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Idiopathic pulmonary fibrosis (IPF) is an irreversible lung condition that progresses over time, which ultimately results in respiratory failure and mortality. In this study, we found that PLAC8 was downregulated in the lungs of IPF patients based on GEO data, in bleomycin (BLM)-induced lungs of mice, and in primary murine alveolar epithelial type II (pmATII) cells and human lung epithelial cell A549 cells. Overexpression of PLAC8 facilitated autophagy and inhibited apoptosis of pmATII cells and A549 cells in vitro. Moreover, inhibition of autophagy or overexpression of p53 partially abolished the effects of PLAC8 on cell apoptosis. ATII cell-specific overexpression of PLAC8 alleviated BLM-induced pulmonary fibrosis in mice. Mechanistically, PLAC8 interacts with VCP-UFD1-NPLOC4 complex to promote p53 degradation and facilitate autophagy, resulting in inhibiting apoptosis of alveolar epithelial cells and attenuating pulmonary fibrosis. In summary, these findings indicate that PLAC8 may be a key target for therapeutic interventions in pulmonary fibrosis., Competing Interests: Competing interests: The authors declare no competing interests. Ethical approval: All animal experiments were conducted under the Guide for the Care and Use of Laboratory Animals and this study was approved by the Ethics Committee of Shengjing Hospital of China Medical University (2023PS005K)., (© 2025. The Author(s).)

A series of novel 2-substituted 2, 3-dihydroquinazolin-4(1H)-one derivatives were designed, synthesized and estimated for their in vitro antiproliferative activities against HepG2, U251, PANC-1, A549 and A375 cell lines. Among them, compound 32 was the most promising candidate, and displayed strong broad-spectrum anticancer activity. The mechanism studies revealed that compound 32 inhibited tubulin polymerization in vitro, disrupted cell microtubule networks, arrested the cell cycle at G2/M phase, and induced apoptosis by up-regulating the expression of cleaved PARP-1 and caspase-3. Furthermore, molecular docking analysis suggested that compound 32 well occupied the binding site of tubulin. In addition, compound 32 exhibited no significant activity against 30 different kinases respectively, indicating considerable selectivity. Moreover, compound 32 significantly inhibited the tumour growth of the HepG2 xenograft in a nude mouse model by oral gavage without apparent toxicity. These results demonstrated that some 2-substituted 2, 3- dihydroquinazolin-4(1H)-one derivatives bearing phenyl, biphenyl, naphthyl or indolyl side chain at C 2 -position might be potentially novel antitumor agents as tubulin polymerization inhibitors., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Dysfunctional renal tubular epithelial cells, induced by high glucose, are commonly observed in the kidney tissues of diabetic nephropathy (DN) patients. The epithelial-mesenchymal transition (EMT) of these cells often leads to renal interstitial fibrosis and kidney damage in DN. High glucose also triggers mitochondrial damage and apoptosis, contributing further to the dysfunction of renal tubular epithelial cells. Cellular senescence, a recognized characteristic of DN, is primarily caused by high glucose. However, it remains unclear whether high glucose-induced cellular senescence in DN exacerbates the functional impairment of tubular epithelial cells. In this study, we examined the relationship between EMT and cellular senescence in kidney tissues from streptozotocin (STZ)-induced DN and HK-2 cells treated with high glucose (HG). We also investigated the impact of HG concentrations on tubular epithelial cells, specifically mitochondrial dysfunction, cellular senescence and apoptosis. These damages were primarily associated with the secretion of cytokines (such as IL-6, and TNF-α), production of reactive oxygen species (ROS), and an increase of intracellular Ca 2+ . Notably, resveratrol, an anti-aging agent, could effectively attenuate the occurrence of EMT, mitochondrial dysfunction, and apoptosis induced by HG. Mechanistically, anti-aging treatment leads to a reduction in cytokine secretion, ROS production, and intracellular Ca 2+ levels., Competing Interests: Declaration of competing interest All authors declare no competing interests. We declare that neither the entire paper nor any part of its content has been published or has been accepted elsewhere. If accepted, it will not be published elsewhere in the same form, in English or in any other languages, including electronic form without the written consent of the copyright-holder., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Several clinical trials have been conducted to evaluate the use of flavopiridol (FP) to treat a variety of cancers, and almost all cancer drugs were found to be associated with toxicity and side effects. It is not clear whether the use of FP will affect the female reproductive system. Granulosa cells, as the important cells that constitute the follicle, play a crucial role in determining the reproductive ability of females. In this study, we investigated whether different concentrations of FP have a toxic effect on the growth of immortalized human ovarian granulosa cells. The results showed that FP had an inhibitory effect on cell proliferation at a level of nanomole concentration. FP reduced cell proliferation and induced apoptosis by inducing mitochondrial dysfunction and oxidative stress, as well as increasing BAX/BCL2 and pCDK1 levels. These results suggest that toxicity to the reproductive system should be considered when FP is used in clinical applications., (© 2024. The Author(s).)

Prenatal exposure to Benzo[a]pyrene (BaP) has been suggested to increase the risk of adverse pregnancy outcomes. However, the role of placental apoptosis on BaP reproductive toxicity is poorly understood. We conducted a maternal animal model of C57BL/6 wild-type (WT) and transformation-related protein 53 (Trp53) heterozygous knockout (p53KO) mice, as well as a nested case-control study involving 83 women with PB and 82 term birth from a birth cohort on prenatal exposure to BaP and preterm birth (PB). Pregnant WT and p53KO mice were randomly allocated to BaP treatment and control groups, intraperitoneally injected of low (7.8 mg/kg), medium (35 mg/kg), and high (78 mg/kg) doses of 3,4-BaP per day and equal volume of vegetable oil, from gestational day 10.5 until delivery. Results show that high-dose BaP treatment increased the incidence of preterm birth in WT mice. The number of fetal deaths and resorptions increased with increasing doses of BaP exposure in mice. Notably, significant reductions in maternal and birth weights, increases in placental weights, and decrease in the number of livebirths were observed in higher-dose BaP groups in dose-dependent manner. We additionally observed elevated p53-mediated placental apoptosis in higher BaP exposure groups, with altered expression levels of p53 and Bax/Bcl-2. In case-control study, the expression level of MMP2 was increased among women with high BaP exposure and associated with the increased risk of all PB and moderate PB. Our study provides the first evidence of BaP-induced reproductive toxicity and its adverse effects on maternal-fetal outcomes in both animal and population studies., (© 2024. The Author(s).)

Objectives: The lung injury is often secondary to severe trauma. In the model of crush syndrome, there may be secondary lung injury. We hypothesize that high-mobility group box 1 (HMGB1), released from muscle tissue, mediates the apoptosis of alveolar epithelial cells (AEC) via HMGB1/Receptor of advanced glycation end-products (RAGE)/c-Jun N-terminal kinase (JNK) pathway. The study aimed to investigate how HMGB1 mediated the apoptosis of AEC in the rat model., Methods: Seventy-five SD male rats were randomly divided into five groups: CS, CS + vehicle, CS + Ethyl pyruvate (EP), CS + FPS-ZM1 group, and CS + SP600125 groups. When the rats CS model were completed after 24 h, the rats were sacrificed. We collected the serum and the whole lung tissues. Inflammatory cytokines were measured in serum samples. Western blot and RT-qPCR were used to quantify the protein and mRNA. Lastly, apoptotic cells were detected by TUNEL. We used SPSS 25.0 for statistical analyses., Results: Nine rats died during the experiments. Dead rats were excluded from further analysis. Compared to the CS group, levels of HMGB1 and inflammatory cytokines in serum were downregulated in CS + EP, CS + FPS-ZM1, and CS + SP600125 groups. Western blot and RT-qPCR analysis revealed a significant downregulation of HMGB1, RAGE, and phosphorylated-JNK in CS + EP, CS + FPS-ZM1, and CS + SP600125 groups, compared with the CS groups, excluding total-JNK mRNA. Apoptosis of AEC was used TUNEL to assess. We found the TUNEL-positive cells were downregulated in CS + EP, CS + FPS-ZM1, and CS + SP600125 groups., Conclusion: The remote lung injury begins early after crush injuries. The HMGB1/RAGE/JNK signaling axis is an attractive target to abrogate the apoptosis of AEC after crush injuries., (© 2022. The Author(s).)

Curcumin is a diketone compound found in turmeric. It is used as food additives and spices, and has anti-proliferation and anti-cancer properties. However, the effect of curcumin on human keratinocytes (KCs) is still unclear. In this study, curcumin dramatically inhibited the cell growth of immortalized human KCs (HaCaT) and arrested the cells at the G2/M phase, with an apoptosis rate of 33.95% after 24 μM curcumin treatment. HaCaT cells showed changes in typical apoptotic morphology and the configuration of nuclear matrix-intermediate filaments (NM-IFs) after treatment with curcumin. We identified 16 differentially expressed nuclear matrix (NM) proteins, including apoptosis inducing factor (AIF) and caspase 3, by 2-DE and MALDI-TOF/TOF mass spectrometry. The expression of AIF decreased in the mitochondria and increased in the nucleus. Immunofluorescence assays showed that AIF was released from the mitochondria to the nucleus. AIF silencing and caspase inhibitor (z-vad-fmk) both lead to HaCaT cells being insensitive to apoptosis induced by curcumin. Meanwhile, after curcumin treatment, mitochondrial membrane depolarization led to cytochrome c release from the mitochondria to the cytoplasm, and the ratio of Bax to Bcl-2 in HaCaT cells was also increased, which subsequently initiated the activation of caspase-3. These results suggest that curcumin-induced apoptosis of HaCaT cells occurs not only through the caspase-dependent pathway but also through the caspase-independent pathway. This discovery enhances the development and utilization of curcumin and provides possible evidence for the treatment of proliferative skin diseases, including skin cancer.

Background Liver X receptor (LXR) belongs to the metabolic nuclear receptor superfamily, which plays a critical regulatory role in vascular physiology/pathology. However, effects of systemic LXR activation on established vulnerable plaques and the potential isotype-specific role involved remain unclear. Methods and Results The 8-week-old male apolipoprotein E -/- mice went through carotid branch ligation and renal artery constriction, combined with a high-fat diet. Plaques in the left carotid artery acquired vulnerable features 4 weeks later, confirmed by magnetic resonance imaging scans and histological analysis. From that time on, mice were injected intraperitoneally daily with PBS or GW3965 (10 mg/kg per day) for an additional 4 weeks. Treatment with LXR agonists reduced the lesion volume by 52.61%, compared with the vehicle group. More important, a profile of less intraplaque hemorrhage detection and necrotic core formation was found. These actions collectively attenuated the incidence of plaque rupture. Mechanistically, reduced lesional apoptosis, enhanced efferocytosis, and alleviated endoplasmic reticulum stress are involved in the process. Furthermore, genetic ablation of LXRα, but not LXRβ, blunted the protective effects of LXR on the endoplasmic reticulum stress-elicited C/EBP-homologous protein pathway in peritoneal macrophages. In concert with the LXRα-predominant role in vitro, activated LXR failed to stabilize vulnerable plaques and correct the acquired cellular anomalies in LXRα -/- apolipoprotein E -/- mice. Conclusions Our results revealed that LXRα mediates the capacity of LXR activation to stabilize vulnerable plaques and prevent plaque rupture via amelioration of macrophage endoplasmic reticulum stress, lesional apoptosis, and defective efferocytosis. These findings might expand the application scenarios of LXR therapeutics for atherosclerosis.

Tyrosinase is considered a molecular marker of melanoma, and few natural antitumor drugs targeting tyrosinase have been identified. In this study, proanthocyanidins (PAs) were isolated from the leaves of Photinia × fraseri and their structures were characterized by high performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI-MS), and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and the effects of antityrosinase activity were investigated. The results showed that the basic structural units of PAs are composed of catechin and epicatechin and that oligomer is the main component. PAs exhibited better antityrosinase activity via chelation of copper ions and by disturbing o-quinone production. Furthermore, analyses of the cell cycle, apoptosis rate, and regulation of melanin protein expression revealed preliminarily that PAs could affect melanin production by downregulating microphthalmia transcription factor (MITF) expression and by inhibiting the activities of tyrosinase and tyrosinase related protein 1 (TRP-1), leading to cell cycle arrest and apoptosis of melanoma cells. Collectively, our study demonstrated that PAs are potential tyrosinase inhibitors and have good antimelanoma effects. These findings provide a theoretical support for the application of tyrosinase inhibitors and for further drug development.

Background: Hyperhomocysteinemia (HHcy) is one of the major risk factors of cardiovascular diseases. Metformin acts as a cardioprotective role in several cardiovascular diseases, including ischemia/reperfusion, atherosclerosis, and myocardial infarction. However, whether metformin protects against HHcy-induced cardiac hypertrophy is unclear., Methods and Results: HHcy model was established in C57BL/6 mice with high L-methionine (L-MET) diet for 12 weeks. AC16 cells were exposed to homocysteine (Hcy) and then intervened with different concentrations of metformin in in vitro studies. The results showed that HHcy was able to induce cardiac hypertrophy, and metformin could abrogate this effect. HHcy increased the fibrosis area and induced apoptosis in the myocardium, whereas metformin could reverse the detrimental effects above. TUNEL assay showed that metformin was able to decrease Hcy-induced apoptosis in AC16 cells. Moreover, western blotting assay revealed that metformin could decrease Hcy-induced expression of Bax and cleaved caspase3, and increase the expression of Bcl-2., Conclusions: This study demonstrates that metformin is able to attenuate HHcy-induced cardiac hypertrophy by decreasing myocardial fibrosis and apoptosis., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

The mechanisms and biological functions of migrating platelets in cancer remain largely unknown. Here, we analyzed platelet infiltration in hepatocellular carcinoma. We detected platelet extravasation in both mouse and human HCC tissues. CX3CL1 directly induced platelet migration, and hypoxia enhanced platelet migration by upregulating CX3CL1 expression. Knocking down CX3CL1 in HCC cells reduced platelet migration in vitro, as well as infiltration of HCC tissue in an orthotopic HCC mouse model. Components of the CX3CR1/Syk/PI3K pathway were essential for CX3CL1-induced platelet migration. Migrating platelets induced HCC cell apoptosis in vitro, as indicated by a reduced mitochondrial membrane potential and an increased percentage of apoptotic cells. In the orthotopic tumor implantation model, decreased platelet infiltration was associated with accelerated tumor growth. Taken together, our findings indicate that HCC cell-derived CX3CL1 contributes to tumor infiltration by platelets, which in turn promotes apoptosis of HCC cells., (© 2020 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

A novel hexanuclear copper(ii)-based complex, [Cu6(tpbb)2(NO3)12] (1), was synthesized, which shows potent cytotoxicity to hepatoma carcinoma cells by inducing apoptosis and apoptosis-related processes. Furthermore, mechanistic investigations based on proteomes revealed that the induced apoptosis was mediated by acting on several targets and multiple pathways in a pleiotropic way.

An extract of Dracocephalum moldevica (DML) was found to exert protective effects on cerebral ischemia-reperfusion injury (CIRI); however, the mechanisms underlying the observed actions of this plant-derived mixture remain to be determined. Thus, the aim of this study was to examine the influence of DML on CIRI rat model induced by middle cerebral artery occlusion (MCAO). The following parameters were measured: (1) viable neurons in the infarcted area using Nissl staining; and (2) immunohistochemistry and Western blot were employed to determine protein expression levels of p53, bcl-2 associated X protein (bax) and B-cell lymphoma-2 (bcl-2), three biomarkers of apoptosis. MCAO significantly decreased the number of viable cortical pyramidal neurons in the infarcted area, while treatment with DML extract significantly elevated the number of viable neurons. MCAO was found to significantly elevate in gene expression levels of p53 and protein expression levels bax accompanied by diminished protein expression levels of bcl-2. Prior administration of DML extract produced marked reduction in gene expression levels of p53 and protein expression levels bax but increased in protein expression levels of bcl-2. Data suggested apoptosis was initiated in MCAO and that DML was effective in treating CIRI via an anti-apoptotic action as evidenced by inhibition of gene expression levels of p53 and protein expression levels of bax with concomitant elevation in protein expression levels of bcl-2. Our findings suggest that extract of DML may prove beneficial in treatment of cerebrovascular disorders.

This study aimed to investigate the role of miR-590-5p in chondrocyte apoptosis and autophagy in response to mechanical pressure injury in vitro, as well as to elucidate its regulatory mechanism in the pathogenesis of osteoarthritis. We applied mechanical pressure of 10 MPa to chondrocytes for 60 minutes to establish the chondrocyte model of experimentally induced mechanical injury. We then investigated the expression of miR-590-5p in the injury model and the effects of miR-590-5p dysregulation on the expression of cell apoptosis-related and autophagy-related proteins. Cell apoptosis was detected by flow cytometry. Moreover, the potential targets of miR-590-5p were investigated. Mechanical pressure injury resulted in a significantly increased expression of miR-590-5p. Suppression of miR-590-5p significantly increased chondrocytes viability, inhibited chondrocytes apoptosis and autophagy in response to mechanical pressure injury. In addition, mechanical pressure injury led to a decreased expression of transforming growth factor β1 (TGFβ1). Moreover, TGFβ1 was confirmed as a direct target of miR-590-5p. Knockdown of TGFβ1 significantly induced chondrocytes apoptosis and autophagy in response to mechanical pressure injury, which was contrary to the effects of miR-590-5p suppression. Furthermore, overexpression of TGFβ1 and miR-590-5p at the same time significantly reversed the effects of overexpression of miR-590-5p alone on chondrocytes apoptosis and autophagy. Our results indicate that upregulation of miR-590-5p may target TGFβ1 to promote chondrocyte apoptosis and autophagy in response to mechanical pressure injury, thus contributing to the pathogenesis of osteoarthritis., (© 2018 Wiley Periodicals, Inc.)

Chemical investigation of Euphorbia helioscopia resulted in the identification of seven new triterpenes named euphorbatrine A-G (1-7), including two (19αH)-lupane (1-2) and three (9βH)-lanostane (4-6), which are very rare metabolites from plants. Their structures were elucidated by extensive spectroscopic analysis, X-ray crystallography and chemical methods. Compounds 5 and 6 triggered apoptosis of Hela -/- cell with EC 50 values of 1.59±0.25 and 26.48±0.78μM, respectively., (Copyright © 2017 Elsevier B.V. All rights reserved.)

BACKGROUND Gremlin has been reported to be up-regulated in glomerular mesangial cells in diabetic nephropathy (DN). However, the regulation of gremlin in podocytes is still rarely reported. This study aimed to investigate the underlying mechanisms by which gremlin mediates the pathogenesis of DN via transforming growth factor-β (TGF-β) signaling pathways. MATERIAL AND METHODS Lentiviral and RNAi transfection were performed to increase and decrease gremlin expression in high-glucose conditions. Expression at the mRNA and protein level was detected by RT-qPCR and Western blotting. RESULTS The expression of gremlin was significantly higher in high-glucose (HG, 30mM) than normal-glucose (NG, 5.5 mM) conditions. The gremlin overexpression significantly suppressed the expression of nephrin and synaptopodin. The phosphorylation of canonical TGF-b signaling pathway components, including Smad2/3 and MKK, was increased in the gremlin-overexpressing group. In addition, the expression levels of Bax and cleaved caspase-3 were also higher in the gremlin-overexpressing group. TGF-β pathway inhibitor (SB505124) significantly inhibited TGF-β pathway activity and enhanced the expression of nephrin and synaptopodin. CONCLUSIONS These results indicate that gremlin can aggravate podocyte lesions through the TGF-β signaling pathway, providing a novel therapeutic target for DN.

Spermatogenesis, the process by which haploid sperm cells are produced from a diploid precursor cell, is essential for sexual reproduction. Here, we report that RING-finger protein 138 (Rnf138) is highly expressed in testes, especially in spermatogonia and spermatocytes. The role of Rnf138 in spermatogenesis was examined using a Rnf138-knockout mouse model. Rnf138 deficiency resulted in increased apoptosis in spermatogenic cells, loss of proliferative spermatogonia, delayed development of spermatozoa and impaired fertility. The proportion of PLZF+Ki67+ cells within the PLZF+ population decreased in the knockout mice. The phenotype was further assessed by RNA-sequencing (RNA-seq), which determined that the expression levels of many genes involved in spermatogenesis were altered in the testis of Rnf138-knockout mice. Thus, Rnf138 deficiency promotes the apoptosis of spermatogenic cells, which may have been caused by the aberrant proliferation of spermatogonia in mouse testis development.

Breast cancer is reported as the most frequent tumor with limited treatments among the female worldwide. Galangin, a natural active compound 3, 5, 7-trihydroxyflavone, is a type of bioflavonoid isolated from the Alpinia galangal root and suggested to induce apoptosis in various cancers. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an effective anti-tumor agent for human breast cancer. Promoted expression of CHOP, a down-streaming transcription factor for endoplasmic reticulum stress (ER stress), enhanced death factor 4 (DR4) activity and accelerated reactive oxygen species (ROS) as well as cell death. Adenosine monophosphate-activated protein kinase (AMPK) is crucial for various cancers mortality. In the present study, galangin regulated ER stress to augment CHOP and DR4 expression levels, sensitizing TRAIL activity, leading to human breast cancer cell apoptosis through Caspase-3 activation, which was associated with AMPK phosphorylation. In addition, AMPK inhibition and silence reduced anti-cancer activity of galangin and TRAIL in combinational treatment. Hence, our study indicated that galangin could effectively stimulate human breast cancer cells to TRAIL-induced apoptosis through TRAIL/Caspase-3/AMPK signaling pathway. AMPK signaling pathway activation by galangin might be of benefit for promoting the effects of TRAIL-regulated anti-tumor therapeutic strategy., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

Trim69 contains the hallmark domains of a tripartite motif (TRIM) protein, including a Ring-finger domain, B-box domain, and coiled-coil domain. Trim69 is structurally and evolutionarily conserved in zebrafish, mouse, rat, human, and chimpanzee. The role of this protein is unclear, however, so we investigated its function in zebrafish development. Trim69 is extensively expressed in zebrafish adults and developing embryos-particularly in the testis, brain, ovary, and heart-and its expression decreases in a time- and stage-dependent manner. Loss of trim69 in zebrafish induces apoptosis and activates apoptosis-related processes; indeed, the tp53 pathway was up-regulated in response to the knockdown. Expression of human trim69 rescued the apoptotic phenotype, while overexpression of trim69 does not increase cellular apoptosis. Taken together, our results suggest that trim69 participates in tp53-mediated apoptosis during zebrafish development. Mol. Reprod. Dev. 83: 442-454, 2016. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Background: Chemotherapeutic resistance is a main problem in clinical breast cancer therapy. The purpose of our study is to investigate whether the combination of neoadjuvant chemotherapy and survivin siRNA treatment could enhance the therapeutic effect of neoadjuvant chemotherapy using paclitaxel or epirubicin., Materials and Methods: The molecular cloning technique was applied to construct the expression vector of siRNA against survivin. Effectene Transfection Reagent was used to transfect plasmids to MCF-7 cells. Survivin expressions were detected by quantitative real-time PCR (qRT-PCR) and Western blot methods. The effect of paclitaxel or epirubicin, with or without the combination of survivin siRNA treatment, on drug susceptibility of MCF-7 cells was detected by CCK-8 assay. MCF-7 cell apoptosis was detected by Flow Cytometry., Results: Survivin siRNA effectively inhibited the expression of Survivin RNA and protein levels (P < 0.05). Both paclitaxel and epirubicin can suppress the proliferation of MCF-7 cells and induce apoptosis to a certain degree respectively. The combination of survivin siRNA with the two chemotherapy drugs significantly enhanced both effects of the two chemotherapeutics respectively (P < 0.05)., Conclusion: Survivin siRNA combined with the neoadjuvant chemotherapy can significantly enhance the sensitivity of MCF-7 cells to chemotherapeutics and cell apoptosis. This technology has important potential value in the therapeutic study of breast cancer.

Lactobacillus paracasei subsp. paracasei M5L (L. paracasei M5L) was isolated and co-cultured with HT-29 colon cancer cells to study its anti-colorectal cancer effects and mechanism. Using the MTT assay we found that L. paracasei M5L significantly inhibits HT-29 cell proliferation. Morphological and biochemical characteristics of apoptosis were observed and confirmed by hematoxylin and eosin (HE) staining and transmission electron microscopy (TEM). Lactobacilli could change the cell cycle distribution and induce calreticulin (CRT) translocation from the endoplasmic reticulum to the surface of the cytomembrane. We also determine that vast reactive oxygen species (ROS) were generated, while the activities of superoxide dismutase (SOD) and catalase (CAT) were noticeably diminished following L. paracasei M5L treatment. This study reveals that L. paracasei M5L induces apoptosis in HT-29 cells through ROS generation followed by CRT accompanied endoplasmic reticulum (ER) stress and S phase arrest. These results provide new insights into the possible molecular mechanism of L. paracasei M5L as a novel probiotic with the potential for further application.

The present study was designed to investigate the effect of tanshinol on hydrogen peroxide (H2O2)‑induced apoptosis in human umbilical vein endothelial cells (HUVECs), and to determine the underlying mechanisms. HUVECs were pre‑incubated with tanshinol (25‑200 µM) for 24 h, followed by an incubation with 600 µM H2O2 for 6 h. The cell viability was assessed using MTT reagent and the level of cell death was determined by measuring lactate dehydrogenase (LDH) activity. Superoxide dismutase (SOD) activity, levels of malondialdehyde (MDA), reactive oxygen species (ROS) production and NADPH oxidase activity were measured spectrophotometrically using commercially available kits. The apoptotic rate of the HUVECs was detected using Annexin‑V/propidium iodide (PI) staining, followed by flow cytometry analysis using a fluorescence microscope. The protein expression of SOD‑1, SOD‑2, B‑cell lymphoma‑2 (Bcl‑2), cytochrome c and caspase‑3 was determined by western blot analysis. Pretreatment with tanshinol resulted in a significant increase in the cellular viability of HUVECs and SOD activity, and a decrease of cell apoptosis, MDA levels and ROS production, induced by H2O2. These findings were accompanied by the upregulation of Bcl‑2 protein expression, reduction in the release of cytochrome c from the mitochondria to the cytosol and a downregulation of caspase‑3 protein expression. This study showed that tanshinol protects against atherosclerosis by preventing H2O2‑induced apoptosis of HUVECs. These effects appear to be mediated by enhancing the antioxidant defenses and preserving the mitochondrial function of the cells.

In this study, a polysaccharide (PS) was successfully extracted from porcine cartilage and its effect on chronic myeloid leukemia was examined using human K562 cells. The results of cell proliferation assays indicated that the PS inhibited cancer cell growth at different concentrations. Morphological and biochemical changes characteristic of apoptosis were observed and confirmed by PI staining and TUNEL assay. The nuclear DNA, RNA and proteins of the cancer cells subjected to PS treatment were irreversibly destroyed by reactive oxygen species (ROS), additionally, the ROS effected on the cells directly. The apoptotic signals altered the permeability of the mitochondrial outer membrane, thereby resulted in the release of apoptotic factors into the cytoplasm that induced apoptosis. As caspase-3/7, 8 and 9 were expressed, it was speculated that both intrinsic and extrinsic pathways were involved in the PS-induced apoptosis.

Apoptosis is a regulated cellular suicide program that is critical for the development and maintenance of healthy tissues. Previous studies have shown that small kinetochore associated protein (SKAP) cooperates with kinetochore and mitotic spindle proteins to regulate mitosis. However, the role of SKAP in apoptosis has not been investigated. We have identified a new interaction involving SKAP, and we propose a mechanism through which SKAP regulates cell apoptosis. Our experiments demonstrate that both overexpression and knockdown of SKAP sensitize cells to UV-induced apoptosis. Further study has revealed that SKAP interacts with Pre-mRNA processing Factor 19 (Prp19). We find that UV-induced apoptosis can be inhibited by ectopic expression of Prp19, whereas silencing Prp19 has the opposite effect. Additionally, SKAP negatively regulates the protein levels of Prp19, whereas Prp19 does not alter SKAP expression. Finally, rescue experiments demonstrate that the pro-apoptotic role of SKAP is executed through Prp19. Taken together, these findings suggest that SKAP promotes UV-induced cell apoptosis by negatively regulating the anti-apoptotic protein Prp19.

Endometrial carcinoma (EC) is the most common gynecologic cancer worldwide and is one of the leading causes of death in women. Therefore, it is urgent to elucidate the pathological mechanisms of EC. SERPINA3 is a member of the serpin super-family of protease inhibitors. Its aberrant expression has been observed in various tumor cells. However, its clinical significance and biological function in endometrial cancer remains unknown. In the present study, we demonstrated that SERPINA3 expression was significantly up-regulated in EC samples and was closely correlated with lower differentiation, higher stage, positive lymph node or vascular thrombosis and negative estrogen receptor (ER), indicating a poor prognosis. We then demonstrated that SERPINA3 promoted EC cells proliferation by regulating G2/M checkpoint in cell cycle and inhibited cells apoptosis, and we further uncovered that the pro-proliferative effect of SERPINA3 on EC was likely ascribed to the activation of MAPK/ERK1/2 and PI3K/AKT signaling. The results of our study may provide insight into the application of SERPINA3 as a novel predictor of clinical outcomes and a potential therapeutic target of EC.

Prohibitin (PHB), also known as inhibin, is important in cell proliferation, differentiation and apoptosis. This protein localizes to the inner membrane of mitochondria, where it acts as a chaperone protein, and is also found in the nucleus, where it negatively regulates transcription. The tumor-suppressive role of PHB in cell proliferation appears to be contradictory. In this study, we investigated the existence, localization and alterations in the expression of PHB in the whole cell and nuclear matrix and analyzed its co-localization with the expression products of related genes. The western blot analysis results revealed that PHB exists in the composition of nuclear matrix proteins and that the expression level of PHB is significantly increased in the whole cell and markedly decreased in the nuclear matrix after curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione) treatment. The laser confocal scanning microscope results demonstrated the co-localization of PHB with p53, c-Myc, Bax, and Fas in HaCaT cells, and this co-localization region was transferred as a result of curcumin treatment. In addition, the results of the GST pull-down assay demonstrated the direct interaction of PHB with p53, c-Myc and Bax but not Fas in vitro. Results of the present study confirmed that the expression and distribution of PHB, which is a nuclear matrix protein, affect the apoptosis of HaCaT cells and its co-localization with specific gene products connected with cell apoptosis.

Increasing evidence shows that abnormal microRNAs (miRNAs) expression is involved in tumorigenesis. They might be the novel biomarkers or therapeutic targets in disease treatment. miR-29 family was previously reported to act as tumor suppressors or oncogenes in diverse cancers. However, their accurate expression, function and mechanism in gastric cancer (GC) are not well known. Here, we found that the expression of miR-29 family members was significantly reduced in GC compared with adjacent controls. Among them, miR-29c had the most reduced percentage in GC and was associated with aggressive and progressive phenotypes of GC. We further demonstrated that miR-29 family acted as tumor suppressors through targeting CCND2 and matrix metalloproteinase-2 genes in GC. Moreover, the inverse relationship between miR-29 family and their targets was verified in patients and xenograft mice. Finally, reintroduction of miR-29 family significantly inhibited tumor formation of GC cells in the xenograft mice. Take together, our finding characterized the expression properties of miR-29 family, contributed to the function and molecular mechanism of miR-29 family in GC and implied that miR-29 family might be employed as novel prognostic markers and therapeutic targets of GC.

In this study, we aimed to investigate the aberrant expression and shift in localization of prohibitin (PHB) during apoptosis of human cholangiocarcinoma cells. Our study demonstrated that PHB was expressed primarily in the cytoplasm and only a little in the nucleus. However, PHB expression significantly decreased, and its localization shifted from the cytoplasm to the nucleus during apoptosis. PHB co-localized with AIF, Rb, p53, and c-Fos, but the region of co-localization was altered after treatment. Meanwhile, we detected a direct interaction between PHB and both p53 and Rb in Mz-ChA-1 cells. These results suggest that the altered localization and expression of PHB, as well as its co-localization with related oncogenes and tumor suppressor genes, can affect the apoptosis of Mz-ChA-1 cells., (Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Three-dimensional (3D) morphometric analysis of cellular and subcellular structures provides an effective method for spatial cell biology. Here, 3D cellular and nuclear morphologies are reconstructed to quantify and compare morphometric differences between normal and apoptotic endothelial cells. Human umbilical vein endothelial cells (HUVECs) are treated with 60 μM H2 O2 to get apoptotic cell model and then a series of sectional images are acquired from laser scanning confocal microscopy. The 3D cell model containing plasma membrane and cell nucleus is reconstructed and fused utilizing three sequential softwares or packages (Mimics, Geomagic, and VTK). The results reveal that H2 O2 can induce apoptosis effectively by regulating the activity of apoptosis-related biomolecules, including pro-apoptotic factors p53 and Bax, and anti-apoptotic factor Bcl-2. Compared with the normal HUVECs, the apoptotic cells exhibit significant 3D morphometric parameters (height, volume and nucleus-to-cytoplasm ratio) variation. The present research provides a new perspective on comparative quantitative analysis associated with cell apoptosis and points to the value of LSCM as an objective tool for 3D cell reconstruction., (Copyright © 2013 Wiley Periodicals, Inc.)

Natural compounds isolated from medicinal plants are invaluable resources for drug discovery. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent unique by its cancer cell-specific proapoptotic action, but its potential is heavily curbed by acquired resistance. We herein reported for the first time the identification of cytotoxic and TRAIL-sensitizing components of Salvia miltiorrhiza (Danshen), a traditional medicinal plant effective for treating cardiovascular disorders. Specifically, we found that the ethanol extract and its group 5 fraction of S. miltiorrhiza showed evident cytotoxicity against the human lung adenocarcinoma cell line A549 and ovarian adenocarcinoma cell line TOV-21G in a concentration-dependent manner. Likewise, a dose-dependent cytotoxicity was exerted by the standard solutions of cryptotanshinone, tanshinone I and tanshinone IIA, the major components of the group 5 fraction, where tanshinone IIA were most potent and displayed an IC₅₀ of 2.00 ± 0.36 μM and 2.75 ± 0.23 μM for A549 and TOV-21G, respectively. Induction of apoptosis represents an essential mechanism underlying tanshinone IIA-mediated cytotoxic action, as evidenced by the proteolytic processing of PARP upon tanshinone IIA stimulation and, importantly, a marked rescue of the viability of tanshinone IIA-treated cells when co-treatment with the pan-caspase inhibitor z-VAD-fmk. Noteworthy, stimulation with cryptotanshinone, tanshinone I or tanshinone IIA all effectively potentiated TRAIL to reduce viability and inhibit the colony formation capacity of TRAIL-resistant TOV-21G and SKOV3. Collectively, we revealed the proapoptotic and TRAIL-sensitizing components of S. miltiorrhiza and further implicated the potential of developing these active compounds as monotherapeutic agent or TRAIL-based therapy for cancer chemoprevention or chemotherapy., (Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

The study aimed to evaluate the effects of deoxycholic acid (DCA) on human gastric carcinoma cell lines and to explore its mechanisms. In the present study, effects of DCA on SGC-7901 cell growth, cell cycle, and apoptosis were investigated by MTT assay, inverted microscopy, fluorescence microscopy, PI single- and FITC/PI double-staining flow cytometry, and western blotting. The study have revealed that DCA significantly inhibited the growth of SGC-7901 cells in a dose- and time-dependent manner and arrested cell cycle at G0/G1 phase. SGC-7901 cells showed typical apoptotic morphological changes after treated with DCA for 48 h. The intensity of typical apoptosis pattern- "ladders" formed by DNA in fragments of multiples of 200 base pairs was also observed. Apoptosis of SGC-7901 cells induced by DCA were associated with collapse of the mitochondrial membrane potential. DCA treatment could also increase the ratio of Bax to Bcl-2 in SGC-7901 cells. Meanwhile, the expression of p53, cyclinD1, and c-Myc were changed after DCA treatment. These results suggest that DCA induces apoptosis of gastric carcinoma cells through an intrinsic mitochondrial-dependent pathway, and the increase in the Bax/Bcl-2 ratio and collapse of the mitochondrial membrane potential may play important roles in DCA-induced apoptosis of gastric carcinoma cells.

We have previously reported that rhomboid domain containing 1 (RHBDD1), a new member of the Rhomboid family, is highly expressed in testis. In the present work, luciferase analyses showed that RHBDD1 enhanced the AP-1 activity in a dose- and activity-dependent manner. RHBDD1 overexpression increased c-Jun activity, and the activity was visibly inhibited when RHBDD1 was knocked down. The suppression of c-Jun by inactivation of RHBDD1 was rescued by overexpressing RHBDD1. However, c-Jun overexpression did not alter the expression level of RHBDD1. Furthermore, knockdown of RHBDD1 led to increased cell apoptosis and reduced expression of Bcl-3. Our findings indicate that RHBDD1 could inhibit cell apoptosis by activating and upregulating c-Jun and its downstream target, Bcl-3., (Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

In the present study, effects of aqueous extracts from Crocodylus siamensis bile (AE-CB) on SMMC-7721 cell growth, cell cycle, and apoptosis were investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, inverted microscopy, fluorescence microscopy, propidium iodide (PI) single- and fluorescein isothiocyanate (FITC)/PI double-staining flow cytometry, and western blotting. Our data have revealed that AE-CB significantly inhibited the growth of SMMC-7721 cell and arrested cell cycle at G0/G1 phase. SMMC-7721 cells showed typical apoptotic morphological changes after treated with AE-CB for 48 h. Cell death assay indicated that SMMC-7721 cells underwent apoptosis in a dose-dependent manner induced by AE-CB. In addition, AE-CB treatment could downregulate the protein level of Bcl-2 and upregulate the Bax, leading to the increase in the ratio of Bax to Bcl-2 in SMMC-7721 cells. Meanwhile, it was observed that the expression of Survivin and c-Myc decreased, but the expression of P53 increased. All these events were associated with increase of reactive oxygen species. The data indicated that mitochondrial pathway might play an important role in bile extract-induced apoptosis in SMMC-7721 cells. These results provide significant insight into the anticarcinogenic action of bile extract on SMMC-7721 cells.

Objective: To reveal the effects of three days' repeated exhausted eccentric exercise on the skeletal muscle apoptosis and proliferation in rats., Methods: Fifty male SD rats aged at 8 week old were randomly divided into control group (C) and training groups (B1, B2, B3, B4) (n = 10), the training groups ran on a treadmill every day till exhausted. After they had been trained repeatedly for three days, their medial head of triceps brachii muscle cell apoptosis was detected in paraffin section by the TUNEL, expression of proliferating cell nuclear antigen (PCNA) protein was examined by immunohistochemistry., Results: (1) The apoptosis appeared sequential change, and it was consistent with the exercise-induced skeletal muscle micro-injury (EIMmI). The apoptosis index in the training group after exercise was much greater than that in the control group (P < 0.05), and it reached the peak at 24 h after exercise, then it reduced at 48 h after exercise. (2) The express of PCNA exhibited a sequential change after exercise, the proliferation index in the training group after exercise was greater than that in the control group (P < 0.05), it increased after exercise immediately, but it reduced at 3 h after exercise, then was reached the peak at 24 h after exercise, the proliferation index was moderately correlated with the apoptosis index (P < 0.05)., Conclusion: (1) Cell apoptosis can induce the delayed skeletal muscle damage. (2) Apoptosis may be a start factor of skeletal muscle regeneration.

YWK-II protein/APLP2 is a member of an evolutionarily conserved protein family that includes amyloid precursor protein (APP) and amyloid precursor-like protein-1 (APLP1). We have previously demonstrated that YWK-II/APLP2 functions as a novel G(0)-protein-coupled receptor for Müllerian inhibiting substance (MIS) in cell survival. However, factors regulating the stability and turnover of YWK-II/APLP2 have not been identified. Here we present evidence that human leukocyte antigen-B-associated transcript 3 (Bat3), an important regulator involved in apoptosis, can interact with YWK-II/APLP2 and enhance its stability by reducing its ubiquitylation and degradation by the ubiquitin-proteasome system. Coexpression of different Bat3 domain deletion constructs with YWK-II/APLP2 reveals that the proline-rich domain of Bat3 is required for its binding to YWK-II/APLP2. In addition, we find that the protein levels of YWK-II/APLP2 could be enhanced by nuclear export of Bat3 under apoptotic stimulation. We also find elevated levels of Bat3 and YWK-II/APLP2 in human colorectal cancer with a positive correlation between the two. Taken together, these results have revealed a previously undefined mechanism regulating cell apoptosis and suggest that aberrant enhancement of YWK-II/APLP2 by nuclear export of Bat3 may play a role in cancer development by inhibiting cell apoptosis.

Aim: To investigate the effects of ESC-3 isolated from crocodile bile on the growth and apoptosis induction of human cholangiocarcinoma cells., Methods: ESC-3 was isolated from crocodile bile by Sephadex LH-20 and RP-18 reversed-phase column. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay was conducted to determine the effects of ESC-3 on the proliferation of human cholangiocarcinoma cell lines (QBC939, Sk-ChA-1 and MZ-ChA-1). Giemsa staining, Hoechst 33258 and acridine orange/ethidium bromide staining showed the morphological changes of Mz-ChA-1 cells exposed to ESC-3 at different concentrations. Flow cytometry with regular propidium iodide (PI) staining was performed to analyze the cell cycle distribution of Mz-ChA-1 cells and to assess apoptosis by annexin v-fluorescein isothiocyanate (V-FITC)/PI staining. Rh123 staining was used to detect the alteration of mitochondrial membrane potential (ΔΨm). The protein levels of Bax, Bcl-2, Cdk2, cytochrome c and caspase-3 were further confirmed by Western blotting., Results: ESC-3 significantly inhibited the growth of three human cholangiocarcinoma cell lines and arrested Mz-ChA-1 cell cycle at G0/G1 phase. Mz-ChA-1 cells showed typical apoptotic morphological changes after treated with ESC-3 (10 μg/mL) for 48 h. Cell death assay indicated that Mz-ChA-1 cells underwent apoptosis in a dose-dependent manner induced by ESC-3. In addition, ESC-3 treatment could downregulate the protein level of Bcl-2 and upregulate the Bax, leading to the increase in the ratio of Bax to Bcl-2 in Mz-ChA-1 cells. Meanwhile, cytochrome c was released from the mitochondria into the cytosol, which subsequently initiated the activation of caspase-3. All these events were associated with the collapse of the mitochondrial membrane potential., Conclusion: ESC-3, the active ingredient of crocodile bile, induced apoptosis in Mz-ChA-1 cells through the mitochondria-dependent pathway and may be a potential chemotherapeutic drug for the treatment of cholangiocarcinoma.

Animal bile is popularly used as a traditional medicine in China, and bile acids are their major bioactive constituents. In the present study, effects of bile extract from crocodile gallbladder on QBC939 cell growth, cell cycle, and apoptosis were investigated by MTT assay, inverted microscopy, fluorescence microscopy, transmission electron microscopy, scanning electron microscopy, PI single- and FITC/PI double-staining flow cytometry, and western blotting. Our data have revealed that bile extract inhibited cells growth significantly, and the cell cycle was arrested in G1 phase. Bile extract induced QBC939 cell apoptosis, which was associated with collapse of the mitochondrial membrane potential and increase of ROS. In bile extract-treated cells, it was observed that the expression of bcl-2 decreased and cytochrome c released to cytosol, but the expression of bax remained unchanged. The data indicated that mitochondrial pathway might play an important role in bile extract-induced apoptosis in QBC939 cells. These results provide significant insight into the anticarcinogenic action of bile extract on cholangiocarcinoma cells.

The COPS3 gene has stimulating effect on cell proliferation and progression of osteosarcomas and related cells. However, the features of COPS3 and its potential application as a therapeutic target in other cancers has not yet been studied. In this study, therefore, the effect of COPS3 silencing via COPS3 siRNA on lung cancer cell proliferation was examined. Expression levels of COPS3 gene in COPS3 siRNA infected cells and control siRNA infected cells were compared with real time PCR and Western blot analysis. Cell proliferation levels were comprehensively analyzed by MTT, BrdU incorporationy, and colony formation assays. For mechanistic assessment the effects of COPS3 silencing on cell cycle and apoptosis were analyzed using flow cytometry. Results showed that successful silencing of the COPS3 gene at both translational and transcriptional levels significantly reduced the proliferation and colony formation by lung cancer cells (p<0.01). Flow cytometry showed cell cycle arrest in the G0/G1 phase after COPS3 silencing, and more importantly, apoptosis was induced as a result of COPS3 knockdown, which negatively affected cell survival. Therefore, these results provide another piece of important evidence that the COPS3 gene expressed in lung cancer cells may play a critical role in stimulating proliferation. Down-regulation of COPS3 could significantly inhibit lung cancer cell growth, which was most likely mediated via induction of cell cycle arrest in G0/G1 phase and apoptosis.

High-mobility group box 1 (HMGB1) is a multifunctional protein with intranuclear and extracellular functions. Although HMGB1 is overexpressed in approximately 85% of gastric cancers, the role of HMGB1 in gastric cancer biology remains unclear. In this study, we investigate the effect of downregulation of HMGB1 on the biological behavior of gastric cancer cells. MGC-803 gastric cancer cells were transduced with HMGB1-specific RNAi lentiviral vectors. Real-time polymerase chain reaction and Western blot analysis of HMGB1 mRNA and protein, respectively, validated the silencing effects. HMGB1-specific silencing significantly decreased cell proliferation. The impact on proliferation was observed at the cell cycle level--the number of cells in the G0/G1 phase increased, whereas that in S and G2/M phases decreased. Cell cycle changes were accompanied by decreases in cyclin D1 expression. Furthermore, HMGB1 silencing sensitized cells to apoptosis that was induced by oxaliplatin and mediated by the caspase-3 pathway. Finally, silencing of HMGB1 expression significantly reduced cellular metastatic ability and MMP-9 expression in MGC-803 cells. In summary, HMGB1 not only plays an essential role in the proliferation and invasion of MGC-803 cells but also represents a potential target for the therapeutic intervention of gastric cancer., (Copyright © 2011 John Wiley & Sons, Ltd.)

Vascular endothelial cell (VEC) apoptosis is the main event occurring during the development of atherosclerosis. Pterostilbene (PT), a natural dimethylated analog of resveratrol, has been the subject of intense research in cancer and inflammation. However, the protective effects of PT against oxidized low-density lipoprotein (oxLDL)-induced apoptosis in VECs have not been clarified. We investigated the anti-apoptotic effects of PT in vitro and in vivo in mice. PT at 0.1-5 μM possessed antioxidant properties comparable to that of trolox in a cell-free system. Exposure of human umbilical vein VECs (HUVECs) to oxLDL (200 μg/ml) induced cell shrinkage, chromatin condensation, nuclear fragmentation, and cell apoptosis, but PT protected against such injuries. In addition, PT injection strongly decreased the number of TUNEL-positive cells in the endothelium of atherosclerotic plaque from apoE(-/-) mice. OxLDL increased reactive oxygen species (ROS) levels, NF-κB activation, p53 accumulation, apoptotic protein levels and caspases-9 and -3 activities and decreased mitochondrial membrane potential (MMP) and cytochrome c release in HUVECs. These alterations were attenuated by pretreatment with PT. PT inhibited the expression of lectin-like oxLDL receptor-1 (LOX-1) expression in vitro and in vivo. Cotreatment with PT and siRNA of LOX-1 synergistically reduced oxLDL-induced apoptosis in HUVECs. Overexpression of LOX-1 attenuated the protection by PT and suppressed the effects of PT on oxLDL-induced oxidative stress. PT may protect HUVECs against oxLDL-induced apoptosis by downregulating LOX-1-mediated activation through a pathway involving oxidative stress, p53, mitochondria, cytochrome c and caspase protease. PT might be a potential natural anti-apoptotic agent for the treatment of atherosclerosis.

The study aimed to evaluate the effects of β-escin on human cholangiocarcinoma cell lines (QBC939, Sk-ChA-1 and MZ-ChA-1) and to explore its mechanisms. Cell growth, cell cycle and apoptosis were investigated, respectively, by MTT assay, single PI and FITC/PI double-staining flow cytometry, and fluorescence microscopy. The protein expression was determined by western blotting. The study revealed that β-escin inhibited cholangiocarcinoma cell growth in a dose- and time-dependent manner, and the cell cycle of QBC939 and Sk-ChA-1 cells was arrested in the G2/M phase, and MZ-ChA-1 cells in G1 phase. Apoptosis of the three cholangiocarcinoma cell lines induced by β-escin was associated with the collapse of the mitochondrial membrane potential and the activation of caspase-3. The apoptotic effect of β-escin was suppressed by pancaspase inhibitor z-VAD-fmk. Molecular dissection revealed that the antiapoptotic protein bcl-2 was down-regulated after cholangiocarcinoma cell lines were treated with β-escin, while the protein levels of bax and p53 were unchanged. Apoptosis was accompanied by an increase in reactive oxygen species (ROS). These results suggest that β-escin induces apoptosis of cholangiocarcinoma cells through an intrinsic mitochondrial caspase-dependent pathway, and the increase in the bax/bcl-2 ratio and ROS may play important roles in β-escin-induced apoptosis of cholangiocarcinoma cells., (Copyright © 2011 John Wiley & Sons, Ltd.)

Regulation of apoptosis at various stages of differentiation plays an important role in spermatogenesis. Therefore, the identification and characterisation of highly expressed genes in the testis that are involved in apoptosis is of great value to delineate the mechanism of spermatogenesis. Here, we reported that Fank1, a novel gene highly expressed in testis, functioned as an anti-apoptotic protein that activated the activator protein 1 (AP-1) pathway. We found that Jab1 (Jun activation domain-binding protein 1), a co-activator of AP-1, specifically interacted with Fank1. Reporter analyses showed that Fank1 activated AP-1 pathway in a Jab1-dependent manner. Fank1 overexpression also increased the expression and activation of endogenous c-Jun. Further study showed that Fank1 inhibited cell apoptosis by upregulating and activating endogenous c-Jun and its downstream target, Bcl-3. This process was shown to be Jab1 dependent. Taken together, our results indicated that by interacting with Jab1, Fank1 could suppress cell apoptosis by activating the AP-1-induced anti-apoptotic pathway.

Background: 5-Aminolevulinic acid (ALA) has been used as a photodynamic sensitizer for cancer treatment using photodynamic therapy. However, the light has markedly limited penetration depth. It was found that ALA also responds to low energy ultrasound, which has the capability to penetrate deep into tissues. Therefore, sonodynamic therapy (SDT) is a promising method for noninvasive treatment of tumors embedded deep in the tissue. It is desirable to kill the cancer cells via apoptosis rather than necrosis, and therefore, it is necessary to gain a better understanding of the mechanisms of treating cancer using SDT., Materials and Methods: The apoptosis of SAS cells induced by pulsed 1.05MHz ultrasound in combination with ALA was investigated in vitro., Results: The cells exposed to SDT with 10 μg/ml ALA displayed significantly higher apoptosis than cells treated by ultrasound alone. There was notably increased reactive oxygen species (ROS) production in the cells treated by SDT with ALA than by ultrasound alone, resulting in higher lipid peroxidation (LPO) level and more cells losing their mitochondrial membrane potential (MMP)., Conclusion: ALA-mediated SDT produced strong apoptotic effects on SAS cells, which were mainly related to the excessive intracellular ROS production followed by LPO increase and MMP decrease.

We investigated the antitumor effect of a short-chain polysaccharide (PS) extracted from porcine cartilage both in L1210 leukemic cells in vitro and in mice bearing L1210 ascites tumors. Our results show that treatment with PS resulted in a significant (P < 0.01) inhibition of cell proliferation and caused apoptotic death in L1210 cells. We also found that PS increased the ratio of Bax/Bcl-2 and activated caspase-9 and caspase-3 but not caspase-8. These results point to the activation of the mitochondrial apoptotic pathway in response to the treatment of L1210 cells with PS. Our results suggest that PS has the potential to provide significant natural defence against leukemias and lymphomas.

Extracellularly added recombinant granulysin was reported to kill mammalian target cells. The sites of actions and molecular mechanisms of granulysin in target cell killing, however, are presently unclear. In order to provide new insights into its potential mechanism of target cell damage, we here constructed recombinant plasmids carrying 9 kDa granulysin cDNA and examined effects of intracellularly expressed granulysin on the target hepatoma SMMC-7721 cells. The localization of intracellularly expressed granulysin was examined by fluorescence microscopy and confocal microscopy. Effects of granulysin on cell proliferation and apoptosis were measured by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromid) assay, flow cytometry and fluorescence microscopy, respectively. Changes of mitochondrial membrane potential were monitored by fluorescence microscopy. On the other hand, mitochondrial release of cytochrome c and apoptosis-inducing factor (AIF) was evaluated by Western blot and confocal microscopy. Intracellularly expressed granulysin was preferentially localized in cytoplasm, noticeably inhibited cell proliferation and induced cell death accompanied by reduced mitochondrial membrane potential, release of AIF and cytochrome c from mitochondria. Taken together, our findings demonstrate for the first time that localization and effect of intracellularly expressed granulysin on non-native cancer cells and indicate its potential utility in gene therapy for cancer.

The purpose of this study was to employ microarray analysis to evaluate differential gene expression in synovial tissue samples obtained from patients with rheumatoid arthritis (RA) or osteoarthritis (OA) to study the expression profile of apoptosis-associated genes in these tissues. Four samples were obtained from RA-affected patients and three from osteoarthritis patients. After total RNA was extracted from synovial tissue, the RNA was processed using two-cycle target labeling, followed by hybridization and scanning procedure. The GeneChip Human Genome U133 Plus 2.0 containing 900471 gene loci was used and eight genes associated with apoptosis were identified with a selected p value<0.05 and a twofold change in expression in rheumatoid samples compared to osteoarthritis tissues. Anti-apoptotic genes were generally upregulated whereas apoptotic genes were downregulated suggesting that these genes may play a role in the pathogenesis of RA. Furthermore, these genes may serve as novel therapeutic targets for the treatment of RA.

To study the function of mitochondrial PTEN in mediation of cellular apoptosis, the adenoviral recombinant of Mito-PTEN, which contains CoxVII (subunit VII of Cytochrome C Oxidase) gene in N-terminus, were generated. Using CoxV II-PTEN-EYFPN1 as a template, Cox VII-PTEN was cloned into the shuttle vector pAdTrack-CMV with the restriction endonuclease sites Xho I and Xba I . The shuttle plasmid was linearize with Pme I and co-transformed with adenoviral backbone vector pAdeasy-1 into E. coli BJ5183. Following selection and identification, the positive recombinant plasmids were transformed into E. coli Dalpha for propagation. To package the adenoviruses, recombinant plasmid candidate was linearize using Pac I and transfected into HEK-293A cells with Lipofectamine 2000. Through freeze-thaw-vortex cycles, recombinant viral particles were collected and harvested, and utilized to infect 293A cells for further amplification. The method of TCID50 was employed to determine virus titers. With green fluorescent protein (GFP) as marker, the efficiency of transfection and infection was monitored by fluorescence microscopy, and the apoptosis of A431 cells after infection of Mito-PTEN-Ad was analyzed by flow cytometry. Adenoviral recombinant of Mito-PTEN was packaged successfully with the TCID50 as 10 pfu/mL and the expressed protein was detected by western blot. In addition, it has been demonstrated that Mito-PTEN promoted apoptosis of A431 cells. Take together, the successful generation of adenoviral recombinant of Mito-PTEN, which could induce apoptosis in A431 cells, sets up a basis for further functional studies of mitochondrial PTEN and provides us a potential tool for cancer treatment in future.

Background: Classical transplantation immunosuppression relies heavily upon the interruption of interleukin-2 (IL-2) signaling by calcineurin inhibition. However, recent evidence in murine models suggests that IL-2 is necessary for activation-induced cell death (AICD) of allograft-specific lymphocytes., Methods: We examined the apoptotic effects of the calcineurin inhibitor cyclosporine A and mTOR inhibitor rapamycin on the apoptotic alterations that occur in allospecifically activated human lymphocytes in a one-way mixed lymphocyte culture (MLC)., Results: Cyclosporine increased caspase-3 activation in MLC, which corresponded with a decrease in lymphocyte apoptosis in MLC. Cyclosporine also reduced apoptosis in the CD4+ helper T cell subset, while CD8+ cells had similar or increased apoptosis when compared to controls. In contrast, rapamycin-treated cultures had normal levels of CD4+ T cell apoptosis when compared to control MLC, with decreases seen in CD8+ T lymphocytes., Conclusions: In humans, blockade of IL-2 receptor signal with rapamycin allows apoptosis of allospecifically activated CD4+ lymphocytes to occur, while blockade of IL-2 production with cyclosporine results in decreased apoptosis in this T cell subset. As helper T cells are integral to the immune response, these results may explain the tolerogenic effects of rapamycin.

Objective: To study the mechanism of cardiovascular disease affected by PM2.5., Methods: ECV304 cells were exposed to PM2.5 of different concentration (50, 200 and 400 microg/ml), after 24h, the viability of cells by MTT, SOD and GSH contents in cells and apoptosis of cells determined by flow cytometer were measured., Results: Viability of ECV304 cells declined and mortality of ECV304 cells increased gradually with increase of concentration, GSH contents in cells (mg/g prot) were 20.643 +/- 2.167, 16.774 +/- 2.911 (P < 0.05), 15.658 +/- 3.471 (P < 0.01), and SOD contents in cells (U/mg prot) were 5.878 +/- 0.401, 5.140 +/- 0.448 (P < 0.01), 4.817 +/- 0.451 (P < 0.01) when the concentration of PM2.5 was 50, 200 and 400 microg/ml., Conclusion: PM2.5 can cause vascular endothelial cells to die by way of oxidative injury, then induce cardiovascular disease.

Aim: To determine the changes of delayed rectifier K(+) currents (I(k)) and inward rectifier K(+) currents (I(k1)) in the ventricular myocytes of guinea pigs during the gradual apoptotic process by the chronic oxidant stress treatment., Methods: H(2)O(2) 50 micromol/L (24 h) was used for inducing apoptosis in the cardiomyocytes culture of neonatal rats and to treat the isolated ventricular myocytes of adult guinea pigs in vitro for 24 h. Apoptosis was evaluated by TUNEL methods and voltage-dependent K(+) currents were recorded by patch-clamp techniques., Results: H(2)O(2) 50 micromol/L (24 h) induced cell apoptosis in the cardiomyocytes culture of neonatal rats. This concentration was used to treat the isolated ventricular myocytes of adult guinea pigs in vitro for 24 h and the voltage-dependent K(+) currents densities (I(k), I(k1)) were down-regulated. The densities of the delayed rectifier K(+) currents (I(k)) in 50 micromol/L H(2)O(2) group were 2.52+/-0.57 pA/pF vs 5.73+/-1.84 pA/pF in the control group at +50 mV (n=8, P<0.01). The densities of the inward rectifier K(+) currents (I(k1)) in 50 micromol/L H(2)O(2) group were -13.9+/-2.70 pA/pF, 2.52+/-0.57 pA/pF vs -59.7+/-11.9 pA/pF, 5.73+/-1.84 pA/pF in the control group at -120 mV (n=8, P<0.01) and -40 mV (n=8, P<0.05), respectively. The extent of inward rectifier property of I(k1) was weakened by 50 micromol/L H(2)O(2) treatment., Conclusion: The densities of I(k), I(k1) in the cardiomyocytes of guinea pigs were down-regulated and the inward rectifier property of I(k1) was weakened during the gradual apoptotic process after 50 micromol/L H(2)O(2) treatment for 24 h.