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In recent years, there has been a growing focus on the toxicity and mortality induced by nanoplastics (NPs) in aquatic organisms. However, studies investigating mechanisms underlying oxidative stress (OS), apoptosis, and inflammation induced by NPs in fish remain limited. This study observed that polystyrene NPs (PS-NPs) were accumulated into zebrafish larvae and zebrafish embryonic fibroblast (ZF4 cells), accompanied by the occurrence of pathological damage both at the cellular and tissue-organ level. Additionally, the transcriptional up-regulation of NADPH oxidases (NOXs) and subsequent excessive generation of reactive oxygen species (ROS) resulted in notable changes in the relative mRNA and protein expression levels associated with antioxidant oxidase systems in larvae. Furthermore, the study identified the impact of NPs on mitochondrial ultrastructural, resulting in mitochondrial depolarization and downregulation of mRNA expression related to the electron transport chain due to excessive ROS generation. Short-term exposure to NPs also triggered apoptosis and inflammation in zebrafish larvae, evident from significant up-regulation in mRNA expressions of proapoptotic factors and NF-κB proinflammatory signaling pathway, as well as increased transcription and protein levels of pro-inflammatory factors in larvae. Inhibition of intracellular excessive ROS effectively reduced the induction of apoptosis, NF-κB P65 nuclear migration levels, and cytokine secretion, underscoring OS as a pivotal factor throughout the process of apoptosis and inflammatory responses induced by NPs. This research significantly advances our comprehension of biological effects and underlying mechanisms of NPs in freshwater fish., 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 B.V. All rights reserved.)

The aim of this study was to explore the role of NADH:ubiquinone oxidoreductase subunit B3 (NDUFB3) in human gynecological malignancies and to screen potential natural compounds targeting it. GEPIA and HPA databases were used to study the expression characteristics of NDUFB3. GO and KEGG enrichment analyses were performed using the R software clusterProfiler package. GSEA for NDUFB3 was performed using the LinkedOmics database. Natural compounds targeting NDUFB3 were screened by virtual screening and molecular docking. After NDUFB3 was depleted or wedelolactone treatment, cell proliferation was detected by CCK-8 assay. The production of reactive oxide species (ROS) in tumor cells was detected by dihydroethidium fluorescent probe. The cell cycle and apoptosis were evaluated by flow cytometry. It was revealed that NDUFB3 was highly expressed in ovarian cancer (OV), uterine corpus endometrial carcinoma (UCEC), and cervical squamous cell carcinoma (CESC). NDUFB3 expression was associated with multiple immunomodulators in CESC, OV, and UCEC. NDUFB3 was predicted to modulate MAPK signaling pathways in CESC, OV, and UCEC. Knocking down NDUFB3 inhibited the proliferation of CESC, OV, and UCEC cells, increased intracellular ROS production, and induced cell cycle arrest and apoptosis. Wedelolactone was a potential small molecule with a strong ability to bind with the active pocket of NDUFB3, and wedelolactone could kill CESC, OV, and UCEC cells partly via NDUFB3. In conclusion, NDUFB3 may be a potential biomarker and a new target for gynecological tumors, and wedelolactone may exert antitumor activity via targeting NDUFB3., (© 2024 John Wiley & Sons Ltd.)

Triple-negative breast cancer (TNBC) represents a significant health concern for women worldwide, and the overproduction of MMP9 and CD151 is associated with various cancers, influencing tumour growth and progression. This study aimed to investigate how CD151 and MMP9 affect TNBC cell migration, apoptosis, proliferation, and invasion. Immunohistochemical experiments revealed that CD151 and MMP9 were positively expressed in triple-negative breast cancer, and lymph node metastasis, the histological grade, and CD151 and MMP9 expression were found to be independent prognostic factors for the survival of patients with triple-negative breast cancer. Cytological experiments indicated that the knockdown of CD151 or MMP9 slowed triple-negative breast cancer cell growth, migration, and invasion and increased the apoptosis rate. Compared with CD151 knockdown, double MMP9 and CD151 knockdown further promoted cell death and inhibited TNBC cell proliferation, migration, and invasion. Moreover, β-catenin and p-GSK-3β were significantly downregulated. In summary, simultaneously silencing CD151 and MMP9 further suppressed the proliferation, migration and invasion of TNBC cells and promoted their apoptosis. One possible strategy for inducing this effect is to block the GSK-3β/β-catenin pathway., (© 2024. The Author(s).)

The pathogenesis and development of Moyamoya disease are still unclear. This study aimed to investigate the effect of desmoglein-2 (DSG2) on Moyamoya disease and determine the inhibitory effect of DSG2 in vascular remodeling in Moyamoya disease.RNA sequencing, immunohistochemistry (IHC), and western blotting were used to detect the expression of DSG2 in the superficial temporal artery (STA) tissues of Moyamoya disease. The association between DSG2 and endothelial cells' biological activities was investigated by cell counting kit-8 (CCK-8), migration assay, tube formation assay, flow cytometry with Annexin V-FITC/PI staining, and TUNEL apoptotic cell detection kit. Pathways affected by overexpression or knockdown of DSG2 were identified in endothelial cells.The expression of DSG2 in the STA tissues of Moyamoya disease was lower than that in normal controls. Overexpression of DSG2 inhibits the proliferation and migration but promotes apoptosis in endothelial cells, and low DSG2 levels result in impaired angiogenesis. In addition, there was an interaction between DSG2 and MMP-9, and DSG2 acted through the PI3K signaling in endothelial cells.Our results indicate that DSG2 affects PI3K signaling in vascular endothelial cells, and MMP-9 is involved in DSG2-mediated vascular changes in Moyamoya disease., (© 2024. The Author(s).)

This paper investigated the effect of total saponins from Rhizoma Panacis Majoris on the proliferation, apoptosis, and autophagy of human cervical carcinoma HeLa cells. The saponin content was detected by ultraviolet-visible spectrophotometry. Cell coun-ting kit-8(CCK-8) assay, 4,6-diamidino-2-phenylindole(DAPI) staining, and flow cytometry were used to detect the effects of total saponins of Panacis Majoris Rhizoma on cell viability, morphology, cell cycle and apoptosis of HeLa cells. Western blot was used to detect the expression of apoptosis-related proteins B cell lymphoma-2(Bcl-2), Bcl-2-associated X protein(Bax), cleaved caspase-9, and cleaved caspase-3, autophagy-related proteins Beclin-1 and SQSTM1(p62), and the proteins related to the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin(PI3K/Akt/mTOR) and mitogen-activated protein kinase(MAPK) signaling pathways. It was found that the yield and saponin content of total saponins from Rhizoma Panacis Majoris were 6.3% and 78.3%, respectively. Total saponins from Rhizoma Panacis Majoris could significantly inhibit the proliferation(P<0.001), effect the nuclear morphology, block the G_0/G_1 cycle, and induce cell apoptosis in HeLa cells with a concentration-dependent manner. In addition, total saponins from Rhizoma Panacis Majoris up-regulated the expression of pro-apoptotic proteins Bax, cleaved caspase-9, and cleaved caspase-3, and autophagy-related protein p62(P<0.05), while down-regulated the expression of anti-apoptotic protein Bcl-2 and autophagy-related protein Beclin-1(P<0.01). Total saponins from Rhizoma Panacis Majoris could promote the expression of p-p38/p38, p-Jun N-terminal kinase(JNK)/JNK, p-PI3K/PI3K, p-Akt/Akt, p-mTOR/mTOR proteins in PI3K/Akt/mTOR and MAPK signaling pathways(P<0.05). In contrast, the effect on p-ERK/ERK expression was not obvious. Therefore, total saponins from Rhizoma Panacis Majoris may inhibit autophagy and promote apoptosis of HeLa cells through the activation of the PI3K/Akt/mTOR, c-JNK, and p38 MAPK signaling pathways, which indicates that total saponins from Rhizoma Panacis Majoris may have a potential role in cervical cancer treatment.

Background: Chemo-photodynamic combination therapy has demonstrated significant potential in the treatment of cancer. Triptolide (TPL), a naturally derived anticancer agent, when combined with the photosensitizer Chlorin e6 (Ce6), has shown to provide enhanced anti-tumor benefits. However, the development of stimuli-responsive nanovehicles for the co-delivery of TPL and Ce6 could further enhance the efficacy of this combination therapy., Methods: In this study, we synthesized a pH/ROS dual-responsive mPEG- TK -PBAE copolymer, which contains a pH-sensitive PBAE moiety and a ROS-sensitive thioketal (TK) linkage. Through a self-assembly process, TPL and Ce6 were successfully co-loaded into mPEG- TK -PBAE nanoparticles, hereafter referred to as TPL/Ce6 NPs. We evaluated the pH- and ROS-sensitive drug release and particle size changes. Furthermore, we investigated both the in vitro suppression of cellular proliferation and induction of apoptosis in HepG2 cells, as well as the in vivo anti-tumor efficacy of TPL/Ce6 NPs in H22 xenograft nude mice., Results: The mPEG- TK -PBAE copolymer was synthesized through a one-pot Michael-addition reaction and successfully co-encapsulated both TPL and Ce6 by self-assembly. Upon exposure to acid pH values and high ROS levels, the payloads in TPL/Ce6 NPs were rapidly released. Notably, the abundant ROS generated by the released Ce6 under laser irradiation further accelerated the degradation of the nanosystem, thereby amplifying the tumor microenvironment-responsive drug release and enhancing anticancer efficacy. Consequently, TPL/Ce6 NPs significantly increased PDT-induced oxidative stress and augmented TPL-induced apoptosis in HepG2 cells, leading to synergistic anticancer effects in vitro. Moreover, administering TPL/Ce6 NPs (containing 0.3 mg/kg of TPL and 4 mg/kg of Ce6) seven times, accompanied by 650 nm laser irradiation, efficiently inhibited tumor growth in H22 tumor-bearing mice, while exhibiting lower systemic toxicity., Conclusion: Overall, we have developed a tumor microenvironment-responsive nanosystem for the co-delivery of TPL and Ce6, demonstrating amplified synergistic effects of chemo-photodynamic therapy (chemo-PDT) for hepatocellular carcinoma (HCC) treatment., Competing Interests: The authors report no conflicts of interest in this work., (© 2024 Huang et al.)

Mitochondrial stress and endoplasmic reticulum stress (ERS) are known to be closely linked. ATF5 is a key regulator of mitochondrial stress and is involved in ERS regulation. Previously, we used a seizure model to demonstrate that ATF5 regulates mitochondrial stress. However, whether ATF5 affects ERS in epilepsy models has yet to be elucidated. In the present study, we investigated the effects of ATF5 on low-magnesium-induced ERS and the potential mechanisms that underlie these effects. We found that lentiviral overexpression of ATF5 significantly improved low-magnesium-induced ERS, as confirmed by the reduced expression levels of GRP78, PERK, ATF4, and CHOP. In addition, ATF5 overexpression reduced reactive oxygen species (ROS) production and elevated superoxide dismutase (SOD) activity, thus demonstrating that ATF5 plays a key role in maintaining redox homeostasis. Furthermore, ATF5 overexpression rescued low-magnesium-induced neuronal apoptosis, as evidenced by the reduced expression levels of Cleaved-caspase-3 and Bax, and the restored levels of Bcl2. However, these effects were significantly eliminated by lentiviral transduction with ATF5 interference. In addition, treatment of neurons with the mitochondrial antioxidant mitoquinone attenuated the onset of oxidative stress caused by ATF5 interference, partially restored the effect on ERS, and rescued cells from apoptosis. Collectively, these data show that ATF5 attenuates low-magnesium-induced neuronal apoptosis by inhibiting ERS through preventing the accumulation of mitochondrial ROS., (Copyright © 2023 IBRO. Published by Elsevier Ltd. All rights reserved.)

Background: Rheumatoid arthritis (RA) is a multifactorial systemic autoimmune disease characterized by ongoing synovial inflammation, leading to the degradation of cartilage. Cuproptosis, as a newly characterized form of cell death, may influence RA progression by regulating immune cells and chondrocytes. This study sets out to identify the hub cuproptosis-related gene (CRG) associated with the pathogenesis of RA., Methods: A series of bioinformatic analyses were performed to evaluate the expression score of CRGs and the immune infiltration landscape between RA and normal samples. The hub gene was screened through the correlation analysis of CRGs, and the interaction network between the hub gene and transcription factors (TFs) was constructed. Finally, the hub gene was validated through quantitative real-time polymerase chain reaction (qRT-PCR) of patient samples and cell experiments., Results: Drolipoamide S-acetyltransferase (DLAT) was screened as the hub gene. Correlation analysis between the hub gene and immune microenvironment demonstrated that DLAT had the highest correlation with T follicular helper cells. Eight pairs of DLAT-TF interaction networks were constructed. Single-cell sequencing showed that CRGs were highly expressed in RA chondrocytes, and chondrocytes could be classified into three different subsets. qRT-PCR was used to validate the above results. Dlat knockdown in immortalized human chondrocytes led to significantly improved mitochondrial membrane potentials and reduced levels of intracellular reactive oxygen species (ROS), mitochondrial ROS and apoptosis., Conclusions: This study rudimentarily demonstrates the correlation between CRGs and immune cell infiltration in RA. The biomarker DLAT may provide comprehensive insights into the pathogenesis and drug targets of RA., (© 2023 John Wiley & Sons Ltd.)

Mitochondrial-associated endoplasmic reticulum (ER) membranes (MAMs) regulate calcium (Ca 2+ ) homeostasis via Ca 2+ transport-related proteins such as inositol-1,4,5-triphosphate receptor (IP3R). FAM134B-mediated ER-phagy plays an important role in ER homeostasis. However, it remains unknown whether FAM134B-mediated ER-phagy affects mitochondrial Ca 2+ homeostasis and cell death through MAMs. In this study, we demonstrated that colocalization degree of FAM134B with LC3 and the LC3-II/LC3-I ratio were elevated in the hippocampal neuronal culture (HNC) model of acquired epilepsy (AE), which indicate an increased level of autophagy. In this model, FAM134B overexpression enhanced ER-phagy, while FAM134B downregulation had the opposite effect. Additionally, FAM134B overexpression significantly reversed the increases in IP3R expression and mitochondrial Ca 2+ concentration and the decrease in the ER Ca 2+ concentration in this model. FAM134B overexpression also ameliorated the AE-induced ultrastructural damage in neuronal mitochondria, decrease in mitochondrial membrane potential (mMP), cytochrome c (CytC) release and caspase-3 activation, while FAM134B downregulation induced the opposite effects. Altogether, our data indicate that FAM134B-mediated ER-phagy can attenuate AE-induced neuronal apoptosis, possibly by modulating the IP3R in MAMs to alter Ca 2+ exchange between ER and mitochondria and thus inhibit mitochondrial structural damage, a decrease in mMP, release of CytC and mitochondrial apoptosis., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Endoplasmic reticulum stress (ER stress) plays a critical role in neuronal apoptosis along with the aggravation of Alzheimer's disease (AD). Nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) is a ligand-activated transcription factor that is involved in regulating ER stress in Alzheimer's disease (AD), therefore, this protein could be a promising therapeutic target for AD. Vanadium compounds, such as vanadyl acetylacetonate, sodium metavanadate and bis(maltolato)oxovanadium, are well-known as puissant PPARγ modulators. Thus, we are curious whether bis(ethylmaltolato)oxidovanadium (IV) (BEOV) can ameliorate ER stress and subsequent neuronal apoptosis by regulating PPARγ in AD models. To this end, we determined the effect of BEOV on behavioral performance, ER stress and neuronal apoptosis in the triple transgenic mouse AD model (3×Tg-AD). Our results showed that BEOV improved cognitive abilities and reduced the ER stress- and apoptosis-associated proteins in the brains of 3×Tg-AD mice. In vitro administration of BEOV in primary hippocampal neurons and N2asw cells achieved similar results in repressing ER stress. In addition, cotreatment with GW9662 (an antagonist of PPARγ) effectively blocked these neuroprotective effects of BEOV, which provided strong evidence that PPARγ-dependent signaling plays a key role in protecting against ER stress and neuronal apoptosis in AD. In conclusion, our data demonstrated that BEOV alleviated neuronal apoptosis triggered by ER stress by regulating PPARγ in a 3×Tg-AD model., (© 2021. Society for Biological Inorganic Chemistry (SBIC).)

Aims: Deep burn-wounds undergo a dynamic progression in the initial or periburn area after insults, and the zone of stasis is the crucial region suffering the deterioration, considered as salvageable. Few studies explored the role of mitochondria in this process. This study is to clarify a possible "built-in" protection of mitophagy., Main Methods: A classic "comb" scald rat model was established. Histological and blood-flow observation were processed based on hematoxylin-eosin staining and laser analysis. Oxidative and apoptotic status were analyzed by commercial kits. Transmission-electron microscope, immunofluorescence staining, and western blot were applied to detect the mitophagy in the zone of stasis and potential regulators. Adenovirus-based gene-silence contributed to determine the role of HIF-1α as a regulatory mediator., Key Findings: We found that burn-caused typical ischemia and histological deterioration in the zone of stasis, in parallel with increases in oxidative stress and apoptosis. Mitochondrial damage was involved in the aforementioned changes. Furthermore, we detected mitophagy in burn-wounds, which was contradictory to the burn-wound conversion. HIF-1α expression was closely related to the level of mitophagy, while BNIP3 and PARKIN are involved downstream., Significance: We demonstrate that burn-induced mitochondrial impairment contributes to the mobilization of injurious mechanisms in the zone of stasis and that mitophagy provides a beneficial way to protect against burn-wound progression via the elimination of damaged mitochondria. Our findings offer insights into mitochondrial quality control in burn-wound progression and suggest the novel concept that HIF-1α may be a therapeutic target due to its possible regulation on BNIP3- or PARKIN-mediated mitophagy., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Neuronal apoptosis caused by amyloid-beta (Aβ) overproduction is one of the most important pathological features in Alzheimer's disease (AD). Endoplasmic reticulum (ER) stress induced by Aβ overload plays a critical role in this process. Bis(ethylmaltolato)oxidovanadium (IV) (BEOV), a vanadium compound which had been regarded as peroxisome proliferator-activated receptor γ (PPARγ) agonist, was reported to exert an antagonistic effect on ER stress. In this study, we tested whether BEOV could ameliorate the Aβ-induced neuronal apoptosis by inhibiting ER stress. It was observed that BEOV treatment ameliorated both tunicamycin-induced and/or Aβ-induced ER stress and neurotoxicity in a dose-dependent manner through downgrading ER stress-associated and apoptosis-associated proteins in primary hippocampal neurons. Consistent with in vitro results, BEOV also reduced ER stress and inhibited neuronal apoptosis in hippocampi and cortexes of transgenic AD model mice. Moreover, by adopting GW9662 and salubrinal, the inhibitor of PPARγ and hyperphosphorylated eukaryotic translation initiation factor 2α, respectively, we further confirmed that BEOV alleviated Aβ-induced ER stress and neuronal apoptosis in primary hippocampal neurons by activating PPARγ. Taken together, these results provided scientific evidences to support the concept that BEOV ameliorates Aβ-induced ER stress and neuronal apoptosis through activating PPARγ., Competing Interests: Declaration of competing interest All authors declare that they have no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Literatures indicate that microRNA-129-5p (miR-129-5p) or Fas-associated death domain (FADD) is related to intervertebral disc degeneration (IDD), but the effect of miR-129-5p/FADD axis on IDD is not studied. The study aimed to investigate whether miR-129-5p influenced immune privilege and nucleus pulposus (NP) cell apoptosis in rats with IDD via regulating FADD.A rat model with caudal IDD was established, and injected with miR-129-5p agomir or miR-129-5p antagomir to figure out the character of miR-129-5p in the cell apoptosis and inflammation in the nucleus pulposus (NP) tissues of IDD rats. NP cells were grouped as the same ways for determining proliferation, apoptosis, and senescence in NP cells of IDD rats. Annexin V-FITC/PI double staining detected the apoptosis of macrophages and CD8 + cells co-cultured via transfected NP cells. Expression of miR-129-5p, FADD, collagen I, collagen II, aggrecan and Sox-9 in NP tissues and cells were determined.Up-regulated miR-129-5p decreased FADD, collagen I and elevated collagen Ⅱ, aggrecan, and Sox-9 in NP tissues and repressed inflammation in serum and NP tissues in IDD rats. Up-regulated miR-129-5p facilitated proliferation, inhibited senescence, apoptosis, and decreased FADD, collagen I and increased collagen Ⅱ, aggrecan, and Sox-9 in NP cells of IDD rats. Elevated miR-129-5p promoted the apoptosis of macrophages and CD8 + cells.We pronounced that up-regulated miR-129-5p inhibited the apoptosis and facilitated the proliferation of NP cells, as well as the apoptosis of macrophages and CD8 + cells via decreased FADD in IDD, suggesting that miR-129-5p had a protective effect on IDD.

Objective: To investigate the regulatory mechanism of miR-204 on proliferation, apoptosis and autophagy of cervical cancer cells. Methods: The expression of miR-204 in cervical cancer tissues and cell lines was detected by real-time PCR. Cell viability and apoptosis were detected by MTT and flow cytometry, respectively. Protein expression of Bcl-2, Bax, Caspase-3 and LC3I/II in C33A cells after transfection was detected by Western blot assay. The interaction between miR-204 and ATF2 was verified by Targetscan online prediction, dual-luciferase assay and Western blot. Results: The expression of miR-204 was significantly down-regulated in cervical cancer tissues and cell lines ( p  < .05). Cell viability was suppressed while apoptosis was enhanced in C33A cells transfected with miR-204. In addition, overexpression of miR-204 reduced protein expression of Bcl-2 and LC3I/II and elevated protein expression of Bax and Caspase-3 in C33A cells ( p  < .05). The results of Targetscan online prediction, dual-luciferase assay and Western blot indicted that miR-204 could regulate the expression of ATF2. Cell viability was increased ( p  < .05) and the expression of ATF2 and LC3I/II was down-regulated ( p  < .05) in C33A cells after silencing of ATF2. Ectopic expression of ATF2 could restore miR-204 mediated inhibition on proliferation of C33A cells. Conclusions: miR-204 could inhibit proliferation and autophagy and induce apoptosis of cervical cancer cells by targeting ATF2, representing promising target for cervical cancer treatment.

Aconitine (AC) and mesaconitine (MA) are major bioactive diterpenoid alkaloids derived from herbal aconitum plants. Emerging evidence indicates that AC plays a pivotal role in the cardiotoxicity for aconite poisoning. However, the cardiotoxicity data of MA, especially those on the difference between AC and MA are quite limited. Zebrafish embryos were used in this study for toxicological screening, and the cardiac morphology and function were observed. Embryos were analyzed by means of high-performance liquid chromatography (HPLC) after exposure and pharmacokinetic behaviors were also investigated. Results showed that 1.5% of the aconitum alkaloids penetrated into the zebrafish embryos. 2.5 μg/L AC and 20 μg/L MA caused a deficient cardiovascular system with yolk sac hemorrhage and early cardiac dysfunctions were observed in 96 h post-fertilization. AC showed greater cardiotoxicity than MA by comparing the EC 50 of pericardium edema. Aconitum alkaloids exposure also resulted in a significant decrease in the expression of cardiac genes (Tbx5, Gata4, and Nkx2.5) from an early stage (12-24 hpf), which may partly explained that the death caused by aconitum is most likely to occur within the first 24 h. In addition, a high percentage of apoptotic cells was observed in the brain region, which identified another potential target of the DDA action in zebrafish embryos., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Diabetic nephropathy results from hyperglycemia‑mediated renal glomerular cell death via mitochondrial apoptosis. There is an emerging requirement for novel approaches with mitochondrial protective effects that alleviate the hyperglycemia‑induced loss of functional cells during diabetic renal damage. Liraglutide, a type of glucagon‑like peptide‑1 agonist, has been suggested to inhibit the progression of obesity and hyperglycemia. However, the contributions and mechanism of action of liraglutide on hyperglycemia‑mediated cell mitochondrial apoptosis in diabetic kidneys have not been illustrated. The present study demonstrated that liraglutide may protect human renal mesangial cells (HRMCs) against hyperglycemia‑induced cell death by inhibiting mitochondrial apoptosis. Liraglutide administration also maintained HRMC viability and promoted HRMC proliferation within a high glucose stress environment. Functional studies demonstrated that hyperglycemia triggered mitochondrial dysfunction, including mitochondrial potential reduction, mitochondrial permeability transition pore opening, reactive oxygen species overproduction and the activation of the mitochondrial apoptotic pathway. However, liraglutide treatment preserved mitochondrial function and prevented activation of mitochondrial apoptosis by upregulating sirtuin 3 (Sirt3) expression. Deletion of Sirt3 abrogated the protective effects of liraglutide on mitochondrial homeostasis following high glucose challenge. In addition, molecular analysis confirmed that liraglutide upregulated Sirt3 via activating the extracellular signal‑regulated kinase‑Yes‑associated protein (ERK‑Yap) signaling pathway. Inhibition of the ERK‑Yap axis negated the action of liraglutide on Sirt3 activation, leading to mitochondrial injury and HRMC apoptosis. Taken together, the present study illustrated that liraglutide protected renal mesangial cells from hyperglycemia‑mediated mitochondrial apoptosis by upregulating Sirt3 expression and activation of the ERK‑Yap signaling pathway.

Bacterial quorum-sensing autoinducers are small chemicals released to control microbial community behaviours. N-(3-oxo-dodecanoyl) homoserine lactone, the autoinducer of the Pseudomonas aeruginosa LasI-LasR circuitry, triggers significant cell death in lymphocytes. We found that this molecule is incorporated into the mammalian plasma membrane and induces dissolution of eukaryotic lipid domains. This event expels tumour necrosis factor receptor 1 into the disordered lipid phase for its spontaneous trimerization without its ligand and drives caspase 3-caspase 8-mediated apoptosis. In vivo, P. aeruginosa releases N-(3-oxo-dodecanoyl) homoserine lactone to suppress host immunity for its own better survival; conversely, blockage of caspases strongly reduces the severity of the infection. This work reveals an unknown communication method between microorganisms and the mammalian host and suggests interventions of bacterial infections by intercepting quorum-sensing signalling.

Objectives: To explore the potential therapeutic effect of Tanshinone IIA against ovarian cancer in vitro and elucidate the underlying molecular mechanism., Methods: The cell survival upon Tanshinone IIA treatment was determined by the clonogenic assay. Cell apoptosis was analysed by Annexin V/propidium iodide double staining. The cleaved caspase-3/poly ADP-ribose polymerase and apoptosis-related factors were quantified by Western blotting. The relative expression of microRNAs (miRs) was determined by real-time polymerase chain reaction., Key Findings: Tanshinone IIA treatment induced significant apoptosis in TOV-21G cells. Tanshinone suppressed survivin expression while not affected Bax, Bcl-2 and Bcl-xL. We further predicted and experimentally confirmed overexpression of miR-205 in TOV-21G, which ectopic significantly inhibited survivin and promoted cell apoptosis. miR-205-specific antagonist completely abrogated the cell suppressive effect of Tanshinone IIA., Conclusions: Our data suggested that Tanshinone IIA induced cell apoptosis in ovarian carcinoma TOV-21G cells via direct upregulation of miR-205. Our study highlighted the potential therapeutic application of Tanshinone IIA against ovarian malignancy., (© 2018 Royal Pharmaceutical Society.)

When orthopedic joints coated by hydroxyapatite (HA) were implanted in the human body, they release wear debris into the surrounding tissues. The generation and accumulation of wear particles will induce aseptic loosening. However, the potential bioeffect and mechanism of HA-coated orthopedic implants on bone cells are poorly understood. In this study, defect-related luminescent bur-like hydroxyapatite (BHA) microspheres with the average diameter of 7-9 μm which are comparable to that of the wear-debris particles from aseptically loosened HA implants or HA debris have been synthesized by hydrothermal synthesis and the MC3T3-E1 cells were set as a cells model to study the potential bioeffect and mechanism of BHA microspheres. The studies demonstrated that BHA microspheres could be taken into MC3T3-E1 cells via endocytosis involved in micropinocytosis- and clathrin-mediated endocytosis process, and exert cytotoxicity effect. BHA microspheres could induce the cell apoptosis by intracellular production of reactive oxygen species (ROS), which led to not only an increase in the permeability of lysosome and release of cathepsins B, but also mitochondrial dysfunction and DNA damage. Our results provide novel evidence to elucidate their toxicity mechanisms and might be helpful for more reasonable applications of HA-based orthopaedic implants in the future.

Myeloid-derived suppressor cells (MDSCs) are thought to play a critical immunosuppressive role in tumorigenesis. In this project, we aimed to investigate subset alteration of MDSCs in gastric cancer (GC), and the effects of epirubicin (EPI) and paclitaxel (TAX) on MDSCs. The frequencies of MDSC subsets in peripheral blood were observed by using flow cytometry after treatment with EPI- or TAX- based chemotherapy in GC patients. After treatment with EPI or TAX in vitro, the subsets, apoptosis, cell cycle, and MAPK and NF-κB protein expressions of mouse bone marrow MDSCs were analyzed. The frequency of MDSCs in the peripheral blood of GC patients was higher than that in healthy controls. Granulocyte-type MDSCs (G-MDSCs) were significantly more than monocyte-type MDSCs (M-MDSCs) in GC patients. The frequencies of MDSC subsets in the peripheral blood decreased after EPI- or TAX-based chemotherapy. High levels of MDSC subsets were correlated with low cancer differentiation degree. High level of M-MDSCs was related to lymph node metastasis, and was negatively correlated with the overall survival of GC patients. After treatment with EPI or TAX, levels of mouse bone marrow MDSC subsets decreased significantly in vitro. Arg-1 secretion and expression of total and phosphorylated MAPK and NF-κB by MDSCs decreased. EPI or TAX decreases the levels of MDSCs, inhibits the proliferation and function of MDSCs in vitro, and induces their apoptosis via the MAPK and NF-κB signaling pathways.

The cytotoxicities of three aconitum alkaloids- aconitine, hypaconitine and mesaconitine, and their abilities to bind DNA have been explored. Rat myocardial cells H9c2 were treated with aconitum alkaloids and assessed the cytotoxicities by using MTT assay and flow cytometry. Apoptosis was evidenced by the results of the annexin V/propidium iodide (PI) assay. Aconitine was found to be the most toxic in rat myocardial cells H9c2 in three aconitum alkaloids. At the same time, DNA adducts were isolated and then analyzed by UV-Vis spectroscopy after exposure to alkaloids, which indicated that three alkaloids could bind to DNA in rat myocardial cells H9c2. Furthermore, their binding modes were investigated by UV-Visible, fluorescence, DNA melting studies and ionic strength effect. Results indicated that the interaction between three alkaloids and DNA were intercalation coupled with electrostatic effect. The estimated binding constants were between 4.83 × 10 5 M -1 to 9.85 × 10 5 M -1 for three alkaloids at 298 K.

Highly efficient target therapy is urgently needed for prostate cancer with overexpression of γ-seminoprotein (γ-SM). Recent studies indicated that mesenchymal stem cells (MSCs) are attractive candidate for cell-based, targeted therapy due to their tumor tropism. Here we designed a dual-target therapeutic system in which MSCs were engineered to produce and deliver scFv-Fdt-tBid, a novel γ-SM-targeted immunoproapoptotic molecule. Such engineered MSCs (MSC.scFv-Fdt-tBid) would home to tumor sites and release the fusion protein to induce the apoptosis of prostate cancer cells. Our data demonstrated that scFv-Fdt-tBid showed a selective, potent and dose-dependent inhibition for γ-SM-positive cells (LNCaP, C4-2, 22Rv1) rather than γ-SM-negative cells and MSCs. Importantly, MSC.scFv-Fdt-tBid caused cell death through an apoptosis-dependent manner. Further, the tropism of MSC.scFv-Fdt-tBid to prostate cancer was verified both in vitro and in vivo. Finally, the in vivo experiments demonstrated that MSC.scFv-Fdt-tBid significantly inhibited γ-SM-positive tumor growth without toxic side effects. Collectively, this study represented a novel immunoproapoptotic molecule scFv-Fdt-tBid for γ-SM-positive tumors and demonstrated the therapeutic efficiency and safety of scFv-Fdt-tBid-modified MSCs against prostate cancers., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Despite accumulating evidence that long noncoding RNAs (lncRNAs) are associated with cancer development in multiple types of cancer, the biological roles of many lncRNAs in human hepatocellular carcinoma (HCC) metastasis have not been well characterized., Methods: A lncRNA+ mRNA human gene expression microarray analysis was used to identify differentially expressed lncRNAs in metastatic HCC tissues compared to non-metastatic tissue., Results: We observed remarkable overexpression of HOXD-AS1 in metastatic cancer tissues. In vitro and in vivo gain- or loss-of-function studies re-affirmed that HOXD-AS1 is able to facilitate cancer metastasis and inhibit apoptosis. Moreover, we identified that HOXD-AS1 upregulated the Rho GTPase activating protein 11A (ARHGAP11A) by competitively binding to microRNA-19a (miR19a), resulting in induced metastasis. Interestingly, the regulator of G-protein signaling 3 (RGS3), a potential inhibitor of the MEK-ERK1/2 signaling axis, was also found to be downregulated by ectopic HOXD-AS1 overexpression, leading to a remarkably reduced apoptotic effect., Conclusions: The present investigation strongly indicates that HOXD-AS1 is an oncogenic lncRNA that promotes HCC metastasis and that its pro-metastatic phenotype can partially be attributed to the HOXD-AS1/miR19a/ARHGAP11A signaling axis.

Autolysis easily happens to sea cucumber (Stichopus japonicus, S. japonicus) for external stimulus like UV exposure causing heavy economic losses. Therefore, it is meaningful to reveal the mechanism of S. japonicas autolysis. In the present study, to examine the involvement of apoptosis induction in UVA-induced autolysis of S. japonicas, we investigated the biochemical events including the DNA fragmentation, caspase-3 activation, mitogen-activated protein kinases (MAPKs) phosphorylation and free radical formation. Substantial morphological changes such as intestine vomiting and dermatolysis were observed in S. japonicus during the incubation after 1-h UVA irradiation (10W/m(2)). The degradation of the structural proteins and enhancement of cathepsin L activity were also detected, suggesting the profound impact of proteolysis caused by the UVA irradiation even for 1h. Furthermore, the DNA fragmentation and specific activity of caspase-3 was increased up to 12h after UVA irradiation. The levels of phosphorylated p38 mitogen activated protein kinase (MAPK) and phosphorylated c-Jun.-N-terminal kinase (JNK) were significantly increased by the UVA irradiation for 1h. An electron spin resonance (ESR) analysis revealed that UVA enhanced the free radical formation in S. japonicas, even through we could not identify the attributed species. These results suggest that UVA-induced autolysis in S. japonicas at least partially involves the oxidative stress-sensitive apoptosis induction pathway. These data present a novel insight into the mechanisms of sea cucumber autolysis induced by external stress., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Three water-soluble ruthenium(II) complexes with chiral 4-(2,3-dihydroxypropyl)-formamide oxoaporphine (FOA) were synthesized and characterized. It was found that these ruthenium(II) complexes exhibited considerable in vitro anticancer activities and that they were the effective stabilizers of telomeric and G-quadruplex-DNA (G4-DNA) in promoter of c-myc, which acted as a telomerase inhibitor targeting G4-DNA and induced cell senescence and apoptosis. Interestingly, the in vitro anticancer activity of 6 (LC-003) was higher than those of 4 (LC-001) and 5 (LC-002), more selective for BEL-7404 cells than for normal HL-7702 cells, and preferred to activate caspases-3/9. The different biological behaviors of the ruthenium complexes could be correlated with the chiral nature of 4-(2,3-dihydroxypropyl)-formamide oxoaporphine. More significantly, 6 exhibited effective inhibitory on tumor growth in BEL-7402 xenograft mouse model and higher in vivo safety than cisplatin. These mechanistic insights indicate that 6 displays low toxicity and can be a novel anticancer drug candidate.

Objective: To investigate the effects of Artesunate(Art) on the LX-2 cell., Methods: The cultured hepatic stellate cells were divided into control group and Art-treated groups with 250,350,450 µmol/L. The rate of cellular proliferation was detected by MIT assay, the content of ceramide (Cer)was determined by HPLC method, the content of hydroxyproline (Hyp) was determined by enzyme digestion method, the expressions of PPAR-γ, p53 and Caspase 3 were detected by Western blot., Results: Compared with control group, IX-2 treated with Art were inhibited in a concentration-dependent manner(P < 0.01). Art could significantly increase the content of cerarnide in LX-2 ( P <0.01), and the content of Hyp was significantly decreased (P <0.05, P <0.01). The expressions of PPAR-γ, p53 and Caspase 3 were increased compared with that of control group(P < 0.01)., Conclusion: Artesunate could inhibit the proliferation and induce apoptosis of hepatic stellate cells through upregulating ceramide.

Angiogenic factor with G patch and FHA domains 1 (AGGF1) is a newly identified proangiogenic protein, which plays an important role in vascular disease and angiogenesis. However, its role in myocardial ischemia/reperfusion (I/R) injury remains unknown. This study investigated whether AGGF1 is involved in the pathogenesis of mouse myocardial I/R injury and the underlying mechanisms. Wild-type (WT) C57BL/6 J mice were treated at 30 min prior to I/R injury with anti-AGGF1 neutralizing antibody (3 mg/kg) or recombinant human AGGF1 (rhAGGF1, 0.25 mg/kg). After I/R injury, the infarct size, the number of TUNEL-positive cardiomyocytes, Bax/Bcl2 ratio, inflammatory cytokine expression and angiogenesis were markedly increased as compared with sham control. Treatment of WT mice with anti-AGGF1 neutralizing antibody resulted in exaggeration of myocardial I/R injury but reducing angiogenesis. In contrast, administration of rhAGGF1 markedly reversed these effects. Furthermore, anti-AGGF1- or rhAGGF1-mediated effects on I/R-induced cardiac apoptosis, inflammation and angiogenesis were dose dependent. In addition, the protective effects of AGGF1 on cardiomyocyte apoptosis and inflammation were confirmed in cultured cardiomyocytes after I/R. Finally, these effects were associated with activation of ERK1/2, Stat3 and HIF-1α/VEGF pathways and inhibition of activation of NF-κB, p53 and JNK1/2 pathways. In conclusion, we report the first in vivo and in vitro evidence that AGGF1 reduces myocardial apoptosis and inflammation and enhances angiogenesis, leading to decreased infarct size after I/R injury. These results may provide a novel therapeutic approach for ischemic heart diseases.

Atherosclerosis is considered a disease of chronic inflammation largely initiated and perpetuated by macrophage-dependent synthesis and release of pro-inflammatory mediators. Class A scavenger receptor (SR-A) expressed on macrophages plays a key role in this process. However, how SR-A-mediated pro-inflammatory response is modulated in macrophages remains ill defined. Here through immunoprecipitation coupled with mass spectrometry, we reported major vault protein (MVP) as a novel binding partner for SR-A. The interaction between SR-A and MVP was confirmed by immunofluorescence staining and chemical cross-linking assay. Treatment of macrophages with fucoidan, a SR-A ligand, led to a marked increase in TNF-α production, which was attenuated by MVP depletion. Further analysis revealed that SR-A stimulated TNF-α synthesis in macrophages via the caveolin- instead of clathrin-mediated endocytic pathway linked to p38 and JNK, but not ERK, signaling pathways. Importantly, fucoidan invoked an enrichment of MVP in lipid raft, a caveolin-reliant membrane structure, and enhanced the interaction among SR-A, caveolin, and MVP. Finally, we demonstrated that MVP elimination ameliorated SR-A-mediated apoptosis in macrophages. As such, MVP may fine-tune SR-A activity in macrophages which contributes to the development of atherosclerosis.

Aim: To observe and verify the benzene-induced cell cycle arrest and apoptosis in human peripheral blood mononuclear cells (PBMCs), and explore its effects on oxidative DNA damage., Methods: PBMCs were isolated, cultivated for 24 h and then divided into 4 groups supplemented with alcohol solvent as a control, low, middle and high concentration benzene (0.5, 5, 50 μmol/L), respectively. Another 24 h later, we assayed the cell growth arrest by MTT, detected cell cycle and apoptosis rate by flow cytometry, the content of reactive oxygen species (ROS) by DCFH-DA assay, superoxide dismutase (SOD) activity by xanthine oxidase test, malondialdelhde (MDA) content by thiobarbituric acid test, glutathione (GSH) content by ELISA, DNA damage by single cell gel electrophoresis (SCGE) technology and micronucleus assay., Results: Compared with the control group, benzene decreased cell viability (P<0.05) and increased cell apoptosis (P<0.05) in a dose-dependent manner, along with induction of S phase and G2/M phase arrest (P<0.05). Meanwhile, benzene induced the accumulation of intracellular ROS and MDA, micronucleus rates, comet rates and comet tail length, which were found dose-dependent and statistically significant compared to the solvent control (P<0.05). The activity of SOD and the contents of GSH decreased significantly (P<0.05)., Conclusion: Benzene can induce cell apoptosis, S+G2/M phase accumulation and change of oxidoreduction status in PBMCs, and strengthen the effects of lipid peroxidation as well as the DNA damage.

The morphological changes of ricin-induced apoptosis in a human cervical cancer cell line were studied. To shed light on the mechanism of action of ricin toxin (RT) at the cellular level, we examined cell growth, apoptosis, changes of mitochondrial membrane potential (MMP) and cytochrome C translocation in HeLa cells by exposing these cells to RT for indicated times. The effect of RT on cell proliferation was measured by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS), inner salt; MTS assay and apoptosis were measured using flow cytometry, fluorescence microscopy and electron microscopy. Changes in MMP were monitored using flow cytometry. Western blot analysis was used to evaluate the release of mitochondrial cytochrome C. RT noticeably inhibited the proliferation of HeLa cells, and the half maximal inhibitory concentration dose was about 100 ng/ml. HeLa cells treated with RT showed typical characteristics of apoptosis rather than necrosis, including phosphatidylserine exposed from the inner to the outer leaflet of the plasma membrane, abnormal cell morphology, chromatin condensation and nuclear fragmentation. In contrast, during the process of cellular apoptosis, the messenger RNA (mRNA) and protein expression of cytochrome C in treated and untreated Hela cells were not significantly changed (data not shown). However, when cells were treated with RT, the massive translocation of cytochrome C to the nucleus was evident. Our results indicate that RT-induced HeLa cell apoptosis, especially for cytochrome C translocation, may play an important role in apoptosis induced by RT.

Ghrelin, a gastric hormone, exerts cardioprotective function by increasing myocardial contractility and vasodilation. Previous studies have reported that angiotensin II (Ang II) production increased in heart failure, which can induce cardiomyocyte apoptosis. In this study, we investigated the effect of ghrelin on Ang II-induced H9c2 cardiomyocyte apoptosis. The results showed that Ang II inhibited H9c2 cell viability, which was blocked by ghrelin. By annexin V-propidium iodide dual staining and 2'-deoxyuridine 5'-triphosphate nick end-labeling analysis, we found that Ang II induced H9c2 cell apoptosis, whereas coincubation of ghrelin with Ang II significantly reduced H9c2 cell apoptosis induced by Ang II. Simultaneously, the results revealed that ghrelin regulated the Ang II-induced imbalance of Bax and Bcl-2 expression and reduced Ang II-induced caspase-3 expression. Moreover, mRNA expressions of endoplasmic reticulum stress-related molecules GRP78, caspase-12, and C/EBP homologous protein were significantly upregulated by Ang II. However, their expressions were significantly inhibited by ghrelin. In addition, we found that ghrelin markedly inhibited Ang II-induced Ang II type 1 receptor expression. These data suggest that ghrelin may play an antagonistic role in Ang II-induced cardiomyocyte apoptosis via decreasing Ang II type 1 receptor expression and inhibiting the activation of endoplasmic reticulum stress pathway.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/policies/article-withdrawal). This article has been retracted at the request of This article has been retracted at the request of the authors and the Editor-in-Chief. The authors contacted the Editors of the European Journal of Cancer regarding oversights or labelling errors introduced in Fig. 2 and Fig.7 during data processing. The authors provided the raw data, however the Editors conclude that the raw data management cannot be relied upon and the article needs to be retracted., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Objective: To observe the affect of Small interfering RNA Rac1b (Si-Rac1b) on the malignant biological behaviors of colorectal cancer cell including the proliferation, migration, invasion and apoptosis of the cells., Methods: Mediated by lipofectamine 2000, Si-Rac1b was transfected into colorectal cancer cell line SW1116 (with overexpression of Rac1b). The expression of Rac1b was detected by Western blotting and RT-PCR. The CCK-8 assay was used to analyze the cell proliferation, the Wound-healing assay and invasion assay were respectively applied to analyze the cell migration and invasion, and the Hoechst 33258 was used to evaluate the apoptotic index., Results: Si-Rac1b can knock down the Rac1b but not Rac1 both in the level of mRNA and protein. In addition, Si-Rac1b could singnificantly facilitate the cell proliferation, migration, invasion and control the cell apoptosis., Conclusion: Si-Rac1b could partically reverse the malignant phenotypes of colorectal cancer cell.

Objective: To study the mechanism of hypoxia inducing factor-1α (HIF-1α) pathway in establishment of hypoxia inducing low endometrial receptivity., Methods: RL95-2 cell lines, the ideal model of study ER, were cultured in hypoxia condition induced by CoCl2, and the expression of mRNA and protein of HIF-1α and tumor necrosis factor like weak inducer of apoptosis (TWEAK) were measured by reverse transcription-PCR and western blot. The apoptosis rate was analyzed by flow cytometry. Then the mechanism confirmed by comparing the two factors in endometrium and the ultra-appearance of inflammatory reaction and apoptosis between recurrent spontaneous abortion women and control women., Results: (1) On different time point (0, 12, 24, 48 hour), mRNA expression of HIF-1α were 0.272 ± 0.010, 0.354 ± 0.020, 0.591 ± 0.020, 0.890 ± 0.020, while the expression of TWEAK were 0.104 ± 0.010, 0.510 ± 0.020, 1.021 ± 0.020, 1.237 ± 0.040, respectively, the expression level between 12, 24, 48 and 0 hour all showed significant differences (P < 0.05). (2) Protein expression of HIF-1α were 0.853 ± 0.010, 0.931 ± 0.030, 1.124 ± 0.010, 1.317 ± 0.020 respectively, while was 0.042 ± 0.010, 0.091 ± 0.010, 0.131 ± 0.020, 0.205 ± 0.030 in TWEAK expression, the different level were statistically significant (P < 0.05). (3) With longer culture under hypoxia, the cell apoptosis rate increased obviously. The apoptosis rate of each time point were (3.2 ± 1.4)%, (16.2 ± 3.2)%, (26.3 ± 3.5)%, (31.8 ± 3.5)%, the differences between 12, 24, 48 and 0 hour had significance (P < 0.05). (4) The positive rate of HIF-1α stained in epithelium cells and stroma cells of test group were 32.3%, 8.4% and 16.7%, 7.3% in control group. The positive rate of TWEAK were 28.3%, 3.9% in recurrent spontaneous abortion group and 11.6%, 2.7% in control group (P < 0.05). The ultra-appearance of inflammatory cell infiltrated and apoptosis were obvious in test group., Conclusions: Cell inflammation reaction and apoptosis induced by HIF-1α pathway may participate the mechanism of hypoxia inducing low endometrial receptivity. HIF-1α might become a novel target for improving poor endometrial receptivity.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) exhibits potent antitumor activity via membrane receptors on cancer cells without deleterious side effects for normal tissue. Unfortunately, breast cancer cells, as many other cancer types, develop resistance to TRAIL; therefore, TRAIL sensitizing agents are currently being explored. 2-Tellurium-bridged β-cyclodextrin (2-TeCD) is a synthetic organotellurium compound, with both glutathione peroxidase-like catalytic ability and thioredoxin reductase inhibitor activity. In the present study, we reported that 2-TeCD sensitized TRAIL-resistant human breast cancer cells and xenograft tumors to undergo apoptosis. In vitro, 2-TeCD efficiently sensitized MDA-MB-468 and T47D cells, but not untransformed human mammary epithelial cells, to TRAIL-mediated apoptosis, as evidenced by enhanced caspase activity and poly (adenosine diphosphate-ribose) polymerase cleavage. From a mechanistic standpoint, we showed that 2-TeCD treatment of breast cancer cells significantly upregulated the messenger RNA and protein levels of TRAIL receptor, death receptor (DR) 5, in a transcription factor Sp1-dependent manner. 2-TeCD treatment also suppressed TRAIL-induced nuclear factor-κB (NF-κB) prosurvival pathways by preventing cytosolic IκBα degradation, as well as p65 nuclear translocation. Consequently, the combined administration suppressed anti-apoptotic molecules that are transcriptionally regulated by NF-κB. In vivo, 2-TeCD and TRAIL were well tolerated in mice and their combination significantly inhibited growth of MDA-MB-468 xenografts and promoted apoptosis. Upregulation of DR5 and downregulation of NF-κB by the dual treatment were also observed in tumor tissues. Overall, 2-TeCD sensitizes resistant breast cancer cells to TRAIL-based apoptosis in vitro and in vivo. These findings provide strong evidence for the therapeutic potential of this combination against breast cancers.

hPEBP4 (human phosphatidylethanolamine-binding protein 4) has been identified to be able to potentiate the resistance of breast, prostate, and ovarian cancers, with the preferential expression of hPEBP4, to tumor necrosis factor-alpha (TNF-alpha) or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis, suggesting that inhibitors targeting the anti-apoptotic protein hPEBP4 may be useful to increase the sensitivity of hPEBP4-expressing cancer cells to TNF-alpha or TRAIL-induced apoptosis. By structure-based virtual screening and following surface plasmon resonance-based binding assay, seven small compounds were found to potently bind with hPEBP4. The hit compounds were further functionally screened for their ability to inhibit cancer cell growth, and one small compound, IOI-42, was identified to be able to promote TNF-alpha-mediated growth inhibition of MCF-7 breast cancer cells. IOI-42 could potentiate TNF-alpha-induced apoptosis of MCF-7 cells by inhibiting hPEBP4 and could suppress anchorage-independent cell growth of MCF-7 cells. We further demonstrated that IOI-42 could reduce the endogenous association of hPEBP4 with Raf-1/MEK1 and enhance the activation of ERK1/2 and JNK while inhibiting Akt activation. Furthermore, IOI-42 also promoted TRAIL-induced cell apoptosis of prostate cancer cells. Taken together, our data suggest that IOI-42, as the first chemical inhibitor of anti-apoptotic protein hPEBP4, may serve as a potential anti-tumor drug by sensitizing tumor cells to apoptotic inducers.

The apoptotic loss of cardiomyocytes contributes to numerous cardiovascular disorders. Evidence suggests that free fatty acids induce cellular apoptosis, and recent studies have shown that free fatty acids dramatically elevate mRNA levels of uncoupling protein 2 (UCP2) in some cell lines. In this study, we investigated the possibility that free fatty acids induce the expression of UCP2 through the peroxisome proliferator-activated receptor pathway, thereby increasing cell apoptosis in adult rat cardiomyocytes. Primary cultured adult rat cardiomyocytes exposed to free fatty acids exhibited a dose-dependent increase in apoptosis. Quantitative real-time reverse transcription-polymerase chain reaction and Western blotting showed significant increases in the level of UCP2 expression at 6, 12, and at 24 hours after treatment of adult rat cardiomyocytes with free fatty acids. Expression of UCP2 was suppressed with RNA interference, and knockdown of UCP2 attenuated free fatty acid-induced apoptosis in the cardiomyocytes. In summary, free fatty acids induced UCP2 expression through peroxisome proliferator-activated receptor alpha in adult rat cardiomyocytes.

Inhibition of calcium/calmodulin-dependent protein kinase II (CaMKII) results in hypophosphorylation of CaMKII substrates and in some cases suppresses cell growth. We previously presented the first report of the human CaMKII inhibitory protein, hCaMKIINbeta. Here we report the functional characterization of hCaMKIINbeta in ovarian cancer cells. We showed that hCaMKIINbeta was highly expressed in normal ovarian tissues but was not detected in human ovarian adenocarcinoma, indicating that decreased expression of hCaMKIINbeta may be involved in the pathogenesis of human ovarian adenocarcinoma. As an endogenous CaMKII inhibitor, hCaMKIINbeta could significantly inhibit the growth of human ovarian cancer cells in vitro. In vivo, hCaMKIINbeta decreased the tumorigenicity and growth of HO-8910PM human ovarian cancer cells and prolonged the survival of tumor-bearing mice. hCaMKIINbeta blocked cell cycle progression and induced apoptosis of HO-8910PM cells, which was correlated with the up-regulation of p21, p53, and Bax and the down-regulation of cyclin A, cyclin D1, cyclin E, CDK2, phosphorylated retinoblastoma, and Bcl-2. We further demonstrated that hCaMKIINbeta-mediated CaMKII inhibition suppressed Akt activation, leading to the down-regulation of HDM2, which was responsible for the up-regulation of p53 and p21 in human ovarian cancer cells. The tumor-suppressive effect and the negative expression in human ovarian cancer tissues suggest that hCaMKIINbeta may play an important role in the regulation of tumor cell growth, possibly contributing to the development of new therapeutic strategies for ovarian cancer.

Endocrine glands-derived vascular endothelial growth factor (EG-VEGF, also termed as Prok1)--a novel cytokine that selectively acts on the endothelial cells of endocrine glands--was recently reported to be involved in the regulation of tumor cell growth and survival. However, its roles in the regulation of pancreatic cancer progression remain unclear. In this report, we investigated the suppressive effects of EG-VEGF on pancreatic cancer cell apoptosis and the relevant mechanisms. By using reverse-transcriptase polymerase chain reaction (RT-PCR) we found that the Mia PaCa II cells of the pancreatic cancer cell line express the mRNAs of both EG-VEGF (Prok1) and its receptors. EG-VEGF protects pancreatic cancer cells from apoptosis through upregulation of myeloid cell leukemia-1 (Mcl-1), an anti-apoptotic protein of the bcl-2 family. Treatment of pancreatic cancer cells with EG-VEGF results in the rapid phosphorylation of mitogen-activated protein kinase (MAPK), STAT3, and AKT, which are involved in the upregulation of Mcl-1 expression. EG-VEGF (Prok1) protects Mia PaCa II cells from apoptosis through G protein-coupled receptor (GPR)-induced activation of multiple signal pathways, and hence can be a novel target for pancreatic cancer therapy.

Notch signaling plays a critical role in regulating cell proliferation, differentiation, and apoptosis. Our previous study showed that overexpression of Notch1 could inhibit human hepatocellular carcinoma (HCC) cell growth by arresting the cell cycle and inducing apoptosis. HCC cells are resistant to apoptotic induction by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), so new therapeutic approaches have been explored to sensitize HCC cells to TRAIL-induced apoptosis. We are wondering whether and how Notch1 signaling can enhance the sensitivity of HCC cells to TRAIL-induced apoptosis. In this study, we found that overexpression of ICN, the constitutive activated form of Notch1, up-regulated p53 protein expression in HCC cells by inhibiting proteasome degradation. p53 up-regulation was further observed in human primary hepatocellular carcinoma cells after activation of Notch signaling. Inhibition of the Akt/Hdm2 pathway by Notch1 signaling was responsible for the suppression of p53 proteasomal degradation, thus contributing to the Notch1 signaling-mediated up-regulation of p53 expression. Accordingly, Notch1 signaling could make HCC cells more sensitive to TRAIL-induced apoptosis, whereas Notch1 signaling lost the synergistic promotion of TRAIL-induced apoptosis in p53-silenced HepG2 HCC cells and p53-defective Hep3B HCC cells. The data suggest that enhancement of TRAIL-induced apoptosis by Notch1 signaling is dependent upon p53 up-regulation. Furthermore, Notch1 signaling could enhance DR5 expression in a p53-dependent manner. Taken together, Notch1 signaling sensitizes TRAIL-induced apoptosis in HCC cells by inhibiting Akt/Hdm2-mediated p53 degradation and up-regulating p53-dependent DR5 expression. Thus, our results suggest that activation of Notch1 signaling may be a promising approach to improve the therapeutic efficacy of TRAIL-resistant HCC.

Background: FBG2 (F-BOX6) gene is an important member in ubiquitin metabolic system F-BOX family, and forms E3 complex with the other members in the family. But its role in gastric cancer is still not clear. In the present study, we intended to investigate the influence of FBG2 on the growth, proliferation, apoptosis, invasion and cell cycle of the gastric cancer line MKN45 and gastric cell line HFE145., Methods: As a critical component of ubiquitin-protein ligase complex, FBG2 cDNA was subcloned into a constitutive vector PCDNA3.1 followed by transfection in MKN45 and HFE145 by using liposome. Then stable transfectants were selected and appraised. The apoptosis and cell cycles of these clones were analyzed by using flow cytometry. The growth and proliferation were analyzed by cell growth curves and colony-forming assay respectively. The invasion of these clones was tested by using cancer cell migration assay. The FBG2 stable expression clones(MKN-FBG2 and HFE-FBG2) and their control groups were detected and compared respectively., Results: MKN-FBG2 grew faster than MKN45 and MKN-PC(MKN45 transfected with PCDNA3.1 vector). HFE-FBG2 grew faster than HFE145 and HFE-PC(HFE145 transfected with PCDNA3.1 vector). The cell counts of MKN-FBG2 in the forth, fifth, sixth and seventh days were significantly more than those of others (P < 0.05). Cell cycle analysis showed that MKN-FBG2 and HFE-FBG2 proliferated faster, proportions of cells in G2-M and S were different significantly with control groups (P < 0.05). Results of colony-forming assay showed that the colony formation rates of MKN-FBG2 and HFE-FBG2 were higher than those of control groups (P < 0.05). The results of cell migration assay were all negative., Conclusion: FBG2 can promote the growth and proliferation of gastric cancer cells and normal gastric cells. It can help tumor cell maintain malignant phenotype too. But it can have a negative influence on the apoptosis or the ability of invasion of gastric cancer cells.

We identified a novel ubiquitin-like molecule DULP from human dendritic cells. DULP contains a domain that shares 26% identity and 34% similarity with ubiquitin, and it possesses the corresponding Ile-44 hydrophobic patch used by mono- or poly-ubiquitin to interact with a ubiquitin-interaction motif (UIM) or ubiquitin-associated domain (UBA). Lysine residue corresponding to 6 of ubiquitin, which is involved in the formation of a multi-ubiquitin chain that can bind proteasomal subunit Rpn10/S5a, is also conserved in its ubiquitin-homology domain. However, DULP does not possess the highly conserved C-terminus Gly-Gly required for ubiquitin conjugation or the Lys-48 required for the formation of polyubiquitin chain to target substrates for degradation, suggesting it might be a novel ubiquitin-domain protein (UDP). DULP was found widely expressed in many cells and the ubiquitin-homology domain was not cleaved. We also confirmed that DULP expression was enriched in the nucleus and much weaker in the cytosol. Besides, we found that overexpression of DULP in 293T cells induced apoptosis, which might not be associated with the mitochondrial or proteasome pathway, with the specific mechanism remaining unclear. Further investigations are needed to identify the precise biological functions of DULP.

We recently identified the Phafin protein family, whose members all contain an N-terminal PH domain (pleckstrin homology) and a C-terminal FYVE (Fab1, YGLO23, Vps27, and EEA1) domain. LAPF (lysosome-associated apoptosis-inducing protein containing PH and FYVE domains, also known as Phafin-1), as one representative member of this new family, has been shown to be able to initiate caspase-independent apoptosis through lysosomal-mitochondrial apoptotic pathway. Here, we describe the cloning and functional characterization of another Phafin member, EAPF (endoplasmic reticulum-associated apoptosis-involved protein containing PH and FYVE domains)/Phafin-2. Overexpression of EAPF/Phafin-2 enhances the sensitivity of L929 and MCF-7 cells to TNF-alpha-induced apoptosis, concomitant with its partial translocation to endoplasmic reticulum (ER). Both the PH and the FYVE domains contribute to the ER translocation of EAPF/Phafin-2 as well as EAPF/Phafin-2-enhanced apoptosis. Knockdown of mouse and human EAPF/Phafin-2 expression protects L929 cells and MCF-7 cells from TNF-alpha-induced apoptosis, respectively. We demonstrate that EAPF/Phafin-2 induces a much sharper and more rapid Ca2+ influx triggered by TNF-alpha and Ca2+ release ER contributes to the enhancement of EAPF/Phafin-2 in TNF-induced apoptosis. EAPF/Phafin-2 increases the activity of caspase 12, suggesting that EAPF/Phafin-2 is involved in ER-related apoptotic pathway. Overexpression of EAPF/Phafin-2 also enhances TNF-alpha-induced activity of caspase 3 (but not caspase 8 or 9), and promotes TNF-alpha-triggered mitochondrial membrane permeabilization (MMP) in L929 cells, including dissipation of mitochondrial membrane potential and release of AIF. Besides, EAPF/Phafin-2 also suppresses the unfolded protein response by inhibiting phosphorylation of eIF2alpha. Therefore, our results demonstrate that EAPF/Phafin-2 facilitates TNF-alpha-induced cellular apoptosis through an ER-mitochondrial apoptotic pathway, which may improve our understanding of drug-induced cancer cell death and cancer chemotherapy.

Objective: To explore the protection on apoptosis and the mechanism of promoting the cytoactive of osteoblast by Morinda Root Polysaccharide through the observations of the cultured osteoblast in vitro., Methods: Prepared blood serum with Morinda Root Polysaccharide and Morinda Root aqueous extract and cultured Osteoblast in vitro with it. The second generation osteoblasts in vitro were separated from the cranium of 24-hours newborn SD rat, which were divided into control group (adding only rat serum during cultivation), induction apoptosis group (adding trans-retinoic acid in control group), Morinda Root aqueous extract group (adding serum prepared by Morinda Root aqueous extract in induction apoptosis group) and Morinda Root Polysaccharide group (adding serum prepared by Morinda Root Polysaccharide in induction apoptosis group). Adopting fluorescence microscope, apoptosis detected by flow cytometry and gene expression of Bcl-2 and Bax detected by RT-PCR, to evaluate the effect of Morinda Root Polysaccharide on the course of osteoblast apoptosis., Results: The apoptotic rate of Morinda Root aqueous extract group and Morinda Root Polysaccharide group were significantly lower than that of induction apoptosis group (P < 0.01). The apoptosis ratio of Morinda Root Polysaccharide group was lower than that of Morinda Root aqueous extract group (P < 0.05). Expression level of Bcl-2 mRNA of apoptosis cell: control group > Morinda Root Polysaccharide group > Morinda Root aqueous extract group > induction apoptosis group (P < 0.01). Expression level of Bax mRNA: induction apoptosis group > Morinda Root aqueous extract group > control group > Morinda Root Polysaccharide group (P < 0.01). Bcl-2/Bax: control group > Morinda Root Polysaccharide group > Morinda Root aqueous extract group > induction apoptosis group (P < 0.01)., Conclusion: Morinda Root can inhibit the apoptosis of osteoblast induced by trans-retinoic acid in some extent. The above role of Morinda Root Polysaccharide is significant better than that of Morinda Root aqueous extract. It is indicated that Morinda Root Polysaccharide is one of the essential component of inhibiting osteoblast apotosis.

Evidence suggests a functional association between the tumor suppressor p53 and apoptosis-involved organelle lysosome; however, the detailed mechanisms remain poorly understood. We recently reported that a lysosome-targeting protein, LAPF (lysosome-associated and apoptosis-inducing protein containing PH and FYVE domains), could initiate apoptosis of L929 cells through a lysosomal-mitochondrial pathway. In this study, we show that LAPF specifically interacted with phosphorylated p53 (Ser(15/18)) both in vitro and in vivo, which could be enhanced by apoptotic stimuli, such as tumor necrosis factor alpha (TNF-alpha) and ionizing irradiation. The PH domain of LAPF and the transactivation domain of p53 mediated the interaction between both molecules. Phosphorylated p53 (Ser(15/18)) could translocate to lysosomes before lysosomal membrane permeabilization (LMP) in LAPF-initiated and TNF-induced apoptosis. Silencing of LAPF expression abrogated lysosomal translocation of phosphorylated p53 (Ser(15/18)), whereas silencing of p53 expression had no effect on lysosomal translocation of LAPF. Similar to that of LAPF silencing, silencing of endogenous p53 expression in L929 cells could significantly impair TNF-alpha-induced LMP and apoptosis. However, reexpression of wild-type p53, p53S15D (substitution of Ser(15) to Asp that mimics a phosphorylated state), and p53R175H (a transcription-deficient mutant) in p53-knockdown L929 cells could rescue the decrease in TNF-induced apoptosis. The data suggest that phosphorylated p53 (Ser(15/18)) might translocate to lysosome via forming complexes with adaptor protein LAPF and subsequently result in LMP and apoptosis, which might be in a transcription-independent manner.

Background: Uncontrolled cell division is one of the hallmarks of tumor growth. Researches have been focused on numerous molecules involved in this process. Cyclins are critical regulatory proteins of cell cycle progression and/or transcription. The present study aimed to investigate the anti-proliferative effect of cyclin L2, and to define its growth regulatory mechanisms using human lung adenocarcinoma cell line A549., Methods: Human cyclin L2 was transfected into human lung adenocarcinoma cells (A549 cell), and was expressed in a mammalian expression vector pcDNA3.1. The effects and mechanisms of the cyclin L2 in cell growth, cell cycle analysis and apoptosis were studied by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), flow cytometry or Western blot, respectively., Results: Overexpression of cyclin L2 inhibited the growth of A549 cells. Cell cycle analysis in cells transfected with pCCNL2 revealed an increment in proportion in G0/G1 phase ((68.07 +/- 4.2)%) in contrast to (60.39 +/- 2.82)% of the cells transfected with mock vector. Apoptosis occurred in (7.25 +/- 0.98)% cells transfected with pCCNL2, as compared with (1.25 +/- 0.21)% of the mock vector control group. Cyclin L2-induced-G0/G1 arrest and apoptosis involved upregulation of caspase-3 and downregulation of Bcl-2 and survivin., Conclusion: The results indicate that overexpression of cyclin L2 protein may promote efficient growth inhibition of human lung adenocarcinoma cells by inducing G0/G1 cell cycle arrest and apoptosis.

The treatment options available for prostate cancer are limited because of its resistance to therapeutic agents. Thus, a better understanding of the underlying mechanisms of the resistance of prostate cancer will facilitate the discovery of more efficient treatment protocols. Human phosphatidylethanolamine-binding protein 4 (hPEBP4) is recently identified by us as an anti-apoptotic molecule and a potential candidate target for breast cancer treatment. Here we found the expression levels of hPEBP4 were positively correlated with the severity of clinical prostate cancer. Furthermore, hPEBP4 was not expressed in TRAIL-sensitive DU145 prostate cancer cells, but was highly expressed in TRAIL-resistant LNCaP cells, which show highly activated Akt. Interestingly, hPEBP4 overexpression in TRAIL-sensitive DU145 cells promoted Akt activation but inhibited ERK1/2 activation. The hPEBP4-overexpressing DU145 cells became resistant to TRAIL-induced apoptosis consequently, which could be reversed by PI3K inhibitors. In contrast, silencing of hPEBP4 in TRAIL-resistant LNCaP cells inhibited Akt activation but increased ERK1/2 activation, resulting in their sensitivity to TRAIL-induced apoptosis that was restored by the MEK1 inhibitor. Therefore, hPEBP4 expression in prostate cancer can activate Akt and deactivate ERK1/2 signaling, leading to TRAIL resistance. We also demonstrated that hPEBP4-mediated resistance to TRAIL-induced apoptosis occurred downstream of caspase-8 and at the level of BID cleavage via the regulation of Akt and ERK pathways, and that hPEBP4-regulated ERK deactivation was upstream of Akt activation in prostate cancer cells. Considering that hPEBP4 confers cellular resistance to TRAIL-induced apoptosis and is abundantly expressed in poorly differentiated prostate cancer, silencing of hPEBP4 suggests a promising approach for prostate cancer treatment.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in a variety of tumor cell lines but not typically in normal or nontransformed cells, which makes TRAIL a desirable therapeutic agent to fight cancer. Human phosphatidylethanolamine-binding protein 4 (hPEBP4) is a recently identified anti-apoptotic molecule and has been shown to be highly expressed in breast and ovarian cancer cells. We demonstrate that silencing of hPEBP4 in CaoV-3 ovarian cancer cells potentiates TRAIL-induced apoptosis. We found that endogenous hPEBP4 interacts with Raf-1 and MEK1 in TRAIL-treated CaoV-3 cells by co-immunoprecipitation analysis. Simultaneously, silencing of hPEBP4 in CaoV-3 cells enhances TRAIL-induced ERK and JNK activation. Moreover, the inhibitors of MEK1 or JNK can reduce hPEBP4-silence-induced TRAIL sensitivity. Therefore, silencing of hPEBP4 in CaoV-3 ovarian cancer promotes TRAIL-induced apoptosis, and the increased MAPK activation is required for the apoptosis sensitization. All these data indicate that silencing of hPEBP4, an important potential target, may be a promising approach for the treatment of ovarian cancer.

Lysosomes have recently been identified as important apoptotic signal integrators in response to various stimuli. Here we report the functional characterization of LAPF, a novel lysosome-associated apoptosis-inducing protein containing PH and FYVE domains. LAPF is a representative of a new protein family, the Phafins (protein containing both PH and FYVE domains), which consists of 14 unidentified proteins from various species. Overexpression of LAPF in L929 cells induces apoptosis and also increases cell sensitivity to TNFalpha-induced apoptosis, concomitant with its translocation to lysosomes. Two mutants of LAPF, either lacking the PH or FYVE domain, failed to induce cell death and translocate to lysosomes, suggesting that both domains are required for its apoptosis-inducing activity and relocation. We demonstrate that LAPF may induce apoptosis via the following steps: LAPF translocation to lysosomes, lysosomal membrane permeabilization (LMP), release of cathepsin (cath) D and L, mitochondrial membrane permeabilization (MMP), release of apoptosis-inducing factor (AIF), and caspase-independent apoptosis. The cath D-specific inhibitor attenuates LAPF-induced apoptosis, indicating a pivotal role of lysosomes in LAPF-initiated apoptosis. We also demonstrate that the lysosomal pathway was employed in the typical apoptotic model in which high dose TNFalpha was used to stimulate L929 cells. Silencing of LAPF expression by small RNA interference protected L929 cells from hTNFalpha-induced apoptosis by impairing hTNFalpha-triggered LMP and MMP. Therefore, LAPF may launch caspase-independent apoptosis through the lysosomal-mitochondrial pathway.

Purpose: The current therapeutic approach is not so effective in breast cancer patients. Alternative treatment protocols aimed at different targets need to be explored. We recently reported a novel phosphatidylethanolamine-binding protein, human phosphatidylethanolamine-binding protein 4 (hPEBP4), as an antiapoptotic molecule. The finding led us to explore a promising approach for breast cancer therapy via silencing the expression of hPEBP4., Experimental Design: hPEBP4 expression in clinical breast specimens was examined by Tissue Microarrays. RNA interference was used to silence hPEBP4 expression in MCF-7 breast carcinoma cells and the effects on cell proliferation, cell cycle progression, apoptosis, as well as underlying mechanisms, were investigated., Results: hPEBP4 was found to be expressed in up to 50% of breast cancers but in only <4% of normal breast tissues. Silencing of hPEBP4 potentiated tumor necrosis factor-alpha (TNF-alpha)-induced apoptosis and cell cycle arrest in MCF-7 cells, which was due to the increased mitogen-activated protein kinase activation and the enhanced phosphatidylethanolamine externalization. Further investigation showed that silencing of hPEBP4 in MCF-7 cells promoted TNF-alpha-induced stability of p53, up-regulation of phospho-p53ser15, p21waf/cip, and Bax, and down-regulation of Bcl-2 and Bcl-xL, which were shown to depend on extracellular signal-regulated kinase 1/2 and c-jun NH2-terminal kinase activation by hPEBP4 silencing. Moreover, the increased proportion of cells in the G0-G1 phase of cell cycle was observed in hPEBP4-silenced MCF-7 cells on TNF-alpha treatment and the expression of cyclin A and cyclin E was down-regulated more significantly., Conclusions: The antiapoptotic effect and the preferential expression pattern in breast cancer tissues make hPEBP4 a new target for breast cancer therapy. Silencing of hPEBP4 expression may be a promising approach for the treatment of breast carcinoma.