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      Targeted inhibition of mammalian target of rapamycin (mTOR) enhances radiosensitivity in pancreatic carcinoma cells

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          Abstract

          The mammalian target of rapamycin (mTOR) is a protein kinase that regulates protein translation, cell growth, and apoptosis. Rapamycin (RPM), a specific inhibitor of mTOR, exhibits potent and broad in vitro and in vivo antitumor activity against leukemia, breast cancer, and melanoma. Recent studies showing that RPM sensitizes cancers to chemotherapy and radiation therapy have attracted considerable attention. This study aimed to examine the radiosensitizing effect of RPM in vitro, as well as its mechanism of action. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and colony formation assay showed that 10 nmol/L to 15 nmol/L of RPM had a radiosensitizing effects on pancreatic carcinoma cells in vitro. Furthermore, a low dose of RPM induced autophagy and reduced the number of S-phase cells. When radiation treatment was combined with RPM, the PC-2 cell cycle arrested in the G2/M phase of the cell cycle. Complementary DNA (cDNA) microarray and reverse transcription polymerase chain reaction (RT-PCR) revealed that the expression of DDB1, RAD51, and XRCC5 were downregulated, whereas the expression of PCNA and ABCC4 were upregulated in PC-2 cells. The results demonstrated that RPM effectively enhanced the radiosensitivity of pancreatic carcinoma cells.

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          Most cited references 50

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          Monodansylcadaverine (MDC) is a specific in vivo marker for autophagic vacuoles.

          We report the use of the autofluorescent compound monodansylcadaverine (MDC) for in vivo labeling of autophagic vacuoles. When applied to various cell types (PaTu 8902, MDCK I, PC12, AR4-2J, WI-38) in culture, spherical structures were observed by fluorescence microscopy, predominantly located in the perinuclear region. Only PC12 and WI-38 cells had some of these labeled structures in their filopodiae. Dose-response experiments with PaTu 8902 showed that the optimal concentration for in vivo labeling was 0.05 to 0.1 mM, while cells detached and disintegrated, when MDC concentration exceeded 0.1 mM. After incubation with MDC and subcellular fractionations of PaTu 8902 cells on sucrose density gradients, a narrow fluorescence peak at 20 to 26% sucrose concentration equal to densities of about 1.081 to 1.108 g/cm3 was observed. Ultrastructural analysis of these fractions revealed autophagic vacuoles in different stages of their development. To investigate whether endosomal compartments were also labeled by MDC, we coincubated PaTu 8902 cells with MDC and the fluid-phase markers, RITC-dextran and ferritin, respectively. Fluorescence measurements after subcellular fractionations as well as fine structural analysis indicated that MDC-labeled autophagic vacuoles did not contain fluid-phase markers and were spatially separated from endosomal compartments. We further could demonstrate, after subcellular fractionation procedures, that MDC-labeled organelles contained the lysosomal enzymes acid phosphatase and the mature form of cathepsin D. Membrane markers of rough endoplasmic reticulum (TRAM and sec61 beta), and for smooth endoplasmic reticulum (cytochrome P450) were not detected in the same fractions. These results indicate that MDC accumulates as a selective fluorescent marker for autophagic vacuoles under in vivo conditions and is not present in the early and late endosome.
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            Inhibition of mammalian target of rapamycin or apoptotic pathway induces autophagy and radiosensitizes PTEN null prostate cancer cells.

            The phosphatidylinositol 3-kinase/Akt pathway plays a critical role in oncogenesis, and dysregulation of this pathway through loss of PTEN suppression is a particularly common phenomenon in aggressive prostate cancers. The mammalian target of rapamycin (mTOR) is a downstream signaling kinase in this pathway, exerting prosurvival influence on cells through the activation of factors involved in protein synthesis. The mTOR inhibitor rapamycin and its derivatives are cytotoxic to a number of cell lines. Recently, mTOR inhibition has also been shown to radiosensitize endothelial and breast cancer cells in vitro. Because radiation is an important modality in the treatment of prostate cancer, we tested the ability of the mTOR inhibitor RAD001 (everolimus) to enhance the cytotoxic effects of radiation on two prostate cancer cell lines, PC-3 and DU145. We found that both cell lines became more vulnerable to irradiation after treatment with RAD001, with the PTEN-deficient PC-3 cell line showing the greater sensitivity. This increased susceptibility to radiation is associated with induction of autophagy. Furthermore, we show that blocking apoptosis with caspase inhibition and Bax/Bak small interfering RNA in these cell lines enhances radiation-induced mortality and induces autophagy. Together, these data highlight the emerging importance of mTOR as a molecular target for therapeutic intervention, and lend support to the idea that nonapoptotic modes of cell death may play a crucial role in improving tumor cell kill.
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              Autophagy upregulation by inhibitors of caspase-3 and mTOR enhances radiotherapy in a mouse model of lung cancer.

              Autophagy has been reported to be increased in irradiated cancer cells resistant to various apoptotic stimuli. We therefore hypothesized that induction of autophagy via mTOR inhibition could enhance radiosensitization in apoptosis-inhibited H460 lung cancer cells in vitro and in a lung cancer xenograft model. To test this hypothesis, combinations of Z-DEVD (caspase-3 inhibitor), RAD001 (mTOR inhibitor) and irradiation were tested in cell and mouse models. The combination of Z-DEVD and RAD001 more potently radiosensitized H460 cells than individual treatment alone. The enhancement in radiation response was not only evident in clonogenic survival assays, but also was demonstrated through markedly reduced tumor growth, cellular proliferation (Ki67 staining), apoptosis (TUNEL staining) and angiogenesis (vWF staining) in vivo. Additionally, upregulation of autophagy as measured by increased GFP-LC3-tagged autophagosome formation accompanied the noted radiosensitization in vitro and in vivo. The greatest induction of autophagy and associated radiation toxicity was exhibited in the tri-modality treatment group. Autophagy marker, LC-3-II, was reduced by 3-methyladenine (3-MA), a known inhibitor of autophagy, but further increased by the addition of lysosomal protease inhibitors (pepstatin A and E64d), demonstrating that there is autophagic induction through type III PI3 kinase during the combined therapy. Knocking down of ATG5 and beclin-1, two essential autophagic molecules, resulted in radiation resistance of lung cancer cells. Our report suggests that combined inhibition of apoptosis and mTOR during radiotherapy is a potential therapeutic strategy to enhance radiation therapy in patients with non-small cell lung cancer.
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                Author and article information

                Journal
                Drug Des Devel Ther
                Drug Des Devel Ther
                Drug Design, Development and Therapy
                Dove Medical Press
                1177-8881
                2013
                19 March 2013
                : 7
                : 149-159
                Affiliations
                [1 ]Department of Oncology, Second Affiliated Hospital, Medical School of Xi’an Jiaotong University, Xi’an, People’s Republic of China
                [2 ]Department of Nephrology, Second Affiliated Hospital, Medical School of Xi’an Jiaotong University, Xi’an, People’s Republic of China
                [3 ]Department of Pharmacology, Second Affiliated Hospital, Medical School of Xi’an Jiaotong University, Xi’an, People’s Republic of China
                Author notes
                Correspondence: Zhi-Jun Dai; Xi-Jing Wang; Department of Oncology, Second Affiliated Hospital, Medical School of Xi’an Jiaotong University, 710004 Xi’an, People’s Republic of China Tel +86 29 8767 9226 Fax +86 29 8767 9282 Email dzj0911@ 123456126.com ; wangxijing@ 12345621cn.com

                *These authors contributed equally to this work

                Article
                dddt-7-149
                10.2147/DDDT.S42390
                3610438
                23662044
                © 2013 Dai et al, publisher and licensee Dove Medical Press Ltd

                This is an Open Access article which permits unrestricted noncommercial use, provided the original work is properly cited.

                Categories
                Original Research

                Pharmacology & Pharmaceutical medicine

                rapamycin, radiation, pancreatic carcinoma, mtor

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