Growth-inhibitory Effects of Epidermal Growth Factor on Human Breast Cancer and Carcinoma of the Esophagus Transplanted into Nude Mice
YOICHI MURAYAMA, M.D.
The present work was performed to clarify the effects of epidermal growth factor (EGF) on the growth of human breast cancer and carcinoma of the esophagus. Human breast cancer MX1, UM-1, and esophageal cancer ES4 were transplanted to the subcutaneous tissue of nude mice. Human EGF (2 IAg) was injected locally into the subcutaneous tissue surrounding the tumor. The tumor growth was followed for 7 days after treatment, and the estimated tumor weight, tumor doubling time, tumor growth curve, tumor growth rate, and results from histologic examination were used to evaluate the effects of EGF on the growth of tumors. We investigated the dose effect on the growth of these tumors using various concentrations of EGF. We also studied the EGF receptor status of these transplanted tumors and its effect on the influence of EGF treatment. A growth-inhibitory effect was noted in these three tumors with 2 isg of EGF. EGF inhibited growth of ES4 tumor in a dose-dependent manner. Treatment with 2 ng of EGF or saline did not inhibit growth. However treatment with 20 ng or 200 ng of EGF inhibited growth in proportion to the concentrations. All tumors were positive by the EGF receptor binding assay. The efficacy of EGF treatment correlated with EGF receptor levels. EGF receptor levels were also influenced by EGF treatment. These results suggest that human EGF showed an anti-tumor effect on MX-1 and UM-1 breast cancer and also on ES4 esophageal cancer.
B
ASED ON THE RESULTS of in vitro' and in vivo
studies,5-7 transforming growth factor a and/or epidermal growth factor (EGF) appears to be a hormonally regulated positive growth factor and transforming growth factor A appears to be a negative growth factor in human breast cancers. In general hormones have multiple modulatory activities according to the concentration. For example, a low concentration of estradiol is a positive growth factor for breast cancers,8 although a high concentration ofestradiol is a negative growth factor for breast cancer.9 From our clinical experience in estrogen therapy9 for recurrent breast cancer using high-dose DES Address reprint requests to Yoichi Murayama, M.D., Second Department of Surgery, School of Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, Japan 113. Accepted for publication July 13, 1989.
263
From the Second Department of Surgery, School of Medicine, Tokyo Medical and Dental University, Tokyo, Japan
or estradiol, and the negative growth effects of EGF in human breast cancer cell tissue cultures,'0 the hypothesis that a high concentration of EGF is a negative growth factor in human breast cancer has been extended to include an in vivo study. On the other hand, EGF is also a growth factor for squamous cells. However, growth-inhibitory effects of EGF and overexpression of its receptor in cultured human squamous cell carcinoma has been reported." Consequently we investigated the effects of human EGF on the growth of human breast cancer and esophageal cancer transplanted into nude mice to clarify the anti-tumor effects of human EGF.
Materials and Methods
Animals Adult athymic nude mice (SPF/VAF Crj: CD-I (IcR)nu; Charles River Japan, Inc., Atugi, Kanagawa, Japan) weighing 30 to 40 g were housed under specific pathogenfree (SPF) conditions. Tumors Two tumors obtained from human breast cancer were used. MX-1 was isolated from a human female breast cancer in 1974 at the National Cancer Institute (NCI) of the United States. MX- 1 does not have estrogen receptors. A second primary tumor, UM- 1, was isolated from a Japanese breast cancer patient in our laboratory. UM- 1 demonstrated resistance to anti-estrogen therapy. UM- 1 is a homogeneous tumor from histologic sections. Histologically MX- 1 is a poorly differentiated duct cell carcinoma and UM- 1 is an invasive ductal carcinoma. The second transplanted generation was used for the experiment.
264
MURAYAMA
Es-4, transplanted in 1975 from a primary esophageal carcinoma of a 54-year-old man, was a moderately differentiated squamous cell carcinoma.'2
Experimental Protocol
Breast cancer. A piece of tumor was transplanted to the subcutus of the back of athymic female nude mice. The transplanted tumor volume was about 27 mm3 (3 mm X 3 mm X 3 mm). Two micrograms of EGF in 0.2 mL of 1/lOON CH3COOH as a solvent or 0.2 mL of the solvent alone was injected subcutaneously around the tumor 13 days after transplantation. The animals were examined every day for tumor growth and the diameter of the tumor was measured with calipers in two perpendicular directions (breadth and width) for 7 days after treatment.
Mice with an MX-1 tumor were divided into three groups. The first group included five mice that received no injections and served as a no-treatment control. The second group included five mice which received an injection of 0.2 mL of solvent. The third group included 7 mice which received an injection of 2 ug of EGF in 0.2 mL of solvent. Mice with an UM-1 tumor were divided into three groups. The first group was a control group that consisted of five mice that received no injections. The second group consisted of five mice that received local injections of 2 ,ug of EGF in 0.2 mL ofsolvent. The third group consisted of four mice that received only local injections of 0.2 mL of solvent. The estimated tumor weight, tumor doubling time, tumor growth curve, tumor growth rate, and histologic findings were recorded to evaluate the effects of EGF on tumor growth in tumors transplanted to nude mice. The estimated tumor weight (W) was calculated as follows: W = long length X short length 2/2. The tumor growth rate was defined as Wb/Wa where Wa is the estimated tumor weight 13 days after transplantation (before treatment), and Wb is the estimated tumor weight 7 days after treatment. The tumor was removed 7 days after treatment and stored at -80 C for histologic examination and EGF-R assay.
Carcinoma of the esophagus. A piece of esophageal cell line Es-4 tumor (3 mm X 3 mm X 3 mm) was transplanted into the subcutaneous tissue of each of the backs of adult athymic male nude mice. The tumors were divided into three groups: the untreated control group, the solvent-injected group, and the EGF-treated group. Human EGF (2 ,g) in 0.2 mL of solvent was subcutaneously injected around the tumor 6 days after transplantation. The solvent was phosphate-buffered saline, pH
Ann. Surg. * March 1990
7.4 (0.16 g of NaH2PO4 plus 0.76 g of Na2HPO4 plus 0.44 g of NaCl/100 mL). After treatment the anti-tumor effect of EGF was investigated by evaluating the tumor growth rate and the tumor growth curve for 7 days. The tumor growth rate was defined as the ratio of Wb/Wa where Wa was the estimated tumor weight 6 days after transplantation (before treatment), and Wb was the estimated tumor weight 7 days after treatment. The dosedependent effects on h-EGF were investigated using 2 ng, 20 ng, and 200 ng of h-EGF. Transplanted tumors were surgically removed 7 days after treatment and were stored at -80 C for EGF-R assay.
EGF-Receptor Assay EGF-receptor (EGF-R) binding capacity was determined according to the method described previously by Fitzpatrick et al.'3 Tumor specimens were minced and homogenized using several intermittent bursts of a Polytron in ice-cold buffer. The homogenate was then centrifuged at 105,000g for 30 minutes, and the pellet was assayed for EGF-R. Membrane pellets were resuspended in phosphate buffer and centrifuged at 2000g for 10 minutes, and the supernatant was assayed for EGF-R. Aliquots of the membranes were incubated with '25I-EGF (1 nM) in the presence (nonspecific binding) or absence (total binding) of unlabeled EGF (100 nM) for 1 hour at 25 C in a final reaction volume of 110 ,uL. The binding was stopped by addition of 2 mL of ice-cold phosphate buffer containing bovine serum albumin (5 mg/mL) and filtered through 0.22-,um pore filters (Gelman GA-8). The incubation tubes and filters were washed twice with 2 mL of phosphate buffer containing bovine serum albumin, and the filters were counted in a Beckman 4000 dual-channel gamma counter. Specific binding was calculated by subtracting nonspecific binding from total binding, and results TABLE 1. Effects ofEpidermal Growth Factor on the Growth of Human Breast Cancer Transplanted into Nude Mice
Growth Rate (Mean ± Standard
Tumors
Treatments (Number of Tumors)
MX-I
None (control) (N = 5)
3.1 ± 0.3
EGF in solvent (N = 7)
1.8 ± 0.5
Solvent alone (N = 5) None (control) (N = 5)
2.7 ± 0.5 3.1 ± 2
EGF in solvent (N = 5)
0.7 ± 0.4
Solvent alone (N = 4)
3.7 ± 1.9
Deviation)
cancer
p < 0.01
UM-I
>
p
YOICHI MURAYAMA, M.D.
The present work was performed to clarify the effects of epidermal growth factor (EGF) on the growth of human breast cancer and carcinoma of the esophagus. Human breast cancer MX1, UM-1, and esophageal cancer ES4 were transplanted to the subcutaneous tissue of nude mice. Human EGF (2 IAg) was injected locally into the subcutaneous tissue surrounding the tumor. The tumor growth was followed for 7 days after treatment, and the estimated tumor weight, tumor doubling time, tumor growth curve, tumor growth rate, and results from histologic examination were used to evaluate the effects of EGF on the growth of tumors. We investigated the dose effect on the growth of these tumors using various concentrations of EGF. We also studied the EGF receptor status of these transplanted tumors and its effect on the influence of EGF treatment. A growth-inhibitory effect was noted in these three tumors with 2 isg of EGF. EGF inhibited growth of ES4 tumor in a dose-dependent manner. Treatment with 2 ng of EGF or saline did not inhibit growth. However treatment with 20 ng or 200 ng of EGF inhibited growth in proportion to the concentrations. All tumors were positive by the EGF receptor binding assay. The efficacy of EGF treatment correlated with EGF receptor levels. EGF receptor levels were also influenced by EGF treatment. These results suggest that human EGF showed an anti-tumor effect on MX-1 and UM-1 breast cancer and also on ES4 esophageal cancer.
B
ASED ON THE RESULTS of in vitro' and in vivo
studies,5-7 transforming growth factor a and/or epidermal growth factor (EGF) appears to be a hormonally regulated positive growth factor and transforming growth factor A appears to be a negative growth factor in human breast cancers. In general hormones have multiple modulatory activities according to the concentration. For example, a low concentration of estradiol is a positive growth factor for breast cancers,8 although a high concentration ofestradiol is a negative growth factor for breast cancer.9 From our clinical experience in estrogen therapy9 for recurrent breast cancer using high-dose DES Address reprint requests to Yoichi Murayama, M.D., Second Department of Surgery, School of Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, Japan 113. Accepted for publication July 13, 1989.
263
From the Second Department of Surgery, School of Medicine, Tokyo Medical and Dental University, Tokyo, Japan
or estradiol, and the negative growth effects of EGF in human breast cancer cell tissue cultures,'0 the hypothesis that a high concentration of EGF is a negative growth factor in human breast cancer has been extended to include an in vivo study. On the other hand, EGF is also a growth factor for squamous cells. However, growth-inhibitory effects of EGF and overexpression of its receptor in cultured human squamous cell carcinoma has been reported." Consequently we investigated the effects of human EGF on the growth of human breast cancer and esophageal cancer transplanted into nude mice to clarify the anti-tumor effects of human EGF.
Materials and Methods
Animals Adult athymic nude mice (SPF/VAF Crj: CD-I (IcR)nu; Charles River Japan, Inc., Atugi, Kanagawa, Japan) weighing 30 to 40 g were housed under specific pathogenfree (SPF) conditions. Tumors Two tumors obtained from human breast cancer were used. MX-1 was isolated from a human female breast cancer in 1974 at the National Cancer Institute (NCI) of the United States. MX- 1 does not have estrogen receptors. A second primary tumor, UM- 1, was isolated from a Japanese breast cancer patient in our laboratory. UM- 1 demonstrated resistance to anti-estrogen therapy. UM- 1 is a homogeneous tumor from histologic sections. Histologically MX- 1 is a poorly differentiated duct cell carcinoma and UM- 1 is an invasive ductal carcinoma. The second transplanted generation was used for the experiment.
264
MURAYAMA
Es-4, transplanted in 1975 from a primary esophageal carcinoma of a 54-year-old man, was a moderately differentiated squamous cell carcinoma.'2
Experimental Protocol
Breast cancer. A piece of tumor was transplanted to the subcutus of the back of athymic female nude mice. The transplanted tumor volume was about 27 mm3 (3 mm X 3 mm X 3 mm). Two micrograms of EGF in 0.2 mL of 1/lOON CH3COOH as a solvent or 0.2 mL of the solvent alone was injected subcutaneously around the tumor 13 days after transplantation. The animals were examined every day for tumor growth and the diameter of the tumor was measured with calipers in two perpendicular directions (breadth and width) for 7 days after treatment.
Mice with an MX-1 tumor were divided into three groups. The first group included five mice that received no injections and served as a no-treatment control. The second group included five mice which received an injection of 0.2 mL of solvent. The third group included 7 mice which received an injection of 2 ug of EGF in 0.2 mL of solvent. Mice with an UM-1 tumor were divided into three groups. The first group was a control group that consisted of five mice that received no injections. The second group consisted of five mice that received local injections of 2 ,ug of EGF in 0.2 mL ofsolvent. The third group consisted of four mice that received only local injections of 0.2 mL of solvent. The estimated tumor weight, tumor doubling time, tumor growth curve, tumor growth rate, and histologic findings were recorded to evaluate the effects of EGF on tumor growth in tumors transplanted to nude mice. The estimated tumor weight (W) was calculated as follows: W = long length X short length 2/2. The tumor growth rate was defined as Wb/Wa where Wa is the estimated tumor weight 13 days after transplantation (before treatment), and Wb is the estimated tumor weight 7 days after treatment. The tumor was removed 7 days after treatment and stored at -80 C for histologic examination and EGF-R assay.
Carcinoma of the esophagus. A piece of esophageal cell line Es-4 tumor (3 mm X 3 mm X 3 mm) was transplanted into the subcutaneous tissue of each of the backs of adult athymic male nude mice. The tumors were divided into three groups: the untreated control group, the solvent-injected group, and the EGF-treated group. Human EGF (2 ,g) in 0.2 mL of solvent was subcutaneously injected around the tumor 6 days after transplantation. The solvent was phosphate-buffered saline, pH
Ann. Surg. * March 1990
7.4 (0.16 g of NaH2PO4 plus 0.76 g of Na2HPO4 plus 0.44 g of NaCl/100 mL). After treatment the anti-tumor effect of EGF was investigated by evaluating the tumor growth rate and the tumor growth curve for 7 days. The tumor growth rate was defined as the ratio of Wb/Wa where Wa was the estimated tumor weight 6 days after transplantation (before treatment), and Wb was the estimated tumor weight 7 days after treatment. The dosedependent effects on h-EGF were investigated using 2 ng, 20 ng, and 200 ng of h-EGF. Transplanted tumors were surgically removed 7 days after treatment and were stored at -80 C for EGF-R assay.
EGF-Receptor Assay EGF-receptor (EGF-R) binding capacity was determined according to the method described previously by Fitzpatrick et al.'3 Tumor specimens were minced and homogenized using several intermittent bursts of a Polytron in ice-cold buffer. The homogenate was then centrifuged at 105,000g for 30 minutes, and the pellet was assayed for EGF-R. Membrane pellets were resuspended in phosphate buffer and centrifuged at 2000g for 10 minutes, and the supernatant was assayed for EGF-R. Aliquots of the membranes were incubated with '25I-EGF (1 nM) in the presence (nonspecific binding) or absence (total binding) of unlabeled EGF (100 nM) for 1 hour at 25 C in a final reaction volume of 110 ,uL. The binding was stopped by addition of 2 mL of ice-cold phosphate buffer containing bovine serum albumin (5 mg/mL) and filtered through 0.22-,um pore filters (Gelman GA-8). The incubation tubes and filters were washed twice with 2 mL of phosphate buffer containing bovine serum albumin, and the filters were counted in a Beckman 4000 dual-channel gamma counter. Specific binding was calculated by subtracting nonspecific binding from total binding, and results TABLE 1. Effects ofEpidermal Growth Factor on the Growth of Human Breast Cancer Transplanted into Nude Mice
Growth Rate (Mean ± Standard
Tumors
Treatments (Number of Tumors)
MX-I
None (control) (N = 5)
3.1 ± 0.3
EGF in solvent (N = 7)
1.8 ± 0.5
Solvent alone (N = 5) None (control) (N = 5)
2.7 ± 0.5 3.1 ± 2
EGF in solvent (N = 5)
0.7 ± 0.4
Solvent alone (N = 4)
3.7 ± 1.9
Deviation)
cancer
p < 0.01
UM-I
>
p
