Expression Of Smac Research Articles

In vivo and in vitro studies on the role of regulating Smac expression in the occurrence and development of colon cancer.

We aimed to explore the role of regulating Smac expression levels in the occurrence and development of colon cancer through in vitro and in vivo experiments. Colon cancer cells HT-29 were cultured and transfected into different groups. qRT-PCR was used to detect the expression level of Smac in cells; Flow cytometry was used to detect the apoptotic ability of each group of cells; Western blot was used to detect the protein expression of Smac and apoptosis-related factors Survivin and Caspase-3; The nude mouse tumorigenesis experiment was conducted to detect the regulatory effect of regulating Smac expression levels on the growth of colon cancer transplanted tumors in vivo. In comparison to the FHC group, the HT-29 group exhibited a decrease in Smac expression. The si-Smac group, when compared with the si-NC group, showed significant reductions in Smac mRNA and protein levels, weaker cell apoptosis, increased Survivin, and decreased Caspase-3 expression. Contrarily, the oe-Smac group, against the oe-NC group, displayed increased Smac mRNA and protein levels, enhanced apoptosis, reduced Survivin, and elevated Caspase-3 expression. In nude mice tumor transplantation experiments, the LV-sh-Smac group, as opposed to the LV-sh-NC group, had tumors with greater volume and weight, reduced Smac and Caspase-3, and increased Survivin expression. In contrast, the LV-oe-Smac group, compared with the LV-oe-NC group, showed tumors with decreased volume and mass, increased expressions of Smac and Caspase-3, and decreased Survivin. Smac is lowly expressed in colon cancer. Upregulation of Smac expression can inhibit the occurrence and development of colon cancer, possibly by inhibiting Survivin expression and promoting Caspase-3 expression, thereby enhancing the pro-apoptotic function.

Mechanistic analyses reveal that Pueraria montana var. lobata (Willd.) is effective in inhibiting ovarian cancer progression.

Ovarian cancer (OC) is a common malignancies of the female genitalia. P. montana var. lobata (Willd.), a herb with anti-tumor effects, is widely used in the clinical treatment of ovarian cancer (OC), but the ingredients and molecular mechanism of action remains to be explored. In this study, we extracted the main active ingredients of P. montana var. lobata (Willd.) from the TCMSP database, and predicted its potential targets of action against OC from the DisGeNET and GeneCards databases. Protein-protein interaction (PPI) was constructed using the STRING database, while pathway enrichment analyses were performed using the DAVID database. Next, we generated an Ingredient-Target-Pathway network using Cytoscape 3.7.2, then processed the key targets of action and main active ingredients for molecular docking.The results showed that seven active ingredients of P montana var. lobata (Willd.) were associated with treating for OC, namely beta-sitosterol, coumestrol, daidzein, formononetin, genistein, puerarin and scoparone, two important targets Casp3 and Jun, and signaling pathways of P. montana var. lobata (Willd.) against the progression of OC. TUNEL staining, enzyme-linked immunosorbent assay (ELISA), and Western blot assays, the pharmacodynamic effect of puerarin in the treatment of OC and the major targets were verified. Animal experiment demonstrated that application of puerarin at different times of modeling not only upregulated expression of Casp3, Smac, and c-jun proteins, but also promoted apoptosis in tumor cells, hence inhibiting progression of OC. This study demonstrates that P. montana var. lobata (Willd.) can thereby induce apoptosis in tumor cells and inhibit malignant progression through activating expression of Casp3, smac, and c-jun proteins to regulate related apoptosis pathways, as validated by network pharmacology predictions and animal experiments, and can be verifed by large-scale clinical trials in the future. This study also provides theoretical support and new research perspectives for this disease.

Effect of astragaloside on expression of smac, cytochrome C and caspase-3 in hepatic tissue of acute hepatic failure induced by D-Gal NLPS

This study assessed astragaloside IV’s effect on liver Smac, Cytochrome C (Cyt-C) and Caspase-3 expression in acute liver failure in mice. Mice were randomly assigned into normal group, model group, model+astragaloside (1%) group and model+astragaloside (5%) group. Mice were given by gavage of astragaloside (50 mg/kg/d, 250 mg/kg/d) for 2 d before making the model in model + astragaloside group and model+astragaloside group. Equal dose of normal saline was administrated to the other groups. Except for the normal group, mice in the other groups were intraperitoneally injected with D-GalN/LPS for 48 h. The mice were at the end killed after the final intraperitoneal injection for 2 hours. Hepatic apoptosis and expressions of hepatic Smac, Cyt-C and caspase-3 were then analyzed. Mice exhibited less hepatocyte apoptosis and lower content of hepatic Cyt-C expression in both the model+astragaloside and model+astragaloside groups (P <0.01). D-GalN/LPS significantly enhanced Caspase-3 and Smac mRNA. Moreover, astragaloside suppressed the activity of Caspase-3 and decreased hepatic Smac mRNA expression in mice (P <0.01). Astragaloside IV can reduce the content of Cyt-C and down-regulate the expression of Smac and caspase-3, thereby protecting acute liver failure.

LncRNA TUG1 regulates Smac/DIABLO expression by competitively inhibiting miR-29b and modulates the apoptosis of lens epithelial cells in age-related cataracts.

As one of the early discovered long non-coding RNAs (lncRNA), taurine upregulation gene 1 (TUG1) has been widely expressed in a variety of tumors. Moreover, it promotes cell proliferation, differentiation, apoptosis, and migration. However, our understanding of its importance in the pathogenesis of cataracts remains limited. This study aimed to explore the mechanism by which lncRNA TUG1 mediates lens epithelial cell apoptosis in age-related cataracts (ARC) by regulating the microRNAs (miR-29b)/second mitochondria-derived activator of caspases axis, and to identify more non-surgical strategies for cataract treatment. The messenger RNA expression levels of TUG1, miR-29b, and Smac were detected using quantitative real-time polymerase chain reaction in vivo and in vitro. The expression of the Smac protein was analyzed by Western blotting and immunofluorescence. Flow cytometry and cell counting kit-8 assays were used to detect the cell apoptosis and proliferation rates, respectively. The targeted regulatory relationship between lncRNA TUG1, miR-29b, and Smac was verified by viral vector construction, co-transfection, nuclear and cytoplasmic separation, luciferase reporter assays, and RNA immunoprecipitation. TUG1 and Smac were expressed at high levels in ARC and HLE-B3 cells treated with 200 μmol/L H2O2, whereas miR-29b expression was decreased. In vitro cell experiments confirmed that down-regulation of TUG1 could inhibit the apoptosis of lens epithelial cells. Mechanistically, Smac expression was negatively regulated by miR-29b. TUG1 competitively inhibited miR-29b expression and caused greater release of Smac. In addition, miR-29b partially reversed the effects of TUG1 on human lens epithelial cell line cells. lncRNA TUG1 increases Smac expression and promotes apoptosis of lens epithelial cells in ARC by competitively inhibiting miR-29b. This mechanism is the cytological basis for ARC formation. Based on these results, the lncRNA TUG1/miR29b/Smac axis may be a new molecular pathway that regulates ARC development.

Effect of Butylphthalide Capsules on Smac and XIAP Expression in Rats after Ischemia Reperfusion

IntroductionLittle is known about the protective effects of butylphthalide on cerebral ischemia-reperfusion injury. This study aims to investigate the impact on the second mitochondrial-derived activator of Caspases (Smac) and X-linked inhibitor of apoptosis protein (XIAP) expression in the ischemic semidark area using a rat model of carotid artery stenosis. MethodsThirty Sprague–Dawley rats were randomly divided into the sham-operated group, carotid stenosis model controls, low-dose (20 mg/kg), medium-dose (40 mg/kg), and high-dose (80 mg/kg) butylphthalide groups. The neurological function was scored by the balance beam test (BBT). The morphological changes of brain tissue were detected by Hematoxylin-eosin (HE) staining, with apoptosis detected by Terminal Deoxynucleotidyl Transferase mediated dUTP Nick-End Labeling (TUNEL) staining. Smac and XIAP protein expression were detected by immunohistochemistry (IHC). The expressions of Smac and XIAP mRNA were detected by real-time quantitative polymerase chain reaction (RT-qPCR). ResultsHE showed that neuronal loss, nuclear consolidation, and vacuolar degeneration were significantly reduced in the medium and high-dose butylphthalide groups compared with the model controls. The BBT scores and apoptotic index were significantly lower in the medium and high doses of butylphthalide compared with the model controls. RT-qPCR and IHC showed that Smac, XIAP mRNA and protein expressions in the ischemic hemispheric region were significantly reduced in low, medium, and high doses of butylphthalide compared with the model controls (P < 0.05), showing some concentration effect. ConclusionsButylphthalide can significantly reduce Smac and XIAP mRNA and protein expression, inhibit neuronal apoptosis induced by ischemia-reperfusion injury in rats with carotid stenosis, and exert neuroprotective effects.

Tenglong Buzhong granules inhibits the growth of SW620 human colon cancer.

To observe the anticancer effects of the granular preparation of Tenglong Buzhong decoction (,TBD), i.e Tenglong Buzhong granules (, TBG), in human SW620 colon cancer. BALB/c nude mice were subcutaneously transplanted with SW620 cells, and treated with TBG (2.56 g/kg, once per day) and/or 5-Fu (104 mg/kg, once per week) for 21 d. Apoptosis, Caspase activities and cellular senescence were measured by commercial kits. The protein expression and phosphorylation were detected by Western blot or immunohistochemistry. TBG and 5-Fu inhibited tumor growth. The tumor inhibition rate of the TBG, 5-Fu, and TBG+5-Fu groups was 42.25%, 51.58%, and 76.08%, respectively. Combination of TBG and 5-Fu showed synergetic anti-cancer effects. TBG and 5-Fu induced apoptosis, activated caspase-3, -8, and -9, increased SMAC expression, inhibited XIAP expression. TBG induced cellular senescence, upregulated cyclin-dependent kinase inhibitor 1a (CDKN1a) and cyclin-dependent kinase inhibitor 2a (CDKN2a) expression, and inhibited phosphorylation of retinoblastoma-associated protein (RB) and expression of cyclin E1 (CCNE1) and cyclin-dependent kinases (CDK) 2. TBG also inhibited angiogenesis accompanied by downregulation of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1α). TBG inhibits SW620 colon cancer growth, induces apoptosis SMAC-XIAP-Caspases signaling, induces cellular senescence through CDKN1a/CDKN2a-RB-E2F signaling, inhibits angiogenesis by down-regulation of HIF-1α and VEGF, and enhances the effects of 5-Fu.

Scrophularıa oxysepala inhibit oral cancer cell line OECM-1 through induction of apoptosis

Oral cancer is a deadly malignancy that indicated high resistance to chemotherapeutic agents. Natural compounds derived from herbal medicine are many popular, and commonly used for treatment of several human disorders and malignancies in the world. In this study we aimed to investigate the anticancer activity of Scrophularıa oxysepala essential oil on oral cancer cell line OECM-1 and underlying mechanisms. The essential oil of S. oxysepala prepared by Clevenger instrument from dried leaves of plant. Cytotoxic effects of S. oxysepala essential oil was investigated by Tetrazolium staining on OECM-1 oral cancer cells. Moreover, the effects of S. oxysepala essential oil was investigated on apoptosis rate in cancer cells. The apoptosis-related genes (BAX, BCL2, SMAC, SURVIVIN) expression in treated cancer cells were evaluated by quantitative Real-Time PCR method. Our results indicated a significant cytotoxic effect of S. oxysepala essential oil against oral cancer cells in a time- and concentration-dependent manner. We observed several morphological changes in treated oral cancer cells by S. oxysepala essential oil that indicate cancer cell death and apoptosis. In addition, S. oxysepala essential oil induced apoptosis rate in treated cancer cells through regulation of BAX, BCL2, SMAC, and SURVIVIN genes expression. In general, our study demonstrated a significant cytotoxic effects of S. oxysepala essential oil against oral cancer cells, which may be due to induction of apoptosis through regulation of apoptosis-related genes expression.

LINC-DUBR Suppresses Malignant Progression of Ovarian Cancer by Downregulating miR-107 to Induce SMAC Expression.

Background LINC-DUBR may be a potential therapeutic target in ovarian cancer (OC). The purpose of this research was to explore the impact of miR-107 on the tumorigenicity of OC and its underlying molecular mechanisms. Methods RT-qPCR was adopted to measure the expression of LINC-DUBR and miR-107 in ovarian cancer tissues and cells. CCK-8 assays and transwell chamber assays were conducted to evaluate the impacts of LINC-DUBR and miR-107 on the proliferation and invasion of human ovarian cancer cells (SKOV3). In addition, we determined the relationship between LINC-DUBR, miR-107, and SMAC using TargetScan and luciferase reporter assay. The protein expression of SMAC was determined by western blot. Results Compared with normal tissues and cells, LINC-DUBR was downregulated and miR-107 was highly expressed in ovarian cancer tissues and cell lines. Overexpression of LINC-DUBR inhibited the cell proliferation and invasive ability in OC cells SKOV3. The luciferase reporter assay proved overexpression of LINC-DUBR repressed cells proliferation and invasion via binding to miR-107 in ovarian cancer. In addition, we found that SMAC was downregulated directly by miR-107 in ovarian cancer. miR-107 mimic significantly increased cell proliferation and invasiveness of SKOV3, while overexpressed SMAC eliminated this effect. Furthermore, miR-107 could regulate the XIAP/caspase-3 signaling pathway in ovarian cancer by targeting SMAC. Conclusion LINC-DUBR suppressed malignant progression of ovarian cancer by downregulating miR-107 to induce SMAC expression and involving in the XIAP/caspase-3 signaling pathway.

Proapoptotic Protein Smac Mediates Apoptosis in Ovarian Cancer Cells When ‎Treated with the Carpachromene ‎

IntroductionWe also investigated the Carpachromene in the cytotoxicity studies against common human ovarian cancer cell ‎line i.e., SW 626, in-vitro.Material and methodsCell viability of Carpachromene was very low against common ‎human ovarian cancer ‎cell line i.e. SW 626 without any cytotoxicity on normal cell line. To compare the ‎biological activities of molecules, the enzymes used are α-glucosidase, acetylcholinesterase, respectively. Finally, ‎calculations were made using the molecular docking method to compare the biological activity of the ‎carpachromene molecule. We then examined whether the release of Smac is necessary for apoptosis in ovarian ‎cancer cells using the SW 626‎ cell line. We first examined mitochondrial and cytosolic Smac levels after ‎Carpachromene treatment. ‎ResultsFollowing the docking calculations, the properties of the carpachromene molecule ‎were examined by ADME/T analysis in order to be used as a drug in the future. In addition, the anti-oxidant ‎properties of the molecules were examined in both gas and water phase with the HF/6-31g basis set with the ‎Gaussian software program. As shown, exposure of ovarian cancer cells to Carpachromene decreased ‎mitochondrial Smac and increased cytosolic Smac levels in a time-dependent fashion. As depicted in results, a ‎decrease in Smac expression was confirmed by Western blot. Silencing of Smac significantly inhibited ‎Carpachromene-induced caspase-3 cleavage and attenuated apoptosis in these cells Moreover, overexpression of ‎a Smac heptapeptide (Smac-N7) enhanced Carpachromene-induced cell deathConclusionsAccording to the above findings, the Carpachromene may be administrated for the treatment of several types of ‎human ovarian cancer in humans. ‎

Overexpression of BIRC6 driven by EGF-JNK-HECTD1 signaling is a potential therapeutic target for triple-negative breast cancer

Triple-negative breast cancer (TNBC) is an aggressive and highly lethal disease. The lack of targeted therapies and poor patient outcome have fostered efforts to discover new molecular targets to treat patients with TNBC. Here, we showed that baculoviral IAP repeat containing 6 (BIRC6) is overexpressed and positively correlated with epidermal growth factor (EGF) receptor (EGFR) in TNBC cells and tissues and that BIRC6 overexpression is associated with poor patient survival. Mechanistic studies revealed that BIRC6 stability is increased by EGF-JNK signaling, which prevents ubiquitination and degradation of BIRC6 mediated by the E3 ubiquitin ligase HECTD1. BIRC6 in turn decreases SMAC expression by inducing the ubiquitin-proteasome pathway, thereby antagonizing apoptosis and promoting the proliferation, colony formation, tumorsphere formation, and tumor growth capacity of TNBC cells. Therapeutically, the PEGylated cationic lipid nanoparticle (pCLN)-assisted delivery of BIRC6 small interfering RNA (siRNA) efficiently silences BIRC6 expression in TNBC cells, thus suppressing TNBC cell growth in vitro and in vivo, and its antitumor activity is significantly superior to that of the EGFR inhibitor gefitinib. Our findings identify an important regulatory mechanism of BIRC6 overexpression and provide a potential therapeutic option for treating TNBC.

Enhancement of Allo-NK Cell Activity in Myeloid Leukemia Cell Line KG-1a after Combined with Aclacinomycin

Abstract Objective: To investigate whether aclacinomycin can enhance the killing effects of allogeneic NK cells on acute myeloid leukemia and the molecular mechanisms, thus providing an experimental basis and a new thinking for the clinical treatment of acute leukemia. Methods: NK cells were isolated from the peripheral blood of healthy people by antibody-labeled immunomagnetic beads, the percentage of NK cells was detected by flow cytometry, and the inhibitory effects of aclacinomycin on the proliferation of KG-1a cells were detected by CCK-8 assay. In vitro killing assay was employed to detect the in vitro killing effects on NK-1a cells by NK cells with different effector/target ratios after treatment. Clone formation assay was used to detect the inhibitory effects of aclacinomycin in combination with allogeneic NK cells on the proliferation of KG-1a cells. Furthermore, the effects of aclacinomycin in combination with allogeneic NK cells in inducing the apoptosis of KG-1a cells was detected by flow cytometry, and changes in the expressions of IAP protein families and proteins associated with both intracellular and extracellular apoptosis signaling pathways after treatment of aclacinomycin in combination with allogeneic NK cells were analyzed by immunoblotting. Meanwhile, the differences in the expression levels of calreticulin, heat shock protein, adenosine triphosphate and high mobility protein-1 were detected. Results: The percentages of NK cells in the peripheral blood before and after isolation with anti-CD3-CD56+ antibody-labeled immunomagnetic beads were (15.2 ± 3.7)% and (84.9 ± 5.5)%, respectively. The results of CCK-8 assay showed that the inhibitory effects of aclacinomycin on the proliferation of KG-1a cells were increased in a time-dependent manner. At the same time, this study showed that after the co-culture of NK cells and KG-1a cells at the effector/target ratios of 10: 1 and 20: 1, the killing effects on KG-1a cells were not significant (P <0.05). After 72 hours of treatment at the ratio of 20: 1, aclacinomycin could significantly increase the killing effects of NK cells on KG-1a cells. The results of clone formation assay showed that at 72 hours after the treatment of acycloemycin in combination with allogeneic NK cells, the number of KG-1a cell clones was (11 ± 3), significantly lower than that in the aclacinomycin-alone group (68 ± 7) and the NK cell group (121 ± 12, P <0.05), the differences were statistically significant (P <0.001). The results of flow cytometry showed that the total apoptotic rate of KG-1a cells was increased significantly at 72 hours after the treatment of aclacinomycin in combination with allogeneic NK cells, and the results of immunoblotting showed that the expression of cIAP, XIAP and Bcl-2 were significantly down-regulated after the treatment of aclacinomycin in combination with NK cells, while the expressions of pro-apoptosis proteins Smac and caspase-3 were significantly up-regulated. Meanwhile, the expressions of immunogenic death-associated calreticulin, heat shock protein, adenosine triphosphate and high mobility protein-1 were significantly up-regulated. Conclusions: Aclacinomycin can significantly enhance the killing effects of allogeneic NK cells on the KG-1a cell line in vitro through such a molecular mechanism in which the expression levels of IAP protein family-related proteins are inhibited. Moreover, the mechanisms are associated closely with the activation of both intracellular and extracellular apoptosis signaling pathways and the activation of immunogenic death-associated proteins. DisclosuresNo relevant conflicts of interest to declare.

DPP3/CDK1 contributes to the progression of colorectal cancer through regulating cell proliferation, cell apoptosis, and cell migration

At present, colorectal cancer (CRC) has become a serious threat to human health in the world. Dipeptidyl peptidase 3 (DPP3) is a zinc-dependent hydrolase that may be involved in several physiological processes. However, whether DPP3 affects the development and progression of CRC remains a mystery. This study is the first to demonstrate the role of DPP3 in CRC. Firstly, the results of immunohistochemistry analysis showed the upregulation of DPP3 in CRC tissues compared with normal tissues, which is statistically analyzed to be positively correlated with lymphatic metastasis, pathological stage, positive number of lymph nodes. Moreover, the high expression of DPP3 predicts poor prognosis in CRC patients. In addition, the results of cell dysfunction experiments clarified that the downregulation of DPP3 significantly inhibited cell proliferation, colony formation, cell migration, and promoted apoptosis in vitro. DPP3 depletion could induce cell apoptosis by upregulating the expression of BID, BIM, Caspase3, Caspase8, HSP60, p21, p27, p53, and SMAC. In addition, downregulation of DPP3 can reduce tumorigenicity of CRC cells in vivo. Furthermore, CDK1 is determined to be a downstream target of DPP3-mediated regulation of CRC by RNA-seq, qPCR, and WB. The interaction between DPP3 and CDK1 shows mutual regulation. Specifically, downregulation of DPP3 can accentuate the effects of CDK1 knockdown on the function of CRC cells. Overexpression of CDK1 alleviates the inhibitory effects of DPP3 knockdown in CRC cells. In summary, DPP3 has oncogene-like functions in the development and progression of CRC by targeting CDK1, which may be an effective molecular target for the prognosis and treatment of CRC.

Melatonin increases the anticancer potential of doxorubicin in Caco-2 colorectal cancer cells.

Colorectal cancer (CC) is an important human malignancy with high cancer related death worldwide. The chemotherapy using doxorubicin hydrochloride is one of the most common cancer therapeutic methods. However, drug resistance lowers the treatment efficacy in CC patients. The combination therapies seem to be more promising by taking the advantage of synergistic effects. The present study aimed to evaluate a new strategy to enhance the anticancer activity of doxorubicin in Caco-2 CC cell line by co-administration of melatonin. The effects of doxorubicin, melatonin, and their combinations (Dox-Mel) were investigated on the proliferation and viability, morphological alterations, and tumor spheroid formation. Flow cytometry was employed to compare the apoptotic situation of the cells in study groups. Changes in metastatic potential of the cells were assessed by wound healing assay and trans-well migration assays. Moreover, expression of BAX, SMAC, BCL-2, SURVIVIN, MMP-2, and MMP-9 genes were evaluated by quantitative real time PCR and western blotting. Our study showed that doxorubicin, melatonin, and Dox-Mel significantly decreased the proliferation and viability, tumor spheroid formation, invasion, and migration. Furthermore, the changes were in a concentration and time dependent manner. There was an increase in apoptosis rate in the treatment groups. Expression of genes involved in apoptosis and cell motility were altered significantly. It was observed that anticancer activity of Dox-Mel combination was significantly more than doxorubicin and melatonin treatments alone. We showed an enhanced apoptotic and anticancer activity of doxorubicin and melatonin combination chemotherapy on CC cell line than doxorubicin or melatonin treatments alone. This combination could promote the treatment efficiency and alleviate the un-intended side effects by lowering the dose of doxorubicin prescription.

Polygoni Multiflori Radix Preparat Delays Skin Aging by Inducing Mitophagy.

Background As the skin is the largest organ of the human body, it is aging inevitably and produces cosmetic and psychological problems, and even disease. Therefore, the molecular mechanisms related to the prevention of skin aging need to be further explored. Methods Aging models were constructed by D-galactose. Mice were administrated with polygoni multiflori radix preparat (PMRP), PMRP and 3-methyladenine, or PMRP and rapamycin intragastrically. The apparent and viscera index of aged rats was measured. Then, the physicochemical property, antioxidant ability, histological structure, mitochondrial membrane potential, ATP and ROS levels, and mitophagy of aged skins were determined. Finally, the expression of PINK1, Parkin, P62, and LC3II/I; apoptosis-related proteins; and the percentage of apoptotic cells were measured. Results PMRP relieved skin aging with reducing of thymus index, improvement of pathological damage and histological structure, increase of the expression area of fibrous tissue, the ratio of type I to type III collagen, and antioxidant ability of aged skins. Importantly, PMRP also improved mitochondrial dysfunction with an increase in the content of mitochondrial membrane potential and ATP and a decrease of ROS levels. Moreover, mitophagy was enhanced with the treatment of PMRP when observed using electron microscopy, and the expression of PINK1, Parkin, and LC3I/II was increased with PMRP treatment but P62 expression was decreased. Meanwhile, PMRP alleviated apoptosis with a decrease of apoptotic cell and the expression of Cleaved-cas3, Bax, Cyt-c, AIF, and Smac as well as an increase of Bcl-2 expression. Conclusion The results demonstrated that the polygoni multiflori radix preparata may delay skin aging by inducing mitophagy.

Effects of Different Degrees of Carotid Artery Stenosis on the Expression of XIAP and Smac in the Ischemic Penumbra of Rats with Cerebral Ischemia-Reperfusion

ObjectiveTo investigate the effects of different degrees of carotid artery stenosis (CAS) on the expression of XIAP and Smac in ischemic penumbra of rats with cerebral ischemia-reperfusion (I/R). Materials and MethodsSamples were collected at 12 h and 24 h after reperfusion, and then the treated groups were divided into the NC-12 group, NC-24 group, MIS-12 group, MIS-24 group, MOS-12 group, MOS-24 group, SES-12 group and SES-24 group. HE staining was used to observe the pathological changes of the brain tissue. TUNEL assay was used to detect the apoptosis in the ischemic penumbra. IHC and RT-qPCR were used to detect the expression of XIAP and Smac in the brain tissue. ResultsBy observing the pathological sections of brain tissue, the rats in MIS, MOS and SES groups showed loose brain tissue on the infarcted side and neuronal pyknosis in the ischemic penumbra. And with the aggravation and prolongation of the degree of stenosis, the degree of brain injury deepened. It was further found that the TUNEL positive rate was significantly increased in the ischemic penumbra in the SES and MOS groups compared with that in the normal control (NC) group. The results of IHC and RT-qPCR showed that the mRNA expression of XIAP and Smac in the ischemic penumbra was significantly up-regulated in the MIS, MOS and SES groups compared with that in the NC group. ConclusionsCAS may activate XIAP/Smac signaling pathway to induce neuronal apoptosis and promote the injury in the ischemic penumbra caused by cerebral I/R.

Influence of high-intensity interval training and intermittent fasting on myocardium apoptosis pathway and cardiac morphology of healthy rats

AimTo evaluate the influence of intermittent fasting and high-intensity interval training (HIIT) on myocardial apoptosis signaling and cardiac morphological characteristics in healthy rats. MethodsMale Wistar rats (n = 60) were divided into four groups: sedentary control (SED-C), intermittent fasting (SED-IF), high-intensity interval training (HIIT-C), and high-intensity interval training plus intermittent fasting (HIIT-IF). SED-C and HIIT-C groups were treated daily with ad libitum chow; SED-IF and HIIT-IF received the same standard chow every other day. HIIT-C and HIIT-IF rats were submitted to an HIIT protocol five times a week for 12 weeks. At the end of the experiment, functional capacity, cardiac morphology, and expression of apoptosis signaling pathways-related proteins were analyzed. Key findingsHIIT increased cardiomyocyte cross-sectional area, collagen interstitial fraction, and the pro-apoptotic proteins AIF and caspase-3 expression, and reduced pro-apoptotic protein CYTC expression and the cleaved-to-non-cleaved PARP-1 ratio in myocardium. Intermittent fasting reduced cardiomyocyte cross-sectional area, collagen interstitial fraction, and expression of Bax, CYTC and cleaved PARP-1, and increased expression of the anti-apoptotic protein BCL-2. SMAC, ARC, and caspase-8 expression was not changed by HIIT or intermittent fasting. SignificanceHIIT promotes cardiomyocyte hypertrophy and interstitial fibrosis, and modulates the apoptosis signaling pathway in healthy rat myocardium. Intermittent fasting reduces pro-apoptotic and increases antiapoptotic signaling, besides attenuating HIIT-induced cardiomyocyte hypertrophy and myocardial interstitial fibrosis.

Circadian gene Clock participates in mitochondrial apoptosis pathways by regulating mitochondrial membrane potential, mitochondria out membrane permeablization and apoptosis factors in AML12 hepatocytes

Circadian rhythms help organisms adapt to changes of external environment by regulating energy metabolism and remaining the balance of homeostasis. Numerous researches have proved that the physiological function of liver was precisely controlled by circadian rhythms. Clock, one of core circadian genes, has been demonstrated to regulate the oxidative phosphorylation process of mitochondrial, which provides energy for living cells and acts as one of the hub for apoptosis. However, whether Clock gene regulates mitochondrial apoptosis pathways in liver cells remains less explored. In the present study, we used lentiviral vector to establish a stable AML12 cell lines which were capable of expressing specific shRNA to interfere the expression of Clock gene and investigated the effect of Clock on mitochondrial apoptosis pathways. Herein, we found that the interference of Clock gene could significantly suppress mitochondrial apoptosis pathways by stabilizing mitochondrial membrane potential and inhibiting mitochondria out membrane permeablization, which might be a result of lower expression of BAD and BIM proteins. Moreover, the interference of Clock gene could downregulate the expression of mitochondrial apoptosis factors, i.e. AIF, CYCS, APAF-1 and SMAC, which will suppress the formation of apoptosome and the process of DNA degradation to further inhibit apoptosis process. This work provides an insight on the important role of Clock gene participating in mitochondrial apoptosis pathways of hepatocytes and unveils a probable pathogenesis of how circadian rhythm regulates liver diseases.

Low expression of pro-apoptotic proteins Bax, Bak and Smac indicates prolonged progression-free survival in chemotherapy-treated metastatic melanoma

Despite the introduction of novel targeted therapies, chemotherapy still remains the primary treatment for metastatic melanoma in poorly funded healthcare environments or in case of disease relapse, with no reliable molecular markers for progression-free survival (PFS) available. As chemotherapy primarily eliminates cancer cells by apoptosis, we here evaluated if the expression of key apoptosis regulators (Bax, Bak, Bcl-2, Bcl-xL, Smac, Procaspase-9, Apaf-1, Procaspase-3 and XIAP) allows prognosticating PFS in stage III/IV melanoma patients. Following antibody validation, marker expression was determined by automated and manual scoring of immunohistochemically stained tissue microarrays (TMAs) constructed from treatment-naive metastatic melanoma biopsies. Interestingly and counter-intuitively, low expression of the pro-apoptotic proteins Bax, Bak and Smac indicated better prognosis (log-rank p < 0.0001, p = 0.0301 and p = 0.0227 for automated and p = 0.0422, p = 0.0410 and p = 0.0073 for manual scoring). These findings were independently validated in the cancer genome atlas (TCGA) metastatic melanoma cohort (TCGA-SKCM) at transcript level (log-rank p = 0.0004, p = 0.0104 and p = 0.0377). Taking expression heterogeneity between the markers in individual tumour samples into account allowed defining combinatorial Bax, Bak, Smac signatures that were associated with significantly increased PFS (p = 0.0002 and p = 0.0028 at protein and transcript level, respectively). Furthermore, combined low expression of Bax, Bak and Smac allowed predicting prolonged PFS (> 12 months) on a case-by-case basis (area under the receiver operating characteristic curve (ROC AUC) = 0.79). Taken together, our results therefore suggest that Bax, Bak and Smac jointly define a signature with potential clinical utility in chemotherapy-treated metastatic melanoma.

Mechanism for ginsenoside Rh2-induced apoptosis of triple-negative breast cancer MDA-MB-231 cells

Objective: The authors aimed to explore the apoptosis of triple-negative breast cancer (TNBC) MDA-MB-231 cells induced by ginsenoside Rh2 and the underlying mechanism. Materials and Methods: Changes in the viability of MDA-MB-231 cells after treatment with 20 (S)-Rh2 and 20 (R)-Rh2 for 48 hours were detected by MTT assay. Changes in the morphology of cell nuclei were observed by DAPI staining. The expressions of caspase-3, caspase-9, cytochrome c, Smac, Bak, and Bax related to the mitochondrial pathway were detected by Western blotting. Results: 20 (S)-Rh2 inhibited the proliferation of MDA-MB-231 cells, but 20 (R)-Rh2 failed to do so. After treatment with 20(S)-Rh2 for two hours under visible light, they shrank and had incomplete morphology, whereas the morphology of control group hardly changed. Under UV light, the nuclei stained with DAPI were blue. After treatment with 20 (S)-Rh2 for one hour, the nuclei shrank and ruptured, and nearly 90% ruptured at two hours. In contrast, the nuclei of PBS control group remained intact. The activity of caspase-9 in MDA-MB-231 cells treated with 7.5 µg/mL 20 (S)-Rh2 was increased at 30 minutes, and gradually increased over extended time. Under identical conditions, the activity of caspase-9 in control group did not change significantly. Cytochrome c and Smac were released from mitochondria to the cytoplasm at one hour after treatment with 7.5 µg/mL 20 (S)-Rh2. However, such release was not detected in the control group. Bax was translocated after 30 minutes of treatment with 7.5 µg/mL 20 (S)-Rh2, which then gradually accumulated in mitochondria over time and peaked at two hours. The Bax expression in the entire cell lysate remained unchanged. The translocation of Bax was not detected in control group. Conclusion: 20 (S)-Rh2 evidently inhibited the proliferation of TNBC cell line MDA-MB-231. It killed the cells by inducing apoptosis, probably by activating the mitochondrial pathway.

Cannabidiol promotes apoptosis via regulation of XIAP/Smac in gastric cancer

According to recent studies, Cannabidiol (CBD), one of the main components of Cannabis sativa, has anticancer effects in several cancers. However, the exact mechanism of CBD action is not currently understood. Here, CBD promoted cell death in gastric cancer. We suggest that CBD induced apoptosis by suppressing X-linked inhibitor apoptosis (XIAP), a member of the IAP protein family. CBD reduced XIAP protein levels while increasing ubiquitination of XIAP. The expression of Smac, a known inhibitor of XIAP, was found to be elevated during CBD treatment. Moreover, CBD treatment increased the interaction between XIAP and Smac by increasing Smac release from mitochondria to the cytosol. CBD has also been shown to affect mitochondrial dysfunction. Taken together, these results suggest that CBD may have potential as a new therapeutic target in gastric cancer.