The ATM inhibitor KU55933 sensitizes radioresistant bladder cancer cells with DAB2IP gene defect

Abstract

Purpose: Our preliminary results showed that differentially expressed in ovarian cancer-2/disabled homolog 2 (DOC-2/DAB2) interactive protein (DAB2IP), a putative tumor suppressor gene, is down-regulated in bladder cancer (BCa) with aggressive phenotypes. In this study, we investigated how DAB2IP knockdown influenced BCa cell response to ionizing radiation (IR) and discussed possible ways to enhance cell radiosensitivity.

Methods and materials: The small interfering RNA (siRNA) system was implemented to inhibit endogenous DAB2IP expression in two human BCa cell lines, T24 and 5637. Cell sensitivity to IR alone or combined treatment was measured by a colony formation assay (CFA). Western blot was used to determine the phosphorylation levels of ataxia-telangiectasia mutated (ATM), catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) and related DNA damage repair (DDR) proteins. Immunofluorescence as well as a flow cytometry assay were employed to detect DNA double-strand break (DSB) repair and cell cycle distribution, respectively.

Results: DAB2IP-knockdown of BCa cells (i.e., siDAB2IP) exhibit increased clonogenic survival in response to IR compared with control cells (i.e., siCON) expressing an endogenous level of DAB2IP. The mechanism in siDAB2IP cells could be explained by elevated ATM expression and activation, increased S phase cell distribution as well as faster DSB repair kinetics. 2-morpholin-4-yl-6-thianthren-1-yl-pyran-4-one (KU55933) significantly sensitized siDAB2IP cells to IR due to inhibition of the phosphorylation of ATM and its downstream targets following IR and slower DSB repair kinetics.

Conclusions: Loss of DAB2IP expression in BCa cells signifies their radioresistance. KU55933, which suppresses ATM phosphorylation upon irradiation, could be applied in the radiotherapy of BCa patients with a DAB2IP gene defect.     

Ionizing radiation causes active degradation and reduces matrix synthesis in articular cartilage

Abstract

Purpose: Little is known regarding radiation effects on adult articular (joint) cartilage, though joint damage has been reported following cancer treatment or occupational exposures. The aim of this study was to determine if radiation can reduce cartilage matrix production, induce cartilage degradation, or interfere with the anabolic effects of IGF-1.

Materials and methods: Isolated chondrocytes cultured in monolayers and whole explants harvested from ankles of human donors and knees of pigs were irradiated with 2 or 10 Gy γ-rays, with or without IGF-1 stimulation. Proteoglycan synthesis and IGF-1 signaling were examined at Day 1; cartilage degradation throughout the first 96 hours.

Results: Human and pig cartilage responded similarly to radiation. Cell viability was unchanged. Basal and IGF-1 stimulated proteoglycan synthesis was reduced following exposure, particularly following 10 Gy. Both doses decreased IGF-induced Akt activation and IGF-1 receptor phosphorylation. Matrix metalloproteinases (ADAMTS5, MMP-1, and MMP-13) and proteoglycans were released into media after 2 and 10 Gy.

Conclusions: Radiation induced an active degradation of cartilage, reduced proteoglycan synthesis, and impaired IGF-1 signaling in human and pig chondrocytes. Lowered Akt activation could account for decreased matrix synthesis. Radiation may cause a functional decline of cartilage health in joints after exposure, contributing to arthropathy.     

PARP inhibitor attenuated colony formation can be restored by MAP kinase inhibitors in different irradiated cancer cell lines

Abstract

Purpose: Sensitizing cancer cells to irradiation is a major challenge in clinical oncology. We aimed to define the signal transduction pathways involved in poly(ADP-ribose) polymerase (PARP) inhibitor-induced radiosensitization in various mammalian cancer lines.

Materials and methods: Clonogenic survival assays and Western blot examinations were performed following telecobalt irradiation of cancer cells in the presence or absence of various combinations of PARP- and selective mitogen-activated protein kinase (MAPK) inhibitors.

Results: HO3089 resulted in significant cytotoxicity when combined with irradiation. In human U251 glioblastoma and A549 lung cancer cell lines, Erk1/2 and JNK/SAPK were found to mediate this effect of HO3089 since inhibitors of these kinases ameliorated it. In murine 4T1 breast cancer cell line, p38 MAPK rather than Erk1/2 or JNK/SAPK was identified as the main mediator of HO3089's radiosensitizing effect. Besides the aforementioned changes in kinase signaling, we detected increased p53, unchanged Bax and decreased Bcl-2 expression in the A549 cell line.

Conclusions: HO3089 sensitizes cancer cells to photon irradiation via proapoptotic processes where p53 plays a crucial role. Activation of MAPK pathways is regarded the consequence of irradiation-induced DNA damage, thus their inhibition can counteract the radiosenzitizing effect of the PARP inhibitor.     

Farnesyltransferase inhibitors as radiation sensitizers

Purpose: The inhibition of activated Ras combined with radiotherapy was identified as a potential method for radiosensitization.

Materials and methods: Immunoblotting was used to control for prenylation inhibition of the respective Ras isoforms and for changes in activity of downstream proteins. Clonogenic assays with human and rodent tumour cell lines and transfected cell lines served for the testing of radiosensitivity. Xenograft tumours were treated with farnesyl transferase inhibitors and radiation and assayed for ex vivo plating efficiency, regrowth of tumours and EF5 staining for detection of hypoxia. Concurrent treatment with L‐778,123 and radiotherapy was performed in non‐small cell lung cancer (NSCLC) and head and neck cancer (HNC) patients.

Results: Blocking the prenylation of Ras proteins in cell lines with Ras activated by mutations or receptor signalling resulted in radiation sensitization in vitro and in vivo. The PI3 kinase downstream pathway was identified as a contributor to Ras‐mediated radiation resistance. Additionally, increased oxygenation of xenograft tumours was observed after FTI treatment. Combined treatment in a phase I study was safe and effective in NSCLC and HNC.

Conclusions: Tumour cells with activated Ras were sensitized to radiation. Unravelling the underlying mechanisms promises to lead to even more specific drugs with higher potency and safety.     

Famitinib enhances nasopharyngeal cancer cell radiosensitivity by attenuating radiation-induced phosphorylation of platelet-derived growth factor receptor and c-kit and inhibiting microvessel formation

Purpose: Famitinib is a novel tyrosine kinase inhibitor. We investigated the effects of famitinib on the radiosensitivity of human nasopharyngeal carcinoma (NPC) cell radiosensitivity in vitro and in vivo, and explored its possible mechanisms.

Materials and methods: Human nasopharyngeal carcinoma cell line (CNE-2) were treated with famitinib and radiation, and analyzed by3-(4,5-dimethylthaizol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), clonogenic survival assay, and Western blot. A xenograft model using CNE-2 cells was established to analyze the effects of famitinib and radiation on tumor volume and microvessel density (MVD).

Results: Famitinib dose-dependently inhibited CNE-2 cells growth and significantly reduced clonogenic survival (p < 0.05), with a sensitivity enhancement ratio (SER) of 1.45. The tumor inhibition rate of the combined treatment group was 91%, which was significantly higher than the radiation group (35%, p < 0.05) and famitinib group (46%, p < 0.05). Famitinib attenuated radiation-induced phosphorylation of the platelet-derived growth factor receptor (PDGFR) and stem cell factor (c-kit) at 0, 30, 60 min after radiation treatment. Furthermore, radiation combined with famitinib decreased tumor MVD (p < 0.05).

Conclusions: Famitinib significantly increased CNE-2 cell radiosensitivity in vitro and in vivo by attenuating radiation-induced PDGFR and c-kit phosphorylation and by inhibiting microvessel formation.     

alpha6 Integrin Cleavage: Sensitizing human prostate cancer to ionizing radiation

Purpose: The goal was to determine if prostate tumor cells containing a mutant α6 integrin would be defective in tumor re-population following clinically relevant fractionated ionizing radiation (IR) treatments.

Material and methods: Human prostate cancer cells derived from PC3N cells were used which conditionally expressed a cleavable, wild type form of α6 integrin (PC3N-α6-WT) or a mutated non-cleavable form of α6 integrin (PC3N-α6-RR). The resulting tumor growth before, during and after fractionated doses of IR (3 Gy×10 days) was analyzed using the endpoints of tumor growth inhibition (T/C), tumor growth delay (T-C), tumor doubling time (Td) and tumor cell kill (Log₁₀ cell kill).

Results: The T/C values were 36.1% and 39.5%, the T-C values were 20.5 days and 28.5 days and the Td values were 5.5 and 10.5 days for the irradiated PC3N-α6-WT and PC3N-α6-RR cells, respectively. The Log₁₀ was 1.1 for the PC3N-α6-WT cells and 0.8 for the PC3N-α6-RR cells. The tumor response to IR was altered in tumors expressing the mutant α6 integrin as indicated by a significant increase in tumor growth inhibition, an increase in tumor growth delay, an increase in tumor doubling time and an increase in tumor cell kill.

Conclusions: Blocking integrin cleavage in vivo may be efficacious for increasing the IR responsiveness of slow growing, pro-metastatic human prostate cancer.     

Valproic acid modulates radiation-enhanced matrix metalloproteinase activity and invasion of breast cancer cells

Abstract

Purpose: To evaluate matrix metalloproteinase (MMP) activity and invasion after ionizing radiation (IR) exposure and to determine whether MMP could be epigenetically modulated by histone deacetylase (HDAC) inhibition.

Material and methods: Two human breast cancer cell lines (MDA-MB-231 and MCF-7) were cultured in monolayer (2D) and in laminin-rich extracellular matrix (3D). Invasion capability, collagenolytic and gelatinolytic activity, MMP and TIMP protein and mRNA expression and clonogenic survival were analyzed after IR exposure, with and without a HDAC inhibition treatment [1.5?mM valproic acid (VA) or 1?μM trichostatin-A (TSA)].

Results: IR exposure resulted in cell line-dependent stimulation of invasion capacity. In contrast to MCF-7 cells, irradiated MDA-MB-231 showed significantly enhanced mRNA expression of mmp-1, mmp-3 and mmp-13 and of their regulators timp-1 and timp-2 relative to unirradiated controls. This translated into increased collagenolytic and gelatinolytic activity and could be reduced after valproic acid (VA) treatment. Additionally, VA also mitigated IR-enhanced mmp and timp mRNA expression as well as IR-increased invasion capability. Finally, our data confirm the radiosensitizing effect of VA.

Conclusion: These results suggest that IR cell line-dependently induces upregulation of MMP mRNA expression, which appears to be mechanistically linked to a higher invasion capability that is modifiable by HDAC inhibition.     

Role of the bradykinin B2 receptor in a rat model of local heart irradiation

Abstract

Purpose: Radiation-induced heart disease (RIHD) is a delayed effect of radiotherapy for cancers of the chest, such as breast, esophageal, and lung. Kinins are small peptides with cardioprotective properties. We previously used a rat model that lacks the precursor kininogen to demonstrate that kinins are involved in RIHD. Here, we examined the role of the kinin B2 receptor (B2R) in early radiation-induced signaling in the heart.

Materials and methods: Male Brown Norway rats received the B2R-selective antagonist HOE-140 (icatibant) via osmotic minipump from 5 days before until 4 weeks after 21 Gy local heart irradiation. At 4 weeks, signaling events were measured in left ventricular homogenates and nuclear extracts using western blotting and real-time polymerase chain reaction. Numbers of CD68-positive (monocytes/macrophages), CD2-positive (T-lymphocytes), and mast cells were measured using immunohistochemistry.

Results: Radiation-induced c-Jun phosphorylation and nuclear translocation were enhanced by HOE-140. HOE-140 did not modify endothelial nitric oxide synthase (eNOS) phosphorylation or alter numbers of CD2-positive or mast cells, but enhanced CD68-positive cell counts in irradiated hearts.

Conclusions: B2R signaling may regulate monocyte/macrophage infiltration and c-Jun signals in the irradiated heart. Although eNOS is a main target for kinins, the B2R may not regulate eNOS phosphorylation in response to radiation.     

New rationales for using TGFbeta inhibitors in radiotherapy

Purpose: The first reports that ionizing radiation (IR) induces rapid and persistent activation of transforming growth factor β1 (TGFβ) were nearly two decades ago. Subsequent studies have shown that TGFβ is a major mediator of cellular and tissue responses to IR and have revealed novel facets of its complex biology.

Results: We and others have recently shown that inhibition of production or signaling of TGFβ in epithelial cells modulates radiosensitivity and impedes activation of the DNA damage response program. The primary transducer of cellular response to DNA damage caused by ionizing radiation is the nuclear protein kinase ataxia telangiectasia mutated, whose activity is severely compromised when TGFβ is inhibited. Thus, in conjunction, with its well-recognized contribution to normal tissue fibrosis, the role of TGFβ in the genotoxic stress program provides a previously unsuspected avenue to modulate radiotherapy.

Conclusions: We hypothesize that identification of the circumstances and tumors in which TGFβ manipulation enhances tumor cell radiosensitivity, while protecting normal tissues, could significantly increase therapeutic index.     

List of reviewers

Abstract

Purpose: Perturbations in protein folding induce endoplasmic reticulum (ER) stress, which elicits coordinated response, namely the unfolded protein response (UPR), to cope with the accumulation of misfolded proteins in ER. In this study, we characterized mechanisms underlying ionizing radiation (IR)-induced UPR signaling pathways.

Materials and methods: We analyzed alterations in UPR signaling pathways in human umbilical vein endothelial cells (HUVEC) and human coronary artery endothelial cells (HCAEC) irradiated with 15 Gy IR.

Results: IR selectively activated the eIF2α/ATF4 branch of the UPR signaling pathway, with no alterations in the IRE1 and ATF6 branches in HUVEC and HCAEC. Phosphorylation of PERK was enhanced in response to IR, and the IR-induced activation of the eIF2α/ATF4 signaling pathway was completely inhibited by PERK knockdown with siRNA. Surprisingly, chemical chaperones, which inhibit the formation of misfolded proteins and sequential protein aggregates to reduce ER stress, failed to prevent the IR-induced phosphorylation of PERK and the subsequent activation of the eIF2α/ATF4 signaling pathway.

Conclusions: PERK mediates the IR-induced selective activation of the eIF2α/ATF4 signaling pathway, and the IR-induced activation of PERK/eIF2α/ATF4 signaling in human vascular endothelial cells is independent of alterations in protein-folding homeostasis in the ER.     

Ionizing radiation modules of the expression and tyrosine phosphorylation of the focal adhesion‐associated proteins focal adhesion kinase (FAK) and its substrates p130cas and paxillin in A549 human lung carcinoma cells in vitro

Purpose: Focal adhesion kinase (FAK) is involved in the regulation of many cellular processes, including cell survival and death, proliferation and migration. The same endpoints are influenced by ionizing radiation (IR). Therefore, study was performed to determine the effect of IR on the expression and phosphorylation of FAK and two of its substrates, p130cas and paxillin, in vitro.

Materials and methods: Exponentially growing A549 lung carcinoma cells were exposed to 6?Gy X‐rays. Protein expression and the extent of tyrosine phosphorylation were investigated by immunoprecipitation experiments and Western blotting analysis using specific or unspecific phosphotyrosine antibodies. Immunofluorescence staining in combination with confocal laser scanning microscopy was done to localize the proteins within the cell.

Results: Tyrosine phosphorylation, of M_r 110?000–150?000 and 65?000–75?000 protein bands, was induced within 30?min after exposure to IR. Three of these proteins were identified as FAK, p130cas and paxillin. IR induced phosphorylation of FAK (tyr397 and tyr925) but did not change FAK expression. Additionally, IR induced phosphorylation of paxillin (tyr31 and tyr181) within 30?min and an up‐regulation of paxillin expression 2–6?h after exposure. Furthermore, a higher amount of phosphorylated p130cas was found in irradiated cells. Immunofluorescence staining demonstrated that in A549 cells, all three proteins colocalize at sites of focal adhesions at the cytoplasmic face of the cell membrane and to lamellopodia.

Conclusions: The data indicate that these focal adhesion‐associated proteins are modulated by IR and thus are likely to play a role in the cellular response to IR. These proteins might represent attractive targets to modulate FAK‐initiated signalling pathways, which may be involved in improved radioresistance and, furthermore, in important pathological phenomena such as tumour growth and metastatic phenotypes.     

Radiation-induced molecular changes in rat mammary tissue: Possible implications for radiation-induced carcinogenesis

Purpose:?Ionizing radiation is a potent mammary gland carcinogen, yet the exact molecular etiology of radiation-induced breast cancer remains unknown.

Materials and methods:?Our study utilized a rat model of breast carcinogenesis to analyse the molecular and epigenetic changes induced in mammary gland tissue upon exposure to ionizing radiation (IR). Using a methylation-sensitive cytosine extension assay we studied the IR-induced changes in DNA methylation. In parallel, we analysed the expression of proteins involved in DNA methylation, DNA repair and cell proliferation control. Molecular changes were related to cellular proliferation and apoptosis.

Results:?We found that IR led to a loss of genomic cytosine methylation in the exposed mammary tissue. Global DNA hypomethylation was paralleled by reduction in the levels of maintenance (DNMT1) and de novo (DNMT3a and 3b) DNA methyltransferases and methyl-binding protein MeCP2. The observed DNA hypomethylation was linked, at least in part, to activation of DNA repair processes. Concurrently, we observed increased levels of phosphorylated extracellular signal-regulated kinase (p-ERK1/2), phosphorylated AKT kinase (p-AKT), cyclin D1 and proliferating cells nuclear antigen (PCNA) proteins, suggesting IR alters intra-cellular signaling and cell cycle control mechanisms in mammary tissue. We also noted a significant induction of apoptosis in the exposed tissue 6 hours after irradiation. The observed apoptosis levels were paralleled by the slight elevation of cellular proliferation.

Conclusions:?We have demonstrated that a single exposure to 5 Gy of X rays leads to noticeable epigenetic changes in the rat mammary gland that occurred in the context of activation of DNA damage repair and alterations in the pro-survival growth-stimulatory cellular signaling pathways. The possible cellular repercussions of the observed changes in relationship to breast carcinogenesis are discussed.     

Association of ribosomal protein S6 kinase 1 with cellular radiosensitivity of non-small lung cancer

Purpose: Ribosomal S6 kinase 1 (S6K1) plays an important role in cell proliferation, protein translation and cell survival. This study investigated the possibility of using S6K1 as a new target in the radiotherapy of non-small cell lung cancer (NSCLC) and its potential mechanism.

Materials and methods: shRNA interference technology was applied to silence the expression of S6K1 in A549 and H460, and clonogenic assay was performed to measure the radiosensitizing effects. DNA repair was monitored by γH2AX foci formation. Cell cycle and apoptosis were measured by flow cytometry assays. Protein expression was analyzed by Western blotting. S6K1 and lentivirus containing programmed cell death 4 (PDCD4), a substrate of S6K1, were co-transfected into cells and radiosensitivity was also detected.

Results: S6K1 knockdown significantly enhanced the radiosensitivity of NSCLC cells. The notable findings in response to this combined treatment were prolonged delay in radiation-induced DNA DSB repair, robust G2/M checkpoint arrest, increased apoptosis and upregulation of PDCD4. Moreover, PDCD4 knockdown reversed the radiosensitizing effects of S6K1 inhibition in NSCLC.

Conclusions: S6K1 is a promising tumor-specific target for the enhancement of NSCLC radiosensitivity and its effects may be mediated by increased expression of PDCD4.     

Pharmacological inhibition of EGFR tyrosine kinase affects ILK-mediated cellular radiosensitization in vitro

Purpose: Integrin-linked kinase (ILK) mediates signals from β integrins and links integrins to epidermal growth factor receptor (EGFR). Previous studies have identified an antisurvival effect of ILK in irradiated cells. The aim of this study was to evaluate the role of EGFR tyrosine kinase (tk) activity for ILK-mediated radiosensitization.

Materials and methods: Human FaDu squamous cell carcinoma (SCC) cells stably transfected with hyperactive ILK (ILK-hk) and ILK^fl/fl and ILK^?/? mouse fibroblasts were treated with the pharmacological EGFR-tk inhibitor BIBX1382BS without or in combination with single doses of X-rays. Clonogenic radiation survival, protein expression and phosphorylation (EGFR, v-akt murine thymoma viral oncogene homolog 1 (Akt), p42/44 mitogen-activated protein kinase), DNA-double strand break (DSB) repair measured by γH2AX foci, cell morphology and cell cycle distribution were examined.

Results: Expression of ILK-hk or ILK^fl/fl status resulted in significant radiosensitization relative to vector controls or ILK^?/?. Following BIBX1382BS, clonogenic survival of normal fibroblasts and vector controls remained unaffected while ILK-hk-related radiosensitization was significantly diminished. In contrast to BIBX1382BS, which did not affect DNA-DSB repair, ILK-hk-mediated radiosensitization was associated with reduced DNA-DSB repair. At 10 days after BIBX1382BS treatment, FaDu transfectants, in contrast to fibroblasts, showed reduced cell size, accumulation of G1 phase cells and reduced Akt-serine(S)473 phosphorylation.

Conclusions: Our findings confirm ILK as a cell type-independent antisurvival factor in irradiated cells, which actions in terms of radiosensitization critically depend on proper EGFR-tk activity.     

Impact of carbon ion irradiation on epidermal growth factor receptor signaling and glioma cell migration in comparison to conventional photon irradiation

Abstract

Purpose: Radiotherapy of malignant gliomas may be limited by an interference of radiation with the migratory potential of tumor cells. Therefore, the influence of conventional photon and modern carbon ion (¹²C) irradiation on glioblastoma cell migration and on epidermal growth factor receptor-related (EGFR) signaling was investigated in vitro.

Materials and methods: EGFR overexpressing glioblastoma cell lines U87 EGFR++ and LN229 EGFR++ were irradiated with 0, 2 or 6 Gy photons or ¹²C heavy ions. Migration was analyzed 24 h after treatment in a standardized Boyden Chamber assay. At different time points EGFR, protein kinase B (PKB/AKT) and extracellular signal-related kinases (ERK1/2) were analyzed by Western blotting.

Results: 2 Gy photon irradiation increased U87 EGFR++ migration and decreased motility of LN229 EGFR++ cells. Heavy ions decreased migration of both cell lines as a function of dose. There was a time-dependent increase of phosphorylation of EGFR, AKT and ERK1/2 in U87 EGFR++ after 2 Gy photon irradiation. After heavy ion irradiation EGFR, AKT or ERK1/2 remained unchanged.

Conclusions: Results suggest that the impact of irradiation on tumor cell migration depends on radiation type and cell line. Photons, but not heavy ions, potentially contribute to treatment failure by increasing EGFR-related tumor cell migration.     

β1 integrin targeting to enhance radiation therapy

Purpose:?Cell adhesion to extracellular matrix (ECM) proteins is mediated by the integrin family and has been known to modify radiation sensitivity and resistance in several cell types, including cancer cells. In particular, β1 integrin signaling has been implicated in the progression and metastasis of various cancers and has been shown to facilitate resistance to radiation therapy.

Conclusion:?In this mini-review, we provide a brief overview of integrin targeting in radiation therapy. We specifically focus on the updated findings of β1 integrin-mediated signaling pathways after exposure to ionising radiation (IR) using in?vitro and in?vivo experimental models, which could represent promising therapeutic targets for breast cancer.     

An essential role of integrin-linked kinase in the cellular radiosensitivity of normal fibroblasts during the process of cell adhesion and spreading

Purpose: In addition to focal adhesion kinase (FAK), Paxillin and p130 Crk-associated substrate (p130Cas), integrin-linked kinase (ILK) mediates signals from β integrins for controlling, e.g., survival, adhesion and spreading. To evaluate the role of ILK in the cellular radiosensitivity at different stages of cell adhesion and spreading, ILK^{floxed/floxed (fl/fl)} and ILK^?/? mouse fibroblasts were examined.

Materials and methods: Cells were irradiated (0 – 4 Gy, X-rays) in suspension, after varying time periods on fibronectin (FN) or after 24 h on different matrix proteins. Irradiation was combined with phosphatidylinositol-3 kinase (PI3K) inhibition using Ly294002. Clonogenic radiation survival, cell adhesion, and kinetics of protein expression and phosphorylation during FN adhesion (ILK, v-akt murine thymoma viral oncogene homolog 1 (AKT), FAK, Paxillin, p130Cas) were examined.

Results: In suspension and during the first hour on FN, irradiated ILK^fl/fl cells survived significantly better than ILK^?/? cells in a PI3K- and serum-dependent manner. 24-h cell cultures on different matrix proteins showed no difference in radiosensitivity. During FN adhesion, which was slightly impaired in ILK^?/? cells, protein kinetics uncovered differences in AKT, FAK, Paxillin and p130Cas phosphorylation in the two cell lines. Phosphorylation of FAK, Paxillin and p130Cas was downregulated upon exposure to ionizing radiation in an ILK-independent manner.

Conclusions: These findings indicate a critical function of ILK in the cellular radiosensitivity during the early stages of adhesion to and spreading on FN. On the basis of the presented data, a precise correlation of adhesion-, serum- and PI3K-mediated changes in PI3K/AKT and FAK/Paxillin/p130Cas signaling cascades was not found. However, identifying the underlying mechanisms of adhesion- and spreading-related changes in the cellular radiosensitivity might be relevant for an optimization of radiotherapeutic strategies specifically targeting cells located at the invasive edge of a malignant tumor.     

Time and dose-dependent activation of p53 serine 15 phosphorylation among cell lines with different radiation sensitivity

Purpose:?Proper detection of DNA damage and signal transduction to other proteins following irradiation (IR) is essential for cellular integrity. The serine 15 (Ser15) on p53 is crucial for p53 stabilization and a requirement for transient and permanent cell cycle arrest. Here, we sought to determine the relationship between p53 serine 15 phosphorylation (p53-p-Ser15) on cellular sensitivity and if this modification is associated with DNA double-strand break (DSB) repair.

Materials and methods:?Eight lymphoblastoid cell lines including ataxia-telangiectasia (A-T), Nijmegen breakage syndrome (NBS) and radiosensitive patient derived cell lines were irradiated with 1 Gy, 2 Gy and 5 Gy. Then growth inhibition, p53 induction and phosphorylation on Ser15 as assessed by immunoblotting and DNA DSB repair as assessed by constant field gel electrophoresis were examined.

Results:?Phosphorylation of p53 at Ser15 in control cells rapidly increased, peaking at 3 – 6 hours and then sustained a low level of phosphorylation for up to 6 days following IR. For these cell lines, the amount of p53-p-Ser15 corresponded to the sensitivity of cells and the amount of DNA DSB. In A-T cells, p53-p-Ser15 was reduced in spite of increased DNA DSB. NBS cells had similar phosphorylation dynamics as the control cell line, which was not consistent with their increased sensitivity. Radiosensitive patients' cell lines differed only slightly from controls.

Conclusions:?Cells that are competent in signal transduction have p53-p-Ser15 kinetics corresponding to cellular radiosensitivity as assessed by clonogenicity and DNA DSB repair, and cells impaired in signal transduction lack this correspondence. Therefore, using p53-p-Ser15 as a general marker of radiation sensitivity has confounding factors which may impair proper radiosensitivity prediction.     

ASPM influences DNA double-strand break repair and represents a potential target for radiotherapy

Abstract

Purpose: In a previous study using HiCEP (High coverage expression profiling), we demonstrated that ASPM (abnormal spindle-like microcephaly-associated) or the most common-type microcephaly (MCPH5) gene was selectively down-regulated by IR (ionizing radiation). The roles of ASPM on radiosensitivity, however, have never been studied.

Materials and methods: Using glioblastoma cell lines and normal human fibroblasts, we investigated how IR sensitivity (survived fraction, DNA repair and chromosome aberration) was affected by the reduction of ASPM by specific siRNA (small interfering RNA).

Results: Down-regulation of ASPM by siRNA enhanced radiosensitivity in three human cell lines examined. Constant-field gel electrophoreses and γ-H2AX (phosphorylated form of Histone H2A variant H2AX) foci analysis showed that ASPM-specific siRNA impaired DNA double-strand breaks (DSB) in irradiated cells. Elevated levels of abnormal chromosomes were also observed following ASPM siRNA. In addition IR-sensitization by ASPM knockdown was not enhanced in DNA-PK (DNA-dependent protein kinase) deficient glioblastoma cells suggesting that ASPM impacts upon a DNA-PK-dependent pathway.

Conclusions: Our results show for the first time that ASPM is required for efficient non-homologous end-joining in mammalian cells. In clinical applications, ASPM could be a novel target for combination therapy with radiation as well as a useful biomarker for tumor prognosis as ever described.     

Demonstration of dose dependent cytotoxic activity in SW480 colon cancer cells by 177Lu-labeled siRNA targeting IGF-1R

PurposeThe radiolabeling of targeting biomolecules with gamma emitter radionuclides for tracing and beta emitters for therapy involves the conjugation of such biomolecules to the chelating agents to form complexes with the radionuclide of interest. In this study, radioconjugate of IGF-1R siRNA with lutetium-177 (¹⁷⁷Lu) was produced, and the anti-proliferation and apoptosis effects elicited by this ¹⁷⁷Lu-siRNA complex in the SW480 colon cancer cells were evaluated.MethodsIGF-1R and Luciferase siRNAs were conjugated with p-SCN-Bn-DTPA, and then radiolabeled with ¹⁷⁷Lu. The effects of labeled and non-labeled IGF-1R siRNAs on IGF-1R expression were assessed with RT-PCR analysis and ELISA assay. IGF-1R siRNAs induced cell death and apoptosis were investigated using MTT assay and Annexin-V/propidium iodide (PI) double staining, respectively.ResultsCombined purification using Vivaspin and PD-10 columns resulted in a radiochemical purity of 97.32% ± 1.97%. Knockdown effect of the labeled IGF-1R siRNA was not significantly different from the non-labeled duplex of the same sequence (P < 0.05), but it was significant compared to the Luciferase siRNAs (P < 0.001). Proliferation decreased significantly, but apoptosis increased in the cells treated with the ¹⁷⁷Lu-IGF-1R siRNA in comparison with either ¹⁷⁷Lu or IGF-1R siRNA (P < 0.001).ConclusionRadioconjugate of IGF-1R siRNA, p-SCN-Bn-DTPA and ¹⁷⁷Lu was successfully produced and characterized as radiopharmaceutical. The present study demonstrates the involvement of ¹⁷⁷Lu-labeled IGF-1R siRNA in the inhibition of cell growth and induction of apoptosis in colon cancer cells.