Enhanced radiosensitization of enzalutamide via schedule dependent administration to androgen-sensitive prostate cancer cells.

BACKGROUND: Prostate cancer (PCa) is a progressive disease and the most diagnosed cancer in men. The current standard of care for high-risk localized PCa is a combination of androgen deprivation therapy (ADT) and radiation (XRT). The majority of these patients however become resistant due to incomplete responses to ADT as a result of selective cells maintaining androgen receptor (AR) activity. Improvement can be made if increasing radiosensitivity is realized. Therefore, the aim of this study is to investigate the efficacy of the next-generation PCa drug Enzalutamide (ENZA), as a radiosensitizer in XRT therapy. METHODS: Using a number of androgen-dependent (LNCaP, PC3-T877A) and androgen-independent (C4-2, 22RV1, PC3, PC3-AR V7) cell lines, the effect of ENZA as a radiosensitizer was studied alone or in combination with ADT and/or XRT. Cell viability and cell survival were assessed, along with determination of cell cycle arrest, DNA damage response and repair, apoptosis and senescence. RESULTS: Our results indicated that either ENZA alone (in AR positive, androgen-dependent PCa cells) or in combination with ADT (in AR positive, hormone-insensitive PCa cells) potentiates radiation response [Dose enhancement factor (DEF) of 1.75 in LNCAP and 1.35 in C4-2] stronger than ADT + XRT conditions. Additionally, ENZA sensitized androgen dependent PCa cells to XRT in a schedule-dependent manner, where concurrent administration of ENZA and radiation lead to a maximal radiosensitization when compared to either drug administration prior or after XRT. In LNCaP cells, ENZA treatment significantly prolonged the presence of XRT-induced phospho-γH2AX up to 24 h after treatment; suggesting enhanced DNA damage. It also significantly increased XRT-induced apoptosis and senescence. CONCLUSIONS: Our data indicates that ENZA acts as a much stronger radiosensitizer compared to ADT. We have also observed that its efficacy is schedule dependent and related to increased levels of DNA damage and a delay of DNA repair processes. Finally, the initial abrogation of DNA-PKcs activity by AR inhibition and its subsequent recovery might represent an important mechanism by which PCa cells acquire resistance to combined anti-androgen and XRT treatment. This work suggests a new use of ENZA in combination with XRT that could be applicable in clinical trial settings for patients with early and intermediate hormone responsive disease.

Adipose mesenchymal stromal cells response to ionizing radiation.

BACKGROUND AIMS: This study evaluates the biological response of adipose tissue-derived mesenchymal stromal cells (aMSCs) to ionizing radiation (IR). METHODS: Irradiated BALB/c mice aMSCs were characterized for functionality and phenotype. The clonogenic capacity of irradiated aMSCs was assessed and compared with those of metastatic breast cancer cell line (4T1) and normal mouse fibroblasts (NIH3T3-wt). We investigated the IR-induced DNA damage response, apoptosis, changes in cell cycle (CC) dynamics and protein and gene expression. RESULTS: Irradiated and non-irradiated aMSCs were able to differentiate into adipocytes, chondrocytes and osteocytes with no significant difference. Irradiated aMSCs maintained the expression of mesenchymal stromal cells (MSCs) surface antigens and, as expected, were negative for hematopoietic stem cells (HSCs) surface antigens when tested up to 7 days after IR for all irradiation doses with no significant difference. Clonogenically, irradiated aMSCs had higher relative survival fraction and plating efficiency than 4T1 and NIH3T3-wt. Irradiated aMSCs expressed higher □H2AX and significantly showed faster and more time-efficient IR-induced DNA damage response evident by up-regulated DNA-PKcs and RAD51. Two hours after IR, most of aMSCs DNA damage/repair-related genes showed up-regulation that disappeared within 6 h after IR. Irradiated aMSCs showed a significant rise and an earlier peak of p-ATM-dependent and -independent (p84/5E10-mediated) G2/M CC arrest compared with 4T1 and NIH3T3-wt. CONCLUSIONS: After IR exposure, aMSCs showed a robust and time-efficient radiation-induced DNA damage repair response, stable phenotypical characteristics and multi-lineage differentiation potential, suggesting they may be reliable candidates for cell therapy in radiation oncology regenerative medicine.

ZRBA1, a Mixed EGFR/DNA Targeting Molecule, Potentiates Radiation Response Through Delayed DNA Damage Repair Process in a Triple Negative Breast Cancer Model.

PURPOSE: ZRBA1 is a combi-molecule designed to induce DNA alkylating lesions and to block epidermal growth factor receptor (EGFR) TK domain. Inasmuch as ZRBA1 downregulates the EGFR TK-mediated antisurvival signaling and induces DNA damage, we postulated that it might be a radiosensitizer. The aim of this study was to further investigate the potentiating effect of ZRBA1 in combination with radiation and to elucidate the possible mechanisms of interaction between these 2 treatment modalities. METHODS AND MATERIALS: The triple negative human breast MDA-MB-468 cancer cell line and mouse mammary cancer 4T1 cell line were used in this study. Clonogenic assay, Western blot analysis, and DNA damage analysis were performed at multiple time points after treatment. To confirm our in vitro findings, in vivo tumor growth delay assay was performed. RESULTS: Our results show that a combination of ZRBA1 and radiation increases the radiation sensitivity of both cell lines significantly with a dose enhancement factor of 1.56, induces significant numbers of DNA strand breaks, prolongs higher DNA damage up to 24 hours after treatment, and significantly increases tumor growth delay in a syngeneic mouse model. CONCLUSIONS: Our data suggest that the higher efficacy of this combination could be partially due to increased DNA damage and delayed DNA repair process and to the inhibition of EGFR. The encouraging results of this combination demonstrated a significant improvement in treatment efficiency and therefore could be applicable in early clinical trial settings.

Sorafenib in combination with ionizing radiation has a greater anti-tumour activity in a breast cancer model.

High expression of vascular endothelial growth factor (VEGF) in patients with breast cancer has been associated with a poor prognosis, indicating that VEGF could be linked to the efficacy of chemotherapy and radiotherapy. It has also been suggested that radiation resistance is partly due to tumour cell production of angiogenic cytokines, particularly VEGF receptor (VEGFR). This evidence indicates that inhibition of VEGFR might enhance the radiation response. Sorafenib tosylate (Bay 54-9085) is an oral, small-molecule multikinase inhibitor of several targets including RAF/MEK/ERK MAP kinase signalling, VEGFR-2, VEGFR-3 and platelet-derived growth factor receptor-beta. Sorafenib has shown clinical efficacy in treating solid tumours such as renal cell and hepatocellular carcinomas. However, strategies are yet to be identified to prolong and maximize the anticancer effect of this multikinase inhibitor. The objective of this study was to determine whether a combination of Sorafenib and radiation will enhance the treatment response in vitro and in vivo. Radio-modulating effect of Sorafenib was assessed by performing clonogenic assays. In addition, cell cycle analyses as well as annexin-V apoptosis assays were performed 24 and 48 h after treatment, respectively. To confirm our in-vitro results, tumour growth delay assays were performed. Our results showed a strong and supra-additive antitumour effect of radiation combined with Sorafenib in vitro (dose enhancement factor of 1.76). The combined therapy demonstrated a strong and significant G2/M cell cycle arrest (combined treatment vs. irradiated alone: P<0.0008). Moreover, annexin-V staining showed a significant increase in the level of apoptosis (combined treatment vs. irradiated alone: P<0.0004). Study of the syngeneic model demonstrated the superior potency of the Sorafenib combined with radiotherapy. Our results demonstrate that higher antitumour activity can be achieved when radiation and Sorafenib are combined.

Brg-1 mediates the constitutive and fenretinide-induced expression of SPARC in mammary carcinoma cells via its interaction with transcription factor Sp1.

BACKGROUND: Secreted protein, acidic and rich in cysteine (SPARC) is a matricellular protein that mediates cell-matrix interactions. It has been shown, depending on the type of cancer, to possess either pro- or anti-tumorigenic properties. The transcriptional regulation of the SPARC gene expression has not been fully elucidated and the effects of anti-cancer drugs on this process have not been explored. RESULTS: In the present study, we demonstrated that chromatin remodeling factor Brg-1 is recruited to the proximal SPARC promoter region (-130/-56) through an interaction with transcription factor Sp1. We identified Brg-1 as a critical regulator for the constitutive expression levels of SPARC mRNA and protein in mammary carcinoma cell lines and for SPARC secretion into culture media. Furthermore, we found that Brg-1 cooperates with Sp1 to enhance SPARC promoter activity. Interestingly, fenretinide [N-4(hydroxyphenyl) retinamide, 4-HPR], a synthetic retinoid with anti-cancer properties, was found to up-regulate the transcription, expression and secretion of SPARC via induction of the Brg-1 in a dose-dependent manner. Finally, our results demonstrated that fenretinide-induced expression of SPARC contributes significantly to a decreased invasion of mammary carcinoma cells. CONCLUSIONS: Overall, our results reveal a novel cooperative role of Brg-1 and Sp1 in mediating the constitutive and fenretinide-induced expression of SPARC, and provide new insights for the understanding of the anti-cancer effects of fenretinide.

MAPKAPK-2 signaling is critical for cutaneous wound healing.

Cutaneous wound healing is a complex process, which is heavily dependent on successful inflammatory action. Mitogen-activated protein kinase (MAPK)-activated protein kinase-2 (MAPKAPK-2 or MK2), a major substrate of p38 MAPK, has been shown to be a major player in multiple inflammatory diseases, but its role in cutaneous wound healing has not yet been explored. In this study, by comparing excisional wounds made on the backs of MK2 knockout (KO) and MK2 wild-type (WT) mice, we found that the kinetics of wound healing are significantly affected by the absence of MK2 (P=0.010 to P<0.001). Histological examination showed a higher level of acanthosis of the migrating wound keratinocyte layer as well as a higher level of collagen deposition in the granulation tissue of the wounds from MK2 WT mice compared with those from MK2 KO mice. Interestingly, although MK2 did not influence macrophage and neutrophil infiltration of the wounds, the expression of many cytokines and chemokines was significantly affected at different days post wounding. Furthermore, the delayed healing rate of wounds in MK2 KO mice can be significantly improved by passive transfer of macrophages with intact MK2. Overall, these results show a critical role for MK2 gene expression in macrophages participating in the process of cutaneous wound healing.

Interaction of ionizing radiation and ZRBA1, a mixed EGFR/DNA-targeting molecule.

ZRBA1 is a molecule termed 'combi-molecule' designed to induce DNA-alkylating lesions and to block epidermal growth factor receptor (EGFR) tyrosine kinase. Owing to its ability to downregulate the EGFR tyrosine kinase-mediated antiapoptotic signaling and DNA repair proteins, we inferred that it could significantly sensitize cells to ionizing radiation. Using the MDA-MB-468 human breast cancer cell line in which ZRBA1 has already been reported to induce significant EGFR/DNA-targeting potency, the results showed that: (i) concurrent administration of ZRBA1 and 4 Gy radiation led to a significant decrease in cell viability, (ii) the greater efficacy of the combination was sequential, being limited to conditions wherein the drug was administered concurrently with radiation or before radiation, and (iii) the efficacy enhancement of the combination was further confirmed by clonogenic assays from which a dose enhancement factor of 1.34 could be observed at survival fraction of 0.01. Flow cytometric analysis showed significant enhancement of cell cycle arrest in G2/M (P<0.046, irradiated cells vs. cells treated with ZRBA1 and radiation) and increased apoptosis when ZRBA1 was combined with radiation. Likewise, significant levels of double-strand breaks were observed for the combination, as determined by neutral comet assay (P<0.045, irradiated cells vs. cells treated with ZRBA1 and radiation). These results in toto suggest that the superior efficacy of the ZRBA1 plus radiation combination may be secondary to the ability of ZRBA1 to arrest the cells in G2/M, a cell cycle phase in which tumor cells are sensitive to radiation. Furthermore, the increased levels of DNA damage, combined with the concomitant downregulation of EGFR-mediated signaling by ZRBA1, may account for the significant levels of cell killing induced by the combination.