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Successful repair and renewal of alveolar epithelial cells (AECs) are critical in prohibiting the accumulation of myofibroblasts in pulmonary fibrogenesis. MicroRNAs (miRNAs) are multifocal regulators involved in lung injury and repair. However, the contribution of miRNAs to AEC2 renewal and apoptosis is incompletely understood. We report that miRNA-29c (miR-29c) expression is lower in AEC2s of individuals with idiopathic pulmonary fibrosis than in healthy lungs. Epithelial cells overexpressing miR-29c show higher proliferative rates and viability. miR-29c protects epithelial cells from apoptosis by targeting forkhead box O3a (Foxo3a). Both overexpression of miR-29c conventionally and AEC2s specifically lead to less fibrosis and better recovery in vivo. Furthermore, deficiency of miR-29c in AEC2s results in higher apoptosis and reduced epithelial renewal. Interestingly, a gene network including a subset of apoptotic genes was coregulated by both Toll-like receptor 4 and miR-29c. Taken together, miR-29c maintains epithelial integrity and promotes recovery from lung injury, thereby attenuating lung fibrosis in mice.

Aim: Rheumatoid arthritis is characterized by type 17 helper T cell (Th17)/regulatory T cell (Treg) imbalance. The objective of this article is to study whether insufficient apoptosis contributes to the imbalance of Th17/Treg in rheumatoid arthritis., Methods: Twenty-one rheumatoid arthritis patients and eight healthy volunteers were involved in this study. The percentage of CD4(+) interleukin (IL)-17(+) T cells and CD4(+) transcription factor-forkhead box protein 3 (Foxp3)(+) T cells were measured by flow cytometry, and active caspase-3 labeling was used to detect early apoptosis. The number of T cell subtypes in peripheral blood between the two groups was compared, as well as the apoptotic ratio., Results: Neither the number of Th17 nor Treg cells was significantly different between rheumatoid arthritis patients and healthy controls. However, the number of regulatory T cells positively correlated with erythrocyte sedimentation rate, Disease Activity Score of 28 joints and rheumatoid factor. For the apoptosis of T cell subtypes, the percentage of apoptotic Th17 cells was higher in peripheral blood of rheumatoid arthritis patients compared to controls. Furthermore, peripheral Th17 cells were more sensitive to apoptosis than Treg cells, but there was no difference between rheumatoid arthritis patients and controls., Conclusion: It seemed that there was no relationship between the number and apoptosis ratio of peripheral Th17/Treg cells. But the number of Treg cells positively correlated with disease activity. Furthermore, Th17 cells are more sensitive to apoptosis after freezing, especially in RA patients. This serendipitous finding may provide new areas for the further study of these two cell populations., (© 2013 Asia Pacific League of Associations for Rheumatology and Wiley Publishing Asia Pty Ltd.)

It is well known that maternal folate deficiency results in adverse pregnancy outcomes. In addition to aspects in embryonic development, maternal uterine receptivity and the decidualization of stromal cells is also very important for a successful pregnancy. In this study, we focused on endometrium decidualization and investigated whether apoptosis, which is essential for decidualization, was impaired. Flow cytometry and TUNEL detection revealed that apoptosis of mouse endometrium decidual cells was suppressed in the dietary folate-deficient group on Days 7 and 8 of pregnancy (Day 1 = vaginal plug) when decidua regression is initiated. The endometrium decidual tissue of the folate deficiency group expressed less Bax compared to the normal diet group while they had nearly equal expression of Bcl2 protein. Further examination revealed that the mitochondrial transmembrane potential (ΔΨm) decreased, and the fluorescence of diffuse cytoplasmic cytochrome c protein was detected using laser confocal microscopy in normal decidual cells. However, no corresponding changes were observed in the folate-deficient group. Western blotting analyses confirmed that more cytochrome c was released from mitochondria in normal decidual cells. Taken together, these results demonstrated that folate deficiency could inhibit apoptosis of decidual cells via the mitochondrial apoptosis pathway, thereby restraining decidualization of the endometrium and further impairing pregnancy.

Background: Heat stress can be acutely cytotoxic, and heat stress-induced apoptosis is a prominent pathological feature of heat-related illnesses, although the precise mechanisms by which heat stress triggers apoptosis are poorly defined., Methods: The percentages of viability and cell death were assessed by WST-1 and LDH release assays. Apoptosis was assayed by DNA fragmentation and caspase activity. Expression of cleaved PARP, Apaf-1, phospho-PERK, Phospho-eIF2a, ATF4, XBP-1s, ATF6, GRP78, phospho-IP3R, RYR and SERCA was estimated by Western blot. The effect of calcium overload was determined using flow cytometric analysis with the fluorescent probe Fluo-3/AM. The generation of ROS (O2-, H2O2, NO) was labeled by confocal laser scanning microscopy images of fluorescently and flow cytometry., Results: In this study, we found that heat stress in HUVEC cells activated initiators of three major unfolded protein response (UPR) signaling transduction pathways: PERK-eIF2a-ATF4, IRE1-XBP-1S and ATF6 to protect against ER stress, although activation declined over time following cessation of heat stress. Furthermore, we show that intense heat stress may induce apoptosis in HUVEC cells through the calcium-mediated mitochondrial apoptotic pathway, as indicated by elevation of cytoplasmic Ca2+, expression of Apaf-1, activation of caspase-9 and caspase-3, PARP cleavage, and ultimately nucleosomal DNA fragmentation; Reactive oxygen species (ROS) appear to act upstream in this process. In addition, we provide evidence that IP3R upregulation may promote influx of Ca2+ into the cytoplasm after heat stress., Conclusion: Our findings describe a novel mechanism for heat stress-induced apoptosis in HUVEC cells: following elevation of cytoplasm Ca2+, activation of the mitochondrial apoptotic pathway via the IP3R upregulation, with ROS acting as an upstream regulator of the process.

Background: Toll-like receptor 4 (TLR4) plays a key role in mediating kidney damage during ischemia/reperfusion (I/R) injury, and its expression is enhanced following renal I/R injury. Our study focused on TLR4 silencing-mediated downstream antiapoptotic pathways during hypoxia/reoxygenation (H/R) and investigated whether TLR4 overexpression exacerbates the renal damage induced by I/R injury., Methods: Proximal tubule epithelial cells (PTECs) were isolated and H/R injury mediated by ATP depletion, and replenishment was performed to mimic in vivo I/R injury. PTECs were transfected with either TLR4 siRNA or TLR4-overexpressing vectors to determine the contribution of TLR4 to H/R injury-induced apoptosis and inflammatory response., Results: H/R injury significantly enhanced PTEC apoptosis (p < 0.01) and the production of tumor necrosis factor (TNF)-α and interleukin (IL)-8; however, TLR4 silencing significantly reversed these effects (p < 0.05). Moreover, compared to PTECs or PTECs-siCon exposed to H/R injury, overexpression of TLR4 further upregulated TNF-α and IL-8 (p < 0.05), but did not enhance apoptosis. The expression of cytochrome C and caspases 3, 8, and 9 was decreased in the siTLR4 group compared to controls after H/R injury, whereas TLR4 silencing did not alter CHOP expression. TLR4 overexpression failed to promote the expression of cytochrome C and caspases 3, 8, and 9, and reduced the expression of CHOP and GPR78., Conclusions: Knockdown of TLR4 could protect PTECs from H/R injury via inhibiting mitochondrial and death receptor pathways. TLR4 overexpression did not increase PTEC apoptosis induced by H/R injury due in part to the downregulation of CHOP.

p21-activated kinase 5 (PAK5) is a recently identified member of the group B PAK family. The PAK proteins are effectors of the small GTPase Cdc42 and Rac1 and are known to regulate cell motility and activate cell-survival signaling pathways. Especially, the mitochondrial localization of PAK5 is vital to its effects on apoptosis and cell survival. Previously, we demonstrated that PAK5 expression increased significantly during the malignant progression of colorectal carcinoma (CRC) and that PAK5 promoted CRC metastasis by regulating CRC cell adhesion and migration. In the present study, we aim to investigate the role of PAK5 in camptothecin-induced apoptosis and its potential mechanism of action. Our results showed that overexpression of PAK5 inhibited camptothecin-induced apoptosis by inhibiting the activity of caspase-8 in CRC cells. Accordingly, knockdown of PAK5 in LoVo cells resulted in increased apoptosis. Mechanistically, we found that PAK5 directly phosphorylated Bad on serine 112 and indirectly led to phosphorylation of serine 136 via the Akt pathway. In conclusion, our study revealed previously unappreciated inhibitory role of PAK5 in camptothecin-induced apoptosis, thus suggesting PAK5 as a novel therapeutic target in CRC.

Background: Reactive oxygen species (ROS) are intricately involved in tumor progression through effects on proliferation, apoptosis and metastasis. But how ROS works is not well understood. In previous study, we found PUMA (p53-upregulated modulator of apoptosis) played an important role in oxaliplatin-induced apoptosis. In the present study, we detect the role of PUMA in H2O2-induced apoptosis in colorectal cancer cells and investigate the potential mechanism., Methods and Results: We showed that H2O2 stimulated the activity of a 493 PUMA promoter reporter gene construct. Suppressing the expression of PUMA abrogated H2O2-induced apoptosis. Deletion of the Sp1-binding sites also decreased the transactivation of PUMA promoter by H2O2. Furthermore, induction of PUMA promoter activity by H2O2 was abrogated by PFT-alpha (a p53 inhibitor) and Mithramycin A (a Sp1 inhibitor), as compared with PFT-alpha alone. To determine the effects of Sp1 on PUMA in H2O2-induced apoptosis, procaspase 3, procaspase 9 and procaspase 8 expression was assessed. Mithramycin A and PFT-alpha also reduced H2O2-induced apoptosis synergistically and abrogated the expression of procaspase 3 and procaspase 9., Conclusion: Our findings suggest that PUMA plays a role in H2O2-induced apoptosis, and that Sp1 works together with p53 in the regulation of H2O2-induced PUMA expression and apoptosis in colorectal cancer cells. This study provides important regulatory insights in the mechanisms of ROS in colorectal cancer.

Our previous work confirmed that Sph2/LA1029 was a sphigomyelinase-like hemolyisn of Leptospira interrogans serogroup Icterohaemorrhagiae serovar Lai. Characteristics of both hemolytic and cytotoxic activities of Sph2 were reported in this paper. Sph2 was a heat-labile neutral hemolysin and had similar hemolytic behavior as the typical sphingomyelinase C of Staphylococcus aureus upon sheep erythrocytes. The cytotoxic activity of Sph2 was shown in mammalian cells such as BALB/C mouse lymphocytes and macrophages, as well as human L-02 liver cells. Transmission electron microscopic observation showed that the Sph2 treated BALB/C mouse lymphocytes were swollen and ruptured with membrane breakage. They also demonstrated condensed chromatin as a high-density area. Cytoskeleton changes were observed via fluorescence confocal microscope in Sph2 treated BALB/C mouse lymphocytes and macrophages, where both cytokine IL-1beta and IL-6 were induced. In addition, typical apoptotic morphological features were observed in Sph2 treated L-02 cells via transmission electron microscope and the percentage of apoptotic cells did increase after the Sph2 treatment detected by flow cytometry. Therefore, Sph2 was likely an apoptosis-inducing factor of human L-02 liver cells.

The isoflavone genistein (GEN), a biologically active component of soy foods, is associated with reduced breast cancer risk in women who consume soy-rich diets. GEN has been reported to influence many biological processes, of which suppression of cell proliferation and stimulation of apoptosis are considered to be the major pathways underlying its inhibition of tumorigenesis. This study evaluated the mechanism by which diets containing GEN promote mammary epithelial cell death. We report that mammary glands of young adult female rats exposed from gestation day 4 to postnatal day 50, to AIN-93G diets containing as sole protein source, casein (CAS) supplemented with GEN, or soy protein isolate (SPI+) had increased apoptosis, relative to rats fed CAS diet devoid of GEN. Mammary gland proliferation was unaffected by diet. The increased apoptotic index in mammary glands of GEN and SPI+ rats was accompanied by increased levels of the tumor suppressor protein PTEN (phosphatase and tensin homolog deleted in chromosome ten), albeit enhanced mammary expression of the pro-apoptotic p21, Bax and Bok genes was observed only in GEN-fed rats. GEN-induced apoptosis in MCF-7 cells was concomitant with increased PTEN expression, and this was abrogated by PTEN siRNA. MCF-7 cells treated with serum from GEN- or SPI(+)-fed rats had increased apoptosis as well as increased levels of the PTEN transcript. PTEN siRNA attenuated the increased apoptotic response of MCF-7 cells to serum from rats fed SPI+ or GEN, although the inhibition to basal (CAS serum) apoptotic levels was achieved only for cells treated with GEN serum. Decreased p21 and Bok gene expression accompanied the inhibition of apoptosis by PTEN siRNA. Data implicate PTEN in the induction of apoptosis by GEN and suggest that the promotion of apoptosis leading to inhibition of tumorigenesis in vivo by diets containing GEN may also involve the distinct activities of yet unknown GEN metabolite(s) and/or other systemic factors induced by GEN.

We have found that AIN-93G diets made with whey protein hydrolysate (WPH) reduce 7,12-dimethyl-benz[a]anthracene (DMBA)-induced tumor incidence in Sprague-Dawley (Harlan) rats relative to those fed a diet with casein (CAS). Herein, we replicated these findings in another Sprague-Dawley substrain (Charles River) and examined whether WPH protective effects were associated with altered mammary gland differentiation status and expression of the tumor suppressor phosphatase and tensin homolog deleted in chromosome ten (PTEN). Mammary tumor incidence was lower in DMBA-treated rats fed WPH than in those fed CAS. Mammary glands of WPH- and CAS-fed rats were isolated at weaning [postnatal day (PND) 21-28] and at an early adult stage (PND 50-53) and analyzed for proliferative (proliferating cell nuclear antigen immunoreactivity), apoptotic (terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick-end labeling), and differentiation (beta-casein) indices, as well as for PTEN mRNA and protein levels. PND 50-53 rats fed WPH showed decreased proliferation and increased apoptosis in mammary structures, coincident with increased mammary beta-casein gene expression, decreased terminal end-bud numbers, and increased ductal lengths, relative to same-age CAS-fed rats. When challenged with DMBA for 24 h, mammary glands of PND 53 CAS-fed rats had decreased cell survival in both terminal end buds and ductal epithelium, while the mammary glands of WPH-fed rats were not altered from pre-DMBA levels. At 7 d post-DMBA, mammary glands of CAS- and WPH-fed rats exhibited comparable apoptotic indices. Mammary PTEN expression was higher in WPH- than in CAS-fed rats at PND 21-28, but was not different in young adults fed either diet. Results demonstrate that dietary WPH advances mammary gland differentiation during neonatal development and suggest that the transiently increased expression of the pro-apoptotic signal PTEN during a sensitive developmental window may partly underlie the cancer protective effects of WPH.

Abstract Heat stroke is a serious pathological condition with high mortality. Vascular endothelial cell injury is a key feature in the pathogenesis of heat stroke, but the specific pathophysiological process whereby this occurs is still unclear. Currently, relevant studies are primarily based upon examination of apoptosis. Recently, pyroptosis, a new form of inflammation-related programmed cell death, was also demonstrated to be involved in heat stroke pathophysiology. Herein, we present evidence that vascular endothelial cell pyroptosis can be induced by heat stress in a time- and temperature-dependent manner. Furthermore, this process can be significantly inhibited by GSDMD siRNA. These findings suggest a new therapeutic target for heat stroke. [ABSTRACT FROM AUTHOR]

The aim of the present study was to examine the effects of alisertib (ALS) on RAS signaling pathways against a panel of colorectal cancer (CRC) cell lines and engineered Flp-In stable cell lines expressing different Kirsten rat sarcoma virus (KRAS) mutants. The viability of Caco-2KRAS wild-type, Colo-678KRAS G12D, SK-CO-1KRAS G12V, HCT116KRAS G13D, CCCL-18KRAS A146T and HT29BRAF V600E cells was examined by Cell Titer-Glo assay, and that of stable cell lines was monitored by IncuCyte. The expression levels of phosphorylated (p-)Akt and p-Erk as RAS signal outputs were measured by western blotting. The results suggested that ALS exhibited different inhibitory effects on cell viability and different regulatory effects on guanosine triphosphate (GTP)-bound RAS in CRC cell lines. ALS also exhibited various regulatory effects on the PI3K/Akt and mitogen-activated protein kinase (MAPK) pathways, the two dominant RAS signaling pathways, and induced apoptosis and autophagy in a RAS allele-specific manner. Combined treatment with ALS and selumetinib enhanced the regulatory effects of ALS on apoptosis and autophagy in CRC cell lines in a RAS allele-specific manner. Notably, combined treatment exhibited a synergistic inhibitory effect on cell proliferation in Flp-In stable cell lines. The results of the present study suggested that ALS differentially regulates RAS signaling pathways. The combined approach of ALS and a MEK inhibitor may represent a new therapeutic strategy for precision therapy for CRC in a KRAS allele-specific manner; however, this effect requires further study in vivo. [ABSTRACT FROM AUTHOR]

Background: Heat stress can be acutely cytotoxic, and heat stress-induced apoptosis is a prominent pathological feature of heat-related illnesses, although the precise mechanisms by which heat stress triggers apoptosis are poorly defined. Methods: The percentages of viability and cell death were assessed by WST-1 and LDH release assays. Apoptosis was assayed by DNA fragmentation and caspase activity. Expression of cleaved PARP, Apaf-1, phospho-PERK, Phospho-eIF2a, ATF4, XBP-1s, ATF6, GRP78, phospho-IP3R, RYR and SERCA was estimated by Western blot. The effect of calcium overload was determined using flow cytometric analysis with the fluorescent probe Fluo-3/AM. The generation of ROS (O2−, H2O2, NO) was labeled by confocal laser scanning microscopy images of fluorescently and flow cytometry. Results: In this study, we found that heat stress in HUVEC cells activated initiators of three major unfolded protein response (UPR) signaling transduction pathways: PERK-eIF2a-ATF4, IRE1-XBP-1S and ATF6 to protect against ER stress, although activation declined over time following cessation of heat stress. Furthermore, we show that intense heat stress may induce apoptosis in HUVEC cells through the calcium-mediated mitochondrial apoptotic pathway, as indicated by elevation of cytoplasmic Ca2+, expression of Apaf-1, activation of caspase-9 and caspase-3, PARP cleavage, and ultimately nucleosomal DNA fragmentation; Reactive oxygen species (ROS) appear to act upstream in this process. In addition, we provide evidence that IP3R upregulation may promote influx of Ca2+ into the cytoplasm after heat stress. Conclusion: Our findings describe a novel mechanism for heat stress-induced apoptosis in HUVEC cells: following elevation of cytoplasm Ca2+, activation of the mitochondrial apoptotic pathway via the IP3R upregulation, with ROS acting as an upstream regulator of the process. [ABSTRACT FROM AUTHOR]

p21-activated kinase 5 (PAK5) is a recently identified member of the group B PAK family. The PAK proteins are effectors of the small GTPase Cdc42 and Rac1 and are known to regulate cell motility and activate cell-survival signaling pathways. Especially, the mitochondrial localization of PAK5 is vital to its effects on apoptosis and cell survival. Previously, we demonstrated that PAK5 expression increased significantly during the malignant progression of colorectal carcinoma (CRC) and that PAK5 promoted CRC metastasis by regulating CRC cell adhesion and migration. In the present study, we aim to investigate the role of PAK5 in camptothecin-induced apoptosis and its potential mechanism of action. Our results showed that overexpression of PAK5 inhibited camptothecin-induced apoptosis by inhibiting the activity of caspase-8 in CRC cells. Accordingly, knockdown of PAK5 in LoVo cells resulted in increased apoptosis. Mechanistically, we found that PAK5 directly phosphorylated Bad on serine 112 and indirectly led to phosphorylation of serine 136 via the Akt pathway. In conclusion, our study revealed previously unappreciated inhibitory role of PAK5 in camptothecin-induced apoptosis, thus suggesting PAK5 as a novel therapeutic target in CRC. [ABSTRACT FROM AUTHOR]

[Display omitted] • Oral bioavailability of COS was improved by nanoencapsulation. • Microfold cell-mediated route was proved to be effective for intestinal absorption of CANs. • CANs enriched in liver improved apoptosis and LSEC capillarization of liver fibrosis. • Antihepatotoxic mechanism of COS was attributed to inhibition of JNK/p38 MAPK pathway. Chitooligosaccharides (COS) significantly attenuates liver dysfunction. However, the conundrum of the oral bioavailability of COS limits their pharmacological effects. Therefore, a strategy of nanoencapsulation was employed to enhance oral bioavailability and tissue-targeted distribution of COS. In this study, nanospheres loaded with COS (CANs) were prepared, their bioavailability, biodistribution, transport mechanism and anti-liver fibrosis effects were explored. Nanoencapsulation improved the oral bioavailability of various COS monomers through microfold cell-mediated absorption route in an indiscriminate manner. CANs were more favorably enriched and protractedly accumulated in the liver. In a liver fibrosis model, CANs ameliorated the pathological state and extracellular matrix deposition. The alleviation of liver fibrosis for COS could be attributed to the inhibition of liver cell apoptosis and liver sinusoidal endothelial cell (LSEC) capillarization. Consequently, this study highlights the improved oral bioavailability of COS and proposes a novel mechanism of COS, for better understanding its hepatoprotective effect. [ABSTRACT FROM AUTHOR]

Abstract: Dietary soy intake is associated with protection from breast cancer, but questions persist on the potential risks of the major soy isoflavone genistein (GEN) on female reproductive health. Here, we evaluated intermediate markers of cancer risk in uteri of cycling, young adult Sprague–Dawley rats lifetime exposed to one of three AIN-93G semipurified diets: casein (CAS), soy protein isolate (SPI+ with 276 mg GEN aglycone equivalents/kg) and CAS+GEN (GEN at 250 mg/kg). Postnatal day 50 (PND50) rats lifetime exposed to GEN or SPI+ had similar uterine luminal epithelium height, myometrial thickness, endometrial gland numbers, endometrial immunoreactive proliferating cell nuclear antigen (PCNA), and serum estrogen and progesterone, as CAS-fed rats. GEN-fed rats showed modestly increased apoptosis in uterine glandular epithelium, compared to those of CAS- or SPI+-fed groups. Diet had no effect on the uterine expression of genes for the tumor suppressors PTEN, p53 and p21, and the apoptotic-associated proteins Bcl2, Bax and progesterone receptor. Uterine tissue and serum concentrations of total GEN were higher in rats fed GEN than in those fed SPI+. Human Ishikawa endocarcinoma cells treated with GEN-fed rat serum tended to exhibit increased apoptotic status than those treated with CAS-fed rat serum. Exogenously added GEN (0.2 and 2 μM) increased, while estradiol-17β (0.1 μM) decreased Ishikawa cell apoptosis, relative to untreated cells. Results suggest that lifetime dietary exposure to soy foods does not alter uterine cell phenotype in young adult rats, while GEN, by enhancing uterine endometrial glandular apoptosis in vivo and in vitro, may confer protection against uterine carcinoma. Given its limited influence on uterine phenotype of young adult females, GEN, when taken as part of soy foods or as supplement, should be favorably considered for other potential health benefits. [Copyright &y& Elsevier]

Abstract: We previously demonstrated that female Sprague–Dawley rats fed AIN-93G diets containing soy protein isolate (SPI+) had lower DMBA-induced mammary tumor incidence than those fed diets containing casein (CAS), due partly to altered Phase I metabolism with soy. Here, we evaluated the tumor protective effects of these same diets to the direct-acting carcinogen N-methyl-nitrosourea (NMU). Tumor incidence was reduced and tumor latency was enhanced, in NMU-administered female rats lifetime exposed to SPI+, relative to the CAS group. Tumor multiplicity did not differ with diet, while tumor grade tended to be more advanced with SPI+. Normal mammary glands of CAS and SPI+ tumor-bearing rats had comparable proliferative and apoptotic status. However, mammary expression of HER-2/neu and progesterone receptor (PR) genes was higher for SPI+ rats. Moreover, tumored SPI+ rats had lower serum progesterone levels than those fed CAS, while serum estrogen did not differ. Serum from tumored SPI+ rats had higher apoptotic activity towards mammary epithelial MCF-7 cells, than CAS serum. Thus, dietary soy protects against mammary tumorigenesis induced by a direct-acting carcinogen and alters signaling pathways involving PR and HER-2/neu. [Copyright &y& Elsevier]