[pemetrexed + sildenafil], via autophagy-dependent HDAC downregulation,enhances the immunotherapy response of NSCLC cells

Pemetrexed is an approved therapeutic in NSCLC and ovarian cancer. Our studies focused on the ability of [pemetrexed + sildenafil] exposure to alter the immunogenicity of lung and ovarian cancer cells. Treatment of lung and ovarian cancer cells with [pemetrexed + sildenafil] in vitro rapidly reduced the expression of PD-L1, PD-L2 and ornithine decarboxylase (ODC), and increased the expression of class I MHCA. In a cell-specific fashion, some cells also released the immunogenic nuclear protein HMGB1 into the extracellular environment. [Pemetrexed + sildenafil] reduced the expression of multiple histone deacetylases that was blocked by knock down of autophagy regulatory proteins. [Pemetrexed + sildenafil] lethality was enhanced by the histone deacetylase inhibitors AR42 and sodium valproate; AR42 and valproate as single agents also rapidly reduced the expression of PD-L1, PD-L2 and ODC, and increased expression of MHCA and CerS6. Nitric oxide and CerS6 signaling was required for drug-induced death receptor activation and tumor cell killing. In vivo, [pemetrexed + sildenafil] lethality against lung cancer cells was enhanced by sodium valproate. Using syngeneic mouse lung cancer cells [pemetrexed + sildenafil] enhanced the anti-tumor effects of antibodies directed to inhibit PD-1 or CTLA4. [Pemetrexed + sildenafil] interacted with the anti-PD-1 antibody to strongly enhance tumor infiltration by M1 macrophages; activated NK cells and activated T cells. Our data demonstrate that treatment of tumor cells with [pemetrexed + sildenafil] results in tumor cell killing and via autophagy-dependent downregulation of HDACs, it opsonizes the remaining tumor cells to anti-tumor immunotherapy antibodies.     

246例乳腺影像报告和数据系统3级评分乳腺病变ER、PR和C-erbB-2表达的临床分析

目的通过对246例乳腺影像报告和数据系统(Breast Imaging Reporting and Data System,BIRADS)3级乳腺肿块的病理、雌激素受体(ER)、孕激素受体(PR)、乳腺癌原癌基因(C-erb B-2)的表达水平进行研究统计,为BI-RADS 3级乳腺肿块是否需要手术活检或治疗提供依据。方法特选取行乳腺B超或高频X线检查BI-RADS 3级有可触及乳腺肿块或局限性增厚腺体,同时进行空芯针穿刺活检的女性患者246例(共246个肿块),对所有标本进行组织病理学检查及ER、PR、Cerb B-2检查。分析患者的影像学结果及其与病理免疫组织化学部分重要指标的关系。结果由病理结果可知,246例乳腺肿块中纤维腺瘤125例,囊肿30例,乳腺癌17例,乳管内乳头状瘤23例,腺病31例,导管扩张症20例。恶性率为6.91%。影像学表现肿块最大径≤20 mm者,其免疫组织化学指标PR阳性率高,统计学上有意义(P<0.05)。影像表现中肿块内有点状钙化,其免疫组织化学指标C-erb B-2、ER阳性率高,统计学上有意义(P<0.05)。结论本研究发现BI-RADS 3级乳腺病变存在恶性率为6.91%,所以影像表现肿块内点状钙化者或肿块最大径≥20 mm的肿块,需积极手术活检或治疗。     

Butyl paraben promotes apoptosis in human trophoblast cells through increased oxidative stress‐induced endoplasmic reticulum stress

Butyl paraben (BP) has antimicrobial effects and is widely used as a preservative in cosmetics, foods, and pharmaceuticals. It is also absorbed into various tissues of the human body. It is known that BP is measurable in maternal and fetal tissues during pregnancy, but the effects of BP on placental development, essential for maintaining normal pregnancy, are unclear. Therefore, we investigated the effect of BP on the proliferation, apoptosis, and invasiveness of human trophoblast cells, using an HTR8/SVneo cell line. BP inhibited cell proliferation and induced both apoptosis and endoplasmic reticulum stress. In addition, BP promoted the production of intracellular reactive oxygen species, increased Ca²⁺ concentration in HTR8/SVneo cells, and induced mitochondrial membrane depolarization. BP also inhibited the activation of PI3K/AKT pathways including AKT, ribosomal protein S6, P70 S6 kinase, and glycogen synthase kinase 3β. Furthermore, pretreatment of cells with LY294002 (an AKT inhibitor) and U0126 (ERK1/2 inhibitor) revealed that ERK1/2 activity is also involved in BP‐mediated signal transduction in HTR8/SVneo cells. We therefore suggest that exposing human trophoblast cells to BP diminishes normal physiological activity, leading to apoptosis and problems with early placental development.     

Characterization of the modes of action of deoxynivalenol (DON) in the human Jurkat T-cell line

Abstract

Deoxynivalenol (DON) is one of the most abundant mycotoxins worldwide and mostly detected in cereals and grains. As such, DON poses a risk for many adverse health effects to human and animals. In particular, immune cells are very sensitive to DON, with the initiating step leading to toxicity being a binding to the eukaryotic 60S ribosomal subunit and induction of ribotoxic stress. The present study aimed to: (1) extend insight into the mechanism of action (MOA) of DON in immune cells; and (2) understand why immune cells are more sensitive to DON than most other cell types. Previously published microarray studies have described the effects of DON on immune cells. To build upon these findings, here, immunocytological and biochemical studies were performed using human T-lymphocyte Jurkat cells that were exposed for 3?h to 0.5?µM DON. Induction of ER stress by DON was confirmed by immunocytology demonstrating increased protein expression of two major ER stress markers ATF3 and DDIT3. T-cell activation was confirmed by induction of phosphorylation of protein kinases JNK and AKT, activation of NF-κB (p65), and increased expression of NFAT target gene NUR77; each of these are known inducers of the T-cell activation response. Induction of an oxidative stress response was also confirmed by monitoring the nuclear translocation of major oxidative stress markers NRF2 and KEAP1, as well as by changes (i.e. decreases) in cell levels of reduced glutathione. Lastly, this study showed that DON induced cleavage of caspase-3, an event known to mediate apoptosis. Taken together, these results allowed us to formulate a potential mechanism of action of DON in immune cells, i.e. binding to eukaryotic 60S ribosomal subunit?→?ribotoxic stress?→?ER stress?→?calcium release from the ER into cytoplasm?→?T-cell activation and oxidative stress?→?apoptosis. It is proposed that immune cells are more sensitive to DON than other cell types due to the induction of a T-cell activation response by increased intracellular calcium levels.     

Relationship between expression of ER,PR, Her-2, Ki-67 and neoadjuvant chemotherapy effect in breast cancer

Objective： The purpose of the study was to investigate the relationship between the expression of estrogen receptor （ER）, progestogen receptor （PR）, human epidermal growth factor receptor （Her-2）, Ki-67 and the effect of neoadjuvant chemotherapy in breast cancer. Methods： The expression of ER, PR, Her-2 and Ki-67 in 45 breast cancers which received neoadjuvant chemotherapy was detected by immunohistochemistry. Results： The effective rates in ER negative and PR negative groups were higher than those in ER positive and PR positive groups （83.3% vs 59. 4%, 82.4% vs 60.6%）. There was no significant difference of the effective rate between Her-2 overexpressed group and Her-2 non-overexpressed group （81.8% vs 64.1%）, and the same thing happened between Ki-67 negative group and Ki-67 positive group （67.7% vs 63.2%）. Conclusion： In the patients with breast cancer, ER, PR negative ones were more sensitive to neoadjuvant chemotherapy. These patients may get more benefits from chemotherapy. ER, PR could be feasible markers for predicting the effective rate of neoadjuvant chemotherapy.     

Effects of ketamine administration on mTOR and reticulum stress signaling pathways in the brain after the infusion of rapamycin into prefrontal cortex

Recent studies show that activation of the mTOR signaling pathway is required for the rapid antidepressant actions of glutamate N-methyl-D-aspartate (NMDA) receptor antagonists. A relationship between mTOR kinase and the endoplasmic reticulum (ER) stress pathway, also known as the unfolded protein response (UPR) has been shown. We evaluate the effects of ketamine administration on the mTOR signaling pathway and proteins of UPR in the prefrontal cortex (PFC), hippocampus, amygdala and nucleus accumbens, after the inhibiton of mTOR signaling in the PFC. Male adult Wistar rats received pharmacological mTOR inhibitor, rapamycin (0.2 nmol), or vehicle into the PFC and then a single dose of ketamine (15 mg/kg, i.p.). The immunocontent of mTOR, eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), eukaryotic elongation factor 2 kinase (eEF2K) homologous protein (CHOP), PKR-like ER kinase (PERK) and inositol-requiring enzyme 1 (IRE1) – alpha were determined in the brain. The mTOR levels were reduced in the rapamycin group treated with saline and ketamine in the PFC; p4EBP1 levels were reduced in the rapamycin group treated with ketamine in the PFC and nucleus accumbens; the levels of peEF2K were increased in the PFC in the vehicle group treated with ketamine and reduced in the rapamycin group treated with ketamine. The PERK and IRE1-alpha levels were decreased in the PFC in the rapamycin group treated with ketamine. Our results suggest that mTOR signaling inhibition by rapamycin could be involved, at least in part, with the mechanism of action of ketamine; and the ketamine antidepressant on ER stress pathway could be also mediated by mTOR signaling pathway in certain brain structures.     

Combined Effects of Multiple Endoplasmic Reticulum Stresses on Cytokine Secretion in Macrophage

Cells show various stress signs when they are challenged with severe physiological problems. Majority of such cellular stresses are conveyed to endoplasmic reticulum (ER) and unfolded protein response (UPR) serves as typical defense mechanism against ER stress. This study investigated an interaction between ER stress agents using macropage cell line Raw 264.7. When activated by lipopolysaccharide (LPS), the cell lines showed typical indicators of ER stress. Along with molecular chaperones, the activation process leads to the production of additional infl ammatory mediators. Following activation, the macrophage cell line was further treated with TUN and characterized in terms of chaperone expression and cytokine secretion. When treated with TUN, the activated macrophage cell leads to increased secretion of IL-6 although expression of ER stress markers, GRP94 and GRP78 increased. The secretion of cytokines continued until the addition of BFA which inhibits protein targeting from ER to Golgi. However, secretion of cytokines was ceased upon dual treatments with BFA and TG. This result strongly implies that cells may differently deal with various polypeptides depending on the urgency in cellular function under ER stress. Considering IL-6 is one of the most important signal molecules in macrophage, the molecule might be able to circumvent ER stress and UPR to reach its targeting site.