Reactive oxygen species‐induced cell death of rat primary astrocytes through mitochondria‐mediated mechanism

Astrocytes, the most abundant glial cell population in the central nervous system (CNS), play physiological roles in neuronal activities. Oxidative insult induced by the injury to the CNS causes neural cell death through extrinsic and intrinsic pathways. This study reports that reactive oxygen species (ROS) generated by exposure to the strong oxidizing agent, hexavalent chromium (Cr(VI)) as a chemical‐induced oxidative stress model, caused astrocytes to undergo an apoptosis‐like cell death through a caspase‐3‐independent mechanism. Although activating protein‐1 (AP‐1) and NF‐κB were activated in Cr(VI)‐primed astrocytes, the inhibition of their activity failed to increase astrocytic cell survival. The results further indicated that the reduction in mitochondrial membrane potential (MMP) was accompanied by an increase in the levels of ROS in Cr(VI)‐primed astrocytes. Moreover, pretreatment of astrocytes with N‐acetylcysteine (NAC), the potent ROS scavenger, attenuated ROS production and MMP loss in Cr(VI)‐primed astrocytes, and significantly increased the survival of astrocytes, implying that the elevated ROS disrupted the mitochondrial function to result in the reduction of astrocytic cell viability. In addition, the nuclear expression of apoptosis‐inducing factor (AIF) and endonuclease G (EndoG) was observed in Cr(VI)‐primed astrocytes. Taken together, evidence shows that astrocytic cell death occurs by ROS‐induced oxidative insult through a caspase‐3‐independent apoptotic mechanism involving the loss of MMP and an increase in the nuclear levels of mitochondrial pro‐apoptosis proteins (AIF/EndoG). This mitochondria‐mediated but caspase‐3‐independent apoptotic pathway may be involved in oxidative stress‐induced astrocytic cell death in the injured CNS. J. Cell. Biochem. 107: 933–943, 2009. © 2009 Wiley‐Liss, Inc.     

Spatiotemporal activation of caspase‐dependent and ‐independent pathways in staurosporine‐induced apoptosis of p53wt and p53mt human cervical carcinoma cells

Background information. Caspase‐dependent and ‐independent death mechanisms are involved in apoptosis in a variety of human carcinoma cells treated with antineoplastic compounds. Our laboratory has reported that p53 is a key contributor of mitochondrial apoptosis in cervical carcinoma cells after staurosporine exposure. However, higher mitochondrial membrane potential dissipation and greater DNA fragmentation were observed in p53^wt (wild‐type p53) HeLa cells compared with p53^mt (mutated p53) C‐33A cells. Here, we have studied events linked to the mitochondrial apoptotic pathway. Results. Staurosporine can induce death of HeLa cells via a cytochrome c/caspase‐9/caspase‐3 mitochondrial‐dependent apoptotic pathway and via a delayed caspase‐independent pathway. In contrast with p53^wt cells, p53^mt C‐33A cells exhibit firstly caspase‐8 activation leading to caspase‐3 activation and Bid cleavage followed by cytochrome c release. Attenuation of PARP‐1 [poly(ADP‐ribose) polymerase‐1] cleavage as well as oligonucleosomal DNA fragmentation in the presence of z‐VAD‐fmk points toward a major involvement of a caspase‐dependent pathway in staurosporine‐induced apoptosis in p53^wt HeLa cells, which is not the case in p53^mt C‐33A cells. Meanwhile, the use of 3‐aminobenzamide, a PARP‐1 inhibitor known to prevent AIF (apoptosis‐inducing factor) release, significantly decreases staurosporine‐induced death in these p53^mt carcinoma cells, suggesting a preferential implication of caspase‐independent apoptosis. On the other hand, we show that p53, whose activity is modulated by pifithrin‐α, isolated as a suppressor of p53‐mediated transactivation, or by PRIMA‐1 (p53 reactivation and induction of massive apoptosis), that reactivates mutant p53, causes cytochrome c release as well as mitochondrio—nuclear AIF translocation in staurosporine‐induced apoptosis of cervical carcinoma cells. Conclusions. The present paper highlights that staurosporine engages the intrinsic mitochondrial apoptotic pathway via caspase‐8 or caspase‐9 signalling cascades and via caspase‐independent cell death, as well as through p53 activity.     

Molecular mechanism of diallyl disulfide in cell cycle arrest and apoptosis in HCT‐116 colon cancer cells

Diallyl disulfide (DADS) is the most prevalent oil‐soluble sulfur compound in garlic and inhibits cell proliferation in many cancer cell lines. Here we examined DADS cytotoxicity in a redox‐mediated process, involving reactive oxygen species (ROS) production. In the present study, p53‐independent cell cycle arrest at G2/M phase was observed with DADS treatment, along with time‐dependent increase of cyclin B1. In addition, apoptosis was also observed upon 24‐h DADS treatment accompanied by activation of p53. In HCT‐116 cells, DADS application induced a dose‐dependent increase and time‐dependent changes in ROS production. Scavenging of DADS‐induced ROS by N‐acetyl cysteine or reduced glutathione inhibited cell cycle arrest, apoptosis and p53 activation by DADS. These results suggest that ROS trigger the DADS‐induced cell cycle arrest and apoptosis and that ROS are involved in stress‐induced signaling upstream of p53 activation. Transfection of p53 small interfering RNA prevents the accumulation of cleaved poly(ADP‐ribose) polymerase and sub‐G1 cell population by 65% and 35%, respectively. Moreover, DADS‐induced apoptosis was also prevented by treatment with oligomycin, which is known to prevent p53‐dependent apoptosis by reducing ROS levels in mitochondria. These results suggest that mitochondrial ROS may serve as second messengers in DADS‐induced apoptosis, which requires activation of p53. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:71–79, 2009; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20266     

Triggering p53 activation is essential in ziyuglycoside I‐induced human retinoblastoma WERI‐Rb‐1 cell apoptosis

Ziyuglycoside I (Ziyu I), one of the major components isolated from the root of Sanguisorba officinalis L., has been proved for the antitumor properties on oral cancer, prostate cancer, and colorectal cancer. However, the effect of Ziyu I on retinoblastoma (RB) is not well understood. In this study, we investigated the inhibitory effect and underlying molecular mechanism of Ziyu I on human RB WERI‐Rb‐1 cells. Our results indicated that Ziyu I could suppress cell viability and induce mitochondrial‐dependent cell apoptosis in WERI‐Rb‐1 cells. Furthermore, Ziyu I treatment increased p53 expression as well as improved p53 stabilization through downregulation of pS166‐Mdm2 and upregulation of phosphorylated‐ and acetylated‐p53. Blockade of p53 significantly attenuated Ziyu I‐induced mitochondrial dysfunction. Our findings demonstrate that Ziyu I exhibits excellent anticancer effect on human RB WERI‐Rb‐1 cells by triggering p53 activation, and imply Ziyu I as a potential compound for chemotherapy of human RB.     

Inhibition of extracellular signal regulated kinase (ERK) leads to apoptosis inducing factor (AIF) mediated apoptosis in epithelial breast cancer cells: the lack of effect of ERK in p53 mediated copper induced apoptosis

Recent studies have shown that MEK/ERK-mediated signals play a major role in regulation of activity of p53 tumor suppressor protein. In this study, we investigated whether or not there is functional interaction between p53 and MEK/ERK pathways in epithelial breast cancer cells exposed to copper or zinc. We demonstrated that expression of wild-type p53 induced by copper or zinc significantly reduced phosphorylation of extracellular signal regulated kinase (ERK) in epithelial breast cancer MCF7 cells. Mutation or suppression of p53 in MDA-MB231 and MCF7-E6 cells, respectively, resulted in a strong ERK phosphorylation in the presence of metals. Weak ERK phosphorylation in MCF7 cells induced by copper or zinc was linked to mitochondrial disruption and apoptosis. Furthermore, inhibition of ERK through addition of PD98059 stimulated p53 activation in MCF7 cells and also led to upregulation of p53 downstream targets, p21 and Bax, which is a proapototic member of Bcl-2 family triggering mitochondrial pore opening. Moreover, blockage of the MEK/ERK pathway caused a breakdown of the mitochondrial membrane potential accompanied by an elevation in the ROS production. Disruption of p53 expression attenuated the depolarization of the mitochondrial membrane and ROS generation. Furthermore, PD98059 initiated apoptosis inducing factor (AIF) translocation from mitochondria to the nucleus in MCF7 cells; which are depleted in caspase 3. Interestingly, repression of MEK/ERK pathway did not intensify the cell stress caused by metal toxicity. Therefore, these findings demonstrate that MEK/ERK pathway plays an important role in downregulation of p53 and cell survival. Inhibition of ERK can lead to apoptosis via nuclear relocation of AIF. However, metal-induced activation of p53 and mitochondrial depolarization appears to be independent of ERK. Our data suggest that copper induces apoptosis through depolarization of mitochondrial membrane with release of AIF, and this process is MEK/ERK independent.     

Phenethyl isothiocyanate induces DNA damage-associated G2/M arrest and subsequent apoptosis in oral cancer cells with varying p53 mutations

Phenethyl isothiocyanate (PEITC) is a naturally occurring cruciferous vegetable-derived compound that inhibits cell growth and induces apoptosis in oral cancer cells. However, the exact mechanism of PEITC action has not been fully elucidated. This study investigated the molecular mechanism and anticancer potential of PEITC in oral squamous cell carcinoma (OSCC) cells with various p53 statuses. PEITC inhibited the growth of OC2, SCC4, and SCC25 cells (functional p53 mutants) in a dose-dependent manner with low toxicity to normal cells. Treatment with PEITC induced reactive oxygen species production, nitric oxide generation, and GSH depletion and triggered DNA damage response as evidenced by flow cytometry, 8-OHdG formation, and comet assay. Furthermore, the subsequent activation of ATM, Chk2, and p53 as well as the increased expression of downstream proteins p21 and Bax resulted in a G2/M phase arrest by inhibiting Cdc25C, Cdc2, and cyclin B1. The PEITC-induced apoptotic cell death, following a diminished mitochondrial transmembrane potential, reduced the expression of Bcl-2 and Mcl-1, released mitochondrial cytochrome c, and activated caspase 3 and PARP cleavage. The p53 inhibitor pifithrin-α and the antioxidants N-acetylcysteine and glutathione (GSH) protected the cells from PEITC-mediated apoptosis. However, mito-TEMPO, catalase, apocynin, and L-NAME did not prevent PEITC-induced cell death, suggesting that PEITC induced G2/M phase arrest and apoptosis in oral cancer cells via a GSH redox stress and oxidative DNA damage-induced ATM–Chk2–p53-related pathway. These results provide new insights into the critical roles of both GSH redox stress and p53 in the regulation of PEITC-induced G2/M cell cycle arrest and apoptosis in OSCCs.     

Pharmacological inhibition of GSK3 attenuates DNA damage-induced apoptosis via reduction of p53 mitochondrial translocation and Bax oligomerization in neuroblastoma SH-SY5Y CELLS

Glycogen synthase kinase-3 (GSK3) and p53 play crucial roles in the mitochondrial apoptotic pathway and are known to interact in the nucleus. However, it is not known if GSK3 has a regulatory role in the mitochondrial translocation of p53 that participates in apoptotic signaling following DNA damage. In this study, we demonstrated that lithium and SB216763, which are pharmacological inhibitors of GSK3, attenuated p53 accumulation and caspase-3 activation, as shown by PARP cleavage induced by the DNA-damaging agents doxorubicin, etoposide and camptothecin. Furthermore, each of these agents induced translocation of p53 to the mitochondria and activated the mitochondrial pathway of apoptosis, as evidenced by the release of cytochrome C from the mitochondria. Both mitochondrial translocation of p53 and mitochondrial release of cytochrome C were attenuated by inhibition of GSK3, indicating that GSK3 promotes the DNA damage-induced mitochondrial translocation of p53 and the mitochondrial apoptosis pathway. Interestingly, the regulation of p53 mitochondrial translocation by GSK3 was only evident with wild-type p53, not with mutated p53. GSK3 inhibition also reduced the phosphorylation of wild-type p53 at serine 33, which is induced by doxorubicin, etoposide and camptothecin in the mitochondria. Moreover, inhibition of GSK3 reduced etoposide-induced association of p53 with Bcl2 and Bax oligomerization. These findings show that GSK3 promotes the mitochondrial translocation of p53, enabling its interaction with Bcl2 to allow Bax oligomerization and the subsequent release of cytochrome C. This leads to caspase activation in the mitochondrial pathway of intrinsic apoptotic signaling.     

P53 activation plays a crucial role in silibinin induced ROS generation via PUMA and JNK

Silibinin is an active constituent extracted from blessed milk thistle (Silybum marianum). Our previous study demonstrated that silibinin induced autophagy and apoptosis via reactive oxygen species (ROS) generation in HeLa cells. In this study, we investigated whether the autophagy- and apoptosis-associated molecules also involved in ROS generation. Silibinin promoted the expression phosphorylated-p53 (p-p53) in a dose-dependent manner. Pifithrin-α (PFT-α), a specific inhibitor of p53, reduced ROS production and reversed silibinin's growth-inhibitory effect. The ROS scavenger N-acetyl cysteine (NAC) attenuated silibinin-induced up-regulation of p-p53 expression, suggesting that p53 might be regulated by ROS and forms a positive feedback loop with ROS. On the other hand, silibinin dose-dependently promoted the expression of phosphorylated-c-Jun N-terminal kinase (p-JNK). Inhibition of JNK by SP600125 decreased ROS generation. NAC down-regulated the expression of p-JNK, indicating that JNK could be activated by ROS. Activation of p53 was suppressed by SP600125 and expression of p-JNK was inhibited by PFT-α, therefore silibinin might activate a ROS-JNK-p53 cycle to induce cell death. Silibinin up-regulated the PUMA and Bax expressions and down-regulated the mitochondrial membrane potential (MMP) level. PFT-α reduced the expression of PUMA and Bax. These results showed that p53 could interfere with mitochondrial functions such as MMP via PUMA pathways, thus resulting in ROS generation. In order to elucidate the functions of p53 in silibinin induced ROS generation, we have chosen the A431 cells (human epithelial carcinoma) because they lack p53 activity (p53His273 mutation). Interestingly, silibinin did not up-regulate the ROS level in A431 cells but lower the ROS level. PFT-α had no influence on ROS level in A431 cells. p53 activation plays a crucial role in silibinin induced ROS generation.     

Mechanisms of ceramide‐induced COX‐2‐dependent apoptosis in human ovarian cancer OVCAR‐3 cells partially overlapped with resveratrol

Ceramide is a member of the sphingolipid family of bioactive molecules demonstrated to have profound, diverse biological activities. Ceramide is a potential chemotherapeutic agent via the induction of apoptosis. Exposure to ceramide activates extracellular‐signal‐regulated kinases (ERK)1/2‐ and p38 kinase‐dependent apoptosis in human ovarian cancer OVCAR‐3 cells, concomitant with an increase in the expression of COX‐2 and p53 phosphorylation. Blockade of cyclooxygenase‐2 (COX‐2) activity by siRNA or NS398 correspondingly inhibited ceramide‐induced p53 Ser‐15 phosphorylation and apoptosis; thus COX‐2 appears at the apex of the p38 kinase‐mediated signaling cascade induced by ceramide. Induction of apoptosis by ceramide or resveratrol was inhibited by the endocytosis inhibitor, cytochalasin D (CytD); however, cells exposed to resveratrol showed greater sensitivity than ceramide‐treated cells. Ceramide‐treated cells underwent a dose‐dependent reduction in trans‐membrane potential. Although both ceramide and resveratrol induced the expressions of caspase‐3 and ‐7, the effect of inducible COX‐2 was different in caspase‐7 expression induced by ceramide compared to resveratrol. In summary, resveratrol and ceramide converge on an endocytosis‐requiring, ERK1/2‐dependent signal transduction pathway and induction of COX‐expression as an essential molecular antecedent for subsequent p53‐dependent apoptosis. In addition, expressions of caspase‐3 and ‐7 are observed. However, a p38 kinase‐dependent signal transduction pathway and change in mitochondrial potential are also involved in ceramide‐induced apoptosis. J. Cell. Biochem. 114: 1940–1954, 2013. © 2013 Wiley Periodicals, Inc.     

Fuziline alleviates isoproterenol‐induced myocardial injury by inhibiting ROS‐triggered endoplasmic reticulum stress via PERK/eIF2α/ATF4/Chop pathway

Fuziline, an aminoalcohol‐diterpenoid alkaloid derived from Aconiti lateralis radix preparata, has been reported to have a cardioprotective activity in vitro. However, the potential mechanism of fuziline on myocardial protection remains unknown. In this study, we aimed to explore the efficacy and mechanism of fuziline on isoproterenol (ISO)‐induced myocardial injury in vitro and in vivo. As a result, fuziline effectively increased cell viability and alleviated ISO‐induced apoptosis. Meanwhile, fuziline significantly decreased the production of ROS, maintained mitochondrial membrane potential (MMP) and blocked the release of cytochrome C, suggesting that fuziline could play the cardioprotective role through restoring the mitochondrial function. Fuziline also could suppress ISO‐induced endoplasmic reticulum (ER) stress via the PERK/eIF2α/ATF4/Chop pathway. In addition, using ROS scavenger NAC could decrease ISO‐induced apoptosis and block ISO‐induced ER stress, while PERK inhibitor GSK2606414 did not reduce the production of ROS, indicating that excess production of ROS induced by ISO triggered ER stress. And fuziline protected against ISO‐induced myocardial injury by inhibiting ROS‐triggered ER stress. Furthermore, fuziline effectively improved cardiac function on ISO‐induced myocardial injury in rats. Western blot analysis also showed that fuziline reduced ER stress‐induced apoptosis in vivo. Above these results demonstrated that fuziline could reduce ISO‐induced myocardial injury in vitro and in vivo by inhibiting ROS‐triggered ER stress via the PERK/eIF2α/ATF4/Chop pathway.     

Astaxanthin prevents pulmonary fibrosis by promoting myofibroblast apoptosis dependent on Drp1‐mediated mitochondrial fission

Promotion of myofibroblast apoptosis is a potential therapeutic strategy for pulmonary fibrosis. This study investigated the antifibrotic effect of astaxanthin on the promotion of myofibroblast apoptosis based on dynamin‐related protein‐1 (Drp1)‐mediated mitochondrial fission in vivo and in vitro. Results showed that astaxanthin can inhibit lung parenchymal distortion and collagen deposition, as well as promote myofibroblast apoptosis. Astaxanthin demonstrated pro‐apoptotic function in myofibroblasts by contributing to mitochondrial fission, thereby leading to apoptosis by increasing the Drp1 expression and enhancing Drp1 translocation into the mitochondria. Two specific siRNAs were used to demonstrate that Drp1 is necessary to promote astaxanthin‐induced mitochondrial fission and apoptosis in myofibroblasts. Drp1‐associated genes, such as Bcl‐2‐associated X protein, cytochrome c, tumour suppressor gene p53 and p53‐up‐regulated modulator of apoptosis, were highly up‐regulated in the astaxanthin group compared with those in the sham group. This study revealed that astaxanthin can prevent pulmonary fibrosis by promoting myofibroblast apoptosis through a Drp1‐dependent molecular pathway. Furthermore, astaxanthin provides a potential therapeutic value in pulmonary fibrosis treatment.     

Arsenic Trioxide Selectively Induces Early and Extensive Apoptosis via the APO2/Caspase-8 Pathway Engaging the Mitochondrial Pathway in Myeloma Cells with Mutant p53

Arsenic trioxide (ATO) is effective in the treatment of acute promyelocytic leukemia (APL) and induces apoptosis in APL cells and in a great variety of other cancer cells. We have previously shown that ATO induces apoptosis in myeloma cells in two different modes depending on p53 status in the cells. In cells expressing mutated p53, ATO induced, G2/M arrest and activation caspase 8 and 3 and rapid and extensive apoptosis. Myeloma cells expressing w.t. p53, ATO induced G1 arrest and delayed apoptosis with activation of caspase 9 and 3. APO2/TRAIL receptor expression was induced in both cell types and APO2/TRAIL synergized with ATO in the induction of apoptosis. Here we tested the effect of ATO on mitochondrial membrane potential (MMP) in myeloma cells expressing mutated or w.t. p53. In myeloma cells expressing mutated p53, depolarization of MMP occurred early, concomitant with induction of APO2/TRAIL, activation of BID and release of AIF, preceding apoptosis. However, in cells expressing w.t. p53, APO2/TRAIL is not induced, BID is not cleaved and depolarization of MMP occurs concurrently with cytochrome c release and apoptosis. These results explain the greater sensitivity to ATO of cells with mutated p53 and suggest perhaps a general mechanism for ATO-induced apoptosis.     

Arsenic trioxide selectively induces early and extensive apoptosis via the APO2/caspase-8 pathway engaging the mitochondrial pathway in myeloma cells with mutant p53

Arsenic trioxide (ATO) is effective in the treatment of acute promyelocytic leukemia (APL) and induces apoptosis in APL cells and in a great variety of other cancer cells. We have previously shown that ATO induces apoptosis in myeloma cells in two different modes depending on p53 status in the cells. In cells expressing mutated p53, ATO induced, G2/M arrest and activation caspase 8 and 3 and rapid and extensive apoptosis. Myeloma cells expressing w.t. p53, ATO induced G1 arrest and delayed apoptosis with activation of caspase 9 and 3. APO2/TRAIL receptor expression was induced in both cell types and APO2/TRAIL synergized with ATO in the induction of apoptosis. Here we tested the effect of ATO on mitochondrial membrane potential (MMP) in myeloma cells expressing mutated or w.t. p53. In myeloma cells expressing mutated p53, depolarization of MMP occurred early, concomitant with induction of APO2/TRAIL, activation of BID and release of AIF, preceding apoptosis. However, in cells expressing w.t. p53, APO2/TRAIL is not induced, BID is not cleaved and depolarization of MMP occurs concurrently with cytochrome c release and apoptosis. These results explain the greater sensitivity to ATO of cells with mutated p53 and suggest perhaps a general mechanism for ATO-induced apoptosis.     

Crosstalk between AMPK activation and angiotensin II‐induced hypertrophy in cardiomyocytes: the role of mitochondria

AMP‐kinase (AMPK) activation reduces cardiac hypertrophy, although underlying molecular mechanisms remain unclear. In this study, we elucidated the anti‐hypertrophic action of metformin, specifically, the role of the AMPK/eNOS/p53 pathway. H9c2 rat cardiomyocytes were treated with angiotensin II (AngII) for 24 hrs in the presence or absence of metformin (AMPK agonist), losartan [AngII type 1 receptor (AT1R) blocker], Nω‐nitro‐L‐arginine methyl ester (L‐NAME, pan‐NOS inhibitor), splitomicin (SIRT1 inhibitor) or pifithrin‐α (p53 inhibitor). Results showed that treatment with metformin significantly attenuated AngII‐induced cell hypertrophy and death. Metformin attenuated AngII‐induced activation (cleavage) of caspase 3, Bcl‐2 down‐regulation and p53 up‐regulation. It also reduced AngII‐induced AT1R up‐regulation by 30% (P < 0.05) and enhanced AMPK phosphorylation by 99% (P < 0.01) and P‐eNOS levels by 3.3‐fold (P < 0.01). Likewise, losartan reduced AT1R up‐regulation and enhanced AMPK phosphorylation by 54% (P < 0.05). The AMPK inhibitor, compound C, prevented AT1R down‐regulation, indicating that metformin mediated its effects via AMPK activation. Beneficial effects of metformin and losartan converged on mitochondria that demonstrated high membrane potential (Δψ_m) and low permeability transition pore opening. Thus, this study demonstrates that the anti‐hypertrophic effects of metformin are associated with AMPK‐induced AT1R down‐regulation and prevention of mitochondrial dysfunction through the SIRT1/eNOS/p53 pathway.     

Gastrodin Inhibits Glutamate-Induced Apoptosis of PC12 Cells via Inhibition of CaMKII/ASK-1/p38 MAPK/p53 Signaling Cascade

Previous research demonstrated that glutamate induces neuronal injury partially by increasing intracellular Ca²⁺ concentrations ([Ca²⁺]_i), and inducing oxidative stress, leading to a neurodegenerative disorder. However, the mechanism of glutamate-induced injury remains elusive. Gastrodin, a major active component of the traditional herbal agent Gastrodia elata (GE) Blume, has been recognized as a potential neuroprotective drug. In the current study, a classical injury model based on glutamate-induced cell death of rat pheochromocytoma (PC12) cells was used to investigate the neuroprotective effect of gastrodin, and its potential mechanisms involved. In this paper, the presence of gastrodin inhibits glutamate-induced oxidative stress as measured by the formation of reactive oxygen species (ROS), the level of malondialdehyde (MDA), mitochondrial membrane potential (MMP), and superoxide dismutase (SOD); gastrodin also prevents glutamate-induced [Ca²⁺]_i influx, blocks the activation of the calmodulin-dependent kinase II (CaMKII) and the apoptosis signaling-regulating kinase-1 (ASK-1), inhibits phosphorylation of p38 mitogen-activated kinase (MAPK). Additionally, gastrodin blocked the expression of p53 phosphorylation, caspase-3 and cytochrome C, reduced bax/bcl-2 ratio induced by glutamate in PC12 cells. All these findings indicate that gastrodin protects PC12 cells from the apoptosis induced by glutamate through a new mechanism of the CaMKII/ASK-1/p38 MAPK/p53-signaling pathway.     

Pterostilbene protects vascular endothelial cells against oxidized low-density lipoprotein-induced apoptosis in vitro and in vivo

Vascular endothelial cell (VEC) apoptosis is the main event occurring during the development of atherosclerosis. Pterostilbene (PT), a natural dimethylated analog of resveratrol, has been the subject of intense research in cancer and inflammation. However, the protective effects of PT against oxidized low-density lipoprotein (oxLDL)-induced apoptosis in VECs have not been clarified. We investigated the anti-apoptotic effects of PT in vitro and in vivo in mice. PT at 0.1–5 μM possessed antioxidant properties comparable to that of trolox in a cell-free system. Exposure of human umbilical vein VECs (HUVECs) to oxLDL (200 μg/ml) induced cell shrinkage, chromatin condensation, nuclear fragmentation, and cell apoptosis, but PT protected against such injuries. In addition, PT injection strongly decreased the number of TUNEL-positive cells in the endothelium of atherosclerotic plaque from apoE^−/− mice. OxLDL increased reactive oxygen species (ROS) levels, NF-κB activation, p53 accumulation, apoptotic protein levels and caspases-9 and -3 activities and decreased mitochondrial membrane potential (MMP) and cytochrome c release in HUVECs. These alterations were attenuated by pretreatment with PT. PT inhibited the expression of lectin-like oxLDL receptor-1 (LOX-1) expression in vitro and in vivo. Cotreatment with PT and siRNA of LOX-1 synergistically reduced oxLDL-induced apoptosis in HUVECs. Overexpression of LOX-1 attenuated the protection by PT and suppressed the effects of PT on oxLDL-induced oxidative stress. PT may protect HUVECs against oxLDL-induced apoptosis by downregulating LOX-1-mediated activation through a pathway involving oxidative stress, p53, mitochondria, cytochrome c and caspase protease. PT might be a potential natural anti-apoptotic agent for the treatment of atherosclerosis.     

Dysfunctional telomeres induce p53‐dependent and independent apoptosis to compromise cellular proliferation and inhibit tumor formation

Aging is associated with progressive telomere shortening, resulting in the formation of dysfunctional telomeres that compromise tissue proliferation. However, dysfunctional telomeres can limit tumorigenesis by activating p53‐dependent cellular senescence and apoptosis. While activation of both senescence and apoptosis is required for repress tumor formation, it is not clear which pathway is the major tumor suppressive pathway in vivo. In this study, we generated Eμ‐myc; Pot1b ^?/? mouse to directly compare tumor formation under conditions in which either p53‐dependent apoptosis or senescence is activated by telomeres devoid of the shelterin component Pot1b. We found that activation of p53‐dependent apoptosis plays a more critical role in suppressing lymphoma formation than p53‐dependent senescence. In addition, we found that telomeres in Pot1b^?/?; p53^?/? mice activate an ATR‐Chk1‐dependent DNA damage response to initiate a robust p53‐independent, p73‐dependent apoptotic pathway that limited stem cell proliferation but suppressed B‐cell lymphomagenesis. Our results demonstrate that in mouse models, both p53‐dependent and p53‐independent apoptosis are important to suppressing tumor formation.