Cytoprotective effects of the lipoidic‐liquiform pro‐vitamin C tetra‐isopalmitoyl‐ascorbate (VC‐IP) against ultraviolet‐A ray‐induced injuries in human skin cells together with collagen retention,MMP inhibition and p53 gene repression

Irradiation with ultraviolet‐A (UVA) ray at doses of 20–100 J/cm² diminished the cell viability of human keratinocytes HaCaT and human melanoma cells HMV‐II, both of which were protected by pre‐irradiational administration with the ascorbic acid (Asc) derivative, VC‐IP (2,3,5,6‐O‐tetra‐2′‐hexyldecanoyl‐L‐ascorbic acid; vitamin C‐isopalmityl tetraester), which is the first lipoidic‐liquiform pro‐vitamin C by itself that is materialized by esterization of all four intramolecular hydroxyl groups of an Asc molecule with branched chain fatty groups, resulting in molecular fluidity higher than that of the corresponding straight chains. Irradiation with UVA to HaCaT keratinocytes was shown to cause the formation of 8‐hydroxydeoxyguanosine (8‐OHdG), translocation of phosphatidylserine in the inner layer into the outer layer of cell membrane, and lowering of a mitochondrial membrane potential, all of which were repressed by pre‐irradiational administration with VC‐IP. Expression of p53 gene, another hallmark of UV‐induced DNA damages, was promoted by UVA irradiation to the keratinocytes but also repressed by VC‐IP. Administration with VC‐IP of 10–50 µM to human fibroblasts NHDF achieved the enhancement of collagen synthesis, repression of matrix metalloprotease‐2/9 activity, and increasing of intracellular Asc contents more markedly than that with Asc itself of the same concentrations. Thus UVA‐induced diverse harmful effects could be prevented by VC‐IP, which was suggested to ensue intrinsically from the persistent enrichment of intracellular Asc, through esterolytic conversion of VC‐IP to a free‐form Asc molecule, resulting in relief to UVA‐caused oxidative stress. J. Cell. Biochem. 106: 589–598, 2009. © 2009 Wiley‐Liss, Inc.     

Surface molecules on HaCaT keratinocytes after interaction with non‐thermal atmospheric pressure plasma

Non‐thermal atmospheric‐pressure plasmas have been developed that will be used in future for several purposes, e.g. medicine. Living tissues and cells are at the focus of plasma treatment, e.g. to improve wound healing, or induce apoptosis and growth arrest in tumour cells. Detailed investigations of plasma‐cell interactions are needed. Cell surface adhesion molecules as integrins, cadherins or the EGFR (epidermal growth factor receptor) are of importance in wound healing and also for development of cancer metastasis. This study has focused on measurement of cell surface molecules on human HaCaT keratinocytes (human adult low calcium temperature keratinocytes) promoting adhesion, migration and proliferation as one important feature of plasma‐cell interactions. HaCaT keratinocytes were treated with plasma by a surface dielectric barrier discharge in air. Cell surface molecules and induction of intracellular ROS (reactive oxygen species) were analysed by flow cytometry 24 h after plasma treatment. Besides a reduction of cell viability a significant down‐regulation of E‐cadherin and the EGFR expression occurred. The influence on α₂‐ and β₁‐integrins was less pronounced, and expression of ICAM‐1 (intercellular adhesion molecule 1) was unaffected. The extent of effects depended on the exposure time of cells to the plasma and the treatment regimen. Intracellular level of ROS detected by the fluorescent dye H₂DCFDA (2′,7′‐dichlorodihydrofluorescein diacetate) increased by plasma treatment, but it was neither dependent on the treatment time nor related to the different treatment regimens. Two‐dimensional cultures of HaCaT keratinocytes appear to be a suitable method of investigating plasma‐cell interactions.     

Distinct Melanogenic Response of Human Melanocytes in Mono‐culture,in Co‐Culture with Keratinocytes and in Reconstructed Epidermis,to UV Exposure

Striking differences are observed in the melanogenic response of normal human melanocytes to UVA and UVB irradiation depending on culture conditions and the presence of keratinocytes. Exposure of melanocytes co‐cultured with keratinocytes to UVB irradiation triggered, already at low doses (5 mJ/cm²), an increase in melanin synthesis whereas in melanocyte mono‐cultures, UVB doses up to 50 mJ/cm² had no melanogenic effect. Unlike UVB, UVA exposure caused the same melanogenic response in both mono‐ and co‐cultures. Removing certain keratinocyte growth factors from the co‐culture medium abolished the melanogenic response to UVB, but not to UVA exposure. When integrated into the basal layer of a reconstructed human epidermis, human melanocytes similarly reacted to UVA and UVB irradiation as in vivo by increasing their production and transfer of melanin to the neighboring keratinocytes which resulted in a noticeable tanning of the reconstructed epidermis. The presence of a dense stratum corneum, known to scatter and absorb UV light, is responsible for higher minimal UVB and UVA doses required to trigger a melanogenic response in the reconstructed epidermis compared to keratinocyte–melanocyte co‐cultures. Furthermore, an immediate tanning response was observed in the pigmented epidermis following UVA irradiation. From these results we conclude that: (i) keratinocytes play an important role in mediating UVB‐induced pigmentation, (ii) UVA‐induced pigmentation is the result of a rather direct effect on melanocytes and (iii) reconstructed pigmented epidermis is the most appropriate model to study UV‐induced pigmentation in vitro.     

Peptidoglycan from Staphylococcus aureus has an anti‐apoptotic effect in HaCaT keratinocytes mediated by the production of the cellular inhibitor of apoptosis protein‐2

Colonization of epithelium by microorganisms leads to inflammatory responses. In some cases an anti‐apoptotic response involving the cellular inhibitor of apoptosis protein‐2 (cIAP‐2) also occurs. Although strong expression of cIAP‐2 has been observed in lesional skin from psoriatic patients and in HaCaT keratinocytes treated with peptidoglycan (PGN) from Staphylococcus aureus, anti‐apoptotic responses induced in the skin by cIAP‐2 have seldom been studied. In this study, the effect of PGN on TNF‐α‐induced apoptotic HaCaT keratinocytes was assessed. Morphological analysis, quantification of cells with DNA fragmentation and active caspase‐3 detection was performed to assess apoptotic cell death. Greater LL‐37 and cIAP‐2 production was found in keratinocytes stimulated with PGN than in non‐treated cells (P < 0.05). In comparison with cells treated with TNF‐α only, a significant reduction in apoptotic cell death was observed when HaCaT were pretreated with PGN before inducing apoptosis with TNF‐α (P < 0.05). In addition, an inhibitor of cIAP‐2 activity (LCL161) stopped the PGN effect. These findings show that PGN from S. aureus has an anti‐apoptotic effect in keratinocytes mediated by cIAP‐2 production, suggesting that this anti‐apoptotic activity could favor proliferation of keratinocytes in psoriasis.     

Role of reduced glutathione efflux in apoptosis of immortalized human keratinocytes induced by UVA

We have investigated the role played by GSH efflux in apoptosis of human HaCaT keratinocytes induced by UVA irradiation. UVA irradiation of HaCaT cells caused a rapid rise in GSH efflux across the intact cell membrane, followed by an increase in apoptosis. GSH efflux was stimulated by glucose and was reduced by the addition of exogenous GSH and intracellular GSH depletion by buthionine sulfoximine, suggesting that GSH transport is active and is influenced by the GSH concentration gradient across the cell membrane. Verapamil and cyclosporin A, blockers of the multidrug resistance-associated protein, decreased UVA-induced GSH efflux. GSH efflux occurred within 2 h of UVA irradiation, suggesting that the stimulation of GSH efflux is due to an increase in the activity of pre-existing multidrug resistance-associated protein transporter carrier. Although inhibition of GSH efflux did not affect caspase activation and DNA fragmentation, it delayed the gradual increase in plasma membrane permeability and reduced phosphatidylserine translocation in HaCaT cells. It is therefore likely that upon UVA irradiation, GSH efflux increased the intracellular oxidative stress without intervention of reactive oxygen species, thus resulting in more phosphatidylserine externalization and membrane rearrangement. These provide targets for macrophage recognition and phagocytosis and thus minimize the potential to invoke inflammation or neoplastic transformation.     

Reactive oxygen species are involved in the IFN‐γ‐stimulated production of Th2 chemokines in HaCaT keratinocytes

The increased generation of reactive oxygen species (ROS) induces inflammation in different cell types. However, it is unclear whether ROS play an essential role in the production of thymus and activation‐regulated chemokine (TARC/CCL17) and macrophage‐derived chemokine (MDC/CCL22) in keratinocytes. Here, we investigated the function of ROS in the production of these two Th2 chemokines in interferon‐gamma (IFN‐γ)‐treated HaCaT keratinocytes. We found that IFN‐γ‐induced production of both chemokines in parallel with the increased generation of intracellular ROS. A ROS scavenger, N‐acetyl cysteine (NAC), significantly inhibited the IFN‐γ‐induced production of chemokines as well as the activation of I kappa‐B (IκB)–nuclear factor‐kappa B (NF‐κB). Inhibitors of Janus family kinases (JAKs), p38 mitogen‐activated kinase (MAPK), and NF‐κB suppressed IFN‐γ‐induced production of TARC and MDC. NF‐κB activation was inhibited by both inhibitors of JAKs and p38 MAPK. Importantly, IFN‐γ‐stimulated phosphorylation of p38 MAPK was significantly suppressed by JAKs inhibitors, but not significantly affected by NAC or L ‐buthionine sulfoximine (L‐BSO). However, IFN‐γ‐stimulated activation of IκB and NF‐κB was suppressed by NAC but enhanced by BSO. Furthermore, inhibition of p38 MAPK and JAKs did not affect ROS generation in IFN‐γ‐stimulated HaCaT cells. These results indicate that intracellular ROS and JAKs/p38 MAPK both contribute independently to IFN‐γ‐stimulated production of TARC and MDC in HaCaT keratinocytes, by increasing NF‐κB activation. J. Cell. Physiol. 226: 58–65, 2010. © 2010 Wiley‐Liss, Inc.     

p35 interacts with α‐tubulin and organelle proteins: Nuclear translocation of p35 in dying cells

We identified heterogeneous nuclear ribonucleoprotein (hnRNP) C1/C2, hnRNP A1, the translocase of the transporter outer membrane 40 (TOM40), and α‐tubulin as new interaction partners of anti‐apoptotic protein p35 using MS‐based functional proteomics with GST‐p35 fusion protein as a bait, and using a pull‐down assay with p35‐6His followed by Western blot analysis. p35 was localized in the cytoplasm and in distinct organelles such as the nucleus and mitochondria. p35 was more abundant in the cytoplasm than it was in the nucleus. It co‐localized with α‐tubulin in the cytoplasm in the absence of a death stimulus. However, while cells were undergoing death induced by actinomycin D, cytoplasmic p35 was translocated into the nucleus; this process was inhibited by deletions of the N‐ and C‐terminal domains containing leucine‐rich motifs. Gene delivery of p35 using recombinant adenoviruses inhibited cytoplasmic compartmentalization of hnRNP C1/C2 and hnRNP A1 in dying cells. This study demonstrated translocation of p35 into the nuclei, as well as protection of the hnRNPs from redistribution in cells undergoing death. We propose an active role for p35 in maintaining the integrity of nuclear proteins during cell death.     

Comparative immunolocalization of keratin‐associated beta‐proteins (beta‐keratins) supports a new explanation for the cyclical process of keratinocyte differentiation in lizard epidermis

Lizard epidermis is made of beta‐ and alpha‐layers. Using Western blot tested antibodies, the ultrastructural immunolocalization of specific keratin‐associated beta‐proteins in the epidermis of different lizard species reveals that glycine‐rich beta‐proteins (HgG5) localize in the beta‐layer, while glycine–cysteine‐medium‐rich beta‐proteins (HgGC10) are present in oberhautchen and alpha‐layers. This suggests a new explanation for the formation of different epidermal layers during the shedding cycle in lepidosaurian epidermis instead of an alternance between beta‐keratins and alpha‐keratins. It is proposed that different sets of genes coding for specific beta‐proteins are activated in keratinocytes during the renewal phase of the shedding cycle. Initially, glycine–cysteine‐medium‐rich beta‐proteins with hydrophilic and elastic properties accumulate over alpha‐keratins in the oberhautchen but are replaced in the next cell layer with glycine‐rich hydrophobic beta‐proteins forming a resistant, stiff, and hydrophobic beta‐layer. The synthesis of glycine‐rich proteins terminates in mesos and alpha‐cells where these proteins are replaced with glycine–cysteine‐rich beta‐proteins. The pattern of beta‐protein deposition onto a scaffold of intermediate filament keratins is typical for keratin‐associated proteins and the association between alpha‐keratins and specific keratin‐associated beta‐proteins during the renewal phase of the shedding cycle gives rise to epidermal layers possessing different structural, mechanical, and texture properties.     

CBL‐mediated targeting of CIPKs facilitates the decoding of calcium signals emanating from distinct cellular stores

During adaptation and developmental processes cells respond through nonlinear calcium‐decoding signaling cascades, the principal components of which have been identified. However, the molecular mechanisms generating specificity of cellular responses remain poorly understood. Calcineurin B‐like (CBL) proteins contribute to decoding calcium signals by specifically interacting with a group of CBL‐interacting protein kinases (CIPKs). Here, we report the subcellular localization of all 10 CBL proteins from Arabidopsis and provide a cellular localization matrix of a plant calcium signaling network. Our findings suggest that individual CBL proteins decode calcium signals not only at the plasma membrane and the tonoplast, but also in the cytoplasm and nucleus. We found that distinct targeting signals located in the N‐terminal domain of CBL proteins determine the spatially discrete localization of CBL/CIPK complexes by COPII‐independent targeting pathways. Our findings establish the CBL/CIPK signaling network as a calcium decoding system that enables the simultaneous specific information processing of calcium signals emanating from different intra‐ and extracellular stores, and thereby provides a mechanism underlying the specificity of cellular responses.     

Immunocytochemistry indicates that glycine‐rich beta‐proteins are present in the beta‐layer,while cysteine‐rich beta‐proteins are present in beta‐ and alpha‐layers of snake epidermis

Immunolocalization of glycine‐rich and cysteine–glycine‐medium‐rich beta‐proteins (Beta‐keratins) in snake epidermis indicates a different distribution between beta‐ and alpha‐layers. Acta Zoologica, Stockholm. The epidermis of snakes consists of hard beta‐keratin layers alternated with softer and pliable alpha‐keratin layers. Using Western blot, light and ultrastructural immunolocalization, we have analyzed the distribution of two specific beta‐proteins (formerly beta‐keratins) in the epidermis of snakes. The study indicates that the antibody HgG5, recognizing glycine‐rich beta‐proteins of 12–15 kDa, is poorly or not reactive with the beta‐layer of snake epidermis. This suggests that glycine‐rich proteins similar to those present in lizards are altered during maturation of the beta‐layer. Conversely, a glycine–cysteine‐medium‐rich beta‐protein (HgGC10) of 10–12 kDa is present in beta‐ and alpha‐layers, but it is reduced or disappears in precorneous and suprabasal cells destined to give rise to beta‐ and alpha‐cells. Together with the previous studies on reptilian epidermis, the present results suggest that beta‐proteins rich in glycine mainly accumulate on a scaffold of alpha‐keratin producing a resistant and hydrophobic beta‐layer. Conversely, beta‐proteins lower in glycine but higher in cysteine accumulate on alpha‐keratin filaments present in beta‐ and alpha‐layers producing resistant but more pliable layers.     

Oncoprotein BMI‐1 induces the malignant transformation of HaCaT cells

BMI‐1 (B‐cell‐specific Moloney murine leukemia virus integration site 1), a novel oncogene, has attracted much attention in recent years for its involvement in the initiation of a variety of tumors. Recent evidence showed that BMI‐1 was highly expressed in neoplastic skin lesions. However, whether dysregulated BMI‐1 expression is causal for the transformation of skin cells remains unknown. In this study, we stably expressed BMI‐1 in a human keratinocyte cell line, HaCaT. The expression of wild‐type BMI‐1 induced the malignant transformation of HaCaT cells in vitro. More importantly, we found that expression of BMI‐1 promoted formation of squamous cell carcinomas in vivo. Furthermore, we showed that BMI‐1 expression led to the downregulation of tumore suppressors, such as p16INK4a and p14ARF, cell adhesion molecules, such as E‐Cadherin, and differentiation related factor, such as KRT6. Therefore, our findings demonstrated that dysregulated BMI‐1 could indeed lead to keratinocytes transformation and tumorigenesis, potentially through promoting cell cycle progression and increasing cell mobility. J. Cell. Biochem. 106: 16–24, 2009. © 2008 Wiley‐Liss, Inc.     

Calycosin alleviates allergic contact dermatitis by repairing epithelial tight junctions via down‐regulating HIF‐1α

Calycosin, a bioactive component derived from Astragali Radix (AR; Huang Qi), has been shown to have an effect of anti‐allergic dermatitis with unknown mechanism. This study aims to investigate the mechanism of calycosin related to tight junctions (TJs) and HIF‐1α both in FITC‐induced mice allergic contact dermatitis and in IL‐1β stimulated HaCaT keratinocytes. Th2 cytokines (IL‐4, IL‐5 and IL‐13) were detected by ELISA. The epithelial TJ proteins (occludin, CLDN1 and ZO‐1), initiative key cytokines (TSLP and IL‐33) and HIF‐1α were assessed by Western blot, real‐time PCR, immunohistochemistry or immunofluorescence. Herein, we have demonstrated that allergic inflammation and the Th2 cytokines in ACD mice were reduced significantly by calycosin treatment. Meanwhile, calycosin obviously decreased the expression of HIF‐1α and repaired TJs both in vivo and in vitro. In HaCaT keratinocytes, we noted that IL‐1β induced the deterioration of TJs, as well as the increased levels of TSLP and IL‐33, which could be reversed by silencing HIF‐1α. In addition, administration of 2‐methoxyestradiolin (2‐ME), a HIF‐1α inhibitor,significantly repaired the TJs and alleviated the allergic inflammation in vivo. Furthermore, TJs were destroyed by DMOG or by overexpressing HIF‐1α in HaCaT keratinocytes, and simultaneously, calycosin down‐regulated the expression of HIF‐1α and repaired the TJs in this process. These results revealed that calycosin may act as a potential anti‐allergy and barrier‐repair agent via regulating HIF‐1α in AD and suggested that HIF‐1α and TJs might be possible therapy targets for allergic dermatitis.     

Photobiological implications of melanin photoprotection after UVB‐induced tanning of human skin but not UVA‐induced tanning

Repetitive suberythemal UVA and/or UVB exposures were used to generate comparable UV‐induced tans in human skin over the course of 2 weeks. To evaluate the potential photoprotective values of those UVA‐ and/or UVB‐ induced tans and to avoid the confounding issue of residual UV‐induced DNA damage, we waited 1 week before challenging those areas with a 1.5 MED of UVA+UVB after which we measure DNA damage. The results show that the type of UV used to induce skin pigmentation affects the redistribution of melanin in the skin and/or de novo melanin synthesis. The UVA‐induced tans failed to even provide a minimal SPF of 1.5, which suggests that producing a tan with UVA‐rich sunlamps prior to a holiday or vacation is completely counterproductive.     

UV light killing efficacy of fluorescent protein‐expressing cancer cells in vitro and in vivo

We investigated the cell‐killing efficacy of UV light on cancer cells expressing GFP in the nucleus and RFP in the cytoplasm (dual‐color cells). After exposure to various doses of UVA, UVB, or UVC, apoptotic and viable cells were quantitated under fluorescence microscopy using dual‐color 143B human osteosarcoma cells, HT‐1080 human fibrosarcoma cells, Lewis lung carcinoma (LLC), and XPA‐1 human pancreatic cancer cells in vitro. UV‐induced cancer cell death was wave‐length and dose dependent, as well as cell‐line dependent. After UVA exposure, most cells were viable even when the UV dose was increased up to 200 J/m². With UVB irradiation, cell death was observed with irradiation at 50 J/m². For UVC, as little as 25 J/m² UVC irradiation killed approximately 70% of the 143B dual‐color cells. This dose of UVB or UVA had almost no effect on the cancer cells. UV‐induced cancer cell death varied among the cell lines. Cell death began about 4 h after irradiation and continued until 10 h after irradiation. UVC exposure also suppressed cancer cell growth in nude mice in a model of minimal residual cancer (MRC). No apparent side effects of UVC exposure were observed. This study opens up the possibility of UVC treatment for MRC after surgical resection. J. Cell. Biochem. 110: 1439–1446, 2010. © 2010 Wiley‐Liss, Inc.     

Topology of Endoplasmic Reticulum‐Associated Cellular and Viral Proteins Determined with Split‐GFP

The split green fluorescent protein (GFP) system was adapted for investigation of the topology of ER‐associated proteins. A 215‐amino acid fragment of GFP (S1–10) was expressed in the cytoplasm as a free protein or fused to the N‐terminus of calnexin and in the ER as an intraluminal protein or fused to the C‐terminus of calnexin. A 16‐amino acid fragment of GFP (S11) was fused to the N‐ or C‐terminus of the target protein. Fluorescence occurred when both GFP fragments were in the same intracellular compartment. After validation with the cellular proteins PDI and tapasin, we investigated two vaccinia virus proteins (L2 and A30.5) of unknown topology that localize to the ER and are required for assembly of the viral membrane. Our results indicated that the N‐ and C‐termini of L2 faced the cytoplasmic and luminal sides of the ER, respectively. In contrast both the N‐ and C‐termini of A30.5 faced the cytoplasm. The system offers advantages for quickly determining the topology of intracellular proteins: the S11 tag is similar in length to commonly used epitope tags; multiple options are available for detecting fluorescence in live or fixed cells; transfection protocols are adaptable to numerous expression systems and can enable high throughput applications.      

UVA influenced the SIRT1‐miR‐27a‐5p‐SMAD2‐MMP1/COL1/BCL2 axis in human skin primary fibroblasts

Both SIRT1 and UVA radiation are involved in cellular damage processes such as apoptosis, senescence and ageing. MicroRNAs (miRNAs) have been reported to be closely related to UV radiation, as well as to SIRT1. In this study, we investigated the connections among SIRT1, UVA and miRNA in human skin primary fibroblasts. Our results showed that UVA altered the protein level of SIRT1 in a time point–dependent manner. Using miRNA microarray, bioinformatics analysis, we found that knocking down SIRT1 could cause up‐regulation of miR‐27a‐5p and the latter could down‐regulate SMAD2, and these results were verified by qRT‐PCR or Western blot. Furthermore, UVA radiation (5 J/cm²), knocking down SIRT1 or overexpression of miR‐27a‐5p led to increased expression of MMP1, and decreased expressions of COL1 and BCL2. We also found additive impacts on MMP1, COL1 and BCL2 under the combination of UVA radiation + Sirtinol (SIRT1 inhibitor), or UVA radiation + miR‐27a‐5p mimic. SIRT1 activator resveratrol could reverse damage changes caused by UVA radiation. Besides, absent of SIRT1 or overexpression of miR‐27a‐5p increased cell apoptosis and induced cell arrest in G2/M phase. Taken together, these results demonstrated that UVA could influence a novel SIRT1‐miR‐27a‐5p‐SMAD2‐MMP1/COL1/BCL2 axis in skin primary fibroblasts, and may provide potential therapeutic targets for UVA‐induced skin damage.     

p8/nupr1 regulates DNA‐repair activity after double‐strand gamma irradiation‐induced DNA damage

The stress protein p8 is a small, highly basic, unfolded, and multifunctional protein. We have previously shown that most of its functions are exerted through interactions with other proteins, whose activities are thereby enhanced or repressed. In this work we describe another example of such mechanism, by which p8 binds and negatively regulates MSL1, a histone acetyl transferase (HAT)‐associated protein, which in turn binds the DNA‐damage‐associated 53BP1 protein to facilitate DNA repair following DNA γ‐irradiation. Contrary to the HAT‐associated activity, MSL1‐dependent DNA‐repair activity is almost completely dependent on 53BP1 expression. The picture that has emerged from our findings is that 53BP1 could be a scaffold that gets the HAT MSL1‐dependent DNA‐repair activity to the sites of DNA damage. Finally, we also found that, although p8 expression is transiently activated after γ‐irradiation, it is eventually submitted to sustained down‐regulation, presumably to allow development of MSL1‐associated DNA‐repair activity. We conclude that interaction of MSL1 with 53BP1 brings MSL1‐dependent HAT activity to the vicinity of damaged DNA. MSL1‐dependent HAT activity, which is negatively regulated by the stress protein p8, induces chromatin remodeling and relaxation allowing access to DNA of the repair machinery. J. Cell. Physiol. 221: 594–602, 2009. © 2009 Wiley‐Liss, Inc.     

Paclitaxel and beta‐lapachone synergistically induce apoptosis in human retinoblastoma Y79 cells by downregulating the levels of phospho‐Akt

Paclitaxel (PTX) and beta‐lapachone (LPC) are naturally occurring compounds that have shown a large spectrum of anticancer activity. In this article we show for the first time that PTX/LPC combination induces potent synergistic apoptotic effects in human retinoblastoma Y79 cells. Combination of suboptimal doses of PTX (0.3 nM) and LPC (1.5 µM) caused biochemical and morphological signs of apoptosis at 48 h of treatment. These effects were accompanied by potent lowering in inhibitor of apoptosis proteins and by activation of Bid and caspases 3 and 6 with lamin B and PARP breakdown. PTX/LPC combination acted by favoring p53 stabilization through a lowering in p‐Akt levels and in ps166‐MDM2, the phosphorylated‐MDM2 form that enters the nucleus and induces p53 export and degradation. Treatment with wortmannin or transfection with a dominant negative form of Akt anticipated at 24 h the effects induced by PTX/LPC, suggesting a protective role against apoptosis played by Akt in Y79 cells. In line with these results, we demonstrated that Y79 cells contain constitutively active Akt, which forms a cytosolic complex with p53 and MDM2 driving p53 degradation. PTX/LPC treatment induced a weakness of Akt–MDM2–p53 complex and increased nuclear p53 levels. Our results suggest that phospho‐Akt lowering is at the root of the apoptotic action exerted by PTX/LPC combination and provide strong validation for a treatment approach that targets survival signals represented by phospho‐Akt and inhibitor of apoptosis proteins. J. Cell. Physiol. 222: 433–443, 2010. © 2009 Wiley‐Liss, Inc.