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Thirty-two diterpenoids including 18 ent-kauranes (1-6, and 12-23), nine 8,9-seco-ent-kauranes (7-8, and 24-30), four ent-abietanes (9-10, and 31-32), and one crotofolane (11) were isolated from the twigs and leaves of Croton kongensis. The structures of previously unreported crokokaugenoids A-H (1-8), crokoabiegenoids A-B (9-10), and crokocrotogenoid A (11) were determined by spectroscopic data analyses, TDDFT-ECD and GIAO NMR calculations, and X-ray crystallographic studies. All compounds were evaluated for their cytotoxic activity against five human tumor cell lines, and the structure-activity relationships were discussed. Biological tests exhibited that compound 1 possessed strong anti-proliferation activity, arrested cell cycle at G2/M phase, and induced cell apoptosis of MDA-MB-231. The mechanism investigation showed that compound 1 can inhibit tumor proliferation and migration by targeting mitochondria to increase intracellular reactive oxygen species (ROS) and regulating STAT3 and FAK signal pathways. Collectively, these findings supported the great potential of compound 1 as a hopeful anticancer agent., 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.)

Acute pancreatitis (AP) is a prevalent gastrointestinal disorder. The immune response plays a crucial role in AP progression. However, the impact of immune regulatory checkpoint PD-L1 on severe acute pancreatitis (SAP) remains uncertain. Hence, this study aimed to examine the influence of PD-L1 on SAP. We assessed PD-L1 expression in neutrophils and monocytes obtained from SAP patients. We induced SAP in C57BL/6J mice, PD-L1 gene-deficient mice, and PD-L1 humanized mice using intraperitoneal injections of cerulein plus lipopolysaccharide. Prior to the initial cerulein injection, a PD-L1 inhibitor was administered. Pancreatic tissues were collected for morphological and immunohistochemical evaluation, and serum levels of amylase, lipase, and cytokines were measured. Flow cytometry analysis was performed using peripheral blood cells. The expression of PD-L1 in neutrophils and monocytes was significantly higher in SAP patients compared to healthy individuals. Likewise, the expression of PD-L1 in inflammatory cells in the peripheral blood of SAP-induced C57BL/6J mice was notably higher than in the control group. In mice with PD-L1 deficiency, SAP model exhibited lower pancreatic pathology scores, amylase, lipase, and cytokine levels compared to wild-type mice. PD-L1 deletion resulted in reduced neutrophil apoptosis, leading to an earlier peak in neutrophil apoptosis. Furthermore, it decreased early monocyte apoptosis and diminished the peak of T lymphocyte apoptosis. Within the SAP model, administration of a PD-L1 inhibitor reduced pancreatic pathology scores, amylase, lipase, and cytokine levels in both C57BL/6J mice and PD-L1 humanized mice. These findings suggest that inhibiting PD-L1 expression can alleviate the severity of SAP., 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. Published by Elsevier B.V.)

Insufficient apoptosis of inflammatory macrophages and osteoclasts (OCs) in rheumatoid arthritis (RA) joints contributes toward the persistent progression of joint inflammation and destruction. Here, we deliver celastrol (CEL) to selectively induce apoptosis of OCs and macrophages in arthritic joints, with enzyme-responsive nanoparticles (termed PRNPs) composed of RGD modified nanoparticles (termed RNPs) covered with cleavable PEG chains. CEL-loaded PRNPs (CEL-PRNPs) dually target OCs and inflammatory macrophages derived from patients with RA via an RGD-αvβ3 integrin interaction after PEG cleavage by matrix metalloprotease 9, leading to increased apoptosis of these cells. In an adjuvant-induced arthritis rat model, PRNPs have an arthritic joint-specific distribution and CEL-PRNPs efficiently reduce the number of OCs and inflammatory macrophages within these joints. Additionally, rats with advanced arthritis go into inflammatory remission with bone erosion repair and negligible side effects after CEL-PRNPs treatment. These findings indicate potential for targeting chemotherapy-induced apoptosis in the treatment of advanced inflammatory arthritis.

Introduction: Nicotiflorin is the main characteristic component of Nymphaea candida, which is a natural product that reportedly ameliorates acute injury of the liver and cerebral cortex, but the effect of nicotiflorin on acute kidney injury (AKI) remains unknown. This study aimed to investigate the effects of nicotiflorin on ischaemia/reperfusion (I/R) AKI and the associated mechanisms., Methods: We performed both (a) in vivo experiments with C57BL/6 mice with bilateral renal pedicles clamped for 45 minutes and (b) in vitro experiments with human kidney epithelial cells (HK-2) exposed to hypoxia/reoxygenation to mimic I/R injury to study the role of nicotiflorin in AKI., Results: In vivo, nicotiflorin administration exerted protective effects on renal injury, as demonstrated by reductions in the levels of caspase3 and Bad (P < .05), the upregulation of Bcl-2 expression (P < .05) and improved renal histologic changes, which suggested that nicotiflorin can alleviate I/R injury and cell apoptosis. In vitro, nicotiflorin at a concentration of 75 μg/mL protected cells from hypoxia, which further confirmed that nicotiflorin exerts beneficial effects on hypoxia/reoxygenation. Through computational molecular docking, we found that activating transcription factor 3 (ATF3) exhibits a robust interaction with nicotiflorin with a simulated binding energy of -9.2°. We verified the interaction of nicotiflorin with ATF3 in HK-2 cells, and found that nicotiflorin reduced the apoptosis of HK-2 through ATF3., Conclusion: Based on the above-described results, nicotiflorin appears to have a beneficial impact on deteriorated renal function, as demonstrated using an experimental I/R model. The underlying mechanisms of nicotiflorin might inhibit HK-2 cell apoptosis through ATF3., (© 2020 Asian Pacific Society of Nephrology.)

Receptor-interacting protein 1 (RIP1, also known as RIPK1) is not only a tumor-promoting factor in several cancers but also mediates either apoptosis or necroptosis in certain circumstances. In this study we investigated what role RIP1 plays in human ovarian cancer cells. We showed that knockout (KO) of RIP1 substantially suppressed cell proliferation, accompanied by the G2/M checkpoint arrest in two human ovarian cancer cell lines SKOV3 and A2780. On the other hand, RIP1 KO remarkably attenuated cisplatin-induced cytotoxicity, which was associated with reduction of the apoptosis markers PARP cleavage and the necroptosis marker phospho-MLKL. We found that RIP1 KO suppressed cisplatin-induced ROS accumulation in both SKOV3 and A2780 cells. ROS scavenger BHA, apoptosis inhibitor Z-VAD or necroptosis inhibitor NSA could effectively suppress cisplatin's cytotoxicity in the control cells, suggesting that ROS-mediated apoptosis and necroptosis were involved in cisplatin-induced cell death. In addition, blocking necroptosis with MLKL siRNA effectively attenuated cisplatin-induced cytotoxicity. In human ovarian cancer A2780 cell line xenograft nude mice, RIP1 KO not only significantly suppressed the tumor growth but also greatly attenuated cisplatin's anticancer activity. Our results demonstrate a dual role of RIP1 in human ovarian cancer: it acts as either a tumor-promoting factor to promote cancer cell proliferation or a tumor-suppressing factor to facilitate anticancer effects of chemotherapeutics such as cisplatin.

The cytotropic heterogeneous molecular lipid (CHML) is a mixture of lipids isolated from natural products. CHML is an effective therapy for various kinds of cancers; however, the effect of CHML on glioma cells was seldom reported. Here, we aim to explore the cytotoxicity of CHML on glioma cells, and analyze the possible mechanisms. U251 glioma cells were cultured with CHML at different concentration, and the growth inhibition was measured by CCK-8 assay. Induced apoptosis were detected by flow cytometry, and the induced autophagies were observed by a transmission electron microscope. The key molecules involved in apoptosis and autophagy were detected by quantitative PCR and western-blot. CHML might inhibit the growth of U251 cells and promote apoptosis by up-regulating the expressions of Caspase-8 and Caspase-3; CHML also induced autophagy of U251 cells by promoting the expressions of MAP LC-3 and Beclin-1. CHML can inhibit proliferation of U251 cells by promoting cell apoptosis and inducing autophagy.

Osteosarcoma is the most common malignant bone tumor in children and adolescents. Aberrant expression of HOXA5 results in various diseases, including cancers. However, the specific function and molecular mechanism of HOXA5 in osteosarcoma is not fully understood. In the present study, we focused on HOXA5 in U2OS and MG63 cells in vitro. We observed lower expression of HOXA5 in U2OS, MG63, and SaOS2 human osteosarcoma cells, compared with hFOB1.19 human osteoblastic cells. HOXA5 overexpression in U2OS and MG63 cells markedly reduced cell survival and proliferation and elevated cell apoptosis and caspase-3 activity. HOXA5 also activated the p38α MAPK pathway by increasing p53. Treating U2OS and MG63 cells with the p53 inhibitor α-pifithrin or the p38α MAPK inhibitor SB203580 led to higher cell survival and proliferation and lower cell apoptosis, compared with the pcDNA3.1-HOXA5 group. In conclusion, our study showed that the p53 and p38α MAPK signal axis facilitated HOXA5's role in inhibiting growth and stimulating apoptosis of osteosarcoma cells., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The brominated flame retardant, 2,2',4,4'-tetrabromodiphenyl ether (BDE-47), is well documented to exert potential negative impacts on different marine biota. However, the responsible mechanism remains unknown. The rainbow trout gonadal cell line RTG-2 was used as a model, and the mechanism and pathway underlying BDE-47 (6, 12.5 and 25 μM)-induced apoptosis and toxicity were examined in vitro. Apoptosis occurred in the RTG-2 cells exposed to BDE-47 in a clear concentration-dependent manner. The morphology of the mitochondrial alterations was observed using transmission electron microscopy. BDE-47 exposure decreased the cellular mitochondrial membrane potential, increased the cytochrome c released into the cytoplasm and elevated Fas protein expression. The mRNA expressions of Fas-associated death domain-containing protein (FADD), CHOP and GRP78 were also elevated, and similar increases were found in the activities of intracellular caspase-8, caspase-12, caspase-9 and caspase-3. These results indicated that the mitochondrial, endoplasmic reticulum and death-receptor pathways were involved in apoptosis in RTG-2 cells following BDE-47 exposure. ROS and Ca 2+ were responsible for these changes because their overproduction was detected prior to apoptosis. However, the addition of the ROS scavenger N-acetyl-l-cysteine (NAC) and the intracellular calcium chelator (acetoxymethyl)-1,2-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM) did not significantly alleviate the apoptosis rate. The results of the present study show that BDE-47 exposure induced apoptosis in RTG-2 cells via ROS- and Ca 2 -mediated mitochondrial, endoplasmic reticulum and death-receptor apoptotic pathways., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

We attempted to explore the possible effects of SOCS3 (suppressor of cytokine signaling 3)-modified bone marrow mesenchymal stem cells (BMSCs) on the hypoxic injury of rat adrenal gland pheochromocytoma (PC-12) cells. PC12 cells were cultured with EGFP (enhanced green fluorescent protein)-BMSCs and SOCS3-BMSCs respectively under hypoxia in vitro and classified into control, hypoxia, EGFP-BMSCs, and SOCS3-BMSC groups. CCK-8, Hoechst 33258 staining, and Annexin V-FITC/PI staining were assessed to measure the viability and apoptosis of hypoxia-induced PC12 cells. The JAK/STAT3 pathway- and apoptosis-related proteins were identified by Western blot. Finally, rat models of permanent middle cerebral artery occlusion (pMCAO) were established to verify the potential influences of SOCS3-BMSCs in vivo. SOCS3-modified BMSCs can stably express SOCS3 protein. EGFP-BMSCs, especially SOCS3-BMSCs, can improve cell viability and SOD content, and reduce cell apoptosis, LDH viability, and MDA content in hypoxia-induced PC12 cells (all P < 0.05). Besides, EGFP-BMSCs and SOCS3-BMSCs decreased cleaved caspase-3 level and increased Bcl-2/Bax of hypoxia-induced PC12 cells, while SOCS3-BMSCs could also elevate SOCS3 protein and reduce p-STAT3 protein level in hypoxia-induced PC12 cells (all P < 0.05). In vivo experiments confirmed that EGFP-BMSCs, particularly SOCS3-BMSCs, could ameliorate infarct size and inhibit neuronal apoptosis of different degrees in pMACO rats (all P < 0.05). SOCS3-modified BMSCs can alleviate oxidative stress, improve cell viability, and reduce neuronal apoptosis by downregulation of JAK/STAT3 pathway, thereby exerting the neuroprotective role in ischemic brain injury.

Background: LncRNA-FENDRR is a kind of endothelial genes critical for vascular development. Moreover, miR-126 and vascular endothelial growth factor A (VEGFA) are also involved in the physiological process of vascular endothelial cells. This study aimed to the underlying mechanism of FENDRR involving miR-126 and VEGFA in hypertensive intracerebral hemorrhage (HICH)., Methods: C57BL/6 mice were chosen to establish HICH model. The expression of FENDRR, miR-126, and VEGFA at mRNA level was determined by qRT-PCR. The protein expression of VEGFA was assessed using Western blot. RIP assay and RNA pull-down assay were used to the relationship between FENDRR and miR-126. Flow cytometry was used to analyze cell apoptosis., Results: The levels of FENDRR and VEGFA were increased, and miR-126 expression was decreased in vascular endothelial cells (VECs) from the right brain of model mice and human brain microvascular endothelial cells (HBMECs) treated by thrombin. Overexpression of FENDRR promoted the apoptosis of HBMECs. FENDRR regulating VEGFA participated in HBMECs apoptosis through targeting miR-126. Downregulation of FENDRR was indicated to relieve the HICH in mice., Conclusions: FENDRR could promote the apoptosis of HBMECs via miR-126 regulating VEGFA in HICH., (© 2018 John Wiley & Sons Ltd.)

BACKGROUND Usnic acid (UA), a secondary metabolite, is mainly derived from certain lichen species. Growing evidence suggests that UA has antitumor, anti-oxidative, anti-inflammatory, and other activities in a variety of cancer cells. However, the antitumor effect of UA in gastric cancer cells (GC) is unclear. The aim of this investigation was to assess the antitumor effect of UA in GC cells in vitro and in vivo, and to explore the underlying mechanisms. MATERIAL AND METHODS Cell proliferation was measured by CCK8 assay, the arrest of cell cycle was assessed by flow cytometry, and cellular apoptosis was observed via Hoechst 33258 staining assay. Expression levels of apoptosis-related proteins (activated caspase-3 and PARP, Bax, Bcl2) and autophagy-associated proteins (LC3-II and p62) were verified through Western blot analysis. H&E staining and immunohistochemistry were carried out in the subcutaneously implanted BGC823 tumor model in a nude mouse experiment. RESULTS In vitro, we demonstrated that UA was significantly effective in inducing morphological changes, inhibiting the cell proliferation dose- and time-dependently, arresting the cell cycle phase, promoting cancer cellular apoptosis, and inducing autophagy activity. In vivo, compared to mice treated with 5-FU alone, UA treatment was significantly more effective in suppressing the tumor growth without affecting body weight, and in regulating the amount of Bax and Bcl2 in tumor tissues. CONCLUSIONS UA induces cell cycle arrest and autophagy and exerts anti-proliferative and apoptotic effects by modulating expression of apoptosis-related proteins in stomach neoplasm cells, and has a better antitumor effect compared to 5-Fu in the xenograft model.

The combination of tumor necrosis factor‑related apoptosis‑inducing ligand (TRAIL) with other agents has been recognized as a promising strategy to overcome TRAIL resistance in cancer cells. Baicalin (5, 6‑dihydroxy‑7‑o‑glucuronide flavone) is a flavonoid from the root of the medicinal herb Scutellaria baicalensis Georgi, which has been reported to exert antioxidant, anti‑inflammatory, antiviral and anticancer activities in vitro. However, the effect of baicalin on TRAIL‑induced cytotoxicity has not been previously reported. In the present study, the effect of combining TRAIL and baicalin was investigated in non‑small cell lung cancer cell lines. The results revealed that baicalin was able to sensitize A549 and H2009 cells to TRAIL‑induced apoptosis. This was detected by the potentiation of poly‑adenosine‑5'‑diphosphate‑ribose polymerase cleavage and Annexin V‑fluorescein isothiocyanate staining of cells co‑treated with baicalin and TRAIL. In addition, p38 mitogen‑activated protein kinase was activated in baicalin and TRAIL co‑treated cancer cells, whereas the p38 inhibitor SB203580 effectively suppressed cell death within the co‑treated cells. Butylated hydroxyanisole and N‑acetyl‑cysteine, known reactive oxygen species (ROS) scavengers, significantly suppressed the potentiated cytotoxicity induced by baicalin and TRAIL co‑treatment. The present study is the first, to the best of our knowledge, to demonstrate that baicalin enhances the anticancer activity of TRAIL via p38 activation and ROS accumulation, and may be exploited for anticancer therapy.

Accumulating evidence suggests that microRNA (miR)-146a functions as an oncogene or tumor suppressor in various cancers. However, the role of miR‑146a in gastric cancer (GC) remains to be elucidated. The present study investigated the function of miR‑146a in GC cells. The results of the present study revealed that miR‑146a modulates GC cell apoptosis. Overexpression of miR‑146a significantly increased apoptosis of SGC‑7901 cells, whereas inhibition of miR‑146a protected cells from apoptosis. miR‑146a expression in GC cells was inversely correlated with transforming growth factor β‑activated kinase 1 (TAK1) expression, at the mRNA and protein levels. Furthermore, small interfering RNA‑mediated silencing of TAK1 enhanced GC cell apoptosis, whereas overexpression of TAK1 promoted survival of GC cells. Overexpression of miR‑146a or knockdown of TAK1 led to a marked increase in inhibitor of κBα (IκBα) and a decrease in B‑cell lymphoma 2 (Bcl‑2) expression levels in SGC‑7901 cells. By contrast, silencing of miR‑146a or TAK1 overexpression downregulated IκBα and upregulated Bcl‑2 expression levels. Therefore, the results of the present study demonstrated a novel negative feedback mechanism to promote GC cell apoptosis involving the miR‑146a/TAK1/nuclear factor-κB axis.

Cervical cancer is one of the most common cancers in females, with a high incidence and mortality around the world. However, the pathogenesis in cervical cancer is not completely known. In the present study, we investigated the role of miR-126-5p and Bcl2l2 in cervical cancer cells. First, miR-126-5p expression was aberrantly downregulated in human cervical cancer tumor tissues in comparison with normal tissues, as evaluated by RT-PCR. Consistently, the levels of miR-126-5p were also significantly reduced in cervical cancer cell lines when compared to normal cervical epithelial cells. Flow cytometric analysis showed that the rate of apoptosis of cervical cancer cells was significantly increased by miR-126-5p overexpression but inhibited by miR-126-5p inhibitor. A similar change pattern was observed in the expression of apoptosis-regulated protein caspase 3 in cervical cancer cells transfected with miR-126-5p mimic or inhibitor. By bioinformatic prediction with online databases and verification using luciferase reporter assay, we then identified that Bcl2l2 is a direct target of miR-126-5p in cervical cancer cells. The expression of Bcl2l2 was strongly downregulated by the miR-126-5p mimic but upregulated by the miR-126-5p inhibitor in cervical cancer cells, and Bcl2l2 expression was significantly increased in human cervical cancer tumor tissues, which was negatively correlated with miR-126-5p levels. Furthermore, we confirmed that the rate of apoptosis was significantly increased by Bcl2l2 silencing in cervical cancer cells, which was not affected by the miR-126-5p inhibitor. In addition, the increased apoptosis of cells by the miR-126-5p mimic was inhibited by Bcl2l2 overexpression. In summary, miR-126-5p plays an inhibitory role in human cervical cancer progression, regulating the apoptosis of cancer cells via directly targeting Bcl2l2. This might provide a potential therapeutic target for cervical cancer.

Background: The limited treatment option for recurrent prostate cancer and eventual resistant to conventional chemotherapy drugs has fueled continued interest in finding new anti-neoplastic agents. WZ35, a chemical analog of curcumin, had been demonstrated to have high chemical stability and potential anticancer effects in gastric cancer cells. The present study aimed to investigate the anti-prostate cancer effects of WZ35 in vitro and in vivo as well as the underlying mechanism., Methods: Two prostate cancer cell lines RM-1 and DU145 were utilized to test the anti-cancer effects of WZ35 and the underlying mechanism. MTT assay was used to assess the cytotoxic effect of WZ35. Cell cycle distribution, apoptosis, alteration of ROS, and [Ca 2+ ] i level were evaluated using flow cytometry. Western blotting assay was applied to measure the levels of proteins associated with apoptosis and cell cycle. Immunofluorescence staining and Electron micrographs were used to evaluate activation of mitochondrial apoptosis pathway. Tumor models in nude mice were induced by injection of RM-1 prostate cancer cells to test the in vivo anticancer action of WZ35., Results: Our results showed that WZ35 treatment induced loss of cell viability, cell apoptosis, and G2/M cycle arrest in both RM-1 and DU145 cells, coupled with ROS overproduction, intracellular calcium surge, and activation of mitochondrial apoptosis pathway in RM-1 cells. Interestingly, all above changes induced by WZ35 were completely reversed by ROS blockage. In addition, prevention of [Ca 2+ ] i elevation by BAPTA/AM also inhibited activation of mitochondrial apoptosis pathway induced by WZ35. In vivo studies, WZ35 treatment significantly inhibited RM-1 homograft tumor growth along with increased ROS accumulation, mitochondrial disruption, and cell apoptosis in tumor tissues., Conclusions: In conclusion, this work provides a novel anticancer candidate for the treatment of prostate cancer and demonstrated that increased ROS mediate the anti-cancer effects of WZ35 via activating mitochondrial apoptosis pathway. Importantly, this work also reveals that targeting ROS generation might be an effective strategy in human androgen-resistant prostate cancer treatment. Prostate 77:489-504, 2017. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Chondrocyte plays a critical role in endochondral ossification and cartilage repair by maintaining the cartilaginous matrix. Statins have been widely used to lower the cholesterol level in patients with cardiovascular disorders. Previous research has demonstrated potential role of statins in chondrocyte proliferation. This study addresses the proliferation-regulatory effect of lovastatin in rabbit chondrocytes as well as the underlying signaling mechanisms, thereby exploring its potential application in chondrocyte-related disorders, such as cartilage damage and osteoarthritis. Rabbit chondrocytes were treated with lovastatin at multiple concentrations, and the proliferation rate was measured by CCK-8 test. The results showed significant increase in chondrocyte proliferation under lovastatin treatment. Using real-time quantitative PCR, it was observed that the expression levels of COL2A1, SOX-9, Caspase-3, and MMP-3 genes were significantly changed by lovastatin treatment. Western blotting analysis showed that the abundance of COL2A1, SOX-9, MEK1/2, p-MEK1/2, ERK1/2, p-ERK1/2, Caspase-3, and MMP-3 proteins was also significantly influenced by lovastatin treatment. Interleukine-1 beta (IL-1β) is involved in the progression of osteoarthritis (OA) by inducing articular cartilage and chondrocyte aging and senescence. In this study, we observed that lovastatin treatment inhibited IL-1β-induced chondrocyte apoptosis, while the combined treatment of lovastatin and U0126 evidently offset the apoptosis-inhibiting effect of lovastatin in chondrocyte proliferation. The expressional level and protein abundance of COL2A1, SOX-9, MEK1/2, p-MEK1/2, ERK1/2, p-ERK1/2, caspase-3, and MMP-3 genes showed significant alterations under the combined treatment. Together, our results suggested that lovastatin significantly promoted proliferation and inhibited the IL-1β-induced apoptosis in rabbit chondrocytes, which was mediated by the MEK/ERK signaling.

While chromatin remodeling mediated by post-translational modification of histone is extensively studied in carcinogenesis and cancer cell's response to chemotherapy and radiotherapy, little is known about the role of histone expression in chemoresistance. Here we report a novel chemoresistance mechanism involving histone H4 expression. Extended from our previous studies showing that concurrent blockage of the NF-κB and Akt signaling pathways sensitizes lung cancer cells to cisplatin-induced apoptosis, we for the first time found that knockdown of Akt1 and the NF-κB-activating kinase IKKβ cooperatively downregulated histone H4 expression, which increased cisplatin-induced apoptosis in lung cancer cells. The enhanced cisplatin cytotoxicity in histone H4 knockdown cells was associated with proteasomal degradation of RIP1, accumulation of cellular ROS and degradation of IAPs (cIAP1 and XIAP). The cisplatin-induced DNA-PK activation was suppressed in histone H4 knockdown cells, and inhibiting DNA-PK reduced expression of RIP1 and IAPs in cisplatin-treated cells. These results establish a novel mechanism by which NF-κB and Akt contribute to chemoresistance involving a signaling pathway consisting of histone H4, DNA-PK, RIP1 and IAPs that attenuates ROS-mediated apoptosis, and targeting this pathway may improve the anticancer efficacy of platinum-based chemotherapy., Competing Interests: The authors declare no competing financial interests.

Background: Necrotizing enterocolitis (NEC) is the leading cause of neonatal gastrointestinal mortality; effective interventions are lacking with limited understanding of the pathogenesis of NEC. The importance of Toll-like receptor 4 (TLR4) signaling in NEC is well documented; however, the potential mechanisms that regulate enterocyte inflammation and apoptosis remain unclear. The aim of this study was to characterize the role of TLR4-mediated inflammation and apoptosis in the development of NEC and to determine the major apoptotic pathways and regulators in the process., Methods: TLR4-deficient C57BL/10ScNJ mice and lentivirus-mediated stable TLR4-silent cell line (IEC-6) were used. NEC was induced by formula gavage, cold, hypoxia, combined with lipopolysaccharide in vivo or lipopolysaccharide stimulation in vitro. Enterocyte apoptosis was evaluated by TUNEL or Annexin analysis. The expression of TLR4, caspase3, caspase8, caspase9, Bip, Bax, Bcl-2, and RIP was detected by Western blot and immunofluorescence. Inflammatory factors such as tumor necrosis factor-α and interleukin-2 were examined by Luminex., Results: Defect of TLR4 led to suppressed enterocytes apoptosis both in vitro and in vivo; the expression of caspase3, caspase8, Bip, and Bax was decreased; and caspase9 and Bcl-2 were increased. NEC severity was attenuated in TLR4-deficient mice compared with wild-type counterparts, and enterocytes apoptosis was correlated with NEC severity. RIP and cytokine level of tumor necrosis factor-α and interleukin-2 were also decreased., Conclusions: TLR4-induced inflammation and apoptosis play a critical role in the pathogenesis of NEC. TLR4 inhibition, combined with extrinsic (caspase8) and/or endoplasmic reticulum stress (Bip) apoptosis signaling blockade could serve as a potential effective treating strategy for NEC.

Objective: To explore the molecular mechanism responsible for apoptosis of PC-12 neuronal cells induced by oxygen-glucose deprivation (OGD)., Methods: PC12 cells were exposed to OGD for 24 h to simulate ischemia-reperfusion injury. Flow cytometry was employed detect the cell apoptosis, and the expresions of TRPM8, UCP4, cAMP and PKA in the exposed cells were detected with RT-PCR and Western blotting. The changes in the expressions of Bax, Bcl-2, cAMP, PKA and UCP4 proteins were detected in the exposed cells in resposne to inhibition of TRPM8 and cAMP-PKA signal or over-expression of UCP4., Results: OGD for 24 induced obvious apoptosis in PC-12 cells and caused TRPM8 over-expression and inhibition of UCP4 and cAMP-PKA signaling. Inhibiting TRPM8 expression reduced the cell apoptosis and up-regulated cAMP, p-PKA and UCP4 in the cells exposed to OGD. In cells exposed to OGD, inhibition of TRPM8 and cAMP-PKA signaling suppressed the expressio of UCP4 and increased the cell apoptosis., Conclusion: TRPM8 mediates OGD-induced PC12 cell apoptosis through cAMP-PKA/UCP4 signaling.

PNAS-4 is a novel pro-apoptosis gene identified latetly. In recent years, there has been a large number of research reports on the basic studies about PNAS-4 in cancer gene therapy and gene therapy of PNAS-4 alone or combined with chemotherapy or radiotherapy manifested a good application prospect, but its molecular mechanisms to promote apoptosis is not clear yet. In this paper, recent research about PNAS-4 in cancer gene therapy is briefly reviewed, and recent hypotheses on its molecular mechanisms to promote apoptosis are especially elucidated. Based on its newly identified characteristics of structural domain, we made a point that PNAS-4 might regulate functions of some target protein related to apoptosis by deSumoylation as a new deSumoylating isopeptidase, and consequently promote apoptosis.

Hyperglycemia is an important initiator of cardiovascular disease, contributing to the development of cardiomyocyte death and diabetic complications. The purpose of the present study was to investigate whether high glucose state could induce apoptosis of rat cardiomyocyte cell line H9c2 through microRNA-mediated Bcl-2 signaling pathway. The expression of miR-34a and Bcl-2 mRNA was detected by using real-time PCR. Western blotting was used to examine the changes in apoptosis-associated protein Bcl-2. Apoptosis of H9c2 cells was tested by using flow cytometry. The results showed that the expression of miR-34a was significantly elevated and that of Bcl-2 was strongly reduced, and apoptosis of cardiomyocytes was apparently increased in the high-glucose-treated H9c2 cells as compared with normal-glucose-treated controls. In addition, we identified Bcl-2 gene was the target of miR-34a. miR-34a mimics reduced the expression of Bcl-2 and increased glucose-induced apoptosis, but miR-34a inhibitor acted as the opposite mediator. Our data demonstrate that miR-34a contributes to high glucose-induced decreases in Bcl-2 expression and subsequent cardiomyocyte apoptosis.

Ziyuglycoside II is one of the major active compounds of Sanguisorba officinalis L., which has a wide range of clinical applications including hemostasis, antibiosis, anti-inflammation and anti-oxidation. This study investigated the effect of ziyuglycoside II on the growth of human breast carcinoma MDA-MB-435 cells for the first time. The results showed that ziyuglycoside II could significantly inhibit the growth of MDA-MB-435 cells through blocking cell cycle progression at G0/G1 and S phase as well as via inducing cell apoptosis. Accumulation of reactive oxygen species (ROS) was observed in the progression of cell cycle arrest, which was associated with the increased expression of cell cycle regulating factors, p53 and p21. Subsequent apoptosis induced by ziyuglycoside II was accompanied with the activation of mitochondrial pathway, in particular a decreased mitochondrial membrane potential (MMP) as well as increased Bax/Bcl-2 ratio, cytochrome c release and the activity of caspase-3 and caspase-9. In conclusion, our study was the first to report that ziyuglycoside II has inhibitory effect on the growth of MDA-MB-435 cells, which might become a potential therapeutic approach of breast cancer in the future.

Curcumin (diferuloylmethane) is a natural polyphenol product of the plant Curcuma longa and has a diversity of antitumor activities. T63, a new 4-arylidene curcumin analogue, was reported to inhibit proliferation of lung cancer cells. However, its precise molecular antitumor mechanisms have not been well elucidated. Here, we showed that T63 could significantly inhibit the proliferation of A549 and H460 human lung cell lines via induction of G0/G1 cell cycle arrest and apoptosis. We found that the reactive oxygen species (ROS)-activated FOXO3a cascade plays a central role in T63-induced cell proliferation inhibition. Mechanistically, enhancement of ROS production by T63 induced FOXO3a expression and nuclear translocation through activation of p38MAPK and inhibition of AKT, subsequently elevating the expression of FOXO3a target genes, including p21, p27, and Bim, and then increased the levels of activated caspase-3 and decreased the levels of cyclin D1. Moreover, the antioxidant N-acetylcysteine markedly blocked the above effects, and small interfering RNA-mediated knockdown of FOXO3a also significantly decreased T63-induced cell cycle arrest and apoptosis. In vivo experiments showed that T63 significantly suppressed the growth of A549 lung cancer xenograft tumors, associated with proliferation suppression and apoptosis induction in tumor tissues, without inducing any notable major organ-related toxicity. These data indicated that the novel curcumin analogue T63 is a potent antitumor agent that induces cell cycle arrest and apoptosis and has significant therapeutic potential for lung cancer., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Intestinal inflammatory disease is a kind of non-specific disease with morbidity increasing yearly. It has been proved that the Toll like receptor 4 (TLR4) signaling pathways are closely related to intestinal inflammatory diseases. Myeloid differentiation protein 88 (Myd88) is a critical adaptor protein of TLR4 signaling and critical for the study of intestinal inflammatory disease, but stable Myd88 knockdown in vitro models of cell line are still very few. In the present study, an HIV-1-based lentivirus three-plamid packaging system was used for the construction of a lentivirual vector mediating RNA interference (RNAi) against Myd88 in intestinal fossae epithelial cell line-6 (IEC-6). Real-time PCR and Western blot were used to detect Myd88 expression. Annexin V staining and flowcytometry (FLM) were applied to detect and evaluate the early apoptosis. The results showed that lentiviral vectors containing the shRNA expression cassette specifically targeting Myd88 were constructed and efficiently stably knocked down Myd88 expression in IEC-6 cell line. Early apoptosis was significantly decreased after Myd88 knockdown. This study successfully constructed a lentivirus-based RNAi for Myd88 and detailed the key technique combined with characteristics of the early apoptosis after the Myd88 knockdown, provided a novel, stable and repeatable in vitro model for the pathogenesis, targeting therapeutic study for the intestinal inflammatory diseases.

Bortezomib therapy has proven successful for the treatment of relapsed/refractory, relapsed, and newly diagnosed multiple myeloma (MM); however, dose-limiting toxicities and the development of resistance limit its long-term utility. Here, we show that P5091 is an inhibitor of deubiquitylating enzyme USP7, which induces apoptosis in MM cells resistant to conventional and bortezomib therapies. Biochemical and genetic studies show that blockade of HDM2 and p21 abrogates P5091-induced cytotoxicity. In animal tumor model studies, P5091 is well tolerated, inhibits tumor growth, and prolongs survival. Combining P5091 with lenalidomide, HDAC inhibitor SAHA, or dexamethasone triggers synergistic anti-MM activity. Our preclinical study therefore supports clinical evaluation of USP7 inhibitor, alone or in combination, as a potential MM therapy., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: Oxymatrine, an isolated extract from traditional Chinese herb Sophora Flavescens Ait, has been traditionally used for therapy of anti-hepatitis B virus, anti-inflammation and anti-anaphylaxis. The present study was to investigate the anti-cancer effect of oxymatrine on human pancreatic cancer PANC-1 cells, and its possible molecular mechanism., Methods: The effect of oxymatrine on the viability and apoptosis was examined by methyl thiazolyl tetrazolium and flow cytometry analysis. The expression of Bax, Bcl-2, Bcl-x (L/S), Bid, Bad, HIAP-1, HIAP-2, XIAP, NAIP, Livin and Survivin genes was accessed by RT-PCR. The levels of cytochrome c and caspase 3 protein were assessed by Western blotting., Results: Oxymatrine inhibited cell viability and induced apoptosis of PANC-1 cells in a time- and dose-dependent manner. This was accompanied by down-regulated expression of Livin and Survivin genes while the Bax/Bcl-2 ratio was upregulated. Furthermore, oxymatrine treatment led to the release of cytochrome c and activation of caspase-3 proteins., Conclusion: Oxymatrine can induce apoptotic cell death of human pancreatic cancer, which might be attributed to the regulation of Bcl-2 and IAP families, release of mitochondrial cytochrome c and activation of caspase-3.

To study the expression of X-linked inhibitor of apoptosis protein (XIAP) and cell apoptosis in vitro model of acute pancreatitis (AP), we carried out experiments to stimulate AR42J cell line with caerulein (10(-8) mol/L) for 12 hours, then collected cells at various time points (0 h, 4 h, 8 h, 12 h, and 24 h, respectively). We then observed the morphologic changes of AR42J cells with the stimulation of caerulein with electronic microscope. The gene expression of XIAP, caspase-3 and caspase-9 was detected using real-time fluorescence quantitative polymerase chain reaction (FQ-PCR), and the protein expression of XIAP was assessed by western blot. The activation of nuclear factor-kappa B (NF-kappaB) was measured by flow cytometry (FCM). With the stimulation of caerulein, the expression of XIAP and the NF-kappaB activation could first decrease and then increase, but the change of caspase-3 and caspase-9 expressions were opposite. XIAP may inhibit the cell apoptosis in rat pancreatic acinus AR42J cell lines at first with the stimulation of caerulein, then NF-kappaB can upgrade the expression of XIAP and increase the cell apoptosis.

Objective: To investigate the expression of apoptosis related gene glycogen synthase kinase-3B (GSK-3B) in acute edematous pancreatitis (AEP), and the relationship between GSK-3B and AEP., Methods: The AEP rat model was established by the injection of caerulein [50 microg/(kg x h)] intraperitoneally per hour for 4 times. There were total 36 rats, every 6 rats were sacrificed at 0 h, 1 h, 3 h, 6 h, 12 h and 18 h after the injection of caerulein. Blood samples were taken from portal vein for ELISA examination of pancreatic amylase and lipase, pancreatic mRNA was extracted for RT-PCR analysis of GSK-3B, caspase-3, 9. The expression of GSK-3B protein was examed by Western blot, and acinar cell apoptosis was measured by TUNEL assay., Results: AEP was successfully induced and pancreatic lesions were most severe at 6 h after the injection of caerulein. Immunohistochemistry assay showed that GSK-3B protein located in cytoplasm of pancreatic acinar cells and endothelial cells, but not in islet cells. The mRNA levels of GSK-3B, caspase-3, 9, the protein level of GSK-3B, as well as cell apoptotic index in AEP rats were significantly increased when compared with the control rats received normal saline. Acinar cell apoptosis index was positively correlated with serum amylase, lipase, ratio of dry/wet of pancreas and GSK-3B mRNA expression, while GSK-3B mRNA level was positively correlated with caspase-3, 9 mRNA expression., Conclusion: The expression levels of GSK-3B mRNA and protein were related to the severity of AEP and acinar cell apoptotic index.

5-Lipoxygenase (5-LOX), which is believed to be a major source of oxidative stress, participates in somatostatin-receptor transmembrane signaling in the central nervous system. We used the Tet-On inducible expression system in PC12 cells to obtain cell lines with reproducible, stable 5-LOX expression levels to study its function. Cell apoptosis rates induced by Aβ(42) were determined using an apo-BrdDU kit. Lipid peroxide, antioxidant enzyme, and caspase-3 activities were evaluated with respective commercial kits. The expression of 5-LOX, bcl-2, and bax were detected by immunoblotting. A subclone of PC18 with Tet-On inducible expression of 5-LOX was selected from PC12 transfectants. Expression of 5-LOX had no significant inhibitory effect on the cell viability of the PC18 clone. In contrast, compared with the control group, the cell viability of clone PC18 was significantly reduced after the induction of 5-LOX during Aβ exposure. The differences in cell viability before and after the induction of 5-LOX during Aβ insult were significantly offset by AA861. Overexpression of 5-LOX only slightly improved the activities of superoxide dismutase (SOD). The levels of intracellular peroxides, SOD and caspase-3 activity, and Bax expression were significantly upregulated, and the levels of glutathione peroxidase and catalase were downregulated correspondingly in clone PC18 during Aβ exposure. These results indicate that constitutive expression of 5-LOX is not detrimental per se, but overexpression of 5-LOX may become problematic during Aβ exposure.

Background: Toll-like receptor 4 (TLR4) plays an important role in the occurrence and development of acute pancreatitis (AP). Apoptosis of pancreatic cells is closely related to the severity of AP. TLR4 is known to induce apoptosis in some cell types and therefore it is of importance to investigate potential associations between TLR4 activity and apoptosis in the setting of AP., Methods: A total of 50 wild-type (C57BL/10J) and TLR4-deficient (C57BL/10ScNJ) mice were divided into three groups: 2-hour, 4-hour, and control groups. Each group was divided into two equal subgroups: TLR4-wild-type mice and TLR4-deficient mice. AP was experimentally induced by 7 intraperitoneal injections of 50 μg/kg cerulein at hourly intervals. Control mice received 7 injections of equal volumes of saline. The severity of pancreatic injury during AP was assessed by serum amylase concentration and histopathology. The level of apoptosis of pancreatic cells in response to AP was evaluated by calculating the apoptotic index (AI) and comparing the expression levels of cytochrome C and Fas-associated protein with death domain (FADD) between TLR4-wild-type and TLR4-deficient mice at 2 time points., Results: The AI was found to be significantly lower in the pancreas of TLR4-deficient mice with AP compared to TLR4-wild-type mice at two hours after the last treatment injection. Enzyme-linked immunosorbent assay and real-time reverse transcription-polymerase chain reaction also revealed significantly lower expression of cytochrome C and FADD in the pancreas of TLR4-deficient mice than in TLR4-wild-type animals at the same time point. Serum amylase concentration and morphological severity of AP in pancreatic tissue were found to be similar in the two strains of mice at both time points., Conclusion: We postulate that TLR4 can mediate apoptosis of pancreatic cells during the early stages of AP, via the activation of both intrinsic and extrinsic apoptotic signaling pathways.

Purpose: The aim of the study is to dissect the cytotoxic mechanisms of 1-(4-hydroxy-3-methoxyphenyl)-7-(3,4-dihydroxyphenyl)-4E-en-3-heptanone (compound 1) in SH-SY5Y cells and therefore to provide new insight into neuroblastoma chemotherapy., Methods: Nine diarylheptanoids were isolated from Alpinia officinarum by chromatography and their cytotoxicity was evaluated by an MTS assay. Flow cytometry, BrdU incorporation assay and fluorescence staining were employed to investigate cytostatic and apoptotic effects induced by the compound 1. In addition, Western blot, qPCR and siRNA techniques were used to elucidate the molecular mechanisms of the cytotoxicity., Results: The study to elucidate the cytotoxic mechanisms of compound 1, the most potent diarylheptanoid showed that cell cycle-related proteins, cyclins, CDKs and CDKIs, as well as two main apoptotic related families, caspase and Bcl 2 were involved in S phase arrest and apoptosis in neuroblastoma cell line SH-SY5Y. Furthermore, following the drug treatment, the protein expression of p53, phospho-p53 (Ser20) as well as the p53 transcriptional activated genes ATF3, puma and Apaf-1 were increased dramatically; MDM2 and Aurora A, the two p53 negative regulators were decreased; the p53 protein stability was enhanced, whereas the p53 mRNA expression level slightly decreased and ATF3 mRNA expression apparently increased. In addition, the knockdown of ATF3 gene by siRNA partially suppressed p53, caspase 3, S phase arrest and apoptosis triggered by compound 1., Conclusion: These results suggest that compound 1 induces S phase arrest and apoptosis via up regulation of ATF3 and stabilization of p53 in SH-SY5Y cell line. Therefore, compound 1 might be a promising lead structure for neuroblastoma therapy.

Background: Hepatocyte apoptosis is a severe form of cell death after hepatic ischemia-reperfusion injury (HIRI), and its relief is an important issue in liver transplantation. Hypoxic preconditioning (HP) is considered to have protective effects on HIRI. This study was designed to explore the impact of HP on apoptosis and its possible mechanism during orthotopic liver autotransplantation., Methods: A modified orthotopic liver autotransplantation model was used to simulate HIRI. Sprague-Dawley rats were randomly divided into normal control, autotransplantation (AT) and HP groups. The HP group was subjected to an 8% oxygen atmosphere for 90 minutes before surgery. At 1, 6 and 24 hours after surgery, the rats were killed and their liver tissue was sampled to assess the expression of Bcl-2 protein. The samples were subjected to blood chemistry study, morphological study under a light or transmission electron microscope, and quantitative study of mitochondria., Results: The serum levels of ALT and AST in the HP group were lower than those in the AT group at 1, 6 and 24 hours after orthotopic liver autotransplantation (P<0.05). Bcl-2 protein expression was increased in the HP group at each measurement point (P<0.05). Light microscopy showed that hepatic injury in the AT group was much more severe than in the HP group. Hepatocytes in the AT group showed typical apoptosis signs under a transmission electron microscope. The ultrastructural appearance of hepatocytes in the HP group was much better than in the AT group, and the area, perimeter and diameter of the mitochondria were smaller in the HP group than in the AT group (P<0.05)., Conclusions: Hepatocytes sense and respond to decreased tissue oxygenation. Stimulation by HP relieves apoptosis by upregulating expression of Bcl-2 protein and its protection of mitochondria after orthotopic liver autotransplantation.

Objective: To investigate the effects of ischemic postconditioning(IPo) on cellular apoptosis in acute renal ischemic-reperfusion injury (IRI) in rats., Methods: Model of acute renal ischemic-reperfusion injury (IRI) was constructed using Wistar rats. Forty healthy male rats were divided into 4 groups randomly,including group C,group IRI, group IPo and group Ado. Rats of group C received right nephronectomy and separation of left renal vessels. Before reperfusion, group IPo was perfused for 10 s, following ischemic reperfusion for another 10 s with 6 cycles. Group Ado was infused with adenosine intravenously 10 min before ischemia. Cellular apoptosis was determined with TUNEL (TdT-mediated dUTP Nick End Labelling), flowcytometry and histopathological staining., Results: Compared with group C, the apoptotic index increased predominatly in group IRI (P<0.05), whereas, apoptosis index decreased obviousely in group IPo, Ado (P<0.05). In addition, the apoptosis index between group IPo and Ado was not statistically different., Conclusion: Ischemic postconditioning could decrease cellular apoptosis induced by IRI in rat kidney.

Self-renewal and multilineage differentiation of hematopoietic stem cell (HSC) are their functional characteristics. The regulation of HSC self-renewal is governed by a balance between positive regulatory signals promoting growth and negative regulatory signals resulting in apoptosis. Among the positive regulatory signals, HOXB4 activates distinct pathways that enhance self-renewal divisions of HSC without overriding the regulatory mechanisms that maintain normal steady-state hemopoiesis. The upregulation of HOXB4 gene expression can greatly promote the HSC self-renewal, but does not affect the HSC differentiation, the morphology and function of linage-specific cells and terminally-differentiated blood cells. Furthermore, HOXB4 can enhance the hematopoietic potential of embryonic stem cell (ESC), promoting the differentiation of ESC into hematopoietic cells. As a consequence, upregulation of HOXB4 expression and/or corresponding HOXB4 target genes can have enormous therapeutical potential for human HSC in the stem cell transplantation and gene therapy. In this review the regulatory role of HOXB4 in HSC self-renewal, "zero" effect of HOXB4 on differentiation specificity of HSC lines and terminal differentiation cells, and molecular mechanisms of regulating HSC self-renewal by HOXB4 are summarised.

Background: Apoptosis resistance of myofibroblasts is critical in pathology of irradiation‐induced fibrosis and osteoradionecrosis of the jaw (ORNJ). However, molecular mechanism of apoptosis resistance induced by irradiation in oral myofibroblasts remains largely obscure. Methods: Matched ORNJ fibroblasts and normal fibroblasts pairs from gingival were primarily cultured, and myofibroblast markers of α‐SMA and FAP were evaluated by qRT‐PCR and western blot. CCK8 assay and flow cytometric analysis were performed to investigate the cell viability and apoptosis under irradiation treatment. Autophagy‐related protein LC3 and ATG7, and punctate distribution of LC3 localization were further detected. After inhibition of autophagy with inhibitor CQ and 3‐MA, as well as transfected ATG7‐siRNA, cell viability and apoptosis of ORNJ and normal fibroblasts were further assessed. Results: Compared with normal fibroblasts, ORNJ fibroblasts exhibited significantly higher α‐SMA and FAP expression, increased cell, viability and decreased apoptosis under irradiation treatment. LC3‐II and ATG7 were up‐regulated in ORNJ fibroblasts with irradiation stimulation. After inhibition of irradiation‐induced autophagic flux with lysosome inhibitor CQ, LC3‐II protein was accumulated and punctate distribution of LC3 localization was increased in ORNJ fibroblasts. Moreover, autophagy inhibitor CQ and 3‐MA enhanced the irradiation‐induced apoptosis but inhibited viability of ORNJ fibroblasts. Silencing ATG7 with siRNA could obviously weaken irradiation‐induced LC3‐II expression, and promoted irradiation‐induced apoptosis of ORNJ fibroblasts. After knockdown of ATG7, finally, p‐AKT(Ser473) and p‐mTOR(Ser2448) levels of ORNJ fibroblasts were significantly increased under irradiation. Conclusion: Compared with normal fibroblasts, human gingival myofibroblasts are resistant to irradiation‐induced apoptosis via autophagy activation. Silencing ATG7 may evidently inhibit activation of autophagy, and promote apoptosis of gingival myofibroblasts via Akt/mTOR pathway. [ABSTRACT FROM AUTHOR]

This present study was conducted to provide evidence and an explanation for the apoptosis that occurs in the marine rotifer Brachionus plicatilis when facing 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47) stress. Metabolomics analysis showed that aminoacyl-tRNA biosynthesis, valine, leucine and isoleucine biosynthesis, and arginine biosynthesis were the top three sensitive pathways to BDE-47 exposure, which resulted in the reduction in the amino acid pool level. Pyrimidine metabolism and purine metabolism pathways were also significantly influenced, and the purine and pyrimidine content were obviously reduced in the low (0.02 mg/L) and middle (0.1 mg/L) concentration groups while increased in the high (0.5 mg/L) concentration group, evidencing the disorder of nucleotide synthesis and decomposition in B. plicatilis. The biochemical detection of the key enzymes in purine metabolism and pyrimidine metabolism showed the downregulation of Glutamine Synthetase (GS) protein expression and the elevation of Xanthine Oxidase (XOD) activity, which suggested the impaired DNA repair and ROS overproduction. The content of DNA damage biomarker (8-OHdG) increased in treatment groups, and the p53 signaling pathway was found to be activated, as indicated by the elevation of the p53 protein expression and Bax/Bcl-2 ratio. The ROS scavenger (N-acetyl-L-cysteine, NAC) addition effectively alleviated not only ROS overproduction but also DNA damage as well as the activation of apoptosis. The combined results backed up the speculation that purine metabolism and pyrimidine metabolism alteration play a pivotal role in BDE-47-induced ROS overproduction and DNA damage, and the consequent activation of the p53 signaling pathway led to the observed apoptosis in B. plicatilis. [ABSTRACT FROM AUTHOR]

Cutaneous melanoma (CM, hereafter referred to as melanoma) is a highly malignant tumor that typically undergoes early metastasis. Pyroptosis, as a special programmed cell death process that releases inflammatory factors and has been widely studied in tumors, but its role in melanoma has not been fully elucidated. In this study, we examined the relationship between pyroptosis and the prognosis of melanoma through bioinformatic analysis of RNA-sequencing data. Our results demonstrated that pyroptosis is a protective factor associated with melanoma prognosis. A higher pyroptosis score was associated with a more favorable overall survival. We used weighted gene co-expression networks analysis (WGCNA) to establish an effective prognosis model based on 12 pyroptosis-related genes. We then validated it in two independent cohorts. Furthermore, a nomogram combining clinicopathological characteristics and a pyroptosis-related gene signature (PGS) score was designed to effectively evaluate the prognosis of melanoma. Additionally, we analyzed the potential roles of pyroptosis in the tumor immune microenvironment and drug response. Interestingly, we found that the elevated infiltration of multiple immune cells, such as CD4+ T cells, CD8+ T cells, dendritic cells, and M1 macrophages, may be associated with the occurrence of pyroptosis. Pyroptosis was also related to a better response of melanoma to interferon-α, paclitaxel, cisplatin and imatinib. Through Spearman correlation analysis of the 12 pyroptosis-related genes and 135 chemotherapeutic agents in the Genomics of Drug Sensitivity in Cancer database, we identified solute carrier family 31 member 2 (SLC31A2) and collagen type 4 alpha 5 chain (COL4A5) as being associated with resistance to most of these drugs. In conclusion, this PGS is an effective and novelty prognostic indicator in melanoma, and also has an association with the melanoma immune microenvironment and melanoma treatment decision-making. [ABSTRACT FROM AUTHOR]

Objective: To examine the effect of Shenmai Injection (SMJ) on ferroptosis during myocardial ischemia reperfusion (I/R) injury in rats and the underlying mechanism. Methods: A total of 120 SPF-grade adult male SD rats, weighing 220–250 g were randomly divided into different groups according to a random number table. Myocardial I/R model was established by occluding the left anterior descending artery for 30 min followed by 120 min of reperfusion. SMJ was injected intraperitoneally at the onset of 120 min of reperfusion, and erastin (an agonist of ferroptosis), ferrostatin-1 (Fer-1, an inhibitor of ferroptosis) and ML385 (an inhibitor of nuclear factor erythroid-2 related factor 2 (Nrf2)) were administered intraperitoneally separately 30 min before myocardial ischemia as different pretreatments. Cardiac function before ischemia, after ischemia and after reperfusion was analysed. Pathological changes in the myocardium and the ultrastructure of cardiomyocytes were observed, and the myocardial infarction area was measured. Additionally, the concentration of Fe2+ in heart tissues and the levels of creatine kinase-MB (CK-MB), troponin I (cTnl), malondialdehyde (MDA) and superoxide dismutase (SOD) in serum were measured using assay kits, and the expressions of Nrf2, glutathione peroxidase 4 (GPX4) and acyl-CoA synthetase long-chain family member 4 (ACSL4) were examined by Western blot. Results: Compared with the sham group, I/R significantly injured heart tissues, as evidenced by the disordered, ruptured and oedematous myocardial fibres; the increases in infarct size, serum CK-MB, cTnI and MDA levels, and myocardial Fe2+ concentrations; and the decreases in SOD activity (P<0.05). These results were accompanied by ultrastructural alterations to the mitochondria, increased expression of ACSL4 and inhibited the activation of Nrf2/GPX4 signalling (P<0.05). Compared with I/R group, pretreatment with 9 mL/kg SMJ and 2 mg/kg Fer-1 significantly reduced myocardial I/R injury, Fe2+ concentrations and ACSL4 expression and attenuated mitochondrial impairment, while 14 mg/kg erastin exacerbated myocardial I/R injury (P<0.05). In addition, cardioprotection provided by 9 mL/kg SMJ was completely reversed by ML385, as evidenced by the increased myocardial infarct size, CK-MB, cTnI, MDA and Fe2+ concentrations, and the decreased SOD activity (P<0.05). Conclusions: Ferroptosis is involved in myocardial I/R injury. Pretreatment with SMJ alleviated myocardial I/R injury by activating Nrf2/GPX4 signalling-mediated ferroptosis, thereby providing a strategy for the prevention and treatment of ischemic heart diseases. [ABSTRACT FROM AUTHOR]

Background: Acute kidney injury (AKI) is a common syndrome impacting about 13.3 million patients per year. Tilianin has been reported to alleviate myocardial ischemia/reperfusion (I/R) injury, while its effect on AKI is unknown; thus, this study aimed to explore if tilianin protects I/R-induced AKI and the underlying mechanisms. Methods: The microarray dataset GSE52004 was downloaded from GEO DataSets (Gene Expression Omnibus). Differential expression analysis and gene-set enrichment analysis (GSEA) were performed by R software to identify apoptosis pathway-related genes. Then, RcisTarget was applied to identify the transcription factor (TF) related to apoptosis. The STRING database was used to construct a protein–protein interaction (PPI) network. Cytoscape software visualized PPI networks, and hub TFs were selected via cytoHubba. AutoDock was used for molecular docking of tilianin and hub gene-encoded proteins. The expression levels of hub genes were assayed and visualized by quantitative real-time PCR, Western blotting, and immunohistochemistry by establishing I/R-induced AKI mouse models. Results: Bioinformatics analysis showed that 34 genes, including FOS, ATF4, and Gadd45g, were involved in the apoptosis pathway. In total, seven hub TFs might play important roles in tilianin-regulating apoptosis pathways. In in vivo , tilianin improved kidney function and reduced the number of TUNEL-positive renal tubular epithelial cells (RTECs) after I/R-induced AKI. Tilianin reduced the activation of the ERK pathway and then downregulated the expression of EGR1. This further ameliorated the expression of anti-apoptotic genes such as BCL2L1 and BCL2, reduced pro-apoptotic genes such as BAD, BAX, and caspase-3, and reduced the release of cytochrome c. Conclusion: Tilianin reduced apoptosis after I/R-induced AKI by the ERK/EGR1/BCL2L1 pathway. Our findings provided novel insights for the first time into the protective effect and underlying molecular mechanisms of tilianin on I/R-induced AKI. [ABSTRACT FROM AUTHOR]

Summary: Camptothecin (CPT) and its derivatives, irinotecan and topotecan are specific topoisomerase I (Top1) inhibitors and potent anticancer drugs. Mechanistically, they induce DNA double-strand breaks (DSBs). Although CPT is an effective chemotherapeutic agent used in the management of advanced colorectal cancer, there exist associated side effects. Herein, we aimed to establish novel drug combinations that can effectively aid in managing the CPT-related side effects. Besides, bromodomain and extra-terminal domain (BET) inhibitors have proved as promising drugs that target epigenetic mechanisms in various cancers, they alter DNA repair processes, hence are a potential candidate for CPT synthetic lethality. A novel BET inhibitor JQ1 synergized with CPT, exerted antiproliferative effects. Through cell cycle analyses and apoptosis assays, we revealed that a combination of CPT and JQ1 induces subG1-phase arrest and enhances cell apoptosis. This combination increased the intensity of γ-H2AX staining, a specific marker of DSBs. Moreover, colorectal cancer cells highly expressing Top1 showed greater sensitivity to JQ1, which was lowered through the lentiviral shRNA-mediated knockdown of Top1. JQ1, combined with CPT, impeded the recruitment of the Mre11-mediated MRN complex. Finally, JQ1 enhanced the in vivo sensitivity of tumors to CPT without inducing toxicity. These results demonstrate that a combination of BET inhibitor with Top1 inhibitor is safe and exerts positive chemotherapeutic effects in colorectal cancer. [ABSTRACT FROM AUTHOR]

Chrysin amino acid derivatives were synthesized to evaluate for their antiproliferative activities. Among them, N-(7-((5-hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)valeryl)-L-leucine (8c) displayed the most remarkable inhibitory activities against MCF-7 cells with IC50 values of 16.6 μM. Preliminary mechanistic studies showed that 8c could inhibit the colony formation and migration of MCF-7 cells. Flow cytometry analysis demonstrated that 8c mediated cell apoptosis and the prolongation of cell cycle progression in G1/S-phase against MCF-7 cells. Besides, 8c displayed the moderate inhibition against EGFR. Western blot assay suggested that 8c significantly inhibited EGFR phosphorylation. Molecular docking showed that 8c can bind the EGFR kinase well. [ABSTRACT FROM AUTHOR]

Background: Pancreatic cancer is one of the most malignant tumors, and gemcitabine has been considered as the standard treatment and been widely utilized as a first-line drug for advanced pancreatic cancer, but gemcitabine-resistance always occurs after a short period of treatment. Methods: Two pancreatic cancer cell lines Panc-1 and MIA-PaCa-2 were used as the study subject and their gemcitabine-resistant cells were established. Both drug-resistant cells were divided into four groups: blank, emodin, gemcitabine, and emodin+gemcitabine. Cell viability was detected by MTT assay. Flow cytometry was performed to detect cell apoptosis rate and P-gp function. Quantitative real-time polymerase chain reaction and Western blotting were used to detect Survivin, XIAP, Caspase-9/3, NF-κB p65, IKKβ and IκB-α mRNA/protein expressions, respectively. Electrophoretic mobility shift assay (EMSA) was performed to detect NF-κB binding activity. Rhodamine 123 efflux assay was used to detect P-gp function. Results: Emodin could inhibit cell activity in all cell lines. Both emodin and gemcitabine can significantly increase the apoptosis rate, and the combination of the two drugs can further significantly increase the apoptosis rate in normal pancreatic cancer cell lines. In both drug-resistant pancreatic cancer cell lines, it can be observed that although gemcitabine can increase the apoptosis rate, the effect of promoting apoptosis is significantly lower than that of emodin; the drug combination can still significantly increase the apoptosis rate on the basis of emodin alone. Emodin can significantly reduce the mRNA and protein expression levels of Survivin, XIAP, NF-κB, and IKKβ, and significantly increase the mRNA and protein expression levels of Caspase-3/9 and IκB-α. Emodin significantly reduced NF-κB activity and emodin significantly promoted P-gp fluorescence intensity from Rhodamine 123 efflux assay. Conclusion: Emodin inhibits the expression of IKKβ, thereby inhibiting the expression and activity of downstream NF-κB, and inhibits P-gp function at the same time, ultimately achieving the purpose of reversing the drug-resistance of pancreatic cancer cell lines. [ABSTRACT FROM AUTHOR]