Knockdown of thioredoxin interacting protein attenuates high glucose-induced apoptosis and activation of ASK1 in mouse mesangial cells

Mesangial cell apoptosis contributes to the pathogenesis of diabetic nephropathy. Here we show that thioredoxin interacting protein (TXNIP) is involved in high glucose (HG)-induced mouse mesangial cell (MMC) apoptosis. HG induced activation of apoptosis signal regulating kinase-1 (ASK1) in a time-dependent manner in MMCs. Treatment with antioxidant, tempol, or knockdown of TXNIP in MMCs reduced HG-mediated apoptosis, expression of cleaved caspase-3, Bax/Bcl-2 ratio and activation of ASK1. These data suggest that knockdown of TXNIP prevented HG-induced cell apoptosis and activation of ASK1 may be via reduction of oxidative stress in MMCs.     

高浓度葡萄糖通过p38MAPK-TXNIP/TRX-ROS通路抑制MC3T3-E1细胞成骨分化

目的：研究高浓度葡萄糖抑制MC3T3-E1细胞成骨分化的机理。方法：建立MC3T3-E1细胞成骨分化诱导体系,观察不同浓度葡萄糖（5.5mM和22mM）对MC3T3-E1细胞成骨分化的影响;用不同浓度的p38 MAPK抑制剂Fr167653（0.1μM、1.0μM和10μM）进行药物干预,观察MC3T3-E1细胞在22mM葡萄糖浓度下成骨分化的变化情况。通过钙含量检测、Real time PCR检测相关分化的变化;用Western Blot方法检测MC3T3-E1细胞分化过程中p38 MAPK磷酸化状态、TXNIP表达水平的变化;使用胰岛素二硫键还原法检测细胞内TRX活性水平;使用活性氧检测试剂盒检测细胞内自由氧生成水平。结果：体外诱导条件下,高浓度（22mM）葡萄糖通过升高p38 MAPK磷酸化水平,上调TXNIP表达水平,同时降低TRX活性,使细胞内自由氧生成增加,抑制MC3T3-E1细胞的成骨分化;Fr167653通过抑制p38 MAPK磷酸化,下调TXNIP表达同时升高TRX活性,抑制细胞内自由氧生成,解除高浓度葡萄糖对细胞成骨分化的抑制作用。结论：高浓度葡萄糖通过p38 MAPK-TXNIP/TRX-ROS信号通路抑制MC3T3-E1细胞成骨分化。     

Vitamin D receptor activation in a diabetic-like environment: potential role in the activity of the endothelial pro-inflammatory and thioredoxin pathways

High blood and tissue concentrations of glucose and advanced glycation end products (AGEs) are thought to play an important role in the development of diabetic vascular complications. Thioredoxin interacting protein (TXNIP) is up-regulated in response to high levels of glucose and is an endogenous inhibitor of thioredoxin (TRX), and may play a contributory role in the occurrence of diabetic-related vascular diseases. Vitamin D inhibits endothelial proliferation and is a cardiovascular protective agent. The present study evaluated the impact of paricalcitol and calcitriol on the endothelial inflammatory and TXNIP pathways in cultured endothelial cells exposed to a diabetic-like environment. Fresh human umbilical vein cord endothelial cells (HUVEC) were treated for 24h with 200 μg/ml AGE-HSA and 250 mg/dl glucose concentrations, with paricalcitol or calcitriol. IL6, IL8, NFκB (p50/p65), receptor of AGE (RAGE), TXNIP, and TRX expressions were evaluated at the levels of mRNA, protein, and TRX activity. Calcitriol and paricalcitol significantly down-regulated the markers involved in the inflammatory responses. Only paricalcitol induced a significant decrease in TXNIP mRNA and protein expressions. Neither paricalcitol nor calcitriol affected TRX reductase activity or TRX mRNA and protein expressions. Our findings indicate that in an endothelial diabetic-like environment, paricalcitol and calcitriol significantly decreased the expression of genes involved in the inflammatory pathway. In this in vitro study, it seems that the TRX antioxidant system was not involved. The different effects found between paricalcitol and calcitriol might reflect the selectivity of vitamin D receptor (VDR) activation.     

Lentinan exerts synergistic apoptotic effects with paclitaxel in A549 cells via activating ROS‐TXNIP‐NLRP3 inflammasome

Paclitaxel is generally used to treat cancers in clinic as an inhibitor of cell division. However, the acquired resistance in tumours limits its clinical efficacy. Therefore, the aim of this study was to detect whether co‐treatment with lentinan enhanced the anti‐cancer effects of paclitaxel in A549 cells. We found that the combination of paclitaxel and lentinan resulted in a significantly stronger inhibition on A549 cell proliferation than paclitaxel treatment alone. Co‐treatment with paclitaxel and lentinan enhanced cell apoptosis rate by inducing caspase‐3 activation. Furthermore, co‐treatment with paclitaxel and lentinan significantly triggered reactive oxygen species (ROS) production, and increased thioredoxin‐interacting protein (TXNIP) expression. Moreover, co‐treatment with paclitaxel and lentinan enhanced TXNIP‐NLRP3 interaction, and activated NLRP3 inflammasome whereat interleukin‐1β levels were increased and cell apoptosis was induced. In addition, combination of paclitaxel and lentinan could activate apoptosis signal regulating kinase‐1 (ASK1)/p38 mitogen‐activated protein kinase (MAPK) signal which also contributed to cell apoptosis. Taken together, co‐treatment with paclitaxel and lentinan exerts synergistic apoptotic effects in A549 cells through inducing ROS production, and activating NLRP3 inflammasome and ASK1/p38 MAPK signal pathway.     

Thioredoxin-interacting protein deficiency alleviates phenotypic alterations of podocytes via inhibition of mTOR activation in diabetic nephropathy

Thioredoxin-interacting protein (TXNIP) is induced by high glucose (HG), whereupon it acts to inhibit thioredoxin, thereby promoting oxidative stress. We have found that TXNIP knockdown in human renal tubular cells helped prevent the epithelial-to-mesenchymal transition (EMT). Here, we studied the potential effect of TXNIP on podocyte phenotypic alterations in diabetic nephropathy (DN) in vivo and in vitro. In conditionally immortalized mouse podocytes under HG conditions, knocking down TXNIP disrupted EMT, reactive oxygen species (ROS) production, and mammalian target of rapamycin (mTOR) pathway activation. Further, Raptor short hairpin RNA (shRNA), Rictor shRNA, and mTOR specific inhibitor KU-0063794 were used to assess if the mTOR signal pathway is involved in HG-induced EMT in podocytes. We found that Raptor shRNA, Rictor shRNA, and KU-0063794 could all restrain HG-induced EMT and ROS production in podocytes. In addition, antioxidant Tempol or N-acetylcysteine presented a prohibitive effect on HG-induced EMT in podocytes. Streptozotocin was utilized to render equally diabetic in wild-type (WT) control and TXNIP ^−/− (TKO) mice. Diabetes did not increase levels of 24-hr urinary protein, serum creatinine, blood urea nitrogen, and triglyceride in TXNIP ^−/− mice. Podocyte phenotypic alterations and podocyte loss were detected in WT but not in TKO diabetic mice. Oxidative stress was also suppressed in diabetic TKO mice relative to WT controls. Also, TXNIP deficiency suppresses the activation of mTOR in glomeruli of streptozotocin-induced diabetic mice. Moreover, TXNIP expression, mTOR activation, Nox1, and Nox4 could be detected in renal biopsy tissues of patients with DN. This suggests that decreased TXNIP could ameliorate phenotypic alterations of podocytes via inhibition of mTOR in DN, highlighting TXNIP as a promising therapeutic target.     

Thioredoxin interacting protein (TXNIP) induces inflammation through chromatin modification in retinal capillary endothelial cells under diabetic conditions

Chronic hyperglycemia and activation of receptor for advanced glycation end products (RAGE) are known risk factors for microvascular disease development in diabetic retinopathy. Thioredoxin‐interacting protein (TXNIP), an endogenous inhibitor of antioxidant thioredoxin (TRX), plays a causative role in diabetes and its vascular complications. Herein we investigate whether HG and RAGE induce inflammation in rat retinal endothelial cells (EC) under diabetic conditions in culture through TXNIP activation and whether epigenetic mechanisms play a role in inflammatory gene expression. We show that RAGE activation by its ligand S100B or HG treatment of retinal EC induces the expression of TXNIP and inflammatory genes such as Cox2, VEGF‐A, and ICAM1. TXNIP silencing by siRNA impedes RAGE and HG effects while stable over‐expression of a cDNA for human TXNIP in EC elevates inflammation. p38 MAPK‐NF‐κB signaling pathway and histone H3 lysine (K) nine modifications are involved in TXNIP‐induced inflammation. Chromatin immunoprecipitation (ChIP) assays reveal that TXNIP over‐expression in EC abolishes H3K9 tri‐methylation, a marker for gene inactivation, and increases H3K9 acetylation, an indicator of gene induction, at proximal Cox2 promoter bearing the NF‐κB‐binding site. These findings have important implications toward understanding the molecular mechanisms of ocular inflammation and endothelial dysfunction in diabetic retinopathy. J. Cell. Physiol. 221: 262–272, 2009. © 2009 Wiley‐Liss, Inc     

Apollon/Bruce is upregulated by Humanin

Hydrogen sulfide (H₂S) protects cardiomyoblasts against high glucose (HG)-induced injury by inhibiting the activation of p38 mitogen-activated protein kinase (MAPK). This study aims to determine whether the leptin–p38 MAPK pathway is involved in HG-induced injury and whether exogenous H₂S prevents the HG-induced insult through inhibition of the leptin–p38 MAPK pathway in H9c2 cells. H9c2 cells were treated with 35 mM glucose (HG) for 24 h to establish a HG-induced cardiomyocyte injury model. Cell viability; mitochondrial membrane potential (ΔΨ _m); apoptosis; reactive oxygen species (ROS) level; and leptin, leptin receptor, and p38 MAPK expression level were measured by the methods indicated. The results showed pretreatment of H9c2 cells with NaHS before exposure to HG led to an increase in cell viability, decrease in apoptotic cells, ROS generation, and a loss of ΔΨ _m. Exposure of H9c2 cells to 35 mM glucose for 24 h significantly upregulated the expression levels of leptin and leptin receptors. The increased expression levels of leptin and leptin receptors were markedly attenuated by pretreatment with 400 μM NaHS. In addition, the HG-induced increase in phosphorylated (p) p38 MAPK expression was ameliorated by pretreatment with 50 ng/ml leptin antagonist. In conclusion, the present study has demonstrated for the first time that the leptin–p38 MAPK pathway contributes to the HG-induced injury in H9c2 cells and that exogenous H₂S protects H9c2 cells against HG-induced injury at least in part by inhibiting the activation of leptin–p38 MAPK pathway.     

Involvement of vesicle-cytoskeleton interaction in AQP5 trafficking in AQP5-gene-transfected HSG cells

We examined the regulatory role of a reduction/oxidation (redox) control protein, thioredoxin (TRX), in tumor necrosis factor-alpha (TNF-alpha)-induced p38 MAP kinase activation and p38 MAP kinase-mediated cytokine expression utilizing TRX-transfected murine L929 cells (TRX14). The results showed that TNF-alpha-induced p38 MAP kinase activation and interleukin-6 (IL-6) production by TRX 14 were less than those by the parental L cells and the control transfected L cells (Neo-1). SB 203580 as the specific inhibitor for p38 MAP kinase activity inhibited TNF-alpha-induced IL-6 production by the parental L cells, indicating that TNF-alpha-activated p38 MAP kinase regulates IL-6 production by the cell lines used in this study. These results showed that overexpression of TRX negatively regulates p38 MAP kinase activation and p38 MAP kinase-mediated IL-6 production by TNF-alpha-stimulated cells, indicating that TRX is critical for p38 MAP kinase activation which regulates cytokine expression.     

Knockdown of thioredoxin-interacting protein ameliorates high glucose-induced epithelial to mesenchymal transition in renal tubular epithelial cells

Epithelial to mesenchymal transition (EMT) of tubular cells contributes to the renal accumulation of matrix protein that is associated with diabetic nephropathy. Both high glucose and transforming growth factor-β (TGF-β) are able to induce EMT in cell culture. In this study, we examined the role of the thioredoxin-interacting protein (TXNIP) on EMT induced by high glucose or TGF-β1 in HK-2 cells. EMT was assessed by the expression of α-smooth muscle actin (α-SMA) and E-cadherin and the induction of a myofibroblastic phenotype. High glucose (30 mM) was shown to induce EMT at 72 h. This was blocked by knockdown of TXNIP or antioxidant NAC. Meanwhile, we also found that knockdown of TXNIP or antioxidant NAC inhibited high glucose-induced generation of reactive oxygen species (ROS), phosphorylation of p38 MAPK and ERK1/2 and expression of TGF-β1. HK-2 cells that were exposed to TGF-β1 (4 ng/ml) also underwent EMT. The expression of TXNIP gene and protein was increased in HK-2 cells treated with TGF-β1. Transfection with TXNIP shRNA was able to attenuate TGF-β1 induced-EMT. These results suggested that knockdown of TXNIP antagonized high glucose-induced EMT by inhibiting ROS production, activation of p38 MAPK and ERK1/2, and expression of TGF-β1, highlighting TXNIP as a potential therapy target for diabetic nephropathy.     

Flavonoids inhibit high glucose-induced up-regulation of ICAM-1 via the p38 MAPK pathway in human vein endothelial cells

Recently, several flavonoids have been shown to have cardioprotective, cancer preventive, or anti-inflammatory properties. However, the specific mechanisms underlying their protective effects remain unclear. We aimed to investigate the different effects of three representative flavonoids—hesperidin, naringin, and resveratrol—on intracellular adhesion molecule-1 (ICAM-1) induction in human umbilical vein endothelial cells (HUVECs) by using high-glucose (HG) concentrations and the possible underlying molecular mechanisms. In HG-induced HUVEC cultures, the effects of three different flavonoids on ICAM-1 production and p38 phosphorylation were examined in the presence or absence of inhibitors targeting the mitogen-activated protein kinase (MAPK) signal transduction pathway. HG stimulation of HUVECs increased the levels of the adhesion molecules ICAM-1 and endothelial selectin (E-selectin). Pretreatment with all the three flavonoids drastically inhibited ICAM-1 expression in a time-dependent manner, but did not alter VCAM-1 and E-selectin expressions. Moreover, we investigated the effects of flavonoids on the MAPK signal transduction pathway, because MAPK families are associated with vascular inflammation under stress. These flavonoids did not block HG-induced phosphorylation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), but completely inhibited the HG-induced phosphorylation of p38 MAPK. SB202190, an inhibitor of p38 MAPK, also inhibited the HG-induced enrichment of ICAM-1. This study demonstrated that hesperidin, naringin, and resveratrol reduced the HG-induced ICAM-1 expression via the p38 MAPK signaling pathway, contributing to the inhibition of monocyte adhesion to endothelial cells.     

硫氧还蛋白相互作用蛋白在糖尿病及其并发症中的作用

硫氧还蛋白相互作用蛋白(thioredoxin-interacting protein,TXNIP)又称维生素D₃上调蛋白1,因其能够与硫氧还蛋白(thioredoxin,Trx)结合并抑制其活性和表达而得名。本文概述了TXNIP的发现与结构,及其自身通过发挥调节糖脂代谢的作用进而影响糖尿病前期的发生发展。并在此基础上总结了TXNIP参与糖尿病发生发展的2条主要途径:TXNIP通过拮抗Trx的抗凋亡作用来激发细胞凋亡信号导致胰岛细胞凋亡;TXNIP过表达促使胰岛细胞磷酸化,进而使抑癌相关蛋白质表达增加,最终引起胰岛细胞衰老。进一步重点阐述了TXNIP在糖尿病心肌病、糖尿病肾病、糖尿病性视网膜病等糖尿病并发症中的作用:TXNIP能通过各种间接途径干预信号通路,进一步参与氧化应激、细胞凋亡、激活炎症、细胞自噬及糖脂代谢等生理生化过程。TXNIP具有极其重要的生物学功能,深入了解TXNIP在糖尿病及其并发症中的影响机制,对糖尿病及其并发症的治疗具有重要意义。最后对TXNIP的研究进行了展望,未来可进一步着手研究TXNIP基因是如何与其他基因或危险因素协同作用,进而共同参与糖尿病及其并发症的发生发展,且TXNIP单个基因甲基化尚不能全面揭示糖尿病及其并发症发生的分子机制,这些后续的深入研究,将为在糖尿病及其并发症的诊断与治疗中作为靶标分子的应用奠定基础。     

All‐trans retinoic acid ameliorates hepatic stellate cell activation via suppression of thioredoxin interacting protein expression

Activation of hepatic stellate cells (HSCs) is the effector factor of hepatic fibrosis and hepatocellular carcinoma (HCC) development. Accumulating evidence suggests that retinoic acids (RAs), derivatives of vitamin A, contribute to prevention of liver fibrosis and carcinogenesis, however, regulatory mechanisms of RAs still remain exclusive. To elucidate RA signaling pathway, we previously performed a genome‐wide screening of RA‐responsive genes by in silico analysis of RA‐response elements, and identified 26 RA‐responsive genes. We found that thioredoxin interacting protein (TXNIP), which inhibits antioxidant activity of thioredoxin (TRX), was downregulated by all‐trans retinoic acid (ATRA). In the present study, we demonstrate that ATRA ameliorates activation of HSCs through TXNIP suppression. HSC activation was attenuated by TXNIP downregulation, whereas potentiated by TXNIP upregulation, indicating that TXNIP plays a crucial role in activation of HSCs. Notably, we showed that TXNIP‐mediated HSC activation was suppressed by antioxidant N‐acetylcysteine. In addition, ATRA treatment or downregulation of TXNIP clearly declined oxidative stress levels in activated HSCs. These data suggest that ATRA plays a key role in inhibition of HSC activation via suppressing TXNIP expression, which reduces oxidative stress levels.     

丁酸钠至少部分通过NF-Y 依赖的TXNIP 表达诱导人肺癌细胞A549死亡

组蛋白去乙酰化酶（HDACs）抑制剂丁酸钠调节细胞分化、增殖和抑制肿瘤发生。硫氧还蛋白相互作用蛋白( thioredoxin-interacting protein,TXNIP)通过负性调控硫氧还蛋白的活性,调控细胞内的氧化还原平衡,抑制细胞生长。本研究证明,丁酸钠可通过激活依赖于转录因子NF-Y的TXNIP 表达,诱导人非小细胞肺癌细胞A549死亡。MTT法显示,5 mmol/L丁酸钠处理A549 细胞72 h可显著诱导其死亡;流式细胞分析发现,其中大部分细胞以凋亡形式死亡。表达芯片分析表明,在丁酸钠处理的A549 细胞中,TXNIP 的mRNA 水平显著提高30～50倍;实时定量PCR、免疫细胞化学和蛋白质印迹结果进一步证明,丁酸钠可显著上调TXNIP 表达。荧光素酶报告基因分析证明,与对照细胞比较,丁酸钠刺激的细胞内报告酶活性可提高约10 倍,提示丁酸钠可激活TXNIP 启动子的转录活性。TXNIP 启动子删除突变分析显示,删除NF-Y 结合的DNA 序列显著降低丁酸钠对TXNIP 启动子的激活能力, 表明NF-Y转录因子参与丁酸钠介导的TXNIP基因转录激活。为分析TXNIP 在A549 细胞中的定位和部分功能,在A549细胞 中过表达GFP TXNIP 融合蛋白及其截短突变体融合蛋白;结果显示,野生型和保留N 端1-281aa的截短突变体定位在细胞核,而删除N 端1-200aa 时,其定位在细胞核和细胞质,提示N 端1 200aa 可调节该蛋白质的定位。然而,丁酸钠刺激未发现表达的GFP TXNIP在细胞内定位改变。以上结果表明,丁酸钠可通过激活转录因子NF YC 依赖的TXNIP激活,诱导A549 细胞死亡,但不能改变TXNIP蛋白在细胞内的定位。上述结果还提示,TXNIP 的N 端1-200aa 可能在调节TXNIP 的细胞定位中发挥作用。是否丁酸钠刺激TXNIP表达导致的细胞死亡系通过改变细胞氧化压力,以及TXNIP在细胞中定位的详尽调节机制尚待进一步研究证明。     

Calreticulin transacetylase mediated upregulation of thioredoxin by 7,8-diacetoxy-4-methylcoumarin enhances the antioxidant potential and the expression of vascular endothelial growth factor in peripheral blood mononuclear cells

Extensive research carried out in our group on polyphenolic acetates (PAs) substantiated the potential role of PAs in causing diverse biological and pharmacological actions. Our earlier investigations firmly established the calreticulin transacetylase (CRTAase) catalyzed activation of nitric oxide synthase (NOS) by PAs. In this report, we have studied the effect of 7,8-diacetoxy-4-methylcoumarin (DAMC, a model PA) and other acetoxy coumarins on the thioredoxin and VEGF expression in human peripheral blood mononuclear cells (PBMCs), with a view to substantiate our earlier observation that DAMC was a superb inducer of angiogenesis. Real time RT-PCR analysis revealed the enhanced expression of thioredoxin reductase (TRXR) and diminished expression of thioredoxin interacting protein (TRXIP) leading to the increased expression and activity of thioredoxin (TRX) in PBMCs due to the the action of DAMC. The fact that TRX activity of PBMCs was enhanced by various acetoxy coumarins in tune with their affinity to CRTAase as substrate, suggested the possible activation of TRX due to acetylation. The overexpression of thioredoxin was found to correlate with that of VEGF as proved by real time RT-PCR and VEGF -ELISA results, apart from the DAMC-caused enhanced production of NO acting as an inducer of VEGF. Moreover, the intracellular ROS levels were also found to be reduced drastically, by DAMC thus reducing the oxidative stress in cells. These observations strongly evidenced the crucial role of TRX in DAMC-induced tissue angiogenesis with the involvement of VEGF.     

Down-regulation of MSH2 expression by an Hsp90 inhibitor enhances pemetrexed-induced cytotoxicity in human non-small-cell lung cancer cells

Elevated heat shock protein 90 (Hsp90) expression has been linked to poor prognosis in patients with non-small cell lung cancer (NSCLC). The multitargeted antifolate pemetrexed has demonstrated certain clinical activities against NSCLC. However, the efficacy of the combination of pemtrexed and Hsp90 inhibitor to prolong the survival of patients with NSCLC still remains unclear. Human MutS homolog 2 (MSH2), a crucial element of the highly conserved DNA mismatch repair system, and defects or polymorphisms of MSH2 have been found in lung cancer. In this study, we evaluated the effects of pemetrexed on NSCLC cell lines (H520 and H1703) and found that treatment with this drug at 20–50 µM increased the MSH2 mRNA and protein levels in a MKK3/6–p38 MAPK signal activation-dependent manner. Furthermore, the knockdown of MSH2 expression by transfection with small interfering RNA of MSH2 or the blockage of p38 MAPK activation by SB202190 enhanced the cytotoxicity of pemetrexed. Combining the drug treatment with an Hsp90 inhibitor resulted in an enhanced pemetrexed-induced cytotoxic effect, accompanied with the reduction of MSH2 protein and mRNA levels. The expression of constitutively active MKK6 (MKK6E) or HA-p38 MAPK vectors significantly rescued the decreased p38 MAPK activity, and restored the MSH2 protein levels and cell survival in NSCLC cells co-treated with pemetrexed and Hsp90 inhibitor. In this study, we have demonstrated that down-regulation of the MKK3/6–p38 MAPK signal with the subsequent reduction of MSH2 enhanced the cytotoxic effect of pemetrexed in H520 and H1703 cells. The results suggest a potential future benefit of combining pemetrexed and the Hsp90 inhibitor to treat lung cancer.     

p38 Mitogen-activated protein kinase (p38 MAPK) and NADPH Oxidase (NOX) are cytoprotective determinants in the trophozoite-induced apoptosis of peripheral blood mononuclear cells

In a host–parasite interaction model, peripheral blood mononuclear cells (PBMCs) were co-incubated with trophozoites of Entamoeba histolytica to determine if the cytotoxic killing of PBMCs involves (NOX)-derived reactive oxygen species (ROS) and p38 mitogen-activated protein kinase (MAPK). Experimental PBMC populations were pre-treated with diphenylene iodonium chloride to inhibit NOX, N-acetylcysteine to inhibit p47^phox (a subunit of NOX), and SB202190 to inhibit p38 MAPK, with co-suppression of caspases. Percentage apoptosis, caspase-3 activity and ROS generation were monitored in all PBMC populations. Pre-treatment significantly raised the proportion of apoptotic PBMCs, but changes in caspase-3 activity and ROS production were relatively negligible. These results indicate that p38 MAPK and NOX were cytoprotective determinants in the trophozoite-induced apoptosis of PBMCs. Further, the programmed cell death herein investigated was independent of both caspases and ROS, and the exact mechanism of cell death remains to be an open question.