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 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

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

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

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

Combination Therapy with the Histone Deacetylase Inhibitor LBH589 and Radiation Is an Effective Regimen for Prostate Cancer Cells

<div><p>Radiation therapy (RT) continues to be one of the most popular treatment options for localized prostate cancer (CaP). The purpose of the study was to investigate the <i>in vitro</i> effect of LBH589 alone and in combination with RT on the growth and survival of CaP cell lines and the possible mechanisms of radiosensitization of this combination therapy. The effect of LBH589 alone or in combination with RT on two CaP cell lines (PC-3 and LNCaP) and a normal prostatic epithelial cell line (RWPE-1) was studied by MTT and clonogenic assays, cell cycle analysis, western blotting of apoptosis-related and cell check point proteins, and DNA double strand break (DSB) repair markers. The immunofluorescence staining was used to further confirm DSB expression in treated CaP cells. Our results indicate that LBH589 inhibited proliferation in both CaP and normal prostatic epithelial cells in a time-and-dose-dependent manner; low-dose of LBH589 (IC₂₀) combined with RT greatly improved efficiency of cell killing in CaP cells; compared to RT alone, the combination treatment with LBH589 and RT induced more apoptosis and led to a steady increase of sub-G1 population and abolishment of RT-induced G2/M arrest, increased and persistent DSB, less activation of non-homologous end joining (NHEJ)/homologous recombination (HR) repair pathways and a panel of cell cycle related proteins. These results suggest that LBH589 is a potential agent to increase radiosensitivity of human CaP cells. LBH589 used either alone, or in combination with RT is an attractive strategy for treating human CaP.</p> </div

Effects of RT or combination treatment (LBH589 and RT) on cell cycle checkpoints, DNA damage and repair proteins in PC-3 and LNCaP CaP cells from pre-RT to 72 h post-RT.

<p>Cell cycle related proteins (p21, p-p53, p53, p-Cdk1, t-Cdk1, p-Chk1, t-Chk1, p-Chk2, t-Chk2, p-Rb795, p-Rb807/811, t-Rb) and DNA damage and repair related proteins (γH2AX, Ku70, Ku80, BRCA1, BRCA2, and RAD51) were determined by western blotting. t: Changes in total protein levels upon DNA damage; p: activation of DNA or cell cycle checkpoint proteins (phosphorylated form). Typical images are shown from three independent experiments (N=3).</p

Quantitative analysis of DSB repair marker in the HR pathway for RAD51 foci at pre-RT, 24 h and 72 h after 2 Gy RT or combination treatment in PC-3 and LNCaP cells.

<p>(<b>A</b>) After 2 Gy RT, average RAD51 foci number increased significantly at 24 and 72 h in both PC-3 and LNCaP cells, while average BRCA1 foci number in combination-treated (LBH589+2 Gy RT) cells was also increased at 24 and 72 h, but was significantly lower than that in RT-treated cells. (<b>B</b>) Representative images of RAD51 staining after 2 Gy RT or combination treatment (LBH589+2 Gy RT) in PC-3 and LNCaP cells. <i>p</i><0.05(†) indicates significant difference between RT-treated cells and combination-treated cells at the same time point. <i>p</i><0.05(*) indicates significant difference between cells at specific time points and cells receiving the same treatment at the “Pre-RT” time point. Points, mean; bars, SD. N=50. Typical images are shown from three independent experiments (N=3). Magnification × 60 in all images.</p