Nm23-H1 homologs suppress tumor cell motility and anchorage independent growth.

Nm23-H1 suppresses metastasis, as well as in vitro cell motility, invasion and anchorage independent growth, in a variety of cancer models. Eight human homologs of Nm23 have been identified that share 26-88% identity with the prototype Nm23-H1. Here, we examine the potential of its homologs, -H2, DR-, -H4 and -H5, to inhibit in vitro correlates of metastasis in two highly metastatic human cell lines, MDA-MB-435 and MDA-MB-231. The metastatic cells were transfected with mammalian expression constructs containing the genes encoding for Nm23-H1, -H2, DR-, -H4 and -H5 and the resultant transfectants were analyzed by Boyden chamber motility and soft agar colonization assays. Nm23-H1 suppressed motility by 3.3- and 1.5-fold in MDA-MB-435 and MDA-MB-231 cells, respectively and inhibited anchorage independent growth in soft agar by 2.9- and 1.9-fold, respectively. None of the -H1 homologs were capable of suppressing motility in MDA-MB-435 cells, but in MDA-MB-231 cells, -H2 inhibited motility by 3-fold upon overexpression. When anchorage independent growth was assessed, -H2, -H4 and -H5 suppressed growth from 1.2- to 2.0-fold in both cell lines. Given their ability to suppress anchorage independent growth, Nm23-H1 homologs -H2, -H4 and -H5 may have some capacity to suppress metastasis. Motility suppression appears to be cell context dependent, but sequence disparities between -H1/H2 and the other family members may reveal regions critical for this inhibitory phenotype. Similarly, sequence differences between DR-Nm23 and its homologs may be important for anchorage independent growth suppression.

Nm23-H1 suppresses metastasis by inhibiting expression of the lysophosphatidic acid receptor EDG2.

Nm23-H1 transcriptionally down-regulates expression of the lysophosphatidic acid receptor EDG2 and this down-regulation is critical for Nm23-H1-mediated motility suppression in vitro. We investigated the effect of altered EDG2 expression on Nm23-H1-mediated metastasis suppression in vivo. Clonal MDA-MB-435-derived tumor cell lines transfected with Nm23-H1 together with either a vector control or EDG2 had similar anchorage-dependent and anchorage-independent growth rates in vitro. However, a 45- and 300-fold inhibition of motility and invasion (P < 0.0001), respectively, was observed in Nm23-H1/vector lines, whereas coexpression of EDG2 restored activity to levels observed in the parental line. Using fluorescently labeled cells and ex vivo microscopy, the capacity of these cells to adhere, arrest, extravasate, and survive in the murine lung over a 24-h time course was measured. Only 5% of Nm23-H1/vector-transfected cells were retained in the murine lung 6 h following tail vein injection; coexpression of EDG2 enhanced retention 8- to 13-fold (P < 0.01). In a spontaneous metastasis assay, the primary tumor size of Nm23-H1/vector and Nm23-H1/EDG2 clones was not significantly different. However, restoration of EDG2 expression augmented the incidence of pulmonary metastasis from 51.9% to 90.4% (P = 2.4 x 10(-5)), comparable with parental MDA-MB-435 cells. To determine the relevance of this model system to human breast cancer, a cohort of breast carcinomas was stained for Nm23-H1 and EDG2 and a statistically significant inverse correlation between these two proteins was revealed (r = -0.73; P = 0.004). The data indicate that Nm23-H1 down-regulation of EDG2 is functionally important to suppression of tumor metastasis.

Platelet rich plasma (PRP) enhances anabolic gene expression patterns in flexor digitorum superficialis tendons.

Platelet rich plasma (PRP) has recently been investigated for use in tissue regeneration studies that seek to utilize the numerous growth factors released from platelet alpha-granules. This study examined gene expression patterns, DNA, and collagen content of equine flexor digitorum superficialis tendon (SDFT) explants cultured in media consisting of PRP and other blood products. Blood and bone marrow aspirate (BMA) were collected from horses and processed to obtain plasma, PRP, and platelet poor plasma (PPP). IGF-I, TGF-beta1, and PDGF-BB were quantified in all blood products using ELISA. Tendons were cultured in explant fashion with blood, plasma, PRP, PPP, or BMA at concentrations of 100%, 50%, or 10% in serum-free DMEM with amino acids. Quantitative RT-PCR for expression of collagen type I (COL1A1), collagen type III (COL3A1), cartilage oligomeric matrix protein (COMP), decorin, matrix metalloproteinase-3 (MMP-3), and matrix metalloproteinase-13 (MMP-13) was performed as were DNA and total soluble collagen assays. TGF-beta1 and PDGF-BB concentrations were higher in PRP compared to all other blood products tested. Tendons cultured in 100% PRP showed enhanced gene expression of the matrix molecules COL1A1, COL3A1, and COMP with no concomitant increase in the catabolic molecules MMP-3 and MMP-13. These findings support in vivo investigation of PRP as an autogenous, patient-side treatment for tendonitis.