(5-(2-4-bis((3s)-3-methylmorpholin-4-yl)pyrido(2-3-d)pyrimidin-7-yl)-2-methoxyphenyl)methanol and cardamonin

A number of mammalian target of rapamycin (mTOR) inhibitors have been approved for the treatment of certain types of cancer or are currently undergoing clinical trials. However, mTOR targeted therapy exerts selective pressure on tumour cells, which leads to the preferential growth of resistant subpopulations. There are two classes of mTOR inhibitors: i) The rapalogs, such as rapamycin, which bind to the 12‑kDa FK506‑binding protein/rapamycin‑binding domain of mTOR; and ii) the ATP‑competitive inhibitors, such as AZD8055, which block the mTOR kinase domain. Cardamonin inhibits mTOR by decreasing the expression of regulatory‑associated protein of mTOR (Raptor), a mechanism of action which differs from the currently available mTOR inhibitors. The present study investigated the inhibitory effects of cardamonin on mTOR inhibitor‑resistant cancer cells. HeLa cervical cancer cells and MCF‑7 breast cancer cells were exposed to high concentrations of mTOR inhibitors, until resistant clones emerged. Cytotoxicity was measured using the MTT and colony forming assays. The inhibitory effect of cardamonin on mTOR signalling was assessed by western blotting. The resistant cells were less sensitive to mTOR inhibitors compared with the parental cells. Consistent with the anti‑proliferation effect, rapamycin and AZD8055 had no effect on the phosphorylation of rapamycin‑sensitive sites on ribosomal protein S6 kinase B1 (S6K1) and AZD8055‑sensitive sites on protein kinase B and eukaryotic translation initiation factor 4E binding protein 1 (Thr 37/46), respectively, in rapamycin‑ and AZD8055‑resistant cells. Cardamonin inhibited cell proliferation and decreased the phosphorylation of mTOR and S6K1, as well as the protein level of raptor, in the mTOR inhibitor‑resistant cells. Therefore, cardamonin may serve as a therapeutic agent for patients with cervical and breast cancer resistant to mTOR inhibitors.

Topics: Cell Proliferation; Chalcones; Drug Resistance, Neoplasm; HeLa Cells; Humans; MCF-7 Cells; Morpholines; Neoplasm Proteins; Neoplasms; Sirolimus; TOR Serine-Threonine Kinases

The mammalian target of rapamycin (mTOR) integrates energy level to modulate cell proliferation and autophagy. Cardamonin exhibits anti-proliferative activity through inhibiting mTOR. In this study, the effect of cardamonin on autophagy and its mechanism on mTOR inhibition were investigated.. Cell viability and proliferation were measured by MTT assay and BrdU incorporation, respectively. Cell apoptosis was assayed by flow cytometry and cell autophagy was detected by electron microscopy and GFP-LC3 fluorescence. The mechanism of cardamonin on mTORC1 inhibition was investigated by Raptor siRNA and Raptor over-expression.. The cell viability and proliferation were inhibited by cardamonin. The autophagosomes and the protein level of LC3-II were increased by cardamonin. Cell apoptosis and the levels of cleaved PARP and Caspase-3 were increased by cardamonin. Cardamonin inhibited the phosphorylation of mTOR and ribosome S6 protein kinase 1 (S6K1) as well as the protein level of regulatory associated protein of mTOR (Raptor). However, cardamonin had no effect on the component of mTORC2 and its downstream substrate Akt. The inhibitory effect of cardamonin on the phosphorylation of mTOR and S6K1 was eliminated by Raptor knockdown with siRNA, whereas this effect of cardamonin was stronger than that of rapamycin and AZD8055 in Raptor over-expression cells. Cell viability was inhibited by cardamonin in both Raptor knockdown and Raptor over-expression cells, which was consistent with the inhibitory effect of cardamonin on mTOR.. These findings demonstrated that the autophagy induced by cardamonin was associated with mTORC1 inhibition through decreasing the protein level of Raptor in SKOV3 cells.

Topics: Apoptosis; Autophagy; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cell Survival; Chalcones; Humans; Mechanistic Target of Rapamycin Complex 1; Microtubule-Associated Proteins; Morpholines; Oncogene Protein v-akt; Phosphorylation; Poly (ADP-Ribose) Polymerase-1; Regulatory-Associated Protein of mTOR; Ribosomal Protein S6 Kinases, 70-kDa; RNA, Small Interfering; Sirolimus; TOR Serine-Threonine Kinases