nu-7441 and Colorectal-Neoplasms

Colorectal cancer (CRC) is one leading contributor of cancer-related mortalities. Mammalian target of rapamycin (mTOR), existing in two complexes (mTORC1/2), is a valuable target for possible CRC interference. In the current study, we showed that WAY-600, a potent mTOR inhibitor, only exerted moderate activity against primary and HT-29 CRC cells. We proposed that DNA-dependent protein kinase catalytic subunit (DNA-PKcs) could be the major resistance factor of WAY-600 in CRC cells. DNA-PKcs inhibitors, including NU7026 and NU7441, dramatically enhanced WAY-600-induced cytotoxic and pro-apoptotic effect against the CRC cells. Further, WAY-600-exerted cytotoxicity was significantly increased in DNA-PKcs-silenced (by targeted siRNA/shRNA) CRC cells, but was attenuated with DNA-PKcs overexpression. Our evidence suggested that DNA-PKcs Thr-2609 phosphorylation might be critical for WAY-600's resistance. Mutation of this site through introducing a dominant negative DNA-PKcs (T2609A) dramatically potentiated WAY-600's sensitivity in HT-29 cells. Meanwhile, overexpression of protein phosphatase 5 (PP5) dephosphorylated DNA-PKcs at Thr-2609, and significantly increased WAY-600's sensitivity in HT-29 cells. In vivo, WAY-600-induced anti-HT-29 xenograft growth activity was significantly potentiated with NU7026 co-administration. These results suggest that DNA-PKcs could be the major resistance factor of WAY-600 in CRC cells.

Topics: Animals; Apoptosis; Chromones; Colorectal Neoplasms; DNA-Activated Protein Kinase; Drug Resistance, Neoplasm; Drug Synergism; Gene Knockdown Techniques; HT29 Cells; Humans; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Morpholines; Nuclear Proteins; Phosphorylation; Protein Kinase Inhibitors; RNA, Small Interfering; TOR Serine-Threonine Kinases; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

DNA double-strand breaks (DSB) are the most cytotoxic lesions induced by ionizing radiation and topoisomerase II poisons, such as etoposide and doxorubicin. A major pathway for the repair of DSB is nonhomologous end joining, which requires DNA-dependent protein kinase (DNA-PK) activity. We investigated the therapeutic use of a potent, specific DNA-PK inhibitor (NU7441) in models of human cancer. We measured chemosensitization by NU7441 of topoisomerase II poisons and radiosensitization in cells deficient and proficient in DNA-PK(CS) (V3 and V3-YAC) and p53 wild type (LoVo) and p53 mutant (SW620) human colon cancer cell lines by clonogenic survival assay. Effects of NU7441 on DSB repair and cell cycle arrest were measured by gammaH2AX foci and flow cytometry. Tissue distribution of NU7441 and potentiation of etoposide activity were determined in mice bearing SW620 tumors. NU7441 increased the cytotoxicity of ionizing radiation and etoposide in SW620, LoVo, and V3-YAC cells but not in V3 cells, confirming that potentiation was due to DNA-PK inhibition. NU7441 substantially retarded the repair of ionizing radiation-induced and etoposide-induced DSB. NU7441 appreciably increased G(2)-M accumulation induced by ionizing radiation, etoposide, and doxorubicin in both SW620 and LoVo cells. In mice bearing SW620 xenografts, NU7441 concentrations in the tumor necessary for chemopotentiation in vitro were maintained for at least 4 hours at nontoxic doses. NU7441 increased etoposide-induced tumor growth delay 2-fold without exacerbating etoposide toxicity to unacceptable levels. In conclusion, NU7441 shows sufficient proof of principle through in vitro and in vivo chemosensitization and radiosensitization to justify further development of DNA-PK inhibitors for clinical use.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Cell Cycle; Cell Line, Tumor; CHO Cells; Chromones; Colorectal Neoplasms; Cricetinae; DNA Damage; DNA-Activated Protein Kinase; Drug Synergism; Etoposide; Female; Histones; Humans; Mice; Mice, Nude; Morpholines; Phosphorylation; Protein Kinase Inhibitors; Radiation-Sensitizing Agents; Tissue Distribution; Xenograft Model Antitumor Assays