A preliminary study of imaging paclitaxel-induced tumor apoptosis with 99Tcm-His10-Annexin V

Backgroud In tumors the process of apoptosis occurs over an interval of time after chemotherapy. It is important to determine the best time for detecting apoptosis by in vivo imaging. In this study, we evaluated the dynamics and feasibility of imaging non-small cell lung cancer (NSCLC) apoptosis induced by paclitaxel treatment using a 99Tcm-labeled Annexin V recombinant with ten consecutive histidines (His10-Annexin V) in a mouse model.

Methods 99Tcm-His10-Annexin V was prepared by one step direct labeling; radio-chemical purity (RCP) and radio-stability was tested. The binding of 99Tcm-His10-Annexin V to apoptotic cells was validated in vitro using camptothecin-induced Jurkat cells. In vivo bio-distribution was determined in mice by dissection. The human H460 NSCLC tumor cell line (H460) tumor-bearing mice were treated with intravenous paclitaxel 24, 48 and 72 hours later. 99Tcm-His10-Annexin V was injected intravenously, and planar images were acquired at 2, 4 and 6 hours post-injection on a dual-head gamma camera fitted with a pinhole collimator. Tumor-to-normal tissue ratios (T/NT) were calculated by ROI analysis and they reflected specific binding of 99Tcm-His10-Annexin V. Mice were sacrificed after imaging. Caspase-3, as the apoptosis detector, was determined by flow cytometry, and DNA fragmentation was analyzed by the terminal deoxynucleotidytransferase mediated dUTP nick-end labeling (TUNEL) assay. Nonspecific accumulation of protein was estimated using bovine serum albumin (BSA). The imaging data were correlated with TUNEL-positive nuclei and caspase-3 activity.

Results 99Tcm-His10-Annexin V had a RCP >98% and high stability 2 hours after radio-labeling, and it could bind to apoptotic cells with high affinity. Bio-distribution of 99Tcm-His10-Annexin V showed predominant uptake in kidney, relatively low uptake in myocardium, liver and gastrointestinal tract, and rapid clearance from blood and kidney was observed. The T/NT was significantly increased after paclitaxel treatment, whereas it was low in untreated tumors (T/NT=1.43±0.18). The %ID/g activity in Group 2 (24 hours), Group 3 (48 hours) and Group 4 (72 hours) after treatment was 2.55±0.73, 3.35±1.10, and 3.4±0.96, respectively. Whereas in the non-treated group, Group 1, %ID/g was 1.10±0.18. The radiotracer uptake was positively correlated to the apoptotic index (r=0.852, P<0.01), as well as caspase-3 activity (r=0.816, P<0.01).

Conclusion This study addresses the dynamics and feasibility of imaging non-small cell lung tumor apoptosis using 99Tcm-His10-Annexin V.

     

Effects of lead exposure on placental cellular apoptosis and endoplasmic reticulum stress in rats

Background Lead exposure during pregnancy contributes to fetal abortion and/or teratogenesis.Endoplasmic reticulum （ER） apoptosis can be induced by various pathological conditions when ER function is disturbed.However,it is unclear whether ER stress and apoptosis play a role in the etiology of lead-exposed disease status.We aimed to investigate whether lead induced placental apoptosis and subsequent toxicity is initiated by ER apoptosis via caspase-12.Methods Sixty-three female Wistar rats were exposed to lead in drinking water during various gestational periods.Blood lead level was determined by atomic absorption spectrophotometry.Placental cytoplasmic organelles were examined by electronic microscopy.Placental caspase-12 mRNA expression was evaluated by qRT-PCR.TUNEL assay was used to determine the placental apoptosis.Results Lead exposure significant induced ER apoptosis compared to that of the controls （P ＜0.05）,accompanied with increased caspase-12 mRNA expression.Significant differences of caspase-12 mRNA expression levels were observed among the four groups （F=13.78,P ＜0.05）.Apoptotic index （AI） was significantly increased in experimental groups compared to that of the controls （F=96.15,P ＜0.05）.In lead-exposed groups,trophoblast cells underwent degeneration and fibrin deposition; Mitochondria were swollen and decreased in number; ER swelling,expansion,and vacuolization were observed.Conclusion Lead exposure contributes to placental apoptosis,as well as increased caspase-12 mRNA expression,which in turn promoted ER stress.     

Protective effects of penehyclidine hydrochloride on acute lung injury caused by severe dichlorvos poisoning in swine

Background Organophosphate poisoning is an important health problem in developing countries which causes death mainly by inducing acute lung injury. In this study, we examined the effects of penehyclidine hydrochloride （PHC）, a selective M-receptor inhibitor, on dichlorvos-induced acute lung injury in swine. Methods Twenty-two female swines were randomly divided into control （n=5）, dichlorvos （n=6）, atropine （n=6）, and PHC （n=5） groups. Hemodynamic data, extravascular lung water index （EVLWI）, and pulmonary vascular permeability index （PVPI） were monitored; blood gas analysis and acetylcholinesterase （AchE） levels were measured. PaO2/FiO2, cardiac index （CI）, and pulmonary vascular resistance indices （PVRI） were calculated. At termination of the study, pulmonary tissue was collected for ATPase activity determination and wet to dry weight ratio （W/D） testing 6 hours post-poisoning. TUNEL assay, and Bax, Bcl-2, and caspase-3 expression were applied to pulmonary tissue, and histopathology was observed. Results After poisoning, PHC markedly decreased PVRI, increased CI more effectively than atropine. Anticholinergic treatment reduced W/D, apoptosis index （AI）, and mitigated injury to the structure of lung; however, PHC reduced AI and caspase-3 expression and improved Bcl-2/Bax more effectively than atropine. Atropine and PHC improved ATPase activities; a significant difference between groups was observed in Ca2＋-ATPase activity, but not Na＋-K＋-ATPase activity. Conclusions The PHC group showed mild impairment in pathology, less apoptotic cells, and little impact on cardiac function compared with the atropine group in dichlorvos-induced acute lung injury.     

Insulin enhances apoptosis induced by cisplatin in human esophageal squamous cell carcinoma EC9706 cells related to inhibition of autophagy

Background Chemoresistance is common among patients with esophageal squamous cell carcinoma （ESCC）.We investigated the effect and mechanism of insulin on enhancing anticancer functions of cisplatin in human esophageal cancer cell line EC9706.Methods The viability of EC9706 cells exposed to cisplatin was assessed using MTT assay.The times T1,when the number of living cells reached a plateau and T2,when the number of living cells reached a new plateau after the addition of insulin were found.T1 and T2 plateau cells were stained by Annexin V-FITC/PI and monodansylcadaverin （MDC）.Fluorescent microscopy was used to observe the expression of apoptosis and autophagy intuitively.Apoptotic ratio and fluorescent intensity were analysed by flow cytometry （FCM） quantitatively.Western blotting analysis was used to estimate the protein expression levels of AKT,mTOR,PI3K,PTEN,autophage related indicator LC3-Ⅱ and autophage related protein Beclin1 changes that occurred in the course of treatment.Results A larger number of typical autophagosomes were detected in EC9706 cells exposed to cisplatin.Insulin can increase the apoptosis induced by cisplatin.Apoptotic ratio of T1 plateau cells （（32.6±4.3）％） is significantly less than T2 plateau （（47.5±5.6）％）.MDC fluorescent intensity at T1 plateau （104.9±13.2） was significantly higher than intensity at T2 plateau （82.6±10.3）.After cotreatment with insulin,the expression level of LC3-Ⅱ,Beclin1 and PTEN in T2 plateau cells were significantly downregulated,but AKT,mTOR and PI3K expressions significantly upregulated compared with T1 plateau.Conclusions Insulin could enhance cisplatin-induced apoptosis in human esophageal squamous cell carcinoma EC9706 cells related to inhibition of autophagy.The activation of PI3K/Akt/mTOR signaling pathway induced by insulin resulted in the suppression of autophagy in EC9706 cells,which may be attributed to the anticancer effects of cisplatin.     

Duodenal-jejunal bypass surgery on type 2 diabetic rats reduces the expression of matrix metalloproteinase-9 and tissue inhibitor of matrix metalloproteinase-1 in the thoracic aorta

Background Bariatric surgery offers a productive resolution of type 2 diabetes mellitus （T2DM）.The development of T2DM vasculopathy is due to chronic inflammation,which increases matrix metalloproteinase-9 （MMP-9） and tissue inhibitor of matrix metalloproteinase-1 （TIMP-1） expression.This study sought to examine MMP-9 and TIMP-1 expression in the thoracic aorta after duodenal-jejunal bypass （DJB） surgery on a T2DM rat model induced by a high-fat diet and low dose streptozotocin （STZ）.Methods Twenty-one T2DM Wistar rats induced by high-fat diet and low dose STZ were randomly divided into DJB and sham duodenal-jejunal bypass （S-DJB） groups.Ten Wistar rats were fed a normal diet as a control.Recovery of gastrointestinal function post-operation and resumption of a normal diet completed the experiment.Body weight,blood glucose,blood lipid levels,and MMP-9 and TIMP-1 expression levels in aortic endothelial cells were measured throughout.Results DJB rats showed significant weight loss 2 weeks post-operation compared with S-DJB rats.After surgery,DJB rats showed significant improvement and steady glycemic control with improved insulin sensitivity and glucose tolerance.They also exhibited improved lipid metabolism with a decrease in fasting free fatty acids （FFAs） and triglycerides （all P ＜0.05）.Immunohistochemistry showed decreased MMP-9 and TIMP-1 expression 12 weeks after surgery （P ＜ 0.01）.Conclusions DJB surgery on an induced T2DM rat model improves blood glucose levels and lipids,following a high-fat diet and low dose STZ treatment.In addition,DJB decreased MMP-9 and TIMP-1 expression in vascular endothelial cells,which may play an important role in delaying the development of T2DM vascular disease.     

Antitumor effects of mutant endostatin are enhanced by Bcl-2 antisense oligonucleotides in UM-UC-3 bladder cancer cell line

Background Endostatin is a potent inhibitor of tumor angiogenesis. In the preliminary studies, we developed a mutant endostatin containing Arg-Gly-Asp-Arg-Gly-Asp (RGDRGD) sequences. In this study, we compared the antitumor effects of mutant endostatin and Bcl-2 antisense oligonucleotides both in combination and individually.

Methods The artificially synthesized Bcl-2 ASODN (antisense oligonucleotides) included a translation-initiation site and was transfected into the bladder cancer cells by Lipofectamine. Cell growth was investigated by the tumor cell growth chart, MTT assay, caspase-3 activity detection assay, AO/EB fluorescein stain, and the annexin V-FITC apoptosis detection assay. In the in vivo study, UM-UC-3 bladder cancer cells were subcutaneously implanted into nude mice and the growth of tumor was examined. The ultrastructure of the tumor tissues in the treated and control groups were observed.

Results The cell growth chart showed that the cell population of the treated combination group decreased by 52.04% compared to the control group. The inhibition rate of the treated combination group was (79.66±6.79)%, whereas those of the individual ASODN and ES groups were (53.39±3.22)% and (50.22±5.46)% respectively. In the caspase-3 activity detection using AO/EB fluorescein stain and annexin V-FITC apoptosis detection assay, the co-inhibitory effect was higher than the individual inhibitory effects (P<0.05). There were significant differences in the inhibition of the solid tumor growth in the in vivo study.

Conclusions Our findings indicated that Bcl-2 antisense oligonucleotides enhance the antitumor effects of mutant endostatin both in vitro and in vivo. We noted the synergistic effects of Bcl-2 antisense oligonucleotides combined with mutant endostatin.

     

Intense exercise can cause excessive apoptosis and synapse plasticity damage in rat hippocampus through Ca2+ overload and endoplasmic reticulum stress-induced apoptosis pathway

Background Intense exercise can cause injury and apoptosis, but few studies have reported its effect on the central nervous system （CNS）. The initial reason for hippocampus injury is the excitotoxicity of glutamate and calcium overload. Intracellular free Ca2＋ （[Ca2＋]i） overload may trigger the apoptosis pathway and neuron damage. The aim of this study was to investigate whether intense exercise could cause hippocampus apoptosis and neuron damage and then to determine which pathway was activated by this apoptosis. Methods We used one bout of swimming exhaustion rats as models. Intracellular [Ca2~]i was measured to estimate the calcium overload by Fura-2/AM immediately after exhaustion; glial fibrillary acidic protein （GFAP） and synaptophysin （SYP） immunofluorescence were performed for estimating astrocyte activation and synapse plasticity 24 hours after exhaustion. Apoptosis cells were displayed using dUTP nick end labelling （TUNEL） stain; endoplasmic reticulum （ER） stress-induced apoptosis pathway and mitochondrial apoptosis pathway were synchronously detected by Western blotting. Results An increasing level of intracellular [Ca2＋]i （P 〈0.01） was found in the hippocampus immediately after exhaustion. GFAP and SYP immunofluorescence showed that the astrocytes are activated, and the synapse plasticity collapsed significantly 24 hours after exhaustion. TUNEL stain showed that the number of apoptosis cells were notably raised （P 〈0.01）; Western blotting of the apoptosis pathway showed increasing levels of caspase-3 cleavage （P 〈0.01）, Bax （P 〈0.01）, caspase-12 cleavage （P 〈0.01）, C/EBP-homologous protein （CHOP） （P 〈0.01）, and phospho-Junamino- terminal kinases （p-JNK; P 〈0.01） and decreasing level of Bcl-2 （P 〈0.01）. Our results proved that exhaustion can induce hippocampus injury and apoptosis by [Ca2＋]i overload, with collapsed synaptic plasticity as the injury pattern and ER stress-induced apoptosis as the activated pathway. Concl     

Ca2+在紫外线诱导成纤维细胞凋亡中的作用

目的了解细胞内外钙离子浓度对紫外线(UVB)辐射诱导成纤维细胞(NIH3T3)凋亡作用的影响.方法 UVB照射成纤维细胞10 min,培养12 h后,用流式细胞仪测定凋亡率. 结果 UVB照射组比UVB不照射组凋亡率有明显增加;UVB照射组内各组间凋亡率因细胞内外Ca2+浓度不同存在明显差异. 结论 UVB辐射诱导成纤维细胞发生凋亡与胞内外Ca2+浓度有关,Ca2+参与了凋亡的信号转导.UVB诱导成纤维细胞发生的凋亡,主要通过Ca2+转导凋亡信号这一途径,还反映出胞浆Ca2+升高既来源于胞内钙池,又来源于胞外游离Ca2+.     

Regularity of ultraviolet (UV)-induced NIH3T3 cell damage and characteristics of DNA cleavage in UV-induced NIH3T3 cells apoptosis

INTRODUCTIONThesolarspectrumonthesurfaceoftheearthcomprisesultraviolet(UV),visibleandinfraredradiation,withrelativeintensityof3%,37%and60%respectively.Inaddition,X--raysandradiowaveshavebeendetected,however,theirintensityisnegligiblylowllj.AlthoughUVradiationisaminorpartofthesolarspectrum,itisconsideredtobeveryeffective.Basedondifferencesinbiologicalresponses,UVradiationissubdividedintothreewavebands:UVC(100urn--280"m),UVB(180urn--320urn)andUVA(320urn--400"m)[ZJ.Inthisstudy,cytotox…     

Detection of atherosclerotic plaque progression in the abdominal aorta of rabbits with 3T magnetic resonance imaging

Background  With features of high tissue contrast, MRI can be used for the qualitative and quantitative evaluation of atherosclerosis plaques. In this study we investigated the development of atherosclerosis plaque with high resolution 3T MRI in a rabbit model and compared the findings with the histopathological results.

Method  Twenty male New Zealand white rabbits were randomly allocated into an experimental group (n=16) and a control group (n=4). Atherosclerotic lesions were induced in the abdominal aorta by balloon injury and cholesterol feeding. Multiple sequences MRI examination (ToF, T1WI, T2WI, and CE T1WI) were performed at the 2nd, 3rd, and 4th months after aortic denudation. Vessel wall thickness, total vessel area, lumen area, and vessel wall area were recorded. Plaque components were analyzed using histological results as a standard reference.

Results  Seventeen rabbits (14 in the experimental group and 3 in the control group) received all three MR examinations. Gradually, from 2 months to 4 months, vessel wall thickness and area in the experimental group increased significantly compared with the control group (P <0.01). In the lumen area progressive stenosis was not found, even a slight dilation had developed in the experimental group. Lipid, fibrotic and calcified plaques can be differentiated by MR image. According to histological results, MRI had good performance in detection of lipid plaque.

Conclusion  MRI can be used to monitor progression of atherosclerosis and differentiate plaque components.

     

Early changes of CD4+CD25+Foxp3+ regulatory T cells and Th1/Th2, Tc1/Tc2 profiles in the peripheral blood of rats with controlled hemorrhagic shock and no fluid resuscitation

Background  Hemorrhagic shock induces immune dysfunction. Regulatory T cells (Tregs), T-helper (Th) cells, and cytotoxic T-lymphocytes (CTLs) can execute many crucial actions in immune and inflammatory responses. This study was conducted to investigate the early pathophysiological changes of CD4⁺CD25⁺Foxp3⁺ Treg and Th1/Th2, Tc1/Tc2 profiles in the peripheral blood of rats with controlled hemorrhagic shock and no fluid resuscitation.

Methods  A rat model of controlled hemorrhagic shock with no fluid resuscitation was established. Peripheral blood samples were taken before and four hours after hemorrhagic shock with no fluid resuscitation. Three color flow cytometry was used to detect Tregs, Th1, Th2, Tc1 and Tc2 cells in the samples.

Results  In the peripheral blood of rats, the percentage of Tregs four hours after hemorrhagic shock was significantly lower than before hemorrhagic shock (P=0.001). The ratios of Th1/Th2 and Tc1/Tc2 were changed from (23.08±8.98)% to (23.91±15.36)%, and from (40.40±21.56)% to (65.48±23.88)%, respectively.

Conclusions  At an early stage, the advent of hemorrhagic shock is related to an early decrease of Tregs, and a mild shift in the Th1/Th2, Tc1/Tc2 balance toward Th1 and Tc1 dominance. These changes are part of a hyper-inflammatory state of the host, and will deteriorate the maintenance of immune balance. Further influences and detailed mechanisms need to be investigated.     

Endoplasmic reticulum stress is involved in podocyte apoptosis induced by saturated fatty acid palmitate

Background  Podocyte apoptosis is recently indicated as an early phenomenon of diabetic nephropathy. Pancreatic b-cells exposed to saturated free fatty acid palmitate undergo irreversible endoplasmic reticulum (ER) stress and consequent apoptosis, contributing to the onset of diabetes. We hypothesized that palmitate could induce podocyte apoptosis via ER stress, which initiates or aggravates proteinuria in diabetic nephropathy.

Methods  Podocyte apoptosis was detected by 4¢,6-diamidio-2-phenylindole (DAPI) stained apoptotic cell count and Annexin V-PI stain. The expressions of ER molecule chaperone glucose-regulated protein 78 (GRP78), indicators of ER-associated apoptosis C/EBP homologous protein (CHOP), and Bcl-2 were assayed by Western blotting and real-time PCR. GRP78 and synaptopodin were co-localized by immunofluorescence stain.

Results  Palmitate significantly increased the percentage of cultured apoptotic murine podocytes time-dependently when loading 0.75 mmol/L (10 hours, 13 hours, and 15 hours compared with 0 hour, P <0.001) and dose-dependently when loading palmitate ranging from 0.25 to 1.00 mmol/L for 15 hours (compared to control, P <0.001). Palmitate time-dependently and dose-dependently increased the protein expression of GRP78 and CHOP, and decreased that of Bcl-2. Palmitate loading ranging from 0.5 to 1.0 mmol/L for 12 hours significantly increased mRNA of GRP78 and CHOP, and decreased that of Bcl-2 compared to control (P <0.001), with the maximum concentration being 0.75 mmol/L. Palmitate 0.5 mmol/L loading for 3 hours, 8 hours, and 12 hours significantly increased mRNA of GRP78 and CHOP, and decreased that of Bcl-2 compared to 0 hour (P <0.001), with the maximum effect at 3 hours. Confocal microscopy demonstrated that GRP78 expression was significantly increased when exposed to 0.5 mmol/L of palmitate for 8 hours compared to control.

Conclusion  Palmitate could induce podocyte apoptosis via ER stress, suggesting podocyte apoptosis and consequent proteinuria caused by lipotoxic free fatty acid could be ameliorated by relief of ER stress.

     

Direct contact with bone marrow stromal cells promotes the invasions of SHI-1 leukemia cells

Background Interactions of tumor cells with the microenvironment were deemed to promote the tumor invasion and metastasis.CXC chemokine receptor 4 (CXCR4) and extracellular matrix metalloproteinase ind...