pki-166 and Carcinoma--Renal-Cell

We have reported that connexin (Cx) 32 acts as a tumor suppressor gene in renal cancer cells partly due to Her-2 inactivation. Here, we determined if a Her-2/Her-1 inhibitor (PKI-166) can enhance the tumor-suppressive effect of Cx32 in Caki-2 cells from human renal cell carcinoma. The expression of Cx32 in Caki-2 cells was required for PKI-166-induced cytotoxic effect at lower doses. The cyctotoxicity was dependent on the occurrence of apoptosis and partly mediated by Cx32-driven gap junction intercellular communications. These results suggest that PKI-166 further supports the tumor-suppressive effect of the Cx32 gene in renal cancer cells through the induction of apoptosis.

Topics: Carcinoma, Renal Cell; Cell Line, Tumor; Connexins; Dose-Response Relationship, Drug; ErbB Receptors; Gap Junction beta-1 Protein; Genes, erbB-1; Growth Inhibitors; Humans; Kidney Neoplasms; Pyrimidines; Pyrroles; Receptor, ErbB-2

Renal cell carcinoma (RCC) frequently produces metastases to the musculoskeletal system that are a major source of morbidity in the form of pain, immobilization, fractures, neurological compromise, and a decreased ability to perform activities of daily living. Patients with metastatic RCC therefore have a dismal prognosis because there is no effective adjuvant treatment for this disease. Because the epidermal growth factor receptor (EGF-R) signaling cascade is important in the growth and metastasis of RCC, its blockade has been hypothesized to inhibit tumor growth and hence prevent resultant bone destruction. We determined whether blockade of EGF-R by the tyrosine kinase inhibitor PKI 166 inhibited the growth of RCC in bone. We use a novel cell line, RBM1-IT4, established from a human RCC bone metastasis. Protein and mRNA expression of the ligands and receptors was assessed by Western and Northern blots. The stimulation of RBM1-IT4 cells with epidermal growth factor or transforming growth factor alpha resulted in increased cellular proliferation and tyrosine kinase autophosphorylation. PKI 166 prevented these effects. First, RBM1-IT4 cells were implanted into the tibia of nude mice, where they established lytic, progressively growing lesions, after which the mice were treated with PKI 166 alone or in combination with paclitaxel (Taxol). Immunohistochemical analysis revealed that tumor cells and tumor-associated endothelial cells in control mice expressed activated EGF-R. Treatment of mice with PKI 166 alone or in combination with Taxol produced a significant decrease in the incidence and size of bone lesions as compared with the results in control or Taxol-treated mice (P < 0.001). Treatment with PKI 166 also decreased the expression of phosphorylated EGF-R by tumor cells and tumor-associated endothelial cells, and this was even more pronounced with PKI 166 plus Taxol treatment. The PKI 166 plus Taxol combination produced apoptosis of tumor cells and tumor-associated endothelial cells. Tumor cell proliferation, shown by proliferating cell nuclear antigen positivity, was decreased in all treatment groups. In addition, the integrity of the bone was maintained in mice treated with PKI 166 or PKI 166 plus Taxol, whereas massive bone destruction was seen in control and Taxol-treated mice. These results suggest that blockade of EGF-R signaling inhibits growth of RCC in the bone by its effect on tumor cells and tumor-associated endothelial cells.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Bone Neoplasms; Carcinoma, Renal Cell; Cell Division; Epidermal Growth Factor; ErbB Receptors; Humans; Kidney Neoplasms; Male; Mice; Mice, Nude; Paclitaxel; Phosphorylation; Pyrimidines; Pyrroles; Recombinant Proteins; Signal Transduction; Transforming Growth Factor alpha; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Renal cell carcinoma frequently metastasizes to the skeleton in the later stages of the disease. Patients with bone metastasis from renal cell carcinoma experience severe pain, neurologic compromise, and frequent pathologic fractures. These tumors are relatively resistant to chemotherapy and radiation, and the 5-year survival of patients is less than 10%. The epidermal growth factor receptor is overexpressed in human renal cell carcinoma and hypothesized to be a potential target in the treatment of bone metastasis. Using in vitro studies, it was shown that blockade of the epidermal growth factor receptor was effective in decreasing proliferation and receptor autophosphorylation of a human bone-derived renal cell carcinoma cell line. In an experimental nude mouse model, treatment with Taxol and protein tyrosine kinase inhibitor 166, a small molecule receptor tyrosine kinase inhibitor, blocked the growth of renal cell carcinoma in the tibia and resulted in decreased bone destruction. The use of protein tyrosine kinase inhibitor 166 and Taxol was cytostatic and nontoxic in long-term animal experiments. Epidermal growth factor receptor blockade is an exciting potential therapy for renal cell carcinoma bone metastasis in humans, but because it is cytostatic rather than cytotoxic, its optimal role may be as a supplement to cytotoxic chemotherapy. It ultimate role and its relationship to other therapeutic interventions remains to be elucidated and validated.

Topics: Animals; Antineoplastic Agents, Phytogenic; Bone Neoplasms; Carcinoma, Renal Cell; ErbB Receptors; Kidney Neoplasms; Mice; Mice, Nude; Paclitaxel; Phosphorylation; Pyrimidines; Pyrroles; Tumor Cells, Cultured

We determined whether blockade of the epidermal growth factor-receptor (EGF-R) signaling pathway by oral administration of the EGF-R tyrosine kinase inhibitor PKI166 can inhibit angiogenesis and growth of SN12PM6 human renal cell carcinoma (HRCC) in the kidney of nude mice and whether gemcitabine can potentiate these effects. In vitro treatment of HRCC cells with PKI166 inhibited EGF-R autophosphorylation, which correlated with a decrease in expression of Bcl-xl protein and phosphorylation of signal transducers and activators of transcription, particularly signal transducers and activators of transcription 3. PKI166 also decreased expression of vascular endothelial growth factor and basic fibroblast growth factor in a dose-dependent manner. Oral administration of PKI166 or PKI166 and injected gemcitabine or gemcitabine alone beginning 7 days after implantation of SN12PM6 cells into the kidney of athymic nude mice reduced the volume of tumors by 26, 61, and 23%, respectively. In another experiment 28 days after the orthotopic implantation of SN12PM6 cells, nephrectomy was performed followed by 4 weeks of treatment. Treatment with PKI166 and, more so, PKI166 plus gemcitabine significantly inhibited lung metastasis, corresponding to a significant increase in overall length of survival. EGF-R activation was significantly blocked by therapy with PKI166 and was associated with a significant reduction in expression of vascular endothelial growth factor and interleukin-8, decreased microvessel density, decreased staining of proliferating cell nuclear antigen, and increased tumor cell apoptosis. Collectively, the data indicate that targeting activation of EGF-R on HRCC produces significant therapeutic benefits.

Topics: Administration, Oral; Animals; Antineoplastic Agents; Apoptosis; Blotting, Western; Carcinoma, Renal Cell; Deoxycytidine; DNA-Binding Proteins; Down-Regulation; Endothelial Growth Factors; Enzyme Inhibitors; ErbB Receptors; Gemcitabine; Immunohistochemistry; In Situ Nick-End Labeling; Intercellular Signaling Peptides and Proteins; Kidney; Lung; Lymphokines; Mice; Mice, Nude; Neoplasm Metastasis; Neovascularization, Pathologic; Phosphorylation; Proliferating Cell Nuclear Antigen; Protein-Tyrosine Kinases; Pyrimidines; Pyrroles; Signal Transduction; STAT3 Transcription Factor; Time Factors; Trans-Activators; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors