Independence of Sialic Acid Levels in Normal and Malignant Growth

[CANCER RESEARCH 35, 1520-1523, June 1975]

S. V. Khadapkar, N. A. Sheth, and S. V. Bhide

Biology Division, CancerResearchInstitute, Tata Memorial Centre, Pare!, Bombay400 012, India

SUMMARY were obtained from the Animal Colony of the Cancer Research Institute, Bombay, India. Sialic acid content in breast or tumor tissue and serum of For sequential metabolic studies, breast or tumor tissue mouse strains that are either susceptible or resistant to and serum of 4-month-old, 12-month-old, and tumor-bear breast cancer was measured at various age periods. Sialic ing virgin C3H/J mice were used. Virgin C57BL mice of acid content was .also studied in normal lung tissue and in corresponding ages were used for comparison. lung adenoma and hepatoma. Sialic acid levels during For studies on female mice under various physiological nonmalignant growth of a tissue were measured in breast conditions, 3-month-old female C3H/J and C57BL mice tissue during pregnancy and lactation, and in regenerating were mated with males of their respective strains. The day a liver, as well as in newborn and postnatal liver. The sialic vaginal plug was observed was considered Day 1. Pregnant acid content, when expressed per mg of protein, increased in mice were killed on the 15th day of pregnancy and lactating mammary tumor, lung adenoma, and hepatoma. It also mice were killed on the 12th day after delivery. Breast tissue increased in nonmalignant growth of breast tissue during and serum of all the groups were studied. pregnancy and lactation and of regenerating liver and For studies on primary hepatomas, 8-week-old male postnatal liver. Increase in sialic acid per mg DNA was Swiss mice were kept on a continuous feeding of 0.03% observed only in lung tumors, regenerating liver, and thioacetamide mixed with a normal diet. Mice developed postnatal liver. It appears that the changes in sialic acid tumors at about 17 months of age (9). Normal mice of the level are independent of the normal or malignant growth same strain, age, and sex, but fed on a stock diet, were used of a tissue and that these changes might be the function of for comparison. Sialic acid was measured in hepatoma and the parameter used to express the sialic acid values, i.e., control liver tissues. either the DNA content or protein content of a given tis For studies on primary lung adenomas, newborn Swiss sue. mice were given 1 s.c. injection of urethan (1 mg/g body weight). Treated mice developed lung adenomas at the age INTRODUCTION of about 12 to 16 months (8). Normal mice of the same age and sex were used as controls. Sialic acid was measured in The role of sialic acid in malignant cells has been widely lung tissue or tumor as well as in serum. studied, although its correlation with cancer is not fully Sialic acid levels were measured in regenerating liver established (4, 6, 22, 29, 3 1). Most data on sialic acids in the tissues. Regenerating liver was obtained from 12-week-old literature are expressed per mg of protein or per g of tissue partially hepatectomized male Swiss mice. Partial hepatec weight. However, both these parameters are themselves tomy was carried out according to the method of Higgins variable in nature. Hence it was thought that if the sialic and Anderson (10) and groups of mice were killed at acid content were expressed per mg of DNA it would intervals of 0, 24, and 72 hr and 10 days after operation. indicate sialic acid per cell and give a better insight as to Normal mice of the same age, sex, and strain were sham whether sialic acid variations are linked with cancer. This operated and killed at the corresponding intervals of time paper is an attempt to understand this relationship in mentioned above. normal and malignant growth. For studies on developing liver tissue, Swiss mice at Because of our long-standing interest in spontaneous newborn and postpartum stages were used. Newborn Swiss mammary carcinogenesis (24â€”27)and chemically induced mice were obtained within 18 hr after birth, and liver tissues hepatocarcinogenesis (2), we measured progressive changes from 4 to 6 mice were pooled. Liver tissue from postpartum in sialic acid levels in mammary carcinogenesis in C3H/i mice was collected at 7, 14, and 21 days and 8 weeks after mice and in chemically induced hepatomas and lung tumors. the litter was born. Each pooled tissue was considered as a For measurements in normal growing tissues we studied single sample; sialic acid was estimated in 4 different breast tissue of pregnant and lactating mice, as well as samples. regenerating liver and postpartum liver. For studies on tumor tissue in transplantation a chemi cally induced fibrosarcoma (generation 154) was trans MATERIALS AND METHODS planted s.c. in 5- to 6-week-old male Swiss mice. Mice were killed at various time intervals from Day 12 after the Chemicals of analytical grade were used. transplantation (when the tumor was palpable) until the Virgin C3H/J and C57BL mice and male Swiss mice 27th day. Tumor and serum were used to measure sialic acid. Animals were sacrificed by cervical dislocation and ReceivedNovember 12, 1973;acceptedMarch 10, 1975. tissue was dissected out, minced with fine scissors, and

1520 CANCER RESEARCH VOL. 35

immediately homogenized in water in a Potter-Elvehjem as the mammary tumor showed remarkably higher levels of homogenizer. An aliquot of tissue homogenate was adjusted sialic acid than did normal mammary tissue, it seemed to 0. 1 N with the addition of sulfuric acid and heated to 80Â° essential to determine whether this increase in sialic acid for 60 mm. For analysis on serum samples, blood was was merely a reflection of increased cell number. For that collected from heart puncture prior to killing and an aliquot purpose sialic acid was measured per mg of DNA.Table 2 of separated serum was diluted to I ml and hydrolyzed as shows the values of sialic acid per mg of protein and DNA mentioned above. Sialic acid was estimated in serum or in the breast tissue of pregnant and lactating mice as well as tissue hydrolyzate according to the method of Warren (30). in primary mammary tumors. It may be observed from Sialic acid content was expressed in terms of @gof Table 2 that the apparent increase in sialic acid content (as N-acetyl neuraminic acid per mg of protein or per mg of expressed per mg protein in Table 1) of proliferating normal DNA in the case of tissue and per ml of serum. The mammary gland in pregnancy and lactation or in tumors adsorption of sialic acid was measured at 549 nm and also at does not exist when the results are expressed per mg of 532 nm to eliminate the contribution of 2-deoxyribose. DNA. It is thus evident that the increase in sialic acid of Finally, the correction factor was applied for calculations as these tissues was a reflection of increase in cell number, suggested by Warren (30). Protein content was measured by while sialic acid content of individual cells does not seem to the method of Lowry et a!. (15). DNA was measured increase. spectrophotometrically by the method of Ogur and Rosen Next, a study was undertaken to compare sialic acid (18). Sialic acid was measured in the tissues or serum of content in tumors of epithelial cell origin. Table 3 shows individual mice unless specified otherwise. Each group that lung tumor and hepatoma had levels of sialic acid that consisted of at least 4 mice. Student's t test was used to were significantly higher as expressed per mg protein than assess the statistical significance. The difference between the that in corresponding normal tissue. The levels of sialic acid 2 values was considered significant only when p < 0.05. expressed per mg of DNA did not show an increase in hepatoma, while lung tumor had a significantly higher value RESULTS than its normal counterpart. There was no significant change in serum sialic acid of mice bearing the above-men Initially, sialic acid content in breast tissue of resistant tioned tumors. and susceptible strains of mice was measured at 4 and 12 Table 2 months of age and results were compared with sialic acid Sialic acid content as expressedper mg ofprotein and mg oJDNA in breast content in tumor tissue. Table 1 shows the sialic acid tissueof normal, pregnant, and lactating mice and in tumor tissueof content in breast tissue of both susceptible C3H/J and C3H/J mice resistant C57BL mice remained comparable at 4 and 12 Sialic acid is expressed in terms of ,ug of N-acetylneuraminic acid. Pregnantmicewerekilled on the 15thday after vaginalplug wasobserved. months. There was no significant difference in sialic acid Lactating mice werekilled on the 12thday of delivery. levels as the age increased, while the tumor tissue had a significantly higher level of sialic acid than did breast tissue. acid/ sialic acid/ DNA/ Subsequently, we measured the sialic acid content in proteinNormal6.6Groupsialic mg protein@g mg DNA@g mg normal physiological growth of breast tissue during preg 3Tumor24.2 Â±G.6a290 Â±2623.5 Â± nancy and lactation. Table 1 shows that sialic acid content 2bPregnant15.4 Â±2b248 Â±1996 Â± increased substantially in breast tissue of pregnant and 3'Lactating29.0 Â±0.6b340 Â±1046.2 Â± lactating mice of both susceptible and resistant strains. Â±4'290 Â±60105 Â±10' Serum sialic acid in different experimental groups did not a Mean Â± S.E. of 3 experiments. vary significantly from the corresponding control value. b Statistical significance when compared with corresponding control Since breast tissue of pregnant and lactating mice as well group.

Table 1 Sialic acidcontentin breastor tumor tissueandin serumin control, tumor-bearing,pregnant.and lactating C3H/J and C57BL mice Sialic acid is expressed in terms of @gofN-acetylneuraminic acid per mg tissue protein or per ml serum. Pregnantmice were killed on the 15thday after vaginal plug was observed.Lactating micewerekilled on the 12thday of delivery.

(mo.)TissuesC3H/JC57BLSerumC3H/JC57BLControl4Breast7.0GroupAge

71Control12Breast7.6 Â±0.6Â°6.7 Â±0.8295 Â±11266 Â± 20Tumor Â±0.48.5 Â±0.5283 Â±20285 Â± 12Tumor21.7Â±2.9'260Â±24bearingPregnant4Breast19.3

28Lactating4Breast22.5 Â±l.8b10.0 Â±l.2b368 Â±29307 Â± Â±[email protected] Â±1.0'281 Â±30258 Â±27

a Mean Â± SE. of 4 experiments.

b Statistical significance when compared with corresponding control group.

JUNE 1975 I 521

Table 3 Sialic acid content in normal and tumor tissues and in serum of mice bearing primary tumors of epithelial cell origin Sialic acid is expressed in terms of@g of N-acetylneuraminic acid per mg tissue protein. per mg tissue DNA. or per ml serum.

DNALiverLungBreastLiverLungBreastTissuesMgsialicacid/mg proteinpg sialic acid/mg

Normal Â±II 1.2 26 248Â±19SerumTumor6.2Â±0.1'11.8Â±0.4'7.3Â±0.612.6Â±1.0'7.0Â±0.621.6Â±2.9'131 124Â± 8144Â±202Â±17'290Â±

Normal Â±7.9369 Â± 7.9 Â±10 Tumor369 413 Â±30283 260 Â±24

a Mean Â±S.E. of 4 experiments. b Statistical significance when compared with corresponding normal values.

When all 3 of these primary tumors were transplanted Table 5 s.c., the lung tumor grew faster than other tumors. Sialic acid content in tumor and serum of mice bearing transplanted Liver tissues with a fast rate of growth, either normal or JIbrosarcoma induced, were studied next. Table 4 shows that in regenerat Sialic acid is expressed in terms of @igof N-acetylneuraminic acid. ing liver tissues the sialic acid per mg of DNA or protein increased significantly at 24 hr and continued to remain high until 10days after operation. The studies on developing Daysafter sialic tissue, like liver, of newborn mice and at postpartum stages trans acid/mg tumor sialic (zg sialic plantationTumorSerumDNA0369Â±9Â°128.3Â±0.364.1serum)pgproteinzg acid/mg acid/ml showed a similar pattern. Sialic acid content per mg of DNA in newborn liver was significantly higher than that in adult liver. The sialic acid content further increased in liver Â±4.4493Â±20'189.5Â±0.3e40.2Â±3.8c2712.9Â±0.6c4l.9Â±4.2c418Â±21â€• of 7-day-old mice. In 14- and 21-day-old mice the sialic acid content was still higher than that in adult liver tissue at 2 months. a Mean Â±S.E. of 4 experiments. We tried to determine whether the increase in tumor mass b Statistical significance when compared with the 0-day value. affected the sialic acid content per Se. For that purpose sialic C Statistical significance when compared with the 12-day value. acid in tumors and serum of mice bearing transplanted fibrosarcoma was studied at different intervals after trans 12 and 18 but increased on the 27th day. Serum sialic acid plantation. Table S shows that sialic acid expressed per mg increased on the 12th day after transplantation and re of DNA decreased from Days 12 to 18 and did not show mained high on the 27th day. further change on the 27th day, whereas sialic acid cx pressed per mg of protein did not vary significantly on Days DISCUSSION Table 4 Sialic acid in regeneratingliver and in newbornand postpartum liver The above data show that sialic acid content in mammary Sialic acid is expressedin terms of @sgofN-acetylneuraminicacid. tumor increases when measured per mg protein. Keenan and Morre ( I3) have also observed an increase in sialic acid 14 sialic acid/ sialic acid! DNARegenerating mg protein@ig mg per mg protein in breast tumors of rats. However, this increase of sialic acid in mammary tumor is not observed liverOhr8.8Â±0.4a131Â± when it is expressed per mg DNA (which is a closer estimate 1124hr13.7Â±0.9â€•381Â±56b72hr14.6Â±0.7'352Â± to expressing results per cell). Similarly, the apparent l4blOdays13.4 increase in sialic acid is nullified in the case of proliferating 9'Newborn Â±1.34'348 Â± normal mammary tissue during pregnancy and lactation. During pregnancy the acini and the ducts of mammary liverNewborn14.3Â±0.3c207Â±and postpartum tissue proliferate in a significant manner (7) and remain lIc7 proliferated and secretory during lactation. It is evident that 4cl4daysl2.8Â±0.4c356Â±days17.4 Â±0.7@373 Â± 8c2ldaysll.2Â±0.08c295Â± total sialic acid per gland may increase during pregnancy 4c2mo.8.8Â±0.4131Â±11 and lactation, yet with the simultaneous increase in cell number, the sialic acid level per cell, i.e., per mg DNA, remains comparable to that of the normal virginal stage. a Mean Â± SE. of 4 experiments. b Statistical significance when compared with the 0-hr value. As in mammary tissue, the sialic acid content of hepa

C Statistical significance when compared with the 2-month value. toma is higher than that of normal liver when expressed per

1522 CANCER RESEARCH VOL. 35

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1975 American Association for Cancer Research. Sialic Acid in Norma! and Malignant Growth mg of protein but does not vary significantly when expressed 8. Gin, C. P., and Bhide, S. V. Metabolic Studies on the Mechanism of per mg of DNA. This anomaly may be explained as follows. Urethan Action. V. SequentialMetabolic Changesin Urethan Treated It is probable that along with some increase in sialic acid Swiss Mice with Reference to Nucleic Acid Metabolism. Indian J. there may be a simultaneous decrease in protein content, Cancer. 7: 184â€”189,1970. 9. Gothoskar, S. V., Talwalkar, G. V., and Bhide, S. V. Tumorigenic which will be reflected in a higher ratio of sialic acid content Effect of Thioacetamide on Swiss Strain Mice. Brit. J. Cancer, per mg protein. On the other hand, with the increase in sialic 24:498-503, 1970. acid, there is a simultaneous increase in DNA content of 10. Higgins, G. M., and Anderson, R. M. Experimental Pathology of hepatoma, with the result that there is no significant Liver of the White Rat following Partial Surgical Removal. Arch. difference observed between normal liver and hepatoma. Pathol., 12: 186-190, 1930. The literature on these aspects supports this contention. I 1. Inbar, M., and Sachs,L. Structural Differencein Siteson the Surface Increase in sialic acid per mg tissue weight has been Membrane of Normal and Transformed Cells. Nature, 223: 7 10-712, reported in hepatoma (12), Walker 256 carcinoma (17), and 1969. cancer of the stomach (1) and pancreas (16). Decrease in 12. Kalant, H., Mons, W., and Guttman, M. Sialic Acid Content of Normal Rat Liver and DAB Induced Hepatomata. Can. J. Physiol. protein content per g weight of hepatoma has been observed Pharmacol.,42: 25-31, 1964. by various workers ( I 1, 28) as well as in our own laboratory 13. Keenan, T. W., and Morre, D. J. Mammary Carcinoma: Enzymic (unpublished data). Increase in DNA content per g weight Block in DisialogangliosideBiosynthesis.Science,182:935-937, 1973. of hepatoma has also been reported (21, 23); hence it is 14. Kimura, A., Nagai, Y., andTurumi, K. I. HexosamineandSialic Acid conceivable that increase in sialic acid per mg DNA might Contents in Cells. Nature, /9!: 596, 1961. not be observed. In fact, Kimura et a!. (14) did not observe 15. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. any change in sialic acid content in tumor cells when Protein Measurement with Folin Phenol Reagent. J. Biol. Chem., 193: measured on a per cell basis. 265â€”275.1951. The contention that the observed changes in sialic acid 16. Mabry, E. W., and Carubelli, R. Sialic Acid in Human Cancer. are more or less a reflection of the parameter that is used to Experientia, 28: 182-183, 1972. express the sialic acid holds true even in case of lung tumor 17. MacBeth, R. A. L., and Bekesi, J. G. Tissue Glycoproteins in Rats and in regenerating liver. We have observed that the protein Bearing Walker 256 Carcinoma. Cancer Res., 24: 614-618, 1964. 18. Munro, H. N., and Fleck, A. The Determination of Nucleic Acids. content per mg tissue in lung tumor is lower than that in Methods Biochem.Anal. 14: 113-176, 1966. normal lung but that the DNA content per mg tissue does 19. Novikoff, A. B., and Potter, V. R. BiochemicalStudieson Regenerat not vary significantly from the normal lung (unpublished ing Liver. J. Biol. Chem., 173: 223â€”232,1948. data). In regenerating liver a decrease in protein nitrogen 20. Price, J. M., and Laird, A. K. A Comparison of the Intracellular has also been observed (20), and it has also been reported Composition of Regenerating Liver and Induced Liver Tumor. Can that the DNA content per mg tissue does not increase in a cerRes.,10:650â€”658,1950. remarkable manner at 72 hr after the operation (3, 5, 19). It 21. Price, J. M., Miller, J. A., Miller, E. C., and Weber, G. M. Studies on is thus possible that in both these instances the actual the Intracellular Composition of Liver and Liver Tumors from Rats increase in sialic acid is sustained even when it is expressed Fed 4-Diemthylaminoazobenzene.CancerRes.,9: 96-102, 1949. per mg DNA or per mg protein. 22. Ray, P. K.. and Simmons, R. L. Differential Release of Sialic Acid from Normal and Malignant Cells by Vibrio cholerae Neuraminidase In conclusion the profile of sialic acid in malignant tissues or Influenza Virus Neuraminidase. Cancer Res., 33: 936-939, 1973. is variable in different types of tumors and it is not possible 23. Rutman, R. J., Cantarow, A., and Paschkis, K. E. Studies in to correlate these changes with growth, either malignant or 2-Acetylaminofluorene Carcinogenesis. Cancer Res., 14: 111- I 14, normal. 1954. 24. Sheth, N. A., and Bhide, S. V. Comparative Study of Protein REFERENCES Biosynthesis in Mice Susceptible and Resistant to Breast Cancer. J. NatI. Cancer Inst., 51: 1977â€”1979,1973. 1. Barker, S. A., Stacey, M., Tipper, D. J., and Kirkhan, J. H. 25. Sheth, N. A., Bhide, S. V., and Ranadive, K. J. Xanthine Oxidase Mucoproteins Containing Neuraminic Acid. Nature, 184: 68-69, Activity in Progressive Spontaneous Mammary Carcinogenesis. Brit. 1959. J. Cancer,22: 833â€”838,1969. 2. Bhide, S. V. Progressive Metabolic Alterations in Thioacetamide 26. Sheth, N. A., Bhide, S. V., and Ranadive, K. J. Behaviour of Adenylic Induced Hepatocarcinogenesis.Brit.J. Cancer,24: 504-509, 1970. Acid and Adenosine Deaminase in Spontaneous Mammary Car 3. Brody, S., and Balis, M. E. Mechanism of Growth. I. Interrelation cinogenesis in Mice. Indian J. Cancer, 7. 274-279, 1970. between Deoxyribonuclease and Deoxyribonucleic Acid Synthesis in 27. Sheth, N. A., Wagle, M. M., Bhide, S. V., and Ranadive, K. J. Studies Nonmalignant Growth. Cancer Res., 19: 538â€”543,1959. on Sequential Metabolic Alterations in Spontaneous Mammary 4. Bryant, M. L., Stoner, G. D., and Metzger, R. P. Protein-bound Carcinogenesis. Brit. J. Cancer, 21: 228-234, 1967. Carbohydrate Content of Normal and Tumorous Human Lung 28. Toennies, G. Proteinâ€”Chemical Aspects of Cancer. Cancer Res., 7: Tissue. Biochim. Biophys. Acta, 343@226-231, 1974. 193-229,1947. 5. Bucher, N. L. R., Scott, T. F., and Aub, J. C. Regeneration of Liver in 29. van Beek,W. P., Smets,L. A., and Emmelot, P. IncreasedSialic Acid Parabiotic Rat. Cancer Res., 11: 457-465, 1951. Density in Surface Glycoproteins of Transformed and Malignant 6. Emmelot, P. Biochemical Propertiesof Normal and Neoplastic Cell Cellsâ€”aGeneralPhenomenon?CancerRes.,33. 2913â€”2922,1973. Surfaces. A Review. European J. Cancer, 9: 319-333, 1973. 30. Warren, L. The Thiobarbituric Acid Assay of Sialic Acids. J. Biol. 7. Engel, S. Human Lactation in Milk. In: S. K. Kon, and A. T. Cowie Chem., 234: 1971-1975,1959. (eds.), â€œMilkâ€•,theMammary Gland and Its Secretion, Vol. 1, pp. 31. Weiss,L. Neuraminidase,Sialic Acids and Cell Interactions.J. NatI. 281â€”320.NewYork: Academic Press,Inc., 1961. Cancer Inst., 50: 3-19, 1973.

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S. V. Khadapkar, N. A. Sheth and S. V. Bhide

Cancer Res 1975;35:1520-1523.

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