most studies focusing on the LDHA isoform. MATERIALS AND METHODS: In this study, LDHA was silenced using long-term MISSION® shRNA lentivirus in human breast cancer MDA-MB-231 cells. Down-regulation of LDHA transcription and protein expression was confirmed by western blot, immunocytochemistry and qPCR. A number of parameters were measured in fully viable vector controls versus knock-down (KD) clones, including levels of lactic acid produced, glucose consumed, ATP and basic metabolic rates. RESULTS: The data show that lentivirus V-165 generated a knock-down clone most effective in reducing both gene and protein levels to less than 1% of vector controls. Stable KD showed absolutely no changes in cell viability, lactic acid production, ATP, glucose consumption or basic metabolic rate. Given the complete absence of impact on any observed parameter by LDH-A KD and this being somewhat contrary to findings in the literature, further analysis was required to determine why. Whole-transcriptome analytic profile on MDA-MB-231 for LDH subtypes using Agilent Human Genome 4×44k microarrays, where the data show the following component breakdown. Transcripts: 30.47 % LDHA, 69.36% LDHB, 0.12% LDHC and 0.05% LDHD. CONCLUSION: These findings underscore the importance of alternative isoforms of LDH in cancer cells to produce lactic acid, when LDHA is silenced or inhibited. LDHA silencing alone is not effective in hampering or inducing changes in survival, metabolism or lactic acid produced in a cell line with high concentrations of LDHB. Future research will be required to confirm effects of dual LDHA/B knockdown and further confirm that the sole source of lactic acid produced occurs through LDH (all isoforms) in breast cancer cells.