Kelly S. Persons

An Evaluation of the Vitamin D3 Content in Fish: Is the Vitamin D Content Adequate to Satisfy the Dietary Requirement for Vitamin D?

It has been suggested that the major source of vitamin D should come from dietary sources and not sun exposure. However, the major fortified dietary source of vitamin D is milk which often does not contain at least 80% of what is stated on the label. Fish has been touted as an excellent source of vitamin D especially oily fish including salmon and mackerel. Little is known about the effect of various cooking conditions on the vitamin D content in fish. We initiated a study and evaluated the vitamin D content in several species of fish and also evaluated the effect of baking and frying on the vitamin D content. Surprisingly, farmed salmon had approximately 25% of the vitamin D content as wild salmon had. The vitamin D content in fish varied widely even within species. These data suggest that the tables that list the vitamin D content are out-of-date and need to be re-evaluated.

Thyroid Hormone Action on Skin: Diverging Effects of Topical versus Intraperitoneal Administration

Previously, we demonstrated stimulation of epidermal proliferation and hair growth in triiodothyronine (T(3)) treated mice. To distinguish skin effects of directly applied T(3) from those of systemic hyperthyroidism, we treated CD-1 mice with either intraperitoneally (IP) or topically administered T(3). Relative to controls, mice receiving T(3) IP had 10% thinner epidermis (p < 0.01) and 48% fewer hairs (p < 0.001). By contrast, mice receiving T(3) topically had 78% thicker epidermis (p < 0.01) and 160% more hairs (p < 0.01). To gain insight into factors responsible for the diverging effects, we contrasted T(3) effect on proliferation of isolated keratinocyte cultures versus keratinocytes cocultured with dermal fibroblasts. For keratinocytes grown in the absence of fibroblasts, T(3) stimulated proliferation in a dose-dependent, biphasic pattern with the peak at 0.5 nM T(3) (84 +/- 30%, p < 0.05). Paradoxically, T(3) inhibited proliferation of keratinocytes cocultured with fibroblasts, the nadir at 0.1 nM T(3) (34% +/- 4%, p < 0.001). These studies are the first describing divergent effects of IP and topically administered thyroid hormone. The data suggest that while T(3) stimulated keratinocyte proliferation, T(3) also stimulated proliferation inhibitory factor(s) from skin fibroblasts. Insight into the interplay among the competing factors will be important in understanding thyroid hormone regulation of skin physiology.

An evaluation of 1,25-dihydroxyvitamin D3 analogues on the proliferation and differentiation of cultured human keratinocytes, calcium metabolism and the differentiation of human HL-60 cells

Abstract Synthetic analogues of 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ) were examined for their biological activities in four assay systems: (a) inhibition of proliferation and stimulation of terminal differentiation in cultured normal human keratinocytes, (b) intestinal calcium absorption and bone calcium mobilization in vitamin D-deficient rats, (c) competitive binding to the rat intestinal 1,25(OH) 2 D 3 receptor, and (d) induction of differentiation of human HL-60 leukemia cells. Six analogues were found to have minimal activity in enhancing intestinal calcium absorption and bone calcium mobilization while retaining at least the same activity as 1,25(OH) 2 D 3 in inhibiting proliferation and inducing terminal differentiation of cultured keratinocytes. Evidence suggests that it may be possible to dissociate antiproliferative activity from differentiation-inducing activity and calcium metabolism by specific modifications of the 1,25(OH) 2 D 3 molecule. The uncoupling of these activities could potentially create an ideal analogue to treat psoriasis that should have potent antiproliferative activity with minimum effects on differentiation and on calcium metabolism.

Evaluation of C-2-substituted 19-nor-1α,25-dihydroxyvitamin D3 analogs as therapeutic agents for prostate cancer

1alpha,25-Dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) is known to inhibit the proliferation and invasiveness of prostate cancer cells. However, 1alpha,25(OH)(2)D(3) can cause hypercalcemia and is not suitable as a therapeutic agent. 19-Nor-vitamin D derivatives are known to be less calcemic when administered systemically. In order to develop more potent anti-cancer agents with less calcemic side effect, we therefore utilized (3)H-thymidine incorporation as an index for cell proliferation and examined the antiproliferative activities of nine C-2-substituted 19-nor-1alpha,25(OH)(2)D(3) analogs in the immortalized PZ-HPV-7 normal prostate cell line. Among the nine analogs we observed that the substitution with 2alpha- or 2beta-hydroxypropyl group produced two analogs having antiproliferative potency that is approximately 500- to 1000-fold higher than 1alpha,25(OH)(2)D(3). The (3)H-thymidine incorporation data were supported by the cell counting data after cells were treated with 1alpha,25(OH)(2)D(3), 19-nor-2alpha-(3-hydroxypropyl)-1alpha,25(OH)(2)D(3) or 19-nor-2beta-(3-hydroxypropyl)-1alpha,25(OH)(2)D(3) for 7 days. 19-Nor-2alpha-(3-hydroxypropyl)-1alpha,25(OH)(2)D(3) and 19-nor-2beta-(3-hydroxypropyl)-1alpha,25(OH)(2)D(3) were also shown to be about 10-fold more active than 1alpha,25(OH)(2)D(3) in cell invasion studies using prostate cancer cells. In conclusion, a substitution at the C-2 position of 19-nor-1alpha,25(OH)(2)D(3) molecule with a hydroxypropyl group greatly increased the antiproliferative and anti-invasion potencies. Thus, these two analogs could be developed to be effective therapeutic agents for treating early and late stages of prostate cancer.

An evaluation of the biologic activity and vitamin D receptor binding affinity of the photoisomers of vitamin D3 and previtamin D3.

Skin is in the site of previtamin D3 and vitamin D3 synthesis and their isomerization in response to ultraviolet irradiation. At present, little is known about the function of the photoisomers of previtamin D3 and the vitamin D3 in skin cells. In this study we investigated the antiproliferative activity of the major photoisomers and their metabolites in the cultured human keratinocytes by determining their influence on 3H-thymidine incorporation into DNA. Our results demonstrated at both 10(-8) and 10(-6) M in a dose-dependent manner. Lumisterol, tachysterol3, 5,6-trans-vitamin D3, and 25-hydroxy-5,6-trans-vitamin D3 only induced significant inhibition at 10(-6) M. 25-Hydroxytachysterol3 was approximately 10- to 100-fold more active than tachysterol3. 7-Dehydrocholesterol was not active even at 10(-6) M. The dissociation constants of vitamin D receptor (VDR) for 25-hydroxytachysterol3, 25-hydroxy-5,6-trans-vitamin D3, and 5,6-trans-vitamin D3 were 22, 58, and 560 nM, respectively. The dissociation constants for 7-dehydrocholesterol, tachysterol, and lumisterol were greater than 20 microM. In conclusion, vitamin D3, its photoisomers and the photoisomers of previtamin D3 have antiproliferative activity in cultured human keratinocytes. However, the antiproliferative activity did not correlate with their binding affinity for VDR. The results suggest that some of the photoproducts may be metabolized to their 25-hydroxylated and 1 alpha,25-dihydroxylated counterparts before acting on VDR. Alternatively, a different receptor may recognize these photoproducts or another mechanism may be involved in modulating the antiproliferative activity of the photoisomers examined.

1α,25-Dihydroxyvitamin D3-3β-(2)-bromoacetate, an affinity labeling derivative of 1α,25-dihydroxyvitamin D3 displays strong antiproliferative and cytotoxic behavior in prostate cancer cells

In this report we describe that 1,25(OH)2D3‐3‐BE, a VDR‐affinity labeling analog of 1,25(OH)2D3, showed strong and dose‐dependent growth‐inhibitory effect in several epithelial cells, i.e., keratinocytes (primary cells), MCF‐7 breast cancer, PC‐3, and LNCaP prostate cancer and PZ‐HPV‐7 immortalized normal prostate cell‐lines. Furthermore, 10−6 M of 1,25(OH)2D3‐3‐BE induced apoptosis specifically in LNCaP and PC‐3 cells; and the effect was much less pronounced at lower doses. We also showed that the effect (of 1,25(OH)2D3‐3‐BE) was not due to probable degradation (hydrolysis) of 1,25(OH)2D3‐3‐BE or random interaction of this molecule with cellular proteins. Tissue‐ or cell‐specific action of 1,25(OH)2D3 and its mimics is not common due to the ubiquitous nature of VDR. Furthermore, variable effects of 1,25(OH)2D3 and its analogs in various cell‐lines potentially limits their application as anticancer agents. We showed that 1,25(OH)2D3‐3‐BE displayed similar growth‐inhibitory and cytotoxic activities towards androgen sensitive LNCaP and androgen‐independent PC‐3 cell‐lines. Therefore, these results raise the possibility that 1,25(OH)2D3‐3‐BE or similar VDR‐cross linking analogs of 1,25(OH)2D3 might be considered for further development as potential candidates for prostate cancer. J. Cell. Biochem. 89: 909–916, 2003.

A VITAMIN D RECEPTOR-ALKYLATING DERIVATIVE OF 1α, 25-DIHYDROXYVITAMIN D3 INHIBITS GROWTH OF HUMAN KIDNEY CANCER CELLS AND SUPPRESSES TUMOR-GROWTH

1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] has shown strong promise as an antiproliferative agent in several malignancies, yet its therapeutic use has been limited by its toxicity leading to search for analogues with antitumor property and low toxicity. In this study, we evaluated the in vitro and in vivo properties of 1,25-dihydroxyvitamin D3-3-bromoacetate [1,25(OH)2D3-3-BE], an alkylating derivative of 1,25(OH)2D3, as a potential therapeutic agent for renal cancer. Dose response of 1,25(OH)2D3-3-BE in 2 kidney cancer cell lines was evaluated for its antiproliferative and apoptotic properties, and mechanisms were evaluated by Western blot and FACS analyses. Therapeutic potential of 1,25(OH)2D3-3-BE was assessed both by determining its stability in human serum and by evaluating its efficacy in a mouse xenograft model of human renal tumor. We observed that 1,25(OH)2D3-3-BE is significantly more potent than an equivalent concentration of 1,25(OH)2D3 in inhibiting growth of A498 and Caki 1 human kidney cancer cells. 1,25(OH)2D3-3-BE–mediated growth inhibition was promoted through inhibition of cell-cycle progression by downregulating cyclin A and induction of apoptosis by stimulating caspase activity. Moreover, 1,25(OH)2D3-3-BE strongly inhibited Akt phosphorylation and phosphorylation of its downstream target, caspase-9. 1,25(OH)2D3-3-BE seemed to be stable in human serum. In xenograft mouse model of human renal tumor, 1,25(OH)2D3-3-BE was more potent at reducing tumor size than 1,25(OH)2D3, which was accompanied by an increase in apopotosis and reduction of cyclin A staining in the tumors. These results suggest a translational potential of this compound as a therapeutic agent in renal cell carcinoma. Data from this study and extensive studies of vitamin D for the prevention of many malignancies support the potential of 1,25(OH)2D3-3-BE for preventing renal cancer and the development of relevant in vivo prevention models for assessing this potential, which do not exist at present. Cancer Prev Res; 3(12); 1596–607. ©2010 AACR.

The Thyroid Hormone Degrading Type 3 Deiodinase Is the Primary Deiodinase Active in Murine Epidermis

INTRODUCTION Many tissues express thyroid hormone metabolizing deiodinases that both activate and inactivate thyroid hormones through conversion processes. Many believe that the primary role of thyroid hormone deiodinases is the activation of the prohormone thyroxine (T(4)) to the active hormone triiodothyronine because athyreotic humans can be treated with T(4) alone. In our hands a nonspecific deiodinase inhibitor (iopanoic acid [IOP]) decreased cutaneous cell proliferation in vitro, so we hypothesized that topical IOP would inhibit epidermal proliferation in vivo. METHODS IOP was applied topically to mice. Treatments were applied daily for 1 week. Skin biopsies were either stained for 5-bromo-2-deoxyuridine or flash-frozen to assay for deiodinase activity. RESULTS Topical IOP resulted in a dose-dependent increase in epidermal proliferation. Assay revealed significant inactivating type 3 deiodinase (Dio3) activity in the epidermis but little or no activating (Dio1 or Dio2) activity. Dio3 activity was decreased 44%±21% in epidermis from mice treated with low-dose IOP and 80%±4% in epidermis from mice treated with high-dose IOP (p<0.001). CONCLUSION We hypothesize that keratinocytes express Dio3 in vivo to maintain cutaneous health and prevent the skin from becoming hyperproliferative. Our data support the developing recognition that the primary role of thyroid hormone deiodinases in some tissues may be the degradation of thyroid hormone to protect the tissue against thyrotoxicosis.

A Thyroid Hormone Deiodinase Inhibitor Can Decrease Cutaneous Cell Proliferation In Vitro

BACKGROUND Although cutaneous manifestations associated with thyroid dysfunction are classic, the potential for thyroid hormone or its antagonists to treat dermatological disease has not been explored with rigor. The predominant circulating thyroid hormone is the pro-hormone, thyroxine (T(4)). Skin, like many tissues, expresses thyroid hormone deiodinases to convert T(4) to the active thyroid hormone, triiodothyronine (T(3)). Previously, we determined that T(3) is necessary for optimal growth of keratinocytes and fibroblasts. The first hypothesis of this experiment was that the deiodinase inhibitor iopanoic acid (IOP) could inhibit cutaneous cell proliferation. The second hypothesis of this experiment was that the action of IOP could be attributed to its inhibition of conversion of T(4) to T(3). Although IOP is known to inhibit T(4) to T(3) conversion, the inhibition of cutaneous cell proliferation by IOP might conceivably result from other properties of IOP. METHODS In separate experiments, primary culture human keratinocytes and dermal fibroblasts were incubated overnight with IOP. RESULTS Proliferation was inhibited in a dose-dependent manner in both cell lines. Overnight incubation with T(3) restored the proliferation but overnight incubation with T(4) did not. CONCLUSION The study is the first to demonstrate that IOP inhibits cutaneous cell proliferation and that supplemental T(3) is sufficient to correct at least part of the inhibition caused by IOP. The data suggest that IOP decreases cutaneous cell proliferation by inhibition of intracellular T(4) to T(3) conversion. The data provide indirect evidence of the presence of type 1 or type 2 iodothyronine deiodinase activity in skin cells. The data support our previous hypothesis that T(3) is necessary for normal cutaneous proliferation.

Internalization of a C17α-alkynylestradiol-porphyrin conjugate into estrogen receptor positive MCF-7 breast cancer cells

We hypothesized that expression of nuclear estrogen receptor (ER) in hormone-sensitive breast cancer cells could be harnessed synergistically with the tumor-accumulating effect of porphyrins to selectively deliver estrogen-porphyrin conjugates into breast tumor cells, and preferentially kill tumor cells upon exposure to visible light. In this study we synthesized a conjugate of C(17α)-alkynylestradiol and pyropheophorbide and demonstrated that this conjugate is internalized by ER-positive MCF-7 cells while pyropheophorbide did not, suggesting an ER-mediated uptake and internalization of the conjugate by incipient nuclear ER in MCF-7 cells. This study is a direct demonstration of our hypothesis about ER-mediated internalization of estrogen-porphyrin conjugates.