1 First Department of Physiology, Shinshu University School of Medicine, and 2 Institute of Organ Transplants, Reconstructive Medicine, and Tissue Engineering, Shinshu University Graduate School of Medicine, Matsumoto 390-8621, Japan
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ABSTRACT |
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We investigated whether
supernatant cultured with melanoma cell lines B16-BL6 and K1735 or the
Lewis lung carcinoma cell line (LLC) can regulate lymphatic pump
activity with bioassay preparations isolated from murine iliac lymph
vessels. B16-BL6 and LLC supernatants caused significant
dilation of lymph microvessels with cessation of pump activity. B16-BL6
supernatant produced dose-related cessation of lymphatic pump activity.
There was no significant tachyphylaxis in the supernatant-mediated
inhibitory response of lymphatic pump activity. Pretreatment with
3 × 105 M
N
-nitro-L-arginine methyl ester
(L-NAME) or 10
7 M or 10
6 M
glibenclamide and 5 × 10
4 M 5-hydroxydecanoic acid
caused significant reduction of supernatant-mediated inhibitory
responses. Simultaneous treatment with 10
3 M
L-arginine and 3 × 10
5 M
L-NAME significantly lessened L-NAME-induced
inhibition of the supernatant-mediated response, suggesting that
endogenous nitric oxide (NO) plays important roles in
supernatant-mediated inhibitory responses. Chemical treatment dialyzed
substances of <1,000 molecular weight (MW), producing complete
reduction of the supernatant-mediated response. In contrast,
pretreatment with heating or digestion with protease had no significant
effect on supernatant-mediated response. These findings suggest that
B16-BL6 cells may release nonpeptide substance(s) of <1,000 MW,
resulting in significant cessation of lymphatic pump activity via
production and release of endogenous NO and activation of mitochondrial
ATP-sensitive K+ channels.
malignant melanoma; active lymph transport; nitric oxide; mitochondrial adenosine 5'-triphosphate-sensitive potassium channel
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INTRODUCTION |
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THERE ARE CERTAIN PATTERNS of melanoma metastasis. Thus the initial site of distant metastasis is most commonly skin, subcutaneous tissues, liver, or lung (2, 3). During metastasis, melanoma cells can also easily penetrate lymph capillaries and spread through the lymphatic system. Tumor emboli may be trapped in the first drainage lymph node, or they may bypass those regional lymph nodes to form distant nodal metastasis (skip metastasis) (4, 13). However, the important clinical question of why melanoma cells can spread more quickly via the hematogenous route still remains unresolved.
The transport of lymph depends on passive and active driving forces as well as on the rate of lymph production in organs and tissues (14). The active driving mechanism plays a pivotal role in the centripetal propulsion of lymph, which is caused by the intrinsic active pump activity of the lymph vessels (6, 14). The rhythm and amplitude of the active pump activity are modified by neural and mechanical factors as well as humoral factors (7, 9, 15).
Little information exists, however, regarding potential effects of chemical substances released from melanoma cells on the active lymph transport mechanisms of lymph vessels. Thus, to understand the factors governing the spread of melanoma cells through the lymphatic system, we examined whether the malignant melanoma cell lines B16-BL6 and K1735 can release chemical substance(s) that modifies intrinsic active pump activity of lymph vessels isolated for bioassay and then investigated physiologically or pharmacologically the chemical properties of the substance(s).
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MATERIALS AND METHODS |
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Five-week-old male ddY mice (body wt ~25 g, n = 62) were used for the present studies. The mice were housed in an environmentally controlled vivarium and fed a standard pellet diet and water ad libitum. All experimental protocols were approved by the Animal Ethics Committee, Shinshu University School of Medicine, in accordance with the principles and guidelines of the American Council on Animal Care.
Cell culture.
B16-BL6 murine melanoma cells, transformed 3Y1 (SR-3Y1-2) cells, and
Lewis murine lung carcinoma cells (LLC) were kindly provided by Dr. S. Taniguchi (Shinshu University School of Medicine). K1735 M2 murine
melanoma cells were donated by Dr. I. J. Fidler (MD Anderson
Cancer Center, University of Texas). In brief, a transformed rat
fibroblastic cell line, SR-3Y1-2, was established from an embryonic
cell line 3Y1-B clone1-6 after being infected with a Raus sarcoma
virus (21). These cells were seeded into 100-mm tissue
culture dishes (Corning) and cultured in Dulbecco's modified Eagle's
medium (DMEM; Dainihonseiyaku) supplemented with 10% fetal bovine
serum (JHR Bioscience), penicillin (100 U/ml; Sanko Junyaku, Tokyo,
Japan), streptomycin (100 µg/ml; Sanko Junyaku), and amphotericin B
(250 µg/l; Sigma, St. Louis, MO). The cells were grown at 37°C in a
humidified incubator with 5% CO2-95% air. The culture
medium was changed every 2 or 3 days. The same number of the cells
(1,000 cells/ml) were cultured to a postconfluent condition in the
dishes, and then the cells were rinsed twice with conditioned phosphate buffer solution (Hanks' buffered salt solution, pH 7.4; Sanko Junyaku). Forty-eight hours after resuspension in 15 ml of nutrient mixture [Ham's F-12 (GIBCO BRL-Life Technologies)-DMEM (1:1) mixture medium without serum], a uniform volume of supernatant (15 ml/dish) was collected from the seven culture dishes and then centrifuged (1,000 rpm for 5 min) and filtered ( = 0.2 µm, Millipore) to remove
cell debris. Ten milliliters of supernatant [from 105 ml (15 ml × 7 dishes)] was then diluted at a concentration of 20% with
Krebs-bicarbonate solution (in mM: 120.0 NaCl, 5.9 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 NaH2PO4, 5.5 glucose, and 25.0 NaHCO3) to normalize the concentration of chemical
substances in the medium. In some experiments, B16-BL6 supernatant
solutions at different concentrations (10.0%, 20.0%, 40.0%, and
100.0%) were constructed with appropriate volumes of Krebs-bicarbonate solution.
Lymphatic bioassay preparation. The mice were anesthetized with pentobarbital sodium (50 mg/kg ip). After an abdominal incision, iliac lymph microvessels with their lymph nodes were excised and placed in a petri dish containing cold (4°C) Krebs-bicarbonate solution. With the use of microsurgical instruments and an operating microscope, the lymph microvessels (n = 62; 101-244 µm in maximum diameter, 3 mm in length) were isolated and then transferred to a 10-ml organ chamber with two glass micropipettes containing Krebs-bicarbonate solution.
After each lymph microvessel was mounted on a pipette (proximal) and secured with several sutures, the perfusion pressure was raised to 4 cmH2O to flush out and clear the vessel. The distal end of the vessel was then mounted onto the outflow micropipette (distal). The proximal and distal micropipettes were connected with Tygon tubing with a 50-ml syringe and a stopcock, respectively. The Krebs-bicarbonate solution, maintaining a PO2 of ~50 mmHg and a pH of 7.4 ± 0.01, bubbled with a gas mixture of 5% CO2 and 95% N2, was perfused extraluminally over the bioassay lymph vessels within the organ bath. The flow rate of the perfused solution was kept at 12 ml/min throughout the experiment. After cannulation of the lymph microvessel, the chamber was transferred to the stage of an intravital microscope (Olympus BH-2). The lymph vessels were then warmed slowly to 37°C and allowed to equilibrate for 60 min.Measurement of diameter in bioassay lymph microvessels. The images of the bioassay lymph microvessels were obtained with the use of an objective lens (×4), a photo-eyepiece lens (×3), and a monochrome charge-coupled device camera (KOKOM KCB-270A). Changes in the diameter of lymph microvessels were manually and automatically measured with a custom-made diameter detection device using an edge-detection method (16). These changes were recorded on both a videocassette recorder (Toshiba) and a direct-writing oscillograph (Sanei-Sokki Recti 8K). The intraluminal pressure in the microvessels was kept at 3-4 cmH2O by elevating a 50-ml syringe connected to the inflow tubing while the outflow tubing was closed with a stopcock throughout the experiments. This pressure was optimal for production of active pump activity of the isolated murine lymph microvessels (10, 12).
Experimental protocol.
To evaluate functional viability of endothelial cells,
107 M acetylcholine (ACh) was first perfused
extraluminally over all of the lymph microvessels before the
experiments started. In the first experimental protocol, the effects of
B16-BL6 supernatant, transformed 3Y1 supernatant, K1735 supernatant,
LLC supernatant, or vehicle (culture medium without any cells) on
active pump activity of the isolated lymph microvessels were evaluated
by 3-min extraluminal perfusion of Krebs-bicarbonate solution
containing the test supernatant or vehicle.
Drugs. Salts (Wako), ACh chloride (Daiichiseiyaku, Tokyo, Japan), glibenclamide (RBI), IBTX (Peptide Institute, Osaka, Japan), 5-HD, L-NAME, L-arginine, and indomethacin (Sigma) were used in the present study. Glibenclamide and indomethacin were diluted with dimethyl sulfoxide (DMSO) and ethanol, respectively. The concentrations of DMSO and ethanol did not exceed 0.036% in the organ chamber; these concentrations did not affect the active pump activity of the isolated lymph microvessels. Concentrations of drugs are expressed as the final concentration in the organ chamber. All salts and drugs were prepared on the day of the experiment.
Statistical analyses. The supernatant-induced inhibitory response is expressed as a percentage of inhibition of active pump activity of the isolated lymph microvessels. Thus the averaged frequency (times/min) of the pump activity during the occurrence of supernatant-induced response was normalized by that obtained before the application of supernatant.
The data are presented as means ± SE, and n indicates the number of vessels. Significant differences (P < 0.05) were determined by one-way analysis of variance (ANOVA), followed by Duncan's post hoc test and paired Student's t-test, as appropriate. ![]() |
RESULTS |
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Isolated bioassay lymph microvessels of mice exhibited regular
active pump activity at an intraluminal pressure of 3-4
cmH2O. The maximum and minimum diameters of lymph
microvessels were calculated to be 177.7 ± 5.5 (n = 62) and 159.2 ± 5.3 (n = 62) µm,
respectively. The frequency of the pump activity of microvessels was
13.8 ± 0.2 min1 (n = 62).
Effects of B16-BL6 supernatant on lymphatic pump activity.
Figure 1 shows representative tracings of
the effects of B16-BL6, transformed 3Y1, LLC, and K1735 supernatants
diluted by 20% and the culture medium itself (vehicle) on the active
pump activity of an isolated lymph microvessel. B16-BL6 and LLC
supernatants caused a significant dilation of the lymph microvessel
with cessation of pump activity (Fig. 1, A and
D). In contrast, both transformed 3Y1 supernatant and
vehicle had no significant effect on lymphatic active pump activity
(Fig. 1, B and C). In 11 of 15 lymph
microvessels, K1735 supernatant also produced no significant effect on
lymphatic pump activity (Fig. 1E). No tachyphylaxis was
observed in the B16-BL6 supernatant-induced inhibitory response of the
lymphatic pump activity. Repeated administrations (3 times) of the
B16-BL6 supernatant into the same bioassay lymph microvessel at 30-min intervals produced a marked inhibition of the pump activity in a
similar manner. The inhibitory responses of lymphatic pump activity induced by the first, second, and third administrations of B16-BL6 supernatant were 30.8 ± 0.3% (n = 3),
35.7 ± 7.9% (n = 3), and
35.7 ± 0.9%
(n = 3), respectively.
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Effects of K+ channel blockers on
B16-BL6 supernatant-mediated inhibitory response of lymphatic pump
activity.
Figure 3B shows the summarized data for the effects of
glibenclamide (107 M and 10
6 M) on B16-BL6
supernatant-mediated inhibitory responses of lymphatic pump activity.
Pretreatment with glibenclamide caused significant dose-related
reduction of B16-BL6 supernatant-mediated inhibitory response
[control,
51.1 ± 3.5% (n = 5);
10
7 M glibenclamide,
11.9 ± 2.5%
(n = 5; P < 0.05 vs. control); 10
6 M glibenclamide, 7.0 ± 9.4% (n = 5; P < 0.05 vs. control)].
Effects of L-NAME, L-NAME and
L-arginine, or indomethacin on B16-BL6 supernatant-mediated
inhibitory response of lymphatic pump activity.
The B16-BL6 supernatant-mediated inhibitory response of lymphatic pump
activity was significantly reduced by pretreatment with 3 × 105 M L-NAME. This
L-NAME-mediated inhibitory effect was significantly lessened by simultaneous treatment with 3 × 10
5 M
L-NAME and 10
3 M L-arginine.
These experimental findings are summarized in Fig. 3E. Thus
the B16-BL6 supernatant-mediated inhibitory responses in the absence of
L-NAME, in the presence of L-NAME (3 × 10
5 M) alone, and in the presence of L-NAME
(3 × 10
5 M) + L-arginine
(10
3 M) were
53.7 ± 13.2% (n = 4),
7.7 ± 7.4% (n = 4; P < 0.05 vs. control), and
27.2 ± 9.8% (n = 4;
P < 0.05 vs. L-NAME alone), respectively.
Effects of heating, enzymatic digestion with protease, or dialysis
of B16-BL6 supernatant on supernatant-mediated inhibitory response of
lymphatic pump activity.
Pretreatment with heating or enzymatic digestion of the B16-BL6
supernatant with protease had no significant effect on the B16-BL6
supernatant-mediated inhibitory response of lymphatic pump activity.
Figure 4, A and B,
shows these summarized data [heated, 54.2 ± 9.0%
(n = 4) vs. control,
40.8 ± 11.5%
(n = 4; not significant); protease,
43.1 ± 10.9% (n = 4) vs. control,
36.4 ± 7.4%
(n = 4; not significant)]. In contrast, pretreatment with dialysis significantly reduced the B16-BL6 supernatant-mediated inhibitory response. Figure 4C shows the summarized data
[control,
43.7 ± 14.9% (n = 4) vs. dialysis,
+3.8 ± 2.4% (n = 4; P < 0.05)].
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DISCUSSION |
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Release of inhibitory substance(s) on lymphatic pump activity by malignant melanoma cell line. The lymphatic system plays an important role in regulating the transport of extracellular fluids and macromolecular substances in tissues. Thus lymph vessels act to return fluid and protein that escape from the capillary blood vessels to the systemic circulation. In the process of the transport, the escaped fluid and protein enter into the initial microlymphatics by a transient pressure gradient between the interstitial space and the initial lymphatics (1, 18). To accomplish these tasks, larger collecting lymph vessels work as a series of lymphatic pumps (lymphatic pump activity) that propel the lymph fluid centripetally by rhythmic constriction and dilation.
Our major findings in this study are summarized as follows. B16-BL6 and LLC supernatants caused a significant dilation of isolated murine iliac lymph microvessels with cessation of lymphatic pump activity. The supernatant cultured with B16-BL6 malignant melanoma cells caused a dose-related reduction of lymphatic pump activity. The B16-BL6 supernatant-mediated inhibitory response was significantly reduced by pretreatment with glibenclamide, 5-HD, or L-NAME. An additional treatment of L-arginine with L-NAME significantly lessened the L-NAME-induced reduction of the B16-BL6 supernatant-mediated inhibitory response. The concentrations of glibenclamide (10 ![]() |
ACKNOWLEDGEMENTS |
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The authors thank Dr. S. Taniguchi, Shinshu University School of Medicine, for the generous donation of the B16-BL6, LLC, and transformed 3Y1 cell lines. We also thank Dr. I. J. Fidler, University of Texas MD Anderson Cancer Center, for the kind gift of the K1735 cell line.
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FOOTNOTES |
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This study was supported by Grants-in-Aid for Scientific Research from the Japanese Ministry of Education, Science, Sports, and Culture (09877008 and 11470010).
Address for reprint requests and other correspondence: T. Ohhashi, 1st Dept. of Physiology, Shinshu Univ. School of Medicine, 3-1-1 Asahi, Matsumoto 390-8621, Japan (E-mail: ohhashi{at}sch.md.shinshu-u.ac.jp).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 28 February 2001; accepted in final form 23 July 2001.
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