sirolimus 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

Autophagy is closely related to the formation and development of multiple human tumors including ovarian cancer. As a major regulator of this process, the role of mTOR (mammalian target of rapamycin) has been well proven. Cardamonin, a kind of flavonoid from plants, has effects on induction of autophagy and thus antiproliferation of cancer cells. However, the detailed mechanism remains unclear. DAP1 (death-associated protein 1) is a proline-rich protein, which is involved in the regulation of cellular growth and programmed cell death including autophagy and apoptosis. The aim of this study was to investigate whether DAP1 is involved in proliferation inhibition and autophagy induced by cardamonin in tumor cells. Using online bioinformatics tools, we found that DAP1 expression is closely related to the survival of patients with ovarian cancer. Our study showed that autophagy induced by cardamonin was associated with mTOR inhibition, and DAP1 was involved in this process. Silence of DAP1 decreased cell proliferation but enhanced the antiproliferative effect of cardamonin in SKOV3 cells. The level of autophagy was elevated by DAP1 silencing in SKOV3 cells. Notably, cardamonin showed higher autophagy flux in the DAP1 small interfering RNA group. Taken together, our results implied that DAP1 negatively regulates autophagy induced by cardamonin, and it may be a potential target for ovarian cancer therapy.

Topics: Apoptosis; Apoptosis Regulatory Proteins; Autophagy; Cell Line, Tumor; Cell Proliferation; Chalcones; Female; Gene Expression; Gene Expression Regulation, Neoplastic; Humans; Ovarian Neoplasms; RNA, Small Interfering; Sirolimus

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

The mammalian target of rapamycin is crucial in the regulation of cell growth and metabolism. Recent studies suggest that the mammalian target of rapamycin and its downstream 70-kDa ribosomal S6 kinase 1 negatively modulate the insulin-signaling pathway, which is considered the main cause of insulin resistance. The aim of this study is to investigate the effects of cardamonin, a potential inhibitor of the mammalian target of the rapamycin, on insulin-resistant vascular smooth muscle cells and the molecular mechanisms involved. Vascular smooth muscle cells were cultured with high glucose and high insulin to induce insulin resistance. The mammalian target of rapamycin was overstimulated in cells that were incubated with high glucose and high insulin, as reflected by the excessive activation of S6 kinase 1. Insulin-resistant vascular smooth muscle cells displayed hyperphosphorylation of insulin receptor substrate-1 at Ser residues 636/639, which decreased the activity of insulin receptor substrate-1. Also, the activation of protein kinase B and phosphorylation of glycogen synthesis kinase-3β were inhibited. Cardamonin increased the 2-deoxyglucose uptake and glycogen concentration, which was reduced by insulin resistance. As with rapamycin, cardamonin inhibited the activity of the mammalian target of rapamycin and S6 kinase 1, decreased the Ser 636/639 phosphorylation of insulin receptor substrate-1 and increased the activation of protein kinase B. Both of them increased the Ser9 phosphorylation of glycogen synthesis kinase-3β and decreased the expression of glycogen synthesis kinase-3β. However, neither cardamonin nor rapamycin increased the expression of glucose transport 4 which decreased in insulin-resistant vascular smooth muscle cells. This study suggests that cardamonin inhibited the activity of the mammalian target of rapamycin and eliminated the negative feedback of the mammalian target of rapamycin and S6 kinase 1 on the insulin-signaling pathway.

Topics: Animals; Blood Glucose; Cells, Cultured; Chalcones; Deoxyglucose; Glycogen; Insulin; Insulin Resistance; Mammals; Phosphorylation; Proto-Oncogene Proteins c-akt; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

The objective of this study was to determine possible effects and potential mechanisms of cardamonin on improving insulin resistance and vascular proliferative lesions in the rat's model system. Fed with 60% fructose-enriched diet for 12 weeks, male Sprague-Dawley (SD) rats developed insulin resistance and hyperinsulinemia. They also showed excessive proliferation of the vascular smooth muscle cells (VSMCs) and activation of the mammalian target of rapamycin (mTOR)/translation control proteins p70 ribosomal S6 kinase (P70S6K1)/eukaryotic initiation factor 4E binding protein 1 (4E-BP1) signaling in the rat thoracic aorta. From weeks 9-12, cardamonin was injected into the peritoneal cavity once daily. Under the detection of microscopy and electron microscopy, cardamonin improved hyperinsulinemia and inhibited proliferation of VSMCs in the thoracic aorta of rats in a dose-dependent manner. By the Real-Time RT-PCR, mRNA expression of mTOR, P70S6K1 and 4E-BP1 was significantly reduced in cardamonin treated rats. Similarly, protein over-expression of mTOR and p-P70S6K1 was obviously inhibited by immunohistochemical analyses. These findings suggest that cardamonin may play a role in ameliorating insulin resistance and smooth muscle hyperplasia of major vessels in fructose-induced rats, possibly via a mechanism that involves the modulation of insulin/mTOR signaling.

Topics: Animals; Aorta, Thoracic; Carrier Proteins; Cell Proliferation; Chalcones; Dose-Response Relationship, Drug; Fructose; Hyperinsulinism; Hypoglycemic Agents; Insulin; Insulin Resistance; Male; Mammals; Molecular Structure; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Random Allocation; Rats; Rats, Sprague-Dawley; Ribosomal Protein S6 Kinases; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases