Ординатура / Офтальмология / Английские материалы / Ocular Oncology_Albert, Polans_2003

J.Effectiveness and Toxicity of 1a-OH-D2 in the Athymic Xenograft Model

Following 5 weeks of treatment with 1a-OH-D2, the size of tumors in animals receiving this analogue in 0.1-, 0.2-, 0.3-, and 0.6-mg doses were compared to controls that received coconut oil via oral gavage. The results are shown in Figure 9A. Survival data are summarized in Figure 9B [16].

K.Effectiveness and Toxicity of 1a-OH-D2 in the LHb-Tag Transgenic Model

Following 5 weeks of treatment with doses of 0.1, 0.3, 0.5, or 1.0 mg/day of 1a-OH- D2, the size of tumors in animals was compared to controls that received coconut oil via oral gavage. The results are shown in Figure 10A [29]. Survival data are summarized in Figure 10B [29].

L.Characterization of the Mechanism of Arrest of Tumor Growth by Vitamin D Analogues

Specimens of Y-79 xenografts taken from athymic mice treated for 5 weeks with either 16,23-D3 or calcitriol were compared with tumors from control animals at the same stage. Paraffin sections of tumors (two mice per treatment) were analyzed for cell death using terminal transferase dUTP-nick end labeling (TUNEL) and cell proliferation using the monoclonal antibody MIB-1. Figure 11 (sections A–C) shows that control tumors and tumors treated with calcitriol exhibit some TUNEL labeling (approximately 0.13 to 0.64% of the cells, respectively), but the level of cell death is more than six times greater in tumors treated with 16,23-D3 (approximately 4.2% of the cells). In addition to an examination of the rate of cell death in these tumors, we also looked for evidence of the type of cell death (apoptosis or necrosis). The morphology of nuclear fragmentation was examined from sections of tumors stained with hematoxylin and eosin (Fig. 11, panel D). All tumors, but especially those treated with 16,23-D3, contained highly pyknotic and fragmented nuclei, both morphologically consistent with apoptotic cell death [30,31]. Dying cells in these tumors also stained using the 30 overhang ligation technique (Fig. 11, panel E). In addition to these results obtained with Y79 xenografts, similar features of apoptotic cell death were observed in dying cells found in spontaneous tumors of transgenic mice treated with vitamin D analogues (data not shown). Thus it is important to note that vitamin D–induced cell death in these tumors appears to be apoptotic and therefore controlled by genes being expressed in the dying cells.

In a similar experiment, specimens of Y-79 xenografts taken from athymic mice treated for 5 weeks with 1a-OH-D2 (2 mg per day) were compared with controls using TUNEL labeling. As with 16,23-D3 and calcitriol, 1a-OH-D2 caused an increase in cell death via apoptotic changes observed in the tumor sections (Fig. 12).

Conversely, all four tumor specimens studied had relatively uniform labeling with the MIB-1 antibody (Fig. 13: 2.3%, 2.6%, and 3.3% for control, 16,23-D3 and calcitriol, respectively; and Fig. 14 for 1a-OH-D2) suggesting that the rate of cell proliferation is unaffected. The combined observations for tumors treated with calcitriol suggest that the mechanism of tumor growth arrest for this analogue is also

Figure 9 (A) Effect of 1a-OH-D2 on growth of Y-79 retinoblastoma xenografts compared to controls following 5 weeks of treatment. The 0.3-mg and 0.2-mg groups were statistically significantly smaller than the control group (p < 0.003 and p <0.004, respectively). (B) Survival of 1a-OH-D2-treated xenograft mice compared to control mice after 5 weeks of treatment.

Figure 11 Cell death in Y79 xenograft tumors after 5 weeks of vitamin D treatment demonstrated by TUNEL staining in (A) untreated tumors, (B) tumors treated with 16,23-D3, and (C) tumors treated with calcitriol. Panels A, B, and C exhibit signs of active cell death. 16,23-D3 tumors had TUNEL staining that was six times greater than control or calcitriol tumors at this stage. D. Higher magnification of an H&E-stained section of a 16,23-D3–treated tumor. Nuclear morphology of the cells is consistent with apoptotic cell death, including highly pyknotic nuclei and cells with extensive nuclear fragmentation. E. Fluorescence micrograph of dying cells in a tumor treated with 16,23-D3 and labeled using the 30 overhang ligation technique [24]. DNA fragments are apoptotic but not necrotic; nuclei containing a high proportion of similar 30 overhanging nucleotides can be covalently linked to biotinlabeled DNA. Ligated DNA is visualized using streptavidin conjugated to Texas red. Morphological evidence combined with this labeling technique indicates that these tumor cells are dying by apoptosis.

mediated by an increase in cell death. It is possible, however, that the time point examined in this preliminary study was too late to observe the period of peak TUNEL activity for the calcitriol treatment.

Morphological and histochemical methods suggest that vitamin D analogues activate apoptotic cell death in retinoblastoma. Since this mechanism of cell death is genetically regulated, the expression patterns of three genes dissociated with cell death were investigated by immunohistochemistry in sections of 5-week-treated tumors treated with calcitriol, 16,23-D3, and corresponding controls. The gene

products examined included the tumor suppressor protein p53, and the gene products of two genes regulated by p53 (p21waf-1/cip-1 and bax).

Nuclear p53 staining was present in the same three treatment groups, although it was most abundant in tumors treated with 16,23-D3. Similarly, both p21 and bax immunoreactivity were strongest in the 16,23-D3 groups. Since the expression of p53, and its downstream target genes, correlates with retinoblastoma cell death [23], it is not surprising to find elevated expression levels in the treatment group undergoing the greatest rate of cell death.

Figure 12 Percentage of TUNEL-positive areas compared to total surface area of tumor section. Specimens treated with 1a-OH-D2 exhibit increased TUNEL staining when compared to controls (N ¼ 5, p < 0.05, unpaired t-test).

IV. CONCLUSIONS

As we noted in the introduction to this chapter, alternatives to the current methods of RB therapies are needed [32]. Vitamin D analogues hold the promise of fulfilling this need. We initially studied ergocalciferol and calcitriol (Fig. 1) in the athymic Y- 79 RB xenograft mouse model. Although these compounds showed impressive reductions in tumor growth as compared to controls, the doses required for this effect caused mortality with rates ranging from 25% to 46% [18,28]. It should be noted, however, that the immunocompromised athymic mouse model has an extremely high sensitivity to calcitriol, vitamin D2, 16,23-D3, and 1a-OH-D2 and that immunocompetent mice, such as the transgenic strain, are a better indicator of actual drug toxicity. In an experiment in which calcitriol was administered to athymic mice in a dose required to reduce tumor growth to 36% of the control (i.e., 0.05 mg), there was still only a 40% survival rate of treated animals [15]. By withholding doses of ergocalciferol from sick animals, the survival rate could be improved to 75% [18]. In all of the experiments, the toxicity appeared to be related to the hypercalcemia induced by these compounds. Consequently, we concluded that ergocalciferol and calcitriol were excessively toxic, and were not suitable for treatment of children with RB.

We then turned our attention to a synthetic analogue of calcitriol, 16,23-D3 (Fig. 1) which has an antineoplastic effect similar to that of calcitriol but with less hypercalcemic activity. In our initial experiments with this compound, animals treated with 0.05 mg of 16,23-D3 were found to have a significantly smaller tumor

Figure 13 Immunohistochemical labeling of Y-79 tumors grown in athymic mice after 5 weeks of vitamin D treatment. Sections of untreated tumors, 16,23-D3-treated tumors, and calcitriol-treated tumors were stained with the following proteins: p53 (monoclonal DO-1), p21 (monoclonal Ab-6), BAX (polyclonal Ab-1), and antibodies directed against the Ki-67 nuclear antigen (MIB-1). Each panel is a Normarski interference image taken near the center of the tumors because they exhibited uniform cell density. p53 and p53-regulated genes (p21 and BAX) are upregulated in tumors treated with 16,23-D3. This change in gene expression mirrors the increased apoptotic activity seen in Fig. 10. MIB-1 staining is relatively equal among all the treatments, suggesting that cell proliferation is unaffected.

cross-sectional area when compared to corresponding controls (p ¼ 0.02) [19]. All animals survived 5 weeks of treatment. In subsequent studies using higher doses (0.2–0.75 mg), the drug was effective in reducing tumor growth and the survival rate was approximately 90% [15,20]. This appears to be a promising drug for use in treatment of RB children. It was not approved by the Food and Drug Administration (FDA) until March 1999 for investigational use in human cancer patients. This led us to investigate an analogue of vitamin D2, 1a-OH-D2.

1a-OH-D2 is a vitamin D analogue that was approved by the FDA in 1999 for oral use in the treatment of secondary hyperparathyroidism due to renal failure. This compound was approved earlier for investigational use in human tumor treatment in 1996 and is currently being used in phase 2 human clinical trials of prostate cancer (George Wilding, personal communication). Like 16,23-D3, 1a-OH-D2 is known to induce low levels of hypercalcemia while providing effective systemic serum drug levels for tumor treatment [33,34]. In a recent study [16], we found that 1a-OH-D2 limited tumor growth of the human Y-79 RB cell line, which was subcutaneously injected in athymic ‘‘nude’’ mice. We reported a dose-response efficacy curve with minimal toxicity in both the athymic and transgenic mouse models of RB [16,29]. Our results indicate that 1a-OH-D2, a compound not known to be a mutagen, can

Figure 14 MIB-1 immunostaining of tumor sections to study cell proliferation. Labeled cells were counted using 620 magnification; 10 fields per section were included in the analysis. No increase in MIB-1 immunostaining was seen after treatment of 1a-OH-D2 (N ¼ 5, p ¼ 0.2495, unpaired t-test).

limit tumor growth in a nontoxic dose range. In comparing our studies [16,29] to a similar study of 16,23-D3 and calcitriol in same mouse models [15,19,20], 1a-OH-D2 appears to be similar in tumor reduction capability at a 0.3-mg dose level when compared to 0.5 mg of 16,23-D3 or 0.05 mg of calcitriol.

It is probable that tumor cells must contain VDR in order for vitamin D analogues to be effective in limiting their growth. Examination of 23 consecutively received RB samples with PCR amplification for the presence of VDR mRNA has been completed. Message encoding receptors were present in all specimens, providing convincing evidence that these tumor cells express receptor protein. The 95% confidence interval for the probability of any RB being positive is (0.85, 1), based on the binomial distribution.

Calcitriol and its analogues have been demonstrated to inhibit cellular proliferation in other malignant cell lines besides RB, including leukemic, breast, colon, renal, and lung carcinomas [35–38]. It is hypothesized that the antiproliferative effect of these compounds is mediated by a vitamin D receptor–linked mechanism, although exceptions exist [39–42]. Considerable evidence exists that the antineoplastic and differentiating effects of vitamin D compounds affect fundamental cellular processes of proliferation, differentiation, and apoptosis. The

resulting key biochemical events are related to activation of cyclin-dependent kinase inhibitors, such as p21waf-1/cip-1, and there are some reports that activated VDR can

directly mediate the expression of this gene [43–47].

In this chapter, we present data showing that tumor growth attenuation of Y- 79 xenografts in athymic mice following treatment with 16,23-D3 is due to apoptotic cell death. Calcitriol and vitamin D analogues can also induce apoptosis in leukemic (HL60) cells as well as human breast cancer and colon cancer cell lines [48–50].

Recent studies have shown that human RB and RB cell lines are extremely susceptible to p53-mediated apoptosis and elevated p21waf-1/cip-1 expression [23,47]. These results indicate that the treatment of xenograft tumors elicits the increased expression of both p53 and p53-regulated genes. These data are consistent with the role of bound VDR in stimulating the activation of apoptotic genes and may account for the decreased toxicity of these compounds without compromised efficacy. It is likely that 1a-OH-D2 has a similar mechanism of action against RB tumors in the athymic mouse.

ACKNOWLEDGMENTS

This research was supported by the Research To Prevent Blindness (RPB) and NIH/ NEI RO1 grant EYO1917. 16,23-D3 was graciously provided to us by Ilex Corporation. 1a-OH-D2 and supplemental funds for animal care costs were graciously provided to us from BoneCare International, Inc. The authors also wish to thank the following physicians and their patients for donating human RB samples for vitamin D receptor analysis: David H. Abramson, Cornell Medical College; Thomas Lee, New York Presbyterian Hospital; J. William Harbour, Washington University Medical School; Jerry Shields, and Carol Shields, Wills Eye Hospital; Timothy Murray, Bascom Palmer Eye Institute; Ted Dryja, Massachusetts Eye & Ear Infirmary; Joan O’Brien, University of California, San Francisco; A. Linn Murphree, and Anita Fisher, PhD, Children’s Hospital of Los Angeles; Monte Mills, Children’s Hospital of Philadelphia; and Mansoor Movaghar, Davis Duehr Dean Eye Clinic.

The authors also wish to thank Drs. Robert Nickells and Cassandra Schlamp for their technical work associated with the mechanism of action of vitamin D analogues and Dr. David Gamm for expertise in the vitamin D receptor assays. The authors also wish to thank Drs. Steven M. Cohen, Amanda M. Saulenas, Ilona Slusker-Shternfeld, Sina Sabet, Craig Wilkerson, Paul J. Bryar, Richard Grostern, Boaz Lissauer, Daniel G. Dawson, Joel Gleiser, as well and Ms. Janice M. Lokken for their technical expertise on the vitamin D2, calcitriol, 16,23-D3, and 1a-OH-D2 drug trials.

Portions of this chapter are reprinted with the permission of the editor and publishers from the article ‘‘Vitamin D analogs, a new treatment for retinoblastoma: The first Ellsworth Lecture,’’ which appeared in Ophthalmic Genetics, Vol. 22, No. 3.

REFERENCES

1.Ferris FL, Chew EY. A new era for the treatment of RB. Arch Ophthalmol 1996;114:1412.

2.Eng C, Li FP, Abramson DH, Ellsworth RM, Wong FL, Goldman MB, Seddon J, Tarbell N, Boice JD Jr. Mortality from second tumors among the long-term survivors of RB. J Natl Cancer Inst 1993;85:1121–1128.

3.Roarty JD, McLean IW, Zimmerman LE. Incidence of second neoplasms in patients with bilateral RB. Ophthalmology 1988;95:1583–1587.

4.Abramson DH, Frank CM. Second nonocular tumors in survivors of bilateral RB. A possible age effect on radiation-related risk. Ophthalmology 1998;105:573–580.

5.Gallie BL, Budning A, DeBoer G, Thiessen JJ, Koren G, Verjee Z, Ling V, Chang H S-L. Chemotherapy with focal therapy can cure intraocular RB without radiotherapy. Arch Ophthalmol 1996;114:1321–1328.

6.Verhoeff R. RB undergoing spontaneous regression: Calcifying agents suggested in treatment of RB. Am J Ophthalmol 1966;62:573–574.

7.Reid TW, Albert DM, Rabson AS, Russell P, Craft J, Chu ES, Tralka TS, Wilcox J. Characteristics of an established cell line of RB. J Natl Cancer Inst 1974;53:347–360.

8.Gallie BL, Albert DM, Wong JJ, Buyukmihci N, Puliafito CA. Heterotransplantation of RB into the athymic ‘‘nude’’ mouse. Invest Ophthalmol Vis Sci 1977;16:256–299.

9.Windle JJ, Albert DM, O’Brien JM, Marcus DM, Disteche CM, Bernards R, Mellon PL. RB in transgenic mice. Nature 1990;343:665–669.

10.O’Brien JM, Marcus DM, Bernards R, Carpenter JL, Windle JJ, Mellon P, Albert DM. A transgenic mouse model for RB. Arch Ophthalmol 1990;108:1145–1151.

11.Koshy KT. Vitamin D: An update. J Pharm Sci 1982;71:137–152.

12.Jones G, Byrnes B, Palma F, Segev D, Mazur Y. Displacement potency of vitamin D2 analogs in competitive protein-binding assays for 25-hydroyxvitamin D3, 24.25dihydroyvitamin D3, and 1,25-idhydroxyvitamin D3. J Clin Endocrinol Metab 1980;50(4):773–775.

13.Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987;162:156–159.

14.Saulenas A, Cohen S, Key L, Winter C, Albert DM. Vitamin D and RB: The presence of receptors and inhibition of growth in vitro. Arch Ophthalmol 1988;106:533–535.

15.Sabet SJ, Darjatmoko SR, Lindstrom MJ, Albert DM. Antineoplastic effect and toxicity of 1,24-dihydroxy-16-cnc-23-ync-vitamin D3 in athymic mice with Y-79 human RB tumors. Arch Ophthalmol 1999;117:365–370.

16.Grostern RJ, Bryar PJ, Zimbric ML, Darjatmoko SR, Lissauer BJ, Lindstrom MJ, Lokken JM, Strugnell SA, Albert DM. Toxicity and dose-response studies of 1ahydroxyvitamin D2 in a retinoblastoma xenograft model. Arch Ophthalmol 2002;120:607–612.

17.Albert DM, Saulenas AM, Cohen SM. Verhoeff’s query: Is vitamin D effective against RB? Arch Ophthalmol 1988;106:536–540.

18.Albert DM, Marcus DM, Gallo JP, O’Brien JM. The antineoplastic effect of vitamin D in transgenic mice with RB. Invest Ophthalmol Vis Sci 1992;33(8):2354–2364.

19.Shternfeld IS, Lasudry JGH, Chappell RJ, Darjatmoko SR, Albert DM. Antineoplastic effect of 1,25-dihydroxy-16-ene-23-yne-Vitamin D3 analogue in transgenic mice with RB. Arch Ophthalmol 1996;114:1396–1401.

20.Wilkerson CL, Darjatmoko SR, Lindstrom MJ, Albert DM. Toxicity and dose-response studies of 1,25-(OH)2-16-ene-23-yne vitamin D3 in transgenic mice. Clin Cancer Res 1998;4:2253–2256.

21.Gavrieli Y, Sherman Y, Ben-Sasson SA. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 1992;119:493–501.

22.Quigley HA, Nickells RW, Kerrigan LA, Pease ME, Thibault DJ, Zack DJ. Retinal ganglion cell death in experimental glaucoma and after axotomy occurs by apoptosis. Invest Ophthalmol Vis Sci 1995;36:774–786.

23.Nork TM, Poulsen G, Millecchia LL, Jantz RG, Nickells RW. p53 regulates apoptosis in human RB. Arch Ophthalmol 1997;115:213–219.

24.Didenko VV, Hornsby PJ. Presence of double-strand breaks with single-ase 30 overhangs in cells undergoing apoptosis but not necrosis. J Cell Biol 1996;135:1369–1376.

25.Reitsma PH, Rothberg PG, Astrin SM, Trial J, Bar-Shavit Z, Hall A, Teitelbaum SL, Kahn AJ. Regulation of myc gene expression in HL-60 leukaemia cells by a vitamin D metabolite. Nature 1983;306:492–494.

26.Mangelsdorf DJ, Koeffler HP, Donaldson CA, Pike JW, Haussler MR. 1,25dihydroxyvitamin D3-induced differentiation in a human promyelocytic leukemia cell line (HL-60): Receptor-mediated maturation to macrophage-like cells. J Cell Biol 1984;98:391–398.

27.Colston RW, Feldman D. Demonstration of a 1,25-dihydroxycholecalciferol cytoplasmic receptor-like binder in mouse kidney. J Clin Endocrinol Metab 1979;49:798–800.

28.Cohen SM, Saulenas AM, Sullivan CR, Albert DM. Further studies on the effect of vitamin D on RB. Arch Ophthalmol 1988; 106:541–543.

29.Dawson DG, Gleiser J, Zimbric ML, Darjatmoko SR, Frisbie JC, Lokken JM, Lindstrom MJ, Audo I, Strugnell SA, Albert DM. Toxcitiy and dose-response studies of 1a-hydroxyvitamin D2 in LHb-Tag transgenic mice. Trans Am Ophthalmol Soc 2002; 100:125–130.

30.Kerr JFR, Wylli AH, Currie AR. Apoptosis: A basic biological phenomenon with wideranging implications in tissue kinetics. J Cell Biol 1972;26:239–257.

31.Nickells RW, Zack DJ. Apoptosis in ocular disease: A molecular overview. Ophthalm Genet 1996;17:145–165.

32.O’Brien JM. Alternative treatment in RB. Ophthalmology 1998;105:571–572.

33.Knutson JC, Hollis BW, LeVan LW, Valliere C, Gould KG, Bishop CW. Metabolism of 1a-hydroxyvitamin D2 to activated dihydroxyvitamin D2 metabolites decreases endogenous 1a-dihydroxyvitamin D3 in rats and monkeys. Endocrinology 1995;136:4749–4753.

34.Strugnell S, Byford V, Makin HLJ, Moraiarty RM, Gilardi R, LeVan LW, Knutson JC, Bishop CW, Jones G. 1a,24S-dihydroxyvitamin D2: A biologically active product of 1ahydroxyvitamin D2 made in the human hepatoma, HEP3B. Biochem J 1995;310:233– 241.

35.Walters M. Newly identified actions of the vitamin D endocrine system. Endocr Rev 1992;13:719–764.

36.Brenner RV, Shabahang M, Schumacker LM, Nauta RJ, Uskokovic MR, Evans SR, Buras RR. The antiproliferative effect of vitamin D analogs on MCF-7 human breast cancer cells. Cancer Lett 1995;92:77–82.

37.Schwartz GG, Oeler TA, Uskokovic MR, Bahnson RR. Human prostate cancer cells: Inhibition of proliferation by vitamin D analogs. Anticancer Res 1994;14:1077–1082.

38.Pakkala S, deVos S, Elstner E, Rude RK, Uskokovic M, Binderup L, Koeffler HP. Vitamin D3 analogs: Effect on leukemic clonal growth, differentiation, and serum calcium. Leuk Res 1995;19:65–72.

39.Inaba M, Okuno S, Koyama H, Nishizawa Y, Morii H. Dibutyryl cAMP enhances the effect of 1,25-dihydroxyvitamin D3 on a human promyclocytic leukemia cell, HL-60, at both the receptor and the postreceptor steps. Arch Biochem Biophys 1992;29:181–186.

40.Kawa S, Yoshizawa K, Tokoo M, Imai H, Oguchi H, Kiyosawa K, Homma T, Nikaido T, Furihata K. Inhibitory effect of 22-oxa-1,25-dihydroxyvitamin D3 on the proliferation of pancreatic cancer cell lines. Gastroenterology 1996;110:1605–1613.

41.Bhatia M, Kirkland JB, Meekling-Gill KA (1995). Monocytic differentiation of acute promyelocytic leukemia cells in response to 1,25-dihydroxyvitamin D3 is independent of nuclear receptor binding. J Biol Chem 1995;270:15.962–15.965.

42.Brown AJ, Dusso A, Slatopolsky E. Selective vitamin D analogs and their therapeutic applications. Semin Nephrol 1974;14:156–174.

43.Frey MR, Zhao X, Evans SS, Black JD. Activation of protein kinase C (PKC) isozymes inhibits cell cycle progression, modulates phosphorylation of the RBs (Rb) protein, and induces expression of p21waf1/cip1 in intestinal epithelial cells. Proc Am Assoc Cancer Res 1996;37:51.