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Cell based immunotherapy is rapidly emerging as a promising cancer treatment. A modest increase in salt (sodium chloride) concentration in immune cell cultures is known to induce inflammatory phenotypic differentiation. In our current study, we analyzed the ability of salt treatment to induce ex vivo expansion of tumor-primed CD4 (cluster of differentiation 4)+T cells to an effector phenotype. CD4+T cells were isolated using immunomagnetic beads from draining lymph nodes and spleens from tumor bearing C57Bl/6 mice, 28 days post-injection of Py230 syngeneic breast cancer cells. CD4+T cells from non-tumor bearing mice were isolated from splenocytes of 12-week-old C57Bl/6 mice. These CD4+T cells were expanded ex vivo with five stimulation cycles, and each cycle comprised of treatment with high salt (Δ0.035 M NaCl) or equimolar mannitol controls along with anti-CD3/CD28 monoclonal antibodies for the first 3 days, followed by the addition of interleukin (IL)-2/IL-7 cytokines and heat killed Py230 for 4 days. Ex vivo high salt treatment induced a two-fold higher Th1 (T helper type 1) expansion and four-fold higher Th17 expansion compared to equimolar mannitol treatment. Importantly, the high salt expanded CD4+T cells retained tumor-specificity, as demonstrated by higher in vitro cytotoxicity against Py230 breast cancer cells and reduced in vivo syngeneic tumor growth. Metabolic studies revealed that high salt treatment enhanced the glycolytic reserve and basal mitochondrial oxidation of CD4+T cells, suggesting a role of high salt in enhanced pro-growth anabolic metabolism needed for inflammatory differentiation. Mechanistic studies demonstrated that the high salt induced switch to the effector phenotype was mediated by tonicity-dependent transcription factor, TonEBP/NFAT5. Using a transgenic murine model, we demonstrated that CD4 specific TonEBP/NFAT5 knock out (CD4 cre/cre NFAT5 flox/flox ) abrogated the induction of the effector phenotype and anti-tumor efficiency of CD4+T cells following high salt treatment. Taken together, our data suggest that high salt-mediated ex vivo expansion of tumor-primed CD4+T cells could induce effective tumor specific anti-cancer responses, which may have a novel cell-based cancer immunotherapeutic application.

Objectives: Studies have shown that human and peripheral blood mononuclear cells (PBMCs) are mostly used for research purposes to study several biochemical endpoints. The effects of the flavonoids, genistein, kaempferol, and quercetin on phospho tensin homolog (PTEN) levels in cancer cells (i.e., breast [BT549], lung [A549]), human embryonic kidney cells (HEK293), and the levels of lipid peroxides (LP) in PBMCs were respectively investigated. Materials and methods: Cancer, kidney, and PBMCs from several donors were each exposed to each of the flavonoids at concentrations of 0, 5, 10, 15, 20, and 25 µM. Our hypotheses were that exposure of cancer and kidney cells to genistein, kaempferol, and quercetin can increase PTEN and decrease lipid peroxides in PBMCs levels respectively to better cope with oxidative stress. Results: The results indicate that the flavonoids increased total PTEN levels in a dose-dependent manner. The effect of quercetin was more pronounced followed by genistein and kaempferol. Furthermore, decreases in lipid peroxides were observed in the PBMCs for the flavonoid-treated samples compared to those exposed to flavonoids and with oxidative stress as described by Fenton's chemistry. Levels of LP in quercetin-treated samples were lower compared to kaempferol and genistein. Conclusions: The findings suggest that the flavonoids play an important role in controlling oxidative stress in several human cells.

Mammaglobin-A (MamA) is overexpressed in 40-80% of all human breast cancers. Recent phase I clinical trials of the MamA DNA vaccine showed encouraging safety outcomes. However, this vaccine elicited only a modest increase in MamA specific CD8+T lymphocyte (CTL) activation. As vaccine adjuvants play a critical role in enhancing the immunotherapeutic efficiency of vaccines, we tested the potential role of three synthetic CpG oligodeoxynucleotides (ODN2216-class A ODN, ODN2006-class B ODN, and ODN M362-class C ODN) to further enhance MamA specific CTL responses. Towards this, naïve CD8+T cells were obtained from healthy HLA-A2+ human donors. The HLA-A2 specific immunodominant epitope of MamA, MamA2.1 (LIYDSSLCDL), was utilized to activate naïve CD8+T cells. The THP-1 (HLA-A2+) cells were used as antigen presenting cells to stimulate naïve CD8+T cells along with (or without) co-treatment of various ODNs mentioned above. Activation of naïve CD8+T cells with the MamA2.1 peptide along with ODNs demonstrated enhanced MamA specific CTL mediated cytotoxicity on AU565 (HLA-A + /MamA + ) breast cancer cells following co-treatment with ODN2006 and M362 compared to ODN2216 or MamA2.1 peptide alone. However, no significant cytotoxicity was noted upon treatment of MamA2.1 activated CTLs on MCF7 (HLA-A + /MamA - ) cells, suggesting that the activation of CTLs is specific to the MamA antigen. Functional characterization studies demonstrated specific IL-12 mediated cross-talk between TLR-6 and -9 in THP-1 cells following stimulation with ODN2006 and M362, which was critical for the final cytotoxic activation of CD8+T lymphocytes. Based on these data, we conclude that ODN2006 and ODN M362 exerted a strong adjuvant effect through induction of the initial innate immune response through TLR9 upregulation followed by enhanced MamA specific CTL dependent adaptive immune responses. Our current data provide evidence for the application of Class-B/-C-CpG-ODNs as potential vaccine adjuvants towards enhancing the success of MamA based breast cancer vaccination.

Chronic inflammation is a well-known precursor for cancer development and proliferation. We have recently demonstrated that high salt (NaCl) synergizes with sub-effective interleukin (IL)-17 to induce breast cancer cell proliferation. However, the exact molecular mechanisms mediating this effect are unclear. In our current study, we adopted a phosphoproteomic-based approach to identify salt modulated kinase-proteome specific molecular targets. The phosphoprotemics based binary comparison between heavy labelled MCF-7 cells treated with high salt (Δ0.05 M NaCl) and light labelled MCF-7 cells cultured under basal conditions demonstrated an enhanced phosphorylation of Serine-493 of SIK3 protein. The mRNA transcript and protein expression analysis of SIK3 in MCF-7 cells demonstrated a synergistic enhancement following co-treatment with high salt and sub-effective IL-17 (0.1 ng/mL), as compared to either treatments alone. A similar increase in SIK3 expression was observed in other breast cancer cell lines, MDA-MB-231, BT20, and AU565, while non-malignant breast epithelial cell line, MCF10A, did not induce SIK3 expression under similar conditions. Biochemical studies revealed mTORC2 acted as upstream mediator of SIK3 phosphorylation. Importantly, cell cycle analysis by flow cytometry demonstrated SIK3 induced G0/G1-phase release mediated cell proliferation, while SIK3 silencing abolished this effect. Also, SIK3 induced pro-inflammatory arginine metabolism, as evidenced by upregulation of the enzymes iNOS and ASS-1, along with downregulation of anti-inflammatory enzymes, arginase-1 and ornithine decarboxylase. Furthermore, gelatin zymography analysis has demonstrated that SIK3 induced expression of tumor metastatic CXCR4 through MMP-9 activation. Taken together, our data suggests a critical role of SIK3 in mediating three important hallmarks of cancer namely, cell proliferation, inflammation and metastasis. These studies provide a mechanistic basis for the future utilization of SIK3 as a key drug discovery target to improve breast cancer therapy.

Chronic inflammation is known to play a critical role in the development of cancer. Recent evidence suggests that high salt in the tissue microenvironment induces chronic inflammatory milieu. In this report, using three breast cancer-related cell lines, we determined the molecular basis of the potential synergistic inflammatory effect of sodium chloride (NaCl) with interleukin-17 (IL-17). Combined treatment of high NaCl (0.15M) with sub-effective IL-17 (0.1 nM) induced enhanced growth in breast cancer cells along with activation of reactive nitrogen and oxygen (RNS/ROS) species known to promote cancer. Similar effect was not observed with equi-molar mannitol. This enhanced of ROS/RNS activity correlates with upregulation of γENaC an inflammatory sodium channel. The similar culture conditions have also induced expression of pro-inflammatory cytokines such as IL-6, TNFα etc. Taken together, these data suggest that high NaCl in the cellular microenvironment induces a γENaC mediated chronic inflammatory response with a potential pro-carcinogenic effect., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Matrix metalloproteinases (MMP-2 and -9) play an important role in the tumor metastasis through cleavage of proinflammatory cytokines. Violacein a small molecule produced by Chromobacterium violaceum and has been implicated with anti-cancer effects. In this study we investigated the molecular basis of violacein mediated downregulation of CXCL12/CXCR4, chemokine-receptor ligand interaction. Zymography analysis demonstrated that violacein significantly inhibited the cytokine (TNFα and TGFβ) mediated MMP-2 activation in MCF-7 breast cancer cell line. MMP-2 plays a critical role in the secretion of inflammatory chemokine, CXCL12, involved in cell migration and cancer metastasis. ELISA analysis demonstrated that violacein inhibited the secretion of CXCL12 from the activated MCF-7 cells. Further, we show that MMP-2/-9 act synergistically at two distinct steps towards the membrane expression of the tumor metastasis chemokine receptor, CXCR4. Violacein efficiently downregulated the CXCR4 membrane expression through MMP-9 inhibition. Taken together, these studies demonstrate a unique anti-tumor mechanism of action of violacein through reduction of CXCL12/CXCR4 interaction. These studies could offer a novel venue for violacein in cancer therapy.

Background: Plants that are used as traditional medicine represent a relevant pool for selecting plant candidates that may have anticancer properties. In this study, the ethnomedicinal approach was used to select several medicinal plants native to Nigeria, on the basis of their local or traditional uses. The collected plants were then evaluated for cytoxicity., Methods: The antitumor activity of methanolic extracts obtained from 24 of the selected plants, were evaluated in vitro on five human cancer cell lines., Results: Results obtained from the plants screened indicate that 18 plant extracts of folk medicine exhibited promising cytotoxic activity against human carcinoma cell lines. Erythrophleum suaveolens (Guill. & Perr.) Brenan was found to demonstrate potent anti-cancer activity in this study exhibiting IC(50) = 0.2-1.3 μg/ml., Conclusions: Based on the significantly potent activity of some plants extracts reported here, further studies aimed at mechanism elucidation and bio-guided isolation of active anticancer compounds is currently underway.

We report on the synthesis of a novel series of fluorinated acridones from 5-trifluoromethyl-1,3-cyclohexanedione. The cytotoxic activities of the compounds were studied in several cancer cells. Compounds 9a, 9c, 9e, 9f, and 9h exhibited significant anticancer activities in selected cell lines. Compound 9c is the most active showing GI(50) that ranged in values from 0.13 to 26 microM, covering a wide range of cancer cell lines.

Protein Kinase C (PKC) exists as one of twelve serine/threonine isoforms and has been found to mediate ethanol-induced activation of the Mitogen-Activated Protein Kinase (MAPK) pathway. The aim of this study was to determine the PKC isoform(s) that are mediators of ethanol-induced MAPK activity (ERK 1 and 2) and to verify the necessity of calcium in this activation process using cell culture in the presence and absence of ethanol, and other agents that modulate PKC expression. Western blotting analysis was used to assess the effect of ethanol on activating classical (alpha/ssII), novel (delta) and atypical (zeta/lambda) PKC isoforms in vascular smooth muscle cells (VSMCs). The results indicate that ethanol treated VSMCs express the classical PKC-alpha/ssII, novel PKC-delta, and atypical PKC-zeta/lambda isoforms. The expression of PKC-alpha/ssII was inhibited within the first two min of stimulation, followed by activation with maximum expression at 10 min. Similarly, PKC-delta and zeta expressions were suppressed during the first two min of ethanol stimulation with maximum increase in expressions at 10 min. The PKC inhibitor GF109203X and the calcium chelating agent BAPTA, enhanced ethanol-induced PKC expression, whereas, diltiazem reduced expression of PKC by 10% of control. On the other hand, BAPTA in the presence of GF10203X inhibited expression of ERK 1 & 2 downstream from the PKC pathway, whereas, BAPTA alone enhanced expression. These results demonstrate also that classical, novel, and atypical PKCs respond to ethanol during the initial phase of activation of ERK 1 & 2.

Histamine (HA) is one of many neurotransmitters that have been implicated in cardiovascular functioning. Alterations in vascular smooth muscle due to the effects of histamine have been suggested. We investigated the modulatory effect of HA on mitogen activated protein kinase (MAPK) expression, specifically extracellular regulating kinase (ERK) 1 & 2 in vascular smooth muscle cells (VSMCs) from both spontaneously hypertensive (SHR) and control Wistar Kyoto (WKY) rats. Cross-talking between calcium (Ca2+) and HA during HA-induced modulatory effect on MAPK expression in SHR VSMCs was also investigated. A stimulatory increase in expression of ERK 1 & 2 was observed to be dose and time dependent with maximum expression occurring within 5 min in both SHR and WKY VSMCs. The stimulatory increase in expression is persistent for 60 min in SHR VSMCs, whereas, in WKY cells the stimulatory effect persists for only 20 min. Mepyramine, the H1 receptor antagonist, reduced the HA-induced increase in ERK 1 & 2 significantly in SHR VSMCs. A reduction in the HA stimulated increase in ERK 1 & 2 expression was observed at 20 min of exposing cells to diltiazem, the calcium channel blocker, whereas, the calcium chelator, BAPTA effect on ERK 1 & 2 expression was observed within 5 min in SHR VSMCs. The data demonstrates that cross-talking occurs between HA stimulation and Ca2+ induction during HA-induced activation of ERK 1 & 2 in VSMCs of both cell types. Although both intracellular calcium ([Ca2+]i) and extracellular Ca2+ maybe involved in the activation of ERK 1 & 2 by HA, the dependence on [Ca2+]i is more dramatic than the dependence on extracellular Ca2+ in hypertensive cells, which may contribute to the role of HA as a risk factor of hypertension in VSMCs of the aorta.

Specific binding of [3H]-N-alpha-methylhistamine to homogenates from cerebral cortex tissue was analyzed in aged Wistar Kyoto (WKY) and Spontaneously Hypertensive rats (SHR). Scatchard plot analysis of [3H]-N-alpha-methylhistamine binding of the H3 receptor in the cerebral cortex from aged (6, 9, 12, and 16 week) SHR animals indicated that Bmax increased, respectively, 38.05 +/- 1.58, 59.63 +/- 2.48, 79.17 +/- 5.02, and 84.41 +/- 3.72 fmol/mg of protein. Binding studies using tissue from WKY rats indicated that maximal binding (Bmax) of the ligand to the receptor was not significantly altered. The analyses also yielded Kd values of 5, 7.2, 6.3 and 3.8 nM in SHR tissue respectively. Primers based on the sequence of the third intracellular loop of the H3 receptor were amplified at 35 cycles yielding several amplicons. These amplicons expressed sizes 875, 485, and 280 bp in 6 and 9 week cortical tissue from WKY animals where as in cortical tissue from 6 and 9 week SHR animals only two amplicons were expressed, 485 and 280 bp, respectively. Differences in gene expression for 12 and 16 week WKY and SHR rats were also compared using identical primers. Five amplicons were expressed in cortical tissue from 12 and 16 week WKY rats with 1000, 900, 821, 485, and 430 bp where as in 12 and 16 week SHR animals only one amplicon was expressed at 485 bp. The present results imply (1) that H3 receptor density in cortical tissue of SHR animals increases with age where as the number of the expressed amplicons of the detected H3 receptor decreases; and (2) even though a decrease in number of expressed amplicons of the H3 receptor were observed, an increase in expression of the larger amplicon (~500 bp) is evident.

Cadmium (Cd) is frequently used in various industrial applications and is a ubiquitous environmental toxicant, also present in tobacco smoke. An important route of exposure is the circulatory system whereas blood vessels are considered to be main stream organs of Cd toxicity. Our previous results indicate that cadmium chloride (CdCl2) affects mean arterial blood pressure in hypertensive rats. We hypothesized that Cd alters the intracellular calcium transient mechanism, by cadmium-induced stimulation of MAPKs (ERK 1 & 2) which is mediated partially through calcium-dependent PKC mechanism. To investigate this hypothesis, we exposed primary cultures of vascular smooth muscle cells (VSMCs) from wistar kyoto (WKY) and spontaneously hypertensive rats (SHR) to increased concentrations of CdCl2 on cell viability, expression of mitogen-activated protein kinases (MAPKs/ERK 1 & 2), and protein kinase C (PKC) which are activated by Cd in several cell types. The results from these studies indicate that CdCl2 decreased cell viability of both SHR and WKY VSMCs in a concentration dependent-manner. Viability of both cell types decreased 33+/-5.3 (SHR) and 39+/-2.3% (WKY) when exposed to 1 microM CdCl2, whereas, 8 and 16 microM reduced viability by 66+/-3.1 and 62+/-4.5% in SHR cells. CdCl2 increased ERK 1 & 2 in a biphasic manner with maximum increase occurring when cells are exposed to 1 and 4 muM in SHR VSMCs, whereas, a reduction in ERK 1 and 2 is observed when WKY cells are treated with 2 microM. The results also indicate that CdCl2 increased PKC a/Beta in both SHR and WKY VSMCs with a greater increase in expression in SHR VSMCs. In addition, the [Ca2+]i chelator, BAPTA, suppressed the CdCl2 effect, whereas, the PKC inhibitor, GF109203X, reduced the CdCl2 induced-effect on PKC expression. The present studies support the hypothesis that Cd can be a risk factor of hypertension through dysfunction of vascular smooth muscle cells under certain conditions.

A young azoospermic patient is described whose spermatogenesis reflected an abnormality of meiosis. A diagnosis of asynapsis of chromosomes during early spermatogenesis was made by cytogenetic examination of a testicular biopsy. Standard histologic examination gave no indication of this abnormality. An incidental history of intrauterine diethylstilbesterol (DES) exposure of the patient was elicited. Attention is called to the dearth of cytogenetic studies of spermatogenesis in DES-treated male progeny and the usefulness in general of meiotic cytogenetic studies for obtaining accurate diagnoses in human infertility.