Curcumin inhibits UV irradiation-induced oxidative stress and apoptotic biochemical changes in human epidermoid carcinoma A431 cells

Ultraviolet (UV) light is a strong apoptotic trigger that induces caspase-dependent biochemical changes in cells. Previously we showed that UV irradiation can activate caspase-3, and the subsequent cleavage and activation of p21(Cdc42/Rac)-activated kinase 2 (PAK2) in human epidermoid carcinoma A431 cells. In this study we demonstrate that curcumin (Cur), the yellow pigment of Curcuma longa with known anti-oxidant and anti-inflammatory properties, can prevent UV irradiation-induced apoptotic changes, including c-Jun N-terminal kinase (JNK) activation, loss of mitochondrial membrane potential (MMP), mitochondrial release of cytochrome C, caspase-3 activation, and cleavage/activation of PAK2 in A431 cells. Flow cytometric analysis using the cell permeable dye 2',7'-dichlorofluorescin diacetate (DCF-DA) as an indicator of reactive oxygen species (ROS) generation revealed that the increase in intracellular oxidative stress caused by UV irradiation could be abolished by Cur. In addition, we found that SP600125, a JNK-specific inhibitor, reduced UV irradiation-induced JNK activation as well as caspase-3 activation, indicating that JNK activity is required for UV irradiation-induced caspase activation. Collectively, our results demonstrate that Cur significantly attenuates UV irradiation-induced ROS formation, and suggest that ROS triggers JNK activation, which in turn causes MMP change, cytochrome C release, caspase activation, and subsequent apoptotic biochemical changes.     

Heat Shock Stress Induces Cleavage and Activation of PAK2 in Apoptotic Cells

Heat shock induces a stress response in mammalian cells and can also lead to apoptotic cell death. Here we report that a 36-kDa myelin basic protein (MBP) kinase detected by an in-gel kinase assay can be drastically activated in several cell types by heat shock. Immunoblot analysis revealed that this 36-kDa MBP kinase can be recognized by an antibody against the C-terminal region of a family of p21^Cdc42/Rac-activated kinases (PAKs). By using this antibody and a PAK2-specific antibody against the N-terminal region of PAK2 as tools, we further demonstrated that heat shock can induce cleavage of PAK2 to generate a 36-kDa C-terminal catalytic fragment in mouse Balb/c 3T3 and human Hep 3B cells. The kinetic profile of appearance of the 36-kDa C-terminal catalytic fragment of PAK2 matched exactly with the activation of the 36-kDa MBP kinase in these cells induced by heat shock. In addition, the heat shock-induced cleavage and activation of PAK2 was found to be closely associated with both DNA fragmentation and activation of an ICE/CED-3 family cysteine protease termed caspase-3 in heat shock-treated Hep 3B cells. Moreover, blockage of the activation of caspase-3 by pretreating the cells with two specific tetrapeptidic inhibitors of caspases (Ac-DEVD-cho and Ac-YVAD-cmk) could substantially diminish the extent of heat shock-induced cleavage/activation of PAK2. Overall, our results point out that PAK2 is cleaved and activated during the heat shock-induced apoptotic cell death process and suggest that caspase-3 is involved in this process.     

Oleanolic Acid Induces Apoptosis in Human Leukemia Cells through Caspase Activation and Poly（ADP-ribose） Polymerase Cleavage

It has been shown that Fructus Ligustri Lucidi （FLL）, a promising traditional Chinese medicine, can inhibit the growth of tumors. However, the effective component and molecular mechanism of FLL act to inhibit tumor proliferation are unclear. In this study, we demonstrated that oleanolic acid （OA）, a principal chemical component of FLL, inhibited the proliferation of human leukemia HL60 cells in culture. MTT assay showed that treatment of HL60 cells with FLL crude extracts or OA dramatically blocked the growth of target tumor cell in a time- and dose-dependent manner. Morphological changes of the nuclei and DNA fragmentation showed that apoptotic cell death occurred in the HL60 cells after treating with FLL extracts （20 mg/ml） or OA （3.65×10^-2 mg/ml）. Furthermore, flow cytometry assay showed that treatment of HL60 cells with FLL or OA caused an increased accumulation of G1 and sub-G1 subpopulations. Western blot analysis showed that caspase-9 and caspase-3 were activated, accompanied by the cleavage of poly （ADP-ribose） polymerase （PARP） in the target cells during FLL- or OA-induced apoptosis, These results suggest that OA acts as the effective component of FLL by exerting its cytotoxicity towards target tumor cells through activation of caspases and cleavage of PARP.     

芳姜黄酮对人皮肤鳞状细胞癌A431细胞迁移、侵袭及凋亡的影响和机制研究

为研究芳姜黄酮(Ar-Turmerone)对人鳞状细胞癌A431细胞增殖、迁移、侵袭和凋亡的影响及机制。实验采用CCK-8法检测抑制率,吉姆萨染色观察细胞形态,划痕实验和Transwell小室实验研究细胞迁移和侵袭能力的变化,流式细胞仪检测细胞凋亡率。此外,通过实时荧光定量聚合酶链反应(Real-time PCR)与蛋白质印迹法(western blot)法检测mRNA和蛋白表达。siRNA阻断Notch1,Hes1和PTEN,检测相应的下游mRNA和蛋白的表达变化,流式细胞仪检测细胞凋亡率。结果发现,芳姜黄酮可以抑制A431细胞增殖,使细胞形态发生改变,抑制细胞体外迁移和侵袭能力,促进细胞凋亡。经过芳姜黄酮处理后,Notch1,Hes1,PTEN的mRNA和蛋白表达升高。沉默Notch1,Hes1 mRNA和蛋白表达低于单纯给药组,而沉默Hes1,PTEN mRNA和蛋白表达也低于单纯给药组;沉默PTEN后,与单纯给药组相比,细胞死亡率降低。总之,芳姜黄酮可以抑制人鳞状细胞癌A431细胞的增殖并促进其凋亡,且具有抑制体外迁移和侵袭的作用,其促进细胞凋亡的机制是通过Notch1/Hes1/PTEN途径实现的。     

Telmisartan induces apoptosis and regulates Bcl-2 in human renal cancer cells

It has been well-characterized that the renin-angiotensin system (RAS) physiologically regulates systemic arterial pressure. However, RAS signaling has also been shown to increase cell proliferation during malignancy, and angiotensin receptor blockers (ARBs) are able to decrease pro-survival signaling by inhibiting anti-apoptotic molecules and suppressing caspase activity. In this study, the apoptotic effects of telmisartan, a type of ARB, was evaluated using a non-cancerous human renal cell line (HEK) and a human renal cell carcinoma (RCC) cell line (786). Both types of cells were treated with telmisartan for 4 h, 24 h, and 48 h, and then were assayed for levels of apoptosis, caspase-3, and Bcl-2 using MTT assays, flow cytometry, and immunostaining studies. Analysis of variance was used to identify significant differences between these data (P < 0.05). Following the treatment of 786 cells with 100 µM and 200 µM telmisartan, a marked inhibition of cell proliferation was observed. 50 µM cisplatin also caused high inhibition of these cells. Moreover, these inhibitions were both concentration- and time-dependent (P < 0.05). Various apoptotic effects were also observed compared with control cells at the 24 h and 48 h timepoints assayed (P < 0.001). Furthermore, positive caspase-3 staining and down-regulation of Bcl-2 were detected, consistent with induction of cell death. In contrast, treatment of HEK cells with telmisartan did not produce an apoptotic effect compared with control cells at the 24 h timepoint (P > 0.05). Treatment with cisplatin promoted in HEK cells high index of apoptosis (P < 0.001). Taken together, these results suggest that telmisartan induces apoptosis via down-regulation of Bcl-2 and involvement of caspase-3 in human RCC cells.     

BimL involvement in Bax activation during UV irradiation-induced apoptosis

Bax, a proapoptotic member of the Bcl-2 family, localizes largely in the cytoplasm but translocates to mitochondria and undergoes oligomerization to induce the release of apoptogenic factors in response to apoptotic stimuli. However, the molecular mechanism of Bax activation is not fully understood. We show here the role of BimL in Bax activation during UV irradiation-induced apoptosis. In this study, GFP-BimL plasmid was constructed. The dynamic interaction between BimL and Bax during UV irradiation-induced apoptosis was observed using fluorescence resonance energy transfer (FRET) technique. Our experimental results showed that BimL translocation to mitochondria occurred before Bax translocation, and that BimL activated Bax indirectly. Moreover, inhibition of c-Jun N-terminal protein kinase (JNK) activation blocked BimL translocation, delayed and attenuated Bax translocation and subsequent apoptosis. These results demonstrate that BimL is involved in UV irradiation-induced apoptosis by indirectly activating Bax.     

Okadaic acid stimulates caspase-like activities and induces apoptosis of cultured rat mesangial cells

Glomerular mesangial cells play an important role in the development of glomerulosclerosis. Mesangial cell apoptosis has been shown to be involved in different stages of development of glomerulonephritis. The aim of the present study was to evaluate the effect of inhibition of serine/threonine phosphatases by okadaic acid, a shell fish toxin, on rat mesangial cell apoptosis and to examine the molecular mechanisms particularly the role of caspases. Okadaic acid significantly induced mesangial cell apoptosis, as measured by an increase in cytoplasmic nucleosome-associated DNA fragmentation. The induction of apoptosis was dependent on protein synthesis, because cyclohexamide, a protein synthesis inhibitor, blocked okadaic acid-induced apoptosis. In addition, okadaic acid stimulated caspase activities (as measured by caspase substrate peptide hydrolysis) in cultured rat mesangial cells at different time points. After 12 h treatment, okadaic acid caused a modest increase in caspase-8 (IETD-pNAse)(159.3 ± 6.7%) activity, while after 18 h treatment, okadaic acid caused a significant increase in caspase-3 (DEVD-pNAse)(906 ± 245%) activity. Okadaic acid-stimulated caspase-3 activity was inhibited by Z-IETD-FMK (caspase-8 inhibitor) suggesting that the caspase-3 activity is downstream of caspase-8 activity. Both caspase-3 and caspase-8 inhibitors blocked okadaic acid-stimulated apoptosis. These data suggest that inhibition of protein phosphatases by okadaic acid induces apoptosis in rat mesangial cells by activating caspase-3- and -8-like activities and that caspase-3-like activity is downstream of caspase-8-like activity.     

Parthenolide protects human lens epithelial cells from oxidative stress-induced apoptosis via inhibition of activation of caspase-3 and caspase-9

The apoptosis of lens epithehal cells has been proposed as the common basis of cataract formation, with oxidative stress as the major cause. This study was performed to investigate the protective effect of the herbal constituent parthenolide against oxidative stress-induced apoptosis of human lens epithelial （HLE） cells and the possible molecular mechanisms involved. HLE cells （SRA01-04） were incubated with 50 μM H2O2 in the absence or presence of different doses of parthenolide （10, 20 and 50 μM）. To study apoptosis, the cells were assessed by morphologic examination and Annexin V-propidium iodide double staining flow cytometry; to investigate the underlying molecular mechanisms, the expression of caspase-3 and caspase-9 were assayed by Western blot and quantitative RT-PCR, and the activities of caspase-3 and caspase-9 were measured by a Chemicon caspase colorimetric activity assay kit. Stimulated with H202 for 18 h, a high fraction of riLE cells underwent apoptosis, while in the presence ofparthenolide of different concentrations, dose-dependent blocking of HLE cell apoptosis was observed. The expression of caspase-3 and caspase-9 induced by H202 in HLE cells was significantly reduced by parthenolide both at the protein and mRNA levels, and the activation ofcaspase-3 and caspase-9 was also suppressed by parthenolide in a dose-dependent manner. In conclusion, parthenolide prevents HLE cells from oxidative stress-induced apoptosis through inhibition of the activation ofcaspase-3 and caspase-9, suggesting a potential protective effect against cataract formation.     

Fluorescence resonance energy transfer analysis of bid activation in living cells during ultraviolet-induced apoptosis

Ultraviolet (UV) irradiation is a DNA-damaging agent that triggers apoptosis through both themembrane death receptor and mitochondrial apoptotic signaling pathways.Bid,a pro-apoptotic Bcl-2family member,is important in most cell types to apoptosis in response to DNA damage.In this study,arecombinant plasmid,YFP-Bid-CFP,comprised of yellow and cyan fluorescent protein and a full length Bid,was used as a fluorescence resonance energy transfer analysis (FRET) probe.Using the FRET techniquebased on YFP-Bid-CFP,we found that Bid activation was initiated at 9±1 h after UV irradiation,and theaverage duration of the activation was 75±10 min.Bid activation coincided with a collapse of the mitochondrialmembrane potential with an average duration of 50±10 min. When cells were pretreated with Z-IETD-fmk(caspase-8 specific inhibitor) the process of Bid activation was completely inhibited,but the apoptosis wasonly partially affected.Z-DEVD-fmk (caspase-3 inhibitor) and Z-FA-fmk (non asp specific inhibitor) didnot block Bid activation.Furthermore,the endogenous Bid activation with or without Z-IETD-fmk in responseto UV irradiation was confirmed by Western blotting.In summary, using the FRET technique,we observedthe dynamics of Bid activation during UV-induced apoptosis and found that it was a caspase-8 dependentevent.     

Cyclophosphamide-induced apoptosis in A431 cells is inhibited by fucosyltransferase IV

Fucosyltransferase IV (FUT4) is an essential enzyme that catalyzes the synthesis of difucosylated oligosaccharide LeY which is overexpressed in the cancers derived from the epithelial tissues. Our previous studies have shown that FUT4 overexpression promotes A431 cell proliferation through the MAPK and PI3K/Akt signaling pathways, but the relationship between FUT4 and apoptosis remained unclear. Here, we investigated the effect of FUT4 overexpression on cyclophosphamide (CPA)-induced apoptosis in A431 cells. Western blot analysis showed that FUT4 overexpression decreased expression of Bax, Caspase 3, and PARP proteins, and increased anti-apoptotic Bcl-2 protein in A431 cells. The anti-apoptosis effect of FUT4 was confirmed both by Annexin-V/PI and JC-1 assays. The results showed that FUT4 overexpression up-regulated phosphorylation of ERK1/2 and Akt which was inhibited by CPA in dose-dependent manner. By blocking the ERK/MAPK and PI3K/Akt pathways with specific inhibitors, we demonstrated that these two pathways were required in mediating the anti-apoptosis effect of FUT4. We concluded that FUT4 inhibited cell apoptosis induced by CPA through decreasing the expression of apoptotic proteins Bax, Caspase 3, and PARP and increasing the expression of anti-apoptotic protein Bcl-2 via the ERK/MAPK and PI3K/Akt signaling pathways in A431 cells.     

ER stress does not cause upregulation and activation of caspase-2 to initiate apoptosis

A recent report claimed that endoplasmic reticulum (ER) stress activates the ER trans-membrane receptor IRE1α, leading to increased caspase-2 levels via degradation of microRNAs, and consequently induction of apoptosis. This observation casts caspase-2 into a central role in the apoptosis triggered by ER stress. We have used multiple cell types from caspase-2-deficient mice to test this hypothesis but failed to find significant impact of loss of caspase-2 on ER-stress-induced apoptosis. Moreover, we did not observe increased expression of caspase-2 protein in response to ER stress. Our data strongly argue against a critical role for caspase-2 in ER-stress-induced apoptosis.     

RGD peptide-induced apoptosis in human leukemia HL-60 cells requires caspase-3 activation

RGD motif-containing peptides have been used in various studies of cell adhesion and growth. We report that RGD triggered apoptosis at a concentration of 1 mmol/L, whereas RAD-containing peptides failed to induce apoptosis in HL-60 cells. RGD-treated cells revealed internucleosomal DNA fragmentation. Western blot reveals caspase-3 activation in RGD peptide-treated cells. A caspase-3 inhibitor z-VAD-FMK completely blocked the apoptosis, but a caspase-1 inhibitor (Ac-YVAD-CMK) and caspase-2 inhibitor (z-VDVAD-FMK) did not block the apoptosis, suggesting that caspase-3 might have a critical role in the execution process of apoptosis induced by RGD. RGD peptides have been used extensively to inhibit tumor metastasis. Our results should help in further understanding the RGD peptide-induced apoptosis, which is important since RGD peptides have a potential role in therapies of the future. This revised version was published online in July 2006 with corrections to the Cover Date.     

Potentiation of Fas- and TRAIL-mediated apoptosis by IFN-gamma in A549 lung epithelial cells: enhancement of caspase-8 expression through IFN-response element

Epithelial cell apoptosis triggered cooperatively by multiple cytokines contributes to the injury induced by inflammatory responses in the lung and elsewhere. Here we show that interferon-gamma (IFN-gamma) sensitizes A549 cells, human lung epithelial cells, to cytokine-mediated apoptosis by upregulating caspase-8 expression. Pretreating the cells with IFN-gamma potentiated Fas- and TNF-related apoptosis inducing ligand (TRAIL)-induced cell death, but other forms of apoptosis, not mediated via receptors, were unaffected. Western blotting and inhibitor assays showed that IFN-gamma selectively increased expression of caspases-7 and -8, but not caspases-2, -3, -9, or -10, as a necessary step leading to apoptosis. Assaying promoter activity using a luciferase reporter gene indicated that an IFN-gamma response element was located in the 5'-flanking region of the caspase-8 gene, spanning positions -227 to -219. Taken together, these findings suggest that IFN-gamma potentiates Fas- and TRAIL-mediated apoptosis by increasing caspase-8 expression via an IFN-gamma response element in A549 cells.     

Contribution of alpha2beta1, alpha3beta1, alpha6beta4 integrins and 67 kDa laminin receptor to the interaction of epidermoid carcinoma A-431 cells with laminin-2/4

Laminin-2/4 is the major laminin isoform of normal muscle and nerve tissues and plays an important role in tumor invasion and metastasis. Despite the fact that laminin-2/4 has been found in the skin basement membrane, insufficient evidence is available on the effect of laminin-2/4 on the behavior of both normal and transformed skin cells. A comparison of the contribution of alpha2beta1, alpha3beta1, alpha6beta4 integrins and 67 kDa laminin receptor on the surface of the human epidermoid carcinoma cell, A-431, to interaction with laminin-2/4 was carried out. The cell interaction with extracellular matrix component is a multistage process. We employed new methods for studying different stages of the interaction of A-431 cells with laminin-2/4. We demonstrated that integrins alpha2beta1, alpha3beta1, alpha6beta4 and 67 kDa laminin receptor are involved in the interaction of A-431 cells with laminin-2/4. We found that contribution of the same receptors to different stages of the interaction with laminin can be different. alpha2beta1 integrins are involved in EGF-induced A-431 cells' migration on laminin-2/4. We demonstrated the cooperation between alpha2beta1 and alpha3beta1 integrins during adhesion and spreading of A-431 cells on laminin-2/4-coated substrate. These results provide information about laminin-2/4 receptors and their contribution to different stages of the interaction with cells.     

Caspase-3 protease activation during the process of genistein-induced apoptosis in TM4 testicular cells

The role of caspase-3 (CPP32) protease in the molecular pathways of genistein-induced cell death in TM4 cells was investigated. Fluorescence microscopy with Hoechst-33258-PI nuclear stain was used to distinguish between apoptosis and necrosis pathways of cell death. The viability of the test cells was assessed with both the trypan blue exclusion and MTT tetrazolium (3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyltetralzolium bromide, 2.5 mg/mL) assays. Caspase-3 enzymatic activity was determined using CasPASE Apoptosis Assay Kit. The overall results from all the data demonstrated that: i) genistein exerts dose- and time-dependent effects on TM4 testis cells; ii) apoptosis is induced by lower concentrations of genistein and necrosis induced by higher concentrations of genistein; iii) genistein induced activation caspase-3 enzymatic activity; iv) genistein-induction of apoptosis and necrosis was significantly inhibited by the caspase-3 inhibitor, z-DEV-FMK; v) sodium azide induced necrosis without activation of CPP32 enzymatic activity, and induction of apoptosis; and vi) genistein-induced apoptosis was associated with activation of CPP32 enzymatic activity in the cells. The overall results indicate a strong evidence of caspase-3 (CPP332) mediation in the molecular pathways of genistein-induced apoptosis in testicular cells. Apoptosis is the physiologically programmed cell death in which intrinsic mechanisms participate in the death of the cell, in contrast to necrosis, which induces inflammatory response in the affected cell. The fact that the chemopreventive role of several cancer drugs is due to induction of apoptosis augments the biotherapeutic potential of genistein for the treatment of malignant diseases including prostate and testicular cancers. It is therefore inevitable that identification of the apoptotic pathways and the points at which regulation occurs could be instrumental in the design of genistein biotherapy for such diseases.     

Tyrosin dephosphorylation and concurrent inactivation of protein kinase FA/GSK-3α by genistein in A431 cells

Modulation of protein Kinase _F/GSK-3α by tyrosine phosphorylation in A431 cells was investigated. Kinase F _A/GSK-3α was found to exist in a highly tyrosine-phosphorylated/activated state in resting cells but could become tyrosine-dephosphorylated and inactivated down to less than 30% of control values in concentration dependent manner by 50-400 μM genistein( a Specific tyrosine kinase inhibitor), as demonstrated by metobolic ³²p-labeling of the cells followed by immunoprecipitation and two-dimensional phosphoamino acid analysis and byimmunodetection in an antikinase F_A/GSK-3α immunoprecipitate kinase assay. Taken together, the results provide evidence that Kinase F_A/GSK-3α may exist in a highly tyrosine-phosphorylated/activated state in resting cells which can by tyrosine-dephosphorylated and nactivated by extracellular stimulus and that tyrosine kinase(s) and /or tyrosine phosphatase(s) may play a role in the modulation of kinse F_A/GSK-3α activity in cells.     

Vanadate-activated calcium influx in A431 cells is dependent on the plasma membrane potential

Vanadate can activate the uptake of Ca in A431 epidermal carcinoma cells by two- to fivefold with no detectable lag period. Preincubation with epidermal growth factor (EGF) to down-regulate the EGF receptor prevents subsequent stimulation by EGF but not that by vanadate. Ca uptake is sodium-independent and is not activated by depolarization in high KCl. On the contrary, vanadate-stimulated uptake is completely inhibited by decreasing the plasma membrane potential from about -65 to -30 mV. These results demonstrate that the EGF receptor is not itself functioning as a Ca channel, that vanadate is not acting at the level of EGF receptor, and that the Ca transport system exhibits an unusual potential sensitivity in that it is inhibited by depolarization of the plasma membrane.