BRIEF COMMUNICATION
Growth Control of Human Colon Cancer Cells by Vitamin D and Calcium In Vitro Heide S. Cross,* Margit Pavelka, Jan Slavik, Meinrad Peterlik
Vol. 84, No. 17, September 2, 1992
1,25-(OH)2D3 {see data above). It also inhibited DNA synthesis under conditions in which 1,25-(OH)2D3 was ineffective, i.e., at 1.80 mM [Ca2 + ] o in preconfluent cells and at 0.25 mM [Ca2 + ] 0 in confluent cells (Fig. 1). Only at 1.80 mM [Ca2 + ] o in confluent cells was Ro 23-7553 not more potent than its natural congener (Fig. 1). In contrast to 1,25-(OH)2D3, Ro 23-7553 exhibited no mitogenic potential in confluent CaCo-2 cells (cf. Fig. 1). Concentrations (means ± SE) of [Ca 2+ ]j in preconfluent CaCo-2 cells were 28 ± 5 nM when the cells were maintained at 0.25 mM [Ca2 + ] 0 and 48 ± 8 nM when they were maintained at 1.80 mM [Ca2 + ] 0 (& 15 determinations). The corresponding values for confluent cells were 50 ± 5 nM and 124 ± 8 nM. Both 1,25-(OH)2D3 and Ro 23-7553 significantly (P^s.OOl) raised the [Ca2+]j levels (up to three times above control levels) after a 5-minute exposure and under any growth condition, but both failed to do so when cells were continuously exposed to the agents from day 1 of culture onward. This observation indicates that the vitamin D compounds do not exert their antiproliferative effect through a change in steady-state [Ca 2+ ]; levels [see also (72)]. As a result of enhanced proliferation, which is observed during logarithmic growth in low [Ca2 + ] 0 concentrations or after confluence in normal [Ca2 + ] o medium, cells grow in highly disordered multilayers (Fig. 2, A and D). The antiproliferative effects of 1,25(OH)2D3 and of Ro 23-7553 (cf. Fig. 1)
Downloaded from http://jnci.oxfordjournals.org/ at Harvard University on July 6, 2015
Vitamin D deficiency and reduced intestinal calcium absorption are considered risk factors for the development of colorectal cancer in humans {1-3). To further test this hypothesis, we examined the effects of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] and of a synthetic, nonhypercalcemic vitamin D analogue, 1,25-dihydroxy-A1623yne-vitamin D 3 (Ro 23-7553), on proliferation of human colon adenocarcinoma-derived CaCo-2 cells grown in different concentrations of extracellular calcium ([Ca2 + ]0). We initiated a series of studies [cf. (4)] to address the following questions: 1) Can growth of the vitamin D receptor-positive (5) CaCo-2 cells be modulated by variations in [Ca2 + ] o and intracellular free calcium concentrations ([Ca2 + ]i)? 2) If so, would the antiproliferative effect of 1,25-(OH)2D3 depend on the different growth conditions induced? 3) To what extent is the growth of CaCo-2 cells affected by Ro 23-7553, which has a high intrinsic antiproliferative potential in other systems [cf. (6,7)]1 Culture of CaCo-2 cells, cell proliferation assays, and alkaline phosphatase assays were done as described previously (4). [Ca 2+ ] o levels were measured by fluorescence titration with a Nova 9 Calcium Analyzer (Nova Biomedical, Waltham, Mass.). [Ca 2+ ]| levels were measured by the fura-2 method [cf. (8)] in CaCo-2 cells grown on glass coverslips in tissue culture dishes (five coverslips per dish).
1,25-(OH),D 3 was a gift of Hoffmann-La Roche (Basel, Switzerland). Ro 23-7553 was provided by Dr. Milan Uskokovic, Hoffmann-La Roche (Nutley, N.J.). The fura-2 acetoxymethylester was obtained from Molecular Probes (Eugene, Ore.). All other reagents were from Sigma Chemical Co. (St. Louis, Mo.). Statistical analysis was performed using Student's unpaired t test. Significance of difference was assumed at P=£.O5. In the absence of any steroid hormone, cell numbers in log-phase cultures between day 2 and day 7 were significantly (P=£.O5) higher at 0.25 mM than at 1.80 mM [Ca2 + ] o (up to 50% on day 6). In contrast, in confluent cultures, growth at 1.80 mM [Ca2 + ] o was increased up to 100% (on day 14, P-
I
120 160
i-
(2) GARLAND C, SHEKELLE RB,
BARRETT-
CONNOR E, ET AL: Dietary vitamin D and calcium and risk of colorectal cancer: A 19year prospective study in men. Lancet 1:307-309, 1985 (3) LIPKIN M, NEWMARK HL, HELLOFF G, EDS:
Calcium, Vitamin D and Prevention of Colon Cancer. Boca Raton, Fla: CRC Press, 1991 (4) CROSS HS, HUBER C, PETERLIK M: Anti-
proliferative effect of 1,25-dihydroxyvitamin D} and its analogs on human colon adenocarcinoma cells (CaCo-2): Influence of extracellular calcium. Biochem Biophys ResCommun 179:57-62, 1991 (5) GIULIANO AR, FRANCESCHI RT, WOOD RJ:
Characterization of the vitamin D receptor from the Caco-2 human colon carcinoma cell line: Effect of cellular differentiation. Arch Biochem Biophys 285:261-269, 1991 (6) ZHOU JY, NORMAN AW, LUBBERT M, ET
14 13 12 11 10 9 8
14 13 12 11 10 9 8
- log [M]
AL: Novel vitamin D analogs that modulate leukemic cell growth and differentiation with little effect on either intestinal calcium absorption or bone calcium mobilization. Blood 74:82-93, 1989 (7) KISTLER A, GALLI B, HORST R, ET AL:
Effects of vitamin D derivatives on soft tissue calcification in neonatal and calcium mobilization in adult rats. Arch Toxicol 63:394-400, 1989
were accompanied by a shift in this growth pattern to a more monolayerlike appearance (Fig. 2, B, C, E, and F). The reversion to a normal growth phenotype suggests that vitamin D not only inhibits growth but also induces differentiation in CaCo-2 cells as in other normal or malignant cells [e.g., {13-15)]. This notion is supported by our observations on the activity of alkaline phosphatase, a differentiation marker for colonic epithelial cells {16). Independent of lCa2 + ] o , the alkaline phosphatase activity amounted to 9 ± 2 and 39 ± 2 U/mg protein (six or more determinations), respectively, in preconfluent and confluent CaCo-2 cells and was significantly {P^.05) augmented by both vitamin D sterols between 0.1 and 10 nM (maximal stimulation, 5=100%). In conclusion, 1,25-(OH),D 3 is a potent modulator of the growth of cultured CaCo-2 cells. The direction of this modulation is related to the proliferative potential of the cells, which largely depends on [Ca2 + ] o levels. Because the antimitogenic potency of the synthetic, nonhypercalcemic vitamin D analogue Ro 23-7553 in general exceeds that of 1,25-(OH),D 3 , Ro 23-7553 might be useful in future vitamin D therapy for colon cancer. 1356
Fig. 2. Influence of vitamin D compounds on growth pattern of CaCo-2 cells. A. B. and C: day 5 of culture at 0.25 mM [Ca2+],,. Additions to cultures: A, none; B, 10 nM 1.25-(OH),D3; C. 10 nM Ro "237553. D, E, and F: day 14 of culture at 1.80 mM [Ca2 + ] o . Additions to cultures: D. none: E. 10 nM I.25-(OH),D,: F. 10 nM Ro 23-7553. For light microscopy, cells were fixed in situ, embedded in Epon 812. stained with toluidine blue in acetone, and examined with a Leitz Orthoplan microscope equipped with a Leitz Orthomat E automatic camera system (original magnification X620).
Journal of the National Cancer Institute
Downloaded from http://jnci.oxfordjournals.org/ at Harvard University on July 6, 2015
E" 200
and vitamin D reduce the likelihood of colon cancer? Int J Epidemiol 9:227-231, 1980
(8) GRYNKIEWICZ G. POENIE M. TSIEN RY: A
keratinocytes grown in serum-free conditions. J Invest Dermatol 86:709-714, 1986
(12) PILLAI S, BIKLE DD: Role of intracellular-
new generation of Ca 2+ indicators with greatly improved fluorescence properties. J Biol Chem 260:3440-3450. 1985
free calcium in the cornified envelope formation of keratinocytes: Differences in the mode of action of extracellular calcium and 1.25-dihydroxyvitamin D,. J Cell Physiol 146:94-100. 1991
(9) BUSET M. LlPKlN M. WINAWER S, ET AL:
Inhibition of human colonic epithelial cell proliferation in vivo and in vitro by calcium. Cancer Res 46:5426-5430, 1986
(15) PILLAI S. BIKLE DD, HINCENBERGS M. ET
AL: Biochemical and morphological characterization of growth and differentiation of normal human neonatal keratinocytes in a serum-free medium. J Cell Physiol 134:229-237. 1988
(13) ABE E. MIYAURA C, SAKAGAMI H: Differen-
tiation of mouse myeloid leukemia cells induced by 1 alpha. 25-dihydroxyvitamin D3. Proc Natl Acad Sci U S A 78:49904994, 1981
(10) LIPKIN M, FRIEDMAN E. WINAWER SJ, ET
AL: Colonic epithelial cell proliferation in responders and nonresponders to supplemental dietary calcium. Cancer Res 49:248-254, 1989 ( / / ) WHITFIELD JF: Calcium, Cell Cycles and Cancer. Boca Raton, Fla: CRC Press, 1990
(16) SCHWARTZ B, AVIVI C, LAMPRECHT SA: Iso-
(14) SMITH EL, WALWORTH NC, HOLICK MF:
lation and characterization of normal and neoplastic colonic epithelial cell populations. Gastroenterology 100:692-702, 1991
Effect of 1 alpha. 25-dihydroxyvitamin D, on the morphologic and biochemical differentiation of cultured human epidermal
1991 ONCOLOGY OVERVIEWS The International Cancer Information Center of the National Cancer Institute announces five new clinical ONCOLOGY OVERVIEWS. ONCOLOGY OVERVIEWS are specialized bibliographies with abstracts, each referencing 200-600 recent publications on a cancer topic of high current interest, drawn from over 4,000 sources. Leading researchers in the field covered by each OVERVIEW review and select the most relevant and significant abstracts for each topic, resulting in a tightly focused, quick reference to the most recent cancer literature.
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Antiangiogenesis: A Potential Therapeutic Strategy, Judah Folkman, MD, editor.
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Current Management of Childhood Leukemia, Alvin M. Mauer, MD, editor.
LJ
017-042-00286-9
$10.00
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017-042-00289-3
$6.00
Therapeutic Use of Interleukin-2, Paul M. Sondel, MD, PhD, editor. Childhood Brain Tumors, R. J. Packer, MD, editor.
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Growth Control of Human Colon Cancer Cells by Vitamin D and Calcium In Vitro Heide S. Cross,* Margit Pavelka, Jan Slavik, Meinrad Peterlik
Vol. 84, No. 17, September 2, 1992
1,25-(OH)2D3 {see data above). It also inhibited DNA synthesis under conditions in which 1,25-(OH)2D3 was ineffective, i.e., at 1.80 mM [Ca2 + ] o in preconfluent cells and at 0.25 mM [Ca2 + ] 0 in confluent cells (Fig. 1). Only at 1.80 mM [Ca2 + ] o in confluent cells was Ro 23-7553 not more potent than its natural congener (Fig. 1). In contrast to 1,25-(OH)2D3, Ro 23-7553 exhibited no mitogenic potential in confluent CaCo-2 cells (cf. Fig. 1). Concentrations (means ± SE) of [Ca 2+ ]j in preconfluent CaCo-2 cells were 28 ± 5 nM when the cells were maintained at 0.25 mM [Ca2 + ] 0 and 48 ± 8 nM when they were maintained at 1.80 mM [Ca2 + ] 0 (& 15 determinations). The corresponding values for confluent cells were 50 ± 5 nM and 124 ± 8 nM. Both 1,25-(OH)2D3 and Ro 23-7553 significantly (P^s.OOl) raised the [Ca2+]j levels (up to three times above control levels) after a 5-minute exposure and under any growth condition, but both failed to do so when cells were continuously exposed to the agents from day 1 of culture onward. This observation indicates that the vitamin D compounds do not exert their antiproliferative effect through a change in steady-state [Ca 2+ ]; levels [see also (72)]. As a result of enhanced proliferation, which is observed during logarithmic growth in low [Ca2 + ] 0 concentrations or after confluence in normal [Ca2 + ] o medium, cells grow in highly disordered multilayers (Fig. 2, A and D). The antiproliferative effects of 1,25(OH)2D3 and of Ro 23-7553 (cf. Fig. 1)
Downloaded from http://jnci.oxfordjournals.org/ at Harvard University on July 6, 2015
Vitamin D deficiency and reduced intestinal calcium absorption are considered risk factors for the development of colorectal cancer in humans {1-3). To further test this hypothesis, we examined the effects of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] and of a synthetic, nonhypercalcemic vitamin D analogue, 1,25-dihydroxy-A1623yne-vitamin D 3 (Ro 23-7553), on proliferation of human colon adenocarcinoma-derived CaCo-2 cells grown in different concentrations of extracellular calcium ([Ca2 + ]0). We initiated a series of studies [cf. (4)] to address the following questions: 1) Can growth of the vitamin D receptor-positive (5) CaCo-2 cells be modulated by variations in [Ca2 + ] o and intracellular free calcium concentrations ([Ca2 + ]i)? 2) If so, would the antiproliferative effect of 1,25-(OH)2D3 depend on the different growth conditions induced? 3) To what extent is the growth of CaCo-2 cells affected by Ro 23-7553, which has a high intrinsic antiproliferative potential in other systems [cf. (6,7)]1 Culture of CaCo-2 cells, cell proliferation assays, and alkaline phosphatase assays were done as described previously (4). [Ca 2+ ] o levels were measured by fluorescence titration with a Nova 9 Calcium Analyzer (Nova Biomedical, Waltham, Mass.). [Ca 2+ ]| levels were measured by the fura-2 method [cf. (8)] in CaCo-2 cells grown on glass coverslips in tissue culture dishes (five coverslips per dish).
1,25-(OH),D 3 was a gift of Hoffmann-La Roche (Basel, Switzerland). Ro 23-7553 was provided by Dr. Milan Uskokovic, Hoffmann-La Roche (Nutley, N.J.). The fura-2 acetoxymethylester was obtained from Molecular Probes (Eugene, Ore.). All other reagents were from Sigma Chemical Co. (St. Louis, Mo.). Statistical analysis was performed using Student's unpaired t test. Significance of difference was assumed at P=£.O5. In the absence of any steroid hormone, cell numbers in log-phase cultures between day 2 and day 7 were significantly (P=£.O5) higher at 0.25 mM than at 1.80 mM [Ca2 + ] o (up to 50% on day 6). In contrast, in confluent cultures, growth at 1.80 mM [Ca2 + ] o was increased up to 100% (on day 14, P-
I
120 160
i-
(2) GARLAND C, SHEKELLE RB,
BARRETT-
CONNOR E, ET AL: Dietary vitamin D and calcium and risk of colorectal cancer: A 19year prospective study in men. Lancet 1:307-309, 1985 (3) LIPKIN M, NEWMARK HL, HELLOFF G, EDS:
Calcium, Vitamin D and Prevention of Colon Cancer. Boca Raton, Fla: CRC Press, 1991 (4) CROSS HS, HUBER C, PETERLIK M: Anti-
proliferative effect of 1,25-dihydroxyvitamin D} and its analogs on human colon adenocarcinoma cells (CaCo-2): Influence of extracellular calcium. Biochem Biophys ResCommun 179:57-62, 1991 (5) GIULIANO AR, FRANCESCHI RT, WOOD RJ:
Characterization of the vitamin D receptor from the Caco-2 human colon carcinoma cell line: Effect of cellular differentiation. Arch Biochem Biophys 285:261-269, 1991 (6) ZHOU JY, NORMAN AW, LUBBERT M, ET
14 13 12 11 10 9 8
14 13 12 11 10 9 8
- log [M]
AL: Novel vitamin D analogs that modulate leukemic cell growth and differentiation with little effect on either intestinal calcium absorption or bone calcium mobilization. Blood 74:82-93, 1989 (7) KISTLER A, GALLI B, HORST R, ET AL:
Effects of vitamin D derivatives on soft tissue calcification in neonatal and calcium mobilization in adult rats. Arch Toxicol 63:394-400, 1989
were accompanied by a shift in this growth pattern to a more monolayerlike appearance (Fig. 2, B, C, E, and F). The reversion to a normal growth phenotype suggests that vitamin D not only inhibits growth but also induces differentiation in CaCo-2 cells as in other normal or malignant cells [e.g., {13-15)]. This notion is supported by our observations on the activity of alkaline phosphatase, a differentiation marker for colonic epithelial cells {16). Independent of lCa2 + ] o , the alkaline phosphatase activity amounted to 9 ± 2 and 39 ± 2 U/mg protein (six or more determinations), respectively, in preconfluent and confluent CaCo-2 cells and was significantly {P^.05) augmented by both vitamin D sterols between 0.1 and 10 nM (maximal stimulation, 5=100%). In conclusion, 1,25-(OH),D 3 is a potent modulator of the growth of cultured CaCo-2 cells. The direction of this modulation is related to the proliferative potential of the cells, which largely depends on [Ca2 + ] o levels. Because the antimitogenic potency of the synthetic, nonhypercalcemic vitamin D analogue Ro 23-7553 in general exceeds that of 1,25-(OH),D 3 , Ro 23-7553 might be useful in future vitamin D therapy for colon cancer. 1356
Fig. 2. Influence of vitamin D compounds on growth pattern of CaCo-2 cells. A. B. and C: day 5 of culture at 0.25 mM [Ca2+],,. Additions to cultures: A, none; B, 10 nM 1.25-(OH),D3; C. 10 nM Ro "237553. D, E, and F: day 14 of culture at 1.80 mM [Ca2 + ] o . Additions to cultures: D. none: E. 10 nM I.25-(OH),D,: F. 10 nM Ro 23-7553. For light microscopy, cells were fixed in situ, embedded in Epon 812. stained with toluidine blue in acetone, and examined with a Leitz Orthoplan microscope equipped with a Leitz Orthomat E automatic camera system (original magnification X620).
Journal of the National Cancer Institute
Downloaded from http://jnci.oxfordjournals.org/ at Harvard University on July 6, 2015
E" 200
and vitamin D reduce the likelihood of colon cancer? Int J Epidemiol 9:227-231, 1980
(8) GRYNKIEWICZ G. POENIE M. TSIEN RY: A
keratinocytes grown in serum-free conditions. J Invest Dermatol 86:709-714, 1986
(12) PILLAI S, BIKLE DD: Role of intracellular-
new generation of Ca 2+ indicators with greatly improved fluorescence properties. J Biol Chem 260:3440-3450. 1985
free calcium in the cornified envelope formation of keratinocytes: Differences in the mode of action of extracellular calcium and 1.25-dihydroxyvitamin D,. J Cell Physiol 146:94-100. 1991
(9) BUSET M. LlPKlN M. WINAWER S, ET AL:
Inhibition of human colonic epithelial cell proliferation in vivo and in vitro by calcium. Cancer Res 46:5426-5430, 1986
(15) PILLAI S. BIKLE DD, HINCENBERGS M. ET
AL: Biochemical and morphological characterization of growth and differentiation of normal human neonatal keratinocytes in a serum-free medium. J Cell Physiol 134:229-237. 1988
(13) ABE E. MIYAURA C, SAKAGAMI H: Differen-
tiation of mouse myeloid leukemia cells induced by 1 alpha. 25-dihydroxyvitamin D3. Proc Natl Acad Sci U S A 78:49904994, 1981
(10) LIPKIN M, FRIEDMAN E. WINAWER SJ, ET
AL: Colonic epithelial cell proliferation in responders and nonresponders to supplemental dietary calcium. Cancer Res 49:248-254, 1989 ( / / ) WHITFIELD JF: Calcium, Cell Cycles and Cancer. Boca Raton, Fla: CRC Press, 1990
(16) SCHWARTZ B, AVIVI C, LAMPRECHT SA: Iso-
(14) SMITH EL, WALWORTH NC, HOLICK MF:
lation and characterization of normal and neoplastic colonic epithelial cell populations. Gastroenterology 100:692-702, 1991
Effect of 1 alpha. 25-dihydroxyvitamin D, on the morphologic and biochemical differentiation of cultured human epidermal
1991 ONCOLOGY OVERVIEWS The International Cancer Information Center of the National Cancer Institute announces five new clinical ONCOLOGY OVERVIEWS. ONCOLOGY OVERVIEWS are specialized bibliographies with abstracts, each referencing 200-600 recent publications on a cancer topic of high current interest, drawn from over 4,000 sources. Leading researchers in the field covered by each OVERVIEW review and select the most relevant and significant abstracts for each topic, resulting in a tightly focused, quick reference to the most recent cancer literature.
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* 6601 I I Y E S , please send me the following:
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017-042-00288-5
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The Cell Cycle in Cancer Prognosis, Arthur B. Pardee, PhD, editor.
•
017-042-00285-1
$5.50
Antiangiogenesis: A Potential Therapeutic Strategy, Judah Folkman, MD, editor.
D
017-042-00287-7
$7.50
Current Management of Childhood Leukemia, Alvin M. Mauer, MD, editor.
LJ
017-042-00286-9
$10.00
•
017-042-00289-3
$6.00
Therapeutic Use of Interleukin-2, Paul M. Sondel, MD, PhD, editor. Childhood Brain Tumors, R. J. Packer, MD, editor.
The total cost of my order is $_ _. International customers please add 25%. Prices include regular domestic postage and handling and are subject to change. Please Choose Method of Payment: (Company or Personal Name)
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Mail To: New Orders, Superintendent of Documents P.O. Box 371954, Pittsburgh, PA 15250-7954 BRIEF COMMUNICATION 1357
Downloaded from http://jnci.oxfordjournals.org/ at Harvard University on July 6, 2015
To purchase an ONCOLOGY OVERVIEW, please check the appropriate box, complete the order form, and mail the entire page with remittance to the address at the bottom of the form.
