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Vitamin D3 analog, 20-hydroxyvitamin D, inhibits cancer cells and is non-toxic – March 2012

3 articles on this page by many of the same authors - third describes the synthesis

patented in 2007

20-hydroxyvitamin D? inhibits proliferation of cancer cells with high efficacy while being non-toxic.

Anticancer Res. 2012 Mar;32(3):739-46.
Wang J, Slominski A, Tuckey RC, Janjetovic Z, Kulkarni A, Chen J, Postlethwaite AE, Miller D, Li W wli at uthsc.edu
Department of Pharmaceutical Sciences, University of Tennessee Health Science Center, Memphis, TN, USA.

AIM:
To define the potential utility of 20-hydroxyvitamin D(3) OH)D(3 as a tumorostatic agent, we assessed its in vitro antiproliferative activity and its in vivo toxicity.
MATERIALS AND METHODS:
The antitumor activity of 20(OH)D(3) was tested against breast and liver cancer cell lines using colony formation assays. To assess in vivo toxicity, mice were injected with 5-30 ?g/kg 20(OH)D(3) intraperitoneally each day for 3 weeks. Blood and organ samples were collected for clinical pathology analyses.
RESULTS:
20(OH)D(3) displays similar tumorostatic activity towards MDA-MB-453 and MCF7 breast carcinomas, and HepG2 hepatocarcinoma, in a dose-dependent manner. This compound is not hypercalcemic, does not cause detectable toxicities in liver, kidney, or blood chemistry in mice at a dose as high as 30 ?g/kg. In contrast, both 25(OH)D(3) and 1,25(OH)(2)D(3) caused severe hypercalcemia at a dose of 2 ?g/kg.
CONCLUSION:
20(OH)D(3) possesses high efficacy for inhibiting cancer cell proliferation in vitro and is non-toxic in vivo, supporting its further development as a potential anticancer therapeutic agent.
PMID: 22399586 PMCID: PMC3312810 [Available on 2013/3/1]

Figure Captions thumbnails of fiqures on the web

Figure 1.
20(OH)D3 inhibits the proliferation of HepG2 hepatocellular carcinoma cells. HepG2 cells were grown in 6-well plates and were treated with different concentrations of 20(OH)D3, or with 1?,25(OH)2D3 as a positive control. 14 days after treatment, cells had formed colonies which were fixed in para-formaldehyde (PFA), stained with crystal violet and counted under a cell counter. The size and number of colonies were scored. Data representing colonies larger than 0.2 mm (A) and larger than 0.5 mm (B) in size following treatment with 20(OH)D3 and 1?,25(OH)2D3 are shown as the mean±SD (n=3). *p<0.05 versus vehicle control. CFU: Colony-forming unit.
Figure 2.
20(OH)D3 inhibits the proliferation of MDA-MB-453 human breast carcinoma cells. MDA-MB-453 cells were grown in 6-well plates and were treated with different concentrations of 20(OH)D3, or with 25(OH)D3 as a positive control. By 14 days after treatment cells had formed colonies which were fixed in PFA, stained with crystal violet and counted under a cell counter. The size and number of colonies were scored. Data representing colonies larger than 0.2 mm (A) and larger than 1.5 mm (B) in size following treatment with 20(OH)D3, and 25(OH)D3 are shown as the mean±SD (n=3). *p<0.05 versus vehicle control. CFU: Colony-forming unit.
Figure 3.
20(OH)D3 inhibits the proliferation of MCF7 breast cancer cells. MCF7 cells were grown in soft agar and were treated with different concentrations of 20(OH)D3, or 1?,25(OH)2D3 as a positive control. By 26 days after treatment cells had formed colonies which were stained with MTT reagent and counted under a microscope. The size and number of colonies were scored. Data representing colonies larger than 0.2 mm in size following treatment with 20(OH)D3 and 1?,25(OH)2D3 are shown as the mean±SD (n=3). *p<0.05 or **p<0.01 versus vehicle control. CFU: Colony-forming unit.
Figure 4.
20(OH)D3 did not show hypercalcemic side effects at different high doses up to 30 ?g/kg. Mice were treated with i.p. injection for 3 weeks (five mice per treatment group). Data are presented as the mean value of serum calcium concentration (mg/l) for each group. There were no significant differences between the vehicle control group and the 20(OH)D3 treatment groups at all doses (p>0.05). There were significant differences between the positive control groups and vehicle control group, and between the positive control groups and the 20(OH)D3 treatment groups (p<0.01). Dotted line: Upper range for normal calcium level in serum (10.5 mg/dl).
Figure 5.
20(OH)D3 did not show toxicity in vivo at different high doses up to 30 ?g/kg. A: Analysis of complete clinical pathology chemistry profiles testing liver/kidney functions and corticosterone levels. B: Complete blood count (CBC) with differential results for all treatment groups in the toxicity study. All data are well within the normal physiological range for mice and there are no statistically significant differences between mice in the vehicle control group and all treatment groups. ALT: Alanine aminotransferase; ALK: alkaline phosphatase; AST: aspartate aminotransferase; TBIL: total bilirubin; ALB: albumin; TPR: total protein; BUN: blood urea nitrogen; CREAT: creatinie; PHOS: inorganic phosphate; WBC: white blood cell count; BA: basophil granulocytes; NE: neutrophil granulocytes; LY: lymphocytes; MO: monocytes; EO: eosinophil granulocytes; RBC: red blood cell count; Hb: hemoglobin; HCT%: hematocrit percentage; MCV: mean corpuscular volume; MCH: mean corpuscular hemoglobin; MCHC: mean corpuscular hemoglobin concentration; RDW: red cell distribution width; PLT: platelet count; MPV: mean platelet volume.
Figure 6.
Histopathological analyses of representative organs taken from mice in the vehicle control group and mice receiving the highest dose of 20(OH)D3. No calcification or other abnormalities were observed in any of these organs. Organs were processed and slides were digitized and analyzed. Photographs of various organs were taken at different magnifications.

Previous article by some of the same authors

PLoS One. 2010 Mar 26;5(3):e9907.

Products of vitamin D3 or 7-dehydrocholesterol metabolism by cytochrome P450scc show anti-leukemia effects, having low or absent calcemic activity.

Slominski AT, Janjetovic Z, Fuller BE, Zmijewski MA, Tuckey RC, Nguyen MN, Sweatman T, Li W, Zjawiony J, Miller D, Chen TC, Lozanski G, Holick MF.
Department of Pathology and Laboratory Medicine, University of Tennessee Health Science Center, Memphis, Tennessee, United States of America. aslominski at uthsc.edu
Image
BACKGROUND:
Cytochrome P450scc metabolizes vitamin D3 to 20-hydroxyvitamin D3 (20(OH)D3) and 20,23(OH)(2)D3, as well as 1-hydroxyvitamin D3 to 1alpha,20-dihydroxyvitamin D3 (1,20(OH)(2)D3). It also cleaves the side chain of 7-dehydrocholesterol producing 7-dehydropregnenolone (7DHP), which can be transformed to 20(OH)7DHP. UVB induces transformation of the steroidal 5,7-dienes to pregnacalciferol (pD) and a lumisterol-like compounds (pL).
METHODS AND FINDINGS:
To define the biological significance of these P450scc-initiated pathways, we tested the effects of their 5,7-diene precursors and secosteroidal products on leukemia cell differentiation and proliferation in comparison to 1alpha,25-dihydroxyvitamin D3 (1,25(OH)(2)D3). These secosteroids inhibited proliferation and induced erythroid differentiation of K562 human chronic myeloid and MEL mouse leukemia cells with 20(OH)D3 and 20,23(OH)(2)D3 being either equipotent or slightly less potent than 1,25(OH)(2)D3, while 1,20(OH)(2)D3, pD and pL compounds were slightly or moderately less potent. The compounds also inhibited proliferation and induced monocytic differentiation of HL-60 promyelocytic and U937 promonocytic human leukemia cells. Among them 1,25(OH)(2)D3 was the most potent, 20(OH)D3, 20,23(OH)(2)D3 and 1,20(OH)(2)D3 were less active, and pD and pL compounds were the least potent. Since it had been previously proven that secosteroids without the side chain (pD) have no effect on systemic calcium levels we performed additional testing in rats and found that 20(OH)D3 had no calcemic activity at concentration as high as 1 microg/kg, whereas, 1,20(OH)(2)D3 was slightly to moderately calcemic and 1,25(OH)(2)D3 had strong calcemic activity.
CONCLUSIONS:
We identified novel secosteroids that are excellent candidates for anti-leukemia therapy with 20(OH)D3 deserving special attention because of its relatively high potency and lack of calcemic activity.
PMID: 20360850
PDF is attached at the bottom of this page

Chemical synthesis of 20S-hydroxyvitamin D3, which shows antiproliferative activity.

Steroids. 2010 Dec;75(12):926-35. Epub 2010 Jun 11.
Li W, Chen J, Janjetovic Z, Kim TK, Sweatman T, Lu Y, Zjawiony J, Tuckey RC, Miller D, Slominski A.
Department of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee Health Science Center, Memphis, TN 38163, USA. wli at uthsc.edu

20S-hydroxyvitamin D3 (20S-(OH)D3), an in vitro product of vitamin D3 metabolism by the cytochrome P450scc, was recently isolated, identified and shown to possess antiproliferative activity without inducing hypercalcemia. The enzymatic production of 20S-(OH)D3 is tedious, expensive, and cannot meet the requirements for extensive chemical and biological studies. Here we report for the first time the chemical synthesis of 20S-(OH)D3 which exhibited biological properties characteristic of the P450scc-generated compound. Specifically, it was hydroxylated to 20,23-dihydroxyvitamin D3 and 17,20-dihydroxyvitamin D3 by P450scc and was converted to 1alpha,20-dihydroxyvitamin D3 by CYP27B1. It inhibited proliferation of human epidermal keratinocytes with lower potency than 1alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) in normal epidermal human keratinocytes, but with equal potency in immortalized HaCaT keratinocytes. It also stimulated VDR gene expression with similar potency to 1,25(OH)2D3, and stimulated involucrin (a marker of differentiation) and CYP24 gene expression, showing a lower potency for the latter gene than 1,25(OH)2D3. Testing performed with hamster melanoma cells demonstrated a dose-dependent inhibition of cell proliferation and colony forming capabilities similar or more pronounced than those of 1,25(OH)2D3. Thus, we have developed a chemical method for the synthesis of 20S-(OH)D3, which will allow the preparation of a series of 20S-(OH)D3 analogs to study structure-activity relationships to further optimize this class of compound for therapeutic use.
Copyright 2010 Elsevier Inc. All rights reserved.
PMID: 20542050
PDF is attached at the bottom of this page

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Clipped from email interaction with the corresponding author May 9, 2012


Question2: Is 20-hydroxyvitamin D? stable - that is, can it be packaged, distributed, sold, etc?
Answer: As far as I know, both of them are stable --- but are UV light sensitive.
This is how the skin produce vitamin D3 from its precursor (7DHC) using the sunlight.
One can package them in amber vials or other ways so that they will not be exposed to excessive light.

If you or anyone are interested in getting more information, please contact Dr. Janet Ralbovsky whose email is jralbovs at uthsc.edu for help.
click here patent details - 2007
2 page PDF summary from the University is attached at the bottom of this page

See also VitaminDWiki

Attached files

ID Name Comment Uploaded Size Downloads
1320 Patent vitamin D Analog.pdf admin 09 May, 2012 17:02 152.37 Kb 950
1317 20S-hydroxyvitamin D3 synthesis.pdf PDF admin 09 May, 2012 14:16 1.02 Mb 772
1316 Analogues1.jpg admin 09 May, 2012 13:54 55.33 Kb 1595
1315 D3 derivatives.pdf admin 09 May, 2012 13:50 995.19 Kb 787
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