Original Article
DOI: 10.1111/vco.12097
Plasma matrix metalloproteinase-9 activity in cats with lymphoma T. Tamamoto, K. Ohno, M. Takahashi, K. Fukushima, H. Kanemoto, Y. Fujino and H. Tsujimoto Department of Veterinary Internal Medicine, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan
Abstract
Keywords cat, lymphoma, matrix metalloproteinase-9 (MMP-9)
In this study, plasma MMP-9 activity was evaluated in cats with lymphoma. Plasma samples were obtained from 26 cats with lymphoma before treatment. From 13 of the included 26 cats, plasma samples were obtained 4 weeks after the initiation of treatment. Plasma samples were also obtained from 10 healthy cats as a control. Plasma MMP-9 activity was examined by gelatin zymography and semi-quantitative value (arbitrary unit; a.u.) for each sample was calculated. Relatively high levels of MMP-9 were observed in cats with lymphoma compared with those in healthy control cats. MMP-9 quantification through zymography showed significantly higher activity in cats with lymphoma (median, 0.63 a.u.; range, 0.23–3.24 a.u.) than in healthy controls (0.22 a.u.; 0.12–0.46 a.u.; P < 0.01). MMP-9 activities were significantly different before (0.73 a.u.; 0.30–3.24 a.u.) and after treatment (0.50 a.u.; 0.14–1.32 a.u.; P = 0.017). Measuring plasma MMP-9 activity in cats with lymphoma may become an appropriate monitoring tool for feline lymphoma.
Introduction
Correspondence address: K. Ohno Department of Veterinary Internal Medicine Graduate School of Agricultural and Life Sciences The University of Tokyo 1-1-1 Yayoi Bunkyo-ku Tokyo 113-8657 Japan e-mail: [email protected]. u-tokyo.ac.jp
Matrix metalloproteinases (MMPs) are a family of extracellular matrix-degrading proteases that are zinc-dependent. MMPs are associated with invasion and metastasis during tumor progression because of their ability to degrade the extracellular matrix (ECM) and basement membrane.1 Matrix metalloproteinase-9 (MMP-9), also known as type IV collagenase, is a member of the MMPs. As type IV collagen is one of the integral components of the basement membrane, MMP-9 is believed to play a key role in tumor invasion and metastasis.2 The strong expression and release of MMP-9 may enable tumor cells to invade through the basement membrane, thereby initiating the spread of tumors.3 MMP-9 is also able to regulate angiogenesis directly.4 Lymphoma is one of the most common neoplasm in cats. Various anatomical locations have been described, and multiple lesions are frequently observed.5 Chemotherapy is most frequently selected for the treatment of lymphoma in cats, and
© 2014 John Wiley & Sons Ltd
a variety of chemotherapeutic protocols have been reported.6 – 10 In humans, elevated serum MMP-9 concentrations were observed in patients with Hodgkin’s disease and non-Hodgkin’s lymphoma (NHL).11 Elevated plasma MMP-9 activities were described in dogs with lymphoma, and decreased following chemotherapy.12 Thus, elevated MMP-9 concentration may be associated with the pathophysiology of lymphoma in both humans and animals, and to detect the changes of MMP-9 concentration may be a useful monitoring tool for lymphoma. In cats with various solid tumors, higher expression of MMP-9 in tumor tissues and elevated serum MMP-9 concentrations before treatment have been also described.13 However, there is scarce information about MMP-9 in cats with lymphoma. The aims of this study were to evaluate whether plasma MMP-9 activities in cats with lymphoma were elevated compared with those in healthy cats, and to examine the changes in plasma MMP-9 activities before and after treatment.
1
2 T. Tamamoto et al.
Plasma samples were obtained before treatment initiation from 26 client-owned cats with lymphoma referred to the Veterinary Medical Center of the University of Tokyo (VMC-UT) from January 2012 to March 2013. Information about signalment, anatomical location, treatment, and response to treatment was collected from medical records. A diagnosis of lymphoma was confirmed in all cats by cytopathological examination or histopathological examination of biopsy samples. Cats diagnosed as low grade lymphoma were excluded from the study. Cats that had serious concurrent diseases were also excluded. Combination chemotherapy, radiation, and/or surgery were selected for each case as treatments. From 13 of the included 26 cats, plasma samples were obtained 4 weeks after the initiation of treatment. Responses to treatment at the time of post-treatment sampling were determined grossly and/or by imaging tests (ultra-sonography or computed tomography). Plasma samples were also obtained from 10 healthy cats kept at VMC-UT as a control. All samples were prepared by centrifugation of whole blood and stored at −20 ∘ C until analysis. Plasma MMP-9 activity was examined by gelatin zymography. Plasma samples were diluted in sample buffer [500 mM Tris-HCl pH 6.8, 20% glycerol, 4% sodium dodecylsulphate (SDS), 0.2% bromophenol blue] and a dilution containing an equal volume of plasma was subjected to electrophoresis on a 10% SDS-PAGE gel co-polymerized with 0.1% gelatin. Following electrophoresis, the gels were rinsed in 2.5% Triton X-100 for 2 hours at room temperature and incubated in enzymatic activation buffer (50 mM Tris-HCl, 200 mM NaCl, 5 mM CaCl2 , pH 7.6) for 22 hours at 37 ∘ C with gentle shaking. The gels were stained with 0.25% Coomassie brilliant blue R-250, 50% methanol, and 5% acetic acid for 30 minutes, and then de-stained in 5% methanol and 7% acetic acid for 1 hour. A commercially available zymography marker (Gelatin Zymo MMP Marker; Life Laboratory, Yamagata, Japan) was run on each gel as a positive control. MMP-9 levels were assessed using gelatinolytic activity, indicated as clear bands against the dark blue background. All gels were analyzed with an imaging analyzer system (Cool Saver version 1.0;
ATTO, Tokyo, Japan) and software (CS Analyzer version 2.0; ATTO). To obtain a semi-quantitative value for each sample, the imaging assessment value of each unknown band was compared with the value of a MMP-9 standard band. The ratio of unknown to standard was calculated, and an arbitrary unit (a.u.) value was assigned to each cat sample. Statistical analyses were performed with a statistical software package (JMP version 5.0.1 J; SAS Institute, Cary, NC, USA). Shapiro–Wilk normality test was performed to determine data were normally-distributed or not. Statistical differences between the independent two groups (healthy control group versus lymphoma group) were examined using a Mann–Whitney U-test and between the paired two groups (before versus after treatment) were examined using a Wilcoxon signed-rank test. A value of P < 0.05 was considered significant. The median age of the cats with lymphoma was 12 years (range, 3–17 years). Sex, breed, anatomical location, MMP-9 activities, treatment, and response to treatment were summarized in Table 1. Variety of locations of lymphoma were observed and gastrointestinal lymphoma was the most common form. Relatively high levels of MMP-9 were observed in cats with lymphoma compared with those in healthy control cats (Figure 1A). The semi-quantification of MMP-9 through zymography showed significantly higher activity in cats with lymphoma (median, 0.63 a.u.; range, 0.23–3.24 a.u.) than in healthy controls (median, 0.22 a.u.; range, 0.12–0.46 a.u.; P < 0.01, Fig. 1B). Among the 26 cats, post-treatment samples were available on 13 cats (cat no. 1, 3, 5, 7, 8, 9, 11, 12, 13, 14, 20, 22, and 24). In 13 cases, MMP-9 activities were significantly different before (median, 0.73 a.u.; range, 0.30–3.24 a.u.) and after treatment (median, 0.50 a.u.; range, 0.14–1.32 a.u.; P = 0.017). Especially, in 10 cats responded to the treatment (cat no. 1, 3, 5, 7, 8, 11, 12, 13, 20, and 22), MMP-9 activities were all decreased by the treatment. In cats that did not respond to the treatment, the change of plasma MMP-9 activities were varied in cases; decreased (cat no.9), slightly increased (cat no. 24), and markedly increased (cat no. 14). The role of MMP-9 in the solid tumors is widely investigated. Although, in the previous study,
© 2014 John Wiley & Sons Ltd, Veterinary and Comparative Oncology, doi: 10.1111/vco.12097
MMP-9 activity in lymphoma cats 3
Table 1. Summary data of 26 cats with lymphoma MMP-9(a.u.)
Cat no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
Age (year)
Sex
Breed
Anatomical location
Before treatment
4 weeks after treatment
Treatment
Response to treatment
12 12 15 11 6 8 13 17 16 3 10 10 8 12 10 15 17 4 10 16 14 15 15 5 11 8
Castrated male Castrated male Castrated male Castrated male Spayed female Spayed female Castrated male Castrated male Spayed female Female Spayed female Female Castrated male Spayed female Spayed female Spayed female Spayed female Female Male Spayed female Castrated male Castrated male Castrated male Spayed female Spayed female Spayed female
DSH DSH DSH DSH DSH DSH DSH DSH SF DSH DSH DSH DSH DSH DSH DSH DSH DSH RB DSH DSH DSH DSH Somali DSH DSH
G K, CNS MC G Mediastinal G K MC G N, K G G N N L Laryngeal N K, tracheal G L, K, CNS G G G G Cutaneous G
3.24 2.92 2.65 2.25 1.35 1.27 1.06 0.87 0.8 0.8 0.73 0.65 0.65 0.61 0.57 0.55 0.49 0.48 0.45 0.42 0.39 0.37 0.31 0.3 0.26 0.23
0.5 NA 0.73 NA 0.62 NA 0.5 0.71 0.33 NA 0.62 0.29 0.14 1.32 NA NA NA NA 0.28 NA NA 0.31 NA 0.35 NA NA
Chemo Chemo Chemo Chemo Chemo Chemo Chemo Chemo Chemo Chemo Chemo Surgery Chemo + Rad Chemo + Rad Chemo Chemo Chemo Chemo Surgery ND Chemo Chemo Chemo Chemo Chemo PDL
R ND R R R ND R R NR ND R R R NR ND ND ND ND R ND NR R ND NR R ND
Chemo, combination chemotherapy; CNS, central nervous system; DSH, domestic short hair; G, gastrointestinal; K, kidney; L, liver; MC, multi-centric; NR, no response; ND, no data; N, nasal; NA, not available; ND, no data; PDL, prednisolone only; R, response; Rad, radiation; RB, Russian Blue; SF, Scottish Fold.
elevated serum MMP-9 activities in cats with various solid tumors were described, only three or less cats with lymphoma were included and there was no individual information about the association between lymphoma and MMP-9 activity in cats.13 This study showed increased plasma MMP-9 activities in cats with lymphoma compared with those in healthy cats. The results of this study were in accordance with elevated serum MMP-9 activities in humans and dogs with lymphoma, as previously described.11,12 However, healthy controls used in this study were not age and sex matched. It is one of the limitations of this study. In this study, changes in MMP-9 activities before and after treatment were examined, and the values decreased particularly in cats that responded to treatment. Similar results were obtained in a
previous study of dogs with lymphoma.12 In the previous study, lymphocytes derived from human NHL patients showed MMP-9 expression at mRNA or protein levels and proteolytic activity to the ECM.14 Moreover, in human patients with mycosis fungoides, the expression of MMP-9 mRNAs was significantly upregulated with advancing stage.15 MMP-9 was also described as one of the important factors for angiogenesis4 and angiogenesis plays a critical role in the development of tumors. From these findings, MMP-9 may be involved in the progression of lymphoma through the degradation of ECM, spread of tumor cells, and angiogenesis. Thus, measurement of the MMP-9 activities is considered to be important in order to evaluate the stage of lymphoma progression. However, in this study, serial measurements of MMP-9 activities
© 2014 John Wiley & Sons Ltd, Veterinary and Comparative Oncology, doi: 10.1111/vco.12097
4 T. Tamamoto et al.
A
Promotion of Science and the Ministry of Education, Culture, Sports, Science and Technology of the Japanese Government.
B
References
Figure 1. Plasma MMP-9 activity in cats with lymphoma (n = 26) and control cats (n = 10) assessed by gelatin zymography. (A) Representative photo from 5 samples. Each sample was analyzed in duplicate. Lane 1 and 2 are healthy control cats. Lane 3, 4, and 5 were lymphoma cats. (B) Semi-quantitative values are expressed as arbitrary units (a.u.) and horizontal lines represent the median value in each group. MMP-9 activity was significantly different between cats with lymphoma and control cats (P < 0.01), as assessed by a Mann–Whitney U-test.
were performed in only 13 cats, and it was not assessed whether MMP-9 activity increases at the time of relapse. As MMP-9 expression in tumor tissues was described as a prognostic marker in human patients with non-Hodgkin’s lymphoma,16 it is considered to be important to evaluate the expression of MMP-9 in lymphoma tissues. However, MMP-9 expression in lymphoma tissue was not examined in this study. In conclusion, MMP-9 activities in plasma were elevated in cats with lymphoma than those in healthy control cats. Measuring MMP-9 activities may become an appropriate monitoring tool for feline lymphoma. Further studies should evaluate the relationship between plasma concentrations of MMP-9 and expression of MMP-9 in tumor tissues, and a future investigation should preferably evaluate the relationship between MMP-9 and the progression of feline lymphoma in a large-scale study.
Acknowledgements This study was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the
1. Stamenkovic I. Matrix metalloproteinases in tumor invasion and metastasis. Seminars in Cancer Biology 2000; 10: 415–433. 2. Himelstein BP, Canete-Soler R, Bernhard EJ, Dilks DW and Muschel RJ. Metalloproteinases in tumor progression: the contribution of MMP-9. Invasion and Metastasis 1994; 14: 246–258. 3. Bogusiewicz M, Stryjecka-Zimmer M, Szymanski M, Rechberger T and Golabek W. Activity of matrix metalloproteinases-2 and -9 in advanced laryngeal cancer. Otolaryngology-Head and Neck Surgery 2003; 128: 132–136. 4. Bergers G, Brekken R, McMahon G, Vu TH, Itoh T, Tamaki K, Tanzawa K, Thorpe P, Itohara S, Werb Z and Hanahan D. Matrix metalloproteinase-9 triggers the angiogenic switch during carcinogenesis. Nature Cell Biology 2000; 2: 737–744. 5. Louwerens M, London CA, Pedersen NC and Lyons LA. Feline lymphoma in the post-feline leukemia virus era. Journal of Veterinary Internal Medicine 2005; 19: 329–335. 6. Teske E, van Straten G, van Noort R and Rutteman GR. Chemotherapy with cyclophosphamide, vincristine, and prednisolone (COP) in cats with malignant lymphoma: new results with an old protocol. Journal of Veterinary Internal Medicine 2002; 16: 179–186. 7. Simon D, Eberle N, Laacke-Singer L and Nolte I. Combination chemotherapy in feline lymphoma: treatment outcome, tolerability, and duration in 23 cats. Journal of Veterinary Internal Medicine 2008; 22: 394–400. 8. Taylor SS, Goodfellow MR, Browne WJ, Walding B, Murphy S, Tzannes S, Gerou-Ferriani M, Schwartz A and Dobson JM. Feline extranodal lymphoma: response to chemotherapy and survival in 110 cats. Journal of Small Animal Practice 2009; 50: 584–592. 9. Moore A. Extranodal lymphoma in the cat: prognostic factors and treatment options. Journal of Feline Medicine and Surgery 2013; 15: 379–390. 10. Waite AH, Jackson K, Gregor TP and Krick EL. Lymphoma in cats treated with a weekly cyclophosphamide-, vincristine-, and prednisone-based protocol: 114 cases (1998-2008). Journal of the American Veterinary Medical Association 2013; 242: 1104–1109.
© 2014 John Wiley & Sons Ltd, Veterinary and Comparative Oncology, doi: 10.1111/vco.12097
MMP-9 activity in lymphoma cats 5
11. Hazar B, Polat G, Seyrek E, Bagdatoglglu O, Kanik A and Tiftik N. Prognostic value of matrix metalloproteinases (MMP-2 and MMP-9) in Hodgkin’s and non-Hodgkin’s lymphoma. International Journal of Clinical Practice 2004; 58: 139–143. 12. Aresu L, Arico A, Comazzi S, Gelain ME, Riondato F, Mortarino M, Morello E, Stefanello D and Castagnaro M. VEGF and MMP-9: biomarkers for canine lymphoma. Veterinary and Comparative Oncology 2014; 12(1): 29–36. 13. Jankowski MK, Ogilvie GK, Lana SE, Fettman MJ, Hansen RA, Powers BE, Mitchener KM, Lovett SD, Richardson KL, Parsley L and Walton JA. Matrix metalloproteinase activity in tumor, stromal tissue, and serum from cats with malignancies. Journal of Veterinary Internal Medicine 2002; 16: 105–108.
14. Kossakowska AE, Hinek A, Edwards DR, Lim MS, Zhang CL, Breitman DR, Prusinkiewicz C, Stabbler AL, Urbanski LS and Urbanski SJ. Proteolytic activity of human non-Hodgkin’s lymphomas. American Journal of Pathology 1998; 152: 565–576. 15. Vacca A, Ribatti D, Ria R, Pellegrino A, Bruno M, Merchionne F and Dammacco F. Proteolytic activity of human lymphoid tumor cells. Correlation with tumor progression. Developmental Immunology 2000; 7: 77–88. 16. Sakata K, Satoh M, Someya M, Asanuma H, Nagakura H, Oouchi A, Nakata K, Kogawa K, Koito K, Hareyama M and Himi T. Expression of matrix metalloproteinase 9 is a prognostic factor in patients with non-Hodgkin lymphoma. Cancer 2004; 100: 356–365.
© 2014 John Wiley & Sons Ltd, Veterinary and Comparative Oncology, doi: 10.1111/vco.12097
DOI: 10.1111/vco.12097
Plasma matrix metalloproteinase-9 activity in cats with lymphoma T. Tamamoto, K. Ohno, M. Takahashi, K. Fukushima, H. Kanemoto, Y. Fujino and H. Tsujimoto Department of Veterinary Internal Medicine, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan
Abstract
Keywords cat, lymphoma, matrix metalloproteinase-9 (MMP-9)
In this study, plasma MMP-9 activity was evaluated in cats with lymphoma. Plasma samples were obtained from 26 cats with lymphoma before treatment. From 13 of the included 26 cats, plasma samples were obtained 4 weeks after the initiation of treatment. Plasma samples were also obtained from 10 healthy cats as a control. Plasma MMP-9 activity was examined by gelatin zymography and semi-quantitative value (arbitrary unit; a.u.) for each sample was calculated. Relatively high levels of MMP-9 were observed in cats with lymphoma compared with those in healthy control cats. MMP-9 quantification through zymography showed significantly higher activity in cats with lymphoma (median, 0.63 a.u.; range, 0.23–3.24 a.u.) than in healthy controls (0.22 a.u.; 0.12–0.46 a.u.; P < 0.01). MMP-9 activities were significantly different before (0.73 a.u.; 0.30–3.24 a.u.) and after treatment (0.50 a.u.; 0.14–1.32 a.u.; P = 0.017). Measuring plasma MMP-9 activity in cats with lymphoma may become an appropriate monitoring tool for feline lymphoma.
Introduction
Correspondence address: K. Ohno Department of Veterinary Internal Medicine Graduate School of Agricultural and Life Sciences The University of Tokyo 1-1-1 Yayoi Bunkyo-ku Tokyo 113-8657 Japan e-mail: [email protected]. u-tokyo.ac.jp
Matrix metalloproteinases (MMPs) are a family of extracellular matrix-degrading proteases that are zinc-dependent. MMPs are associated with invasion and metastasis during tumor progression because of their ability to degrade the extracellular matrix (ECM) and basement membrane.1 Matrix metalloproteinase-9 (MMP-9), also known as type IV collagenase, is a member of the MMPs. As type IV collagen is one of the integral components of the basement membrane, MMP-9 is believed to play a key role in tumor invasion and metastasis.2 The strong expression and release of MMP-9 may enable tumor cells to invade through the basement membrane, thereby initiating the spread of tumors.3 MMP-9 is also able to regulate angiogenesis directly.4 Lymphoma is one of the most common neoplasm in cats. Various anatomical locations have been described, and multiple lesions are frequently observed.5 Chemotherapy is most frequently selected for the treatment of lymphoma in cats, and
© 2014 John Wiley & Sons Ltd
a variety of chemotherapeutic protocols have been reported.6 – 10 In humans, elevated serum MMP-9 concentrations were observed in patients with Hodgkin’s disease and non-Hodgkin’s lymphoma (NHL).11 Elevated plasma MMP-9 activities were described in dogs with lymphoma, and decreased following chemotherapy.12 Thus, elevated MMP-9 concentration may be associated with the pathophysiology of lymphoma in both humans and animals, and to detect the changes of MMP-9 concentration may be a useful monitoring tool for lymphoma. In cats with various solid tumors, higher expression of MMP-9 in tumor tissues and elevated serum MMP-9 concentrations before treatment have been also described.13 However, there is scarce information about MMP-9 in cats with lymphoma. The aims of this study were to evaluate whether plasma MMP-9 activities in cats with lymphoma were elevated compared with those in healthy cats, and to examine the changes in plasma MMP-9 activities before and after treatment.
1
2 T. Tamamoto et al.
Plasma samples were obtained before treatment initiation from 26 client-owned cats with lymphoma referred to the Veterinary Medical Center of the University of Tokyo (VMC-UT) from January 2012 to March 2013. Information about signalment, anatomical location, treatment, and response to treatment was collected from medical records. A diagnosis of lymphoma was confirmed in all cats by cytopathological examination or histopathological examination of biopsy samples. Cats diagnosed as low grade lymphoma were excluded from the study. Cats that had serious concurrent diseases were also excluded. Combination chemotherapy, radiation, and/or surgery were selected for each case as treatments. From 13 of the included 26 cats, plasma samples were obtained 4 weeks after the initiation of treatment. Responses to treatment at the time of post-treatment sampling were determined grossly and/or by imaging tests (ultra-sonography or computed tomography). Plasma samples were also obtained from 10 healthy cats kept at VMC-UT as a control. All samples were prepared by centrifugation of whole blood and stored at −20 ∘ C until analysis. Plasma MMP-9 activity was examined by gelatin zymography. Plasma samples were diluted in sample buffer [500 mM Tris-HCl pH 6.8, 20% glycerol, 4% sodium dodecylsulphate (SDS), 0.2% bromophenol blue] and a dilution containing an equal volume of plasma was subjected to electrophoresis on a 10% SDS-PAGE gel co-polymerized with 0.1% gelatin. Following electrophoresis, the gels were rinsed in 2.5% Triton X-100 for 2 hours at room temperature and incubated in enzymatic activation buffer (50 mM Tris-HCl, 200 mM NaCl, 5 mM CaCl2 , pH 7.6) for 22 hours at 37 ∘ C with gentle shaking. The gels were stained with 0.25% Coomassie brilliant blue R-250, 50% methanol, and 5% acetic acid for 30 minutes, and then de-stained in 5% methanol and 7% acetic acid for 1 hour. A commercially available zymography marker (Gelatin Zymo MMP Marker; Life Laboratory, Yamagata, Japan) was run on each gel as a positive control. MMP-9 levels were assessed using gelatinolytic activity, indicated as clear bands against the dark blue background. All gels were analyzed with an imaging analyzer system (Cool Saver version 1.0;
ATTO, Tokyo, Japan) and software (CS Analyzer version 2.0; ATTO). To obtain a semi-quantitative value for each sample, the imaging assessment value of each unknown band was compared with the value of a MMP-9 standard band. The ratio of unknown to standard was calculated, and an arbitrary unit (a.u.) value was assigned to each cat sample. Statistical analyses were performed with a statistical software package (JMP version 5.0.1 J; SAS Institute, Cary, NC, USA). Shapiro–Wilk normality test was performed to determine data were normally-distributed or not. Statistical differences between the independent two groups (healthy control group versus lymphoma group) were examined using a Mann–Whitney U-test and between the paired two groups (before versus after treatment) were examined using a Wilcoxon signed-rank test. A value of P < 0.05 was considered significant. The median age of the cats with lymphoma was 12 years (range, 3–17 years). Sex, breed, anatomical location, MMP-9 activities, treatment, and response to treatment were summarized in Table 1. Variety of locations of lymphoma were observed and gastrointestinal lymphoma was the most common form. Relatively high levels of MMP-9 were observed in cats with lymphoma compared with those in healthy control cats (Figure 1A). The semi-quantification of MMP-9 through zymography showed significantly higher activity in cats with lymphoma (median, 0.63 a.u.; range, 0.23–3.24 a.u.) than in healthy controls (median, 0.22 a.u.; range, 0.12–0.46 a.u.; P < 0.01, Fig. 1B). Among the 26 cats, post-treatment samples were available on 13 cats (cat no. 1, 3, 5, 7, 8, 9, 11, 12, 13, 14, 20, 22, and 24). In 13 cases, MMP-9 activities were significantly different before (median, 0.73 a.u.; range, 0.30–3.24 a.u.) and after treatment (median, 0.50 a.u.; range, 0.14–1.32 a.u.; P = 0.017). Especially, in 10 cats responded to the treatment (cat no. 1, 3, 5, 7, 8, 11, 12, 13, 20, and 22), MMP-9 activities were all decreased by the treatment. In cats that did not respond to the treatment, the change of plasma MMP-9 activities were varied in cases; decreased (cat no.9), slightly increased (cat no. 24), and markedly increased (cat no. 14). The role of MMP-9 in the solid tumors is widely investigated. Although, in the previous study,
© 2014 John Wiley & Sons Ltd, Veterinary and Comparative Oncology, doi: 10.1111/vco.12097
MMP-9 activity in lymphoma cats 3
Table 1. Summary data of 26 cats with lymphoma MMP-9(a.u.)
Cat no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
Age (year)
Sex
Breed
Anatomical location
Before treatment
4 weeks after treatment
Treatment
Response to treatment
12 12 15 11 6 8 13 17 16 3 10 10 8 12 10 15 17 4 10 16 14 15 15 5 11 8
Castrated male Castrated male Castrated male Castrated male Spayed female Spayed female Castrated male Castrated male Spayed female Female Spayed female Female Castrated male Spayed female Spayed female Spayed female Spayed female Female Male Spayed female Castrated male Castrated male Castrated male Spayed female Spayed female Spayed female
DSH DSH DSH DSH DSH DSH DSH DSH SF DSH DSH DSH DSH DSH DSH DSH DSH DSH RB DSH DSH DSH DSH Somali DSH DSH
G K, CNS MC G Mediastinal G K MC G N, K G G N N L Laryngeal N K, tracheal G L, K, CNS G G G G Cutaneous G
3.24 2.92 2.65 2.25 1.35 1.27 1.06 0.87 0.8 0.8 0.73 0.65 0.65 0.61 0.57 0.55 0.49 0.48 0.45 0.42 0.39 0.37 0.31 0.3 0.26 0.23
0.5 NA 0.73 NA 0.62 NA 0.5 0.71 0.33 NA 0.62 0.29 0.14 1.32 NA NA NA NA 0.28 NA NA 0.31 NA 0.35 NA NA
Chemo Chemo Chemo Chemo Chemo Chemo Chemo Chemo Chemo Chemo Chemo Surgery Chemo + Rad Chemo + Rad Chemo Chemo Chemo Chemo Surgery ND Chemo Chemo Chemo Chemo Chemo PDL
R ND R R R ND R R NR ND R R R NR ND ND ND ND R ND NR R ND NR R ND
Chemo, combination chemotherapy; CNS, central nervous system; DSH, domestic short hair; G, gastrointestinal; K, kidney; L, liver; MC, multi-centric; NR, no response; ND, no data; N, nasal; NA, not available; ND, no data; PDL, prednisolone only; R, response; Rad, radiation; RB, Russian Blue; SF, Scottish Fold.
elevated serum MMP-9 activities in cats with various solid tumors were described, only three or less cats with lymphoma were included and there was no individual information about the association between lymphoma and MMP-9 activity in cats.13 This study showed increased plasma MMP-9 activities in cats with lymphoma compared with those in healthy cats. The results of this study were in accordance with elevated serum MMP-9 activities in humans and dogs with lymphoma, as previously described.11,12 However, healthy controls used in this study were not age and sex matched. It is one of the limitations of this study. In this study, changes in MMP-9 activities before and after treatment were examined, and the values decreased particularly in cats that responded to treatment. Similar results were obtained in a
previous study of dogs with lymphoma.12 In the previous study, lymphocytes derived from human NHL patients showed MMP-9 expression at mRNA or protein levels and proteolytic activity to the ECM.14 Moreover, in human patients with mycosis fungoides, the expression of MMP-9 mRNAs was significantly upregulated with advancing stage.15 MMP-9 was also described as one of the important factors for angiogenesis4 and angiogenesis plays a critical role in the development of tumors. From these findings, MMP-9 may be involved in the progression of lymphoma through the degradation of ECM, spread of tumor cells, and angiogenesis. Thus, measurement of the MMP-9 activities is considered to be important in order to evaluate the stage of lymphoma progression. However, in this study, serial measurements of MMP-9 activities
© 2014 John Wiley & Sons Ltd, Veterinary and Comparative Oncology, doi: 10.1111/vco.12097
4 T. Tamamoto et al.
A
Promotion of Science and the Ministry of Education, Culture, Sports, Science and Technology of the Japanese Government.
B
References
Figure 1. Plasma MMP-9 activity in cats with lymphoma (n = 26) and control cats (n = 10) assessed by gelatin zymography. (A) Representative photo from 5 samples. Each sample was analyzed in duplicate. Lane 1 and 2 are healthy control cats. Lane 3, 4, and 5 were lymphoma cats. (B) Semi-quantitative values are expressed as arbitrary units (a.u.) and horizontal lines represent the median value in each group. MMP-9 activity was significantly different between cats with lymphoma and control cats (P < 0.01), as assessed by a Mann–Whitney U-test.
were performed in only 13 cats, and it was not assessed whether MMP-9 activity increases at the time of relapse. As MMP-9 expression in tumor tissues was described as a prognostic marker in human patients with non-Hodgkin’s lymphoma,16 it is considered to be important to evaluate the expression of MMP-9 in lymphoma tissues. However, MMP-9 expression in lymphoma tissue was not examined in this study. In conclusion, MMP-9 activities in plasma were elevated in cats with lymphoma than those in healthy control cats. Measuring MMP-9 activities may become an appropriate monitoring tool for feline lymphoma. Further studies should evaluate the relationship between plasma concentrations of MMP-9 and expression of MMP-9 in tumor tissues, and a future investigation should preferably evaluate the relationship between MMP-9 and the progression of feline lymphoma in a large-scale study.
Acknowledgements This study was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the
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