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laminin coated surface
Colloids and Surfaces B: Biointerfaces 19 (2000) 55 – 59 www.elsevier.nl/locate/colsurfb
Effects of colchicine and cytochalasin D on the adhesion properties of the HCC onto the collagen IV/laminin coated surface Kai-Feng Shao, Ze-Zhi Wu, Bo-Chu Wang *, Mian Long, Shao-Xi Cai Bioengineering College, Chongqing Uni6ersity, Chongqing, 400044, PR China Received 11 November 1999; accepted 6 December 1999
Abstract The adhesion properties of both hepatocytes and hepatocellular carcinoma (HCC) cells onto the Coll IV/Laminin coated surface were measured by means of micropipette aspiration technique. Further cytoskeletal agents of colchicine and cytochalasin D were tested for their effects on adherence to Coll IV/Laminin coated dishes. The results showed: cytochalasin D, which inhibits microfilament polymerization, had great inhibitory effect on adherence of both kinds cells to Coll IV/Laminin substratum (about 70 – 90%). At the same time, HCCs extension obviously decreased and focal contacts almost vanished after being treated with cytochalasin D, in contrast, there was no significant effect on the formation of extention of hepatocytes. Colchicine, which inhibits microtubular polymerization, had different effects on both cells: Compared with untreated groups, the adhesion forces of HCC cells decreased and those of hepatocytes increased. These data suggested that, in these tumor cells, microfilaments are crucial for adherence, and abnormal cytoskeletons of tumor cells may be basis of their abnormal adhesion properties. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Hepatocellular carcinoma; Adhesion; Colchicine; Cytochalasin D
1. Introduction Tumor invasion is clearly an active process undergoing three stages: attachment, migration and metastasis [1]. In facts, tumor invasion could be considered a dynamic adhesive process, which contained much complex and subtle physiological and biochemical interactions between the tumor cells and extracellular matrix. * Corresponding author. E-mail address: [email protected] (B.-C. Wang).
In the past several years, studies [1,2] were merely focused on the effects of matrix macromolecules such as laminin, fibronectin and collagen IV on the adhesion properties of tumor cells. Little, however, is known about what role cytoskeletal system play in the process of tumor cells attachment to the extracellular matrix. The association of cytoskeletal system with certain membrane proteins and extracellular matrix points to the wide variety of cell functions that are possibly modulated via the cytoskeletal system [3]. So understanding the adhesion properties of
0927-7765/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 7 - 7 7 6 5 ( 9 9 ) 0 0 1 7 0 - 8
56
K.-F. Shao et al. / Colloids and Surfaces B: Biointerfaces 19 (2000) 55–59
tumor cell would be a key to investigate tumor invasion at cell level. In this article, employing the hepatocellular carcinoma cells as a model, we have attempted to utilize the cytoskeletal agents, colchicine and cytochalasin D, as tools in order to assess the relative contribution of microtubules and microfilaments to components of the invasion process.
2. Materials and methods
2.1. Cell lines Hepatocellular carcinoma (HCCs) cells, named SMMC-7721, were purchased from the second Military Medical University. Human hepatocytes (HHCs) were derived from primary liver which obtained from patient because of accidental death, they were isolated by 0.05% collagenase separated by speed gradient centrifugal method [4], and determined by trypan blue dye exclusion test. Both of cells were routinely cultured in our lab. RPMI-1640 Supplemented with 10% fetal bovine serum, glutamine, penicillin (10 unit%), and streptomysin (10 mg/ml).
2.2. Cytoskeletal agents Colchicine and cytochalasin D were purchased from Sigma chemical Co. (St. Louis, Missouri, USA). Colchicine was diluted to the stock concentrations of 1, 15, 30 and 60 mg/ml in RPMI1640 medium. And, cytochalasin D was dissolved in dimethyl sulfoxide (DMSO) and diluted to the concentrations of 0.025, 0.05, 2.5 and 5 mg/ml in RPMI1640 medium.
2.3. Preparation of substrate Collagen IV was purchased from Sigma Company dissolved in 0.1 M acetic acid and diluted to a stock concentration of 400 mg/ml in PBS solution. Laminin was purchased with the stock concentration of 500 mg/ml from Sigma Company. The final concentrations of substrate were adjusted to 2 mg/ml of collagen IV and 1.25 mg/ml of laminin. Poly-D-lysine (PDL) was purchased from
Sigma company, diluted to a concentration of 2 mg/ml. Bovine serum albumin (BSA) was purchased from Hua-Mei company, diluted to a concentration of 0.5% in PBS (pH 7.4) Solution.
2.4. Substrate-coating technique The artificial basement membrane was made by coating glass surface with PDL, collagen IV, laminin and BSA. At first, 200 ml of PDL (2 mg/ml) was coated in the marked circle area of chamber to help the collagen IV adhesion to the glass floor. After incubation for 30 min at 37°C, the PDL solution was removed from chamber, PBS solution washed chamber twice. Then, 200 ml of the solution containing 2 mg/ml of collagen IV and 1.25 mg/ml of laminin were coated onto the same area of the chamber. After incubation for 40 min at 37°C, the rest solution were removed, PBS solution washed in same manner. After that, 200 ml of 0.5% BSA solution was coated onto the same area to block non-specific binding of cells to the plastic [5], and washed twice.
2.5. Attachment assay The attachment assay was adapted from that described by Wu Ze-Zhi [6]. The whole experiment system includes micropipette puller system, micromanipulation system and data processing system (Fig. 1). Micropipettes with an internal radius (Rp) of 5.5–6.5 mm were prepared with the use of a micropipette puller and filled with culture medium. The micropipette was mounted on a hydraulic micromanipulator, with the wide end of the pipette connected to a pressure regulation system. The pressure regulation system used had an accuracy of better than 2 N/m2 and a time constant of approximately 20 ms. These studies were performed at room temperature. The experiment was recorded on videotape. The arrangement has been described in detail elsewhere [4]. The minimum aspiration pressure that led to the total separation of a cell and coated surface is referred to as the critical separation pressure. The critical separation force (F) was calculated as
K.-F. Shao et al. / Colloids and Surfaces B: Biointerfaces 19 (2000) 55–59
F = Dp × p × (Rp)2
57
3.1. Effect of cytochalasin D on adherence
where Rp is the internal radius of a pipette and Dp is the critical separation pressure.
2.6. Statistics All data were represented as mean 9 S.E.M., the significance of the agent effects were determined using the Student’s t-test for paired values, as appropriate.
3. Results We employed the hepatocellular carcinoma cells, SMMC-7721, as a model for tumor cell adherence. In employing this model, cells were pretreated for half an hour with cytoskeletal agents at different concentrations and then their adherence was compared to that of untreated control cells.
On the collagen IV/laminin coated surface, the properties of both kinds of cells show the significance of the difference. The adhesion force of the HCC cells was twice as much as that of the hepatocyte, and morphologic observation indicated adherent HCC cells had well-spread appearance, easier to form the membrane tether. After being treated with cytochalasin D (0.025–5 mg/ ml), adhesion forces of both cells fell down greatly (70–90%). HCC cells maintained a rounded morphology characteristic of non-adherent ones, their extension obviously decreased and focal contacts almost vanished. The fact that those distinct adhesion features of HCC cells nearly disappeared showed that, cytochalasin D, which is more specific for microfilaments, was inhibitory at very low concentrations. It suggested that microfilaments might be crucial for adherence in tumor cells (Table 1).
Fig. 1. Schematic block diagram of the micropipette aspiration system. Table 1 Effects of cytochalasin D on the adhesion forces of HCC cells and hepatocytes in a concentration range of 0.25–5 mg/ml on the coll IV/laminin coated surfacesa
Hepatocytes HCC cells
a
0 mg/ml (control)
0.25 mg/ml
0.5 mg/ml
2.5 mg/ml
5 mg/ml
4329 203 (n= 67) 9599 362+++ (n =80)
1339 65*** (n=63) 1119 67*** (n= 83)
39 9 14*** (n = 70) 148 974+++*** (n =73)
120 981** (n =63) 93 947*** (n =91)
51 9 23*** (n =63) 158 9 62+++*** (n =81)
10−10 N; n, number of cells measured. Compared with control: ***PB0.001, **PB0.01. HCC cells compared with hepatocytes: PB0.001, ++PB0.01.
+++
K.-F. Shao et al. / Colloids and Surfaces B: Biointerfaces 19 (2000) 55–59
58
Table 2 Effects of colchicine on the adhesion forces of HCC cells and hepatocytes in a concentration range of 1–60 mg/ml on the coll IV/laminin coated surfacesa
Hepatocytes HCC cells
a
0 mg/ml
1 mg/ml
15 mg/ml
30 mg/ml
60 mg/ml
4329 203 (n=67) 9599 362+++ (n= 80)
9689327*** (n =66)
2241 9 923*** (n = 59) 979 9474+++*** (n =63)
615 9 298*** (n =56) 534 9308*** (n =72)
1651 9652** (n = 62) 428 9 185+++** (n = 76)
4079190+++*** (n= 74)
10−10 N; n, number of cells measured. Compared with control: ***PB0.001, **PB0.01. HCC cells compared with hepatocytes: PB0.001, ++PB0.01.
+++
3.2. Effects of colchicine on adherence Colchicine, which inhibits microtubular polymerization, had different effects on both cells. Compared with untreated groups respectively, the adhesion forces of HCC cells decreased and those of hepatocytes increased. At the same time, morphologic observation showed that both kinds of cells, extending peripherad, had larger rounded morphology after being treated with colchicine. At this point, it is completely different from tumor cells in untreated groups (Table 2).
4. Discussion As the goal of this study was to know what roles the cytoskdeton system play in the adhesion process of tumor cells to the basement membrane, we have studied in vitro how cytoskeletal agents, which inhibit the cytoskeleton, affect the adhesion properties of hepatocytes and hepatocellular carcinama (HCC) cells onto the collagen IV and laminin coated surface. Many reports have converged to study the biological properties of cytoskeleton by using cytoskeletal agents. Agents such as colchicine, which inhibits mircotubular polymerization, have been showed to inhibit motility in fibroblast [7]. Further, Mary L. Stracke [5] has employed the two stereoisomeric forms of tubolozole whose cis-isomer, like colchicine, inhibits microtubular polymerization and whose trans-isomer has no inhibitory effects on microtubular, and found that microtubular agent inhibits motility of a cell by disturbing the microtubular polymerization, but
not by the agents itself. Similarly, the cytochalasin B and D, by inhibiting actin polymerization [8,9], can abolish motility and adherence in fibroblasts and neutrophils, respectively. So, using the cytoskeletal agents to study the cell biological effect was reasonable. In our experiments, colchicine and cytochalasin D have been employed as the anti-microtubular agent and the anti-microfilament agent, separately. The results showed colchicine had different effect on hepatocytes and HCC cells: compared with untreated groups, the adhesion forces of HCC cells decreased and those of Hepatocytes inereased. Cytochalasin D had great inhibitory effect on adherence of both kinds of cells to collagen IV/laminin substratum (about 70–90%). These data showed that tumor cells have distinct adhesion behaviors from the normal cells and the adhesion properties were relevant to cytoskeleton system. Previous work with neutrophils and fibroblasts [10] had indicated that cytoskeleton system, especial microfilaments were necessary to form the focal contacts, which mediated the interactions among the cytoskeleton system, the cell membrane and the extra cellular matrix. Earlier studies [11] pointed that focal contact could result in the rearrangement of cytoskeleton system. The more direct experiment found [12] that while pulling the micropipette outward, which linked an adhesion molecule receptor of a live cell, the rearrangement of cytoskeleton system occurred. These results have great uniformity with those we got above. It suggested that, in tumor cells, microfilaments are crucial for adherence.
K.-F. Shao et al. / Colloids and Surfaces B: Biointerfaces 19 (2000) 55–59
As described above, compared with normal cells, tumor cells show the distinct adhesion properties, i.e. on the collagen IV/laminin coated surface, the adhesion forces of HCC cells were obviously higher than Hepatoytes, the adherent HCC cells had good tension, easier to form membrane tether [13] than hepatocytes under the suction force. But, those distinct features disappeared after being treated with cytochalasin D. This fact supported the concept that those distinct adhesion features of HCC cells caused by their microfilament system. As many labs reported that, cytoskeleton system of tumor cells were different completely from normal cells [14], in one hand, much decrease in the number of microtubules and loss of microfilaments in tumor cells, on the other hand, the tumor cells could express some new cytoskeletal components. Taking together, the above results suggested that the distinct adhesion behaviors of tumor cells are related to changes in the cytoskeleton system, and abnormal cytoskeleton of tumor cells may be basis of their abnormal adhesion properties.
Acknowledgements Program supported by the National Natural Science Foundation of China (No. 39500037).
References [1] L.A. Liotta, C.N. Rao, S.H. Barsky, Tumor invasion and extracellular matrix, Lab. Invest. 49 (1983) 636.
.
59
[2] Terranova, V.P., Diflorio, R., Hujanen, E., et al., 1986. Laminin promotes rabbit neutrophil motility and attachment. J. Clin. Invest. 77, 1180 – 1186. [3] A. Ben-Ze’ev, A. Duerr, F. Solomon, et al., Cell 17 (1979) 859 – 865. [4] F. Braet, R.D. Zanger, M. Sasaoki, et al., Assessment of method of isolation, purification and cultivation of rat liver endothelial cells, Lab. Invest. 70 (1994) 957– 994. [5] Mary, L., Soroush, M., Liotta, L., et al., 1993. Cytoskeletal agents inhibit motility and adherence of human tumor cells. Kidney Intl. 43, 151 – 157. [6] Ze-Zhi, W., Zhi-Qing, L., Mian, L., et al., 1996. Investigation on the mechanics of adhesion between granulocytes and endothelial cells in burned rats. J. Biomed. Eng. (Chinese edition with English abstract) 13 (3), 219 – 222. [7] J.M. Vasiliev, I.M. Gelfand, L.A. Domnina, et al., Effect of colchimid on the locomotory behavior of fibroblasts, J. Embryol. Exp. Morph. 24 (1970) 625 – 640. [8] S.H. Zigmond, J.G. Hirsch, Effects of cytochalasin B on polymorphonuclear leokocyte locomotion, phagocytosis and glycolysis, Exp. Cell. Res. 73 (1972) 383 –393. [9] S.B. Carter, Effects of cytochalasins on mammalian cells, Nature 213 (1967) 261 – 264. [10] G. Rinnerthaler, B. Geiger, J.V. Small, Contact formation during fibroblast locomotion: Involvement of membrane rullfules and microtubules, J. Cell Biol. 106 (1988) 747 – 760. [11] Horwitz, A.F. 1997. Integrins and healthy, Sci. Am. 7, 20 – 27. [12] Wang, N., Ingber, J.P., Ingber, D.E., 1993. Mechanotransduction across the cell surface and through the cytoskeleton, Science 260, 1124 – 1127. [13] Shao, J.-Y., Hochmuth, R., 1996. Micropipette suction for measuring piconewton forces of adhesion and tether formation from neutrophil membranes, Biophys. J. 71, 2892 – 2901. [14] A. Ben-Ze’ev, The cytoskeleton in cancer cells, Biochem. Biophys. Acta 780 (1985) 197 – 212.
Effects of colchicine and cytochalasin D on the adhesion properties of the HCC onto the collagen IV/laminin coated surface Kai-Feng Shao, Ze-Zhi Wu, Bo-Chu Wang *, Mian Long, Shao-Xi Cai Bioengineering College, Chongqing Uni6ersity, Chongqing, 400044, PR China Received 11 November 1999; accepted 6 December 1999
Abstract The adhesion properties of both hepatocytes and hepatocellular carcinoma (HCC) cells onto the Coll IV/Laminin coated surface were measured by means of micropipette aspiration technique. Further cytoskeletal agents of colchicine and cytochalasin D were tested for their effects on adherence to Coll IV/Laminin coated dishes. The results showed: cytochalasin D, which inhibits microfilament polymerization, had great inhibitory effect on adherence of both kinds cells to Coll IV/Laminin substratum (about 70 – 90%). At the same time, HCCs extension obviously decreased and focal contacts almost vanished after being treated with cytochalasin D, in contrast, there was no significant effect on the formation of extention of hepatocytes. Colchicine, which inhibits microtubular polymerization, had different effects on both cells: Compared with untreated groups, the adhesion forces of HCC cells decreased and those of hepatocytes increased. These data suggested that, in these tumor cells, microfilaments are crucial for adherence, and abnormal cytoskeletons of tumor cells may be basis of their abnormal adhesion properties. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Hepatocellular carcinoma; Adhesion; Colchicine; Cytochalasin D
1. Introduction Tumor invasion is clearly an active process undergoing three stages: attachment, migration and metastasis [1]. In facts, tumor invasion could be considered a dynamic adhesive process, which contained much complex and subtle physiological and biochemical interactions between the tumor cells and extracellular matrix. * Corresponding author. E-mail address: [email protected] (B.-C. Wang).
In the past several years, studies [1,2] were merely focused on the effects of matrix macromolecules such as laminin, fibronectin and collagen IV on the adhesion properties of tumor cells. Little, however, is known about what role cytoskeletal system play in the process of tumor cells attachment to the extracellular matrix. The association of cytoskeletal system with certain membrane proteins and extracellular matrix points to the wide variety of cell functions that are possibly modulated via the cytoskeletal system [3]. So understanding the adhesion properties of
0927-7765/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 7 - 7 7 6 5 ( 9 9 ) 0 0 1 7 0 - 8
56
K.-F. Shao et al. / Colloids and Surfaces B: Biointerfaces 19 (2000) 55–59
tumor cell would be a key to investigate tumor invasion at cell level. In this article, employing the hepatocellular carcinoma cells as a model, we have attempted to utilize the cytoskeletal agents, colchicine and cytochalasin D, as tools in order to assess the relative contribution of microtubules and microfilaments to components of the invasion process.
2. Materials and methods
2.1. Cell lines Hepatocellular carcinoma (HCCs) cells, named SMMC-7721, were purchased from the second Military Medical University. Human hepatocytes (HHCs) were derived from primary liver which obtained from patient because of accidental death, they were isolated by 0.05% collagenase separated by speed gradient centrifugal method [4], and determined by trypan blue dye exclusion test. Both of cells were routinely cultured in our lab. RPMI-1640 Supplemented with 10% fetal bovine serum, glutamine, penicillin (10 unit%), and streptomysin (10 mg/ml).
2.2. Cytoskeletal agents Colchicine and cytochalasin D were purchased from Sigma chemical Co. (St. Louis, Missouri, USA). Colchicine was diluted to the stock concentrations of 1, 15, 30 and 60 mg/ml in RPMI1640 medium. And, cytochalasin D was dissolved in dimethyl sulfoxide (DMSO) and diluted to the concentrations of 0.025, 0.05, 2.5 and 5 mg/ml in RPMI1640 medium.
2.3. Preparation of substrate Collagen IV was purchased from Sigma Company dissolved in 0.1 M acetic acid and diluted to a stock concentration of 400 mg/ml in PBS solution. Laminin was purchased with the stock concentration of 500 mg/ml from Sigma Company. The final concentrations of substrate were adjusted to 2 mg/ml of collagen IV and 1.25 mg/ml of laminin. Poly-D-lysine (PDL) was purchased from
Sigma company, diluted to a concentration of 2 mg/ml. Bovine serum albumin (BSA) was purchased from Hua-Mei company, diluted to a concentration of 0.5% in PBS (pH 7.4) Solution.
2.4. Substrate-coating technique The artificial basement membrane was made by coating glass surface with PDL, collagen IV, laminin and BSA. At first, 200 ml of PDL (2 mg/ml) was coated in the marked circle area of chamber to help the collagen IV adhesion to the glass floor. After incubation for 30 min at 37°C, the PDL solution was removed from chamber, PBS solution washed chamber twice. Then, 200 ml of the solution containing 2 mg/ml of collagen IV and 1.25 mg/ml of laminin were coated onto the same area of the chamber. After incubation for 40 min at 37°C, the rest solution were removed, PBS solution washed in same manner. After that, 200 ml of 0.5% BSA solution was coated onto the same area to block non-specific binding of cells to the plastic [5], and washed twice.
2.5. Attachment assay The attachment assay was adapted from that described by Wu Ze-Zhi [6]. The whole experiment system includes micropipette puller system, micromanipulation system and data processing system (Fig. 1). Micropipettes with an internal radius (Rp) of 5.5–6.5 mm were prepared with the use of a micropipette puller and filled with culture medium. The micropipette was mounted on a hydraulic micromanipulator, with the wide end of the pipette connected to a pressure regulation system. The pressure regulation system used had an accuracy of better than 2 N/m2 and a time constant of approximately 20 ms. These studies were performed at room temperature. The experiment was recorded on videotape. The arrangement has been described in detail elsewhere [4]. The minimum aspiration pressure that led to the total separation of a cell and coated surface is referred to as the critical separation pressure. The critical separation force (F) was calculated as
K.-F. Shao et al. / Colloids and Surfaces B: Biointerfaces 19 (2000) 55–59
F = Dp × p × (Rp)2
57
3.1. Effect of cytochalasin D on adherence
where Rp is the internal radius of a pipette and Dp is the critical separation pressure.
2.6. Statistics All data were represented as mean 9 S.E.M., the significance of the agent effects were determined using the Student’s t-test for paired values, as appropriate.
3. Results We employed the hepatocellular carcinoma cells, SMMC-7721, as a model for tumor cell adherence. In employing this model, cells were pretreated for half an hour with cytoskeletal agents at different concentrations and then their adherence was compared to that of untreated control cells.
On the collagen IV/laminin coated surface, the properties of both kinds of cells show the significance of the difference. The adhesion force of the HCC cells was twice as much as that of the hepatocyte, and morphologic observation indicated adherent HCC cells had well-spread appearance, easier to form the membrane tether. After being treated with cytochalasin D (0.025–5 mg/ ml), adhesion forces of both cells fell down greatly (70–90%). HCC cells maintained a rounded morphology characteristic of non-adherent ones, their extension obviously decreased and focal contacts almost vanished. The fact that those distinct adhesion features of HCC cells nearly disappeared showed that, cytochalasin D, which is more specific for microfilaments, was inhibitory at very low concentrations. It suggested that microfilaments might be crucial for adherence in tumor cells (Table 1).
Fig. 1. Schematic block diagram of the micropipette aspiration system. Table 1 Effects of cytochalasin D on the adhesion forces of HCC cells and hepatocytes in a concentration range of 0.25–5 mg/ml on the coll IV/laminin coated surfacesa
Hepatocytes HCC cells
a
0 mg/ml (control)
0.25 mg/ml
0.5 mg/ml
2.5 mg/ml
5 mg/ml
4329 203 (n= 67) 9599 362+++ (n =80)
1339 65*** (n=63) 1119 67*** (n= 83)
39 9 14*** (n = 70) 148 974+++*** (n =73)
120 981** (n =63) 93 947*** (n =91)
51 9 23*** (n =63) 158 9 62+++*** (n =81)
10−10 N; n, number of cells measured. Compared with control: ***PB0.001, **PB0.01. HCC cells compared with hepatocytes: PB0.001, ++PB0.01.
+++
K.-F. Shao et al. / Colloids and Surfaces B: Biointerfaces 19 (2000) 55–59
58
Table 2 Effects of colchicine on the adhesion forces of HCC cells and hepatocytes in a concentration range of 1–60 mg/ml on the coll IV/laminin coated surfacesa
Hepatocytes HCC cells
a
0 mg/ml
1 mg/ml
15 mg/ml
30 mg/ml
60 mg/ml
4329 203 (n=67) 9599 362+++ (n= 80)
9689327*** (n =66)
2241 9 923*** (n = 59) 979 9474+++*** (n =63)
615 9 298*** (n =56) 534 9308*** (n =72)
1651 9652** (n = 62) 428 9 185+++** (n = 76)
4079190+++*** (n= 74)
10−10 N; n, number of cells measured. Compared with control: ***PB0.001, **PB0.01. HCC cells compared with hepatocytes: PB0.001, ++PB0.01.
+++
3.2. Effects of colchicine on adherence Colchicine, which inhibits microtubular polymerization, had different effects on both cells. Compared with untreated groups respectively, the adhesion forces of HCC cells decreased and those of hepatocytes increased. At the same time, morphologic observation showed that both kinds of cells, extending peripherad, had larger rounded morphology after being treated with colchicine. At this point, it is completely different from tumor cells in untreated groups (Table 2).
4. Discussion As the goal of this study was to know what roles the cytoskdeton system play in the adhesion process of tumor cells to the basement membrane, we have studied in vitro how cytoskeletal agents, which inhibit the cytoskeleton, affect the adhesion properties of hepatocytes and hepatocellular carcinama (HCC) cells onto the collagen IV and laminin coated surface. Many reports have converged to study the biological properties of cytoskeleton by using cytoskeletal agents. Agents such as colchicine, which inhibits mircotubular polymerization, have been showed to inhibit motility in fibroblast [7]. Further, Mary L. Stracke [5] has employed the two stereoisomeric forms of tubolozole whose cis-isomer, like colchicine, inhibits microtubular polymerization and whose trans-isomer has no inhibitory effects on microtubular, and found that microtubular agent inhibits motility of a cell by disturbing the microtubular polymerization, but
not by the agents itself. Similarly, the cytochalasin B and D, by inhibiting actin polymerization [8,9], can abolish motility and adherence in fibroblasts and neutrophils, respectively. So, using the cytoskeletal agents to study the cell biological effect was reasonable. In our experiments, colchicine and cytochalasin D have been employed as the anti-microtubular agent and the anti-microfilament agent, separately. The results showed colchicine had different effect on hepatocytes and HCC cells: compared with untreated groups, the adhesion forces of HCC cells decreased and those of Hepatocytes inereased. Cytochalasin D had great inhibitory effect on adherence of both kinds of cells to collagen IV/laminin substratum (about 70–90%). These data showed that tumor cells have distinct adhesion behaviors from the normal cells and the adhesion properties were relevant to cytoskeleton system. Previous work with neutrophils and fibroblasts [10] had indicated that cytoskeleton system, especial microfilaments were necessary to form the focal contacts, which mediated the interactions among the cytoskeleton system, the cell membrane and the extra cellular matrix. Earlier studies [11] pointed that focal contact could result in the rearrangement of cytoskeleton system. The more direct experiment found [12] that while pulling the micropipette outward, which linked an adhesion molecule receptor of a live cell, the rearrangement of cytoskeleton system occurred. These results have great uniformity with those we got above. It suggested that, in tumor cells, microfilaments are crucial for adherence.
K.-F. Shao et al. / Colloids and Surfaces B: Biointerfaces 19 (2000) 55–59
As described above, compared with normal cells, tumor cells show the distinct adhesion properties, i.e. on the collagen IV/laminin coated surface, the adhesion forces of HCC cells were obviously higher than Hepatoytes, the adherent HCC cells had good tension, easier to form membrane tether [13] than hepatocytes under the suction force. But, those distinct features disappeared after being treated with cytochalasin D. This fact supported the concept that those distinct adhesion features of HCC cells caused by their microfilament system. As many labs reported that, cytoskeleton system of tumor cells were different completely from normal cells [14], in one hand, much decrease in the number of microtubules and loss of microfilaments in tumor cells, on the other hand, the tumor cells could express some new cytoskeletal components. Taking together, the above results suggested that the distinct adhesion behaviors of tumor cells are related to changes in the cytoskeleton system, and abnormal cytoskeleton of tumor cells may be basis of their abnormal adhesion properties.
Acknowledgements Program supported by the National Natural Science Foundation of China (No. 39500037).
References [1] L.A. Liotta, C.N. Rao, S.H. Barsky, Tumor invasion and extracellular matrix, Lab. Invest. 49 (1983) 636.
.
59
[2] Terranova, V.P., Diflorio, R., Hujanen, E., et al., 1986. Laminin promotes rabbit neutrophil motility and attachment. J. Clin. Invest. 77, 1180 – 1186. [3] A. Ben-Ze’ev, A. Duerr, F. Solomon, et al., Cell 17 (1979) 859 – 865. [4] F. Braet, R.D. Zanger, M. Sasaoki, et al., Assessment of method of isolation, purification and cultivation of rat liver endothelial cells, Lab. Invest. 70 (1994) 957– 994. [5] Mary, L., Soroush, M., Liotta, L., et al., 1993. Cytoskeletal agents inhibit motility and adherence of human tumor cells. Kidney Intl. 43, 151 – 157. [6] Ze-Zhi, W., Zhi-Qing, L., Mian, L., et al., 1996. Investigation on the mechanics of adhesion between granulocytes and endothelial cells in burned rats. J. Biomed. Eng. (Chinese edition with English abstract) 13 (3), 219 – 222. [7] J.M. Vasiliev, I.M. Gelfand, L.A. Domnina, et al., Effect of colchimid on the locomotory behavior of fibroblasts, J. Embryol. Exp. Morph. 24 (1970) 625 – 640. [8] S.H. Zigmond, J.G. Hirsch, Effects of cytochalasin B on polymorphonuclear leokocyte locomotion, phagocytosis and glycolysis, Exp. Cell. Res. 73 (1972) 383 –393. [9] S.B. Carter, Effects of cytochalasins on mammalian cells, Nature 213 (1967) 261 – 264. [10] G. Rinnerthaler, B. Geiger, J.V. Small, Contact formation during fibroblast locomotion: Involvement of membrane rullfules and microtubules, J. Cell Biol. 106 (1988) 747 – 760. [11] Horwitz, A.F. 1997. Integrins and healthy, Sci. Am. 7, 20 – 27. [12] Wang, N., Ingber, J.P., Ingber, D.E., 1993. Mechanotransduction across the cell surface and through the cytoskeleton, Science 260, 1124 – 1127. [13] Shao, J.-Y., Hochmuth, R., 1996. Micropipette suction for measuring piconewton forces of adhesion and tether formation from neutrophil membranes, Biophys. J. 71, 2892 – 2901. [14] A. Ben-Ze’ev, The cytoskeleton in cancer cells, Biochem. Biophys. Acta 780 (1985) 197 – 212.