Selective Cyclooxygenase-2 Inhibitor Etoricoxib Attenuated Hypoxic Cancer Milieu induced M2-Polarization of Macrophages and Acquisition of Pro-angiogenic and Pro-invasive Attributes

 

Nem Kumar Jain^1,2,3, Khemraj Singh Baghel¹

¹Division of Toxicology, Central Drug Research Institute (CSIR), Lucknow, Uttar Pradesh 226031, India

²National Institute of Pharmaceutical Education and Research (NIPER), Raebareli, Uttar Pradesh 229010, India

³ School of Pharmacy, ITM University Gwalior, Gwalior, Madhya Pradesh, 475001

 

ABSTRACT:

Cyclooxygenase-2 (COX-2) inhibitors have been found to reduce the relative risk of breast cancer in several experimental and clinical studies. Various mechanisms of anti-tumor activity of COX-2 inhibition have been reported viz. anti-proliferation, apoptosis, preventing invasion and metastasis, and potentiation of anti-tumor immune responses. In addition to this, previous work indicated that attenuation of pro-tumoral M2 polarization of macrophages could be another mechanism of anti-cancer activity of COX-2 inhibitors. This prompted our investigation of the pro-tumoral M2 polarization inhibitory potential of Etoricoxib, a selective COX-2 blocker. Treatment with Etoricoxib markedly inhibited hypoxic breast cancer cell induced macrophage M2 polarization to a greater extent compared to Flunixin meglumine, a preferential COX-2 inhibitor. Moreover, we found that Etoricoxib treatment results in suppression of pro-angiogenic and pro-invasive functions of tumor associated macrophages (TAMs). Our study provides evidence for potential applicability of Etoricoxib as an anti-breast cancer modality via targeting M2 polarization of macrophages.

 

 

INTRODUCTION:

Macrophages represent the most abundant subpopulation of infiltrating immune cells of tumor stroma. They constitute up to 70% of tumor mass and are often termed as tumor associated macrophages (TAMs) (1,2). Macrophages exhibit two distinct phenotypes viz. classically activated, anti-tumor M1-macrophages and alternatively activated, pro-tumoral M2-macrophages (3, 4). The TAMs exhibit marker profile characteristics of alternatively activated M2 macrophages (2). Experimental studies employing mice models indicate critical role of TAMs during tumor progression via potentiation of inflammation, angiogenesis, invasion and metastasis (5-8). Consequently, TAMs have emerged as potential target for devising anti-cancer immunotherapy.

 

TAMs are also instrumental in determining response/resistance to therapeutics particularly anti-angiogenic therapeutics. TAMs driven reparative mechanisms markedly compromise the efficacy of various anti-angiogenic therapeutics and lead to development of drug refractory/resistant cancer (9). Thus, a successful anti-cancer strategy should also target TAMs. In fact, simultaneous targeting of TAMs mechanisms has been suggested as an effective means to counter cancer progression as well as improve the efficacy of anti-angiogenic therapy. One of the key TAMs related phenomenon that are amenable to therapeutic intervention is their phenotype switching into alternatively activated M2-polarized macrophages. It has been hypothesized that hindering macrophage polarization toward pro-tumor M2 macrophage could be possible anticancer strategy for preventing resistance to cancer therapeutics particularly anti-angiogenic agents. In accordance, hindering macrophage M2 polarization via Oncostatin M blockade resulted in significant tumor regression 4T1/BALB/c-syngenic-mice model of breast cancer (10). An important and definitive marker of M2 polarization is expression of scavenger receptor CD206 and CD163. Therefore in our study, expression levels of CD206 were employed as a marker for M2 polarized macrophages. Expression of M2-polarized macrophages phenotype i.e. expression of CD163 has been linked to decreased progression free survival and overall survival in ovarian cancer (11). Clinically it has been found that increase in M2 macrophages has been associated with fast proliferation, poor differentiation, estrogen receptor negative, histological ductal type and reduced recurrence free survival (12).

 

Over the past decade, a multitude of studies have pointed towards cyclooxygenase-2 (COX-2) inhibition as a potential anti-cancer modality. COX-2 is highly inducible isoform of COX enzymes, induced by various factors. It is associated with carcinogenesis, neo-angiogenesis, immune-suppression and prevention of apoptosis in several cancers (13). Various Non-steroidal anti-inflammatory drugs (NSAIDS), particularly selective COX-2 inhibitors viz. Celecoxib, Rofecoxib, and Meloxicam have shown anti-tumor properties against breast, lung and colorectal cancers in several experimental, epidemiological and clinical studies (14). Taken together, these studies strongly suggest that COX-2 represents a key therapeutic target for cancer intervention (13). One of the proposed mechanism underlying anti-cancer effects of COX-2 inhibitors is attenuation of pro-tumoral M2 polarization of macrophages. Previous studies indicate that COX-2 participates in macrophage polarization and inhibition of COX-2 is associated with impaired macrophage M2 polarization (15). Celecoxib, a selective COX-2 inhibitor has been shown to alter the phenotype of TAMs from pro-tumor M2 macrophages to anti-tumor M1 macrophages in ApcMin/+ mouse polyps (15). Etodolac, a preferential COX-2 inhibitor has been shown to impede polarization of macrophages to an M2-skewed phenotype and reduced lung metastasis in an experimentally induced breast cancer model (16). Considering pivotal role of pro-tumoral M2 skewed macrophages in cancer progression and emerging role of COX-2 in polarization of macrophages towards a pro-tumoral M2 skewed phenotype, the current study was planned to evaluate effect of selective COX-2 inhibitor Etoricoxib on macrophages with special reference to their phenotype and pro-tumoral functions. Special emphasis was laid on hypoxic microenvironment as the hypoxic tumor milieu has been proposed as one of the key micro-environmental determinant governing macrophage phenotype switching as well as cancer progression. The study is likely to substantiate the applicability of selective COX-2 inhibitors as anti- cancer modality.

 

 

 

MATERIALS AND METHODS:

Antibodies and Reagents:

Anti-VEGF, anti-CD206 and anti- β-actin monoclonal antibodies were purchased from Santacruz (USA). FITC conjugate of anti-CD206 antibody was procured from BD Biosciences. Alexafluor 555 conjugates of anti-mouse and anti-rabbit IgG were procured from Invitrogen (USA). HRP conjugates of rabbit and mouse IgG were purchased from Cell Signaling Technology (USA). RPMI 1640, fetal bovine serum (FBS), DMEM medium were purchased from GIBCO-Invitrogen Corporation (USA). Antibiotics were purchased from in vitro gen (USA). 8.0μm polycarbonate (PCF) cell culture inserts and Amicon ultracentrifugal filters were purchased from Millipore (USA). Etoricoxib was purchased from Sigma Aldrich (USA). Phorbol 12-myristate 13-acetate (PMA) was procured from Calbiochem (USA).

 

Cell culture and In- vitro Differentiation:

Human leukemia monocyte THP-1 cells and human mammary cancer-derived MDA-MB-231 cells were procured from ATCC. Cells were maintained at 37ºC with a 5% CO₂ in humidified atmosphere (95% humidity) in RPMI-1640 or DMEM supplemented with 10% FBS and antibiotics (100μg/ml penicillin, 0.25μg/ml amphotericin-B and 100μg/ml streptomycin). THP-1 cells were differentiated to macrophages according to Dockrell et al (17). The differentiation was initiated by adding 30nM PMA to the cells. After 3 days cells were switched to PMA free media for further 5 days so as to ensure maximal dif­ferentiation.

 

Hypoxia Treatment:

Cells were exposed to hypoxic environment within the hypoxia chamber (Stem cell technologies, USA) maintained at low oxygen tension (1% O₂, 5% CO₂ and 94% N₂) and 37ºC. The oxygen concentration in the hypoxic chamber and the exposure medium was moni­tored by using an oxygen indicator (Forma Scientific, USA).

 

Cell Invasion Assay:

For assessing invasive potential, 2x10⁴ THP-1 derived macrophages were seeded in transwell cell culture inserts (24 well, 8mm pore size; Corning) which were coated with matrigel and were primed with normoxic and hypoxic cancer cell conditioned media (CM) with or without treatment. After 24 h, cells on the upper surface of the insert membrane were removed with a cotton plug and invaded cells were fixed with 3.7% paraformaldehyde. Finally, invaded cells were mounted with prolong gold antifade-DAPI aqueous mounting media and visualized (200X) using Leica DCF 450C florescence microscope in five different fields for each inserts.

 

Immunoblotting:

Cells were lysed and the protein samples were analyzed by the Lowry’s Folin method (18) and the equal amounts of protein sample were resolved on SDS-PAGE. Gels were transferred on to PVDF and the membranes were probed with antibodies. Membranes were further incubated with secondary antibodies conjugated to HRP which were detected by ECL reagent. The expression level of various proteins was quantified by measuring the intensity of respective bands using ImageJ software (ImageJ, National Institute of Health, Bethesda, MD). Intensity of loading control i.e. β-actin bands (in cell lysate) was used for normalizing the expression levels.

 

Fluorescence Immunocytochemistry and Flow Cyto­metry:

The presence of M2-macrophage specific cell sur­face marker (CD206) was detected using fluorescence immunocytochemistry and flow cytometry as described previously (10). For fluorescence im­munocytochemistry, cells were fixed and washed, further the specimen were blocked with 5% BSA for 1 h. Thereafter, cells were incubated overnight with anti-human CD206 (1:100) at 4°C. Specimens were then incu­bated with Alexafluor 555 conjugated anti-mice IgG (1:100) for 1h. Finally cells were mounted in prolong gold antifade-DAPI aqueous mounting media and vi­sualized (200X) using Leica DCF 450C florescence mi­croscope. For flow cytometry based detection of CD206 positive M2-macrophges, the control and experimental macrophage cultures were fixed with 3.7% parafor­maldehyde and washed. Thereafter, cells were suspended in PBS and incubated with FITC conju­gated anti- CD206 antibody for 1 h at 4°C. Finally, 10,000 viable cells were analyzed using FACS Calibur flow cytometer (BD Biosciences, USA).

 

Zymography Assay:

THP-1 derived macrophages were incubated with culture supernatant harvested from normoxic and hypoxic cancer cells. A fraction was utilized for assessing initial levels of MMP-9 activities and remaining fraction was introduced to THP-1 derived macrophages in presence or absence of stipulated treatments. After 24 h the media was collected and concentrated 10X using Amicon Ultra centrifugal filters. Protein estimation of concentrated media was done by Lowry’s Folin method (18). Concentrated conditioned media equivalent to 20µg of each sample were mixed with Non-reducing sample buffer, and resolved through 8% polyacrylamide-0.1% gelatin gel at 90 V for 0.5 h after dye front disappears. After washing with 2.5% Triton X-100 (300-500ml) for 40 min at room temperature gel was incubated for 20 h at 37^oC with incubation buffer. Gel was then stained with 0.5% Coomassie Brilliant Blue R-250 on shaker for 1- 2 h, and destained in 20% methanol and 10% acetic acid. Gelatinolytic activity was detected as transparent bands against the blue background of Coomassie brilliant blue stained gelatin.

 

Chick Chorioallantoic Membrane Assay:

To detect in vivo angiogenesis, we conducted Chick Chorioallantoic Membrane (CAM) assays. Control and experimental THP-1 derived macrophages (5x10³) were loaded on sterile gelatin sponge (4x4mm) which in turn was implanted onto the CAM at day 8 of fertilization. At day 12, CAMs were fixed with 10% formalin. Angiogenesis was quantified by counting the blood vessel branch points under a M205 FA Leica ste­reozoom microscope.

 

Real-Time PCR:

Total RNA was isolated with TRI reagent (Molecular Research Center), and cDNA was generated from 2μg of total RNA using High Capacity cDNA Reverse Transcription Kit. Quantitative PCR was performed on a Light Cycler 480 System (Roche) 96-well plates using SYBR Green qPCR Master Mix in accordance with manufacturer’s protocol. β-actin was used as an internal control. The relative expression ratio of a target gene was calculated as described by Pfaffi (2001) (19). The primers used were: human MMP-9 forward, 5'-GATGCGTGGAGAGTCGAAAT -3'; human MMP-9 reverse, 5'- CACCAAACTGGATGACGATG-3'.

 

Statistical analysis:

All data was analyzed by One Way Analysis of Variance (ANOVA) followed by Bonferroni post hoc test. A value of p < 0.05 was considered as statistically significant.

 

RESULTS:

M2-polarization of THP-1 derived macrophages following their incubation with hypoxic breast cancer cell conditioned media (CM) was attenuated in presence of Etoricoxib We previously demonstrated that THP-1 derived macrophages, when incubated with culture supernatant of hypoxic breast cancer cell, exhibited increased levels of M2-specific attributes such as CD206 and CD163. Taking cue from this, in the current study culture supernatant of hypoxic breast cancer cell was employed as stimulus for M2 polarization and inhibitory effect of Etoricoxib on M2 polarization was evaluated. We evaluated the presence of M2 specific surface marker viz. CD206 on macrophages that were incubated with hypoxic MDA-MB-231 cells conditioned media for 24 h in ab­sence or presence of Etoricoxib (10μM). The cells incubated with culture supernatant of normoxic cancer cells served as negative control while treatment with equimolar concentration of Flunixin meglumine (10μM), a well known preferential COX-2 inhibitor served as positive control. Macrophages incubated with hypoxic breast cancer cell CM underwent M2-polarization to a greater extent that the macrophages that were incubated with hypoxic breast cancer cell CM in presence of Etoricoxib (10μM) and Flunixin meglumine (10μM) (Fig.1A). Flow cytometric detection for CD206 revealed that amongst macrophages incubated with conditioned media (CM) harvested from normoxic MDA-MB-231 cells; only 4.2% underwent M2 polarization. However, when incubated with CM harvested from hypoxic (6h) MDA-MB-231 CM, 36.32% macrophages underwent M2 polarization. This exhibited marked decline to 9.06% in presence of Flunixin meglumine (10μM) and to 6.12% in presence of Etoricoxib (10μM) (Fig. 1B). The results indica­ted attenuation of hypoxic breast cancer cell CM induced polarization of macrophages towards an M2 skewed phenotype by Etoricoxib (Fig.1).

 

 

Figure 01. Pronounced Attenuation of Hypoxic Breast Cancer Cells Induced M2- Polarization of Macrophages by Etoricoxib.

Representative immunocytochemistry (A) and flow cytometry analysis (B) depicting marked suppression of hypoxic breast cancer cells CM induced alternate M2 macrophages polarization in the presence of Etoricoxib compared to Flunixin meglumine. Data were expressed as Mean ± SEM (n=3) of the results from three separate experiments. * indicates a p < 0.05 as compared to hypoxia primed MDA-MB-231 CM treated macrophages.

 

Acquisition of pro-angiogenic functions by macrophages following their incubation with hypoxic breast cancer CM was attenuated in presence of Etoricoxib.

A key functional attribute of M2 polarized macrophages is their ability to promote angiogenesis. Having established that, treatment with COX-2 inhibitors (Etoricoxib and Flunixin meglumine) impaired M2-polarization, we next decided to study the effect of Etoricoxib on acquisition of pro-angiogenic potential by macrophages. Expression level of VEGF was employed as an indirect measure of angiogenic potential. Compared to macrophages incubated with normoxic breast cancer CM, the hypoxic condition media (6h) primed macrophages exhibited upre­gulated VEGF levels which showed marked decline in presence of Flunixin meglumine (10μM) and Etoricoxib (10μM) (Fig. 2A  and 2B). Furthermore Chic chorioal­lantoic membrane (CAM) assay was carried out as a direct measure of angiogenic potential of macrophages exposed to different experimental conditions. Results revealed that compared to macrophages incubated with culture supernatant of normoxic breast cancer cells, the ones that were incubated with culture supernatant of hy­poxic breast cancer exhibited much higher angiogenesis index which were markedly reduced in pres­ence of Flunixin meglumine (10μM) and Etoricoxib (10μM) (Fig. 2C and 2D).

 

Figure 02. Etoricoxib Inhibited hypoxic MDA-MB-231 cells CM induced Expression of Pro-angiogenic Phenotype by Macrophages via downregulating VEGF expression within Macrophages.

1. THP-1 derived Macrophages normoxic MDA-MB-231 CM. 2. THP-1 derived Macrophages hypoxic (6 h) MDA-MB-231 CM. 3. THP-1 derived Macrophages hypoxic (6 h) MDA-MB-231 CM + Flunixin meglumine (10µM). 4. THP-1 derived Macrophages hypoxic (6 h) MDA-MB-231 CM + Etoricoxib (10µM). Data were expressed as Mean ± SEM (n=3) of the results from three separate experiments. *** indicates a p < 0.0001 as compared to hypoxia primed MDA-MB-231 CM treated macrophages.

 

 

Etoricoxib abated hypoxic breast cancer CM induced pro-invasive attributes of macrophages M2 macrophages posses enhanced invasive attributes. They are known to secrete elevated levels of MMPs so as to facilitate local invasion by cancer cells. We next studied if Etoricoxib treatment could impede acquisition of this key functional attribute. Culture supernatant was harvested from normoxic or hypoxic breast cancer cells. Simultaneously, one fraction was utilized for assessing initial levels of MMP-9 activities (Fig.3A), and remaining fraction was introduced to THP-1 derived macrophages in absence or presence of Flunixin meglumine (10μM) and Etoricoxib (10μM) for 24 h. After 24h culture supernatant was harvested and subjected to gelatin zymography for detecting secreted MMP-9 levels and cell lysate was subjected to qRT-PCR assay for assessing mRNA expression levels of MMP-9. Distinct gelatinolytic bands were observed at calculated 84kDa corresponding to the active form of type IV collagenase (MMP-9) activity which was much more intensified in macrophages incubated with hypoxic breast cancer cell CM. Treatment with Etoricoxib and Flunixin meglumine resulted in diminished levels of secreted MMP-9 levels (Fig.3B). Compared to macrophages incubated with normoxic breast cancer cells CM, the macrophages incubated with hypoxic breast cancer CM exhibited markedly upregulated mRNA expression levels of MMP-9. Concomitant exposure to flunixin meglumine (10μM) and Etoricoxib (10μM) exposure reversed the hypoxic cancer cell CM induced MMP-9 upregulation (Fig.3C). Results indicated that Etoricoxib, prevented macrophages from acquiring a pro-invasion function. In agreement with this, these macrophages exhibited a proportionate invasive potential as evaluated through matrigel transvasation assay (Fig.3D and 3E). Collectively these findings indicate that Etoricoxib can effectively hinder macrophage M2 polarization.

 

Figure 03. Etoricoxib Compromised Hypoxic Breast Cancer Cells CM Induced Pro-invasive Functions of M2 Skewed Macrophage.

E) Bars represent mean invaded cells per field±SEM (n=5) from three separate experiments.1.THP-1 derived Macrophages co-cultured with normoxic MDA-MB-231 CM. 2. THP-1 derived Macrophages co-cultured with hypoxic (6 h) MDA-MB-231 CM. 3. THP-1 derived Macrophages co-cultured hypoxic (6 h) MDA-MB-231 CM + Flunixin meglumine (10µM). 4. THP-1 derived Macrophages Co-cultured with hypoxic (6 h) MDA-MB-231 CM + Etoricoxib (10µM). *** indicates a p < 0.0001 as compared to hypoxia primed MDA-MB-231 CM treated macrophages.

 

DISCUSSION:

TAMs have recently emerged as important anti-cancer targets (20). Multiple TAMs related phenomenon such as their intra-tumoral infiltration, phenotype switching is being considered as key steps amenable to therapeutic intervention (21). Within tumor microenvironment, phenotype switching of macrophages to M2-polarized subtype (2) is a critical phenomenon because the M2-polarized macrophages subsequently facilitate cancer cell invasion (22), tumor neo-vasculogenesis (23) and thereby account for adverse prognosis (24).

 

A sizeable body of data indicates that COX-2 participates in macrophage phenotype switching and inhibition of COX-2 is associated with impaired macrophage M2-polarization. Various other preferential and/or selective COX-2 inhibitors viz. Celecoxib (15), Etodolac (16) and Flunixin meglumine (25) have been reported to attenuate Macrophage M2 polarization, establishing COX-2 as key target for impeding pro-tumoral M2 skewed TAMs polarization. Prompted by this in the current study we evaluated if Etoricoxib, a selective COX-2 inhibitor could impede macrophage M2-polarization and minimize their pro-tumoral function. Etoricoxib has shown potent anti-inflammatory activity against osteoarthritis, rheumatoid arthritis, acute gouty arthritis, ankylosing spondylitis, low back pain, acute postoperative pain and primary dysmenorrheal (26).

 

Using immunocytochemical and flowcytometry based detection of M2 macrophage specific surface marker viz. CD-206, we have successfully demonstrated that hypoxic breast cancer cells induced alternate activation of THP-1 derived macrophages is markedly hampered by Etoricoxib, a selective COX-2 inhibitor. It is well documented that M2 skewed TAMs promotes angiogenesis through VEGF, and promote invasion through release of metalloproteinases (27). Because COX-2 is prerequisite for differentiating monocyte to M2 polarized macrophages, COX-2 inhibition may impede subsequent pro-tumoral functions of TAMs. We observed diminished VEGF expression in macrophages that was simultaneously incubated with hypoxic breast cancer CM and Etoricoxib/Flunixin meglumine as compared to hypoxic breast cancer cells CM primed macrophages alone. In addition, these macrophages exhibited diminished angiogenic potential during in vivo CAM assay. In agreement with this COX-2 inhibition through Etodolac resulted in reduced expression of VEGF, and MMP-9 in TAMs (16). Subsequently, we investigated the effect of COX-2 inhibition on acquisition of pro-invasive phenotype by THP-1 derived M2 macrophages in reference to hypoxic microenvironment. Quantitative RT-PCR analysis revealed markedly downregulated mRNA expression levels of MMP-9 in Etoricoxib treated TAMs. Etoricoxib is well studied for its chemopreventive activity in several animal models of colon and lung carcinoma. The anticancer activity of Etoricoxib has been associated with induction of apoptosis in 1, 2-dimethylhydrazine (DMH) induced colon carcinogenesis in rat model (28) and reduction in pro-angiogenic mediators viz. COX-2, MMP-2, MMP-9, MCP-1, MIP-1β and VEGF in 9, 10-dimethylbenz (a)anthracene (DMBA) induced lung carcinoma (29).

 

In conclusion, our study highlights the ability of Etoricoxib to inhibit macrophage M2 polarization, a key phenomenon for cancer progression. Thus, our data further substantiates anti-cancer effects of Etoricoxib and indicate for its potential role as anti-cancer modality.

 

ACKNOWLEDGEMENT:

The authors are thankful to the Director CSIR-CDRI for infrastructure/facility support, Division of Sophisticated Analytical Instruments Facility (SAIF) for Flowcytometric Analysis and Tissue and cell culture unit for providing cell lines. This work was supported by grants from CSIR India (CSIR-Network Project (UNDO): BSC0103) and NIPER fellowship.

 

REFERENCES:

1.      P.M. Kelly, R.S. Davison, E. Bliss, J.O. McGee, Macrophages in human breast disease: a quantitative immunohistochemical study, Br. J. Cancer. 57 (1988) 174–177.

2.      A. Sica, T. Schioppa, A. Mantovani, P. Allavena, Tumor associated macrophages are a distinct M2 polarized population promoting tumor progression: potential targets of anti-cancer therapy, Eur. J. Cancer. 42 (2006) 717-727.

3.      G.J. Gordon, G.N. Rockwell, R.V. Jensen, J.G. Rheinwald, J.N. Glickman, J.P. Aronson, B.J. Pottorf M.D. Nitz, W.G. Richards, D.J. Sugarbaker, R. Bueno, Identification of novel candidate oncogenes and tumor suppressors in malignant pleural mesothelioma using large-scale transcriptional profiling, Am. J. Pathol. 166 (2005) 1827-1840.

4.      A. Mantovani, A. Sica, S. Sozzani, P. Allavena, A. Vecchi, M. Locati, The chemokine system in diverse forms of macrophage activation and polarization, Trends Immunol. 25 (2004) 677-686.

5.      A. Mantovani, P. Allavena, A. Sica, F. Balkwill, Cancer related inflammation, Nature. 454 (2008) 436-444.

6.      S.I. Grivennikov, F.R. Greten, M. Karin, Immunity, inflammation, and cancer, Cell. 140 (2010) 883-899.

7.      B.Z. Qian, J.W. Pollard, Macrophage diversity enhances tumor progression and metastasis, Cell. 141 (2010) 39-51.

8.      D. Hanahan, L.M. Coussens, Accessories to the crime: functions of cells recruited to the tumor microenvironment, Cancer Cell. 21 (2012) 309-322.

9.      W. Zhang, X.D. Zhu, H.C. Sun, Y.Q. Xiong, P.Y. Zhuang, H.X. Xu, L.Q. Kong, L. Wang, W.Z. Wu, Z.Y. Tang, Depletion of tumor-associated macrophages enhances the effect of sorafenib in metastatic liver cancer models by antimetastatic and antiangiogenic effects, Clin. Cancer Res. 16 (2010) 3420-3430.

10.   C. Tripathi, B.N. Tewari, R.K. Kanchan, K.S. Baghel, N. Nautiyal, R. Shrivastava, H. Kaur, M.L.B. Bhatt, S. Bhadauria, Macrophages are recruited to hypoxic tumor areas and acquire a Pro-angiogenic M2-Polarized phenotype via hypoxic cancer cell derived cytokines Oncostatin M and Eotaxin, Oncotarget. 5 (2014) 5350-5368.

11.   C. Lan, X. Huang, S. Lin, H. Huang, Q. Cai, T. Wan, J. Lu, J. Liu, Expression of M2-polarized macrophages is associated with poor prognosis for advanced epithelial ovarian cancer, Technol. Cancer Res. Treat. 12 (2013) 259-267.

12.   S. Sousa, R. Brion, M. Lintunen, P. Kronqvist, J. Sandholm, J. Mönkkönen, P. Kellokumpu-Lehtinen, S. Lauttia, O. Tynninen, H. Joensuu, D. Heymann, J.A. Määttä, Human breast cancer cells educate macrophages toward the M2 activation status. Breast Cancer Res. 17 (2015) 1-14.

13.   A.J. Dannenberg, N.K. Altorki, J.O. Boyle, C. Dang, L.R. Howe, B.B. Weksler, K. Subbaramaiah, Cyclooxygenase 2: A pharmacological target for the prevention of cancer, Lancet Oncol. 2 (2001) 544–551.

14.   X. Dong, R. Li, P. Xiu, X. Dong, Z. Xu, B. Zhai, F. Liu, H. Jiang, H. Sun, J. Li, H. Qiao, Meloxicam executes its antitumor effects against hepatocellular carcinoma in COX-2- dependent and -independent pathways, PLoS One. 9 (2014) e92864.

15.   Y. Nakanishi, M. Nakatsuji, H. Seno, S. Ishizu, R. Akitake-Kawano, K. Kanda, T. Ueo, H. Komekado, M. Kawado, M. Minami, T. Chiba, COX-2 inhibition alters the phenotype of tumor-associated macrophages from M2 to M1 in ApcMin/+ mouse polyps, Carcinogenesis. 32 (2011) 1333–1339.

16.   Y.R. Na, Y.N. Yoon, D.I. Son, S.H. Seok, Cyclooxygenase-2 inhibition blocks M2 macrophage differentiation and suppresses metastasis in murine breast cancer model, PLoS One. 8 (2013) e63451.

17.   M. Daigneault, J.A. Preston, H.M. Marriott, M.K.B. Whyte, D.H. Dockrell, The identification of markers of macrophage differentiation in PMA-stimulated THP-1 cells and monocyte-derived macrophages, PloS One. 5 (2010) e8668.

18.   O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193 (1951) 265-275.

19.   M.W. Pfaffl, A new mathematical model for relative quantification in real-time RT–PCR, Nucleic Acids Res. 29 (2001) e45.

20.   A. Mantovani, C. Porta, L. Rubino, P. Allavena, A. Sica, Tumor-associated macrophages (TAMs) as new target in anticancer therapy, Drug Discov. Today Ther. Strateg. 3 (2006) 361-366.

21.   R. Noy, J.W. Pollard, Tumor-associated macrophages: from mechanisms to therapy, Immunity. 41 (2014), 49-61.

22.   J.W. Tiu, J.S. Chen, C.T. Shun, S.J. Lin, Y.H. Liao, C.Y. Chu, T.F. Tsai, H.C. Chiu, Y.S. Dai, H. Inoue, P.C. Yang, M.L. Kuo, S.H. Jee, Tumor-associated macrophage-induced invasion and angiogenesis of human basal cell carcinoma cells by cyclooxygenase-2 induction, J. Invest. Dermatol. 129 (2009) 1016-1025.

23.   C. Tataroglu, A. Kargi, S. Ozkal, N. Eşrefoglu, A. Akkoçlu, Association of macrophages, mast cells and eosinophil leukocytes with angiogenesis and tumor stage in non-small cell lung carcinomas (NSCLC), Lung Cancer. 43 (2004) 47-54.

24.   R.D. Leek, C.E. Lewis, R. Whitehouse, M. Greenall, J. Clarke, A.L. Harris, Association of macrophage infiltration with angiogenesis and prognosis in invasive breast carcinoma, Cancer Res. 56 (1996) 4625-4629.

25.   P. Dubey, R. Shrivastava, C. Tripathi, N.K. Jain, B.N. Tewari, M.U.D. Lone, K.S. Baghel, V. Kumar, S. Mishra, S. Bhadauria, M.L.B. Bhatt, Cyclooxygenase-2 inhibition attenuates hypoxic cancer cells induced M2-polarization of macrophages, Cell. Mol. Biol. (Noisy-le-grand) 60 (2014) 10-15.

26.   P. Brooks, P. Kubler, Etoricoxib for arthritis and pain management, Ther. Clin. Risk Manag. 2 (2006) 45-57.

27.   V. Gocheva, H.W. Wang, B.B. Gadea, T. Shree, K.E. Hunter, A.L. Garfall, T. Berman, J.A. Joyce, IL-4 induces cathepsin protease activity in tumor-associated macrophages to promote cancer growth and invasion, Genes Dev. 24 (2010) 241-255.

28.   P. Sharma, J. Kaur, S.N. Sanyal, Effect of Etoricoxib, a cyclooxygenase-2 selective inhibitor on aberrant crypt formation and apoptosis in 1,2 dimethyl hydrazine induced colon carcinogenesis in rat model. Nutr. Hosp. 25 (2010) 39-48.

29.   N. Nadda, V. Vaish, S. Setia, S.N. Sanyal, Angiostatic role of the selective cyclooxygenase-2 inhibitor etoricoxib (MK0663) in experimental lung cancer, Biomed Pharmacother. 66 (2012) 474-83.

 

 

 

 

 

Received on 18.05.2019           Modified on 21.06.2019

Accepted on 30.07.2019         © RJPT All right reserved