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Increased secretory leukocyte protease inhibitor (SLPI) production by highly metastatic mouse breast cancer cells.

Sayers KT, Brooks AD, Sayers TJ, Chertov O - PLoS ONE (2014)

Bottom Line: Additionally higher levels of SLPI were also observed in 4T1.2 breast tumors in vivo following immunohistochemical staining.A comparison of SLPI mRNA levels by gene profiling using microarrays and RT-PCR did not detect major differences in SLPI gene expression between the 4T1 and 4T1.2 cells indicating that SLPI secretion is regulated at the protein level.Our results demonstrate that secretion of SLPI is drastically increased in highly metastatic cells, suggesting a possible role for SLPI in enhancing the metastatic behavior of breast cancer cell line 4T1.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research Technology Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, Maryland, United States of America.

ABSTRACT
The precise molecular mechanisms enabling cancer cells to metastasize from the primary tumor to different tissue locations are still largely unknown. Secretion of some proteins by metastatic cells could facilitate metastasis formation. The comparison of secreted proteins from cancer cells with different metastatic capabilities in vivo might provide insight into proteins involved in the metastatic process. Comparison of the secreted proteins from the mouse breast cancer cell line 4T1 and its highly metastatic 4T1.2 clone revealed a prominent differentially secreted protein which was identified as SLPI (secretory leukocyte protease inhibitor). Western blotting indicated higher levels of the protein in both conditioned media and whole cell lysates of 4T1.2 cells. Additionally higher levels of SLPI were also observed in 4T1.2 breast tumors in vivo following immunohistochemical staining. A comparison of SLPI mRNA levels by gene profiling using microarrays and RT-PCR did not detect major differences in SLPI gene expression between the 4T1 and 4T1.2 cells indicating that SLPI secretion is regulated at the protein level. Our results demonstrate that secretion of SLPI is drastically increased in highly metastatic cells, suggesting a possible role for SLPI in enhancing the metastatic behavior of breast cancer cell line 4T1.

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Proteins secreted by 4T1 and 4T1.2 cells.Cell conditioned media generated under serum-free conditions was fractionated on DEAE column and proteins were eluted step-wise by solutions containing 0.1 M, 0.2 M and 0.5 M of sodium chloride. After precipitation aliquots of fractions were separated on SDS gel and stained with Coomassie Brilliant blue for visual comparison of protein bands. Selected differences observed between the two cell lines are highlighted.
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pone-0104223-g001: Proteins secreted by 4T1 and 4T1.2 cells.Cell conditioned media generated under serum-free conditions was fractionated on DEAE column and proteins were eluted step-wise by solutions containing 0.1 M, 0.2 M and 0.5 M of sodium chloride. After precipitation aliquots of fractions were separated on SDS gel and stained with Coomassie Brilliant blue for visual comparison of protein bands. Selected differences observed between the two cell lines are highlighted.

Mentions: A comparison of the secreted proteins from the mouse breast cancer cell lines 4T1 and 4T1.2 was carried out to identify proteins associated with the highly metastatic 4T1.2 phenotype. Proteins secreted by 4T1 and 4T1.2 were fractionated using anion exchange chromatography on DEAE-650 with step-wise elution by 0.1, 0.2 and 0.5 M NaCl. The eluted proteins were separated by SDS gel electrophoresis. The Coomassie stained gel was used to visually compare protein bands. Most protein bands observed from CM of 4T1 and 4T1.2 cells were identical and of similar intensity, but one standout protein band was predominantly present in 0.1 M eluate of 4T1.2 cell supernatant (Fig.1 and Fig.2A). The band on a Coomassie stained gel was digested with trypsin and the obtained peptides analyzed by MALDI-TOF MS/MS. The spectrum of a peptide with m/z of 1620.914 Da was searched using MASCOT MS/MS Ion Search which resulted in confident identification of the peptide CVNPVPIRKPVWR of SLPI using both SwissProt (e = 1.7×10−3) and NCBInr (e = 2.7×10−4) databases (Fig. 2B). Analysis of m/z 1276.572 allowed identification of sequence TDWECPGKQR of SLPI (not shown).


Increased secretory leukocyte protease inhibitor (SLPI) production by highly metastatic mouse breast cancer cells.

Sayers KT, Brooks AD, Sayers TJ, Chertov O - PLoS ONE (2014)

Proteins secreted by 4T1 and 4T1.2 cells.Cell conditioned media generated under serum-free conditions was fractionated on DEAE column and proteins were eluted step-wise by solutions containing 0.1 M, 0.2 M and 0.5 M of sodium chloride. After precipitation aliquots of fractions were separated on SDS gel and stained with Coomassie Brilliant blue for visual comparison of protein bands. Selected differences observed between the two cell lines are highlighted.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4128660&req=5

pone-0104223-g001: Proteins secreted by 4T1 and 4T1.2 cells.Cell conditioned media generated under serum-free conditions was fractionated on DEAE column and proteins were eluted step-wise by solutions containing 0.1 M, 0.2 M and 0.5 M of sodium chloride. After precipitation aliquots of fractions were separated on SDS gel and stained with Coomassie Brilliant blue for visual comparison of protein bands. Selected differences observed between the two cell lines are highlighted.
Mentions: A comparison of the secreted proteins from the mouse breast cancer cell lines 4T1 and 4T1.2 was carried out to identify proteins associated with the highly metastatic 4T1.2 phenotype. Proteins secreted by 4T1 and 4T1.2 were fractionated using anion exchange chromatography on DEAE-650 with step-wise elution by 0.1, 0.2 and 0.5 M NaCl. The eluted proteins were separated by SDS gel electrophoresis. The Coomassie stained gel was used to visually compare protein bands. Most protein bands observed from CM of 4T1 and 4T1.2 cells were identical and of similar intensity, but one standout protein band was predominantly present in 0.1 M eluate of 4T1.2 cell supernatant (Fig.1 and Fig.2A). The band on a Coomassie stained gel was digested with trypsin and the obtained peptides analyzed by MALDI-TOF MS/MS. The spectrum of a peptide with m/z of 1620.914 Da was searched using MASCOT MS/MS Ion Search which resulted in confident identification of the peptide CVNPVPIRKPVWR of SLPI using both SwissProt (e = 1.7×10−3) and NCBInr (e = 2.7×10−4) databases (Fig. 2B). Analysis of m/z 1276.572 allowed identification of sequence TDWECPGKQR of SLPI (not shown).

Bottom Line: Additionally higher levels of SLPI were also observed in 4T1.2 breast tumors in vivo following immunohistochemical staining.A comparison of SLPI mRNA levels by gene profiling using microarrays and RT-PCR did not detect major differences in SLPI gene expression between the 4T1 and 4T1.2 cells indicating that SLPI secretion is regulated at the protein level.Our results demonstrate that secretion of SLPI is drastically increased in highly metastatic cells, suggesting a possible role for SLPI in enhancing the metastatic behavior of breast cancer cell line 4T1.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research Technology Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, Maryland, United States of America.

ABSTRACT
The precise molecular mechanisms enabling cancer cells to metastasize from the primary tumor to different tissue locations are still largely unknown. Secretion of some proteins by metastatic cells could facilitate metastasis formation. The comparison of secreted proteins from cancer cells with different metastatic capabilities in vivo might provide insight into proteins involved in the metastatic process. Comparison of the secreted proteins from the mouse breast cancer cell line 4T1 and its highly metastatic 4T1.2 clone revealed a prominent differentially secreted protein which was identified as SLPI (secretory leukocyte protease inhibitor). Western blotting indicated higher levels of the protein in both conditioned media and whole cell lysates of 4T1.2 cells. Additionally higher levels of SLPI were also observed in 4T1.2 breast tumors in vivo following immunohistochemical staining. A comparison of SLPI mRNA levels by gene profiling using microarrays and RT-PCR did not detect major differences in SLPI gene expression between the 4T1 and 4T1.2 cells indicating that SLPI secretion is regulated at the protein level. Our results demonstrate that secretion of SLPI is drastically increased in highly metastatic cells, suggesting a possible role for SLPI in enhancing the metastatic behavior of breast cancer cell line 4T1.

Show MeSH
Related in: MedlinePlus