Regression of advanced rat and human gliomas by local or systemic treatment with oncolytic parvovirus H-1 in rat models.

Oncolytic virotherapy is a potential treatment modality under investigation for various malignancies including malignant brain tumors. Unlike some other natural or modified viruses that show oncolytic activity against cerebral neoplasms, the rodent parvovirus H-1 (H-1PV) is completely apathogenic in humans. H-1PV efficiently kills a number of tumor cells without harm to corresponding normal ones. In this study, the concept of H-1PV-based virotherapy of glioma was tested for rat (RG-2 cell-derived) and for human (U87 cell-derived) gliomas in immunocompetent and immunodeficient rat models, respectively. Large orthotopic rat and human glioma cell-derived tumors were treated with either single stereotactic intratumoral or multiple intravenous (iv) H-1PV injections. Oncolysis was monitored by magnetic resonance imaging and proven by histology. Virus distribution and replication were determined in brain and organs. In immunocompetent rats bearing RG-2-derived tumors, a single stereotactic intratumoral injection of H-1PV and multiple systemic (iv) applications of the virus were sufficient for remission of advanced and even symptomatic intracranial gliomas without damaging normal brain tissue or other organs. H-1PV therapy resulted in significantly improved survival (Kaplan-Meier analysis) in both the rat and human glioma models. Virus replication in tumors indicated a contribution of secondary infection by progeny virus to the efficiency of oncolysis. Virus replication was restricted to tumors, although H-1PV DNA could be detected transiently in adjacent or remote normal brain tissue and in noncerebral tissues. The results presented here and the innocuousness of H-1PV for humans argue for the use of H-1PV as a powerful means to perform oncolytic therapy of malignant gliomas.

Cytosolic activation of cathepsins mediates parvovirus H-1-induced killing of cisplatin and TRAIL-resistant glioma cells.

Gliomas are often resistant to the induction of apoptotic cell death as a result of the development of survival mechanisms during astrocyte malignant transformation. In particular, the overexpression of Bcl-2-family members interferes with apoptosis initiation by DNA-damaging agents (e.g., cisplatin) or soluble death ligands (e.g., TRAIL). Using low-passage-number cultures of glioma cells, we have shown that parvovirus H-1 is able to induce death in cells resistant to TRAIL, cisplatin, or both, even when Bcl-2 is overexpressed. Parvovirus H-1 triggers cell death through both the accumulation of lysosomal cathepsins B and L in the cytosol of infected cells and the reduction of the levels of cystatin B and C, two cathepsin inhibitors. The impairment of either of these effects protects glioma cells from the viral lytic effect. In normal human astrocytes, parvovirus H-1 fails to induce a killing mechanism. In vivo, parvovirus H-1 infection of rat glioma cells intracranially implanted into recipient animals triggers cathepsin B activation as well. This report identifies for the first time cellular effectors of the killing activity of parvovirus H-1 against malignant brain cells and opens up a therapeutic approach which circumvents their frequent resistance to other death inducers.

Parvovirus H-1 infection of human glioma cells leads to complete viral replication and efficient cell killing.

The extremely poor prognosis of malignant gliomas requires the investigation of other than standard therapies, i.e., the application of oncolytic viruses. In our study, we evaluated the effects of the oncosuppressive parvovirus H-1 on different established glioblastoma cell lines of rat and human origin and on short-term/low-passage cultures of human glioblastoma cells. We observed an efficient and dose-dependent killing of all glioma cell cultures at low multiplicities of infectious particles (MOI) per cell. Southern blot analysis of viral DNA amplification, RT-PCR analysis of viral RNA expression and Western blot analysis of the expression of viral structural (VP-1/VP-2) and nonstructural (NS-1) proteins demonstrated the biosynthesis of these viral macromolecular components in all of the cultures. Moreover, all the glioma cells were proficient for the production of infectious H-1 virus particles. The amount of virus production differed between a several fold increase of the input virus titer in most of the short-term/low-passage cultures up to 1,000-fold in one short-term glioma and in the rat cells. Glioma cells lines and, more importantly, short-term/low-passage cultures of human glioblastomas were found to be highly susceptible target cells for H-1 virus mediated cytotoxicity. The formation of fully infectious progeny particles in infected glioma cells offers the chance for the induction of secondary rounds of infection resulting in an advanced cytotoxic effect. These advantageous characteristics of H-1 virus infection of glioma cells, combined with the known low toxicity of H-1 virus in nontransformed cells, make parvovirus H-1 a promising candidate for oncolytic glioma therapy.