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NEOPLASIA
From the Division of Hematology/Oncology, Cedars-Sinai
Medical Center, UCLA School of Medicine, Los Angeles, CA; Hoffmann
LaRoche, Nutley, NJ; and Department of Hematology, Showa University
School of Medicine, Tokyo, Japan.
The active form of vitamin D3,
1,25(OH)2D3, inhibits proliferation and induces
differentiation of a variety of malignant cells. A new class of vitamin
D3 analogs, having 2 identical side chains attached to
carbon-20, was synthesized and the anticancer effects evaluated. Four
analogs were evaluated for their ability to inhibit growth of myeloid
leukemia (NB4, HL-60), breast (MCF-7), and prostate (LNCaP) cancer
cells. All 4 analogs inhibited growth in a dose-dependent manner. Most
effective was 21-(3-methyl-3-hydroxy-butyl)-19-nor D3
(Gemini-19-nor), which has 2 side chains and removal of the C-19.
Gemini-19-nor was approximately 40 625-, 70-, 23-, and 380-fold more
potent than 1,25(OH)2D3 in inhibiting 50%
clonal growth (ED50) of NB4, HL-60, MCF-7, and LNCaP cells,
respectively. Gemini-19-nor (10 The present chemotherapy of cancer uses agents that
are usually toxic to normal cells. On the other hand, induction of
cellular differentiation may supplement the use of cytotoxic drugs in
several forms of neoplasia, like the successful use of
all-trans-retinoic acid (ATRA) in the treatment of acute
promyelocytic leukemia. The physiologically active form of vitamin
D3, 1,25(OH)2D3, is a member of the
secosteroid hormone family, which controls calcium homeostasis and bone
metabolism. 1,25(OH)2D3 can induce
differentiation and inhibit the growth of a number of malignant cell
types, including myeloid leukemia, breast, prostate, colon, skin, and
brain. Several studies suggested that growth inhibition by
1,25(OH)2D3 may be attributed to inhibition of
the G1 to S transition in the cell cycle, which probably is
due at least in part to stimulation of expression of the
cyclin-dependent kinase inhibitors (CDKIs), p21waf1 and
p27kip1 as well as induction of programmed cell
death.1-4 In a clinical study, oral administration of
1,25(OH)2D3 to preleukemic patients was only
partially effective5; calcemic side effects prevented the
administration of the dosage of the compound needed to achieve the
concentration of 1,25(OH)2D3 in vivo, which was
known to be necessary from our in vitro studies.5,6
Therefore, synthesis of vitamin D3 analogs with
potent antiproliferative and differentiation activity against cancer
cells with decreased risk of hypercalcemia has received considerable
attention.7-14
Recently, PTEN/MMAC1/TEP1, a tyrosine phosphatase, was identified and
mapped to chromosome 10q23.3.15-17 PTEN gene
mutations have been observed in a variety of human cancers including
breast, prostate, brain, lymphoma, and leukemia.15,18-23
Germline deletion of PTEN in the mouse resulted in early embryonic
lethality, and heterozygous mice developed malignant
neoplasms.24,25 These findings strongly suggested that
PTEN is a candidate tumor suppressor.
In this study, a class of newly synthesized vitamin D3
analogs having 2 identical side chains attached to carbon-20 was
analyzed. We focused particularly on the most active analog, which has
a deletion of C-19, 21-(3-methyl-3-hydroxy-butyl)-19-nor D3
(Gemini-19-nor). This new vitamin D3 analog was more potent
than 1,25(OH)2D3 in mediating growth
inhibition, differentiation, apoptosis, G1/G0 arrest of the cell cycle, and expression of
p27kip1. Furthermore, we observed that this
compound induced the expression of PTEN in myeloid leukemic cells as
the cells underwent differentiation.
Cells and compounds
All vitamin D3 analogs were synthesized by Milan R. Uskokovic (Hoffmann-La Roche, Nutley, NJ). The analogs are shown in
Figure 1. The vitamin D3
compounds were dissolved in absolute ethanol at 10
Soft agar colony assay
Analysis of differentiation Expression of cell surface antigens was determined by flow cytometry. HL-60 cells were cultured with either 1,25(OH)2D3 or Gemini-19-nor (10 8
and 107 M) for 4 days. After twice washing with
phosphate-buffered saline (PBS), cells were incubated for 30 minutes
with fluorescein isothiocyanate (FITC)-conjugated murine antihuman
CD11b or antihuman CD14 antibody (DAKO, Carpinteria, CA). Murine IgG1
antibody (DAKO) was used as negative control. Cells were analyzed by a
FACScan (Becton Dickinson, Mountain View, CA). HL-60 cells were
assessed for their ability to produce superoxide as measured by
reduction of nitroblue tetrazolium (NBT), by morphology as detected on
cytospin preparations stained with Diff-Quick Stain Set (Baxter
Healthcare, Miami, FL). All experiments were independently done at
least 3 times. All data were statistically analyzed by Student
t test.
Cell cycle analysis Cell cycle analysis was performed on HL-60 cells incubated for 4 days with either 1,25(OH)2D3 or Gemini-19-nor at either 108 or 107 M. The cells were
fixed in chilled methanol overnight before staining with 50 µg/mL
propidium iodide (PI), 1 mg/mL RNase, and 0.1% NP40. Analysis was
performed immediately after staining using a FACScan (Becton Dickinson)
and CELLFit program (Becton Dickinson). All experiments were
independently performed at least 3 times. All data were statistically
analyzed by Student t test.
Apoptosis analysis To study induction of apoptosis by vitamin D3 analogs, annexin V assay (Annexin V-FITC Apoptosis Detection Kit; Pharmingen, San Diego, CA) was performed according to the manufacturer's instructions. Briefly, cells were harvested after exposure with either 1,25(OH)2D3 or Gemini-19-nor (108 and 107 M), washed twice
with PBS, incubated with FITC-conjugated annexin V and PI for 15 minutes, and measured by FACScan (Becton Dickinson). All experiments
were independently done at least 3 times. All data were statistically
analyzed by Student t test.
Western blot analysis Cells were washed twice in PBS, suspended in lysis buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 0.1% sodium dodecyl sulfate [SDS], 0.5% sodium deoxycholate, 1% NP40, 100 µg/mL phenylmethylsulfonyl fluoride, 2 µg/mL aprotinin, 1µg/mL pepstatin, and 10 µg/mL leupeptin), and placed on ice for 30 minutes. After centrifugation at 15 000g for 20 minutes at 4°C, the supernatant was collected. Protein concentrations were quantitated using the Bio-Rad assay (Bio-Rad Laboratories, Hercules, CA). Whole lysates (40 µg) were resolved by 4% to 15% SDS-polyacrylamide gel, transferred to an immobilon polyvinylidene difuride membrane (Amersham, Arlington Heights, IL) and probed with anti-p27kip1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA), anti-PTEN antibody (Santa Cruz Biotechnology), and anti-GAPDH antibody (Research Diagnostics, Flanders, NJ). The blots were developed using the enhanced chemoluminescence (ECL) kit (Amersham). Band intensity was measured using a densitometer and fold increase in expression as compared to control, untreated cells was calculated.
Effect of vitamin D3 analogs on clonal proliferation The myeloid leukemia (HL-60 and NB4), breast cancer (MCF-7), and prostate cancer (LNCaP) cells were cloned in soft agar in the presence of various concentrations of vitamin D3 analogs. All 4 Gemini vitamin D3 analogs and 1,25(OH)2D3 inhibited clonal growth of all 4 cell lines in a dose-dependent manner (Figure 2). The effective dose that inhibited 50% colony formation (ED50) was determined (Table 1). Gemini-19-nor was approximately 70-, 40 625-, 23-, and 380-fold more potent than 1,25(OH)2D3 in mediating clonal growth inhibition of HL-60, NB4, MCF-7, and LNCaP cells, respectively. Gemini 1-F-25-OH was 3- to 6-fold stronger than 1,25(OH)2D3 in suppressing clonal growth of the myeloid leukemia cell lines (HL-60 and NB4), but it had the same potency as 1,25(OH)2D3 against MCF-7 and LNCaP cells. Furthermore, the potency of Gemini 5,6-trans and Gemini 3-epi were nearly equivalent to 1,25(OH)2D3 for all 4 of the cell lines. Because Gemini-19-nor was found to be the most potent compound, all additional experiments focused on this analog.
Clonal growth of normal bone marrow-committed myeloid stem cells
(colony-forming units-granulocyte/macrophage [CFU-GM]) were not
inhibited by either 1,25(OH)2D3 or
Gemini-19-nor over a concentration range of 10
Effect of Gemini-19-nor on differentiation of leukemia cell lines The induction of expression of the cell surface antigens CD11b and CD14 occurs on HL-60 cells as they undergo differentiation. The ability of Gemini-19-nor and 1,25(OH)2D3 to induce CD11b and CD14 was analyzed using flow cytometry (Figure 4). A 4-day exposure of HL-60 cells to either 1,25(OH)2D3 (107 M) or
Gemini-19-nor (107 M) resulted in both producing nearly
90% and 80% CD11b+ and CD14+ cells,
respectively. At 108 M, Gemini-19-nor and
1,25(OH)2D3 induced 90% and 50%
CD11b+ cells, and 75% and 30% CD14+ cells,
respectively.
The capacity of HL-60 cells to produce superoxide as measured by the
reduction of NBT was another marker of differentiation that was used.
Gemini-19-nor was more potent than 1,25(OH)2D3 with either Gemini-19-nor or 1,25(OH)2D3
(10
Effect of Gemini-19-nor on induction of apoptosis Several vitamin D3 analogs and 1,25(OH)2D3 have been shown to cause apoptosis of several type of cancer cells.27,28 Gemini-19-nor and 1,25(OH)2D3 (108 M, 4 days)
induced 20% and 6% of HL-60 cells, respectively, to undergo apoptosis
(Figure 6). At a higher concentration
(107 M, 4 days), both compounds produced apoptosis of
about 20% of HL-60 cells (Figure 6).
Analysis of the cell cycle and expression of p27kip1 The effect of Gemini-19-nor and 1,25(OH)2D3 on the cell cycle of the HL-60 cells was determined. A significant accumulation (P < .05) of cells in the G1/G0 and G2/M phases of the cell cycle occurred, with a concomitant decrease in the proportion of those in S phase after 4 days of culture with either 1,25(OH)2D3 (107 M) or Gemini-19-nor (108 and
107 M) (Figure 7).
The cyclin-dependent kinase inhibitor, p27kip1, may
act as a key regulator of G1/G0 accumulation
induced by vitamin D3. Both
1,25(OH)2D3 and Gemini-19-nor induced
expression of p27kip1 in a dose-dependent manner at
4 days of exposure of HL-60 cells as determined by Western blot
analysis (Figure 8A). Consistent with the
cell cycle results, Gemini-19-nor strongly induced expression of
p27kip1 at a lower concentration (10
Induction of expression of PTEN Recent studies showed that the tumor suppressor PTEN was mutated or otherwise dysregulated in several types of human tumors. It is involved in the normal regulation of cell growth, the cell cycle, and apoptosis.29,30 We evaluated the effect of vitamin D3 compounds on protein expression of PTEN in HL-60 cells that have no mutation, deletion, or methylation of this gene.22 The nontreated, control HL-60 cells had a very low level of expression of PTEN as determined by Western blot analysis (Figures 8A,B). Expression markedly increased by 16-fold in HL-60 cells at 4 days of culture with 109 M Gemini-19-nor; under the
same culture conditions, the 1,25(OH)2D3 (109 M) did not induce detectable levels of PTEN (Figure
8A). At 107 M, Gemini-19-nor and
1,25(OH)2D3 increased the expression of PTEN by
about 32-fold and 14-fold, respectively. A time course study showed
that Gemini-19-nor enhanced expression of PTEN by 6-fold at 0.5 days
and about 36-fold at 3 days of exposure (Figure 8B).
We also examined if other inducers of differentiation of HL-60 cells
could up-regulate expression of PTEN. The phorbol diester, TPA, induces
macrophage-like differentiation31 and ATRA induces granulocyte-like differentiation of HL-60 cells. Four days of culture
with either TPA (10
A recent study revealed that a vitamin D3 compound that has 2 side chains on C-20 (Gemini) was more active than 1,25(OH)2D3 in its ability to inhibit clonal growth of malignant cells.32 Therefore, we synthesized additional novel Gemini compounds and examined their biologic effects on cancer cells. In this study, we evaluated 4 compounds from the newly synthesized family of Gemini. The Gemini-19-nor, which has 2 side chains on C-20 and the removal of the C-19, was the most potent inhibitor of clonal proliferation of myeloid leukemia, breast, and prostate cancer cells. It was more active than 1,25(OH)2D3 in these 3 types of cancers. In particular, the analog showed marked activity with the NB4 acute promyelocyte leukemia cells and was 40 625-fold more potent than 1,25(OH)2D3. Therefore, we focused on the activity of this analog compared with 1,25(OH)2D3. Previously, we reported that analogs of
1,25(OH)2D3 that had removal of their C-19
moiety (19-nor 1,25D3 analogs) were active against
prostate, breast, and myeloid leukemia cells.12,13,33,34 The 19-nor analog with the code name LH
(1,25[OH]2-16-ene-23-yne-26,27-F6-19-nor-D3) was most potent against cancer cells of breast (ED50:
MCF-7, 8 × 10 Exposure of HL-60 myeloid leukemia cells to either
1,25(OH)2D3 or Gemini-19-nor induced the
expression of the cell surface markers, CD11b and CD14. Gemini-19-nor
(10 Gemini-19-nor also mediated apoptosis. Previously, we reported that vitamin D3 analogs in concert with a RXR ligand induced apoptosis of myeloid leukemia cells and caused levels of expression of Bcl-2 to decrease suggesting an association between the 2 events.3,4,35 However, other experiments found that a vitamin D3 analog could induce apoptosis of an HL-60 variant without a reduction of cellular levels of Bcl-2.4 Another group reported that vitamin D3 compounds induced apoptosis via a novel caspase- and p53-independent pathway, and apoptosis was inhibited by forced expression of Bcl-2.36 These findings suggested that vitamin D3 might use several pathways to induce apoptosis. Previous studies showed that vitamin D3 analogs caused accumulation at the G1/G0 phase of the cell cycle, and this block may be mediated by p21waf1 and p27kip1 CDKIs.2,26,37 Gemini-19-nor D3 also produced G1- to S-phase block of the cell cycle and induced p27kip1 expression. These results support the hypothesis that CDKIs mediated at least in part the antiproliferative affects of the vitamin D3 compounds by induction of a G1/G0 accumulation. A block in the G2/M checkpoint has also been previously observed in HL-60 cells treated with 1,25(OH)2D3,38 and Gemini-19-nor was able to more potently induce a G2/M block compared to 1,25(OH)2D3. This effect has been attributed to a decrease in levels of p34(cdc),39 a protein kinase which associates with B-type cyclins and controls transition through G2/M. Therefore Gemini-19-nor may be able to decrease levels of this protein to a greater extent than 1,25(OH)2D3. Recent studies indicated that the candidate tumor suppressor,
PTEN, could block the phosphatidylinositol 3'-kinase (PI3K)/Akt signaling pathway, resulting in cell death or inhibition of growth or
both.29,30 Activated Akt mediates cell survival by
inhibition of mitochondrial release of cytochrome c, inactivation of
Forkhead transcription factors (FKHR), and phosphorylation of BAD
and caspase-9.40 The overexpression of exogenous
PTEN induced apoptosis of malignant cells.41-45 Moreover,
a genetic link between the Fas proapoptotic pathway and PTEN was
suggested, because Fas-mediated apoptosis was impaired in the germline
heterozygous PTEN+/ Several studies indicated that PTEN induced cell differentiation in glioma cells.54,55 Therefore, we analyzed inducers of myeloid differentiation other than vitamin D3. We choose TPA, a stimulator of the protein kinase C pathway that induces macrophage-like differentiation, and ATRA, which binds the nuclear hormone receptor, retinoic acid receptor and induces granulocytic differentiation. As shown in Figure 6C, TPA and ATRA significantly induced PTEN expression. These observations suggest that PTEN expression is associated with monocytic and granulocytic differentiation. We do not know if this marked increase in PTEN expression is the cause or the effect of terminal differentiation of HL-60 cells. Further studies are required to define the role PTEN plays in this process of myeloid differentiation. Taken together, the new vitamin D3 analog Gemini-19-nor D3 strongly inhibited growth of transformed cells, and produced myeloid differentiation, apoptosis, and G1/G0 arrest associated with elevated levels of p27kip1. Moreover, the vitamin D3 analog induced expression of PTEN. These observations suggest that the anticancer effects of vitamin D3 might be regulated in part via PTEN. This analog may provide an adjuvant therapy for myeloid leukemia, especially acute promyelocytic leukemia, and may be effective in other types of cancers.
Submitted March 21, 2000; accepted December 12, 2000.
Supported by US Defense and National Institutes of Health grants as well as the Lymphoma Foundation, Parker Hughes Trust, Horn Foundation, and the C. and H. Koeffler Fund.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: H. Phillip Koeffler, Division of Hematology/Oncology, Cedars-Sinai Medical Center, UCLA School of Medicine, 8700 Beverly Blvd, B-208, Los Angeles, CA 90048.
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© 2001 by The American Society of Hematology.
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  H.-J. Choi, T.-W. Chung, S.-J. Kim, S.-Y. Cho, Y.-S. Lee, Y.-C. Lee, J.-H. Ko, and C.-H. Kim The AP-2{alpha} transcription factor is required for the ganglioside GM3-stimulated transcriptional regulation of a PTEN gene Glycobiology, May 1, 2008; 18(5): 395 - 407. [Abstract] [Full Text] [PDF]
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 S. Sundaram, M. J. Beckman, A. Bajwa, J. Wei, K. M. Smith, G. H. Posner, and D. A. Gewirtz QW-1624F2-2, a synthetic analogue of 1,25-dihydroxyvitamin D3, enhances the response to other deltanoids and suppresses the invasiveness of human metastatic breast tumor cells. Mol. Cancer Ther., November 1, 2006; 5(11): 2806 - 2814. [Abstract] [Full Text] [PDF]
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I. Samudio, M. Konopleva, S. Safe, T. McQueen, and M. Andreeff Guggulsterones induce apoptosis and differentiation in acute myeloid leukemia: identification of isomer-specific antileukemic activities of the pregnadienedione structure Mol. Cancer Ther., December 1, 2005; 4(12): 1982 - 1992. [Abstract] [Full Text] [PDF]
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K. Nakagawa, Y. Sasaki, S. Kato, N. Kubodera, and T. Okano 22-Oxa-1{alpha},25-dihydroxyvitamin D3 inhibits metastasis and angiogenesis in lung cancer Carcinogenesis, June 1, 2005; 26(6): 1044 - 1054. [Abstract] [Full Text] [PDF]
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 T. Kumagai, L.-Y. Shih, S. V. Hughes, J. C. Desmond, J. O'Kelly, M. Hewison, and H. P. Koeffler 19-Nor-1,25(OH)2D2 (a Novel, Noncalcemic Vitamin D Analogue), Combined with Arsenic Trioxide, Has Potent Antitumor Activity against Myeloid Leukemia Cancer Res., March 15, 2005; 65(6): 2488 - 2497. [Abstract] [Full Text] [PDF]
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 L. V. Stewart and N. L. Weigel Vitamin D and Prostate Cancer Experimental Biology and Medicine, April 1, 2004; 229(4): 277 - 284. [Abstract] [Full Text] [PDF]
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M. Danilenko, Q. Wang, X. Wang, J. Levy, Y. Sharoni, and G. P. Studzinski Carnosic Acid Potentiates the Antioxidant and Prodifferentiation Effects of 1{alpha},25-Dihydroxyvitamin D3 in Leukemia Cells but Does Not Promote Elevation of Basal Levels of Intracellular Calcium Cancer Res., March 15, 2003; 63(6): 1325 - 1332. [Abstract] [Full Text] [PDF]
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 V. Vegesna, J. O'Kelly, M. Uskokovic, J. Said, N. Lemp, T. Saitoh, T. Ikezoe, L. Binderup, and H. P. Koeffler Vitamin D3 Analogs Stimulate Hair Growth in Nude Mice Endocrinology, November 1, 2002; 143(11): 4389 - 4396. [Abstract] [Full Text] [PDF]
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 R. J. Bernardi, D. L. Trump, W.-D. Yu, T. F. McGuire, P. A. Hershberger, and C. S. Johnson Combination of 1{alpha},25-Dihydroxyvitamin D3 with Dexamethasone Enhances Cell Cycle Arrest and Apoptosis: Role of Nuclear Receptor Cross-Talk and Erk/Akt Signaling Clin. Cancer Res., December 1, 2001; 7(12): 4164 - 4173. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
20°C and protected from light.
12-O-Tetradecanoylphorbol 13-acetate (TPA) and ATRA were purchased from
Sigma (St Louis, MO).
,
Gemini-3-epi; O, Gemini-19-nor; X, Gemini-1-F-25-OH. Each point
represents a mean of 3 independent experiments with triplicate
dishes.
