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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]

Background and Aim: Hydronidone (HDD) is a novel pirfenidone derivative developed initially to reduce hepatotoxicity. Our previous studies in animals and humans have demonstrated that HDD treatment effectively attenuates liver fibrosis, yet the underlying mechanism remains unclear. This study aimed to investigate whether HDD exerts its anti‐fibrotic effect by inducing apoptosis in activated hepatic stellate cells (aHSCs) through the endoplasmic reticulum stress (ERS)‐associated mitochondrial apoptotic pathway. Methods: The carbon tetrachloride (CCl4)‐ and 3,5‐diethoxycarbonyl‐1,4‐dihydrocollidine (DDC)‐induced liver fibrosis models were used for in vivo studies. In vitro studies were conducted using the human hepatic stellate cell line LX‐2. The apoptotic effect of HDD on aHSCs was examined using TUNEL and flow cytometry assays. The small interfering RNA (siRNA) technique was employed to downregulate the expression of interest genes. Results: HDD treatment significantly promoted apoptosis in aHSCs in both the CCl4‐ and DDC‐induced liver fibrosis in mice and LX‐2 cells. Mechanistic studies revealed that HDD triggered ERS and subsequently activated the IRE1α‐ASK1‐JNK pathway. Furthermore, the influx of cytochrome c from the mitochondria into the cytoplasm was increased, leading to mitochondrial dysfunction and ultimately triggering apoptosis in aHSCs. Notably, inhibition of IRE1α or ASK1 by siRNA partially abrogated the pro‐apoptotic effect of HDD in aHSCs. Conclusions: The findings of both in vivo and in vitro studies suggest that HDD induces apoptosis in aHSCs via the ERS‐associated mitochondrial apoptotic pathway, potentially contributing to the amelioration of liver fibrosis. [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.

Background: Nasopharyngeal carcinoma (NPC), predominant in Southern China and Southeast Asia, is a malignant tumor that arises from the epithelial lining of the nasopharynx. Current NPC treatments result in unfavorable side effects. Natural compounds with anti-proliferative capabilities are gaining popularity as a way to mitigate the toxicity of radiation and synthetic antitumor drugs. Ponicidin, an ent-kaurane diterpenoid, has been demonstrated to possess several pharmacological activities, including antitumor, antibacterial, immunoregulatory, antiviral, and anti-inflammatory properties. Materials and Methods: In the current investigation, the anti-carcinogenic activity of Ponicidin against nasopharyngeal cell line C666-1, has been investigated. The influence of Ponicidin on cell viability, apoptotic induction, mitochondrial membrane potential (MMP), reactive oxygen species (ROS) generation, and apoptotic markers has been examined. Doxorubicin was selected as the positive control for the experiments. Results: The effect of Ponicidin on the viability of C666-1 cells revealed that its cytotoxic potential increased in a dose-dependent pattern and the optimum concentration chosen for further experiments was 7.5 µM. The Ponicidin-treated cells demonstrated a significant increase in the amount of TBARS, whereas it reduced the activity of superoxide dismutase (SOD) and glutathione (GSH) levels, indicating augmented oxidative stress and lower antioxidant activity in the C666-1 cells. The acridine orange/ethidium bromide (AO/EtBr) staining technique was used to assess morphological changes with respect to apoptosis, which confirmed its apoptotic nature. Furthermore, Ponicidin elevated the ROS generation, modified the mitochondrial membrane permeability, elevated the apoptotic marker levels (caspase-3, caspase-9, and Bax) and reduced the Bcl-2 expression in C666-1 cell lines. Conclusion: The findings suggest that Ponicidin successfully inhibited cancer cell proliferation by following the mitochondrial apoptotic pathway and thus could be effectively utilized as a potent anti-cancer agent for the treatment of nasopharyngeal cancer. [ABSTRACT FROM AUTHOR]

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]

Penthorum chinense Pursh has been widely used as a Chinese traditional functional food for liver disease prevention and therapy. A novel cold-water soluble polysaccharide (PCP-4) has been prepared from Penthorum chinense Pursh at 4 °C. PCP-4 contained both α and β configurations and mainly consisted of galactose, arabinose, galacturonic acid, rhamnose, and glucose (molar ratio: 6.38:5.26:1.69:1:0.87) with an average molecular weight of 1.83 × 106 Da. In vitro experiments results demonstrated that PCP-4 effectively inhibits the proliferation of H22 cells and blocks cells at S phase in a dose-dependent manner. Otherwise, PCP-4-treated cells exhibited typical morphological features of apoptotic cells. Combined the results from western blot analysis, Annexin V-FITC/PI staining, Rhodamine 123 staining and DCFH-DA staining, PCP-4 was found to induce H22 cell apoptosis in vitro via the mitochondrial signaling pathway. The results of in vivo experiments suggested that PCP-4 significantly suppresses xenograft tumor growth by protecting immune organs, enhancing immune cells functionality, and inducing apoptosis. Overall, these results clearly show that Penthorum chinense Pursh polysaccharide serves as a valuable natural product for hepatocellular carcinoma therapy. • A novel polysaccharide (PCP-4) was isolated from Penthorum chinense Pursh. • PCP-4 suppressed H22 cell proliferation and blocked cell at the S phase in vitro. • PCP-4 activated mitochondrial pathway to promote H22 cell apoptosis in vitro. • PCP-4 could significantly inhibit tumor growth in H22 tumor-bearing mice. • PCP-4 exerted inhibitory effect by enhancing immunity and inducing tumor apoptosis. [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]

Diaphorina citri is a serious citrus pest. Dinotefuran is highly insecticidal against D. citri . To analyze the sublethal effects of dinotefuran on D. citri adults, an indoor toxicity test was performed, which revealed that the lethal concentration 50 (LC 50 ) values were 4.23 and 0.50 μg/mL for 24 and 48 h treatments, respectively. RNA-Seq led to the identification of 71 and 231 differentially expressed genes (DEGs) after dinotefuran treatments with LC 20 and LC 50 doses, respectively. Many of the DEGs are significantly enriched in the apoptosis pathway. Dinotefuran-induced apoptosis in the gut cells was confirmed through independent assays of 4',6-diamidino-2-phenylindole (DAPI) and TdT-mediated dUTP nick end labeling (TUNEL) staining. Increased levels of reactive oxygen species (ROS) and a loss of mitochondrial membrane potential were observed. Four caspase genes were identified, and dinotefuran treatments resulted in increased mRNA levels of DcCasp1 and DcCasp3a . These findings shed light on the sublethal effects of dinotefuran on D. citri .

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.

Although many studies have reported toxic effects of cadmium (Cd) and lead (Pb) in the central nervous system, few studies have investigated the combined toxicity of Cd and Pb. The mechanisms by which these combined heavy metals induce toxicity, as well as effective means to exert neuroprotection from these agents, remain poorly understood. To investigate the protective effects of alpha-lipoic acid (α-LA) on Cd- and/or Pb-induced cortical damage in rats, 48 Sprague-Dawley rats were exposed to drinking water containing 50 mg/L of Cd and/or 300 mg/L of Pb for 12 weeks, in the presence or absence of α-LA co-treatment (50 mg/kg) via gavage. We observed that exposure to Cd and/or Pb decreased the brain weight/body weight ratio and increased Cd and/or Pb contents as well as ultrastructural damage to the cerebral cortex. Cd and/or Pb also induced endoplasmic-reticulum (ER) stress and activated Fas (CD95/APO-1)/Fas ligand (FasL) and mitochondrial apoptotic pathways. Furthermore, co-treatment of Cd and Pb further exacerbated part of these phenotypes than treatment of Cd or Pb alone. However, simultaneous supplementation with α-LA attenuated Cd and/or Pb-induced neurotoxicity by increasing the brain weight/body weight ratio, reducing Cd and/or Pb contents, ameliorating both nuclear/mitochondrial damage and ER stress, and attenuating activation of Fas/FasL and mitochondrial apoptotic pathways. Collectively, our results indicate that the accumulation of Cd and/or Pb causes cortical damage and that α-LA exerts protection against Cd- and/or Pb-induced neurotoxicity. These findings highlight that α-LA may be exploited for the treatment and prevention of Cd- and/or Pb-induced neurotoxicity. [ABSTRACT FROM AUTHOR]

The potential toxicity of Zinc oxide nanoparticles (ZnO NPs) to human beings has become a widespread concern. This study explored the reproductive toxicity and the mechanism of toxicity of ZnO NPs in early pregnant mice. The results showed that abnormal weight changes, induced inflammation, reduced level of serum sex hormones, damaged uterus, increased abortion, and abnormal development of fetus. In the uterus, the transcription levels of ZnT‐1, HO‐1, Bax, Bax/Bcl‐2, JNK, and Caspase‐3 were significantly up‐regulated while Bcl‐2, ER‐1 and PR were significantly down‐regulated. The TUNEL‐positive cells increased that were exposed to high levels of ZnO NPs. In summary, those results indicated that Zn from high levels of exposure to ZnO NPs accumulated in the uterus that could have caused the formation of ROS that led to oxidative stress, which might have activated the mitochondrial apoptotic pathway that could have caused the uterine injury which induced the observed reproductive toxicity. [ABSTRACT FROM AUTHOR]

A great number of studies have shown the pivotal role of mitochondria in cancer progression and numerous studies indicated that lncRNAs are involved in tumor metabolism. However, the relationship between UCA1 and mitochondria in PDAC remains unclear. Here, we reported for the first time that UCA1-driven change in mitochondrial dynamics induced mitochondrial apoptotic pathway in PDAC. In this research, data mining revealed that upregulated UCA1 occurred in PDAC patients, which meant a high likelihood of a poor prognosis. Following, UCA1 silencing could notably decrease cell viability and induce cell apoptosis. Further research revealed that UCA1 silencing could induce more cytochrome c localization in the cytosol, which triggered the mitochondrial apoptotic pathway in PDAC cell lines. Meanwhile, the morphological analysis showed significantly enhanced mitochondrial fragmentation presented in UCA1 knockdown cells, coupled with increased expression of Drp1 and Fis1, together with an activation form of Drp1, which would promote mitochondria fission. Additional, mitochondrial fission inhibitor Mdivi1 markedly reversed the effects of the UCA1 knockdown on cell apoptosis in PDAC. Collectively, we deduce that UCA1-driven change in mitochondrial dynamics induced the mitochondrial apoptotic pathway in PDAC. Therefore, lncRNA UCA1 could be considered as a promising therapeutic target for the poor prognosis in PDAC. [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.

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]

Bisphenol‐A‐glycidyldimethacrylate (BisGMA) is a resin monomer frequently used in dentin restorative treatments. The leakage of BisGMA monomer from BisGMA‐based polymeric resins can lead to cytotoxicity in macrophages. Rutin has various beneficial bioeffects, including antioxidation and antiinflammation. In this study, we found that pretreatment of RAW264.7 macrophages with rutin‐inhibited cytotoxicity induced by BisGMA in a concentration‐dependent manner. BisGMA‐induced apoptosis, which was detected by levels of phosphatidylserine from the internal to the external membrane and formation of sub‐G1, and genotoxicity, which was detected by cytokinesis‐blocked micronucleus and single‐cell gel electrophoresis assays, were inhibited by rutin in a concentration‐dependent manner. Rutin suppressed the BisGMA‐induced activation of caspase‐3 and ‐9 rather than caspase‐8. Rutin inhibited the activation of the mitochondrial apoptotic pathway, including cytochrome C release and mitochondria disruption, after macrophages were treated with BisGMA. Finally, BisGMA‐induced reactive oxygen species (ROS) generation and antioxidant enzyme (AOE) deactivation could be reversed by rutin. Parallel trends were observed in the elevation of AOE activation and inhibition of ROS generation, caspase‐3 activity, mitochondrial apoptotic pathway activation, and genotoxicity. These results suggested that rutin suppressed BisGMA‐induced cytotoxicity through genotoxicity, the mitochondrial apoptotic pathway, and relatively upstream factors, including reduction of ROS generation and induction of AOE. [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.

Mitochondrial dysfunction is becoming one of the most emerging pathological process in the etiology of neurological disorders. Other common etiologies of the neurological disorders are aging and oxidative stress. Neurodegenerative disorders for instance Huntington's disease, Parkinson's disease, Amyotrophic lateral sclerosis, Epilepsy, Schizophrenia, Multiple sclerosis, Neuropathic pain and Alzheimer's disease involves mitochondrial dysfunction and is regarded as the core of their pathological processes. Most central pathological feature of the neurodegenerative diseases is apoptosis which is regulated by mitochondria. Altered signaling of the apoptotic mechanisms are involved in neurodegeneration. Abnormal levels of these molecular apoptotic proteins promotes the pathogenesis of neurological disorders. Mitochondria are also implicated in the production of reactive oxygen species (ROS). Raised ROS levels initiates the cascade leading to the non-apoptotic death of cells. ROS produced in cells acts as signaling molecules, but when produced in abundance will result in cellular consequences to deoxyribonucleic acid, proteins and lipids, decreased effectiveness of cellular mechanisms, initiation of inflammatory pathways, excitotoxicity, protein agglomeration and apoptosis. Protecting mitochondrial function has been identified as the most effective therapeutic approach to attenuate the pathogenesis of neurodegenerative diseases. This review aims to provide an insight into the mitochondrial dysfunction in the pathogenesis of neurological disorders, alteration in signaling cascades of apoptosis in mitochondrial dysfunction and the therapeutic strategies (both natural and synthetic drugs) targeting these mitochondrial apoptotic pathways and oxidative stress that holds great promise. • Mitochondrial dysfunction is one of the most emerging pathological process in neurological disorders. • Most central pathological feature of the neurodegenerative diseases is apoptosis which is regulated by mitochondria. • Alteration in the apoptotic signaling in mitochondrial dysfunction is an insight of therapeutic strategies. [ABSTRACT FROM AUTHOR]

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]

Spinal cord injury (SCI) induces aberrant expression of microRNAs (miRNAs), causing various secondary injury responses, including inflammation, apoptosis and oxidative stress. However, the mechanisms underlying miRNA-mediated apoptosis have not been fully elucidated. In the present study, a rat SCI model was established and a miRNA microarray was analyzed to detect miRNA expression profiles at different times post-SCI. The Basso, Beattie and Bresnahan score, cresyl violet staining and terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling staining were used to evaluate locomotor activity, lesion volume and neuronal cell apoptosis, respectively, at different time points post-SCI. It was observed that numerous miRNAs were altered at 14 days post-SCI and miR-124 was one of the most notably downregulated miRNAs. The present results demonstrated that overexpression of miR-124 by agomir-124 improves functional recovery, decreases lesion size and suppresses neuronal cell apoptosis in a rat SCI model. Luciferase reporter assay demonstrated that miR-124 inhibited apoptosis regulator BAX (Bax) expression, a key molecule in the activation of the mitochondrial apoptotic pathway, by targeting its 3′-untranslated region in BV-2 cells. Furthermore, restoration of Bax by pc-DNA-Bax inhibits the protective effect of miR-124 in H2O2-treated BV-2 cells. Notably, the present results demonstrated that miR-124 may block the mitochondrial apoptotic pathway by inhibiting Bax, cleaved-caspase-9 and cleaved-caspase-3 expression in rats following SCI. Collectively, the present results suggested that miR-124 may improve functional recovery and supress neuronal cell apoptosis by blocking the mitochondrial apoptotic pathway in SCI rats, suggesting that miR-124 may serve as a potential therapeutic target in SCI treatment. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Effects of the pyrethroid insecticide LCT on Ca2+-related physiology were explored in Gibco® Human Astrocytes (GHA cells). • In GHA cells, LCT induced Ca2+-associated cytotoxicity that involved Ca2+ entry via store-operated Ca2+ channels and Ca2+ release from the endoplasmic reticulum. • In GHA cells, LCT-activated mitochondrial apoptotic pathway was significantly inhibited by prechelating cytosolic Ca2+ with BAPTA-AM. Abstract Exposure to insecticides has been found to have deleterious effects on human health. Lambda-cyhalothrin (LCT), a mixture of isomers of cyhalothrin, is a pyrethroid insecticide routinely used in pest control programs. LCT was reported to cause neurotoxic effects in various models. However, the mechanism of underlying effect of LCT on cytotoxicity in normal human brain cells is still elusive. This study examined whether LCT affected Ca2+ homeostasis and Ca2+-related physiology in Gibco® Human Astrocytes (GHA cells), and explored whether BAPTA-AM (1,2-bis(2-aminophenoxy)ethane-N,N,N'N'-tetraacetic acid), a selective Ca2+ chelator, has protective effects on LCT-treated GHA cells. The data show that LCT (10–15 μM) concentration-dependently induced cytotoxicity in both GHA cells and DI TNC1 normal rat astrocytes but only induced intracellular Ca2+ concentration ([Ca2+] i) rises in GHA cells. In terms of Ca2+ signaling in GHA cells, LCT-induced [Ca2+] i rises were reduced by removing extracellular Ca2+ and were inhibited by store-operated Ca2+ channel modulators (2-APB, econazole or SKF96365). In Ca2+-free medium, pretreatment with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin abolished LCT-induced [Ca2+] i rises. Conversely, incubation with LCT abolished thapsigargin-induced [Ca2+] i rises. Regarding cytotoxicity, LCT evoked apoptosis by regulating apoptotic protein expressions (Bax, BCl-2, cleaved caspase-9/-3). This apoptotic response was significantly inhibited by prechelating cytosolic Ca2+ with BAPTA-AM. Together, in GHA cells, LCT induced [Ca2+] i rises by inducing Ca2+ entry via store-operated Ca2+ channels and Ca2+ release from the endoplasmic reticulum. Moreover, BAPTA-AM has a protective effect on inhibiting LCT-activated mitochondrial apoptotic pathway. This study provided new insights into the molecular protective mechanism of LCT-induced cytotoxicity in normal human astrocytes. [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]

Our previous study showed that docosahexaenoic acid (DHA) plays an important role in decreasing lipid accumulation by inducing apoptosis of the adipocytes in grass carp. However, the mechanism involved remains unclear. DHA has been reported as the natural ligand of PPARγ. The present study aimed to assess whether PPARγ mediates the pro-apoptotic effects by DHA. Adipocytes of grass carp were cultured until 2 days post-confluence and were treated with DHA at various concentrations—0, 25, 50, 100, 200, and 400 μmol/L for 24 h and at 200 μmol/L for various time periods (0, 12, 24, and 48 h, respectively). Besides, the adipocytes were exposed to 200 μM DHA and PPARγ antagonist or inhibitor of certain key enzymes of apoptosis, following which the expression levels of key genes of the cell apoptotic and mitochondrial apoptotic pathways were detected. We found that DHA induced apoptosis of grass carp adipocytes in a time- and dose-dependent manner (P < 0.05). In addition, DHA treatment significantly increased the protein and gene expression levels of PPARγ (P < 0.05), but the PPARγ antagonist significantly abolished this effect and the DHA pro-apoptotic effect (P < 0.05). Moreover, treatment with caspase 9 inhibitor significantly attenuated the DHA-induced preadipocytes apoptosis effects, while treatment with caspase 8 inhibitor showed no influence. These observations suggest that the DHA-induced apoptosis in adipocytes might be mediated by PPARγ and via the intrinsic apoptotic pathway in grass carp. [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]

Choriocarcinoma is a malignant gestational trophoblastic disease and relapse or drug resistance occurs in ~25% of gestational trophoblastic tumors. Cell apoptosis serves a role in the progression from hydatidiform mole to persistent gestational trophoblastic disease. It has been demonstrated that daidzein [7‑hydroxy‑3‑(4‑hydroxyphenyl)‑4H‑chromen‑4‑one] may induce apoptosis in a number of cancer types via the mitochondrial apoptotic pathway by altering the B‑cell lymphoma (Bcl)‑2/Bcl‑2 associated X, apoptosis regulator (Bax) ratio, and activating the caspase cascade. Daidzein also serves a role in regulation of production of human chorionic gonadotropin in trophoblast cells and inhibition of cell proliferation. However, few reports have been published regarding the effect of daidzein on apoptosis in choriocarcinoma. Therefore, in the present study, JAR and JEG‑3 human gestational choriocarcinoma cells were used to investigate the effect of daidzein on apoptosis of choriocarcinoma cells. Treatment with daidzein for 48 h reduced cell viability in a dose‑dependent manner. The percentages of early and late apoptotic cells also increased following treatment with daidzein in a dose‑dependent manner, with the number of late apoptotic cells increasing more prominently. Furthermore, treatment with daidzein led to apoptosis‑associated alterations in nuclear morphology of JAR and JEG-3 cells. Expression levels of cleaved poly(ADP‑ribose) polymerase, cleaved caspase‑3 and cleaved caspase‑9 increased following treatment with daidzein, whereas the Bcl‑2/Bax ratio decreased in a dose‑dependent manner. In conclusion, the results of the present study demonstrate that daidzein may induce apoptosis of choriocarcinoma cells in a dose‑dependent manner via the mitochondrial apoptotic pathway. [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.

Oleanolic acid derivatives exhibit potent anticancer activities against numerous types of cancer. However, the antitumor activity of oleanolic acid methylester (OAME), an oleanolic acid derivative, against prostate cancer has not been studied. Hence, the present work was conducted to study the anticancer activities of OAME. Viability assay showed that treatment of cancer cells with OAME induced a significant cell death in concentration- and time-dependent manner. Of note, OAME displayed a selective cytotoxicity against cancer cells compared to normal epithelial cells. Cells treated with OAME exhibited cell cycle arrest at both G1 and G2. Apoptotic induction potential of OAME was demonstrated using Annexin V assay, caspase activation, and DNA fragmentation methods Mechanistically, the results revealed that OAME strongly impacted the intrinsic apoptotic pathway in a concentration-dependent manner, as demonstrated by loss of mitochondrial membrane potential and release cytochrome c into the cytosol. ROS scavenger completely abrogated OAME-induced cell death. In vivo, OAME exerted concentration- dependent antiproliferative effect, associated with a significant level of apoptosis, potent antiangiogenic activity, and downregulation of survivin. This study provides significant insight into the therapeutic activities of OAME against prostate cancer in vitro and in vivo, suggesting that OAME might serve as a promising lead compound to treat hormonal-resistant prostate cancer. [ABSTRACT FROM AUTHOR]

Because of irregular dietary habits and lifestyle in Taiwan, the incidence and mortality rate of colorectal cancer have been increasing rapidly these years. This study investigated the inhibitory activity against the proliferation of human colorectal cancer HCT-116 cells by Inonotus obliquus extracts obtained from submerged fermentation. Cell viability was measured by the reduction of MTT and cell membrane integrity was determined by lactic dehydrogenase (LDH) release. The mRNA expression of proapoptosis and antiapoptosis mediators was assayed by real-time PCR, and the levels of p53 and NF-κB p65 were assessed using Western blot analysis. Furthermore, the influences of I. obliquus extracts to HCT-116 cells were evaluated by caspase-3 activity. The results can be summarized as, for the mitochondrial apoptotic pathway, quantitative RT-PCR data showed up-regulation of proapoptotic genes (Bax, bad, and caspase-3) and increased Bax/bcl-2 ratio by I. obliquus extracts. Moreover, treating with 20 mg/mL I. obliquus extracts augmented caspase-3 activity in HCT-116 cells. Induction of cell cycle G0/G1 phase arrest: I. obliquus extracts up-regulated the mRNA expression of proapoptotic genes (p53, p21WAF1/CIP1) and down-regulated antiapoptotic gene (CyclinD1), while extracts of I. obliquus mycelia increased the expressions of p53 protein in HCT-116 cells. I. obliquus extracts decreased the expression of NF-κB p65 protein and COX-2 gene in HCT-116 cells. Taking together, I. obliquus extracts may be used as a potentially novel food material for health care to improve the treatment of colorectal cancer. [ABSTRACT FROM AUTHOR]

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]

Aims Nicotinamide participates in energy metabolism and influences cellular redox status and modulates multiple pathways related with both cellular survival and death. Recent studies have shown that it induced proliferation inhibition and apoptosis in many cancer cells. However, little is known about the effects of nicotinamide on human cervical cancer cells. We aimed to evaluate the effects of the indicated concentrations nicotinamide on cell proliferation, apoptosis and redox-related parameters in HeLa cells and investigated the apoptotic mechanism. Materials and methods After the treatment of the indicated concentrations nicotinamide, HeLa cell proliferation was evaluated by the CCK-8 assay and the production of ROS (reactive oxygen species) was measured using 2′,7′-Dichlorofluorescin diacetate. The apoptotic effect was confirmed by observing the cellular and nuclear morphologies with fluorescence microscope and apoptotic rate of HeLa cell apoptosis was measured by flow cytometry using Annexin-V method. Moreover, we examined the mitochondrial membrane potential by JC-1 method and measured the expression of apoptosis related genes using qRT-PCR and immunoblotting. Key findings Nicotinamide restrained the HeLa cell proliferation and significantly increased the accumulation of ROS and depletion of GSH at relatively high concentrations. Furthermore, nicotinamide promoted HeLa cell apoptosis via the intrinsic mitochondrial apoptotic pathway. Significance Our study revealed that nicotinamide induced the apoptosis through oxidative stress and intrinsic mitochondrial apoptotic pathways in HeLa cell. The results emerge that nicotinamide may be an inexpensive, safe and promising therapeutic agent or a neoadjuvant chemotherapy for cervical cancer patients, as well useful to find new drugs for cervical cancer therapy. [ABSTRACT FROM AUTHOR]

The present study investigated the effects and mechanisms of demethoxycurcumin (DMC) on a human skin squamous cell carcinoma cell line, A431, and a human keratinocyte cell line, HaCaT. A431 and HaCaT cells were cultured in vitro. The effects of DMC treatment on cell viability were analyzed using the Cell Counting kit-8 (CCK-8) assay; cell cycle distribution was analyzed by flow cytometry; apoptosis was assessed by flow cytometry and Hoechst 33258 staining; and the protein expression levels of cytochrome c, B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (BAX), caspase-9 and caspase-3 were evaluated by western blotting. CCK-8 assay results demonstrated that DMC treatment significantly inhibited viability of A431 and HaCaT cells in a dose-dependent manner. Flow cytometric analysis confirmed that DMC treatment induced apoptosis in a dose-dependent manner, and significantly increased the proportion of cells in G2/M phase. Western blot analysis indicated that the protein expression levels of Bcl-2 were decreased, whereas the expression levels of BAX, caspase-9, caspase-3 and cytochrome c were increased following DMC treatment compared with in untreated cells. In conclusion, DMC treatment significantly inhibited viability of A431 and HaCaT cells, and induced cell cycle arrest in G2/M phase. The present study indicated that DMC may induce apoptosis of skin cancer cells through a caspase-dependent pathway. [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]