Histological analysis of the effects of zoledronic acid-treatment on normal and tumour-bearing bones.

<p>H&E stained sections of the femoral head at 40X (A, C, E and G) and 120X (B, D, F and H) magnification showing normal (A&B) and RM1(BM) tumour bearing bones (C–H); Typical examples of bones from untreated mice (A–D) and mice treated with 20 µg/kg ZOL (E&F) or 100 µg/kg ZOL (G&H) are shown. T = tumour, Arrow = Trabecular bone.</p

Histological analysis with tetrachrome stain for effects of zoledronic acid-treatment on normal and tumour-bearing bones.

<p>Tetrachrome stain for differentiation of mineralised and unmineralised bone at 120x magnification Normal (A), or RM1(BM) tumour bearing bones (B–D). (A&B) Untreated, (C) 20 µg/kg ZOL (D) 100 µg/kg ZOL. T = tumour, Arrow = Trabecular bone.</p

Zoledronic acid treatment did not reduce establishment of metastases in RM1(BM) injected mice.

<p>(A) Total number of metastases, (B) bone-metastases, or (C) soft tissue metastases as assessed by one-way ANOVA. Each point represents the number of metastases in an individual mouse, bars indicate the median within the group.</p

Zoledronic acid preserves bone structure.

<p>MicroCT scans of mouse femur and tibia; normal control (A), RM1(BM) tumour-bearing (B), RM1(BM) tumour-bearing treated with the 20 µg/kg ZOL regimen (C), RM1(BM) tumour-bearing treated with the 100 µg/kg ZOL regimen (D).</p

Zoledronic acid treatment alters bone volume and the bone surface-area:volume ratio in normal and tumour-bearing bones.

<p>There was no significant change in bone surface area (A) induced by the presence of a tumour or treatment with zoledronic acid. However, bone volume was dramatically reduced in RM1(BM) containing bones and the treatment with ZOL prevented this loss in a dose-dependent manner (B), resulting in lower surface area/volume ratio (increased bone density) compared to normal or tumor-bearing bones (C). Results are based on measurements obtained from CT scans as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019389#s4" target="_blank">Materials and Methods</a> section. Statistical analysis was performed by one-way ANOVA followed by Tukey's post test, * p<0.05, ** p<0.01, *** p<0.001.</p

Zoledronic acid treatment improves mouse survival but does not reduce the number of metastases.

<p>(A) Kaplan-Meier survival plot of mice given an intra-arterial injection of RM1(BM) cells followed by treatment with the 100 µg/kg or 20 µg/kg zoledronic dosing regimens or vehicle only as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019389#s4" target="_blank">Materials and Methods</a>. The log rank test for trend indicates a trend of increased survival with increasing dose (p = 0.012), and a significant difference in survival between mice in the 100 µg/kg dose group vs vehicle control groups (p = 0.031, Kaplan-Meier followed by Breslow pair-wise comparison using SPSS 17.0).</p

Surrogate markers of bone metabolism in serum of normal, tumor-bearing and zoledronic acid treated mice.

<p>(A) Levels of the osteoblast marker, osteocalcin were reduced in serum of zoledronic acid treated mice. (B<i>)</i> Tartrate-resistant acid phosphatase 5b, an indicator of osteoclast activity, was also reduced with treatment. Points on the graph represent serum levels for individual mice while bars show the position of the means within each group. Statistical analysis was performed using one-way ANOVA for analysis followed by Tukey's post test. *** p<0.001.</p

<em>In Vitro</em> and <em>In Vivo</em> Prostate Cancer Metastasis and Chemoresistance Can Be Modulated by Expression of either CD44 or CD147

<div><p>CD44 and CD147 are associated with cancer metastasis and progression. Our purpose in the study was to investigate the effects of down-regulation of CD44 or CD147 on the metastatic ability of prostate cancer (CaP) cells, their docetaxel (DTX) responsiveness and potential mechanisms involved <em>in vitro</em> and <em>in vivo</em>. CD44 and CD147 were knocked down (KD) in PC-3M-luc CaP cells using short hairpin RNA (shRNA). Expression of CD44, CD147, MRP2 (multi-drug resistance protein-2) and MCT4 (monocarboxylate tranporter-4) was evaluated using immunofluorescence and Western blotting. The DTX dose-response and proliferation was measured by MTT and colony assays, respectively. The invasive potential was assessed using a matrigel chamber assay. Signal transduction proteins in PI3K/Akt and MAPK/Erk pathways were assessed by Western blotting. An <em>in vivo</em> subcutaneous (s.c.) xenograft model was established to assess CaP tumorigenecity, lymph node metastases and DTX response. Our results indicated that KD of CD44 or CD147 decreased MCT4 and MRP2 expression, reduced CaP proliferation and invasive potential and enhanced DTX sensitivity; and KD of CD44 or CD147 down-regulated p-Akt and p-Erk, the main signal modulators associated with cell growth and survival. <em>In vivo</em>, CD44 or CD147-KD PC-3M-luc xenografts displayed suppressed tumor growth with increased DTX responsiveness compared to control xenografts. Both CD44 and CD147 enhance metastatic capacity and chemoresistance of CaP cells, potentially mediated by activation of the PI3K and MAPK pathways. Selective targeting of CD44/CD147 alone or combined with DTX may limit CaP metastasis and increase chemosensitivity, with promise for future CaP treatment.</p> </div

Immunostaining for CD44, CD147, MCT4 and MRP2 in KD and control prostate cancer (CaP) cell lines.

<p><b>Notes:</b><b>Immunofluorescence staining:</b> 0 =  negative; 1 =  weak; 2 =  moderate; 3 =  strong.</p><p>KD: knock down; scr: scrambled shRNA control.</p

Histology and CD31, CD44 and CD147 expression of s.c. xenografts.

<p>Morphological changes are shown in PC-3M-luc-CD44-KD and PC-3M-luc-CD147-KD xenografts with VC treatment (<b>A</b>). Obvious targeted lesions are shown in PC-3M-luc-CD44-KD and PC-3M-luc-CD147-KD xenografts with DTX treatment compared to the xenografts from PC-3M-luc and PC-3M-luc-scr cell lines (<b>B</b>). Low to medium CD31 expression is shown in PC-3M-luc-CD44-KD and PC-3M-luc-CD147-KD treated with VC and high CD31 expression is shown in PC-3M-luc and PC-3M-luc-src xenografts treated with VC (<i>P</i><0.05) (<b>C</b>). Markedly reduced CD31 expression is shown in PC-3M-luc-CD44-KD and PC-3M-luc-CD147-KD xenografts treated with DTX and medium CD31 expression is shown in PC-3M-luc and PC-3M-luc-scr xenografts treated with DTX (<i>P</i><0.05) (<b>D</b>). High CD44 expression is shown in PC-3M-luc and PC-3M-luc-scr xenografts; medium CD44 expression is seen in PC-3M-luc-CD147-KD xenografts; low CD44 expression is seen in PC-3M-luc-CD44-KD xenografts (<i>P</i><0.05) (<b>E</b>). High CD147 expression is shown in PC-3M-luc and PC-3M-luc-scr xenografts; low CD147 expression is seen in PC-3M-luc-CD44-KD xenografts; very low CD147 expression is seen in PC-3M-luc-CD147-KD xenografts (<i>P</i><0.05) (<b>F</b>). Brown color staining indicates positive while blue hematoxylin stains nuclei. Magnification ×400 in all images. DTX: docetaxel; KD: knock down; scr: scrambled shRNA control; VC: vehicle control.</p