Studies on Biological and Molecular Effects of Small-Molecule Kinase Inhibitors on Human Glioblastoma Cells and Organotypic Brain Slices.

Glioblastoma is the most common and aggressive primary brain tumor. Multiple genetic and epigenetic alterations in several major signaling pathways-including the phosphoinositide 3-kinases (PI3K)/AKT/mTOR and the Raf/MEK/ERK pathway-could be found. We therefore aimed to investigate the biological and molecular effects of small-molecule kinase inhibitors that may interfere with those pathways. For this purpose, patient-derived glioblastoma cells were challenged with dactolisib, ipatasertib, MK-2206, regorafenib, or trametinib. To determine the effects of the small-molecule kinase inhibitors, assays of cell proliferation and apoptosis and immunoblot analyses were performed. To further investigate the effects of ipatasertib on organotypic brain slices harboring glioblastoma cells, the tumor growth was estimated. In addition, the network activity in brain slices was assessed by electrophysiological field potential recordings. Multi-kinase inhibitor regorafenib and both MK-2206 and dactolisib were very effective in all preclinical tumor models, while with respect to trametinib, two cell lines were found to be highly resistant. Only in HROG05 cells, ipatasertib showed anti-tumoral effects in vitro and in organotypic brain slices. Additionally, ipatasertib diminished synchronous network activity in organotypic brain slices. Overall, our data suggest that ipatasertib was only effective in selected tumor models, while especially regorafenib and MK-2206 presented a uniform response pattern.

Unraveling the therapeutic potential of GANT61/Dactolisib combination as a novel prostate cancer modality.

Aberrant activation of several signaling pathways has been implicated in prostate cancer (PCa) progression to castrate-resistant prostate cancer (CRPC). Phosphoinositide-3-kinase/Protein Kinase B/mechanistic Target of Rapamycin (PI3K/AKT/mTOR) and Hedgehog/GLI (Hh/GLI) pathways are major participants in progression to CRPC. In this sense, the current work aims to assess the potential antitumor effects resulting from co-targeting the aforementioned pathways in PC3 cells with Dactolisib as a dual PI3K/mTOR inhibitor and GANT61 as a GLI1 antagonist. Three replica of PC3 cells were assigned for four treatment groups; vehicle control, Dactolisib-treated, GANT61-treated, and combination-treated groups. GLI1 gene expression was determined by quantitative real-time PCR while active caspase-3 was determined colorimetrically. P-AKT, p70 ribosomal s6 protein kinase 1 (pS6K1), cyclin D1, vascular endothelial growth factor 1 (VEGF1), and Microtubule-associated proteins 1A/1B light chain 3 (LC3) protein levels were determined by ELISA technique. GLI1 gene expression was down-regulated as a result of Dactolisib, GANT61, and their combination. Additionally, both drugs significantly reduced p-AKT, pS6K1, cyclin D1, and VEGF1 protein levels. Dactolisib elevated LC3 protein levels and GANT61 augmented Dactolisib effect on LC3. Moreover, only Dactolisib/GANT61combination significantly increased active caspase-3 level. To sum up, Dactolisib/GANT61 combination was shown to be promising in PCa treatment. Further in-vitro and in-vivo studies are warranted to support our findings.

Targeting PI3K/AKT/mTOR pathway to enhance the anti-leukemia efficacy of venetoclax.

BACKGROUND: The treatment of acute myeloid leukemia (AML) is developing towards "targeted therapy", which faces challenges such as low sensitivity and drug resistance. Therefore, targeted drugs need to be used in combination with other drugs to overcome clinical problems. OBJECTIVE: AML cells and animal models were used to determine the synergistic anti-leukemic effect of Dactolisib (BEZ235) and Venetoclax (ABT199) and explore its mechanism. METHODS: In vitro experiments, we used cell counting kit-8 (CCK8), flow cytometry, real-time quantitative PCR (qPCR), and Western blot to detect the anti-leukemic effects of ABT199 and BEZ235. In vivo experiments, female nude mice were injected subcutaneously with THP-1 cells to form tumors, evaluate the combined effect of ABT199 and BEZ235 by indicators such as tumor size, tumor weight, Ki67 and cleaved-Caspase3 staining. The mice's body weight and HE staining were used to evaluate the liver injury and adverse drug reactions. RESULTS: The combination of BEZ235 and ABT199 has a synergistic effect through promoting apoptosis and inhibiting proliferation. The BEZ235 increased the drug sensitivity of ABT199 by reducing the MCL-1 protein synthesis and promoted the degradation of MCL-1 protein, which is considered as the mechanism of reversing ABT199 resistance. Furthermore, the BEZ235 and ABT199 can synergistically enhance the inhibition of PI3K/AKT/mTOR pathway. CONCLUSION: The combination of BEZ235 and ABT199 exhibits a synergistic anti-tumor effect in AML by down-regulating MCL-1 protein.

Suppression of MYC by PI3K/AKT/mTOR pathway inhibition in combination with all-trans retinoic acid treatment for therapeutic gain in acute myeloid leukaemia.

Aberrant activity of the phosphatidylinositol-3 kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR [PAM]) pathway, as well as suppressed retinoic acid signalling, contribute to enhanced proliferation and the differentiation blockade of immature myeloid cells in acute myeloid leukaemia (AML). Inhibition of the PAM pathway was shown to affect especially mixed-lineage leukaemia-rearranged AML. Here, we sought to test a combined strategy using small molecule inhibitors against members of the PAM signalling pathway in conjunction with all-trans retinoic acid (ATRA) to target a larger group of different AML subtypes. We find that ATRA treatment in combination with inhibition of PI3K (ZSTK474), mTOR (WYE132) or PI3K/mTOR (BEZ235, dactolisib) drastically reduces protein levels of the proto-oncogene MYC. In combination with BEZ235, ATRA treatment led to almost complete eradication of cellular MYC, G1 arrest, loss of clonal capacity and terminal granulocytic differentiation. We demonstrate that PAM inhibitor/ATRA treatment targets MYC via independent mechanisms. While inhibition of the PAM pathway causes MYC phosphorylation at threonine 58 via glycogen synthase kinase 3 beta and subsequent degradation, ATRA reduces its expression. Here, we present an approach using a combination of known drugs to synergistically reduce aberrant MYC levels, thereby effectively blocking proliferation and enabling differentiation in various AML subtypes.

The Anti-Proliferative Effect of PI3K/mTOR and ERK Inhibition in Monolayer and Three-Dimensional Ovarian Cancer Cell Models.

Most ovarian cancer patients are diagnosed with advanced stage disease, which becomes unresponsive to chemotherapeutic treatments. The PI3K/AKT/mTOR and the RAS/RAF/MEK/ERK kinase signaling pathways are attractive targets for potential therapeutic inhibitors, due to the high frequency of mutations to PTEN, PIK3CA, KRAS and BRAF in several ovarian cancer subtypes. However, monotherapies targeting one of these pathways have shown modest effects in clinical trials. This limited efficacy of the agents could be due to upregulation and increased signaling via the adjacent alternative pathway. In this study, the efficacy of combined PI3K/mTOR (BEZ235) and ERK inhibition (SCH772984) was investigated in four human ovarian cancer cell lines, grown as monolayer and three-dimensional cell aggregates. The inhibitor combination reduced cellular proliferation in a synergistic manner in OV-90 and OVCAR8 monolayers and in OV-90, OVCAR5 and SKOV3 aggregates. Sensitivity to the inhibitors was reduced in three-dimensional cell aggregates in comparison to monolayers. OV-90 cells cultured in large spheroids were sensitive to the inhibitors and displayed a robust synergistic antiproliferative response to the inhibitor combination. In contrast, OVCAR8 spheroids were resistant to the inhibitors. These findings suggest that combined PI3K/mTOR and ERK inhibition could be a useful strategy for overcoming treatment resistance in ovarian cancer and warrants further preclinical investigation. Additionally, in some cell lines the use of different three-dimensional models can influence cell line sensitivity to PI3K/mTOR and RAS/RAF/MEK/ERK pathway inhibitors.

Differential Targeting of Gr-MDSCs, T Cells and Prostate Cancer Cells by Dactolisib and Dasatinib.

Granulocytic myeloid-derived suppressor cells (Gr-MDSCs) promote immune evasion and resistance to immunotherapeutics in a variety of malignancies. Our previous study showed that dual PI3K/mTOR inhibitor Dactolisib impaired the viability and immunosuppressive function of Gr-MDSCs, and significantly synergized with immune checkpoint blockade (ICB) antibodies targeting PD1 and CTLA4 to eradicate metastatic castration-resistant prostate cancer (CRPC) in a preclinical transgenic mouse model. On the contrary, tyrosine kinase inhibitor Dasatinib diminished tumor-infiltrating T lymphocytes and showed no synergic activity with ICB. The understanding of the distinct effects of Dactolisib and Dasatinib on Gr-MDSCs, T cells and prostate neoplastic cells is inadequate, limiting the clinical translation of the combination immunotherapy. To address this question, we applied Reverse Phase Protein Array (RPPA) to profile 297 proteins and protein phosphorylation sites of Gr-MDSCs, T cells and prostate cancer cells isolated from the CRPC model. We found cell type-specific protein expression patterns and highly selective targets by the two drugs, including preferential inhibition of phospho-4E-BP1 in Gr-MDSCs by Dactolisib and preferential suppression of phospho-Src and phospho-p38 MAPK in T cells. Furthermore, transcriptomic profiling of Gr-MDSCs treated with the two inhibitors revealed downregulation of mitochondrial respiration pathways by Dactolisib but not Dasatinib. Overall, these results provide important mechanistic insight into the efficacious combination of Dactolisib and ICB as well as the detrimental effect of Dasatinib on anti-tumor immunity.

Folate decorated polymeric micelles for targeted delivery of the kinase inhibitor dactolisib to cancer cells.

One of the main challenges in clinical translation of polymeric micelles is retention of the drug in the nanocarrier system upon its systemic administration. Core crosslinking and coupling of the drug to the micellar backbone are common strategies to overcome these issues. In the present study, polymeric micelles were prepared for tumor cell targeting of the kinase inhibitor dactolisib which inhibits both the mammalian Target of Rapamycin (mTOR) kinase and phosphatidylinositol-3-kinase (PI3K). We employed platinum(II)-based linker chemistry to couple dactolisib to the core of poly(ethylene glycol)-b-poly(acrylic acid) (PEG-b-PAA) polymeric micelles. The formed dactolisib-PEG-PAA unimers are amphiphilic and self-assemble in an aqueous milieu into core-shell polymeric micelles. Folate was conjugated onto the surface of the micelles to yield folate-decorated polymeric micelles which can target folate receptor over-expressing tumor cells. Fluorescently labeled polymeric micelles were prepared using a lissamine-platinum complex linked in a similar manner as dactolisib. Dactolisib polymeric micelles showed good colloidal stability in water and released the coupled drug in buffers containing chloride or glutathione. Folate decorated micelles were avidly internalized by folate-receptor-positive KB cells and displayed targeted cellular cytotoxicity at 50-75 nM IC50. In conclusion, we have prepared a novel type of folate-receptor targeted polymeric micelles in which platinum(II) linker chemistry modulates drug retention and sustained release of the coupled inhibitor dactolisib.

The PI3K/mTOR dual inhibitor BEZ235 nanoparticles improve radiosensitization of hepatoma cells through apoptosis and regulation DNA repair pathway.

Polymer materials encapsulating drugs have broad prospects for drug delivery. We evaluated the effectiveness of polyethylene glycol-poly (lactic-co-glycolic acid) (PLGA-PEG) encapsulation and release characteristics of PI3K/mTOR inhibitor NVP-BEZ235 (BEZ235). We proposed a strategy for targeting radiosensitization of liver cancer cells. The biocompatibility, cell interaction, and internalization of Glypican-3 (GPC3) antibody-modified, BEZ235-loaded PLGA-PEG nanoparticles (NP-BEZ235-Ab) in hepatoma cells in vitro were studied. Also, the cell killing effect of NP-BEZ235-Ab combined with γ-ray cell was evaluated. We used confocal microscopy to monitor nanoparticle-cell interactions and cellular uptake, conducted focus-formation experiments to analyze the synergistic biological effects of NP-BEZ235-Ab and priming, and studied synergy in liver cancer cells using molecular biological methods such as western blotting. We found that PLGA-PEG has good loading efficiency for BEZ235 and high selectivity to GPC3-positive HepG2 liver cancer cells, thus documenting that NP-BEZ235-Ab acts as a small-molecule drug delivery nanocarrier. At the nominal concentration, the NP-BEZ235-Ab nanoformulation synergistically kills liver cancer cells with significantly higher efficiency than does the free drug. Thus, NP-BEZ235-Ab is a potential radiosensitizer.

NVP-BEZ235 (Dactolisib) Has Protective Effects in a Transgenic Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) is a neurodegenerative disease and the main cause of dementia. Its major symptom is memory loss, which is a result of neuronal cell death, which is accompanied by neuroinflammation. Some studies indicate the overactivation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mechanistic target of rapamycin (mTOR) pathway in this disease, being, thus, a potential target for pharmacological treatment. Here, we used a transgenic mouse model of AD that expresses a mutant amyloid-ß precursor protein (T41 mice) to investigate the effects of dactolisib (alternative name: NVP-BEZ235, abbreviation BEZ), a dual PI3K/mTOR inhibitor. Ten-months-old T41 animals were treated for 14 days with BEZ or vehicle via oral gavage and then submitted to social memory, open field and contextual conditioned fear tests. Hippocampal slices were prepared and Aß1-42 content, NeuN, Iba-1, CD68 and GFAP were evaluated. Tissues were further processed to evaluate cytokines levels through cytometric bead array. The treatment with BEZ (5 mg/kg) reduced social memory impairment in T41 mice. However, BEZ did not have any effect on altered Aß levels, NeuN, or GFAP staining. The drug reduced the CD68/Iba-1 ratio in CA3 region of hippocampus. Finally, BEZ diminished IL-10 levels in T41 mice. Thus, although its mechanisms are not clear, BEZ protects against memory impairment, reduces microglial activation and reestablishes IL-10 levels, revealing beneficial effects, which should be further investigated for the treatment of AD.

In silico Analysis and Molecular Docking Studies of Novel 4-Amino-3- (Isoquinolin-4-yl)-1H-Pyrazolo[3,4-d]Pyrimidine Derivatives as Dual PI3-K/mTOR Inhibitors.

BACKGROUND: mTORC1/ PI3K control multiple anabolic pathways, including protein synthesis, ribosome production, lipogenesis, and nucleotide synthesis, are all important for cell and tissue growth. Sapanisertib and Dactolisib inhibit PI3K/AKT/mTOR pathway, an important signaling pathway for many cellular functions such as growth control, metabolism and translation initiation. METHODS: Dactolisib contains quinolin-3-yl-2,3-dihydroimidazo[4,5-c]quinolin scaffold and Sapanisertib contains benzo[d]oxazol-5-yl-1-ethyl-1H-pyrazolo[3,4-d]pyrimidinnucleous. From the reference to both of drug novel series of 4-Amino-3-(isoquinolin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin was developed by molecular docking. In sillico analysis was done with SWISSADME online tools. RESULTS: Among all the designed derivatives, compounds 6(-10.6 kcal/mol) , 12( -10.7 kcal/mol), 14( -10.2 kcal/mol), and 16(-10.2 kcal/mol) have a good binding affinity than others. Biological activity was predicted by Molinspirationonline software tool showing that all compounds are active on G- protein coupled receptor. In silico toxicity profile of designed compounds was performed using the SWISSADME program, indicating that all the compounds follow the Lipinski rule of five and do not penetrate Blood brain barrier. CONCLUSION: Series of pyrazolo[3,4-d]pyrimidin derivatives gives good binding affinity with pan- PI3-Kinese/Mtor inhibitors. The present study provided a better understanding of the molecular modeling requisite for maintaining and/or improving PI3K/mTOR inhibitors.

Investigational chemotherapy and novel pharmacokinetic mechanisms for the treatment of breast cancer brain metastases.

In women, breast cancer is the most common cancer diagnosis and second most common cause of cancer death. More than half of breast cancer patients will develop metastases to the bone, liver, lung, or brain. Breast cancer brain metastases (BCBM) confers a poor prognosis, as current therapeutic options of surgery, radiation, and chemotherapy rarely significantly extend life and are considered palliative. Within the realm of chemotherapy, the last decade has seen an explosion of novel chemotherapeutics involving targeting agents and unique dosage forms. We provide a historical overview of BCBM chemotherapy, review the mechanisms of new agents such as poly-ADP ribose polymerase inhibitors, cyclin-dependent kinase 4/6 inhibitors, phosphatidyl inositol 3-kinaseinhibitors, estrogen pathway antagonists for hormone-receptor positive BCBM; tyrosine kinase inhibitors, antibodies, and conjugates for HER2+ BCBM; repurposed cytotoxic chemotherapy for triple negative BCBM; and the utilization of these new agents and formulations in ongoing clinical trials. The mechanisms of novel dosage formulations such as nanoparticles, liposomes, pegylation, the concepts of enhanced permeation and retention, and drugs utilizing these concepts involved in clinical trials are also discussed. These new treatments provide a promising outlook in the treatment of BCBM.

The dual PI3K/mTOR inhibitor dactolisib elicits anti-tumor activity in vitro and in vivo.

Glioblastomas (GBMs) are among the most malignant of all human tumors and have poor prognosis. The current standard of care (SOC) includes maximal surgical tumor resection followed by adjuvant temozolomide (TMZ) and concomitant radiotherapy (RT). However, even with this treatment, the 5-year survival rate is less than 10%, and thus, follow-up treatment is required to improve efficacy. In GBMs as well as many other solid cancers, PI3K/mTOR signaling is overactivated. Therefore, multiple tumor-based PI3K inhibitors have been studied in various cancers. In the current study, we investigated the effect of the dual PI3K/mTOR inhibitor dactolisib on TMZ+RT treatment in three human GBM cell lines and a orthotopic xenograft model. Dactolisib alone induced cytotoxicity and pro-apoptotic effects, which act as antitumor factors. Combined with SOC treatment, dactolisib inhibited cell viability, induced enhanced pro-apoptotic effect, and attenuated migration/invasion in all three cell lines, thereby enhancing the SOC therapeutic effect. Protein microarray analysis showed that A172 cells treated with TMZ+RT+dactolisib had higher p27 and lower Bcl-2 expression than other groups. Moreover, in the xenograft model, oral dactolisib combined with TMZ+RT inhibited tumor growth and prolonged survival. Thus, SOC combined with dactolisib shows potent anti-tumor activity and has promising potential for solid tumor treatment.

Phase Ib dose-finding study of abiraterone acetate plus buparlisib (BKM120) or dactolisib (BEZ235) in patients with castration-resistant prostate cancer.

BACKGROUND: The phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt) signalling axis and androgen receptor (AR) pathways exhibit reciprocal feedback regulation in phosphatase and tensin homologue (PTEN)-deficient metastatic castration-resistant prostate cancer (CRPC) in preclinical models. This phase Ib study evaluated the pan-PI3K inhibitor buparlisib (BKM120) and the dual pan-PI3K/ mammalian target of rapamycin (mTOR) inhibitor dactolisib (BEZ235) in combination with abiraterone acetate (AA) in patients with CRPC. MATERIALS AND METHODS: Patients with CRPC who had progressed on AA therapy received escalating doses of either buparlisib or dactolisib, along with fixed doses of AA (1000 mg once daily (qd)) and prednisone (5 mg twice daily (bid)). The primary objective was to define the maximum tolerated dose (MTD) and/or the recommended dose for expansion (RDE) of either buparlisib or dactolisib in combination with AA. Secondary objectives included safety, antitumour activity (Prostate Cancer Working Group 2 (PCWG2) criteria; 30% of prostate-specific antigen (PSA) decline at ≥week 12) and pharmacokinetic (PK) profile. RESULTS: In buparlisib + AA arm, 25 patients received buparlisib + AA (median age, 67 years; Eastern Cooperative Oncology Group performance status (ECOG PS) of 0/1/2 for 7/17/1 patients, respectively). At 100 mg qd; two patients experienced dose-limiting toxicities (DLTs) (grade 3 hyperglycaemia; grade 2 asthenia), and this was the maximum buparlisib dose explored. Buparlisib + AA showed a 26% lower median area under the curve from time zero to 24°h (AUC0-24) and 48% lower median maximum serum concentration (Cmax) versus the single-agent buparlisib assessed in first-in-human study. No objective response and few PSA decreases were reported. In dactolisib + AA arm, 18 patients (median age, 71 years; ECOG PS of 0/1 for 6/12 patients, respectively) received dactolisib + AA at the first dose level (200 mg bid). Five patients had 9 DLTs (grades 2&3 stomatitis; grade 3 hyperglycaemia; grades 2& 3 diarrhoea; grades 1& 2 pyrexia, grade 2 vomiting, and grade 2 chills). CONCLUSIONS: Based on the assessment of available pharmacokinetics, safety, and efficacy data, no further study is planned for either buparlisib or dactolisib in combination with AA in CRPC.

Dactolisib (NVP-BEZ235) toxicity in murine brain tumour models.

BACKGROUND: Glioblastomas (GBMs) are highly malignant brain tumours with a poor prognosis, and current cytotoxic regimens provide only a limited survival benefit. The PI3K/Akt/mTOR pathway has been an attractive target for therapy due to its high activation in GBMs as well as other cancers. The dual pan-PI3K/mTOR kinase inhibitor dactolisib (NVP-BEZ235) is an anti-neoplastic compound currently under investigation. However, little is known about its efficacy in human GBMs. We aimed at evaluating the efficacy of dactolisib in human glioblastoma cells, as well as in murine models carrying human GBM xenografts. METHODS: To assess the effect of dactolisib in vitro, MTS assay, manual cell count, BrdU incorporation and Annexin V staining experiments were used to observe growth and apoptosis. Furthermore, Akt phosphorylation (S473), a downstream target of PI3K, was explored by western blotting. Animal studies utilizing orthotopic xenograft models of glioblastoma were performed in nude rats and NOD/SCID mice to monitor survival benefit or inhibition of tumor growth. RESULTS: We found that dactolisib in vitro shows excellent dose dependent anti-growth properties and increase in apoptosis. Moreover, dose dependent inhibition of Akt phosphorylation (S473), a downstream effect of PI3K, was observed by western blotting. However, in two independent animal studies utilizing nude rats and NOD/SCID mice in orthotopic xenograft models of glioblastoma, we observed no survival benefit or inhibition of tumour growth. Severe side effects were observed, such as elevated levels of blood glucose and the liver enzyme alanine transaminase (ALT), in addition to diarrhoea, hair loss (alopecia), skin rash and accumulation of saliva in the oral cavity. CONCLUSION: Taken together, our results suggest that despite the anti-neoplastic efficacy of dactolisib in glioma treatment in vitro, its utility in vivo is questionable due to toxicity.

Synergistic targeted inhibition of MEK and dual PI3K/mTOR diminishes viability and inhibits tumor growth of canine melanoma underscoring its utility as a preclinical model for human mucosal melanoma.

Human mucosal melanoma (MM), an uncommon, aggressive and diverse subtype, shares characteristics with spontaneous MM in dogs. Although BRAF and N-RAS mutations are uncommon in MM in both species, the majority of human and canine MM evaluated exhibited RAS/ERK and/or PI3K/mTOR signaling pathway activation. Canine MM cell lines, with varying ERK and AKT/mTOR activation levels reflective of naturally occurring differences in dogs, were sensitive to the MEK inhibitor GSK1120212 and dual PI3K/mTOR inhibitor NVP-BEZ235. The two-drug combination synergistically decreased cell survival in association with caspase 3/7 activation, as well as altered expression of cell cycle regulatory proteins and Bcl-2 family proteins. In combination, the two drugs targeted their respective signaling pathways, potentiating reduction of pathway mediators p-ERK, p-AKT, p-S6, and 4E-BP1 in vitro, and in association with significantly inhibited solid tumor growth in MM xenografts in mice. These findings provide evidence of synergistic therapeutic efficacy when simultaneously targeting multiple mediators in melanoma with Ras/ERK and PI3K/mTOR pathway activation.

Dual PI3K/mTOR inhibitor NVP-BEZ235 sensitizes docetaxel in castration resistant prostate cancer.

PURPOSE: Effective therapeutic strategies that can achieve long-term improvement in patients with castration resistant prostate cancer are urgently needed. We recently reported that the activated PI3K/Akt/mTOR signaling pathway induced by docetaxel explains resistance to docetaxel in castration resistant prostate cancer. In this study we explored the efficacy of NVP-BEZ235, a dual PI3K and mTORC1/2 inhibitor, for docetaxel resistant castration resistant prostate cancer. MATERIALS AND METHODS: We used the 2 human castration resistant prostate cancer cell lines C4-2 and C4-2AT6. At our laboratory C4-2AT6 cells were established from C4-2 under androgen ablated treatment for 6 months. We investigated the efficacy of NVP-BEZ235 monotherapy and NVP-BEZ235 combined with docetaxel in vitro and in vivo. RESULTS: Increased phosphorylated Akt in C4-2AT6 cells was significantly inhibited by NVP-BEZ235 in a dose and time dependent manner. WST cell proliferation assay results in C4-2AT6 cells revealed that combined administration of NVP-BEZ235 and docetaxel had significant, synergistically greater cytotoxicity than NVP-BEZ235 or docetaxel monotherapy. Combined NVP-BEZ235 (40 mg/kg) and docetaxel (4 mg/kg) in vivo in a castrated mouse xenograft model inhibited C4-2AT6 tumor growth to a greater degree than in the monotherapy groups. Also, NVP-BEZ235 showed significant efficacy with docetaxel at a low concentration in vivo, suggesting that NVP-BEZ235 effectively decreased resistance to docetaxel. CONCLUSIONS: Results suggest that inhibition of the PI3K/Akt/mTOR signaling pathway by NVP-BEZ235 can overcome docetaxel resistance in human castration resistant prostate cancer. Our findings provide a molecular basis for the clinical use of combined administration of NVP-BEZ235 and docetaxel in patients with castration resistant prostate cancer.