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Adenovirus-mediated thymidine kinase gene therapy for recurrent ovarian cancer: expression of coxsackie-adenovirus receptor and integrins alphavbeta3 and alphavbeta5
Abstract
Ten patients with recurrent ovarian cancer received a combined treatment of optimal tumor debulking, adenovirus-mediated herpes simplex virus thymidine kinase gene therapy (GT), and systemic application of acyclovir or valacyclovir and topotecan. Biopsies were taken at the time of secondary debulking about 1 month after the application of GT and chemotherapy and were analyzed for expression of coxsackie-adenovirus receptor (CAR) and integrins alphavbeta3 and alphavbeta5 with respect to treatment response.
Treatment modalities and study design have been described recently. Immunohistochemistry was used to visualize expression of CAR and integrins alphavbeta3 and alphavbeta5 in tumor samples taken before and after application of GT.
Before GT six of ten patients presented with CAR-positive and four with CAR-negative tumors. After GT all tumors showed CAR expression. Integrin alphavbeta3 was found in all tumors before and after GT. Expression of integrin alphavbeta5 was seen in eight of ten tumor samples before GT and in all samples after GT.
Despite the importance of CAR and integrin expression for successful adenovirus internalization, other cell surface receptors might be involved in this process. It is too early to decide whether expressions of CAR and integrin alphavbeta3/alphavbeta5 on tumor cells are appropriate additional inclusion criteria for the enrollment of patients in GT trials. Further research is necessary to evaluate the effect of GT plus chemotherapy on CAR and integrin expression.
... Despite the known importance of CAR for successful transduction of cells, other unknown mechanism of cell virus interaction may also be important. variable CAR expressions in tumors may interfere with the interpretation of results of clinical trials, but it is too early to determine whether expressions of CAR in tumor cells are appropriate additional criteria for the enrollment of patients in adenovirus gene therapy trials (25). ...
The adenovirus vector-based cancer gene therapy is controversial. Low transduction efficacy is believed to be one of the main barriers for the decreased expression of coxsackie and adenovirus receptor (CAR) on tumor cells. However, the expression of CAR on primary tumor tissue and tumor tissue survived from treatment has still been not extensively studied. The present study analyzed the adenovirus infection rates and CAR expression in human lung adenocarcinoma cell line A549 and its cisplatin-resistant subline A549/DDP. The results showed that although the CAR expression in A549 and A549/DDP was not different, compared with the A549, A549/DDP appeared obviously to reject adenovirus infection. Moreover, we modified CAR expression in the two cell lines with proteasome inhibitor MG-132 and histone deacetylase inhibitor trichostatin A (TSA), and analyzed the adenovirus infection rates after modifying agent treatments. Both TSA and MG-132 pretreatments could increase the CAR expression in the two cell lines, but the drug pretreatments could only make A549 cells more susceptible to adenovirus infectivity.
... HSV-1 TK / GCV has been used successful for gene therapy in a wide variety of animal tumor models, and is currently in clinical trials for human cancers (Floeth et al. 2001;Hasenburg et al. 2002;Voges et al. 2003). These trials have indicated the need for optimization of this strategy of combined gene/chemotherapy of cancer (Fillat et al. 2003). ...
... Efforts to develop efficient Ad vectors for gene delivery in humans have centered primarily on human Ad serotype 5, owing to its well-elucidated biology. However, testing of Ad5 vectors in preclinical studies and gene therapy clinical trials identified several important drawbacks: widespread preexisting anti-Ad5 immunity in humans (12,55), an unacceptably large amount of damage to normal nontarget tissues, which causes severe toxicity (28,37), and low levels of target-specific gene delivery (15,18,19). Collectively, these findings warranted the search for Ad vector prototypes better suited for gene transfer in humans. ...
Efforts to develop adenovirus vectors suitable for genetic interventions in humans have identified three major limitations of the most frequently used vector prototype, human adenovirus serotype 5 (Ad5). These limitations--widespread preexisting anti-Ad5 immunity in humans, the high rate of transduction of normal nontarget tissues, and the lack of target-specific gene delivery--justify the exploration of other Ad serotypes as vector prototypes. In this paper, we describe the development of an alternative vector platform using simian Ad serotype 24 (sAd24). We found that sAd24 virions formed unstable complexes with blood coagulation factor X and, because of that, transduced the liver and other organs at low levels when administered intravenously. The overall pattern of biodistribution of sAd24 particles was similar, however, to that of Ad5, and the intravenously injected sAd24 was cleared by Kupffer cells, leading to their depletion. We modified the virus's fiber protein to design a Her2-specific derivative of sAd24 capable of infecting target human tumor cells in vitro. In the presence of neutralizing anti-Ad5 antibodies, Her2-mediated infection with targeted sAd24 compared favorably to that with the Ad5-derived vector. When used to target Her2-expressing tumors in animals, this fiber-modified vector achieved a higher level of gene transfer to metastasis-containing murine lungs than to tumor-free lungs. In aggregate, these studies provide important insights into sAd24 biology, identify its advantages and limitations as a vector prototype, and are thus essential for further development of an sAd24-based gene delivery platform.
... The transduction of solid tumor specimens obtained during surgery shows that they can be easily transduced by adenovirus, particularly cells at the periphery, using a replication-defective adenovirus. Consistent with this observation, clear expression of CAR has been shown by immunohistochemistry of solid ovarian tumor 13,25 and is also shown here. A replication-competent adenovirus is, therefore, needed to allow penetration and spread within the solid tumor as replication-incompetent viruses are limited to cells at the periphery of a solid tumor mass. ...
Adenovirus is the most frequently used virus in gene therapy clinical trials. There have been conflicting reports on the ability of adenovirus to transduce primary ovarian cancer samples and the expression of relevant cell surface molecules. These factors were examined using primary ovarian cancer cells cultured from ascites and solid tumor to gain insights into the clinical use of adenovirus in ovarian cancer. The level of transduction of primary cultures was much higher than uncultured cells and established cell lines, and correlated with higher levels of coxsackie-adenovirus receptor (CAR) and integrin expression. Growth of primary cultures in autologous ascitic fluid prevented an increase in CAR expression and inhibited transduction compared with cells treated in supplemented RPMI. Cells at the periphery of solid tumor samples were transduced using a replication-incompetent virus and correlated with CAR expression. However, transduction was abolished by autologous ascitic fluid, despite the expression of CAR. We conclude that the use of adenoviruses for ovarian cancer gene therapy will require testing in the presence of inhibitory factors in ascitic fluid. The clinical use of adenoviral vectors may require circumvention of such inhibitory factors and the use of replication competent adenovirus to enable efficient viral penetration of the cancer.
... Advantages include the potential lack of cross-resistance with standard therapies, and the ability of Ads to infect both proliferating and quiescent cells. In the context of ovarian cancer gene therapy, many investigators have focused on intraperitoneally administered gene therapy vectors (Bauerschmitz et al., 2002;Hasenburg et al., 2002). However, systemic metastasis remains an unsolved problem in a significant number of patients with advanced or recurrent ovarian cancer. ...
Approaches to alter the native tropism of adenoviruses (Ads) are beneficial to increase their efficacy and safety profile. Liver tropism is important with regard to potential clinical toxicity in humans. Ad5/3 chimeras in which the Ad5 knob is substituted by the Ad3 knob, such as Ad5/3luc1, have been recently shown to increase infectivity of ovarian cancer cell lines and primary tumor cells, which express low levels of the coxsackie-adenovirus receptor (CAR), without increasing infectivity of liver cells. A novel strategy to address the problem of liver uptake and improve the tumor/liver ratio is genetic replacement of the Ad fiber shaft. Ad5.Ad3.SH.luc1 is an Ad5-based vector that contains the fiber shaft from Ad serotype 3 but the fiber knob from Ad serotype 5. To compare tumor/liver of Ad5.Ad3.SH.luc1 and Ad5/3luc1 in vivo, we created three different tumor and treatment models of ovarian cancer in mice, simulating intraperitoneal and intravenous administration of tumors. Ad5.Ad3.SH.luc1 displayed the lowest liver tropism of all viruses in all models tested. Intravenous administration of all viruses resulted in higher tumor transduction rates compared to intraperitoneal administration. Genetic shortening of the Ad5 fiber shaft significantly increases relative tumor/liver gene transfer. This could improve the effective tumor dose and reduce side effects, thereby increasing the bioavailability of therapeutic agents.
Multidrug resistance gene 1 (MDR1) mediated resistance to chemotherapeutic agents is a major obstacle for the therapy of various cancer types. The use of conditionally replicating adenoviruses (CRAds) is dependent on molecular differences between tumor cells and non tumor cells. Transcriptional targeting of CRAd replication is an effective way to control replication regulation. The aim of this study was to evaluate the effect of a MDR1 targeted fiber-modified CRAd against chemotherapy resistant ovarian cancer.
MDR1 expression was evaluated in chemotherapy naïve and pretreated ovarian cancer cells and various control cells. We constructed 2 variants of a fiber-modified CRAd, Ad5/3MDR1E1 and Ad5/3MDR1E1∆24 containing the MDR1 promoter to control viral replication via the E1A gene. The MDR promoter activity and cell killing efficacy were evaluated in vitro. Orthotopic murine models of peritoneally disseminated ovarian cancer were utilized to evaluate the preclinical efficacy of MDR targeted CRAds in vivo. To evaluate the liver toxicity of MDR1 targeted CRAds, we compared Ad5/3MDR1E1 with Ad5/3∆24, a CRAd that replicates in cancer cells inactive in the Rb/p16 pathway by use of an in vivo hepatotoxicity model.
We demonstrate efficient oncolysis of Ad5/3MDR1E1 in both chemotherapy resistant ovarian cancer cell lines and in primary tumor cells from pretreated patients as well as therapeutic efficacy in an orthotopic mouse model. Ad5/3MDR1E1 demonstrated significantly decreased liver toxicity compared to other 5/3-fiber modified control vectors examined.
In summary, Ad5/3MDR1E1 is an efficient and safe gene therapy approach for specific targeting of chemotherapy resistant cancer cells.
Nederlandse samenvatting Eierstokkanker (ovariumcarcinoom) heeft het hoogste sterftecijfer van alle gynaecologische kankers. Traditionele therapieën zoals operatieve verwijdering van de tumor, al dan niet in combinatie met chemotherapie, hebben helaas niet tot een verbetering van de levensverwachting voor deze ziekte geleid. Nieuwe benaderingen voor de therapie van eierstokkanker zijn dus zeer noodzakelijk. Eierstokkanker is het gevolg van een opeenstapeling van veranderingen in het genetische materiaal in de cel. Dit, tezamen met het feit dat de ziekte meestal beperkt blijft tot de buikholte, maakt het gebruik van gentherapie een aantrekkelijk alternatief. Dit proefschrift beschrijft derhalve nieuwe gentherapie benaderingen voor de behandeling van eierstokkanker. In Hoofdstuk 1 wordt een overzicht gegeven van deze benaderingen, en ook worden de verschillende vectoren die worden gebruikt in gentherapie beschreven. Tenslotte worden verschillende mechanismen behandeld die er voor zorgen dat de gentherapie alleen actief is in eierstokkanker cellen, en niet elders in het lichaam. In Hoofdstuk 2 wordt het doel van het beschreven onderzoek uiteengezet. In Hoofdstuk 3 wordt vervolgens de ontwikkeling beschreven van een nieuw soort gentherapie voor eierstokkanker, waarbij het ontstaan van nieuwe bloedvaten in de tumor wordt tegengegaan. Deze nieuwe bloedvaten zijn cruciaal voor het groeien en uitzaaien van een eierstoktumor. Het molecuul ‘vascular endothelial growth factor’ (VEGF) speelt een grote rol in dit proces, en een verhoogde productie van dit molecuul gaat derhalve samen met versnelde tumorgroei, uitzaaiingen en een kortere levensverwachting. Een methode om VEGF uit te schakelen is het tot expressie brengen van een bepaalde receptor voor dit molecuul: deze receptor (genaamd ‘sflt-1’) vangt het teveel aan VEGF weg, zodat het zijn functie niet meer kan uitoefenen. In de experimenten beschreven in dit hoofdstuk wordt een vector gebruikt gebaseerd op het adenovirus om de sflt-1 receptor tot expressie te brengen. Dit virus is zodanig veranderd dat het efficiënt genen kan afleveren in tumor cellen. Dit is gedaan door extra aminozuren (een zogenaamde RGD sequentie) in te bouwen in de buitenkant van het virus (het capside). Het afleveren van het sflt-1 gen door middel van dit virus was in celkweek experimenten in staat celgroei te remmen van endotheelcellen, de cellen die de bloedvatwand bekleden. Vervolgens werd dit virus in diermodellen getest, en het bleek ook in muizen de tumorgroei van menselijke eierstokkanker cellen te kunnen remmen. In dit model werd het virus met het sflt-1 gen aan muizen toegediend in de buikholte, waar ook de tumorgroei plaatsvindt, en dit is dus een voorbeeld van een lokale behandeling. Aangezien ook uitzaaiingen van eierstokkanker behandeld moeten worden, wordt in Hoofdstuk 4 getest of intraveneuze toediening van het adenovirus met het sflt-1 gen deze uitzaaiingen kan verhinderen. Dit blijkt inderdaad het geval te zijn, maar dit gaat gepaard met toxiciteit in de lever. Dit is het gevolg van de ophoping van het virus en expressie van het sflt-1 gen in de lever na intraveneuze toediening, zodat daar lokaal de balans tussen bloedvatafbraak en –herstel wordt verstoord. Dit hoofdstuk geeft dus duidelijk aan dat therapeutische genen alleen op de plek van werking moeten worden afgeleverd, omdat anders schade aan normaal weefsel wordt toegebracht. Een van de manieren om genen alleen in de tumor tot expressie te brengen is ‘transcriptional targeting’ – hierbij wordt gebruikt gemaakt van tumor specifieke genregulatie mechanismen die niet actief zijn elders in het lichaam, zodat toxiciteit in normaal weefsel wordt vermeden. Deze genregulatie mechanismen worden ‘promoters’ genoemd, en in Hoofdstuk 5 worden verschillende van deze promoters geanalyseerd voor hun selectiviteit voor eierstokkanker. De ‘midkine’ promoter en twee verschillende ‘cyclooxygenase-2’ (cox-2) promoters werden getest voor hun selectiviteit in zowel celkweek experimenten alsook in experimenten met proefdieren, en het bleek dat de cox-2M promoter het meest selectief was, met hoge expressie in eierstokkanker en lage expressie in de lever. Een proefdierexperiment waarin de expressie van een toxisch gen werd gecontroleerd door deze cox-2M promoter liet zien dat toxiciteit in de lever inderdaad op deze manier kon worden vermeden. Een alternatief voor transcriptional targeting is het gebruik van een gen dat niet toxisch is voor normale weefsels, maar wel in staat is tumorgroei te remmen. In Hoofdstuk 6 wordt een voorbeeld van een dergelijk gen geanalyseerd, te weten ‘melanoma-differentiation associated gene-7’ (mda-7). Mda-7 heeft de eigenschap celdood te kunnen induceren in tumorcellen maar niet in normale cellen in het lichaam. Celkweek experimenten met een adenovirus dat mda-7 in de cel aflevert lieten zien dat dit gen inderdaad specifiek celdood kan induceren in eierstokkanker cellen, en niet in normale cellen. Helaas is het adenovirus niet erg efficiënt in het infecteren van tumor cellen, zodat het gen niet z’n optimale werking kan uitoefenen. Om dit probleem op te lossen werd het adenovirus specifiek gestuurd naar de eierstokkanker cellen om het infectie proces efficiënter te laten verlopen, een proces dat ‘transductional targeting’ wordt genoemd. Het bleek dat deze ‘gestuurde’ virussen inderdaad de effectiviteit van mda-7 verbeterden, al moeten proefdierexperimenten daarover meer uitsluitsel geven. In Hoofdstuk 7 wordt daarom de productie van meerdere virussen beschreven die het mda-7 gen in tumor cellen kunnen afleveren. Om de virussen naar de tumor te sturen werden verschillende extra aminozuren in de buitenkant van het virus ingebouwd. Ten eerste werd een virus met de eerder genoemde ‘RGD sequentie’ gebruikt, ten tweede een virus met extra lysine aminozuren, en ten derde een virus met beide aanpassingen: zowel RGD als extra lysines op het oppervlak. Zowel in celkweek experimenten als in proefdieren was het dubbel veranderde virus het meest effectief in het afleveren van het mda-7 gen, en het remde de tumorgroei en verlengde daarmee de levensverwachting van de muizen. Samenvattend worden in dit proefschrift voorbeelden gegeven van nieuwe therapeutische strategieën voor eierstokkanker met behulp van gentherapie. Het blijkt dat remming van de aanmaak van nieuwe bloedvaten tumorgroei kan remmen, maar dat deze therapie lokaal moet worden toegepast om toxiciteit elders in het lichaam te vermijden. Verder is aangetoond dat zowel ‘transcriptional’ als ‘transductional targeting’ de selectiviteit en effectiviteit van gentherapie kan verbeteren. Tenslotte zijn de voordelen aangetoond van het gebruik van het mda-7 gen dat alleen toxisch is voor tumorcellen, en niet voor normale weefsels. Gentherapie met tumor-gestuurde virussen die mda-7 tot expressie brengen lijkt dus veelbelovend voor de behandeling van eierstokkanker, maar verder onderzoek nodig is voordat het in patiënten kan worden toegepast.
Unzureichende Möglichkeiten in Diagnose und Behandlung von epithelialen Ovartumoren führen zu einer niedrigen Überlebensrate der Patientinnen. Die molekularen Zusammenhänge, die der Progression dieser Krankheit zugrunde liegen, sind noch weitgehend unbekannt und erschweren Verbesserungen in der Früherkennung und Therapie.
In der vorliegenden Arbeit wurden mit Hilfe der Affymetrix Genchip-Technologie die Genexpressionsprofile von elf Ovartumor-Zelllinien mit denen von zwei IOSE-Zelllinien, die aus normalen Epithelzellen des Ovars etabliert wurden, verglichen. So sollten Gene identifiziert werden, welche die Tumorzellen von den normalen Zellen unterscheiden und eventuell als diagnostischer Marker oder als Zielgen für therapeutische Zwecke dienen können.
Mit besonderem Augenmerk auf Transmembran- bzw. sezernierte Proteine wurden zunächst 21 bzw. sieben Gene in den beiden Gruppen identifiziert, deren Überexpression in Karzinomen des Ovars zum ersten Mal ermittelt wurde. Die Ergebnisse der Affymetrix-Analyse wurden mittels RT-PCR in sieben ausgewählten Genen bestätigt. Die Expressionsanalysen von drei ausgewählten Genen, NMU, JAG2 und L1CAM, wurden auf Ovartumor-Gewebeproben ausgeweitet und eine mögliche Rolle in der Tumorgenese des Ovarkarzinoms diskutiert.
Da für L1CAM spezifische Antikörper zur Verfügung standen, konnte gezeigt werden, dass die Abundanz der L1CAM-mRNA mit der Protein-Abundanz korrelierte und die Lokalisierung in der Zelle wie erwartet in der Plasmamembran erfolgte. Die Expression von L1CAM konnte immunhistochemisch in Paraffinschnitten von Ovarkarzinomen nachgewiesen werden. Zu einem geringeren Prozentsatz konnte das Zelladhäsionsmolekül auch in Borderline-Tumoren, die in der Regel durch eine gute Prognose gekennzeichnet sind, und Fibromen identifiziert werden. In malignen Ovartumoren nicht epithelialer Herkunft konnte keine L1CAM-Expression festgestellt werden. Zeitgleich wurde die Überexpression von L1CAM in Ovarkarzinomen beschrieben und das Molekül als prognostischer Marker in Karzinomen des Ovars, Uterus und des Endometriums identifiziert.
Durch funktionelle Analysen sollte die Funktion von L1CAM in Ovartumorzellen untersucht werden. Bei der Klonierung von L1CAM wurde festgestellt, dass aus den verwendeten Ovartumor-Zelllinien nur eine Isoform des Gens isoliert werden konnte, in der die Exons 2 und 27 deletiert sind. In stabilen L1CAMΔ2,27-Transfektanten konnte gezeigt werden, das die Expression dieser Isoform zu erhöhter Adhäsion an Laminin im Vergleich zu Vektor-Transfektanten führt. Die Invasionsfähigkeit der Transfektanten wurde durch die ektopische L1CAMΔ2,27-Expression nicht verändert.
Zusammenfassend wurden in dieser Arbeit unter der Verwendung der Genchip-Technologie die zwei potenziellen Markergene NMU und JAG2 identifiziert, die in weiteren Analysen auf ihre Bedeutung in Ovarkarzinomen untersucht werden müssen. L1CAM wurde in immunhistochemischen Studien als Marker identifiziert, dessen prognostischer Wert in Zukunft die Diagnose und Therapie dieser aggressiven Erkrankung verbessern könnte. Zudem bieten die vielfältigen Funktionen von L1CAM Möglichkeiten bei der therapeutischen Intervention durch monoklonale Antikörper, die durch die Expression einer Spleißvariante in Ovartumorzellen spezifiziert werden könnte.
The purpose of the study was to determine the capability of the midkine (MK) and cycooxygenase-2 (cox-2) gene promoter regions to function as tumor-specific promoters for use in targeted gene therapy of ovarian cancer.
Established and primary ovarian cancer and mesothelial cells were transduced by adenoviral vectors containing a reporter or thymidine kinase gene expressed under the control of the MK, cox-2, or cytomegalovirus (CMV) promoters. SCID or C57BL/6 mice were injected i.p. with these same vectors. In vitro reporter gene expression and cellular cytotoxicity was determined using luciferase and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, respectively. Acute toxicity in vivo was assessed by histological evaluation of harvested tissues.
Consistent activation of the MK and cox-2 promoters was noted in all of the ovarian cancer cell lines in addition to primary ovarian cancer cells. In contrast, reduced reporter activity was reported in mesothelial cells transduced with adenoviruses containing the test promoters, which was especially apparent for the cox-2 promoter. Additionally, the cox-2 promoter exhibited significantly lower reporter gene levels in liver and peritoneum than the control promoter in in vivo experiments. Tumor-cell killing induced by Adcox-2 MTK was comparable to that observed with AdCMVTK. However, a clear differential toxicity pattern was observed in favor of animals treated with Adcox-2 MTK when compared with controls.
These data clearly demonstrate that the transcriptional control afforded by the cox-2 promoter is tumor-specific and is able to mitigate associated toxicity in normal tissue while maintaining therapeutic efficacy in the context of an ovarian cancer molecular chemotherapeutic approach.
Gene therapy remains a promising therapeutic modality for ovarian cancer. Yet much work remains to be done to see gene therapy realize its full potential in elucidating the complex genetic interactions of delivered genes within target cancer cells and in the development of improved vector systems. Because most neoplasms involve multiple mutations, the targeting of a single mutation is unlikely to achieve total tumor control: gene therapy strategies that target multiple cellular processes or invoke various antitumor approaches need to be investigated. Additionally, current vector systems do not transduce ovarian cancer cells efficiently and are hampered by immune responses that further limit their efficacy. Additionally, limitations in vector specificity lead to transduction of normal cells and subsequent toxicity. Investigators are developing refinements to current gene therapy approaches that would address these limitations and that are soon to be incorporated into clinical trials. It is hoped that these advances will lead to improvements in the therapeutic index for ovarian cancer gene therapy and provide another effective therapeutic tool for this deadly disease.
To create tumor-targeted Ad vectors, ablation of native CAR and integrin receptor binding is crucial to enhance the specificity of tumor transduction. Toward this aim, we have previously created base vectors in which binding to CAR (single-ablated) or to both CAR and integrins (double-ablated) has been ablated. In this study, the biodistribution of the conventional (CAR and integrin binding intact), single-ablated, and double-ablated vectors was evaluated following intraperitoneal administration. The mesothelial lining of the peritoneal organs was the principle site of CAR-dependent gene transfer by the conventional vector. Surprisingly, the single-ablated vector strongly transduced the liver parenchyma rather than the mesothelium, while the double-ablated vector did not significantly transduce the parenchyma or mesothelium. The high level of parenchymal transduction by the single-ablated vector suggested that it efficiently entered the bloodstream from the peritoneal cavity. Consistent with this hypothesis, a large proportion of active particles distributed and persisted in the bloodstream following intraperitoneal administration of either the single- or the double-ablated vector. The above results suggest that the double-ablated vector backbone may not only significantly improve targeting to cancers located in the peritoneal cavity, but may also significantly improve targeting to metastatic tumors located throughout the body by virtue of its enhanced bloodstream persistence.
Melanoma differentiation associated gene-7 [mda-7/Interleukin (IL)-24] has been identified as a novel anti-cancer agent, which specifically induces apoptosis in cancer cells but not in normal epithelial, endothelial and fibroblast cells. The objective of this study was to evaluate the anti-tumor effect of adenovirus-mediated mda-7/IL-24 (Ad.mda-7) gene therapy in ovarian carcinoma and further improve anti-tumor effect by enhancing infectivity of Ad.mda-7.
A panel of human ovarian carcinoma cells, OV-4, HEY, SKOV3, SKOV3.ip1 and control normal human mesothelial cells, were infected by a replication deficient recombinant adenovirus encoding mda-7/IL-24 and control virus Ad.CMV.Luc. After 72 h, apoptosis was evaluated by TUNEL and Hoechst staining and further quantified by fluorescent activated cell sorter (FACS) analysis. Infectivity of Ad.mda-7 was enhanced by retargeting it to CD40 or EGF receptors overexpressed on ovarian cancer cells. Subsequently, enhancement in apoptosis of CD40- or epidermal growth factor receptor (EGFR)-retargeted Ad.mda-7 was evaluated.
Adenoviral-mediated delivery of mda-7 induces apoptosis ranging from 10-23% in human ovarian cancer cells tested with the highest percentage of apoptosis noted in SKOV3 cells. Minimal apoptosis was noted in normal mesothelial cells. CD40- or EGFR-retargeted Ad.mda-7 increased apoptosis by 10-32% when compared to that achieved with untargeted Ad.mda-7.
Ad.mda-7 exhibits ovarian cancer-specific apoptosis, but does not affect normal human mesothelial cells. Infectivity enhanced CD40- and EGFR-retargeted Ad.mda-7 augments apoptosis induction, thus increasing the therapeutic index and translational potential of Ad.mda-7 gene therapy.
Gene-directed enzyme-prodrug therapy (GDEPT) aims to improve the therapeutic ratio (benefit versus toxic side-effects) of cancer chemotherapy. A gene encoding a 'suicide' enzyme is introduced into the tumour to convert a subsequently administered non-toxic prodrug into an active drug selectively in the tumour, but not in normal tissues. Significant effects can now be achieved in vitro and in targeted experimental models, and GDEPT therapies are entering the clinic. Our group has developed a GDEPT system that uses the bacterial enzyme carboxypeptidase G2 to convert nitrogen mustard prodrugs into potent DNA crosslinking agents, and a clinical trial of this system is pending.
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