|Cancer||Cell death||Cell cycle||Cytoskeleton||Exo/endocytosis||Differentiation||Division||Organelles||Signalling||Stem cells||Trafficking|
Lentivirus-mediated overexpression of TGF-β inducible early gene 1 inhibits SW1990 pancreatic cancer cell growth
Lei Jiang1, Fule Wang, Feiyan Lin, Shen‑Meng Gao, Yingxia Tan, Yixiang Han, Chiqi Chen and Jianbo Wu
Laboratory of Internal Medicine, The First Affiliated Hospital of Wenzhou Medical College, Wenzhou, Zhejiang 325000, Peoples Republic of China
TIEG1 (TGF-β inducible early gene 1) plays a significant role in regulating cell proliferation and apoptosis in various cell types. Previous studies have shown a close relationship between the expression level of TIEG1 and various cancers, including breast, prostate, colorectal and pancreatic cancer. In this study, we up-regulated the gene expression of TIEG1 in SW1990 pancreatic cancer cell line by a lentivirus transfection system and investigated its potential as a therapeutic target for pancreatic cancer. The results showed that lentivirus-mediated overexpression of TIEG1 gene inhibited human pancreatic cancer SW1990 cell proliferation and caused the cell cycle arrest at the G1-phase in vitro. SW1990 cells transduced with lenti-TIEG1 showed significant inhibition of colony formation and cancer cell growth in 3-D culture model. Moreover, overexpression of TIEG1 gene significantly slowed the growth of SW1990 xenografts in nude mice. Taken together, these data provided evidence that overexpression of TIEG1 gene by a lentivirus transfection system led to suppressed human pancreatic cancer cell growth and might therefore be a feasible approach in the clinical management of pancreatic cancer.
Key words: lentivirus, pancreatic cancer, TIEG1, tumourigenicity
Abbreviations: 3-D, three dimensional, EGFP, enhanced green fluorescent protein, TGF-β, transforming growth factor-β, TIEG1, TGF-β inducible early gene 1
1To whom correspondence should be addressed (email email@example.com).
Pancreatic cancer is one of the most malignant tumours in the world and possesses the highest fatality rate among all cancers (Jemal et al., 2006). Pancreatic cancer typically spreads quickly and is rarely detected in its early stages, which is a major reason why it is one of the leading causes of cancer death. Since only 20% of the pancreatic tumour cases could submit to surgical treatment (Wade et al., 1996) and most of the others are highly resistant to chemotherapy and radiation protocols (Masui et al., 2006; Reni et al., 2006), the prognosis of pancreatic tumour is generally poor. Pancreatic cancer is notorious for late presentation, with a median survival rate of less than 6 months (Sener et al., 1999). Systemic recurrence including peritoneal dissemination and the high rate of local invasion also compromised the treatment outcome (Niederhuber et al., 1995; Duffy et al., 2003). Because this disease is basically a drug-resistant tumour, new therapeutic strategy (e.g. gene therapy) is pressingly needed to promote the treatment outcome and improve the survival rate of pancreatic cancer patients.
In previous years, multiple genes have been found to undergo genetic alternations during the development of pancreatic cancer (Mimeault et al., 2005). Deregulation of TGF-β (transforming growth factor-β) signalling appears to be an important step in the development of pancreatic carcinogenesis (Rane et al., 2006). TIEG1 (TGF-β inducible early gene 1) encodes 480 amino acids and belongs to the Krüppel-like family of transcription factors (Fautsch et al., 1998; Chrisman and Tindall, 2003).The TIEG1 gene is a primary response gene induced by TGF-β and is involved in TGF-β signal transduction (Cook and Urrutia, 2000). TIEG1 was shown to play a significant role in regulating cell proliferation and apoptosis in various cell types (Chalaux et al., 1999; Ribeiro et al., 1999; Tachibana et al., 1997). Overexpression of TIEG1 was found to mimic TGF-β action and induce apoptosis in several cell types, including human osteoblast cells (Subramaniam et al., 1995), pancreatic carcinoma cells (Jiang et al., 2009; Tachibana et al., 1997), mink lung epithelial cells (Chalaux et al., 1999) and liver cancer cells (Ribeiro et al., 1999). These data suggest that TIEG1 might be a tumour suppressor.
We previously reported that TIEG1 inhibited pancreatic cancer cell proliferation and promoted the gemcitabine chemosensitivity of cancer in vitro, through down-regulation of stathmin expression. In the present study, we employed our established lentiviral system to overexpress TIEG1 gene in pancreatic cancer cell; the roles of TIEG1 in pancreatic cancer cell growth in vitro and in vivo conditions were examined.
2. Materials and methods
2.1. Cell culture
Human pancreatic cancer SW1990 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and 100 U/ml penicillin/streptomycin (Invitrogen). The modified human embryonic kidney 293T cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 100 U/ml penicillin/streptomycin (Invitrogen). All cultures were maintained in humidified 37°C incubator with 5% CO2.
2.2. Lentiviral transduction
Lentiviral vectors expressing TIEG1 or EGFP (enhanced green fluorescent protein) was constructed as previously described (Jiang et al., 2009). The VSV-G pseudotyped lentiviral particles were produced by co-transfection of the transfer vector and three packaging vectors into 293T cells. Subsequent purification was performed using ultracentrifugation. The cells were transduced with lentivirus as described (Jiang et al., 2009).
2.3. Western blotting
After 72 h of lentiviral infection, cells were homogenized with lysis buffer containing a protease inhibitor cocktail (Sigma–Aldrich). Proteins were subjected to SDS/PAGE, and Western blot analysis was performed according to previously published protocols (Jiang et al., 2009). Briefly, cell lysates were separated by 12% SDS/PAGE and transferred to PVDF membranes. The membranes were probed for TIEG1 or actin protein using specific antibodies per the manufacturer's instruction. Signals were detected using the enhanced chemiluminescence substrate kit (Amersham Biosciences).
2.4. Cell growth
Triplicate samples of log phase infected cells (1×104) were seeded onto six-well plates. Viable cells were counted by 0.4% Trypan Blue exclusion daily for 3 days; then, the mean cell number for each group was calculated.
2.5. Cell cycle analysis
SW1990 cells transduced with lenti-EGFP or lenti-TIEG1 were seeded onto six-well plates at a density of 2×105 per well. Cells were harvested after 48 h and fixed in 70% ethanol for at least 1 h at 4°C. The cells were then stained with 50 mg/l propidium iodide which contain 0.1% Triton X-100 and RNase (100 mg/l). Cell cycle phase distribution was analysed with the use of ModFit LT 2.0 software using data obtained from triplicate experiments.
2.6. Colony formation assay
For colony formation assay, 5000 cells in complete RPMI 1640 medium were seeded onto a 10-cm culture dish and allowed to grow for 14 days to form colonies. Results were observed and photographed after Coomassie Blue staining.
2.7. Culture of SW1990 cells in 3-D (three dimensional) collagen gels
SW1990 cells transduced with lenti-EGFP or lenti-TIEG1 were collected and resuspended in complete RPMI 1640 and type I collagen gel (BD Biosciences) solution, which supported the growth of cells in a 3-D matrix (Wozniak and Keely, 2005). The solution containing 5000 cells was added into six-well tissue culture plates and was allowed to solidify at 37°C. Then, RPMI 1640 medium was added to the space surrounding collagen I gel embedded with cells and replenished every 2 days. After 14 days, the number of spheroids was counted at 20 random fields under a light microscope with magnification of ×40.
2.8. Tumour xenografts in nude mice
Six-week-old male athymic nude mice were injected subcutaneously with 1×106 SW1990 cells transduced with lenti-EGFP or lenti-TIEG1. The volumes of tumours were monitored at the indicated times and calculated according to the formula: 0.5×length×width2. All animal experiments were approved by the Animal Experimental Ethics Committee of Wenzhou Medical College.
2.9. Statistical analysis
Data were expressed as the mean±S.E.M. Statistical differences between groups were compared using the Student's t test. P≤0.05 was considered to be statistically significant.
3.1. Lentivirus-mediated overexpression of TIEG1 significantly inhibited pancreatic cancer cell growth in vitro
To examine the effect of TIEG1 overexpression, Western blot analysis was performed on SW1990 pancreatic cancer cells after lentivirus infection. As shown, TIEG1 protein overexpression was verified by Western blot analysis in SW1990 cells (Figure 1A). Overexpression of TIEG1 caused a dramatic reduction in the proliferation of SW1990 cells (Figure 1B). Moreover, a significant decrease was observed in the colony formation for SW1990 cells transduced with lenti-TIEG1 when compared with controls Figure 1C).
3.2. Overexpression of TIEG1-induced G1-phase arrest in SW1990 cells
TIEG1 overexpression-induced cell cycle arrest in SW1990 cells was investigated (Figure 2). A significant increase in the number of G1-phase cells was seen in SW1990 cells with lenti-TIEG1 infection (72.49% of the cell population compared with control value 45.75%, P<0.05), and the percentage of cells in S-phase and in G2-/M-phase were statistically significantly reduced, indicating that the growth-inhibiting effect of lenti-TIEG1 occurs via arrest at the G1- to S-phase transition.
3.3. Overexpression of TIEG1 gene inhibited both spheroid size and number in 3-D SW1990 cell culture
The images of SW1990 cell growth in 3-D culture model are shown (Figure 3A). Lentivirus-mediated overexpression of TIEG1 significantly decreased both spheroids size and number (Figures 3A and 3B) in 3-D culture model. This indicates a negative correlation between the expression of TIEG1 and the rate of pancreatic cancer SW1990 cell growth.
3.4. Decrease of human pancreatic cancer cell tumourigenicity by lenti-TIEG1 in nude mice xenografts
The effects of TIEG1 overexpression on the tumourigenicity of human pancreatic cancer cells were investigated. Nude mice were injected subcutaneously with SW1990 transduced with lenti-EGFP or lenti-TIEG1 (groups of seven mice each), and the volumes of tumours were monitored at the indicated times. By 6 weeks after cell injection, the mean tumour volumes of mice injected with the lenti-TIEG1-tranduced cells were statistically significantly smaller than those of mice injected with control cells. Overexpression of TIEG1 led to a marked decrease of tumourigenicity in human pancreatic cancer SW1990 cells (Figures 4A and 4B).
The present study is a study that thoroughly illustrates the effect of TIEG1 on pancreatic cancer SW1990 cell growth in vitro and in vivo. TIEG1 was successfully overexpressed in pancreatic cancer cells by our newly developed lentivirus-mediated gene transfer system (Jiang et al., 2009). TIEG1 is a recently characterized transcription factor regulated by TGF-β that induces growth inhibition and apoptosis when overexpressed in pancreatic carcinoma cells (Tachibana et al., 1997; Jiang et al., 2009) and hepatocellular carcinoma cells (Ribeiro et al., 1999). Subramaniam et al. (1998) have reported that TIEG1 expression correlates inversely with the stage of breast cancer. TIEG1 protein levels were shown to be markedly reduced in metastatic breast cancer tissues compared with normal breast epithelia. TIEG mRNA levels accurately discriminated between normal breast tissue and primary tumours with a sensitivity and specificity of 96 and 93%, respectively (Reinholz et al., 2004). Besides, TIEG1 is believed to play a crucial role in inhibiting cancer cell proliferation and promoting chemosensitivity of cancer, which made it a potential agent to try in gene therapy (Jiang et al., 2009). On the other hand, TIEG1 is regarded as an important factor for mediating TGF-β signalling, and deregulation of TGF-β signalling pathway is known to be associated with neoplastic transformation (Taipale et al., 1998; Massague et al., 2000).
Lentiviral vectors show exceptional promise as gene transfer agents and have been proven to be effective vehicles for transduction of cancer cells of various organs (Copreni et al., 2004). Gene therapy vectors derived from lentiviruses offer many potentially unique advantages over more conventional gene delivery systems (Connolly, 2002). Lentiviral vectors have recently been approved in many human diseases as the effective tool of human gene therapy (DiGiusto et al., 2010; Sumimoto and Kawakami, 2010).
In this study, lentivirus-mediated overexpression of TIEG1 was found to inhibit human pancreatic cancer SW1990 cell proliferation and induce G1-phase arrest in vitro. Lenti-TIEG1 significantly reduced SW1990 cell colony formation and inhibited cancer cell growth in 3-D cell culture system. Moreover, overexpression of TIEG1 gene by a lentivirus transfection system led to suppression of SW1990 xenografts growth in nude mice. Taken together, our study provided evidence that overexpression of TIEG1 induced by lentivirus system inhibited the pancreatic cancer cell growth in vitro and in vivo, which made it a candidate target for further study in gene therapy.
Lei Jiang, Fule Wang, Feiyan Lin, Yixiang Han and Chiqi Chen performed the research. Lei Jiang, Yingxia Tan, Shen-Meng Gao and Jianbo Wu participated in the study design and data discussion. Lei Jiang, Yingxia Tan and Jianbo Wu participated in the design of the study and coordination and helped to draft the manuscript.
The project was supported by the
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Received 12 December 2010/27 January 2011; accepted 28 April 2011
Published as Cell Biology International Immediate Publication 28 April 2011, doi:10.1042/CBI20100896
© The Author(s) Journal compilation © 2011 Portland Press Limited
ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)
Figure 1 Overexpression of TIEG1 by lentivirus-mediated transfection-induced growth inhibition in SW1990 pancreatic cancer cells
Figure 3 Overexpression of TIEG1 gene inhibited both spheroid size and number in 3-D SW1990 cell culture