Target Of Rapamycin Inhibitor RAD001 Research Articles

Mammalian target of rapamycin inhibitor RAD001 sensitizes endometrial cancer cells to paclitaxel-induced apoptosis via the induction of autophagy.

The aim of the present study was to investigate the effects of the mammalian target of rapamycin (mTOR) inhibitor, RAD001, on the growth of human endometrial cancer cells. The effects of RAD001 on human endometrial cancer Ishikawa and HEC-1A cell proliferation were determined by MTT assay. Green fluorescent protein microtubule-associated protein 1 light chain 3α (GFP-LC3) protein aggregates were observed under a confocal microscope, and Ishikawa and HEC-1A cell apoptosis was detected using flow cytometry. The expression levels of LC3-I, LC3-II and mTOR proteins were detected by western blot analysis. The results showed that RAD001 effectively inhibited human endometrial cancer Ishikawa and HEC-1A cell proliferation via downregulation of AKT/mTOR phosphorylation. Moreover, RAD001 induced autophagic cell death and a higher sensitivity to paclitaxel-induced apoptosis. These results indicate that RAD001 could have therapeutic potential in human endometrial cancer with hyperactivated AKT/mTOR signaling.

The mammalian target of rapamycin inhibitor RAD001 (everolimus) synergizes with chemotherapeutic agents, ionizing radiation and proteasome inhibitors in pre-B acute lymphocytic leukemia

Despite incremental improvements in outcomes for patients with acute lymphoblastic leukemia, significant numbers of patients still die from this disease. Mammalian target of rapamycin inhibitors have shown potential in vitro and in vivo as therapeutic agents against a range of tumors including acute lymphoblastic leukemia. Flow cytometry was used to evaluate drug-induced cell death in acute lymphoblastic leukemia cell lines and patients' samples. Human xenografts in immunocompromised mice were used to assess the in vivo effects of selected combinations. Pharmacological inhibitors and lentiviral small interfering ribonucleic acid knock-down of p53 were used to investigate the mechanism of cell killing involved. Synergistic interactions between RAD001 and cytotoxic agents were demonstrated in vitro and in vivo, with increased caspase-dependent killing. RAD001 suppressed p53 and p21 responses, while suppression of p53 did not prevent killing, indicating p53 independence. RAD001 and cytotoxic agents activated the JUN N-terminal kinase pathway and the combination further increased JUN N-terminal kinase activation. JUN N-terminal kinase inhibition reduced synergistic cell killing by cytotoxic agents and RAD001 in pre-B acute lymphoblastic leukemia cell lines and patients' samples. Bortezomib and MG132, which activate the JUN N-terminal kinase pathway, also synergized with RAD001 in killing pre-B acute lymphoblastic leukemia cells. Killing was greater when RAD001 was combined with proteasome inhibitors than with cytotoxic drugs. These observations suggest that combining mammalian target of rapamycin inhibitors with conventional chemotherapy or selected novel agents has the potential to improve clinical responses in patients with pre-B acute lymphoblastic leukemia.

Antiangiogenic Therapy with Mammalian Target of Rapamycin Inhibitor RAD001 (Everolimus) Increases Radiosensitivity in Solid Cancer

Radiotherapy exerts direct antivascular effects in tumors and also induces a proangiogenic stress response in tumor cells via the phosphoinositide 3-kinase/Akt/mammalian target of rapamycin (mTOR) pathway. Therefore, the combination of radiotherapy and antiangiogenic therapy with mTOR inhibitor RAD001 (Everolimus) might exert additive/synergistic effects on tumor growth. Effects of radiation combined with mTOR inhibitor RAD001 were studied on proliferation of murine colon cancer CT-26, human pancreatic cancer L3.6pl, and human umbilical vascular endothelial cells in vitro. In vivo tumor growth of subcutaneous colon cancer CT 26 and orthotopic pancreatic cancer L3.6pl was assessed after fractionated radiotherapy (5 x 2 or 5 x 4 Gy) with or without the addition of the mTOR inhibitor RAD001. RAD001 (1.5 mg/kg/d) was administered until the end of experiments beginning before or after radiotherapy. A single dose of 2 Gy reduced in vitro proliferation of L3.6pl (-16%), CT-26 (-70%), and human umbilical vascular endothelial cells (HUVEC; -72%). The mTOR inhibitor RAD001 (10 ng/mL) suppressed proliferation of HUVEC (-83%), L3.6pl (-8%), and CT-26 (-82%). Combination of even low concentrations of 0.01 ng/mL RAD001 and 0.25 Gy radiation significantly reduced proliferation of HUVECs (-57%), whereas additive effects of RAD001 and radiation on tumor cells were seen only at the highest concentrations tested. In vivo, RAD001 introduced before radiotherapy (5 x 2 Gy) improved tumor growth control in mice (L3.6pl: 326 mm(3) versus 1144 mm(3); CT-26: 210 mm(3) versus 636 mm(3); P < 0.05 versus control). RAD001 turned out to possess a dose-modifying effect on radiotherapy. Endothelial cells seem to be most sensitive to combination of mTOR inhibition and radiotherapy. Additive tumor growth delay using the mTOR inhibitor RAD001 and radiotherapy in vivo therefore might rely on combined antiangiogenic and antivascular effects.

Cancer-specific mutations in PIK3CA are oncogenic in vivo

The PIK3CA gene, coding for the catalytic subunit p110alpha of class IA phosphatidylinositol 3-kinases (PI3Ks), is frequently mutated in human cancer. Mutated p110alpha proteins show a gain of enzymatic function in vitro and are oncogenic in cell culture. Here, we show that three prevalent mutants of p110alpha, E542K, E545K, and H1047R, are oncogenic in vivo. They induce tumors in the chorioallantoic membrane of the chicken embryo and cause hemangiosarcomas in the animal. These tumors are marked by increased angiogenesis and an activation of the Akt pathway. The target of rapamycin inhibitor RAD001 blocks tumor growth induced by the H1047R p110alpha mutant. The in vivo oncogenicity of PIK3CA mutants in an avian species strongly suggests a critical role for these mutated proteins in human malignancies.