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The inhibitory effects of bryostatin 1 administration on the growth of rabbit papillomas
Cancer Letters 136 (1999) 67±74
The inhibitory effects of bryostatin 1 administration on the growth of rabbit papillomas Jason M. Bodily a, David J. Hoopes a, Beverly L. Roeder b, Sharon G. Gilbert a, George R. Pettit c, Cherry L. Herald c, Darin N. Rollins d, Richard A. Robison a,* a Department of Microbiology, Brigham Young University, 791 WIDB, PO Box 25133, Provo, UT 84602-5133, USA Department of Animal Science, Brigham Young University, 791 WIDB, PO Box 25133, Provo, UT 84602-5133, USA c Cancer Research Institute, Arizona State University, Tempe, Arizona, USA d Department of Statistics, Brigham Young University, 791 WIDB, PO Box 25133, Provo, UT 84602-5133, USA
b
Received 6 July 1998; received in revised form 18 September 1998; accepted 18 September 1998
Abstract Bryostatin 1 is a protein kinase C modulator that shows antineoplastic activity in a variety of tumor systems. This study examined the effects of bryostatin 1 administration on papilloma growth in rabbits. Investigations of optimal route, dose, and schedule were performed. Several groups of rabbits were inoculated with cottontail rabbit papillomavirus (CRPV) DNA. Bryostatin 1 was administered i.v., both daily and weekly, and intralesionally both weekly and bi-weekly. Intralesionally dosed papillomas were examined histologically for immune cell in®ltration. In weekly and daily i.v. trials, 2.5 and 1.0 mg/kg, respectively, showed the greatest overall reduction in tumor size. Bryostatin 1 administered intralesionally also slowed papilloma growth. Treated lesions had signi®cantly higher numbers of heterophils and eosinophils. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Bryostatin 1; Papillomavirus; Cancer therapeutics; PKC; Cervical cancer
1. Introduction The bryostatins are a unique family of 20 naturally occurring macrocyclic lactones which have demonstrated considerable antineoplastic activity. Bryostatin 1 was ®rst isolated from the marine bryozoan Bugula neritina collected off the California coast. B. neritina is a colonial ®lter-feeder commonly known as a sea-mat or false coral and often grows on docks, pilings, rocks, and ship hulls in temperate ocean areas [1]. Several teams of chemists have succeeded in synthesizing bryostatin 7 and portions of bryostatin * Corresponding author. Tel.: 1 1-801-378-2416; fax: 1 1-801378-9197; e-mail: [email protected].
1 [2], but so far the bryozoan is the only source of bryostatin 1 [3]. Both in vitro and in vivo studies have demonstrated the anti-tumor activity of bryostatin 1. For example, bryostatin 1 treatment inhibited the in vitro growth of human lung tumor cells [4], murine reticular cell sarcoma and lymphoma cells [5], and U937 human leukemia cell lines [6]. Bryostatin 1 also induced the differentiation of HL-60 cells [7] and myeloid [8,9] and lymphocytic [10] human leukemias. The in vivo antineoplastic activity of bryostatin 1 has been shown by its ability to cure mice with ovarian sarcomas [11], P388 lymphocytic leukemia [12], and malignant melanoma [13]. In combination with other therapeutic agents, it has also been shown to cure human Walden-
0304-3835/99/$ - see front matter q 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(98)00310-3
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J.M. Bodily et al. / Cancer Letters 136 (1999) 67±74
stroÈm's macroglobulinemia in SCID mice [14] and to sensitize HeLa cells to cisplatin [15]. Several phase I human clinical trials of bryostatin 1 against a range of cancers have been completed [16±18], revealing some bene®cial effects against malignant melanoma, ovarian carcinoma, non-Hodgkin's lymphoma, and cervical cancer (unpublished). Although the list of transformed cells against which bryostatin 1 has been tested is extensive, its activity against virus-induced tumors has not been investigated. The discovery that certain urogenital cancers, such as cervical cancer, are highly linked to infection by some types of human papillomaviruses (HPV) has provided an impetus to study these types of transformed cells [19,20]. HPV DNA is present in 93% of all cervical cancers. Worldwide, cervical cancer ranks second only to breast cancer in both mortality and incidence as the most common human female malignancy with 47 000 new cases diagnosed each year [21]. Of the current therapies available to treat HPV infections, none are capable of eliminating the virus completely, and recurrence rates are high [22]. A more effective treatment for HPV-induced tumors would therefore be of tremendous bene®t in reducing the incidence of urogenital cancers. The cottontail rabbit papillomavirus (CRPV) system is recognized as a good model for human papillomavirus-induced transformation [23]. The present study was undertaken to examine the effects of bryostatin 1 administration on experimentally induced rabbit papillomas. Different routes, doses, and treatment schedules were evaluated in an effort
to de®ne optimal therapeutic conditions. We report that bryostatin 1 slows the growth of papillomas when administered either intravenously or intralesionally 2. Materials and methods 2.1. Isolation and puri®cation of CRPV-DNA The plasmid CRPV-pLAII contains the genome of the CRPV Washington B strain. Plasmid was isolated by modi®cation of a method previously described by Cattaneo et al. [24]. Escherichia coli HB101 cells containing CRPV-pLAII (kindly provided by Dr. Janet Brandsma, Yale University) were grown in Terri®c broth (1.2% tryptone (Difco, Detroit, MI), 2.4% yeast extract (Difco), 0.4% glycerol) for 18 h at 378C, shaking at 250 rev./min. The cells were pelleted and resuspended in TEG buffer (25 mM Tris±HCl (pH 8.0), 10 mM EDTA (pH 8.0), 50 mM glucose) incubated with lysozyme, and treated with lysis solution (2% SDS, 0.5 M NaOH) and put on ice for 15 min. Potassium acetate (5 M) was added, the mixture shaken mildly, and again placed on ice for 15 min. Precipitated cell walls, membranes, and genomic DNA were pelleted, and the supernatant containing plasmid DNA was ®ltered and precipitated with 100% ethanol overnight at 2 208C. The precipitate was pelleted and resuspended in a solution containing 100 mg/ml RNase A and incubated for 30 min at 308C. Suf®cient 5 M NaCl and 30% polyethylene glycol were added to precipitate the plasmid. The
Table 1 A summary of experiments involving intravenous bryostatin 1 treatments Dosing schedule
Number of rabbits
Dose of bryostatin 1 (mg/kg)
Treatment initiation (days post CRPV DNA inoculation)
Dosing duration (days)
Slowed papilloma growth
Weekly
4 4 4 4 4
0.0 2.5 5.0 10.0 20
0 0 0 0 0
70 70 70 70 ±a
No Yes (P , 0:05) No No ±a
Daily
4 3 5
0.0 0.1 1.0
15 15 15
38 38 38
No Yes (P , 0:0002) Yes (P , 0:05)
a
This dose was lethal to all rabbits within 12 h of ®rst dose.
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2.3. Catheter implantation
Fig. 1. Final mean papilloma volumes after 70 days of weekly i.v. administration at doses of 2.5, 5.0, 10.0 and 0.0 mg/kg. Bryostatin 1 treatment was initiated immediately after CRPV DNA inoculation. Each group consisted of four rabbits. Bars represent ^ one standard error of the mean.
solution was put on ice for 2 h after which the plasmid DNA was pelleted. After resuspension in TE buffer, the DNA was incubated for 30 min in Proteinase K solution (0.05 mg/ml Proteinase K, 1% SDS) at 608C. An equal volume of 50 mM NaCl/40 mM NaOH solution was added, incubated for 15 min at room temperature, and neutralized with 2.0 M Tris (pH 7.5). The plasmid was puri®ed with two to three phenol/chloroform extractions and one chloroform extraction. The plasmid was precipitated with 300 mM sodium acetate and two volumes of 95% ethanol and then pelleted. Plasmid was resuspended in TE buffer at a concentration of , 1 mg DNA/ml. 2.2. Experimental animals and induction of papillomas Random-bred 2-kg female New Zealand White rabbits (Oryctolagus cuniculus, Western Oregon Rabbit Company, Philomath, OR) served as subjects in this study. Papillomas were induced following methods described by Brandsma et. al. [25]. Brie¯y, the animals' backs were shaved and, under anesthesia, 100 sites on each animal were inoculated with 50 ml CRPV DNA in TE buffer (1 mg DNA/ml) using a PedO-Jet air-injector (Stirn Industries, Dayton, NJ).
Rabbits that were to receive daily i.v. treatments were catheterized to avoid repeated venipuncture. Indwelling catheters were surgically implanted in the right jugular vein on day 14±16 post CRPV DNA inoculation. The procedure outlined by PerryClark and Meunier [26] was used with the following modi®cations: Venocath-18 catheters (Abbott Ireland, Sligo, Ireland) were used in place of vascular access ports. A screw-in male adapter plug (Abbott Laboratories, North Chicago, IL) was attached to the exteriorized hub of the Venocath to allow daily drug injection. The catheter hub was sutured to the skin at the exit site to minimize the risk of disturbance. The entire area was protected with a canvas/Velcro patch glued to the rabbit's fur with Kamar adhesive (Kamar Inc, Steamboat Springs, CO). For those animals receiving weekly treatment, i.v. drug was administered using a 24 gauge 3/4 inch Angiocath (Becton Dickinson Vascular Access, Sandy, UT) via the marginal ear vein. 2.4. Drug administration 2.4.1. Intravenous administration Bryostatin 1 was isolated as described (12), and solutions of bryostatin 1 were formulated in a 60% ethanol/saline carrier solution (0.9% NaCl) to produce solutions corresponding to each of the dosage groups. The volumes of solution administered were determined for each rabbit to assure proper dosage according to rabbit mass; administered volumes ranged from 70 to 110 ml. The control rabbits were given comparable volumes of carrier solution without bryostatin 1. Treatments were chased with either 5.0 ml of Lactated Ringers Solution (weekly treatments) or 1.5 ml of 100 units/ml heparin sodium in 0.9% saline (daily treatments). Doses were administered as listed in Table 1. 2.4.2. Intralesional administration After serving as controls in the i.v. study, eight rabbits with matched pairs of equal-sized papillomas (approximately 150 mm 3) were selected as subjects in the intralesional study. One papilloma in each pair received 0.5±5 mg of bryostatin 1 in 25 ml 60% ethanol/saline solution while the companion lesion
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Fig. 2. The growth of rabbit papillomas during daily i.v. bryostatin 1 treatment. Values represent mean volumes of all papillomas on n rabbits. Treatment groups were: W: 1.0 mg/kg, n 5; B: 0.1 mg/kg, n 3; O: control, n 4. Bars represent ^ one standard error of the mean.
received an equal volume of carrier solution with no bryostatin 1. Doses were injected into the dermal layer immediately beneath the papilloma using a 26 gauge needle. Treatments continued weekly for 10 weeks. At 1 and 2 weeks post treatment initiation, one half of the papillomas were biopsied. Rabbits were anesthe-
Fig. 4. Immune cell in®ltrates into bryostatin 1-treated (A) and control (B) papillomas. Values are mean scores from biopsies taken at 10 and 20 weeks (n 16 biopsies per treatment group). Bars represent ^ one standard error of the mean.
tized, and biopsies of the lesions were taken using a 1.5 mm dermal punch (Acuderm Inc, Ft. Lauderdale, FL). Samples were ®xed in 10% formaldehyde and imbedded in paraf®n. Sections were cut with a microtome and subjected to sequential washes with xylene, 100% ethanol, 70% ethanol, and distilled water. A standard tissue stain was performed using hematoxylin and eosin. An experienced pathologist examined the entirety of two sections from each biopsy (250 £ magni®cation) and used a semi-quantitative assessment method to determine the extent of immune cell in®ltration. Speci®c immune cells were identi®ed by their characteristic staining and morphology. The sections were dehydrated with washes of 95% ethanol, 100% ethanol, and xylene. The presence of eosinophils, monocytes, and heterophils was quantitated with scores ranging from 0 to 3 (0, absence of cells; 3, abundant in®ltration). After 10 weeks, six pairs of papillomas were further treated bi-weekly with 0.5 mg bryostatin 1 for 10 additional weeks. 2.5. Statistical analysis
Fig. 3. Growth of rabbit papillomas during intralesional bryostatin 1 treatment. O: control, n 8; X: treatment, n 8. From 0 to 10 weeks, bryostatin 1 was administered weekly. From 10 to 20 weeks, administration was bi-weekly. Treatment doses ranged from 0.5 to 5.0 mg/kg, but since there was no signi®cant difference in effect between doses, the data were combined for simplicity. Bars represent ^ one standard error of the mean.
Statistical analyses were performed with SAS software (Cary, North Carolina) using a mixed model analysis of variance. Individual dosage levels were compared with controls using standard contrasts.
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3. Results 3.1. Intravenous bryostatin 1 administration Table 1 summarizes the experiments evaluating intravenous bryostatin 1 administration. No dose, route, or schedule of bryostatin 1 administration produced a signi®cant difference in the total number of papillomas formed. Fig. 1 shows that weekly i.v. administration of bryostatin 1 diminished the size of developing papillomas. The average papilloma volume per rabbit in the 2.5 mg/kg group was 92.0% smaller than that of the controls after 70 days (P , 0:05) while the volumes in the other two treatment groups (5 mg/kg, 10 mg/kg) did not differ significantly from the controls. Daily i.v. treatment with bryostatin 1 at 0.1 mg/kg (Fig. 2) slowed papilloma development by about 66% relative to the controls after 60 days (P , 0:0002). The 1.0 mg/kg daily dose increased the size of papillomas in the initial weeks but led to a reduction by the end with a ®nal reduction of 72% as compared to the controls. The 20 mg/kg i.v. dose proved lethal to all rabbits. This is signi®cantly lower than the previously reported LD10 (29 mg/kg) and LD50 (68 mg/kg) in rodents (15). However, at lower doses, no adverse effects of bryostatin 1 administration were observed on the behavior of the animals (drinking, eating, urinating, etc.). In animals that received injections via the ear veins, slight endothelial cell toxicity was observed in some rabbits. 3.2. Intralesional bryostatin 1 administration Intralesional administration of bryostatin 1 both retarded papilloma growth and promoted tumor regression. Fig. 3 depicts the total papilloma volume for treated papillomas versus matched controls on the same animal. No signi®cant difference was noted between the various doses of bryostatin (0.5±5.0 mg/ kg) administered. Analysis revealed that the treated papillomas were signi®cantly smaller than the controls both at 10 weeks (P , 0:05) and at 20 weeks (P , 0:01). During the ®rst 10 weeks (weekly doses), the treated papillomas increased in volume but at a slower rate than the controls. However, during the second 10 weeks (bi-weekly doses) the treated papillomas showed a marked decrease in volume, leading
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to a reduction of about 68% by the end of the 20 week test period. Fig. 4 shows the histological effects of the intralesional administration of bryostatin 1. Immune cell in®ltration into treatment and control papillomas differed signi®cantly at the level of P , 0:05 (eosinophil) and P , 0:08 (heterophil). There was no significant difference in the numbers of mononuclear cells. Lesions that were regressing by the time of biopsy were found to have a higher heterophil and eosinophil in®ltration as compared to those that were not regressing (data not shown). 4. Discussion Figs. 3 and 4 show that injections of bryostatin 1 lead to papilloma regression with a concomitant in®ltration of heterophils and eosinophils. At least some of bryostatin 1's antineoplastic activity can be attributed to its ability to activate immune cells and stimulate cytokine production. It has been proven to enhance production of hematopoietic growth factors, increase plasma interleukin-6 and TNF-a concentrations, and activate polymorphonuclear leukocytes [16,27,28]. Working synergistically with the immune modulator AS101, it was shown to induce the secretion of tumor necrosis factor (TNF), gamma interferon (IFN-g) and interleukin-2 (IL-2) by human mononuclear cells and TNF and IL-2 by mouse lymphoid cells [29]. In conjunction with IFN-g, bryostatin 1 has been shown to up-regulate the inducible NO synthase gene and induce the production of NO in murine macrophages [30]. In this study, the in®ltration of heterophils and eosinophils was signi®cantly increased in treated lesions relative to controls (Fig. 4). Treatment also correlated with a signi®cantly smaller papilloma volume (Fig. 3). It was noted that individual lesions that were regressing, regardless of whether they had received treatment or not, had elevated levels of heterophils and eosinophils when compared to lesions that were not regressing (data not shown). Further, elevated levels of neutrophils were also found in regressing lesions in the bovine papilloma system using a different modulator [31]. Neutrophils have been shown to possess cytotoxic activity [32]. Taken together, these results suggest that the regression of papillomas injected with bryostatin 1 may be due, at
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least in part, to the action of these in¯ammatory cells, which are either directly activated by bryostatin 1 or by bryostatin 1-induced cytokines. The data in Fig. 3 indicate further that local injections of bryostatin 1 may have a slight systemic effect: control papillomas which received no bryostatin 1 showed a reduction in growth rate when another lesion on the same animal was treated. Whether this result is due to systemic immune stimulation by locally produced in¯ammatory factors or to some other mechanism remains to be determined. As shown in Figs. 1 and 2, the growth of rabbit papillomas shows an unusual biphasic response to bryostatin 1; the lower doses administered showed a reduction in papilloma size, while the higher doses showed either no reduction or some enhancement at various stages. Bryostatin 1 has been shown to differentially down-regulate the protein kinase C (PKC) isoform delta in a biphasic manner: at low concentrations, bryostatin 1 down-regulated PKC-d analogously to the phorbol ester, phorbol 12-myristate13-acetate (PMA), but at higher concentrations, bryostatin 1 failed to do so. Further, at the higher concentrations, bryostatin 1 protected PKC-d from down-regulation by PMA [33]. This biphasic effect of bryostatin 1 is seen in a range of experimental systems and parallels its biological activities [34], suggesting that the differential down-regulation of some PKC isoforms, particularly PKC-d, may be responsible for at least some of the effects described here. There is some evidence that a variety of viral protein functions may depend on PKC activity. Jones and co-workers showed that HPV-immortalized keratinocytes were resistant to a variety of differentiation stimulators but growth was inhibited by staurosporine, a PKC inactivator [35]. The papillomavirus E7 gene product has been shown to be a substrate of PKC in vitro [36], and PKC has also been shown to regulate some of the papillomavirus late gene products [37]. Furthermore, the papillomavirus E5 oncoprotein activates c-jun via a PKC-dependent pathway [38]. Chronic exposure to bryostatin 1 may directly interfere with the viral life cycle by reducing the levels or ratios of active PKC isoforms. This would interfere with the tumor-inducing capacity of the virus and the subsequent development of a papilloma. As very little detailed work has been done to examine the functional
roles of various PKC isoforms in papillomas, further biochemical studies are needed to con®rm this possibility. The weekly intravenous administration of 2.5 mg/ kg of bryostatin 1 produced the maximum observed reduction in papilloma growth. This re¯ects the extreme potency of bryostatin1 in vivo, with observed activity at nanomolar concentrations. At higher concentrations, bryostatin 1 did not display inhibitory effects on the growth of papillomas. This stands in contrast to the results of some of the ®rst human clinical trials on other types of tumors where bryostatin1 administration was recommended at its maximum tolerated dose [18]. The optimum therapeutic dose may well be a function of the speci®c type of tumors involved. Future studies are needed to de®ne these critical parameters for other tumor systems. In summary, bryostatin 1, when administered both intralesionally and intravenously, signi®cantly slowed the growth of rabbit papillomas, but no schedule or dose reduced the total number of papillomas formed. Finally, intralesional administration of bryostatin 1 promoted the in®ltration of both heterophils and eosinophils. Future studies should shed light on the potential use of bryostatin 1 in the modulation of HPVmediated human disease. Acknowledgements We would like to thank Steve Moss, Steve Baldwin, David Eldredge, Brett Cherry, and Mata Cain for their assistance in the lab. This study was funded in part by grants from The Brigham Young University Professional Development Committee and the BYU Cancer Research Center. References [1] G.R. Pettit, The Bryostatins, in: W. Herz, G.W. Kirby, W. Steglick, C. Tamm (Eds.), Progress in the Chemistry of Organic Natural Products, Vol. 57, Springer, Vienna, 1991, pp. 154±195. [2] K.J. Hale, J.A. Lennon, S. Manaviazar, M.H. Javaid, C.J. Hobbs, Asymmetric synthesis of the C(17)-C(27) segment of the antineoplastic macrolide bryostatin 1, Tetrahedron Lett. 36 (1995) 1359±1362. [3] J.H. Beijnen, K.P. Flora, G.W. Halbert, R.E.C. Henrar, J.A. Slack, CRC/ EORTC/NCI Joint Formulation Working Party:
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The inhibitory effects of bryostatin 1 administration on the growth of rabbit papillomas Jason M. Bodily a, David J. Hoopes a, Beverly L. Roeder b, Sharon G. Gilbert a, George R. Pettit c, Cherry L. Herald c, Darin N. Rollins d, Richard A. Robison a,* a Department of Microbiology, Brigham Young University, 791 WIDB, PO Box 25133, Provo, UT 84602-5133, USA Department of Animal Science, Brigham Young University, 791 WIDB, PO Box 25133, Provo, UT 84602-5133, USA c Cancer Research Institute, Arizona State University, Tempe, Arizona, USA d Department of Statistics, Brigham Young University, 791 WIDB, PO Box 25133, Provo, UT 84602-5133, USA
b
Received 6 July 1998; received in revised form 18 September 1998; accepted 18 September 1998
Abstract Bryostatin 1 is a protein kinase C modulator that shows antineoplastic activity in a variety of tumor systems. This study examined the effects of bryostatin 1 administration on papilloma growth in rabbits. Investigations of optimal route, dose, and schedule were performed. Several groups of rabbits were inoculated with cottontail rabbit papillomavirus (CRPV) DNA. Bryostatin 1 was administered i.v., both daily and weekly, and intralesionally both weekly and bi-weekly. Intralesionally dosed papillomas were examined histologically for immune cell in®ltration. In weekly and daily i.v. trials, 2.5 and 1.0 mg/kg, respectively, showed the greatest overall reduction in tumor size. Bryostatin 1 administered intralesionally also slowed papilloma growth. Treated lesions had signi®cantly higher numbers of heterophils and eosinophils. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Bryostatin 1; Papillomavirus; Cancer therapeutics; PKC; Cervical cancer
1. Introduction The bryostatins are a unique family of 20 naturally occurring macrocyclic lactones which have demonstrated considerable antineoplastic activity. Bryostatin 1 was ®rst isolated from the marine bryozoan Bugula neritina collected off the California coast. B. neritina is a colonial ®lter-feeder commonly known as a sea-mat or false coral and often grows on docks, pilings, rocks, and ship hulls in temperate ocean areas [1]. Several teams of chemists have succeeded in synthesizing bryostatin 7 and portions of bryostatin * Corresponding author. Tel.: 1 1-801-378-2416; fax: 1 1-801378-9197; e-mail: [email protected].
1 [2], but so far the bryozoan is the only source of bryostatin 1 [3]. Both in vitro and in vivo studies have demonstrated the anti-tumor activity of bryostatin 1. For example, bryostatin 1 treatment inhibited the in vitro growth of human lung tumor cells [4], murine reticular cell sarcoma and lymphoma cells [5], and U937 human leukemia cell lines [6]. Bryostatin 1 also induced the differentiation of HL-60 cells [7] and myeloid [8,9] and lymphocytic [10] human leukemias. The in vivo antineoplastic activity of bryostatin 1 has been shown by its ability to cure mice with ovarian sarcomas [11], P388 lymphocytic leukemia [12], and malignant melanoma [13]. In combination with other therapeutic agents, it has also been shown to cure human Walden-
0304-3835/99/$ - see front matter q 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(98)00310-3
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stroÈm's macroglobulinemia in SCID mice [14] and to sensitize HeLa cells to cisplatin [15]. Several phase I human clinical trials of bryostatin 1 against a range of cancers have been completed [16±18], revealing some bene®cial effects against malignant melanoma, ovarian carcinoma, non-Hodgkin's lymphoma, and cervical cancer (unpublished). Although the list of transformed cells against which bryostatin 1 has been tested is extensive, its activity against virus-induced tumors has not been investigated. The discovery that certain urogenital cancers, such as cervical cancer, are highly linked to infection by some types of human papillomaviruses (HPV) has provided an impetus to study these types of transformed cells [19,20]. HPV DNA is present in 93% of all cervical cancers. Worldwide, cervical cancer ranks second only to breast cancer in both mortality and incidence as the most common human female malignancy with 47 000 new cases diagnosed each year [21]. Of the current therapies available to treat HPV infections, none are capable of eliminating the virus completely, and recurrence rates are high [22]. A more effective treatment for HPV-induced tumors would therefore be of tremendous bene®t in reducing the incidence of urogenital cancers. The cottontail rabbit papillomavirus (CRPV) system is recognized as a good model for human papillomavirus-induced transformation [23]. The present study was undertaken to examine the effects of bryostatin 1 administration on experimentally induced rabbit papillomas. Different routes, doses, and treatment schedules were evaluated in an effort
to de®ne optimal therapeutic conditions. We report that bryostatin 1 slows the growth of papillomas when administered either intravenously or intralesionally 2. Materials and methods 2.1. Isolation and puri®cation of CRPV-DNA The plasmid CRPV-pLAII contains the genome of the CRPV Washington B strain. Plasmid was isolated by modi®cation of a method previously described by Cattaneo et al. [24]. Escherichia coli HB101 cells containing CRPV-pLAII (kindly provided by Dr. Janet Brandsma, Yale University) were grown in Terri®c broth (1.2% tryptone (Difco, Detroit, MI), 2.4% yeast extract (Difco), 0.4% glycerol) for 18 h at 378C, shaking at 250 rev./min. The cells were pelleted and resuspended in TEG buffer (25 mM Tris±HCl (pH 8.0), 10 mM EDTA (pH 8.0), 50 mM glucose) incubated with lysozyme, and treated with lysis solution (2% SDS, 0.5 M NaOH) and put on ice for 15 min. Potassium acetate (5 M) was added, the mixture shaken mildly, and again placed on ice for 15 min. Precipitated cell walls, membranes, and genomic DNA were pelleted, and the supernatant containing plasmid DNA was ®ltered and precipitated with 100% ethanol overnight at 2 208C. The precipitate was pelleted and resuspended in a solution containing 100 mg/ml RNase A and incubated for 30 min at 308C. Suf®cient 5 M NaCl and 30% polyethylene glycol were added to precipitate the plasmid. The
Table 1 A summary of experiments involving intravenous bryostatin 1 treatments Dosing schedule
Number of rabbits
Dose of bryostatin 1 (mg/kg)
Treatment initiation (days post CRPV DNA inoculation)
Dosing duration (days)
Slowed papilloma growth
Weekly
4 4 4 4 4
0.0 2.5 5.0 10.0 20
0 0 0 0 0
70 70 70 70 ±a
No Yes (P , 0:05) No No ±a
Daily
4 3 5
0.0 0.1 1.0
15 15 15
38 38 38
No Yes (P , 0:0002) Yes (P , 0:05)
a
This dose was lethal to all rabbits within 12 h of ®rst dose.
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2.3. Catheter implantation
Fig. 1. Final mean papilloma volumes after 70 days of weekly i.v. administration at doses of 2.5, 5.0, 10.0 and 0.0 mg/kg. Bryostatin 1 treatment was initiated immediately after CRPV DNA inoculation. Each group consisted of four rabbits. Bars represent ^ one standard error of the mean.
solution was put on ice for 2 h after which the plasmid DNA was pelleted. After resuspension in TE buffer, the DNA was incubated for 30 min in Proteinase K solution (0.05 mg/ml Proteinase K, 1% SDS) at 608C. An equal volume of 50 mM NaCl/40 mM NaOH solution was added, incubated for 15 min at room temperature, and neutralized with 2.0 M Tris (pH 7.5). The plasmid was puri®ed with two to three phenol/chloroform extractions and one chloroform extraction. The plasmid was precipitated with 300 mM sodium acetate and two volumes of 95% ethanol and then pelleted. Plasmid was resuspended in TE buffer at a concentration of , 1 mg DNA/ml. 2.2. Experimental animals and induction of papillomas Random-bred 2-kg female New Zealand White rabbits (Oryctolagus cuniculus, Western Oregon Rabbit Company, Philomath, OR) served as subjects in this study. Papillomas were induced following methods described by Brandsma et. al. [25]. Brie¯y, the animals' backs were shaved and, under anesthesia, 100 sites on each animal were inoculated with 50 ml CRPV DNA in TE buffer (1 mg DNA/ml) using a PedO-Jet air-injector (Stirn Industries, Dayton, NJ).
Rabbits that were to receive daily i.v. treatments were catheterized to avoid repeated venipuncture. Indwelling catheters were surgically implanted in the right jugular vein on day 14±16 post CRPV DNA inoculation. The procedure outlined by PerryClark and Meunier [26] was used with the following modi®cations: Venocath-18 catheters (Abbott Ireland, Sligo, Ireland) were used in place of vascular access ports. A screw-in male adapter plug (Abbott Laboratories, North Chicago, IL) was attached to the exteriorized hub of the Venocath to allow daily drug injection. The catheter hub was sutured to the skin at the exit site to minimize the risk of disturbance. The entire area was protected with a canvas/Velcro patch glued to the rabbit's fur with Kamar adhesive (Kamar Inc, Steamboat Springs, CO). For those animals receiving weekly treatment, i.v. drug was administered using a 24 gauge 3/4 inch Angiocath (Becton Dickinson Vascular Access, Sandy, UT) via the marginal ear vein. 2.4. Drug administration 2.4.1. Intravenous administration Bryostatin 1 was isolated as described (12), and solutions of bryostatin 1 were formulated in a 60% ethanol/saline carrier solution (0.9% NaCl) to produce solutions corresponding to each of the dosage groups. The volumes of solution administered were determined for each rabbit to assure proper dosage according to rabbit mass; administered volumes ranged from 70 to 110 ml. The control rabbits were given comparable volumes of carrier solution without bryostatin 1. Treatments were chased with either 5.0 ml of Lactated Ringers Solution (weekly treatments) or 1.5 ml of 100 units/ml heparin sodium in 0.9% saline (daily treatments). Doses were administered as listed in Table 1. 2.4.2. Intralesional administration After serving as controls in the i.v. study, eight rabbits with matched pairs of equal-sized papillomas (approximately 150 mm 3) were selected as subjects in the intralesional study. One papilloma in each pair received 0.5±5 mg of bryostatin 1 in 25 ml 60% ethanol/saline solution while the companion lesion
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Fig. 2. The growth of rabbit papillomas during daily i.v. bryostatin 1 treatment. Values represent mean volumes of all papillomas on n rabbits. Treatment groups were: W: 1.0 mg/kg, n 5; B: 0.1 mg/kg, n 3; O: control, n 4. Bars represent ^ one standard error of the mean.
received an equal volume of carrier solution with no bryostatin 1. Doses were injected into the dermal layer immediately beneath the papilloma using a 26 gauge needle. Treatments continued weekly for 10 weeks. At 1 and 2 weeks post treatment initiation, one half of the papillomas were biopsied. Rabbits were anesthe-
Fig. 4. Immune cell in®ltrates into bryostatin 1-treated (A) and control (B) papillomas. Values are mean scores from biopsies taken at 10 and 20 weeks (n 16 biopsies per treatment group). Bars represent ^ one standard error of the mean.
tized, and biopsies of the lesions were taken using a 1.5 mm dermal punch (Acuderm Inc, Ft. Lauderdale, FL). Samples were ®xed in 10% formaldehyde and imbedded in paraf®n. Sections were cut with a microtome and subjected to sequential washes with xylene, 100% ethanol, 70% ethanol, and distilled water. A standard tissue stain was performed using hematoxylin and eosin. An experienced pathologist examined the entirety of two sections from each biopsy (250 £ magni®cation) and used a semi-quantitative assessment method to determine the extent of immune cell in®ltration. Speci®c immune cells were identi®ed by their characteristic staining and morphology. The sections were dehydrated with washes of 95% ethanol, 100% ethanol, and xylene. The presence of eosinophils, monocytes, and heterophils was quantitated with scores ranging from 0 to 3 (0, absence of cells; 3, abundant in®ltration). After 10 weeks, six pairs of papillomas were further treated bi-weekly with 0.5 mg bryostatin 1 for 10 additional weeks. 2.5. Statistical analysis
Fig. 3. Growth of rabbit papillomas during intralesional bryostatin 1 treatment. O: control, n 8; X: treatment, n 8. From 0 to 10 weeks, bryostatin 1 was administered weekly. From 10 to 20 weeks, administration was bi-weekly. Treatment doses ranged from 0.5 to 5.0 mg/kg, but since there was no signi®cant difference in effect between doses, the data were combined for simplicity. Bars represent ^ one standard error of the mean.
Statistical analyses were performed with SAS software (Cary, North Carolina) using a mixed model analysis of variance. Individual dosage levels were compared with controls using standard contrasts.
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3. Results 3.1. Intravenous bryostatin 1 administration Table 1 summarizes the experiments evaluating intravenous bryostatin 1 administration. No dose, route, or schedule of bryostatin 1 administration produced a signi®cant difference in the total number of papillomas formed. Fig. 1 shows that weekly i.v. administration of bryostatin 1 diminished the size of developing papillomas. The average papilloma volume per rabbit in the 2.5 mg/kg group was 92.0% smaller than that of the controls after 70 days (P , 0:05) while the volumes in the other two treatment groups (5 mg/kg, 10 mg/kg) did not differ significantly from the controls. Daily i.v. treatment with bryostatin 1 at 0.1 mg/kg (Fig. 2) slowed papilloma development by about 66% relative to the controls after 60 days (P , 0:0002). The 1.0 mg/kg daily dose increased the size of papillomas in the initial weeks but led to a reduction by the end with a ®nal reduction of 72% as compared to the controls. The 20 mg/kg i.v. dose proved lethal to all rabbits. This is signi®cantly lower than the previously reported LD10 (29 mg/kg) and LD50 (68 mg/kg) in rodents (15). However, at lower doses, no adverse effects of bryostatin 1 administration were observed on the behavior of the animals (drinking, eating, urinating, etc.). In animals that received injections via the ear veins, slight endothelial cell toxicity was observed in some rabbits. 3.2. Intralesional bryostatin 1 administration Intralesional administration of bryostatin 1 both retarded papilloma growth and promoted tumor regression. Fig. 3 depicts the total papilloma volume for treated papillomas versus matched controls on the same animal. No signi®cant difference was noted between the various doses of bryostatin (0.5±5.0 mg/ kg) administered. Analysis revealed that the treated papillomas were signi®cantly smaller than the controls both at 10 weeks (P , 0:05) and at 20 weeks (P , 0:01). During the ®rst 10 weeks (weekly doses), the treated papillomas increased in volume but at a slower rate than the controls. However, during the second 10 weeks (bi-weekly doses) the treated papillomas showed a marked decrease in volume, leading
71
to a reduction of about 68% by the end of the 20 week test period. Fig. 4 shows the histological effects of the intralesional administration of bryostatin 1. Immune cell in®ltration into treatment and control papillomas differed signi®cantly at the level of P , 0:05 (eosinophil) and P , 0:08 (heterophil). There was no significant difference in the numbers of mononuclear cells. Lesions that were regressing by the time of biopsy were found to have a higher heterophil and eosinophil in®ltration as compared to those that were not regressing (data not shown). 4. Discussion Figs. 3 and 4 show that injections of bryostatin 1 lead to papilloma regression with a concomitant in®ltration of heterophils and eosinophils. At least some of bryostatin 1's antineoplastic activity can be attributed to its ability to activate immune cells and stimulate cytokine production. It has been proven to enhance production of hematopoietic growth factors, increase plasma interleukin-6 and TNF-a concentrations, and activate polymorphonuclear leukocytes [16,27,28]. Working synergistically with the immune modulator AS101, it was shown to induce the secretion of tumor necrosis factor (TNF), gamma interferon (IFN-g) and interleukin-2 (IL-2) by human mononuclear cells and TNF and IL-2 by mouse lymphoid cells [29]. In conjunction with IFN-g, bryostatin 1 has been shown to up-regulate the inducible NO synthase gene and induce the production of NO in murine macrophages [30]. In this study, the in®ltration of heterophils and eosinophils was signi®cantly increased in treated lesions relative to controls (Fig. 4). Treatment also correlated with a signi®cantly smaller papilloma volume (Fig. 3). It was noted that individual lesions that were regressing, regardless of whether they had received treatment or not, had elevated levels of heterophils and eosinophils when compared to lesions that were not regressing (data not shown). Further, elevated levels of neutrophils were also found in regressing lesions in the bovine papilloma system using a different modulator [31]. Neutrophils have been shown to possess cytotoxic activity [32]. Taken together, these results suggest that the regression of papillomas injected with bryostatin 1 may be due, at
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least in part, to the action of these in¯ammatory cells, which are either directly activated by bryostatin 1 or by bryostatin 1-induced cytokines. The data in Fig. 3 indicate further that local injections of bryostatin 1 may have a slight systemic effect: control papillomas which received no bryostatin 1 showed a reduction in growth rate when another lesion on the same animal was treated. Whether this result is due to systemic immune stimulation by locally produced in¯ammatory factors or to some other mechanism remains to be determined. As shown in Figs. 1 and 2, the growth of rabbit papillomas shows an unusual biphasic response to bryostatin 1; the lower doses administered showed a reduction in papilloma size, while the higher doses showed either no reduction or some enhancement at various stages. Bryostatin 1 has been shown to differentially down-regulate the protein kinase C (PKC) isoform delta in a biphasic manner: at low concentrations, bryostatin 1 down-regulated PKC-d analogously to the phorbol ester, phorbol 12-myristate13-acetate (PMA), but at higher concentrations, bryostatin 1 failed to do so. Further, at the higher concentrations, bryostatin 1 protected PKC-d from down-regulation by PMA [33]. This biphasic effect of bryostatin 1 is seen in a range of experimental systems and parallels its biological activities [34], suggesting that the differential down-regulation of some PKC isoforms, particularly PKC-d, may be responsible for at least some of the effects described here. There is some evidence that a variety of viral protein functions may depend on PKC activity. Jones and co-workers showed that HPV-immortalized keratinocytes were resistant to a variety of differentiation stimulators but growth was inhibited by staurosporine, a PKC inactivator [35]. The papillomavirus E7 gene product has been shown to be a substrate of PKC in vitro [36], and PKC has also been shown to regulate some of the papillomavirus late gene products [37]. Furthermore, the papillomavirus E5 oncoprotein activates c-jun via a PKC-dependent pathway [38]. Chronic exposure to bryostatin 1 may directly interfere with the viral life cycle by reducing the levels or ratios of active PKC isoforms. This would interfere with the tumor-inducing capacity of the virus and the subsequent development of a papilloma. As very little detailed work has been done to examine the functional
roles of various PKC isoforms in papillomas, further biochemical studies are needed to con®rm this possibility. The weekly intravenous administration of 2.5 mg/ kg of bryostatin 1 produced the maximum observed reduction in papilloma growth. This re¯ects the extreme potency of bryostatin1 in vivo, with observed activity at nanomolar concentrations. At higher concentrations, bryostatin 1 did not display inhibitory effects on the growth of papillomas. This stands in contrast to the results of some of the ®rst human clinical trials on other types of tumors where bryostatin1 administration was recommended at its maximum tolerated dose [18]. The optimum therapeutic dose may well be a function of the speci®c type of tumors involved. Future studies are needed to de®ne these critical parameters for other tumor systems. In summary, bryostatin 1, when administered both intralesionally and intravenously, signi®cantly slowed the growth of rabbit papillomas, but no schedule or dose reduced the total number of papillomas formed. Finally, intralesional administration of bryostatin 1 promoted the in®ltration of both heterophils and eosinophils. Future studies should shed light on the potential use of bryostatin 1 in the modulation of HPVmediated human disease. Acknowledgements We would like to thank Steve Moss, Steve Baldwin, David Eldredge, Brett Cherry, and Mata Cain for their assistance in the lab. This study was funded in part by grants from The Brigham Young University Professional Development Committee and the BYU Cancer Research Center. References [1] G.R. Pettit, The Bryostatins, in: W. Herz, G.W. Kirby, W. Steglick, C. Tamm (Eds.), Progress in the Chemistry of Organic Natural Products, Vol. 57, Springer, Vienna, 1991, pp. 154±195. [2] K.J. Hale, J.A. Lennon, S. Manaviazar, M.H. Javaid, C.J. Hobbs, Asymmetric synthesis of the C(17)-C(27) segment of the antineoplastic macrolide bryostatin 1, Tetrahedron Lett. 36 (1995) 1359±1362. [3] J.H. Beijnen, K.P. Flora, G.W. Halbert, R.E.C. Henrar, J.A. Slack, CRC/ EORTC/NCI Joint Formulation Working Party:
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