TMZ In Combination With Other Drugs And Efficacy Of BNCT For Refractory Pituitary Adenomas

Current status of therapy for refractory Pituitary adenomasPituitary adenomas (PAs) account for approximately15%of intracranial tumors and are the second most common intracranial neoplasm after gliomas. Although most PAs are benign and noninvasive tumors that show expansive growth to surrounding tissues or remain within the sella, approximately35%of them are locally invasive and aggressive pituitary tumors with massive invasion of bone, dura, and/or adjacent structures, and undergo rapid growth. Another subset of non-metastatic invasive pituitary tumors displaying more aggressive behavior are called atypical PAs, and are characterized by fast invasive growth, increased mitotic activity, a Ki-67labeling index greater than3%, and positive P53immunoreactivity. Although repeated surgeries, radiosurgery and alternative medical therapies are routinely used for invasive PAs and atypical PAs, they tend to recur quickly after initial treatments, and are generally unresponsive to conventional therapy including surgeries, radiotherapy and pharmacological treatment. These invasive PAs refractory to standard treatments and tend to recur quickly after initial treatments are defined "Refractory pituitary adenomas". Refractory PAs are notoriously difficult to manager and are associated with poor prognosis and some even have a fatal outcome. Therefore, it is urgent to identify new therapeutic targets and develop new treatment strategies for these refractory PAs.Section one:Inhibition of PI3K/mTOR Pathway Enhances Temozolomide-Induced Cytotoxicity in Pituitary Adenoma Cell Lines in vitro and Xenografted Pituitary Adenoma in Female Nude MiceObject:Invasive pituitary adenomas (PAs) are often refractory to standard therapy and a salvage treatment with temozolomide (TMZ). Hyperactivation of the phosphoinositide3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway contributes to chemotherapy resistance in many cancers. Hyperactive PI3K/AKT/mTOR pathway frequently occurs in invasive PAs. XL765, a novel dual PI3K/mTOR inhibitor, has recently shown its efficacy as a monotherapy and in combination with conventional therapeutics in many cancers. The aim of the present study was to investigate whether XL765sensitizes PAs cells to TMZ in vitro and in vivo.Methods:Experiments were carried out to evaluate the effect of XL765and TMZ alone or in combination on cell proliferation and apoptosis of PAs cell lines (aT3-1, GH3and MMQ) in vitro as well as the tumor growth and serum growth hormone (GH) and prolactin (PRL) secretions in a GH3xenograft tumor model of female nude mice.Results:XL765and TMZ synergistically inhibited the growth of PAs cell lines and induced apoptosis. Combination of XL765and TMZ synergistically inhibited tumor growth, decreased serum GH and PRL levels, and prolonged survival of GH3xenograft tumor models without increased systemic side effects. In addition, XL765in combination with TMZ dramatically decreased phosphorylation of AKT and mTOR as well as the expression of Bcl-2. The increased expression of cleaved PARP and Bax along with elevated Caspase-3/7activity was also observed in combination group.Conclusions:We report here that XL765exerts its antitumor effects in pituitary adenomas by inhibiting the PI3K/mTOR pathway, we futher emphasize that it could enhance the efficacy of TMZ in in pituitary adenoma cell Lines in vitro and a xenografted pituitary adenoma model. Therefore dual inhibitors of PI3K/mTOR may enhance alkylating agent mediated cytotoxicity in the treatment of invasive PAs. Section two:Pyrimethamine sensitizes pituitary adenomas cells to temozolomide through cathepsin B-dependent and caspase-dependent apoptotic pathwaysObject:Invasive pituitary adenomas (PAs) are generally refractory to conventional therapy and salvage treatment with temozolomide (TMZ). In addition to antiprotozoan effects, pyrimethamine (PYR) has recently shown its strong antitumor activity as an antineoplastic agent or in combination with TMZ in metastatic melanoma cells. In this study, we aim to investigate whether PYR could enhace the efficacy of TMZ against pituitary adenomas.Methods:The effects of TMZ, PYR or TMZ/PYR combination on rat/mouse PA cell lines aT3-1, GH3, MMQ and ATt-20as well as GH3xenograft tumor model were evaluated. The cell viability, cell invasiveness, cell cycles, cell apoptosis, caspase activities of PA cell lines were assessed after incubation with TMZ, PYR alone or TMZ/PYR combination. The expression of Bcl-2, Bax, PARP and cathepsin B were analysed to investigate the mechanisms of apoptosis induced by TMZ, PYR alone or TMZ/PYR combination. A GH3xenograft tumor model was used to evaluate the antitumor effects of TMZ, PYR alone or TMZ/PYR combination. The serum GH and PRL of mice harboring GH3xenograft tumor also were tested after treatmet with TMZ, PYR alone or TMZ/PYR combination.Results:TMZ/PYR combination synergistically inhibited proliferation, invasion and induced apoptosis of PA cell lines in vitro. Strikingly, combination treatment with TMZ and PYR produced synergistic antitumor activity and enhanced the survival rate of GH3xenograft tumor models without increasing systemic side effects. In addition, TMZ/PYR induced cell cycle arrest, increased DNA damage, upregulated the expression of cathepsin B, BAX, cleaved PARP and phosphorylated histone H2AX as well as elevated caspase3/7,8,9activities. The decreased expression of Bcl-2, MMP-2and MMP-9alone with cytochrome c release from mitochondria into the cytosol were also observed in the TMZ/PYR combination group. The increase in cell apoptosis due to combination with PYR was rescued by leucovorin. These data suggest that PYR may enhance the efficacy of TMZ via triggering both cathepsin B-dependent and caspase-dependent apoptotic pathways.Conclusions:These results suggest that PYR could be effective to treat PAs as an antineoplastic agent, as well as a potent enhancer of efficacy of TMZ for refractory PAs. Therefore, combination of PYR and TMZ may provide a novel regimen for PAs refractory to standard therapy and TMZ. Section three:Folate receptor-mediated boron-10containing carbon nanoparticles as potential delivery vehicles for boron neutron capture therapy of nonfunctional pituitary adenomasObject:Invasive nonfunctional pituitary adenomas(NFPAs)are difficult to completely resect and often develop tumor recurrence after initial surgery.Currently,nomedications are clinically effective in the control of NFPA. Althoug radiation therapy and radiosurgery are useful to prevent tumor regrowth, they are frequently withheld because of severe complications. Boron neutron capture therapy (BNCT) is a binary radiotherapy that selectively and maximally damages tumor cells without harming the surrounding normal tissue.Folate receptor(FP)-targeted boron-10containing carbon nanoparticles is a novel boron delivery agent that can be selectively taken up by FR-expressing cells via FR-mediated endocytosis. In our previous studies we have proved that FR is is overexpressed in NFPAs but not in hormone-secreting adenomas or normal pituitary.In this paper, we intent to investigate that whether the FR-targeted boron delivery agent could be selectively uptaken by NFPA cells,and wthether the FR-mediated BNCT have therapeutic effects on NFPAs.Methods:The FR expression of primary cultured NFPA cells was determined by immunofluorescence analysis. After incubation with FR-targeted boron-10containing carbon nanoparticles,the uptake of these boron agents by NFPA cells was examined using confocal scanning laser microscope. The Intracellular boron concentration of NFPA cells after incubation with FR-targeted boron-10containing carbon nanoparticles was accurately determined by inductively coupled plasma-atomic emission spectrum. After incubation with FR-targeted boron-10containing carbon nanoparticles and irradiation at a neutron flux, the cell viability of NFPA cells was determined by CCK-8, and the apoptosis was detected by Annexin V/PI analysis. The expression of Bax and Bcl-2in NFPA cells after BNCT was examined by western blotting as well.Results: In this study, FR-targeted boron-10con-taining carbon nanoparticles were selectively uptaken by NFPAs cells expressing FR but not other types of non-FR expressing pituitary adenomas. After incubation with boron-10containing carbon nanoparticles and following irradiation with thermal neutrons, the cell viability of NFPAs was significantly decreased, while apoptotic cells were simultaneously increased. However, cells administered the same dose of FR-targeted boron-10 containing carbon nanoparticles without neutron irradiation or received the same neutron irradiation alone did not show significant decrease in cell viability or increase in apoptotic cells. The expression of Bcl-2was down regulated and the expression of Bax was up regulated in NFPAs after treatment with FR-mediated BNCT.Conclusions:In summary, FR-targeted boron-10containing carbon nanoparticles may be an ideal de livery system of boron to NFPAs cells for BNCT. Furthermore, our study also provides a novel insight in to therapeutic strategies for invasive NFPA refractory to conventional therapy, while exploring these new applications of BNCT for tumors, especially benign tumors.