Mapping glycine uptake and its metabolic conversion to glutathione in mouse mammary tumors using functional mass spectrometry imaging.

Although glutathione plays a key role in cancer cell viability and therapy response there is no clear trend in relating the level of this antioxidant to clinical stage, histological grade, or therapy response in patient tumors. The likely reason is that static levels of glutathione are not a good indicator of how a tissue deals with oxidative stress. A better indicator is the functional capacity of the tissue to maintain glutathione levels in response to this stress. However, there are few methods to assess glutathione metabolic function in tissue. We have developed a novel functional mass spectrometry imaging (fMSI) method that can map the variations in the conversion of glycine to glutathione metabolic activity across tumor tissue sections by tracking the fate of three glycine isotopologues administered in a timed sequence to tumor-bearing anesthetized mice. This fMSI method generates multiple time point kinetic data for substrate uptake and glutathione production from each spatial location in the tissue. As expected, the fMSI data shows glutathione metabolic activity varies across the murine 4T1 mammary tumor. Although glutathione levels are highest at the tumor periphery there are regions of high content but low metabolic activity. The timed infusion method also detects variations in delivery of the glycine isotopologues thereby providing a measure of tissue perfusion, including evidence of intermittent perfusion, that contributes to the observed differences in metabolic activity. We believe this new approach will be an asset to linking molecular content to tissue function.

Multiple Infusion Start Time Mass Spectrometry Imaging of Dynamic SIL-Glutathione Biosynthesis Using Infrared Matrix-Assisted Laser Desorption Electrospray Ionization.

Due to the high association of glutathione metabolism perturbation with a variety of disease states, there is a dire need for analytical techniques to study glutathione kinetics. Additionally, the elucidation of microenvironmental effects on changes in glutathione metabolism would significantly improve our understanding of the role of glutathione in disease. We therefore present a study combining a multiple infusion start time protocol, stable isotope labeling technology, infrared matrix-assisted laser desorption electrospray ionization, and high-resolution accurate mass-mass spectrometry imaging to study spatial changes in glutathione kinetics across in sectioned mouse liver tissues. After injecting a mouse with the isotopologues [2-13C,15N]-glycine, [1,2-13C2]-glycine, and [1,2-13C2,15N]-glycine at three different time points, we were able to fully resolve and spatially map their metabolism into three isotopologues of glutathione and calculate their isotopic enrichment in glutathione. We created a tool in the open-source mass spectrometry imaging software MSiReader to accurately compute the percent isotope enrichment (PIE) of these labels in glutathione and visualize them in heat-maps of the tissue sections. In areas of high flux, we found that each label enriched an approximate median of 1.6%, 1.8%, and 1.5%, respectively, of the glutathione product pool measured in each voxel. This method may be adapted to study the heterogeneity of glutathione flux in diseased versus healthy tissues.

Flow-Encoded Oxygen Control to Track the Time-Dependence of Molecular Changes Induced by Static or Cycling Hypoxia.

Detecting the effects of low oxygen on cell function is often dependent on monitoring the expression of a number of hypoxia markers. The time dependence of the appearance and stability of these markers varies between cell lines. Assessing cellular marker dynamics is also critical to determining how quickly cells respond to transient changes in oxygen levels that occurs with cycling hypoxia. We fabricated a manifold designed to use flow-encoding to produce sequential changes in gas mixtures delivered to a permeable-bottom 96-well plate. We show how this manifold and plate design can be used to expose cells to either static or cycling hypoxic conditions for eight different time periods thereby facilitating the study of the time-response of cells to altered oxygen environments. Using this device, we monitored the time-dependence of molecular changes in human PANC-1 pancreatic carcinoma and Caco-2 colon adenocarcinoma cells exposed to increasing periods of static or cycling hypoxia. Using immunohistochemistry, both cell lines show detectable levels of the marker protein hypoxia-inducible factor-1α (HIF-1α) after 3 h of exposure to static hypoxia. Cycling hypoxia increased the expression level of HIF-1α compared to static hypoxia. Both static and cycling hypoxia also increased glucose uptake and aldehyde dehydrogenase activity. This new device offers a facile screening approach to determine the kinetics of cellular alterations under varying oxygen conditions.

Mixing and delivery of multiple controlled oxygen environments to a single multiwell culture plate.

Precise oxygen control is critical to evaluating cell growth, molecular content, and stress response in cultured cells. We have designed, fabricated, and characterized a 96-well plate-based device that is capable of delivering eight static or dynamically changing oxygen environments to different rows on a single plate. The device incorporates a gas-mixing tree that combines two input gases to generate the eight gas mixtures that supply each row of the plate with a different gas atmosphere via a removable manifold. Using air and nitrogen as feed gases, a single 96-well plate can culture cells in applied gas atmospheres with Po2 levels ranging from 1 to 135 mmHg. Human cancer cell lines MCF-7, PANC-1, and Caco-2 were grown on a single plate under this range of oxygen levels. Only cells grown in wells exposed to Po2 ≤37 mmHg express the endogenous hypoxia markers hypoxia-inducible factor-1α and carbonic anhydrase IX. This design is amenable to multiwell plate-based molecular assays or drug dose-response studies in static or cycling hypoxia conditions.

Subcutaneous administration of bovine superoxide dismutase protects lungs from radiation-induced lung injury.

BACKGROUND: The objective of the present study was to determine whether single administration of the antioxidant enzyme bovine superoxide dismutase (bSOD) after radiation therapy (RT) mitigates development of pulmonary toxicity in rats. METHODS: Female F344 rats (n = 60) were divided among six experimental groups: (1) RT, single dose of 21 Gy to the right hemithorax; (2) RT + 5 mg/kg bSOD; (3) RT + 15 mg/kg bSOD; (4) No RT; (5) sham RT + 5 mg/kg bSOD; and (6) sham RT + 15 mg/kg bSOD. A single subcutaneous injection of bSOD (5 or 15 mg/kg) was administered 24 h post-radiation. The effects of bSOD on radiation-induced lung injury were assessed by measurement of body weight, breathing frequency, and histopathological changes. Immunohistochemistry was used to evaluate oxidative stress (8-OHdG(+), NOX4(+), nitrotyrosine(+), and 4HNE(+) cells), macrophage activation (ED1(+)), and expression of profibrotic transforming growth factor-ß or TGF-ß in irradiated tissue. RESULTS: Radiation led to an increase in all the evaluated parameters. Treatment with 15 mg/kg bSOD significantly decreased levels of all the evaluated parameters including tissue damage and breathing frequency starting 6 weeks post-radiation. Animals treated with 5 mg/kg bSOD trended toward a suppression of radiation-induced lung damage but did not reach statistical significance. CONCLUSIONS: The single application of bSOD (15 mg/kg) ameliorates radiation-induced lung injury through suppression of reactive oxygen species/reactive nitrogen species or ROS/RNS-dependent tissue damage.

Sickle erythrocytes target cytotoxics to hypoxic tumor microvessels and potentiate a tumoricidal response.

Resistance of hypoxic solid tumor niches to chemotherapy and radiotherapy remains a major scientific challenge that calls for conceptually new approaches. Here we exploit a hitherto unrecognized ability of sickled erythrocytes (SSRBCs) but not normal RBCs (NLRBCs) to selectively target hypoxic tumor vascular microenviroment and induce diffuse vaso-occlusion. Within minutes after injection SSRBCs, but not NLRBCs, home and adhere to hypoxic 4T1 tumor vasculature with hemoglobin saturation levels at or below 10% that are distributed over 70% of the tumor space. The bound SSRBCs thereupon form microaggregates that obstruct/occlude up to 88% of tumor microvessels. Importantly, SSRBCs, but not normal RBCs, combined with exogenous prooxidant zinc protoporphyrin (ZnPP) induce a potent tumoricidal response via a mutual potentiating mechanism. In a clonogenic tumor cell survival assay, SSRBC surrogate hemin, along with H(2)O(2) and ZnPP demonstrate a similar mutual potentiation and tumoricidal effect. In contrast to existing treatments directed only to the hypoxic tumor cell, the present approach targets the hypoxic tumor vascular environment and induces injury to both tumor microvessels and tumor cells using intrinsic SSRBC-derived oxidants and locally generated ROS. Thus, the SSRBC appears to be a potent new tool for treatment of hypoxic solid tumors, which are notable for their resistance to existing cancer treatments.

Temporal expression of hypoxia-regulated genes is associated with early changes in redox status in irradiated lung.

The development of normal lung tissue toxicity after radiation exposure results from multiple changes in cell signaling and communication initiated at the time of the ionizing event. The onset of gross pulmonary injury is preceded by tissue hypoxia and chronic oxidative stress. We have previously shown that development of debilitating lung injury can be mitigated or prevented by administration of AEOL10150, a potent catalytic antioxidant, 24h after radiation. This suggests that hypoxia-mediated signaling pathways may play a role in late radiation injury, but the exact mechanism remains unclear. The purpose of this study was to evaluate changes in the temporal expression of hypoxia-associated genes in irradiated mouse lung and determine whether AEOL10150 alters expression of these genes. A focused oligo array was used to establish a hypoxia-associated gene expression signature for lung tissue from sham-irradiated or irradiated mice treated with or without AEOL10150. Results were further verified by RT-PCR. Forty-four genes associated with metabolism, cell growth, apoptosis, inflammation, oxidative stress, and extracellular matrix synthesis were upregulated after radiation. Elevated expression of 31 of these genes was attenuated in animals treated with AEOL10150, suggesting that expression of a number of hypoxia-associated genes is regulated by early development of oxidative stress after radiation. Genes identified herein could provide insight into the role of hypoxic signaling in radiation lung injury, suggesting novel therapeutic targets, as well as clues to the mechanism by which AEOL10150 confers pulmonary radioprotection.

Role of oxidative stress in a rat model of radiation-induced erectile dysfunction.

INTRODUCTION: Chronic oxidative stress is one of the major factors playing an important role in radiation-induced normal tissue injury. However, the role of oxidative stress in radiation-induced erectile dysfunction (ED) has not been fully investigated. Aims. To investigate role of oxidative stress after prostate-confined irradiation in a rat model of radiation-induced ED. METHODS: Fifty-four young adult male rats (10-12 weeks of age) were divided into age-matched sham radiotherapy (RT) and RT groups. Irradiated animals received prostate-confined radiation in a single 20 Gy fraction. MAIN OUTCOME MEASURES: Intracavernous pressure (ICP) measurements with cavernous nerve electrical stimulation were conducted at 2, 4, and 9 weeks following RT. The protein expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits (Nox4 and gp91(phox)), markers of oxidative DNA damage (8-hydroxy-2'-deoxyguanosine [8-OHdG]), lipid peroxidation (4-hydroxynonenal [4HNE]), and inflammatory response including inducible nitric oxide synthase, macrophage activation (ED-1), and nitrotyrosine, and endogenous antioxidant defense by nuclear factor erythroid 2-related factor (Nrf2) were evaluated in irradiated prostate tissue and corpora cavernosa (CC). In addition, we investigated the relationships between results of ICP/mean arterial pressure (MAP) ratios and expression level of oxidative stress markers. RESULTS: In the RT group, hemodynamic functional studies demonstrated a significant time-dependent decrease in ICP. Increased expression of Nox4, gp91(phox), 8-OHdG, and 4HNE were observed in the prostate and CC after RT. Similarly, expressions of inflammatory markers were significantly increased. There was a trend for increased Nrf2 after 4 weeks. ICP/MAP ratio negatively correlated with higher expression level of oxidative markers. CONCLUSION: NADPH oxidase activation and chronic oxidative stress were observed in irradiated prostate tissue and CC, which correlated with lower ICP/MAP ratio. Persistent inflammatory responses were also found in both tissues after RT. These findings suggest that oxidative stress plays a crucial role in the development of radiation-induced ED.

Oxidative stress mediates radiation lung injury by inducing apoptosis.

PURPOSE: Apoptosis in irradiated normal lung tissue has been observed several weeks after radiation. However, the signaling pathway propagating cell death after radiation remains unknown. METHODS AND MATERIALS: C57BL/6J mice were irradiated with 15 Gy to the whole thorax. Pro-apoptotic signaling was evaluated 6 weeks after radiation with or without administration of AEOL10150, a potent catalytic scavenger of reactive oxygen and nitrogen species. RESULTS: Apoptosis was observed primarily in type I and type II pneumocytes and endothelium. Apoptosis correlated with increased PTEN expression, inhibition of downstream PI3K/AKT signaling, and increased p53 and Bax protein levels. Transforming growth factor-ß1, Nox4, and oxidative stress were also increased 6 weeks after radiation. Therapeutic administration of AEOL10150 suppressed pro-apoptotic signaling and dramatically reduced the number of apoptotic cells. CONCLUSION: Increased PTEN signaling after radiation results in apoptosis of lung parenchymal cells. We hypothesize that upregulation of PTEN is influenced by Nox4-derived oxidative stress. To our knowledge, this is the first study to highlight the role of PTEN in radiation-induced pulmonary toxicity.

In vivo MR studies of glycine and glutathione metabolism in a rat mammary tumor.

The metabolism of glycine into glutathione was monitored noninvasively in vivo in intact rat mammary adenocarcinomas (R3230Ac) by MRI and MRS. Metabolism was tracked by following the isotope label from intravenously infused [2-(13)C]-glycine into the glycinyl residue of glutathione. Signals from [2-(13)C]-glycine and γ-glutamylcysteinyl-[2-(13)C]-glycine ((13)C-glutathione) were detected by nonlocalized (13)C spectroscopy, as these resonances are distinct from background signals. In addition, using spectroscopic imaging methods, heterogeneity in the in vivo tumor distribution of glutathione was observed. In vivo spectroscopy also detected isotope incorporation from [2-(13)C]-glycine into both the 2- and 3-carbons of serine. Analyses of tumor tissue extracts showed single- and multiple-label incorporation from [2-(13)C]-glycine into serine from metabolism through the serine hydroxymethyltransferase and glycine cleavage system pathways. Mass spectrometric analysis of extracts also showed that isotope-labeled serine is further metabolized via the trans-sulfuration pathway, as (13)C isotope labels appear in both the glycinyl and cysteinyl residues of glutathione. Our studies demonstrate the use of MRI and MRS for the monitoring of tumor metabolic processes central to oxidative stress defense.

Radiation-induced erectile dysfunction using prostate-confined modern radiotherapy in a rat model.

INTRODUCTION: The mechanisms of radiation-induced erectile dysfunction (ED) are unclear, as clinical studies are limited, and previous animal models were based on wide-field irradiation, which does not model current radiotherapy (RT) techniques. AIMS: To perform functional and morphological analyses of erectile function (EF) utilizing image-guided stereotactic prostate-confined RT in a rat model. METHODS: Sixty young adult male rats aged 10-12 weeks old were divided into age-matched sham and RT groups. A single 20-Gy fraction to the prostate was delivered to RT animals. Penile bulb, shaft, and testes were excluded from treatment fields. MAIN OUTCOME MEASURES: Bioassay and intracavernous pressure (ICP) measurements were conducted at 2, 4, and 9 weeks following RT. Perfusion analysis of the corpora cavernosa (CC) was conducted using Hoechst injected prior to sacrifice. Penile shaft and cavernous nerve (CN) were evaluated by immunohistochemistry. Plasma testosterone level was analyzed using a testosterone enzyme-linked immunosorbent assay (ELISA) assay kit. RESULTS: Irradiated animals demonstrated statistically significant time-dependent functional impairment of EF by bioassay and ICP measurement from 4 weeks. Neuronal nitric oxide synthase (NOS) expression was decreased in CN by 4 weeks. In CC, expression levels of anti-alpha smooth muscle actin and endothelial NOS were significantly decreased at 9 weeks. In penile dorsal vessels, smooth muscle/collagen ratio was significantly decreased at 4 and 9 weeks. Additionally, Hoechst perfusion showed time-dependent decrease in CC of RT animals, whereas CD31 expression was not affected. No toxicities were noted; testosterone levels were similar in both groups. CONCLUSION: We demonstrated time-dependent ED following image-guided stereotactic RT. Our results imply that reduction of neuronal NOS expression in cavernous nerve could trigger consecutive reduction of smooth muscle content as well as blood perfusion in CC that resulted in corporal veno-occlusive dysfunction. Present study could be a cornerstone to future research that may bring comprehensive scientific understanding of radiation-induced ED.

Prognostic significance of carbonic anhydrase IX (CA-IX), endoglin (CD105) and 8-hydroxy-2'-deoxyguanosine (8-OHdG) in breast cancer patients.

The aim of this study was to examine the prognostic significance of carbonic anhydrase IX (CA-IX), an endogenous marker for tumor hypoxia; endoglin (CD105), a proliferation-associated and hypoxia-inducible glycoprotein and 8-hydroxy-2'-deoxyguanosine (8-OHdG), an oxidative DNA lesion, in breast cancer patients. Immunohistochemical expressions of CA-IX, CD105 and 8-OHdG, analyzed on paraffin-embedded tumor tissues from forty female breast cancer patients, were used to assess their prognostic implication on overall survival (OS) and relapse-free survival (RFS). Patients with high CA-IX expression (above cut-off value) had a higher occurrence of relapse (P = = 0.002). High CA-IX expression was significantly associated with shorter RFS (P < 0.001, hazard ratio (HR) 0.21) and shorter OS (P < 0.001, HR 0.19). Lymph node negative patients with high CA-IX expression had worse RFS (P = 0.031, HR 0.14) and OS (P = 0.005, HR 0.05). Patients with grade I&II tumors and high CA-IX expression showed shorter RFS (P = 0.028, HR 0.28) and OS (P = 0.008, HR 0.20). Worse OS (P = 0.046, HR 0.28) was found in subgroup of patients with grade II tumors and high CA-IX expression. Among all three markers, only high CA-IX expression was strong independent prognostic indicator for shorter OS (HR 4.14, 95% CI 1.28-13.35, P = 0.018) and shorter RFS (HR 3.99, 95% CI 1.38-11.59, P = 0.011). Elevated expression of CA-IX was an independent prognostic factor for decreased RFS and OS and a significant marker for tumor aggressiveness. CD105 had week prognostic value; whereas, 8-OHdG, in this study, did not provide sufficient evidence as a prognostic indicator in breast cancer patients.

Morphology of hypoxia following cryoablation in a prostate cancer murine model: its relationship to necrosis, apoptosis and, microvessel density.

The aim of this study is to investigate the tumor tissue changes in terms of hypoxia and demonstrate its relationship to vascularity and apoptosis following therapeutic cryoablation in a prostate tumor murine model. Total 67 male C57BL/J6 mice were assigned into sham-operation group and cryoablation group. Murine prostate tumors (RM-9) were inoculated subcutaneously in a right hind leg and treated with cryotherapy. Of 30 mice, tumor volumes were measured for 12 days following operation. Of 37 mice, tumor tissues were harvested in 24h following operation, and histological/molecular changes were analyzed. Hematoxylin and eosin or immunohistochemical staining were utilized to quantify tumor necrosis, hypoxia (pimonidazole), vascularization (CD31), and apoptosis (cleaved caspase-3). The results showed that cryoablated tumors demonstrated significant delayed growth following treatment compared to controls. Pathological analysis revealed that the severity of hypoxia increased in the cryoablation arm compared to controls. Necrotic and apoptotic populations were also found to be increased in the cryoablation arm (P=0.028 and 0.021). Hypoxia demonstrated a positive correlation with necrosis (r=0.520, P=0.001) and apoptosis (r=0.474, P=0.003), while showing negative correlation with microvessel density (MVD) (r=-0.361, P=0.021). We concluded that in the peripheral areas from the cryoneedle impact site, strong hypoxic responses were found, which may play important role in tumor freezing injury. To our knowledge, this is the first report describing cryoablation-mediated changes of hypoxia at a molecular level in the prostate cancer murine model.

Role of vitamin D(3) as a sensitizer to cryoablation in a murine prostate cancer model: preliminary in vivo study.

OBJECTIVES: Calcitriol has been reported to have antitumor efficacy in several cancers. In this study, we hypothesized that calcitriol may potentially function as a cryosensitizer that can enhance cryoablation, and we investigated several molecular marker changes in a murine model of prostate cancer. METHODS: Murine prostate tumors (RM-9) were grown in male C57BL/6J mice subcutaneously with neoadjuvant intratumoral injection of calcitriol followed by cryoablation. The microenvironmental changes after cryoablation alone and in combination with calcitriol were analyzed in a comparative fashion using immunohistochemistry and Western blot analyses. RESULTS: Both cryoablation and the combination group could suppress tumor growth after treatment compared with the control. At final pathologic assessment, a larger necrotic area was seen in the combination group (P = .026). Although microvessel density (CD31) and the area of hypoxia (pimonidazole) was not different between the control and combination groups, cell proliferation (Ki-67) significantly decreased in the combination treatment (P = .035). In Western blot analyses, several markers for apoptosis were expressed significantly higher with the combination treatment. CONCLUSIONS: The synergistic effect of calcitriol with cryoablation was demonstrated because of enhanced antitumor efficacy by increasing necrosis and apoptosis and reduced cell proliferation. This study suggests that calcitriol is a potentially applicable reagent as a freeze sensitizer to cryoablation.

Phosphorylated epidermal growth factor receptor and cyclooxygenase-2 expression in localized non-small cell lung cancer.

PURPOSE: The prognostic significance of epidermal growth factor receptor (EGFR) and cyclooxygenase-2 (COX-2) expression remains unestablished, although EGFR and COX-2 are frequently overexpressed in non-small cell lung cancer (NSCLC). Considering the importance of EGFR activation after ligand binding, however, the expression of phosphorylated EGFR (p-EGFR) may have more significance in predicting tumor aggressiveness in NSCLC than either EGFR or COX-2 expression. PATIENTS AND METHODS: We studied the relationships between p-EGFR, EGFR, and COX-2 overexpression and examined their association with prognosis in localized NSCLC. The expression of p-EGFR, EGFR, and COX-2 was studied by immunohistochemistry in 77 surgically-resected stage I/II NSCLC cases. EGFR mutational status was determined by sequencing exons 18-21. Correlation of expression with clinical outcome and other biomarkers, including Ki-67 and microvessel density (MVD), was also examined. RESULTS: Out of the 77 patients, EGFR overexpression was observed in 37 (48.1%), p-EGFR expression was found in 22 (28.6%), and COX-2 overexpression was seen in 45 (58.4%). Expression of p-EGFR was associated with COX-2 overexpression (P = 0.047), but not EGFR overexpression or high Ki-67 (P = 0.087 and P = 0.092, respectively). COX-2 overexpression was significantly associated with high Ki-67 (P = 0.011). Expression of p-EGFR correlated with lower disease-free survival (P = 0.045), but not overall survival. Neither EGFR nor COX-2 overexpression was associated with prognosis. CONCLUSION: p-EGFR appears to be a better indicator for lower disease-free survival than EGFR overexpression itself in localized NSCLC. Pathways other than EGFR activation may influence COX-2 overexpression.

In vivo selection of tumor-targeting RNA motifs.

In an effort to target the in vivo context of tumor-specific moieties, we screened a large library of nuclease-resistant RNA oligonucleotides in tumor-bearing mice to identify candidate molecules with the ability to localize to hepatic colon cancer metastases. One of the selected molecules is an RNA aptamer that binds to p68, an RNA helicase that has been shown to be upregulated in colorectal cancer.

Antiangiogenic action of redox-modulating Mn(III) meso-tetrakis(N-ethylpyridinium-2-yl)porphyrin, MnTE-2-PyP(5+), via suppression of oxidative stress in a mouse model of breast tumor.

MnTE-2-PyP(5+) is a potent catalytic scavenger of reactive oxygen and nitrogen species, primarily superoxide and peroxynitrite. It therefore not only attenuates primary oxidative damage, but was found to modulate redox-based signaling pathways (HIF-1alpha, NF-kappaB, SP-1, and AP-1) and thus, in turn, secondary oxidative injury also. Cancer has been widely considered an oxidative stress condition. The goal of this study was to prove if and why a catalytic SOD mimic/peroxynitrite scavenger would exert anti-cancer effects, i.e., to evaluate whether the attenuation of the oxidative stress by MnTE-2-PyP(5+) could suppress tumor growth in a 4T1 mouse breast tumor model. Tumor cells were implanted into Balb/C mouse flanks. Three groups of mice (n=25) were studied: control (PBS) and 2 and 15 mg/kg/day of MnTE-2-PyP(5+) given subcutaneously twice daily starting when the tumors averaged 200 mm(3) (until they reached approximately 5-fold the initial volume). Intratumoral hypoxia (pimonidazole, carbonic anhydrase), HIF-1alpha, VEGF, proliferating capillary index (CD105), microvessel density (CD31), protein nitration, DNA oxidation (8-OHdG), NADPH oxidase (Nox-4), apoptosis (CD31), macrophage infiltration (CD68), and tumor drug levels were assessed. With 2 mg/kg/day a trend toward tumor growth delay was observed, and a significant trend was observed with 15 mg/kg/day. The 7.5-fold increase in drug dose was accompanied by a similar (6-fold) increase in tumor drug levels. Oxidative stress was largely attenuated as observed through the decreased levels of DNA damage, protein 3-nitrotyrosine, macrophage infiltration, and NADPH oxidase. Further, hypoxia was significantly decreased as were the levels of HIF-1alpha and VEGF. Consequently, suppression of angiogenesis was observed; both the microvessel density and the endothelial cell proliferation were markedly decreased. Our study indicates for the first time that MnTE-2-PyP(5+) has anti-cancer activity in its own right. The anti-cancer activity via HIF/VEGF pathways probably arises from the impact of the drug on the oxidative stress. Therefore, the catalytic scavenging of ROS/RNS by antioxidants, which in turn suppresses cellular transcriptional activity, could be an appropriate strategy for anti-cancer therapy. Enhancement of the anti-cancer effects may be achieved by optimizing the dosing regime, utilizing more bioavailable Mn porphyrins (MnP), and combining MnP treatment with irradiation, hyperthermia, and chemotherapy. Mn porphyrins may be advantageous compared to other anti-cancer drugs, owing to their radioprotection of normal tissue and the ability to afford pain management in cancer patients via prevention of chronic morphine tolerance.

Her2/neu signaling blockade improves tumor oxygenation in a multifactorial fashion in Her2/neu+ tumors.

PURPOSE: Tumor hypoxia reduces the efficacy of radiation and chemotherapy as well as altering gene expression that promotes cell survival and metastasis. The growth factor receptor, Her2/neu, is overexpressed in 25-30% of breast tumors. Tumors that are Her2(+) may have an altered state of oxygenation, relative to Her2(-) tumors, due to differences in tumor growth rate and angiogenesis. METHODS: Her2 blockade was accomplished using an antibody to the receptor (trastuzumab; Herceptin). This study examined the effects of Her2 blockade on tumor angiogenesis, vascular architecture, and hypoxia in Her2(+) and Her2(-) MCF7 xenograft tumors. RESULTS: Treatment with trastuzumab in Her2(+) tumors significantly improved tumor oxygenation, increased microvessel density, and improved vascular architecture compared with the control-treated Her2(+) tumors. The Her2(+) xenografts treated with trastuzumab also demonstrated decreased proliferation indices when compared with control-treated xenografts. These results indicate that Her2 blockade can improve tumor oxygenation by decreasing oxygen consumption (reducing tumor cell proliferation and inducing necrosis) and increasing oxygen delivery (vascular density and architecture). CONCLUSIONS: These results support the use of trastuzumab as an adjunct in the treatment of breast tumors with chemotherapy or radiotherapy, as improvements in tumor oxygenation should translate into improved treatment response.

Targeting lactate-fueled respiration selectively kills hypoxic tumor cells in mice.

Tumors contain oxygenated and hypoxic regions, so the tumor cell population is heterogeneous. Hypoxic tumor cells primarily use glucose for glycolytic energy production and release lactic acid, creating a lactate gradient that mirrors the oxygen gradient in the tumor. By contrast, oxygenated tumor cells have been thought to primarily use glucose for oxidative energy production. Although lactate is generally considered a waste product, we now show that it is a prominent substrate that fuels the oxidative metabolism of oxygenated tumor cells. There is therefore a symbiosis in which glycolytic and oxidative tumor cells mutually regulate their access to energy metabolites. We identified monocarboxylate transporter 1 (MCT1) as the prominent path for lactate uptake by a human cervix squamous carcinoma cell line that preferentially utilized lactate for oxidative metabolism. Inhibiting MCT1 with alpha-cyano-4-hydroxycinnamate (CHC) or siRNA in these cells induced a switch from lactate-fueled respiration to glycolysis. A similar switch from lactate-fueled respiration to glycolysis by oxygenated tumor cells in both a mouse model of lung carcinoma and xenotransplanted human colorectal adenocarcinoma cells was observed after administration of CHC. This retarded tumor growth, as the hypoxic/glycolytic tumor cells died from glucose starvation, and rendered the remaining cells sensitive to irradiation. As MCT1 was found to be expressed by an array of primary human tumors, we suggest that MCT1 inhibition has clinical antitumor potential.

Small molecular inhibitor of transforming growth factor-beta protects against development of radiation-induced lung injury.

PURPOSE: To determine whether an anti-transforming growth factor-beta (TGF-beta) type 1 receptor inhibitor (SM16) can prevent radiation-induced lung injury. METHODS AND MATERIALS: One fraction of 28 Gy or sham radiotherapy (RT) was administered to the right hemithorax of Sprague-Dawley rats. SM16 was administered in the rat chow (0.07 g/kg or 0.15 g/kg) beginning 7 days before RT. The rats were divided into eight groups: group 1, control chow; group 2, SM16, 0.07 g/kg; group 3, SM16, 0.15 g/kg; group 4, RT plus control chow; group 5, RT plus SM16, 0.07 g/kg; group 6, RT plus SM16, 0.15 g/kg; group 7, RT plus 3 weeks of SM16 0.07 g/kg followed by control chow; and group 8, RT plus 3 weeks of SM16 0.15 g/kg followed by control chow. The breathing frequencies, presence of inflammation/fibrosis, activation of macrophages, and expression/activation of TGF-beta were assessed. RESULTS: The breathing frequencies in the RT plus SM16 0.15 g/kg were significantly lower than the RT plus control chow from Weeks 10-22 (p <0.05). The breathing frequencies in the RT plus SM16 0.07 g/kg group were significantly lower only at Weeks 10, 14, and 20. At 26 weeks after RT, the RT plus SM16 0.15 g/kg group experienced a significant decrease in lung fibrosis (p = 0.016), inflammatory response (p = 0.006), and TGF-beta1 activity (p = 0.011). No significant reduction was found in these measures of lung injury in the group that received SM16 0.7 g/kg nor for the short-course (3 weeks) SM16 at either dose level. CONCLUSION: SM16 at a dose of 0.15 g/kg reduced functional lung damage, morphologic changes, inflammatory response, and activation of TGF-beta at 26 weeks after RT. The data suggest a dose response and also suggest the superiority of long-term vs. short-term dosing.