In vivo knockdown of the androgen receptor results in growth inhibition and regression of well-established, castration-resistant prostate tumours

Purpose: Progression to the castration-resistant state is the incurable and lethal end stage of prostate cancer, and there is strong evidence that androgen receptor (AR) still plays a central role in this process. We hypothesize that knocking down AR will have a major effect on inhibiting growth of castration-resistant tumors. Experimental Design: Castration-resistant C4-2 human prostate cancer cells stably expressing a tetracycline-inducible AR-targeted short hairpin RNA (shRNA) were generated to directly test the effects of AR knockdown in C4-2 human prostate cancer cells and tumors. Results:In vitro expression of AR shRNA resulted in decreased levels of AR mRNA and protein, decreased expression of prostate-specific antigen (PSA), reduced activation of the PSA-luciferase reporter, and growth inhibition of C4-2 cells. Gene microarray analyses revealed that AR knockdown under hormone-deprived conditions resulted in activation of genes involved in apoptosis, cell cycle regulation, protein synthesis, and tumorigenesis. To ensure that tumors were truly castration-resistant in vivo, inducible AR shRNA expressing C4-2 tumors were grown in castrated mice to an average volume of 450 mm3. In all of the animals, serum PSA decreased, and in 50% of them, there was complete tumor regression and disappearance of serum PSA. Conclusions: Whereas castration is ineffective in castration-resistant prostate tumors, knockdown of AR can decrease serum PSA, inhibit tumor growth, and frequently cause tumor regression. This study is the first direct evidence that knockdown of AR is a viable therapeutic strategy for treatment of prostate tumors that have already progressed to the castration-resistant state.

Interactions between human osteoblasts and prostate cancer cells in a novel 3D in vitro model

Cell-cell and cell-matrix interactions play a major role in tumor morphogenesis and cancer metastasis. Therefore, it is crucial to create a model with a biomimetic microenvironment that allows such interactions to fully represent the pathophysiology of a disease for an in vitro study. This is achievable by using three-dimensional (3D) models instead of conventional two-dimensional (2D) cultures with the aid of tissue engineering technology. We are now able to better address the complex intercellular interactions underlying prostate cancer (CaP) bone metastasis through such models. In this study, we assessed the interaction of CaP cells and human osteoblasts (hOBs) within a tissue engineered bone (TEB) construct. Consistent with other in vivo studies, our findings show that intercellular and CaP cell-bone matrix interactions lead to elevated levels of matrix metalloproteinases, steroidogenic enzymes and the CaP biomarker, prostate specific antigen (PSA); all associated with CaP metastasis. Hence, it highlights the physiological relevance of this model. We believe that this model will provide new insights for understanding of the previously poorly understood molecular mechanisms of bone metastasis, which will foster further translational studies, and ultimately offer a potential tool for drug screening. © 2010 Landes Bioscience.

Engineered silk fibroin protein 3D matrices for in vitro tumor model

3D in vitro model systems that are able to mimic the in vivo microenvironment are now highly sought after in cancer research. Antheraea mylitta silk fibroin protein matrices were investigated as potential biomaterial for in vitro tumor modeling. We compared the characteristics of MDA-MB-231 cells on A. mylitta, Bombyx mori silk matrices, Matrigel, and tissue culture plates. The attachment and morphology of the MDA-MB-231 cell line on A. mylitta silk matrices was found to be better than on B. mori matrices and comparable to Matrigel and tissue culture plates. The cells grown in all 3D cultures showed more MMP-9 activity, indicating a more invasive potential. In comparison to B. mori fibroin, A. mylitta fibroin not only provided better cell adhesion, but also improved cell viability and proliferation. Yield coefficient of glucose consumed to lactate produced by cells on 3D A. mylitta fibroin was found to be similar to that of cancer cells in vivo. LNCaP prostate cancer cells were also cultured on 3D A. mylitta fibroin and they grew as clumps in long term culture. The results indicate that A. mylitta fibroin scaffold can provide an easily manipulated microenvironment system to investigate individual factors such as growth factors and signaling peptides, as well as evaluation of anticancer drugs.

The severity of chorioamnionitis in pregnant sheep is associated with in vivo variation of the surface-exposed multiple-banded antigen/gene of ureaplasma parvum

Ureaplasma species are the bacteria most frequently isolated from human amniotic fluid in asymptomatic pregnancies and placental infections. Ureaplasma parvum serovars 3 and 6 are the most prevalent serovars isolated from men and women. We hypothesized that the effects on the fetus and chorioamnion of chronic ureaplasma infection in amniotic fluid are dependent on the serovar, dose, and variation of the ureaplasma multiple banded antigen (MBA) and mba gene. We injected high- or low dose U. parvum serovar 3, serovar 6, or vehicle intra-amniotically into pregnant ewes at 55 days of gestation (term = 150 days) and examined the chorioamnion, amniotic fluid, and fetal lung tissue of animals delivered by cesarean section at 125 days of gestation. Variation of the multiple banded antigen/mba generated by serovar 3 and serovar 6 ureaplasmas in vivo were compared by PCR assay and Western blot. Ureaplasma inoculums demonstrated only one (serovar 3) or two (serovar 6) MBA variants in vitro, but numerous antigenic variants were generated in vivo: serovar 6 passage 1 amniotic fluid cultures contained more MBA size variants than serovar 3 (P = 0.005),and ureaplasma titers were inversely related to the number of variants (P = 0.025). The severity of chorioamnionitis varied between animals. Low numbers of mba size variants (five or fewer) within amniotic fluid were associated with severe inflammation, whereas the chorioamnion from animals with nine or more mba variants showed little or no inflammation. These differences in chorioamnion inflammation may explain why not all women with in utero Ureaplasma spp. experience adverse pregnancy outcomes.