Increased production of SLPI by 4T1.2 cells.

<p>(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.</p

Identification of SLPI as a protein secreted at higher levels by 4T1.2 cells.

<p>(A). Coomassie stained SDS-PAGE gel of 0.1 M eluate from DEAE column with a difference in a protein of approximately 12 kDa indicated. (B). The band was excised and in-gel trypsin digestion was performed, a tryptic peptide with m/z of 1620.914 Da was analyzed using MALDI-TOF MS/MS and the resulting peptide mass spectrum was searched using MASCOT giving an identification of the peptide CVNPVPIRKPVWR from SLPI. (C). N-terminal Edman sequencing of the first 10 residues when searched with BLAST also indicated the band was SLPI.</p

Proteins secreted by 4T1 and 4T1.2 cells.

<p>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.</p

Staining of 4T1 and 4T1.2 cells for SLPI.

<p>Immunohistochemical staining also indicates increased production of SLPI by 4T1.2 cells. (A). Staining of cell pellets of 4T1 and 4T1.2 cells grown in vitro shows that almost all 4T1.2 cells produce SLPI whereas production by 4T1 cells is confined to a subpopulation of cells. (B). Staining of breast tumors derived from the injection of 4T1 and 4T1.2 cells also demonstrates a stronger and more uniform staining of SLPI in the 4T1.2 tumors.</p

17β-Hydroxywithanolides as Sensitizers of Renal Carcinoma Cells to Tumor Necrosis Factor‑α Related Apoptosis Inducing Ligand (TRAIL) Mediated Apoptosis: Structure–Activity Relationships

Renal cell carcinoma (RCC) is a cancer with poor prognosis, and the 5-year survival rate of patients with metastatic RCC is 5–10%. Consequently, treatment of metastatic RCC represents an unmet clinical need. Screening of a 50 000-member library of natural and synthetic compounds for sensitizers of RCC cells to TRAIL-mediated apoptosis led to identification of the 17β-hydroxywithanolide (17-BHW), withanolide E (<b>1</b>), as a promising lead. To explore structure–activity relationships, we obtained natural and semisynthetic withanolides <b>1</b>, <b>2a</b>, <b>2c</b>, and <b>3</b>–<b>36</b> and compared their ability to sensitize TRAIL-mediated apoptosis in a panel of renal carcinoma cells. Our findings revealed that 17-BHWs with a α-oriented side chain are superior to known TRAIL-sensitizing withanolides belonging to withaferin A class with a β-oriented side chain and demonstrated that the 17-BHW scaffold can be modified to enhance sensitization of RCCs to TRAIL-mediated apoptosis, thereby assisting development of natural-product-inspired drugs to treat metastatic RCC