Effect of estrogen on growth and apoptosis in esophageal adenocarcinoma cells.

The epidemiology of esophageal adenocarcinoma demonstrates a strong gender bias with a sex ratio of 8-9:1 in favor of males. A potential explanation for this is that estrogen might protect against esophageal adenocarcinoma. Estrogen has previously been shown to stimulate apoptosis in esophageal squamous cancer cells. However, the effect of estrogen on esophageal adenocarcinoma cells has not been determined. We used immunoblotting analysis to determine the expression of estrogen receptors, cell adhesion marker E-cadherin, and proliferation marker Ki-67 in cell lines derived from esophageal adenocarcinoma (OE-19, OE-33) and Barrett's esophagus (QhTRT, ChTRT, GihTRT). Estrogen and selective estrogen receptor modulator (SERM)-dependent effects on cell growth were determined by the CellTiter-96 Aqueous Proliferation Assay. Apoptosis was determined by Annexin V/Propidium Iodide cell labeling and flow cytometry. We detected that physiological and supra-physiological concentrations of 17ß-estradiol and SERM decreased cell growth in esophageal adenocarcinoma cells. In Barrett's esophagus cells (QhTRT, ChTRT), decreased growth was also detected in response to estrogen/SERM. The level of estrogen receptor expression in the cell lines correlated with the level of anti-growth effects induced by the receptor agonists. Flow cytometry analysis confirmed estrogen/SERM stimulated apoptosis in esophageal adenocarcinoma cells. Estrogen/SERM treatments were associated with a decrease in the expression of Ki-67 and an increase in E-cadherin expression in esophageal adenocarcinoma cells. This study suggests that esophageal adenocarcinoma and Barrett's esophagus cells respond to treatment with selective estrogen receptor ligands, resulting in decreased cell growth and apoptosis. Further research to explore potential therapeutic applications is warranted.

Estrogen and progesterone interactive effects in postconfluent MCF-7 cell culture.

17beta-Estradiol (E2) stimulates morphological differentiation of an MCF-7 human mammary carcinoma cell line resulting in the development of multicellular rounded nodules (foci) above the epithelial monolayer. Examining the combined effect of progesterone (P4) and E2 on foci formation we detected P4-dependent foci enlargement and phenotypic modification. Notably, P4 dose-dependently potentiated lower dose E2-induced increases in foci numbers. We detected P4-dependent changes in cytoskeleton protein expression levels and accelerated cell division. P4 alone or in combination with E2 additively modified the expression of adhesion proteins and stimulated expression of tropomyosin (Tm). Antiprogestin and antiestrogen pretreatment abrogated P4-dependent increases in foci number and stimulation of Tm expression, indicating involvement of both E2 and P4 receptor signaling. Novel aspects of endocrine-regulated changes in microfilament and adhesion protein composition are discussed in association with tumorigenesis and metastatic capability in breast carcinoma cells.

Anti-apoptotic effects of 3,5,3'-tri-iodothyronine in mouse hepatocytes.

The present study demonstrates that 3,5,3'-tri-iodothyronine (T3) in physiological dose range inhibits tumor necrosis factor alpha(TNFalpha)/Fas-induced apoptosis in mouse hepatocytes. T3 pretreatment prevented Fas-induced early stage of apoptosis signs assessed by flow cytometry analysis of the annexin V positive cell population. T3 attenuated TNFalpha/Fas-induced cleavage of caspase-8 and DNA fragmentation. We found that T3 exerted its anti-apoptotic effects by mobilization of several non-genomic mechanisms independent of transcriptional activity. Inhibition of protein kinase A (PKA), extracellular signal-regulated kinase (ERK), and Na+/H+ exchanger blocked T3-dependent anti-apoptotic effects indicating an involvement of these intracellular targets into T3-induced signaling cascade. Furthermore, physiological concentrations of T3, but not reverse T3, caused increases in intracellular cAMP content and activated PKA. T3 markedly induced phosphorylation of ERK. We also detected T3-dependent intracellular alkalinization that abolished TNFalpha-induced acidification. PKA inhibitor KT-5720 blocked T3-induced activation of ERK and intracellular alkalinization confirming the upstream position of PKA signaling. We further detected that hepatocytes from hypothyroid mice are more sensitive to TNFalpha/Fas-induced apoptosis than euthyroid animals in vivo. Together, these findings imply that T3 triggers PKA- and ERK-regulated intracellular pathways capable of driving and ensuring hepatocytes survival in the presence of death receptor ligand-induced damage under chronic inflammatory conditions.

Effects of chemotherapy agents on Sphingosine-1-Phosphate receptors expression in MCF-7 mammary cancer cells.

Sphingosine-1-phosphate (S1P) is a potent bioactive sphingolipid involved in the regulation of cell proliferation and cancer progression. Increased expression of S1P receptors has been detected in advanced breast tumours with poor prognosis suggesting that S1P receptors might control tumour response to chemotherapy. However, it remains unclear how the levels of S1P receptor expression are influenced by chemotherapy agents. Western immunoblotting, PCR analysis and fluorescent microscopy techniques were used in this study to analyze expression patterns of S1P receptors 2 and 3 (S1P2/S1P3) in MCF-7 breast adenocarcinoma cells treated by Tamoxifen (TAM) and/or Medroxyprogesterone acetate (MPA). We found that TAM/MPA induce downregulation of S1P3 receptors, but stimulate expression of S1P2. According to cell viability and caspase activity analyses, as expected, TAM activated apoptosis. We also detected TAM/MPA-induced autophagy marked by formation of macroautophagosomes and increased level of Beclin 1. Combined application of TAM and MPA resulted in synergistic apoptosis- and autophagy-stimulating effects. Assessed by fluorescent microscopy with autophagosome marker LAMP-2, changes in S1P receptor expression coincided with activation of autophagy, suggestively, directing breast cancer cells towards death. Further studies are warranted to explore the utility of manipulation of S1P2 and S1P3 receptor expression as a novel treatment approach.

Tamoxifen enhances the cytotoxicity of conventional chemotherapy in esophageal adenocarcinoma cells.

INTRODUCTION: Esophageal adenocarcinoma is a lethal malignancy which is increasing in incidence, and many patients receive chemotherapy as part of their treatment. We have previously demonstrated that esophageal adenocarcinoma-derived cell lines respond to treatment with estrogen receptor modulators, such as tamoxifen. Reports from breast cancer suggest that tamoxifen may attenuate the efficacy of other chemotherapeutic agents. We have therefore assessed the response of esophageal adenocarcinoma cell lines to tamoxifen therapy when given in combination with conventional agents. METHODS: Two estrogen receptor (ER)-positive esophageal adenocarcinoma cell lines (OE-19 and OE-33) were treated with combinations of tamoxifen, cisplatin and 5-fluorouracil (5-FU). Effects on cell viability were measured using an MTS assay, and cell death was detected with annexin V/propidium iodide flow cytometry. To assess whether the efficacy of tamoxifen in these cell lines might be relevant to the clinical setting, we analyzed ER status in 10 esophageal adenocarcinoma tissue specimens by immunohistochemistry. RESULTS: IC50 values (µM) for OE-19 and OE-33 were 11.2 and 7.1 for tamoxifen, 19.6 and 4.7 for cisplatin, and 1.7 and 5.9 for 5-FU, respectively. Cell death was detected in 11.9% and 15.8% of cells treated with tamoxifen, 7.9% and 8.7% cells treated with cisplatin, and 3.6% and 8.6% cells treated with 5-FU at their IC50s. The addition of tamoxifen to cisplatin increased cell death by 11.4% in OE-19 (p < 0.0001) and 16.3% in OE-33 (p < 0.0001). Similarly, the addition of tamoxifen to 5-FU increased cell death by 11.6% in OE-19 (p < 0.0001) and 15.9% in OE-33 (p < 0.0001). Eight of 10 tissue specimens showed positive staining for ERα and 7 of 10 for ERß. CONCLUSIONS: In a cell culture model the addition of tamoxifen to conventional chemotherapy appears to be both feasible and beneficial. Expression of ERα and ERß was also confirmed in esophageal adenocarcinoma tissues.

Galanin receptor 3--a potential target for acute pancreatitis therapy.

BACKGROUND: Galanin participates in the pathogenesis of acute pancreatitis (AP). The galanin receptor (GALR) sub-types involved, however, are unclear. We aimed to determine GALRs messenger RNA (mRNA) expression in mouse pancreas, describe their localization, and ascertain if GALR2 and GALR3 are involved in AP. METHODS: Galanin receptor expression in murine whole pancreas, acinar, and islet cells was quantified by polymerase chain reaction amplification of reverse-transcribed RNA for mRNA, Western blot analysis for protein and in situ hybridization for GALR localization. Isolated acinar cells were used to determine galanin's effect on amylase secretion. Acute pancreatitis was induced in mice by caerulein injections. Mice, with and without AP, were treated with the highly selective GALR2 antagonist M871, or the specific GALR3 antagonist SNAP-37889. Indices of AP were measured at 12 h. KEY RESULTS: Murine pancreas expresses mRNA for GALRs. In islets the expression of all GALR are comparable, whereas in acinar cells GALR3 is predominantly expressed. Western blot analysis confirmed that the GALR proteins are expressed by acinar cells. In situ hybridization analysis confirmed that GALR3 mRNA is present in islet and acinar cells, while mRNA for GALR1 and 2 is confined to islets. Galanin did not influence basal and caerulein-stimulated amylase release from acinar cells. M871 treatment reduced some, whereas SNAP-37889 treatment reduced all indices of AP (by 40-80%). CONCLUSIONS & INFERENCES: Galanin receptor mRNA and protein are expressed in mouse pancreas, with GALR3 mRNA predominating. GALR3 antagonism reduced the severity of AP whereas GALR2 antagonism was less effective. GALR3 is a potential target for treatment of AP.

Long-term persistence after acute Q fever of non-infective Coxiella burnetii cell components, including antigens.

BACKGROUND: Previous studies of inciting factors for a prolonged post-infection fatigue syndrome after Q fever (variously termed QFS or Q fever associated CFS/ME in the literature) showed that after the acute infection a high proportion of asymptomatic and QFS patients had Q fever antibody and also low levels in PBMC and bone marrow of Coxiella burnetii (C.b.) DNA with PCR assays directed against three different target sequences in different parts of the coxiella genome. Attempts to isolate a strain of C.b. in A/J mice, and cell culture from PCR positive PBMC and bone marrow were consistently negative. The detailed composition of the persisting coxiella residues remains to be defined. AIM: To retest and provide detailed results on selected PCR positive samples from the Birmingham Q fever outbreak patients tested by a highly sensitive method to detect viable organisms and to determine the nature of the residual coxiella cell components. DESIGN: Laboratory case study. METHODS: NOD/SCID mice were inoculated with samples from the 1989 Q fever outbreak in Birmingham and followed for evidence of infection and the presence of coxiella DNA and specific antigens in spleen and liver macrophages. A significant, unexpected finding of specific antigen was followed by assessment of its ability to provoke production of inflammatory and non-inflammatory cytokines in mice, in THP-1 human macrophage cell cultures and to induce inflammatory lesions in the skin of guinea pigs hyperimmunized against Q fever vaccine. RESULTS: Culture of samples from 10 Birmingham Q fever patients in NOD/SCID mice, 12 years from infection did not yield viable Coxiella burnetii, as shown earlier. However complexes of material with coxiella antigens were found in mouse spleens in all cases but in significantly greater amounts in samples from those with post Q fever fatigue syndrome. The antigenic complexes [now designated 'immunomodulatory complexes' (IMC)] were shown to stimulate cytokine release in the mice and in the THP-1 macrophages and to provoke an inflammatory reaction on intradermal injection into the skin of Q fever hyperimmunized guinea pigs. CONCLUSION: The study identifies a non-infective complex of C.b. antigens able to survive in the host and provoke aberrant humoral and cell medicated immunity responses - a possible pathogenic link between initial infection and a subsequent long-term post Q fever fatigue syndrome.

In vitro effect of hydrocortisone on calcium distribution in rat thymocytes.

Chlorotetracycline (CTC) was used as a fluorescence indicator to investigate the intracellular membrane-bound Ca2+ redistribution in the hydrocortisone (HC)-treated rat thymocytes. The effect of HC (0.1-1 microM) on the thymocytes incubated in Ca2+ containing media for 60-120 min increased 1.5-fold the quantity of membrane-bound Ca2+ in the plasma membrane. When thymocytes were incubated in Ca(2+)-free media, no HC-induced changes in the Ca2+ pool of plasma membrane were revealed. In the presence of inhibitors of respiration and oxidative phosphorylation, the CTC fluorescence intensity in the HC-treated cells decreased to a greater extent than in the control, thereby indicating an increase in the mitochondrial Ca2+ pool. Using inhibitors of endoplasmic reticulum Ca(2+)-ATPase (vanadate, BHQ). HC was shown to decrease the non-mitochondrial Ca2+ pool. Calmodulin blockers (triphthasine and R24) slightly decreased the CTC fluorescence intensity in the HC-treated cells as compared to the control. HC was found to inhibit the calmodulin-mediated Ca2+ accumulation in the thymocyte membrane. Based on the experiments using AlF4, we concluded that HC stimulates the activity of G-proteins through the receptor-mediated mechanism. A possible role of the observed Ca2+ redistribution between the thymocyte intracellular compartments, specifically between mitochondria and reticulum, is discussed.

Effect of thyroxine on calcium distribution in rat thymocytes in vitro.

Chlorotetracycline (CTC) was used as a fluorescent Ca2+-sensitive probe to study the redistribution of intracellular membrane-bound Ca2+ in thyroxine (T4)-treated rat thymocytes. Incubation of thymocytes in the Ca2+-supplemented medium in the presence of 1-100 nM T4 for 30 min resulted in a twofold increase in the amount of EGTA-accessible plasma membrane-bound Ca2+ as compared to that in the Ca2+-free medium. The induced decrease in CTC fluorescence was more pronounced with the occurrence of respiration and oxidative phosphorylation in inhibitors. The mitochondrial Ca2+ pool was shown to increase. The nonmitochondrial Ca2+ pool decreased after a 30-min incubation in the presence of 1 nM T4 and increased when 100 nM T4 was used under the same conditions. Without incubation, different concentrations of T4 stimulated the decrease in the Ca2+ pool of the endoplasmic reticulum (ER) compared to the control cells, which was demonstrated using inhibitors of the ER Ca2+-ATPase (vanadate, BHQ). Calmodulin blockers (triftazin and R24) caused a significant decrease (over 50%) in CTC fluorescence in the T4-treated thymocytes. This suggests that T4 can act as an in vitro stimulator of calmodulin-dependent Ca2+ accumulation in thymocyte membranes. The results of our experiments with AlF4- suggest that T4 stimulates the activity of G-proteins by a receptor-mediated mechanism.

[Dose-dependent character of the effect of hydrocortisone on energy metabolism of rat thymocytes]. / Dozozavisimyi kharakter vliianiia gidrokortizona na énergeticheskii metabolizm timotsitov krys.

The influence of the hydrocortisone (GC) different doses on rat's thymocytes energetic metabolism was investigated. It has been demonstrated that as the prolonged action of GC in vivo (20 micrograms/kg of mass during 6 d. daily) as the preincubation of thymocytes with 10 microM GC in vitro are cytotoxic, leading to absolute uncoupling of oxidative phosphorylation (OP) and decreasing the rate of DNP-stimulated respiration speed. The injection of GC (20 micrograms/kg mass) at once before 72 hours to killing resulted the OP uncoupling almost on 2,5 time. Short-time action of GC at the same dose before 3 hours to drilling and so the thymocytes preincubation with 1 microM GC stimulated the transport processes on the level of substrate oxidation without the OP uncoupling. Short-time action of GC in vivo caused the inhibition of respiration, if we used glucose as the oxidative substrate, and stimulated the respiration in a presence of pyruvate Na in incubation media.

Modulation of intracellular Ca(2+) concentration by vitamin B12 in rat thymocytes.

We have studied several novel effects of vitamin B12 (cyanocobalamin) on cellular Ca(2+) homeostasis in rat thymocytes. We determined the effect of various concentrations of vitamin B12 on intracellular Ca(2+) concentration ([Ca(2+)]i) and parameters of Ca(2+)in signaling using the fluorescent dye Fura-2. The basal [Ca(2+)]i in Ca(2+)-containing media was 115 +/- 5 nM but in vitamin B12 (10 nM)-treated thymocytes [Ca(2+)]i was decreased to 60 +/- 15 nM (mean +/- SEM) during the first 5 min. The decline in [Ca(2+)]i was accompanied by an increase in the endoplasmic reticulum Ca(2+) store, presumably as a result of Ca-ATPase activation. At the same time 100 nM-10 mM B12 induced the accumulation of Ca(2+) in mitochondria. Somewhat higher concentrations of B12 (1-10 microM) had no effect on [Ca(2+)]i. A further increase in B12 concentration with range from 50 microM to 1 mM caused a dose-dependent elevation of [Ca(2+)]i from the basal level (115 +/- 5 nM) up to 200 +/- 50 nM in thymocytes, and this elevation was partially blocked in Ca(2+)-free media. This high concentration of vitamin B12 caused a gradual decrease of endoplasmic reticulum Ca(2+) stores by means of Ca-ATPase inhibition. The B12-induced increase in [Ca(2+)]i was not observed after depletion of intracellular Ca(2+) stores, induced by addition of 2',5'-di(tert-butyl)-1,4-benzohydroquinone (BHQ), an inhibitor of endoplasmic reticulum Ca (2+)-ATPase, concanavalin A, or arachidonic acid. These studies show that vitamin B12 regulates [Ca(2+)]i via several different mechanisms at different B12 concentrations. Participation of G proteins and calmodulin activity in B12-mediated [Ca(2+)]i increase is discussed.