SUPPLEMENTAL DATA
Cucurbitacins sensitize renal carcinoma cells to TRAIL-induced apoptosis by a STAT3-independent mechanism
Curtis J. Henrich,1,2 Cheryl L. Thomas,1 Alan D. Brooks, 2,3 Nancy Lynn Booth,1,4 Evan M. Lowery,3 Richard J. Pompei,3 James B. McMahon,1 and Thomas J. Sayers2,3
1Molecular Targets Laboratory
NCI-Frederick
Frederick, Maryland
2Basic Research Program
SAIC-Frederick, Inc.
NCI-Frederick
Frederick, Maryland
3Laboratory for Experimental Immunology and Cancer Inflammation Program
NCI-Frederick
Frederick, Maryland
4present address:
Spherix, Incorporated
Bethesda, Maryland
In order to assess the broader utility of cucurbitacins as TRAIL sensitizers as well as to compare them to other, known, TRAIL sensitizers, the most active cucurbitacin, cucurbitacin B was tested for its ability to sensitize a number of other cancer cell lines to TRAIL-induced apoptosis. The assays were similar to those described in the main text except that estimation of viable cell numbers utilized a commercially available MTS assay (Promega, Madison, WI) rather than the XTT assay. Cells were plated at 5000 per well in 96-well plates. After overnight attachment, cells were treated for 4 h in the presence or absence of cucurbitacin B (100 nM final concentration) followed by overnight (18-20 h) incubation with or without TRAIL (50 ng/ml final). Cucurbitacin B was not removed in these experiments. Data are presented as average ± standard deviation (n = 3). The following categories of cell lines are shown Figure 1: renal carcinoma, Figure 2: breast carcinoma, Figure 3: colon carcinoma, Figure 4: melanoma.
Of the 16 substantially TRAIL-resistant cell lines (operationally defined as > 50% cell survival after treatment by TRAIL alone, 10 were sensitized by cucurbitacin B (i.e. greater than additive reduction in cell number in the presence of both agents compared to each alone). Comparison of sensitization of these cell lines by cucurbitacin to the sensitization by bortezomib, a well-established TRAIL sensitizing drug gives an interesting comparative pattern (see Brooks et al. [22] for bortezomib data). The patterns significantly diverge for renal carcinoma cell lines. SN12C, Caki-1, and 786-O are not sensitized by bortezomib, but are by cucurbitacin B. By contrast, all of the other TRAIL-resistant cell lines sensitized by cucurbitacin B (with the exception of Colo-205 colon carcinoma) are also sensitized by bortezomib. Different patterns of sensitization across multiple cell lines is consistent with different mechanisms of action for the two agents, a conclusion also supported by differences in N-acetyl cysteine effects (see main text).
Given that cucurbitacins have been reported to affect STAT3 signaling, the effects of each cucurbitacin on extent of STAT3 phosphorylation and on levels of the STAT3-induced apoptotic protein Bcl-xL were investigated (Figure 5 – below).
Figure 1: Effect of cucurbitacin B on TRAIL-induced apoptosis: renal carcinoma cell lines
Figure 2: Effect of cucurbitacin B on TRAIL-induced apoptosis: breast carcinoma cell lines
Figure 3: Effect of cucurbitacin B on TRAIL-induced apoptosis: colon carcinoma cell lines
Figure 4: Effect of cucurbitacin B on TRAIL-induced apoptosis: melanoma cell lines
Figure 5: Effects of cucurbitacins on levels of STAT3 phosphorylation and Bcl-xL.
(A) ACHN cells were plated at 2.5 x 106 cells/well in 6-well plates and allowed to attach overnight. After 4 h treatment with the indicated cucurbitacin (B, C, D, E, and I: 1 mM, cucurbitacins K and P: 10 mM), cells were rinsed (ice cold buffer) and extracted in lysis buffer containing protease and phosphatase inhibitors. Protein was measured using BCA assay and separated by electrophoresis and western blotting for STAT3 and phosphoSTAT3 was performed as described in Materials and Methods. (B) Extractions performed as above and western blotting was performed for STAT3, phosphoSTAT3 and Bcl-xL.
Anti-STAT3 (79D7 rabbit monoclonal), anti-phosphoSTAT3 (M9C6 mouse monoclonal, recognizing phosphorylation at tyr705), anti-Bcl-xL (54H6 rabbit monoclonal) were obtained from Cell Signaling Technology (Danvers, MA).