Accelerated mammary tumor development in adiponectin haplodeficient MMTV-PyVT mice.

<p>Tumor growth in <i>PyVT(+/−)/ADN(+/+)</i> and <i>PyVT(+/−)/ADN(+/−)</i> mice were monitored starting from 6 and 11 wks, up to 14 and 28 wks for female (left panel) and male (right panel) mice respectively. Tumor sizes were measured using vernier calipers and tumor volume calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004968#s4" target="_blank">Methods</a>. Each group contained 13–20 mice, and the mean tumor volume ±SD was presented.</p

Mammary tumor cells derived from adiponectin haplodeficient mice were more aggressive.

<p>Primary mammary tumor cells were isolated from FVB/N PyVT mice with normal [<i>PyVT(+/−)/ADN(+/+)</i>] or reduced [<i>PyVT(+/−)/ADN(+/−)</i>] adiponectin expressions, and implanted into nude mice for assessing their tumor development <i>in vivo</i> (A and B), or subjected to culture and [³H]-thymidine incorporation assays for evaluating their proliferations <i>in vitro</i> (C and D). The comparison between <i>PyVT(+/−)/ADN(+/+)</i> and <i>PyVT(+/−)/ADN(+/−)</i> groups were performed for tumor cells derived from both female (A and C) and male (B and D) mice. Tumor <i>g</i>rowth was presented as the fold changes of tumor volume against the first measurement at day 4 (A and B). DNA synthesis was monitored in 0.5% and 10% FBS culture conditions at 24 and 48 hrs after seeding (C and D). CPM, counts per minute. *, P<0.05 and **, P<0.01 vs corresponding groups (n = 13–18).</p

Serum adiponectin distributions in wildtype and PyVT mice.

<p>The serum adiponectin concentrations were measured by an in-house sandwich ELISA assay using blood samples collected from the tail vein of FVB/N and C57BL/6J mice. The median and mean values were calculated and displayed in the table.</p

Tumor cells derived from male <i>PyVT(+/−)/ADN(+/−)</i> mice show increased metastatic capacities in nude mice comparing with those of <i>PyVT(+/−)/ADN(+/+)</i> mice.

<p>Both hematoxylin and eosin staining (upper panel) and the morphological evaluations (bottom panel) were performed to evaluate metastasis of the lung tissues.</p

Reduced tumor latency in adiponectin haplodeficient MMTV-PyVT mice of both FVB/N and C57BL/6J genetic backgrounds.

<p>The tumor onset was closely monitored by visual inspection and palpation every 2–3 days. Latency of mammary tumors was defined as the age when a palpable lump was first detected in the mammary gland. Kaplan-Meier estimates of the tumor-free survival curves were calculated and plotted. Median value represents the time point when 50% of animals developed palpable tumor masses. The significance of differences in latency was analyzed by the Log-rank test. The comparisons were performed between <i>ADN(+/+)</i> and <i>ADN(+/−)</i> female (left panel) and male (right panel) animals in FVB/N and C57BL/6J genetic backgrounds. CI, confidence interval.</p

List of primers used for real time quantitative PCR analysis.

<p>List of primers used for real time quantitative PCR analysis.</p

A basal-like subtype of mammary tumors derived from adiponectin haplodeficient MMTV-PyVT mice.

<p><i>A</i>, Quantitative RT-PCR analysis of the expression levels of gene markers associated with different subtypes of breast tumors. The RNA was extracted from cultured primary tumor cells isolated from 14-week old <i>PyVT(+/−)/ADN(+/+)</i> and <i>PyVT(+/−)/ADN(+/−)</i> female FVB/N mice and quantitative PCR analysis performed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004968#s4" target="_blank">Methods</a>. *, P<0.01 vs <i>PyVT(+/−)/ADN(+/+)</i> group, n = 10. <i>B</i>, Morphological features of tumors derived from 14-week old female <i>PyVT(+/−)/ADN(+/+)</i> and <i>PyVT(+/−)/ADN(+/−)</i> mice of the FVB/N background. Distinct morphologies were observed between tumors collected from mice with normal and reduced adiponectin levels. Note that central necrosis and geographic tumor necrosis, as well as stromal lymphocytic responses represented the typical basal-like subtype of breast tumors in <i>PyVT(+/−)/ADN(+/−)</i> mice. <i>C</i>, The protein levels of p53 were much higher in tumors derived from <i>PyVT(+/−)/ADN(+/+)</i> mice compared to those of the <i>PyVT(+/−)/ADN(+/−)</i> mice as measured by Western Blotting using specific antibodies purchased from Cell Signaling Biotechnology.</p

List of primers used for genotyping.

<p>List of primers used for genotyping.</p

Inactivation of PTEN was at least partially caused by the augmented Trx1/TrxR1 redox activities in <i>PyVT(+/−)/ADN(+/−)</i> tumor cells.

<p><i>A</i>, activities of PTEN, TrxR1 and Trx were evaluated in the lysates derived from <i>PyVT(+/−)/ADN(+/+)</i> and <i>PyVT(+/−)/ADN(+/−)</i> tumor cells using colorimetric assays. Briefly, cells were treated with vehicle control (CON), 10 µM curcumin (CUR) or 15 µg/ml adiponectin (ADN) for 24 hrs. Immunoprecipitation and phosphatase assay were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004968#s4" target="_blank">Methods</a>. Results were expressed as fold changes relative to the enzyme activities in <i>PyVT(+/−)/ADN(+/+)</i> tumor cells treated with vehicle control. <i>B</i>, total protein levels of PTEN, TrxR1 and Trx1 in cell lysates from <i>PyVT(+/−)/ADN(+/+)</i> and <i>PyVT(+/−)/ADN(+/−)</i> tumors were analyzed by Western Blotting. <i>C</i>, co-immunoprecipitations were performed with the specific antibodies against PTEN or Trx1 in both <i>PyVT(+/−)/ADN(+/+)</i> and <i>PyVT(+/−)/ADN(+/−)</i> tumor cell lysates. The immune-complexes were analyzed by SDS-PAGE and Western Blotting using antibodies as indicated. <i>D</i>, intrinsic transcriptional activities of TrxR1 promoter (left panel) and the mRNA levels of TrxR1 and Trx1 (right panel) were evaluated in <i>PyVT(+/−)/ADN(+/+)</i> and <i>PyVT(+/−)/ADN(+/−)</i> tumor cells treated without (CON) or with adiponectin (ADN, 15 µg/ml) using TrxR1 reporter assay (left panel) and quantitative RT-PCR respectively (right panel). *, P<0.05 vs <i>PyVT(+/−)/ADN(+/+)</i> cell control; #, P<0.05 vs <i>PyVT(+/−)/ADN(+/−)</i> cell control (n = 3, from three independent experiments).</p

Total net weights (g) of tumor and lung tissues collected from nude mice implanted with primary tumor cells isolated from female and male <i>PyVT(+/−)/ADN(+/+)</i> and <i>PyVT(+/−)/ADN(+/−)</i> mice.

*<p>p<0.05 vs the corresponding <i>PyVT(+/−)/ADN(+/+)</i> mice group.</p