(Plates LXXVI-LXXVII)

Transcription

1 [GANN, 63, ; October, 1972] UDC SERUM GLYCOPROTEIN LEVELS IN THE RAT BEARING ASCITES HEPATOMA 109A (Plates LXXVI-LXXVII) Yoshito HAYASHI (Department of Clinical Cancer Chemotherapy, Research Institute for Tuberculosis, Leprosy and Cancer, Tohoku University*1) Synopsis As a relatively simplified model for the study of glycoproteins in relation to experimental cancer from a point of tumor-host relationship, experiments were carried out with Donryu rats bearing subcutaneous AH-109A tumor and basic data were obtained. In rats given implants of AH-109A cells, significant increases in serum protein-bound hexoses, hexosamines, and sialic acid occurred in parallel with tumor growth. These changes could be accounted for almost exclusively by the elevation of seromucoid-bound carbohydrate level. Significant increase of ulin occurred during tumor growth. It was suggested that an increase of greatly contributed to an increase of total serum protein-bound carbohydrates. As although there was no change in the quantity of protein moiety, there was a relative increase of carbohydrate-rich proteins. Autopsy confirmed that liver metastasis was negligible. In addition, daily cycle of seromucoid level and the effect of fasting and refeeding on seromucoid level in normal rats were observed. INTRODUCTION The elevation of serum glycoprotein level in animals with cancer has been studied by many investigators.2,6,13,20,23) The seromucoid fraction, which is itself a complex mixture of different glycoproteins, has drawn particular attention of many clinicians, because sequential estimation of the changes in seromucoid levels is a very useful means for predicting the prognosis and for evaluating the effect of cancer chemotherapy. Saito et al.16) confirmed its value experimentally using the rats bearing ascitic Yoshida sarcoma. It is therefore very important for cancer chemotherapeutists to continue a basic and detailed study of serum glycoproteins from the viewpoint of tumor-host relationship. This paper deals with the changes appearing in serum glycoprotein level of the rat when AH-109A cells were transplanted subcutaneously. Subcutaneous transplantation possesses many advantages over intraperitoneal one in obtaining the basic data on serum glycoproteins in tumor-host relationship. MATERIALS AND METHODS Animals Healthy adult male Donryu rats weighing from 115 to 150g at the time of transplantation were selected for this study. They were housed five to a cage and maintained on a standard solid diet (MF, Oriental Yeast Ind. Co., Tokyo) with free access to tap water. 63(5) 591

2 Y. HAYASHI Tumor Source and Inoculation Ascites hepatoma 109A cells*2 (AH-109A) which had been maintained in animals by serial intraperitoneal transplantation, were used. Three male Donryu rats bearing 4- or 5-day-old AH-109A cells were selected as donors. The ascitic tumor cells in the so-called nearly pure culture state (often hemorrhagic) were withdrawn aseptically with a glass pipette, and transferred into a heparinized glass flask and suspended in cold sterile physiological saline. When examined microscopically, the suspension contained 104 tumor cells/mm3. One ml of the suspension that contained approximately 107 tumor cells was then inoculated subcutaneously on the left dorsal region of recipient rats. Five animals served as untreated controls, while 65 animals were given inoculation of tumor cells. A set of control animals was placed under the same environmental conditions. The animals which died during the course of the experiment were omitted. Exsanguination Test animals in 8 separate groups, each consisting of 5 or 10 rats, were sacrificed at 3-day intervals starting on the 3rd day after inoculation by exsanguination from the abdominal aorta under light ether anesthesia between 6:00am and 7:00am. Control animals were also bled at the same clock time. The technique devised for bleeding was as follows: From the abdominal aorta blood was collected as much as possible by applying a needle (0.8mm in internal diameter, 7.5cm in length) connected with a polyethylene capillary tube (10cm in length), and the dry end of the tube was placed into a siliconized glass tube without anticoagulant in which the blood was clotted and centrifuged down. After being left at room temperature for 1hr, sera were separated from the clot by lysis in sera was never observed. Transplanted Tumor Studies The animals were weighed before exsanguination. Then the entire blocks of tumor tissue were extirpated free from fat and fascia, and weighed. The tumor growth (%) was then calculated from: tumor weight /residual body weight (obtained by subtracting tumor weight from total body weight) Histological Examination of Liver The occurrence of liver metastasis was checked by histological studies on a piece of liver tissue from 17 rats killed in later stages on the 15th, 18th, 21st, and 24th post-implant days. Chemical Analysis Chemical analyses of defibrinated sera were initiated 1 week after the last sample was taken. It should be mentioned that, in order to evaluate the data clearly, all the determinations including those of the controls were performed under the same conditions simultaneously. All colorimetric readings were made on a Hitachi photoelectric spectrophotometer. Total serum protein was estimated by the phenol reagent of Lowry,11) with bovine serum albumin as a standard. In each determination, 0.2ml of a 1:200 dilution of serum in 0.85M NaCl solution was used. Serum protein-bound carbohydrate was determined on protein fractions after precipitation with 95% ethanol. *2 Obtained through the courtesy of Professor Haruo Sato, Director of Cancer Research Laboratory of this Institute. 592 GANN

3 SERUM GLYCOPROTEIN IN RATS BEARING AH-109A For the estimation of serum protein-bound hexoses (galactose and mannose), the serum proteins were precipitated with ethanol as described by Weimer and Moshin,22) and the hexose content of the precipitated protein was estimated by the colorimetric method of Lustig and Langer,12) as employed by Weimer and Moshin,22) using an equimolar mixture of galactose and mannose as a standard. The separation of serum protein-bound hexosamines (glucosamine and galactosamine) hydrolytic product thus obtained was subsequently analyzed for hexosamine by the method of Elson and Morgan,8) using glucosamine-hcl as a standard. Treatment with cation-exchange resin employed by Boas5) was omitted. Estimation of the serum protein-bound sialic acid was carried out by the method of change chromatography as employed by Svennerholm21) was omitted. N-Acetylneuraminic acid (Sigma, crystalline from egg) was used as a standard. Sialic acid species of Donryu rat serum protein was not determined in the present study. Fucose content of the serum proteins was determined by the method of Dische and Shettles,7) as modified by Gyorky and Houck,9) with pure L-fucose as a standard. In this method, 1ml of serum sample was used and the interference by chromogens due to protein was minimized by precipitation of protein with 3% trichloroacetic acid after Seromucoid isolated by the method of Weimer and Moshin22) was analyzed for its hexose content by a procedure similar to that described for serum protein-bound hexoses. Electrophoresis All sera were analyzed by cellulose acetate zone electrophoresis using barbital-barbituric acid buffer at ph 8.6 (ionic strength, 0.05) under a constant level of 0.8mA/cm for 60-70min. Serum sample (0.004ml/1.5cm) was applied with a micropipette on a 2.4-cm wide Oxoid strip (Oxoid Ltd., London) on the midline. After electrophoretic separation, the strip was cut into two pieces with a pair of scissors, into the upper and the lower ones. The upper one was stained for protein with Ponceau 3R Extra Pure (Tokushu Chemical Co. Ltd., Tokyo), and the lower one for glycoprotein with Schiff's reagent. The procedure for staining of glycoproteins after electrophoresis in this study was a modified PAS staining procedure by Nagase.15) These air-dried strips were made permeable to light with decahydronaphthalene and then the strips were scanned with a Yamato-Asuka model UV51 densitometer (Yamato-Asuka Industrial Co. Ltd., Tokyo) equipped with interference filters at 500 4mm). All electrophoretic analyses were carried out on sera pooled for 2 months. Determination of Daily Cycle in Seromucoid Prior to this study, an experiment was carried out in which daily (24-hr) change in seromucoid level was determined. Twenty normal adult male rats which weighed 180 to 200g and had previously been maintained for 1 week under the same environmental conditions as described above were used for this purpose. In the experiment, the rats were kept away from light between 6:00pm and 6:00am. Groups of 3 to 5 rats were sacrificed at intervals. Fasting and Re-feeding Fifty-five normal male rats of 150 to 200g in weight were used in this study. They were housed 5 to a cage and maintained on a standard solid 63(5) 593

4 Y. HAYASHI am. After being maintained for 9 days under the same environmental conditions, the rats were not fed, only tap water being given freely, and were then fed. They were sacrificed at intervals, in group of 3 to 5. RESULTS History of Rats and Tumor Growth A successful transplantation rate of 100% was obtained. The tumor grew readily in all implanted rats and no regression was recorded. The rats given AH-109A cells subcutaneously could survive as long as 20 to 24 days after transplantation. These values were close to those previously reported by Sato et al.,17) who recorded 100% for transplantability and 20 to 38 days of survival. By autopsy, neither ascites nor metastasis to the abdominal organs was detected throughout the period of observation in any individual rat given AH-109A cells. In some rats enlarged inguinal or axillary lymph node metastatic tumors were found. In such cases, tumor weight was calculated as the sum of the weight of primary and metastatic tumors. A photomicrograph of the tumor on the 22nd day is shown in Photo 1. The results of histological findings of the liver are listed in Table I, and their micrographs are shown in Photos 2-7. As can be seen from these data, only a little or no liver metastases became apparent throughout the period of the experiment, and these never progressed to the extent that the liver tissue is replaced by metastatic lesions. Jaundice was never observed in any serum. Table I. Histological Examination of the Liver in Rats Bearing Subcutaneous AH-109A * Size of tumor nodule: L, relatively large; M, moderate; S, relatively small; M, minute. Figures in parentheses represent the count of nodule/30mm2 of liver tissue section. 594 GANN

5 SERUM GLYCOPROTEIN IN RATS BEARING AH-109A Fig. 1. Growth curve for tumor and host The results of tumor growth are shown in Fig. 1. The growth curve shows three phases of growth. After inoculation, there was a period of slow growth corresponding to the establishment of the tumor graft. This was followed by a period of rapid growth starting between the 9th and 12th day. Finally as the nodes became large (by the 21st day a mass weighing 65.1g on an average was formed), there was a gradual decrease in the rate of growth. Chemical Studies The results of chemical analysis are summarized in Table II. The normal level of total serum protein was 6.33g/dl. In normal controls, the amount of protein-bound hexoses was 110.3mg/dl, hexosamines 114.9mg/dl, sialic acid 94.7mg/dl, and fucose 8.49mg/dl. Total carbohydrates in normal rats expressed as the sum of four carbohydrate species was thus 328.4mg/dl. Usually the level of seromucoid in terms of hexose content was 17.3mg/dl, and constituted 14.8% of the total serum protein-bound hexoses. A progressive decrease in total serum protein level from 6.33g/dl to some 5.50g/dl occurred in tumor-bearing rats, and the decrease was most remarkable on and after the 15th day. Significant elevation of hexoses, hexosamines, sialic acid, and seromucoid be came apparent first between the 9th and 12th day. The maximum value of hexoses was observed on the 21st, that of hexosamines on the 15th, that of sialic acid on the 21st, and that of seromucoid occurred on the 21st day. Comparison of Fig. 1 and Table II revealed that there was a close correlationship between the extent of tumor growth and the levels of these sugars. In addition it was found that the level of seromucoid was closely correlated with the extent of tumor growth. On the other hand, the concentration of fucose appeared to be relatively constant until the 15th day and decreased thereafter. 63(5) 595

6 Y. HAYASHI 596 GANN

7 SERUM GLYCOPROTEIN IN RATS BEARING AH-109A Tumor wt. Fig. 2. Seromucoid level and tumor growth The values obtained for a group sacrificed on the same day are connected with a solid line. It can readily be seen that the seromucoid manifested a continuous rise after transplantation and reached a maximum of 67.5mg/dl on the 21st day. The data calculated by subtracting seromucoid-bound hexoses from total serum protein-bound hexoses showed no significant change during tumor growth (normal level was 93mg/dl). Fig. 2 shows that, even when tumors were comparatively small in size (3 to 9% of residual body weight on the 9th post-implant day), the level of seromucoid was already found to be significantly elevated. It is of interest to review the ratios of the three carbohydrates to hexoses after transplantation. The hexosamine/hexose ratio and sialic acid/hexose ratio were somdwhat elevated on the 9th day to about 1.09 (1.132/1.042) and 1.17 (1.003/0.859) of their normal values, respectively, and thereafter they remained relatively constant, whereas the fucose/hexose ratio continued to decrease on and after the 12th day, and reached the value approximately 5/7 of the normal on the 21st day. Electrophoretic Studies The results of zone electrophoretic analysis on cellulose acetate are presented in Table III. Various electrophoretic protein fractions were expressed in terms of percent 63(5) 597

8 Y. HAYASHI Table III. Serum Protein and Glycoprotein Changes during Growth of AH-109A standard deviation b) Expressed as % of total protein c) Expressed as % of total glycoprotein d) Total protein percentage of electrophoretic fraction (g/dl) e) Total carbohydrate percentage of electrophoretic fraction (mg/dl) f) n, number of samples protein fraction; g/dl). The calculation, although it may be incorrect to a certain extent in principle because the dye-combining capacity of albumin is larger than that of globulins, would be a useful adjunct to find the relative changes of each fraction through the course of tumor growth. The electrophoretic glycoprotein fractions were expressed as percentage of total glycoprotein, and also expressed in relation to the total serum protein-bound carbohydrate (mg/dl) for the evaluation of the actual amount of carbohydrate in each fraction, under the assumption that individual 598 GANN

9 SERUM GLYCOPROTEIN IN RATS BEARING AH-109A Tumor, in the Rat could be separated only with difficulty, they were calculated simply Albumin continued to decrease markedly throughout the period of observation, whereas an elevation of became strikingly apparent by the 9th or 12th day after transplantation. It is noteworthy that an elevation relative to protein (on a basis of serum concentration; g/dl) became manifest only on the 12th to 15th day, whereas that of relative to glycoprotein (on a basis of serum concentration; mg/dl) became progressively elevated throughout the period of study. The degree of elevation of relative to glycoprotein was significantly high as compared with that relative to protein. The fluctuation of level was in sharp contrast to that Significant and progressive decrease was 63(5) 599

10 Y. HAYASHI observed throughout the entire course of the experiment and its level reached finally the level about one-half of its normal value. The ratio of the carbohydrate level (g/dl) to protein level (g/dl), is shown on the righthand side of Table III. It is evident that the elevation of the ratio for total serum is due to a marked increase in carbohydrate-rich proteins. The carbohydra ratio showed a marked deviation from the normal throughout the period of observation. It is therefore suggested that the fraction contains some carbohydrate-rich proteins, the level of which became markedly elevated during the course of the experiment. The ratios related to albumin, were not very different from the respective control values as compared with that of Daily Cycle in Seromucoid The results obtained from the observation on fluctuation of seromucoid levels (hexose content) during 24hr in normal rats are shown graphically in Fig. 3, which indicates that the level of seromucoid changed significantly in a daily cycle, being highest at midnight (19.2mg/dl) and returning gradually to the levels of daytime (15.2mg/dl). Fig. 3. Fluctuation of seromucoid levels during 24hr in normal rats Effect of Fasting and Re-feeding on Seromucoid Level The results of the effect of fasting and re-feeding on seromucoid level (hexose content) in normal rats are shown in Fig. 4. The level of seromucoid was 15.7mg/dl before fasting. The level became lower gradually in parallel with the loss of weight until the 2nd to 4th day of fasting, and remained constant on and after the 4th day (12.0 mg/dl). The loss of body weight was 14.4% on the 2nd, 22.5% on the 4th, and 36.5% on the 6th day after fasting. On the other hand, in the group which was fed after fasting, the level became rapidly elevated after re-feeding and reached the maximum level (20.0mg/dl) on the 2nd or 3rd day after re-feeding. It then became normal (16.3mg/dl) by the 4th day after refeeding, when the body weight returned to that before fasting. 600 GANN

11 SERUM GLYCOPROTEIN IN RATS BEARING AH-109A No. of days Fig. 4. Effect of fasting and re-feeding on seromucoid levels in normal rats DISCUSSION The effect of tumor growth on the levels of various carbohydrate components of serum proteins was studied on the rat bearing AH-109A which was transplanted subcutaneously. The subcutaneous transplantation possesses a number of advantages over the intraperitoneal transplantation; (1) reduction in frequency of liver and other metastasis, (2) the absence of ascites, (3) the ease with which tumor growth can be observed, (4) longer survival time sufficient to follow up the pathophysiological findings, and (5) the possibility that the cause of death can be found easily. The changes in glycoprotein as revealed in the present work therefore are purely the results of humoral effect of tumor growth. In the present work, all the animals were exsanguinated at a definite clock time of 6:00 to 7:00am. The seromucoid level was found to fluctuate remarkably in diurnal cycle, being highest at midnight and returning gradually to the level of day time. Since a progressive decrease (about 10% of initial body weight by the 21st day after transplantation) in residual body weight was noted with the tumor-bearing rats, it was of interest to examine how the level of seromucoid changed in rats which have lost weight by fasting. 63(5) 601

12 Y. HAYASHI It was thought that the level of seromucoid in the emaciated rats would serve as another control (i.e., as one control of rat losing weight), because at one point, the level of seromucoid is influenced by the loss in weight even though changes of the level were mainly due to the direct effect of fasting. It was found that fasting resulted in a decrease in seromucoid, which returned to the level before fasting by re-feeding. However, elevation of the seromucoid level observed in the tumor-bearing rat exceeded by far the range of these "physiological" fluctuations. The changes in glycoprotein in the tumorbearing rat were not the direct result of the loss in body weight. A few comments may be necessary concerning the methods of chemical analysis employed. It has been pointed out that abnormally high hexosamine values are obtained by applying the Elson-Morgan method directly to tissue proteins.3,18) According to Boas,5) however, when serum hydrolysate was the test material, its passage through a cation exchanger did not improve the results significantly. Accordingly, this step was omitted. The reliability of the data on sialic acid as obtained by the thiobarbituric acid method was tested by comparing them with the data obtained by the direct Ehrlich method.24) The course of change as revealed by the two methods was essentially the same, but the latter method was not suitable for determining the actual concentration of sialic acid, because of the development of interfering purple color. In conclusion, all of the chemical methods employed in the present work were accurate enough for determination of the carbohydrate levels. The present work revealed that change in seromucoid level in AH-109A-bearing rats was very closely in parallel with the rate of tumor growth. A similar conclusion has been reached by Baldwin et al.2) on sarcoma-66-bearing rats. Seromucoid thus has the closest relation to the rate of tumor growth, although the underlying mechanism has not yet been elucidated. As already noted by Baldwin et al.2) for sarcoma-66 tumor, the elevation of total protein-bound hexoses could be accounted for entirely by an increase in the quantity of seromucoid. Furthermore, in the present work, different carbohydrate components became elevated concurrently. These results suggest strongly that the elevation of all the carbohydrate components tested can be almost exclusively attributed to the elevation of seromucoid. The fact that fucose level is little affected by tumor growth also supports this possibility, since seromucoid is known to contain little fucose.25) is probably the major source of protein-bound fucose. Recently, Nagase et al.15) studied the carbohydrate levels and electrophoretic pattern of serum glycoproteins in rats bearing intraperitoneally transplanted AH-109A. Although the results with subcutaneous AH-109A were similar to theirs in principle, gave no well-separated subfractions. The discrepancy may be due to the fact that the sera used in the present work were stored for a long time and thawed repeatedly. Re-examination with freshly prepared sera is therefore desirable. Seromucoid is by definition a fraction of serum protein that is soluble in 0.6N HClO4 but insoluble in 1M phosphotungstic acid.26) The fraction is heterogeneous in nature and contains glycoproteins such as orosomucoid tein, hemopexin, haptoglobins, and 3.5S components.19) 602 GANN

13 SERUM GLYCOPROTEIN IN RATS BEARING AH-109A There have been some reports which suggest that the seromucoid produced in response to tumor growth has a different composition from its normal counterpart.10,14) The present author has examined the sera from normal and Yoshida sarcoma-bearing rats by DEAE-cellulose column chromatography and preliminary data seem to confirm the above observation. Prior to advanced investigations as to how serum glycoprotein levels are regulated, there is therefore an urgent need to obtain more precise informations concerning the character and level of individual glycoproteins in seromucoid fraction under pathological as well as normal conditions in experimental animals. Polyacrylamide-gel disc electrophoresis which was recently developed and, the method of immunodiffusion will be useful for this purpose, since they have been used successfully to separate, to identify, and to determine human plasma protein molecules. This work was supported by a Grant-in-Aid for Cancer Research from the Ministry of Education. The author expresses his thanks to Professor Shigeru Tsuiki, Director of the Department of Biochemistry of this Institute, for his helpful criticism and advice, to Dr. Sumi Nagase, Chief, Department of Chemistry, Sasaki Institute, Tokyo, for her valuable technical advice in connection with this study, and to Assistant Professor Maroh Suzuki, Cancer Research Laboratory of this Institute, for his kind guidance on histological examination. He also expresses sincere thanks to Professor Tatuo Saito, Director of this Department, for his help, advice, and encouragement. (Received May 19, 1972) REFERENCES 1) Aminoff, D., Biochem. J., 81, 384 (1961). 2) Baldwin, R. W., Harries, H. J., Brit. J. Cancer, 12, 99 (1958). 3) Blix, G., Acta Chem. Scand., 2, 467 (1948). 4) Bjornesjo, K. B., Scand. J. Clin. Lab. Invest., 7, 153 (1955). 5) Boas, N. F., J. Biol. Chem., 204, 553 (1953). 6) Catchpole, H. R., Proc. Soc. Exp. Biol. Med., 75, 221 (1950). 7) Dische, Z., Shettles, L. B., J. Biol. Chem., 175, 595 (1948). 8) Elson, L. A., Morgan, W. T. J., Biochem. J., 27, 1824 (1933). 9) Gyorky, G., Houck, J. C., Can. J. Biochem., 43, 1807 (1965). 10) Harshman, S., Bryant, G., Cancer Res., 24, 1626 (1964). 11) Lowry, O. H., Rosebrough, N. J., Farr, A. L., Randal, R. J., J. Biol. Chem., 193, 265 (1951). 12) Lustig, B., Langer, A., Biochem. Z., 242, 320 (1931). 13) Macbeth, R. A. L., Bekesi, J. G., Tuba, J., Cancer Res., 23, 938 (1963). 14) Macbeth, R. A. L., Bekesi, J. G., ibid., 24, 2044 (1964). 15) Nagase, S., Satoh, H., Nippon Rinsho, 28, 417 (1970). 16) Saito, T., Ohira, S., Wakui, A., Yokoyama, M., Takahashi, H., Himori, T., Kaneko, T., Kobayashi, Y., Kanamaru, R., Maeda, S., Tohoku Igaku Zasshi, 75, 198 (1967). 17) Sato, H., Satoh, H., Isaka, H., Yoshida, T., Igaku-no-Ayumi, 68, 521 (1969). 18) Schloss, B., Anal. Chem., 23, 1321 (1951). 19) Schultze, H. E., Heide, K., Haupt, H., Clin. Chim. Acta, 7, 854 (1962). 20) Shetlar, M. R., Erwin, C. P., Everett, M. R., Cancer Res., 10, 445 (1950). 21) Svennerholm, E., Svennerholm, L., Acta Chem. Scand., 12, 547 (1958). 22) Weimer, H. E., Moshin, J. R., Am. Rev. Tuberc., 68, 594 (1953). 23) Weimer, H. E., Quinn, F. A., Redlich-Moshin, J., Nishihara, H., J. Natl. Cancer Inst., 19, 409 (1957). 24) Werner, I., Odin, L., Acta Soc. Med. Upsalien., 57, 230 (1952). 25) Winzler, R. J., "The Plasma Proteins", Vol. 1, p. 309 (1960). Academic Press Inc., New York. 63(5) 603

14 Y. HAYASHI 26) Winzler, R. J., "Methods of Biochemical Analysis", Vol. 2, p. 279 (1961). Interscience Publishers Inc., New York. EXPLANATION OF PLATES LXXVI-LXXVII Photo 1. AH-109A tumor on the 22nd day. H-E. Photos 2-7. Histological pictures of the liver. H-E. Photo 2. (Table I. Rat No. 5-4) and Photo 3. (Table I. Rat No. 7-3). No liver metastases are found. Photo 4. (Table I. Rat No. 5-7). A moderate-sized nodule of tumor is seen. Photo 5. Photo 6. Photo 7. (Table I. Rat No. 5-3). A small-sized nodule of tumor is seen. (Table I. Rat No. 7-1). Two moderate-sized nodules of tumor are seen. (Table I. Rat No. 7-1). A large-sized nodule of tumor is seen. H-E=Hematoxylin and Eosin stain. 604 GANN

15 GANN, Vol. 63 PLATE LXXVI

16 GANN, Vol. 63 PLATE LXXVII