Anti-angiogenic action of redox modulating Mn(III) ortho tetrakis N-ethylpyridylporphyrin, MnTE-2-PyP⁵⁺via suppression of oxidative stress in a mouse model of breast tumor

Abstract

MnTE-2-PyP⁵⁺ 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-1α, NF-κB, SP-1 and AP-1), and thus in turn secondary oxidative injury also. Cancer has been widely considered as an oxidative stress condition. The goal of the present 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-PyP⁵⁺ 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), 2 mg/kg/day and 15 mg/kg/day of MnTE-2-PyP⁵⁺ given subcutaneously twice daily starting when the tumors averaged 200 mm³ (until they reached ~5-fold of initial volum). Intratumoral hypoxia (pimonidazole, carbonic anhydrase, CAIX), HIF-1α, 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 was significant with 15 mg/kg/day. The 7.5-fold increase in drug dose was accompanied by similar (6-fold) increase in drug tumor levels. Oxidative stress was largely attenuated as was 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-1α 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-PyP⁵⁺ 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, due to their radioprotection of normal tissue and the ability to afford pain management via prevention of chronic morphine tolerance.

Introduction

Cationic Mn porphyrins (MnP) bearing electron-withdrawing positively charged groups close to the metal center exert both thermodynamic and electrostatic facilitation of the reaction with anionic superoxide and peroxynitrite and are thus among the most potent scavengers of those species [1–8]. They have been originally developed based on structure-activity relationships between the catalytic rate constant for O₂^.− dismutation and the metal-centered redox potential [2, 6, 8]. It has been later shown that their ability to scavenge O₂^.− parallels their efficacy in reducing ONOO⁻, both being catalytic in nature [9, 10]. The most potent cationic beta and meso substituted quaternized Mn(III) N-pyridylporphyrins have metal-centered reduction potential around the midway potential for the O₂^.− reduction and oxidation similar to SOD enzymes (E_1/2 = ~ + 300 mV vs. NHE). In addition, those Mn porphyrins afford electrostatic facilitation for the reaction with O₂^{. −} which results in k_cat that approaches or equals the k_cat of the SOD enzymes themselves [6, 8, 11]. Such positive potentials make Mn^IIIP readily reducible with cellular reductants such as ascorbate and tetrahydrobiopterin [9, 10, 12]. Via one-or two-electron oxidation with O₂⁻ or ONOO⁻, respectively, Mn porphyrin in its reduced state (Mn^IIP) would in vivo behave as a superoxide [12], or peroxynitrite reductase, rather than as an SOD mimic.

The most potent among Mn porphyrins, MnTE-2-PyP⁵⁺ (Figure 1), MnTnHex-2-PyP⁵⁺, and MnTDE-2-ImP⁵⁺ [2, 4, 5] proved effective in different animal models of oxidative stress [13–25]. As our insight into the efficacy of these compounds and their mechanism of action in vivo increases along with our understanding that oxidative stress is a factor in cancer, aging, central nervous system disorders and other diseases, it became obvious that Mn porphyrins (a) decrease the primary oxidative damage to biological targets and (b) suppress the redox-based cellular transcriptional activity which would otherwise result in activation of inflammatory and immune responses that would amplify the primary oxidative event. Both actions likely stem from the elimination of the signaling ROS/RNS and are based on favorable redox properties of Mn porphyrins. We have shown significant to full (brought to control levels) suppression of the major transcription factors AP-1 [22], SP-1 [21], NF-κB [14, 21] and HIF-1α [18] by MnTE-2-PyP⁵⁺ (Figure 1), which was accompanied with decreased levels of related inflammatory cytokines. Suppression of all four pathways is relevant in tumor growth as we have already shown with skin cancerogenesis (AP-1) and tumor radiosensitization studies (HIF-1α). Activation of HIF-1α by reactive species H₂O₂, .NO and/or ONOO⁻ is fully prevented by MnTE-2-PyP⁵⁺, which in turn results in suppression of VEGF and bFGF [18]. Suppression of HIF-1α stabilization by .NO [26] may also play a role. In a 4T1 breast tumor mouse model, MnTE-2-PyP⁵⁺ was further reported to be an effective tumor radiosensitizer, augmenting the radiation-induced damage to the tumor vasculature [17, 27]. When irradiated, due to enhanced production of reactive species, tumors “in their efforts to survive”, upregulate HIF-1α and VEGF to support new vasculature and thus tumor growth [17, 27]. Again, by removing reactive species, MnTE-2-PyP⁵⁺ inhibits HIF-1α activation and VEGF expression, consequently causing a significant suppression of tumor angiogenesis/growth [17, 18].

Figure 1.

Structure of MnTE-2-PyP⁵⁺

It is well-documented that tumors are under oxidative stress which is a driving force that promotes tumor growth [28]. The major cellular antioxidant defense, mitochondrial MnSOD, was shown to modulate tumor growth [29–32]. The mechanistic explanations of such effects that have been offered, including those by the Oberly and Melendez groups are complex [33–40]. When tumor is irradiated, one would expect that Mn porphyrin, if any, would protect it via its basic antioxidant function whereby decreasing levels of ROS/RNS, suppressing tumor oxidative stress and in turn preventing tumor growth. While this likely occurs to some degree, the other, anti-angiogenic action of MnP via suppression of transcriptional activity i.e. suppression of vasculature growth prevails. Consequently MnTE-2-PyP⁵⁺ is not tumor-protective [17, 27].

The radioprotection of normal tissue by Mn porphyrins [17, 18, 26, 29], along with anti-cancer effects observed in this study, would allow these drugs to exert dual actions in tumor therapy. The efficacy in preventing chronic morphine tolerance was recently reported [25]. It would add another dimension to the use of Mn porphyrins in tumor therapy – pain management, which along with radioprotectiveness would make these drugs advantageous over other anti-cancer agents.

Our goal herein was to further our understanding of the impact of MnTE-2-PyP⁵⁺, in its own right, on tumor growth suppression through modulation of induction of HIF-1α/VEGF pathways on murine breast tumor.

Materials and Methods

Mouse tumor model and treatment

Balb/C mice (Charles River Laboratories, Wilmington, MA) were housed and treated in accordance with approved guidelines from the Duke University Institutional Animal Care and Use Committee. The animals were allowed to adapt to the laboratory environment for 7 days before experiments.

4T1 cells were routinely cultured in Dulbecco’s Modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS). 4T1 tumors were grown in the flank of mice by injecting a suspension of 10⁶ tumor cells/mouse. Tumor volumes were measured with calipers and calculated according to two diameters with the formula: V = (a² × b)/2, where V is the volume, a is the short diameter, and b is the long diameter.

MnTE-2-PyP⁵⁺ was synthesized as previously described [2]. The compound was prepared in a sterile phosphate-buffered saline (PBS) and administered twice daily by subcutaneous (s.c.) injections (low dose, 2 mg/kg/day and high dose, 15 mg/kg/day). Animals were randomly assigned to three treatment groups with 25 mice per group: control (PBS), low dose and high dose of MnTE-2-PyP⁵⁺. Treatment started when tumors size averaged 200 mm³ (day 0). Mice received on average 80 μL of the solution, the volume adjusted to the increasing weight of the animal. No sign of toxicity was observed with either dose. The tumor growth and animal weight were followed on alternate days. When the tumor volume reached 5 fold of initial volume, the animals were injected with pimonidazole and sacrificed three hours later. The tumors were harvested (snap frozen) and stored at −80 °C for further processing.

Immunohistochemistry

Using a LEICA CM 1850 cryotome (Meyer Instruments Inc., Houston, TX), tumors were cut into 10–12 μm frozen sections, and adhered to Poly-L-Lysine coated slides. The sections were fixed in 4% paraformaldehyde in PBS (pH 7.2) for 45 minutes, then blocked with 10 % normal serum. The following primary antibodies were applied: rabbit polyclonal anti-VEGF (Santa Cruz Biotechnology, Santa Cruz, CA), mouse monoclonal anti-carbonic anhydrase IX (CAIX; gift from Dr. Oosterwijk), mouse monoclonal anti-HIF-1α (Novus Biological, Littleton, CO, USA) rabbit polyclonal pimonidazole (gift from Dr. James Raleigh), rat monoclonal anti-CD31, CD105 and CD68 (BD Pharmingen, San Jose, CA, USA), goat polyclonal against homologue of gp91^phox catalytic subunit of phagocyte NAD(P)H oxidase (Nox-4), mouse monoclonal anti 8-OHdG (1:2000, JaICA, Shizuoka, Japan), mouse monoclonal anti 3-nitrotyrosine (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit monoclonal anti-caspase 3 (Cell Signaling Technology Inc., Danvers, MA, USA) and rabbit polyclonal Ki-67 (Vector Laboratories, Burlingame, CA, USA). Omission of the primary antibody served as negative control. Biotinylated donkey anti-rabbit, goat or anti-mouse secondary antibodies were used where relevant. The ABC kit and Nova-Red solution (Vector Laboratories, Burlingame, CA, USA) were used according to manufacturer’s recommendations. Slides were counterstained with Harris hematoxylin and mounted with coverslips.

The relative expressions of VEGF, CAIX, pimonidazole, Nox-4, and nitrotyrosine, in treated versus control tumors were calculated from the quantification of digitized images [positively-stained area (brown staining) and total area per low power field (Adobe Photoshop)]. Microvessel density (MVD) and proliferating capillary index (PCI) was calculated at lower magnification and CD31/105-positive vessels were counted in each tumor xenograft from 4–5 random fields. Vascular hot spots were also identified, and individual microvessel counts were made on a 100X field. Any highlighted endothelial cell or cell group occurring closely together and distinctly apart from microvessels, tumor cells, or other near-lying connective tissue morphology, was defined as a singular microvessel count. Results were expressed as the highest number of microvessels in any single 100X field. Tumor cells with intranuclear staining were considered positive for HIF-1α. After the slides were scanned at low magnification (40X), positive cells were counted in 4–5 random fields at a magnification of 400X (NIH Image J). The total number of cells in the respective fields was also determined to derive labeling index for HIF-1α.

Double immunofluorescent immunohistochemistry

The slides were incubated sequentially with the following primary antibodies: CD31 (1:200, rat anti mouse, BD Pharmingen), CD68 (1:200, rat anti mouse, BD Pharmingen), nitrotyrosine and Nox-4 (1:250 and 1:200, Santa Cruz biotechnology). FITC and TRITC labeled secondary antibodies (Jackson Immuno-research) were applied respectively. The tumor sections were digitized with a computerized digital image analysis system. A high resolution, intensified solid state camera on a fluorescence microscope (Axioskop, Zeiss) was used. Each tumor field was sequentially scanned two times at 100 magnification, using two different filters for the FITC (green), and Texas red (red) signals to identify double staining. Then respective stains were overlayed by using Metamorph software (Universal Imaging Corporation).

Protein Extraction and Western Blot

The snap frozen tumor tissues were homogenized in 500 μl ice-cold buffer containing 1% sodium dodecylsulfate, 5 mM Tris-HCl (pH 7.4), 2 mM EDTA, 10μg/ml aprotinin, and 0.5 mM phenylmethylsulfonyl fluoride. Samples were sonicated on ice for 15 sec and centrifuged at 12,000 × g for 5 min at 4°C to remove large tissue debris. Protein concentrations were determined by the Bradford assay [41]. In preparation for Western blotting, the lysate was diluted 6-fold with sample buffer [480 ml/L glycerol, 60g/L SDS, 17.28 mM β-mercaptoethanol, 12 mM EDTA, 300 mmol/L Tris-HCl (pH 6.8), 0.1g/L bromophenol blue], and boiled for 5 min. After running, PAGE gels were incubated with anti-HIF-1α antibody (1:500 dilution; Novus Biological, Littleton, CO) for 3 hrs, and blots were washed, and then incubated with peroxidase-labeled anti-mouse antibody (1:5,000; y) for 2 hrs. The blots were developed with Chemiluminescent substrate (Pierce Chemical Company, Rockford, IL, USA). To control for loading efficiency, blots were re-probed for α-tubulin. Western blot data were quantified as described [42].

Determination of MnTE-2-PyP⁵⁺ levels in tumor

MnTE-2-PyP⁵⁺ was determined as previously described for the analysis of its mitochondrial levels and pharmacokinetic study [43, 44]. After tumors were homogenized with 1 + 2 volumes of water, and proteins removed, MnTE-2-PyP⁵⁺ was reduced to MnTE-2-PyP⁴⁺ by ascorbate. The exchange of Mn site by Zn was followed by HPLC/fluorescence analysis of ZnTE-2-PyP⁵⁺. Three tumors from low-dose and high-dose groups were analyzed.

Statistics

Results are expressed as means ± SEM Student’s t test. P<0.05 was considered statistically significant.

Results

MnTE-2-PyP⁵⁺ has anti-tumor effects

There was a trend towards tumor growth delay by this compound with low dose (p=0.06), but was significant with high dose (p=0.002) (Figure 2). The suppression of tumor growth became obvious at later time points when tumor vasculature starts growing to support exponential tumor growth. At a high dose tumor growth ceased at 14 days. No differences in weight gain among groups were observed (data not shown).

Figure 2. Anticancer activity of Mn(III) tetrakis(N-ethylpyridinium-2-yl) porphyrin (MnTE-2-PyP⁵⁺.

4T1 murine breast tumors were grown in Balb/C mice, allowed to reach ≥200 mm³ in size, and randomized to one of three treatment groups: (1) phosphate-buffered saline (PBS, control), (2) MnTE-2-PyP⁵⁺ (2mg/kg/day given in two daily increments), (3) MnTE-2-PyP⁵⁺ (15 mg/kg/day given in two daily increments). The high dose-treatment group had significant effects on tumor growth delay starting at day 12, as compared to control (n = 25 per group)(*p < 0.02 vs. Control).

Levels of MnTE-2-PyP⁵⁺ in tumors

Tumor accumulation of MnTE-2-PyP ⁵⁺ occurred in a dose dependent manner (Table 1). The 7.5-fold increase in drug dose was paralleled by 6-fold increase in drug tumor levels.

Table 1.

Levels of MnTE-2-PyP⁵⁺ in tumors after subcutaneous administration to Balb/C mice for 13 days at 2mg/kg/day and 15 mg/kg/day (given subcutaneously in two daily increments).

MnTE-2-PyP5+	tumor (μg/g wet tissue)
2 mg/kg/day	536 (SEM ± 43.58)
15 mg/kg/day	3139 (SEM ± 235.4)

MnTE-2-PyP⁵⁺ decreases tumor oxidative stress and inflammatory response

NADPH oxidase (Nox-4) was significantly reduced in low-and high dose-groups (p=0.01 and p=0.003) (Figure 3). In comparison, PBS-treated tumors displayed strong immunoreactivity. DNA oxidation assessed by 8-OHdG immunohistochemistry, revealed strong staining in control tumors (Figure 3). A significant reduction in DNA oxidation was seen in low and high dose groups (p=0.01and p=0.009) (Figure 3). Tumor sections from PBS-treated mice displayed strong immunoreactivity for the presence of protein 3-nitrotyrosine. Thus, diffusely positive cytoplasmic immunostaining for nitrotyrosine was evident in the tumor epithelial/endothelial and inflammatory cells. In contrast, only mild immunoreactivity for nitrotyrosine was detected in low and high dose groups (p=0.01 and p=0.002)(Figure 3). Staining for macrophages (CD-68) demonstrated an increase in both the number and the activity of macrophages in PBS-treated tumors (Figure 3). A significant decrease in the macrophage count was found in low and high dose groups (p=0.03 and p=0.01) (Figure 3).

Figure 3. (A)Immunohistochemistry of Nox-4, 8-OHdG, 3-Nitrotyrosine and CD68.

The tumors treated with low and high dose of MnTE-2-PyP⁵⁺ exhibited much fewer activated macrophages and lower level of oxidative stress. These photomicrographs are representative of results obtained from 5–6 animals in each group. (B). Semiquantitative anlysis for Nox-4 displayed an intense expression in control tumors. A significant decrease in the Nox-4 was found in the groups receiving MnTE-2-PyP⁵⁺ treatment compared with the control alone. (C). PBS-treated tumor sections displayed strong immunoreactivity for the presence of 3-nitrotyrosine. Significantly reduced immunoreactivity was detected in MnTE-2-PyP⁵⁺ treated groups. (D). 8-OHdG expression revealed strong nuclear oxidation in control tumors. A significant reduction in DNA oxidation was found with MnTE-2-PyP⁵⁺ treatment. (E). Semiquantitative anlysis for macrophages demonstrated an increase in number of macrophages in control tumors. A significant decrease in the macrophage count was found in the groups receiving MnTE-2-PyP⁵⁺ treatment compared with the PBS group.

MnTE-2-PyP⁵⁺ increases tumor oxygenation

PBS-treated tumors exhibited strong HIF-1 α nuclear staining, (Figure 4). In a low dose group, the immunoreactivity of HIF-1α displayed trends towards significant reduction, but was significantly reduced in a high dose group (5 mg/kg, twice daily), (p=0.02) (Figure 4E). Western blot analysis further confirmed the HIF-1α results (Figure 4E). CAIX immunoreactivity (p=0.02 and p=0.006) and pimonidazole staining (p=0.03 and p=0.02) was significantly reduced in both low and high dose groups when compared to PBS group (Figure 4).

Figure 4. (A)Immunohistochemistry of HIF-1 α, CAIX, and pimonidazole.

HIF-1α, positive nuclear staining (brown) was most intense in control tumors, less so in the low- and significantly less in high dose treatment groups. CA IX and pimonidazole were significantly lowered in low and high treatment groups. These photomicrographs are representative of results obtained from five to six animals in each group. Semi-quantitative analysis of (B) HIF-1α, (C) CAIX and (D) pimonidazole.

(E) HIF-1α western blot expression: (I), Representative Western blots for HIF-1α and α-tubulin. (II) densitometric readings (HIF-1α/α-tubulin) of the Western blot expressed as percent control (control set to 100%). High dose group significantly inhibited HIF-1α expression. *p < 0.05.

MnTE-2-PyP⁵⁺ decreases the angiogenic profile of tumors

Lower levels of VEGF expression were observed in tumors from mice treated with both low and high dose of MnTE-2-PyP⁵⁺ than with PBS (p=0.04 and p=0.01) (Figure 5). CD31 also showed a high density of microvessels (MVD) in saline-treated tumors. We observed a decrease in MVD when comparing tumors of mice treated with PBS versus low dose (p=0.06) and it becomes significant when mice were treated with high dose (p-0.02) (Figure 5). When proliferating capillaries index (PCI) was analyzed with CD105 staining (Figure 5), it also displayed a high endothelial proliferation in PBS-treated tumors. We observed a significant decrease in PCI when comparing tumors treated with PBS versus low (p=0.008) or high dose (p=0.03) (Figure 5).

Figure 5. (A)Immunohistochemistry of VEGF, CD31 and CD105.

Effect of MnTE-2-PyP⁵⁺ on angiogenic profile of 4T1 tumors. For VEGF, positive staining (brown) was most intense in control tumors with increased MVD and PCI. However, VEGF expression was significantly lowered after treatment with MnTE-2-PyP⁵⁺, compared to control tumors. Similarly less number of blood vessels (MVD) and endothelial proliferation (PCI) were documented in groups treated with MnTE-2-PyP⁵⁺. These photomicrographs are representative of results obtained from five to six animals in each group. Semiquantitative analysis of (B) VEGF (C) Microvessel Density (MVD) (D) Proliferating Capillary Index (PCI) in the tumors with or without MnTE-2-PyP⁵⁺ treatment.

Co-localization of blood vessels with Nox-4 and nitrotyrosine

Representative images of 4T1 tumors stained for CD31 pairing with Nox-4 and nitrotyrosine are shown in Figure 6. Co-localization of Nox-4 and nitrotyrosine with CD31 displayed higher expression in angiogenic areas.

Figure 6. Co-localization of blood vessels with Nox-4 and 3-nitrotyrosine.

Representative images of 4T1 stained either for CD31 pairing with Nox-4 and nitrotyrosine. Co-localization of Nox-4 and nitrotyrosine with CD31 displayed higher expression in angiogenic areas.

Co-localization of macrophages with Nox-4 and nitrotyrosine

Representative images of 4T1 tumors stained for CD68 pairing with Nox-4 and nitrotyrosine are shown in Figure 7. Co-localization of Nox-4 and nitrotyrosine with CD68 displayed higher expression in inflammatory spots.

Figure 7. Co-localization of macrophages with Nox-4 and 3-nitrotyrosine.

Representative images of 4T1 tumors stained for CD68 pairing with Nox-4 and nitrotyrosine. Co-localization of Nox-4 and nitrotyrosine with CD68 displayed higher expression in inflammatory spots.

MnTE-2-PyP⁵⁺ does not affect tumor cell proliferation and apoptosis (data not shown)

To demonstrate the tumor proliferative capacity, we counted cells with Ki-67 positive nuclei in random fields for each treatment arm. Quantification confirmed that, compared to PBS, low and high doses did not significantly reduce cell proliferation. We next checked the other side of the proliferation: “apoptosis” balance by labeling cells for activated caspase 3 (aCASP-3), a marker of apoptosis, on slices from treated tumors. Interestingly, these tumors also did not display any difference. Such data also support the notion that the anti-tumor effects are primarily on the levels of endothelial cells.

Discussion

Tumors are under persistent oxidative stress and are thus constantly inflamed [28, 35, 45–47]. Several studies reported that increased oxidant/antioxidant ratio is directly related to tumor progression, angiogenesis and migration/invasion [35]. Tumor cells produce ROS/RNS at a far greater rate than do the non-transformed cell lines [48]; markers of constitutive oxidative stress have been detected in samples from in vivo breast carcinomas [49, 50]. 8-Hydroxy-2′-deoxyguanosine, oxidatively modified DNA base product, are almost 10 times more prevalent in invasive ductal breast carcinoma cells than in normal control samples from the same patient [50]. Reactive oxygen and nitrogen species, have a potent stabilizing effects on HIF-1α, and therefore on tumor angiogenesis [18, 51–56]. Angiogenesis is critical for tumor growth and invasiveness [57]. Recently, DPI (diphenyleneiodonium chloride, NADPH oxidase inhibitor) and rotenone (inhibitor of complex I of mitochondrial respiration) that decreases levels of endogenous reactive oxygen species, were reported to inhibit angiogenesis and tumor growth [58].

Macrophages are prominent inflammatory cells in tumor tissues, comprising up to 80% of the cell mass in breast carcinoma [59]. They are major producers of ROS/RNS and play an important part in promoting tumor growth and progression [60, 61]. Tumor-associated macrophages have been shown to deliver a sublethal oxidative stress to murine mammary carcinoma [62]. Tumor necrosis factor-a secreted by tumor-associated macrophages is shown to induce cellular oxidative stress [63]. Macrophages are generally at higher levels in more hypoxic tumors as seen in primary human breast carcinomas and in various animal tumors [47, 64–67]. In response to low oxygen tension prevalent in tumors, macrophages are recruited from the circulation, where they are activated to undergo the respiratory burst releasing superoxide and nitric oxide [65–68], which affects signaling events that in turn increases leukocyte migration and vascular permeability, tumorgrowth, invasion, metastasis, cytokine production and angiogenesis. We have previously found that if macrophages were co-cultured under hypoxia with MnTE-2-PyP⁵⁺, superoxide production was suppressed to baseline levels [69]. Attempts have been made to reduce the number of macrophages in tumors. In a rat model of prostate cancer, anti-angiogenic drugs markedly reduced tumor associated macrophage numbers (<50% of controls), along with tumor growth [70].

Our data show that MnTE-2-PyP⁵⁺ accumulates in tumor dose dependently; 7.5-fold increase in dose resulted in 6-fold increase in tumor drug level. Data also show that drug in tumors did reach saturation levels, which allows for further enhancement of anti-angiogenesis with increased dosing. Our data further clearly show that tumor oxidative stress was greatly suppressed by MnTE-2-PyP⁵⁺ in a dose dependent manner. Macrophage infiltration, NADHP oxidase levels, 3-nitrotyrosine formation (an indicator of ONOO⁻ formation) [71, 72] and DNA oxidation were all lowered in tumors in drug-treated groups and most so with high dose of MnTE-2-PyP⁵⁺ (Figure 3). NADPH oxidases appear to be the major components of tumor oxidative stress [73–76] and Nox-4-generated O₂^.−/H₂O₂ were shown to stabilize and/or increase expression of HIF-1α [77, 78]. Because MnTE-2-PyP⁵⁺ diminished levels of ROS/RNS, as expected (based on our previous findings [18, 40] hypoxia was reduced (pimonidazole), as well as HIF-1α and carbonic anhydrase IX expressions (Figure 4). Consequently the expression of the corresponding VEGF protein was diminished resulting in significant suppression of angiogenesis as observed through decrease in microvessel density and endothelial proliferation (Figure 5). Immunofluorescent immunohistochemistry displayed Nox-4/nitrotyrosine co-localization with angiogenic hot spots in tumor (Figure 6). Co-localization of Nox-4/nitrotyrosine with macrophages was also seen (Figure 7).

When the tumor is small, and existing tumor vasculature can still support its growth, the anti-angiogenic effect of MnTE-2-PyP⁵⁺ was small, but became significant once the tumor growth accelerates, and new vasculature as well as vasculature remodeling occurs. The latter was accompanied by cycling hypoxia//reoxygenation which further enhances ROS/RNS production (Figure 2). Tumors cease to grow at ~day 14. The pharmacokinetic studies [43] indicate that a loading dose of MnTE-2-PyP⁵⁺ may be necessary for a certain time period, followed by a maintenance dosing. Further studies will optimize the dosing regime whereby anti-cancer effects may be enhanced. Our data are in agreement with the trend of the tumor growth/suppression observed when NOS inhibitor L-NAME was tested in a same 4T1 murine breast tumor model in Balb/C mice where the tumor suppression effect becomes obvious only after ~15^th day.

Two previous studies on 4T1 tumor growth in Balb/C mice utilizing MnTE-2-PyP⁵⁺ were performed. In a 4T1 window chamber study on BALB/c mice, the very first encouraging data on tumor suppression by MnTE-2-PyP⁵⁺ were provided although the mechanism of action was at that point still not understood. In that study a CMV-RFP construct was introduced into tumors that allows expression of red fluoresecence protein (RFP) controlled by cytomegalovirus (CMV) promoter so that tumor cells can be tracked. Details are given in ref 18. The 4T1 line also expresses hypoxia responsive green fluorescent protein serving as a reporter for HIF-1α, which allows a serial monitoring of HIF-1 activity levels by intravital fluorescence activity. The data indicate that anti-angiogenic effects became obvious at ~ day 10, and becomes dramatic at 2 weeks after transplantation of tumors on mouse back (Figure 8A) whereupon the tumors become necrotic and start to shrink (Figure 8B and 8C). It was also obvious that angiogenic suppression by MnTE-2-PyP⁵⁺ begins early on while the tumors are still similar to the size of the control PBS-treated tumors. Such data are consistent with the finding in this study. In a subsequent (2^nd) study [17,18] MnTE-2-PyP was given to BALB/c mouse in 3 doses only, either every 12 hours within first 36 hours [17], or every 24 hours within first 74 hours after tumors reached volume of ~200 mm³ [18]. At the time of drug administration, tumors had been in a slow initial stage of its growth. The pharmacokinetic study [43] suggests that such drug delivery could not have provided the tumor levels of MnTE-2-PyP⁵⁺ required throughout its aggressive growth and explains the absence of effects in a second study. However, more recent data keep showing that MnTE-2-PyP prevents significantly-to-fully the activation of major transcription factors [16–18, 21,22,24,69] involved in cell proliferation and apoptosis and angiogenesis, and clearly indicate that the very first data should not be overlooked and that there is a rational behind the MnP anti-cancer activity which relates the ROS/RNS scavenging ability of Mn porphyrin directly to the redox-sensitive cellular transcriptional activity. Therefore the cancer research continued and eventually, this 3^rd study was performed with a comprehensive insight into tumor angiogenesis and oxidative stress, and with the goal of explaining if and why a catalytic SOD mimic/peroxynitrite scavenger would exert anti-cancer effects. Data for the first time show clearly that MnTE-2-PyP⁵⁺ has anti-cancer effect in its own right, due at least in part to the suppression of oxidative stress (thus levels of signaling reactive species), which in turn suppresses cellular transcriptional activity and leads to the suppression of tumor vasculature growth. We have not observed a significant effect of MnTE-2-PyP⁵⁺ on proliferation and apoptosis of tumor cells, per se, which further supports the notion that the major anti-tumor impact occurs on the level of tumor vasculature/endothelial cells [46, 79]. Also, Moeller et al [17] and our preliminary experiments on different tumor cell lines including CaCo, 4T1, HCT116, and HeLa (Ye X, Batinic-Haberle, I et al, unpublished) indicate the lack of cytotoxicity of MnTE-2-PyP⁵⁺ at up to mM levels.

Figure 8. Intravital fluorescent monitoring of HIF-1 activity and tumor angiogenesis and tumor growth in a 4T1 window chamber study on BALB/c mouse with MnTE-2-PyP⁵⁺ (AEOL10113).

Drug was given at 6 mg/kg/day ip with 5 mice per group. A CMV-RFP construct was introduced into tumor that allowed expression of red fluoresecence protein (RFP) of a cytomegalovirus (CMV) promoter so that tumor cells can be tracked. Details are given in ref 18. The 4T1 line expresses also hypoxia responsive green fluorescent protein serving as a reporter for HIF-1α, which in turn allows a serial monitoring of HIF-1 activity levels by intravital fluorescence activity (Figure 8A and B). The growth curves (Figure 8C): green diamonds, MnTE-2-PyP⁵⁺-treatment (AEOL10113), and the red square, PBS treatment.

Gao et al recently reported that a stoichiometric antioxidant, N-acetylcysteine, exerts anti-tumor effects which are HIF-dependent [80]. Our data support their findings. Therefore, the scavenging of ROS/RNS by antioxidants, which suppresses cellular transcriptional activity, could be an appropriate strategy for anticancer therapy. The catalytical scavengers would be advantageous. The effects may be increased with more cell permeable compounds such as the 4 orders of magnitude more lipophilic hexyl analogue, MnTnHex-2-PyP⁵⁺. This analogue has been up to 120-fold more effective in several animal models of oxidative stress injuries [16, 19, 25], due predominantly to its enhanced lipohilicity [81]. A 3000-fold enhancement in an oxygen/glucose deprivation neuronal model as a result of increased lipophilicity has also been observed with the octyl analogue, MnTnOct-2-PyP⁵⁺ when compared to MnTE-2-PyP⁵⁺ [82]. The anti-cancer effects may further be increased if the drug is combined with radiation and hyperthermia [17, 40]. Preliminary data from our group (Ye X et al, unpublished) indicate that Mn porphyrins enhance chemotherapy also.

Conclusions

Our study indicates for the first time that a catalytic scavenger of superoxide and peroxyntrite, MnTE-2-PyP⁵⁺ has anti-cancer activity in its own right. This effect is the result of the redox-based impact of the drug on the oxidative stress whereby decreased levels of reactive species lead to the suppression of HIF/VEGF pathways and therefore suppression of tumor angiogenesis. Our work has important implication for follow up studies aimed at enhancing tumor therapy via: (1) exploring the possible enhancement of anti-cancer effect utilizing more bioavailable drugs; (2) optimizing drug dosing regimes and (3) combining catalytic antioxidants with irradiation, hyperthermia and chemotherapy. Given the additional ability to radioprotect normal tissue and its potential in maintaining analgesic effects when morphine is given chronically, MnTE-2-PyP⁵⁺ can exert triple action in cancer treatment, being therefore advantageous over other anti-cancer drugs.

Abbreviations

SOD

superoxide dismutase

MnP

any porphyrin

Mn^IIIP/Mn^IIP

oxidized(III)/reduced(II) porphyrin at Mn site

MnTE-2-PyP⁵⁺

Mn(III) meso-tetrakis(N-ethylpyridinium-2-yl)porphyrin (AEOL10113)

MnTnHex-2-PyP⁵⁺

Mn(III) meso-tetrakis(N-n-hexylpyridinium-2-yl)porphyrin

MnTnOct-2-PyP⁵⁺

Mn(III) meso-tetrakis (N-n-octylpyridinium-2-yl)porphyrin

MnTDE-2-ImP⁵⁺

Mn(III) meso-tetrakis (2-diethylimidazolium)porphyrin (AEOL10150)

HIF-1α

hypoxia inducible factor-1α

NF-κB

nuclear factor κB

AP-1

activator protein-1

CD31

marker for microvessel density

CD105

marker for proliferative capillary index

CD68

marker of macrophage infiltration

CAIX

carbonic anhydrase IX

Nox-4

homologue of gp91^phox catalytic subunit of phagocyte NAD(P)H oxidase

8-OHdG

8-hydroxy-2′-deoxyguanosine

DPI

diphenyleneiodonium chloride