RESUMO
BACKGROUND: Osteosarcoma (OSA) is the most frequent type of primary malignant bone tumor and is apt to occur in children and young adults. Pulmonary metastasis (OSPM) is the major reason for its fatal outcome. Osteocalcin (OC) is a major noncollagenous bone protein whose expression is limited almost exclusively to bone marrow and osteotropic tumors. OC is also known to express in cell lines with bone metastasis feathers. Gene therapy strategies with the OC promoter directing the replication of adenovirus in a tumor-specific manner are a potential modality for OSPM therapy. METHODS: We detected OC mRNA expression by RNA in situ hybridization in OSA and OSPM samples from patients, and tested OC promoter transcriptional activity in OSA and non-OSA cell lines. Then we used a transcriptional replication-competent adenovirus, Ad-OC-E1a, to treat OSPM, and evaluated its tumor-specific replication and killing activities in vitro as well as anti-OSPM efficacy in vivo via systemic delivery. RESULTS: OC mRNA was detected in all types of OSA tissues, including OSPM tissues. The transcriptional activity of the OC promoter was much higher in a OSPM cell line SAOS-2LM7 and primary OSA cell line MG63 than in non-OSA cell lines, including cell lines from breast cancer, colon cancer, and liver cancer. Ad-OC-E1a expressed E1a protein only in MG63 and SAOS-2LM7, which indicated that adenovirus E1a was under strict control by the OC promoter. Ad-OC-E1a demonstrated killing and viral replication activity close to wild-type adenovirus levels in MG63 and SAOS-2LM7, but the killing and viral replication activities were attenuated significantly in cells expressing low OC transcriptional activity. To test whether Ad-OC-E1a could be used to target human OSPM in vivo, SAOS-2LM7 pulmonary metastasis models in nude mice were induced and treated by tail-vein injection with Ad-OC-E1a. Compared to tumor nodules in the lung in groups treated with PBS or control virus, the quantity of metastasized tumor nodules decreased significantly. Adenovirus-infected cells were stained immunohistochemically only inside and around the OSPM nodules but spared normal lung tissue and other organs. CONCLUSIONS: These data demonstrated that OC promoter could direct adenovirus replication by controlling the E1a gene to target human OSPM in a tumor-specific manner, providing an efficient tool to develop a feasible therapeutic modality for OSPM.
Assuntos
Adenoviridae/genética , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/terapia , Osteossarcoma/patologia , Osteossarcoma/terapia , Transcrição Gênica/genética , Replicação Viral/genética , Proteínas E1A de Adenovirus/genética , Animais , Linhagem Celular Tumoral , Citotoxicidade Imunológica , Regulação Neoplásica da Expressão Gênica , Terapia Genética/métodos , Humanos , Neoplasias Pulmonares/genética , Camundongos , Camundongos Nus , Metástase Neoplásica/genética , Metástase Neoplásica/terapia , Especificidade de Órgãos , Osteocalcina/genética , Osteossarcoma/genética , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Resultado do Tratamento , Células Tumorais CultivadasRESUMO
The breast-specific antigen alpha-lactalbumin is expressed in >60% of breast cancer tissues. To evaluate the effect of gene therapy for breast cancer by controlling adenovirus replication with human alpha-lactalbumin promoter, we investigated the activity of a 762-bp human alpha-lactalbumin promoter. Alpha-lactalbumin promoter showed significantly higher activity in MDA-MB-435S and T47D breast cancer cells than in normal breast cell lines or other tumor cell lines. We then developed two novel breast cancer-restricted replicative adenoviruses, AdALAE1a and AdE1aALAE1b. In AdALAE1a, expression of adenoviral E1a gene is under the control of alpha-lactalbumin promoter, and in AdE1aALAE1b, expression of both E1a and E1b genes is under the control of a single alpha-lactalbumin promoter. Both breast cancer-restricted replicative adenoviruses showed viral replication efficiency and tumor cell-killing capability similar to wild-type adenovirus in MDA-MB-435S and T47D cells. The replication efficiency and tumor cell-killing capability of both viruses were attenuated significantly in cells that did not support alpha-lactalbumin promoter. AdE1aALAE1b showed better breast cancer-restricted replication than AdALAE1a, suggesting that a transcriptional targeting modality with alpha-lactalbumin promoter controlling both E1a and E1b gene expression is superior to alpha-lactalbumin promoter controlling only E1a gene expression. Importantly, we found that AdE1aALAE1b could be used to target hormone-independent breast tumors in vivo by inhibiting the growth of MDA-MB-435S s.c. tumors. These data showed that alpha-lactalbumin promoter could regulate the replication of adenovirus to target hormone-independent breast cancers, suggesting that alpha-lactalbumin promoter can be used to develop a novel therapeutic modality for hormone-independent breast cancer.
