John A. Yee

Dietary supplementation with secoisolariciresinol diglycoside (SDG) reduces experimental metastasis of melanoma cells in mice.

We investigated the effect of dietary supplementation with secoisolariciresinol diglycoside (SDG), a lignan precursor isolated from flaxseed, on experimental metastasis of B16BL6 murine melanoma cells in C57BL/6 mice. Four diets were compared: a basal diet (control group) and the basal diet supplemented with SDG at 73, 147 or 293 micromol/kg (equivalent to SDG provided in the 2.5, 5 or 10% flaxseed diet). Mice were fed the diet for 2 weeks before and after an intravenous injection of 0.6 x 10(5) tumor cells. At necropsy, the number and size of tumors that formed in the lungs were determined. The median number of tumors in the control group was 62, and those in the SDG-supplemented groups were 38, 36 and 29, respectively. The last was significantly different from the control (P < 0.01). Dietary supplementation with SDG at 73, 147 and 293 micromol/kg also decreased tumor size (tumor cross-sectional area and volume) in a dose-dependent manner compared with the control values. These results show that SDG reduced pulmonary metastasis of melanoma cells and inhibited the growth of metastatic tumors that formed in the lungs. It is concluded that dietary supplementation with SDG reduces experimental metastasis of melanoma cells in mice.

Dietary flaxseed supplementation and experimental metastasis of melanoma cells in mice

The present study investigated the effect of dietary supplementation of flaxseed, the richest source of lignans, on experimental metastasis of B16BL6 murine melanoma cells in C57BL/6 mice. Mice were fed a basal diet or the basal diet supplemented with 2.5, 5 or 10% flaxseed for 2 weeks before and after the intravenous injection of 0.75 x 10(5) melanoma cells. At necropsy, the number of tumors that developed in the lungs was counted, the cross-sectional area of tumors was measured and the volumes of tumors were calculated. The median number of tumors in mice fed the 2.5, 5 and 10% flaxseed-supplemented diets was 32, 54 and 63% lower than that of the controls, respectively. The addition of flaxseed to the diet also caused a dose-dependent decrease in the tumor cross-sectional area and the tumor volume. These results provide the first experimental evidence that flaxseed reduces metastasis and inhibits the growth of the metastatic secondary tumors in animals. It is concluded that flaxseed may be a useful nutritional adjuvant to prevent metastasis in cancer patients.

NADPH-diaphorase-positive nerves and the role of nitric oxide in CGRP relaxation of uterine contraction

We previously demonstrated calcitonin gene-related peptide (CGRP) immunoreactivity in sensory nerves in the rat uterus and that CGRP inhibits stimulated uterine contraction in vitro. The present study was undertaken to: 1) examine possible roles nitric oxide (NO) may have in the inhibitory action of CGRP on uterine contraction and 2) identify sites where NO may be synthesized. The relaxing effect of CGRP on SP-stimulated uterine contraction was established in vitro on uterine horns from diethylstilbestrol-treated rats. These experiments were repeated with or without an arginine analog [NG-monomethyl-L-arginine (L-NMMA)] that inhibits NO formation. The localization of the synthetic enzyme for NO production, NO synthase, was accomplished by histochemically staining for NADPH-diaphorase. Calcitonin gene-related peptide (10(-7) M) significantly reduced SP (10(-5) or 10(-6) M)-stimulated uterine contraction. The L-NMMA (10(-3) M) blocked the relaxing action of CGRP on SP-stimulated uterine contraction. The L-NMMA alone had no effect on SP-stimulated uterine contraction. NADPH-diaphorase-positive nerve fibers were located in the myometrium, endometrium, and adjacent to the vasculature. These data demonstrate that: 1) L-NMMA suppresses the relaxant effect of CGRP on myometrial activity and 2) NADPH-diaphorase (indicative of NO synthase) is localized in uterine nerve fibers. These data suggest that the inhibitory action of CGRP may be dependent on NO formation and that the enzyme necessary for NO production is present in nerves in areas optimal to affect myometrial activity.

Effect of dietary supplementation of selenite on pulmonary metastasis of melanoma cells in mice

The purpose of the present study was to determine the effect of dietary supplementation of selenite on experimental pulmonary metastasis of B16BL6 murine melanoma cells in C57BL/6 mice by means of an intravenous injection model. Three groups of mice were fed a basal AIN-93G diet containing 0.1 ppm selenium (control group) or the basal diet supplemented with 2 or 4 ppm selenium as selenite (experimental groups). Mice were fed the diet for two weeks before and after the intravenous injection of 0.75 x 10(5) viable tumor cells. At necropsy the number of tumors that developed in the lungs and their cross-sectional area were determined, and tumor volume was calculated. In the control group, 12 of the 15 mice had > or = 1 lung tumors. In contrast, only 4 of the 15 mice in each of the selenite-supplemented groups had > or = 11 tumors. The incidence of metastasis in mice fed the control and the 2- and 4-ppm selenium diets was 93%, 73%, and 53%, respectively. The median number of lung tumors was 53, 1, and 1 in mice fed the basal and the 2- and 4-ppm selenium diets, respectively. Tumor cross-sectional area and tumor volume were significantly decreased in selenite-supplemented groups. These results demonstrate that dietary supplementation of selenite reduced pulmonary metastasis of B16BL6 melanoma cells in C57BL/6 mice and also inhibited the growth of the metastatic tumors that developed in the lungs. It is concluded that selenite may be a useful adjuvant to prevent metastatic diseases in cancer patients.

Functional characterization of prostaglandin E2 inducible osteogenic colony forming units in cultures of cells isolated from the neonatal rat calvarium.

Prostaglandin E2 (PGE2) increases the number of mineralized nodules that form in cultures of rat calvarial (RC) cells. The purpose of our study was to characterize PGE2‐inducible osteogenic colony forming units (CFU‐Os) by determining their number, the cell populations from which they were released, their specific responsive period to PGE2, and their proliferating and differentiating characteristics under the stimulation of PGE2. Limiting dilution analysis was used to determine the number of PGE2‐inducible CFU‐Os. Sequential digestion of intact rat parietal bones with collagenase isolated 5 subpopulations of RC cells that were used to estimate the cell populations where PGE2‐inducible CFU‐Os resided. The responsive period of PGE2‐inducible CFU‐Os to PGE2 was evaluated by treating cultures of mixed RC cells for all possible combinations of days 1–10, 11–20, and 21–30. PGE2 effects on proliferation and differentiation of CFU‐Os were evaluated by comparing the DNA synthesis and AP activity in subpopulations I and IV on days 3, 6, and 9. Results showed: (1) PGE2‐inducible CFU‐Os represent 0.27% of cells in the mixed RC population, (2) the majority of determined and PGE2‐inducible CFU‐Os were found in the subpopulations released during the 60–100 min digestion periods, (3) the response of PGE2‐inducible CFU‐Os is limited to the first 10 days of culture, and (4) PGE2‐stimulated nodule formation is associated with an early increase in DNA synthesis and a sustained increase in alkaline phosphatase activity. We conclude that, functionally, PGE2‐inducible CFU‐Os are slowly proliferating AP negative cells primarily found in the subpopulations III‐V. PGE2 stimulates them to proliferate and become AP+, and function as determined CFU‐Os to form mineralized nodules in vitro.

Expression of parathyroid hormone-related peptide (PthrP) and its receptor (PTH1R) during the histogenesis of cartilage and bone in the chicken mandibular process.

The purpose of this study was to examine the expression and actions of parathyroid hormone‐related protein (PTHrP) when skeletal histogenesis occurs in the chicken mandible. Prior to the appearance of skeletal tissues, PTHrP and PTH1R were co‐expressed by cells in the ectoderm, skeletal muscle, peripheral nerve and mesenchyme. Hyaline cartilage was first observed at HH stage 27 when many but not all chondroblasts expressed PTHrP and PTH1R. By stage 34, PTHrP and PTH1R were not detected in chondrocytes but were expressed in the perichondrium. Alkaline phosphatase (AP)‐positive preosteoblasts and woven bone appeared at stages 31 and 34, respectively. Preosteoblasts, osteoblasts and osteocytes co‐expressed PTHrP and PTH1R. Treatment with chicken PTHrP (1–36) increased cAMP in mesenchyme from stage 26 embryos. Continuous exposure to chicken PTHrP (1–36) for 14 days increased cartilage nodule number and decreased AP while intermittent exposure did not affect cartilage nodule number and increased AP in cultures of stage 26 mesenchymal cells. Adding a neutralizing anti‐PTHrP antibody to the cultures reduced cartilage nodule number and did not affect AP. These findings show that PTHrP and PTH1R are co‐expressed by extraskeletal and skeletal cells before and during skeletal tissue histogenesis, and that PTHrP may influence skeletal tissue histogenesis by affecting the differentiation of mandibular mesenchymal cells into chondroblasts and osteoblasts.

Effect of dietary supplementation of soybeans on experimental metastasis of melanoma cells in mice.

The purpose of the present study was to determine the effect of dietary supplementation of soybean protein isolate (SPI) on experimental metastasis of B16BL6 murine melanoma cells in C57BL/6 mice. Four groups of mice were fed a basal AIN-93G diet or the basal diet supplemented with 10%, 15%, or 20% SPI for two weeks before and after the intravenous injection of 0.75 x 10(5) cells. At necropsy the number of tumors that developed in the lungs and their cross-sectional area were determined, and tumor volume was calculated. In the control group, 12 of the 15 mice had > or = 11 lung tumors. In contrast, only 3 or 4 of the 15 mice fed the SPI diets had > or = 11 tumors. The incidence of metastasis was 93%, 60%, 53%, and 53%, and the median number of lung tumors was 53, 2, 2, and 1 in mice fed the basal, 10%, 15%, and 20% SPI diets, respectively. Tumor cross-sectional area and tumor volume of SPI groups were significantly decreased compared with the controls. These results demonstrate that dietary supplementation of SPI reduced pulmonary metastasis of B16BL6 cells in mice and inhibited the growth of tumors that developed in the lungs. It is concluded that soybeans may be a useful adjuvant for preventing metastatic diseases in cancer patients.

Biphasic effects of interleukin‐1β on osteoblast differentiation in vitro

A rat calvarial cell model of osteoblast differentiation using the formation of bone nodules in vitro as an endpoint was used to assess the effects of IL‐1β on osteoblast differentiation. Short‐term treatment (2 days) with IL‐1β early in culture resulted in increased nodule number and size as well as calcium content in contrast to long‐term treatment (6 days) in cultures assessed at 10–12 days. This increase in bone formation was blocked by IL‐1 receptor antagonists. Short‐term treatment increased COX‐2, prostaglandin (PGE2), and iNOS production. Exogenous PGE2 with IL‐1β enhanced this effect. COX‐2 inhibitors, indomethacin and N‐39, blocked 50% of nodule formation. NO donor did not modify effects of IL‐1β, but iNOS inhibitor (1400W) partially blocked the effects. However, PGE2 and NO donors could not rescue the decreased nodule number resulting from long‐term IL‐1β treatment. The results of this study suggest a biphasic effect of IL‐1β on bone nodule formation activated by IL‐1β binding with IL‐1 receptors, and the anabolic effect of early short‐term treatment with IL‐1β is likely mediated by PGE without ruling out nitric oxide.

Continuous and intermittent exposure of neonatal rat calvarial cells to PTHrP (1-36) inhibits bone nodule mineralization in vitro by downregulating bone sialoprotein expression via the cAMP signaling pathway

The development and growth of the skeleton in the absence of parathyroid-hormone-related protein (PTHrP) is abnormal. The shortening of appendicular bones in PTHrP gene null mice is explained by an effect of PTHrP on endochondral bone growth. Whether or not PTHrP influences intramembranous ossification is less clear. The purpose of this study was to determine the effect of exogenous PTHrP on intramembranous ossification in vitro. Neonatal rat calvarial cells maintained in primary cell culture conditions that permit spontaneous formation of woven bone nodules by intramembranous ossification were studied. The expression of PTHrP, parathyroid hormone 1 receptor (PTH1R), and alkaline phosphatase (AP) by osteogenic cells in developing nodules and the effects of PTHrP (1-36) on nodule development was determined over 3-18 days. PTHrP and PTH1R were detected colonies of osteogenic cells on culture day three, and AP was detected on day six. PTHrP and its receptor were localized in pre-osteoblasts, osteoblasts, and osteocytes, and AP activity was detected in pre-osteoblasts and osteoblasts but not osteocytes. Continuous and intermittent exposure to PTHrP (1-36) decreased the number of mineralized bone nodules and bone sialoprotein (BSP) mRNA and protein, but had no effect on the number of AP-positive osteogenic cell colonies, cell proliferation, apoptosis, or osteopontin (OPN) mRNA. These results demonstrate that osteogenic cells that participate in the formation of woven bone nodules in vitro exhibit PTHrP and PTH1R before they demonstrate AP activity. Exogenous PTHrP (1-36) inhibits the mineralization of woven bone deposited during bone nodule formation in vitro, possibly by reducing the expression of BSP.