1660 Gut 1992; 33: 1660-1663 Vitamin D and its metabolites inhibit cell
proliferation in human rectal mucosa and a colon Gut: first published as 10.1136/gut.33.12.1660 on 1 December 1992. Downloaded from cancer cell line
M G Thomas, S Tebbutt, R C N Williamson
Abstract because ofits ability to switch cellular activity (in Like calcium, vitamin D may protect against various cancer cell lines) from proliferation to colorectal neoplasia as it reduces epithelial cell differentiation.'>6 In particular, the human proliferation and induces differentiation. colon cancer cell lines HT-29'7 '1 and LOVO'4 Although its therapeutic use is limited by its possess high affinity receptors for the active effects on calcium metabolism, analogues metabolite ofvitamin D3 (1,25 (OH)2 D3), which such as calcipotriol produce little hyper- suppresses cell growth and induces changes calcaemia. Stathmokinetic and immuno- indicative of differentiation. Receptors for 1,25 histochemical techniques were used to study (OH)2 D3 have also been found in normal human the effect of 1,25 (OH)2 D3 and its analogues on colon.'9 Although the therapeutic use of 1,25 cell proliferation in human rectal mucosa and a (OH)2 D3 and 1-alpha hydroxycholecalciferol is colon cancer cell line. Paired sigmoidoscopic limited by their profound effects on calcium biopsy specimens were obtained from 17 con- metabolism,20 analogues such as calcipotriol trol patients and five patients with familial (MC-903, a secosterol)2' 23 are now available adenomatous polyposis. Explants were estab- which have limited hypercalcaemic and hyper- lished in organ culture, with or without the calciuric effects. addition of vitamin D. Proliferation was asses- We have used stathmokinetic and immuno- sed using (1) metaphase arrest to determine histochemical techniques to study the effect of the crypt cell production rate (CCPR) and (2) three agents - 1,25 (OH)2 D3, vitamin D2, and Ki-67 monoclonal antibody directed against an calcipotriol - on cell proliferation in human antigen present in proliferating cells. 1,25 rectal epithelium. We have studied both macro- (OH)2 D3 in concentrations of 1 tM-100 pM scopically normal rectal tissue in control patients (10--10-1o M) reduced the CCPR (cells/ and those with familial adenomatous polyposis crypt/hour) from 4.74 to 2.15-2.67 (p<0.001), (at increased risk ofneoplasia) and HT-29 cells in
and the Ki-67 labelling index from 7.28-3.74 culture. http://gut.bmj.com/ (p (10-5 M to 10-9 M) produced a clearcut dose 17 patients (mean age 60 years, range 37-80 on October 2, 2021 by guest. Protected copyright. response inhibition of HT-29 cell growth. years) with macroscopically normal rectal Thus, vitamin D and its metabolites inhibit mucosa who were attending the outpatient clinic proliferation in normal and premalignant rectal with incidental anal conditions. Biopsy speci- epithelium and suppress growth in a colorectal mens were also obtained from five patients (mean cancer cell line. age 38 years, range 35-40 years) with familial (Gut 1992; 33: 1660-1663) adenomatous polyposis who had previously undergone total abdominal colectomy with ileorectal anastomosis and were attending There is a clear link between the intensity of cell regularly for follow up. One specimen from each proliferation and susceptibility to neoplasia.' 2 In patient was examined histologically to exclude the large intestine, surgical or dietary manipula- mucosal disease, notably microadenoma in tions that stimulate cell growth generally pro- familial adenomatous polyposis.. The other mote experimental colorectal carcinogenesis,3-5 biopsy specimen was maintained in organ whereas mucosal hypoplasia (for example, by culture with or without the addition ofvitamin D Department of Surgery, Royal Postgraduate defunctioning colostomy) has a protective metabolites. In four control patients, tissue was Medical School, effect.6 Patients with colonic adenoma or frozen to - 80°C (after organ culture) for subse- Hammersmith Hospital, carcinoma show increased labelling indices in quent immunohistochemistry. London ethical committee was M G Thomas 'normal' biopsy specimens taken from multiple Local approval S Tebbutt sites,7 and those with ulcerative proctocolitis obtained, and all patients had given fully R C N Williamson have increased crypt cell proliferation even in informed consent to the procedures. Correspondence to: quiescent disease.8 Thus, the cytokinetic status Professor R C N Williamson, Director of Surgery, Royal of the epithelium9'0 could reflect a subject's Postgraduate Medical School, susceptibility to colorectal neoplasia. VITAMIN D PREPARATIONS Hammersmith Hospital, Du Cane Road, London. Calcium may protect against colorectal cancer 1,25 (OH)2 D3 and ergocalciferol (vitamin D2) Accepted for publication by reducing epithelial cell turnover."12 Vitamin were donated by Roche Products Ltd (Welwyn 31 May 1992 D could be another chemopreventative agent'3 Garden City, UK). The secosterol calcipotriol Vitamin D and its metabolites inhibitcellproliferation in human rectal mucosa and a colon cancercell line 1661 (MC-903) was supplied by Leo Pharmaceutical Ki-67 antibody is directed against an antigen Products Ltd (Ballerup, Denmark). expressed in proliferating cells.2627 Slides were Stock solutions of test compounds were pre- developed in diamino benzadine-hydrogen pared in absolute alcohol and stored at -200C peroxide (DAB) in Tris buffer for 5 minutes, Gut: first published as 10.1136/gut.33.12.1660 on 1 December 1992. Downloaded from until use. Control medium was prepared with a then washed in tap water. Haematoxylin was similar dilution of alcohol and in a pilot study used as a counterstain. had no obvious effect on proliferation. To assess rectal epithelial proliferation, the labelling index was determined in at least 15 crypts per section.28 The labelling index was ORGAN CULTURE calculated as the ratio of Ki-67 positive to Rectal biopsy specimens were divided into tiny negative cells per crypt. The mean values of explants and orientated mucosal surface upper- these counts were compared using a paired most on a metal grid within an organ culture dish Student's t test (each case acting as its own (Lux Laboratories). Explants were cultured as control). paired samples in standard culture medium (CMRL 1066, Gibco, Paisley, UK) or in standard culture medium to which vitamin D CELL CULTURE metabolites had been added: 1,25 (OH)2 D3 at HT-29 cells were maintained as a monolayer of concentrations of 1 F,M (10-6 M), 10 nM (10-8 cells in Dulbecco's modified Eagle's medium M), or 100 pM (10- '0 M); ergocalciferol (vitamin (DMEM, Flow Laboratories, High Wycombe, D2) at 10 nM (10-8 M); and calcipotriol (MC- UK) with 10% fetal calf serum, 100 U/ml 903) at 100 mM (10 M). Thus each biopsy penicillin, and 100 ,ug/ml streptomycin specimen acted as its own control. The concen- (Serolabs, Crawley, UK). Cells were incubated trations of vitamin D chosen were in a similar at 37°C in 95% 02 and 5% C02, and the medium range to previous studies,"'8 and the lowest dose was changed every 2 days. At 80-90% visual of 1,25 (OH)2 D3 is probably within the human confluence, the cells were trypsinised with physiological range. In total, 34 biopsy speci- 0.25% trypsin and EDTA (Flow Laboratories). mens were obtained from controls and 10 from After washing in phosphate buffer saline (PBS), familial adenomatous polyposis patients, and aliquots of 1x 10 cells were added to a six well each biopsy was divided into several explants tissue culture plate (Gibco). The cells were (between six and 15). cultured in DMEM, with or without the addition The concentration of 1,25 (OH)2 D3 in the of calcipotriol (MC-903) at a final concentration standard culture medium was 6 pM (6x 10-12 of 10- to 10-9 M. All plates were set up in M), as determined by batch testing the fetal calf quadruplicate. serum. The organ culture dishes were sealed in After viability assessment using trypan blue an atmosphere of 95% 02 and 5% CO2 at a exclusion, the total cell number was determined temperature of37°C and were then gently rocked at 7, 14, and 21 days by counting at least three http://gut.bmj.com/ at 5 cycles per minute.24 After 15 hours, samples from each concentration using a haemo- vincristine 0.6 ,ug/ml (Oncovin, Eli Lilly, cytometer. Results were analysed using one way Basingstoke, UK) was added to the culture analysis of variance and the Mann-Whitney U medium to induce metaphase arrest within the test. colonic crypts.824 Explants were removed one, two, and three hours later, fixed in Carnoy's fluid, and stored in 70% alcohol. Biopsy tissues Results on October 2, 2021 by guest. Protected copyright. were rehydrated later in successive solutions of 50%, 25%, and 10% alcohol. After acid hydro- ORGAN CULTURE lysis in 1 M HC1 at 60°C for 6 minutes, explants The overall mean (SEM) crypt cell production were stained with Schiff's reagent. At least 20 rate (CCPR) in 17 normal patients (Figure) was crypts were microdissected from each speci- 4.74 (0.25) cells/crypt/hour, with a range of men,25 and the number of arrested metaphases 2.85-7.07. This value is similar to but slightly per crypt was plotted against time from lower than our previously reported results.824 vincristine administration. The slope of this line Explants showed excellent preservation of crypt (determined by least squares linear regression architecture, with an infection rate and crypt analysis) gave a value for the crypt cell produc- necrosis rate of less than 1%. The active form of tion rate in cells/crypt/hour." vitamin D3 (1,25 (OH)2 D3) consistently halved the overall CCPR in normal tissue irrespective of the dose used (analysis of variance). Thus, at IMMUNOHISTOCHEMISTRY 1 FiM the CCPR was reduced from 4.96-2-15 After 18 hours' organ culture in medium with or cells/crypt/hour, at 10 nM it was reduced from without the addition of 1,25 (OH)2 D3 (100 pM), 4.71-2. 10 cells/crypt/hour, and at 100 pM it was paired mucosal explants were mounted and then reduced from 4.862-67 cells/crypt/hour. The 3 [im cryostat sections were cut and air dried data showed a dose-response trend when indi- before blocking in H202 (0 22%) and methanol vidual results were compared with their own for 5 minutes. After washing in tap water and paired controls (as opposed to the overall control then Tris buffer (pH 7.3) for 5 minutes (x3), values), percentage reductions being 57%, 55%, sections were stained using a three stage peroxi- and 45% with diminishing doses of 1,25 (OH)2 dase procedure in which Ki-67 monoclonal anti- D3 (57% v 45%: p<0 05). Ergocalciferol body (1:50 in Tris buffer), biotinylated rabbit (vitamin D2) at a dose of 10 nM reduced CCPR in anti-mouse (1:300 in Tris buffer), and avidin- the normal rectal tissue from 5 27-2.74 cells/ biotin complex (Dakopatts) were applied. The crypt/hour. Calcipotriol (10-7 M) also reduced 1662 Thomas, Tebbutt, Williamson 10- p< 005 in vitro CCPR in normal and premalignant 0 Control human rectal epithelium by vitamin D and its 8 Test group metabolites. The data have been verified Gut: first published as 10.1136/gut.33.12.1660 on 1 December 1992. Downloaded from 8- immunohistochemically using the monoclonal antibody Ki-67 to show a reduction in the -c p<005 p<0-05 labelling index. The presence of vitamin D3 D 6 p<0-001 receptors in normal and malignant colorectal tissue,'9 together with the increased colonic U) T T absorption of calcium after small bowel resec- 0 tion,29 had previously suggested that the colon a-CC 4 could be a target organ for 1,25 (OH)2 D3. We have now shown an in vitro physiological 2 1 the crypt labelling index (measured using Ki-67) the fact would require correlation with changes http://gut.bmj.com/ from a control value of7-28 (0 68) (mean (SEM)) in a measurable gene product.'4 The almost equal to 3-74 (0-64) (n=4, p<001, paired Student's t inhibition of CCPR by ergocalciferol compared test) with the active metabolite (1,25 (OH)2 D3) argues against a receptor mediated effect, since non-hydroxylated vitamin D2 binds poorly to the CELL CULTURE vitamin D receptor. An HT-29 cells in control media showed a rapid log alternative hypothesis is that our observed on October 2, 2021 by guest. Protected copyright. phase growth in the first 14 days of culture. effect on proliferation may be a non-genomic Thereafter cells continued to proliferate effect, possibly related to calcium ion transport but at a diminished rate (possibly because (as suggested by the presence of cytosolic of cell to cell contact). After 21 days' culture calcium binding proteins in the colonic mucosa there were 8-29 (0 74)x 106 cells in control of short bowel syndrome)34 or to a post-receptor cultures (mean (SEM)). Calcipotriol produced a binding effect.20 In support of this, both clearcut dose dependent inhibition of prolifera- verapamil and glucocorticoids (which influence tion. After 21 days' culture calcipotriol reduced calcium transport) affect the morphological the number of cells to 0-06% of the control value changes induced by 1,25 (OH)2 D3 in LOVO at 10-5 M (p<0 01), to 0-11% at 10-6 M cells. '4 (p<0 01), to 8-45% at 10-7 M (p<0 001), and Irrespective of the mode of action, vitamin D, to 94 10% at 1-8 M (see Table). its metabolites and analogues inhibit colonic epithelial proliferation, at least in vitro. In premalignant conditions associated with an Discussion accelerated epithelial cell proliferation, a reduc- We have shown for the first time an inhibition of tion in CCPR might be beneficial. Further studies to evaluate the mode of action and The effect ofcalcipotriol on HT-29 cell growth. Values are total cell numbers (mean (SEM)) possible therapeutic use of vitamin D meta- Concentration bolites and analogues are obviously required. ofcalcipotriol The authors thank Dr Lise Binderup from Leo Pharmaceuticals, (M) Day 7 Day 14 Day 21 who provided us with the secosterol Calcipotriol (MC 903). Financial assistance for this work was provided by the Nutritional Control 12 84 (0 88)x 105 64-75 (3 97)x 105 82-92 (1 45)x 105 consultative Panel. I x 10- 3-34 (1l09)x 104** 4 40 (0 53)x 103** 4 70 (1-37)x 103** lx lo-6 6-40 (I 00)x 104** 10-90 (3 48)x 104** 9-00 (1-50)x 103** 1 Lipkin M. Biomarkers of increased susceptability to gastro- l X l0-7 1 97 (0-73)x 105*** 4-44 (1 80)x IO5*** 7 01 (2 92)x lI5*** intestinal cancer: new applications of studies of cancer 1xl0-8 4.50 (0.23)x 105* 48-70 (144)x 105* 78-00 (2 49)x105 prevention in human subjects. CancerRes 1988; 48: 235-45. I x io- 8-00 (0 41)x 105* 45.00 (0 81)x 105* 1 10.00 (001)x 105 2 Rainey JB, Davies PW, Williamson RCN. Relative effects of ileal resection and bypass on intestinal adaptation and Significance v control (analysis of variance): *p<005; **p<0o01; ***p<0.0001. carcinogenesis. BrjSurg 1984; 71: 197-202. Vitamin D and its metabolites inhibit cellproliferation in human rectalmucosa anda colon cancercell line 1663 3 Williamson RCN, Bauer FLR, Ross JS, Watkins JB, Malt Chipponi J, et al. 1,25 dihydroxy vitamin D3 receptors and RR. Enhanced colonic carcinogenesis with azoxymethane in human colon adenocarcinoma. BrJ Surg 1991; 78: 435-9. rats after pancreatobiliary diversion to mid small bowel. 20 Binderup L, Latini S, Binderup E, Bretting C, Calverley M, Gastroenterology 1976; 76: 1386-92. Hansen K. 20-Epi-vitamin D3 analogues: a novel class of Gut: first published as 10.1136/gut.33.12.1660 on 1 December 1992. Downloaded from 4 Reddy BS, Maeura Y. Tumour promotion by dietary fat in potential regulators of cell growth and immune responses. azoxymethane-induced colon carcinogenesis in female F344 BiochemPharmacol 1991; 42: 1569-75. rats: influence of amount and source of dietary fat. JNCL 21 Keragballe K, Beck H, Segaard H. Improvement of psoriasis 1984; 72: 745-50. by a topical vitamin D3 analogue (MC-903) in a double blind S Rainey JB, Davies PW, Bristol JB, Williamson RCN. study. BrJ Dermatol 1988; 119: 223-30. Adaptation and carcinogenesis in defunctioned rat colon: 22 Binderup L, Bramm E. Effects of a novel vitamin D analogue divergent effects of faeces and bile acids. Brj Cancer 1983; MC-903 on cell proliferation and differentiation in-vitro and 48:477-84. on calcium metabolism in-vivo. Biochem Pharmacol 1988; 6 Terpstra OT, Dahl EP, Williamson RCN, Ross JS, Malt RA. 37: 889-95. Colostomy closure promotes cell proliferation and dimethyl- 23 Kragballe K. Treatment of psoriasis by the application of the hydrazine-induced carcinogenesis in rat distal colon. Gastro- novel cholecalciferol analogue calcipotriol (MC 903). Arch enterology 1981; 81: 475-80. Dermnatol 1989; 125: 1647-52. 7 Terpstra OT, Van Blankstein M, Dees J, Eilers GAM. 24 Appleton GVN, Wheeler EE, Al-Mufti R, Challacombe DN, Abnormal pattern of cell proliferation in the entire colonic Williamson RCN. Rectal hyperplasia after jejunoileal bypass mucosa of patients with colon adenoma or cancer. Gastro- for morbid obesity. Gut 1988; 29: 1544-8. enterology 1987; 92: 704-8. 25 Fergusson A, Sutherland A, MacDonald TT, Allen F. Tech- 8 Allan A, Bristol JB, Williamson RCN. Crypt cell production nique for microdissection and measurement in biopsies of rate in ulcerative proctocolitis: differential increments in human small intestine. I Clin Pathol 1977; 30: 1068-73. remission and relapse. Gut 1985; 26: 999-1003. 26 Gerdes J, Schwab U, Lemeke H, Stein H. Production of a 9 Wright NA, Appleton DR. The metaphase arrest technique - mouse monoclonal antibody reactive with a human nuclear a critical review. Cell Tissue Kinet 1980; 13: 643-63. antigen associated with cell proliferation. IntJ Cancer 1983; 10 Wright NA, Goodlod RA. Measurement of cell proliferation. 31: 13-20. Gastroenterology 1986; 1: 216-7. 27 Gerdes J, Lemeke H, Baisch H, Wacker HH, Schwab U, Stein 11 Appleton GVN, Davies PW, Bristol JB, Williamson RCN. H. Cell cycle analysis of a cell proliferation-associated Inhibition ofintestinal carcinogenesis by dietary supplemen- human nuclear antigen defined by the monoclonal antibody tation with calcium. BrJ Surg 1987; 74: 523-5. Ki-67. J Immunol 1984, 133: 1710-5. 12 Appleton GVN, Bristol JB, Williamson RCN. Increased 28 Franklin WA, McDonald GB, Stein HO, Gatter KC, Jewell dietary calcium and small bowel resection have opposite DP, Clarke LC, et al. Immunohistological demonstration of effects on colonic cell turnover. BrISurg 1986; 73: 1018- abnormal colonic crypt kinetics in ulcerative colitis. Human 21. Pathol 1985; 16: 1129-32. 13 Garland C, Barrett-Connor ER, Shekelle RB, Crique MM, 29 Hylander E, Ladefoged K, Jarnum S. The importance of the PaulO. Dietary vitamin D and calcium and risk ofcolorectal colon in calcium absorption following small intestinal cancer. Lancet 1985; i: 307-9. resection. Scandj Gastroenterol 1980; 15: 55-60. 14 Lointier P, Wargovich MI, Saez S, Levin B, Wildrick DM, 30 Studzinski GP, Bhandal AK, Brelvi ZS. Cell cycle sensitivity Boman BM. The role of vitamin D3 in the proliferation of a of HL-60 cells to the differentiation-inducing effects of 1,25 human colon cancer cell line lovo in-vitro. Anticancer Res dihydroxy vitamin D3. CancerRes 1985; 45: 3898-905. 1987; 7: 817-22. 31 Bhalla AK, Amento EP, Clemens TL, Holick MF, Krane SM. 15 Tanakar M, Abe E, Miyaura C, Shina Y, Suda T. 1,25- Specific high affinity receptors for 1,25 dihydroxy vitamin dihydroxy vitamin D3 induces differentiation of human D3 in human peripheral blood mononuclear cells: presence promyelocytic leukaemic cell HL-60 into monocyte- in monocytes and induction in T-lymphocytes following macrophage but not granulocytes. Biochem Biophy Res activation.J Clin EndocrinolMetab 1983; 57: 1308. Commun 1983; 117: 86-92. 32 Provvedini DM, Deftos LJ, Manolagos SC. 1,25 dihydroxy 16 Colston KW, Berger U, Coombes RC. Possible role ofvitamin vitamin D3 receptors in a subset of mitotically active D in controlling breast cancer proliferation. Lancet 1989; 38: lymphocytes from rat thymus. Biochem Biophys Res Commun 188-91. 1984; 121:277. 17 Brehier A, Thomasset M. Human colon cell line HT-29 33 Magnelsodorf DJ, Koeffher HP, Donaldson CA, Pike JW, characterisation of 1,25 dihydroxy vitamin D3 receptor and Haussler MR. 1,25 dihydroxyvitamin D3-induced differen- induction of differentiation by the hormone. J Steroid tiation in a human promyelocytic leukaemia cell line (HL- Biochem 1988; 29: 265-70. 60): receptor-mediated maturation to macrophage-like cells. http://gut.bmj.com/ 18 Harper KD, Iozzo RV, Haddard JG. Receptors for and J Cell Biol 1984; 98: 391. bioresponses to 1,25 dihydroxyvitamin D in a human colon 34 Staun M. Distribution of the 10000 molecular weight calcium carcinoma cell line (HT-29). Metabolism 1989; 28: 1062-9. binding protein along the small and large intestine of man. 19 Lointier P, Meggouh F, Dechelotte P, Pezet D, Ferrier Ch, Gut 1987; 28: 878-82. on October 2, 2021 by guest. Protected copyright.