MDA-7/IL-24 suppresses human ovarian carcinoma growth in vitro and in vivo
© Gopalan et al; licensee BioMed Central Ltd. 2007
Received: 07 December 2006
Accepted: 02 February 2007
Published: 02 February 2007
Previous studies showed that the human melanoma differentiation-associated gene-7 (mda-7), also known as interleukin-24 (IL-24), has potent antitumor activity against human and murine cancer cells. However, the majority of these studies were limited to in vitro testing. In the present study, we investigated the antitumor activity of mda-7/IL-24 against human ovarian cancer cells both in vitro and in vivo.
In vitro, treatment of ovarian cancer cells with an adenoviral vector carrying the mda-7 gene (Ad-mda7) resulted in inhibition of cell proliferation and induction of cell cycle arrest, leading to apoptosis. We did not observe inhibitory activity in Ad-mda7-treated normal cells. In vivo, treatment of subcutaneous tumor xenografts with Ad-mda7 resulted in significant tumor growth inhibition when compared with that in control groups (p < 0.001). Molecular analysis of ovarian tumor tissue lysates treated with Ad-mda7 showed that MDA-7 protein expression was associated with activation of the caspase cascade.
Our results show that treatment of ovarian cancer cells with mda-7/IL-24 results in growth suppression both in vitro and in vivo.
Ovarian cancer is the second most common gynecological malignancy in the United States and is the fifth leading cause of death in women worldwide . An estimated 25,000 women have been diagnosed with ovarian cancer in 2005, and 50% of these patients have died of the disease. Despite advances in the treatment of ovarian cancer, the overall long-term survival rate of this disease has not improved significantly. Hence, developing and testing new therapeutic agents and strategies for it are warranted.
The human melanoma differentiation-associated gene-7 (mda-7), also known as interleukin-24 (IL-24), encodes a protein of 206 amino acids with a predicted molecular mass of 23.8 kDa . Gene transfer studies have demonstrated that mda-7 exerts its antitumor activity in a spectrum of cancer cells via multiple cell-type-dependent signaling pathways, resulting in apoptosis [2, 3]. Recent in vitro studies from our laboratory and those performed by others showed that mda-7/IL-24 can suppress ovarian cancer cell growth in vitro and is dependent on the Fas/FasL or stress activated p38 mitogen-activated protein kinase (MAPK) pathway [4, 5]. Additionally, investigators have shown that an adenoviral vector carrying the mda-7 gene (Ad-mda7) radiosensitizes ovarian cancer cells in vitro . Although mda-7/IL-24 has displayed antitumor activity against ovarian cancer cells in vitro, the documented evidence of this activity in vivo is limited.
In the present study, we investigated the antitumor properties of Ad-mda7 against human ovarian cancer cells both in vitro and in vivo in a mouse model. We demonstrated that Ad-mda7 selectively exerts its antitumor effects against ovarian cancer cells, leading to suppression of tumor growth in vivo.
Ad-mda7 inhibits ovarian cancer cell proliferation, induces cell cycle arrest, and regulates signaling molecules associated with apoptosis
Prior to the start of our study, we determined the transduction efficiency of an adenoviral vector carrying green fluorescent protein in MDAH2774, OVCA420, and IOSE-80 cells at different doses. We observed a dose-dependent increase in transduction of both tumor and normal cells, as more than 60% of the cells were transduced at 3000 vp/cell (data not shown). Based on this result, we used Ad-luc and Ad-mda7 at 3000 vp/cell in all subsequent experiments.
Cell cycle distribution in ovarian cancer and normal cells treated with PBS, Ad-luc or Ad-mda7
MDAH 2774 (%)
OVCA 420 (%)
IOSE 80 (%)
72.2 ± 10.5
20.4 ± 9.3
7.3 ± 1.5
68.2 ± 4.9
21.7 ± 5.2
10.0 ± 1.6
40.2 ± 1.4
42.0 ± 2.6
18.0 ± 1.6
52.5 ± 6.2
25.2 ± 3.0
22.2 ± 3.3
68.2 ± 6.3
13.1 ± 2.3
18.2 ± 4.6
55.3 ± 2.2
26.3 ± 1.2
19.0 ± 1.5
243 ± 38
27.0 ± 14.6
50.2 ± 16.4
57.0 ± 4.8
14.4 ± 2.4
29.0 ± 4.2
48.8 ± 3.6
28.6 ± 2.1
23.0 ± 2.5
Ad-mda7 inhibits ovarian tumor xenografts in vivo
Previous studies from our laboratory showed that intratumoral administration of Ad-mda7 inhibits the growth of lung tumor xenografts [13, 14]. Additionally, we showed that systemic nanoparticle-based delivery of mda-7 inhibits experimental lung metastasis . Based on these reports and our present observation of the ability of Ad-mda7 to inhibit ovarian tumor cell proliferation in vitro, we tested the growth inhibitory effects of Ad-mda7 in vivo
In the present study we investigated the antitumor activity of mda-7/IL-24 against human ovarian cancer cells and compared to normal ovarian epithelial cells in vitro and the mda-7-mediated growth inhibitory effects on human ovarian tumor xenograft mouse models in vivo. For this purpose we used an adenoviral vector carrying the mda-7 gene (Ad-mda7). In vitro studies showed Ad-mda7 selectively inhibited the growth of ovarian tumor cells but not normal ovarian epithelial cells. Associated with Ad-mda7-mediated growth inhibition was the marked increase in the number of tumor cells in the G2/M phase of cell cycle indicating cell cycle arrest. The ability of Ad-mda7 to selectively inhibit ovarian tumor cell growth in the G2/M phase with no effect on normal cells concurs with our previous reports on the inhibitory effects of mda-7 on other human tumor cell types [2, 4, 8, 12, 15].
Studies in lung cancer and melanoma have previously shown Ad-mda7-mediated tumor cell death occurs by activation of PKR, p38MAPK and pJNK leading to activation of the caspase cascade [7, 9–11]. Analysis for activation of these molecular markers in the present study showed increased PKR, p38MAPK and pJNK expression in Ad-mda7-treated ovarian tumor cells but not in normal cells. Furthermore, activation of caspase-9, -3 and PARP was also observed in Ad-mda7-treated ovarian tumor cells but not in normal cells. Our results show that Ad-mda7 induces cell death of ovarian cancer cells by activation of the cell death signaling pathways, an observation that has not previously been reported for ovarian cancer.
Since in vitro results do not always correlated with in vivo studies, we investigated the growth inhibitory effects of Ad-mda7 using ovarian tumor xenografts established in nude mice. We observed Ad-mda7 significantly inhibited the growth of subcutaneous MDAH2774 tumors. MDA-7-mediated growth inhibition in vivo correlated with activation of molecular markers such as caspases that was also observed in vitro in Ad-mda7-treated tumor cells. The ability of Ad-mda7 to inhibit the growth of ovarian tumor xenografts is not surprising and concurs with our previous findings on lung tumor xenografts . However, the Ad-mda7-mediated inhibitory effect on ovarian tumors observed in the present study was more significant than that observed previously in lung tumor xenografts .
Although we showed in this study that mda-7 gene delivery using an adenoviral vector can inhibit subcutaneous ovarian tumor growth and established a proof of concept, the reality is that ovarian tumors grow inside the abdomen, so the therapeutic effect of Ad-mda7 must be tested in an intraperitoneal tumor model. However, reports have shown that ovarian tumor cells growing in the peritoneal cavity are not efficiently infected with adenoviral vectors carrying therapeutic genes for several reasons, including low surface expression of the adenoviral receptors by tumor cells, interference between the adenovirus and tumor cells by ascitic fluid, rapid clearance of the adenovirus, and induction of host immunity against the viral proteins [16, 17]. To overcome some of these limitations, investigators have used targeting strategies that have shown limited success [18, 19]. A recent study by Mahasreshti et al.  showed that targeted Ad-mda7 delivery can inhibit ovarian cancer growth and prolong the duration of animal survival when compared with that using a nontargeted adenoviral vector carrying the mda-7 gene. However, in that study, the animals received treatment only twice, raising the possibility that when administered repeatedly, a targeted adenoviral vector can become ineffective because of clearance of it by the host immune system. Additionally, in the same study, the researchers administered mda-7 to tumor-bearing mice at earlier time points (days 2 and 14 after tumor cell inoculation) at which time there may not have been enough ascitic fluid to interfere with adenoviral infection.
Based on these reports demonstrating the potential problems of using adenovirus-based therapy for ovarian cancer, an alternate is the use of lipid-based nanoparticles for the treatment of intraperitoneal ovarian tumor-bearing mice. The rationale for testing nanoparticle-based mda-7 therapy are that they are less immunogenic and stable for extended periods of time (6-24 h) in vivo compared to adenovirus-based therapy. Furthermore, studies from our laboratory have shown that the lipid-based nanoparticles are effective gene delivery vehicles when administered systemically [14, 15]. We in the laboratory are currently testing the antitumor properties of mda-7 contained in lipid-based nanoparticles for the treatment of intraperitoneal ovarian tumor-bearing mice. It is anticipated that these studies will provide a basis for future preclinical studies.
Our study demonstrates Ad-mda7 can selectively and effectively inhibit ovarian cancer both in vitro and in vivo and is therapeutic agent for ovarian cancer. However, delivery of mda-7 using alternate gene delivery vector systems such as nanoparticles is required to achieve effective control of ovarian tumor growth in the abdominal cavity.
Cell lines and cell culture
The human ovarian cancer cell lines MDAH2774 and OVCA420 were grown as described previously . IOSE-80 human normal ovarian epithelial cells were provided by Dr Gordon Mills (The University of Texas M. D. Anderson Cancer Center, Houston, TX).
The replication-defective Ad-mda7 vector was constructed and purified as previously described [8, 13]. Briefly, replication-deficient human type 5 adenoviral vectors were constructed to express either mda-7 (Ad-mda7) or luciferase (Ad-luc) genes linked to an internal cytomegalovirus immediated-early promoter and followed by an SV40 polyadenylation signal. The viruses were propagated in HEK293 human embryonic kidney cells and purified by column chromatography.
Cell viability and cell cycle assay
Tumor and normal cells (105) treated with phosphate-buffered saline (PBS), an adenoviral vector carrying the luciferase gene (Ad-luc), or Ad-mda7 (3000 vp/cell) were subjected to cell viability and cell cycle analysis as described previously [8, 12, 13]. Cell viability analysis was performed on day 3 and day 5 after treatment, whereas cell cycle analysis was performed on day 3 after treatment. Treatment was performed in triplicate, and experiments were repeated at least twice to ensure reproducibility and statistical significance. The results presented are representative of one experiment.
Western blot analysis
Tumor and normal cells (105) treated with PBS, Ad-luc, or Ad-mda7 were harvested at 24, 48, and 72 h after treatment and subjected to Western blotting [8, 12, 13]. The following primary antibodies were used in the Western blot analysis: total double-stranded RNA-dependent protein kinase (PKR), p38MAPK, phospho-specific c-Jun N-terminal kinase (pJNK), and caspase-9 (Cell Signaling, Boston, MA); caspase-3, poly(ADP-ribose) polymerase (PARP), and β-actin (Sigma Chemical Co., St. Louis, MO); and MDA-7 (Introgen Therapeutics, Inc., Houston, TX).
In vivo studies
Female athymic BALB/c female nude mice of 4–6 weeks age were purchased from Charles River Laboratories (Willimington, MA). Mice were housed in sterile pathogen free environment and fed ad libitum.
Prior to start of the experiment mice were subjected to whole body cesium radiation (350 rads) to enhance uptake of human xenograft tumor cells. Twenty-four after radiation the mice were injected with MDAH2774 tumor cells (5 × 106) subcutaneously into the lower right flank (n = 18). The mice were separated into groups by treatment (six each for PBS, Ad-luc, and Ad-mda7) and administered treatment when their tumors reached 40 to 50 mm3 in volume. Treatments were administered intratumorally for all groups on days 1, 3, 5, 7, 17, 20, 24, 26, and 32. Ad-luc and Ad-mda7 doses administered were 5 × 109 vp/dose. Tumor growth was measured two to three times per week as described previously [13, 15]. The animals were killed by CO2 inhalation at the end of the experiment per institutional approved guidelines. The experiments were carried out twice; data from one representative study are presented. All of the in vivo studies conducted were approved by the institutional animal care and welfare committee and performed according to N.I.H. guidelines.
For molecular analysis of MDA-7, procaspase-3, procaspase-9, and PARP expression in Ad-mda7-treated tumor tissues, subcutaneous ovarian tumor tissue samples were harvested from mice upon termination of the study and snap-frozen in dry ice. Samples were subsequently ground using a homogenizer, and tissue lysates were prepared and analyzed for expression of the indicated molecular markers using Western blot analysis as described previously .
All of the experiments were performed twice, and experimental results were analyzed for statistical significance using Student's t-test and analysis of variance. Values of p < 0.05 were considered statistically significant.
We thank Dr Judith K. Wolf (M. D. Anderson) for providing the human ovarian cancer cell lines. Don Norwood for editorial assistance, and Debbie M. Smith for help in the preparation of the manuscript. This work was supported in part by CA89778, CA88421, and CA097598 (S.C.), and Cancer Center Support (Core) Grant CA16672 (M. D. Anderson) from the National Cancer Institute; ATP/ARP grant 003657-0078-2001 from the Texas Higher Education Coordinating Board (R.R.); and a sponsored research agreement with Introgen Therapeutics, Inc.
- Greenlee R, Murray T, Bolden S, Wingo PA: Cancer Statistics 2000. CA Cancer J Clin. 2000, 50: 7-33.View ArticlePubMedGoogle Scholar
- Chada S, Sutton RB, Ekmekcioglu S, Ellerhorst J, Muhm JB, Leitner WW, Yang HY, Sahin AA, Hunt KK, Fuson KL, Poindexter N, Roth JA, Ramesh R, Grimm EA, Mhashilkar AM: MDA-7/IL-24 is a unique cytokine–tumor suppressor in the IL-10 family. Int Immunopharmacol. 2004, 4: 649-667. 10.1016/j.intimp.2004.01.017View ArticlePubMedGoogle Scholar
- Inoue S, Shanker M, Miyahara R, Gopalan B, Oida Y, Branch CD, Munshi A, Meyn RE, Andreeff M, Tanaka F, Mhashilkar AM, Chada S, Ramesh R: MDA-7/IL-24 based cancer gene therapy: translation from the laboratory to the clinic. Curr Gene Ther. 2006, 6: 73-91. 10.2174/156652306775515574View ArticlePubMedGoogle Scholar
- Gopalan B, Litvak A, Sharma C, Mhashilkar AM, Chada S, Ramesh R: Activation of the Fas-FasL signaling pathway by MDA-7/IL-24 kills human ovarian cancer cells. Cancer Res. 2005, 65: 3017-3024.PubMedGoogle Scholar
- Mahasreshti PJ, Kataram M, Wu H, Yalavarthy LP, Carey D, Fisher PB, Chada S, Alvarez RD, Haisma HJ, Dent P, Curiel DT: Ovarian cancer targeted adenoviral-mediated mda-7/IL-24 gene therapy. Gynecol Oncol. 2006, 100: 521-532. 10.1016/j.ygyno.2005.08.042View ArticlePubMedGoogle Scholar
- Emdad L, Sarkar D, Lebedeva IV, Su ZZ, Gupta P, Mahasreshti PJ, Dent P, Curiel DT, Fisher PB: Ionizing radiation enhances adenoviral vector expressing mda-7/IL-24-mediated apoptosis in human ovarian cancer. Cell Physiol. 2006, 208: 298-306. 10.1002/jcp.20663.View ArticleGoogle Scholar
- Fisher PB, Gopalkrishnan RV, Chada S, Ramesh R, Grimm EA, Rosenfeld MR, Curiel DT, Dent P: mda-7/IL-24, a novel cancer selective apoptosis inducing cytokine gene: from the laboratory into the clinic. Cancer Biol Ther. 2003, 2: S23-S37.View ArticlePubMedGoogle Scholar
- Mhashilkar AM, Schrock RD, Hindi M, Liao J, Sieger K, Kourouma F, Zou-Yang H, Onishi E, Takh O, Vedvick TS, Stewart L, Watson GJ, Snary D, Fisher PB, Ramesh R, Roth JA, Chada S: Melanoma-differentiation associated gene-7 (mda-7): a novel antitumor gene for cancer gene therapy. Mol Med. 2001, 7: 271-282.PubMed CentralPubMedGoogle Scholar
- Pataer A, Vorburger SA, Barber GN, Chada S, Mhashilkar AM, Zou-Yang H, Stewart AL, Balachandran S, Roth JA, Hunt KK, Swisher SG: Adenoviral transfer of the melanoma differentiation-associated gene 7 (mda7) induces apoptosis of lung cancer cells via up-regulation of the double-stranded RNA-dependent protein kinase (PKR). Cancer Res. 2002, 62: 2239-2243.PubMedGoogle Scholar
- Kawabe S, Nishikawa T, Munshi A, Roth JA, Chada S, Meyn RE: Adenovirus-mediated mda-7 gene expression radiosensitizes non-small cell lung cancer cells via TP53-independent mechanisms. Mol Ther. 2002, 6: 637-644. 10.1016/S1525-0016(02)90714-8View ArticlePubMedGoogle Scholar
- Sarkar D, Su ZZ, Lebedeva IV, Sauane M, Gopalkrishnan RV, Valerie K, Dent P, Fisher PB: mda-7 (IL-24) mediates selective apoptosis in human melanoma cells by inducing the coordinated overexpression of the GADD family of genes by means of p38 MAPK. Proc Natl Acad Sci USA. 2002, 99: 10054-10059. 10.1073/pnas.152327199PubMed CentralView ArticlePubMedGoogle Scholar
- Saeki T, Mhashilkar A, Chada S, Branch C, Roth JA, Ramesh R: Tumor-suppressive effects by adenovirus-mediated mda-7 gene transfer in non-small cell lung cancer cell in vitro. Gene Ther. 2000, 7: 2051-2057. 10.1038/sj.gt.3301330View ArticlePubMedGoogle Scholar
- Saeki T, Mhashilkar A, Swanson X, Zou-Yang XH, Sieger K, Kawabe S, Branch CD, Zumstein L, Meyn RE, Roth JA, Chada S, Ramesh R: Inhibition of human lung cancer growth following adenovirus-mediated mda-7 gene expression in vivo. Oncogene. 2002, 21: 4558-4566. 10.1038/sj.onc.1205553View ArticlePubMedGoogle Scholar
- Ramesh R, Ito I, Saito Y, Wu Z, Mhashikar AM, Wilson DR, Branch CD, Roth JA, Chada S: Local and systemic inhibition of lung tumor growth after nanoparticle-mediated mda-7/IL-24 gene delivery. DNA Cell Biol. 2004, 23: 850-857.View ArticlePubMedGoogle Scholar
- Ramesh R, Saeki T, Templeton NS, Ji L, Stephens LC, Ito I, Wilson DR, Wu Z, Branch CD, Minna JD, Roth JA: Successful treatment of primary and disseminated human lung cancers by systemic delivery of tumor suppressor genes using an improved liposome vector. Mol Ther. 2001, 3: 337-350. 10.1006/mthe.2001.0266View ArticlePubMedGoogle Scholar
- Yang Y, Li Q, Ertl HCJ, Wilson JM: Cellular and humoral immune responses to viral antigens create barriers to lung-directed gene therapy with recombinant adenoviruses. J Virol. 1995, 69: 2004-2015.PubMed CentralPubMedGoogle Scholar
- Yang Y, Ertl HCJ, Wilson JM: MHC class I-restricted cytotoxic T lymphocytes to viral antigens destroy hepatocytes in mice infected with E1-deleted recombinant adenoviruses. Immunity. 1994, 1: 433-442. 10.1016/1074-7613(94)90074-4View ArticlePubMedGoogle Scholar
- Blackwell JL, Li H, Gomez-Navarro J, Dmitriev I, Krasnykh V, Richter CA, Shaw DR, Alvarez RD, Curiel DT, Strong TV: Using a tropism-modified adenoviral vector to circumvent inhibitory factors in ascites fluid. Hum Gene Ther. 2000, 11: 1657-1669. 10.1089/10430340050111313View ArticlePubMedGoogle Scholar
- Raki M, Kanerva A, Ristimaki A, Desmond RA, Chen DT, Ranki T, Sarkioja M, Kangasniemi L, Hemminki A: Combination of gemcitabine and Ad5/3-Delta24, a tropism modified conditionally replicating adenovirus, for the treatment of ovarian cance. Gene Ther. 2005, 12: 1198-1205. 10.1038/sj.gt.3302517View ArticlePubMedGoogle Scholar
- Santoso JT, Tang DC, Lane SB, Hung J, Reed DJ, Muller CY, Carbone DP, Lucci JA, Miller DS, Mathis JM: Adenovirus-based p53 gene therapy in ovarian cancer. Gynecol Oncol. 1995, 59: 171-178. 10.1006/gyno.1995.0002View ArticlePubMedGoogle Scholar
- Inoue S, Hartman A, Branch CD, Bucana CD, Bekele BN, Stephens LC, Chada S, Ramesh R: Combination treatment of Bevacizumab and Ad-mda7 produces a synergistic and complete inhibitory effect on lung tumor xenograft. Mol Ther. 2007, 287-294.Google Scholar
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