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Experimental Therapeutics |
Departments of Molecular and Cellular Oncology [Y. W., D-H. Y., B. S., Y. D., R. S., Y. Z., M-C. H.], Surgical Oncology [D-H. Y., M-C. H.], Gastrointestinal Medical Oncology [B. W., K. X.], The University of Texas, M. D. Anderson Cancer Center, Houston, Texas 77030
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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p202 is an IFN-inducible protein, and its expression is associated with growth inhibition (4
, 5)
. The findings that p202 interacts with cell cycle transcriptional regulators, such as E2F-1/DP-1, E2F-4/DP-1, AP-1 (c-Fos/c-Jun), and c-Myc, and represses their transcriptional activities have provided insight into the molecular mechanism by which p202 mediates growth inhibition (6, 7, 8, 9, 10)
. We have documented previously that p202 inhibits human cancer cell growth in vitro and suppresses tumor growth in vivo (11
, 12)
. Furthermore, we showed that p202 expression sensitizes breast cancer cells to TNF-
-induced apoptosis. The mechanism responsible for the p202-mediated sensitization is likely attributable to the inactivation of TNF--induced NF-
B4
by p202 (12)
. In light of the antiapoptotic role of NF-B in TNF--mediated apoptosis (13)
, we have hypothesized that NF-B inactivation by p202 leads to the abolishment of the antiapoptotic process and results in sensitizing cancer cells to TNF--induced apoptosis. On the other hand, the aberrant NF-B activity has been implicated, at least in part, in tumorigenesis and the chemoresistant phenotype of certain human cancers including pancreatic cancer (14
, 15)
. Thus, it is likely that p202-based gene therapy may be particularly useful in targeting tumors that contain such aberrant NF-B activity. In this study, we tested that possibility in pancreatic cancer cells that possess the constitutively active NF-B (16)
. We showed that, in addition to the growth inhibition in vitro and tumor suppression in vivo, p202 expression was found associated with suppression of metastasis and angiogenesis in an orthotopic pancreatic cancer xenograft model. Importantly, we demonstrated a treatment efficacy of using p202/liposome gene therapy in a pancreatic cancer xenograft model. Together, our results raise a possibility of using p202-based gene therapy strategy in pancreatic cancer treatment.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Colony-forming Assay.
Cells were transfected with a p202 expression vector (CMV-p202; in which p202 cDNA is driven by CMV promoter) or an control vector (pcDNA3). Both plasmids contain the neomycin-resistant gene. Three weeks after transfection and G418 selection, cell colonies were stained by 0.5% crystal violet containing 20% ethanol.
Western Blot Analysis.
Protein lysate was prepared with RIPA-B cell lysis buffer containing 20 mM Na2PO4 (pH 7.4), 150 mM NaCl, 1% Triton X-100, 100 mM NaF, 2 mM Na3VO4, 5 mM phenylmethylsulfonyl fluoride, 1% aprotinin, and 10 µg/ml leupeptin. Rabbit anti-p202 polyclonal antibody was kindly provided by Dr. Divaker Choubey (Loyola University, Chicago, IL). Donkey antirabbit IgG peroxidase (Jackson) was used as secondary antibody. Western blots were developed by enhanced chemiluminescence (ECL; Amersham).
Northern Blot Analysis.
Total RNA was isolated from PANC-1, pcDNA3, p202-1, and p202-2 cells using a TRIZOL RNA isolation kit (Life Technologies, Inc.). Twenty µg of total RNA were separated by electrophoresis under denaturing conditions and then transferred to a Hybond N+ membrane. Full-length p202 cDNA isolated from CMV-p202 plasmid by BamHI digest was gel-purified and 32P-labeled by using a random-labeling kit. Hybridization was performed at 65°C overnight in solution containing 1% BSA (w/v), 0.2 M sodium phosphate, 1 mM EDTA, 7% SDS (w/v), 15% formamide, and 40 µg/ml salmon testes DNA (Sigma Chemical Co.). The blot was subsequently washed three times in 40 mM sodium phosphate, 1 mM EDTA, 1% SDS (w/v) at 65°C for 5 min/wash and twice at 70°C for 10 min/wash. The p202 RNA (1.8 kb) was visualized by using a PhosphorImager.
Transfection and luc Assays.
PANC-1, pcDNA3, p202-1, and p202-2 cells were transfected with 0.5 µg of B-luc construct and 0.1 µg of the internal transfection control (pRL-TK; Promega). Forty-eight h after transfection, cells were harvested, and luc activity was measured using the dual luc assay system (Promega) according to the protocol supplied by the manufacturer. The B-luc activity was normalized by the internal control luc activity of pRL-TK. To determine the p202 dose effect, PANC-1 cells were cotransfected with 50 ng of CMV-luc and an increasing amount (0, 0.5, or 2 µg) of CMV-p202. The total amount of DNA transfected at each p202 dose was kept constant (2.05 µg) by adding an appropriate amount of pcDNA3 vector. luc activity was measured 48 h after transfection. The relative activities were calculated by setting the luc activities obtained from transfections without CMV-p202 (0 µg) at 100%. The data represent mean ± SD of two independent experiments.
Soft Agar Assay.
Aliquots of cells (1 x 104) were mixed at 37°C with 0.5% agarose (Sea Plaque, low gelling temperature; FMC Bioproducts, Rockland, ME) in complete medium and gelled at 4°C for 15 min over a previously gelled layer of 1% agarose in complete medium in six-well dishes. After incubation for 3 weeks, 200 µl of 1 mg/ml p-iodonitrotetrazolium violet were added and incubated for an additional 24 h. Colonies were photographed using a Zeiss microscope and counted using computer software associated with the microscope.
Ectopic Tumorigenicity Assays in Nude Mice.
Aliquots of cells (1 x 106) in 200 µl of PBS were injected s.c. on both sides of the abdomen of female nude mice, 4–5 weeks of age. Tumor sizes were measured with a caliper every week. The tumor volume was calculated using the formula: Volume = S x S x L/2, where S is the short length of the tumor in cm and L is the long length of the tumor in cm.
Orthotopic Tumorigenicity and Survival Assays in Nude Mice.
Aliquots of cells (1 x 106) were suspended in 50 µl of PBS as single-cell suspensions. Nude mice were anesthetized with methoxyflurane and placed in the supine position. An upper midline abdomen incision was made, and the pancreas was exteriorized. Tumor cells were injected into the tail of the pancreas, and the abdomen was closed using wound clips. Animals were sacrificed 3 months after tumor inoculation. Tumors in the pancreas were harvested and weighed. Livers were fixed in Bouin’s solution for 24 h to differentiate the neoplastic lesions from the organ parenchyma, and the metastases on the surface of liver were counted with the aid of a dissecting microscope. For survival assays, daily survival of mice was monitored and recorded as dead or euthanized when the animals reached the moribund stage.
In Vitro Invasion Assay.
The procedure was followed as described previously (17)
, except for the following modification. The 24-well chamber with an 8-µm pore size polycarbonate filter (Costar Co., Cambridge, MA) was coated with Matrigel (Becton Dickinson Labware, Bedford, MA) according to the manufacturer’s protocol.
Zymography.
Cells were grown to 70% confluency in DMEM/F12 medium containing 10% FBS and switched to serum-free medium (17)
. After 2 days of incubation, the conditioned medium was collected, passed through a 0.22 µm filter, and then concentrated to a small volume on Centricon YM 30 filter units. Four µg of each sample were loaded on the gel. For the positive control, 0.25 µl of FCS was used. Zymography was performed using gelatin-embedded SDS gels as described previously (17)
.
Immunohistochemistry.
Tumor tissue sections (5 µm thick) of the formalin-fixed, paraffin-embedded specimens were deparaffinized in xylene and rehydrated in graded alcohol. The endogenous peroxidase was blocked by the use of 3% hydrogen peroxide in PBS for 12 min. The samples were incubated for 20 min at room temperature with a protein blocking solution containing 5% normal horse serum and 1% normal goat serum and in PBS. Samples were then incubated at 4°C in a 1:50 dilution of rabbit polyclonal anti-IL-8 antibody (Biosource International, Camarillo, CA) or 1:50 dilution of rabbit anti-VEGF antibody, followed by the incubation with peroxidase-conjugated antirabbit IgG at room temperature for 1 h, and with diaminobenzidene (Research Genetics) for 5 min. The sections were counterstained with Mayer’s hematoxylin (Biogenex Laboratories, San Ramon, CA) and mounted with a Universal mount (Research Genetics). When examined under a microscope, a positive reaction was indicated by a reddish-brown precipitate in the cytoplasm or the nucleus. Tissue sections vessels in solid tumors growing in the pancreas of nude mice were determined under light microscope after immune staining of sections with anti-CD31 antibodies. Cryostat sections of tumors were fixed with 2% paraformaldehyde in PBS (pH 7.5) for 10 min at room temperature and processed for immunostaining as described above for paraffin-embedded tissues.
p202 Gene Therapy Treatment in a Human Pancreatic Cancer Xenograft Model.
Aliquots of PANC-1 cells (1 x 106) in 200 µl of PBS were injected (per site) s.c. on both sides of the abdomen of female nude mice, 4–5 weeks of age. After tumors reached 5 mm in diameter, mice received treatment with the p202/SN2 complex through intratumor injection. For each injection, 15 µg of CMV-p202 or the control vector, i.e., a luc cDNA driven by a CMV promoter (CMV-luc), was complexed with 30 µg of SN2. CMV-p202/SN2 complex was administrated twice a week. Mice in the control group were injected with either CMV-luc/SN2 complex or SN2 alone. Tumor size and treatment related side effect were monitored twice a week. SN2 is a liposome-based, nonviral delivery system developed recently by our group.5
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B (16)
.] As shown in Table 1
, with all cell lines tested, the number of G418-resistant colonies in the p202-transfected cells was consistently fewer than that of the pcDNA3-transfected cells with a reduction ranged from 75% (BxPC-3) to 100% (CFPAC-1). This result suggests that p202 possesses a strong growth-inhibitory activity in human pancreatic cancer cells. Consistent with that observation, our effort to isolate p202 stable pancreatic cancer cell lines has yielded only 2 p202-expressing PANC-1 clones (i.e., p202-1 and p202-2) of 20 G418-resistant clones screened by Western analysis. p202-1 expresses a higher level of p202 protein than p202-2 (Fig. 1a)
, and that correlates well with the levels of p202 mRNA expression of these clones as determined by a Northern blot analysis (Fig. 1b)
. To test whether p202 expression inhibits NF-B activity in the p202-expressing cells (12)
, we performed a gel-shift assay and found that the p202 expression level in p202-1 and p202-2 cells is efficient to abolish the DNA binding activity of NF-B (data not shown). The reduced NF-B DNA binding activity in p202-expressing cells was further confirmed by the reduced NF-B-mediated promoter activity in p202-1 and p202-2 cells as compared with that in the control cells (pcDNA3; Fig. 1c
). As shown in Fig. 2a
, the growth rate of p202-1 cells is the slowest as compared with that of PANC-1, vector control (pcDNA3), and the low p202 expressor, p202-2 cells. The different levels of p202 expression may account for the different growth rates seen between p202-1 and p202-2 cells. This result is consistent with our previous observation that the extent of growth inhibition is p202 dose dependent (11)
. To further confirm that observation, we performed a transient transfection assay in which a fixed amount (50 ng) of CMV-luc was cotransfected with an increasing amount (0, 0.5, and 2 µg) of CMV-p202 in PANC-1 cells. Because the apparent luc activity is indicative of living cells, we showed that p202 expression caused overall growth inhibition in a dose-dependent manner (Fig. 2b)
. Under the same condition, no apparent apoptosis was observed as determined by flow cytometry analysis to detect sub-G1 apoptotic cells (data not shown). Thus, our results support the idea that the level of p202 expression is proportional to its growth-inhibitory activity. Consistent with the ability of p202 to suppress transformation phenotype (11
, 12)
, both p202-1 and p202-2 cells showed a significant reduction in the number of soft-agar colonies as compared with that of the control cell lines, i.e., PANC-1 and pcDNA3 (Fig. 2c)
. Our results thus indicate that p202 is a potent growth inhibitor in suppressing pancreatic cancer cell growth.
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, p202-1 tumors grew significantly slower than the vector-transfected PANC-1 tumors. p202-2 tumors (which express less p202 than p202-1 tumors) on the other hand started to show a modest growth reduction 12 weeks after injection as compared with that of the control tumors. Similar to p202-mediated growth inhibition, the extent of antitumor activity observed here appears to be dependent on the level of p202 expression. To further examine the p202-mediated antitumor activity in an organ (pancreas)-specific environment, we injected p202-1 and p202-2 cells directly into mouse pancreas. Three months after injection, mice were sacrificed, and tumor growth was measured. We found that although PANC-1 tumors and vector control tumors grew readily in mouse pancreas at 100% frequency (five of five), p202-1 and p202-2 cells are tumorigenic at a much lower frequency, i.e., 20% (one of five) and 40% (two of five), respectively (Table 2)
. Furthermore, the average tumor size (measured by weight) of the control tumors was about five times that of p202-1 or p202-2 tumors. When the survival rate was measured, we observed that mice bearing p202 tumors had a longer survival than those bearing control tumors without p202 expression (Fig. 3b)
. In particular, mice bearing p202-1 or p202-2 tumors had 50% survival at 90 days after implantation as opposed to the mice bearing either PANC-1 or vector control tumors that showed 0% survival at the same time. Our results clearly demonstrated a potent antitumor activity of p202 in orthotopic pancreatic cancer xenograft models but to a lesser extent in the ectopic environment.
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. This result suggests a possible antimetastasis function of p202 in pancreatic cancer cells. To test that possibility in vitro, we used a double-chamber assay (17)
in which the test cells were grown in the top chamber, and the bottom chamber was filled with conditioned medium containing a chemoattractant, e.g., laminin. A Matrigel-coated membrane was used to separate the two chambers. To migrate from the top chamber to the bottom chamber, cells must digest away the reconstituted basement membrane matrix by producing secretory proteases, such as MMPs, and then penetrate through pores on the membrane. Thus, this assay somewhat mimics a typical metastatic process, and the number of cells found on the bottom side of the membrane [which can be visualized by Giemsa staining, i.e., blue cells (Fig. 4a)
] is indicative of the metastatic potential of the test cells. On the basis of these criteria, we found that p202-1 possesses the least metastatic potential among the cell lines tested (Fig. 4b)
. p202-2 and p202-pool cells (i.e., the pooled p202-transfected clones) have slightly lower metastatic potential than that of PANC-1 or vector control cells (Fig. 4, a and b)
. Because MMP-2 (Mr 72,000) is one of the important MMPs secreted by cancer cells during the metastatic process (18)
, we examined whether MMP-2 expression is altered in p202-1 and p202-2 cells by using a zymography to analyze the MMP-2 activity in each cultured medium. As shown in Fig. 4c
, the level of MMP-2 secreted by p202-1 cells is greatly reduced, but PANC-1, vector control (pcDNA3-1 and pcDNA3-2), and p202-2 cells maintain a high level of secreted MMP-2. Although the MMP-2 level is not significantly reduced in p202-2 cells, it is likely that the low p202 expression level accounts for the difference in both the MMP-2 activity and the in vitro invasiveness between p202-1 and p202-2 cells. The MMP-2 activity in serum serves as a positive control. Together, our in vivo and in vitro results support the idea that p202 expression suppresses metastatic potential of pancreatic cancer cells.
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shows that the number of blood vessels [stained by antibody against a blood vessel marker, i.e., CD31 (20)
] was significantly reduced in p202-1 tumor as compared with PANC-1 tumor. Because the expression of angiogenic factors such as IL-8 and VEGF are critical for the onset of angiogenesis (19)
, we examined a possible correlation between the expression of these proteins and the reduced angiogenesis in p202-1 tumors. Using immunohistochemical analysis with antibody specific to IL-8 or VEGF, we showed that p202-1 tumor has much reduced IL-8 and VEGF protein staining (dark gray color) as compared with that of PANC-1 tumor (Fig. 5)
. These results strongly suggest that p202 expression in pancreatic tumors is associated with suppression of angiogenesis.
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and Table 2
), we tested a potential therapeutic effect of p202 gene therapy treatment in a s.c. pancreatic cancer model. Briefly, the mice bearing s.c. PANC-1 tumors were treated by intratumor injection of CMV-p202/SN2 complex twice a week for 8 weeks. (SN2 is a lipid formula developed in our laboratory, and when complexed with DNA, it enhances in vivo and in vitro transfection efficiency.5
) The control groups consisted of tumor-bearing mice treated with SN2 alone (SN2) or CMV-luc/SN2 complex. As shown in Fig. 6
, although there was no significant difference in tumor growth between SN2 and CMV-luc/SN2 complex treatment groups, the CMV-p202/SN2-treated tumors exhibited a slower growth rate than that of the control treatments. This proof-of-concept experiment clearly shows a feasibility of using CMV-p202/SN2 complex to achieve a therapeutic effect on an ectopic pancreatic cancer xenograft model.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B. This observation is significant because persistent NF-B activity was associated with antiapoptosis and chemoresistance in human cancers (14
, 15)
, and that sets the stage for us to test whether p202 expression can sensitize these pancreatic cancer cells to chemotherapy treatment. To date, very few chemo-drugs are found beneficial in treating pancreatic cancer (3)
, and the constitutive activation of NF-B is likely to contribute to such chemoresistant phenotype. It is therefore possible that a p202-based gene therapy may restore chemosensitivity and lead to a potential p202/chemo-drug combined treatment for pancreatic cancer. Experiments are under way to test this hypothesis. We observed a more pronounced p202-mediated antitumor activity (i.e., reduced tumor growth and prolonged survival) in the orthotopic pancreatic cancer xenograft model than that in a s.c. ectopic xenograft model. Although the reason for the differential therapeutic effect remains unknown, it does indicate that the orthotopic xenograft model is not only a more relevant but also a better model to evaluate future treatment efficacy of p202-based gene therapy than the s.c. ectopic model. We also observed a decreased vascularity (CD31 staining) and a decreased level of angiogenic factors, e.g., IL-8 and VEGF, in p202-expressing tumors. Thus, our results suggest that p202 inhibits the expression of angiogenic factors and that in turn leads to suppression of angiogenesis. Remarkably, we observed that mice bearing p202-expressing tumors apparently lacked liver metastasis, and this result correlates with a reduced invasiveness and a reduction in MMP-2 expression in vitro. Together, our data present the first evidence to suggest that p202 expression is associated with suppression of both angiogenesis and metastasis.
Finally, we showed a significant therapeutic effect using CMV-p202/SN2 treatment in an s.c. pancreatic cancer xenograft model. It nevertheless implicates a feasibility of using a p202-based gene therapy treatment for pancreatic cancer. Given that the orthotopic model is a better system to study the efficacy of p202 antitumor activity as indicated in this report, it is possible that CMV-p202/SN2 treatment may yield even better antitumor activity in an orthotopic xenograft model. Success of a p202-based gene therapy in orthotopic model will pave the way for future experiments to determine the efficacy p202/chemo-drug combined treatment in preclinical gene therapy settings.
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FOOTNOTES |
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1 Funded by Grant CA77858 from the NIH and Contract DAMD17-00-1-0312 from the Department of Defense (both to M-C. H.), Grant 003657-0082-1999 (to D-H. Y.) from the University Cancer Foundation at the University of Texas M. D. Anderson Cancer Center and Texas Advanced Technology Program, Grant RPG-00-054-01-CMS from the American Cancer Society (to K. X.), and Cancer Center Core Grant 16672. 
2 These authors contributed equally to this work.
3 To whom requests for reprints should be addressed, at Department of Molecular and Cellular Oncology, The University of Texas, M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: (713) 792-3668; Fax: (713) 794-0209; E-mail: mchung{at}mdanderson.org
4 The abbreviations used are: NF-B, nuclear factor-B; CMV, cytomegalovirus; luc, luciferase; VEGF, vascular endothelial growth factor; MMP, matrix metalloproteinase; IL, interleukin.
5 Y. Zou and M-C. Hung, unpublished results.
Received 2/28/01. Accepted 7/26/01.
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