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The Japanese encephalitis virus (JEV), a zoonotic flavivirus, is Asia's primary cause of viral encephalitis. JEV induces apoptosis in a variety of cells; however, the precise mechanisms underlying this apoptosis resulting from JEV infection remain to be elucidated. Our previous studies showed that the proapoptosis gene BAX may have a role in JEV proliferation. In this study, we constructed a PK-15 cell line (BAX.KO) with a knockout of the BAX gene using CRISPR/Cas9. The knockout of the BAX gene effectively inhibited the proliferation of JEV, resulting in a 39.9% decrease in viral protein levels, while BAX overexpression produced the opposite effect. We confirmed that JEV induces apoptosis of PK-15 using 4′,6-diamidino-2-phenylindole (DAPI) staining and Annexin V-FITC/PI staining. Furthermore, we found that the phosphorylation of P53 and the expression levels of BAX, NOXA, PUMA, and cleaved-caspase-3/9 were significantly upregulated after JEV infection. Moreover, we found that JEV infection not only caused mitochondrial damage, the release of mitochondrial cytochrome C (Cyt C), and the downregulation of the apoptosis-inhibiting protein BCL-2 but also reduced the mitochondrial membrane potential (MOMP) and the accumulation of intracellular reactive oxygen species (ROS). These factors collectively encourage the activation of the mitochondrial apoptosis pathway. In contrast, BAX gene knockout significantly reduces the apoptotic changes caused by JEV infection. Treatment with the caspase3 inhibitor attenuated JEV-induced viral proliferation and release, leading to a decrease in viral protein levels of 46% in PK-15 cells and 30% in BAX.KO cells. In conclusion, this study clarified the molecular mechanisms of JEV-induced apoptosis and provided a theoretical basis for revealing the pathogenic mechanisms of JEV infection. [ABSTRACT FROM AUTHOR]

Breast cancer is the most prevalent form of cancer in women worldwide. Triple-negative breast cancer is the most unfavorable for patients, but it is also the most sensitive to chemotherapy. Triterpene glycosides from sea cucumbers possess a high therapeutic potential as anticancer agents. This study aimed to identify the pathways triggered and regulated in MDA-MB-231 cells (triple-negative breast cancer cell line) by the glycosides cucumarioside A0-1 (Cuc A0-1) and djakonovioside A (Dj A), isolated from the sea cucumber Cucumaria djakonovi. Using flow cytometry, fluorescence microscopy, immunoblotting, and ELISA, the effects of micromolar concentrations of the compounds on cell cycle arrest, induction of apoptosis, the level of reactive oxygen species (ROS), mitochondrial membrane potential (Δψm), and expression of anti- and pro-apoptotic proteins were investigated. The glycosides caused cell cycle arrest, stimulated an increase in ROS production, and decreased Δψm in MDA-MB-231 cells. The depolarization of the mitochondrial membrane caused by cucumarioside A0-1 and djakonovioside A led to an increase in the levels of APAF-1 and cytochrome C. This, in turn, resulted in the activation of caspase-9 and caspase-3 and an increase in the level of their cleaved forms. Glycosides also affected the expression of Bax and Bcl-2 proteins, which are associated with mitochondria-mediated apoptosis in MDA-MB-231 cells. These results indicate that cucumarioside A0-1 and djakonovioside A activate the intrinsic apoptotic pathway in triple-negative breast cancer cells. Additionally, it was found that treatment with Cuc A0-1 resulted in in vivo inhibition of tumor growth and metastasis of murine solid Ehrlich adenocarcinoma. [ABSTRACT FROM AUTHOR]

The Japanese encephalitis virus (JEV), a zoonotic flavivirus, is Asia’s primary cause of viral encephalitis. JEV induces apoptosis in a variety of cells; however, the precise mechanisms underlying this apoptosis resulting from JEV infection remain to be elucidated. Our previous studies showed that the proapoptosis gene BAX may have a role in JEV proliferation. In this study, we constructed a PK-15 cell line (BAX.KO) with a knockout of the BAX gene using CRISPR/Cas9. The knockout of the BAX gene effectively inhibited the proliferation of JEV, resulting in a 39.9% decrease in viral protein levels, while BAX overexpression produced the opposite effect. We confirmed that JEV induces apoptosis of PK-15 using 4′,6-diamidino-2-phenylindole (DAPI) staining and Annexin V-FITC/PI staining. Furthermore, we found that the phosphorylation of P53 and the expression levels of BAX, NOXA, PUMA, and cleaved-caspase-3/9 were significantly upregulated after JEV infection. Moreover, we found that JEV infection not only caused mitochondrial damage, the release of mitochondrial cytochrome C (Cyt C), and the downregulation of the apoptosis-inhibiting protein BCL-2 but also reduced the mitochondrial membrane potential (MOMP) and the accumulation of intracellular reactive oxygen species (ROS). These factors collectively encourage the activation of the mitochondrial apoptosis pathway. In contrast, BAX gene knockout significantly reduces the apoptotic changes caused by JEV infection. Treatment with the caspase3 inhibitor attenuated JEV-induced viral proliferation and release, leading to a decrease in viral protein levels of 46% in PK-15 cells and 30% in BAX.KO cells. In conclusion, this study clarified the molecular mechanisms of JEV-induced apoptosis and provided a theoretical basis for revealing the pathogenic mechanisms of JEV infection.

Breast cancer is the most prevalent form of cancer in women worldwide. Triple-negative breast cancer is the most unfavorable for patients, but it is also the most sensitive to chemotherapy. Triterpene glycosides from sea cucumbers possess a high therapeutic potential as anticancer agents. This study aimed to identify the pathways triggered and regulated in MDA-MB-231 cells (triple-negative breast cancer cell line) by the glycosides cucumarioside A0-1 (Cuc A0-1) and djakonovioside A (Dj A), isolated from the sea cucumber Cucumaria djakonovi. Using flow cytometry, fluorescence microscopy, immunoblotting, and ELISA, the effects of micromolar concentrations of the compounds on cell cycle arrest, induction of apoptosis, the level of reactive oxygen species (ROS), mitochondrial membrane potential (Δψm), and expression of anti- and pro-apoptotic proteins were investigated. The glycosides caused cell cycle arrest, stimulated an increase in ROS production, and decreased Δψm in MDA-MB-231 cells. The depolarization of the mitochondrial membrane caused by cucumarioside A0-1 and djakonovioside A led to an increase in the levels of APAF-1 and cytochrome C. This, in turn, resulted in the activation of caspase-9 and caspase-3 and an increase in the level of their cleaved forms. Glycosides also affected the expression of Bax and Bcl-2 proteins, which are associated with mitochondria-mediated apoptosis in MDA-MB-231 cells. These results indicate that cucumarioside A0-1 and djakonovioside A activate the intrinsic apoptotic pathway in triple-negative breast cancer cells. Additionally, it was found that treatment with Cuc A0-1 resulted in in vivo inhibition of tumor growth and metastasis of murine solid Ehrlich adenocarcinoma.

Su-Jun Sun,1 Peng Deng,1 Chun-E Peng,1 Hai-Yu Ji,2 Long-Fei Mao,1 Li-Zeng Peng1 1Key Laboratory of Agro-Products Processing Technology of Shandong Province, Key Laboratory of Novel Food Resources Processing Ministry of Agriculture, Institute of Agro-Food Science and Technology, Shandong Academy of Agricultural Sciences, Jinan, People’s Republic of China; 2Center for Mitochondria and Healthy Aging, College of Life Sciences, Yantai University, Yantai, People’s Republic of ChinaCorrespondence: Li-Zeng Peng, Tel +86-159-5412-8918, Email penglizeng@sdnu.edu.cnIntroduction: Chitosan is the product of the natural polysaccharide chitin removing part of the acetyl group, and exhibits various physiological and bioactive functions. Selenium modification has been proved to further enhance the chitosan bioactivities, and has been a hot topic recently.Methods: The present study aimed to investigate the potential inhibitory mechanism of selenium-modified chitosan (SMC) on HepG2 cells through MTT assays, morphological observation, annexin V–FITC/PI double staining, mitochondrial membrane potential determination, cell-cycle detection, Western blotting, and two-dimensional gel electrophoresis (2-DE).Results: The results indicated that SMC can induce HepG2 cell apoptosis with the cell cycle arrested in the S and G2/M phases and gradual disruption of mitochondrial membrane potential, reduce the expression of Bcl2, and improve the expression of Bax, cytochrome C, cleaved caspase 9, and cleaved caspase 3. Also, 2-DE results showed that tubulin α1 B chain, myosin regulatory light chain 12A, calmodulin, UPF0568 protein chromosome 14 open reading frame 166, and the cytochrome C oxidase subunit 5B of HepG2 cells were downregulated in HepG2 cells after SMC treatment.Discussion: These data suggested that HepG2 cells induced apoptosis after SMC treatment via blocking the cell cycle in the S and G2/M phases, which might be mediated through the mitochondrial apoptotic pathway. These results could be of benefit to future practical applications of SMC in the food and drug fields.Keywords: selenium-modified chitosan, antihepatoma activity, mitochondrial apoptotic pathway, differential protein analysis

Tris(1,3-dichloro-2-propyl) phosphate (TDCPP) is an organophosphorus flame retardant that has been utilized in recent years as a primary replacement for polybrominated diphenyl ethers (PBDEs) in a wide variety of fire-sensitive applications. However, the impact of TDCPP on the immune system has not been fully determined. As the largest secondary immune organ in the body, the spleen is considered to be an important study endpoint for determining immune defects in the body. The aim of this study is to investigate the effect of TDCPP toxicity on the spleen and its possible molecular mechanisms. In this study, for 28 consecutive days, TDCPP was administered intragastrically (i.g), and we assessed the general condition of mice by evaluating their 24 h water and food intake. Pathological changes in spleen tissues were also evaluated at the end of the 28-day exposure. To measure the TDCPP-induced inflammatory response in the spleen and its consequences, the expression of the critical players in the NF-κB pathway and mitochondrial apoptosis were detected. Lastly, RNA-seq was performed to identify the crucial signaling pathways of TDCPP-induced splenic injury. The results showed that TDCPP intragastric exposure triggered an inflammatory response in the spleen, likely through activating the NF-κB/IFN-γ/TNF-α/IL-1β pathway. TDCPP also led to mitochondrial-related apoptosis in the spleen. Further RNA-seq analysis suggested that the TDCPP-mediated immunosuppressive effect is associated with the inhibition of chemokines and the expression of their receptor genes in the cytokine–cytokine receptor interaction pathway, including four genes of the CC subfamily, four genes of the CXC subfamily, and one gene of the C subfamily. Taken together, the present study identifies the sub-chronic splenic toxicity of TDCPP and provides insights on the potential mechanisms of TDCPP-induced splenic injury and immune suppression. [ABSTRACT FROM AUTHOR]

Abstract Duck Tembusu virus (DTMUV) is a pathogenic flavivirus that has caused enormous economic losses in Southeast Asia. Our previous study showed that DTMUV could induce duck embryo fibroblast (DEF) apoptosis, but the specific mechanism was not clear. In this study, we confirmed that DTMUV could induce the apoptosis of DEFs by DAPI staining and TUNEL staining. Furthermore, we found that the expression levels of cleaved-caspase-3/7/8/9 were significantly upregulated after DTMUV infection. After treatment of cells with an inhibitor of caspase-8 or caspase-9, DTMUV-induced apoptosis rates were significantly decreased, indicating that the caspase-8-mediated death receptor apoptotic pathway and caspase-9-mediated mitochondrial apoptotic pathway were involved in DTMUV-induced apoptosis. Moreover, we found that DTMUV infection not only caused the release of mitochondrial cytochrome C (Cyt C) and the downregulation of the apoptosis-inhibiting protein Bcl-2 but also reduced the mitochondrial membrane potential (MMP) and the accumulation of intracellular reactive oxygen species (ROS). Key genes in the mitochondrial apoptotic pathway and death receptor apoptotic pathway were upregulated to varying degrees, indicating the activation of the mitochondrial apoptosis pathway and death receptor apoptosis pathway. In conclusion, this study clarifies the molecular mechanism of DTMUV-induced apoptosis and provides a theoretical basis for revealing the pathogenic mechanism of DTMUV infection.

Mitochondria play a crucial role in upholding metabolic homeostasis. Mitochondrial damage closely associated with the pathogenesis of fatty liver hemorrhagic syndrome (FLHS), while mitophagy being among the most effective methods for eliminating the damaged mitochondria. Dioscin, a natural extract, can activate autophagy; however, its effects on FLHS regarding mitophagy regulation remain unelucidated. We explored the impact of dioscin on FLHS induced by a high-energy and low-protein (HELP) diet in laying hens, mainly focused the protective effects of dioscin on mitochondrial injury. To investigate the impact of dioscin on fatty liver syndrome in laying hens, we first induced the condition by feeding them a high-energy and low-protein diet. Then, we assessed lipid metabolism-related markers using oil red staining and a commercial detection kit. In addition, the role of dioscin on fatty liver syndrome in laying hens was confirmed by assessing the activation of hepatocyte fat deposition and hepatocyte apoptosis; and the mechanism of dioscin in FLHS was investigated through LMH cell experiment in vitro. Furthermore, CETSA and molecular docking were conducted for additional confirmation. The results showed that dioscin alleviated mitochondrial damage, relieved the excessive deposition of hepatic lipid droplets and oxidative stress induced by HELP diet in laying hens. Furthermore, dioscin regulated the mitophagy by activating the estrogen receptor α (ERα)/adenosine 5′-monophosphate-activated protein kinase (AMPK) signaling pathway, thus mitigating mitochondria injury and apoptosis in hepatocytes. In addition, we found that dioscin promoted the translocation of nuclear transcription factor into nucleus by activating ERα-AMPK signaling, facilitating autophagic flux in the liver of laying hens and LMH cells. Furthermore, cells pretreated with the lysosomal acidification inhibitor bafilomycin A1 blocked the inhibitory effect of dioscin on the apoptosis induced by palmitic acid (PA)-stimulation in LMH cells, suggesting that dioscin reduces PA-induced apoptosis by activating mitophagy. Moreover, dioscin-induced lysosomal acidification and mitochondrial biogenesis were reversed in PA-induced LMH cells pretreated with ERα-specific inhibitor methylpiperidino pyrazole. This study firstly demonstrated that dioscin alleviates fatty liver syndrome induced by HELP diet in laying hens. The findings from this study illustrated that dioscin plays a significant role in reducing mitochondrial damage and apoptosis, and these beneficial effects mainly achieve through promotion of ERα-AMPK signaling, which mediates autophagy within the liver of laying hens fed a HELP-diets. These findings provide a theoretical basis for considering dioscin as a possible treatment option for mitigating FLHS in egg-laying hens. [Display omitted] [ABSTRACT FROM AUTHOR]

Dibutyl phthalate (DBP) is a typical phthalate (PAEs). The environmental health risks of DBP have gradually attracted attention due to the common use in the production of plastics, cosmetics and skin care products. DBP was associated with diabetes, but its mechanism is not clear. In this study, an in vitro culture system of rat insulinoma (INS-1) cells was established to explore the effect of DBP on insulin synthesis and secretion and the potential mechanisms. INS-1 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum and treated with 15, 30, 60 and 120 μmol/L of DBP and dimethyl sulfoxide (vehicle, < 0.1%) for 24 h. The contents of insulin in the intracellular fluid and the extracellular fluid of the cells were measured. The results showed that insulin synthesis and secretion in INS-1 cells were significantly decreased in 120 μmol/L DBP group. The apoptosis rate and mitochondrial membrane potential of INS-1 cells were measured by flow cytometry with annexin V-FITC conjugate and PI, and JC-1, respectively. The results showed that DBP caused an increase in the apoptosis rate and a significant decrease in the mitochondrial membrane potential in INS-1 cells in 60 μmol/L and 120 μmol/L DBP group. The results of western blot showed that the expression of Bax/Bcl-2, caspase-3, caspase-9 and Cyt-C were significantly increased. Meanwhile, the level of oxidative stress in INS-1 cells was detected by fluorescent probes DCFH-DA and western blot. With the increase of DBP exposure, the oxidative stress levels (MDA, GSH/GSSG) were increased; and the antioxidant index (SOD) levels were decreased. Our experimental results provide reliable evidence that DBP induced apoptosis and functional impairment in INS-1 cells through the mitochondrial apoptotic pathway and oxidative stress. Therefore, we hypothesized that interference with these two pathways could be considered in the development of preventive protection measures.

Furanocoumarins, the secondary metabolites of plants, are considered to be natural insecticides and fungicides because they prevent the invasion of plant pathogenic microorganisms and the predation of herbivorous insects. In this study, novel 2-arylfuranocoumarin derivatives were designed to synthesize by condensation, esterification, bromination, and Wittig reaction. The results showed an excellent photosensitive activity of 2-thiophenylfuranocoumarin (I34). Cell Counting Kit-8 detected that I34 could inhibit the proliferation of Spodoptera frugiperda (Sf9) cells in a time- and concentration-dependent manner under ultraviolet A (UV-A) light for 3 min. The inverted microscope revealed that cells treated with I34 swelled, the membrane was ruptured, and apoptotic bodies appeared. The flow cytometry detected that I34 could induce apoptosis of Sf9 cells, increase the level of intracellular reactive oxygen species (ROS), decrease the mitochondrial membrane potential, and block cell cycle arrest in the G2/M phase. Transmission electron microscopy detected cell mitochondrial cristae damage, matrix degradation, and mitochondrial vacuolation. Further enzyme activity detection revealed that the enzyme activities of apoptosis-related proteins caspase-3 and caspase-9 increased significantly (p < 0.05). Finally, Western blotting analysis detected that the phosphorylation level of Akt and Bad and the expression of the apoptosis inhibitor protein Bcl-XL were inhibited, cleaved-PARP and P53 were increased, and cytochrome C was released from the mitochondria into the cytoplasm. Moreover, under UV-A irradiation, I34 promoted the increase in ROS in Sf9 cells, activated the mitochondrial apoptotic signal transduction pathway, and finally, inhibited cell proliferation. Thus, novel furanocoumarins exhibit a potential application prospect as a biochemical pesticide. [ABSTRACT FROM AUTHOR]

Apoptosis plays a key role in the effective removal of excessive and defective germ cells, which is essential for sequential hermaphroditism and sex change in vertebrates. The ricefield eel, Monopterus albus is a protogynous hermaphroditic fish that undergoes a sequential sex change from female to male. Previous studies have demonstrated that apoptosis is involved in sex change in M. albus. However, the apoptotic signaling pathway is unclear. In the current study, we explored the underlying mechanism of apoptosis during gonadal development and focused on the role of the mitochondrial apoptosis signaling pathway in sex change in M. albus. Flow cytometry was performed to detect apoptosis in gonads at five sexual stages and ovary tissues exposed to hydrogen peroxide (H2O2) in vitro. Then the expression patterns of key genes and proteins in the mitochondrial pathway, death receptor pathway and endoplasmic reticulum (ER) pathway were examined. The results showed that the apoptosis rate was significantly increased in the early intersexual stage and then decreased with the natural sex change from female to male. Quantitative real-time PCR revealed that bax, tnfr1, and calpain were mainly expressed in the five stages. ELISA demonstrated that the relative content of cytochrome-c (cyt-c) in the mitochondrial pathway was significantly higher than that of caspase8 and caspase12, with a peak in the early intersexual stage, while the levels of caspase8 and caspase12 peaked in the late intersexual stage. Interestingly, the Pearson's coefficient between cyt-c and the apoptosis rate was 0.705, which suggests that these factors are closely related during the gonadal development of M. albus. Furthermore, the cyt-c signal was found to be increased in the intersexual stage by immunohistochemistry. After incubation with H2O2, the mRNA expression of mitochondrial pathway molecules such as bax, apaf-1, and caspase3 increased in ovary tissues. In conclusion, the present results suggest that the mitochondrial apoptotic pathway may play a more important role than the other apoptotic pathways in sex change in M. albus. [ABSTRACT FROM AUTHOR]

Chelerythrine (CHE) is a majorly harmful isoquinoline alkaloid ingredient in Chelidonium majus that could trigger potential hepatotoxicity, but the pivotal molecular mechanisms remain largely unknown. In the present study, CHE-induced cytotoxicity and the underlying toxic mechanisms were investigated using human HepG2 cells in vitro. Data showed that CHE treatment (at 1.25–10 μM)-induced cytotoxicity in HepG2 cells is dose-dependent. CHE treatment increased the production of ROS and induced oxidative stress in HepG2 cells. Additionally, CHE treatment triggered the loss of mitochondrial membrane potential, decreased the expression of mitochondrial complexes, upregulated the expression of Bax, CytC, and cleaved-PARP1 proteins and the activities of caspase-9 and caspase-3, and downregulated the expression of Bcl-XL, and HO-1 proteins, finally resulting in cell apoptosis. N-acetylcysteine supplementation significantly inhibited CHE-induced ROS production and apoptosis. Furthermore, CHE treatment significantly downregulated the expression of phosphorylation (p)-Akt (Ser473), p-mTOR (Ser2448), and p-AMPK (Thr172) proteins in HepG2 cells. Pharmacology inhibition of Akt promoted CHE-induced the downregulation of HO-1 protein, caspase activation, and apoptosis. In conclusion, CHE-induced cytotoxicity may involve the inhibition of Akt pathway and the activation of oxidative stress-mediated mitochondrial apoptotic pathway in HepG2 cells. This study sheds new insights into understanding the toxic mechanisms and health risks of CHE. [ABSTRACT FROM AUTHOR]

Betulinic acid (BA), an occurring pentacyclic triterpenoid, has various biological activities, such as anti-inflammation and antioxidation. Previous studies found that BA attenuated cyclophosphamide (CYP)-induced intestinal mucosal damage by inhibiting intestinal mucosal barrier dysfunctions and cell apoptosis. However, the effects and regulation mechanisms of BA on CYP-induced renal damage has not been reported in literature. Here, we found that BA pretreatment alleviated the elevation of serum urea level and inhibited the increase in serum neutrophil gelatinase-associated lipocalin level induced by CYP. Meanwhile, BA ameliorated renal tubular epithelial cell edema, and vacuolization of renal cortical tubular and renal glomerulus. Moreover, pretreatment with BA inhibited the mRNA expressions of pro-inflammatory cytokines interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α, and increased mRNA expressions of anti-inflammatory cytokines such as IL-10 and transforming growth factor-β by inactivation nuclear factor kappa-B. Simultaneously, BA decreased the accumulation of reactive oxygen species and malondialdehyde, and lowered the levels of superoxide dismutase and glutathione, while increased the activity of glutathione peroxidase in CYP-induced kidney damage mice. Besides, BA reduced the phosphorylation of extracellular signal-regulated kinases (ERK), inhibited the ratio of Bcl-2/Bax and cell apoptosis in CYP-triggered kidney damage. Furthermore, BA and/or PD98059 (an inhibitor of ERK) regulated mitigation of CYP-elicited renal injury and deactivation of the ERK pathway and mitochondrial apoptotic pathway, indicating that the protective effect of BA on CYP-induced renal damage may be associated with the down-regulation of ERK-mediated mitochondrial apoptotic pathway. Thus, BA could be a candidate agent against chemotherapy drug-induced nephrotoxicity by reducing inflammation and oxidative stress through suppression of ERK-mediated mitochondrial apoptotic pathway.

T-2 toxin exposure could cause neurotoxicity; however, the precise molecular mechanisms remain unclear. In the present study, we investigated T-2 toxin-induced cytotoxicity and underlying molecular mechanisms using a mouse microglia BV2 cell line. The results show that T-2 toxin treatment-induced cytotoxicity of BV2 cells was dose- and time-dependent. Compared to the control, T-2 toxin treatment at 1.25–5 ng/mL significantly increased reactive oxygen species (ROS) production and triggered oxidative stress. T-2 toxin treatment also caused mitochondrial dysfunction in BV2 cells, which was evidenced by decreased mitochondrial transmembrane potential, upregulated expression of Bax protein, and decreased expression of Bcl-2 protein. Meanwhile, T-2 toxin treatment upregulated the expression of cleaved-caspase-3, cleaved-PARP-1 proteins, and downregulated the expression of HO-1 and nuclear Nrf2 proteins, finally inducing cell apoptosis in BV2 cells. N-acetylcysteine (NAC) supplementation significantly attenuated T-2 toxin-induced cytotoxicity. Moreover, T-2 toxin treatment activated autophagy and upregulated autophagy flux, and the inhibition of autophagy significantly promoted T-2 toxin-induced cell apoptosis. Taken together, our results reveal that T-2 toxin-induced cytotoxicity in BV2 cells involves the production of ROS, the activation of the mitochondrial apoptotic pathway, and the inhibition of the Nrf2/HO-1 pathway. Our study offers new insight into the underlying molecular mechanisms in T-2 toxin-mediated neurotoxicity. [ABSTRACT FROM AUTHOR]

Duck Tembusu virus (DTMUV) is a pathogenic flavivirus that has caused enormous economic losses in Southeast Asia. Our previous study showed that DTMUV could induce duck embryo fibroblast (DEF) apoptosis, but the specific mechanism was not clear. In this study, we confirmed that DTMUV could induce the apoptosis of DEFs by DAPI staining and TUNEL staining. Furthermore, we found that the expression levels of cleaved-caspase-3/7/8/9 were significantly upregulated after DTMUV infection. After treatment of cells with an inhibitor of caspase-8 or caspase-9, DTMUV-induced apoptosis rates were significantly decreased, indicating that the caspase-8-mediated death receptor apoptotic pathway and caspase-9-mediated mitochondrial apoptotic pathway were involved in DTMUV-induced apoptosis. Moreover, we found that DTMUV infection not only caused the release of mitochondrial cytochrome C (Cyt C) and the downregulation of the apoptosis-inhibiting protein Bcl-2 but also reduced the mitochondrial membrane potential (MMP) and the accumulation of intracellular reactive oxygen species (ROS). Key genes in the mitochondrial apoptotic pathway and death receptor apoptotic pathway were upregulated to varying degrees, indicating the activation of the mitochondrial apoptosis pathway and death receptor apoptosis pathway. In conclusion, this study clarifies the molecular mechanism of DTMUV-induced apoptosis and provides a theoretical basis for revealing the pathogenic mechanism of DTMUV infection. [ABSTRACT FROM AUTHOR]

Tris(1,3-dichloro-2-propyl) phosphate (TDCPP) is an organophosphorus flame retardant that has been utilized in recent years as a primary replacement for polybrominated diphenyl ethers (PBDEs) in a wide variety of fire-sensitive applications. However, the impact of TDCPP on the immune system has not been fully determined. As the largest secondary immune organ in the body, the spleen is considered to be an important study endpoint for determining immune defects in the body. The aim of this study is to investigate the effect of TDCPP toxicity on the spleen and its possible molecular mechanisms. In this study, for 28 consecutive days, TDCPP was administered intragastrically (i.g), and we assessed the general condition of mice by evaluating their 24 h water and food intake. Pathological changes in spleen tissues were also evaluated at the end of the 28-day exposure. To measure the TDCPP-induced inflammatory response in the spleen and its consequences, the expression of the critical players in the NF-κB pathway and mitochondrial apoptosis were detected. Lastly, RNA-seq was performed to identify the crucial signaling pathways of TDCPP-induced splenic injury. The results showed that TDCPP intragastric exposure triggered an inflammatory response in the spleen, likely through activating the NF-κB/IFN-γ/TNF-α/IL-1β pathway. TDCPP also led to mitochondrial-related apoptosis in the spleen. Further RNA-seq analysis suggested that the TDCPP-mediated immunosuppressive effect is associated with the inhibition of chemokines and the expression of their receptor genes in the cytokine–cytokine receptor interaction pathway, including four genes of the CC subfamily, four genes of the CXC subfamily, and one gene of the C subfamily. Taken together, the present study identifies the sub-chronic splenic toxicity of TDCPP and provides insights on the potential mechanisms of TDCPP-induced splenic injury and immune suppression.

Ershiwuwei Zhenzhu pills was originally recorded in the Tibetan medical book Si Bu Yi Dian in the 8th century AD and is now included in the Pharmacopoeia of the People's Republic of China (2020). The pills can calm the nerves and open the mind as well as treat cerebral ischemia reperfusion injury, stroke, hemiplegia. However, its quality standards have not yet been established, and the therapeutic effect on cerebral ischemia by regulating the mitochondrial apoptosis pathway has not been elucidated. LC-MS was used to establish quality standards for Ershiwuwei Zhenzhu pills. Metabonomics, molecular docking, neuroethology, cerebral infarction ratio, pathological detection of diencephalon, cortex, and hippocampus, and molecular biology techniques were used to reveal the mechanism of the pills in regulating the mitochondrial apoptosis pathway to treat cerebral ischemia. The contents of 20 chemical components in Ershiwuwei Zhenzhu pills from 12 batches and 8 manufacturers was determined for the first time. Eleven differential metabolites and three metabolic pathways, namely, fructose and mannose metabolism, glycerophospholipid metabolism, and purine metabolism, were identified by metabonomics. The pills improved the neuroethology abnormalities of MCAO rats and the pathological damage in the diencephalon and decreased the ratio of cerebral infarction. It also significantly reduced the mRNA expression of AIF, Apaf-1, cleared caspase8, CytC, and P53 mRNA in the brain tissue and the protein expression of Apaf-1 and CYTC and increased the protein expression of NDRG4. In vitro quantitative analysis of the in vitro chemical components of Ershiwuwei Zhenzhu pills has laid the foundation for improving its quality control. The potential mechanism of the pills in treating cerebral ischemia may be related to the Apaf-1/CYTC/NDRG4 apoptosis pathway. This work provides guidance for clinical drug use for patients. [Display omitted] [ABSTRACT FROM AUTHOR]

Chelerythrine (CHE) is a majorly harmful isoquinoline alkaloid ingredient in Chelidonium majus that could trigger potential hepatotoxicity, but the pivotal molecular mechanisms remain largely unknown. In the present study, CHE-induced cytotoxicity and the underlying toxic mechanisms were investigated using human HepG2 cells in vitro. Data showed that CHE treatment (at 1.25–10 μM)-induced cytotoxicity in HepG2 cells is dose-dependent. CHE treatment increased the production of ROS and induced oxidative stress in HepG2 cells. Additionally, CHE treatment triggered the loss of mitochondrial membrane potential, decreased the expression of mitochondrial complexes, upregulated the expression of Bax, CytC, and cleaved-PARP1 proteins and the activities of caspase-9 and caspase-3, and downregulated the expression of Bcl-XL, and HO-1 proteins, finally resulting in cell apoptosis. N-acetylcysteine supplementation significantly inhibited CHE-induced ROS production and apoptosis. Furthermore, CHE treatment significantly downregulated the expression of phosphorylation (p)-Akt (Ser473), p-mTOR (Ser2448), and p-AMPK (Thr172) proteins in HepG2 cells. Pharmacology inhibition of Akt promoted CHE-induced the downregulation of HO-1 protein, caspase activation, and apoptosis. In conclusion, CHE-induced cytotoxicity may involve the inhibition of Akt pathway and the activation of oxidative stress-mediated mitochondrial apoptotic pathway in HepG2 cells. This study sheds new insights into understanding the toxic mechanisms and health risks of CHE.

Abstract Malignant glioma is one of the most challenging central nervous system diseases to treat and has high rates of recurrence and mortality. Current therapies often fail to control tumor progression or improve patient survival. Marinobufagenin (MBG) is an endogenous mammalian cardiotonic steroid involved in sodium pump inhibition. Currently, various studies have indicated the potential of MBG in cancer treatments; however, the precise mechanisms are poorly understood. The functions of MBG were examined using colony formation, migration, cell cycle, and apoptosis assays in glioma cells. A mitochondrial membrane potential assay was performed to determine the mitochondrial transmembrane potential change, and cytochrome c release from mitochondria was assayed by fluorescence microscopy. An immunofluorescence assay was performed, and the nuclear translocation of NF‐κB in glioma cells was confirmed by confocal microscopy. Western blotting and RT‐qPCR were used to detect the protein and gene expression levels, respectively. In addition, transfection experiment of ATP1A1‐siRNA was further carried out to confirm the role of sodium pump α1 subunit in the anticancer effect of MBG in human glioma. The apoptosis‐promoting and anti‐inflammatory effects of MBG were further investigated, and the sodium pump α1 subunit and the ERK signaling pathway were found to be involved in the anticancer effect of MBG. The in vivo anticancer efficacy of MBG was also tested in xenografts in nude mice. Thus, therapies targeting the ERK signaling‐mediated mitochondrial apoptotic pathways regulated by MBG might represent potential treatments for human glioma, and this study could accelerate the finding of newer therapeutic approaches for malignant glioma treatment.

Heatstroke is a life-threatening illness that is characterised by a core body temperature >40°C and central nervous system dysfunction. Acute kidney injury (AKI) is a common complication of heatstroke, and the mitochondrial apoptotic pathway has been demonstrated to be one of the leading causes of tissue damage and cell death in AKI. Curcumin is a phenol that is extracted from turmeric and demonstrates anti-apoptotic properties. To test if curcumin can protect the kidney from injury caused by heat stress, the effect of curcumin administration on renal injury and apoptosis of renal tissue was examined in a rat model of dry-heat environment. A total of 50 Sprague-Dawley rats were randomly divided into five groups (n=10): Standard temperature control, dry-heat control and curcumin treatment groups (50, 100 and 200 mg/kg groups). After exposure to a dry-heat environment for 150 min, the rats were anesthetized and euthanized. Blood, urine and renal tissue were collected to quantify the expression of specific mitochondrial apoptosis-related molecules. Curcumin pre-treatment decreased blood urea nitrogen and serum creatinine, urinary kidney injury molecule-1, and neutrophil gelatinase-associated lipocalin levels compared with the dry-heat control group. Curcumin was also revealed to downregulate c-Jun N-terminal kinases (JNK), cytochrome c, caspase-3 and caspase-9 expression upon treatment with 100 and 200 mg/kg curcumin, which may result in inhibition of the mitochondrial apoptotic pathway in renal cells. The current study revealed that Curcumin may to have potential for preventing heatstroke-induced AKI. [ABSTRACT FROM AUTHOR]

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Abstract Background Cardol is a major bioactive constituent in the Trigona incisa propolis from Indonesia, with a strong in vitro antiproliferative activity against the SW620 colorectal adenocarcinoma cell line (IC50 of 4.51 ± 0.76 μg/mL). Cardol induced G0/G1 cell cycle arrest and apoptotic cell death. The present study was designed to reveal the mechanism of cardol’s antiproliferative effect and induction of apoptosis. Methods Changes in cell morphology were observed by light microscopy. To determine whether the mitochondrial apoptotic pathway was involved in cell death, caspase-3 and caspase-9 activities, western blot analysis, mitochondrial membrane potential, and intracellular reactive oxygen species (ROS) levels were assayed. Results Changes in the cell morphology and the significantly increased caspase-3 and caspase-9 activities, plus the cleavage of pro-caspase-3, pro-caspase-9 and PARP, supported that cardol caused apoptosis in SW620 cells within 2 h after treatment by cardol. In addition, cardol decreased the mitochondrial membrane potential while increasing the intracellular ROS levels in a time- and dose-dependent manner. Antioxidant treatment supported that the cardol-induced cell death was dependent on ROS production. Conclusion Cardol induced cell death in SW620 cells was mediated by oxidative stress elevation and the mitochondrial apoptotic pathway, and these could be the potential molecular mechanism for the antiproliferative effect of cardol.

Glutathione S‐transferase omega 1 (GSTO1) contributes to the inactivation of a wide range of drug compounds via conjugation to glutathione during phase reactions. Chemotherapy‐induced GSTO1 expression in breast cancer cells leads to chemoresistance and promotes metastasis. In search of novel GSTO1 inhibitors, we identified S2E, a thia‐Michael adduct of sulfonamide chalcone with low LC50 (3.75 ± 0.73 μm) that binds to the active site of GSTO1, as revealed by molecular docking (glide score: −8.1), cellular thermal shift assay and fluorescence quenching assay (Kb ≈ 10 × 105 mol·L−1). Docking studies confirmed molecular interactions between GSTO1 and S2E, and identified the hydrogen bond donor Val‐72 (2.14 Å) and hydrogen bond acceptor Ser‐86 (2.77 Å). Best pharmacophore hypotheses could effectively map S2E and identified the 4‐methyl group of the benzene sulfonamide ring as crucial to its anti‐cancer activity. Lack of a thiophenyl group in another analog, 2e, reduced its efficacy as observed by cytotoxicity and pharmacophore matching. Furthermore, GSTO1 inhibition by S2E, along with tamoxifen, led to a significant increase in apoptosis and decreased migration of aggressive MDA‐MB‐231 cells, as well as significantly decreased migration, invasion and mammosphere formation in sorted breast cancer stem cells (CSCs, CD24−/CD44+). GSTO1 silencing in breast CSCs also significantly increased apoptosis and decreased migration. Mechanistically, GSTO1 inhibition activated the c‐Jun N‐terminal kinase stress kinase, inducing a mitochondrial apoptosis signaling pathway in breast CSCs via the pro‐apoptotic proteins BAX, cytochrome c and cleaved caspase 3. Our study elucidated the role of the GSTO1 inhibitor S2E as a potential therapeutic strategy for preventing chemotherapy‐induced breast CSC‐mediated cancer metastasis and recurrence. [ABSTRACT FROM AUTHOR]

In the study, effects of waterborne zinc (Zn) exposure on apoptosis were investigated, and the potential mechanism of apoptosis participating in the Zn-induced variations of lipid metabolism was explored in a low vertebrate, yellow catfish Pelteobagrus fulvidraco . We found that Zn induced occurrence of apoptosis of livers and hepatocytes in yellow catfish. Waterborne Zn also increased hepatic transcriptional levels of p53, cytochrome c (Cycs), caspase 3a (Casp3a) and caspase 3b (Casp3b) of yellow catfish. Zn increased caspase 3 activity and reduced the mitochondrial permeability transition (MTP) in yellow catfish hepatocytes. Z-VAD-fmk (caspase inhibitor) and CsA pretreatment (MTP inhibitor) attenuated the Zn-induced apoptosis and reduction in MTP. Z-VAD-fmk pretreatments attenuated the Zn-induced increase in transcriptional levels of p53, Cycs and Casp3b although the differences were not statistically significant between the Zn group and Zn + Z-VAD-fmk group. In contrast, Zn and N-acetylcysteine (NAC) did not significantly influence the reactive oxygen species (ROS) production. Zn significantly reduced triglyceride (TG) content, increased the activities of carnitine palmitoyltransferase 1 (CPT I), hormone-sensitive lipase (HSL) and adipose TAG lipase (ATGL), and the transcriptional levels of p53, Cycs and caspase 3b of the hepatocytes; these Zn-induced effects on TG contents, activities of CPT I, HSL and ATGL, and mRNA levels of p53, Cycs and caspase 3b could partly be reversed by Z-VAD-fmk, suggesting that Zn induced the mitochondrial-mediated apoptosis and reduced lipid accumulation. Taken together, our study demonstrated the importance of mitochondria-mediated apoptosis in Zn-induced lipolysis, which suggested a new mechanism for elucidating metal element influencing lipid metabolism. [ABSTRACT FROM AUTHOR]

Abstract: Objectives: Our previous in vitro study showed that 5‐(3, 4, 5‐trimethoxybenzoyl)‐4‐methyl‐2‐(p‐tolyl) imidazol (BZML) is a novel colchicine binding site inhibitor with potent anti‐cancer activity against apoptosis resistance in A549/Taxol cells through mitotic catastrophe (MC). However, the mechanisms underlying apoptosis resistance in A549/Taxol cells remain unknown. To clarify these mechanisms, in the present study, we investigated the molecular mechanisms of apoptosis and autophagy, which are closely associated with MC in BZML‐treated A549 and A549/Taxol cells. Methods: Xenograft NSCLC models induced by A549 and A549/Taxol cells were used to evaluate the efficacy of BZML in vivo. The activation of the mitochondrial apoptotic pathway was assessed using JC‐1 staining, Annexin V‐FITC/PI double‐staining, a caspase‐9 fluorescence metric assay kit and western blot. The different functional forms of autophagy were distinguished by determining the impact of autophagy inhibition on drug sensitivity. Results: Our data showed that BZML also exhibited desirable anti‐cancer activity against drug‐resistant NSCLC in vivo. Moreover, BZML caused ROS generation and MMP loss followed by the release of cytochrome c from mitochondria to cytosol in both A549 and A549/Taxol cells. However, the ROS‐mediated apoptotic pathway involving the mitochondria that is induced by BZML was only fully activated in A549 cells but not in A549/Taxol cells. Importantly, we found that autophagy acted as a non‐protective type of autophagy during BZML‐induced apoptosis in A549 cells, whereas it acted as a type of cytoprotective autophagy against BZML‐induced MC in A549/Taxol cells. Conclusions: Our data suggest that the anti‐apoptosis property of A549/Taxol cells originates from a defect in activation of the mitochondrial apoptotic pathway, and autophagy inhibitors can potentiate BZML‐induced MC to overcome resistance to mitochondrial apoptosis. [ABSTRACT FROM AUTHOR]

Abstract: Malignant glioma is one of the most challenging central nervous system diseases to treat and has high rates of recurrence and mortality. Current therapies often fail to control tumor progression or improve patient survival. Marinobufagenin (MBG) is an endogenous mammalian cardiotonic steroid involved in sodium pump inhibition. Currently, various studies have indicated the potential of MBG in cancer treatments; however, the precise mechanisms are poorly understood. The functions of MBG were examined using colony formation, migration, cell cycle, and apoptosis assays in glioma cells. A mitochondrial membrane potential assay was performed to determine the mitochondrial transmembrane potential change, and cytochrome c release from mitochondria was assayed by fluorescence microscopy. An immunofluorescence assay was performed, and the nuclear translocation of NF‐κB in glioma cells was confirmed by confocal microscopy. Western blotting and RT‐qPCR were used to detect the protein and gene expression levels, respectively. In addition, transfection experiment of ATP1A1‐siRNA was further carried out to confirm the role of sodium pump α1 subunit in the anticancer effect of MBG in human glioma. The apoptosis‐promoting and anti‐inflammatory effects of MBG were further investigated, and the sodium pump α1 subunit and the ERK signaling pathway were found to be involved in the anticancer effect of MBG. The in vivo anticancer efficacy of MBG was also tested in xenografts in nude mice. Thus, therapies targeting the ERK signaling‐mediated mitochondrial apoptotic pathways regulated by MBG might represent potential treatments for human glioma, and this study could accelerate the finding of newer therapeutic approaches for malignant glioma treatment. [ABSTRACT FROM AUTHOR]

Background/Aims: Cajaninstilbene acid (3-hydroxy-4-prenyl-5-methoxystilben-2 -carboxylic acid, CSA), a natural stilbene isolated from the leaves of Cajanus cajan, has attracted considerable attention for its wide range of pharmacological activities. This study investigated whether CSA protects against corticosterone (CORT)-induced injury in PC12 cells and examined the potential mechanisms underlying this protective effect. Methods: Cell viability and cytotoxicity were detected using a 3-(4,5-desethyithiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and a lactate dehydrogenase (LDH) assay kit, respectively. PC12 cell apoptosis was measured using Hoechst 33342 staining and a DNA fragmentation assay kit, and intracellular Ca2+ concentrations were assessed by fluorescent labelling. Next, the mitochondrial permeability transition pores (mPTPs) and mitochondrial membrane potentials (∆Ψm) were detected using a colorimetric mPTP detection kit and a 5,5',6,6'-tetrachloro-1,1',3,3'- tetraethylbenzimidazolyl-carbocyanine iodide (JC-1) kit, respectively. Finally, cytochrome c, caspase-3 and inhibitor of caspase-activated deoxyribonuclease (ICAD) expression levels were monitored by western blot analysis. Results: Treatment with 100 µmol/l CORT induced cytotoxicity in PC12 cells. However, CSA dose-dependently increased cell viability and decreased LDH release as well as CORT-induced apoptosis. Mechanistically, compared with the CORT-treated group, CSA strongly attenuated intracellular Ca2+ overload and restored mitochondrial functions, including mPTPs and ∆Ψm. Furthermore, the down-regulation of cytochrome c and ICAD protein expression and the blockage of caspase-3 activity were observed upon CSA treatment. Conclusions: In summary, our data are the first to show that the in vitro antidepressant-like effect of CSA may be attributed to the cytoprotection of neurons and that such neuroprotective mechanisms are correlated with intracellular Ca2+ homeostasis and mitochondrial apoptotic pathways.

Previous studies have demonstrated that microRNA-10a (miR-10a) regulates various opposing biological functions in breast cancer. The aim of the present study was to investigate the exact functions of miR-10a in the pathogenesis of breast cancer. miR-10a expression was initially detected in two human breast cancer cell lines, MCF-7 and MDA-MB-231 and a normal human mammary epithelial cell line MCF-10A. The proliferation, migration and apoptosis of breast cancer cells were analyzed using MTT assays, Transwell assays and flow cytometry, respectively, following transfection of MCF-7 and MDA-MB-231 cells with an miR-10a mimic or anti-miR-10a. The expression of phosphorylated (p-)protein kinase B (Akt), p-mammalian target of rapamycin (p-mTOR), p-ribosomal protein S6 kinase β-1 (p-p70S6K), phosphatidylinositol- 4,5-bisphosphate 3-kinase catalytic subunit α (PIK3CA), Cytochrome C (Cyt C), B-cell lymphoma 2 (Bcl-2), BCL-2 associated X, apoptosis regulator (Bax), and cleaved caspase-3 were analyzed by western blotting. The migration of MCF-7 cells pretreated with an mTOR inhibitor CCI-779, was detected using a Transwell assay. Relative miR-10a expression was significantly elevated in MDA-MB-231 breast cancer cells and was at its highest levels in MCF-7 cells. Transfection with the miR-10a mimic significantly inhibited proliferation and migration, and promoted the apoptosis of breast cancer cells. Furthermore, upregulation of miR-10a markedly suppressed the levels of p-Akt, p-mTOR, p-p70S6K, and PIK3CA, and increased the expression of Cyt C, cleaved caspase-3, and the ratio of Bax/Bcl-2. Anti-miR-10a had the opposite effects. In addition, CCI-779 reversed the effect of anti-miR-10a on the migration of MCF-7 cells in a dose-dependent manner. In conclusion, miR-10a is downregulated in high aggressive breast cancer cells. miR-10a inhibited the proliferation and migration, and promoted apoptosis of breast cancer cells via phosphoinositide/Akt/mTOR signaling, and the mitochondrial apoptotic pathway. [ABSTRACT FROM AUTHOR]

Researches have been conducted to explore the biological effect of gastrodin, a natural compound extracted from the rhizome of Gastrodia elata Blume, in different models. However, the effects of gastrodin on cytotoxicity, cell cycle distribution and oxidative stress in glia cells have not been explored. The aim of this study was to investigate the cytotoxic effect of gastrodin and its mechanisms in DBTRG-05MG human glioblastoma cells and CTX TNA2 rat astrocytes. In DBTRG-05MG cells but not in CTX TNA2 cells, gastrodin (20-30 μM) induced cytotoxicity, G2/M phase cell cycle arrest and apoptosis. Regarding oxidative stress, gastrodin (20-30 μM) elevated intracellular ROS levels but reduced GSH levels. Treatment with the antioxidant NAC (10 μM) partially reversed gastrodin-altered antioxidant enzymes levels. Furthermore, gastrodin induced mitochondria-associated apoptosis. The apoptotic effects evoked by gastrodin were partially inhibited by the antioxidant NAC and the pancaspase inhibitor Z-VAD-FMK. Together, in DBTRG-05MG cells, but not in CTX TNA2 cells, gastrodin activated ROS-associated mitochondrial apoptotic pathways that involved cell cycle arrest. These data provide insight into the molecular mechanisms governing the ability of gastrodin to induce cytotoxicity in human glioblastoma cells and further suggest that gastrodin is a new potential agent for the treatment of human gliblasoma. [ABSTRACT FROM AUTHOR]

Background: Cardol is a major bioactive constituent in the propolis from Indonesia, with a strong in vitro antiproliferative activity against the SW620 colorectal adenocarcinoma cell line (IC50 of 4.51 ± 0.76 µg/mL). Cardol induced G0/G1 cell cycle arrest and apoptotic cell death. The present study was designed to reveal the mechanism of cardol's antiproliferative effect and induction of apoptosis. Methods: Changes in cell morphology were observed by light microscopy. To determine whether the mitochondrial apoptotic pathway was involved in cell death, caspase-3 and caspase-9 activities, western blot analysis, mitochondrial membrane potential, and intracellular reactive oxygen species (ROS) levels were assayed. Results: Changes in the cell morphology and the significantly increased caspase-3 and caspase-9 activities, plus the cleavage of pro-caspase-3, pro-caspase-9 and PARP, supported that cardol caused apoptosis in SW620 cells within 2 h after treatment by cardol. In addition, cardol decreased the mitochondrial membrane potential while increasing the intracellular ROS levels in a time- and dose-dependent manner. Antioxidant treatment supported that the cardol-induced cell death was dependent on ROS production. Conclusion: Cardol induced cell death in SW620 cells was mediated by oxidative stress elevation and the mitochondrial apoptotic pathway, and these could be the potential molecular mechanism for the antiproliferative effect of cardol. [ABSTRACT FROM AUTHOR]

Background: Lung cancer is one of the leading causes of cancer related deaths worldwide. Marine microalgae are a source of biologically active compounds and are widely consumed as a nutritional supplement in East Asian countries. It has been reported that Chlorella or Chlorella extracts have various beneficial pharmacological compounds that modulate immune responses; however, no studies have investigated the anti-cancer effects of Chlorella sorokiniana (CS) on non-small cell lung cancer (NSCLC). Methods: In this study, we evaluated the anti-cancer effects of CS in two human NSCLC cell lines (A549 and CL1-5 human lung adenocarcinoma cells), and its effects on tumor growth in a subcutaneous xenograft tumor model. We also investigated the possible molecular mechanisms governing the pharmacological function of CS. Results: Our results showed that exposure of the two cell lines to CS resulted in a concentration-dependent reduction in cell viability. In addition, the percentage of apoptotic cells increased in a dose-dependent manner, suggesting that CS might induce apoptosis in human NSCLC cells. Western blot analysis revealed that exposure to CS resulted in increased protein expression of the cleaved/activated forms of caspase-3, caspase-9, and PARP, except caspase-8. ZDEVD (caspase-3 inhibitor) and Z-LEHD (caspase-9 inhibitor) were sufficient at preventing apoptosis in both A549 and CL1-5 cells, proving that CS induced cell death via the mitochondria-mediated apoptotic pathway. Exposure of A549 and CL1-5 cells to CS for 24 h resulted in decreased expression of Bcl-2 protein and increased expression of Bax protein as well as decreased expression of two IAP family proteins, survivin and XIAP. Conclusions: We demonstrated that CS induces mitochondrial-mediated apoptosis in NSCLC cells via downregulation of Bcl-2, XIAP and survivin. In addition, we also found that the tumors growth of subcutaneous xenograft in vivo was markedly inhibited after oral intake of CS. [ABSTRACT FROM AUTHOR]

Gallic acid, a polyhydroxylphenolic compound, is widely distributed in various plants, fruits and foods. It has been shown that gallic acid passes into blood brain barrier and reaches the brain tissue of middle cerebral artery occlusion rats. However, the effect of gallic acid on Ca 2+ signaling in glia cells is unknown. This study explored whether gallic acid affected Ca 2+ homeostasis and induced Ca 2+ -associated cytotoxicity in DBTRG-05MG human glioblastoma cells and CTX TNA2 rat astrocytes. Gallic acid (20–40 μM) concentration-dependently induced cytotoxicity and intracellular Ca 2+ level ([Ca 2+ ] i ) increases in DBTRG-05MG cells but not in CTX TNA2 cells. In DBTRG-05MG cells, the Ca 2+ response was decreased by half by removal of extracellular Ca 2+ . In Ca 2+ -containing medium, gallic acid-induced Ca 2+ entry was inhibited by store-operated Ca 2+ channel inhibitors (2-APB, econazole and SKF96365). In Ca 2+ -free medium, pretreatment with the endoplasmic reticulum Ca 2+ pump inhibitor thapsigargin abolished gallic acid-induced [Ca 2+ ] i increases. Conversely, incubation with gallic acid also abolished thapsigargin-induced [Ca 2+ ] i increases. Inhibition of phospholipase C with U73122 abolished gallic acid-induced [Ca 2+ ] i increases. Gallic acid significantly caused cytotoxicity in DBTRG-05MG cells, which was partially prevented by prechelating cytosolic Ca 2+ with BAPTA-AM. Moreover, gallic acid activated mitochondrial apoptotic pathways that involved ROS production. Together, in DBTRG-05MG cells but not in CTX TNA2 cells, gallic acid induced [Ca 2+ ] i increases by causing Ca 2+ entry via 2-APB, econazole and SKF96365-sensitive store-operated Ca 2+ entry, and phospholipase C-dependent release from the endoplasmic reticulum. This Ca 2+ signal subsequently evoked mitochondrial pathways of apoptosis that involved ROS production. [ABSTRACT FROM AUTHOR]

Dibutyl phthalate (DBP) is a typical phthalate (PAEs). The environmental health risks of DBP have gradually attracted attention due to the common use in the production of plastics, cosmetics and skin care products. DBP was associated with diabetes, but its mechanism is not clear. In this study, an in vitro culture system of rat insulinoma (INS-1) cells was established to explore the effect of DBP on insulin synthesis and secretion and the potential mechanisms. INS-1 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum and treated with 15, 30, 60 and 120 μmol/L of DBP and dimethyl sulfoxide (vehicle, < 0.1%) for 24 h. The contents of insulin in the intracellular fluid and the extracellular fluid of the cells were measured. The results showed that insulin synthesis and secretion in INS-1 cells were significantly decreased in 120 μmol/L DBP group. The apoptosis rate and mitochondrial membrane potential of INS-1 cells were measured by flow cytometry with annexin V-FITC conjugate and PI, and JC-1, respectively. The results showed that DBP caused an increase in the apoptosis rate and a significant decrease in the mitochondrial membrane potential in INS-1 cells in 60 μmol/L and 120 μmol/L DBP group. The results of western blot showed that the expression of Bax/Bcl-2, caspase-3, caspase-9 and Cyt-C were significantly increased. Meanwhile, the level of oxidative stress in INS-1 cells was detected by fluorescent probes DCFH-DA and western blot. With the increase of DBP exposure, the oxidative stress levels (MDA, GSH/GSSG) were increased; and the antioxidant index (SOD) levels were decreased. Our experimental results provide reliable evidence that DBP induced apoptosis and functional impairment in INS-1 cells through the mitochondrial apoptotic pathway and oxidative stress. Therefore, we hypothesized that interference with these two pathways could be considered in the development of preventive protection measures. [Display omitted] • DBP reduced the insulin synthesis and secretion of INS-1 cells. • DBP altered the expression of GLUT2 and PDX-1 associated with insulin synthesis and secretion in INS-1 cells. • DBP induced the mitochondrial apoptotic pathway and oxidative stress in INS-1 cells. [ABSTRACT FROM AUTHOR]

Objective To examine the role of Cd-induced reactive oxygen species (ROS) generation in the apoptosis of neuronal cells. Methods Neuronal cells (primary rat cerebral cortical neurons and PC12 cells) were incubated with or without Cd post-pretreatment with rapamycin (Rap) or N-acetyl- l -cysteine (NAC). Cell viability was determined by MTT assay, apoptosis was examined using flow cytometry and fluorescence microscopy, and the activation of phosphoinositide 3′-kinase/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) and mitochondrial apoptotic pathways were measured by western blotting or immunofluorescence assays. Results Cd-induced activation of Akt/mTOR signaling, including Akt, mTOR, p70 S6 kinase (p70 S6K), and eukaryotic initiation factor 4E binding protein 1 (4E-BP1). Rap, an mTOR inhibitor and NAC, a ROS scavenger, blocked Cd-induced activation of Akt/mTOR signaling and apoptosis of neuronal cells. Furthermore, NAC blocked the decrease of B-cell lymphoma 2/Bcl-2 associated X protein (Bcl-2/Bax) ratio, release of cytochrome c, cleavage of caspase-3 and poly(ADP-ribose) polymerase (PARP), and nuclear translocation of apoptosis-inducing factor (AIF) and endonuclease G (Endo G). Conclusion Cd-induced ROS generation activates Akt/mTOR and mitochondrial pathways, leading to apoptosis of neuronal cells. Our findings suggest that mTOR inhibitors or antioxidants have potential for preventing Cd-induced neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

Betulinic acid (BA), an occurring pentacyclic triterpenoid, has various biological activities, such as anti-inflammation and antioxidation. Previous studies found that BA attenuated cyclophosphamide (CYP)-induced intestinal mucosal damage by inhibiting intestinal mucosal barrier dysfunctions and cell apoptosis. However, the effects and regulation mechanisms of BA on CYP-induced renal damage has not been reported in literature. Here, we found that BA pretreatment alleviated the elevation of serum urea level and inhibited the increase in serum neutrophil gelatinase-associated lipocalin level induced by CYP. Meanwhile, BA ameliorated renal tubular epithelial cell edema, and vacuolization of renal cortical tubular and renal glomerulus. Moreover, pretreatment with BA inhibited the mRNA expressions of pro-inflammatory cytokines interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α, and increased mRNA expressions of anti-inflammatory cytokines such as IL-10 and transforming growth factor-β by inactivation nuclear factor kappa-B. Simultaneously, BA decreased the accumulation of reactive oxygen species and malondialdehyde, and lowered the levels of superoxide dismutase and glutathione, while increased the activity of glutathione peroxidase in CYP-induced kidney damage mice. Besides, BA reduced the phosphorylation of extracellular signal-regulated kinases (ERK), inhibited the ratio of Bcl-2/Bax and cell apoptosis in CYP-triggered kidney damage. Furthermore, BA and/or PD98059 (an inhibitor of ERK) regulated mitigation of CYP-elicited renal injury and deactivation of the ERK pathway and mitochondrial apoptotic pathway, indicating that the protective effect of BA on CYP-induced renal damage may be associated with the down-regulation of ERK-mediated mitochondrial apoptotic pathway. Thus, BA could be a candidate agent against chemotherapy drug-induced nephrotoxicity by reducing inflammation and oxidative stress through suppression of ERK-mediated mitochondrial apoptotic pathway. [Display omitted] • BA alleviated CYP-induced renal damage by suppressing inflammation and oxidative stress. • BA reduced inflammation by inactivating NF-κB pathway. • BA reduced oxidative stress by inhibiting ERK-mediated mitochondrial apoptotic pathway. [ABSTRACT FROM AUTHOR]

Quinocetone, a new quinoxaline 1,4-dioxide derivative used in food-producing animals in China, exerts genotoxic effects on HepG2 cells. It triggers significant cytotoxicity and genotoxicity in vitro, but the detailed mechanism by which quinocetone induces adverse biological effects is not yet known. We analyzed the mechanisms behind quinocetone intoxication by investigating oxidative stress based on non-enzymatic and enzymatic antioxidant activities, and by identifying differentially regulated genes of HepG2 cells exposed to quinocetone using polymerase chain reaction (PCR)-based suppression subtractive hybridization to illustrate the toxicity mechanism of quinocetone. Meanwhile, the characteristics of oxidative stress and differentially regulated genes induced by quinocetone metabolites, 1,4-bisdesoxyquinocetone and 3-methylquinoxaline-2-carboxylic acid, were investigated too. Results showed that quinocetone damaged the antioxidant defense abilities of HepG2 cells by reducing the activities of endogenous antioxidant enzymes, lowering glutathione concentration, and elevating malondialdehyde level. We identified 160 quinocetone-responsive genes that were associated with cell proliferation, glucose metabolism, oxidative stress, and apoptosis, such as NAD(P)H dehydrogenase, quinone 1; and prolyl 4-hydroxylase, beta polypeptide. The expressions of some differentially regulated genes were confirmed by real-time reverse transcription-polymerase chain reaction. However, quinocetone metabolites showed little effects on HepG2 cells. These results showed that reactive oxygen species were the key mediators of quinocetone cytotoxicity in HepG2 cells and that c-MYC-dependent activation of the mitochondrial apoptotic pathway may be associated with quinocetone-induced toxicity. [ABSTRACT FROM AUTHOR]

Background/Aims: Cajaninstilbene acid (3-hydroxy-4-prenyl-5-methoxystilben-2 -carboxylic acid, CSA), a natural stilbene isolated from the leaves of Cajanus cajan, has attracted considerable attention for its wide range of pharmacological activities. This study investigated whether CSA protects against corticosterone (CORT)-induced injury in PC12 cells and examined the potential mechanisms underlying this protective effect. Methods: Cell viability and cytotoxicity were detected using a 3-(4,5-desethyithiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and a lactate dehydrogenase (LDH) assay kit, respectively. PC12 cell apoptosis was measured using Hoechst 33342 staining and a DNA fragmentation assay kit, and intracellular Ca2+ concentrations were assessed by fluorescent labelling. Next, the mitochondrial permeability transition pores (mPTPs) and mitochondrial membrane potentials (∆Ψm) were detected using a colorimetric mPTP detection kit and a 5,5',6,6'-tetrachloro-1,1',3,3'- tetraethylbenzimidazolyl-carbocyanine iodide (JC-1) kit, respectively. Finally, cytochrome c, caspase-3 and inhibitor of caspase-activated deoxyribonuclease (ICAD) expression levels were monitored by western blot analysis. Results: Treatment with 100 µmol/l CORT induced cytotoxicity in PC12 cells. However, CSA dose-dependently increased cell viability and decreased LDH release as well as CORT-induced apoptosis. Mechanistically, compared with the CORT-treated group, CSA strongly attenuated intracellular Ca2+ overload and restored mitochondrial functions, including mPTPs and ∆Ψm. Furthermore, the down-regulation of cytochrome c and ICAD protein expression and the blockage of caspase-3 activity were observed upon CSA treatment. Conclusions: In summary, our data are the first to show that the in vitro antidepressant-like effect of CSA may be attributed to the cytoprotection of neurons and that such neuroprotective mechanisms are correlated with intracellular Ca2+ homeostasis and mitochondrial apoptotic pathways. © 2014 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background: The 3β, 6β, 16β-trihydroxylup-20(29)-ene (TTHL) is a pentacyclic triterpene obtained from the medicinal plant Combretum leprosum Mart. In folk medicine, this plant is popularly known as mofumbo, cipoaba or mufumbo, and is used to treat several diseases associated with inflammation and pain. Methods: We investigated the antitumor efficacy of TTHL isolated from C. leprosum. The TTHL cytotoxic effect was investigated in MRC5, MCF-7, HepG2, T24, HCT116, HT29, and CACO-2 cells after 24, 48, 72 and 120 h of treatment. The mechanisms of cell death and DNA damage induction were investigated by flow cytometry and comet assay, respectively. Results: The results indicated that TTHL induced a time- and concentration-dependent growth inhibition in all human cancer cell lines. The cytotoxicity was more pronounced in MCF-7 breast cancer cells, with an IC50 of 0.30 µg/mL at 120 h. We therefore evaluated the cell death mechanism induced by TTHL (IC20, IC50, and IC80) in MCF-7 cells at 24 h. We found that the treatment with IC50 and IC80 TTHL for 24 h induced apoptosis in 14% (IC50) and 52% (IC80) of MCF-7 cells. The apoptosis induced by TTHL was accompanied by increased levels of both cleaved caspase-9 and intracellular ROS. In order to further understand the biological mechanism of TTHL-induced cytotoxicity, we have also investigated its effect on different Saccharomyces cerevisiae yeast strains. The mutant strains sod1Δ, sod2Δ, and sod1Δsod2Δ, which are deficient in superoxide dismutase antioxidant defenses, were hypersensitive to TTHL, suggesting that its capacity to disturb cellular redox balance plays a role in drug toxicity. Moreover, TTHL induced mutagenicity in the yeast strain XV185-14c. Conclusions: Taken together, the results suggest that TTHL forms covalent adducts with cellular macromolecules, potentially disrupting cellular function and triggering apoptosis. [ABSTRACT FROM AUTHOR]

Canstatin, the non-collagenous domain of collagen type IV α-chains, belongs to a series of collagen-derived angiogenic inhibitors. In this study, the inhibitory effect of recombinant canstatin on tumour growth was investigated using a gastric cancer xenograft model. The volume and weight of tumours in mice treated with canstatin were lower than that in mice treated with PBS. Accordingly, the survival rate of these mice was significantly higher than that of mice bearing tumours treated with PBS. Moreover, valuable insight into the mechanisms mediated by canstatin was obtained. [ABSTRACT FROM AUTHOR]

Abnormalities in the JAK2/STAT3 pathway are involved in the pathogenesis of colorectal cancer (CRC), including apoptosis. However, the exact mechanism by which dysregulated JAK2/STAT3 signalling contributes to the apoptosis has not been clarified. To investigate the role of both JAK2 and STAT3 in the mechanism underlying CRC apoptosis, we inhibited JAK2 with AG490 and depleted STAT3 with a small interfering RNA. Our data showed that inhibition of JAK2/STAT3 signalling induced CRC cellular apoptosis via modulating the Bcl-2 gene family, promoting the loss of mitochondrial transmembrane potential (Δψm) and the increase of reactive oxygen species. In addition, our results demonstrated that the translocation of cytochrome c (Cyt c), caspase activation and cleavage of poly (ADP-ribose) polymerase (PARP) were present in apoptotic CRC cells after down-regulation of JAK2/STAT3 signalling. Moreover, inhibition of JAK2/STAT3 signalling suppressed CRC xenograft tumour growth. We found that JAK2/STAT3 target genes were decreased; meanwhile caspase cascade was activated in xenograft tumours. Our findings illustrated the biological significance of JAK2/STAT3 signalling in CRC apoptosis, and provided novel evidence that inhibition of JAK2/STAT3 induced apoptosis via the mitochondrial apoptotic pathway. Therefore, JAK2/STAT3 signalling may be a potential target for therapy of CRC. [ABSTRACT FROM AUTHOR]

Abstract: Rosemary (Rosmarinus officinalis), a culinary spice and medicinal herb, has been widely used in European folk medicine to treat numerous ailments. Many studies have shown that rosemary extracts play important roles in anti-inflammation, anti-tumor, and anti-proliferation in various in vitro and in vivo settings. The roles of tumor suppression of rosemary have been attributed to the major components, including carnosic acid, carnosol, and rosmarinic acid, rosmanol, and ursolic acid. This study was to explore the effect of rosmanol on the growth of COLO 205 human colorectal adenocarcinoma cells and to delineate the underlying mechanisms. When treated with 50μM of rosmanol for 24h, COLO 205 cells displayed a strong apoptosis-inducing response with a 51% apoptotic ratio (IC50 ∼42μM). Rosmanol increased the expression of Fas and FasL, led to the cleavage and activation of pro-caspase-8 and Bid, and mobilized Bax from cytosol into mitochondria. The mutual activation between tBid and Bad decreased the mitochondrial membrane potential and released cytochrome c and apoptosis-inducing factor (AIF) to cytosol. In turn, cytochrome c induced the processing of pro-caspase-9 and pro-caspase-3, followed by the cleavage of poly-(ADP-ribose) polymerase (PARP) and DNA fragmentation factor (DFF-45). These results demonstrate that the rosmanol-induced apoptosis in COLO 205 cells is involvement of caspase activation and involving complicated regulation of both the mitochondrial apoptotic pathway and death receptor pathway. [ABSTRACT FROM AUTHOR]

Background: Maslinic acid, a pentacyclic triterpene found in the protective wax-like coating of the leaves and fruit of Olea europaea L., is a promising agent for the prevention of colon cancer. We have shown elsewhere that maslinic acid inhibits cell proliferation to a significant extent and activates mitochondrial apoptosis in colon cancer cells. In our latest work we have investigated further this compound's apoptotic molecular mechanism. Methods: We used HT29 adenocarcinoma cells. Changes genotoxicity were analyzed by single-cell gel electrophoresis (comet assay). The cell cycle was determined by flow cytometry. Finally, changes in protein expression were examined by western blotting. Student's t-test was used for statistical comparison. Results: HT29 cells treated with maslinic acid showed significant increases in genotoxicity and cell-cycle arrest during the G0/G1 phase after 72 hours' treatment and an apoptotic sub-G0/G1 peak after 96 hours. Nevertheless, the molecular mechanism for this cytotoxic effect of maslinic acid has never been properly explored. We show here that the anti-tumoral activity of maslinic acid might proceed via p53-mediated apoptosis by acting upon the main signaling components that lead to an increase in p53 activity and the induction of the rest of the factors that participate in the apoptotic pathway. We found that in HT29 cells maslinic acid activated the expression of c-Jun NH2-terminal kinase (JNK), thus inducing p53. Treatment of tumor cells with maslinic acid also resulted in an increase in the expression of Bid and Bax, repression of Bcl-2, release of cytochrome-c and an increase in the expression of caspases -9, -3, and -7. Moreover, maslinic acid produced belated caspase-8 activity, thus amplifying the initial mitochondrial apoptotic signaling. Conclusion: All these results suggest that maslinic acid induces apoptosis in human HT29 colon-cancer cells through the JNK-Bid-mediated mitochondrial apoptotic pathway via the activation of p53. Thus we propose a plausible sequential molecular mechanism for the expression of the different proteins responsible for the intrinsic mitochondrial apoptotic pathway. Further studies with other cell lines will be needed to confirm the general nature of these findings. [ABSTRACT FROM AUTHOR]