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Background: Xin-Ke-Shu (XKS), a patent medicine consisting of five commonly used traditional Chinese herbs, is used for the treatment of coronary heart diseases. A previous study showed that XKS has protective effects for ameliorating myocardial ischemia/reperfusion (I/R) injury., Purpose: This study was aimed to deeply understand the mechanisms and compatible principle of XKS against hypoxia/reoxygenation (H/R) injury and the contribution of each single herb to the efficacy of XKS., Methods: An H/R model in H9c2 cardiomyocytes was applied to mimic I/R injury observed in vivo. The cell viability, the levels of LDH, MDA, SOD, and apoptosis were determined to evaluate the cardioprotection of XKS and its subtracted formula (knocked out one herb) in H/R injury. Cell metabolomics, combined with western blot analysis, was performed to uncover the inert molecular mechanism of XKS against H/R injury., Results: Significant protective effects of XKS against oxidative stress and apoptosis induced by H/R injury were found in the pharmacodynamic evaluation. Moreover, the metabolic profile deviation of the H/R group from the control group was mainly ascribed to thirteen metabolites involved in four aberrant pathways, in which sphingolipid metabolism was revealed as the most relevant pathway involved in H/R injury (impact > 0.1). Notably, the accumulation of phytosphingosine (VIP = 5.84) was considered the most likely characteristic in H/R injury, which is well known to promote the opening of the mitochondrial permeability transition pore (mPTP) and activate cell apoptosis. Furthermore, XKS ameliorated all the abnormalities of the metabolic network in response to H/R injury. In agreement with this, a western blot analysis showed that XKS markedly regulated the over-expression of CaMK II and cleaved caspase-3. However, the subtracted formula showed no significant difference in comparison with the XKS group on protecting H/R injury except for QDS (subtracted Dan-Shen from XKS)., Conclusion: The roots of Salvia miltiorrhiza Bge. (Dan-Shen) play an important role in the regulation of Ca 2+ overloading, oxidative stress and apoptosis in H/R injury. Our study enabled information from holistic cell metabolomics to be used for mechanism and compatibility rule elucidations of TCMs., (Copyright © 2018. Published by Elsevier GmbH.)

New Findings: What is the central question of this study? What are the ultrastructural changes of the caecal mucosa and the status of epithelial cellular apoptosis and oxidative reactions in lactating goats after prolonged feeding with a high-concentrate diet? What is the main finding and its importance? High-concentrate diet results in ultrastructural damage to the caprine caecal epithelium. Increased oxidative and decreased antioxidative reactions are involved in the process of activating epithelial apoptosis in the caecal epithelium of goats fed a high-concentrate diet. Our results provide new insight into the relationship between abnormal fermentation in the hindgut and damage to the intestinal mucosal barrier. The effect of feeding a high-concentrate diet (HC) to lactating ruminants on their hindgut epithelial structure remains unknown. In this study, 12 lactating goats were randomly assigned to either HC (65% of dry matter as concentrate; n = 6) or a low-concentrate diet (LC; 35% of dry matter as concentrate; n = 6). After 10 weeks, the epithelial ultrastructure and cell apoptotic status in the caecal mucosa were determined by transmission electron microscopy and TUNEL, respectively. The results showed that the level of free lipopolysaccharide (P < 0.05), total volatile fatty acid concentrations (P < 0.1) and starch content (P < 0.05) in the caecal digesta were significantly increased in HC- compared with LC-fed goats. The HC-fed goats exhibited obvious epithelial cellular damage, with widened tight junction spaces, nuclear breakdown and mitochondrial swelling. Compared with their LC-fed counterparts, HC-fed goats showed greater apoptosis in the caecal epithelium, as evidenced by more TUNEL-positive apoptotic cells. Western blot analysis showed that there was no significant difference in activated caspase-3, Bax protein expression in caecal epithelial mucosa between HC- and LC-fed goats (P > 0.05). However, the level of malondialdehyde content in the caecal epithelium from HC-fed goats was markedly higher than that in LC-fed goats (P < 0.05), whereas the level of glutathione peroxidase and the superoxide dismutase activity were significantly decreased. Gene expressions of cytokines, including interleukin-1β, interleukin-6, interleukin-10, tumour necrosis factor-α and interferon-γ, as well as myeloperoxidase activity in the caecal mucosa did not show any significant difference between HC- and LC-fed goats. These results indicate that feeding a high-concentrate diet to lactating goats for a prolonged period results in abnormal fermentation and structural disruption in the hindgut, which is accompanied by greater cellular apoptosis and an enhanced oxidative stress response., (© 2015 The Authors. Experimental Physiology © 2015 The Physiological Society.)

The activation of group I metabotropic glutamate receptor (group I mGlus) has been shown to produce neuroprotective or neurotoxic effects. In this study, we investigated the effects of N-acetylcysteine (NAC), a precursor of the antioxidant glutathione, on group I mGlus activation in apoptosis of glial C6 and MN9D cell lines, and a rat model of Parkinson's disease (PD). We demonstrated that NAC protected against apoptosis through modulation of group I mGlus activity. In glial C6 cells, NAC promoted phosphorylation of ERK induced by (s)-3,5-dihydroxy-phenylglycine (DHPG), an agonist of group I mGlus. NAC enhanced the group I mGlus-mediated protection from staurosporine (STS)-induced apoptosis following DHPG treatment. Moreover, in rotenone-treated MN9D cells and PD rat model, NAC protected against group I mGlus-induced toxicity by compromising the decrease in phosphorylation of ERK, phosphorylation or expression level of TH. Furthermore, the results showed that NAC prohibited the level of ROS and oxidation of cellular GSH/GSSG (E(h)) accompanied by activated group I mGlus in the experimental models. Our results suggest that NAC might act as a regulator of group I mGlus-mediated activities in both neuroprotection and neurotoxicity via reducing the oxidative stress, eventually to protect cell survival. The study also suggests that NAC might be a potential therapeutics targeting for group I mGlus activation in the treatment of PD.

Objective: To investigate the anti-tumor effects and the mechanism of the recombinant fowlpox virus expressing Apoptin gene on human laryngeal carcinoma Hep-2., Method: Hep-2 cells cultured in vitro were infected with vFVApoptin. The anti-tumor effects on Hep-2 cells were measured through MTT staining and, the mitochondrial trans-membrane potential (delta psi m) and reactive oxygen species (ROS) were analyzed by flow cytometry. Western blot was used to detect the release of cytochrome c (Cyto c). Caspase-3/9 activities were measured by colorimetric assay., Result: vFVApoptin could restrain Hep-2 cells significantly and, had the function of down-regulating delta psi m, up-regulating ROS, promoting Cyto c release and activating Caspase-3/9., Conclusion: Cyto c were released from mitochondria by the function of up-regulating ROS of vFVApoptin. Cyto c triggered Caspase-9 and, after the activation of Caspase-9, downstream apoptotic factors, such as caspase-3, were activated. Eventually, Hep-2 cells were suppressed by mitochondrial pathway apoptosis induced by vFVApoptin.

Objective: To research the antitumor efficacy on mice model bearing H22 tumor with application of pVVP3IL-18HN and its mechanism of the antitumor effect on Hep-2., Method: pVVP3IL-18HN was introduced into Hep-2 cells by liposome, then cellular morphology was observed through AO/EB stain. To evaluate the mechanism of the antitumor effect on Hep-2 of pVVP3IL-18HN and its effect on tumor immunogenicity, flow cytometer (FCM) was used to detect the variation of mitochondrial trans-membrane potential,reactive oxygen species (ROS) and the MHC-I. C57BL/6 mice were subcutaneously inoculated with 2 X 10(5) H22 tumor cells in the right hindlimb, and 7, 14 and 21 day post-inoculation, pVVP3IL-18HN was administrated intratumorally. The pVVP3, pVIL-18, PVHN and PBS control group were treated as above. Then the tumor suppression and specific cytotoxic T lymphocyte (CTL) activity was detected., Result: After the transfection of pVVP3IL-18HN, tumor cells crenated and stained by AO/EB. In the research pVVP3IL-18HN was found to up-regulate the value of ROS and the expression of MHC-I, and down-regulate the value of mitochondrial trans-membrane potential. The tumor suppression rate of pVVP3IL-18HN was 60.5% and a dramatically increased tumor suppression was revealed in the pVVP3IL-18HN treated mice as compared with control mice treated with PBS (P < 0.05)., Conclusion: pVVP3IL-18HN kill the tumor cells by apoptosis and have notable antitumor effect in vivo.

Apoptin can specifically kill cancer cells but has no toxicity to normal cells. Human telomerase reverse transcriptase (hTERT) can act as a tumour‐specific promoter by triggering the expression of certain genes in tumour cells. This study aims to investigate the inhibitory effects and to explore the inhibitory pathway of a dual cancer‐specific recombinant adenovirus (Ad‐apoptin‐hTERTp‐E1a, Ad‐VT) on breast cancer stem cells. Breast cancer cell spheres were obtained from MCF‐7 cells through serum‐free suspension culture. The cell spheres were detected by flow cytometry for CD44+ CD24− cell subsets. The stemness of MCF‐7‐CSC cells was confirmed by in vivo tumorigenesis experiments. The inhibitory effect of the recombinant adenoviruses on MCF‐7‐CSC cells was evaluated by CCK‐8 assay. In addition, the stemness of adenovirus‐infected MCF‐7‐CSC cells was analysed by testing the presence of CD44+ CD24− cell subsets. The ability of the recombinant adenovirus to induce MCF‐7‐CSC cell apoptosis was detected by staining JC‐1, TMRM and Annexin V. Our results showed that a significantly higher proportion of the CD44+ CD24− cell subsets was present in MCF‐7‐CSC cells with a significantly increased expression of stem cell marker proteins. The MCF‐7‐CSC cells, whlist exhibited a strong tumorigenic ability with a certain degree of stemness in mice, were shown to be strongly inhibited by recombinant adenovirus Ad‐VT through cell apoptosis. In addition, Ad‐VT was shown to exert a killing effect on BCSCs. These results provide a new theoretical basis for the future treatment of breast cancer. [ABSTRACT FROM AUTHOR]

Apoptin is a protein that specifically induces apoptosis in tumor cells. The anti-tumorigenic functions of Apoptin, including autophagy activation and its interaction with apoptosis, have not been precisely elucidated. Here we investigate the main pathways of apoptin-mediated killing of human liver cancer cells, as well as its putative role in autophagy and apoptosis. The anti-proliferative effect of apoptin in liver cancer cells was analyzed in vitro by crystal violet staining and MTS detection, and also in vivo using a tumor-based model. The main pathway related to apoptin-induced growth inhibition in vitro was evaluated by flow cytometry and fluorescence staining. The relationship between apoptosis and autophagy on apoptin-treating cells was analyzed using apoptosis and autophagy inhibitors, mitochondrial staining, Annexin V-FITC/PI flow detection, LC3 staining, and western blotting. The effect of ROS toward the apoptosis and autophagy of apoptin-treating cells was also evaluated by ROS detection, Annexin V-FITC/PI flow detection, LC3 staining, and western blotting. Inhibition of apoptosis in apoptin-treating liver cancer cells significantly reduced the autophagy levels in vitro. The overall inhibition increased from 12 h and the effect was most obvious at 48 h. Inhibition of autophagy could increase apoptin-induced apoptosis of cells in a time-dependent manner, reaching its peak at 24 h. Apoptin significantly alters ROS levels in liver cancer cells, and this effect is directly related to apoptosis and autophagy. ROS appears to be the key factor linking apoptin-induced autophagy and apoptosis through the mitochondria in liver cancer cells. Therefore, evaluating the interaction between apoptin-induced apoptosis and autophagy is a promising step for the development of alternate tumor therapies. [ABSTRACT FROM AUTHOR]

Stachydrine, a constituent of Leonurus japonicus Houtt which also called Japanese motherwort has been shown to improve vascular microcirculation and ameliorate endothelial dysfunction. This study investigated the neuroprotective effect of stachydrine. Male Sprague-Dawley (SD) rats were randomly divided into sham, control, and stachydrine groups. The neurological deficit score was evaluated and the infarct size of the brain was measured using 2,3,5-triphenyltetra-zolium (TTC) chloride staining assay, and the pathological changes in the brain tissues were examined by HE staining. Nissl and terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling (TUNEL) staining were performed to assess the numbers of Nissl bodies and the levels of apoptosis in the neurons. The activities of superoxide dismutase (SOD) and the levels of malondialdehyde (MDA) were also measured. The release of inflammatory factors IL-1β and TNF-α were detected by Enzyme-linked immunosorbent assay (ELISA). Compared with the control group, the stachydrine group showed a significant prevention of neurological deficit, as indicated by the reduced infarct volume in the brain. Moreover, the stachydrine treatment reduced the activities of SOD, the levels of MDA and decreased the amount of IL-1β, and TNF-α, indicating that it could function to decrease the level of inflammation, thus reducing brain damage. The ischemic stroke model of PC12 cells was prepared via oxygen-glucose deprivation (OGD) protocol for 6 h. The expression of P65 and JAK2/STAT3 signaling pathway related proteins was measured by western blot. The treatment group was found to have the survival rate of PC12 cells improved and the release of inflammatory factors reduced when compared with the OGD group. This study demonstrated that stachydrine could improve nerve function by inhibiting the phosphorylation of P65/JAK2 and STAT3. [ABSTRACT FROM AUTHOR]

Aim: This study aimed to explore the miR‐342‐3p expression in pre‐eclampsia (PE) placentas and confirm whether miR‐342‐3p exerts effects on proliferation and migration of HTR‐8/SVneo trophoblastic cells. Methods: The PE placentas (n = 8) were taken from gravidas complicated by PE and delivered after 34 weeks. The chorionic plates and the basal plates were separately taken from the placenta disc near the position of umbilical cord insertion. RT‐qPCR was used to measure the expression of miR‐342‐3p in the chorionic plates and the basal plates. Cell invasion assay and MMT assay were used to assess the effects of miR‐342‐3p on proliferation and migration of HTR‐8/SVneo trophoblastic cells. Luciferase reporter assay and Western blotting were used to analyze the target of miR‐342‐3p and investigate the detailed mechanisms. Results: The expression of miR‐342‐3p was upregulated in both basal plates and chorionic plates in patients with PE compared with healthy pregnant individuals. MiR‐342‐3p inhibitor suppressed the cell viability and invasion, and induced apoptosis in trophoblast cells. Furthermore, inhibitor of DNA binding (ID)‐4 (ID4) was a direct target of miR‐342‐3p, and knockdown of ID4 abrogated the regulation effect of miR‐342‐3p on cell viability, apoptosis and invasion. Conclusion: Inhibition of miR‐342‐3p expression may suppress the occurrence of PE by targeting ID4 in vitro. [ABSTRACT FROM AUTHOR]

Although the generation of BCR-ABL is the molecular hallmark of chronic myeloid leukemia (CML), the comprehensive molecular mechanisms of the disease remain unclear yet. Growth arrest specific 2 (GAS2) regulates multiple cellular functions including cell cycle, apoptosis and calpain activities. In the present study, we found GAS2 was up-regulated in CML cells including CD34+ progenitor cells compared to their normal counterparts. We utilized RNAi and the expression of dominant negative form of GAS2 (GAS2DN) to target GAS2, which resulted in calpain activity enhancement and growth inhibition of both K562 and MEG-01 cells. Targeting GAS2 also sensitized K562 cells to Imatinib mesylate (IM). GAS2DN suppressed the tumorigenic ability of MEG-01 cells and impaired the tumour growth as well. Moreover, the CD34+ cells from CML patients and healthy donors were transduced with control and GAS2DN lentiviral vectors, and the CD34+ transduced (YFP+) progeny cells (CD34+YFP+) were plated for colony-forming cell (CFC) assay. The results showed that GAS2DN inhibited the CFC production of CML cells by 57±3% (n = 3), while affected those of normal hematopoietic cells by 31±1% (n = 2). Next, we found the inhibition of CML cells by GAS2DN was dependent on calpain activity but not the degradation of beta-catenin. Lastly, we generated microarray data to identify the differentially expressed genes upon GAS2DN and validated that the expression of HNRPDL, PTK7 and UCHL5 was suppressed by GAS2DN. These 3 genes were up-regulated in CML cells compared to normal control cells and the growth of K562 cells was inhibited upon HNRPDL silence. Taken together, we have demonstrated that GAS2 is up-regulated in CML cells and the inhibition of GAS2 impairs the growth of CML cells, which indicates GAS2 is a novel regulator of CML cells and a potential therapeutic target of this disease. [ABSTRACT FROM AUTHOR]

Background: Oncolytic virotherapy is an attractive drug platform of cancer gene therapy, but efficacy and specificity are important prerequisites for success of such strategies. Previous studies determined that Apoptin is a p53 independent, bcl-2 insensitive apoptotic protein with the ability to specifically induce apoptosis in tumor cells. Here, we generated a conditional replication-competent adenovirus (CRCA), designated Ad-hTERT-E1a-Apoptin, and investigated the effectiveness of the CRCA a gene therapy agent for further clinical trials. Results: The observation that infection with Ad-hTERT-E1a-Apoptin significantly inhibited growth of the melanoma cells, protecting normal human epidermal melanocytes from growth inhibition confirmed cancer cell selective adenoviral replication, growth inhibition, and apoptosis induction of this therapeutic approach. The in vivo assays performed by using C57BL/6 mice containing established primary or metastatic tumors expanded the in vitro studies. When treated with Ad-hTERT-E1a-Apoptin, the subcutaneous primary tumor volume reduction was not only observed in intratumoral injection group but in systemic delivery mice. In the lung metastasis model, AdhTERT- E1a-Apoptin effectively suppressed pulmonary metastatic lesions. Furthermore, treatment of primary and metastatic models with Ad-hTERT-E1a-Apoptin increased mice survival. Conclusions: These data further reinforce the previously research showing that an adenovirus expressing Apoptin is more effective and advocate the potential applications of Ad-hTERT-E1a-Apoptin in the treatment of neoplastic diseases in future clinical trials. [ABSTRACT FROM AUTHOR]

Immunization strategies using plasmid DNA can potentially improve humoral and cellular immune responses that protect against cancer and infectious diseases. The chicken anemia virus-derived Apoptin protein exhibits remarkable specificity in its ability to induce apoptosis in tumor cells, but not in normal diploid cells. Interleukin-18 (IL-18) is a Th1-type cytokine that has demonstrated potential as a biological adjuvant in murine tumor models. In this study, we analyzed the anti-tumor potential and mechanism of action of simultaneous Apoptin and IL-18 gene transfer in C57BL/6 mice bearing Lewis lung carcinoma (LLC). Here we report that the growth of established tumors in mice immunized with pAPOPTIN in conjunction with pIL-18 was significantly inhibited compared with the growth of tumors in mice immunized with the empty vector (EV) or pAPOPTIN alone. Furthermore, the immunization of mice with pAPOPTIN in conjunction with pIL-18 elicited strong natural killer activity and LLC tumor-specific cytotoxic T lymphocyte (CTL) responses in vitro. In addition, T cells from lymph nodes of mice vaccinated with pIL-18 or pAPOPTIN + pIL-18 secreted high levels of the Th1 cytokine IL-2 and IFN-γ, indicating that the regression of tumor cells is related to a Th1-type dominant immune response. These results demonstrate that vaccination with Apoptin together with IL-18 may be a novel and powerful strategy for cancer immunotherapy. [ABSTRACT FROM AUTHOR]

Clinical studies have demonstrated that conditionally replicating adenovirus is safe. We constructed an oncolytic adenovirus, Ad-hTERT-E1a-Apoptin, using a cancer-specific promoter (human telomerase reverse transcriptase promoter, hTERTp) and a cancer cell-selective apoptosis-inducing gene (Apoptin). Ad-hTERT-E1a-Apoptin was proven effective both in vitro and in vivo in our previous study. In this study, the preclinical safety profiles of Ad-hTERT-E1a-Apoptin in animal models were investigated. At doses of 5.0 × 10 8 , 2.5 × 10 9 , and 1.25 × 10 10 viral particles (VP)/kg, Ad-hTERT-E1a-Apoptin had no adverse effects on mouse behavior, muscle cooperation, sedative effect, digestive system, and nervous systems, or on beagle cardiovascular and respiratory systems at 5.0 × 10 8 , 2.5 × 10 9 , and 1.25 × 10 10 VP/kg doses. In acute toxicity tests in mice, the maximum tolerated dose > 5 × 10 10 VP/kg. There was no inflammation or ulceration at the injection sites within two weeks. In repeat-dose toxicological studies, the no observable adverse effect levels of Ad-hTERT-E1a-Apoptin in rats (1.25 × 10 10 VP/kg) and beagles (2.5 × 10 9 VP/kg) were 62.5- and 12.5-fold of the proposed clinical dose, respectively. The anti-virus antibody was produced in animal sera. Bone marrow examination revealed no histopathological changes. Guinea pigs sensitized by three repeated intraperitoneal injections of 1.35 × 10 10 VP/mL Ad-hTERT-E1a-Apoptin each and challenged by one intravenous injection of 1.67 × 10 8 VP/kg Ad-hTERT-E1a-Apoptin did not exhibit any sign of systemic anaphylaxis. Our data from different animal models suggest that Ad-hTERT-E1a-Apoptin is a safe anti-tumor therapeutic agent. [ABSTRACT FROM AUTHOR]

Kinesin family protein 2A (KIF2A), an M-type nonmotile microtubule depolymerase, plays essential roles in development and progression of various human cancers. However, its exact function and the underlying mechanism in tumorigenesis of gastric cancer (GC) haven't been fully elucidated. In the present study, KIF2A was overexpressed in human GC and predicted poor prognosis according to the results of GEPIA analysis. KIF2A was also observed to be upregulated in 82 GC samples compared with paired pericarcinoma tissues. Its overexpression was associated with tumor metastasis (P = 0.047) and Ⅲ stage GC (P = 0.0267). The mRNA and protein expression levels of KIF2A were significantly suppressed in KIF2A specific siRNA transfected GC cells compared with the wild-type and negative control (NC) siRNA transfected cells. Furthermore, the effects of KIF2A on the growth, migration, invasion, and apoptosis of GC cell were evaluated in vitro and the underlying mechanisms were explored. It was found that silencing KIF2A effectively induced the apoptosis, and inhibited the proliferation, migration and invasion capacities of GC cells. Western blot analysis demonstrated that silencing of KIF2A significantly decreased the expression levels of AKT, Cyclin D1 and S6K. Moreover, bioinformatics analysis showed that the promoter (from -414 to -407bp) of KIF2A has the ability to bind to transcription factor ETV4, which was confirmed by bi-luciferase reporter assay using 293T cells. The level of ETV4 was upregulated and positively correlated with KIF2A in human GC tissues. Our results also proved that ETV4 upregulated the expression of KIF2A and blocked the decline of proliferation induced by KIF2A knockdown in MKN-45 and AGS cells. In summary, KIF2A is upregulated by transcription factor ETV4, and its knockdown can effectively inhibit the proliferation and induce the apoptosis of GC cells through the AKT signaling pathway in GC cells, implying that the inhibition of KIF2A expression is a potential target for GC therapy. [ABSTRACT FROM AUTHOR]