The Proliferation and Migration-Enhancing Effects of Vitronectin in SMMC 7721 Cells: A Pilot Study.
Abstract: To understand the effects of Vitronectin on cell proliferation and migration in the cell line of hepatocellular carcinoma SMMC 7721 the effects of Vitronectin on cell proliferation rate or on the prevention of the cells from the apoptotic stimuli were appraised with WST-1 assay; and the morphology of A3/4-Tubulin was observed with con-focal microscope. The effect on migration was detected with transwell chamber. The results show that Vitronectin helps the cells adhere to Petri dish as well as the sustaining of the morphology of A3/4-Tubulin. Vitronectin could enhance the proliferation rate of SMMC 7721 with the concentration-effect mode and could protect the cells from the stimuli of apoptosis inducer. As to cell migration the results show that Vitronectin enhance cell migration across the membrane of transwell chamber. According the results the conclusion could be made that Vitronectin might play important roles in the following biological effects such as sustaining the morphology of the tumor
cells enhancing the proliferation as well as the migration.
Keywords: Vitronectin Hepatocellular Carcinoma Proliferation Tumor Cell Migration.
Hepatocellular carcinoma (HCC) is one of the major diseases causing serious health problems in the world. According to the statistics almost 0.626 million new cases suffering from liver cancer with 0.598 million deaths each year worldwide ranking sixth for the cancer incidence and third for cancer mortality in the world [1 2]. At present no effective preventive and treatment measures for advanced liver cancer postoperative recurrence and metastasis of the early liver cancer. Therefore early diagnosis and treatment of liver cancer are of great significant. Early diagnosis of cancer depends on effective biological markers currently the most common biological marker associated with HCC is serum alpha-fetoprotein (AFP) level . However previous studies show that AFP has various limitations such as a low sensitivity and a high false negative rate resulting in high unreliability in identifying liver cancer and other liver diseases . Thus to find better biomarkers became a research hotspot.
Vitronectin (VTN) also called serum spreading factor or complements S-protein is a cell adhesion and spreading factor found in serum and tissues . One of the most important roles of VTN is to participate in the formation of extracellular matrix. In normal liver cells VTN expresses only in portal system and central vein subendothelial matrix. But when HCC occurs VTN becomes the main component of the extracellular matrix  and has a high level of expression especially in the cavernous-like vascular . The dramatic difference in VTN expression suggests that it may have important significance in cancer . However there are few reports about effects of VTN on liver cancer cell lines. Therefore we simulate the actual action mode of VTN in physiological processes  trying to observe the growth and the metastasis ability of SMMC 7721 cells when cultured in VTN-coating Petri dishes in order to understand whether VTN has biological significances such as
promoting tumor cell growth metastasis as well as protecting tumor cells from apoptotic stimuli.
MATERIALS AND METHODS ReagentsVTN was purchased from Invitrogen (Grand Island NY). arginine-glycine-asparginine peptide(RGD) was supplied by Sigma-Aldrich (St Louis MO). DIM was purchased from LKT (St. Paul MN) and was prepared in dimethyl sulfoxide (DMSO; Sigma-Aldrich MO). Penicillin streptomycin RPMI 1640 Insulin- Transferrin-Selenium (ITS) and EDTA were purchased from Gibco (Grand Island NY). Cell proliferation reagent (WST-1) was supplied by Roche (Germany). Antibodies against A3/4-Tubulin were purchased from Cell Signaling Technology (Beverly MA).
The human hepatocellular carcinoma cell lines SMMC 7721 was obtained from the Type Culture Collection of the Chinese Academy of Science (Shanghai China). The passage numbers of the cells were from 6~30. The cells were cultured in RPMI 1640 medium with 3% ITS a standard additive for cells grown in serum-free conditions and supplemented with 100 U/mL penicillin and 100 g/mL streptomycin at 37C in the present of 5% CO2.
For cell growth experiments 500 l of VTN solutions diluted in PBS were aliquoted into the dishes (diameter 3 cm NUNC Rochester NY) or 100 l into the 96-well dishes left at room temperature for 18 h and then at 4C for another 18 h. Then the dishes were incubated with 0.1% BSA in PBS for 30 min at 37C to block non-coated areas of plastic. After that the dishes were washed several times with PBS and air-dried for using [9 10].
Cell Attachment and Cytoskeletal Morphology
Dishes were coated with VTN and 10000 cells were inoculated into each dish and incubated at 37 C for 24 h. After wash with PBS for 5 min the cells were fixed for 10 min with methanol-acetic acid (3:1) fixative and blocked with BSA for 20 min. After addition of 2 ml 0.5% Triton X-100 at room temperature for 20 min 50 l rabbit anti-human A3/4-Tubulin monoclonal antibody was added and incubated at 4 C overnight. After wash with PBS the cells were incubated with fluorescent- labeled secondary antibody at room temperature for 20 min followed by DAPI staining and wash with PBS. The cells were observed with a laser con-focal microscope.
About 10000 cells were incubated in a 96-well culture dish coated with VTN solution at 37 C for 24 h. WST-1 a cell proliferation reagent was added into every well and cultured for 2 h. Colorimetric analysis was performed in 450 nm (measure wavelength) and 690 nm (reference wavelength) after full mixing.
Abate Apoptotic Stimuli
About 10000 cells were incubated in a 96-well culture dish coated with VTN solution at 37 C for 24 h followed by treating with various concentration of DIM (5 10 15 20 30 40 and 50 M) for 24 h. WST-1 was added into every well and cultured for 2 h. Colorimetric analysis was performed after full mixing.
Migration assay were performed essentially as described by Leavesley et al. Briefly 1X10 cells cultured in serum-free medium for 4 h were seeded into the upper chamber of an 8.0-m-pore Costar Transwell (24-well plate format). VTN was added into the lower chamber. After 12 h of incubation at 37 C in 5% CO2 Cells that migrated to the lower chamber were fixed then stained with Trypan Blue. The number of cells migrated was estimated by cell counting [11 12].
Data was expressed as mean standard deviation and SPSS 11.0 software was used for statistical analysis. The data of cell attachment and migration rate were analyzed using a one-way ANOVA and post hoc analysis was done using S-N-K t test. The a priori significance level was set at p less than 0.05.
Effects of VTN-Coating on Cell Adherence and the Morphology of A3/4 -Tubulin
Our results showed that adherent cells in VTN- coated group grew well. The spreading cell presented spindle or polygonal shape. Round-shrink cells were rare in the dishes coated with VTN. But the round- shrink cells increased when the dilution ratio of VTN increases. However RGD-coating had no effect on cell adherence. The cells were round-shaped shrinkage and the same as the control group (1% BSA coated group) (Figure 1).
VTN solutions allowed the cells adhere to the Petri dish in 2 h and fully extended into the normal shape of spindle or polygonal. Compared with the control group the difference in cell adhesion rate was statistically significant (F = 37.526 p less than 0.01). However RGD- coating was not helpful for cell adherence. The cell adhesion rates in RGD solutions of various dilution were close to those of their control group (1% BSA coated group) (F = 8.221 p = 0.762). S-N-K test showed that when compared with the corresponding RGD-coated groups the increase in cell adherent rates of VTN-coated cells was statistically significant (Figure 2).
As compared with un-coated cells VTN-coating was good for adherence and the cells had more typical spindle or polygonal structure. In contrast the spindle or polygonal shape of the non-coated cultured cells was not typical and poor stretching. Spindle cells were rare and most cells presented short spindle or cone (Figure 3). Accordingly A3/4-Tubulin which plays an important role in cell morphology movement of cells and organelles as well as material transportation was changed in some extent. A3/4-Tubulin has a network and filamentous structure arranging from perinuclear to the surrounding. As indicated in Figure 2G-I VTN-coating favored the maintenance of normal A3/4-Tubulin structure. Under the general culture conditions without VTN coating the normal filamentous structure of microtubules is not obvious and presents form of pellets or frosted glass with a less regular stretching radial structure (Figure 3).
Effects of VTN on Cell Growth
Figure 4A indicated that VTN-coating plays a certain role in promoting cell growth (F = 61.224 p less than 0.01) and that cell proliferation was inversely proportional to the VTN dilution ratio i.e. the smaller the dilution ratio the levels of cell proliferation were higher. The cell proliferation level under the lowest dilution (1:100) was 1.7-fold higher than that of the control group (1% BSA). In addition RGD-coating has no promoting effect on cell growth (F = 18.370 P=0.813).
Protective Effects of VTN on Liver Cancer Cell Growth Induced by Different Concentrations of DIM
To observe the protective effects of VTN on the cells which were pretreated with apoptotic stimuli the fixed concentration of VTN-coating (1:200) were used. The results showed that VTN coating could reduce the inhibitory effect of apoptosis-inducing agent DIM on SMMC 7721 cell proliferation (F=106.192 Pless than 0.001). In addition RGD-coating had no significant protective effect on DIM inhibition of SMMC 7721 cell protection (F=12.167 P=0.979) (Figure 4B).
Effect of VTN-Coating on the Migration of SMMC 7721 Cells
SMMC 7721 cells were inoculated in the upper chambers of Transwell chamber. Serum-free medium with different concentrations of VTN solution were added in the lower chambers and incubated for 12 h. The number of cells transferring from upper chambers to lower chambers through the membrane was counted. As indicated in Figure 5 different concentrations of VTN RGD or BSA had a different promoting effect on SMMC 7721 cell migration from the upper chamber into lower chamber (F = 197.647 p less than 0.001). One-way ANOVA analysis showed that compared with the BSA group various concentration of VTN was capable of promoting SMMC 7721 cell migration whereas RGD has no similar effect.
It is well known that malignant transformed cells cannot properly undergo apoptosis [13 14] is an important reason of tumor development however uncontrolled cell proliferation and cell differentiation abnormalities play significant roles in the process of neoplasia. In glioma cells abnormal proliferation could be induced when coated with VTN and the highest protective effect could be find when the coating concentration is 500 ng/cm2 . At present the effect of VTN and whether it produces similar inhibition of apoptosis-inducing effects of agents on human hepatocellular cells have not been reported.
Tumor cell migration is a prerequisite of tumor invasion and metastasis. As an extracellular matrix molecule VTN is involved in extracellular matrix composition and has a potential impact on tumor cell metastasis. Pervious studies show that VTN promotes tumor cell metastasis through its receptor molecule Alpha V integrin and other signaling molecules. The most important active sites in the molecular structure of the VTN are located in its N-terminal three peptide structure Arg-Gly-Asp namely RGD sequence . Interaction of VTN and its main cell surface receptor Alpha V integrin depends on this structure site . A variety of extracellular matrix components including fibronectin laminin VTN and collagen contain RGD sequence which is very widely distributed in human body . In this study in order to determine that promotion of cell adhesion cell growth cell transfer and inhibition of tumor cell apoptosis are attributed to VTN RGD-coating was used as a control in the studies. The results demonstrate that RGD-coating has no impact on any effect mentioned above suggesting that the phenomena observed in this study are induced by VTN itself.
The main active components of cruciferous plants indole-3-carbinol (I3C) and its derivatives such as 33'- Diindolylmethane (DIM) can inhibit cell proliferation and induce apoptosis of various tumor cells. In vitro studies indicate that I3C and DIM affect activation of carcinogens and initiation and progress of tumor via a variety of ways inducing apoptosis in various tumor cells such as breast cancer prostate cancer endometrial cancer colorectal cancer and leukemia cells etc. [18-21]. Previous studies by our group show that I3C and DIM can inhibit proliferation of poorly differentiated nasopharyngeal carcinoma cell line CNE1 human hepatoma cell lines HepG2 and SMMC 7721 cell as well as induce apoptosis of these types of cells  in a concentration-dependent manner and time-dependent manner. In the present study we employed DIM as an apoptosis-inducing agent to observe the impact of VTN-coating on its role. Our results show that DIM inhibits proliferation of SMMC 7721 cell and induces apoptosis in a concentration- dependent manner. VTN-coating inhibits apoptosis suggesting that the extracellular VTN can promote tumor cell growth and inhibit effect of apoptotic stimuli on tumor cells.
VTN is a normal component of serum. To avoid the impact of VTN in fetal bovine serum on the experimental results and to ensure nutritional factors required for cell growth we employed Insulin- Transferrin-Selenium (ITS) a basic nutritional support material  to domesticate cells by gradually reducing the serum concentration with gradual addition of ITS. In addition to prevent cell matrix proteins including VTN from hydrolysis by trypsin we used a non-enzymatic digestion chelating agent-EDTA in this study to digest the cells and used low-speed centrifugation to remove EDTA in the cell suspension preventing cell damage induced by intracellular calcium and magnesium ions in the EDTA chelation cells.
Furthermore the current studies show that VTN can promote cell adhesion and contribute to the maintenance of Tubulin structures. Since Tubulin plays extremely important roles in the constitution of the cell stents maintaining cell shape displacement of organelles and participating in material transport therefore we speculate some biological roles of VTN such as promoting cell growth and metastasis of tumor cells may be achieved by regulating Tubulin structure.
In conclusion VTN might function importantly in the growth anti-apoptosis and migration of hepatocellular carcinoma cell line SMMC 7721. The mechanism underlying the above effects may attribute to the sustaining the morphology of cytoskeleton as well as the attachment-helping roles.
DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
No potential conflicts of interest were disclosed.
This work was supported by grants from: 1. The National Natural Science Foundation of China (81101562); 2.The Natural Science Foundation of Guangdong Province (S2012010009633); 3.The Project of Science and Technology of Guangdong Province (2012B060300005); 4.Key Project Guangzhou Medical and Health Science and Technology (20121A021018); 5.The Project for Key The Proliferation and Migration-Enhancing Effects of Vitronectin Journal of Basic and Applied Sciences 2014 Volume 10 211
Medicine Discipline Construction of Guangzhou Municipality (2013201507).
The funders had no role in study design data collection and analysis decision to publish or preparation of the manuscript.
 Ascha MS Hanouneh IA Lopez R Tamimi TA Feldstein AF Zein NN. The incidence and risk factors of hepatocellular carcinoma in patients with nonalcoholic steatohepatitis. Hepatology 2010; 51: 1972-78. http://dx.doi.org/10.1002/hep.23527
 Xu Y Liu L Liu J et al. A potentially functional polymorphism in the promoter region of miR-34b/c is associated with an increased risk for primary hepatocellular carcinoma. International journal of cancer. J Int du Cancer 2011; 128: 412-17. Liu JM Li FM Liu ZB et al. 8-Quinolineboronic acid as a potential phosphorescent molecular switch for the determination of alpha-fetoprotein variant for the prediction of primary hepatocellular carcinoma. Anal Chim Acta 2010; 663: 184-89. http://dx.doi.org/10.1016/j.aca.2010.01.030
 Cui Z Yu X Guo L et al. Combined analysis of serum alpha- fetoprotein and MAGE-A3-specific cytotoxic T lymphocytes in peripheral blood for diagnosis of hepatocellular carcinoma. Disease Markers 2013; 35: 915-23. http://dx.doi.org/10.1155/2013/907394
 Barnes D Wolfe R Serrero G McClure D Sato G. Effects of a serum spreading factor on growth and morphology of cells in serum-free medium. J Supramol Struct 1980; 14: 47-63. http://dx.doi.org/10.1002/jss.400140106 Jaskiewicz K Chasen MR Robson SC. Differential expression of extracellular matrix proteins and integrins in hepatocellular carcinoma and chronic liver disease. Anticancer Res 1993; 13: 2229-37.
 Edwards S Lalor PF Tuncer C Adams DH. Vitronectin in human hepatic tumours contributes to the recruitment of lymphocytes in an alpha v beta3-independent manner. Br J Cancer 2006; 95: 1545-54. http://dx.doi.org/10.1038/sj.bjc.6603467 Nejjari M Hafdi Z Gouysse G et al. Expression regulation function of alpha V integrins in hepatocellular carcinoma: an in vivo and in vitro study. Hepatology 2002; 36: 418-26. http://dx.doi.org/10.1053/jhep.2002.34611
 Zhong Wu X Honke K Long Zhang Y Liang Zha X Taniguchi N. Lactosylsulfatide expression in hepatocellular carcinoma cells enhances cell adhesion to vitronectin and intrahepatic metastasis in nude mice. Int J Cancer 2004; 110: 504-10.
 Uhm JH Dooley NP Kyritsis AP Rao JS Gladson CL. Vitronectin a glioma-derived extracellular matrix protein protects tumor cells from apoptotic death. Clinical Cancer Research: An Official Journal of the American Association for Cancer Research 1999; 5: 1587-94.
 Shih YT Wang MC Peng HH et al. Modulation of chemotactic and pro-inflammatory activities of endothelial progenitor cells by hepatocellular carcinoma. Cell Signal 2012; 24: 779-93. http://dx.doi.org/10.1016/j.cellsig.2011.11.013
 Primiceri E Chiriaco MS Dioguardi F et al. Automatic transwell assay by an EIS cell chip to monitor cell migration. Lab Chip 2011; 11: 4081-86. http://dx.doi.org/10.1039/c1lc20540d
 Serini S Piccioni E Merendino N Calviello G. Dietary polyunsaturated fatty acids as inducers of apoptosis: implications for cancer. Apoptosis: An International Journal on Programmed Cell Death 2009; 14: 135-52.
 Wang Y Lee CG. MicroRNA and cancer--focus on apoptosis. J Cell Mol Med 2009; 13: 12-23. http://dx.doi.org/10.1111/j.1582-4934.2008.00510.x
 Kim J Hong SY Park HS Kim DS Lee W. Structure and function of RGD peptides derived from disintegrin proteins. Mol Cells 2005; 19: 205-11. Bellone M Cocco E Varughese J et al. Expression of alphaV-integrins in uterine serous papillary carcinomas; implications for targeted therapy with intetumumab (CNTO 95) a fully human antagonist anti-alphaV-integrin antibody. International Journal of Gynecological Cancer: Official Journal of the International Gynecological Cancer Society 2011; 21: 1084-90
 Mooney A Jackson K Bacon R et al. Type IV collagen and laminin regulate glomerular mesangial cell susceptibility to apoptosis via beta(1) integrin-mediated survival signals. Am J Pathol 1999; 155: 599-606. http://dx.doi.org/10.1016/S0002-9440(10)65155-3
 Fan S Meng Q Xu J et al. DIM (33'-diindolylmethane) confers protection against ionizing radiation by a unique mechanism. Proc Natl Acad Sci USA 2013; 110: 18650-55. http://dx.doi.org/10.1073/pnas.1308206110
 Wong CP Hsu A Buchanan A et al. Effects of sulforaphane and 33'-diindolylmethane on genome-wide promoter methylation in normal prostate epithelial cells and prostate cancer cells. PloS One 2014; 9: e86787. http://dx.doi.org/10.1371/journal.pone.0086787 Lee SH Min KW Zhang X Baek SJ. 33'-diindolylmethane induces activating transcription factor 3 (ATF3) via ATF4 in human colorectal cancer cells. J Nutr Biochem 2013; 24: 664-71. http://dx.doi.org/10.1016/j.jnutbio.2012.03.016
 Shorey LE Hagman AM Williams DE Ho E Dashwood RH Benninghoff AD. 33'-Diindolylmethane induces G1 arrest and apoptosis in human acute T-cell lymphoblastic leukemia cells. PloS One 2012; 7: e34975. http://dx.doi.org/10.1371/journal.pone.0034975
 Zhu W Li W Yang G Zhang Q Li M Yang X. Indole-3- carbinol inhibits nasopharyngeal carcinoma. Int J Toxicol 2010; 29: 185-92. http://dx.doi.org/10.1177/1091581809356481
 Kisiday JD Kurz B DiMicco MA Grodzinsky AJ. Evaluation of medium supplemented with insulin-transferrin-selenium for culture of primary bovine calf chondrocytes in three- dimensional hydrogel scaffolds. Tissue Eng 2005; 11: 141- 51. http://dx.doi.org/10.1089/ten.2005.11.14
|Printer friendly Cite/link Email Feedback|
|Publication:||Journal of Basic & Applied Sciences|
|Date:||Dec 31, 2014|
|Previous Article:||Identifying Coping Profiles and Profile Differences in Role Engagement and Subjective Well-Being.|
|Next Article:||Autonomic Dysfunction in Asian Indian T2DM Patients is Related to Body Fat Content Instead of Insulin Resistance: A DEXA Study.|