The destruction of extracellular matrix by matrix metalloproteinases is a key event in cancer progression. The tissue inhibitors of metalloproteinases can restrain tumor growth by inhibiting these enzymes. We sought to determine whether overexpression of tissue inhibitor of metalloproteinase-3 (TIMP-3) could suppress the malignant phenotype of human prostate cancer cell line PC-3M. Stable overexpression of TIMP-3 inhibited cell proliferation significantly by MTT assay. Both early and late apoptosis were observed in TIMP-3 overexpressing cells, and flow cytometry analysis showed S-phase blocking of the cell cycle. Monolayer invasion assay and transwell invasion assay showed significantly decreased invasive potential in TIMP-3 overexpressing cells compared with control cells. Cell adhesion and motility were also lower after TIMP-3 was overexpressed. In vivo, cells stably overexpressing TIMP-3 completely lost the ability to form tumors after injection into nude mice. Transfection of TIMP-3 into established tumors by electroporation also had a significant antitumor effect. TIMP-3-treated tumor tissues had significant apoptosis by TUNEL assay. These results showed that overexpression of TIMP-3 inhibits invasion and proliferation of prostate cancer cells in vitro and inhibits tumor growth in vivo. The experiments suggest a potential use for TIMP-3 in the gene therapy of prostate cancer.
Matrix metalloproteinases are a large family of zinc-dependent endopeptidases.1 These enzymes can degrade the extracellular matrix,2 and contribute to the invasion and metastasis of malignant tumors as well as to physiologic processes of embryogenesis and wound repair. Within the extracellular matrix, matrix metalloproteinases are inhibited by a family of proteins known as tissue inhibitors of metalloproteinase, which have four family members.3 Tissue inhibitor of metalloproteinase-3 (TIMP-3) is a unique member of this family. It shows only a 25% homology with the three other members of this family.4 Earlier data have shown that the absence of TIMP-3 in the host enhances tumor growth and angiogenesis.5 The TIMP-3 protein can inhibit invasion and only TIMP-3 has the ability to induce apoptosis in select cell lines such as melanoma cells,6 hepatocellular carcinoma,7 breast cancer cells,8 and lung cancer cells.9 It has also been reported to suppress tumor growth through inhibition of endothelial cell migration and angiogenesis.10 Recently, it has been reported that prostatic tumor cell lines have no TIMP-3 expression, whereas TIMP-3 expression was found in 82% of benign prostatic hypertrophy samples.11 Although the mechanism for the inhibition of TIMP-3 has not been fully characterized, it seems possible that use of TIMP-3 protein as an inhibitor of tumor growth might provide a theoretically novel therapeutic strategy for prostate cancer.
To investigate the potential use of TIMP-3 for gene therapy of prostate cancer, we have used a plasmid expressing the TIMP-3 gene and have sought to examine the antitumor effect of enforced expression of TIMP-3 by gene-modified tumor cells in vitro and in vivo. Our results indicate that overexpression of the TIMP-3 gene is able to achieve significant inhibitory effects on local tumor growth and tumor invasion both in vitro and in vivo.
Materials and methods
Thirty primary prostate tumor samples and 30 adjacent normal prostate tissues were collected from patients who had taken the total prostatectomy for determination of TIMP-3 expression. Paraffin-embedded tissue sections of primary prostate tumors and the adjacent normal prostate tissues were used for immunohistochemical studies. Immunostaining was carried out using Vectastain Elite ABC avidin–biotin staining kit (Vector Laboratories, Inc. Burlingame, CA). Antibodies specific for TIMP-3 was obtained from Santa Cruz Biotech, Santa Cruz, CA. The criteria for this assay results are as follows: the percentage of cells positively stained in each section were categorized as follows: negative (−, samples with ⩽5% positive cells), low (+, 5–25% positive cells), moderate (++, 25–50% positive cells), and strong (+++, 50–100% positive), respectively. Human tissue samples were obtained from archival pathology specimens and all were made anonymous. Patient consents for use of tissue samples for research were obtained according to policies of the ethics committees of Jilin University and the China–Japan Union Hospital of Jilin University.
The human prostate cancer cell line PC-3M and DU14512 was obtained from the American Type Culture Collection (Manassas, VA). Cells were grown in Iscove's-modified Dulbecco's medium supplemented with 10% fetal bovine serum, 100 U ml–1 penicillin, and 100 μg ml–1 streptomycin and maintained in a humidified atmosphere at 37 °C in 5% CO2. (All culture media were purchased from Life Technologies, Inc., Gaithersburg, MD).
Construction of the TIMP-3 recombinant plasmid
Total TIMP-3 RNA was isolated from fresh human placental tissue by Trizol reagent (Gibco BRL, Gaithersburg, MD) as described by the manufacturer. We obtained the full length TIMP-3 cDNA by RT–PCR (Genbank accession no. NM000362). The primers were 5′-IndexTermAAGAATTCATGACCCCTTGGCTCGGG-3′ (sense) and 5′-IndexTermGGTCTAGATCAGGGGTCTGTGGCATT-3′ (antisense), which provided us with the EcoR I and Xba I (Promega, Madison, WI) restriction site (underlined) in both primers. The PCR products were sequenced and then cloned into pcDNA3.1 vector (Invitrogen, Carlsbad, CA) through the EcoR I and Xba I restriction sites to obtain recombinant plasmid pcDNA-TIMP-3.
Gene transfection and generation of stable cell lines
Transfection of PC-3M cells with pcDNA-vector and pcDNA-TIMP-3 plasmids was performed using LipofectAMINE 2000 (Invitrogen) according to the manufacturer's instructions. Cells were cultured for 18 h and then replaced with fresh medium supplemented with 10% fetal bovine serum. For stable transfection, the neomycin analog G418 (800 μg ml–1) was added to the culture for selection. The TIMP-3 overexpressing PC-3M cell clone (PC-3M-TIMP-3), the vector-transfected control clone (PC-3M-vector), and the blank control without transfection (mock) cells were screened and further confirmed by RT–PCR and western blot. Stably transfected PC-3M cells were maintained in media with 0.2 mg ml–1 of G418.
Total RNA was isolated from the transfected cells and control cells using Trizol reagent (Gibco BRL) as described by the manufacturer. The cDNA was synthesized using a Takara RNA PCR Kit (Takara, Dalian, China). The primers for TIMP-3 have been described above. The PCR product contained 633 bp. The primers for β-actin were 5′-IndexTermCTTCTACAATGAGCTGCGTG-3′ (sense), and 5′-IndexTermTCATGAGGTAGCAGTCAGG-3′ (antisense). The PCR product contained 305 bp. The PCR reaction was performed as follows: initial denaturation at 94 °C for 5 min, 25 cycles of 94 °C for 45 s, 55 °C for 60 s, 72 °C for 90 s, and a final extension for 10 min at 72 °C. The PCR products were analyzed by standard agarose gel electrophophoresis, and the bands were quantified with BandLeader 3.0 software.
Cell lysis, protein quantification, and western blot analyses were carried out as described earlier.13 Anti-TIMP-3 and anti-β-actin antibodies were obtained from Chemicon International (Temecula, CA). Protein bands were detected with enhanced chemiluminescence (ECL) western blotting reagents (Amersham, Buckinghamshire, UK) and Hyperfilm-enhanced chemiluminescence (Amersham).
In vitro assays of cell proliferation, apoptosis, and cell cycle
Cell proliferation was assayed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) staining kit (Sigma Chemical, St Louis, MO) as per the manufacturer's protocol. The results were obtained by measuring the absorbance at a wavelength of 550 nm using a Microplate Reader (BioRad, Richmond, CA). The rate of proliferation inhibition (%)=(1−absorbance of experimental group/absorbance of control group) × 100%. The test was repeated three times. To detect apoptosis, cells were stained with 0.1% acridine orange/ethidium bromide (Sigma, Poole, UK) and observed by fluorescence microscopy. For fluorescence-activated cell sorting analysis of apoptosis and cell cycle, stably transfected cells were collected and washed with cold PBS containing 4 mmol l–1 EDTA. After washing, cells were fixed with 70% cold ethanol, collected by centrifugation, and washed once with PBS. They were then suspended in PBS containing 20 μl ml–1 of propidium iodide (Sigma Chemical), 0.2% Triton X-100, and 40 μg ml–1 RNase A, and incubated for at least 30 min at 4 °C. The cells were then analyzed by flow cytometry (FACScan, Becton Dickinson, Franklin Lakes, NJ) using CellQuest software (Becton Dickinson).
Cell adhesion assays
For cell adhesion assays, 96-well culture dishes were precoated with 100 μl of 4 mg ml–1 fibronectin (Sigma Chemical) in 4 °C for 16 h. Stably transfected cells and control cells (2 × I04) were allowed to attach to the coated dishes for 90 min in IMDM supplemented with 0.1% BSA. The cells were washed twice with 1 × PBS, fixed in 2% paraformaldehyde, and the number of attached cells was determined by crystal violet staining.
Cell wound healing assay
To measure cell motility, 2 × 105 cells were seeded in 24-well plates and grown to confluence to form a monolayer. The growth of the cells was arrested for 24 h in 0.4% FCS/IMDM medium. During the last 4 h of this treatment, mitomycin-C was added at a final concentration of 4 mM to prevent cancer cell proliferation. A wound was created by scraping the cell monolayer with a micropipette tip (200 μl). After being washed once with PBS, the culture wells were refilled with IMDM medium with 0.1% BSA and incubated at 37 °C. Migration of cells into the denuded areas in the damaged region was measured at the beginning, and then at 12 and 24 h. ‘Average gap’ (average gap, percentage) was used to quantify the data. The wound at 0 h was considered 100% of average gap.
Monolayer invasion assay
NIH3T3 (target cells) were seeded into 35 mm dishes and when the cells had become confluent, 3 × 105 PC-3M-TIMP-3, PC-3M-vector, and mock cells (Attack cells) were overlaid on the confluent cells. Attack cells that went straight through the NIH3T3 monolayer were counted under a phase contrast microscope. Cell numbers in 10 fields (0.44 mm2 per sight) were counted and converted to the values (X) in 1 CM2, and invasion index of the cells was represented by log X.
Transwell invasion assay
Cell migration through transwell filters was analyzed as described earlier.14 Transfected and control cells were harvested from tissue culture flasks by Trypsin/EDTA and washed three times with IMDM media containing 1% fetal bovine serum. The cells were then resuspended in media containing 1% fetal bovine serum at a density of 106 cells ml–1, and 100 μl of the cell suspension was added to the upper well of the chamber. After 4 h of incubation, the cells, which had migrated to the lower surface of the membranes, were counted under a light microscope. The average number of cells in three replicate wells was determined for each group.
Antitumor effect in vivo
Male BALB/c nude mice, weighing 18–22 g, were purchased from the Beijing Institute for Experimental Animals. All animals were housed and experiments were performed according to the guidelines established by Jilin University for the ethical use of animals in research. For analyzing the tumor growth, 2 × 106 PC-3M-TIMP-3, PC-3M-vector, and untreated cells were inoculated subcutaneously into the right flank of nude mice (five mice per group), respectively. After 60 days, the animals were killed and the tumor sizes were measured. For analyzing the antitumor effect of TIMP-3 plasmid, mice were transplanted with 3 × 106/150 μl PC-3M cells into the right flank to generate a primary tumor. After the formation of palpable tumors (∼5 mm by day 15), the mice were randomized into three experimental groups (five mice per group). These were tested as follows: (a) mock transfection (PBS buffer alone), (b) pcDNA-vector control (20 μg per mouse), and (c) pcDNA-TIMP-3 (20 μg per mouse). The treatment was carried out as described earlier.13 After injection with 20 μg/50 μl of the different plasmids, tumors were pulsed with an electroporation generator (ECM 830, BTX). Mice were killed on day 30, and the tumors treated with either pcDNA-vector vector control or pcDNA-TIMP-3 were processed for section. These were then examined by hematoxylin and eosin staining, terminal deoxynucleotidyl transferase-mediated nick end-labeling (TUNEL) assay, immunohistochemical staining, western blot, RT–PCR, and apoptosis analyses.
Histochemistry and TUNEL assay
Blocks of tumor tissue were fixed in formalin, stained with hematoxylin and eosin, and processed for routine histologic examination. The TUNEL assay was performed with the in situ Cell Death Detection kit (Roche, Inc., Indianapolis, IN) as described earlier.15 With this method, biotin-labeled cleavage sites are detected by reaction with horseradish peroxidase-HRP-conjugated streptavidin and TUNEL-positive cells are visualized by diameno-benzidine (Sigma, St Louis, MO). The apoptotic index was calculated as follows: apoptosis index=(number of apoptotic cells/total cell number counted) × 100%.
The significance of the differences between various samples was determined using Student's two-tailed t-test. The significance of the differences between the median values of the data was determined using the two-tailed Mann–Whitney test. For all analyses, the level of significance was set at P<0.05. All statistical calculations were carried out using the SigmaStat statistical software package (SPSS, Chicago, IL). Data are presented as the mean±s.e. and represent three independent experiments.
Expression of TIMP-3 in prostate cancer cell lines and prostate cancer tissues
To determine whether TIMP-3 is expressed at the low levels in prostate cancer tissue, we compared the level of TIMP-3 expression in normal prostate tissues, prostate cancer tissues, and prostate cancer cell lines (PC-3M, DU145) using western blot and immunochemical analyses with an anti-TIMP-3 antibody. As summarized in Table 1 and shown in Figure 1, low TIMP-3 levels were found in both prostate cancer tissues and cell lines compared with normal prostate tissue (P<0.01).
Transfection of TIMP-3 increases the expression of TIMP-3 at both mRNA and protein levels
After transfection with pcDNA-vector and pcDNA-TIMP-3 vectors, stable TIMP-3 overexpressing cell clones (PC-3M-TIMP-3A and PC-3M-TIMP-3B) and empty vector-transfected cell clones (PC-3M-vector) were selected. To analyze the expression of TIMP-3 mRNA and protein, semi-quantitative RT–PCR and western blots were performed. As shown in Figure 2a and, the expression of TIMP-3 was increased sharply in PC-3M-TIMP-3A and PC-3M-TIMP-3B cells compared with PC-3M-vector and mock control cells (control cells without transfection) (P<0.01). Western blot showed a similar and statistically significant increase (Figure 2c and d).
TIMP-3 overexpression inhibits proliferation and induces apoptosis
We used an MTT assay to monitor the growth of the TIMP-3 overexpressing cells. Stably transfected TIMP-3 overexpressing cell clones showed significantly inhibited cell proliferation after 3 days in vitro (Figure 3a). To detect the apoptosis of the TIMP-3 overexpressing cells, the cells were stained with acridine orange/ethidium bromide staining and the results showed that both early stage apoptotic and late stage apoptotic cells were seen in PC-3M-TIMP group (Figure 3b). Early apoptotic cells were stained green and contained bright green dots in the nuclei as a consequence of chromatin condensation and nuclear fragmentation. Late apoptotic cells were stained orange and showed condensed and fragmented nuclei. Flow cytometry analysis showed that TIMP-3 induced significant apoptosis (∼16.7-fold) in PC-3M-TIMP-3 cells compared with the vector controls (Figure 3c; Table 2; Supplementary Table 1). A further analysis of the flow cytometric data showed that the TIMP-3 overexpressing cells accumulated significantly in S phase compared with the control (Table 2; Supplementary Table 1).
TIMP-3 overexpression inhibited invasion and adhesion of PC-3M cells
To examine the effect of TIMP-3 on cell motility, an in vitro wound-healing assay was performed, which measures the averaged and directional migration of a cell population. The results showed that the cells overexpressing TIMP-3 had a reduced migration rate compared with the control groups at 12 h (P<0.05) and 24 h (P<0.01) (Figure 4a). To further investigate the effect of TIMP-3 overexpression on cell invasion, we determined the ability of PC-3M-TIMP-3 and PC-3M-vector cells to invade through an NIH3T3 cell monolayer and Matrigel in transwell chambers. The results of the monolayer invasion assay are shown in Table 3 and Supplementary Table 1. Overexpression of TIMP-3 significantly inhibited invasion of PC-3M cells compared with control groups (P<0.05). The transwell invasion assay seen in Figure 4b also showed the similar inhibition (P<0.01). To elucidate the mechanism of the inhibition of invasion, we examined the effect of TIMP-3 on the adhesion of the cells. The results show that PC-3M-TIMP-3-transfected cells had reduced adhesive capacity compared with the control groups (Table 3; Supplementary Table 1).
Antitumor activity of TIMP-3 in vivo
To evaluate the tumorigenic properties of the TIMP-3 overexpressing cells in vivo, PC-3M-TIMP-3, PC-3M-vector, and untransfected cells were transplanted into nude mice, and the tumor growth was observed for 60 days. The mice that had been injected with untransfected and PC-3M-vector-transfected cells developed tumors at the sites of injection after 25±2.9 days. In contrast, no tumors were detected in mice injected with PC-3M-TIMP-3 cells (data not shown).
To determine whether TIMP-3 could inhibit tumor growth using a xenograft tumor model, parental PC-3M cells were injected into the right flank of mice. After palpable tumors had formed, the tumors were injected with the pcDNA-vector and pcDNA-TIMP-3 vectors and transfection was then enhanced with an electroporator.13 On day 40, the animals were killed and final tumor weights and volumes were determined. The pcDNA-TIMP-3 vector had a remarkably strong effect on tumor growth compared with the pcDNA-vector and mock group (P<0.01, Table 4; Supplementary Table 1). In addition, the tumors of pcDNA-TIMP-3 group were paler and had fewer tumor vessels compared with the pcDNA-vector group and mock group (Figure 5a). Immunohistochemical analysis showed that the levels of TIMP-3 were increased by treatment with TIMP-3 compared with the control groups (Figure 5b). Hematoxylin and eosin staining and TUNEL assays showed that tumors in the pcDNA-TIMP-3 group had undergone massive apoptosis and necrosis (Figure 5c). The apoptosis index was 42.3±2.8% in the pcDNA-TIMP-3 group, significantly higher than pcDNA-vector group (6.3±1.9%) and mock group (5.9±1.7%), P<0.01.
Destruction of the extracellular matrix is associated with cancer progression including invasion and metastasis.16 The family of matrix metalloproteinases has a major function in these processes.17 In the extracellular matrix, the activity of matrix metalloproteinases can be inhibited by a family of natural inhibitors, which is termed as tissue inhibitors of metalloproteinase.18 The balance between matrix metalloproteinases and tissue inhibitors of metalloproteinase has been implicated as a regulator of cancer invasion and metastasis.19 There are four family members in tissue inhibitors of metalloproteinase and TIMP-3 is different from TIMP-1, -2, and -4 because, except for inhibition of malignant cell invasion, it can also directly promote the apoptosis of cancer cells.20
In this study, we confirmed that TIMP-3 has a key function in promoting the proliferation and invasion of a prostate cancer cell line in vitro and in vivo. Western blot and immunohistochemical assays showed that TIMP-3 is expressed at low levels both in human primary prostate cancer tissues and in prostate cancer cell lines. These data are consistent with other studies7, 11, 21 that have shown loss or low expression of TIMP-3 in a variety of human tumors including prostate cancer. RT–PCR and western blot analyses showed that TIMP-3 is expressed at low levels in prostate cancer cell line PC-3M. Our results show that after stable transfection with a TIMP-3 vector, TIMP-3 overexpression was able to suppress the growth of these cells. These data are consistent with other studies in malignant melanoma6 and breast cancer.22 Our results also show that TIMP-3 overexpression was able to induce apoptosis, proved by acridine orange/ethidium bromide staining and flow cytometry assays. Acridine orange/ethidium bromide staining revealed that the stably transfected cell populations included both early apoptotic and late apoptotic PC-3M cells. Early apoptotic cells were stained green and contain bright green dots in the nuclei as a consequence of chromatin condensation and nuclear fragmentation. Late apoptotic cells were stained orange and showed condensed and fragmented nuclei.23 These results suggested that TIMP-3 had not only an effect on induction of early apoptosis, but also late apoptosis in these cells. Moreover, flow cytometry analysis showed that the rate of apoptosis in the TIMP-3 overexpression group was significantly higher than that in the untreated PC-3M cells and the vector-treated PC-3M cells. Ahonen et al.6 found that TIMP-3 promotes apoptosis through stabilization of distinct death receptors and activation of their apoptotic signaling cascade through caspase-8 in melanoma cells. Bond et al.24 showed that TIMP-3 overexpression induces an apoptotic pathway initiated through an Fas-associated death domain-dependent mechanism, which involves mitochondrial activation in rat smooth muscle cells. Differing from our research, Bian et al.25 showed that TIMP-3 may function as a cell death inducer instead of inhibiting MMPs and have identified this cell death as necrotic death rather than apoptosis. In our study, induction of cell death was associated with blocking of the S phase of the cell cycle. This observation is consistent with findings by Yang and Hawkes26 who reported that inducing DNA synthesis of cells by TIMP-3 leads to detachment of the cells from the extracellular matrix. Our findings differ from the research, which has shown an elevation in the number of cells in S and G2/M phases of the cell cycle.27 The mechanisms by which TIMP-3 induces cell death and affects the cell cycle remain to be fully determined further.
In this study, we also showed an inhibitory effect of TIMP-3 overexpression on cell invasion and adhesion, a finding that has been corroborated by many studies.22, 28, 29 There seem to be some exceptions, for example, in JB6 tumor cells TIMP-3 overexpression failed to inhibit invasion through reconstituted basement membranes,30 suggesting a complex function for TIMP-3 in tumorigenesis.
Prostate cancer is the most common cancer in American men.31 In China, most prostate cancers are diagnosed in an advanced stage when there is little chance of cure by radical prostatectomy.23, 32, 33, 34 Thus, it has become important to develop new treatment strategies for metastasic prostate cancer.35 Invasion and metastasis of tumors cells, including prostate cancer cells, is dependent on their ability to destroy the extracellular matrix.36 Thus, inhibition of these processes may lead to effective treatments for prostate cancer.
It has been suggested that gene therapy strategies could be applied to the treatment of prostate cancer.37, 38 Most of them use recombinant adenoviruses or lentiviruses,39, 40 but because of the possibility of recombination with wild-type adenovirus and the antiviral immune response, these approaches have been limited to research use.41 More recently, electroporation has been applied in vivo and shown to be potentially safe for clinical use.42, 43 Electroporation is able to cause a transient permeabilization of the plasma membrane, which allows exogenous macromolecules to enter the cells. Our data here and elsewhere13, 15 indicate that electroporation has been shown to be useful to transfer vector DNA into target cancer tissues and may allow repeated administrations without immune response. With this approach, the pcDNA-TIMP-3 vector was found to have a significant inhibitory effect on established prostate tumor growth in an in vivo xenograft mouse model through both growth inhibition and increased apoptosis. We also found inhibition of tumor-associated angiogenesis in a TIMP-3 treatment group. The mechanism may be mediated through inhibition of functional capillary morphogenesis.21 Measurements of tumorigenicity in vivo study showed that TIMP-3 overexpressing cells completely lost the ability to form tumors. This finding is similar with the result of Bian et al.25 which showed that although TIMP-3 overexpressing cells can grow in soft agar, they failed to grow in nude mice, and Lam et al.44 who showed growth inhibition of human glioma tumor xenografts in immunodeficient mice. It has also been shown that TIMP-3 overexpression in human colon carcinoma cells induces cell death by stabilizing TNF-alpha receptors.45 These data together show that TIMP-3 has a tumor suppressing activity and that cell–cell interaction is very important in the process of tumorigenicity.
In summary, our results have important implications for the potential use of TIMP-3 gene overexpression on prostate cancer gene therapy. Recently, we have had notable success in a mouse tumor treatment by using attenuated Salmonella enterica serovar typhimurium as a potential gene delivery system.46 Our future studies are now aimed at using Salmonella tumor-targeting delivery systems containing combined antitumor therapies to elucidate the effect of TIMP-3-based gene therapy of prostate cancer.
Conflict of interest
The authors declare no conflict of interest.
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This work was funded by National Natural Science Foundation of China (No. 30801354) and Jilin Provincial Science & Technology Department (No. 20080154). We thank Professor Frederick William Orr for his critical reading and revising our paper.
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Strong association of tissue inhibitor of metalloproteinase (TIMP)-2 and -3 promoter single nucleotide polymorphisms with risk of colorectal cancer in ethnic Kashmiri population – a case control study
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