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Obeticholic acid (OCA) was approved for the treatment of primary biliary cholangitis (PBC) patients. However, it can cause severe drug-induced liver injury (DILI), which may put PBC patients at risk of acute-on-chronic liver failure (ACLF) and even death. Farnesoid X receptor (FXR) is considered as the target of OCA for cholestasis, but there is still a lack of research on whether hepatic and ileal FXR have different effects after OCA treatment. The aim of this study was to investigate the mechanism of OCA aggravating liver fibrosis in cholestasis. The results showed that 40 mg/kg OCA elevated serum AST, ALT, ALP and γ-GT levels in bile duct ligation (BDL) mice. Besides, severe fibrosis and necrosis were observed in the OCA-treated BDL mice, which was related to hepatic apoptosis pathway activation. Both hepatic and ileal FXR signaling could be significantly activated by OCA. However, ileum-specific knockout of Fxr aggravated OCA-induced liver injury in BDL mice. On the contrary, hepatic-specific knockout of Fxr structurally and functionally ameliorated liver pathological processes in the OCA-treated BDL mice, which was due to the blockade of hepatic FXR-induced apoptosis. In conclusion, the mechanism of OCA aggravating liver fibrosis in cholestasis was based on the activation of hepatic FXR-induced apoptosis. It was also indicated ileal FXR might be a safer pharmacological target for bile acids regulation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Previous research suggests mitochondrial apoptosis alleviates rheumatoid arthritis (RA), but the role of mitochondrial apoptosis-related genes (MARGs) is unclear. Urgent exploration of RA-related mitochondrial apoptosis biomarkers is needed. Gene Expression Ontology (GEO)-derived RA datasets were used to identify differentially expressed genes (DEGs) compared to normal controls, intersected with MARGs to obtain differentially expressed mitochondrial apoptosis-related genes (DE-MARGs). Three ML algorithms screened diagnostic biomarkers. A nomogram was built and validated by receiver operating characteristic (ROC) analysis. Gene Set Enrichment Analysis (GSEA), regulatory network, and drug prediction explored biomarker mechanisms. Finally, key cells analysis included clustering, type annotation, pseudo-temporal study, and interaction, focusing on validated biomarker expression in those cells. A total of 147 DE-MARGs linked to energy & ROS metabolism were identified. Four validated biomarkers (MRPS10, EEF2, HSPA9, TUFM) formed a new RA diagnostic model. Moreover, GSEA linked them to oxidative phosphorylation. YY1 regulates EEF2, HSPA9, MRPS10; FOXO3 regulates EEF2, TUFM. Drugs like Nonoxynol-9, Nedocromil, Gadobutrol target these biomarkers. In addition, biomarkers are expressed in plasmablasts, with CD74 as a key receptor binding multiple ligands. RA biomarkers (MRPS10, EEF2, HSPA9, TUFM) linked to energy & ROS, progression tied to AMPK/mTOR, CD74-MIF crucial. Study advances RA pathogenesis knowledge, supporting clinical diagnosis., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Epidermal growth factor receptor (EGFR) is a validated target for non-small-cell lung cancer (NSCLC). However, the treatment for EGFR-C797S mutation induced by third-generation EGFR inhibitors remains a concern. Therefore, the development of the fourth-generation EGFR inhibitors to overcome the EGFR-C797S mutation has great potential for clinical treatment. In this article, we designed and synthesized a series of diphenyl ether substituted quinazolin-4-amine derivatives that simultaneously occupy the ATP binding pocket and the allosteric site of EGFR. Among the newly synthesized compounds, 9d displayed excellent kinase activity against EGFR L858R/T790M/C797S with an IC 50 value of 0.005 μM, and exhibited anti-proliferation activity in BaF3-EGFR L858R/T790M/C797S cells with the IC 50 value of 0.865 μM. Furthermore, 9d could suppress phosphorylation of EGFR and induce cell apoptosis and cycle arrest at G2 phase in a dose-dependent manner in BaF3-EGFR L858R/T790M/C797S cells. More importantly, 9d displayed significant antitumor effects in BaF3-EGFR L858R/T790M/C797S xenograft mouse model (30 mg/kg, TGI = 71.14 %). All the results indicated compound 9d might be a novel fourth-generation EGFR inhibitor for further development in overcoming the EGFR-C797S resistance mutation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Increasing the levels of antiapoptotic Bcl-2 proteins is an important way that cancer cells utilize to get out of apoptosis, underscoring their significance as promising targets for anticancer therapies. Lately, a primary compound 1 bearing thiazolidine-2,4-dione was discovered to exhibit comparable Mcl-1 inhibitory activity in comparison to WL-276. Herein, thirty-nine thiazolidine-2,4-dione analogs were yielded through incorporating different biphenyl moieties (R 1 ), amino acid side chains (R 2 ) and sulfonamides (R 3 ) on 1. The findings indicated that certain compounds exhibited favorable inhibitory effects against Bcl-2/Mcl-1, while demonstrating limited or negligible binding affinity towards Bcl-xL. In particular, compounds 16 and 20 exhibited greater Bcl-2/Mcl-1 inhibition compared to AT-101, WL-276 and 1. Moreover, they demonstrated notable antiproliferative effects and significantly induced apoptosis in U937 cells. The western blot and co-immunoprecipitation assays confirmed that 20 could induce alterations in the expression of apoptosis-associated proteins to result in apoptosis through on-target Bcl-2 and Mcl-1 inhibition. In addition, 20 exhibited favorable stability profiles in both rat plasma and rat liver microsomes. In total, 20 could be used as a promising compound to discover Bcl-2/Mcl-1 dual inhibitors with favorable therapeutic properties., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Sepsis-induced cardiomyopathy (SIC) is described as a reversible myocardial depression that occurs in patients with septic shock. Increasing evidence shows that microRNA-194-5p (miR-194-5p) participates in the regulation of oxidative stress, mitochondrial dysfunction, and apoptosis and its expression is associated with the occurrence and progression of cardiovascular disease; however, the effects of miR-194-5p in SIC are still unclear. This study explores whether miR-194-5p could modulate SIC by affecting oxidative stress, mitochondrial function, and apoptosis. Experimental septic mice were induced by intraperitoneal injection of lipopolysaccharide (LPS) in C57BL/6J mice. The biological role of miR-194-5p in SIC in vivo was investigated using cardiac echocardiography, ELISA, western blot, qRT-PCR, transmission electron microscopy, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, bioinformatics analysis, and dual-luciferase reporter gene assay. Our major finding is that miR-194-5p antagomir mitigates sepsis-induced cardiac dysfunction, inflammation, oxidative stress, apoptosis and mitochondrial dysfunction in the hearts of septic mice, while miR-194-5p agomir triggers the opposite effects. Furthermore, dual-specificity phosphatase 9 (DUSP9) is a direct target of miR-194-5p and the cardioprotective effects of miR-194-5p antagomir on cardiac dysfunction, inflammation, apoptosis, mitochondrial dysfunction and oxidative stress are abolished through inhibiting DUSP9. Therefore, miR-194-5p inhibition could mitigate SIC via DUSP9 in vivo and the novel miR-194-5p/DUSP9 axis might be the potential treatment targets for SIC patients., (© 2024. The Author(s).)

Malignant ascites is a common complication of advanced cancers, which reduces survival rates and diminishes patients' quality of life. Intraperitoneal chemotherapy is a conventional method for treating cancer-related ascites, but the poor drug retention of conventional drugs requires frequent administration to maintain sustained anti-tumor effects. In this study, we encapsulated doxorubicin (DOX) into Brucea javanica oil (BJO) to develop a water-in-oil (W/O) nanoemulsion called BJO@DOX for the treatment of malignant ascites through in-situ intraperitoneal administration. BJO significantly induced apoptosis of S180 cells by upregulating the expression of p53 and caspase-3 (cleaved). Additionally, BJO notably downregulated the expression of Bcl-2, further promoting apoptosis of S180 cells. Cell apoptosis significantly inhibited ascites formation and tumor cell proliferation in a mouse model. The combination of DOX and BJO exhibited satisfactory synergistic effects, consequently prolonging the survival period of mice. Histological examination of major organs indicated that the nanoemulsion had excellent biosafety in vivo. The BJO@DOX nanoemulsion represents a promising platform for in-situ chemotherapy of malignant ascites., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Membranous nephropathy (MN) is a common pathological type of adult nephrotic syndrome. Up to 20% of patients with MN develop end-stage renal disease (ESRD). Podocytes have an important function in maintaining the glomerular filtration barrier and play a crucial role in the occurrence and development of proteinuria and MN. PI3K/AKT signaling pathway is involved in the entire process of podocyte growth, differentiation, and apoptosis. Kemeng Fang (KMF) is a traditional Chinese medicine formula that has been used to delay kidney injury. However, the therapeutic mechanism of KMF in MN is unclear. Here, the MN rat model was established by axillary, inguinal, and tail vein injections of cationized bovine serum albumin (C-BSA), and then KMF and PI3K inhibitor (LY294002) were administered. The data of liver function, kidney function, blood lipid, renal pathology, podocyte function, expression level of PI3K/AKT signaling pathway, and transcriptomics of rats demonstrated that KMF has a protective effect on the podocytes of MN rats by activating the PI3K/AKT signaling pathway, and it can effectively prevent the progression of MN.

Degradation of target proteins has been considered to be a promising therapeutic approach, but the rational design of compounds for degradation remains a challenge. In this study, we reasonably designed and synthesized only 10 compounds to discover effective CDK4/6 protein degraders. Among the newly synthesized compounds, 7f achieved dual degradation of CDK4/6 protein, with DC 50 values of 10.5 and 2.5 nM, respectively. Compound 7f also exhibited inhibitory proliferative activity against Jurkat cells with an IC 50 value of 0.18 μM. Furthermore, 7f induced cell apoptosis and G1 phase cell cycle arrest in a dose-dependent manner in Jurkat cells. In conclusion, these findings demonstrate the potential of 7f as a CDK4/6 degrader and a potential therapeutic strategy against cancer, thereby expanding the potential of CDK4/6 dual PROTACs.

Macroautophagy/autophagy has both negative and positive aspects in the development of many diseases. Yet, its exact role and specific mechanism in the onset of medication-related osteonecrosis of the jaw (MRONJ) is still not fully understood. Retarded gingiva healing is the primary clinical manifestation in patients with MRONJ. In this study, we aimed to explore the relationship between autophagy and apoptosis in MRONJ gingival epithelium and search for a method to prevent this disease. First, we examined clinical samples from patients diagnosed with MRONJ and healthy controls, finding that autophagy-related markers MAP1LC3/LC3 and SQSTM1/p62 synchronously increased, thus suggesting that autophagic flux was suppressed in MRONJ. Moreover, mRNA sequencing analysis and TUNEL assay showed that the process of apoptosis was upregulated in patients and animals with MRONJ, indicating autophagy and apoptosis participate in the development of MRONJ. Furthermore, the level of autophagy and apoptosis in zoledronic acid (ZA)-treated human keratinocytes cell lines (HaCaT cells) was concentration dependent in vitro . In addition, we also found that RAB7 (RAB7, member RAS oncogene family) activator ML098 could rescue MRONJ gingival lesions in mice by activating the autophagic flux and downregulating apoptosis. To sum up, this study demonstrated that autophagic flux is impaired in the gingival epithelium during MRONJ, and the rescued autophagic flux could prevent the occurrence of MRONJ. Abbreviations: ACTB: actin beta; Baf-A1: bafilomycin A 1 ; CASP3: caspase 3; CASP8: caspase 8; CT: computed tomography; DMSO: dimethyl sulfoxide; GFP: green fluorescent protein; HaCaT cells: human keratinocytes cell lines; H&E: hematoxylin and eosin; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MRONJ: medication-related osteonecrosis of the jaw; PARP: poly(ADP-ribose) polymerase; RAB7: RAB7, member RAS oncogene family; RFP: red fluorescent protein; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscopy; ZA: zoledronic acid.

Context: Cyanidin has been shown to have therapeutic potential in osteoarthritis. However, it is unclear whether cyanidin prevents the progression of intervertebral disc degeneration (IVDD)., Objective: This study evaluates the effects of cyanidin on IVDD in vitro and in vivo ., Materials and Methods: Nucleus pulposus cells (NPCs) isolated from lumbar IVD of 4-week-old male Sprague-Dawley (SD) rats were exposed to 20 ng/mL IL-1β, and then treated with different doses (0-120 µM) of cyanidin for 24 h. SD rats were classified into three groups ( n  = 8) and treated as follows: control (normal saline), IVDD (vehicle), IVDD + cyanidin (50 mg/kg). Cyanidin was administered intraperitoneally for 8 weeks., Results: The IC 50 of cyanidin for NPCs was 94.78 µM, and cyanidin had no toxicity at concentrations up to 500 mg/kg in SD rats. Cyanidin inhibited the apoptosis of NPCs induced by IL-1β (12.73 ± 0.61% vs. 18.54 ± 0.60%), promoted collagen II (0.82-fold) and aggrecan (0.81-fold) expression, while reducing MMP-13 (1.02-fold) and ADAMTS-5 (1.40-fold) expression. Cyanidin increased the formation of autophagosomes in IL-1β-induced NPCs, and promoted LC3II/LC3I (0.83-fold) and beclin-1 (0.85-fold) expression, which could be reversed by chloroquine. Cyanidin inhibited the phosphorylation of JAK2 (0.47-fold) and STAT3 (0.53-fold) in IL-1β-induced NPCs. The effects of cyanidin could be enhanced by AG490. Furthermore, cyanidin mitigated disc degeneration in IVDD rats in vivo ., Discussion and Conclusions: Cyanidin improved the function of NPCs in IVDD by regulating the JAK2/STAT3 pathway, which may provide a novel alternative strategy for IVDD. The mechanism of cyanidin improving IVDD still needs further work for in-depth investigation.

Cold storage for organ preservation in kidney transplantation is a core predisposing factor for delayed graft function and the long-term outcome of transplanted kidneys. Hydroxysafflor yellow A (HSYA) is the most effective water-soluble active monomer in Safflower with a strong property of inhibiting hypoxia and reoxygenation (H/R). However, the evidence concerning the effect of HSYA on H/R in kidney transplantation is limited. To investigate whether HSYA has a protective effect on cold H/R injury，we investigated the possible protective mechanism. Here, we incubated HK-2 cells to establish a cold H/R model and observed HSYA activation in an in vitro model of cold-storage rewarming which included the cell survival rate, cell morphology and ultrastructure, protein expression of Bcl-2, Bax, CytC, Apaf-1, and caspase-3, and status of mitochondrial permeability transformation pores (MPTPs). Our data showed that HSYA pretreatment increased the survival rate of the cells, alleviated mitochondrial damage, decreased the expression of apoptosis-related proteins and inhibited the openness of mitochondrial permeability transformation pores. Our findings suggested that HSYA may be a major predisposing mediator of mitochondrial apoptosis and renal tubular injury in cold storage-associated transplantation and may be an effective therapeutic target for improving graft function and graft survival., (Copyright © 2022. Published by Elsevier B.V.)

Actin-like 6A (ACTL6A) is a core subunit of the SWI/SNF chromatin remodeling complex and is highly expressed in several types of human cancers including glioblastoma. Recent studies verified that ACTL6A regulates the proliferation, differentiation, and migration of cancer cells. In this study, we identified ACTL6A as an important regulator of DNA replication. ACTL6A knockdown could impair the DNA replication initiation in glioblastoma cells. The regulation of DNA replication by ACTL6A was mediated through regulating the expression of the CDC45-MCM-GINS (CMG) complex genes. Further investigation revealed that ACTL6A transcriptionally regulates MCM5 expression. Furthermore, ACTL6A knockdown induced DNA damage and diminished the activity of the ATR-Chk1 pathway, which ultimately led glioblastoma cells to apoptosis and death. Taken together, our findings highlight the critical role of ACTL6A in DNA replication and ATR-Chk1 pathway, and reveal a potential target for therapeutic intervention in glioblastoma., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Objective: Human adipose-derived mesenchymal stem cells (ADSCs) have the potential to be applied to solid organ treatments. However, tissue regeneration is limited by the death of transplanted cells. Ischemia is the main cause of the poor outcome. This study aimed to investigate the effect of berberine (BBR) on ADSCs after fat grafting., Methods: The antioxidant BBR on apoptosis and autophagy of ADSCs in vitro ischemia model was induced by hypoxia and serum deprivation (HY/SD). The autophagy promoter rapamycin and autophagy inhibitor 3-MA were incubated separately to investigate the crosstalk between autophagy and apoptosis. Pathway inhibitors further verified whether the autophagy and apoptosis were regulated by AMPK/mTor signaling pathway. Fat survival, fibrosis, level of inflammatory cell infiltration, and the effect of angiogenesis after BBR treatment were observed in vivo., Results: BBR could reduce ROS production and reverse the decreasing cell survival rate. HY/SD would induce apoptosis and autophagy in ADSCs, and BBR could alleviate these processes. After interfering with the level of autophagy, we also proved that apoptosis was regulated by autophagy and changed accordingly. The results also indicated that BBR could protect against autophagy and apoptosis of ADSCs through AMPK/mTor pathway. The treated human-derived adipose tissue was transplanted into BALB/c nude mice, and with the intervention of BBR, the fat grafting had a higher survival rate, lower inflammatory cell infiltration and fibrosis level., Conclusion: Our present study revealed that BBR was a promising anti-autophagy and apoptosis agent for improving the survival rate of ADSCs during cell transplantation., Competing Interests: The authors declare that they have no competing interests., (© 2021 Pang et al.)

Caspase-8 is a unique member of caspases with a dual role in cell death and survival. Caspase-8 expression is often lost in some tumors, but increased in others, indicating a potential pro-survival function in cancer. By analyzing transcriptome of enzalutamide-resistant prostate cancer cells, we found that resistance was conferred by a mild caspase-8 upregulation that in turn led to NF-κB activation and the subsequent upregulation of the downstream IL-8. Mechanistically, we found that the pro-survival and enzalutamide-resistance-promoting features of caspase-8 were independent of its proteolytic activity, using a catalytically-inactive caspase-8 mutant. We further demonstrated that caspase-8 pro-apoptotic function was inhibited via cFLIP binding. Moreover, high caspase-8 expression was correlated with a worse prognosis in prostate cancer patients. Collectively, our work demonstrates that enzalutamide-resistance is mediated by caspase-8 upregulation and the consequent increase in NF-κB/IL-8 mediated survival signaling, highlighting caspase-8 and NF-κB as potential therapeutic targets to overcome enzalutamide-resistance in CRPC., (© 2021. The Author(s).)

Chondrocyte apoptosis contributes to osteoarthritis, while miR-146a is a critical player in chondrocyte apoptosis. Our bioinformatics analysis showed that miR-146a may bind with long non-coding RNA (lncRNA) CALML3 antisense RNA 1 (CALML3-AS1). Our study was therefore carried out to investigate the interactions between lncRNA CALML3-AS1 and miR-146a in osteoarthritis. This study included 66 osteoarthritis patients who were admitted at Shanxi People's Hospital from July 2016 to June 2019. Transfections were performed to analyse gene interactions. RT-qPCR and Western blot were performed to determine the expression levels of gene and protein, respectively. Cell apoptosis of chondrocytes induced by lipopolysaccharide (LPS) was analysed by cell apoptosis assay. We found that CALML3-AS1 was downregulated, while miR-146a was upregulated in osteoarthritis. However, no significant correlation was found between them. In addition, overexpression of CALML3-AS1 or miR-146a did not affect the expression of each other. However, overexpression of CALML3-AS1 resulted in the upregulation of Smad family member 4 (Smad4), a downstream target of miR-146a. We also found that the expression of miR-146a and Smad4 were negatively correlated, while the correlation between CALML3-AS1 and smad4 was not significant. In cell apoptosis assay, overexpression of CALML3-AS1 and Smad4 resulted in decreased proliferation of chondrocytes. MiR-146a played an opposite role and reduced the effects of overexpression of CALML3-AS1 and Smad4. Therefore, CALML3-AS1 may regulate chondrocyte apoptosis by acting as a sponge for miR-146a to upregulate Smad4.

Background: Human adipose mesenchymal stem cells (hADSCs) show poor survival after transplantation, limiting their clinical application. Tissue regeneration resulting from stem cell treatment may be caused by attenuation of hypoxia-inducible factor-1α (HIF-1α). In this study, we constructed hADSCs stably expressing HIF-1α and investigated the potential effects of HIF-1α expression in the ischemic microenvironment on mitochondrial apoptosis and survival of hADSCs, and studied the mechanisms involved., Method: Apoptosis was induced by an ischemic microenvironment in vitro. ADSCs with stable HIF-1α expression were established. Cell survival and apoptosis were observed by CCK-8 assay, western blotting, flow cytometry, and fluorescence staining. ADSCs were subcutaneously transplanted into nude mice in the location where a hypoxia ischemic microenvironment was simulated in vivo. After 1, 3, and 7 d, mitochondrial apoptotic proteins were evaluated by immunohistochemistry and immunofluorescence staining., Results: Exogenous HIF-1α downregulated mitochondrial reactive oxygen species, cytochrome c, caspase-9, and caspase-3, but inhibited mitochondrial membrane potential depolarization and increased the Bcl-2/bax ratio. HIF-1α prevented apoptosis and promoted vascular endothelial growth factor (VEGF) secretion as demonstrated by enzyme-linked immunosorbent assay (ELISA), terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining, and flow cytometry analysis. HIF-1α enhanced the survival of transplanted ADSCs in nude mice., Conclusion: We have shown that through inhibition of the mitochondria-mediated apoptotic pathway and promotion of VEGF secretion in hADSCs in an ischemic microenvironment, HIF-1α may potentially be applied in clinical therapy and as an alternative strategy for improving hADSC therapy., Competing Interests: Declaration of competing Interest The authors declare that they have no competing interests, (Copyright © 2020 British Association of Plastic, Reconstructive and Aesthetic Surgeons. Published by Elsevier Ltd. All rights reserved.)

Rheumatoid arthritis (RA), recognized as a common chronic autoimmune disease, is characterized by the excessive proliferation and inflammatory infiltration of fibroblast-like synoviocytes (FLS). In this study, our purpose is to elucidate the mechanisms of triptolide (TPL) in the treatment of RA by regulating the long non-coding RNA (lncRNA) ENST00000619282, which promoted apoptosis and reduced inflammatory infiltration of FLS in RA (RA-FLS). RA-FLS was treated with different concentrations of TPL at different time points. CCK-8 assay, ELISA, RT-qPCR, immunofluorescence, TUNEL assay, and the transmission electron microscopy were used to measure the changes of cell viability, apoptosis, and the release of inflammatory cytokines. Next, the involvement of ENST00000619282 in TPL-mediated protection against RA was explored. ENST00000619282 expression was significantly increased in the peripheral blood mononuclear cells (PBMCs) of RA patients. ENST0000061928 expression in RA PBMCs was positively associated with ESR, RF, CCP, and DAS28, while TPL treatment led to a downregulation of ENST00000619282. In addition, ENST00000619282 was significantly increased in RA-FLS. Furthermore, overexpression of ENST00000619282 elevated the levels of pro-apoptotic and pro-inflammatory factors, while reduced the levels of anti-apoptotic proteins and antiinflammatory factors. Besides, TPL treatment could reverse these effects by ENST00000619282 overexpression. The anti-RA potential of TPL might be achieved by downregulating ENST00000619282, thereby promoting apoptosis, and reducing the inflammatory response in RA., (© 2021 John Wiley & Sons Ltd.)

Non-small-cell lung cancer (NSCLC) remains the most common malignancy with the highest morbidity and mortality worldwide. In our previous study, we found that a classic traditional Chinese medicine (TCM) formula Ze-Qi-Tang (ZQT), which has been used in the treatment of respiratory diseases for thousands of years, could directly inhibit the growth of human NSCLC cells via the p53 signaling pathway. In this study, we explored the immunomodulatory functions of ZQT. We found that ZQT significantly prolonged the survival of orthotopic lung cancer model mice by modulating the tumor microenvironment (TME). ZQT remarkably reduced the number of MDSCs (especially G-MDSCs) and inhibited their immunosuppressive activity by inducing apoptosis in these cells via the STAT3/S100A9/Bcl-2/caspase-3 signaling pathway. When G-MDSCs were depleted, the survival promotion effect of ZQT and its inhibitory effect on lung luminescence signal disappeared in tumor-bearing mice. This is the first study to illustrate the immunomodulatory effect of ZQT in NSCLC and the underlying molecular mechanism., Competing Interests: The authors declare no conflict of interest in this study., (Copyright © 2021 Zi-hang Xu et al.)

Objective: This report was designed to assess the functional role of miR-218/dachshund family transcription factor 1 (DACH1) in diabetic kidney disease (DKD) and investigate its possible molecular mechanism. Materials and Methods: From the GEO database, we downloaded different datasets for analyzing the expression of miR-218 and DACH1 in DKD. TargetScan was adopted to predict the binding sites between miR-218 and DACH1, which was further verified by dual-luciferase reporter assays. The renal proximal tubule cells (HK-2) treated with high glucose (HG) were used as an in vitro model. QRT-PCR and western blot were used to determine the expression of DACH1 and other relative factors. Cell counting kit-8 and flow cytometer were applied to detect cell viability and apoptosis. The levels of inflammatory cytokines were determined by an ELISA assay. Results: A prominent raise of miR-218 was observed in DKD through bioinformatics analysis, which was further confirmed in the HG-induced model. DACH1 is a target of miR-218. miR-218 reduced cell viability and induced apoptosis by negatively regulating DACH1. Moreover, upregulating miR-218 in HG models increased the concentrations of pro-inflammatory cytokines TNF-α and IL-1β, reduced the level of anti-inflammatory cytokine IL-10, and promoted the epithelial-mesenchymal transition (EMT) process, which is possibly achieved by targeting DACH1. While downregulating miR-218 showed the opposite results. Conclusion: These data demonstrated that, under an in vitro HG environment, miR-218 suppressed the HK-2 cells proliferation, promoted apoptosis, caused an inflammatory response, and facilitated the EMT process largely by targeting DACH1, providing an insight into the therapeutic intervention of DKD.

Importance: Programmed cell death 1 (PD-1) antibodies have shown substantial survival benefit in patients with advanced non-small cell lung cancer (NSCLC). Toripalimab is a promising and practicable PD-1 antibody; however, its performance in NSCLC has not been established., Objectives: To assess the safety, antitumor activity, and pharmacokinetics of toripalimab in patients with advanced NSCLC and to evaluate the utility of JS311, a novel PD ligand 1 (PD-L1) immunohistochemistry (IHC) assay., Design, Setting, and Participants: This single-arm open-label phase 1 trial enrolled 41 patients with advanced NSCLC that had progressed after at least 3 lines of therapy between September 21, 2017, and June 5, 2018, with a median (interquartile range) follow-up of 14.9 (3.2-22.5) months and included a cohort study comparing JS311 with other PD-L1 IHC assays that included 280 NSCLC specimens collected from January 1, 2016, to May 21, 2018. Data collection was conducted from September 21, 2017, to September 27, 2019, and analysis was conducted from September 27, 2019, to December 30, 2019., Exposure: Enrolled patients were administered a single dose of toripalimab, under 2 manufacturing processes and scales (200 L and 500 L), for safety and pharmacokinetic analysis within 28 days, followed by subsequent multidose infusions every 2 weeks. PD-L1 expression was determined by IHC with JS311, comparing its results with results from 22C3, 28-8, and SP263 simultaneously., Main Outcomes and Measures: Progression-free survival (PFS) and overall survival (OS), estimated by Kaplan-Meier curves, and continuous variables compared by t test or Mann-Whitney test. Correlations between PD-L1 IHC antibodies were evaluated by Spearman correlation test., Results: A total of 41 patients (29 [70.7%] men) with a median (interquartile range) age of 59 (53 to 63) years who experienced disease progression following chemotherapy were included. The most common treatment-related adverse events were rash (6 [14.6%]), increased amylase level (5 [12.2%]), and increased aspartate aminotransferase level (5 [12.2%]). In 35 patients included in the pharmacokinetic analysis, drug exposure and area under curve after 1 dose was similar under both manufacturing processes and scales (mean [SD] for 200-L group: 12 465.28 [4128.17] hour × μg/mL; for 500-L group: 12 331.42 [2472.58] hour × μg/ml). In 28 patients included in the response and survival analysis, the median PFS and OS were 2.8 (95% CI, 2.7 to 4.6) months and 13.8 months (95% CI, 10.0 months to not reached), respectively. Stratified by PD-L1 tumor proportion score of at least 50%, 1% to 49%, and less 1%, median PFS rates were 11.2 months (95% CI, 2.3 months to not evaluable), 2.3 (95% CI, 1.7 to 2.7) months, and 2.8 (95% CI, 2.7 to 4.6) months, respectively. A total of 4 anti-PD-L1 IHC antibodies were compared during PD-L1 staining, using 280 NSCLC specimens. The consistency rates between the 4 antibodies were 80.8% to 89.5% (ρ, 0.619 to 0.790) and 93.3% to 95.5% (ρ, 0.691 to 0.773), with PD-L1 tumor proportion scores of 1% and 50% as cut points, respectively., Conclusions and Relevance: In this study, toripalimab exhibited encouraging antitumor activity and manageable safety profiles among patients with heavily treated NSCLC. The novel PD-L1 IHC antibody JS311 was highly consistent with previously verified PD-L1 IHC assays.

Long noncoding RNAs (lncRNAs) are >200-bp molecules that do not generally code for proteins. Human lncRNAs have well-characterized roles in gene expression regulation, particularly with regard to protein-coding genes, and their dysregulation has been linked to disease. Here, we set out to investigate changes in the expression of lncRNAs related to apoptosis and autophagy in the peripheral blood mononuclear cells (PBMCs) of rheumatoid arthritis (RA). In addition, we aimed to correlate lncRNA expression profiles with clinical indexes and self-perception of patients (SPP). To this end, we employed RNA sequencing of lncRNAs in PBMCs from three patients with RA and three healthy controls. We used bioinformatics to screen several dysregulated lncRNAs related to apoptosis and autophagy. To validate key lncRNA candidates, we performed quantitative reverse transcriptase-PCR on 20 patients with RA and 20 healthy controls. We found the expression of seven lncRNAs (MAPKAPK5-AS1, ENST00000619282, C5orf17, LINC01189, LINC01006, DSCR9 and MIR22HG) was significantly altered in PBMCs of patients with RA. Receiver operating characteristic curve analysis suggested that MIR22HG [area under the curve (AUC) = 0.846, P = 0.000], DSCR9 (AUC = 0.783, P = 0.005), LINC01189 (AUC = 0.677, P = 0.034), MAPKAPK5-AS1 (AUC = 0.644, P = 0.025) and ENST00000619282 (AUC = 0.636, P = 0.043) are potential biomarkers of RA. Spearman's correlation analysis revealed selected lncRNAs correlated with clinical indexes and SPP. Therefore, we highlight that some lncRNAs related to apoptosis and autophagy may serve as potential biomarkers for diagnosis and monitoring of RA progression, which also correlate with several clinical indexes and SPP., (© 2020 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

Histone demethylase KDM7A regulates neuronal differentiation and development in mammals. In this study, we found that KDM7A was also required for breast cancer stem cells (BCSCs) maintenance. Silencing KDM7A significantly reduced the BCSCs population and mamosphere formation in vitro, and inhibited breast tumor growth in vivo. Restoring KDM7A expression rescued the defect in stem cell maintenance. Our mechanism analysis suggested that KDM7A upregulated the stemness-associated factors KLF4 and c-MYC for BCSCs maintenance. In addition, KDM7A knockdown promoted apoptosis through decreasing BCL2 expression and BAD phosphorylation in breast cancer (BrCa). Furthermore, restoring KDM7A and BCL2 expression rescued apoptosis inhibition in breast cancer, suggesting that KDM7A inhibited apoptosis by upregulating the BCL2 level in breast cancer. In conclusion, KDM7A promotes cancer stem cell maintenance and apoptosis inhibition in breast cancer., (© 2019 Wiley Periodicals, Inc.)

Apoptosis induced by oxidative stress is one of the most important cardiomyocytes losses during ischemia-reperfusion (I/R). Catestatin (CST) has been demonstrated to have the anti-oxidative capacity in vitro. We hypothesized that CST intervention could reduce apoptosis of cardiomyocytes induced by oxidative stress in I/R. In Langendorff-perfused rat heart global I/R model, CST was introduced at the reperfusion stage. In comparison to the control group, CST led to preservation on activities of superoxide dismutase and glutathione peroxidase, improvement of hemodynamics, and reduced infarction area in reperfused myocardium. The protection of CST was also shown by less apoptotic cardiomyocytes in TUNEL staining, less caspase-3 activation, and increased phosphorylation of protein kinase B (PKB/Akt) in Western blot. To further demonstrate the benefits of CST and explore the possible underlying mechanism, H 2 O 2 -challenged primary-cultured neonatal rat cardiomyocytes were used to simulate the oxidative-stressed scenario. CST incubation with the H 2 O 2 -challenged cardiomyocytes led to reduction of apoptosis, which was demonstrated by less Hoechst 33342 positive staining of nuclei, less caspase-3 activation, and DNA fragmentation. The effect of CST was abrogated by pretreatment of the cardiomyocytes with the PI3K inhibitor LY294002. Furthermore, Akt activation and the anti-apoptosis effect of CST were abolished by pretreatment of the cardiomyocytes with β2 receptor inhibitor ICI118551. Thus, the salvage of oxidative-stress-induced apoptotic cardiomyocytes in I/R by CST might involve activation β2 receptor and regulation of PI3K/Akt signaling in reperfusion injury salvage kinase (RISK) pathway., (Copyright © 2019. Published by Elsevier Inc.)

Coronary heart disease (CHD) is a common heart disease and the leading cause of cardiovascular death. Apoptosis and autophagy are two forms of programmed cell deaths which participate in the pathogenesis, development and prognosis of CHD. They are activated by several different pathways respectively and can interact with each other through the Beclin 1-Bcl-2/Bcl-xL complex, mTOR, TRAIL, TNF-α, ER stress and nucleus p53 pathways. Excessive apoptosis can promote myocardial ischemia, ischemia/reperfusion (I/R) injury, post-ischemia cardiac remodeling and coronary atherosclerosis except for VSMC-induced atherosclerosis progress. In contrast, activated autophagy protects heart from myocardial ischemia injury and post-ischemia cardiac remodeling, but can exert controversial effects on I/R injury and coronary atherosclerosis. Therefore, considering the pathological significance and mechanisms of apoptosis and autophagy underlying CHD, therapeutic implication of targeting apoptosis and autophagy is obvious. Fortunately, some therapeutic drugs and pharmacologic compounds involving mTOR inhibitor and AMPK activator have been reported to regulate apoptosis and autophagy. Although recent studies are limited and insufficient, they have pointed out the complex interplay between apoptosis and autophagy and further provided treatment concept for CHD by balancing the switch between the two responses., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Phospholipase C (PLC) can participate in cell proliferation, differentiation and aging. However, whether it has a function in apoptosis in porcine primary granulosa cells is largely uncertain. The objective of this study was to examine the effects of PLC on apoptosis of porcine primary granulosa cells cultured in vitro. The mRNA expression of BAK, BAX and CASP3, were upregulated in the cells treated with U73122 (the PLC inhibitor). The abundance of BCL2 mRNA, was upregulated, while BAX and CASP3 mRNA expression was decreased after treatment with m-3M3FBS (the PLC activator). Both the early and late apoptosis rate were maximized with 0.5 μM U73122 for 4 h. The rate of early apoptosis was the highest at 4 h and the rate of late apoptosis was the highest at 12 h in the m-3M3FBS group. The protein abundance of PLCβ1, protein kinase C β (PKCβ), calmodulin-dependent protein kinaseII α (CAMKIIα) and calcineurinA (CalnA) were decreased by U73122, and CAMKIIα protein abundance was increased by m-3M3FBS. The mRNA expression of several downstream genes (CDC42, NFATc1, and NFκB) was upregulated by PLC. Our results demonstrated that apoptosis can be inhibited by altering PLC signaling in porcine primary granulosa cells cultured in vitro, and several calcium - sensitive targets and several downstream genes might take part in the processes.

Methamphetamine (METH) is an amphetamine-type drug that is highly addictive and widely abused. Many studies have shown that METH exposure causes severe damage not only to the nervous system but also to the cardiovascular system. Melusin protein is a mechanotransducer that plays an important role in maintaining normal heart function. However, the role of melusin in METH-induced cardiotoxicity has not yet been reported. We hypothesized that methamphetamine can produce cardiac damage and apoptosis by decreasing the quantity of melusin. To test this hypothesis, we determined the protein expression of melusin and apoptosis markers in METH-treated rats and primary rat cardiomyocytes. We also established a melusin-overexpressing cell model to assess the importance of melusin in maintaining antiapoptotic pathways. To confirm our findings from the in vitro and animal models, we also evaluated the apoptotic index of cardiomyocytes and the protein expression of apoptotic markers in postmortem heart tissues from deceased METH abusers and age-matched control subjects. The results showed that the apoptosis of cardiomyocytes was increased significantly and that the protein expression of melusin was decreased after exposure to METH in primary rat cardiomyocytes, in rats and in humans. METH treatment also decreased the expression of the downstream proteins FAK, IQGAP1, p-AKT, p-GSK3β, and p-ERK in primary rat cardiomyocytes and in vivo. After overexpression of melusin, the above effects were partially reversed in primary rat cardiomyocytes. We conclude that METH can produce cardiac damage and apoptosis by decreasing melusin, while melusin-activated signaling by phosphorylated AKT, phosphorylated GSK3β, and ERK may be resistant to methamphetamine-induced myocardial apoptosis., (Copyright © 2019. Published by Elsevier Inc.)

Serratia marcescens are considered to be abundant and optimal resources for obtaining prodigiosin, which can be isolated from soil, water, plants and air but rarely from insects. In the present study, a strain of Serratia marcescens named WA12‑1‑18 was isolated from the gut of Periplaneta americana, which was capable of producing high levels of pigment reaching 2.77 g/l via solid fermentation and was identified as prodigiosin by ultraviolet, high performance liquid chromatography (LC), Fourier‑transform infrared spectroscopy, LC‑mass spectroscopy and nuclear magnetic resonance. The apoptotic tumor cells treated with prodigiosin were examined by 4',6‑diamidino‑2‑phenylindole (DAPI) staining assays and transmission electron microscopy. Flow cytometry (FCM) was utilized to measure the apoptotic rate with Annexin V staining and the expression levels of proteins involved in apoptosis, including B‑cell lymphoma 2 (Bcl‑2), Bcl‑2‑associated X (Bax) and caspase‑3 were determined by western blot analysis and reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR). The experimental results revealed that prodigiosin could inhibit the proliferation of HeLa cells and the half‑maximal inhibitory concentration values of prodigiosin in HeLa were 2.1, 1.2 and 0.5 µg/ml over 24, 48 and 72 h, respectively. Furthermore, DAPI staining assays and transmission electron microscopy clearly demonstrated that prodigiosin could induce HeLa cell apoptosis. FCM results revealed that the cell apoptotic rates were 19.7±1.4, 23.7±2.4 and 26.2±2.3% following the treatment with 0.5, 1.0 and 2.0 µg/ml prodigiosin for 48 h, respectively. Western blot analysis and RT‑qPCR revealed that prodigiosin could activate apoptosis‑associated molecules including Bcl‑2, Bax and caspase‑3. Therefore, the results of the present study demonstrated that the prodigiosin could induce apoptosis in HeLa cells, which may be associated with the upregulation of Bax and caspase‑3, the concomitant downregulation of Bcl‑2 levels and also triggering the extrinsic apoptotic signaling pathway.

Mycobacterium bovis ( M. bovis ) is a member of the Mycobacterium tuberculosis ( Mtb ) complex causing bovine tuberculosis (TB) and imposing a high zoonotic threat to human health. Kallikreins (KLKs) belong to a subgroup of secreted serine proteases. As their role is established in various physiological and pathological processes, it is likely that KLKs expression may mediate a host immune response against the M. bovis infection. In the current study, we report in vivo and in vitro upregulation of KLK12 in the M. bovis infection. To define the role of KLK12 in immune response regulation of murine macrophages, we produced KLK12 knockdown bone marrow derived macrophages (BMDMs) by using siRNA transfection. Interestingly, the knockdown of KLK12 resulted in a significant downregulation of autophagy and apoptosis in M. bovis infected BMDMs. Furthermore, we demonstrated that this KLK12 mediated regulation of autophagy and apoptosis involves mTOR/AMPK/TSC2 and BAX/Bcl-2/Cytochrome c/Caspase 3 pathways, respectively. Similarly, inflammatory cytokines IL-1β, IL-6, IL-12 and TNF-α were significantly downregulated in KLK12 knockdown macrophages but the difference in IL-10 and IFN-β expression was non-significant. Taken together, these findings suggest that upregulation of KLK12 in M. bovis infected murine macrophages plays a substantial role in the protective immune response regulation by modulating autophagy, apoptosis and pro-inflammatory pathways. To our knowledge, this is the first report on expression and the role of KLK12 in the M. bovis infection and the data may contribute to a new paradigm for diagnosis and treatment of bovine TB.

Prostate cancer is a common type of malignancy. Given the complexity of prostate cancer and the pressing challenge of chemoresistance, the current study was conducted to investigate the effect of docetaxel (Doc) on androgen receptor (AR)‑dependent and AR‑independent prostate cancers cells. Subsequent experiments were designed to explore the mechanism underlying the Doc‑induced apoptosis. Three different human prostate cancer cell lines, namely PC‑3, LNCaP and DU‑145, were exposed to various concentrations of Doc. The cytotoxic effects of Doc were evaluated by an MTT assay, while apoptosis and cell cycle distribution were determined by flow cytometric analysis of cells stained with Annexin V‑FITC and propidium iodide. Western blot assay was also used to measure the protein levels of B‑cell lymphoma 2 (Bcl‑2), Bcl‑2‑associated death promoter (Bad), total protein kinase B (Akt), phospho‑Akt and caspase‑3/9. Doc induced cytotoxicity in all three cell lines in a dose‑dependent manner. The half maximal inhibitory concentration values for the effect of Doc on PC‑3, DU‑145 and LNCaP cells were 3.72, 4.46 and 1.13 nM, respectively. Furthermore, the results indicated a significant difference in Doc sensitivity between AR‑dependent and AR‑independent prostate cancer cells. Evaluation of key gene expression at protein levels revealed a notable decrease in antiapoptotic Bcl‑2 and p‑Akt levels, along with a significant increase in pro‑apoptotic Bad, caspase‑3 and caspase‑9 levels. Therefore, Doc may induce cell apoptosis in prostate cancer via various pathways.

Chronic rhinosinusitis without nasal polyps (CRSsNP) is the main type of Chronic rhinosinusitis (CRS) and is a common otorhinolaryngologic disease worldwide. However, the mechanisms of CRSsNP remain poorly understood. In this study, C57BL/6J wild-type and urokinase-type plasminogen activator (uPA) gene knockout (uPA -/- ) mice were used to construct the CRSsNP model. Primary human nasal epithelial cells (HNEC) were isolated from CRSsNP patient and treated with uPA knockdown/overexpression lentivirus. CCK-8 and Annexin-V/PI staining were used to detected cell proliferation and apoptosis. In vivo, we found that uPA depletion alleviated mucosal inflammation in the CRSsNP mice model. Wnt inhibitory factor 1 (WIF1) was upregulated in the uPA -/- CRSsNP mice model. In vitro, inhibition of uPA increased cell proliferation and decreased cell apoptosis. Mechanistically, uPA depletion upregulated WIF1 and BCL2 expression, and reduced the expression level of BAX in CRSsNP HNEC. In contrast, decreased cell proliferation and increased cell apoptosis were observed after uPA overexpression. Consistently, a reduction in WIF1 and BCL2 expression levels and an increase in the BAX expression level were observed upon uPA ectopic expression. Furthermore, WIF1 overexpression rescued the effects caused by uPA overexpression in vitro. In conclusion, uPA affects the CRSsNP nasal mucosal epithelium cell apoptosis by upregulating WIF1. To our knowledge, this is the first study to explore the role of uPA in CRSsNP to date., (Copyright © 2018. Published by Elsevier Inc.)

Thioredoxin reductase (TrxR), a major component of the thioredoxin system, makes a critical role in regulating cellular redox signaling and is found to be overexpressed in many human cancer cells. TrxR has become an attractive target for anticancer agents. In this work, three Ru(II) complexes with salicylate as ligand, [Ru(phen) 2 (SA)] (phen = 1,10-phenanthroline, SA = salicylate, 1), [Ru(dmb) 2 (SA)] (dmb = 4,4'-dimethyl-2,2'-bipyridine, 2) and [Ru(bpy) 2 (SA)] (bpy = 2,2'-bipyridine, 3), were synthesized and characterized. The anticancer effect exerted by them was evaluated. Complex 1 was found to exhibit obvious anticancer activity, in comparison with cisplatin, against cancer cell lines, while displaying low toxicity to the normal cell line BEAS-2B. The mechanism of complex 1 cancer cell growth suppress was investigated in A549 cells. Complex 1 exerted its anticancer through inducing apoptosis and triggering cell cycle arrest at the G0/G1 phase. Complex 1 can selectively inhibit TrxR activity and thus promote the generation and accumulation of reactive oxygen species (ROS), which subsequently trigger mitochondrial dysfunction and DNA damage, activate oxidative stress-sensitive mitogen activated protein kinase (MAPK), and suppress the protein kinase B (PKB or AKT) signal pathway, resulting in apoptosis in A549 cells., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Two 8-O-4'-type neolignan epimers erythro-guaiacylglycerol-β-coniferyl aldehyde ether (1) and threo-guaiacylglycerol-β-coniferyl aldehyde ether (2) were isolated from the stems of Picrasma quassioides. Further chiral separation gave two pairs of enantiomers 1a/1b and 2a/2b. The cytotoxicity assay against hepatocellular carcinoma Hep3B and HepG2 cells was evaluated by MTT assay. The results showed that 1b (IC 50  = 45.56 μM) and 2b (IC 50  = 39.02 μM) had more cytotoxic effect than its enantiomers 1a (IC 50  = 82.66 μM) and 2a (IC 50  = 67.97 μM) in Hep3B cells, respectively. Moreover, 1b and 2b could induce more apoptotic cells as well as higher reactive oxygen species (ROS) generation than 1a and 2a at 50 μM. In addition, a further study on the phosphoinositide 3-kinase (PI3K)/AKT and mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathways was investigated. The results revealed that all compounds had no significant effect on PI3K/AKT pathway, however, 1b and 2b attenuated the relative levels of p-MEK and p-ERK when compared with 1a and 2a. Taken together, the absolute configurations of guaiacylglycerol-β-coniferyl aldehyde ether had an impact on the inhibitory effect on Hep3B cells. The inactivation of MEK/ERK signaling pathway might contribute to apoptosis induction and ROS generation in 1b- and 2b-treated cells., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Heat stress affects milk yield and quality in lactating dairy cows in summer. Bovine mammary epithelial cells (bMECs) play a key role in milk secretion, and microRNAs (miRNAs) regulate numerous functions of bMEC. Previous reports have verified that miR-216b regulated cell apoptosis through repressing target genes in several cancer cells. So, our purpose was to explore the potential involvement of miR-216b in heat stress-induced cell apoptosis in bMECs. Firstly, the heat stress model was constructed and we found that apoptotic rates of bMECs significantly increased under heat stress. The expression of miR-216b, Bax mRNA, and caspase-3 mRNA was upregulated. However, Bcl-2 mRNA level was detected to differentially downregulated. Overexpression of miR-216b remarkably downregulated the expression of caspase-3 and Bax mRNA and protein, and the mRNA and protein level of Bcl-2 was increased. Inhibition of miR-216b increased the activity of caspase-3 and Bax, and the level of Bcl-2 was inhibited. Moreover, Fas was identified as a target gene of miR-216b through bioinformatic analysis and dual-luciferase reporter assay. Fas activity was significantly inhibited and enhanced respectively after transfecting miRNA mimics and inhibitor. Finally, inhibition of Fas via the small interfering RNA (siRNA) also inhibited cell apoptosis induced by heat stress. Taken together, our results indicated that miR-216b exerted as an anti-apoptotic effect under heat stress in bMECs by targeting Fas.

Circulating tumor cells (CTCs) are known as the root cause of cancer metastasis that accounts for 90% of cancer death. Owing to the rarity of blood CTCs and their microenvironmental complexity, the existing biotechnology could not precisely capture and apoptosize CTCs in vivo for cancer metastasis prevention. Here, we designed two double strand circular aptamers aimed to simultaneously target MUC1 and HER2 surface biomarkers on mesenchymal cancer cells. The circular aptamers are composed of a capture arm for binding and seizing CTCs and a circular body for resisting degradation by exonucleases. We conjugated the two circular aptamers onto dendrimer PAMAM G4.5 (dcAp1-G-dcAp2), and the conjugate entity showed both significantly-enhanced biostability in serum for days compared with their linear counterparts and capture specificity in RBC (1:10 8 ) compared with their single circular aptamers. dcAp1-G-dcAp2 apoptosized the targeted cells and inhibited their bioenergetic activities significantly by lowing △Ψm, ATP and lactate productions while increasing ROS production. dcAp1-G-dcAp2 captured CTCs in mice in vivo and in patient blood. This study lays the foundation for developing multiple biostable circular aptamers and conjugating them together to precisely capture and apoptosize mesenchymal CTCs in vivo., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Female germline stem cells (FGSCs) are proposed to be a key factor for ameliorating female infertility. Previously we have shown that neonatal and adult FGSCs could be isolated and purified from mouse ovarian tissues. The long noncoding (lnc) RNA growth arrest-specific 5 sequence (GAS5) transcribed from mammalian genomes plays important regulatory roles in various developmental processes. However, there is no study on the relationship between GAS5 and FGSC development in vitro. In this study, we showed that GAS5 was highly expressed in the neonatal mouse ovary and was located in both FGSCs and oocytes. GAS5 facilitated FGSC proliferation and promoted their survival in vitro. Moreover, GAS5 also inhibited apoptosis of cultured FGSCs. These findings indicate that GAS5 is a crucial regulator of FGSC development. This might serve as a foundation for a strategy of lncRNA-directed diagnosis or treatment of female infertility., (Copyright © 2018. Published by Elsevier B.V.)

Three pairs of enantiomeric neolignans 1a/1b-3a/3b were isolated from the stems of Picrasma quassioides, and separated successfully by chiral-phase HPLC. Their structures were established by comprehensive spectroscopic analyses as well as ECD spectroscopy. The in vitro cytotoxicity of the isolates was evaluated against human hepatocellular carcinoma HepG2 and Hep3B cells. Among them, 1 and its enantiomers 1a/1b, 3 and 3a/3b displayed similar cytotoxicity in pair-wise comparison against HepG2 and Hep3B cells, and the similar effects of 2 and 2a/2b were found in Hep3B cells. Interestingly, 2a and 2b had different cytotoxic activities on HepG2 cells with IC 50 values of 35.6 μM and 104.4 μM, respectively. In addition, 2 exerted middle cytotoxicity against HepG2 cells with an IC 50 value of 78.6 μM. The different cytotoxicity between enantiomers 2a and 2b attracted our interest. To investigate the underlying mechanisms responsible for the distinct cytotoxicity, we further assessed the effects of 2a and 2b on cell cycle distribution, cell apoptosis and reactive oxygen species (ROS) generation. The results indicated that 2a had more significant effect than 2b on apoptosis induction and ROS generation, but both had no obvious effect on cell cycle of HepG2 cells. It is concluded that the different configurations of 2a/2b determined the enantioselective cytotoxicity on HepG2 cells through apoptosis induction and ROS generation., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Bone marrow-derived progenitor cells (BMPCs) are potential candidates for autologous cell therapy in tissue repair and regeneration because of their high angiogenic potential. However, increased progenitor cell apoptosis in diabetes directly limits their success in the clinic. MicroRNAs are endogenous noncoding RNAs that regulate gene expression at the posttranscriptional level, but their roles in BMPC-mediated angiogenesis are incompletely understood. In the present study, we tested the hypothesis that the proangiogenic miR-27b inhibits BMPC apoptosis in Type 2 diabetes. Bone marrow-derived EPCs from adult male Type 2 diabetic db/db mice and their normal littermates db /+ mice were used. MiR-27b expression (real-time PCR) in EPCs was decreased after 24 h of exposure to methylglyoxal (MGO) or oxidized low-density lipoprotein but not high glucose, advanced glycation end products, the reactive oxygen species generator LY83583, or H 2 O 2 The increase in BMPC apoptosis in the diabetic mice was rescued following transfection with a miR-27b mimic, and the increased apoptosis induced by MGO was also rescued by the miR-27b mimic. p53 protein expression and the Bax/Bcl-2 ratio in EPCs (Western blot analyses) were significantly higher in db/db mice, both of which were suppressed by miR-27b. Furthermore, mitochondrial respiration, as measured by oxygen consumption rate, was enhanced by miR-27b in diabetic BMPCs, with concomitant decrease of mitochondrial Bax/Bcl-2 ratio. The 3' UTR binding assays revealed that both Bax, and its activator RUNX1, were direct targets of miR-27b, suggesting that miR-27b inhibits Bax expression in both direct and indirect manners. miR-27b prevents EPC apoptosis in Type 2 diabetic mice, at least in part, by suppressing p53 and the Bax/Bcl-2 ratio. These findings may provide a mechanistic basis for rescuing BMPC dysfunction in diabetes for successful autologous cell therapy.

Trauma-induced secondary cardiac injury (TISCI) is associated with increased adverse cardiac events and death. We have previously reported that TISCI results in myocardial apoptosis and secondary cardiac dysfunction. However, the underlying mechanism is unclear. To identify the time course of trauma-induced cardiomyocyte apoptosis and possible apoptotic pathway, traumatic rat models were built with Noble-Collip drum. Meanwhile, normal rat cardiomyocytes were cultured with traumatic plasma (TP) for 48 h. Cardiomyocyte apoptosis, cardiac function and the apoptosis related enzymes, including caspase-3, -8, -9, and -12, were determined. The results showed that there was no direct injury of rat hearts immediately after trauma. However, compared with hearts from the sham rats, hearts isolated from traumatic rats exhibited reduced +dP/dT max and -dP/dT max 24 h after trauma. In traumatic rats, myocardial apoptotic index and caspase-3 activity obviously increased 6 h after trauma, and achieved the maximal value 12 h after trauma. The activity and expression of caspase-12, an endoplasmic reticulum (ER) stress-specific caspase, elevated markedly 3 h after trauma and reached its peak 6 h after trauma. Otherwise, caspase-8 (extrinsic apoptotic pathway) and caspase-9 (intrinsic apoptotic pathway) in the myocardial tissue of traumatic rats were activated 24 h after trauma. Meanwhile, incubation of normal rat cardiomyocytes with TP increased caspase-12 activity at 6 h, caspase-3 activity at 12 h, caspase-8 and -9 activities at 24 h, respectively. TP-induced cardiomyocyte apoptosis was virtually abolished by Z-ATAD-FMK (a caspase-12 specific inhibitor). In addition, there was a significant negative correlation between myocardial caspase-12 activity and trauma-induced secondary cardiac dysfunction. Our present study demonstrated that caspase-12 is firstly activated and plays an important role in TISCI rats. Inhibition of caspase-12 mediated apoptosis may be a novel strategy in ameliorating posttraumatic cardiomyocyte apoptosis and secondary cardiac injury.

This manuscript describes a synergistic therapy for mastocarcinoma by pH and temperature dual-sensitive nanogel, and effects of microstructure, composition and properties of nanogel on the cellular response mechanism. The extracellular internalization of nanogels was obviously enhanced, due to the passive targeting function at T>VPTT. Interestingly, the increased cytotoxicity was further synergistically enhanced by an unexpected apoptosis as evoked by the 5-fluorouracil loaded nanogel (FLNG). The systemically evaluation of the effectors generated from different sub-cellular organelles including endosome, lysosome, autophagosome confirmed that it was a lysomal dependent apoptosis. Such specific apoptosis was mainly attributed to its activatable protonated PEI at low pH, which caused lysosomal membrane destruction and lysosomal enzyme cathepsin B (Cat B) leakage. This Cat B was then translocated to the mitochondria resulting in mitochondrial membrane permeability increase and mitochondrial membrane potential (MMP) decrease, followed by cytochrome c (Cyt C) release. Cyt C was the main molecule that evoked apoptosis as reflected by overexpression of caspase 9. Additionally, such lysosome dependent, apoptosis was further enhanced by the passive cellular targeting at T>VPTT. Thus, the tumor growth inhibition was synergistically enhanced by the extracellular temperature dependent passive targeting and intracellular pH activatable lysosomal dependent apoptosis., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Sulphated lentinan (sLTN) is known to act as a resistance inducer by causing programmed cell death (PCD) in tobacco suspension cells. However, the underlying mechanism of this effect is largely unknown. Using tobacco BY-2 cell model, morphological and biochemical studies revealed that mitochondrial reactive oxygen species (ROS) production and mitochondrial dysfunction contribute to sLNT induced PCD. Cell viability, and HO/PI fluorescence imaging and TUNEL assays confirmed a typical cell death process caused by sLNT. Acetylsalicylic acid (an ROS scavenger), diphenylene iodonium (an inhibitor of NADPH oxidases) and protonophore carbonyl cyanide p-trifluoromethoxyphenyl hydrazone (a protonophore and an uncoupler of mitochondrial oxidative phosphorylation) inhibited sLNT-induced H 2 O 2 generation and cell death, suggesting that ROS generation linked, at least partly, to a mitochondrial dysfunction and caspase-like activation. This conclusion was further confirmed by double-stained cells with the mitochondria-specific marker MitoTracker RedCMXRos and the ROS probe H 2 DCFDA. Moreover, the sLNT-induced PCD of BY-2 cells required cellular metabolism as up-regulation of the AOX family gene transcripts and induction of the SA biosynthesis, the TCA cycle, and miETC related genes were observed. It is concluded that mitochondria play an essential role in the signaling pathway of sLNT-induced ROS generation, which possibly provided new insight into the sLNT-mediated antiviral response, including PCD., (Copyright © 2016. Published by Elsevier Inc.)

The present study was aimed at investigating the effect of amifostine on myocardial ischemia/reperfusion (I/R) injury of mice and H9c2 cells cultured with TBHP (tert-butyl hydroperoxide). The results showed that pretreatment with amifostine significantly attenuated cell apoptosis and death, accompanied by decreased reactive oxygen species (ROS) production and lower mitochondrial potential (ΔΨm). In vivo, amifostine pretreatment alleviated I/R injury and decreased myocardial apoptosis and infarct area, which was paralleled by increased superoxide dismutase (SOD) and reduced malondialdehyde (MDA) in myocardial tissues, increased Bcl2 expression, decreased Bax expression, lower cleaved caspase-3 level, fewer TUNEL positive cells, and fewer DHE-positive cells in heart. Our results indicate that amifostine pretreatment has a protective effect against myocardial I/R injury via scavenging ROS.

Background/aims: The effects of zinc signaling on proliferation or apoptosis of leukemia cells remain elusive. In the present study, we used N, N, N', N'-tetrakis-(2-pyridylmethyl)-ethylene-diamine (TPEN), a membrane-permeable zinc chelator, to evaluate the effect of zinc depletion on survival and apoptosis of NB4 acute promyelocytic leukemia (APL) cells., Methods: The pro-apoptotic effects of TPEN on NB4 cells were examined by flow cytometry, and observed using an optical microscope. Intracellular labile zinc, nitric oxide (NO) or reactive oxygen species (ROS) changes caused by TPEN were measured by flow cytometry. We then explored possible roles of the crosstalk between intracellular labile zinc signaling and nitric oxide signaling in TPEN-triggered apoptosis., Results: we found that TPEN induced apoptosis in NB4 APL cells in a dosage-dependent manner. We further demonstrated that TPEN triggered apoptosis by attenuating intracellular zinc and nitric oxide signaling in NB4 cells. Both exogenous zinc supplement and the nitric donor sodium nitroprusside (SNP) pre-incubation reversed TPEN-mediated inhibition of intracellular NO and Zn2+ signaling, and rescued NB4 cells from apoptosis., Conclusion: These results suggest for the first time that crosstalk between zinc signaling and nitric oxide pathway is essential for the survival of NB4 cells. TPEN induces apoptosis in NB4 cells via negatively regulating intracellular NO and Zn2+ signaling. Our in vitro data suggest that zinc depletion by TPEN may be a potential therapeutic strategy for APL., (© 2017 The Author(s). Published by S. Karger AG, Basel.)

Chinese medicines are emerging as an attractive new generation of anticancer drugs. Here, we explored the impact of salvianolic acid B (Sal B), the major water-soluble compounds of Danshen, on apoptosis and autophagy of human hepatocellular carcinoma cells (HCC). We also investigated the related molecular mechanisms. We found that Sal B exhibits potent ability to inhibit HCC cells viability in a concentration-dependent manner, and to induce apoptosis via the mitochondrial apoptosis pathway. Additionally, Sal B could also induce autophagy. Furthermore, pretreatment with the autophagy inhibitor chloroquine or 3-methyladenine showed the potential in attenuating the apoptosis rate induced by Sal B. Mechanistically, Sal B treatment inhibited the AKT/mTOR signaling cascade in vitro. Overexpression of AKT abolished the effects of Sal B on HCC cells, suggesting a critical role of the AKT/mTOR signaling pathway in Sal B-induced biological effects. Our results indicated that the mitochondrial pathway was involved in Sal B-induced apoptosis of HCC cells. Moreover, the AKT/mTOR signaling pathway was involved in Sal B-induced autophagy, which promoted apoptosis. This study may provide a promising strategy for using Sal B as a chemotherapeutic agent for patients with HCC.

Aim: The main aim of this article is to explain the apoptosis mechanisms of cancer cells specifically triggered by gold nanorods (GNRs)., Materials & Methods: GNRs were synthesized and optimized, the lysosome damage, cathepsin D, mitochondrial membrane potential, caspase-9, cleaved caspase-9, caspase-3 and intracellular GNRs location related to apoptosis was systematically evaluated., Results: GNRs specifically induce cancer cell apoptosis while posing a negligible impact on normal cells. After incubation with GNRs, the lysosomal permeability in cancer cells as indicated by cathepsin D was markedly higher than that in normal cells and resulted in an obvious decrease in mitochondrial membrane potential. Western blot analysis further confirmed that apoptosis occurred through caspase-9 and caspase-3 activation following mitochondrial damage. Transmission electron microscope images showed that GNRs did not appear in most of the damaged mitochondria but mainly accumulated in lysosomes., Conclusion: These findings indicated that GNR-induced apoptosis specifically in cancer cells by affecting lysosomes and mitochondria.

Oxalicumone A (POA1), a novel dihydrothiophene-condensed sulfur chromone isolated from the marine fungus Penicillium oxalicum SCSGAF 0023, showed cytotoxicity against several cancer cells previously. In this study, its anti-cancer activity and underlying mechanism of this action were investigated in leukemia cells like KG-1a, HL60, U937, and K562. The results showed that POA1 inhibited dose-/time-dependently cell growth and induced apoptosis in leukemia cells. Also, POA1 caused cleavages of caspase-3, 8, 9 and PARP1, loss of mitochondrial membrane potential, up-regulations of phosphorylated p38 and JNK, and activation of endoplasmic reticulum stress (ER stress). Furthermore, 4-PBA (an ER stress inhibitor) but not SP600125 and SB203580 (JNK and p38 inhibitor, respectively) could largely inhibit POA1-induced growth suppression. Additionally, 4-PBA obstructed mitochondrial depolarization and cleavage of PARP1. These data suggested that ER stress pathway might be an important mediator in POA1-induced apoptosis. In conclusion, POA1 may have antitumor effects in leukemia cells through the induction of ER stress pathway., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The apoptosis plays a critical role in a number of inflammatory disorders. Bacterial infection is one of the causes inducing apoptosis. This study aims to investigate the mechanism by which activation of TLR5 induces podocyte apoptosis. In this study, a podocyte cell line was cultured in RPMI1640 medium. The expression of TLR5 was assessed by real-time PCR and Western blotting. The Fas ligand gene transcription was assessed by immunoprecipitation and chromatin immunoprecipitation assay. The results showed that the expression of TLR5 was observed in the podocytes at both mRNA and protein levels. Exposure to TLR5 ligand, flagellin, induced Fas ligand expression and podocyte apoptosis. p300, one of the histone acetyltransferases, mediated the Fas ligand gene transcription in podocytes. In conclusion, TLR5 activation plays an important role in the induction of podocyte apoptosis., (Copyright © 2016 John Wiley & Sons, Ltd.)

Background: Breast cancer is characterized by a distinct metastatic pattern involving the regional lymph nodes, bone marrow, lung and liver. This study is aimed to investigate the effects of silencing the HAS-2 gene on the proliferation and apoptosis of human breast cancer cells., Methods: MCF-7 cells were collected and assigned into control, scrambled siRNA and HAS-2- siRNA groups. After transfection, the morphological changes in the MCF-7 cells were observed using phase contrast microscopy. qRT-PCR and Western blot assays were used to detect the mRNA and protein expression of apoptosis-related proteins. CCK-8 and flow cytometry were performed to evaluate cell proliferation, the cell cycle and apoptosis., Results: In the control and the scrambled siRNA groups, cells grew adhered to the wall and mainly showed a spindle shape with a clear nucleolus. Compared with the control and scrambled siRNA groups, increases in the number of cells in early apoptosis and metaphase cells in apoptosis were observed in the HAS-2-siRNA group. The HAS-2-siRNA group showed decreased expression of HAS-2 relative to that in the control and scrambled siRNA groups. No significant differences in cell proliferation, cell cycle distribution or apoptosis were noted between the control and scrambled siRNA groups. In the HAS-2-siRNA group, the cell proliferation ability decreased significantly, but the number of cells in the G0/G1 stage, the number of apoptotic cells and the expression of caspase-3 and caspase-9 increased significantly., Conclusion: Our findings indicate that HAS-2 gene silencing may inhibit proliferation and promote apoptosis in the MCF-7 human breast cancer cell line., (© 2016 The Author(s) Published by S. Karger AG, Basel.)

Doxorubicin (DOX) is an effective chemotherapeutic agent; however; its use is limited by some side effects; such as cardiotoxicity and thrombocytopenia. DOX-induced cardiotoxicity has been intensively investigated; however; DOX-induced thrombocytopenia has not been clearly elucidated. Here we show that DOX-induced mitochondria-mediated intrinsic apoptosis and glycoprotein (GP)Ibα shedding in platelets. DOX did not induce platelet activation; whereas; DOX obviously reduced adenosine diphosphate (ADP)- and thrombin-induced platelet aggregation; and impaired platelet adhesion on the von Willebrand factor (vWF) surface. In addition; we also show that DOX induced intracellular reactive oxygen species (ROS) production and mitochondrial ROS generation in a dose-dependent manner. The mitochondria-targeted ROS scavenger Mito-TEMPO blocked intracellular ROS and mitochondrial ROS generation. Furthermore; Mito-TEMPO reduced DOX-induced platelet apoptosis and GPIbα shedding. These data indicate that DOX induces platelet apoptosis; and impairs platelet function. Mitochondrial ROS play a pivotal role in DOX-induced platelet apoptosis and GPIbα shedding. Therefore; DOX-induced platelet apoptosis might contribute to DOX-triggered thrombocytopenia; and mitochondria-targeted ROS scavenger would have potential clinical utility in platelet-associated disorders involving mitochondrial oxidative damage.

We have previously reported that the danshensu-cysteine conjugate N-((R)-3-benzylthio-1-methoxy-1-oxo-2-propanyl)-2-acetoxy-3-(3,4-diacetoxyphenyl) propanamide (DSC) is a potent anti-oxidative and anti-apoptotic agent. Herein, we further design and asymmetrically synthesize two diastereoisomers of DSC and explore their potential bioactivities. Our results show that DSC and its two diastereoisomers exert similar protective effects in hydrogen peroxide (H2O2)-induced cellular injury in SH-SY5Y cells, as evidenced by the increase of cell viability, superoxide dismutase (SOD), and reduced glutathione (GSH) activity, and glutathione peroxidase (GPx) expression, and the decrease of cellular morphological changes and nuclear condensation, lactate dehydrogenase (LDH) release, and malondialdehyde (MDA) production. In H2O2-stimulated human umbilical vein endothelial cells (HUVEC), DSC concentration-dependently attenuates H2O2-induced cell death, LDH release, mitochondrial membrane potential collapse, and modulates the expression of apoptosis-related proteins (Bcl-2, Bax, caspase-3, and caspase-9). Our results provide strong evidence that DSC and its two diastereoisomers have similar anti-oxidative activity and that DSC exerts significant vascular-protective effects, at least in part, through inhibition of apoptosis and modulation of endogenous antioxidant enzymes.

The tumor suppressor gene p53 is often inactivated in breast cancer cells due to gene mutation or overexpression of its repressors (such as murine double minute 2 and murine double minute X). Inhibitors of murine double minute 2 (MDM2) and murine double minute X (MDMX) could lead to tumor suppression by restoration of p53 activity and such an approach is a promising strategy for future control of breast cancer. This study aimed to investigate the feasibility of the recombinant MDM2 and MDMX inhibitory protein in control of breast cancer in vitro. A cell-permeable dual-target MDM2/MDMX inhibitory protein was expressed in E. coli and incubated with p53 wild-type breast cancer cells. The data showed that this recombinant MDM2/MDMX inhibitory protein reduced the viability of MCF-7 and ZR-75-30 breast cancer cell lines and promoted cell cycle arrest and apoptosis by activation and stabilization of the p53 protein. Mechanistically, this MDM2/MDMX inhibitory protein increased the expression of p21, Bax and puma proteins, and inhibitory expression of MDM2 and MDMX proteins. This recombinant protein showed a better in vitro effect than that of nutlin-3α, a small molecule MDM2 inhibitor. The data further support the hypothesis that targeting of the p53 gene pathway could effectively control breast cancer.