Kalun Law

Activated macrophages as a novel determinant of tumor cell radioresponse: the role of nitric oxide-mediated inhibition of cellular respiration and oxygen sparing

PURPOSEnNitric oxide (NO), synthesized by the inducible nitric oxide synthase (iNOS), is known to inhibit metabolic oxygen consumption because of interference with mitochondrial respiratory activity. This study examined whether activation of iNOS (a) directly in tumor cells or (b) in bystander macrophages may improve radioresponse through sparing of oxygen.nnnMETHODS AND MATERIALSnEMT-6 tumor cells and RAW 264.7 macrophages were exposed to bacterial lipopolysaccharide plus interferon-gamma, and examined for iNOS expression by reverse transcription polymerase chain reaction, Western blotting and enzymatic activity. Tumor cells alone, or combined with macrophages were subjected to metabolic hypoxia and analyzed for radiosensitivity by clonogenic assay, and for oxygen consumption by electron paramagnetic resonance and a Clark-type electrode.nnnRESULTSnBoth tumor cells and macrophages displayed a coherent picture of iNOS induction at transcriptional/translational levels and NO/nitrite production, whereas macrophages showed also co-induction of the inducible heme oxygenase-1, which is associated with carbon monoxide (CO) and bilirubin production. Activation of iNOS in tumor cells resulted in a profound oxygen sparing and a 2.3-fold radiosensitization. Bystander NO-producing, but not CO-producing, macrophages were able to block oxygen consumption by 1.9-fold and to radiosensitize tumor cells by 2.2-fold. Both effects could be neutralized by aminoguanidine, a metabolic iNOS inhibitor. An improved radioresponse was clearly observed at macrophages to tumor cells ratios ranging between 1:16 to 1:1.nnnCONCLUSIONSnOur study is the first, as far as we are aware, to provide evidence that iNOS may induce radiosensitization through oxygen sparing, and illuminates NO-producing macrophages as a novel determinant of tumor cell radioresponse within the hypoxic tumor microenvironment.

IFN-γ+ CD8+ T Lymphocytes: Possible Link Between Immune and Radiation Responses in Tumor-Relevant Hypoxia

Activated T lymphocytes are known to kill tumor cells by triggering cytolytic mechanisms; however, their ability to enhance radiation responses remains unclear. This study examined the radiosensitizing potential of mouse CD8+ T cells, obtained by T-cell-tailored expansion and immunomagnetic purification. Activated CD8+ T cells displayed an interferon (IFN)-gamma+ phenotype and enhanced by 1.8-fold the radiosensitivity of EMT-6 tumor cells in 1% oxygen, which modeled tumor-relevant hypoxia. Radiosensitization was counteracted by neutralizing IFN-gamma or by blocking the inducible isoform of nitric oxide synthase, thus delineating the immune-tumor cell interaction through the IFN-gamma secretion pathway. Reverse transcriptase-polymerase chain reaction, enzyme-linked immunosorbent assay, and fluorescence-activated cell sorter data in agreement detected downregulation of the IFN-gamma gene by hypoxia, which caused IFN-gamma deficiency next to radioresistance. Therefore, immune and radiation responses are likely to be allied in the hypoxic tumor microenvironment, and CD8+ T cells may bridge immunostimulatory and radiosensitizing strategies.

Auranofin radiosensitizes tumor cells through targeting thioredoxin reductase and resulting overproduction of reactive oxygen species.

Auranofin (AF) is an anti-arthritic drug considered for combined chemotherapy due to its ability to impair the redox homeostasis in tumor cells. In this study, we asked whether AF may in addition radiosensitize tumor cells by targeting thioredoxin reductase (TrxR), a critical enzyme in the antioxidant defense system operating through the reductive protein thioredoxin. Our principal findings in murine 4T1 and EMT6 tumor cells are that AF at 3–10 μM is a potent radiosensitizer in vitro, and that at least two mechanisms are involved in TrxR-mediated radiosensitization. The first one is linked to an oxidative stress, as scavenging of reactive oxygen species (ROS) by N-acetyl cysteine counteracted radiosensitization. We also observed a decrease in mitochondrial oxygen consumption with spared oxygen acting as a radiosensitizer under hypoxic conditions. Overall, radiosensitization was accompanied by ROS overproduction, mitochondrial dysfunction, DNA damage and apoptosis, a common mechanism underlying both cytotoxic and antitumor effects of AF. In tumor-bearing mice, a simultaneous disruption of the thioredoxin and glutathione systems by the combination of AF and buthionine sulfoximine was shown to significantly improve tumor radioresponse. In conclusion, our findings illuminate TrxR in cancer cells as an exploitable radiobiological target and warrant further validation of AF in combination with radiotherapy.

Myeloid-derived suppressor cells reveal radioprotective properties through arginase-induced l-arginine depletion

BACKGROUND AND PURPOSEnHigh arginase-1 (Arg) expression by myeloid-derived suppressor cells (MDSC) is known to inhibit antitumor T-cell responses through depletion of l-arginine. We have previously shown that nitric oxide (NO), an immune mediator produced from l-arginine, is a potent radiosensitizer of hypoxic tumor cells. This study therefore examines whether Arg(+) overexpressing MDSC may confer radioresistance through depleting the substrate for NO synthesis.nnnMATERIAL AND METHODSnMDSC and Arg expression were studied in preclinical mouse CT26 and 4T1 tumor models and further validated in rectal cancer patients in comparison with healthy donors. The radioprotective effect of MDSC was analyzed in hypoxic tumor cells with regard to l-arginine depletion.nnnRESULTSnIn both mouse tumors and cancer patients, MDSC expansion was associated with Arg activation causing accelerated l-arginine consumption. l-Arginine depletion in turn profoundly suppressed the capacity of classically activated macrophages to synthesize NO resulting in impaired tumor cell radiosensitivity. In advanced cT3-4 rectal cancer, circulating neutrophils revealed Arg overexpression approaching that in MDSC, therefore mounting a protumor compartment wherein Arg(+) neutrophils increased from 17% to over 90%.nnnCONCLUSIONSnProtumor Arg(+) MDSC reveal a unique ability to radioprotect tumor cells through l-arginine depletion, a common mechanism behind both T-cell and macrophage inhibition.

Antidiabetic Biguanides Radiosensitize Hypoxic Colorectal Cancer Cells Through a Decrease in Oxygen Consumption

Background and Purpose: The anti-diabetic biguanide drugs metformin and phenformin exhibit antitumor activity in various models. However, their radiomodulatory effect under hypoxic conditions, particularly for phenformin, is largely unknown. This study therefore examines whether metformin and phenformin as mitochondrial complex I blockades could overcome hypoxic radioresistance through inhibition of oxygen consumption. Materials and Methods: A panel of colorectal cancer cells (HCT116, DLD-1, HT29, SW480, and CT26) was exposed to metformin or phenformin for 16 h at indicated concentrations. Afterward, cell viability was measured by MTT and colony formation assays. Apoptosis and reactive oxygen species (ROS) were detected by flow cytometry. Phosphorylation of AMP-activated protein kinase (AMPK) was examined by western blot. Mitochondria complexes activity and oxygen consumption rate (OCR) were measured by seahorse analyzer. The radiosensitivity of tumor cells was assessed by colony formation assay under aerobic and hypoxic conditions. The in vitro findings were further validated in colorectal CT26 tumor model. Results: Metformin and phenformin inhibited mitochondrial complex I activity and subsequently reduced OCR in a dose-dependent manner starting at 3 mM and 30 μM, respectively. As a result, the hypoxic radioresistance of tumor cells was counteracted by metformin and phenformin with an enhancement ratio about 2 at 9 mM and 100 μM, respectively. Regarding intrinsic radioresistance, both of them did not exhibit any effect although there was an increase of phosphorylation of AMPK and ROS production. In tumor-bearing mice, metformin or phenformin alone did not show any anti-tumor effect. While in combination with radiation, both of them substantially delayed tumor growth and enhanced radioresponse, respectively, by 1.3 and 1.5-fold. Conclusion: Our results demonstrate that metformin and phenformin overcome hypoxic radioresistance through inhibition of mitochondrial respiration, and provide a rationale to explore metformin and phenformin as hypoxic radiosensitizers.

Abstract 4986: Myeloid-derived suppressor cells as a biomarker of tumor growth and radiosensitivity: Role of hypoxia-inducible arginase-1.

Introduction: Recent evidence suggests that tumor growth and metastases are sustained by myeloid-derived suppressor cells (MDSC), which over-express arginase-1 (ARG) and cause T-cell suppression. We hypothesize that MDSC impair tumor cell radiosensitivity through ARG-mediated depletion of L-arginine, an essential substrate for the biosynthesis of the endogenous radiosensitizer nitric oxide (NO). Aim: We explored whether MDSC and ARG can be used as biomarkers for tumor growth in experimental mouse models and in the clinical setting of colorectal cancer (CRC). We further examined whether low L-arginine levels are associated with an impaired generation of NO by inducible NO synthase (iNOS) and a reduced radiosensitivity of hypoxic tumor cells. Materials & methods: MDSC (CD11b+Gr-1+) and ARG levels in mouse colon CT26 and mammary 4T1 models were analyzed by flow cytometry (FCM) and RT-PCR. Mice were inoculated I.M. with either CT26 or 4T1 carcinoma cells. After 17 days, MDSC were quantified in the spleen by FCM. In addition, ARG levels were analyzed by RT-PCR and FCM. Similar analyses were performed on human blood MDSC (CD14+HLA-DRlow) derived from CRC patients. To model the hypoxic tumor microenvironment, mouse splenocytes were cultured in 1% oxygen. Activation of iNOS was induced by LPS/IFN-γ and analysed by RT-PCR, Western blot and Griess assay. Radiosensitivity was analysed in a model of metabolic hypoxia by colony formation assay Results: The growth of both CT26 and 4T1 tumors in BALB/c mice was associated with an accumulation of MDSC in the spleen from 2-4% (in naive mice) to 8 and 35% respectively. Freshly isolated splenocytes and MDSC showed low ARG levels. Contrasting, hypoxia-conditioned splenocytes from tumor-bearing mice revealed a drastic transcriptional activation of ARG (up to 525.000-fold), and ARG+ MDSC. On the other hand splenocytes from tumor-free mice did not display ARG activation in hypoxic conditions. The iNOS/NO-mediated radiosensitization of mammary EMT6 tumor cells was strictly dependent on L-arginine levels, with a marginal effect of 1.2-fold at 10-30 μM while approaching a 2.4-fold effect at a physiological concentration of 125 μM. MDSC levels in CRC patients were increased, as compared with healthy donors (8-12% versus 4-5%). Patients with locally advanced CRC had 16-fold higher ARG expression in blood monocytes, as compared to healthy donors. 4 weeks after pre-operative radiotherapy the ARG levels decreased by 2-fold. Conclusions: Although tumor progression is clearly associated with the expansion of MDSC, their hypoxic conditioning appears to be crucial to uncover ARG as a tumor biomarker in vivo. iNOS/NO mediated radiosensitisation of hypoxic tumor cells was drastically impaired at low L-arginine levels which could be depleted by ARG+ MDSC. In a clinical setting, we consider ARG a potential biomarker in locally advanced rectal cancer. Citation Format: Wim Leonard, Ines Dufait, Heng Jiang, Femke Steenbeke, Marieke Vermeersch, Kalun Law, Guy Storme, Joeri Aerts, Valeri Verovski, Mark De Ridder. Myeloid-derived suppressor cells as a biomarker of tumor growth and radiosensitivity: Role of hypoxia-inducible arginase-1. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4986. doi:10.1158/1538-7445.AM2013-4986