N-acetylcysteine counteracts oxidative stress and prevents hCG-induced apoptosis in rat Leydig cells through down regulation of caspase-8 and JNK

Abstract Multiple Myeloma (MM) cells are extremely resistant to apoptosis and currently new potential drug combinations are under investigation. We have shown that the combined treatment with the MEK1/2 inhibitor PD184352 (PD) and Arsenic Trioxide (ATO) resulted in the synergistic (Combination Index <1.0) induction of apoptosis in 7 human myeloma cell lines (HMCLs: XG1, XG6, OPM2, JJN3, RPMI, H929, Sultan) analyzed, irrespective of their p53 status. The combined treatment was also a highly potent inducer of apoptosis and mitochondrial damage in the majority of the primary multiple myeloma (MM) cell samples ex vivo analyzed at different disease stage (9 out of 12). Growth factors, IL-6 or insulin-like growth factor 1 (IGF-1), or a co-culture system with bone marrow stromal cells (BMSCs) failed to confer resistance to this combination regimen. The combination PD/ATO had a minimal effect on normal B cells in vitro. By investigating the molecular mechanisms involved in MM cells PD/ATO-induced apoptosis, we found that co-treatment with PD strikingly elevated the (DR4+DR5)/(DcR1+DcR2) tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) receptors ratio, caspase-8 activation, Bid fragmentation, mitochondrial depolarization and caspase-9 activation of ATO-treated HMCLs that do not have a functional p53 pathway. In HMCLs carrying a functional p53 pathway, the treatment with PD greatly enhanced the ATO-induced p53 accumulation (two fold increase) and p73, a p53 paralogue, cooperated with p53 in the pro-apoptotic p53/p73 target genes up regulation, caspase-9, -3 activation and apoptosis induction; in these HMCLs the selective down-regulation of p53 or p73 demonstrated that both have a biological relevance in PD/ATO-induced caspase-3 activation, PARP fragmentation and apoptosis. In HMCLs carrying a functional p53 the extrinsic caspase-8 mediated pathway was partially activated by PD/ATO treatment. We also demonstrated that, in MM cells carrying or not a functional p53 pathway, the combined treatment PD/ATO increased the level of the pro-apoptotic Bim (PD-mediated) and decreased its neutralizing anti-apoptotic protein Mcl-1 (ATO-mediated). The selective down-regulation of Bim significantly diminished caspase-8/-9/-3 cleavage/activation, PARP fragmentation and apoptosis of PD/ATO-treated MM cells, thereby indicating that Bim can play an important role not only in the intrinsic mitochondrial programmed cell death but also in the extrinsic caspase-8 mediated pathway. Accordingly, a physical interaction between Bim and DR4/DR5 TRAIL receptors in PD/ATO-treated MM cells carrying a non functional p53 was found by coimmunoprecipitation and Western blot studies. Our experiments have enlightened some relevant mechanisms that explain the apoptotic response of myeloma cells to ATO plus MEK inhibitor combination.

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MON-027 Activation of Endoplasmic Reticulum Stress Mediates Oxidative Stress-Induced Apoptosis of Granulosa Cells in Ovaries Affected by Endometrioma

Journal of the Endocrine Society ◽

10.1210/jendso/bvaa046.346 ◽

2020 ◽

Vol 4 (Supplement_1) ◽

Author(s):

Chisato Kunitomi ◽

Miyuki Harada ◽

Jerilee Mariam Khong Azahry

Keyword(s):

Oxidative Stress ◽

Er Stress ◽

Granulosa Cells ◽

Caspase 3 ◽

Caspase 8 ◽

Ovarian Dysfunction ◽

High Oxidative Stress ◽

Induced Apoptosis ◽

Sensor Proteins ◽

Apoptotic Factors

Abstract Endometriosis exerts detrimental effects on ovarian physiology and compromises follicular health. Granulosa cells of endometriosis patients are characterized by increased apoptosis, as well as high oxidative stress. Among several pathophysiologic factors associated with endometriosis, it is expected that oxidative stress contributes to the induction of apoptosis in granulosa cells, although the underlying mechanism remains unclear. Endoplasmic reticulum (ER) stress, a local factor closely associated with oxidative stress, has emerged as a critical regulator of ovarian function. We hypothesized that ER stress is activated by high oxidative stress in granulosa cells in ovaries with endometrioma and mediates oxidative stress-induced apoptosis. Ovaries from patients with endometrioma and control were collected to determine apoptosis, oxidative stress and ER stress by TUNEL, immunohistochemical staining of 8-OHdG and ER stress sensors, respectively. Human granulosa-lutein cells (GLCs) obtained from IVF patients were cultured with H2O2 (an oxidative stress inducer) or tauroursodeoxycholic acid (TUDCA, an ER stress inhibitor in clinical use) to assess apoptosis and ER stress by quantitative PCR and FACS. Activity of pro-apoptotic factors was determined by caspase-8 activity assay and western blotting for cleaved caspase-3. Human GLCs from patients with endometrioma expressed up to two times higher level of mRNAs associated with the unfolded protein response (UPR), including ATF4, ATF6, the spliced form of XBP1, HSPA5, and CHOP. In addition, the levels of phosphorylated ER stress sensor proteins, IRE1 and PERK, were elevated. Given that ER stress results in phosphorylation of ER stress sensor proteins and induces UPR factors, these findings indicate that these cells were under ER stress. H2O2 increased expression of UPR-associated mRNAs in cultured human GLCs, and this effect was abrogated by pre-treatment with TUDCA. Treatment with H2O2 increased apoptosis and the activity of pro-apoptotic factors caspase-8 and caspase-3, both of which were attenuated by TUDCA. Our findings suggest that activated ER stress induced by high oxidative stress in granulosa cells in ovaries with endometrioma mediates apoptosis of these cells, leading to ovarian dysfunction in endometriosis patients. Targeting ER stress with currently clinically available ER stress inhibitors, or with these agents in combination with antioxidants, may serve as a novel strategy for rescuing endometriosis-associated ovarian dysfunction.

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The Role of Peroxiredoxin III and Sulfiredoxin for Mitochondrial ROS Production to Arsenic Trioxide in Acute Promyelocytic Leukemia Cells

Blood ◽

10.1182/blood.v124.21.5217.5217 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5217-5217

Author(s):

Yeung-Chul Mun ◽

Jee-Young Ahn ◽

Eun-Sun Yoo ◽

Kyoung Min Cho ◽

Kyoung Eun Lee ◽

...

Keyword(s):

Oxidative Stress ◽

Arsenic Trioxide ◽

Acute Promyelocytic Leukemia ◽

Caspase 3 ◽

Promyelocytic Leukemia ◽

Ros Generation ◽

Down Regulation ◽

Nb4 Cells ◽

Mitochondrial Ros ◽

Induced Apoptosis

Abstract Backgrounds: The Arsenic trioxide (ATO) is an effective cancer therapeutic drug for acute promyelocytic leukemia (APL), but in some cases, APL cells are resistant to ATO treatment. ATO exerts its effect mainly raising oxidative stress. However, not only the mechanisms of reactive oxygen species (ROS) generation by ATO but involvement of redox enzymes including peroxiredoxin (PRX) during ATO-induced apoptosis and its resistance remain elusive. Recently, Rhee et al had reported that PRX III and sufiredoxin together protect mice from pyrazole-induced oxidative liver injury was found (Antioxid & Redox Signal, 2012:17:1351-1361). Aims of current study are to elucidate that the changes of redox enzyme could be a mechanism of anti-leukemia effect in APL-derived NB4 cells during ATO treatment and to find ways to potentiate the anti-leukemic effects of ATO on APL cells. Methods: NB4, one of the human acute promyelocytic leukemia cell lines, was treated with 0~10 μM arsenic trioxide to induce apoptosis for 16-48 hours in RPMI-1640 medium supplemented with 10% FBS in CO2humidified atmosphere at 37°C. Apoptosis was measured by staining with 7-amino-actinomycin D (7-AAD) with flow cytometry. 2, 7-dichlrodihydro-fluorescein-diacetate (H2DCF-DA) and MitoSOX Red was used to detect cellular and mitochondrial ROS. SO2 form for PRX I, PRX II, and PRX III was detected by western blot assay using PRX SO2 form-specific antibody. Sulfiredoxin (SRX) and caspase 3, 9 were also detected by western blot analysis. To evaluate the effect of SRX depletion, NB4 cells were transfected with small interfering RNA (siRNA). Results: Intracellular ROS of NB4 cells was increased significantly after 16 hour of ATO treatment but decreased after 24 hour of ATO treatment. Mitochondrial ROS of NB4 cells was increased significantly after 39 hour of ATO treatment. Apoptosis of NB4 cell after ATO treatment was increased as time elapsed (24% on 16hr, 26% on 24hr, 48% on 39hr, and 60% on 48hr). Increased cysteine sulfinic acid (Cys–SO2H) PRX III, inactive and oxidized form, was observed as a hyperoxidation reaction in NB4 cells after ATO treatment in concordance with mitochondrial ROS increment of NB4 cells. Increased expressions of cleaved caspase-9 and cleaved caspase-3 were also observed during NB4 cell apoptosis by ATO treatment. Meanwhile, SRX expression was increased in NB4 cells after ATO treatment. Down regulation of SRX by siRNA promoted ROS generation and apoptosis in ATO-treated NB4 cells. Conclusions: Our data showed inactivation of PRX III by Cys–SO2H formation as hyperoxidation is developed during ATO-induced mitochondrial ROS generation and apoptosis process in APL cells. In addition, ATO promotes expression of SRX, which is known as reducing enzyme of Cys–SO2H PRX and which leads to down regulation of ROS accumulation in APL cells. These findings might be due to protective effect of SRX from ATO on mitochondrial oxidative stress. These findings suggest ATO-induced anti-leukemic activity could be down regulated by an enhancing PRX III reduction after ATO-induced SRX activation. Currently, the effect of down regulation of SRX by siRNA are being investigated to amplify the apoptosis in ATO-treated NB4 cells. Our study may provide the insights for finding novel targets in the development of new therapies, which potentiate ATO-induced apoptosis in APL cells. Disclosures No relevant conflicts of interest to declare.

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Role of Autophagy in Zinc Oxide Nanoparticles-Induced Apoptosis of Mouse LEYDIG Cells

International Journal of Molecular Sciences ◽

10.3390/ijms20164042 ◽

2019 ◽

Vol 20 (16) ◽

pp. 4042 ◽

Cited By ~ 8

Author(s):

Jingcao Shen ◽

Dan Yang ◽

Xingfan Zhou ◽

Yuqian Wang ◽

Shichuan Tang ◽

...

Keyword(s):

Oxidative Stress ◽

Zinc Oxide ◽

Zinc Oxide Nanoparticles ◽

Leydig Cells ◽

Oxide Nanoparticles ◽

Zno Nps ◽

Sperm Density ◽

Induced Apoptosis

Zinc oxide nanoparticles (ZnO NPs) have shown adverse health impact on the human male reproductive system, with evidence of inducing apoptosis. However, whether or not ZnO NPs could promote autophagy, and the possible role of autophagy in the progress of apoptosis, remain unclear. In the current study, in vitro and in vivo toxicological responses of ZnO NPs were explored by using a mouse model and mouse Leydig cell line. It was found that intragastrical exposure of ZnO NPs to mice for 28 days at the concentrations of 100, 200, and 400 mg/kg/day disrupted the seminiferous epithelium of the testis and decreased the sperm density in the epididymis. Furthermore, serum testosterone levels were markedly reduced. The induction of apoptosis and autophagy in the testis tissues was disclosed by up-regulating the protein levels of cleaved Caspase-8, cleaved Caspase-3, Bax, LC3-II, Atg 5, and Beclin 1, accompanied by down-regulation of Bcl 2. In vitro tests showed that ZnO NPs could induce apoptosis and autophagy with the generation of oxidative stress. Specific inhibition of autophagy pathway significantly decreased the cell viability and up-regulated the apoptosis level in mouse Leydig TM3 cells. In summary, ZnO NPs can induce apoptosis and autophagy via oxidative stress, and autophagy might play a protective role in ZnO NPs-induced apoptosis of mouse Leydig cells.

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