Peritumoral administration of granulocyte colony ...

[Cancer Biology & Therapy 8:18, 1737-1743; 15 September 2009]; ©2009 In Landes vivo antitumor Bioscienceeffect of G-CSF

Research Paper

Peritumoral administration of granulocyte colony-stimulating factor induces an apoptotic response on a murine mammary adenocarcinoma Julieta Marino,1 Verónica A. Furmento,1 Elsa Zotta2 and Leonor P. Roguin1,* 1Instituto

de Química y Fisicoquímica Biológicas (UBA-CONICET); Facultad de Farmacia y Bioquímica; Buenos Aires, Argentina; 2Departamento de Fisiología; Facultad de Medicina; Buenos Aires, Argentina

Abbreviations: Fas-L, Fas ligand; FBS, fetal bovine serum; G-CSF, granulocyte colony-stimulating factor; IFN, interferon; IL, interleukins; PAS, periodic acid schiff; PMSF, phenylmethanesulfonyl fluoride; TNF, tumor necrosis factor; TRAIL, TNF-related apoptosis-inducing ligand receptor; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labelling; VEGF, vascular endothelial growth factor Key words: granulocyte colony-stimulating factor, mammary adenocarcinoma, apoptosis, antitumor response, mouse tumor model

The aim of the present study was to evaluate the in vivo effect of granulocyte colony-stimulating factor (G-CSF) on LM3 murine mammary adenocarcinoma cells subcutaneously implanted in Balb/c mice as experimental models. We showed that the peritumoral administration of 100 μg/kg of G-CSF diminished tumor progression, while no cytokine effect on LM3 cell proliferation was observed in vitro. Histological examination of G-CSF-treated tumors revealed infiltration of neutrophils and mononuclear cells. Apoptotic cells were identified by TUNEL assays. Western blot analysis of tumor lysates showed that G-CSF treatment increased the amount of Fas-L, TRAIL and Bax proteins, whereas decreased the expression of procaspase 3 and Bcl-2. In addition, cytokine arrays showed an increment in the amount of IL-12, IL-13 and TNFα. Our results suggest that the presence of G-CSF within tumor microenvironment would induce an immune response which eliminates tumor cells by apoptosis. Both death receptor and mitochondrial pathways would be involved in LM3 tumor cell death. We believe that the final local G-CSF concentration at the tumor site and each particular type of tumor should be carefully taken into account in order to evaluate the effect of the cytokine on tumor progression.

Introduction The granulocyte colony-stimulating factor (G-CSF) is a cytokine that induces proliferation and differentiation of myeloid precursor *Correspondence to: Leonor P. Roguin; IQUIFIB; Facultad de Farmacia y Bioquímica; Universidad de Buenos Aires; Junín 956; Buenos Aires C1113AAD Argentina; Tel.: +54.11.49648290; Fax: +54.11.4962.5457; Email: rvroguin@ qb.ffyb.uba.ar Submitted: 05/28/09; Accepted: 06/06/09 Previously published online as a Cancer Biology & Therapy E-publication: http://www.landesbioscience.com/journals/cbt/article/9210

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cells, and also stimulates the survival and functional activation of mature neutrophils.1,2 The role of G-CSF as a regulator of neutrophils production has promoted its clinical use in patients with neutropenia resulting from cancer chemotherapy.3-5 Remarkably, some studies have reported the inhibitory effect of G-CSF on solid tumor growth.6-8 In this sense, the G-CSF-mediated neutrophilia could contribute to eliminate tumor cells through the generation of cytotoxic factors that induce cell death. Thus, H2O2 and superoxide, membrane-perforating agents, Fas ligand (Fas-L), TNF-related apoptosis-inducing ligand (TRAIL), TNFα, IL-1β, IFNs and antibody-dependent cell cytotoxic effectors might be responsible of cell killing.9-12 However, some mediators, such as H2O2 have been related to cancer progression,13,14 and it has also been reported that G-CSF can support tumor progression.15-18 In this study we first decided to evaluate the effects of G-CSF on tumor progression in vivo using a syngeneic BALB/c mouse mammary tumor model. Since we demonstrated that G-CSF effectively diminished tumor growth, we then characterized the mechanism responsible of tumor inhibition by analyzing the cytokine profile and the expression levels of apoptosis-related proteins in tumor lysates.

Results Effect of G-CSF on tumor growth. In order to examine the effect of the peritumoral injection of G-CSF on tumor growth, mice were inoculated with murine mammary carcinoma LM3 cells and 7 d after, PBS or different doses of G-CSF were administered subcutaneously daily for 3 w. The administration of 100 μg/kg of G-CSF reduced tumor volume and weight 70% (p < 0.01) and 40% (p < 0.05), respectively, whereas no significant effect was obtained at 10 μg/kg (Fig. 1). When in vitro assays were performed to evaluate a direct action of G-CSF on LM3 cell growth, no cytokine effect was evident. Thus, the number of cells obtained after incubating 20,000 LM3 cells/well for 72 h at 37°C

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In vivo antitumor effect of G-CSF

Figure 1. In vivo effect of G-CSF treatment on tumor growth. (A) LM3 cells (3 x 105) were injected subcutaneously in the right flank of BALB/c mice. Mice received 0.2 ml of PBS (●), 10 (□) or 100 (▲) μg/kg/day of G-CSF, via s.c. for 3 w. Tumor sizes were measured with a caliper twice weekly. Tumor volume was calculated by the formula V = (D x d2)/2. (B) Photographs of tumors were obtained at the end of the experiment. Scale bar, 1 cm. (C) At the end of the treatment, tumors were excised and the wet weights were measured. Results represent mean values ± SE. Multiple comparison Dunnett’s test was applied after one way ANOVA (*p < 0.05, **p < 0.01).

in 96-well culture microplates with 1 μg/ml of G-CSF (316,000 ± 5,600 cells/well) was similar to that obtained in the absence of cytokine (313,000 ± 1,200 cells/well). Histological analysis of tumors. Examination of tissue sections of G-CSF-treated tumors revealed the presence of polymorphonuclear cells barrier between tumor interfaces with normal muscle tissue (Fig. 2B). This inflammatory infiltrate is also observed surrounding perivascular areas (Fig. 2D). Furthermore, an important mononuclear inflammatory infiltrate was observed in the tumor mass (Fig. 2B). The examination of G-CSF-treated tumors by PAS staining showed no difference in vascularization compared to non-treated tumor (Fig. 2E and F). Effect of G-CSF on the expression of apoptosis-related proteins in tumors. G-CSF effect on induction of apoptosis was evaluated by TUNEL assay in tumors from non-treated mice and 100 μg/kg/ day G-CSF-treated mice. As shown in Figure 3A, the presence of dark apoptotic nuclei was evident in tumor sections from treated mice. The expression of Bcl-2 family proteins was also evaluated 1738

in tumor lysates from mice treated with 100 μg/kg/day G-CSF or PBS. Results obtained by western blot showed a significant increment in the expression levels of the pro-apoptotic protein Bax, no change in the amount of Bcl-XL and a decrease of the anti-apoptotic Bcl-2 protein (Fig. 3B and C). We also found higher levels of ligands involved in the death receptor apoptotic pathway, such as Fas-L and TRAIL, but no modification in the amount of Fas receptor (Fig. 3B and C). In addition, caspase 3 activation was also demonstrated in tumors from G-CSF-treated mice, since the expression of procaspase 3 diminished approximately 40% (Fig. 3B and C). Cytokine expression profile. A murine cytokine array approach was employed to detect the expression of 22 cytokines in tumor lysates from treated and non-treated mice. As shown in Figure 4, the proteins most significantly upregulated in G-CSF-treated tumors were the bioactive form of IL-12 (IL-12p70), IL-13 and TNFα. Thus, densitometric quantification of membranes showed increments of 2–3 fold in the expression levels of these cytokines (Fig. 4B). No difference in the amount of other cytokines was observed.


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Figure 2. Histology of tumors sections. Tumors from PBS- (A, C and E) or 100 μg/kg/day G-CSF-treated (B, D and F) mice were fixed in formol buffer 10% in PBS and embedded in paraffin. Tumor sections were then stained with hematoxylin and eosin [(A and B) x400; (C and D) x1,000] or PAS [(E and F) x200]. M, mononuclear cells; PMN, polymorphonuclear leukocytes; V, blood vessels.

Discussion We herein demonstrated that the in vivo peritumoral administration of G-CSF effectively inhibited LM3 murine mammary adenocarcinoma growth by activating the migration of neutrophils and mononuclear cells which would induce an apoptotic effect responsible of tumor cell death. Since G-CSF had no effect on LM3 cell proliferation in vitro, the antitumor response would not be the result of a cytokine direct action on tumor cells. As it was previously reported,20 we observed that LM3 tumors induced leukocytosis and a reversal of lymphocyte/granulocyte ratio in peripheral blood, although no differences were found between www.landesbioscience.com

G-CSF-treated and non-treated mice (data not shown). In spite of this finding, an inflammatory infiltrate of neutrophils and mononuclear cells was evident in the border and inside the mass of G-CSF-treated tumors. It has been proposed that the localization of neutrophils around the tumor would inhibit tumor growth by a mechanical effect that restricts cell diffusion on the tumor border.21 However, several reports have emphasized the role of neutrophils as mediators of antitumor effects.9-12,22 In particular, it has been reported that the transfer of G-CSF gene to colon adenocarcinoma C-26 cells inhibited tumor development, suggesting that the local release of G-CSF induces an antitumor response by recruitment of neutrophils and other mononuclear


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Figure 3. In vivo induction of apoptosis. (A) Representative TUNEL results from control (PBS) and 100 μg/kg/day G-CSF-treated mice. Dark nuclei correspond to apoptotic cells; counterstaining was performed with hematoxylin (Magnification x1,000). (B) Tumor lysates (n = 7) were processed for western blot analysis as described in Materials and Methods. Actin was employed as loading control. Results from one representative experiment are shown. (C) Quantification of band intensity was performed by using a densitometer (Gel-Pro Analyzer). Control tumors (white bars) G-CSF-treated tumors (black bars). Results are expressed as mean ± SE of three different experiments (*p < 0.05, **p < 0.005).

Figure 4. Mouse cytokines upregulated with G-CSF treatment. (A) Mouse protein array analysis was used to determine the differences in the release of 22 cytokines from non-treated and 100 μg/kg/day G-CSF-treated mice. Membranes were incubated with tumor lysates as described in Materials and Methods. Autoradiographs of the arrays were scanned to determine the density of each protein represented by duplicate spots. Values from scans were adjusted based on the intensity of control spots on the membrane corners. One representative experiment is shown. Spots from the upper rectangle correspond to IL-12p40p70, IL-12p70 and IL-13. The lower rectangle represents TNFα. (B) Results of the modified cytokines are expressed as the relative intensity and represent the mean ± SE of three different experiments (*p < 0.05, **p < 0.01). Control tumors (white bars), G-CSF-treated tumors (black bars). 1740


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cells, such as macrophages and T-cells, into the tumor.8,23 However, authors claimed that inhibition of C-26 tumor growth by G-CSF would be restricted to cells secreting G-CSF, since the injection of 400 μg/day of G-CSF twice a day for 20 d at the site of the tumor was unable to inhibit tumor formation. In contrast, we did demonstrate that the peritumoral administration of a lower dose of G-CSF (100 μg/kg/day, i.e., 2–2.5 μg/day) once a day for 21 d effectively decreased LM3 tumor development and induced an apoptotic response. The difference in tumor cells could possibly explain this discrepancy, supporting the hypothesis that G-CSF effects on tumor progression should be evaluated on each particular tumor. The increment of the local amount of G-CSF after cytokine injection at the LM3 tumor site would be responsible of attracting polymorphonuclear leukocytes and changing the cytokine profile into the microenvironment of the tumor. As a result, tumor cells would be eliminated by the induction of an apoptotic response. Accordingly, apoptotic tumor cells were detected by TUNEL assay in tumors from G-CSF-treated mice. It is known that apoptotic cell death is mediated through the activation of either death receptor or mitochondrial pathways, both regulated by aspartate specific cysteine proteases named caspases.24-29 The interaction of members of the death receptors family (Fas, TNF or TRAIL) with their corresponding ligands in the cell surface leads to the formation of a death-inducing signaling complex that activates the initiator caspase-8. The mitochondrial pathway involves the release of multiple polypeptides from mitochondria, including cytochrome C, which induces the formation of the apoptosome and the activation of caspase-9. The initiator caspases can proteolytically activate the effector caspase-3, which induce the cleavage of cellular substrates that are finally responsible of the execution of cell death.24-29 The involvement of the apoptotic pathway mediated by death receptors was herein indicated by the increment in the levels of Fas-L and TRAIL molecules found in tumor lysates from mice treated with 100 μg/kg/day G-CSF.11,24,28,30-32 In addition, cytokine arrays also showed an increment in the amount of TNFα, a death factor belonging to the TNF family.28,30-32 Various members of the Bcl-2 family proteins may also play a role in the mitochondria-mediated cell death.24,25,28,33,34 Western blot analysis of tumor lysates supernatants from G-CSF treated mice showed an increment of the amount of the pro-apoptotic protein Bax and a decrease of the anti-apoptotic protein Bcl-2, suggesting the involvement of the mitochondrial pathway.24,25,28,33,34 Thus, we demonstrated that both pathways would contribute to the activation of caspase 3, since a significant decreased in the amount of procaspase 3 was observed. In summary, G-CSF effect on polymorphonuclear leukocytes would induce an apoptotic response on tumor cells through the activation of death receptor and mitochondrial pathways, both leading to the activation of the executioner caspase 3. Furthermore, cytokine array assays showed that G-CSF treatment induced higher levels of IL-12, a cytokine previously evaluated as an effective antitumor agent that enhances the cytotoxic activity of immune cells.35,36 An increment in the amount of IL-13 was also evident. Although IL-13 has been formerly related to allergic inflammation and fibrosis formation, it has been recently reported that IL-13 promotes the apoptosis of endothelial cells and www.landesbioscience.com

impairs angiogenesis.37,38 Thus, we hypothesised that both IL-12 and IL-13 could contribute to exert an inhibitory role on tumor development. Even though we demonstrated that G-CSF inhibited tumor growth, other authors have shown that G-CSF promotes tumor growth by stimulating tumor angiogenesis.17,18 These results could reflect the response of a particular type of tumor that releases angiogenic factors which contribute to vessel formation, and/or could be the consequence of the systemic administration of G-CSF. Under our experimental conditions, the histological examination of tumor slices from treated and non-treated mice did not reveal any difference in tumor vascularization. In addition, we did not find a change in the expression level of an angiogenic factor, such as VEGF, either when it was tested by cytokine array or when tumor lysates were examined by western blot with an specific anti-VEGF antibody (data not shown). In conclusion, we demonstrated that the local administration of G-CSF at the tumor site inhibited tumor growth and induced an immune response which eliminated tumor cells by apoptosis. Both death receptor and mitochondrial pathways regulate tumor cell death. Each particular tumor and the local amount of G-CSF within tumor microenvironment should be carefully considered in the evaluation of G-CSF effects on tumor progression.

Materials and Methods Chemicals. Recombinant human G-CSF expressed in Chinese hamster ovary cells was supplied by BIO SIDUS S.A., Buenos Aires, Argentina. Monoclonal anti-Bax, anti-Fas and anti-Fas-L antibodies and polyclonal anti-Bcl-2, anti-Bcl-XL and anti-TRAIL antibodies were from Santa Cruz Biotechnology, Inc., CA USA. Monoclonal antibody against procaspase 3 was purchased from Oncogene Research Products, CA USA. Polyclonal anti-actin antibody was from Sigma-Aldrich, Inc., MO USA. Cell culture. LM3 murine mammary adenocarcinoma cell line,19 kindly provided by the Institute of Oncology Angel H. Roffo (Buenos Aires, Argentina), were grown at 37°C under 5% CO2 atmosphere in DMEM-F12 (Gibco, NY USA) supplemented with 10% FBS, 2 mM L-glutamine, 0.6% Hepes, 50 U/ml penicillin and 50 μg/ml streptomycin. For harvesting, cells were treated with 0.05% trypsin/EDTA using standard procedures. In vivo experiments. All experiments were carried out in accordance with the National Institute of Health (NIH) Guide for the Care and the Use of Laboratory Animals. Female BALB/c mice, obtained from the Animal Care Facility of the School of Pharmacy and Biochemistry, University of Buenos Aires, were housed under controlled conditions and were routinely used at 10–12 w old (approximate weight, 20–25 g). Food and water were administered ad libitum. LM3 cells (3 x 105) diluted in 200 μl of DMEM-F12 were injected subcutaneously in the right flank of each mouse. Seven days after cell inoculation, mice were divided into three groups (n = 7): group I (control) received 0.2 ml of PBS; group II and III received 10 or 100 μg/kg/day of G-CSF, respectively, via s.c. for 3 w. Animals were monitored daily and their body weights were recorded weekly throughout the study. Tumor sizes were measured with a caliper twice a week and tumor volumes were


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calculated using the formula: V = (D x d2)/2, where D is the larger diameter and d is the smaller. At the end of the study, mice were anesthetized via i.p. with 100 μg ketamine and 10 μg diazepam/g of body weight, and tumors were excised, weighed and measured. Tumors were fixed in formol buffer 10% in PBS 0.1 M, pH 7.4, and then dehydrated and included in paraffin. Cuts of 5 μm were made in microtome (Leica RM 2125. Wetzlar, Germany) and mounted on 2% silane-coated slides. Sections were stained with hematoxylin-eosin or Periodic Acid Schiff (PAS) for histological analysis. TUNEL assay. The apoptotic effect of G-CSF administration was detected in tissue sections of tumors by TUNEL assay. Sections of 5 μm from control and 100 μg/kg/day G-CSF treated-mice were used to determine the DNA strand breaks by the DeadEndTM Colorimetric TUNEL System (Promega Corporation, WI USA) according to the manufacturer’s instructions. Hematoxylin staining was used to counterstain tissue sections. Apoptosis-related protein expression in tumors. To evaluate the expression of apoptosis-related proteins, tumors were excised and lysed in a buffer solution containing 0.5% Triton X-100, 1 μg/ml aprotinin, 1 μg/ml trypsin inhibitor, 1 μg/ml leupeptin, 10 mM Na4P2O7, 10 mM NaF, 1 mM Na3VO4, 1 mM EDTA, 1 mM PMSF, 150 mM NaCl, 50 mM Tris, pH 7.4. Clear tumor lysate supernatants were prepared by centrifugation and aliquots containing 100 μg of protein were resuspended in 0.063 M Tris/ HCl, pH 6.8, 2% SDS, 10% glycerol, 0.05% bromophenol blue, 5% 2-ME, loaded onto a 14% SDS-PAGE and then transferred onto nitrocellulose membranes (Amersham Biosciences, Piscataway, NY) for 1 h at 100 V in 25 mM Tris, 195 mM glycine, 20% methanol, pH 8.2. Non-specific antibody binding sites were blocked by incubating membranes for 1 h at room temperature with 10 mM Tris, 130 mM NaCl and 0.05% Tween-20, pH 7.4, (TBS-T), containing 3% BSA. Membranes were then incubated overnight at 4°C with anti-Bax, Bcl-2, Bcl-XL, Fas, Fas-L, TRAIL and procaspase 3 antibodies diluted in TBS-T, containing 1% BSA. After three to four washes with TBS-T, membranes were incubated for 1 h at room temperature with anti-mouse IgG (horseradish peroxidase-conjugated goat IgG from Jackson ImmunoResearch Labs, West Grove, PA) or anti-rabbit IgG (horseradish peroxidaseconjugated goat IgG from Santa Cruz Biotechnology, Inc.,) diluted in TBS-T, 1% BSA. The immunoreactive proteins were visualized using the ECL detection system (Amersham Biosciences) according to the manufacturer’s instructions. For quantification of band intensity, blots were scanned using a densitometer (GelPro Analyzer). Equal protein loading was confirmed by reprobing membranes with a rabbit anti-actin antibody (Sigma-Aldrich, Inc., MI USA). Mouse cytokine protein array. The secretion of various cytokines in tumors lysates was evaluated using Mouse Cytokine Antibody Array I obtained from RayBiotech, Inc., (Norcross, GA) according to the manufacturer’s protocol. This assay consisted of 22 different cytokine antibodies spotted in duplicate onto a membrane. The following cytokines were included in the analysis: G-CSF, GM-CSF, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12 p40p70, IL-12p70, IL-13, IL-17, IFNγ, MCP-1, MCP-5, 1742

RANTES, SCF, sTNFRI, TNFα, thrombopoietin, VEGF. The membranes were blocked and then 1 ml of lysates from either PBSor G-CSF-treated tumors containing 300 μg of proteins was added and incubated at room temperature for 2 h. After washing, 1 ml of primary biotin-conjugated antibody was added and incubated overnight at room temperature. The membranes were then incubated with 2 ml of horseradish peroxidase-conjugated streptavidin at room temperature for 2 h and developed by using enhanced chemiluminescence-type solution. Integrated density values were calculated for each spot using a densitometer (Gel-Pro Analyzer). Positive control signals on each membrane were used to normalize cytokine signal intensities. Statistical analysis. All values are expressed as mean ± SE. Statistical analysis of in vitro data was performed by using the Student’s t-test. ANOVA and Dunnett multiple comparison tests were used to establish the statistical significance of difference in tumor volume and tumor weight between control and treatedmice. All analyses were performed using the statistical software GraphPad Prism. Acknowledgements

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