Silencing of SmgGDS, a Novel mTORC1 Inducer That Binds to RHEBs, Inhibits Malignant Mesothelioma Cell Proliferation.

Malignant mesothelioma (MM) is an aggressive tumor that typically develops after a long latency following asbestos exposure. Although mechanistic target of rapamycin complex 1 (mTORC1) activation enhances MM cell growth, the mTORC1 inhibitor everolimus has shown limited efficacy in clinical trials of MM patients. We explored the mechanism underlying mTORC1 activation in MM cells and its effects on cell proliferation and progression. Analysis of the expression profiles of 87 MMs from The Cancer Genome Atlas revealed that 40 samples (46%) displayed altered expression of RPTOR (mTORC1 component) and genes immediately upstream that activate mTORC1. Among them, we focused on RHEB and RHEBL1, which encode direct activators of mTORC1. Exogenous RHEBL1 expression enhanced MM cell growth, indicating that RHEB-mTORC1 signaling acts as a pro-oncogenic cascade. We investigated molecules that directly activate RHEBs, identifying SmgGDS as a novel RHEB-binding protein. SmgGDS knockdown reduced mTORC1 activation and inhibited the proliferation of MM cells with mTORC1 activation. Interestingly, SmgGDS displayed high binding affinity with inactive GDP-bound RHEBL1, and its knockdown reduced cytosolic RHEBL1 without affecting its activation. These findings suggest that SmgGDS retains GDP-bound RHEBs in the cytosol, whereas GTP-bound RHEBs are localized on intracellular membranes to promote mTORC1 activation. We revealed a novel role for SmgGDS in the RHEB-mTORC1 pathway and its potential as a therapeutic target in MM with aberrant mTORC1 activation. IMPLICATIONS: Our data showing that SmgGDS regulates RHEB localization to activate mTORC1 indicate that SmgGDS can be used as a new therapeutic target for MM exhibiting mTORC1 activation.

SGO1 is involved in the DNA damage response in MYCN-amplified neuroblastoma cells.

Shugoshin 1 (SGO1) is required for accurate chromosome segregation during mitosis and meiosis; however, its other functions, especially at interphase, are not clearly understood. Here, we found that downregulation of SGO1 caused a synergistic phenotype in cells overexpressing MYCN. Downregulation of SGO1 impaired proliferation and induced DNA damage followed by a senescence-like phenotype only in MYCN-overexpressing neuroblastoma cells. In these cells, SGO1 knockdown induced DNA damage, even during interphase, and this effect was independent of cohesin. Furthermore, MYCN-promoted SGO1 transcription and SGO1 expression tended to be higher in MYCN- or MYC-overexpressing cancers. Together, these findings indicate that SGO1 plays a role in the DNA damage response in interphase. Therefore, we propose that SGO1 represents a potential molecular target for treatment of MYCN-amplified neuroblastoma.

Improvement of Pharmaceutical Properties of Isoprenoid Compounds through the Formation of Cyclodextrin Pseudorotaxane-Like Supramolecules.

The purpose of this study was to design cyclodextrin (CyD)-based pseudorotaxane-like supramolecular complexes with various isoprenoid compounds, such as reduced coenzyme Q10 (R-CoQ10), squalene, tocotrienol, and teprenone, and to evaluate their pharmaceutical properties. Squalene, tocotrienol, and teprenone formed precipitates with ß-CyD and γ-CyD in aqueous solution, whereas R-CoQ10 formed precipitates with γ-CyD aqueous solution. The results of powder X-ray diffraction and (1)H-NMR analyses indicated that these precipitates are derived from pseudorotaxane-like supramolecular complexes. The photostability of teprenone was markedly improved by complexation with CyDs, especially in the γ-CyD system. In addition, the dispersion rates of teprenone in the γ-CyD system were higher than those in the ß-CyD system, compared with the corresponding physical mixtures. In conclusion, pharmaceutical properties such as photostability and dispersion rates of isoprenoid compounds were improved by the formation of pseudorotaxane-like supramolecular complexes with ß-CyD and/or γ-CyD.

Folate-PEG-appended dendrimer conjugate with α-cyclodextrin as a novel cancer cell-selective siRNA delivery carrier.

We previously reported that of the various polyamidoamine (PAMAM) STARBURST dendrimer (generation 3, G3) (dendrimer) conjugates with cyclodextrins (CyDs), the dendrimer (G3) conjugate with α-CyD having an average degree of substitution of 2.4 (α-CDE (G3)) has the greatest potential for a novel carrier for siRNA in vitro and in vivo. To improve the siRNA transfer activity and the lack of target specificity of α-CDE (G3), we prepared folate-polyethylene glycol (PEG)-appended α-CDEs (G3) (Fol-PαCs) with various degrees of substitution of folate (DSF) and evaluated their siRNA transfer activity to folate receptor (FR)-overexpressing cancer cells in vitro and in vivo. Of the three Fol-PαCs (G3, DSF 2, 4 and 7), Fol-PαC (G3, DSF 4) had the highest siRNA transfer activity in KB cells (FR-positive). Fol-PαC (G3, DSF 4) was endocytosed into KB cells through FR. No cytotoxicity of the siRNA complex with Fol-PαC (G3, DSF 4) was observed in KB cells (FR-positive) or A549 cells (FR-negative) up to the charge ratio of 100/1 (carrier/siRNA). In addition, the siRNA complex with Fol-PαC (G3, DSF 4) showed neither interferon response nor inflammatory response. Importantly, the siRNA complex with Fol-PαC (G3, DSF 4) tended to show the in vivo RNAi effects after intratumoral injection and intravenous injection in tumor cells-bearing mice. The FITC-labeled siRNA and TRITC-labeled Fol-PαC (G3, DSF 4) were actually accumulated in tumor tissues after intravenous injection in the mice. In conclusion, the present results suggest that Fol-PαC (G3, DSF 4) could potentially be used as a FR-overexpressing cancer cell-selective siRNA delivery carrier in vitro and in vivo.

Inhibitory effect of siRNA complexes with polyamidoamine dendrimer/α-cyclodextrin conjugate (generation 3, G3) on endogenous gene expression.

In the present study, we prepared the small interfering RNA (siRNA) complexes with polyamidoamine (PAMAM) dendrimer (G3) conjugate with α-cyclodextrin (α-CDE (G3)), and examined the physicochemical properties, serum resistance, in vitro RNAi effects on endogenous gene expression, cytotoxicity, interferon response, hemolytic activity, cellular association and intracellular distribution. In addition, these results were compared to the siRNA complexes with the commercial transfection reagents such as linear polyethyleneimine (PEI), Lipofectamine™2000 (L2) and RNAiFect™ (RF). α-CDE (G3) interacted with siRNA, and suppressed siRNA degradation by serum. The siRNA complex with α-CDE (G3) showed the potent RNAi effects against Lamin A/C and Fas expression with negligible cytotoxicity and hemolytic activity, compared to those of the transfection reagents in Colon-26-luc cells and NIH3T3-luc cells. Cell-death patterns induced by siRNA polyplexes with α-CDE (G3) and PEI were different from siRNA lipoplexes with L2 and RF. α-CDE (G3) delivered fluorescent-labeled siRNA to cytoplasm, not nucleus, after transfection in NIH3T3-luc cells. Taken together, α-CDE (G3) could be potentially used as a siRNA carrier to provide the RNAi effect on endogenous gene expression with negligible cytotoxicity.

Preparation of four types of coenzyme Q10/gamma-cyclodextrin supramolecular complexes and comparison of their pharmaceutical properties.

In this study, we prepared four kinds of complexes of Coenzyme Q10 (CoQ10) with gamma-cyclodextrin (gamma-CyD) by the kneading methods and the solubility methods with or without heating, and compared their pharmaceutical properties. Differential scanning calorimetrical curves and powder X-ray diffraction patterns showed that the complexes formed pseudorotaxane-like supramolecular structure, although included free gamma-CyD and CoQ10, when prepared by the kneading method and solubility method without heating. At the preparation process, a heating improved the complexation of CoQ10 with gamma-CyD in the both methods. The dispersion rate of CoQ10 in water increased in the order of CoQ10 alone approximately physical mixture with gamma-CyD<