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Acute pulmonary embolism (APE) with cardiac arrest (CA) is associated with a high mortality rate. Even upon return of the spontaneous circulation (ROSC), APE‑CA survivors are prone to myocardial cell apoptosis, a key cellular mechanism that induces heart failure. A recent study by our group discovered a post‑resuscitation imbalance in the serum angiotensin‑converting enzyme (ACE)2/ACE axis of the renin‑angiotensin system (RAS), as well as regressive cardiac function in a porcine model of APE‑CA. However, it has remained elusive how this imbalance in the ACE2/ACE axis affects myocardial cell apoptosis. In the present study, western blot and immunohistochemical analyses demonstrated that the RAS was only activated in the left myocardium, as evidenced by a decreased ACE2/ACE ratio following APE‑CA and ROSC, but not the right myocardium. Ultrastructural analysis confirmed myocardial apoptosis in the left and right myocardium. Furthermore, B‑cell lymphoma 2 (Bcl‑2)‑associated X protein (Bax) and caspase‑3 levels were elevated and Bcl‑2 levels were decreased in the left myocardium following APE‑CA and ROSC. Treatment with the ACE inhibitor captopril for 30 min after initiation of ROSC prevented the increase in Bax and the decrease in Bcl‑2 in the left myocardium compared with that in saline‑treated pigs. Captopril also inhibited the activation of extracellular signal‑regulated kinase (ERK)1/2 in the left myocardium. The results of the present study suggest that an imbalance in the ACE2/ACE axis has an important role in myocardial apoptosis following APE‑CA, which may be attributed to decreased ERK1/2 activation. In addition, it was indicated that captopril prevents apoptosis in the left myocardium after ROSC.

Background and Aims: Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. Aberrant activation of sonic hedgehog (Shh) signaling pathway plays important roles in tumorigenesis and progression of several tumors. Cyclopamine, an important inhibitor of Shh signaling pathway, can induce cell apoptosis. However, the mechanisms underlying cyclopamine-induced apoptosis are not well understood. The aim of this study is to determine the expression of the Shh signaling pathway components in HCC and to investigate the mechanisms underlying cyclopamine-induced apoptosis in HCC cells., Methods: Shh signaling components (Shh, Ptch, Smo and Gli-1) expression levels were evaluated by immunohistochemistry on tissue microarrays containing 98 HCCs with paired adjacent noncancerous liver tissues. The relationships between sonic hedgehog signal pathway and clinicopathological factors were analyzed in HCC. Cell viability was analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Apoptosis was detected by flow cytometry. mRNA and protein levels were analyzed by RT-PCR and Western blot, respectively., Results: Shh, Ptch, Smo and Gli-1 were overexpressed in HCC tissues compared with paired adjacent noncancerous liver tissue. Activated Shh signaling pathway was associated with tumor size, capsular invasion and vascular invasion in HCC. Cyclopamine remarkably decreased cell viability, induced apoptosis and downregulated Bcl-2 expression in HCC cells., Conclusions: Shh signaling pathway plays an important role in HCC tumorigenesis and progression, indicating that Shh signaling pathway is a potential therapeutic target for HCC. Cyclopamine induces apoptosis through downregulating Bcl-2 in HCC., (Copyright © 2010 IMSS. Published by Elsevier Inc. All rights reserved.)

Background & Objective: Repair of DNA damage is important to cell survival. Our previous study showed DNA damage response induced by manumycin in cancer cells. We hypothesized that methoxyamine, an inhibitor of base-excision repair, can enhance the antineoplastic effect of manumycin. This study was to investigate apoptosis induced by manumycin combined with methoxyamine in myeloid leukemia cell line U937, and to explore the role of mitochondrial apoptotic pathway in apoptosis induction of the two drugs., Methods: U937 cells were treated with various concentrations of manumycin and/or methoxyamine for 48 h. The cell viability was analyzed by MTT assay. Colony formation was evaluated by soft agar clonogenic assay. Cell apoptosis was investigated by flow cytometry. Protein expressions of cytochrome c, caspase-9, and poly ADP-ribose polymerase (PARP) were determined by Western blot., Results: The dose-response curve of manumycin was shifted to the left after addition of methoxyamine. The combination index (CI) was less than 1 (P<0.05) in U937 cells (P<0.05), indicating a synergistic effect of manumycin and methoxyamine. Rates of colony formation of U937 cells treated with 1 micromol/L manumycin, or 5 mmol/L methoxyamine, or the combination of the two were 0.3641+/-0.0463, 0.7541+/-0.0379, and 0.0473+/-0.0024, respectively compared with that of control cells (P<0.05). Moreover, the drug combination resulted in enhanced apoptosis in U937 cells. The apoptotic rates of the control, manumycin, methoxyamine and combination group were (2.34+/-0.30)%, (8.80+/-0.95)%, (2.21+/-0.19)%, and (13.37+/-0.91)%, respectively. The combination of manumycin with methoxyamine also promoted the release of cytochrome c from mitochondria into the cytosol, activated caspase-9, and led appearance of specific cleavage of PARP in U937 cells., Conclusion: Methoxyamine enhances manumycin-induced apoptosis in U937 myeloid leukemia cells.

Aim: To investigate the effects of Gli-1 small interference RNA (siRNA) on Huh7 cells, and the change of Bcl-2 expression in Huh7 cells., Methods: Human hepatocellular carcinoma cells Huh7 were used. Cell viability was analyzed by 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. The expressions of Gli-1 and Bcl-2 family members were detected by RT-PCR and Western blot. Apoptosis was detected by Flow cytometry using propidium iodide, measured by Hoechst 33258 staining using Advanced Fluorescence Microscopy and caspase-3 enzymatic assay. Cell growth was analyzed after treatment with Gli-1 siRNA and 5-fluorouracil (5-Fu)., Results: Inhibition of Gli-1 mRNA in Huh7 cells through Gli-1 siRNA reduced cell viability. Gli-1 siRNA treatment also induced apoptosis by three criteria, increase in the sub-G1 cell cycle fraction, nuclear condensation, a morphologic change typical of apoptosis, and activation of caspase-3. Gli-1 siRNA was also able to down-regulate Bcl-2. However, Gli-1 siRNA resulted in no significant changes in Bcl-xl, Bax, Bad, and Bid. Furthermore, Gli-1 siRNA increased the cytotoxic effect of 5-Fu on Huh7 cell., Conclusion: Down-regulation of Bcl-2 plays an important role in apoptosis induced by Gli-1 siRNA in HCC cells. Combination Gli-1 siRNA with chemotherapeutic drug could represent a more promising strategy against HCC. The effects of the strategies need further investigation in vivo and may have potential clinical application.

Aim: To investigate the effects of 2-methoxyestradiol (2-ME) on 2 myeloid leukemia cell lines HL-60 and U937, and to explore its mechanisms., Methods: Human myeloid leukemia cells HL-60 and U937 were used. Measurement of mitochondrial membrane potential (Dym) was performed using 5,5',6,6'-Tetrachloro-1,1',3,3'- tetraethylbenzimidazolylcarbocyanine iodide ( JC-1). Apoptosis and cellular nitric oxide (NO) were detected by flow cytometry using Annexin V and NO sensor dye. Superoxide anion was measured with a fluorescent plate reader by dihydroethidium (DHE). Cytotoxicity was analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assay., Results: 2-ME resulted in viability decrease in a dose-dependent manner. 2-ME treatment also generated reactive oxygen species (ROS), including NO and superoxide anions, which resulted in mitochondria damage. 2-ME-induced apoptosis was correlated with an increase in ROS. The quenching of ROS with N-acetyl-L-cysteine protected leukemia cells from 2-ME cytotoxicity and prevented apoptosis induction by 2-ME. Furthermore, the addition of manumycin, a farnesyltransferase inhibitor, significantly enhanced apoptosis induced by 2-ME., Conclusion: Cellular ROS generation plays an important role in the cytotoxic effect of 2-ME. It is possible to use ROS generation agents, such as manumycin, to enhance the antileukemic effect. The combination strategy needs further in vivo justification and may have potential clinical application.

Objective: To investigate the apoptosis induced by manumycin in U937 and HL-60 cell lines, and to explore the role of mitochondria apoptotic pathway in manumycin-inducing apoptosis., Methods: Leukemic cells line U937 and HL-60 were treated by manumycin at 2 micromol/L for different time. Apoptosis of leukemia cells was detected by flow cytometry. The cytosolic proteins were extracted using a digitonin buffer. The protein expression of cytochrome C, caspase-9, caspase-8, and caspase-3 were determined by western blot. Mitochondrial membrane potential was detected by JC-1., Results: In U937 and HL-60 cells, manumycin induced mitochondrial depolarization after 6 h treatment. The average red/green fluorescence ratios at 6 h were significantly (P < 0.01) lower than those at time 0, being 0.51 +/- 0.07 and 0.41 +/- 0.06 for control group respectively. Manumycin induced cytochrome C release from the mitochondria into the cytosol after 6 h treatment, and activated caspase-9, caspase-8, and caspase-3 after a 16h treatment. The broad-spectrum caspase-inhibitor Z-VAD-fmk at 50 micromol/L was able to inhibit caspase cleavage completely, but only reduced the manumycin-induced apoptosis rates by 51.69% and 56.47% in U937 and HL-60, respectively., Conclusion: Manumycin induced apoptosis in U937 and HL-60 cell lines via mitochondria apoptotic pathway.

OBJECTIVE: To explore the ability of MALAT1 to influence non-small cell lung cancer (NSCLC) A549 cells in vitro and tumor xenograft growth in vivo by modulating autophagy. METHODS: LncRNA MALAT-1 in normal HBE cells and human NSCLC cells was measured. A549 cells were treated with si-MALAT-1, negative control and si-MALAT-1 + rapamycin. The mRNA levels of MALAT-1, P62 and LC3 was determined by the qRT-PCR and the protein levels of autophagy-related proteins by the western blotting. The CCK8 assay was performed for cell proliferation, the scratch test for cell migration, the Transwell assay for cell invasion, and the flow cytometry for cell cycle and apoptosis. Tumor xenograft in nude mice is performed to test tumorigenesis of the transfected A549 cells. RESULTS: The expression level of MALAT-1 in A549, SPC-A-1 and NCI-H460 cells was increased compared to HBE cells. And A549 with a high expression level of MALAT-1 were selected for cell transfection. si-MALAT-1 decreased cell proliferation, migration, invasion, and LC3-II/LC3-I ratio, reduced cell cycle progression, and increased cell apoptosis and P62 protein expression. No significant difference was found between A549 cells and A549 cells transfected with si-MALAT-1 + RAPA, A549 cells transfected with NC and A549 cells transfected with si-MALAT-1 + RAPA. Nude mice injected with A549 cells transfected with si-MALAT-1 had smallest tumor on size and weight among other nude mice. CONCLUSION: Downregulation of MALAT1 may promote apoptosis and suppress proliferation, migration and invasion of human NSCLC A549 cells by inhibiting autophagy, thereby suppressing the development of NSCLC. [ABSTRACT FROM AUTHOR]

Acute pulmonary embolism (APE) is frequently reported in patients with cardiac arrest (CA) in emergency care. Pneumocyte apoptosis is commonly observed in the lungs following an APE. An important pathological mechanism evoking apoptosis during a lipopolysaccharide‑induced acute lung injury is the angiotensin‑converting enzyme 2 (ACE2)/ACE imbalance. The present study uses a porcine model to examine the anti‑apoptotic effects of captopril on APE‑CA and the return of spontaneous circulation (ROSC). Pigs were randomly assigned into four groups: Control, APE‑CA, ROSC‑saline, and ROSC‑captopril. Surviving pigs were euthanized at 6 h and lungs were isolated for analysis using several biochemical assays. Compared with the control group, the ACE2/ACE ratio was lower in the APE‑CA and ROSC pigs. In addition, APE‑CA pigs had higher Bcl‑2‑associated X protein (Bax) and cleaved caspase‑3 levels, and lower B‑cell lymphoma‑2 (Bcl‑2) level compared to control pigs. Captopril treatment reduced lung apoptosis, as demonstrated by lower TUNEL‑positive cells, higher Bcl‑2, and lower cleaved caspase‑3 protein levels in the lung. Notably, the ACE2/ACE ratio was positively correlated with Bcl‑2 protein levels and Bcl‑2/Bax ratio. In conclusion, captopril has a protective effect against lung apoptosis following ROSC and that maintaining the balance of the ACE2/ACE axis is important for inhibiting pulmonary apoptosis during APE. [ABSTRACT FROM AUTHOR]

Background: Manumycin exhibits an antitumor effect in a variety of cancer cell lines, including prostate cancer cell lines (DU145 and PC-3). Our previous studies demonstrated that manumycin induced the apoptosis of anaplastic thyroid cancer cells and leukemia cells via the intrinsic apoptosis pathway. In the current study, we further evaluated the effect of manumycin in two prostate cancer cell lines (LNCaP and 22Rv1), and here we elucidate some of the underlying mechanisms. Materials and methods: The cell viability of prostate cancer cells was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay after treatment with manumycin for 48 hours. Apoptosis was detected by flow cytometry using annexin V and propidium iodide. The expressions of B-cell lymphoma (Bcl)-2 family members and the activations of caspase-9 and caspase-3 were detected by Western blotting. Results: Manumycin treatment resulted in significant decreases in the viabilities of the two prostate cancer cell lines in a dose-dependent manner through apoptosis, and this apoptosis involved caspase-9 activation. A specific inhibitor of caspase-9 protected cells from caspase-3 activation, apoptosis, and cytotoxicity induced by manumycin. We also found that manumycin downregulated Bcl-2 expression and upregulated Bax expression. Conclusion: Our data suggest that manumycin induces apoptosis in prostate cancer cells through regulation of the Bcl-2 family involving caspase-9 activation. These results suggest that manumycin may be beneficial for the treatment of prostate cancer. [ABSTRACT FROM AUTHOR]