Platelet-Derived Growth Factor Receptor and Ionizing Radiation in High Grade Glioma Cell Lines.

Treatment of high grade gliomas (HGGs) has remained elusive due to their high heterogeneity and aggressiveness. Surgery followed by radiotherapy represents the mainstay of treatment for HGG. However, the unfavorable location of the tumor that usually limits total resection and the resistance to radiation therapy are the major therapeutic problems. Chemotherapy with DNA alkylating agent temozolomide is also used to treat HGG, despite modest effects on survival. Disregulation of several growth factor receptors (GFRs) were detected in HGG and receptor amplification in glioblastoma has been suggested to be responsible for heterogeneity propagation through clonal evolution. Molecularly targeted agents inhibiting these membrane proteins have demonstrated significant cytotoxicity in several types of cancer cells when tested in preclinical models. Platelet-derived growth factor receptors (PDGFRs) and associated signaling were found to be implicated in gliomagenesis, moreover, HGG commonly display a Platelet-derived growth factor (PDGF) autocrine pathway that is not present in normal brain tissues. We have previously shown that both the susceptibility towards PDGFR and the impact of the PDGFR inactivation on the radiation response were different in different HGG cell lines. Therefore, we decided to extend our investigation, using two other HGG cell lines that express PDGFR at the cell surface. Here, we investigated the effect of PDGFR inhibition alone or in combination with gamma radiation in 11 and 15 HGG cell lines. Our results showed that while targeting the PDGFR represents a good means of treatment in HGG, the combination of receptor inhibition with gamma radiation did not result in any discernable difference compared to the single treatment. The PI3K/PTEN/Akt/mTOR and Ras/Raf/MEK/ERK pathways are the major signaling pathways emerging from the GFRs, including PDGFR. Decreased sensitivity to radiation-induced cell death are often associated with redundancy in these pro-survival signaling pathways. Here we found that Phosphoinositide 3-kinases (PI3K), Extracellular-signal-regulated kinase 1/2 (ERK1/2), or c-Jun N-terminal kinase 1/2 (JNK1/2) inactivation induced radiosensitivity in HGG cells.

Shock wave treatment enhances endothelial proliferation via autocrine vascular endothelial growth factor.

Extracorporeal cardiac shock wave (SW) therapy is an effective, safe, and non-invasive therapeutic strategy for severe coronary artery disease. Shock wave therapy might affect cardiac tissues because of its ability to promote angiogenesis. In this report, we investigated if the up-regulation of vascular endothelial growth factor (VEGF) by SW therapy is involved in cell proliferation in cultured endothelial cells. After human umbilical vein endothelial cells were treated with SW, the expression and secretion of VEGF as well as cell proliferation were analyzed. We also determined the mechanism underlying SW-induced the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) using western blotting. Our results demonstrated that SW treatment induced VEGF expression in endothelial cells in a hypoxia-inducible factor 1-independent manner. Up-regulation of VEGF expression led to an increase in its concentration in the cultured medium. The autocrine VEGF in the medium activated the ERK MAPK signaling, which in turn enhanced cell proliferation. Therefore, we concluded that VEGF mediates SW application-induced endothelial cell proliferation in a cell-autonomous manner.

Ultraviolet B radiation-stimulated urocortin 1 is involved in tyrosinase-related protein 1 production in human melanoma HMV-II cells.

Ultraviolet B (UVB) radiation stimulates cutaneous melanin pigmentation. The melanosomal enzyme tyrosinase-related protein 1 (TRP1) is involved in the modulation of pigment production in response to this stressor. Recent molecular and biochemical analyses have revealed the presence of corticotropin-releasing factor (CRF) and urocortin 1 (Ucn1), together with their corresponding receptors, in mammalian skin. Although CRF and Ucn1 are thought to have potent effects on the skin system, their possible roles and regulations have yet to be determined fully. Our previous findings in human melanoma HMV-II cells suggest that both CRF and Ucn1 regulate TRP1 gene expression via Nurr-1/Nur77, transcription factors that constitute the nuclear receptor 4a subgroup of orphan nuclear receptors. HMV-II cells were found to express mainly Ucn1 mRNA. This study aimed to explore the effects of UVB on Ucn1 mRNA and TRP1 protein levels in HMV-II cells. UVB (30 mJ/cm(2)) increased Nurr-1, Nur77, and Ucn1 mRNA levels. UVB also increased TRP1 protein levels. Ucn1 knockdown inhibited the UVB-induced increases in TRP1 protein levels. These data suggest that UVB-stimulated Ucn1 contributes to TRP1 production via the transcription of both Nurr-1 and Nur77. Ucn1, produced in melanoma cells, acts on melanoma cells themselves in an autocrine manner.

Differential roles of ATF-2 in survival and DNA repair contributing to radioresistance induced by autocrine soluble factors in A549 lung cancer cells.

Radioresistance is one of the obstacles to the effective radiotherapy for non-small cell lung cancer. Soluble factors in the tumour microenvironment are often implicated in radioresistance but the underpinning mechanism(s) remain largely elusive. We herein studied the wholesome effect of autocrine cytokines and growth factors in the form of self-conditioned medium (CM) on the radiosensitivity of A549 cells. A549 cells grown in CM exhibited radioresistance which was associated with increased survival and DNA repair. CM induced pro-survival pathways through increased intracellular cAMP and phosphorylation of JNK and p38. Downstream to JNK/p38 signalling, ATF-2 phosphorylated at Thr69/71 was accompanied with its increased transcriptional activity in CM treated cells. Pre-treatment with cAMP inhibitor and silencing of ATF-2 abrogated the CM-induced survival. Interestingly, in cells treated with CM followed by radiation, ATF-2 was found to be switched over from transcription factor to DNA damage response protein. In CM treated cells, after γ-radiation p-ATF-2(Thr69/71) and subsequently the transcriptional activity of ATF-2 were declined with simultaneous rise in p-ATF-2(Ser490/498). Immunoprecipitation/immunoblotting and inhibitor studies showed that phosphorylation of ATF-2 at Ser490/498 was mediated by ATM. Moreover, p-ATF-2(Ser490/498) was found to be co-localised with γ-H2AX in DNA repair foci in CM-treated cells. The DNA repair activity of ATF-2 was assisted with higher activity MRN complex in cells grown in CM. Our study revealed that, autocrine soluble factors regulate dual but differential role of ATF-2 as a transcription factor or DNA repair protein, which collectively culminate in radioresistance of A549 cells.

Mathematical model of heterogeneous cancer growth with an autocrine signalling pathway.

OBJECTIVES: Cancer is a complex biological occurrence which is difficult to describe clearly and explain its growth development. As such, novel concepts, such as of heterogeneity and signalling pathways, grow exponentially and many mathematical models accommodating the latest knowledge have been proposed. Here, we present a simple mathematical model that exhibits many characteristics of experimental data, using prostate carcinoma cell spheroids under treatment. MATERIALS AND METHODS: We have modelled cancer as a two-subpopulation system, with one subpopulation representing a cancer stem cell state, and the other a normal cancer cell state. As a first approximation, these follow a logistical growth model with self and competing capacities, but they can transform into each other by using an autocrine signalling pathway. RESULTS AND CONCLUSION: By analysing regulation behaviour of each of the system parameters, we show that the model exhibits many characteristics of actual cancer growth curves. Features reproduced in this model include delayed phase of evolving cancer under 17AAG treatment, and bi-stable behaviour under treatment by irradiation. In addition, our interpretation of the system parameters corresponds well with known facts involving 17AAG treatment. This model may thus provide insight into some of the mechanisms behind cancer.

RXRα ablation in epidermal keratinocytes enhances UVR-induced DNA damage, apoptosis, and proliferation of keratinocytes and melanocytes.

We show here that keratinocytic nuclear receptor retinoid X receptor-α (RXRα) regulates mouse keratinocyte and melanocyte homeostasis following acute UVR. Keratinocytic RXRα has a protective role in UVR-induced keratinocyte and melanocyte proliferation/differentiation, oxidative stress-mediated DNA damage, and cellular apoptosis. We discovered that keratinocytic RXRα, in a cell-autonomous manner, regulates mitogenic growth responses in skin epidermis through secretion of heparin-binding EGF-like growth factor, GM-CSF, IL-1α, and cyclooxygenase-2 and activation of mitogen-activated protein kinase pathways. We identified altered expression of several keratinocyte-derived mitogenic paracrine growth factors such as endothelin 1, hepatocyte growth factor, α-melanocyte stimulating hormone, stem cell factor, and fibroblast growth factor-2 in skin of mice lacking RXRα in epidermal keratinocytes (RXRα(ep-/-) mice), which in a non-cell-autonomous manner modulated melanocyte proliferation and activation after UVR. RXRα(ep-/-) mice represent a unique animal model in which UVR induces melanocyte proliferation/activation in both epidermis and dermis. Considered together, the results of our study suggest that RXR antagonists, together with inhibitors of cell proliferation, can be effective in preventing solar UVR-induced photocarcinogenesis.

PGE(2) is a UVR-inducible autocrine factor for human melanocytes that stimulates tyrosinase activation.

Prostaglandins activate signalling pathways involved in growth, differentiation and apoptosis. Prostaglandin E(2) (PGE(2)) is released by keratinocytes following ultraviolet irradiation (UVR) and stimulates the formation of dendrites in melanocytes. We show that multiple irradiations of human melanocytes with UVR-activated cPLA(2), the rate-limiting enzyme in eicosanoid synthesis and stimulated PGE(2) secretion. PGE(2) increased cAMP production, tyrosinase activity and proliferation in melanocytes. PGE(2) binds to four distinct G-protein coupled receptors (EP(1-4)). We show that PGE(2) stimulates EP(4) receptor signalling in melanocytes, resulting in cAMP production. Conversely, PGE(2) also stimulated the EP(3) receptor in melanocytes, resulting in lowered basal cAMP levels. These data suggest that relative levels or activity of these receptors controls effects of PGE(2) on cAMP in melanocytes. The data are the first to identify PGE(2) as an UVR-inducible autocrine factor for melanocytes. These data also show that PGE(2) activates EP(3) and EP(4) receptor signalling, resulting in opposing effects on cAMP production, a critical signalling pathway that regulates proliferation and melanogenesis in melanocytes.

Involvement of purinergic signaling in cellular response to gamma radiation.

Recent studies have suggested a bystander effect in nonirradiated cells adjacent to irradiated cells; however, the mechanism is poorly understood. In this study, we investigated the involvement of both extracellular nucleotides and activation of P2 receptors in cellular responses to gamma radiation using human HaCaT keratinocytes. The concentration of ATP in culture medium was increased after gamma irradiation (0.1-1.0 Gy), suggesting that radiation induces ATP release from cells. Intracellular Ca(2+) concentration was elevated when conditioned medium from irradiated cells was transferred to nonirradiated cells, and this elevation was suppressed by apyrase (ecto-nucleotidase), indicating the involvement of extracellular nucleotides in this event. Further, we examined the activation of ERK1/2 by gamma radiation and nucleotides (ATP and UTP). Both gamma radiation and nucleotides induced activation of ERK1/2. Next, the effect of inhibitors of P2 receptors on radiation-induced activation of ERK1/2 was examined. The activation of ERK1/2 was blocked by suramin (P2Y inhibitor), MRS2578 (P2Y(6) antagonist) and apyrase. These results suggest that both released nucleotides and activation of P2Y receptors are involved in gamma-radiation-induced activation of ERK1/2. We conclude that ionizing radiation induces release of nucleotides from cells, leading to activation of P2Y receptors, which in turn would result in a variety of biological effects.

Retinal neuroprotection by hypoxic preconditioning is independent of hypoxia-inducible factor-1 alpha expression in photoreceptors.

Hypoxic preconditioning stabilizes hypoxia-inducible factor (HIF) 1 alpha in the retina and protects photoreceptors against light-induced cell death. HIF-1 alpha is one of the major transcription factors responding to low oxygen tension and can differentially regulate a large number of target genes. To analyse whether photoreceptor-specific expression of HIF-1 alpha is essential to protect photoreceptors by hypoxic preconditioning, we knocked down expression of HIF-1 alpha specifically in photoreceptor cells, using the cyclization recombinase (Cre)-lox system. The Cre-mediated knockdown caused a 20-fold reduced expression of Hif-1 alpha in the photoreceptor cell layer. In the total retina, RNA expression was reduced by 65%, and hypoxic preconditioning led to only a small increase in HIF-1 alpha protein levels. Accordingly, HIF-1 target gene expression after hypoxia was significantly diminished. Retinas of Hif-1 alpha knockdown animals did not show any pathological alterations, and tolerated hypoxic exposure in a comparable way to wild-type retinas. Importantly, the strong neuroprotective effect of hypoxic preconditioning against light-induced photoreceptor degeneration persisted in knockdown mice, suggesting that hypoxia-mediated survival of light exposure does not depend on an autocrine action of HIF-1 alpha in photoreceptor cells. Hypoxia-mediated stabilization of HIF-2 alpha and phosphorylation of signal transducer and activator of transcription 3 (STAT 3) were not affected in the retinas of Hif-1 alpha knockdown mice. Thus, these factors are candidates for regulating the resistance of photoreceptors to light damage after hypoxic preconditioning, along with several potentially neuroprotective genes that were similarly induced in hypoxic knockdown and control mice.

A 60-Hz sinusoidal magnetic field induces apoptosis of prostate cancer cells through reactive oxygen species.

PURPOSE: To explore the effects of power frequency magnetic fields (MF) on cell growth in prostate cancer, DU145, PC3, and LNCaP cells were examined in vitro. MATERIALS AND METHODS: The cells were exposed to various intensities and durations of 60-Hz sinusoidal MF in combination with various serum concentrations in the media. To analyze MF effects on cell growth, cell counting, trypan blue exclusion assay, Western blot analysis, flow cytometry, enzyme-linked immunosorbent assay (ELISA), semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), fluorescence microscopy, and spectrofluorometry were used. RESULTS: MF exposure induced significant cell growth inhibition and apoptosis in an intensity- and time-dependent manner, in which cell cycle arrest, cleaved Caspase-3, and reactive oxygen species (ROS) increased. Pretreatment with a Caspase-3 inhibitor or antioxidant, N-acetyl-L-cysteine (NAC), significantly attenuated MF-induced cell growth inhibition and cell death. Media replacement experiments failed to show any notable change in the MF effects. CONCLUSIONS: These results demonstrate 60-Hz sinusoidal MF-activated cell growth inhibition of prostate cancer in vitro. Apoptosis together with cell cycle arrest were the dominant causes of the MF-elicited cell growth inhibition, mediated by MF-induced ROS. These results suggest that a possibility of using 60-Hz MF in radiation therapy of prostate cancer could usefully be investigated.

Identification of PP2A as a crucial regulator of the NF-kappaB feedback loop: its inhibition by UVB turns NF-kappaB into a pro-apoptotic factor.

Although nuclear factor-kappaB (NF-kappaB) usually exerts anti-apoptotic activity, upon activation by interleukin-1 (IL-1) it enhances ultraviolet-B radiation (UVB)-induced apoptosis. This paradoxical effect is associated with NF-kappaB-dependent pronounced secretion of tumour necrosis factor-alpha (TNF) which activates TNF-R1 in an autocrine fashion to enhance UVB-induced apoptosis. We demonstrate that sustained TNF transcription in UVB+IL-1-treated cells involves complete abrogation of the negative feedback loop of NF-kappaB preventing IkappaBalpha resynthesis, hence allowing uncontrolled NF-kappaB activity. We show that IkappaBalpha is not transcriptionally inhibited but resynthesized protein is immediately marked for degradation due to persistent inhibitor of kappaB kinasebeta (IKKbeta) activity. Continuous IKKbeta phosphorylation and activation is caused by UVB-mediated inhibition of the phosphatase PP2A. This study demonstrates that the cellular response to different NF-kappaB activators may be converted to the opposite reaction when both stimuli act in concert. Our data shed new light on the significance of negative feedback regulation of NF-kappaB and identifies PP2A as the key regulator of this process.

Low dose radiation and intercellular induction of apoptosis: potential implications for the control of oncogenesis.

PURPOSE: This review is focused on the potential impact of low dose radiation effects on intercellular induction of apoptosis and the underlying reactive-oxygen species (ROS)-mediated signaling pathways. RESULTS: Transformed cells are subject to ROS-mediated apoptosis induction by non-transformed cells ('intercellular induction of apoptosis') and by ROS-mediated autocrine self-destruction. Sensitivity to intercellular induction of apoptosis and autocrine self-destruction are strictly correlated to the expression of the transformed state. Extracellular superoxide anions generated by transformed target cells drive the selectivity and sensitivity of this signaling system which is based on four different signaling pathways. Low dose irradiation of non-transformed cells enhances intercellular induction of apoptosis in transformed cells. This process is controlled by TGF-beta and seems to depend on the induction of peroxidase release. In addition, low dose radiation enhances superoxide anion generation of transformed target cells. CONCLUSIONS: Low dose radiation-triggered enhancement of intercellular induction of apoptosis and autocrine self-destruction might represent a potential control system during carcinogenesis. It might be the underlying mechanism for the well-known inhibitory effect of low dose radiation on detectable transformation events. However, modifications of the complex intercellular ROS-based signaling system may also lead to configurations in which low dose radiation attenuates ROS-mediated apoptosis induction.

Ultraviolet-B irradiation enhances melanoma cell motility via induction of autocrine interleukin 8 secretion.

Ultraviolet radiation (UVR) is known to be involved in the initiation and progression of malignant melanoma. Many studies have focused on the initiation of melanoma, but less is known about the effect of UVR on established tumor cells. Here, we show that after ultraviolet-B (UVB) irradiation, melanoma cells (MM) are able to secrete autocrine factors that enhance their motility. Time-lapse videomicroscopy of UVB irradiated (15 or 30 mJ/cm(2)) MM showed an initial decrease in MM cell motility one hour after irradiation, with subsequent increase 24 h after UV-B treatment. Conditioned media harvested from MM 24 h following UV-B irradiation specifically enhanced the motility of un-irradiated MM, suggesting that a newly synthesized soluble factor released by UVB MM is involved. As interleukin 8 (IL-8) is known to be up-regulated by different cell types after UV-B irradiation, we investigated IL-8 expression after UVB exposure. Quantitative RT-PCR and ELISA demonstrated an induction of IL-8 in MM by UVB (15 or 30 mJ/cm(2)), and addition of recombinant IL-8 to cell cultures enhanced cell motility to a similar degree than UVB. Importantly, blocking IL-8 activity by a neutralizing anti IL-8 antibody inhibited the up-regulation of MM motility after UVB treatment. We conclude that UVB enhances MM motility and that this effect is mediated at least in part by IL-8 released by MM in an autocrine fashion. Our findings are consistent with the hypothesis that UVB is not only involved in the initiation of melanoma, but may also be important for some aspects of tumor progression.

Effects of PGF2alpha on human melanocytes and regulation of the FP receptor by ultraviolet radiation.

Prostaglandins are potent lipid hormones that activate multiple signaling pathways resulting in regulation of cellular growth, differentiation, and apoptosis. In the skin, prostaglandins are rapidly released by keratinocytes following ultraviolet radiation and are chronically present in inflammatory skin lesions. We have shown previously that melanocytes, which provide photoprotection to keratinocytes through the production of melanin, express several receptors for prostaglandins, including the PGE2 receptors EP1 and EP3 and the PGF2alpha receptor FP, and that PGF2alpha stimulates melanocyte dendricity. We now show that PGF2alpha stimulates the activity and expression of tyrosinase, the rate-limiting enzyme in melanin synthesis. Analysis of FP receptor regulation showed that the FP receptor is regulated by ultraviolet radiation in melanocytes in vitro and in human skin in vivo. We also show that ultraviolet irradiation stimulates production of PGF2alpha by melanocytes. These results show that PGF2alpha binding to the FP receptor activates signals that stimulate a differentiated phenotype (dendricity and pigmentation) in melanocytes. The regulation of the FP receptor and the stimulation of production of PGF2alpha in melanocytes in response to ultraviolet radiation suggest that PGF2alpha could act as an autocrine factor for melanocyte differentiation.

Autocrine pathways of the vascular endothelial growth factor (VEGF) in glioblastoma multiforme: clinical relevance of radiation-induced increase of VEGF levels.

In tumour-induced angiogenesis of gliomas, vascular endothelial growth factor (VEGF) and its receptors fms-like tyrosine kinase (Flt-1) and kinase-insert-domain-containing receptor (KDR) play a major role and are promising targets for tumour therapy. Nevertheless, preliminary results of such therapies could not prove clinical efficacy and thus make a profound knowledge of VEGF regulation essential. Based on earlier results, which demonstrated an inhibitory influence of VEGF on Flt-1-expressing glioblastoma cells, in the present study we focused on the extent of VEGF and VEGF receptor coexpression and possible therapeutical consequences. Protein expression of VEGF, Flt-1 and KDR was analysed by immunohistochemistry in native tumour tissues of 63 glioblastomas. VEGF could be detected in all glioblastomas. Additionally and independently to the expected Flt-1 and KDR expression in tumour endothelia, we found a coexpression of VEGF with Flt-1 in tumour cells of 46 and with KDR in 45 glioblastomas. After exposure of glioblastoma cells to X-ray radiation we observed a strong dose-dependent increase of VEGF secretion in two glioblastoma cell cultures by up to 46% and 96%, respectively that originated from an increased VEGF mRNA expression. In contrast, under the same conditions secretion of HGF/SF was only slightly elevated and bFGF despite being strongly increased remained at very low overall amounts compared to VEGF. Based on previous data on an autocrine function of VEGF in Flt-1-expressing glioblastoma cells we hypothesise that the X-ray radiation induced upregulation of VEGF might result in a downregulation of tumour cell proliferation and thus lead to a reduced sensitivity to radiation therapy. Therefore our results support the idea that a combination of anti-VEGF and radiation therapy might prove a promising new option in fighting against one of the most fatal tumour types.

The UV (Ribotoxic) stress response of human keratinocytes involves the unexpected uncoupling of the Ras-extracellular signal-regulated kinase signaling cascade from the activated epidermal growth factor receptor.

In mammals, UVB radiation is of biological relevance primarily for the cells of the epidermis. We report here the existence of a UVB response that is specific for proliferating human epidermal keratinocytes. Unlike other cell types that also display a UVB response, keratinocytes respond to UVB irradiation with a transient but potent downregulation of the Ras-extracellular signal-regulated kinase (ERK) signaling cascade. The downregulation of ERK precedes a profound decrease in the steady-state levels of cyclin D1, a mediator of the proliferative action of ERK. Keratinocytes exhibit high constitutive activity of the Ras-ERK signaling cascade even in culture medium lacking supplemental growth factors. The increased activity of Ras and phosphorylation of ERK in these cells are maintained by the autocrine production of secreted molecules that activate the epidermal growth factor receptor (EGFR). Irradiation of keratinocytes increases the phosphorylation of EGFR on tyrosine residues Y845, Y992, Y1045, Y1068, Y1086, Y1148, and Y1173 above the basal levels and leads to the increased recruitment of the adaptor proteins Grb2 and ShcA and of a p55 form of the regulatory subunit of the phosphatidylinositide 3-kinase to the UVB-activated EGFR. Paradoxically, however, UVB causes, at the same time, the inactivation of Ras and a subsequent dephosphorylation of ERK. By contrast, the signaling pathway leading from the activated EGFR to the phosphorylation of PKB/Akt1 is potentiated by UVB. The UVB response of keratinocytes appeared to be a manifestation of the more general ribotoxic stress response inasmuch as the transduction of the UVB-generated inhibitory signal to Ras and ERK required the presence of active ribosomes at the time of irradiation.

The first peak of the UVB irradiation-dependent biphasic induction of vascular endothelial growth factor (VEGF) is due to phosphorylation of the epidermal growth factor receptor and independent of autocrine transforming growth factor alpha.

Ultraviolet B (UVB) irradiation, the major damaging component of sunlight, has earlier been reported to enhance cutaneous angiogenesis in chronically sun-exposed skin. We herein provide first evidence for a biphasic induction of the vascular endothelial growth factor (VEGF) following UVB irradiation of the human epidermal cell line HaCaT. The first VEGF peak occurred on mRNA level at 1 h and on protein level at 4 h postirradiation and is fully mediated by the UVB-dependent phosphorylation of the epidermal growth factor receptor, which subsequent to its phosphorylation also initiates at least in part the synthesis of transforming growth factor alpha that confers as shown previously the second late VEGF peak at 8 h on mRNA and at 24 h on protein level.