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Inhibitory Effect of Minocycline on in Vitro Invasion and Experimental Metastasis of Mouse Renal Adenocarcinoma
0022-5347/94/1515-1400$03.00/0 THE JOURNAL OF UROLOGY Copyright © 1994 by AMERICAN UROLOGICAL ASSOCIATION, INC.
Vol. 151, 1400-1404, May 1994
Printed in U. S.A.
INHIBITORY EFFECT OF MINOCYCLINE ON IN VITRO INVASION AND EXPERIMENTAL METASTASIS OF MOUSE RENAL ADENOCARCINOMA NAOYA MASUMORI, TAIJI TSUKAMOTO,* NORIOMI MIYAO, YOSHIAKI KUMAMOTO, IKUO SAIKI AND JUNYA YONEDA From the Department of Urology, Sapporo Medical College and the Institute of Immunological Science, Hokkaido University, Sapporo, Japan
ABSTRACT
Degradation of the extracellular matrix by metalloproteinases is a critical phenomenon in cancer invasion and metastasis. Recent studies have revealed that minocycline (minocycline hydrochloride, a tetracycline) suppresses in vivo and in vitro mammalian collagenolytic activity. We investigated whether minocycline inhibited in vitro invasion and experimental pulmonary metastasis in subline2 of streptozotocin-induced mouse renal adenocarcinoma (MRAC-PM2) cells. In vitro invasion assay demonstrated that treatment with 0.5 ILg.jml. or 5.0 ILg.jml. minocycline significantly inhibited the invasion of MRAC-PM2 cells. In addition, intraperitoneal administration of 0.5 mg. per mouse minocycline reduced the number of metastatic nodules in the lung when MRAC-PM2 cells were injected intravenously. Minocycline also suppressed type IV collagenolytic activity of the cells. However, the drug did not affect [3H]-thymidine uptake, growth of subcutaneously inoculated cells, attachment to the extracellular matrices, or haptotactic migration of the cells. These results indicated that the inhibitory action of type IV collagen degradation by minocycline can contribute, in part, to suppression of the in vitro invasion and metastatic potential of MRAC-PM2 cells. KEY WORDS:
minocyc1ine, adenocarcinoma, collagen, neoplasm invasiveness, neoplasm metastasis
Homeostasis in the remodeling of the basement membrane and the interstitial tissue in vivo depends on the subtle balance between metalloproteinases and their inhibitors.l Cancer cells can affect this balance by secreting and/or activating metalloproteinases. Thus, the cells can degrade the basement membrane and the interstitial tissue during the invasion process. Many studies have demonstrated that metalloproteinase activity, particularly type IV collagenase activity, closely correlates with the invasive or metastatic potential of cancer cells. 2 • 3 The excess secretion or activation of the enzymes promotes the invasion and metastasis of cancer cells. Minocycline hydrochloride (minocycline) is a semisynthetic tetracycline that has been widely used as an antimicrobial agent in the treatment of infectious diseases. Recent studies have revealed that minocycline suppresses in vivo and in vitro mammalian collagenolytic activity. 4, 5 If the inhibitory effect of minocycline on collagenolytic activity extends to cancer cellderived metalloproteinases or tissue-derived metalloproteinases induced by cancer cells, the drug might inhibit cancer cell metastasis as well as invasion. To examine this hypothesis, we investigated the effect of this drug on the in vitro invasiveness and experimental pulmonary metastasis of streptozotocininduced mouse renal adenocarcinoma (MRAC). MATERIALS AND METHODS
Mouse renal adenocarcinoma and its pulmonary metastatic subline. The original MRAC was induced by a single intraperitoneal injection of streptozotocin (Wako Junyaku Co. Ltd., Tokyo, Japan) in the kidney in CBA/H/T6J mice (Jackson Laboratories, Bar Harbor, Massachusetts) and maintained by subcutaneous passage in syngeneic mice. The details of the MRAC were described previously.e,7 In brief, the adenocarcinoma of the kidney showed mitotic activity, nuclear atypia and atypical nucleoli, and the cytoplasm contained eosinophilic and fine granular structures, all of which indicate histopathological Accepted for publication November 4, 1993. * Requests for reprints: Department of Urology, Sapporo Medical College, Sapporo 060, Japan.
features similar to the granular cell type of human renal cell carcinoma. Pulmonary metastatic subline-2 (MRAC-PM2) was selected by an intravenous injection of the MRAC cells and then by injection of cells from its pulmonary metastatic nodules. 7 The MRAC-PM2 was cultured and maintained in MEM D-valine modification medium (DV-MEM; Sigma Chemical Co., St. Louis, Missouri) supplemented with 10% fetal bovine serum (FBS; Flow Laboratories Inc., North Ryde, Australia) and antibiotics in a humidified atmosphere of 5% CO 2 at 37C. In vitro invasion assay. Details of the method were described previously.8,9 In brief, a polyvinylpyrrolidone-free polycarbonate filter with an 8.0 ,um. pore size (Nucleopore Corp., Pleasanton, California) was attached to the bottom of the inner chamber in a Transwell cell culture plate (Costar No. 3422, Cambridge, Massachusetts). The filter was coated on its upper surface with 5 ,ug. (15 ,ug./cm. 2 ) of Matrigel (Becton Dickinson Labware, Bedford, Massachusetts) and on its lower surface with 5 ,ug. (15 ,ug./cm. 2 ) offibronectin (Biomedical Technologies Inc., Stoughton, Massachusetts). The MRAC-PM2 cells with confluent growth were harvested, washed several times with serum-free DV-MEM with 0.1% bovine serum albumin (BSA) and suspended in the same medium. The suspension, 100 ,ul. containing 1 X 105 cells, was applied to the inner chamber. To the outer chamber, 600,ul. of serum-free DV-MEM containing 0.1 % BSA was added. Minocycline (Lederle Ltd., Tokyo, Japan) with a final concentration of 5.0 ,ug. or 0.5 ,ug./ml. in the same medium was applied in the inner chamber. After the cells were incubated for 6 to 8 hours at 37C in a 5% CO 2 atmosphere under 100% humidity, the filter was fixed and stained. Those with invasion onto the lower surface of the filter were counted under microscopy at a magnification of 400. In vitro invasion of MRAC-PM2 cells in each concentration of minocycline was assayed in triplicate. Type IV collagenolytic activity. Type IV collagenolytic activity was determined by the method described previously.lO Briefly, [3H]-labelled type IV collagen solution (3 X 105 cpm/1.6 ,ug./ ml.) in 0.5 M. acetic acid was placed at a volume of 100 ,ul. per well in a tissue culture plate and left overnight in a laminar air
1400
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INmmTION OF IVIUIUNE RENAL ADENOCARCnWiVIA BY MINOCYCLINE
flow hood to allow collagen solutions to dry to a film. Then 100 Ill. of serum-free Dulbecco's modified Eagle's medium and Ham's F12 medium (DME:F12, 1:1 ratio, GIBCO, Grand Island, New York) was added over the dried type IV collagen film and incubated overnight at 37C. In the assay for cell-mediated type IV collagenolytic activity, 1 X 105 MRAC-PM2 cells suspended in 2 ml. of D ME:F12 were added to the precoated wells. Minocycline with a concentration of 0.1 j1.g., 1.0 f.lg., or 10.0 f.lg./ml. in the same medium was applied to the precoated welL After a 48-hour incubation period, 200 ,ul. of the supernatant was withdrawn and mixed with 50 f.lL of ice-cold 50% trichloroacetic acid and centrifuged at 18,000 g for 10 minutes at 4C to precipitate undigested materials. Type IV collagenolytic activity was calculated from the [3HJ-activity in the supernatant and expressed as the nanogram amount of type IV collagen degradation. Microassay for cell adhesion. Binding capacity of MRACPM2 cells to the various extracellular matrices was measured according to the method employed by Saiki et al. IO In brief, MRAC-PM2 cells were incubated for 24 hours in DV-MEM containing 10% FBS supplemented with 1 f.lCi/ml. of 5-[125 I]iododeoxyuridine (specific activity, 2200 Ci/mmol.; New England Nuclear, Boston, Massachusetts). The cells were washed twice in warm Ca 2+- and Ma 2+-free phosphate-buffered saline (PBS) to remove the unbound radiolabel, harvested by 0.05% trypsin and 0.01 % EDTA for 10 minutes at 37C and resuspended in cold serum-free DV-MEM. [125IJ-iododeoxyuridinelabelled MRAC-PM2 cells, 2 X 104 /0.05 m!. of DV-MEM, were added to each well in a 24-well tissue culture plate precoated with 5 ,ug. (2.5 ,ug./cm. 2 ) of fibronectin, 5 Ilg. (2.5 ,ug./cm. 2 ) of laminin (Biomedical Technologies Inc.), or 5 jJ.g. (2.5 ,ug./cm02) of mouse type IV collagen (Becton Dickinson Labware). Minocycline at a concentration of 5.0 f.lg. or 0.5 f.lg./ml. was added to each well. The cultures were incubated at 37C for 30 minutes and then washed 4 times with PBS to remove any unattached cells. The remaining substrate-bound tumor cells were lysed with 0.1 mL of 0.1 N NaOH. The lysate was absorbed by cotton swabs and monitored for radioactivity by gamma counting. The binding capacity was expressed as follows: binding capacity = cpm of targets bound to substrate X total number of tumor cells added/cpm of total tumor cells added. Haptotactic migration assay. A polyvinylpyrrolidone-free polycarbonate filter with an 8.0 !lm. pore size was coated on its lower surface with 5 f.lg. (15 ,ug./cm. 2 ) of fibronectin.lO The following procedures were the same as those for the in vitro invasion assay. The cells migrating onto the lower surface of the filter were counted under microscopy. Serum concentration of minocycline. The serum concentration of minocycline was measured according to the following method. Minocycline, 0.5 mg. diluted in 0.1 ml. of PBS, was intraperitoneally injected into six CBA/H/T6J mice. The blood, which was collected intracardiac puncture 3 hours after injection of the drug, was centrifuged at 3,000 g for 10 minutes. The serum concentration was primarily determined the thin -layer disk method using Bacillus subtilis ATCC 6633 as a test organismY Experimental pulmonary metastasis. Pulmonary metastasis was experimentally induced by an intravenous injection of 5 X 105 MRAC-PM2 cells suspended in 0.1 m!. of PBS into the tail vein in 8-week-old CBA/H/T6J mice. Mice were sacrificed 14 days after injection of the cells, and metastatic nodules in the lung were macroscopically counted. Minocycline, 0.5 mg. or 0.05 mg. diluted in 0.1 m!. of PBS, was intraperitoneally administered according to the following treatment schedules. In treatment 1, minocycline was given 3 hours before and 1 hour after the intravenous injection of the cells. Treatment 2 consisted of minocycline administration, which was started on the day after the cell injection and consecutively given once each day for another 9 days. In treatment 3, minocycline was given according to a schedule combin-
ing treatments 1 and 2. Control mice were given 0.1 ml. of PBS alone intraperitoneally according to the schedule of treatment 30 Assessment of DNA synthesis. The effect of minocycline on DNA synthesis of MRAC-PM2 cells was evaluated with a modification of the method of Hosaka et al. I2 After 1 m!. of 0.6% agarose-DV-MEM solution with 15% FBS was plated as the feeder layer in a 24-well tissue culture plate, 0.5 mL of 0.4 % agarose-DV-MEM solution with 12.5% FBS containing 1 X 105 MRAC-PM2 cells was placed on the feeder layer. Then 50 f.ll. of PBS containing minocycline at a final concentration of 500, 50, or 5 ,ug./ml. was added onto the double-layered agarose. For control, the same volume of PBS alone was added. The cells were incubated for 72 hours under the conditions described earlier. Then 5 f.lCi of [3H]-thymidine was applied on the agarose in each well, and the cells were incubated for an additional 24 hours. After terminating [3H]-thymidine incorporation, the cells were recovered and radioactivity was counted as described previouslyY Subcutaneous growth of MRAC-PM2. MRAC-PM2 cells,S X 105 /0.1 mL of PBS, were subcutaneously inoculated into the flank in 8-week-old CBA/H/T6J mice. When the tumor reached a size of 5 mm. in diameter (approximately 2 weeks after the inoculation), 0.5 mg. or 0.05 mg. of minocycline dissolved in 0.1 mL of PBS, or PBS alone, was given intraperitoneally for 10 consecutive days. The tumor size was measured every 5 days for 30 days from the start of minocycline treatment. In measuring tumor size, the product of the minor diameter and the major diameter was calculated and the ratio of the products at each given time to that at the start of the treatment was determined as follows: tumor growth ratio = minor diameter X major diameter at the given time/minor diameter X major diameter at the start of treatment. Statistical analysis. The statistical difference between the groups was determined by the nonparametric Mann-Whitney U test. RESULTS
Inhibitory effect of minocycline on in vitro invasiveness of MRAC-PM2. The number of invading MRAC-PM2 cells was significantly dependent on incubation time when they were treated with serum-free DV-MEM containing 0.1 % BSA alone (control). Treatment with minocycline at each concentration reduced the number of MRAC-PM2 cells that invaded at each incubation time more than the control did (P < 0.05, table 1). Although not significant, 5.0 f.lg./ml. of minocycline tended to have a more pronounced effect in suppressing invasiveness than did the other concentration of the drug at the 8th hour. Minocycline treatment and type IV collagenolytic activity. Type IV collagenolytic activity was enhanced by the addition of MRAC-PM2 cells alone, suggesting that the cells surely had collagenolytic activity. Although 0.1 Ilg./ml. of minocycline did not suppress type IV collagenolytic activity, higher concentrations of the drug suppressed the activity more than the control (table 2). Suppression ofthe activity tended to be dose dependent. Treatment with a concentration of 10 l.lg./ml. of minocycline reduced the activity by approximately 45% compared to that of the cells alone. TABLE
1. Influence of minocycline on in uitro inuasiueness of MRAC-PM2 cells No. of invading cells/filter (mean± SD)
Concentration of minocycline (I'g./ml.)
6 hours
8 hours
o (control)
6.3 ± 2.11
0.5 5.0
1.0 ± 1.02 1.3 ± 0.6 3
42.3 ± 5.5' 14.0 ± 6.1' 9.3 ± 3.5 6
1-6 Statistical analysis 1 versus 2 or 3, P <0.05 hy Mann-Whitney U-test; 4 versus 5 or 6, P <0.05 by Mann-Whitney U-test.
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INHIBITION OF MURINE RENAL ADENOCARCINOMA BY MINOCYCLINE
TABLE 2.
Inhibitory effect of minocycline on type IV collagenolytic activity Type IV collagenolytic activity" (ng.)
Concentration of minocycline (I'g./ml.)
Treatment DME:F12 alone DME:F12 + cells
17.9 ± 99.1 ± 101.6 ± 78.6 ± 54.4 ±
o 0.1 1.0 10.0
2.9 ' 3.7 2 0.8 13.73 9.8'
• Mean ± SD in triplicate. 1-' Statistical analysis: 1 versus 2, P <0.05 by Mann-Whitney U-test; 2 versus 3 or 4, P <0.05 by Mann-Whitney U-test.
6
.2 iii ...
5
Intraperitoneal administration of minocycline or PBS
- UUUUU ...
.c
:s: 0
"-
4
OJ
0
E
3
::l
Influence of minocycline on attachment of MRAC-PM2 cells
TABLE 3.
Concentration of minocycline (I'g./ml.)
Binding capacity (No. of cells bound/substrate)"
o 0.5 5.0
Fibronectinb
Lamininb
Type IV collagenb
8960 ± 671 10003 ± 760 9938 ± 779
7765 ± 2089 7769 ± 799 6691 ± 369
10674 ± 3962 7718 ± 878 8271 ± 503
I-
2
o
10
• Mean ± SD in triplicate. b Precoated substrate.
Concentration of minocycline (I'g./ml.)
o (control) 0.5 5.0
TABLE 5.
6 hours
8 hours
314.0 ± 63.3 324.0 ± 89.1 323.7 ± 56.7
475.9 ± 58.3 502.7 ± 122.9 526.7 ± 107.2
Minocycline (mg'; mouse)
No. of mice
0 0.05 0.5 0.05 0.5 0.05 0.5
30 6 7 7 7 6 7
Control 1 2 3 1-5
No. of migrating cells/filter (mean ±SD)
Inhibitory effect of minocycline on experimental pulmonary metastasis
Treatment
No. of pulmonary metastatic nodules (mean± SD) 100.4 ± 79.7 ± 39.9 ± 100.4 ± 52.9 ± 45.8 ± 27.3 ±
52.2' 46.4 12.82 45.3 33.3 3 18.5' 17.35
Statistical analysis: 1 versus 2, 3, 4, or 5: P <0.05 by Mann-Whitney U-
test. TABLE 6.
Influence of minocycline on DNA synthesis of MRAC-PM2 determined by f HJ -thymidine incorporation assay
Concentration of minocycline (I'g./ml.)
o 5 50 500
30
Days after the start of treatment
Influence of minocycline on haptotactic migration of MRACPM2
TABLE 4.
20
Uptake of [3H)-thymidine" (DPM) 852.1 714.2 1091.5 844.6
± ± ± ±
377.1 366.0 1029.5 643.3
• Mean ± SD in triplicate.
Influence of minocycline on attachment of MRAC-PM2 cells to the extracellular matrices. Approximately 40% or more of the applied cells attached to the various substrates in the controls. However, the two concentrations of minocycline did not interfere with the binding capacity of the cells to each substrate (table 3). Influence of minocycline on haptotactic migration of MRACPM2 cells. The number of cells that migrated to the lower surface of the filter was increased compared with the in vitro invasion assay for each concentration of minocycline (table 4). Minocycline did not show any inhibitory influence on haptotactic activity of MRAC-PM2 cells at any concentration tested.
105
FIG. 1. 5 X MRAC-PM2 cells were inoculated on flank in CBA/ H/T6J mice. When tumor reached 5 mm. diameter, 0.5 mg. or 0.05 mg. of minocycline was given intraperitoneally for 10 consecutive days. In control mice, 0.1 mL of PBS was given. Tumor growth ratio was determined as follows: tumor growth ratio = minor diameter x major diameter at given time/minor diameter x major diameter at start of treatment. Symbols indicate: open circles (0): control (n = 5); closed circles (e): 0.5 mg. of minocycline per mouse (n = 5) and open squares (0): 0.05 mg. per mouse of minocycline (n = 5). Vertical bar indicates one standard deviation.
Serum concentration of minocycline in CBA/H/T6J mice. The intraperitoneal injection of 0.5 mg. of minocycline produced serum concentrations of 1.17 ± 0.36 /Lg.jml. Suppression by minocycline of experimental pulmonary metastasis of MRAC-PM2. Intravenous injection of MRAC-PM2 cells yielded many visible metastatic nodules in the lungs of mice, which were sacrificed on day 14. No mice died of the procedure during the experiment. When minocycline was given before and after the intravenous injection of MRAC-PM2 cells (treatment 1), 0.5 mg. of the drug significantly suppressed metastatic nodule formation in the lung (P < 0.05, table 5). In treatment 2, which consisted of drug administration for 10 consecutive days started on the day after the intravenous cell injection, a 0.5 mg. dose of minocycline markedly reduced the number of metastatic nodules (P < 0.05). In treatment 3, even treatment with the lower dose of the drug was able to suppress metastatic nodule formation in the lungs of mice (P < 0.05) when it was given for 10 consecutive days as well as around the time of cell injection, as shown in table 5. The most prominent effect of suppression of metastasis was found with 0.5 mg. of minocycline in treatment 3. Influence of minocycline on DNA synthesis of MRAC-PM2 cells. The DNA synthesis of MRAC-PM2 cells was determined by [3HJ-thymidine incorporation assay. We examined three different concentrations, 5 /Lg./ml., 50 /Lg./ml. and 500 /Lg.jml., in the [3HJ-thymidine incorporation assay (table 6). Although [3HJ-thymidine uptake varied widely with each concentration, no significant difference in uptake was found among these concentrations of the drug, indicating a negligible influence of minocycline on DNA synthesis of the cells. Minocycline treatment and subcutaneous growth of MRACPM2 cells. In mice treated with PBS alone (control), tumors inoculated subcutaneously grew 6-fold in size by the end of the experiment (day 30), compared with the initial size on day 1. Minocycline treatment, either 0.5 mg. or 0.05 mg., was unable to retard the growth of the subcutaneous tumors of MRACPM2 (fig. 1). At each measurement, mean tumor sizes were
INHIBITION OF MURI:NE RENAL ADENOCARCINOIvl:A BY MINOCYCUNE
almost equal among the control and the minocycline-treatment groups. Neither significant loss of body weight nor serious inflammation of the peritoneal cavity was found during the experiment. DISCUSSION
Cancer metastasis requires a complex multistep process. 13 After the proliferation of cancer cells in the primary lesion, they degrade the interstitial tissue and the basement membrane and enter the vascular circulation (intravasation). The cells then attach to the endothelial cells of a target organ with subsequent invasion into the basement membrane and, once again, the interstitial tissue (extravasation). Only cells adapted to the organ microenvironment can survive, proliferate and eventually develop metastatic nodules. For full development of metastasis, cancer cells must complete all these steps. In the multistep process, invasion is the most important phenomenon since it is required for both intravasation and extravasion of the cells. Invasion consists ofthree biochemical events: attachment of cancer cells to the extracellular matrix, degradation of the extracellular matrix by metalloproteinases and migration of the cells. 2 Of the metalloproteinases, type IV collagenase is the enzyme most responsible for the process of invasion. 2 Golub et a1. 4,5 have indicated that minocycline suppresses the collagenolytic activity ofthe skin and the gingiva in diabetic rats with associated hypercollagenolytic status, as well as the activity of rat polymorphonuclear leukocytes and rabbit chondrocytes. Moreover, the drug was shown to suppress the collagenolytic activity of Bl6 melanoma cells. 14 Thus, it is valid to speculate that minocycline can suppress the activity of cancer cells as well as that of normal cells. We used minocycline concentrations of 5.0 f.lg. and 0.5 f.lg./ m!. in the in vitro invasion assay because minocycline at a concentration of 5.0 f.lg./ml. was shown to suppress collagenase activity of human rheumatoid synovium to 48% of the control level. 15 In addition, the peak serum concentration of the drug is approximately 6 /-tg./mL, when 200 mg. of it is intravenously given to healthy adult male subjects. 16 Thus, the 5.0 f.lg./ml. used in the in vitro study is within the range of clinically achievable serum concentrations. For in vivo study, we determined the maximum dosage of the drug to be 0.5 mg. per mouse, taking into consideration the LD50 of the mouse (equivalent to 5 mg.) and the 10-day treatment schedule (in treatments 2 and 3)Y In our study, the serum concentration was approximately 1.2 f.lg./ml., when 0.5 mg. of minocycline per mouse was intraperitoneally injected. Therefore, the serum concentration in the in vivo study was included in the range of drug concentrations of 0.5 /-tg./ml. to 5 /-tg./ml. that were used in the in vitro study. In this study, minocycline suppressed type IV collagenolytic activity of cell line MRAC-PM2. Moreover, the drug inhibited the in vitro invasion of MRAC-PM2 cells. In vitro invasiveness, which is evaluated by in vitro invasion assay, closely correlates with collagenolytic activity, particularly type IV collagenolytic activity. Nakajima et aP8 have demonstrated that a decrease in type IV collagenolytic activity eventually results in a decrease of in vitro invasion. The results in our experiment showing that minocycline affected neither attachment to the extracellular matrix nor migration of MRAC-PM2 cells suggested that the drug could inhibit in vitro invasiveness through its suppressive effect on type IV collagenolytic activity. In addition, we demonstrated that minocycline also inhibited experimental pulmonary metastasis of MRAC-PM2 cells. Since type IV collagenolytic activity correlates not only with in vitro invasiveness but also with metastatic potential,19 it was suggested that minocycline blocked extravasation of MRAC-PM2 cells through its suppression of collagenolytic activity, and then eventually inhibited development of pulmonary metastasis. Previous studies1,20 have demonstrated that degradation of the extracellular matrix by metalloproteinases secreted from
1403
endothelial cells is the first step leading to completion of angiogenesis. Thus, minocycline may inhibit the angiogenesis required for proliferation of metastatic nodules through the same action as the inhibition of the collagenolytic activity, although further study will be necessary to confirm this. Van den Bogert et al. 21 have reported that low concentrations of tetracyclines suppress proliferation of several tumor cells, such as ascitically growing hepatomas, solidly growing Leydig cell tumors, mammary gland tumors, T -type leukemia, prostatic carcinoma and renal cell carcinoma. They indicated that tetracyclines inhibit mitochondrial protein synthesis. This inhibition decreases the oxidative ATP -generating capacity needed for cell division, finally leading to proliferation arrest of cells. They have also reported that each cell line has different permeability barriers for tetracyclines and different intervals until proliferation arrest of cells by the drug. Since in vitro proliferation arrest by continuous application of tetracyclines for prostatic carcinoma and renal cell carcinoma does not occur until the 9th and 4th days, respectively, they concluded that tetracyclines inhibit proliferation following 2 cell cycles. Our results, showing that minocycline neither affected the uptake of [3H]_ thymidine nor suppressed tumor proliferation in vivo, indicated that MRAC-PM2 cells had comparatively stronger barriers against minocycline or that the drug did not affect the cells within the time period used in our experiment. Thus, the cytostatic effect of this drug did not contribute to type IV collagenolytic activity, in vitro invasiveness, or experimental metastasis. In terms of the action sites of metalloproteinases, these enzymes are demonstrated to be activated and to degrade the extracellular matrix in the extracellular space following secretion from cells. This suggests that minocycline seems to suppress the activity of metalloproteinases in the extracellular space rather than their synthesis and secretion. Malis and Cooper22 have reported that tetracycline fluorescence observed under an ultraviolet microscope is commonly seen in collagenous fibers within the stroma of bladder carcinoma but not in the cells. Golub et a1. 4 have suggested that the inhibitory effect of minocycline on collagenolytic activity is probably due to its chelation of Ca 2+, since the inhibitory effect is diminished by the addition of CaCh, Not only minocycline, but all tetracyclines, can chelate Ca2+. A recent study has demonstrated that metalloproteinases, including collagenase, are metalloenzymes which have Zn 2+ in the center of the active site 23 and that the presence of Zn 2+ is required for activation of metalloproteinases. To stabilize the tertiary structure of metalloproteinases, Ca 2+ is needed as a cofactor.24 In other words, Zn 2+ acts as an intrinsic metal cation, and Ca2+ acts as an extrinsic one, each of which participates in expressing and maintaining metalloproteinase activity.25,26 Assuming that the inhibitory effect of minocydine on collagenolytic activity is derived from a suppression of collagenase activity by chelation of Ca 2 +, minocycline may suppress not only the activity of interstitial collagenase but also that of other metalloproteinases, such as type IV collagenase and stromelysin, since Ca 2 + is a common extrinsic cofactor for all metalloproteinases. Minocycline did not affect attachment of cancer cells to the extracellular matrix in this study. Cell-substrate adhesion molecules, especially integrins which bind to fibronectin and laminin, require divalent cations, including Ca2+ for function. 27 Although the extent of the Ca2+ requirement for activity may be different between metalloproteinases and cell adhesion molecules, our results would appear to support the idea that not only Ca2+ chelation but also other actions of minocycline, such as direct binding to metalloproteinases, are involved in its inhibition of collagenolytic activity. This direct action of the drug may be supported by the finding that minocycline has been shown to suppress collagenase activity in vivo for more than 19 weeks after the drug was discontinued. 5 Greenwald et
1404
INHIBITION OF MURINE RENAL ADENOCARCINOMA BY MINOCYCLINE
a1,28 have speculated that binding of minocycline to metalloproteinase directly results in a loss of enzymatic activity and that slow clearance of minocycline from such a complex provides the prolonged effect in vivo. In summary, our study clearly demonstrated that the suppressive action of minocycline on type IV collagenolytic activity can contribute to inhibition of the in vitro invasiveness of MRAC-PM2 cells. However, the drug was not shown to have any cytotoxic effect on the cells. In addition, it neither affected attachment of the cells to the extracellular matrix nor their migration. From these results, we conclude that minocycline suppressed experimental pulmonary metastasis, in part, through its inhibitory action on type IV collagenolytic activity of the cells. Further investigation is necessary to clarify the action of minocycline and which metalloproteinase activity the drug really suppresses. REFERENCES
1. Liotta, L. A., Steeg, P. S. and Stetler-Stevenson, W. G.: Cancer metastasis and angiogenesis: an imbalance of positive and negative regulation. Cell, 64: 327, 1991. 2. Liotta, L. A.: Tumor invasion and metastases- role of the extracellular matrix. Cancer Res., 46: 1, 1986. 3. Stetler-Stevenson, W. G.: Type IV collagenases in tumor invasion and metastasis. Cancer Metastasis Rev., 9: 289, 1990. 4. Golub, L. M., Lee, H. M., Lehrer, G., Nemiroff, A., McNamara, T. F., Kaplan, Rand Ramamurthy, N. S.: Minocycline reduces gingival collagenolytic activity during diabetes. J. Periodontal Res., 18: 516, 1983. 5. Golub, L. M., Wolff, M., Lee, H. M., McNamara, T. F., Ramamurthy, N. S., 2mbon, J. and Ciancio, S.: Further evidence that tetracyclines inhibit collagenase activity in human crevicular fluid and from other mammalian sources. J. Peridontal Res., 20: 12, 1985. 6. Miyao, N., Kumamoto, Y. and Tsukamoto, T.: Histopathological analysis of chemical carcinogenesis process by streptozotocin in the mouse kidney. Jpn. J. Urol., 82: 1399, 1991. 7. Miyao, N., Kumamoto, Y., Tsukamoto, T. and Otani, N.: Selection of highly metastatic lines from streptozotocin-induced mouse renal adenocarcinoma and their in vivo metastatic or in vitro invasive potential. Jpn. J. Urol., 82: 1408, 1991. 8. Saiki, I., Murata, J., Watanabe, K, Fujii, H., Abe, F. and Azuma, I.: Inhibition of tumor cell invasion by Ubenimex (Bestatin) in vitro. Jpn. J. Cancer Res., 80: 873, 1989. 9. Otani, N., Tsukamoto, T., Saiki, I., Yoneda, J., Mitaka, T. and Kumamoto, Y.: In vitro invasive potential and type IV collagenolytic activity of human renal cell carcinoma cells derived from the primary and metastatic lesions. J. Urol., 149: 1182, 1993. 10. Saiki, I., Murata, J., Nakajima, M., Tokura, S. and Azuma, I.: Inhibition by sulfated chitin derivatives of invasion through extracellular matrix and enzymatic degradation by metastatic melanoma cells. Cancer Res., 50: 3631, 1990. 11. Edberg, S. C.: The measurement of antibiotics in human body fluids: techniques and significance. In: Antibiotics in Laboratory Medicine. Edited by V. Lorian. Baltimore: Williams and Wilkins, chapt. 12,pp. 381-476, 1986.
12. Hosaka, Y., Tsukamoto, T. and Lieber, M. M.: Soft agarose colony formation assay for human renal cell carcinoma: comparison of optical colony counting versus tritiated thymidine incorporation. J. Urol., 136: 1102, 1986. 13. Fidler, I. J.: Critical factors in the biology of human cancer metastasis. Cancer Res., 50: 6130, 1990. 14. Zucker, S., Lysik, R M., Ramamurthy, N. S., Golub, L. M., Wieman, J. M. and Wilkie, D. P.: Diversity of melanoma plasma membrane proteinases: inhibition of collagenolytic and cytolytic activities by minocycline. J. Natl. Cancer Inst., 75: 517, 1985. 15. Golub, L. M., McNamara, T. F., D'Angelo, G., Greenwald, R A. and Ramamurthy, N. S.: A non-antibacterial chemically-modified tetracycline inhibits mammalian collagenase activity. J Dent. Res., 66: 1310, 1987. 16. Matsumoto, K, Kimura, H., Noguchi, Y., Uzuka, Y., Honda, I. and Nishioka, K: Experimental studies on minocycline intravenous infusion. Jpn. J. Antibiotics, 27: 302, 1974. 17. Nakazawa, S.: The progress of tetracyclines. J. Jpn. Pharm. Assoc., 21: 29, 1969. 18. Nakajima, M., Lotan, D., Baig, M. M., Carralero, R M., Wood, W. R, Hendrix, M. J. C. and Lotan, R: Inhibition by retinoic acid of type IV collagenolysis and invasion through reconstituted basement membrane by metastatic rat mammary adenocarcinoma cells. Cancer Res., 49: 1698, 1989. 19. Nakajima, M., Welch, D. R, Belloni, P. N. and Nicolson, G. L.: Degradation of basement membrane type IV collagen and lung subendothelial matrix by rat mammary adenocarcinoma cell clones of differing metastatic potentials. Cancer Res., 47: 4869, 1987. 20. Kalebic, T., Garbisa, S., Glaser, B. and Liotta, L. A.: Basement membrane collagen: degradation by migrating endothelial cells. Science, 221: 281, 1983. 21. Van den Bogert, C., Dontji, B. H. J., Holtrop., M., Melis, T. E., Romijn, J. C., Van Dongen, J. W. and Kroon, A. M.: Arrest of the proliferation of renal and prostate carcinomas of human origin by mitochondrial protein synthesis. Cancer Res., 46: 3283, 1986. 22. Malis, I. and Cooper, J. F.: The localization of tetracycline microfluorescence in tumors of the urinary tract. J. Urol., 99: 183, 1968. 23. Vallee, B. L. and Auld, D. S.: Zinc coordination, function, and structure of zinc enzymes and other proteins. Biochemistry, 29: 5647,1990. 24. Seltzer, J. L., Welgus, H. G., Jeffrey, J. J. and Eisen, A. Z.: The function of Ca2+ in the action of mammalian collagenases. Arch. Biochem. Biophys., 173: 355, 1976. 25. Seltzer, J. L., Jeffrey, J. J. and Eisen, A. Z.: Evidence for mammalian collagenases as zinc ion metalloenzymes. Biochim. Biophys. Acta, 485: 179, 1977. 26. Macartney, H. W. and Tschesche, H.: The metal ion requirement for activation of latent collagenase from human polymorphonuclear leucocytes. Hoppe-Seylers Z. Physiol. Chern., 362: 1523, 1981. 27. Springer, T. A.: Adhesion receptors of the immune system. Nature, 346: 425, 1990. 28. Greenwald, R. A., Golub, L. M., Lavietes, B., Ramamurthy, N. S., Gruber, B., Laskin, R S. and McNamara, T. F.: Tetracyclines inhibit human synovial collagenase in vivo and in vitro. J. Rheumatol., 14: 28, 1987.
Vol. 151, 1400-1404, May 1994
Printed in U. S.A.
INHIBITORY EFFECT OF MINOCYCLINE ON IN VITRO INVASION AND EXPERIMENTAL METASTASIS OF MOUSE RENAL ADENOCARCINOMA NAOYA MASUMORI, TAIJI TSUKAMOTO,* NORIOMI MIYAO, YOSHIAKI KUMAMOTO, IKUO SAIKI AND JUNYA YONEDA From the Department of Urology, Sapporo Medical College and the Institute of Immunological Science, Hokkaido University, Sapporo, Japan
ABSTRACT
Degradation of the extracellular matrix by metalloproteinases is a critical phenomenon in cancer invasion and metastasis. Recent studies have revealed that minocycline (minocycline hydrochloride, a tetracycline) suppresses in vivo and in vitro mammalian collagenolytic activity. We investigated whether minocycline inhibited in vitro invasion and experimental pulmonary metastasis in subline2 of streptozotocin-induced mouse renal adenocarcinoma (MRAC-PM2) cells. In vitro invasion assay demonstrated that treatment with 0.5 ILg.jml. or 5.0 ILg.jml. minocycline significantly inhibited the invasion of MRAC-PM2 cells. In addition, intraperitoneal administration of 0.5 mg. per mouse minocycline reduced the number of metastatic nodules in the lung when MRAC-PM2 cells were injected intravenously. Minocycline also suppressed type IV collagenolytic activity of the cells. However, the drug did not affect [3H]-thymidine uptake, growth of subcutaneously inoculated cells, attachment to the extracellular matrices, or haptotactic migration of the cells. These results indicated that the inhibitory action of type IV collagen degradation by minocycline can contribute, in part, to suppression of the in vitro invasion and metastatic potential of MRAC-PM2 cells. KEY WORDS:
minocyc1ine, adenocarcinoma, collagen, neoplasm invasiveness, neoplasm metastasis
Homeostasis in the remodeling of the basement membrane and the interstitial tissue in vivo depends on the subtle balance between metalloproteinases and their inhibitors.l Cancer cells can affect this balance by secreting and/or activating metalloproteinases. Thus, the cells can degrade the basement membrane and the interstitial tissue during the invasion process. Many studies have demonstrated that metalloproteinase activity, particularly type IV collagenase activity, closely correlates with the invasive or metastatic potential of cancer cells. 2 • 3 The excess secretion or activation of the enzymes promotes the invasion and metastasis of cancer cells. Minocycline hydrochloride (minocycline) is a semisynthetic tetracycline that has been widely used as an antimicrobial agent in the treatment of infectious diseases. Recent studies have revealed that minocycline suppresses in vivo and in vitro mammalian collagenolytic activity. 4, 5 If the inhibitory effect of minocycline on collagenolytic activity extends to cancer cellderived metalloproteinases or tissue-derived metalloproteinases induced by cancer cells, the drug might inhibit cancer cell metastasis as well as invasion. To examine this hypothesis, we investigated the effect of this drug on the in vitro invasiveness and experimental pulmonary metastasis of streptozotocininduced mouse renal adenocarcinoma (MRAC). MATERIALS AND METHODS
Mouse renal adenocarcinoma and its pulmonary metastatic subline. The original MRAC was induced by a single intraperitoneal injection of streptozotocin (Wako Junyaku Co. Ltd., Tokyo, Japan) in the kidney in CBA/H/T6J mice (Jackson Laboratories, Bar Harbor, Massachusetts) and maintained by subcutaneous passage in syngeneic mice. The details of the MRAC were described previously.e,7 In brief, the adenocarcinoma of the kidney showed mitotic activity, nuclear atypia and atypical nucleoli, and the cytoplasm contained eosinophilic and fine granular structures, all of which indicate histopathological Accepted for publication November 4, 1993. * Requests for reprints: Department of Urology, Sapporo Medical College, Sapporo 060, Japan.
features similar to the granular cell type of human renal cell carcinoma. Pulmonary metastatic subline-2 (MRAC-PM2) was selected by an intravenous injection of the MRAC cells and then by injection of cells from its pulmonary metastatic nodules. 7 The MRAC-PM2 was cultured and maintained in MEM D-valine modification medium (DV-MEM; Sigma Chemical Co., St. Louis, Missouri) supplemented with 10% fetal bovine serum (FBS; Flow Laboratories Inc., North Ryde, Australia) and antibiotics in a humidified atmosphere of 5% CO 2 at 37C. In vitro invasion assay. Details of the method were described previously.8,9 In brief, a polyvinylpyrrolidone-free polycarbonate filter with an 8.0 ,um. pore size (Nucleopore Corp., Pleasanton, California) was attached to the bottom of the inner chamber in a Transwell cell culture plate (Costar No. 3422, Cambridge, Massachusetts). The filter was coated on its upper surface with 5 ,ug. (15 ,ug./cm. 2 ) of Matrigel (Becton Dickinson Labware, Bedford, Massachusetts) and on its lower surface with 5 ,ug. (15 ,ug./cm. 2 ) offibronectin (Biomedical Technologies Inc., Stoughton, Massachusetts). The MRAC-PM2 cells with confluent growth were harvested, washed several times with serum-free DV-MEM with 0.1% bovine serum albumin (BSA) and suspended in the same medium. The suspension, 100 ,ul. containing 1 X 105 cells, was applied to the inner chamber. To the outer chamber, 600,ul. of serum-free DV-MEM containing 0.1 % BSA was added. Minocycline (Lederle Ltd., Tokyo, Japan) with a final concentration of 5.0 ,ug. or 0.5 ,ug./ml. in the same medium was applied in the inner chamber. After the cells were incubated for 6 to 8 hours at 37C in a 5% CO 2 atmosphere under 100% humidity, the filter was fixed and stained. Those with invasion onto the lower surface of the filter were counted under microscopy at a magnification of 400. In vitro invasion of MRAC-PM2 cells in each concentration of minocycline was assayed in triplicate. Type IV collagenolytic activity. Type IV collagenolytic activity was determined by the method described previously.lO Briefly, [3H]-labelled type IV collagen solution (3 X 105 cpm/1.6 ,ug./ ml.) in 0.5 M. acetic acid was placed at a volume of 100 ,ul. per well in a tissue culture plate and left overnight in a laminar air
1400
1401
INmmTION OF IVIUIUNE RENAL ADENOCARCnWiVIA BY MINOCYCLINE
flow hood to allow collagen solutions to dry to a film. Then 100 Ill. of serum-free Dulbecco's modified Eagle's medium and Ham's F12 medium (DME:F12, 1:1 ratio, GIBCO, Grand Island, New York) was added over the dried type IV collagen film and incubated overnight at 37C. In the assay for cell-mediated type IV collagenolytic activity, 1 X 105 MRAC-PM2 cells suspended in 2 ml. of D ME:F12 were added to the precoated wells. Minocycline with a concentration of 0.1 j1.g., 1.0 f.lg., or 10.0 f.lg./ml. in the same medium was applied to the precoated welL After a 48-hour incubation period, 200 ,ul. of the supernatant was withdrawn and mixed with 50 f.lL of ice-cold 50% trichloroacetic acid and centrifuged at 18,000 g for 10 minutes at 4C to precipitate undigested materials. Type IV collagenolytic activity was calculated from the [3HJ-activity in the supernatant and expressed as the nanogram amount of type IV collagen degradation. Microassay for cell adhesion. Binding capacity of MRACPM2 cells to the various extracellular matrices was measured according to the method employed by Saiki et al. IO In brief, MRAC-PM2 cells were incubated for 24 hours in DV-MEM containing 10% FBS supplemented with 1 f.lCi/ml. of 5-[125 I]iododeoxyuridine (specific activity, 2200 Ci/mmol.; New England Nuclear, Boston, Massachusetts). The cells were washed twice in warm Ca 2+- and Ma 2+-free phosphate-buffered saline (PBS) to remove the unbound radiolabel, harvested by 0.05% trypsin and 0.01 % EDTA for 10 minutes at 37C and resuspended in cold serum-free DV-MEM. [125IJ-iododeoxyuridinelabelled MRAC-PM2 cells, 2 X 104 /0.05 m!. of DV-MEM, were added to each well in a 24-well tissue culture plate precoated with 5 ,ug. (2.5 ,ug./cm. 2 ) of fibronectin, 5 Ilg. (2.5 ,ug./cm. 2 ) of laminin (Biomedical Technologies Inc.), or 5 jJ.g. (2.5 ,ug./cm02) of mouse type IV collagen (Becton Dickinson Labware). Minocycline at a concentration of 5.0 f.lg. or 0.5 f.lg./ml. was added to each well. The cultures were incubated at 37C for 30 minutes and then washed 4 times with PBS to remove any unattached cells. The remaining substrate-bound tumor cells were lysed with 0.1 mL of 0.1 N NaOH. The lysate was absorbed by cotton swabs and monitored for radioactivity by gamma counting. The binding capacity was expressed as follows: binding capacity = cpm of targets bound to substrate X total number of tumor cells added/cpm of total tumor cells added. Haptotactic migration assay. A polyvinylpyrrolidone-free polycarbonate filter with an 8.0 !lm. pore size was coated on its lower surface with 5 f.lg. (15 ,ug./cm. 2 ) of fibronectin.lO The following procedures were the same as those for the in vitro invasion assay. The cells migrating onto the lower surface of the filter were counted under microscopy. Serum concentration of minocycline. The serum concentration of minocycline was measured according to the following method. Minocycline, 0.5 mg. diluted in 0.1 ml. of PBS, was intraperitoneally injected into six CBA/H/T6J mice. The blood, which was collected intracardiac puncture 3 hours after injection of the drug, was centrifuged at 3,000 g for 10 minutes. The serum concentration was primarily determined the thin -layer disk method using Bacillus subtilis ATCC 6633 as a test organismY Experimental pulmonary metastasis. Pulmonary metastasis was experimentally induced by an intravenous injection of 5 X 105 MRAC-PM2 cells suspended in 0.1 m!. of PBS into the tail vein in 8-week-old CBA/H/T6J mice. Mice were sacrificed 14 days after injection of the cells, and metastatic nodules in the lung were macroscopically counted. Minocycline, 0.5 mg. or 0.05 mg. diluted in 0.1 m!. of PBS, was intraperitoneally administered according to the following treatment schedules. In treatment 1, minocycline was given 3 hours before and 1 hour after the intravenous injection of the cells. Treatment 2 consisted of minocycline administration, which was started on the day after the cell injection and consecutively given once each day for another 9 days. In treatment 3, minocycline was given according to a schedule combin-
ing treatments 1 and 2. Control mice were given 0.1 ml. of PBS alone intraperitoneally according to the schedule of treatment 30 Assessment of DNA synthesis. The effect of minocycline on DNA synthesis of MRAC-PM2 cells was evaluated with a modification of the method of Hosaka et al. I2 After 1 m!. of 0.6% agarose-DV-MEM solution with 15% FBS was plated as the feeder layer in a 24-well tissue culture plate, 0.5 mL of 0.4 % agarose-DV-MEM solution with 12.5% FBS containing 1 X 105 MRAC-PM2 cells was placed on the feeder layer. Then 50 f.ll. of PBS containing minocycline at a final concentration of 500, 50, or 5 ,ug./ml. was added onto the double-layered agarose. For control, the same volume of PBS alone was added. The cells were incubated for 72 hours under the conditions described earlier. Then 5 f.lCi of [3H]-thymidine was applied on the agarose in each well, and the cells were incubated for an additional 24 hours. After terminating [3H]-thymidine incorporation, the cells were recovered and radioactivity was counted as described previouslyY Subcutaneous growth of MRAC-PM2. MRAC-PM2 cells,S X 105 /0.1 mL of PBS, were subcutaneously inoculated into the flank in 8-week-old CBA/H/T6J mice. When the tumor reached a size of 5 mm. in diameter (approximately 2 weeks after the inoculation), 0.5 mg. or 0.05 mg. of minocycline dissolved in 0.1 mL of PBS, or PBS alone, was given intraperitoneally for 10 consecutive days. The tumor size was measured every 5 days for 30 days from the start of minocycline treatment. In measuring tumor size, the product of the minor diameter and the major diameter was calculated and the ratio of the products at each given time to that at the start of the treatment was determined as follows: tumor growth ratio = minor diameter X major diameter at the given time/minor diameter X major diameter at the start of treatment. Statistical analysis. The statistical difference between the groups was determined by the nonparametric Mann-Whitney U test. RESULTS
Inhibitory effect of minocycline on in vitro invasiveness of MRAC-PM2. The number of invading MRAC-PM2 cells was significantly dependent on incubation time when they were treated with serum-free DV-MEM containing 0.1 % BSA alone (control). Treatment with minocycline at each concentration reduced the number of MRAC-PM2 cells that invaded at each incubation time more than the control did (P < 0.05, table 1). Although not significant, 5.0 f.lg./ml. of minocycline tended to have a more pronounced effect in suppressing invasiveness than did the other concentration of the drug at the 8th hour. Minocycline treatment and type IV collagenolytic activity. Type IV collagenolytic activity was enhanced by the addition of MRAC-PM2 cells alone, suggesting that the cells surely had collagenolytic activity. Although 0.1 Ilg./ml. of minocycline did not suppress type IV collagenolytic activity, higher concentrations of the drug suppressed the activity more than the control (table 2). Suppression ofthe activity tended to be dose dependent. Treatment with a concentration of 10 l.lg./ml. of minocycline reduced the activity by approximately 45% compared to that of the cells alone. TABLE
1. Influence of minocycline on in uitro inuasiueness of MRAC-PM2 cells No. of invading cells/filter (mean± SD)
Concentration of minocycline (I'g./ml.)
6 hours
8 hours
o (control)
6.3 ± 2.11
0.5 5.0
1.0 ± 1.02 1.3 ± 0.6 3
42.3 ± 5.5' 14.0 ± 6.1' 9.3 ± 3.5 6
1-6 Statistical analysis 1 versus 2 or 3, P <0.05 hy Mann-Whitney U-test; 4 versus 5 or 6, P <0.05 by Mann-Whitney U-test.
1402
INHIBITION OF MURINE RENAL ADENOCARCINOMA BY MINOCYCLINE
TABLE 2.
Inhibitory effect of minocycline on type IV collagenolytic activity Type IV collagenolytic activity" (ng.)
Concentration of minocycline (I'g./ml.)
Treatment DME:F12 alone DME:F12 + cells
17.9 ± 99.1 ± 101.6 ± 78.6 ± 54.4 ±
o 0.1 1.0 10.0
2.9 ' 3.7 2 0.8 13.73 9.8'
• Mean ± SD in triplicate. 1-' Statistical analysis: 1 versus 2, P <0.05 by Mann-Whitney U-test; 2 versus 3 or 4, P <0.05 by Mann-Whitney U-test.
6
.2 iii ...
5
Intraperitoneal administration of minocycline or PBS
- UUUUU ...
.c
:s: 0
"-
4
OJ
0
E
3
::l
Influence of minocycline on attachment of MRAC-PM2 cells
TABLE 3.
Concentration of minocycline (I'g./ml.)
Binding capacity (No. of cells bound/substrate)"
o 0.5 5.0
Fibronectinb
Lamininb
Type IV collagenb
8960 ± 671 10003 ± 760 9938 ± 779
7765 ± 2089 7769 ± 799 6691 ± 369
10674 ± 3962 7718 ± 878 8271 ± 503
I-
2
o
10
• Mean ± SD in triplicate. b Precoated substrate.
Concentration of minocycline (I'g./ml.)
o (control) 0.5 5.0
TABLE 5.
6 hours
8 hours
314.0 ± 63.3 324.0 ± 89.1 323.7 ± 56.7
475.9 ± 58.3 502.7 ± 122.9 526.7 ± 107.2
Minocycline (mg'; mouse)
No. of mice
0 0.05 0.5 0.05 0.5 0.05 0.5
30 6 7 7 7 6 7
Control 1 2 3 1-5
No. of migrating cells/filter (mean ±SD)
Inhibitory effect of minocycline on experimental pulmonary metastasis
Treatment
No. of pulmonary metastatic nodules (mean± SD) 100.4 ± 79.7 ± 39.9 ± 100.4 ± 52.9 ± 45.8 ± 27.3 ±
52.2' 46.4 12.82 45.3 33.3 3 18.5' 17.35
Statistical analysis: 1 versus 2, 3, 4, or 5: P <0.05 by Mann-Whitney U-
test. TABLE 6.
Influence of minocycline on DNA synthesis of MRAC-PM2 determined by f HJ -thymidine incorporation assay
Concentration of minocycline (I'g./ml.)
o 5 50 500
30
Days after the start of treatment
Influence of minocycline on haptotactic migration of MRACPM2
TABLE 4.
20
Uptake of [3H)-thymidine" (DPM) 852.1 714.2 1091.5 844.6
± ± ± ±
377.1 366.0 1029.5 643.3
• Mean ± SD in triplicate.
Influence of minocycline on attachment of MRAC-PM2 cells to the extracellular matrices. Approximately 40% or more of the applied cells attached to the various substrates in the controls. However, the two concentrations of minocycline did not interfere with the binding capacity of the cells to each substrate (table 3). Influence of minocycline on haptotactic migration of MRACPM2 cells. The number of cells that migrated to the lower surface of the filter was increased compared with the in vitro invasion assay for each concentration of minocycline (table 4). Minocycline did not show any inhibitory influence on haptotactic activity of MRAC-PM2 cells at any concentration tested.
105
FIG. 1. 5 X MRAC-PM2 cells were inoculated on flank in CBA/ H/T6J mice. When tumor reached 5 mm. diameter, 0.5 mg. or 0.05 mg. of minocycline was given intraperitoneally for 10 consecutive days. In control mice, 0.1 mL of PBS was given. Tumor growth ratio was determined as follows: tumor growth ratio = minor diameter x major diameter at given time/minor diameter x major diameter at start of treatment. Symbols indicate: open circles (0): control (n = 5); closed circles (e): 0.5 mg. of minocycline per mouse (n = 5) and open squares (0): 0.05 mg. per mouse of minocycline (n = 5). Vertical bar indicates one standard deviation.
Serum concentration of minocycline in CBA/H/T6J mice. The intraperitoneal injection of 0.5 mg. of minocycline produced serum concentrations of 1.17 ± 0.36 /Lg.jml. Suppression by minocycline of experimental pulmonary metastasis of MRAC-PM2. Intravenous injection of MRAC-PM2 cells yielded many visible metastatic nodules in the lungs of mice, which were sacrificed on day 14. No mice died of the procedure during the experiment. When minocycline was given before and after the intravenous injection of MRAC-PM2 cells (treatment 1), 0.5 mg. of the drug significantly suppressed metastatic nodule formation in the lung (P < 0.05, table 5). In treatment 2, which consisted of drug administration for 10 consecutive days started on the day after the intravenous cell injection, a 0.5 mg. dose of minocycline markedly reduced the number of metastatic nodules (P < 0.05). In treatment 3, even treatment with the lower dose of the drug was able to suppress metastatic nodule formation in the lungs of mice (P < 0.05) when it was given for 10 consecutive days as well as around the time of cell injection, as shown in table 5. The most prominent effect of suppression of metastasis was found with 0.5 mg. of minocycline in treatment 3. Influence of minocycline on DNA synthesis of MRAC-PM2 cells. The DNA synthesis of MRAC-PM2 cells was determined by [3HJ-thymidine incorporation assay. We examined three different concentrations, 5 /Lg./ml., 50 /Lg./ml. and 500 /Lg.jml., in the [3HJ-thymidine incorporation assay (table 6). Although [3HJ-thymidine uptake varied widely with each concentration, no significant difference in uptake was found among these concentrations of the drug, indicating a negligible influence of minocycline on DNA synthesis of the cells. Minocycline treatment and subcutaneous growth of MRACPM2 cells. In mice treated with PBS alone (control), tumors inoculated subcutaneously grew 6-fold in size by the end of the experiment (day 30), compared with the initial size on day 1. Minocycline treatment, either 0.5 mg. or 0.05 mg., was unable to retard the growth of the subcutaneous tumors of MRACPM2 (fig. 1). At each measurement, mean tumor sizes were
INHIBITION OF MURI:NE RENAL ADENOCARCINOIvl:A BY MINOCYCUNE
almost equal among the control and the minocycline-treatment groups. Neither significant loss of body weight nor serious inflammation of the peritoneal cavity was found during the experiment. DISCUSSION
Cancer metastasis requires a complex multistep process. 13 After the proliferation of cancer cells in the primary lesion, they degrade the interstitial tissue and the basement membrane and enter the vascular circulation (intravasation). The cells then attach to the endothelial cells of a target organ with subsequent invasion into the basement membrane and, once again, the interstitial tissue (extravasation). Only cells adapted to the organ microenvironment can survive, proliferate and eventually develop metastatic nodules. For full development of metastasis, cancer cells must complete all these steps. In the multistep process, invasion is the most important phenomenon since it is required for both intravasation and extravasion of the cells. Invasion consists ofthree biochemical events: attachment of cancer cells to the extracellular matrix, degradation of the extracellular matrix by metalloproteinases and migration of the cells. 2 Of the metalloproteinases, type IV collagenase is the enzyme most responsible for the process of invasion. 2 Golub et a1. 4,5 have indicated that minocycline suppresses the collagenolytic activity ofthe skin and the gingiva in diabetic rats with associated hypercollagenolytic status, as well as the activity of rat polymorphonuclear leukocytes and rabbit chondrocytes. Moreover, the drug was shown to suppress the collagenolytic activity of Bl6 melanoma cells. 14 Thus, it is valid to speculate that minocycline can suppress the activity of cancer cells as well as that of normal cells. We used minocycline concentrations of 5.0 f.lg. and 0.5 f.lg./ m!. in the in vitro invasion assay because minocycline at a concentration of 5.0 f.lg./ml. was shown to suppress collagenase activity of human rheumatoid synovium to 48% of the control level. 15 In addition, the peak serum concentration of the drug is approximately 6 /-tg./mL, when 200 mg. of it is intravenously given to healthy adult male subjects. 16 Thus, the 5.0 f.lg./ml. used in the in vitro study is within the range of clinically achievable serum concentrations. For in vivo study, we determined the maximum dosage of the drug to be 0.5 mg. per mouse, taking into consideration the LD50 of the mouse (equivalent to 5 mg.) and the 10-day treatment schedule (in treatments 2 and 3)Y In our study, the serum concentration was approximately 1.2 f.lg./ml., when 0.5 mg. of minocycline per mouse was intraperitoneally injected. Therefore, the serum concentration in the in vivo study was included in the range of drug concentrations of 0.5 /-tg./ml. to 5 /-tg./ml. that were used in the in vitro study. In this study, minocycline suppressed type IV collagenolytic activity of cell line MRAC-PM2. Moreover, the drug inhibited the in vitro invasion of MRAC-PM2 cells. In vitro invasiveness, which is evaluated by in vitro invasion assay, closely correlates with collagenolytic activity, particularly type IV collagenolytic activity. Nakajima et aP8 have demonstrated that a decrease in type IV collagenolytic activity eventually results in a decrease of in vitro invasion. The results in our experiment showing that minocycline affected neither attachment to the extracellular matrix nor migration of MRAC-PM2 cells suggested that the drug could inhibit in vitro invasiveness through its suppressive effect on type IV collagenolytic activity. In addition, we demonstrated that minocycline also inhibited experimental pulmonary metastasis of MRAC-PM2 cells. Since type IV collagenolytic activity correlates not only with in vitro invasiveness but also with metastatic potential,19 it was suggested that minocycline blocked extravasation of MRAC-PM2 cells through its suppression of collagenolytic activity, and then eventually inhibited development of pulmonary metastasis. Previous studies1,20 have demonstrated that degradation of the extracellular matrix by metalloproteinases secreted from
1403
endothelial cells is the first step leading to completion of angiogenesis. Thus, minocycline may inhibit the angiogenesis required for proliferation of metastatic nodules through the same action as the inhibition of the collagenolytic activity, although further study will be necessary to confirm this. Van den Bogert et al. 21 have reported that low concentrations of tetracyclines suppress proliferation of several tumor cells, such as ascitically growing hepatomas, solidly growing Leydig cell tumors, mammary gland tumors, T -type leukemia, prostatic carcinoma and renal cell carcinoma. They indicated that tetracyclines inhibit mitochondrial protein synthesis. This inhibition decreases the oxidative ATP -generating capacity needed for cell division, finally leading to proliferation arrest of cells. They have also reported that each cell line has different permeability barriers for tetracyclines and different intervals until proliferation arrest of cells by the drug. Since in vitro proliferation arrest by continuous application of tetracyclines for prostatic carcinoma and renal cell carcinoma does not occur until the 9th and 4th days, respectively, they concluded that tetracyclines inhibit proliferation following 2 cell cycles. Our results, showing that minocycline neither affected the uptake of [3H]_ thymidine nor suppressed tumor proliferation in vivo, indicated that MRAC-PM2 cells had comparatively stronger barriers against minocycline or that the drug did not affect the cells within the time period used in our experiment. Thus, the cytostatic effect of this drug did not contribute to type IV collagenolytic activity, in vitro invasiveness, or experimental metastasis. In terms of the action sites of metalloproteinases, these enzymes are demonstrated to be activated and to degrade the extracellular matrix in the extracellular space following secretion from cells. This suggests that minocycline seems to suppress the activity of metalloproteinases in the extracellular space rather than their synthesis and secretion. Malis and Cooper22 have reported that tetracycline fluorescence observed under an ultraviolet microscope is commonly seen in collagenous fibers within the stroma of bladder carcinoma but not in the cells. Golub et a1. 4 have suggested that the inhibitory effect of minocycline on collagenolytic activity is probably due to its chelation of Ca 2+, since the inhibitory effect is diminished by the addition of CaCh, Not only minocycline, but all tetracyclines, can chelate Ca2+. A recent study has demonstrated that metalloproteinases, including collagenase, are metalloenzymes which have Zn 2+ in the center of the active site 23 and that the presence of Zn 2+ is required for activation of metalloproteinases. To stabilize the tertiary structure of metalloproteinases, Ca 2+ is needed as a cofactor.24 In other words, Zn 2+ acts as an intrinsic metal cation, and Ca2+ acts as an extrinsic one, each of which participates in expressing and maintaining metalloproteinase activity.25,26 Assuming that the inhibitory effect of minocydine on collagenolytic activity is derived from a suppression of collagenase activity by chelation of Ca 2 +, minocycline may suppress not only the activity of interstitial collagenase but also that of other metalloproteinases, such as type IV collagenase and stromelysin, since Ca 2 + is a common extrinsic cofactor for all metalloproteinases. Minocycline did not affect attachment of cancer cells to the extracellular matrix in this study. Cell-substrate adhesion molecules, especially integrins which bind to fibronectin and laminin, require divalent cations, including Ca2+ for function. 27 Although the extent of the Ca2+ requirement for activity may be different between metalloproteinases and cell adhesion molecules, our results would appear to support the idea that not only Ca2+ chelation but also other actions of minocycline, such as direct binding to metalloproteinases, are involved in its inhibition of collagenolytic activity. This direct action of the drug may be supported by the finding that minocycline has been shown to suppress collagenase activity in vivo for more than 19 weeks after the drug was discontinued. 5 Greenwald et
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INHIBITION OF MURINE RENAL ADENOCARCINOMA BY MINOCYCLINE
a1,28 have speculated that binding of minocycline to metalloproteinase directly results in a loss of enzymatic activity and that slow clearance of minocycline from such a complex provides the prolonged effect in vivo. In summary, our study clearly demonstrated that the suppressive action of minocycline on type IV collagenolytic activity can contribute to inhibition of the in vitro invasiveness of MRAC-PM2 cells. However, the drug was not shown to have any cytotoxic effect on the cells. In addition, it neither affected attachment of the cells to the extracellular matrix nor their migration. From these results, we conclude that minocycline suppressed experimental pulmonary metastasis, in part, through its inhibitory action on type IV collagenolytic activity of the cells. Further investigation is necessary to clarify the action of minocycline and which metalloproteinase activity the drug really suppresses. REFERENCES
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