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Effect of PKC412, a selective inhibitor of protein kinase C, on lung metastasis in mice injected with B16 melanoma cells
Life Sciences 72 (2003) 1377 – 1387 www.elsevier.com/locate/lifescie
Effect of PKC412, a selective inhibitor of protein kinase C, on lung metastasis in mice injected with B16 melanoma cells Noriko Yoshikawa a, Kazuki Nakamura a,*, Yu Yamaguchi a, Satomi Kagota a, Kazumasa Shinozuka a,b, Masaru Kunitomo a,b a
Department of Pharmacology, Faculty of Pharmaceutical Sciences, Mukogawa Women’s University, 11-68, Koshien Kyuban-cho, Nishinomiya, Hyogo 663-8179, Japan b Institute for Biosciences, Mukogawa Women’s University, Nishinomiya, Hyogo, Japan Received 7 June 2002; accepted 18 October 2002
Abstract PKC412, a selective inhibitor of protein kinase C (PKC), is currently in clinical trials as an anti-tumor drug. In the present study, we investigated the anti-metastatic effect of PKC412 using an experimental metastatic mouse model intravenously injected with melanoma cells. One-hour exposure to various concentrations of PKC412 (0.5, 5 and 50 AM) dose-dependently reduced the lung-metastatic potential of highly metastatic B16F10 and -BL6 mouse melanoma cells in syngeneic mice. Following the exposure, PKC activities in B16-F10 and -BL6 cells were significantly decreased, but growth curves were not influenced. To elucidate the mechanism of the anti-metastatic effect of PKC412, we examined the activity to invade the extracellular matrix and the platelet-aggregating activity of the melanoma cells incubated with PKC412 (0.5, 5 and 50 AM) for 1 hour. PKC412 significantly reduced both the invasive and platelet-aggregating activities. These results suggest that PKC412 shows an anti-metastatic function through the inhibition of the invasive and/or plateletaggregating activities of melanoma cells. PKC412 is potentially a promising candidate for an anti-metastatic agent. D 2002 Elsevier Science Inc. All rights reserved. Keywords: PKC inhibitor; Anti-metastasis; B16 mouse melanoma cells; Chemo-invasion; Platelet aggregation
* Corresponding author. Tel./fax: +81-798-45-9945. E-mail address: [email protected] (K. Nakamura). 0024-3205/02/$ - see front matter D 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 2 ) 0 2 4 0 7 - 4
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Introduction Protein kinase C (PKC) is one of the key enzymes in cellular signal transduction pathways and controls cell proliferation and differentiation. PKC is a family of serine/threonine kinases and consists of at least 12 isoforms. These isoforms are divided into three groups based on their structure and substrate requirements [1–3]. The first group is the conventional PKCs, dependent on Ca2 + and activated by phosphatidylserine and diacylglycerol. The second group is the novel PKCs which act independent of Ca2 + and are regulated by phosphatidylserine and diacylglycerol. The third group is the atypical PKCs which act independent of Ca2 + but do not require diacylglycerol for their activation although phosphatidylserine regulates their activity. O’Brian et al. showed that levels of PKC expression were elevated in human breast tumor biopsies compared with normal breast tissues [4]. Benzil et al. also reported that the expression of PKC-a in human astrocytomas may serve as a direct biological marker of malignancy [5]. PKC-a is known to be mainly expressed in highly metastatic B16 mouse melanoma cells, and the activity of PKC-a in B16 cells is higher than that in weakly metastatic amelanotic B78-H1 melanoma cells without changes in PKC-a expression [6]. PKC412 (4V-N-benzoyl staurosporine) is a more specific but less potent PKC inhibitor than staurosporine [7]. PKC412 potently inhibits the binding of ATP to PKC, especially conventional PKCs; a, hI, hII and g subtypes. PKC412 also inhibits other kinases including tyrosine protein kinases in the receptors for vascular endothelial growth factor (VEGF), platelet derived growth factor (PDGF) and the stem cell factor, c-kit [8]. Accordingly, PKC412 seems likely to suppress tumor growth by the inhibition of conventional PKCs together with an angiogenic response to VEGF instead of basic fibroblast growth factor (bFGF) or hepatocyte growth factor (HGF). At present, PKC412 is undergoing clinical trials as an anti-tumor agent. It is demonstrated that PKC412 can be safely administered by chronic oral therapy and a suitable daily dose for phase II studies is 150 mg given the results of the phase I study [9]. In this study, we focused on the anti-metastatic effect of PKC412 instead of the anti-tumor effect. First, we investigated the effect of PKC412 using a hematogenic lung metastatic mouse model injected with highly metastatic melanoma cells. Second, to elucidate the anti-metastatic mechanism of PKC412, we examined the effect of PKC412 on the activity to invade the extracellular matrix and the plateletaggregating activity of melanoma cells in vitro.
Methods Materials PKC412 (powder) was kindly donated by Novartis Pharma AG (Basel, Switzerland). Stock solution was prepared in dimethylsulfoxide (DMSO) and diluted to the required concentrations in culture medium before use. The EDTA trypsin solution (EDTA; 0.02%, trypsin; 0.1%) and penicillin/ streptomycin solution (penicillin; 50000 U/ml, streptomycin; 50 mg/ml) were purchased from Cosmo Bio Co., LTD (Tokyo, Japan). Dulbecco’s modified Eagle’s medium (DMEM) with glutamine was obtained from ICN Biomedicals, Inc. (Aurora, OH). Dulbecco’s phosphate-buffered saline without calcium and magnesium (DPBS) was from Nissui Pharmaceutical Co., LTD (Tokyo, Japan). Fetal bovine serum (FBS) was from Sigma Chemical Co. (St. Louis, MO). Pierce Colorimetric PKC Assay
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Kit, SpinZyme Format was from Pierce (Rockford, IL). Growth factor reduced MATRIGEL matrix and BIOCOAT cell culture inserts were from Becton Dickinson Labware (Bedford, MA). Animals For the metastatic melanoma syngeneic animals, specific pathogen-free female C57BL/6Cr mice (6 weeks old) were purchased from Japan SLC, Inc (Hamamatsu, Japan). For the platelet aggregation experiments, male Japanese white rabbits (2–3 kg) were purchased from Shimizu Laboratory Animals (Kyoto, Japan). The mice and rabbits were maintained in an air-conditioned room (23 F 2 jC and 60 F 10% humidity) under a 12 hour light/dark cycle (7:00 a.m.–7:00 p.m.). Food and water were given ad libitum during the experiment. All procedures followed the Guiding Principles for the Care and Use of Laboratory Animals approved by The Japanese Pharmacological Society. Cells A mouse epithelial-like melanoma cell line, B16-F0, was obtained from the American Type Culture Collection (Rockville, MD). The B16-F1 cell line was derived from pulmonary metastases produced by the intravenous injection of B16-F0 cells into a syngeneic C57BL/6Cr mouse and the B16-F10 cell line was selected repeatedly ten times by successive passage of lung colonies; the in vivo-in vitro selections based on Fidler’s method [10]. B16-F10 cells were injected into the urinary bladder of male C57BL/6 mice via the vas deferens and the bladder was ligated, excised and maintained on semi-solid agar. Tumor cells that migrated through the wall of the bladder were recovered from the agar, cultured and repassaged. This process was repeated six times and the resulting variant was designated B16-BL6 [11]. The B16-BL6 cell line was kindly donated by Dr. Futoshi Okada at Hokkaido University (Sapporo, Japan). B16 cells passaged less than 25 times were used in all experiments. The doubling times of B16F10 and -BL6 were 13.1 and 12.4 hours, respectively. Cells were cultured in DMEM containing 10% FBS and the penicillin/streptomycin solution. Assay of experimental metastasis of tumor cells Sub-confluent B16-F10 and -BL6 cells were incubated with PKC412 (0, 0.5, 5 and 50 AM) for 1 hour at 37 jC, harvested with EDTA trypsin solution and resuspended to appropriate concentrations in DPBS. Cells (1 105/0.2 ml) were injected into the tail vein of syngeneic C57BL/6Cr mice. Mice were anesthetized with pentobarbital and sacrificed at 15 days after tumor injection. The lung was excised and fixed in a formaldehyde neutral buffer solution. Nodules visible as black forms in the lung were enumerated with the aid of a magnifying glass. Growth curves for tumor cells in vitro Sub-confluent B16-F10 and -BL6 cells were incubated with PKC412 (0, 0.5, 5 and 50 AM) for 1 hour at 37 jC, harvested with EDTA trypsin solution, and resuspended to appropriate concentrations in DMEM containing 10% FBS. Then, 1 105 cells/2 ml in each well of a 12-well culture plate were incubated for 24, 48 and 72 hours in a CO2 incubator at 37 jC. Viable cells of triplicate samples were enumerated with a Coulter counter (Coulter Z1, Beckman Coulter, Inc., Tokyo, Japan).
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Assay of PKC activity in tumor cells using the colorimetric method The assay of PKC activity was carried out according to the kit supplier’s instructions. Briefly, subconfluent B16-F10 and -BL6 cells were exposed to PKC412 (F10: 0, 5 and 50 AM; BL6: 0, 0.5, 5 and 50 AM) for 1 hour at 37 jC, rinsed three times with ice cold DPBS, scraped off, and centrifuged at 200 g for 8 minutes at 4 jC. Cell pellets were lysed in 0.5 ml of homogenization buffer (pH7.5; 20 mM Tris-HCl, 1.0 mM EDTA-2Na, 0.1 AM Okadaic acid, 10.0 Ag/ml of leupeptin, and 0.5% Triton X100, w/v). After centrifugation at 1500 g for 10 minutes at 4 jC, the supernatant, designed as crude enzyme, was used as a source of cellular PKC. Each sample (25 Ag protein) was mixed with dyelabeled substrate, phosphatidylserine, and reaction buffer containing ATP, MgCl2, CaCl2, Tris-amino methane and 0.01% surfactant. After incubation at 30 jC for 30 minutes, the mixtures were mounted on affinity membranes to isolate the PKC-catalyzed products. The absorbance of these products at 570 nm was determined with a spectrophotometer (UV-2100, Shimadzu Co., Kyoto, Japan). Invasion assay for tumor cells in vitro The assay of the invasiveness of B16-F10 and -BL6 cells in vitro was carried out as described by Albini [12]. Briefly, 6.4 mm diameter Transwells were used with tracked-etched polyethylene terephthalate (PET) membrane filters (8 Am pore size) coated 25 Ag/filter MATRIGEL basement membrane matrix extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma. Sub-confluent cells were exposed to PKC412 (0, 0.5, 5 and 50 AM) for 1 hour at 37 jC, harvested with EDTA trypsin solution, and resuspended to appropriate concentrations in DMEM containing 0.1% bovine serum albumin. Five hundred microliter samples of 5 104 cells were placed in the upper chamber compartments. The lower chambers contained 750 Al of serum-free conditioned medium from cultures of 3T3 cells for 24 hours as a chemoattractant. After 24 hours of incubation in a tissue culture incubator, non-invading cells on the upper side of the filter were completely removed by wiping with a cotton swab. Cells that had penetrated through the matrix protein and adhered to the lower surface of the filter were counted microscopically after fixing with methanol and staining with 3% Giemsa in DPBS. Platelet aggregation induced by tumor cells in vitro Blood was collected from the rabbit marginal vein, anti-coagulated with sodium citrate and centrifuged for 10 minutes at 160 g and room temperature to obtain platelet-rich plasma (PRP). Platelet numbers in PRP were counted with a Sysmex K-4500 hematology analyzer (Sysmex Inc., Kobe, Japan). After removal of the upper layer (PRP), the lower layer was centrifuged for 10 minutes at 1000 g and room temperature to obtain platelet-poor plasma (PPP). Platelet aggregation was measured in a cuvette ( = absorption tube) at 37 jC using an aggregometer (Aggrecorder PA-3210, ARKRAY Inc., Kyoto, Japan). Sub-confluent B16-F10 cells were exposed to PKC412 (0, 0.5, 5 and 50 AM) for 1 hour at 37 jC, harvested with EDTA trypsin solution, and resuspended to appropriate concentrations in DPBS. A 1 106/50 Al cell suspension was added to a cuvette containing 0.45 ml of PRP preincubated with 2 mM CaCl2 and the reaction started. The maximum percentage of aggregation for 5 minutes was calculated with the difference in the absorbance of the melanoma cell suspension (1 106) in PRP taken as 0% aggregation and that in PPP as 100% aggregation.
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Statistical analyses The data are expressed as the mean F S.E.M. of 3–7 animals. Statistical analyses were performed by ANOVA followed by Fisher’s PLSD (protected least significant difference) test using the Stat View software package (Abacus Concepts Inc., Berkeley, CA). A difference was considered significant at P < 0.05.
Results Effect of PKC412 on the hematogenic lung metastatic mouse model using B16-F10 and -BL6 cells The mice injected with the tumor cells after pre-incubation with 0, 0.5, 5 and 50 AM PKC412 for 1 hour displayed visible lung nodules 15 days after the injection. Fig. 1 shows a representative photograph of a typical lung with metastatic melanoma nodules.
Fig. 1. Appearance of lungs from C57BL/6Cr mice injected intravenously with highly metastatic B16-BL6 melanoma cells (1 105), after pre-treatment with 0 (control), 0.5, 5 and 50 AM PKC412 for 1 hour at 37 jC. Fifteen days later, mice were anesthetized with pentobarbital and lungs were excised. Each photograph shows a representative specimen from each group and the normal sample is a lung from an age-matched mouse injected intravenously with the same volume of DPBS.
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In the experiment using B16-F10 cells, the mean number of lung nodules was 153 F 39, 123 F 23, 82 F 14 and 53 F 17, respectively. The mean number of nodules after pre-incubation with 5 and 50 AM PKC412 decreased significantly 46 and 65% compared to the control (0 AM PKC412) in a dose-dependent manner (Fig. 2a). In the experiment using B16-BL6 cells, the number of lung nodules following pretreatment with 0, 0.5, 5 and 50 AM PKC412 was 275 F 50, 213 F 23, 198 F 46 and 148 F 30, respectively. The number of nodules after pre-incubation with 50 AM PKC412 showed a significant decrease of 46% compared to the control (0 AM PKC412) mice (Fig. 2b). Effect of PKC412 on the growth curves for B16-F10 and -BL6 cells The growth curves for both B16-F10 and -BL6 cells pretreated with PKC412 (0.5, 5 and 50 AM) for 1 hour at 37 jC did not change compared with the control (0 AM PKC412) (Fig. 3a and b). Effect of PKC412 on the PKC activity in B16-F10 and -BL6 cells The PKC activity in both B16-F10 and -BL6 cells pretreated with 50 AM PKC412 was significantly reduced 24% compared with the control (0 AM PKC412) (Fig. 4a and b). Effect of PKC412 on the invasion assay for B16-F10 and -BL6 cells In the experiment using B16-F10 cells, the number of the invading cells on pretreatment with 0, 0.5, 5 and 50 AM PKC412 was 145 F 5, 106 F 11, 84 F 14 and 103 F 12, respectively. The invasive
Fig. 2. Inhibitory effect of PKC412 on the hematogenic lung metastasis in C57BL/6Cr mice. Highly metastatic B16-F10 (a) and -BL6 (b) mouse melanoma cells (1 105), which had been treated with 0 (control), 0.5, 5 and 50 AM PKC412 for 1 hour at 37 jC, were injected into the tail vein of syngeneic C57BL/6Cr mice. Fifteen days later, visible nodules metastasized in the lung were enumerated with the aid of a magnifying glass. Data are expressed as the mean F S.E.M. of 5 – 7 mice. **P < 0.01, *P < 0.05 vs. control.
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Fig. 3. Effects of PKC412 on growth curves for B16-F10 (a) and -BL6 (b) cells. Sub-confluent cells were treated with PKC412 (0, 0.5, 5 and 50 AM) for 1 hour at 37 jC. Then, at time 0, 1 105 cells in 2 ml of medium per well obtained as a monodisperse suspension by trypsinization, were seeded into a 12-well culture plate. At the time indicated, triplicate cultures were trypsinized and viable cells of samples were enumerated using a Coulter counter.
ability at 0.5, 5 and 50 AM PKC412 showed a significant decrease of 27, 42 and 29% compared to the control (0 AM PKC412) (Fig. 5a). In the experiment using B16-BL6 cells, the number of the invading cells after pretreatment with 0, 0.5, 5 and 50 AM PKC412 was 144 F 26, 129 F 12, 86 F 17 and 83 F 13, respectively. The invasive ability at 5 and 50 AM PKC412 significantly decreased 40 and 42% compared with the control (0 AM PKC412), respectively (Fig. 5b).
Fig. 4. Inhibitory effects of PKC412 on PKC activities in B16-F10 (a) and -BL6 (b) cells. The cells were treated with PKC412 (a: 0, 5 and 50 AM; b: 0, 0.5, 5 and 50 AM) for 1 hour at 37 jC, and then were scraped off, lysed and centrifuged. After centrifugation, the upper layer (sample: 25 Ag protein) was analyzed for PKC activity using a colorimetric method. Data are expressed as the mean F S.E.M. of 3 – 9 samples. **P < 0.01 vs. control.
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Fig. 5. Inhibitory effects of PKC412 on B16-F10 (a) and -BL6 (b) cell invasion of Matrigel-coated filters. Sub-confluent cells were treated with PKC412 (0, 0.5, 5 and 50 AM) for 1 hour at 37 jC. Then, 5 104 cells were seeded into the upper compartment of Transwell chambers. Filters were precoated with basement membrane Matrigel (25 Ag) on the upper surface. The lower chambers contained serum-free conditioned media from cultures of 3T3 cells for 24 hours as a chemoattractant. After incubation for 24 hours, the invading cells on the lower surface were counted microscopically. Data are expressed as the mean F S.E.M. of 5 – 6 samples. *P < 0.05 vs. control.
Effect of PKC412 on the platelet-aggregating activity of B16-F10 cells The platelet-aggregating activity of B16-F10 cells was significantly reduced 28, 56 and 47% respectively by pre-incubation with 0.5, 5 and 50 AM PKC412 compared with the control (0 AM PKC412) (Fig. 6).
Fig. 6. Inhibitory effects of PKC412 on B16-F10 cell-induced platelet aggregation. Sub-confluent cells were treated with PKC412 (0, 0.5, 5 and 50 AM) for 1 hour at 37 jC. Then, cell suspensions (1 106/50 Al) were added to PRP preincubated with 2 mM CaCl2. Data are expressed as the mean F S.E.M. of 3 platelets from rabbits. **P < 0.01, *P < 0.05 vs. control.
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Discussion Since PKC is ubiquitously expressed in human tissues and plays a pivotal role in cellular signal transduction pathways, there is some concern about the side effects of PKC inhibitors in medicine. But, if one chooses diseases caused by the overexpression of PKC and reasonable doses, PKC inhibitors should be promising agents for clinical use. Furthermore, PKCs (a and h) have been known to phosphorylate and activate P-glycoprotein (P-gp) associated with the multidrug resistance (MDR) phenotype of tumor cells on serine/threonine residues [13,14]. Therefore, it is noteworthy that PKC412 inhibits the MDR phenotype by decreasing the phosphorylation of P-gp without reducing the level of MDR mRNA and protein [15,16]. In a phase I and pharmacokinetic study of PKC412 (12.5 to 300 mg/day, p. o.), Propper et al. demonstrated that mean plasma concentrations after the administration were in the range 0.3 to 7 AM and 150 mg is a suitable daily dose for phase II studies [9]. We surmised, therefore, that the metastatic ability of the melanoma cells, B16-F10 and -BL6, is decreased by contact with PKC412 in the blood. In the present experiment, the tumor cells were treated with various concentrations of PKC412 (0.5–50 AM) for 1 hour at 37 jC with due regard to the pharmacokinetic study by 30 patients. [The concentrations of PKC412 were chosen by the fact that mean plasma concentrations of PKC412 during the first 24 hours of treatment (12.5–300 mg/day) were in the range of 0.3 to 7 AM]. It was found that PKC412 reduced the metastatic ability of B16-F10 and -BL6 cells in a dose-dependent manner using the hematogenic lung metastatic mouse model. In our previous paper, we investigated the anti-metastatic effect of other PKC inhibitors (H7 and bisindolylmaleimide) on the hematogenic lung metastatic mouse model using B16-F10 cells [17]. PKC412 showed similar anti-metastatic effect with bisindolylmaleimide and a little bit stronger effect than H7. To clarify whether this anti-metastatic effect is based on the cytotoxicity of PKC412, we tested the effect of PKC412 on the growth of B16-F10 and -BL6 cells. Incubation of these tumor cells for one hour with PKC412 did not affect the rate of growth. So, PKC412 (0.5–50 AM) did not show any cytotoxicity to the melanoma cells. In this case, the cellular PKC activity was weak but significantly inhibited. Thus, it is suggested that PKC412 shows an anti-metastatic effect under conditions that do not result in cell death. The inhibition of PKC activity may be involved in the anti-metastatic effect. Accordingly, we tried to elucidate the mechanism behind the anti-metastatic action of PKC412. The metastasis of tumor cells has several steps including angiogenesis, invasion through the extracellular matrix, blood-clotting for trapping cells or facilitating adherence of cells to capillary walls from the bloodstream, and so forth. Since PKC412 can inhibit VEGF-dependent angiogenesis based on the inhibition of the VEGF receptor kinase insert domain-containing receptor [8,18], we targeted the effects of PKC412 on the invasiveness of melanoma cells in the extracellular matrix and the plateletaggregating activity of melanoma cells in vitro. We indicated the anti-metastatic effect of PKC412 using the hematogenic lung metastatic mouse model. In this model, the platelet-aggregating activity of melanoma cells is the first serious event to facilitate adherence of cells from the bloodstream to vessel walls. The invasiveness of melanoma cells in the extracellular matrix after extravasation from the vessel is the second important step. In B16-F10 cells, PKC412 significantly suppressed both the invasiveness and platelet-aggregating activity. In B16-BL6 cells, PKC412 significantly reduced the invasiveness of the cells.
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In the present study, we could not determine the effect of PKC412 on the platelet-aggregating activity of B16-BL6 cells, because it was much weaker than that of B16-F10 cells (data not shown). Koike et al. reported that platelet aggregation is not essential for blood-borne metastasis and B16-BL6 cell arrest in targets [19]. This is fairly consistent with our result. Sullivan et al. demonstrated that genetically engineered expression of a kinase-defective mutant of PKC-a produced significant decreases in B16F10 cell adhesion and motility [20]. According to their report, it is possible that the effectiveness of PKC412 was partly due to the inhibition of PKC-a. However, since PKC412 also inhibits other kinases (tyrosine protein kinases in the receptors for VEGF, PDGF and the stem cell factor, c-kit), other mechanisms could be involved in the anti-metastatic effect of PKC412. Further study is needed to clarify it. Most recently, we tried to investigate the anti-metastatic effect of PKC412 by the oral administration on the spontaneous metastatic mouse model using B16-BL6 cells as the next step of this experiment. PKC412 significantly prolonged the survival of mice (Nakamura et al., submitted). Conclusion We demonstrated that PKC412, a PKC inhibitor, exhibits anti-metastatic action without causing cell death using a hematogenic lung metastatic mouse model with melanoma cells. Further, the antimetastatic effect is suggested to be due to the inhibition of the invasiveness and platelet-aggregating activity of melanoma cells. These results indicate that PKC412 is a promising candidate for an antimetastatic agent. Acknowledgements This research was supported, in part, by a Special Grant from the Smoking Research Foundation for Biomedical Research of Japan and a Grant-in-Aid for Scientific Research (C) from the Ministry of Education, Science, Sports and Culture of Japan (14572087).
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Effect of PKC412, a selective inhibitor of protein kinase C, on lung metastasis in mice injected with B16 melanoma cells Noriko Yoshikawa a, Kazuki Nakamura a,*, Yu Yamaguchi a, Satomi Kagota a, Kazumasa Shinozuka a,b, Masaru Kunitomo a,b a
Department of Pharmacology, Faculty of Pharmaceutical Sciences, Mukogawa Women’s University, 11-68, Koshien Kyuban-cho, Nishinomiya, Hyogo 663-8179, Japan b Institute for Biosciences, Mukogawa Women’s University, Nishinomiya, Hyogo, Japan Received 7 June 2002; accepted 18 October 2002
Abstract PKC412, a selective inhibitor of protein kinase C (PKC), is currently in clinical trials as an anti-tumor drug. In the present study, we investigated the anti-metastatic effect of PKC412 using an experimental metastatic mouse model intravenously injected with melanoma cells. One-hour exposure to various concentrations of PKC412 (0.5, 5 and 50 AM) dose-dependently reduced the lung-metastatic potential of highly metastatic B16F10 and -BL6 mouse melanoma cells in syngeneic mice. Following the exposure, PKC activities in B16-F10 and -BL6 cells were significantly decreased, but growth curves were not influenced. To elucidate the mechanism of the anti-metastatic effect of PKC412, we examined the activity to invade the extracellular matrix and the platelet-aggregating activity of the melanoma cells incubated with PKC412 (0.5, 5 and 50 AM) for 1 hour. PKC412 significantly reduced both the invasive and platelet-aggregating activities. These results suggest that PKC412 shows an anti-metastatic function through the inhibition of the invasive and/or plateletaggregating activities of melanoma cells. PKC412 is potentially a promising candidate for an anti-metastatic agent. D 2002 Elsevier Science Inc. All rights reserved. Keywords: PKC inhibitor; Anti-metastasis; B16 mouse melanoma cells; Chemo-invasion; Platelet aggregation
* Corresponding author. Tel./fax: +81-798-45-9945. E-mail address: [email protected] (K. Nakamura). 0024-3205/02/$ - see front matter D 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 2 ) 0 2 4 0 7 - 4
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Introduction Protein kinase C (PKC) is one of the key enzymes in cellular signal transduction pathways and controls cell proliferation and differentiation. PKC is a family of serine/threonine kinases and consists of at least 12 isoforms. These isoforms are divided into three groups based on their structure and substrate requirements [1–3]. The first group is the conventional PKCs, dependent on Ca2 + and activated by phosphatidylserine and diacylglycerol. The second group is the novel PKCs which act independent of Ca2 + and are regulated by phosphatidylserine and diacylglycerol. The third group is the atypical PKCs which act independent of Ca2 + but do not require diacylglycerol for their activation although phosphatidylserine regulates their activity. O’Brian et al. showed that levels of PKC expression were elevated in human breast tumor biopsies compared with normal breast tissues [4]. Benzil et al. also reported that the expression of PKC-a in human astrocytomas may serve as a direct biological marker of malignancy [5]. PKC-a is known to be mainly expressed in highly metastatic B16 mouse melanoma cells, and the activity of PKC-a in B16 cells is higher than that in weakly metastatic amelanotic B78-H1 melanoma cells without changes in PKC-a expression [6]. PKC412 (4V-N-benzoyl staurosporine) is a more specific but less potent PKC inhibitor than staurosporine [7]. PKC412 potently inhibits the binding of ATP to PKC, especially conventional PKCs; a, hI, hII and g subtypes. PKC412 also inhibits other kinases including tyrosine protein kinases in the receptors for vascular endothelial growth factor (VEGF), platelet derived growth factor (PDGF) and the stem cell factor, c-kit [8]. Accordingly, PKC412 seems likely to suppress tumor growth by the inhibition of conventional PKCs together with an angiogenic response to VEGF instead of basic fibroblast growth factor (bFGF) or hepatocyte growth factor (HGF). At present, PKC412 is undergoing clinical trials as an anti-tumor agent. It is demonstrated that PKC412 can be safely administered by chronic oral therapy and a suitable daily dose for phase II studies is 150 mg given the results of the phase I study [9]. In this study, we focused on the anti-metastatic effect of PKC412 instead of the anti-tumor effect. First, we investigated the effect of PKC412 using a hematogenic lung metastatic mouse model injected with highly metastatic melanoma cells. Second, to elucidate the anti-metastatic mechanism of PKC412, we examined the effect of PKC412 on the activity to invade the extracellular matrix and the plateletaggregating activity of melanoma cells in vitro.
Methods Materials PKC412 (powder) was kindly donated by Novartis Pharma AG (Basel, Switzerland). Stock solution was prepared in dimethylsulfoxide (DMSO) and diluted to the required concentrations in culture medium before use. The EDTA trypsin solution (EDTA; 0.02%, trypsin; 0.1%) and penicillin/ streptomycin solution (penicillin; 50000 U/ml, streptomycin; 50 mg/ml) were purchased from Cosmo Bio Co., LTD (Tokyo, Japan). Dulbecco’s modified Eagle’s medium (DMEM) with glutamine was obtained from ICN Biomedicals, Inc. (Aurora, OH). Dulbecco’s phosphate-buffered saline without calcium and magnesium (DPBS) was from Nissui Pharmaceutical Co., LTD (Tokyo, Japan). Fetal bovine serum (FBS) was from Sigma Chemical Co. (St. Louis, MO). Pierce Colorimetric PKC Assay
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Kit, SpinZyme Format was from Pierce (Rockford, IL). Growth factor reduced MATRIGEL matrix and BIOCOAT cell culture inserts were from Becton Dickinson Labware (Bedford, MA). Animals For the metastatic melanoma syngeneic animals, specific pathogen-free female C57BL/6Cr mice (6 weeks old) were purchased from Japan SLC, Inc (Hamamatsu, Japan). For the platelet aggregation experiments, male Japanese white rabbits (2–3 kg) were purchased from Shimizu Laboratory Animals (Kyoto, Japan). The mice and rabbits were maintained in an air-conditioned room (23 F 2 jC and 60 F 10% humidity) under a 12 hour light/dark cycle (7:00 a.m.–7:00 p.m.). Food and water were given ad libitum during the experiment. All procedures followed the Guiding Principles for the Care and Use of Laboratory Animals approved by The Japanese Pharmacological Society. Cells A mouse epithelial-like melanoma cell line, B16-F0, was obtained from the American Type Culture Collection (Rockville, MD). The B16-F1 cell line was derived from pulmonary metastases produced by the intravenous injection of B16-F0 cells into a syngeneic C57BL/6Cr mouse and the B16-F10 cell line was selected repeatedly ten times by successive passage of lung colonies; the in vivo-in vitro selections based on Fidler’s method [10]. B16-F10 cells were injected into the urinary bladder of male C57BL/6 mice via the vas deferens and the bladder was ligated, excised and maintained on semi-solid agar. Tumor cells that migrated through the wall of the bladder were recovered from the agar, cultured and repassaged. This process was repeated six times and the resulting variant was designated B16-BL6 [11]. The B16-BL6 cell line was kindly donated by Dr. Futoshi Okada at Hokkaido University (Sapporo, Japan). B16 cells passaged less than 25 times were used in all experiments. The doubling times of B16F10 and -BL6 were 13.1 and 12.4 hours, respectively. Cells were cultured in DMEM containing 10% FBS and the penicillin/streptomycin solution. Assay of experimental metastasis of tumor cells Sub-confluent B16-F10 and -BL6 cells were incubated with PKC412 (0, 0.5, 5 and 50 AM) for 1 hour at 37 jC, harvested with EDTA trypsin solution and resuspended to appropriate concentrations in DPBS. Cells (1 105/0.2 ml) were injected into the tail vein of syngeneic C57BL/6Cr mice. Mice were anesthetized with pentobarbital and sacrificed at 15 days after tumor injection. The lung was excised and fixed in a formaldehyde neutral buffer solution. Nodules visible as black forms in the lung were enumerated with the aid of a magnifying glass. Growth curves for tumor cells in vitro Sub-confluent B16-F10 and -BL6 cells were incubated with PKC412 (0, 0.5, 5 and 50 AM) for 1 hour at 37 jC, harvested with EDTA trypsin solution, and resuspended to appropriate concentrations in DMEM containing 10% FBS. Then, 1 105 cells/2 ml in each well of a 12-well culture plate were incubated for 24, 48 and 72 hours in a CO2 incubator at 37 jC. Viable cells of triplicate samples were enumerated with a Coulter counter (Coulter Z1, Beckman Coulter, Inc., Tokyo, Japan).
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Assay of PKC activity in tumor cells using the colorimetric method The assay of PKC activity was carried out according to the kit supplier’s instructions. Briefly, subconfluent B16-F10 and -BL6 cells were exposed to PKC412 (F10: 0, 5 and 50 AM; BL6: 0, 0.5, 5 and 50 AM) for 1 hour at 37 jC, rinsed three times with ice cold DPBS, scraped off, and centrifuged at 200 g for 8 minutes at 4 jC. Cell pellets were lysed in 0.5 ml of homogenization buffer (pH7.5; 20 mM Tris-HCl, 1.0 mM EDTA-2Na, 0.1 AM Okadaic acid, 10.0 Ag/ml of leupeptin, and 0.5% Triton X100, w/v). After centrifugation at 1500 g for 10 minutes at 4 jC, the supernatant, designed as crude enzyme, was used as a source of cellular PKC. Each sample (25 Ag protein) was mixed with dyelabeled substrate, phosphatidylserine, and reaction buffer containing ATP, MgCl2, CaCl2, Tris-amino methane and 0.01% surfactant. After incubation at 30 jC for 30 minutes, the mixtures were mounted on affinity membranes to isolate the PKC-catalyzed products. The absorbance of these products at 570 nm was determined with a spectrophotometer (UV-2100, Shimadzu Co., Kyoto, Japan). Invasion assay for tumor cells in vitro The assay of the invasiveness of B16-F10 and -BL6 cells in vitro was carried out as described by Albini [12]. Briefly, 6.4 mm diameter Transwells were used with tracked-etched polyethylene terephthalate (PET) membrane filters (8 Am pore size) coated 25 Ag/filter MATRIGEL basement membrane matrix extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma. Sub-confluent cells were exposed to PKC412 (0, 0.5, 5 and 50 AM) for 1 hour at 37 jC, harvested with EDTA trypsin solution, and resuspended to appropriate concentrations in DMEM containing 0.1% bovine serum albumin. Five hundred microliter samples of 5 104 cells were placed in the upper chamber compartments. The lower chambers contained 750 Al of serum-free conditioned medium from cultures of 3T3 cells for 24 hours as a chemoattractant. After 24 hours of incubation in a tissue culture incubator, non-invading cells on the upper side of the filter were completely removed by wiping with a cotton swab. Cells that had penetrated through the matrix protein and adhered to the lower surface of the filter were counted microscopically after fixing with methanol and staining with 3% Giemsa in DPBS. Platelet aggregation induced by tumor cells in vitro Blood was collected from the rabbit marginal vein, anti-coagulated with sodium citrate and centrifuged for 10 minutes at 160 g and room temperature to obtain platelet-rich plasma (PRP). Platelet numbers in PRP were counted with a Sysmex K-4500 hematology analyzer (Sysmex Inc., Kobe, Japan). After removal of the upper layer (PRP), the lower layer was centrifuged for 10 minutes at 1000 g and room temperature to obtain platelet-poor plasma (PPP). Platelet aggregation was measured in a cuvette ( = absorption tube) at 37 jC using an aggregometer (Aggrecorder PA-3210, ARKRAY Inc., Kyoto, Japan). Sub-confluent B16-F10 cells were exposed to PKC412 (0, 0.5, 5 and 50 AM) for 1 hour at 37 jC, harvested with EDTA trypsin solution, and resuspended to appropriate concentrations in DPBS. A 1 106/50 Al cell suspension was added to a cuvette containing 0.45 ml of PRP preincubated with 2 mM CaCl2 and the reaction started. The maximum percentage of aggregation for 5 minutes was calculated with the difference in the absorbance of the melanoma cell suspension (1 106) in PRP taken as 0% aggregation and that in PPP as 100% aggregation.
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Statistical analyses The data are expressed as the mean F S.E.M. of 3–7 animals. Statistical analyses were performed by ANOVA followed by Fisher’s PLSD (protected least significant difference) test using the Stat View software package (Abacus Concepts Inc., Berkeley, CA). A difference was considered significant at P < 0.05.
Results Effect of PKC412 on the hematogenic lung metastatic mouse model using B16-F10 and -BL6 cells The mice injected with the tumor cells after pre-incubation with 0, 0.5, 5 and 50 AM PKC412 for 1 hour displayed visible lung nodules 15 days after the injection. Fig. 1 shows a representative photograph of a typical lung with metastatic melanoma nodules.
Fig. 1. Appearance of lungs from C57BL/6Cr mice injected intravenously with highly metastatic B16-BL6 melanoma cells (1 105), after pre-treatment with 0 (control), 0.5, 5 and 50 AM PKC412 for 1 hour at 37 jC. Fifteen days later, mice were anesthetized with pentobarbital and lungs were excised. Each photograph shows a representative specimen from each group and the normal sample is a lung from an age-matched mouse injected intravenously with the same volume of DPBS.
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In the experiment using B16-F10 cells, the mean number of lung nodules was 153 F 39, 123 F 23, 82 F 14 and 53 F 17, respectively. The mean number of nodules after pre-incubation with 5 and 50 AM PKC412 decreased significantly 46 and 65% compared to the control (0 AM PKC412) in a dose-dependent manner (Fig. 2a). In the experiment using B16-BL6 cells, the number of lung nodules following pretreatment with 0, 0.5, 5 and 50 AM PKC412 was 275 F 50, 213 F 23, 198 F 46 and 148 F 30, respectively. The number of nodules after pre-incubation with 50 AM PKC412 showed a significant decrease of 46% compared to the control (0 AM PKC412) mice (Fig. 2b). Effect of PKC412 on the growth curves for B16-F10 and -BL6 cells The growth curves for both B16-F10 and -BL6 cells pretreated with PKC412 (0.5, 5 and 50 AM) for 1 hour at 37 jC did not change compared with the control (0 AM PKC412) (Fig. 3a and b). Effect of PKC412 on the PKC activity in B16-F10 and -BL6 cells The PKC activity in both B16-F10 and -BL6 cells pretreated with 50 AM PKC412 was significantly reduced 24% compared with the control (0 AM PKC412) (Fig. 4a and b). Effect of PKC412 on the invasion assay for B16-F10 and -BL6 cells In the experiment using B16-F10 cells, the number of the invading cells on pretreatment with 0, 0.5, 5 and 50 AM PKC412 was 145 F 5, 106 F 11, 84 F 14 and 103 F 12, respectively. The invasive
Fig. 2. Inhibitory effect of PKC412 on the hematogenic lung metastasis in C57BL/6Cr mice. Highly metastatic B16-F10 (a) and -BL6 (b) mouse melanoma cells (1 105), which had been treated with 0 (control), 0.5, 5 and 50 AM PKC412 for 1 hour at 37 jC, were injected into the tail vein of syngeneic C57BL/6Cr mice. Fifteen days later, visible nodules metastasized in the lung were enumerated with the aid of a magnifying glass. Data are expressed as the mean F S.E.M. of 5 – 7 mice. **P < 0.01, *P < 0.05 vs. control.
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Fig. 3. Effects of PKC412 on growth curves for B16-F10 (a) and -BL6 (b) cells. Sub-confluent cells were treated with PKC412 (0, 0.5, 5 and 50 AM) for 1 hour at 37 jC. Then, at time 0, 1 105 cells in 2 ml of medium per well obtained as a monodisperse suspension by trypsinization, were seeded into a 12-well culture plate. At the time indicated, triplicate cultures were trypsinized and viable cells of samples were enumerated using a Coulter counter.
ability at 0.5, 5 and 50 AM PKC412 showed a significant decrease of 27, 42 and 29% compared to the control (0 AM PKC412) (Fig. 5a). In the experiment using B16-BL6 cells, the number of the invading cells after pretreatment with 0, 0.5, 5 and 50 AM PKC412 was 144 F 26, 129 F 12, 86 F 17 and 83 F 13, respectively. The invasive ability at 5 and 50 AM PKC412 significantly decreased 40 and 42% compared with the control (0 AM PKC412), respectively (Fig. 5b).
Fig. 4. Inhibitory effects of PKC412 on PKC activities in B16-F10 (a) and -BL6 (b) cells. The cells were treated with PKC412 (a: 0, 5 and 50 AM; b: 0, 0.5, 5 and 50 AM) for 1 hour at 37 jC, and then were scraped off, lysed and centrifuged. After centrifugation, the upper layer (sample: 25 Ag protein) was analyzed for PKC activity using a colorimetric method. Data are expressed as the mean F S.E.M. of 3 – 9 samples. **P < 0.01 vs. control.
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Fig. 5. Inhibitory effects of PKC412 on B16-F10 (a) and -BL6 (b) cell invasion of Matrigel-coated filters. Sub-confluent cells were treated with PKC412 (0, 0.5, 5 and 50 AM) for 1 hour at 37 jC. Then, 5 104 cells were seeded into the upper compartment of Transwell chambers. Filters were precoated with basement membrane Matrigel (25 Ag) on the upper surface. The lower chambers contained serum-free conditioned media from cultures of 3T3 cells for 24 hours as a chemoattractant. After incubation for 24 hours, the invading cells on the lower surface were counted microscopically. Data are expressed as the mean F S.E.M. of 5 – 6 samples. *P < 0.05 vs. control.
Effect of PKC412 on the platelet-aggregating activity of B16-F10 cells The platelet-aggregating activity of B16-F10 cells was significantly reduced 28, 56 and 47% respectively by pre-incubation with 0.5, 5 and 50 AM PKC412 compared with the control (0 AM PKC412) (Fig. 6).
Fig. 6. Inhibitory effects of PKC412 on B16-F10 cell-induced platelet aggregation. Sub-confluent cells were treated with PKC412 (0, 0.5, 5 and 50 AM) for 1 hour at 37 jC. Then, cell suspensions (1 106/50 Al) were added to PRP preincubated with 2 mM CaCl2. Data are expressed as the mean F S.E.M. of 3 platelets from rabbits. **P < 0.01, *P < 0.05 vs. control.
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Discussion Since PKC is ubiquitously expressed in human tissues and plays a pivotal role in cellular signal transduction pathways, there is some concern about the side effects of PKC inhibitors in medicine. But, if one chooses diseases caused by the overexpression of PKC and reasonable doses, PKC inhibitors should be promising agents for clinical use. Furthermore, PKCs (a and h) have been known to phosphorylate and activate P-glycoprotein (P-gp) associated with the multidrug resistance (MDR) phenotype of tumor cells on serine/threonine residues [13,14]. Therefore, it is noteworthy that PKC412 inhibits the MDR phenotype by decreasing the phosphorylation of P-gp without reducing the level of MDR mRNA and protein [15,16]. In a phase I and pharmacokinetic study of PKC412 (12.5 to 300 mg/day, p. o.), Propper et al. demonstrated that mean plasma concentrations after the administration were in the range 0.3 to 7 AM and 150 mg is a suitable daily dose for phase II studies [9]. We surmised, therefore, that the metastatic ability of the melanoma cells, B16-F10 and -BL6, is decreased by contact with PKC412 in the blood. In the present experiment, the tumor cells were treated with various concentrations of PKC412 (0.5–50 AM) for 1 hour at 37 jC with due regard to the pharmacokinetic study by 30 patients. [The concentrations of PKC412 were chosen by the fact that mean plasma concentrations of PKC412 during the first 24 hours of treatment (12.5–300 mg/day) were in the range of 0.3 to 7 AM]. It was found that PKC412 reduced the metastatic ability of B16-F10 and -BL6 cells in a dose-dependent manner using the hematogenic lung metastatic mouse model. In our previous paper, we investigated the anti-metastatic effect of other PKC inhibitors (H7 and bisindolylmaleimide) on the hematogenic lung metastatic mouse model using B16-F10 cells [17]. PKC412 showed similar anti-metastatic effect with bisindolylmaleimide and a little bit stronger effect than H7. To clarify whether this anti-metastatic effect is based on the cytotoxicity of PKC412, we tested the effect of PKC412 on the growth of B16-F10 and -BL6 cells. Incubation of these tumor cells for one hour with PKC412 did not affect the rate of growth. So, PKC412 (0.5–50 AM) did not show any cytotoxicity to the melanoma cells. In this case, the cellular PKC activity was weak but significantly inhibited. Thus, it is suggested that PKC412 shows an anti-metastatic effect under conditions that do not result in cell death. The inhibition of PKC activity may be involved in the anti-metastatic effect. Accordingly, we tried to elucidate the mechanism behind the anti-metastatic action of PKC412. The metastasis of tumor cells has several steps including angiogenesis, invasion through the extracellular matrix, blood-clotting for trapping cells or facilitating adherence of cells to capillary walls from the bloodstream, and so forth. Since PKC412 can inhibit VEGF-dependent angiogenesis based on the inhibition of the VEGF receptor kinase insert domain-containing receptor [8,18], we targeted the effects of PKC412 on the invasiveness of melanoma cells in the extracellular matrix and the plateletaggregating activity of melanoma cells in vitro. We indicated the anti-metastatic effect of PKC412 using the hematogenic lung metastatic mouse model. In this model, the platelet-aggregating activity of melanoma cells is the first serious event to facilitate adherence of cells from the bloodstream to vessel walls. The invasiveness of melanoma cells in the extracellular matrix after extravasation from the vessel is the second important step. In B16-F10 cells, PKC412 significantly suppressed both the invasiveness and platelet-aggregating activity. In B16-BL6 cells, PKC412 significantly reduced the invasiveness of the cells.
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In the present study, we could not determine the effect of PKC412 on the platelet-aggregating activity of B16-BL6 cells, because it was much weaker than that of B16-F10 cells (data not shown). Koike et al. reported that platelet aggregation is not essential for blood-borne metastasis and B16-BL6 cell arrest in targets [19]. This is fairly consistent with our result. Sullivan et al. demonstrated that genetically engineered expression of a kinase-defective mutant of PKC-a produced significant decreases in B16F10 cell adhesion and motility [20]. According to their report, it is possible that the effectiveness of PKC412 was partly due to the inhibition of PKC-a. However, since PKC412 also inhibits other kinases (tyrosine protein kinases in the receptors for VEGF, PDGF and the stem cell factor, c-kit), other mechanisms could be involved in the anti-metastatic effect of PKC412. Further study is needed to clarify it. Most recently, we tried to investigate the anti-metastatic effect of PKC412 by the oral administration on the spontaneous metastatic mouse model using B16-BL6 cells as the next step of this experiment. PKC412 significantly prolonged the survival of mice (Nakamura et al., submitted). Conclusion We demonstrated that PKC412, a PKC inhibitor, exhibits anti-metastatic action without causing cell death using a hematogenic lung metastatic mouse model with melanoma cells. Further, the antimetastatic effect is suggested to be due to the inhibition of the invasiveness and platelet-aggregating activity of melanoma cells. These results indicate that PKC412 is a promising candidate for an antimetastatic agent. Acknowledgements This research was supported, in part, by a Special Grant from the Smoking Research Foundation for Biomedical Research of Japan and a Grant-in-Aid for Scientific Research (C) from the Ministry of Education, Science, Sports and Culture of Japan (14572087).
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