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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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Increased production of SLPI by 4T1.2 cells.(A). Initial Western blot of CM generated in serum-free media to confirm differences in SLPI levels. Cell conditioned media preparations for both 4T1 and 4T1.2 from 2 separate experiments were loaded on the gel. SLPI levels were higher in 4T1.2 conditioned media. (B). Western blot for SLPI of CM from cells grown in media containing FBS. The 4T1.2 CM clearly had increased levels of SLPI as compared to the 4T1 conditioned media. Also observed was a high molecular weight non-specific band believed to be BSA which indicates comparable overall protein loading of CM on these gels. (C). Western blot of cell lysates showing intracellular SLPI was also highly elevated in 4T1.2 cells. Reprobing of the cell lysate blot with an antibody to actin confirms equivalent loading of proteins on the gel.
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pone-0104223-g003: Increased production of SLPI by 4T1.2 cells.(A). Initial Western blot of CM generated in serum-free media to confirm differences in SLPI levels. Cell conditioned media preparations for both 4T1 and 4T1.2 from 2 separate experiments were loaded on the gel. SLPI levels were higher in 4T1.2 conditioned media. (B). Western blot for SLPI of CM from cells grown in media containing FBS. The 4T1.2 CM clearly had increased levels of SLPI as compared to the 4T1 conditioned media. Also observed was a high molecular weight non-specific band believed to be BSA which indicates comparable overall protein loading of CM on these gels. (C). Western blot of cell lysates showing intracellular SLPI was also highly elevated in 4T1.2 cells. Reprobing of the cell lysate blot with an antibody to actin confirms equivalent loading of proteins on the gel.

Mentions: Confirmation of the differences in SLPI secretion between the 4T1 and 4T1.2 cell lines was performed using Western Blotting with an SLPI antibody. Comparison of band intensities between the 4T1 and 4T1.2 samples indicated that SLPI was present at much higher levels in the 4T1.2 CM (Fig. 3B). This increased intensity of the SLPI band in 4T1.2 CM samples was consistently observed using multiple CM preparations. The Western blots were repeated with CM of cells cultured with serum-containing media, and under these conditions even greater differences in SLPI levels between the 4T1 and 4T1.2 CM were observed (Fig. 3B). In an attempt to determine if the higher level of SLPI present in the CM was due to differential secretion of SLPI or overall higher total protein levels, analysis of the cell lysates was also carried out. Western blots of cell lysates mirrored what was observed for the CM, with 4T1.2 lysates having significantly higher levels of SLPI (Fig. 3C). The cell lysate blot was re-probed for actin to show that the amount of protein present on the blot was comparable (Fig. 3C). Since Western blotting of the cell lysates for intracellular SLPI between 4T1 and 4T1.2 showed similar differences as was observed for secreted proteins, the results indicated that much higher overall levels of SLPI were produced by the 4T1.2 cell line as compared with 4T1.


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)

Increased production of SLPI by 4T1.2 cells.(A). Initial Western blot of CM generated in serum-free media to confirm differences in SLPI levels. Cell conditioned media preparations for both 4T1 and 4T1.2 from 2 separate experiments were loaded on the gel. SLPI levels were higher in 4T1.2 conditioned media. (B). Western blot for SLPI of CM from cells grown in media containing FBS. The 4T1.2 CM clearly had increased levels of SLPI as compared to the 4T1 conditioned media. Also observed was a high molecular weight non-specific band believed to be BSA which indicates comparable overall protein loading of CM on these gels. (C). Western blot of cell lysates showing intracellular SLPI was also highly elevated in 4T1.2 cells. Reprobing of the cell lysate blot with an antibody to actin confirms equivalent loading of proteins on the gel.
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pone-0104223-g003: Increased production of SLPI by 4T1.2 cells.(A). Initial Western blot of CM generated in serum-free media to confirm differences in SLPI levels. Cell conditioned media preparations for both 4T1 and 4T1.2 from 2 separate experiments were loaded on the gel. SLPI levels were higher in 4T1.2 conditioned media. (B). Western blot for SLPI of CM from cells grown in media containing FBS. The 4T1.2 CM clearly had increased levels of SLPI as compared to the 4T1 conditioned media. Also observed was a high molecular weight non-specific band believed to be BSA which indicates comparable overall protein loading of CM on these gels. (C). Western blot of cell lysates showing intracellular SLPI was also highly elevated in 4T1.2 cells. Reprobing of the cell lysate blot with an antibody to actin confirms equivalent loading of proteins on the gel.
Mentions: Confirmation of the differences in SLPI secretion between the 4T1 and 4T1.2 cell lines was performed using Western Blotting with an SLPI antibody. Comparison of band intensities between the 4T1 and 4T1.2 samples indicated that SLPI was present at much higher levels in the 4T1.2 CM (Fig. 3B). This increased intensity of the SLPI band in 4T1.2 CM samples was consistently observed using multiple CM preparations. The Western blots were repeated with CM of cells cultured with serum-containing media, and under these conditions even greater differences in SLPI levels between the 4T1 and 4T1.2 CM were observed (Fig. 3B). In an attempt to determine if the higher level of SLPI present in the CM was due to differential secretion of SLPI or overall higher total protein levels, analysis of the cell lysates was also carried out. Western blots of cell lysates mirrored what was observed for the CM, with 4T1.2 lysates having significantly higher levels of SLPI (Fig. 3C). The cell lysate blot was re-probed for actin to show that the amount of protein present on the blot was comparable (Fig. 3C). Since Western blotting of the cell lysates for intracellular SLPI between 4T1 and 4T1.2 showed similar differences as was observed for secreted proteins, the results indicated that much higher overall levels of SLPI were produced by the 4T1.2 cell line as compared with 4T1.

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