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Differential responses of lignin derivatives between tumor and normal tissue derived cell lines; Effects on cellular adhesion and cell growth
Cell Biology International Reports, VoL 16, No. 3, 1992
249
DIFFERENTIAL RESPONSES OF LIGNIN DERIVATIVES BETWEEN TDI~DR AND NORMAL TISSUE DERIVED ('~-3.]. LINES; EFFECTS ON (P~.IIFLARADHESION AND (~-3J. ( ~ Kenji Sori-mchi Department of Microbiology, Dokkyo University School of Medicine, Mibu, Tochigi 321-02, Japan ABSTRACT When cell lines derived from tumor tissues were plated simultaneously with the lignin derivative or dextran sulfate in plastic culture dishes, the cells did not completely adhere to dishes and/or the shape of even the adhering cells changed significantly. On the other hand, using cell lines derived from normal tissues, no significant effect of the polyanions was observed. The present study revealed that the lignin derivatives demonstrated differential responses between cells derived from tumor tissues and from normal tissues, and that the lignin derivatives strongly inhibited the growth of mouse sarcoma cells (FRUKTO) and rat foetal cells (AdI2-3YI-ZI9) transformed with adeno virus type 12. IN'I~OI)UCTION
It is known that cellular transformation may be accompanied by morphological changes at the cellular level. At the molecular level, the transformation induces a change in the carbohydrate and/or in the protein content, and specific markers are produced by some transformed cells. However, we have not yet found a change common to every transformed cell. In pursuing this goal, we investigated the characteristics of insulin receptors (Sorimachi et al., 1985; 1987) and alkaline phosphatase (Sorimachi et al., 1982) among various cell lines, but could not find common changes accompanying cellular transformation. Recently, we found in separate experiments that water-solubilized lignin derivatives (EP3 and LS) (Sorimachi et al., 199Oa), and dextran sulfate (Sorimachi et al., 1991) induced multinucleation due to cell fusion in macrophages in vitro, and that they had anti-viral activity for some viruses, including DNA and RNA viruses, in vitro (Sorimachi et al., 1990b). Quite recently, we showed that these polyanions inhibited fibroblast growth (Sorimachi et al., in press). It is known that LS inhibits the growth of sarcoma cells in vivo (Murakami et al., 1978). These results led me to investigate whether or not lignin derivatives and dextran sulfate show different effects on normal cells and transformed cells. 1 ~ ~
AND I~--~-IODS
0309-16511921030249-9/$03.00/0
© 1992 Academic PressLtd
Cell Biology International Reports, Vol. 16, No. 3, 1992
250 Chemicals.
Acetyl lignin (EP3) was chromatographically purified from the water extract of the culture medium of Lentinus edodes mycellia, LEM, (Suzuki et al., 1989b) and sulfonyl lignin (LS) was prepared by ethanol precipitation from the waste liquor of the acid-sulfite pulping process of wood (Suzuki et al., 1989a). These lignin derivatives were kindly supplied by Prof. Sunao Yamazaki of Tokyo University. 3,5-diaminobenzoic acid dihydrochloride was purchased from Tokyo Kassei Co., Ltd. (Tokyo, Japan). De×tran sulfate (M.W. 8,000) was obtained from Sigma Chemical Company (St. Louis, MO, U.S.A.) Cell culture. The origins of cells are shown in Table i. Cells were cultured in Eagle's minimum essential medium supplemented with 10% fetal bovine serum. To investigate the effects of polyanions on the cell attachment, the cells were plated in the Table i. Origins of cells. Cell origin
Inducer or Transformer
Human fetal liver Human urinary bladder carcinoma " " " Monkey kidney Monkey hepatocarcinoma N-nitrosodiethylamine Rat liver Rat hepatoma N-2-fluorenyldiacetamide Mouse adrenal tumor Mouse sarcoma Hamster tumor SV40 " SV40 Rat foetus " " " " " " "
Nitrosoguanidine " Rous sarcoma virus Adeno virus type 12 Adeno virus DNA containing EIA region Polyoma virus v-H-ras cDNA SV40
Cell line
HuL-I-317 HUB-4 HUB-6 HUB-15 HUB-31 VERO NCLP-6E HEPRO R-YI21B Y-I FRUKT0 HAVITO HAVITO-A 3YI-BI-6 NG-3YI-TI5L NG-3YI-T308 SR-3YI-2 AdI2-3YI-Z19 EIA-3YI-I Py-3YI-S2 HR-3YI-2 SV-3YI-C66
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24-well culture plates in the presence or absence of polyanions, and fixed with ethanol after 10-24 hr. The inoculum size of cells (1-5) x 10 5 was determined according to the growth rate and the DNA content that was measurable by the method of Hinegardner (1971). To investigate the effects of polyanions on the cell growth, the cells were plated and cultured for 10-24 hr, and then further cultured 2 days in the presnce or absence of the poylanions. DNA contents. The DNA content of cultured cells was measured by the method of Hinegardner (1971). After the cells fixed with ethanol were treated with 0.2 ml of 3,5-diaminobenzoic acid solution (0.4 mg/ml) in H20 at 60°C for 45 min, 1.3 ml of 1 N HCI was added to the cells and the solution was processed by fluorometry. RESULTS AND DISCUSSION A human urinary bladder carcinoma cell line (HUB-15) (Kakuya et ai.,1983) showed an epithelial shape (Fig. I-A). When
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Fig. i. Phase contrast micrographs of human urinary bladder carcinoma (HUB-15) cells in the presence and absence of EP3. HUB-15 cells were inoculated simultaneously without (A) and with (B) EP3 at i00 ~g/ml in plastic culture dishes. The photos were taken after 24 hr of incubation at 37°C. The magnification was x i00.
252
Cell Biology International Reports, Vol. 16, No. 3, 1992
the cells were inoculated simultaneously with EP3, a marked cellular morphological change was observed after 24 hr at 37 ° C (Fig. l-B). In order to express quantitatively the effects of the lignin derivatives and dextran sulfate, the DNA content of cells in culture well was measured in human urinary bladder carcinoma cells (HUB-4 and HUB-15) that were plated simultaneously with the polyanion at various concentrations. In HUB-4 cells (Kakuya et al., 1983), simultaneous inoculation of the cells with EP3, LS, or dextran sulfate at 5 ~g/ml almost completely interfered with cellular adhesion to plastic culture dishes. On the other hand, in HUB-13 cells a higher concentration, i00 ~g/ml, was needed to show significant effects of EP3 and dextran-sulfate, while LS showed a significant effect at 50 ~g/ml. Thus, in order to investigate the acute effect of the lignin derivative or dextran sulfate, cells were inoculated simultaneously with the polyanion at I00 ~g/ml and the effects of the drug were investigated after 10-24 hr throughout subsequent experiments. In order to investigate the effects of the polyanions on cells derived from normal tissues, a fetal human liver cell line (HuL-I-317) was used. This cell line showed a round shape in the absence of serum, but the addition of serum to the culture medium induced cell flattening (Sorimachi et al., 1988; 1989). The inoculation of HuL-1-317 cells with the polyanion did not show either a significant morphological change, or a decrease in cellular adhesion (Fig. 2). According to these results, it was assumed that the cellular response to the polyanions might differ between cell lines derived from tumor tissues and normal tissues. Therefore, various cell lines were examined. The cell lines, derived from rat liver (HEPRO) and African green monkey kidney (VERO) (Yasumura et al., 1978) were almost independent of the polyanions for their morphology, and the decreases in cellular adhesion were less than 20% (Fig. 2). Two human urinary bladder carcinoma cell lines (HUB-6 and HUB-31) (Kakuya et al., 1983) responded to the polyanions. The cellular adhesion of HUB-31 cells decreased and the shape of HUB-6 cells changed markedly. In particular, the effects of LS was the strongest among three polyanions, and its effects on the human urinary bladder carcinoma cells were more than 80%. In addition, two cell lines, one derived from rat hepatoma (R-YI21B) (Niwa et al., 1980) by induction with N-2-fluorenyldiacetamide (Reuber, 1961) and a monkey hepatocarcinoma (NCLP-6E) (Dawe et al., 1968) induced with N-nitrosodiethylamine in vivo, also responded to the polyanions, and their cell shape changed slightly and the cellular adhesion decreased. All the other cell lines, derived from mouse adrenal tumor (Y-l) (Yasumura et al., 1968), mouse sarcoma (FRUKTO) (Yasumura and Kuwata, 1968) and hamster tumor induced with SV40 (HAVITO and its 8-azaguanine resistant variant (HAVITO-A) (Yasumura, unpublished), strongly responded to the polyanions. However, the effects of the polyanions on cellular adhesion of
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253
HAVITO-A cells were smaller than those of HAVITO cells, although both cell lines were derived from hamster tumor induced with SV40. Thus, it is likely that there are differences in the structure of plasma membrane between these two cell lines. Evidently, all cell lines here examined that were derived from tumor tissues responded to the lignin derivatives and resulted in decreases in their adhesion to the plastic surface with or without morphological changes, but these effects were not significant in the cell lines derived from normal tissues (Fig. 2). OHuL-1-317OHuL't'317 ONG-3Y1-T15L
120
ONG-3Ylo OPy'3YI"S2opy . 3Y1- $2 T308
100
........................... OHEPRO OHEPRO • VERO
OVERO ,,--I
~80 Z
HAViTO.A ~NCLP.6E 0 HUB-15 ~HUB-31 R-Y121B
0 S.R_-_3Y_1_-2_. . . . . . . . . ~HAVlTQ-A UHUB-15 jHuL.I-317 ~HEPRO gD VERO ~ R-YI21B OYl
(~)~P3~'~I11"~203Y1 B16 oSV'3YI'C66 ""SV 3Y1 " " g3Y~,-gl-8 " " .SV-3YI-CG§ U C6o (~DSR-3Y1-2 E1A-3Y1-t ONG-3Y1-T3O8 OHR-3Y1-2 ONG-3Y1T308
ONCLP-6E
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OHUB-6
o 60
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40
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ONG-3Y1T15L
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OSR-3Y1-2 ONG-3Y1-T15L HUB-6 Y1 ~,R-Y121B J~,HAYITO ""FRUKTO OHUB-31
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OAd12-3Y1-OHR.3Y1-2 Z19 OHU§-4
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LS
DS
EP3
LS
DS
20 OHAVI10 OHUB-4
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Fig. 2. Inhibitory effects of polyanions on cellular adhesion. DS, dextran sulfate. The symbols are : • , derived from normal tissues; O , derived from tumor tissues or tansformed. The cells presented on the left side were derived from normal tissues or tumor tissues, and the cells presented on the right side were transformed from 3YI-BI-6 cells. The values are the means for 48 wells. In particular, the effects of lignosulfonate largest among three polyanions.
(LS)
were
the
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Cell Biology International Reports, Vol. 16, No. 3, 1992
To investigate whether these different responses of cells to the lignin derivatives are based on differences in the transformation of cells, we used various cell lines, transformed in vitro with chemical carcinogens, viruses and oncogenes from the same original cell line (3YI-BI-6) (Kimura et al., 1975) derived from whole rat foetus. The original cell line (3YI-BI-6) was not affected by the polyanions either in their adhesion (Fig. 2) or their morphology (data not shown). EP3 showed significant inhibitory effects on the cellular adhesion of only AdI2-3Y1-ZI9 cells (Zaitsu et al., 1988) transformed with adeno virus type 12 and HR-3YI-2 cells (Shimura et al., 1990) transformed with v-Hras cDNA among 8 cell lines derived from 3YI-B1-6 cells. In addition to these two cell lines, NG-3YI-T308 and NG-3YI-T15L cells (Ohno et al., 1987) transformed with nitrosoguanidine, and EIA-3YI-I cells transformed with adeno virus DNA containing EIA region responded to LS. Adl2-3YI-ZI9 and HR-3YI-2 cells strongly responded not only to the lignin derivatives but also to dextran sulfate. Furthermore, NG-3YI-TI5L cells, and SR-3YI-2 cells (Zaitsu et al., 1988) transformed with Rous sarcoma virus also strongly responded to dextran sulfate. No significant effect of the polyanions was observed in Py-3YI-S2 cells (Zaitsu et al., 1988) transformed with polyoma virus, and SV-3Y1-C66 cells (Zaitsu et al., 1988) transformed with SV40. On the other hand, AdI2-3YI-ZI9 cells formed large aggregates in the presence of the polyanion, and the cellular adhesion extremely decreased (Fig. 2). Therefore, not all cell lines transformed in vitro responded to the drugs. As regards virus transformations, the cells derived from hamster tumor induced with SV40 (HAVITO) strongly responded to the polyanions in cellular adhesion, but the SV-3YI-C66 cell line obtained from transformation of 3YI-BI-6 cell line with SV40 did not appear to respond to the polyanions. This may be due to a difference between in vivo tumor induction and in vitro transformation. Thus, the cell lines (HuL-1-317, VERO, HEPRO and 3Yi-B1-6) derived from normal tissues did not respond to the lignin derivative or dextran sulfate, even though these cell lines cultured in vitro for long periods might already be transformed. In fact, HuL-I-317 and HEPRO cells injected into nude mice formed tumors, but VERO cells did not. To investigate the effects of the polyanions on cell growth, the polyanion at i00 >g/ml was added later to the culture medium in the cells were growing. When cells were cultured in the presence of the polyanion, the lignin derivatives and dextran sulfate inhibited almost completely the growth of Adl2-3YI-ZI9 cells. In addition, EP3 strongly inhibited the growth of FRUKTO, 3YI-B1-6, and NG-3YI-TISL cells. LS showed more than 50% of growth inhibition in VERO, HuL-I-317, R-Y121B, FRUKTO, HAVITO, 3YI-BI-6, NG-3YI-T15L, and Py-3YI-S2 cells. Furthermore, the lignin derivatives and dextran sulfate strongly inhibited the growth of the primary culture of chick embryo fibroblasts
Cell Biology International Reports, VoL 16, No. 3, 1992
255
(Sorimachi et al., in press). In general, the lignin derivatives and dextran sulfate inhibit the growth of sarcoma cells and fibroblasts. Judging from the present results, the lignin derivatives (acetyl lignin and sulfonyl lignin) clearly demonstrated differential responses between cell lines derived from tumor tissues and normal tissues (Fig. 2). These differences in the cellular response to the polyanions might reflect on different mechanisms of cellular transformation. Although, at present, the reason for t~s difference in cellular response to the lignin derivatives and dextran sulfate is not known, the elucidation of this phenomenon would be helpful for the development of anti-cancer drugs. Acknowledg~pnts. The author is indepted to Professor Yosihiro Yasumura for the facilities that were made available in his laboratory. He also gratefully acknowledges the help of Dr. Jacob Robbins of the National Institutes of Health, U.S.A. in preparing this paper, and Professor Akira Niwa of this laboratory in obtaining 3YI cells and thier sublines that were supplied from Japanese Cancer Research Resources Bank (JCRB). References. Dawe, C.J., Whang-Peng, J., Morgan, W.D., O'Gara, R.W. and Kelly, M.G. (1968). Culture of a cell line (NCLP-6) derived from a hepatocarcinoma induced in Macaca mulatta by n-nitrosodiethylamine. J. Natl. Cancer Inst., 40, 1167-1193. Hinegardner, R.T. (1971). An improved fluorometric assay for DNA. Anal. Biochem., 39, 197-201. Kakuya, T., Yamada, T., Yokokawa, M. and Ueda, T. (1983). Establishment of cell strains from human urothelial carcinoma and their morphological characterization. In Vitro, 19, 591-599. Kimura, G., Itagaki, A. and Summers, J. (1975). Rat cell line 3YI and its virogenic polyoma- and SV40-transformed derivatives. Int. J. Cancer, 15, 694-706. Murakami, H., Takeuchi, Y. and Kudo, M. (1978). Antitumor substance consisting of purified lignosulfonate. In Kokai Tokkyo Koho (in Japanese), the Japan Patent Bureau, p. 61. Niwa, A., Yamamoto, K., Sorimachi, K. and Yasumura, Y. (1980). Continuous culture of Reuber hepatoma cells in serum-free, arginine-, glutamine- and tyrosine-deprived chemically defined medium. In Vitro, 16, 987-993.
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Ohno, K., Zaitsu, H. and Kimura, G. (1987). Maintenance of postconfluence stationary cell density by transient increase and decrease in cell number upon medium renewals in rat 3YI fibroblasts: Diminution of the decrease in the cell number after cell transformation by N-methyl-N'-nitro-Nnitrosoguanidine. Fukuoka Acta Medica, 78, 569-577. Reuber, M.D. (1961). A transplantable bile-secreting hepatocellular carcinoma in the rat. J. Natl. Cancer Inst., 26, 891-897. Shimura, H., Mitsudomi, T., Mastuzaki, A., Kabemura, H., Okuda, A. and Kimura, G. (1990). Transformation by v-H-ras does not restore proliferation of a set of temperature-sensitive cell cycle mutants of rat 3YI fibroblasts. Cell Struct. Funct., 15, 211-219. Shimura, H., Ohtsu, M., Matsuzaki, A., Mitsudomi, A., Onodera, K. and Kimura, G. (1988). Selective cytotoxicity of phospholipids and diacylglycerols to rat 3Y1 fibroblasts transformed by adenovirus type 12 or its EIA gene. Cancer Rec., 48, 578-583. Sorimachi, K., Akimoto, K., Yamazaki, S., Niwa, A., Toda, S. and Yasumura, Y. (1990a). Multinucleation of macrophages with water-solubilized lignin derivatives in vitro. Cell Struct. Funct., 15, 317-322. Sorimachi, K., Akimoto, K., Yamazaki, S., Toda, S., Niwa, A. and Yasumura, Y. (1991). Multinucleation of macrophages with dextran sulfate in vitro. Agric. Biol. Chem., 55, 241-242. Sorimachi, K., Hayashi, T., Takaoka, T., Niwa, A. and Yasumura, Y. (1988). Requirement of serum pretreatment for induction of alkaline phosphatase activity with prednisolone, butyrate, dibutyryl cyclic adenosine monophosphate and NaCI in human liver cells continuously cultured in serum-free medium. Cell Struct. Funct., 13, i-ii. Sorimachi, K., leiri, T., Kobayashi, M., Horiuchi, R. and Yasumura, Y. (1989). Inhibitory effect of dibutyryl cyclic adenosine monophosphate on the induction of alkaline phosphatase in human fetal liver cell line. Biochim. Biophys. Acta, 1010, 72-77. Sorimachi, K., Kakuya, T., Niwa, A., Yamada, T. and.Yasumura, Y. (1982). Characterization of insulin receptors in various cultured cells derived from normal or malignant tissues. Dokkyo J. Med. Sci., 9, 21-27. Sorimachi, K., Niwa, A., Yamazaki, S., Toda, S. and Yasumura, Y. (1990b). Anti-viral activity of water-solubilized lignin derivatives in vitro. Agric. Biol. Chem., 54, 1337-1339. Sorimachi, K., Niwa, A., and Yasumura, Y. (1987). Induction of alkaline phosphatase activity by dibutryrl cyclic adenosine monophosphate, prednisolone, butyrate and sodium chloride in various cell lines and the partial charaterization of the enzyme. Dokkyo J. Med. Sci., 14, 27-33.
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Sorimachi, K., Watanabe, K., Yamazaki, S. and Yasumura, Y. Inhibition of fibroblast growth by polyanions: Effects of dextran sulfate and lignin derivatives. Cell Biol. Int. Repts., in press. Sorimachi, K., Yasumura, Y. and Kakuya, T. (1985). Characterization of alkaline phosphatase in human urinary bladder carcinoma cell lines and enzyme regulation with prednisolone or sodium chloride. Jpn. J. Exp. Med., 55, 185-192. Suzuki, H., Iiyama, K., Okubo, A., Yamazaki, S. and Toda, S. (1989a). Lignosulfonate from waste liquor of pulping process activates murine macrophages and causes proliferation of bone marrow cells. Agric. Biol. Chem., 53, 1197-1199. Suzuki, H., Okubo, A., Yamazaki, S., Suzuki, K., Mitsuya, H. and Toda, S. (1989b). Inhibition of the infectivity and cytopathic effect of human immunodeficiency virus by watersoluble lignin in an extract of the culture medium of Lentinus edodes mycelia (LEM). Biochem. Biophys. Res. Commun., 160. 367-373. Yasumura, Y., Buonassisi, V. and Sato, G. (1966). Clonal analysis of differentiated function in animal cell cultures. I. Possible correlated maintenance of differentiated function and the diploid karyotype. Cancer Res., 26 (Part I), 529-535. Yasumura, Y. and Kuwata, T. (1959). Kontinua kulturado de celoj de musa furktoza sarkomo (Takizawa) in vitro (in Esperanto). Gann, 50 (suppl.), 182. Yasumura, Y., Niwa, A. and Yamamoto, K. (1978). Phenotypic requirement for glutamine of kidney cells and for glutamine and arginine of liver cells in culture, in Nutritional Requirements of Cultured Cells, Katsuta, H. (ed), pp. 225257, Japan Scientific Societies Press, Tokyo and University Park ~ress, Baltimore. Zaitsu, H., Tanaka, H., Mitsudomi, T., Matsuzaki, A., Ohtsu, M. and Kimura, G. (1988). Differences in proliferation properties among sublines of rat 3YI fibroblasts transformed by various agents in vitro. Biomed. Res., 9, 181-197. Paper received 20.11.91.
Revised paper accepted 25.01.92.
249
DIFFERENTIAL RESPONSES OF LIGNIN DERIVATIVES BETWEEN TDI~DR AND NORMAL TISSUE DERIVED ('~-3.]. LINES; EFFECTS ON (P~.IIFLARADHESION AND (~-3J. ( ~ Kenji Sori-mchi Department of Microbiology, Dokkyo University School of Medicine, Mibu, Tochigi 321-02, Japan ABSTRACT When cell lines derived from tumor tissues were plated simultaneously with the lignin derivative or dextran sulfate in plastic culture dishes, the cells did not completely adhere to dishes and/or the shape of even the adhering cells changed significantly. On the other hand, using cell lines derived from normal tissues, no significant effect of the polyanions was observed. The present study revealed that the lignin derivatives demonstrated differential responses between cells derived from tumor tissues and from normal tissues, and that the lignin derivatives strongly inhibited the growth of mouse sarcoma cells (FRUKTO) and rat foetal cells (AdI2-3YI-ZI9) transformed with adeno virus type 12. IN'I~OI)UCTION
It is known that cellular transformation may be accompanied by morphological changes at the cellular level. At the molecular level, the transformation induces a change in the carbohydrate and/or in the protein content, and specific markers are produced by some transformed cells. However, we have not yet found a change common to every transformed cell. In pursuing this goal, we investigated the characteristics of insulin receptors (Sorimachi et al., 1985; 1987) and alkaline phosphatase (Sorimachi et al., 1982) among various cell lines, but could not find common changes accompanying cellular transformation. Recently, we found in separate experiments that water-solubilized lignin derivatives (EP3 and LS) (Sorimachi et al., 199Oa), and dextran sulfate (Sorimachi et al., 1991) induced multinucleation due to cell fusion in macrophages in vitro, and that they had anti-viral activity for some viruses, including DNA and RNA viruses, in vitro (Sorimachi et al., 1990b). Quite recently, we showed that these polyanions inhibited fibroblast growth (Sorimachi et al., in press). It is known that LS inhibits the growth of sarcoma cells in vivo (Murakami et al., 1978). These results led me to investigate whether or not lignin derivatives and dextran sulfate show different effects on normal cells and transformed cells. 1 ~ ~
AND I~--~-IODS
0309-16511921030249-9/$03.00/0
© 1992 Academic PressLtd
Cell Biology International Reports, Vol. 16, No. 3, 1992
250 Chemicals.
Acetyl lignin (EP3) was chromatographically purified from the water extract of the culture medium of Lentinus edodes mycellia, LEM, (Suzuki et al., 1989b) and sulfonyl lignin (LS) was prepared by ethanol precipitation from the waste liquor of the acid-sulfite pulping process of wood (Suzuki et al., 1989a). These lignin derivatives were kindly supplied by Prof. Sunao Yamazaki of Tokyo University. 3,5-diaminobenzoic acid dihydrochloride was purchased from Tokyo Kassei Co., Ltd. (Tokyo, Japan). De×tran sulfate (M.W. 8,000) was obtained from Sigma Chemical Company (St. Louis, MO, U.S.A.) Cell culture. The origins of cells are shown in Table i. Cells were cultured in Eagle's minimum essential medium supplemented with 10% fetal bovine serum. To investigate the effects of polyanions on the cell attachment, the cells were plated in the Table i. Origins of cells. Cell origin
Inducer or Transformer
Human fetal liver Human urinary bladder carcinoma " " " Monkey kidney Monkey hepatocarcinoma N-nitrosodiethylamine Rat liver Rat hepatoma N-2-fluorenyldiacetamide Mouse adrenal tumor Mouse sarcoma Hamster tumor SV40 " SV40 Rat foetus " " " " " " "
Nitrosoguanidine " Rous sarcoma virus Adeno virus type 12 Adeno virus DNA containing EIA region Polyoma virus v-H-ras cDNA SV40
Cell line
HuL-I-317 HUB-4 HUB-6 HUB-15 HUB-31 VERO NCLP-6E HEPRO R-YI21B Y-I FRUKT0 HAVITO HAVITO-A 3YI-BI-6 NG-3YI-TI5L NG-3YI-T308 SR-3YI-2 AdI2-3YI-Z19 EIA-3YI-I Py-3YI-S2 HR-3YI-2 SV-3YI-C66
Cell Biology International Reports, Vol. 16, No. 3, 1992
251
24-well culture plates in the presence or absence of polyanions, and fixed with ethanol after 10-24 hr. The inoculum size of cells (1-5) x 10 5 was determined according to the growth rate and the DNA content that was measurable by the method of Hinegardner (1971). To investigate the effects of polyanions on the cell growth, the cells were plated and cultured for 10-24 hr, and then further cultured 2 days in the presnce or absence of the poylanions. DNA contents. The DNA content of cultured cells was measured by the method of Hinegardner (1971). After the cells fixed with ethanol were treated with 0.2 ml of 3,5-diaminobenzoic acid solution (0.4 mg/ml) in H20 at 60°C for 45 min, 1.3 ml of 1 N HCI was added to the cells and the solution was processed by fluorometry. RESULTS AND DISCUSSION A human urinary bladder carcinoma cell line (HUB-15) (Kakuya et ai.,1983) showed an epithelial shape (Fig. I-A). When
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-
~
0
0
0
~.;
~
.
•
9
0
---.
~,4 ....
Fig. i. Phase contrast micrographs of human urinary bladder carcinoma (HUB-15) cells in the presence and absence of EP3. HUB-15 cells were inoculated simultaneously without (A) and with (B) EP3 at i00 ~g/ml in plastic culture dishes. The photos were taken after 24 hr of incubation at 37°C. The magnification was x i00.
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Cell Biology International Reports, Vol. 16, No. 3, 1992
the cells were inoculated simultaneously with EP3, a marked cellular morphological change was observed after 24 hr at 37 ° C (Fig. l-B). In order to express quantitatively the effects of the lignin derivatives and dextran sulfate, the DNA content of cells in culture well was measured in human urinary bladder carcinoma cells (HUB-4 and HUB-15) that were plated simultaneously with the polyanion at various concentrations. In HUB-4 cells (Kakuya et al., 1983), simultaneous inoculation of the cells with EP3, LS, or dextran sulfate at 5 ~g/ml almost completely interfered with cellular adhesion to plastic culture dishes. On the other hand, in HUB-13 cells a higher concentration, i00 ~g/ml, was needed to show significant effects of EP3 and dextran-sulfate, while LS showed a significant effect at 50 ~g/ml. Thus, in order to investigate the acute effect of the lignin derivative or dextran sulfate, cells were inoculated simultaneously with the polyanion at I00 ~g/ml and the effects of the drug were investigated after 10-24 hr throughout subsequent experiments. In order to investigate the effects of the polyanions on cells derived from normal tissues, a fetal human liver cell line (HuL-I-317) was used. This cell line showed a round shape in the absence of serum, but the addition of serum to the culture medium induced cell flattening (Sorimachi et al., 1988; 1989). The inoculation of HuL-1-317 cells with the polyanion did not show either a significant morphological change, or a decrease in cellular adhesion (Fig. 2). According to these results, it was assumed that the cellular response to the polyanions might differ between cell lines derived from tumor tissues and normal tissues. Therefore, various cell lines were examined. The cell lines, derived from rat liver (HEPRO) and African green monkey kidney (VERO) (Yasumura et al., 1978) were almost independent of the polyanions for their morphology, and the decreases in cellular adhesion were less than 20% (Fig. 2). Two human urinary bladder carcinoma cell lines (HUB-6 and HUB-31) (Kakuya et al., 1983) responded to the polyanions. The cellular adhesion of HUB-31 cells decreased and the shape of HUB-6 cells changed markedly. In particular, the effects of LS was the strongest among three polyanions, and its effects on the human urinary bladder carcinoma cells were more than 80%. In addition, two cell lines, one derived from rat hepatoma (R-YI21B) (Niwa et al., 1980) by induction with N-2-fluorenyldiacetamide (Reuber, 1961) and a monkey hepatocarcinoma (NCLP-6E) (Dawe et al., 1968) induced with N-nitrosodiethylamine in vivo, also responded to the polyanions, and their cell shape changed slightly and the cellular adhesion decreased. All the other cell lines, derived from mouse adrenal tumor (Y-l) (Yasumura et al., 1968), mouse sarcoma (FRUKTO) (Yasumura and Kuwata, 1968) and hamster tumor induced with SV40 (HAVITO and its 8-azaguanine resistant variant (HAVITO-A) (Yasumura, unpublished), strongly responded to the polyanions. However, the effects of the polyanions on cellular adhesion of
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HAVITO-A cells were smaller than those of HAVITO cells, although both cell lines were derived from hamster tumor induced with SV40. Thus, it is likely that there are differences in the structure of plasma membrane between these two cell lines. Evidently, all cell lines here examined that were derived from tumor tissues responded to the lignin derivatives and resulted in decreases in their adhesion to the plastic surface with or without morphological changes, but these effects were not significant in the cell lines derived from normal tissues (Fig. 2). OHuL-1-317OHuL't'317 ONG-3Y1-T15L
120
ONG-3Ylo OPy'3YI"S2opy . 3Y1- $2 T308
100
........................... OHEPRO OHEPRO • VERO
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HAViTO.A ~NCLP.6E 0 HUB-15 ~HUB-31 R-Y121B
0 S.R_-_3Y_1_-2_. . . . . . . . . ~HAVlTQ-A UHUB-15 jHuL.I-317 ~HEPRO gD VERO ~ R-YI21B OYl
(~)~P3~'~I11"~203Y1 B16 oSV'3YI'C66 ""SV 3Y1 " " g3Y~,-gl-8 " " .SV-3YI-CG§ U C6o (~DSR-3Y1-2 E1A-3Y1-t ONG-3Y1-T3O8 OHR-3Y1-2 ONG-3Y1T308
ONCLP-6E
I,J,...
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o 60
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OHAVITO
OE1A.3Yl-1
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40
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0 E_I_A"...3_Y_1-1_.
ONG-3Y1T15L
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OSR-3Y1-2 ONG-3Y1-T15L HUB-6 Y1 ~,R-Y121B J~,HAYITO ""FRUKTO OHUB-31
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!
1
1
t
!
t
EP3
LS
DS
EP3
LS
DS
20 OHAVI10 OHUB-4
0
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Fig. 2. Inhibitory effects of polyanions on cellular adhesion. DS, dextran sulfate. The symbols are : • , derived from normal tissues; O , derived from tumor tissues or tansformed. The cells presented on the left side were derived from normal tissues or tumor tissues, and the cells presented on the right side were transformed from 3YI-BI-6 cells. The values are the means for 48 wells. In particular, the effects of lignosulfonate largest among three polyanions.
(LS)
were
the
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To investigate whether these different responses of cells to the lignin derivatives are based on differences in the transformation of cells, we used various cell lines, transformed in vitro with chemical carcinogens, viruses and oncogenes from the same original cell line (3YI-BI-6) (Kimura et al., 1975) derived from whole rat foetus. The original cell line (3YI-BI-6) was not affected by the polyanions either in their adhesion (Fig. 2) or their morphology (data not shown). EP3 showed significant inhibitory effects on the cellular adhesion of only AdI2-3Y1-ZI9 cells (Zaitsu et al., 1988) transformed with adeno virus type 12 and HR-3YI-2 cells (Shimura et al., 1990) transformed with v-Hras cDNA among 8 cell lines derived from 3YI-B1-6 cells. In addition to these two cell lines, NG-3YI-T308 and NG-3YI-T15L cells (Ohno et al., 1987) transformed with nitrosoguanidine, and EIA-3YI-I cells transformed with adeno virus DNA containing EIA region responded to LS. Adl2-3YI-ZI9 and HR-3YI-2 cells strongly responded not only to the lignin derivatives but also to dextran sulfate. Furthermore, NG-3YI-TI5L cells, and SR-3YI-2 cells (Zaitsu et al., 1988) transformed with Rous sarcoma virus also strongly responded to dextran sulfate. No significant effect of the polyanions was observed in Py-3YI-S2 cells (Zaitsu et al., 1988) transformed with polyoma virus, and SV-3Y1-C66 cells (Zaitsu et al., 1988) transformed with SV40. On the other hand, AdI2-3YI-ZI9 cells formed large aggregates in the presence of the polyanion, and the cellular adhesion extremely decreased (Fig. 2). Therefore, not all cell lines transformed in vitro responded to the drugs. As regards virus transformations, the cells derived from hamster tumor induced with SV40 (HAVITO) strongly responded to the polyanions in cellular adhesion, but the SV-3YI-C66 cell line obtained from transformation of 3YI-BI-6 cell line with SV40 did not appear to respond to the polyanions. This may be due to a difference between in vivo tumor induction and in vitro transformation. Thus, the cell lines (HuL-1-317, VERO, HEPRO and 3Yi-B1-6) derived from normal tissues did not respond to the lignin derivative or dextran sulfate, even though these cell lines cultured in vitro for long periods might already be transformed. In fact, HuL-I-317 and HEPRO cells injected into nude mice formed tumors, but VERO cells did not. To investigate the effects of the polyanions on cell growth, the polyanion at i00 >g/ml was added later to the culture medium in the cells were growing. When cells were cultured in the presence of the polyanion, the lignin derivatives and dextran sulfate inhibited almost completely the growth of Adl2-3YI-ZI9 cells. In addition, EP3 strongly inhibited the growth of FRUKTO, 3YI-B1-6, and NG-3YI-TISL cells. LS showed more than 50% of growth inhibition in VERO, HuL-I-317, R-Y121B, FRUKTO, HAVITO, 3YI-BI-6, NG-3YI-T15L, and Py-3YI-S2 cells. Furthermore, the lignin derivatives and dextran sulfate strongly inhibited the growth of the primary culture of chick embryo fibroblasts
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(Sorimachi et al., in press). In general, the lignin derivatives and dextran sulfate inhibit the growth of sarcoma cells and fibroblasts. Judging from the present results, the lignin derivatives (acetyl lignin and sulfonyl lignin) clearly demonstrated differential responses between cell lines derived from tumor tissues and normal tissues (Fig. 2). These differences in the cellular response to the polyanions might reflect on different mechanisms of cellular transformation. Although, at present, the reason for t~s difference in cellular response to the lignin derivatives and dextran sulfate is not known, the elucidation of this phenomenon would be helpful for the development of anti-cancer drugs. Acknowledg~pnts. The author is indepted to Professor Yosihiro Yasumura for the facilities that were made available in his laboratory. He also gratefully acknowledges the help of Dr. Jacob Robbins of the National Institutes of Health, U.S.A. in preparing this paper, and Professor Akira Niwa of this laboratory in obtaining 3YI cells and thier sublines that were supplied from Japanese Cancer Research Resources Bank (JCRB). References. Dawe, C.J., Whang-Peng, J., Morgan, W.D., O'Gara, R.W. and Kelly, M.G. (1968). Culture of a cell line (NCLP-6) derived from a hepatocarcinoma induced in Macaca mulatta by n-nitrosodiethylamine. J. Natl. Cancer Inst., 40, 1167-1193. Hinegardner, R.T. (1971). An improved fluorometric assay for DNA. Anal. Biochem., 39, 197-201. Kakuya, T., Yamada, T., Yokokawa, M. and Ueda, T. (1983). Establishment of cell strains from human urothelial carcinoma and their morphological characterization. In Vitro, 19, 591-599. Kimura, G., Itagaki, A. and Summers, J. (1975). Rat cell line 3YI and its virogenic polyoma- and SV40-transformed derivatives. Int. J. Cancer, 15, 694-706. Murakami, H., Takeuchi, Y. and Kudo, M. (1978). Antitumor substance consisting of purified lignosulfonate. In Kokai Tokkyo Koho (in Japanese), the Japan Patent Bureau, p. 61. Niwa, A., Yamamoto, K., Sorimachi, K. and Yasumura, Y. (1980). Continuous culture of Reuber hepatoma cells in serum-free, arginine-, glutamine- and tyrosine-deprived chemically defined medium. In Vitro, 16, 987-993.
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Ohno, K., Zaitsu, H. and Kimura, G. (1987). Maintenance of postconfluence stationary cell density by transient increase and decrease in cell number upon medium renewals in rat 3YI fibroblasts: Diminution of the decrease in the cell number after cell transformation by N-methyl-N'-nitro-Nnitrosoguanidine. Fukuoka Acta Medica, 78, 569-577. Reuber, M.D. (1961). A transplantable bile-secreting hepatocellular carcinoma in the rat. J. Natl. Cancer Inst., 26, 891-897. Shimura, H., Mitsudomi, T., Mastuzaki, A., Kabemura, H., Okuda, A. and Kimura, G. (1990). Transformation by v-H-ras does not restore proliferation of a set of temperature-sensitive cell cycle mutants of rat 3YI fibroblasts. Cell Struct. Funct., 15, 211-219. Shimura, H., Ohtsu, M., Matsuzaki, A., Mitsudomi, A., Onodera, K. and Kimura, G. (1988). Selective cytotoxicity of phospholipids and diacylglycerols to rat 3Y1 fibroblasts transformed by adenovirus type 12 or its EIA gene. Cancer Rec., 48, 578-583. Sorimachi, K., Akimoto, K., Yamazaki, S., Niwa, A., Toda, S. and Yasumura, Y. (1990a). Multinucleation of macrophages with water-solubilized lignin derivatives in vitro. Cell Struct. Funct., 15, 317-322. Sorimachi, K., Akimoto, K., Yamazaki, S., Toda, S., Niwa, A. and Yasumura, Y. (1991). Multinucleation of macrophages with dextran sulfate in vitro. Agric. Biol. Chem., 55, 241-242. Sorimachi, K., Hayashi, T., Takaoka, T., Niwa, A. and Yasumura, Y. (1988). Requirement of serum pretreatment for induction of alkaline phosphatase activity with prednisolone, butyrate, dibutyryl cyclic adenosine monophosphate and NaCI in human liver cells continuously cultured in serum-free medium. Cell Struct. Funct., 13, i-ii. Sorimachi, K., leiri, T., Kobayashi, M., Horiuchi, R. and Yasumura, Y. (1989). Inhibitory effect of dibutyryl cyclic adenosine monophosphate on the induction of alkaline phosphatase in human fetal liver cell line. Biochim. Biophys. Acta, 1010, 72-77. Sorimachi, K., Kakuya, T., Niwa, A., Yamada, T. and.Yasumura, Y. (1982). Characterization of insulin receptors in various cultured cells derived from normal or malignant tissues. Dokkyo J. Med. Sci., 9, 21-27. Sorimachi, K., Niwa, A., Yamazaki, S., Toda, S. and Yasumura, Y. (1990b). Anti-viral activity of water-solubilized lignin derivatives in vitro. Agric. Biol. Chem., 54, 1337-1339. Sorimachi, K., Niwa, A., and Yasumura, Y. (1987). Induction of alkaline phosphatase activity by dibutryrl cyclic adenosine monophosphate, prednisolone, butyrate and sodium chloride in various cell lines and the partial charaterization of the enzyme. Dokkyo J. Med. Sci., 14, 27-33.
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Sorimachi, K., Watanabe, K., Yamazaki, S. and Yasumura, Y. Inhibition of fibroblast growth by polyanions: Effects of dextran sulfate and lignin derivatives. Cell Biol. Int. Repts., in press. Sorimachi, K., Yasumura, Y. and Kakuya, T. (1985). Characterization of alkaline phosphatase in human urinary bladder carcinoma cell lines and enzyme regulation with prednisolone or sodium chloride. Jpn. J. Exp. Med., 55, 185-192. Suzuki, H., Iiyama, K., Okubo, A., Yamazaki, S. and Toda, S. (1989a). Lignosulfonate from waste liquor of pulping process activates murine macrophages and causes proliferation of bone marrow cells. Agric. Biol. Chem., 53, 1197-1199. Suzuki, H., Okubo, A., Yamazaki, S., Suzuki, K., Mitsuya, H. and Toda, S. (1989b). Inhibition of the infectivity and cytopathic effect of human immunodeficiency virus by watersoluble lignin in an extract of the culture medium of Lentinus edodes mycelia (LEM). Biochem. Biophys. Res. Commun., 160. 367-373. Yasumura, Y., Buonassisi, V. and Sato, G. (1966). Clonal analysis of differentiated function in animal cell cultures. I. Possible correlated maintenance of differentiated function and the diploid karyotype. Cancer Res., 26 (Part I), 529-535. Yasumura, Y. and Kuwata, T. (1959). Kontinua kulturado de celoj de musa furktoza sarkomo (Takizawa) in vitro (in Esperanto). Gann, 50 (suppl.), 182. Yasumura, Y., Niwa, A. and Yamamoto, K. (1978). Phenotypic requirement for glutamine of kidney cells and for glutamine and arginine of liver cells in culture, in Nutritional Requirements of Cultured Cells, Katsuta, H. (ed), pp. 225257, Japan Scientific Societies Press, Tokyo and University Park ~ress, Baltimore. Zaitsu, H., Tanaka, H., Mitsudomi, T., Matsuzaki, A., Ohtsu, M. and Kimura, G. (1988). Differences in proliferation properties among sublines of rat 3YI fibroblasts transformed by various agents in vitro. Biomed. Res., 9, 181-197. Paper received 20.11.91.
Revised paper accepted 25.01.92.