seocalcitol and Skin-Neoplasms

We studied effects of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], its analog seocalcitol (EB 1089), and 25-hydroxyvitamin D3 [25(OH)D3], on the growth of seven melanoma cell lines. While three cell lines (MeWo, SK-Mel-28, SM) responded to antiproliferative effects of active vitamin D analogs, the others (SK-Mel-5, SK-Mel-25, IGR, MeUuso) were resistant. A strong induction (7000-fold) of 1,25-dihydroxyvitamin D-24-hydroxylase (24OHase, CYP24) mRNA was detected in responsive cell lines along with 1,25(OH)2D3-treatment, indicating functional integrity of vitamin D receptor (VDR)-mediated transcription. In contrast, induction of 24OHase was much lower in resistant melanoma cells (70-fold). VDR mRNA was induced up to 3-fold both in responsive and resistant cell lines along with 1,25(OH)2D3-treatment. RNA for vitamin D-activating enzymes vitamin D-25-hydroxylase (25OHase, CYP27A1) and 25-hydroxyvitamin D-lalpha-hydroxylase (lalphaOHase, CYP27B1) was detected in all melanoma cell lines analyzed, additionally we show splicing variants of lalphaOHase in SK-Mel-28 cells. Expression of 250Hase and laOHase was marginally modulated along with treatment. Proliferation of melanoma cells was not inhibited by treatment with 25(OH)D3, indicating no significant stimulation of endogeneous production of antiproliferative acting 1,25(OH)2D3. In conclusion, we characterize the vitamin D endocrine system in melanoma cells and demonstrate that the majority of melanoma cell lines analyzed is resistant to antiproliferative effects of 1,25(OH)2D3. It can be speculated that these alterations are of importance for pathogenesis and growth of metastasizing malignant melanoma. Additionally, our findings indicate that only a minority of cases with metastasizing melanoma may represent a promising target for palliative treatment with new vitamin D analogs that exert little calcemic side effects or for pharmacological modulation of 1,25(OH)2D3-synthesis/metabolism.

Topics: 25-Hydroxyvitamin D3 1-alpha-Hydroxylase; Alternative Splicing; Antineoplastic Agents; Calcitriol; Cell Line, Tumor; Cell Proliferation; Cholestanetriol 26-Monooxygenase; Drug Resistance, Neoplasm; Endocrine System; Humans; Melanoma; Receptors, Calcitriol; Skin Neoplasms; Steroid Hydroxylases; Vitamin D; Vitamin D3 24-Hydroxylase

We examined parathyroid hormone-related peptide (PTHrP) production and regulation in both normal human melanocytes and in a human amelanotic melanoma cell line (A375). Northern blot and immunocytochemical analysis demonstrated that both cultured A375 cells and normal human melanocytes express PTHrP, but A375 cells expressed much higher levels of the peptide. PTHrP secretory rate increased at least 10-fold after treatment with 10% fetal bovine serum (100.2 +/- 2.8 pmol/10(6) cells vs. basal <15 pmol/10(6) cells) in proliferating A375 cells but only twofold in confluent cells. Treatment of A375 cells with increasing concentrations of 1, 25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)] or its low-calcemic analog EB-1089 revealed that EB-1089 was 10-fold more potent than 1, 25-(OH)(2)D(3) on inhibition of both cell proliferation and PTHrP expression. Furthermore, inoculation of A375 cells into the mammary fat pad of female severe combined immunodeficiency mice resulted in the development of hypercalcemia and elevated concentrations of plasma immunoreactive PTHrP in the absence of detectable skeletal metastases. Our study, therefore, demonstrates a stepwise increase in PTHrP expression when cells progress from normal to malignant phenotype and suggests that EB-1089 should be further evaluated as a therapeutic agent in human melanoma.

Topics: Animals; Antineoplastic Agents; Blotting, Northern; Body Weight; Calcitriol; Calcium; Cell Division; Cell Line; Drug Implants; Female; Fibroblast Growth Factor 2; Humans; Hypercalcemia; Immunohistochemistry; Insulin; Melanocytes; Melanoma, Amelanotic; Mice; Mice, SCID; Neoplasm Transplantation; Parathyroid Hormone-Related Protein; Proteins; RNA, Messenger; Skin Neoplasms

EB1089, an analogue of 1,25 dihydroxyvitamin D with low calcemic activity is a potent inhibitor of parathyroid hormone-related peptide (PTHRP) production in vitro. The purpose of the present study was to determine whether EB1089 could reverse established hypercalcemia in BALB C nude mice implanted s.c. with a human epithelial cancer previously shown to produce high levels of PTHRP in vitro. Total plasma calcium was monitored before and after tumor development and increased steadily when the tumor reached > or =0.5 cm3. When total calcium was 22.85 mmol/liter, animals were treated with a constant infusion of EB1089 or vehicle alone for a period of 2 weeks. A significant and sustained reduction of plasma calcium from 3.2+/-0.1 to 2.7+/-0.08 (P < 0.01) mmol/liter was observed during infusion with EB1089. In contrast, calcium levels in vehicle-treated animals continued to rise during the infusion period. Tumor growth velocity also slowed significantly after the administration of EB1089 as compared with vehicle-treated animals. Plasma PTHRP levels measured at the end of the 2 weeks' infusion period were significantly lower in animals treated with EB1089 as compared with animals treated with vehicle alone (44+/-8 pg/ml versus 194+/-35 pg/ml, P < 0.001). These results, therefore, demonstrate that EB1089 can reverse established hypercalcemia in a human model of squamous cancer.

Topics: Animals; Antineoplastic Agents; Calcitriol; Carcinoma, Squamous Cell; Cell Division; Cell Line, Transformed; Cell Transformation, Viral; Drug Screening Assays, Antitumor; Genes, ras; Humans; Hypercalcemia; Keratinocytes; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Proteins; Neoplasm Transplantation; Papillomaviridae; Parathyroid Hormone-Related Protein; Peptide Fragments; Skin Neoplasms

The antitumoral potency of 1,25-dihydroxyvitamin D3 and its synthetic derivatives MC903, EB1089, and KH1060 was investigated on a tumoral Bowen-like epidermis reconstructed from an immortalized human keratinocyte cell line transfected by expression vectors coding for E6 and E7 of human papillomavirus 16. Treatment of skin equivalents by vitamin D derivatives (10(-9) M or 10(-12) M) was performed during (from day 1 to day 15 of culture) or after tissue reconstruction (from day 15 to day 30). Pharmacologic effects were evaluated by morphologic and immunohistologic analysis and compared with those of controls (vehicle alone) and with treatment of skin equivalents derived from normal keratinocytes. When performed during epidermal reconstruction, treatment of tumoral skin equivalents induced only minor morphologic and immunohistologic changes. Conversely, when performed after epidermal reconstruction, treatment with 1,25-dihydroxyvitamin D3, MC903, and EB1089 clearly improved the phenotype of treated tissues. Morphologic analysis showed reorganization of epidermal layers with the appearance of a distinct basal layer and of stratified orthokeratotic stratum corneum. Immunohistochemical analysis demonstrated that the terminal differentiation markers profilaggrin and cytokeratin 10 were re-expressed in the treated tissues while absent in controls. Overall, the results indicate that 1,25-dihydroxyvitamin D3, MC903, and EB1089 can induce a partial reversion of the tumoral phenotype in this in vitro model.

Topics: Antineoplastic Agents; Calcitriol; Humans; Phenotype; Skin; Skin Neoplasms

We have examined the in vitro effects of 1,25 dihydroxy-vitamin D3 [1,25(OH)2D3] and of two side-chain modified analogs of 1,25(OH)2D3 (EB1089 and MC903) on cell growth and parathyroid hormone related peptide (PTHRP) production in immortalized (HPK1A) and neoplastic (HPK1A-ras) keratinocytes. Cell proliferation was strongly inhibited by 1,25(OH)2D3 and its analogs in HPK1A cells, and in this system EB1089 was 10-100 times more potent than 1,25(OH)2D3 or MC903. A similar effect on cell proliferation was observed in HPK1A-ras cells; however, 10-fold higher concentrations of 1,25(OH)2D3 or its analogs were required. We also observed a strong and dose-dependent inhibitory effect of these compounds on PTHRP secretion and gene expression. In both immortalized and neoplastic keratinocytes, EB1089 was 10-100 times more potent than 1,25(OH)2D3 or MC903 on inhibiting PTHRP production. However, although effective in HPK1A-ras cells, 10-fold higher concentrations of 1,25(OH)2D3 or its analogs were required to produce similar actions in this neoplastic model. These studies therefore demonstrate that a 1,25(OH)2D3 analog with low calcemic potency in vivo (EB1089) can inhibit keratinocyte proliferation and PTHRP production by such cells with greater potency than 1,25(OH)2D3. The observed effects of such analogs in neoplastic keratinocytes predicts their potential usefulness in vivo in inhibiting squamous cancer growth and its associated hypercalcemia.

Topics: Animals; Antineoplastic Agents; Calcitriol; Carcinoma, Squamous Cell; Cell Division; Cell Line, Transformed; Cell Transformation, Viral; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Gene Expression Regulation, Neoplastic; Humans; Hypercalcemia; Keratinocytes; Mice; Mice, Nude; Neoplasm Proteins; Neoplasm Transplantation; Parathyroid Hormone-Related Protein; Protein Biosynthesis; Skin Neoplasms; Tumor Cells, Cultured