Evaluation of a Ultraviolet B Light Emitting Diode (LED) for Producing Vitamin D3 in Human Skin.

AIM: A commercially available light emitting diode (LED) that transmitted narrow band ultraviolet B (UVB) radiation was evaluated for its efficacy and efficiency to produce vitamin D3 in human skin. MATERIALS AND METHODS: Human skin samples were obtained from surgical procedures. The LED had peak emission wavelength of 295 nm. Skin samples were exposed to the UVB-LED for varying times and then were analyzed by high-pressure liquid chromatography (HPLC) to determine the vitamin D3 content. RESULTS: There was a statistically significant time- and dose-dependent increase in the percent of 7-dehydrocholesterol that was converted to vitamin D3 in the skin type II samples; 1.3%±0.5, 2.3%±0.6 and 4.5%±1.67 after exposure to 0.75 (11.7 mJ/cm2), 1.5 (23.4 mJ/cm2) and 3 (46.8 mJ/cm2) minimal erythemal doses (MEDs), respectively. CONCLUSION: The UVB-LED was effective and efficient in generating vitamin D3 in human skin, in vitro. The amount of vitamin D3 production increased in a dose-dependent fashion with increased UVB energy. UVB-LEDs can be developed for devices that can efficiently produce vitamin D3 in human skin.

Evaluation of Effectiveness of Ultraviolet Emitting Lamps on the Cutaneous Production of Vitamin D3: Relationship of the Lamps Vitamin D3 Producing Potential to the Production of 8-Hydroxy-2'-Deoxyguanosine and Nitric Oxide.

BACKGROUND/AIM: To assess the effectiveness of three UV emitting lamps on the cutaneous production of vitamin D3, a marker of DNA damage and nitric oxide production in human skin. MATERIALS AND METHODS: Human skin samples (skin types II, III and IV) obtained from surgery were exposed to three different UV emitting lamps for varying times and then extracted and chromatographed to determine the vitamin D3 content. The skin samples exposed to the 3 UV emitting lamps were also evaluated for 8-hydroxy-2'-deoxyguanosine (a marker of DNA damage) and nitric oxide production. RESULTS: It was observed that the spectral output of the 3 lamps had different effects on the cutaneous production of vitamin D3, 8-hydroxy-2'-deoxyguanosine and nitric oxide production. One lamp demonstrated optimal production of vitamin D3 with the least amount of DNA damage and intermediate production of nitric oxide suggesting that it could be developed into a device for treating vitamin D deficiency. CONCLUSION: The spectral output of the experimental UVB emitting lamps significantly influenced the cutaneous production of vitamin D3 8-hydroxy-2'-deoxyguanosine and nitric oxide.

Effect of dietary vitamin D and calcium on the growth of androgen-insensitive human prostate tumor in a murine model.

Vitamin D deficiency has been associated with increased risk of prostate cancer (PC) in epidemiologic and prospective studies. An association has also been made between high dietary calcium and increased PC risk. In this study, we evaluated the effect of dietary vitamin D and calcium on the growth of human androgen-insensitive prostate tumor in an athymic mouse model. We observed highest tumor growth in the normal calcium - vitamin D-deficient group, while tumor growth between the normal calcium - vitamin D-sufficient, high calcium - vitamin D-sufficient and high calcium - vitamin D-deficient diet-groups did not significantly differ but was significantly lower than that in the normal calcium - vitamin D-deficient group. Our results suggest an important role of dietary vitamin D as a preventive agent in androgen-insensitive PC.

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.

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.

Anti-growth effect of 1,25-dihydroxyvitamin D3-3-bromoacetate alone or in combination with 5-amino-imidazole-4-carboxamide-1-beta-4-ribofuranoside in pancreatic cancer cells.

1,25-Dihydroxyvitamin D(3)-3-bromoacetate (1,25(OH)(2)D(3)-3-BE) is a vitamin D receptor-alkylating derivative of 1,25(OH)(2)D(3). The strong dose-dependent antiproliferative and apoptotic effects of this compound in androgen-sensitive and androgen-insensitive prostate cancer cells have been reported. In this communication, it is reported that 1,25(OH)(2)D(3)-3-BE strongly inhibits the growth of several pancreatic cancer cell lines. This effect is further accentuated by combination with 5-amino-imidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR), an activator of AMP-activated protein kinase (AMPK)/acetyl-Co-enzyme A carboxylase (ACC) phosphorylation pathways and an inhibitor of Akt phosphorylation. It was observed that the anti-growth property of 1,25(OH)(2)D(3)-3-BE, either alone or in combination with AICAR resulted in the inhibition of Akt phosphorylation in BxPC-3 cells. In conclusion, 1,25(OH)(2)D(3)-3-BE displays a strong therapeutic potential, alone and in combination with AICAR, in pancreatic cancer.

Evaluation of 19-nor-2alpha-(3-hydroxypropyl)-1alpha,25-dihydroxyvitamin D3 as a therapeutic agent for androgen-dependent prostate cancer.

The high incidence of prostate cancer and lack of an effective, long-term treatment for metastatic disease highlights the need for more potent non-calcemic vitamin D analogs as potential alternative or combinational prostate cancer therapies. Among the analogs, 19-nor-1alpha,25-dihydroxyvitamin D2 (19-nor-1alpha,25(OH)2D2) known as paricalcitol or Zempler, has less calcemic effects and an equipotential activity as 1alpha,25-dihydroxyvitamin D3 (1alpha,25(OH)2D3) in several in vivo and in vitro systems. It was recently demonstrated that a modified analog of paricalcitol, 19-nor-2alpha-(3-hydroxypropyl)-1alpha,25-dihydroxyvitamin D3 (MART-10) compared to 1alpha,25(OH)2D3 was more effective in inhibiting proliferation of an immortalized normal prostate cell line (PZ-HPV-7) (1,000-fold) and invasion of PC-3 prostate cancer cells (10-fold). In this study, the effects of MART-10 and 1alpha,25(OH)2D3 on proliferation, vitamin D receptor transactivation, vitamin D-binding protein (DBP) binding, CYP24A1 (24-OHase) substrate hydroxylation kinetics, and induction of CYP24A1 gene expression were compared in an androgen-dependent prostate cancer cell model, LNCaP. The results demonstrated that MART-10 was 1,000-fold more active than 1alpha,25(OH)2D3 in inhibiting LNCaP cell proliferation. MART-10 was more active than 1alpha,25(OH)2D3 in up-regulating a vitamin D receptor-responsive Luciferase construct and inducing CYP24A1 gene expression in LNCaP prostate cancer cells. In addition, MART-10 has a lower affinity for DBP and less substrate degradation by CYP24A1 compared to 1alpha,25(OH)2D3, indicating that MART-10 has more bioavailability and a longer half-life. Thus, these data suggest that MART-10 may be a potential candidate as a therapeutic agent for prostate cancer, especially for patients who fail in conventional therapies.

Fish oil enhances the antiproliferative effect of 1alpha,25-dihydroxyvitamin D3 on liver cancer cells.

BACKGROUND: Laboratory and epidemiological studies have indicated that 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] and dietary omega 3 (omega3)-polyunsaturated fatty acids (PUFAs) are capable of inhibiting the proliferation of various cancer cells. MATERIALS AND METHODS: Human hepatoblastoma cells (HepG2) were treated with 1alpha,25(OH)2D3 and fish oil alone and in combination. Cell proliferation was measured either by the uptake of [3H]-thymidine into DNA or by counting the cell numbers using a hemocytometer. RESULTS: The HepG2 cell proliferation was inhibited by 1alpha,25(OH)2D3 and fish oil in a dose-dependent manner. The lowest effective concentration of 1alpha,25(OH)2D3 was 10(-7) M and 10(-8) M using the [3H]-thymidine incorporation method and the cell counting method, respectively. Fish oil also caused a significant inhibition in HepG2 cell proliferation at 25 microg/mL. When HepG2 cells were treated with 1alpha,25(OH)2D3 in combination with fish oil, it was found that fish oil increased the antiproliferative effect of 1alpha,25(OH)2D3 on HepG2 cell growth compared to treatment with 1alpha,25(OH)2D3 alone. CONCLUSION: 1alpha,25(OH)2D3 could be used to treat hepatocellular carcinoma (HCC). However, the major side-effect of hypercalcemia limits its use. An enhanced 1alpha,25(OH)2D3-induced inhibition of HepG2 cell proliferation in the presence of PUFAs in the form of fish oil suggests that a lower concentration of 1alpha,25(OH)2D3 could be used to treat hepatocellular carcinoma in the presence of PUFAs to decrease the risk of hypercalcemia caused by high concentrations of 1alpha,25(OH)2D3.

Covalent labeling of nuclear vitamin D receptor with affinity labeling reagents containing a cross-linking probe at three different positions of the parent ligand: structural and biochemical implications.

Structure-functional characterization of vitamin D receptor (VDR) requires identification of structurally distinct areas of VDR-ligand-binding domain (VDR-LBD) important for biological properties of 1alpha,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)). We hypothesized that covalent attachment of the ligand into VDR-LBD might alter 'surface structure' of that area influencing biological activity of the ligand. We compared anti-proliferative activity of three affinity alkylating derivatives of 1,25(OH)(2)D(3) containing an alkylating probe at 1,3 and 11 positions. These compounds possessed high-affinity binding for VDR; and affinity labeled VDR-LBD. But, only the analog with probe at 3-position significantly altered growth in keratinocytes, compared with 1,25(OH)(2)D(3). Molecular models of these analogs, docked inside VDR-LBD tentatively identified Ser237 (helix-3: 1,25(OH)(2)D(3)-1-BE), Cys288 (beta-hairpin region: 1,25(OH)(2)D(3)-3-BE,) and Tyr295 (helix-6: 1,25(OH)(2)D(3)-11-BE,) as amino acids that are potentially modified by these reagents. Therefore, we conclude that the beta-hairpin region (modified by 1,25(OH)(2)D(3)-3-BE) is most important for growth inhibition by 1,25(OH)(2)D(3), while helices 3 and 6 are less important for such activity.

Prostate 25-hydroxyvitamin D-1alpha-hydroxylase is up-regulated by suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor.

Prostatic 25-hydroxyvitamin D-1alpha-hydroxylase (1alpha-OHase) is up-regulated by epidermal growth factor (EGF) and down-regulated by 1alpha,25-dihydroxyvitamin D [1alpha,25(OH)2D] at the promoter level in an autocrine/paracrine fashion, suggesting that local production of 1alpha,25(OH)2D could provide an important cell growth regulatory mechanism. Gene expressions depend on the acetylation status of the histone tails of chromatin, which is regulated by histone acetyltransferases and histone deacetylases (HDAC). A number of HDAC inhibitors, including suberolylanilide hydroxamic acid (SAHA), can inhibit tumor growth in vitro and in vivo. Moreover, SAHA increases the expression of genes which modulate cell cycle progression, tumor suppression, differentiation and apoptosis. Therefore, whether SAHA might also regulate 1alpha-OHase activity in PZ-HPV-7 prostate cells was investigated. SAHA at 10 microM up-regulated 1alpha-OHase activity approximately two-fold as analyzed by the formation of 3H-1alpha,25(OH)2D3 from 3H-25-hydroxyvitamin D3 using high performance liquid chromatography. SAHA (10 microM) also stimulated 1alpha-OHase mRNA expression as measured by real-time polymerase chair reaction, and promoter activity determined by luciferase reporter gene assay. The findings suggest that another important action of SAHA may be to up-regulate the expression of the 1alpha-OHase gene that controls the synthesis of 1alpha,25(OH)2D which in turn regulates prostate growth and differentiation in an autocrine/paracrine fashion.

Mechanistic and pharmacodynamic studies of a 25-hydroxyvitamin D3 derivative in prostate cancer cells.

1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), the biologically active form of vitamin D has strong antiproliferative effects in cancer cells. But it is highly toxic at therapeutic doses. We have observed that 25-hydroxyvitamin D(3)-3-bromoacetate (25-OH-D(3)-3-BE), a derivative of 25-hydroxyvitamin D(3), the pro-hormonal form of 1,25(OH)(2)D(3) has strong growth-inhibitory and proapoptotic properties in hormone-sensitive and hormone-refractory prostate cancer cells. In the present investigation we demonstrate that the antiproliferative effect of 25-OH-D(3)-3-BE is predominantly mediated by VDR in ALVA-31 prostate cancer cells. In other mechanistic studies we show that the proapoptotic property of 25-OH-D(3)-3-BE is related to the inhibition of phosphorylation of Akt, a pro-survival protein. Furthermore, we carried out cellular uptake and serum stability studies of 25-OH-D(3)-3-BE to demonstrate potential therapeutic applicability of 25-OH-D(3)-3-BE in hormone-sensitive and hormone-insensitive prostate cancer.

Vitamin D metabolism in human prostate cells: implications for prostate cancer chemoprevention by vitamin D.

BACKGROUND: Prostate cells can produce 1alpha,25-dihydroxyvitamin D3 (1alpha,25(OH)2D3) from 25-hydroxyvitamin D3 (25(OH)D3) to regulate their own growth. Here, the questions of whether prostate cells express vitamin D-25-hydroxylase (25-OHase) and can convert vitamin D3 to 1alpha,25(OH)2D3 were investigated. MATERIALS AND METHODS: Protein and receptor binding assays were used to determine 25(OH)D3 and 1alpha,25(OH)2D3, respectively. Measurements of proliferation by 3H-thymidine incorporation, and 1alpha,25(OH)2D-responsive gene expression by real-time qPCR and by Western blot were used as functional assays for the presence of 25-OHase activity. RESULTS: Prostate cells metabolized vitamin D3 to 1alpha,25(OH)2D3. Vitamin D3 up-regulated 25(OH)D-24R-hydroxylase and IGFBP3, two 1alpha,25(OH)2D-responsive genes, in prostate cells. CYP2R1 was the major form of 25-OHase expressed in normal and cancerous prostate cells as determined by qPCR. CONCLUSION: The autocrine synthesis of 1alpha,25(OH)2D3 from vitamin D3 suggests that maintaining adequate levels of serum vitamin D could be a safe and effective chemo-preventive measure to decrease the risk of prostate cancer.

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

In this report we describe that 1,25(OH)(2)D(3)-3-BE, a VDR-affinity labeling analog of 1,25(OH)(2)D(3), 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)(2)D(3)-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)(2)D(3)-3-BE) was not due to probable degradation (hydrolysis) of 1,25(OH)(2)D(3)-3-BE or random interaction of this molecule with cellular proteins. Tissue- or cell-specific action of 1,25(OH)(2)D(3) and its mimics is not common due to the ubiquitous nature of VDR. Furthermore, variable effects of 1,25(OH)(2)D(3) and its analogs in various cell-lines potentially limits their application as anticancer agents. We showed that 1,25(OH)(2)D(3)-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)(2)D(3)-3-BE or similar VDR-cross linking analogs of 1,25(OH)(2)D(3) might be considered for further development as potential candidates for prostate cancer.