Search

Objective: To investigate the effect of dihydroartemisinin (DHA) for enhancing the inhibitory effect of cisplatin (DDP) on DDP-resistant nasopharyngeal carcinoma cell line HNE1/DDP and explore the mechanism., Methods: CCK-8 method was used to assess the survival rate of HNE1/DDP cells treated with DHA (0, 5, 10, 20, 40, 80, and 160 μmol/L) and DDP (0, 4, 8, 16, 32, 64, 128 μmol/L) for 24 or 48 h, and the combination index of DHA and DDP was calculated using Compusyn software. HNE1/DDP cells treated with DHA, DDP, or their combination for 24 h were examined for cell viability, proliferation and colony formation ability using CCK-8, EdU and colony-forming assays. Flow cytometry was used to detect cell apoptosis and intracellular reactive oxygen species (ROS). The expression levels of apoptosis-related proteins cleaved PARP, cleaved caspase-9 and cleaved caspase-3 were detected by Western blotting. The effects of N-acetyl-cysteine (a ROS inhibitor) on proliferation and apoptosis of HNE1/DDP cells with combined treatment with DHA and DDP were analyzed., Results: Different concentrations of DHA and DDP alone both significantly inhibited the viability of HNE1/DDP cells. The combination index of DHA (5 μmol/L) combined with DDP (8, 16, 32, 64, 128 μmol/L) were all below 1. Compared with DHA or DDP alone, their combined treatment more potently decreased the cell viability, colony-forming ability and the number of EdU-positive cells, and significantly increased the apoptotic rate, intracellular ROS level, and the expression levels of cleaved PARP, cleaved caspase-9 and cleaved caspase-3 in HNE1/DDP cells. N-acetyl-cysteine pretreatment obviously attenuated the inhibitory effect on proliferation and apoptosis-inducing effect of DHA combined with DDP in HNE1/DDP cells ( P <0.01)., Conclusion: DHA enhances the growth-inhibitory and apoptosis-inducing effect of DDP on HNE1/DDP cells possibly by promoting accumulation of intracellular ROS.

Objective: To explore the effect of pristimerin combined with cisplatin on proliferation and apoptosis of nasopharyngeal carcinoma cells., Methods: CCK-8 assay was used to examine the survival rate of HNE-1 and CNE-2Z cells following treatment for 24 h with different concentrations of pristimerin, cisplatin or their combination. The changes in colony formation ability, apoptosis, and intracellular reactive oxygen species (ROS) levels of the treated cells were analyzed using colony formation assay and flow cytometry. Western blotting was performed to detect the changes in protein expressions in the cells. The effects of pre-treatment with NAC on proliferation, apoptosis, and PI3K/AKT signaling pathway were observed in pristimerin- and/or cisplatin-treated cells., Results: Both pristimerin and cisplatin significantly lowered the survival rate of HNE-1 and CNE-2Z cells ( P < 0.05). Compared with pristimerin or cisplatin alone, their combination more strongly inhibited survival and colony formation ability of the cells, increased cell apoptosis rate and intracellular ROS levels, upregulated the protein expressions of Bax, cleaved caspase-3, and cleaved PARP, and downregulated the protein expressions of Bcl-2, Mcl-1, PARP and p-PI3K and p-AKT ( P < 0.05). NAC pretreatment significantly attenuated proliferation inhibition and apoptosis-promoting effects of pristimerin combined with cisplatin, and partially restored the downregulated protein expressions of p-PI3K and p-AKT in HNE-1 and CNE-2Z cells with the combined treatment ( P < 0.05)., Conclusion: Pristimerin can enhance cisplatin-induced proliferation inhibition and apoptosis in nasopharyngeal carcinoma cells, the mechanism of which may involve ROS-mediated deactivation of the PI3K/AKT signaling pathway.

We have previously identified that PPPDE1 is a deubiquitinase (DUB) belonging to a cysteine isopeptidase family. Here we sought to explore the biological significance of PPPDE1 in hepatocellular carcinoma and its underlying molecular mechanism. In the present study, we found that amplification and overexpression of PPPDE1 were associated with poor prognosis in hepatocellular carcinoma (HCC). We also demonstrated that knocking down of PPPDE1 could significantly block the clonal growth and tumorigenicity of human HCC cells, which revealed a critical role for PPPDE1 in HCC development. Furthermore, we proved that PPPDE1 is a key modulator of p53 protein level and its down stream apoptosis pathway. Taken together, these results suggested that PPPDE1 is a putative HCC driver gene and extensive studies should be conducted in the future to investigate the role of PPPDE1 in HCC and other tumors.

Presently, developing effective anti-colon cancer drugs still remains to be important. Ginkgolic acids (GA), as a botanical drug extracted from the seed coat of Ginkgo biloba L., possess various bioactive properties. Our findings, for the first time, indicated that GA suppressed colon cancer cell proliferation, migration and invasion. GA led to cell death through G0/G1 phase arrest. In addition, apoptosis was significantly induced by GA treatment. The intrinsic apoptosis pathway was included, proved by the release of cytochrome c (Cyto-c) from the mitochondria into the cytosol. GA-induced autophagy was supported by the dose-dependent increase of LC3BII, autophagy-related gene-5 (ATG-5) and Beclin-1. Notably, silencing ATG-5 further reduced the cell viability and enhanced apoptosis in GA-treated colon cancer cells, indicating that GA-induced apoptosis rather than autophagy contributes to colon cancer cell death. And mammalian target of rapamycin complex 1 (mTORC1) was dose-dependently reduced by GA, evidenced by the reduction of p-mTOR, p-p70 ribosomal S6 kinase (p70s6k) and p-pras40. Moreover, GA markedly resulted in reactive oxygen species (ROS) generation, along with increased H 2 O 2 and O 2 . However, blocking ROS generation using its scavenger, NAC, significantly recovered GA-induced cells death, supported by the increase of cell viability, and the decrease of apoptosis. The expressions of autophagy- and cell cycle arrest-related molecules, as well as mTORC1 were also reversed by N-acetyl-l-cysteine (NAC) in GA-treated cells. In vivo, GA reduced tumor growth without toxicity to animals. In conclusion, our study illustrated that GA caused G0/G1 phase arrest and triggered intrinsic apoptosis and autophagy modulated by ROS generation in human colon cancer, elucidating that GA might be considered as a potential agent for colon cancer therapy.- . However, blocking ROS generation using its scavenger, NAC, significantly recovered GA-induced cells death, supported by the increase of cell viability, and the decrease of apoptosis. The expressions of autophagy- and cell cycle arrest-related molecules, as well as mTORC1 were also reversed by N-acetyl-l-cysteine (NAC) in GA-treated cells. In vivo, GA reduced tumor growth without toxicity to animals. In conclusion, our study illustrated that GA caused G0/G1 phase arrest and triggered intrinsic apoptosis and autophagy modulated by ROS generation in human colon cancer, elucidating that GA might be considered as a potential agent for colon cancer therapy., (Copyright © 2018. Published by Elsevier Inc.)

Baicalin, an active flavone isolated from Scutellaria baicalensis Georgi, has been demonstrated to induce various beneficial biochemical effects such as anti‑inflammatory, anti‑viral, and antitumor effects. However, the antitumor mechanism of baicalin is not well understood. In the present study, baicalin was demonstrated to inhibit the viability and migration of a widely used ovarian cancer cell line, A2780, in a dose‑dependent manner. MTT assays revealed that cell viability significantly decreased in ovarian cancer cells treated with baicalin compared with untreated cells, without effect on normal ovarian cells. Flow cytometric analysis indicated that baicalin suppressed cell proliferation by inducing apoptosis. The underlying mechanisms involved were indicated to be downregulation of the anti‑apoptotic protein B‑cell lymphoma 2 apoptosis regulator and activation of caspase‑3 and ‑9. In addition, wound healing and transwell assays revealed that cell migratory potential and expression of matrix metallopeptidase (MMP)‑2 and MMP‑9 were significantly inhibited when cells were exposed to baicalin, compared with untreated cells. The present study therefore suggested that baicalin has the potential to be used in novel anti‑cancer therapeutic formulations for treatment of ovarian cancer.

Epithelial barrier disruption is a major cause of inflammatory bowel disease (IBD); however, the mechanism through which epigenetic regulation modulates intestinal epithelial integrity remains largely undefined. Here we show that EZH2, the catalytic subunit of polycomb repressive complex (PRC2), is indispensable for maintaining epithelial cell barrier integrity and homeostasis under inflammatory conditions. In accordance with reduced EZH2 expression in patients, the inactivation of EZH2 in IECs sensitizes mice to DSS- and TNBS-induced experimental colitis. Conversely, EZH2 overexpression in the intestinal epithelium renders mice more resistant to colitis. Mechanistically, the genes encoding TRAF2/5 are held in a finely tuned bivalent status under inflammatory conditions. EZH2 deficiency potentiates the expression of these genes to enhance TNFα-induced NF-κB signaling, thereby leading to uncontrolled inflammation. More importantly, we show that EZH2 depletion compromises the protective role of NF-κB signaling in cell survival by directly up-regulating ITCH, a well-known E3 ligase that degrades the c-FLIP protein. Thus, our findings highlight an epigenetic mechanism by which EZH2 integrates the multifaceted effects of TNFα signaling to promote the inflammatory response and apoptosis in colitis., Competing Interests: The authors declare no conflict of interest.

Puerarin, a bioactive isoflavone glucoside extracted from radix Puerariae, has been proven to possess many biological activities. However, the role of puerarin in protecting bovine Sertoli cells (bSCs) under heat stress conditions remains to be clarified. The present study aimed to explore the possible protective mechanism of puerarin for primary cultured bSCs subjected to heat stress. Bovine Sertoli cells were treated with 15 μM of puerarin before they were exposed to 42 °C for 1 hour. The dose of puerarin (15 μM) was determined on the basis of cell viability. The results showed that puerarin treatment suppressed the production of reactive oxygen species and decreased the oxidative damage of the bSCs subjected to heat stress, as indicated by changes in superoxide dismutase, catalase, and glutathione peroxidase activities and malondialdehyde content. Moreover, puerarin treatment also suppressed the initiation of mitochondria-dependent apoptotic pathway, as revealed by changes in Bax to Bcl-2 ratio, mitochondrial membrane potential, cytochrome C release, caspase-3 activation, and apoptotic rate compared with the heat stress group. In addition, puerarin treatment increased Hsp72 expression in the bSCs with no apparent cellular cytotoxicity compared with the control group. Furthermore, increased Hsp72 was detected in the heat stress plus puerarin group compared with the heat stress group. In conclusion, puerarin attenuates heat stress-induced oxidative damage and apoptosis of bSCs by suppressing reactive oxygen species production and upregulating Hsp72 expression., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Scutellaria baicalensis has been effectively used in Chinese traditional medicine to prevent miscarriages. However, little information is available on its mechanism of action. This study is designed specifically to reveal how baicalin, the main effective ingredient of S. baicalensis, improves developmental competence of embryos in vitro, using the mouse as a model. Mouse pronuclear embryos were cultured in KSOM medium supplemented with (0, 2, 4 and 8 μg/ml) baicalin. The results demonstrated that in vitro culture conditions significantly decreased the blastocyst developmental rate and blastocyst quality, possibly due to increased cellular stress and apoptosis. Baicalin (4 µg/ml) significantly increased 2- and 4-cell cleavage rates, morula developmental rate, and blastocyst developmental rate and cell number of in vitro-cultured mouse embryos. Moreover, baicalin increased the expression of Gja1, Cdh1, Bcl-2, and Dnmt3a genes, decreased the expression of Dnmt1 gene, and decreased cellular stress and apoptosis as it decreased the expression of HSP70, CASP3, and BAX and increased BCL-2 expression in blastocysts cultured in vitro. In conclusion, baicalin improves developmental competence of in vitro-cultured mouse embryos through inhibition of cellular apoptosis and HSP70 expression, and improvement of DNA methylation.

Diabetes-induced testicular cell death is due predominantly to oxidative stress. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is an important transcription factor in controlling the antioxidative system and is inducible by sulforaphane (SFN). To test whether SFN prevents diabetes-induced testicular cell death, an insulin-defective stage of type 2 diabetes (IDS-T2DM) was induced in mice. This was accomplished by feeding them a high-fat diet (HFD) for 3 mo to induce insulin resistance and then giving one intraperitoneal injection of streptozotocin to induce hyperglycemia while age-matched control mice were fed a normal diet (ND). IDS-T2DM and ND-fed control mice were then further subdivided into those with or without 4-mo SFN treatment. IDS-T2DM induced significant increases in testicular cell death presumably through receptor and mitochondrial pathways, shown by increased ratio of Bax/Bcl2 expression and cleavage of caspase-3 and caspase-8 without significant change of endoplasmic reticulum stress. Diabetes also significantly increased testicular oxidative damage and inflammation. All of these diabetic effects were significantly prevented by SFN treatment with upregulated Nrf2 expression. These results suggest that IDS-T2DM induces testicular cell death presumably through caspase-8 activation and mitochondria-mediated cell death pathways and also by significantly downregulating testicular Nrf2 expression and function. SFN upregulates testicular Nrf2 expression and its target antioxidant expression, which was associated with significant protection of the testis from IDS-T2DM-induced germ cell death., (Copyright © 2014 the American Physiological Society.)

Background: Ischemic preconditioning (IPC) can reduce ischemia/reperfusion (I/R) injury in multiple organs and species. However, the effect of IPC on transplanted submandibular glands remains unknown. We explored the protection of IPC in transplanted submandibular glands in the rabbit and the underlying mechanism., Methods: IPC was performed by clamping the lingual artery for 10 min, with 10 min of reperfusion before transplantation. Male rabbits were randomly divided into control, transplantation, and IPC groups (n = 6 each). Saliva secretion, oxidative stress, pro-inflammatory cytokine levels, and apoptosis-related protein levels were determined at 1, 12, and 24 h after reperfusion., Results: Salivary flow was significantly increased at 12 h and decreased at 24 h in the transplanted glands. IPC treatment prevented the reduced saliva secretion at 24 h after reperfusion (P < 0.01). The mRNA levels of tumor necrosis factor-α, interleukin-1β, and reactive oxygen species, as well as malondialodehyde (MDA) and myeloperoxidase activity, were significantly increased and superoxide dismutase activity was decreased in the transplanted glands. However, these changes were all attenuated with IPC treatment (all P < 0.05). Also, acinar cell apoptosis and Bax protein expression were decreased and Bcl-2 protein expression was increased in the IPC-treated glands at 1 and 12 h after reperfusion (all P < 0.05)., Conclusions: IPC protects the secretory function of transplanted submandibular gland in the rabbit by reducing the inflammatory response, attenuating oxidative stress, and an anti-apoptosis process., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Poor survival of mesenchymal stem cells (MSCs) compromised the efficacy of stem cell therapy for myocardial infarction. The increase of exogenous reactive oxygen species (ROS) in infracted heart is one of the important factors that challenged the survival of donor MSCs. In the study we aimed to evaluate the effect of oxidative stress on the cell death of MSCs and investigate its mechanisms in order to help with the identification of new biological compounds to reduce donor cells damage. Apoptosis of MSCs were evaluated with Hoechst 33342 staining and flow cytometry analysis. The mitochondrial membrane potential of MSCs was analyzed with JC-1 staining. Signaling pathways involved in H(2)O(2) induced apoptosis were analyzed with Western blot. H(2)O(2) induced apoptosis of MSCs in a dose- and time-dependent manner. H(2)O(2) induced apoptosis of MSCs via both endoplasmic reticulum (ER) and mitochondrial pathways rather than extrinsic apoptosis pathway. H(2)O(2) caused transient rather than sustained activation of p38 and JNK with no effect on ERK1/2 pathway. P38 was involved in the regulation of early apoptosis of MSCs while JNK was involved in the late apoptosis. P38 directed both ER stress and mitochondria death pathway in the early apoptosis. In conclusion, exogenous ROS was a major factor to induce apoptosis of MSCs. Both ER stress and mitochondria death pathway were involved in the apoptosis of MSCs. H(2)O(2) activated p38 that directed the above two pathways in the regulation of early apoptosis of MSCs while JNK was involved in the late apoptosis of MSCs., (© 2010 Wiley-Liss, Inc.)

Poor viability of transplanted mesenchymal stem cells (MSCs) in the infracted heart has limited their therapeutic efficacy in cardiac repair after myocardial infarction. We previously demonstrated that hypoxia and serum deprivation (hypoxia/SD) induced mitochondria-dependent apoptosis in MSCs, while lysophosphatidic acid (LPA) could almost completely block this apoptotic process. However, the role of endoplasmic reticulum (ER) stress and its upstream signaling events in hypoxia/SD-induced MSC apoptosis remain largely unknown. Here we found that hypoxia/SD-induced MSC apoptosis was associated with ER stress, as shown by the induction of CHOP expression and procaspase-12 cleavage, while the effects were abrogated by LPA treatment, suggesting ER stress is also a target of LPA. Furthermore, hypoxia/SD induced p38 activation, inhibition of which resulted in decreases of apoptotic cells, procaspase-12 cleavage and mitochondrial cytochrome c release that function in parallel in MSC apoptosis. Unexpectedly, p38 inhibition enhanced hypoxia/SD-induced CHOP expression. Interestingly, p38 activation, a common process mediating various biological effects of LPA, was inhibited by LPA in this study, and the regulation of p38 pathway by LPA was dependent on LPA(1/3)/Gi/ERK1/2 pathway-mediated MKP-1 induction but independent of PI3K/Akt pathway. Collectively, our findings indicate that ER stress is a target of LPA to antagonize hypoxia/SD-induced MSC apoptosis, and the modulation of mitochondrial and ER stress-associated apoptotic pathways by LPA is at least partly dependent on LPA(1/3)/Gi/ERK/MKP-1 pathway-mediated p38 inhibition. This study may provide new anti-apoptotic targets for elevating the viability of MSCs for therapeutic potential of cardiac repair., (© 2010 Wiley-Liss, Inc.)

Objective: to explore whether there is an interaction between Helicobacter pylori (H. pylori), omeprazole and gastrin on the proliferation and apoptosis of gastric epithelial cells., Methods: With omeprazole, H. pylori and gastrin as treatment factors, the in vitro changes of gastric epithelial cells AGS and GES-1 were detected by MTS, Hoechst33342 and flow cytometry., Results: (1) for a single factor, the gastric mucosal cells (AGS and GES-1) treated by H. pylori or omeprazole show significantly higher apoptosis than that of control group [H. pylori and 104 nmol/L omeprazole group: (17.20 ± 0.90)%, (12.81 ± 0.78)% vs (7.96 ± 1.25)%; (4.60 ± 0.34)%, (6.60 ± 0.76)% vs (3.52 ± 0.16)%; all P < 0.05]. The gastric mucosal cells treated by H. pylori or omeprazole show significantly lower proliferation than that of control group (H. pylori and 104 nmol/L omeprazole group: 13.35 ± 0.55 vs 37.78 ± 1.98, 47.62 ± 2.40 vs 62.44 ± 4.46; 27.15 ± 1.64 vs 32.76 ± 1.57, 29.42 ± 1.44 vs 48.86 ± 4.95; all P < 0.05). The differences were statistically significant and the effects were correlated with the concentration of omeprazole. Gastrin had no direct effect on cellular proliferation and apoptosis (all P > 0.05). (2) For a combinations of factors, the GES-1 cells treated with H. pylori and 500 ng/L gastrin had a significantly higher ratio [(7.25 ± 0.54)% vs (4.60 ± 0.34)%, P < 0.05], while the other groups just showed a rising trend in the proportion of apoptosis cells, but there was no statistical significance. Flow cytometry analysis showed that combinations of H. pylori, omeprazole and gastrin caused lower proliferation than that of H. pylori group, and all changes were statistically significant (AGS: 12.68 ± 0.09, 12.28 ± 0.31 vs 13.35 ± 0.55; GES-1: 22.06 ± 1.61, 18.59 ± 0.09 vs 27.15 ± 1.64; all P < 0.05). And combinations of 104 nmol/L omeprazole and 500 ng/L gastrin caused lower proliferation than that of 104 nmol/L omeprazole group (33.23 ± 5.98 vs 47.62 ± 2.40 and 25.97 ± 0.74 vs 29.42 ± 1.44, both P < 0.05). (3) When comparing the apoptosis and proliferation changes of two cell lines, gastric cancer cells AGS had a more marked changes than immortalized cells GES-1., Conclusion: gastrin significantly enhances the effects of omeprazole and H. pylori on gastric epithelial cells. It is speculated that the factors of omeprazole, H. pylori and gastrin may have a synergistic effect of decreased proliferation and increased apoptosis of gastric mucosal cells. Thus a long-term proton pump inhibitor treatment of H. pylori patients may carry a higher risk of atrophic gastritis. But it needs to be confirmed by further experiments.

Background and Objectives: Mesenchymal stem cells (MSC) are multipotent progenitor cells that are have found use in regenerative medicine. We have previously observed that aspirin, a widely used anti-inflammatory drug, inhibits MSC proliferation. Here we have aimed to elucidate whether aspirin induces MSC apoptosis and whether this is modulated through the Wnt/beta-catenin pathway., Materials and Methods: Apoptosis of MSCs was assessed using Hoechst 33342 dye and an Annexin V-FITC/PI Apoptosis Kit. Expression of protein and protein phosphorylation were investigated using Western blot analysis. Caspase-3 activity was detected by applying a caspase-3/CPP32 Colorimetric Assay Kit., Results: In these MSCs, aspirin induced morphological changes characteristic of apoptosis, cytochrome c release from mitochondria, and caspase-3 activation. Stimulating the Wnt/beta-catenin pathway by both Wnt 3a and GSK-3beta inhibitors (LiCl and SB 216763), blocked aspirin-induced apoptosis and protected mitochondrial function, as demonstrated by decreased cytochrome c release and caspase-3 activity. Aspirin initially caused a time-dependent decrease in COX-2 expression but subsequently, and unexpectedly, elevated the latter. Stimulation of COX-2 expression by aspirin was further enhanced following stimulation of the Wnt/beta-catenin pathway. Application of the COX-2 inhibitor NS-398 suppressed elevated COX-2 expression and promoted aspirin-induced apoptosis., Conclusion: These results demonstrate that the Wnt/beta-catenin pathway is a key modulator of aspirin-induced apoptosis in MSCs by regulation of mitochrondrial/caspase-3 function. More importantly, our findings suggest that aspirin may influence MSC survival under certain conditions; therefore, it should be used with caution when considering regenerative MSC transplantation in patients with concomitant chronic inflammatory diseases such as arthritis.

Lysophosphatidic acid (LPA), as an endogenous lipid mediator, has been revealed to regulate many important biological and pathophysiological processes via specific G-protein-coupled receptors termed LPA1-5. We have previously shown that LPA antagonized the apoptosis of mesenchymal stem cells (MSCs) induced by hypoxia and serum deprivation (hypoxia/SD), mimicking ischemic myocardium microenvironment. Whether LPA has the same potentially beneficial effect on MSCs in vivo is unknown. Here we demonstrated that LPA treatment improved graft MSC survival in ischemic myocardium assessed in a gender-mismatched transplantation model by real-time PCR, as well as by TUNEL assay. Moreover, transplantation of LPA-treated MSCs enhanced capillary density determined by immunostaining for platelet endothelial cell adhesion molecule (PECAM)-1, and it is also found that LPA enhanced vascular endothelial growth factor (VEGF) release from MSCs under hypoxia/SD in vitro. We did not get any improvement in left ventricular (LV) function at 1 week after transplantation of LPA-treated MSCs. These data suggest that LPA exerts both protective actions on MSC survival and enhancement on MSC paracrine in vivo and may represent a novel and effective treatment strategy in cell transplantation.

Bone marrow-derived mesenchymal stem cells (MSCs) have shown great promise for cardiac repair. However, poor viability of transplanted MSCs within the ischemic heart has limited their therapeutic potential. Our previous studies have documented that hypoxia and serum deprivation (hypoxia/SD), induced MSCs apoptosis through the mitochondrial apoptotic pathway. Since serum lysophosphatidic acid (LPA) levels are known to be significantly elevated after acute myocardial infarction and that LPA enhanced survival of other cell systems, we embarked on determining whether LPA protects MSCs against hypoxia/SD-induced apoptosis. We have also investigated the potential mechanism(s) that may mediate such actions of LPA. All experiments were carried out on rat bone marrow MSCs. Apoptosis was induced by exposure of cells to hypoxia/SD in a sealed GENbox hypoxic chamber. Effects of LPA were investigated in the absence and presence of inhibitors that target either G(i)proteins, the mitogen activated protein kinases ERK1/2, or phosphoinositide 3-kinase (PI3K). The data obtained showed that hypoxia/SD-induced apoptosis was significantly attenuated by LPA through Gi-coupled LPA(1) receptors linked to the downstream ERK1/2 and PI3K/Akt signaling pathways that function in parallel. Additional studies have demonstrated that hypoxia/SD-induced activation of mitochondrial dysfunction was virtually abolished by LPA treatment and that inhibition of the LPA(1) receptor, Gi proteins, the PI3K/Akt pathway, or ERKs effectively reversed this protective action of LPA. Taken together, our findings indicate that LPA is a novel, potent survival factor for MSCs and this may prove to be of considerable therapeutic significance in terms of exploiting MSC-based therapy in the infracted myocardium.

Cell therapy with bone marrow-derived mesenchymal stem cells (MSCs) has been shown to have great promises in cardiac repair after myocardial infarction. However, poor viability of transplanted MSCs in the infracted heart has limited the therapeutic efficacy. Our previous studies have shown in vitro that rat MSCs undergo caspase-dependent apoptosis in response to hypoxia and serum deprivation (Hypoxia/SD). Recent findings have implicated statins, an established class of cholesterol-lowering drugs, enhance the survival of cells under various conditions. In this study, we investigated the effect of lovastatin on rat MSCs apoptosis induced by Hypoxia/SD, focusing in particular on regulation of mitochondrial apoptotic pathway and the survival signaling pathways. We demonstrated that lovastatin (0.01-1 microM) remarkably prevented MSCs from Hypoxia/SD-induced apoptosis through inhibition of the mitochondrial apoptotic pathway, leading to attenuation of caspase-3 activation. The loss of mitochondrial membrane potential and cytochrome-c release from mitochondria to cytosol were significantly inhibited by lovastatin. Furthermore, the antiapoptotic effect of lovastatin on mitochondrial apoptotic pathway was effectively abrogated by both PI3K inhibitor, LY294002 and ERK1/2 inhibitor, U0126. The phosphorylations of Akt/GSK3 beta and ERK1/2 stimulated by lovastatin were detected. The activation of ERK1/2 was inhibited by a PI3K inhibitor, LY294002, but U0126, a ERK1/2 inhibitor did not inhibit phosphorylation of Akt and GSK3 beta. These data demonstrate that lovastatin protects MSCs from Hypoxia/SD-induced apoptosis via PI3K/Akt and MEK/ERK1/2 pathways, suggesting that it may prove a useful therapeutic adjunct for transplanting MSCs into damaged heart after myocardial infarction., (Copyright (c) 2007 Wiley-Liss, Inc.)

To investigate the apoptosis effect of SARS coronavirus nucleocapsid protein on cultured cell lines and to explore the possible pathway of apoptosis. pCDNA3.1(-)/his-myc vector containing the SARS coronavirus nucleocapsid gene (N), matric gene (M), spike gene (S) were transfected into COS-1, Huh-7 and HepG2 cells. Apoptosis induced by SARS coronavirus N protein under starvation of serum of COS-1 cells was monitored by Annexin V and electron microscopy assays. Intracellular reactive oxygen species (ROS) and mitochondrial membrane potential (DeltaPsim) were determined by flow cytometric assay. Cytochrome C, cleaved caspase (cysteine aspartic acid protease)-3, 9, and poly (ADP-ribose) polymerase (PARP) were detected by Western blot. After removal of serum in COS-1 cells, we observed the loss of DeltaPsim, the increase of ROS and cytochrome C release into cytosol and subsequent activation of caspase-3 and PARP cleavage. The pan-caspase inhibitor z-VAD-fmk can block the activation of caspase 3, 9 and PARP cleavage. In conclusion, SARS coronavirus N protein can induce apoptosis of COS-1 cells by activating mitochondrial pathway. SARS coronavirus M, S protein can not induce apoptosis in COS-1, HepG2 and Huh-7 and SARS coronavirus N protein can not induce apoptosis in HepG2 and Huh-7 by methods used in this study.

In recent years, the understanding that regeneration progresses at the level of the myocardium has placed stem cell research at the center stage in cardiology. Despite an increasing interest in cell transplant research, relatively little is known about the biochemical regulation of the stem cell itself after transplantation into an ischemic heart. We demonstrated here, using rat mesenchymal stem cells (MSCs), that cells undergo caspase-dependent apoptosis in response to hypoxia and serum deprivation (SD), which are both components of ischemia in vivo. In particular, the treated cells exhibited mitochondrial dysfunction, including cytochrome C release, loss in DeltaPsim, and Bax accumulation, but in a p53-independent manner. Although the cells treated by hypoxia/SD possess the activity of caspase-8, zIEDT-fmk, a specific caspase-8 inhibitor, failed to inhibit cell apoptosis induced in our system. Taken together, our findings indicate that MSCs are sensitive to hypoxia/SD stimuli that involve changes in mitochondrial integrity and function but are potentially independent of caspase-8.

Objective To investigate a possible role of adiponectin in the pathogenesis of autoimmune sialoadenitis in non-obese diabetic ( NOD) mouse model of Sjögren's syndrome. Materials and Methods Expression of adiponectin and its receptors (AdipoR1/2) was detected by PCR, immunoblotting, or immunofluorescence. The level of adiponectin was quantified by ELISA. Adiponectin-related signaling molecules and pro-inflammatory cytokines were examined by PCR or immunoblotting. Apoptosis was evaluated by TUNEL staining, flow cytometry, and caspase 3 activation. Results Adiponectin and AdipoR1/2 mRNA and protein were expressed in submandibular glands. Adiponectin immunostaining was widely diffused in the cytoplasm of acinar and ductal cells. AdipoR1 was mainly distributed in acinar cytoplasm, while AdipoR2 was predominantly located at acinar cell membrane. Submandibular adiponectin levels were reduced during the progression of autoimmune sialoadenitis in 7-, 14-, and 21-week-old NOD mice, while AdipoR1/2 levels were unchanged. The levels of phosphorylated adenosine monophosphate-activated protein kinase, extracellular signal-regulated kinase 1/2, and p38 mitogen-activated protein kinase were decreased, while interferon (IFN)- γ and glandular apoptosis were temporally increased at all time points. Moreover, exogenous adiponectin supplement inhibited, whereas neutralizing endogenous adiponectin by its antibody promoted IFN- γ-induced apoptosis and caspase 3 activation in cultured submandibular acinar cells. Conclusions Adiponectin plays a protective role on submandibular cells. Decreased adiponectin might promote glandular destruction in autoimmune sialoadenitis. [ABSTRACT FROM AUTHOR]

Oncogenic mutations in components of the JAK/STAT pathway, including those in cytokine receptors and JAKs, lead to increased activity of downstream signaling and are frequently found in leukemia and other hematological disorders. Thus, small-molecule inhibitors of this pathway have been the focus of targeted therapy in these hematological diseases. We previously showed that t(8;21) fusion protein acute myeloid leukemia (AML)1-ETO and its alternatively spliced variant AML1-ETO9a (AE9a) enhance the JAK/STAT pathway via downregulation of CD45, a negative regulator of this pathway. To investigate the therapeutic potential of targeting JAK/STAT in t(8;21) leukemia, we examined the effects of a JAK2-selective inhibitor TG101209 and a JAK1/2-selective inhibitor INCB18424 on t(8;21) leukemia cells. TG101209 and INCB18424 inhibited proliferation and promoted apoptosis of these cells. Furthermore, TG101209 treatment in AE9a leukemia mice reduced tumor burden and significantly prolonged survival. TG101209 also significantly impaired the leukemia-initiating potential of AE9a leukemia cells in secondary recipient mice. These results demonstrate the potential therapeutic efficacy of JAK inhibitors in treating t(8;21) AML. [ABSTRACT FROM AUTHOR]

An antisense strategy by targeting both bcr3/abl2 and VEGF was designed to suppress the growth of Philadephia1 leukemia cells in vitro and in vivo in mice. In vitro, although bcr3/abl2 or VEGF antisense oligodeoxyribonucleotides (AS-ODNs) alone was able to inhibit the proliferation of K562 cells, the combination of bcr3/abl2 and VEGF AS-ODNs produced an additive inhibitory effect on the growth of K562 cells and significantly enhanced the sensibility of K562 cells to apoptosis-inducing stimuli including STI571. In vivo, the nude mice xenografted with K562 cells received intratumoral injections of bcr3/abl2 and VEGF AS-ODNs showed a significant reduction in leukemia tumor size and microvessel density and an increase of apoptosis in the tumors when compared to the mice that received an individual agent. These results demonstrate that targeting both bcr3/abl2 and VEGF can result in an additive tumor-suppressive action and may represent an excellent strategy to augment the efficacy of chemotherapy in CML.Leukemia (2005) 19, 1517–1524. doi:10.1038/sj.leu.2403851; published online 21 July 2005 [ABSTRACT FROM AUTHOR]