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Immunolocalization of MMP9 and MMP2 in osteolytic metastasis originating from MDA-MB-231 human breast cancer cells.

Liu B, Cui J, Sun J, Li J, Han X, Guo J, Yi M, Amizuka N, Xu X, Li M - Mol Med Rep (2016)

Bottom Line: Numerous tartrate-resistant acid phosphatase-positive osteoclasts were found in the metastasized lesions.The invaded tumor cells positive for mammaglobin 1 exhibited different proliferation activities and apoptosis between the metaphysis and diaphysis.Of note, MMP9 was expressed predominantly in the proliferating cell nuclear antigen‑positive area, whereas the expression of MMP2 was observed predominantly in the diaphysis, which had more TUNEL‑positive cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Bone Metabolism, School of Stomatology, Shandong University, Shandong Provincial Key Laboratory of Oral Tissue Regeneration, Jinan, Shandong 250012, P.R. China.

ABSTRACT
The aim of the present study was to investigate the expression of matrix metalloproteinase (MMP)9 and MMP2, and their potential roles in bone metastasis nests using a well-standardized model of breast cancer bone metastasis in nude mice. BALB/c nu/nu mice (5-week-old; n=10) were subjected to intracardiac injection of MDA-MB-231 human breast cancer cells. After 4 weeks, the mice exhibiting radiolucent lesions in tibiae were sacrificed, and the tibiae were removed for histochemical analysis. The gene expression of MMP2 and MMP9 in the tumor cells, metaphysis and diaphysis of normal BALB/c nu/nu mice were determined using reverse transcription-polymerase chain reaction analysis. The metastatic tumor tissue occupied almost the entire bone marrow cavity. Numerous tartrate-resistant acid phosphatase-positive osteoclasts were found in the metastasized lesions. The invaded tumor cells positive for mammaglobin 1 exhibited different proliferation activities and apoptosis between the metaphysis and diaphysis. Proliferating cell nuclear antigen was expressed at high levels in the metaphyseal area, whereas TdT-mediated dUTP nick-end labeling (TUNEL)-positive cells were more evident in the diaphysis area. Of note, MMP9 was expressed predominantly in the proliferating cell nuclear antigen‑positive area, whereas the expression of MMP2 was observed predominantly in the diaphysis, which had more TUNEL‑positive cells. Taken together, the results suggested that MMP9 and MMP2 may have their own importance in extracellular matrix degradation and trabecular bone damage in different zones of bone metastasis, including the metaphysis and diaphysis.

No MeSH data available.


Related in: MedlinePlus

Immunohistochemical and statistical analyses of PCNA and TUNEL staining for apoptosis. (A) Immunohistochemical analysis of PCNA in the (a) metaphysis and (b) diaphysis in the normal bone marrow of the control group. Immunohistochemistry for PCNA in the (c) metaphyseal and (d) diaphyseal tumor metastases. More PCNA-positive tumor cells (brown color) were detected in the metaphyseal tumor tissue, compared with the diaphyseal tumor tissue. (e and f) Higher magnification of c and d, respectively. (B) TUNEL staining for apoptotic tumor cells in the (a) metaphysis and (b) diaphysis in the normal bone marrow tissue of the control group. TUNEL staining for apoptotic tumor cells in the (c) metaphyseal and (d) diaphyseal tumor metastases. More TUNEL-positive apoptotic tumor cells (blue color) were observed in the diaphyseal tumor tissue, compared with the metaphyseal tumor tissue. (e and f) High magnifications of c and d, respectively. (C) Statistical analyses of the numbers of PCNA/TUNEL-positive tumor cells in the metaphysis and diaphysis. *P<0.01. Scale bar=50 µm in Aa-d and Ba-d; 25 µm in Ae and f, and Be and f. TUNEL, TdT-mediated dUTP nick-end labeling; Meta, metaphysis; Dia, diaphysis; T, tumor cells; CB, cortical bone; *, normal bone tissue.
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f2-mmr-14-02-1099: Immunohistochemical and statistical analyses of PCNA and TUNEL staining for apoptosis. (A) Immunohistochemical analysis of PCNA in the (a) metaphysis and (b) diaphysis in the normal bone marrow of the control group. Immunohistochemistry for PCNA in the (c) metaphyseal and (d) diaphyseal tumor metastases. More PCNA-positive tumor cells (brown color) were detected in the metaphyseal tumor tissue, compared with the diaphyseal tumor tissue. (e and f) Higher magnification of c and d, respectively. (B) TUNEL staining for apoptotic tumor cells in the (a) metaphysis and (b) diaphysis in the normal bone marrow tissue of the control group. TUNEL staining for apoptotic tumor cells in the (c) metaphyseal and (d) diaphyseal tumor metastases. More TUNEL-positive apoptotic tumor cells (blue color) were observed in the diaphyseal tumor tissue, compared with the metaphyseal tumor tissue. (e and f) High magnifications of c and d, respectively. (C) Statistical analyses of the numbers of PCNA/TUNEL-positive tumor cells in the metaphysis and diaphysis. *P<0.01. Scale bar=50 µm in Aa-d and Ba-d; 25 µm in Ae and f, and Be and f. TUNEL, TdT-mediated dUTP nick-end labeling; Meta, metaphysis; Dia, diaphysis; T, tumor cells; CB, cortical bone; *, normal bone tissue.

Mentions: PCNA-positive cells were observed in the metaphyseal area (Fig. 2Ab and c), but few were observed in the diaphyseal area (Fig. 2Ad and f). In the metaphyseal tumor nest, a few scattered TUNEL-positive apoptotic cells were observed (Fig. 2Bb and c). However, in the diaphyseal tumor nest, a higher number of TUNEL-positive tumor cells were present, compared with that in metaphyseal area (Fig. 2Be and f). ANOVA revealed that, in the metaphysis, the number of PCNA-positive cells was significantly higher, compared with the number of TUNEL-positive cells (307.78±27.04, vs. 61.12±7.59 cells/mm2, respectively; P<0.01; n=7; Fig. 2C). In the diaphysis, the number of TUNEL-positive cells was significantly higher, compared with the number of PCNA-positive cells (291.96±20.78, vs. 8.04±1.09 cells/mm2, respectively; P<0.01; n=7; Fig. 2C).


Immunolocalization of MMP9 and MMP2 in osteolytic metastasis originating from MDA-MB-231 human breast cancer cells.

Liu B, Cui J, Sun J, Li J, Han X, Guo J, Yi M, Amizuka N, Xu X, Li M - Mol Med Rep (2016)

Immunohistochemical and statistical analyses of PCNA and TUNEL staining for apoptosis. (A) Immunohistochemical analysis of PCNA in the (a) metaphysis and (b) diaphysis in the normal bone marrow of the control group. Immunohistochemistry for PCNA in the (c) metaphyseal and (d) diaphyseal tumor metastases. More PCNA-positive tumor cells (brown color) were detected in the metaphyseal tumor tissue, compared with the diaphyseal tumor tissue. (e and f) Higher magnification of c and d, respectively. (B) TUNEL staining for apoptotic tumor cells in the (a) metaphysis and (b) diaphysis in the normal bone marrow tissue of the control group. TUNEL staining for apoptotic tumor cells in the (c) metaphyseal and (d) diaphyseal tumor metastases. More TUNEL-positive apoptotic tumor cells (blue color) were observed in the diaphyseal tumor tissue, compared with the metaphyseal tumor tissue. (e and f) High magnifications of c and d, respectively. (C) Statistical analyses of the numbers of PCNA/TUNEL-positive tumor cells in the metaphysis and diaphysis. *P<0.01. Scale bar=50 µm in Aa-d and Ba-d; 25 µm in Ae and f, and Be and f. TUNEL, TdT-mediated dUTP nick-end labeling; Meta, metaphysis; Dia, diaphysis; T, tumor cells; CB, cortical bone; *, normal bone tissue.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4940081&req=5

f2-mmr-14-02-1099: Immunohistochemical and statistical analyses of PCNA and TUNEL staining for apoptosis. (A) Immunohistochemical analysis of PCNA in the (a) metaphysis and (b) diaphysis in the normal bone marrow of the control group. Immunohistochemistry for PCNA in the (c) metaphyseal and (d) diaphyseal tumor metastases. More PCNA-positive tumor cells (brown color) were detected in the metaphyseal tumor tissue, compared with the diaphyseal tumor tissue. (e and f) Higher magnification of c and d, respectively. (B) TUNEL staining for apoptotic tumor cells in the (a) metaphysis and (b) diaphysis in the normal bone marrow tissue of the control group. TUNEL staining for apoptotic tumor cells in the (c) metaphyseal and (d) diaphyseal tumor metastases. More TUNEL-positive apoptotic tumor cells (blue color) were observed in the diaphyseal tumor tissue, compared with the metaphyseal tumor tissue. (e and f) High magnifications of c and d, respectively. (C) Statistical analyses of the numbers of PCNA/TUNEL-positive tumor cells in the metaphysis and diaphysis. *P<0.01. Scale bar=50 µm in Aa-d and Ba-d; 25 µm in Ae and f, and Be and f. TUNEL, TdT-mediated dUTP nick-end labeling; Meta, metaphysis; Dia, diaphysis; T, tumor cells; CB, cortical bone; *, normal bone tissue.
Mentions: PCNA-positive cells were observed in the metaphyseal area (Fig. 2Ab and c), but few were observed in the diaphyseal area (Fig. 2Ad and f). In the metaphyseal tumor nest, a few scattered TUNEL-positive apoptotic cells were observed (Fig. 2Bb and c). However, in the diaphyseal tumor nest, a higher number of TUNEL-positive tumor cells were present, compared with that in metaphyseal area (Fig. 2Be and f). ANOVA revealed that, in the metaphysis, the number of PCNA-positive cells was significantly higher, compared with the number of TUNEL-positive cells (307.78±27.04, vs. 61.12±7.59 cells/mm2, respectively; P<0.01; n=7; Fig. 2C). In the diaphysis, the number of TUNEL-positive cells was significantly higher, compared with the number of PCNA-positive cells (291.96±20.78, vs. 8.04±1.09 cells/mm2, respectively; P<0.01; n=7; Fig. 2C).

Bottom Line: Numerous tartrate-resistant acid phosphatase-positive osteoclasts were found in the metastasized lesions.The invaded tumor cells positive for mammaglobin 1 exhibited different proliferation activities and apoptosis between the metaphysis and diaphysis.Of note, MMP9 was expressed predominantly in the proliferating cell nuclear antigen‑positive area, whereas the expression of MMP2 was observed predominantly in the diaphysis, which had more TUNEL‑positive cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Bone Metabolism, School of Stomatology, Shandong University, Shandong Provincial Key Laboratory of Oral Tissue Regeneration, Jinan, Shandong 250012, P.R. China.

ABSTRACT
The aim of the present study was to investigate the expression of matrix metalloproteinase (MMP)9 and MMP2, and their potential roles in bone metastasis nests using a well-standardized model of breast cancer bone metastasis in nude mice. BALB/c nu/nu mice (5-week-old; n=10) were subjected to intracardiac injection of MDA-MB-231 human breast cancer cells. After 4 weeks, the mice exhibiting radiolucent lesions in tibiae were sacrificed, and the tibiae were removed for histochemical analysis. The gene expression of MMP2 and MMP9 in the tumor cells, metaphysis and diaphysis of normal BALB/c nu/nu mice were determined using reverse transcription-polymerase chain reaction analysis. The metastatic tumor tissue occupied almost the entire bone marrow cavity. Numerous tartrate-resistant acid phosphatase-positive osteoclasts were found in the metastasized lesions. The invaded tumor cells positive for mammaglobin 1 exhibited different proliferation activities and apoptosis between the metaphysis and diaphysis. Proliferating cell nuclear antigen was expressed at high levels in the metaphyseal area, whereas TdT-mediated dUTP nick-end labeling (TUNEL)-positive cells were more evident in the diaphysis area. Of note, MMP9 was expressed predominantly in the proliferating cell nuclear antigen‑positive area, whereas the expression of MMP2 was observed predominantly in the diaphysis, which had more TUNEL‑positive cells. Taken together, the results suggested that MMP9 and MMP2 may have their own importance in extracellular matrix degradation and trabecular bone damage in different zones of bone metastasis, including the metaphysis and diaphysis.

No MeSH data available.


Related in: MedlinePlus