Mitochondrial Damage Pathway Research Articles

#2344 Empagliflozin attenuates renal fibrosis through the DsbA-L-CAS-STING pathway in unilateral ureteral obstruction model

Abstract Background and Aims Chronic kidney disease (CKD) is associated with an increase in morbidity and mortality. Renal fibrosis is a common pathway leading to the progression of CKD. Recent studies have reported an improvement of CKD with use of sodium glucose cotransport 2 inhibitors (SGLT2i) in both patients with DM and those without it. However, the mechanism of SGLT2is effect upon CKD has not been elucidated. In current study, we examined the effect of SGLT2i on renal fibrosis in rats caused by unilateral ureteral obstruction (UUO). Methods Male Sprague Dawley rats (9-10 weeks, weighing 320-360 g) were randomly assigned to two groups (n=8 each). One group received empagliflozin after UUO induction while the other group was left untreated. Kidneys were harvested two weeks after UUO. We evaluated the mitochondrial damage pathway presumed to contribute to the inflammation. Results UUO has resulted in marked renal fibrosis and triggered the activation of the cGAS-STING pathway. Kidneys in the empagliflozin treated group exhibited reduced size compare to the untreated group. Creatinine levels were lower in the empagliflozin group. Empagliflozin has been shown to attenuate renal fibrosis in histopathology. Western blot analysis revealed increased expression of disulfide-bond A oxidoreductase-like proteins (DsbA-L) was decreased cGAS-STING-NFκB in the empagliflozin group compared to the non-empagliflozin treated group (Fig. 1). Conclusion SGLT2i mitigated the progression of renal damage and fibrosis. This reno-protective effect is associated with increased DsbA-L and decreased of cGAS and STING pathway. These findings suggest that SGLT2i may alleviate mitochondrial damage in the UUO model. In addition, SGLT2i could be a promising medication to treat various forms of CKD.

Varying the hydrophobic core composition of polymeric nanoparticles affects NLRP3 inflammasome activation.

Understanding the interactions of nanoparticle carriers with innate immune cells is crucial for informing the design and efficacy of future nano-immunotherapies. An intriguing aspect of their interaction with the immune system has recently emerged, i.e., their ability to activate the NLRP3 inflammasome, a key component of the innate immune response. While the effect of the surface properties of nanoparticles has been extensively investigated in the context of nanoparticle-immune cell interactions, the influence of core composition remains largely unexplored, particularly regarding its impact on inflammasome activation. To shed light on these interactions, we developed a library of supramolecular polymer nanoparticles (SNPs) with different core compositions, varying their hydrophobic quotient by virtue of the side chain length and the repeating units in the polymer construct. The impact of modulating SNP core hydrophobic properties was investigated in macrophages by evaluating their cellular internalization, cytokine release, lysosomal rupture-calcium signaling, calcium flux-mitochondrial ROS production and their ability to activate the NLRP3 inflammasome, providing mechanistic insights into inflammasome activation. We established a direct correlation between increasing the side chain length of the polymer construct, thereby increasing the core hydrophobicity of SNPs and enhanced NLRP3 complex formation, as indicated by ASC speck imaging analysis and the elevated 1L-1β expression. Furthermore, the results demonstrated that the inflammasome signaling cascades and kinetics varied based on the SNP's hydrophobic side chain length and repeating units. Specifically, the nanoparticle with the longest alkyl side chain effectuated NLRP3 activation preferentially through the mitochondrial damage pathway. In vivo evaluation of SNPs in C57BL/6 mice confirmed elevated proinflammatory cytokines, notably with the SNP having the longest C12-alkyl side chain. This confirms that the higher core hydrophobicity composition of the SNP results in inflammasome activation in vivo. In summary, this study established SNP core composition as a novel nanoparticle-associated molecular pattern (NAMP) responsible for NLRP3 inflammasome activation, shedding light on intricate cellular pathways for informed nanoparticle design in immunotherapy and vaccine applications.

Synthesis, characterization and discovery of multiple anticancer mechanisms of dibutyltin complexes based on salen-like ligands

A series of novel dibutyltin complexes based on salen-like ligands (S01–S03) were synthesized and characterized using ultraviolet-visible spectra，infrared spectra, 1H, 13C, and 119Sn nuclear magnetic resonance, high-resolution mass spectrometry, X-ray crystallography, and thermogravimetric analysis. Complex S03 had excellent anticancer activity in vitro (IC50 = 1.5 ± 0.2 μM in CAL-27 cell lines), which highly activated ROS expression levels and induced apoptosis and cell cycle arrest at the G2/M phase. Interestingly, complex S03 induced cancer cell death through multiple mechanisms (mitochondrial pathway, ER-stress pathway, and DNA damage pathway). This study reveals new mechanisms of organotin complexes and provides new insights into the development of organotin metal complexes as anticancer drugs in the future, and compounds with multiple anticancer mechanisms may be a new strategy for delaying or overcoming drug resistance to chemotherapy and target therapy.

GSDME in Endothelial Cells: Inducing Vascular Inflammation and Atherosclerosis via Mitochondrial Damage and STING Pathway Activation.

The initiation of atherosclerotic plaque is characterized by endothelial cell inflammation. In light of gasdermin E's (GSDME) role in pyroptosis and inflammation, this study elucidates its function in atherosclerosis onset. Employing Gsdme- and apolipoprotein E-deficient (Gsdme-/-/ApoE-/-) and ApoE-/- mice, an atherosclerosis model was created on a Western diet (WD). In vitro examinations with human umbilical vein endothelial cells (HUVECs) included oxidized low-density lipoprotein (ox-LDL) exposure. To explore the downstream mechanisms linked to GSDME, we utilized an agonist targeting the stimulator of the interferon genes (STING) pathway. The results showed significant GSDME activation in ApoE-/- mice arterial tissues, corresponding with atherogenesis. Gsdme-/-/ApoE-/- mice displayed fewer plaques and decreased vascular inflammation. Meanwhile, GSDME's presence was confirmed in endothelial cells. GSDME inhibition reduced the endothelial inflammation induced by ox-LDL. GSDME was linked to mitochondrial damage in endothelial cells, leading to an increase in cytoplasmic double-stranded DNA (dsDNA). Notably, STING activation partially offset the effects of GSDME inhibition in both in vivo and in vitro settings. Our findings underscore the pivotal role of GSDME in endothelial cells during atherogenesis and vascular inflammation, highlighting its influence on mitochondrial damage and the STING pathway, suggesting a potential therapeutic target for vascular pathologies.

DBP induced autophagy and necrotic apoptosis in HepG2 cells via the mitochondrial damage pathway.

Phthalate esters (PAEs) are widely present in human tissues and pose significant health risks. In this study, HepG2 cells were treated with 0.0625, 0.125, 0.25, 0.5 and 1mM Dibutyl phthalate (DBP) for 48h to investigate mitochondrial toxicity. The results showed that DBP caused mitochondrial damage, autophagy, apoptosis and necroptosis; Transcriptomics analysis identified that MAPK and PI3K were significant factors in the cytotoxic changes induced by DBP; N-Acetyl-L-cysteine (NAC), SIRT1 activator, ERK inhibitor, p38 inhibitor and ERK siRNA treatments counteracted the changes of SIRT1/PGC-1α and Nrf2 pathway-related proteins, autophagy and necroptotic apoptosis proteins induced by DBP. While PI3K and Nrf2 inhibitors exacerbated the changes in SIRT1/PGC-1α, Nrf2-associated proteins and autophagy and necroptosis proteins induced by DBP. In addition, the autophagy inhibitor 3-MA alleviated the increase in DBP-induced necroptosis proteins. These results suggested that DBP-induced oxidative stress activated the MAPK pathway, inhibited the PI3K pathway, which in turn inhibited the SIRT1/PGC-1α pathway and Nrf2 pathway, thereby causing cell autophagy and necroptosis.

Human health risk assessment of cinnamate UV absorbers: In vitro and in silico investigations

Organic UV absorbers (UVAs) are contaminants of emerging concern. Environmental persistence and potential toxicological enrichment studies of UVAs have attracted international concern. It is important to study the toxicity mechanism of UVAs. This study is the first to report the toxicological mechanism of two cinnamate UV absorbers (CUVAs), 2-ethyl 4-methoxycinnamate (OMC) and isoamyl 4-methoxycinnamate (IMC) based on cellular models and molecular models. Cellular models demonstrated that the CUVAs-induced apoptosis might be associated with cellular mitochondrial damage pathways. The results of molecular models showed that OMC and IMC could affect the binding between major proteins and enzymes in the mitochondrial damage pathway and contaminants, ultimately leading to apoptosis. The cellular-molecular models showed that IMC and OMC have dose–effect relationships on cytotoxicity. The composite model is more informative than a single model. This study further indicate that UVAs causes toxicology effects that have implications for the environment and human health.

Endothelial cell‐specific inducible G2APOL1 risk variant induces hypertension and hypertensive kidney disease in uni‐nephrectomy and high‐salt mice model

Hypertension affects 108 million in the United States, which is more common in African American (AA) population (54%). Several Genome‐Wide Association Studies (GWAS) showed a strong association between the apolipoprotein1 (apol1) gene and hypertension in AA adults. Nearly 45% of AAs carry a coding variant of the APOL1 gene either G1APOL1 or G2APOL1. The disease phenotypes associated with APOL1 RV are dependent on the cell type‐specific expression and toxicity of APOL1. Recently, we have reported that APOL1 is highly expressed in endothelial cells (EC) of the human kidneys using single‐cell analysis, single nuclei analysis, and in‐situ hybridization. People who carry G1 or G2 APOL1 RV have a high risk of chronic kidney disease and hypertensive kidney disease. However, the direct role of APOL1 in the development of hypertension and hypertensive kidney disease is clear. Therefore, the objective of the current study was to examine the role of endothelial‐specific inducible G2APOL1 risk variants in the development of hypertension and hypertensive kidney disease. To understand the role of APOL1 in endothelial cells, we generated mice with endothelial‐specific inducible expression of APOL1 (EC/G2‐APOL1) by crossing the TRE‐G2APOL1 mice with the Cdh5tTA animals. Cadherin 5 (VE‐cadherin or endothelial cadherin) is an endothelial‐specific gene, removal of doxycycline from the diet led to a significant expression of APOL1 in different vascular beds such as the lung, heart, and kidney, which was confirmed both by in situ hybridization and qRT‐PCR. After collecting the baseline data, we performed UNX surgery on both WT control and Cdh5tTA/TRE‐G2APOL1 five‐week‐old mice and kept them in 4% salt diet for 12 weeks after UNX surgery. Systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean arterial pressure (MAP) (measured by tail‐cuff method) were significantly higher in Cdh5tTA/TRE‐G2APOL1 mice compared to control WT mice after 6 weeks of UNX surgery on a high salt diet. The significantly higher blood pressure in Cdh5tTA/TRE‐G2APOL1 mice was further associated with the increased urinary albumin/creatinine ratio and renal fibrosis. Interestingly, the blood pressure parameters SBP, DBP, and MAP were significantly reduced on STING, NLRP3, and gasdermin knock‐out mice on Cdh5tTA/TRE‐G2APOL1 background compared to Cdh5tTA/TRE‐G2APOL1 mice upon UNX‐surgery and 4% salt diet. Furthermore, the expression of the transcript of the mitochondrial gene including COX1, COX2, ATP6, and ND6 in the cytoplasm from kidney tissue obtained from Cdh5tTA/TRE‐G2APOL1 mice was significantly higher compared to control‐WT, which indicate that mitochondrial damage and leakage of the mitochondrial gene into the cytoplasm take place in the Cdh5tTA/TRE‐G2APOL1 mice upon UNX‐surgery and 4% salt‐diet. Therefore, EC specific apol1 gene induces mitochondrial damage, upregulates inflammasome and cGAS‐STING pathway to develop hypertension leading to hypertensive kidney disease. This project was supported by DK105821.

Roxadustat (FG-4592) protects against ischaemia/reperfusion-induced acute kidney injury through inhibiting the mitochondrial damage pathway in mice.

Ischaemia-reperfusion (I/R) is one of the main factors of acute kidney injury (AKI). mitochondrial damage pathway are important features of I/R induced-acute kidney injury (IRI-AKI). Hypoxia-inducible factor (HIF) expression in renal tubule segments is up-regulated during AKI. Herein, we investigated the role of FG-4592 in a mouse model of IRI-AKI to confirm whether FG-4592 is beneficial in AKI. We found that pretreatment with FG-4592significantly ameliorated renal function and renal histological damage in mice after IRI. Furthermore, these results suggest that pretreatment with FG-4592significantly reduced the tubular cells apoptosis (decreased TUNEL-positive cells, Bax, caspase12levels), attenuated mitochondrial damage (increased ATPβ, PPARγ, mitochondrial DNA copy number, and decreased cytoplasmic cytochrome C), and alleviated DNA damage after IRI. In conclusion, pretreatment with FG-4592may effectively prevent kidney from IRI possibly by via diminishing tubular cells injuries and protection of mitochondrial damage pathway. These results further validate that FG-4592may be an effective drug in the clinical treatment of IRI-AKI.

Nao-Fu-Cong ameliorates diabetic cognitive dysfunction by inhibition of JNK/CHOP/Bcl2-mediated apoptosis in vivo and in vitro

Chinese herbal compound Nao-Fu-Cong (NFC) has been mainly used to treat cognitive disorders in Traditional Chinese Medicine (TCM). The present study aimed to investigate whether its neuroprotective effects might be related to the inhibition of JNK/CHOP/Bcl2-mediated apoptosis pathway or not. We randomly assigned STZ (60 mg·kg-1)-induced diabetic rats into control group, diabetic model group and NFC groups (low-dose, medium-dose and high-dose). The primary culture of hippocampal neurons were transferred into different culture media on the third day. The cells were then divided into control group, high-glucose group, NFC (low-dose, medium-dose and high-dose) groups, CHOP si-RNA intervention group, JNK pathway inhibitor SP600125 group and oxidative stress inhibitor N-acetylcysteine (NAC) group. NFC significantly improved the cognitive function of diabetic rats, and had neuroprotective effect on hippocampal neurons cultured in high glucose. Further research results showed that NFC could reduce the apoptosis of hippocampal neurons in rats with diabetic cognitive dysfunction. NFC had inhibitory effects on CHOP/JNK apoptosis pathway induced by high glucose, and also decreased the levels of ROS and increased the mitochondrial membrane potential. These suggested that the neuroprotective effect of NFC might be related to the inhibition of CHOP and JNK apoptotic signaling pathways, and the cross pathway between oxidative stress and mitochondrial damage pathway.

2-Benzhydrylsulfinyl-N-hydroxyacetamide-Na extracted from fig as a novel cytotoxic and apoptosis inducer in SKOV-3 and AMJ-13 cell lines via P53 and caspase-8 pathway

In this study, the fruits of both mature and air-dried figs were exposed to selective sequential extracting processes using soxhlet. Different polarity and non-polarity solvents were used to increase the yield of the isolated extracts. Methanol, ethyl acetate, chloroform, and n-hexane were used to precipitate and isolate the effective compound 2-benzhydrylsulfinyl-n-hydroxyacetamide-Na from 11 extracts and 7 compounds extracted from the organic layer. Various spectral techniques were applied, including UV-spectroscopy, FT-IR, GC-Mass, 1H-NMR, and 13C-NMR for the detection of the precipitate. The aim of current work is to deal with the synthesis of a novel compound 2-benzhydrylsulfinyl-n-hydroxyacetamide-Na from fig fruit and study its effect as anticancer and anti-proliferative agent against SKOV-3, and AMJ-13 cells. The effect of the active compound on breast cancer cells, ovarian cancer cell proliferation was measured by the MTT assay, while its ability to induce apoptosis was detected using DAPI, acridine orange/ethidium bromide (AO/EtBr) staining, flow cytometry assay and finally mitochondrial membrane potential (MMP). Real-Time PCR was used to detect changes in the expression of mRNA for Bax and Bcl-2, P53, caspase-8, and caspase-9. Treated cancer cells with 2-benzhydrylsulfinyl-n-hydroxyacetamide-Na significantly increased ROS synthesis, with subsequent reduction of the MMP through mechanisms that included Bax upregulation P53, Caspase-8, and Caspase-9 and Bcl-2 downregulation. The outcomes of the current study show that 2-benzhydrylsulfinyl-n-hydroxyacetamide-Na extracted from fig fruit suppressed cancers cells’ proliferation, resulting in apoptosis as a novel pathway that involves mitochondrial mechanism via activated P53 and caspase-8 signaling. In addition, the extract exerted no toxic effects, neither on serum levels of liver functional enzymes nor on the normal histological architecture of the lungs and spleen. We demonstrated how the 2-benzhydrylsulfinyl-n-hydroxyacetamide-Na affects cancer cells along with a study the possible mechanisms involved in this effect which is mitochondrial damage and P53 pathway. We conclude that 2-benzhydrylsulfinyl-n-hydroxyacetamide-Na holds a promising potential as an anticancer therapeutic agent.

ZnO Nanoparticles Induced Caspase-Dependent Apoptosis in Gingival Squamous Cell Carcinoma through Mitochondrial Dysfunction and p70S6K Signaling Pathway

Zinc oxide nanoparticles (ZnO-NPs) are increasingly used in sunscreens, food additives, pigments, rubber manufacture, and electronic materials. Several studies have shown that ZnO-NPs inhibit cell growth and induce apoptosis by the production of oxidative stress in a variety of human cancer cells. However, the anti-cancer property and molecular mechanism of ZnO-NPs in human gingival squamous cell carcinoma (GSCC) are not fully understood. In this study, we found that ZnO-NPs induced growth inhibition of GSCC (Ca9-22 and OECM-1 cells), but no damage in human normal keratinocytes (HaCaT cells) and gingival fibroblasts (HGF-1 cells). ZnO-NPs caused apoptotic cell death of GSCC in a concentration-dependent manner by the quantitative assessment of oligonucleosomal DNA fragmentation. Flow cytometric analysis of cell cycle progression revealed that sub-G1 phase accumulation was dramatically induced by ZnO-NPs. In addition, ZnO-NPs increased the intracellular reactive oxygen species and specifically superoxide levels, and also decreased the mitochondrial membrane potential. ZnO-NPs further activated apoptotic cell death via the caspase cascades. Importantly, anti-oxidant and caspase inhibitor clearly prevented ZnO-NP-induced cell death, indicating the fact that superoxide-induced mitochondrial dysfunction is associated with the ZnO-NP-mediated caspase-dependent apoptosis in human GSCC. Moreover, ZnO-NPs significantly inhibited the phosphorylation of ribosomal protein S6 kinase (p70S6K kinase). In a corollary in vivo study, our results demonstrated that ZnO-NPs possessed an anti-cancer effect in a zebrafish xenograft model. Collectively, these results suggest that ZnO-NPs induce apoptosis through the mitochondrial oxidative damage and p70S6K signaling pathway in human GSCC. The present study may provide an experimental basis for ZnO-NPs to be considered as a promising novel anti-tumor agent for the treatment of gingival cancer.

Graphene nanoparticles induces apoptosis in MCF-7 cells through mitochondrial damage and NF-KB pathway

In the present study, the synthesis of reduced graphene oxide (rGO) was performed via 20 kHz frequency ultrasonic solution processing technique. Synthesis of rGo was confirmed by various techniques including color changes, UV–vis spectra, x-ray analysis, infrared spectrophotometry, scanning electron microscopy, and dynamic light-scattering. The cytotoxicity of rGO was examined against human breast cancer MCF-7 cells by measuring different parameters including MTT, suppression of NF-κB translocation, mitochondrial membrane potential (MMP), reactive oxygen species (ROS) production, acridine orange-ethidium bromide staining, and single cell gel electrophoresis. Also, the gene expressions of Bax and Bcl-2 proteins were measured using quantitative PCR analysis as well as florescence microscopy and indicated that rGO induces cell death using apoptosis as an exclusive mechanism. Our results shows for the first time that the rGO inhibited the proliferation of MCF-7 cells, leading to programmed cell death through activation of the mitochondrial-mediated signaling pathway with the involvement of the NF-kB signalling pathway. Taken together the present data suggest that rGO can be possibly employed in the treatment of breast cancer with the function of a potent synergistic agent added to anticancer therapy protocols.

Astragalus polysaccharides suppresses high glucose-induced metabolic memory in retinal pigment epithelial cells through inhibiting mitochondrial dysfunction-induced apoptosis by regulating miR-195

BackgroundMetabolic memory contributes to the development of diabetic retinopathy (DR), which is the complication of diabetes. But it’s still unknown how to prevent the metabolic memory to treat the DR. In our study, we want to examine the function of Astragalus polysaccharides (APS) in the metabolic memory of retinal pigment epithelium (RPE) pretreated with high glucose (HG).MethodsARPE-19 and PRPE cells were exposed to HG followed by normal glucose (NG) treatment with or without APS. QPCR was used to examine the levels of miR-195 and Bcl-2. MDA and SOD detection assays were used to examine the oxidative stress level. Western blotting and immunostaining were applied to detect the protein level of mitochondrial damage and apoptotic signaling pathway. Flow cytometry and TUNEL staining were used to analyze cell apoptosis. Luciferase assay was used to examine the direct target of miR-195.ResultsAPS treatment significantly decreased the expression of miR-195, while increased the expression of Bcl-2 with optimized dosages which were induced by HG treatment, even after replacing the HG with NG. And we found Bcl-2 was the direct target of miR-195. APS alleviated the oxidative stress, mitochondrial damage and cell apoptosis induced by HG and HG + NG treatments in RPE cells via regulating miR-195. Furthermore, we found overexpression of miR-195 abolished the alleviated effects of APS on the HG-treated RPE cells.ConclusionsAPS suppressed high glucose-induced metabolic memory in retinal pigment epithelial cells through inhibiting mitochondrial dysfunction-induced apoptosis by regulating miR-195.

Cytotoxicity of nucleus-targeting fluorescent gold nanoclusters.

Gold nanoclusters (AuNCs) with ultra small sizes and unique fluorescence properties have shown promising potential for imaging the nuclei of living cells. However, little is known regarding the potential cytotoxicity of AuNCs after they enter the cell nucleus. The aim of this study is to investigate whether and how nucleus-targeting AuNCs affect the normal functioning of cells. Highly stable, water-soluble and bright fluorescent Au25NCs (the core of each nanocluster is composed of 25 gold atoms) were synthesized. Specific targeting of Au25NCs to the cell nucleus was achieved by conjugating the TAT peptide to the Au25NCs. Cell viability, cell morphology, cell apoptosis/necrosis, reactive oxygen species (ROS) level and mitochondrial membrane potential examinations were performed on different cell lines exposed to the nucleus-targeting Au25NCs. We found that the nucleus-targeting Au25NCs caused cell apoptosis in a dose-dependent manner. A possible mechanism for the cytotoxicity of the nucleus-targeting Au25NCs was proposed as follows: the nucleus-targeting Au25NCs induce the production of ROS, resulting in the oxidative degradation of mitochondrial components, in turn leading to apoptosis via a mitochondrial damage pathway. This work facilitates a better understanding of the toxicity of AuNCs, especially nucleus-targeting AuNCs.

The mechanism of immunosuppression by perfluorooctanoic acid in BALB/c mice

Perfluorooctanoic acid (PFOA) has been demonstrated to decrease immunity of mice, but little is known about the mechanisms of its immunotoxicity. In order to determine whether the immunotoxicity of PFOA is associated with lipid metabolism, male BALB/c mice were fed with either a regular (RD) or high fat (HFD) diet, and exposed to PFOA at doses of 0, 5, 10, and 20 mg kg−1 per day for 14 days. Following exposure, the body weights of RD-fed mice treated with PFOA were significantly decreased, and the immune system organs showed serious atrophy. Histopathological and ultrastructural changes were also detected. At the same time, the gene expressions of peroxisome proliferator-activated receptor (PPAR) α and γ were also up-regulated in the thymus and the spleen. The percentage of apoptotic cells increased with increasing doses of PFOA, and a larger number of lymphocytes underwent apoptosis within the thymus than the spleen. In the HFD exposure groups, similar phenomena were still observed. HFD feeding caused up-regulation of PPARγ but not PPARα within the thymus of PFOA groups. These results suggest that an excess of dietary lipids does not prevent PFOA-induced immune suppression caused by peroxisome proliferators, and immunomodulation by PFOA is via the PPAR pathway, and the induction of mitochondrial damage and lymphocyte apoptosis pathway.

Suppression of urinary bladder urothelial carcinoma cell by the ethanol extract of pomegranate fruit through cell cycle arrest and apoptosis

BackgroundPomegranate possesses many medicinal properties such as antioxidant, anti-inflammation and antitumor. It has been extensively used as a folk medicine by many cultures. Pomegranate fruit has been shown to have the inhibitory efficacy against prostate cancer and lung cancer in vitro and in vivo. It can be exploited in chemoprevention and chemotherapy of prostate cancer. In this study we examined the anti-cancer efficacy of pomegranate fruit grown in Taiwan against urinary bladder urothelial carcinoma (UBUC) and its mechanism of action.MethodsEdible portion of Taiwanese pomegranate was extracted using ethanol and the anti-cancer effectiveness of ethanol extract was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Flow cytometry and western immunoblotting were exploited to uncover the molecular pathways underlying anti-UBUC activity of Taiwanese pomegranate ethanol extract.ResultsThis study demonstrated that Taiwanese pomegranate fruit ethanol extract (PEE) could effectively restrict the proliferation of UBUC T24 and J82 cells. Cell cycle analyses indicated that the S phase arrest induced by PEE treatment might be caused by an increase in cyclin A protein level and a decrease in the expression of cyclin-dependent kinase 1. The results of western immunoblotting demonstrated that PEE treatment could not only evoke the activation of pro-caspase-3, -8,-9 but also increase Bax/Bcl-2 ratio in T24 cells. The above observations implicated that PEE administration might trigger the apoptosis in T24 cells through death receptor signaling and mitochondrial damage pathway. Besides we found that PEE exposure to T24 cells could provoke intensive activation of procaspase-12 and enhance the expressions of CHOP and Bip, endoplasmic reticulum (ER) stress marker, suggesting that ER stress might be the cardinal apoptotic mechanism of PEE-induced inhibition of bladder cancer cell.ConclusionsThe analytical results of this study help to provide insight into the molecular mechanism of induced bladder cancer cell apoptosis by pomegranate and to develop novel mechanism-based chemopreventive strategy for bladder cancer.

Protective Effect of Melatonin on Methamphetamine-Induced Apoptosis in Glioma Cell Line

Methamphetamine (METH) is a highly addictive drug causing neurodegenerative diseases. METH has been known to be neurotoxic by inducing oxidative stress, free radical, and pro-inflammatory cytokines. Previous studies have shown that METH could induce neuron and glial cell death, especially inducing glial cell-mediated neurotoxicity that plays a critical role in stress-induced central nervous system damage. Therefore, the aim of the present study is to explore the mechanisms of METH-induced cell death in the glial cell. METH-induced glial cells death is mediated via mitochondrial damage pathway. METH activates the upregulation of the Bax, cytochrome c, cleavage caspase 9 and 3 proteins, and downregulation of Bcl-XL protein in cascade. Pretreatment with melatonin, a neurohormone secreted by the pineal gland, effectively reduced glial cell death. Moreover, melatonin increased the Bcl-XL/Bax ratio but reduced the level of cytochrome c, cleavage caspase 9 and 3 proteins. Therefore, these results demonstrated that melatonin could reduce the cytotoxic effect of METH by decreasing the mitochondrial death pathway activation in glial cells. This outcome suggests that melatonin might be beneficial as the neuroprotection in neurodegenerative diseases caused by METH or other pathogens.

DNA Damaging Drugs in the Treatment of Glioblastoma: HR, Apoptosis, Autophagy and Senescence

Alkylating agents, notably temozolomide, are 1st line therapeutics for the treatment of glioma. These agents induce different DNA lesions. One of the DNA adducts is O6-methylguanine (O6MeG), which is a powerful cytotoxic lesion. The damage is repaired by the suicide enzyme alkyltransferase (MGMT), which is a very important defense mechanism and marker of alkylating drug resistance. We have studied in detail how apoptosis is induced following O6MeG in glioma cells. We have shown that O6MeG triggered cell death is executed via the death receptor and the mitochondrial damage pathway, involving DNA double-strand breaks (DSB). The major pathway of repairing O6MeG induced DSB is homologous recombination (HR). Therefore, players involved in DSB recognition and HR are potential targets for therapy, such as NBS-1, ATM, Rad51 and XRCC2. In glioma cells, the efficiency of O6MeG to trigger the p53 dependent death receptor pathway is higher than the p53 independent mitochondrial pathway, which explains why p53 wt glioma cells are more sensitive to temozolomide than p53 mutated cells. Interestingly, p53 wt glioma cells are more resistant than p53 mutant glioma cells to chloroethylating agents (CCNU, ACNU), which are also applied in glioma therapy. This indicates that p53 has a dual role: one is up-regulation of the death receptor thus sensitizing to methylating agents, the other is upregulation of DNA repair thus protecting against O6chloroethylguanine-induced apoptosis (and necrosis). Data will be shown providing evidence that O6MeG induces not only apoptosis, but also autophagy and senescence. The interrelationship of these endpoints will be discussed. Work was supported by DFG KA724 and Deutsche Krebshilfe.

Proteomic exploration of the impacts of pomegranate fruit juice on the global gene expression of prostate cancer cell

Prostate cancer has been known to be the second highest cause of death in cancer among men. Pomegranate is rich in polyphenols with the potent antioxidant activity and inhibits cell proliferation, invasion, and promotes apoptosis in various cancer cells. This study demonstrated that pomegranate fruit juice could effectively hinder the proliferation of human prostate cancer DU145 cell. The results of apoptotic analyses implicated that fruit juice might trigger the apoptosis in DU145 cells via death receptor signaling and mitochondrial damage pathway. In this study, we exploited 2DE-based proteomics to compare nine pairs of the proteome maps collected from untreated and treated DU145 cells to identify the differentially expressed proteins. Comparative proteomics indicated that 11 proteins were deregulated in affected DU145 cells with three upregulated and eight downregulated proteins. These dys-regulated proteins participated in cytoskeletal functions, antiapoptosis, proteasome activity, NF-κB signaling, cancer cell proliferation, invasion, and angiogenesis. Western immunoblotting were implemented to confirm the deregulated proteins and the downstream signaling proteins. The analytical results of this study help to provide insight into the molecular mechanism of inducing prostate cancer cell apoptosis by pomegranate fruit juice and to develop a novel mechanism-based chemopreventive strategy for prostate cancer.

CR‐108, a novel vitamin K derivative induces apoptosis through ROS and mitochondrial dysfunction in breast cancer

Vitamin K derivatives exert anticancer activities. Here, we found a novel vitamin K derivative, CR‐108, which induced apoptosis in human breast cancer cells. CR‐108 reduced the cell viability in both the non‐HER‐2‐overexpressed MCF‐7 and HER‐2‐ overexpressed BT‐474 breast cancer cells. Interestingly, CR‐108 highly elicited the intracellular reactive oxygen species (ROS) in breast cancer cells. CR‐108 markedly induced the loss of mitochondrial membrane potential analyzed by fluorescence intensities of JC‐1 and DiOC6. Moreover, CR‐108 induced cytochrome c release from mitochondria to cytosol, elevated the levels of cleaved PARP proteins, and increased apoptotic induction. A ROS scavenger NAC completely blocked the CR‐ 108‐induced ROS, mitochondrial damage, and apoptosis. Meantime, CR‐108 increased the phosphorylated p38 kinase but conversely inhibited the survivin protein expression. NAC prevented the activation of p38 kinase and restored the survivin protein levels. In addition, a specific p38 kinase inhibitor SB202190 reduced the survivin protein level and attenuated the CR‐108‐induced cytotoxicity. Taken together, CR‐108 induces apoptosis through ROS and mitochondrial damage pathway associated with p38 kinase and survivin in the human breast cancer.