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The antimetastatic activity of enkephalin-like peptides
Cancer Letters, 35 (1987) 133-138 Elsevier Scientific Publishers Ireland Ltd.
THE ANTIMETASTATIC PEPTIDES E.M. SCHOLAR, Department
133
ACTIVITY
OF ENKEPHALIN-LIKE
L. VIOL1 and T.D. HEXUM
of Pharmacology,
University
(Received 12 November 1986) (Revised version received 19 January (Accepted 21 January 1987)
of Nebraska
Medical
School,
Omaha,
NE 68105 (U.S.A).
1987)
SUMMARY
[Met’] Enkephalin and [Leu5] enkephalin, two naturally occurring opioidlike peptides were found to decrease the number of spontaneous pulmonary metastases in C57BL/6J mice implanted in the foot pad with the B16-BL6 melanoma. The number of such metastatic nodules were reduced more than 2-fold. This effect was noted after several different injection schedules. In addition to their effect on metastasis the growth rate of the primary tumor was also decreased by these compounds.
INTRODUCTION
It has recently been shown by several investigators that the enkephalinlike peptides can alter several components of the immune system including lymphocyte proliferation and natural killer (NK) cell cytotoxicity [l-d]. The particular component of the immune system affected seems to vary with the individual enkephalin-like peptide used [2]. Although the role of these endogenous opioid peptides on the immune system remains unclear, there is evidence that they may be involved in the communication between the immune and neuroendocrine systems [2]. Owing to the importance of the immune system in tumor cell proliferation it is likely that these peptides may affect the growth and spread of tumors. Recently, several drugs that stimulate the host’s immune response have been found to be useful as antineoplastic agents [5,6]. The enkephalin-like peptides may also fit into this category. In support of this, Murgo has recently reported that [Met’lenkephalin inhibited the growth of B16-BL6 melanoma cells inoculated in C57BL/6 mice [7]. This antitumor effect was inhibited by the administration of the opiate receptor antagonist naloxone ]71. 0304.3835/87/$03.50 (: 1987 Elsevier Scientific Published and Printed in Ireland
Publishers
Ireland Ltd.
134
Despite advances in surgical technique and the development of aggressive chemotherapy for treatment of the primary tumor, most deaths in cancer patients are caused by metastases. Thus any compound that will prevent or inhibit metastatic growth would be potentially valuable for the treatment of cancer. It is not known at this time what effect the enkephalins and other opioid peptides have on the metastatic process. Murgo in his study with [Met’] enkephalin did not determine the effect this peptide had on tumor metastasis [7]. In this study we chose to determine the effect of the enkephalin-like peptides on the growth and metastasis of the B16-BL6 melanoma. MATERIALS
AND METHODS
Peptides [ Met51 Enkephalin, [ Leu’] enkephalin and [ D-Ala’,N-MePhe4,Met5( O)011enkephalin were purchased from Peninsula Labs, Belmont, CA. They were diluted in Hanks Balanced Salt Solution, stored at - 70’ C and injected S.C. into the dorsal cervico-thoracic region. Control mice were injected with Hanks Balanced Salt Solution. Several different injection schedules were utilized for these peptides including daily, twice weekly, and before and after primary tumor removal. Tumor lines The B16-BL6 melanoma was obtained from the DCT Tumor Bank, National Cancer Institute, Frederick, MD. This is a highly invasive and metastatic subline of the B16 melanoma that was originally isolated by Fidler [B]. This tumor was maintained in plastic tissue culture flasks in a humidified atmosphere containing 5% CO,. The culture medium was minimal essential medium supplemented with 10% fetal calf serum, 2 mM glutamine, 0.1 mM non-essential amino acids, vitamins, a mixture of 100 U/ml penicillin and 100 pg/ml streptomycin and designated complete minimal essential medium (CMEM). Tumor growth Each C57BL/6J mouse was given a S.C. injection of 5 x lo* viable tumor cells (0.1 ml) into the right flank. The subcutaneous tumors were measured with a vernier caliper. Measurements were calculated as the geometric mean of 2 diameters, the first being the longest diameter and the second being measured perpendicular to the first diameter. Tumor growth was followed over a period of approximately 21 days. Spontaneous metastasis Viable B16-BL6 cells (5 x 104) in 0.05 ml were injected into the hind footpad of syngeneic C57BL/6J mice and growth was measured with a vernier
135
caliper. When the primary tumor reached an average diameter of 1.0 x 1.0 cm, the mice were anesthetized with ether and the tumor bearing leg was surgically removed at midfemur so as to include the popliteal lymph node. Three weeks later the animals were killed and the lungs put into Bouins fixative. Twenty-four hours later the lungs were examined for metastatic modules with a dissecting microscope. RESULTS
[Mets] Enkephalin and [Let?] enkephalin were administered daily at a dose of 3.0 mg/kg for 7 days, beginning the day following tumor inoculation. Figure 1 shows the effect of [ Met51 enkephalin and [ Leu5] enkephalin on the subcutaneous growth of the B16-BL6 tumor. Both peptides decreased the rate of tumor growth as compared to growth in the control group. [Met5]Enkephalin also prolonged the time to tumor appearance while [ Leu”] enkephalin did not affect this parameter. Tables 1 and 2 show the effect of [Met5]enkephalin, [Leu’] enkephalin and [ D-Ala”,N-MePhe4,Met5( 0)-ol] enkephalin on the number of pulmonary metastases produced after injection of 5 x lo4 viable B16-BL6 tumor cells into the foot pad. These peptides were all injected at a dose of 3 mg/kg but several different injection schedules were utilized. [Met’] Enkephalin and [D-Ala’,N-MePhe4,Met5(0)-ollenkephalin were injected daily for seven days after amputation of the leg bearing the primary tumor (Table 1). [Met’] enkephalin significantly decreased the number of metastases produced while
W x
0
19 DRYS FOLLOWING
TUMOR
L
20 21 22 INOCULRTION
Fig. 1. The effect of enkephalin-like peptides on growth of the B16-B6 tumor. C57BL/6J mice were injected with 5 x lo4 B16-BL6 melanoma cells into the right flank. Tumor growth was followed until the tumor reached a size of approximately 1 cm in diameter. Control mice received Hank’s balanced salt solution. Controls (0); [Met’] enkephalin ( 3); [Leu5] enkephalin ( A).
136 TABLE
1
EFFECT OF ENKEPHALIN-LIKE CELLS IN C57BL/6 MICE
PEPTIDES
ON THE METASTASIS
OF B16-BL6
C57BL/6 mice were injected with 5 x 10 4 B16BL6 melanoma cells into the hind footpad. When the tumor reached 1.0 cm in diameter the foot was amputated and 3 weeks later the mice were sacrificed and their lungs were examined for the presence of metastases. Group”
No. of mice
Median
Range
Control [Mets] Enkephalin [ D-Ala’,N-MePhe4,Met”( Enkephalin
5 6 7
9.0 5.5h 12.0
662 i&g 5523
0)ol]
_
“Enkephalin-like peptides injected for 7 consecutive days following tumor amputation. Controls received Hank’s balanced salt solution. %ignificant difference in the number of lung metastases compared with the saline control [P < 0.05 by the Mann-Whitney U-test].
TABLE
2
EFFECT OF ENKEPHALIN-LIKE CELLS IN C57BL/6 MICE”
PEPTIDES
ON THE METASTASIS
OF B16-BL6
C57BL/6 mice were injected with 5 x 10 ’ B16-BL6 melanoma cells into the hind footpad. When the tumor reached 1.0 cm in diameter the foot was amputated and 3 weeks later the mice were sacrificed and their lungs were examined for the presence of metastases. Group”
Injection
Control
Biweekly pre- and postamputation Biweekly post-amputation Biweekly post-amputation Biweekly pre- and postamputation
[Leu’] Enkephalin
[Met”] Enkephalin [Met”] Enkephalin
schedule”
No. of mice
Median
Range
10
35.5s
3 63
7
10.Ob
3 -22
12
13.0”
1.~50
11
12.0b
0 32
“Enkephalin-like peptides were injected biweekly either post tumor amputation (6 total injections) or pre- and post-amputation (12 total injections). “Significant difference in the number of lung metastases compared with the saline control [P < 0.05 by the Mann-Whitney U Test].
137
[D-Ala’,N-MePhe4,Met5(0)-ol] enkephalin did not have a significant effect. In another experiment the peptides were given twice weekly, either pre- and post-amputation or post-amputation alone (Table 2). Both [ Leu5] enkephalin and [Met’] enkephalin significantly reduced the number of metastases. [Met5]enkephalin had a similar effect regardless of the injection schedule (Tables 1 and 2). DISCUSSION
Several compounds interacting at opioid receptors, e.g., heroin and naloxone have been shown to influence both the immune system and tumor growth and proliferation [9]. Although several recent studies have shown that the endogenous opioid peptides also influence various immune functions their role in tumor growth and metastasis is unclear [l-5]. Murgo recently demonstrated that [ Met51 enkephalin decreased the growth rate of the BL6 murine melanoma [7]. Our results also confirmed the initial observations made by Murgo that [Met5]enkephalin inhibited the growth rate of the B16-BL6 tumor. In addition [Leu5] enkephalin had a similar although not as significant an effect. The results in this paper demonstrate for the first time that [ Met51 enkephalin and [ Leu5] enkephalin significantly decrease the number of metastases produced by this same tumor. [Leu5]Enkephalin had an equal or greater effect on the number of metastases as compared to [Met’] enkephalin. Why the [ D-Ala”-,N-MePhe4,Met5(0)-ol] -enkephalin did not have this effect is not known at this time. It may be a result of a different receptor preference than [Met5]enkephalin or it may be related to the fact that this compound is an analog of [Met51 enkephalin that resists the action of various endogenous proteases. The results show that decreases in the number of metastases produced occur after any one of several schedules of administration of [ Met51 enkephalin and [ Leu”] enkephalin. It is interesting that these effects are produced despite the fact that these peptides have short plasma half lives. It is not unusual to see a large variability between experiments in the number of metastases produced (Tables 1 and 2). This is especially true when using the spontaneous metastasis system. Other investigators have obtained similar results [9,10]. Although these results show significant effects of these natural opioid peptides to inhibit the development of metastases the mechanism of this effect is not known at the present time. These peptides and especially [ Met51 enkephalin have recently been shown to affect several different immune functions including NK cytotoxicity [l-4]. However, [Met51 enkephalin has also been shown to effect other systems that might influence tumor metastasis. For example, [Met’lenkephalin has recently been found to increase prostaglandin production [ll] and prostaglandins are known to alter metastasis in several systems [ 121. We are currently investigating the mechanism of how the enkephalin-like peptides may effect the metastatic process.
138 ACKNOWLEDGMENTS
The authors would like to thank Linda Russett,for her excellent technical assistance. This work was supported in part by a seed grant from the University of Nebraska Medical Center (E.M.S.) and by a grant-in-aid from the American Heart Association Inc. with funds contributed by the Nebraska Affiliate (T.D.H.).
REFERENCES 1 Koff, W.C. and Dunegan, M.A. (1985) Modulation of macrophage-mediated tumoricidal activity by neuropeptides and neurohormones. J. Immunol. 135, 350-354. 2 Smith, E.M., Harbour-McMenamin, D. and Blalock, J.E. (1985) Lymphocyte production of endorphins and endorphin-mediated immunoregulatory activity. J. Immunol., 135, 7795~7825. 3 Carr, D.J.J. and Klimpel, G.R. (1986) Enhancement of the generation of cytotoxic T. cells by endogenous opiates. J. Neuroimmunol., 12, 7587. 4 Zagon, IS. and Mclaughlin, P.J. (1986) Endogenous opioid systems, stress, and cancer. In: (same as Ref. 10) pp. 81-100. 5 Oldham, R.K., Thurman, G.B., Talmadge, J.E., Stevenson, H.C. and Foon, K.A. (1984) Lymphokines, monoclonal antibodies and other biological response modifiers in the treatment of cancer. Cancer, 54, 27952806. 6 Oldham, R.K. (1984) Biologicals and biological response modifiers: fourth modality of cancer treatment. Cancer Treat. Rep., 68, 221-232. 7 Murgo, A.J. (1985) Inhibition of B16-BL6 melanoma growth in mice by methionineenkephalin. J. Natl. Cancer Inst., 75, 341-344. in uivo and in vitro of 8 Fidler, I.J., Gersten, D.J. and Budmen, M.B. (1976) Characterization tumor cells selected for resistance to syngeneic lymphocyte-mediated cytotoxicity. Cancer Res., 36, 316&3165. 9 Murgo, A.J., Faith, R.E. and Plotnikoff, N.P. (1986) Enkephalins: mediators of stressinduced immunomodulation. In: Enkephalins and Endorphins-Stress and the Immune System, pp. 221-239. Editors: N.P. Plotnikoff, R.E. Faith, A.J. Murgo and R.A. Good. Plenum Press, N.Y. of low 10 Joshi, S.S., Sharp. J.G. and Brunson, K.W. (1987) Differential growth characteristics and high metastatic variants of RAW117 murine lymphosarcoma cells. Oncology, in press. of an invasive variant of the B16 11 Hart, I.R. (1979) The selection and characterization melanoma. Am. J. Pathol., 97, 587600. 12 Wiederhold, M.D. and Ou, D.W. (1986) Increased synthesis of eicosanoids by human monocytes following leucine and methionine enkephalin administration. Fed. Proc., 45, 918. 13 Tisdale, M.J. (1983) Role of prostaglandins in metastatic dissemination of cancer. Exp. Cell. Biol., 51, 25p256.
THE ANTIMETASTATIC PEPTIDES E.M. SCHOLAR, Department
133
ACTIVITY
OF ENKEPHALIN-LIKE
L. VIOL1 and T.D. HEXUM
of Pharmacology,
University
(Received 12 November 1986) (Revised version received 19 January (Accepted 21 January 1987)
of Nebraska
Medical
School,
Omaha,
NE 68105 (U.S.A).
1987)
SUMMARY
[Met’] Enkephalin and [Leu5] enkephalin, two naturally occurring opioidlike peptides were found to decrease the number of spontaneous pulmonary metastases in C57BL/6J mice implanted in the foot pad with the B16-BL6 melanoma. The number of such metastatic nodules were reduced more than 2-fold. This effect was noted after several different injection schedules. In addition to their effect on metastasis the growth rate of the primary tumor was also decreased by these compounds.
INTRODUCTION
It has recently been shown by several investigators that the enkephalinlike peptides can alter several components of the immune system including lymphocyte proliferation and natural killer (NK) cell cytotoxicity [l-d]. The particular component of the immune system affected seems to vary with the individual enkephalin-like peptide used [2]. Although the role of these endogenous opioid peptides on the immune system remains unclear, there is evidence that they may be involved in the communication between the immune and neuroendocrine systems [2]. Owing to the importance of the immune system in tumor cell proliferation it is likely that these peptides may affect the growth and spread of tumors. Recently, several drugs that stimulate the host’s immune response have been found to be useful as antineoplastic agents [5,6]. The enkephalin-like peptides may also fit into this category. In support of this, Murgo has recently reported that [Met’lenkephalin inhibited the growth of B16-BL6 melanoma cells inoculated in C57BL/6 mice [7]. This antitumor effect was inhibited by the administration of the opiate receptor antagonist naloxone ]71. 0304.3835/87/$03.50 (: 1987 Elsevier Scientific Published and Printed in Ireland
Publishers
Ireland Ltd.
134
Despite advances in surgical technique and the development of aggressive chemotherapy for treatment of the primary tumor, most deaths in cancer patients are caused by metastases. Thus any compound that will prevent or inhibit metastatic growth would be potentially valuable for the treatment of cancer. It is not known at this time what effect the enkephalins and other opioid peptides have on the metastatic process. Murgo in his study with [Met’] enkephalin did not determine the effect this peptide had on tumor metastasis [7]. In this study we chose to determine the effect of the enkephalin-like peptides on the growth and metastasis of the B16-BL6 melanoma. MATERIALS
AND METHODS
Peptides [ Met51 Enkephalin, [ Leu’] enkephalin and [ D-Ala’,N-MePhe4,Met5( O)011enkephalin were purchased from Peninsula Labs, Belmont, CA. They were diluted in Hanks Balanced Salt Solution, stored at - 70’ C and injected S.C. into the dorsal cervico-thoracic region. Control mice were injected with Hanks Balanced Salt Solution. Several different injection schedules were utilized for these peptides including daily, twice weekly, and before and after primary tumor removal. Tumor lines The B16-BL6 melanoma was obtained from the DCT Tumor Bank, National Cancer Institute, Frederick, MD. This is a highly invasive and metastatic subline of the B16 melanoma that was originally isolated by Fidler [B]. This tumor was maintained in plastic tissue culture flasks in a humidified atmosphere containing 5% CO,. The culture medium was minimal essential medium supplemented with 10% fetal calf serum, 2 mM glutamine, 0.1 mM non-essential amino acids, vitamins, a mixture of 100 U/ml penicillin and 100 pg/ml streptomycin and designated complete minimal essential medium (CMEM). Tumor growth Each C57BL/6J mouse was given a S.C. injection of 5 x lo* viable tumor cells (0.1 ml) into the right flank. The subcutaneous tumors were measured with a vernier caliper. Measurements were calculated as the geometric mean of 2 diameters, the first being the longest diameter and the second being measured perpendicular to the first diameter. Tumor growth was followed over a period of approximately 21 days. Spontaneous metastasis Viable B16-BL6 cells (5 x 104) in 0.05 ml were injected into the hind footpad of syngeneic C57BL/6J mice and growth was measured with a vernier
135
caliper. When the primary tumor reached an average diameter of 1.0 x 1.0 cm, the mice were anesthetized with ether and the tumor bearing leg was surgically removed at midfemur so as to include the popliteal lymph node. Three weeks later the animals were killed and the lungs put into Bouins fixative. Twenty-four hours later the lungs were examined for metastatic modules with a dissecting microscope. RESULTS
[Mets] Enkephalin and [Let?] enkephalin were administered daily at a dose of 3.0 mg/kg for 7 days, beginning the day following tumor inoculation. Figure 1 shows the effect of [ Met51 enkephalin and [ Leu5] enkephalin on the subcutaneous growth of the B16-BL6 tumor. Both peptides decreased the rate of tumor growth as compared to growth in the control group. [Met5]Enkephalin also prolonged the time to tumor appearance while [ Leu”] enkephalin did not affect this parameter. Tables 1 and 2 show the effect of [Met5]enkephalin, [Leu’] enkephalin and [ D-Ala”,N-MePhe4,Met5( 0)-ol] enkephalin on the number of pulmonary metastases produced after injection of 5 x lo4 viable B16-BL6 tumor cells into the foot pad. These peptides were all injected at a dose of 3 mg/kg but several different injection schedules were utilized. [Met’] Enkephalin and [D-Ala’,N-MePhe4,Met5(0)-ollenkephalin were injected daily for seven days after amputation of the leg bearing the primary tumor (Table 1). [Met’] enkephalin significantly decreased the number of metastases produced while
W x
0
19 DRYS FOLLOWING
TUMOR
L
20 21 22 INOCULRTION
Fig. 1. The effect of enkephalin-like peptides on growth of the B16-B6 tumor. C57BL/6J mice were injected with 5 x lo4 B16-BL6 melanoma cells into the right flank. Tumor growth was followed until the tumor reached a size of approximately 1 cm in diameter. Control mice received Hank’s balanced salt solution. Controls (0); [Met’] enkephalin ( 3); [Leu5] enkephalin ( A).
136 TABLE
1
EFFECT OF ENKEPHALIN-LIKE CELLS IN C57BL/6 MICE
PEPTIDES
ON THE METASTASIS
OF B16-BL6
C57BL/6 mice were injected with 5 x 10 4 B16BL6 melanoma cells into the hind footpad. When the tumor reached 1.0 cm in diameter the foot was amputated and 3 weeks later the mice were sacrificed and their lungs were examined for the presence of metastases. Group”
No. of mice
Median
Range
Control [Mets] Enkephalin [ D-Ala’,N-MePhe4,Met”( Enkephalin
5 6 7
9.0 5.5h 12.0
662 i&g 5523
0)ol]
_
“Enkephalin-like peptides injected for 7 consecutive days following tumor amputation. Controls received Hank’s balanced salt solution. %ignificant difference in the number of lung metastases compared with the saline control [P < 0.05 by the Mann-Whitney U-test].
TABLE
2
EFFECT OF ENKEPHALIN-LIKE CELLS IN C57BL/6 MICE”
PEPTIDES
ON THE METASTASIS
OF B16-BL6
C57BL/6 mice were injected with 5 x 10 ’ B16-BL6 melanoma cells into the hind footpad. When the tumor reached 1.0 cm in diameter the foot was amputated and 3 weeks later the mice were sacrificed and their lungs were examined for the presence of metastases. Group”
Injection
Control
Biweekly pre- and postamputation Biweekly post-amputation Biweekly post-amputation Biweekly pre- and postamputation
[Leu’] Enkephalin
[Met”] Enkephalin [Met”] Enkephalin
schedule”
No. of mice
Median
Range
10
35.5s
3 63
7
10.Ob
3 -22
12
13.0”
1.~50
11
12.0b
0 32
“Enkephalin-like peptides were injected biweekly either post tumor amputation (6 total injections) or pre- and post-amputation (12 total injections). “Significant difference in the number of lung metastases compared with the saline control [P < 0.05 by the Mann-Whitney U Test].
137
[D-Ala’,N-MePhe4,Met5(0)-ol] enkephalin did not have a significant effect. In another experiment the peptides were given twice weekly, either pre- and post-amputation or post-amputation alone (Table 2). Both [ Leu5] enkephalin and [Met’] enkephalin significantly reduced the number of metastases. [Met5]enkephalin had a similar effect regardless of the injection schedule (Tables 1 and 2). DISCUSSION
Several compounds interacting at opioid receptors, e.g., heroin and naloxone have been shown to influence both the immune system and tumor growth and proliferation [9]. Although several recent studies have shown that the endogenous opioid peptides also influence various immune functions their role in tumor growth and metastasis is unclear [l-5]. Murgo recently demonstrated that [ Met51 enkephalin decreased the growth rate of the BL6 murine melanoma [7]. Our results also confirmed the initial observations made by Murgo that [Met5]enkephalin inhibited the growth rate of the B16-BL6 tumor. In addition [Leu5] enkephalin had a similar although not as significant an effect. The results in this paper demonstrate for the first time that [ Met51 enkephalin and [ Leu5] enkephalin significantly decrease the number of metastases produced by this same tumor. [Leu5]Enkephalin had an equal or greater effect on the number of metastases as compared to [Met’] enkephalin. Why the [ D-Ala”-,N-MePhe4,Met5(0)-ol] -enkephalin did not have this effect is not known at this time. It may be a result of a different receptor preference than [Met5]enkephalin or it may be related to the fact that this compound is an analog of [Met51 enkephalin that resists the action of various endogenous proteases. The results show that decreases in the number of metastases produced occur after any one of several schedules of administration of [ Met51 enkephalin and [ Leu”] enkephalin. It is interesting that these effects are produced despite the fact that these peptides have short plasma half lives. It is not unusual to see a large variability between experiments in the number of metastases produced (Tables 1 and 2). This is especially true when using the spontaneous metastasis system. Other investigators have obtained similar results [9,10]. Although these results show significant effects of these natural opioid peptides to inhibit the development of metastases the mechanism of this effect is not known at the present time. These peptides and especially [ Met51 enkephalin have recently been shown to affect several different immune functions including NK cytotoxicity [l-4]. However, [Met51 enkephalin has also been shown to effect other systems that might influence tumor metastasis. For example, [Met’lenkephalin has recently been found to increase prostaglandin production [ll] and prostaglandins are known to alter metastasis in several systems [ 121. We are currently investigating the mechanism of how the enkephalin-like peptides may effect the metastatic process.
138 ACKNOWLEDGMENTS
The authors would like to thank Linda Russett,for her excellent technical assistance. This work was supported in part by a seed grant from the University of Nebraska Medical Center (E.M.S.) and by a grant-in-aid from the American Heart Association Inc. with funds contributed by the Nebraska Affiliate (T.D.H.).
REFERENCES 1 Koff, W.C. and Dunegan, M.A. (1985) Modulation of macrophage-mediated tumoricidal activity by neuropeptides and neurohormones. J. Immunol. 135, 350-354. 2 Smith, E.M., Harbour-McMenamin, D. and Blalock, J.E. (1985) Lymphocyte production of endorphins and endorphin-mediated immunoregulatory activity. J. Immunol., 135, 7795~7825. 3 Carr, D.J.J. and Klimpel, G.R. (1986) Enhancement of the generation of cytotoxic T. cells by endogenous opiates. J. Neuroimmunol., 12, 7587. 4 Zagon, IS. and Mclaughlin, P.J. (1986) Endogenous opioid systems, stress, and cancer. In: (same as Ref. 10) pp. 81-100. 5 Oldham, R.K., Thurman, G.B., Talmadge, J.E., Stevenson, H.C. and Foon, K.A. (1984) Lymphokines, monoclonal antibodies and other biological response modifiers in the treatment of cancer. Cancer, 54, 27952806. 6 Oldham, R.K. (1984) Biologicals and biological response modifiers: fourth modality of cancer treatment. Cancer Treat. Rep., 68, 221-232. 7 Murgo, A.J. (1985) Inhibition of B16-BL6 melanoma growth in mice by methionineenkephalin. J. Natl. Cancer Inst., 75, 341-344. in uivo and in vitro of 8 Fidler, I.J., Gersten, D.J. and Budmen, M.B. (1976) Characterization tumor cells selected for resistance to syngeneic lymphocyte-mediated cytotoxicity. Cancer Res., 36, 316&3165. 9 Murgo, A.J., Faith, R.E. and Plotnikoff, N.P. (1986) Enkephalins: mediators of stressinduced immunomodulation. In: Enkephalins and Endorphins-Stress and the Immune System, pp. 221-239. Editors: N.P. Plotnikoff, R.E. Faith, A.J. Murgo and R.A. Good. Plenum Press, N.Y. of low 10 Joshi, S.S., Sharp. J.G. and Brunson, K.W. (1987) Differential growth characteristics and high metastatic variants of RAW117 murine lymphosarcoma cells. Oncology, in press. of an invasive variant of the B16 11 Hart, I.R. (1979) The selection and characterization melanoma. Am. J. Pathol., 97, 587600. 12 Wiederhold, M.D. and Ou, D.W. (1986) Increased synthesis of eicosanoids by human monocytes following leucine and methionine enkephalin administration. Fed. Proc., 45, 918. 13 Tisdale, M.J. (1983) Role of prostaglandins in metastatic dissemination of cancer. Exp. Cell. Biol., 51, 25p256.