RESUMEN
Reliable preclinical models are needed for screening new cancer drugs. Thus, we developed an improved 3D tumor organoid model termed "organoid raft cultures" (ORCs). Development of ORCs involved culturing tumors ex vivo on collagen beds (boats) with grid supports to maintain their morphological structure. The ORCs were developed from patient-derived xenografts (PDXs) of colon cancers excised from immune-deficient mice (NOD/SCID/IL2Rgammanull). We utilized these new models to evaluate the efficacy of an investigational drug, Navitoclax (ABT-263). We tested the efficacy of ABT-263, an inhibitor of BCL-2 family proteins, in these ORCs derived from a PDX that showed high expression of antiapoptotic BCL2 family proteins (BCL-2, BCL-XL, and BCL-W). Hematoxylin and eosin staining evaluation of PDXs and corresponding ORCs indicated the retention of morphological and other histological integrity of ORCs. ORCs treated with ABT-263 showed decreased expression of antiapoptotic proteins (BCL2, BCL-XL and BCL-W) and increased proapoptotic proteins (BAX and PUMA), with concomitant activation of caspase 3. These studies support the usefulness of the ORCs, developed from PDXs, as an alternative to PDXs and as faster screening models.
Asunto(s)
Neoplasias , Organoides , Ratones , Humanos , Animales , Organoides/metabolismo , Ratones SCID , Ratones Endogámicos NOD , Navíos , Xenoinjertos , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteína bcl-X/metabolismo , Modelos Animales de Enfermedad , Neoplasias/patología , Proteínas Reguladoras de la ApoptosisAsunto(s)
Antivirales/farmacología , Proteínas de Unión al ADN/metabolismo , Papillomavirus Humano 18/efectos de los fármacos , Óxido Nítrico/metabolismo , Proteínas Oncogénicas Virales/metabolismo , Proteínas E7 de Papillomavirus/metabolismo , Siloxanos/farmacología , Células Cultivadas , Ensayos Clínicos Fase II como Asunto , Replicación del ADN , Proteínas de Unión al ADN/genética , Prepucio/citología , Papillomavirus Humano 18/genética , Papillomavirus Humano 18/fisiología , Humanos , Queratinocitos/efectos de los fármacos , Queratinocitos/virología , Masculino , Proteínas Oncogénicas Virales/genética , Proteínas E7 de Papillomavirus/genética , Replicación ViralRESUMEN
Human papillomaviruses (HPVs) cause epithelial proliferative diseases. Persistent infection of the mucosal epithelia by the high-risk genotypes can progress to high-grade dysplasia and cancers. Viral transcription and protein activities are intimately linked to regulation by histone acetyltransferases and histone deacetylases (HDACs) that remodel chromatin and regulate gene expression. HDACs are also essential to remodel and repair replicating chromatin to enable the progression of replication forks. As such, Vorinostat (suberoylanilide hydroximic acid), and other pan-HDAC inhibitors, are used to treat lymphomas. Here, we investigated the effects of Vorinostat on productive infection of the high-risk HPV-18 in organotypic cultures of primary human keratinocytes. HPV DNA amplifies in the postmitotic, differentiated cells of squamous epithelia, in which the viral oncoproteins E7 and E6 establish a permissive milieu by destabilizing major tumor suppressors, the pRB family proteins and p53, respectively. We showed that Vorinostat significantly reduced these E6 and E7 activities, abrogated viral DNA amplification, and inhibited host DNA replication. The E7-induced DNA damage response, which is critical for both events, was also compromised. Consequently, Vorinostat exposure led to DNA damage and triggered apoptosis in HPV-infected, differentiated cells, whereas uninfected tissues were spared. Apoptosis was attributed to highly elevated proapoptotic Bim isoforms that are known to be repressed by EZH2 in a repressor complex containing HDACs. Two other HDAC inhibitors, Belinostat and Panobinostat, also inhibited viral DNA amplification and cause apoptosis. We suggest that HDAC inhibitors are promising therapeutic agents to treat benign HPV infections, abrogate progeny virus production, and hence interrupt transmission.