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Backgrounds: Transforming growth factor (TGF)-β is shown to play a critical role in cancer progression by inducing epithelial mesenchymal transition (EMT). Polyinosinic-polycytidylic acid (polyI:C), a synthetic agonist for toll-like receptor (TLR) 3, has been successfully used to treat some cancer patients as a vaccine adjuvant, but its direct action on the proliferation or migration of cancer cells, such as lung cancer cells, undergoing EMT remains unknown. Methods and results: By an in vitro cell proliferation assay, polyI:C showed no effect on the growth of TGF-β1-treated A549 human lung cancer cells at the concentration range up to 10 mg/ml; however, it markedly suppressed the motility in a cell scratch and a cell invasion assay. By Western blotting, polyI:C dramatically decreased TGF-β1-induced Ak strain transforming (Akt) phosphorylation and increased phosphatase and tensin homologue (PTEN) expression without affecting the Son of mothers against decapentaplegic (Smad) 3 phosphorylation or the expression level of E-cadherin, N-cadherin or Snail, indicating that polyI:C suppressed cell motility independently of the ‘cadherin switching’. The Akt inhibitor perifosine inhibited TGF-β1-induced cell invasion, and the PTEN-specific inhibitor VO-OHpic appeared to reverse the inhibitory effect of polyI:C. Conclusion: Our results indicate that polyI:C has the capacity to suppress the motility of TGF-β1-treated A549 cells by targeting the phosphatidylinositol 3-kinase /Akt pathway partly via PTEN and suggest that polyI:C may be used to prevent or reduce the metastasis of lung cancer cells.

Aim. To research the molecular mechanism of ghrelin in apoptosis, migratory, and invasion of gastric cancer (GC) cells. Methods. After GC AGS cells were handled with ghrelin (10–8 M), cyclooxygenase-2 inhibitor NS398 (100 μM), and Akt inhibitor perifosine (10uM), the rates of apoptosis were detected by TUNEL assay and flow cytometry assay. We assessed the expressions of PI3K, p-Akt, and COX-2 proteins by making use of Western blot analysis. The cell migratory and invasion were detected by using wound-healing and transwell analysis. Results. The migratory and invasion were increased in ghrelin-treated cells, while the rates of apoptosis were decreased. GC AGS cells treated with ghrelin showed an increase in protein expression of p-Akt, PI3K, and COX-2. After cells were treated with Akt inhibitor perifosine, the protein expression of p-Akt, PI3K, and COX-2 and the cell migratory, invasion, and apoptosis were partly recovered. After cells were treated with cyclooxygenase-2 inhibitor NS398, the protein expression of COX-2 and the cell migratory and invasion were decreased, while the rates of apoptosis were increased. Conclusion. Ghrelin regulates cell migration, invasion, and apoptosis in GC cells through targeting PI3K/Akt/COX-2. Ghrelin increases the expression of COX-2 in GC cells by targeting PI3K/Akt. Ghrelin is suggested to be one of the molecular targets in GC.

MicroRNAs (miRNAs) play a vital role in many biological processes, including cell growth, differentiation, apoptosis, development, differentiation, and carcinogenesis. Since miRNAs might play a part in cancer initiation and progression, they comprise an original class of promising diagnostic and prognostic molecular markers. In order to systematically understand the regulation of miRNA expression in cancers, the current study analyzed the miRNA expression profile in NCI-60 human cancer cell lines. Over 300 miRNAs exhibited unique expression profiles in cell lines derived from the same lineage. This study identified 9 lineage-specific miRNA expression patterns. Moreover, results indicated that miR-720 and miR-887 are expressed at relatively high levels in breast cancer cell lines compared to other types of cancer. Ultimately, matching NCI-60 drug response data to miR-720 and miR-887 expression profiles revealed that several FDA-approved drugs were inversely related to miR-720 and miR-887. Furthermore, the anti-cancer effect of perifosine was significantly enhanced by inhibiting miR-720 and decreased by miR-720 precursor treatment in breast cancer cell lines. 5-Fu treatment was enhanced by inhibiting miR-887 and decreased by miR-887 precursor treatment. The current results offer insight into the relationship between miRNA expression and their lineage types, and the approach used here represents a potential cancer therapy with the help of miRNAs.

Cancer is a devastating disease that has plagued humans from ancient times to this day. After decades of slow research progress, promising drug development, and the identification of new targets, the war on cancer was launched, in 1972. The P13K/Akt pathway is a growth-regulating cellular signaling pathway, which in many human cancers is over-activated. Studies have demonstrated that a decrease in Akt activity by Akt inhibitors is associated with a reduction in tumor cell proliferation. There have been several promising drug candidates that have been studied, including but not limited to ipatasertib (RG7440), 1; afuresertib (GSK2110183), 2; uprosertib (GSK2141795), 3; capivasertib (AZD5363), 4; which reportedly bind to the ATP active site and inhibit Akt activity, thus exerting cytotoxic and antiproliferative activities against human cancer cells. For most of the compounds discussed in this review, data from preclinical studies in various cancers suggest a mechanistic basis involving hyperactivated Akt signaling. Allosteric inhibitors are also known to alter the activity of kinases. Perifosine (KRX- 0401), 5, an alkylphospholipid, is known as the first allosteric Akt inhibitor to enter clinical development and is mechanistically characterized as a PH-domain dependent inhibitor, non-competitive with ATP. This results in a reduction in Akt enzymatic and cellular activities. Other small molecule (MK- 2206, 6, PHT-427, Akti-1/2) inhibitors with a similar mechanism of action, alter Akt activity through the suppression of cell growth mediated by the inhibition of Akt membrane localization and subsequent activation. The natural product solenopsin has been identified as an inhibitor of Akt. A few promising solenopsin derivatives have emerged through pharmacophore modeling, energy-based calculations, and property predictions.

Vascular complications are the primary reason for disability and mortality associated with diabetes mellitus (DM), and numerous microRNAs (miRNAs/miRs) are involved in the process, such as miR‑122, miR‑24 and miR‑423. It has been reported that miR‑328 regulates DM and cardiovascular disease; however, the role and mechanism of action underlying miR‑328 in HUVECs is not completely understood. The present study aimed to investigate the role and mechanism of action underlying the effects of miR‑328 on the functions of HUVECs. To simulate hyperglycemia combined with ischemia‑induced tissue starvation, HUVECs were cultured in endothelial cell medium with 25 mmol/l D‑glucose and 2% FBS for 24 h [high glucose (HG) + 2% FBS group]. HUVEC miR‑328 expression levels were detected by reverse transcription‑quantitative PCR. Cell migration, cytotoxicity and tube‑like structure formation were analyzed using wound healing, Cell Counting Kit‑8 and tube formation assays, respectively. Following transfection with miR‑328 inhibitor, miR‑328 expression was downregulated in HUVECs. Protein expression levels were determined by western blotting. Compared with the control group, the migration and tube‑like structure formation of HUVECs were decreased, and cell cytotoxicity was increased in the HG + 2% FBS group. The protein expression levels of vascular endothelial growth factor were also decreased, and the expression levels of miRNA‑328 in the HG + 2% FBS group were increased compared with the control group. However, miRNA‑328 downregulation reversed the aforementioned effects. Further experiments indicated that the AKT signaling pathway was inhibited in the HG + 2% FBS group; however, miR‑328 downregulation activated the AKT/mTOR signaling pathway, which was blocked by the AKT signaling pathway inhibitor, perifosine. Gene prediction and western blotting demonstrated that miR‑328 displayed a regulatory role via Pim‑1 proto‑oncogene, serine/threonine kinase (PIM1). In conclusion, miR‑328 expression was upregulated and angiogenesis was inhibited when HUVECs were subjected to high glucose and low serum conditions. miR‑328 downregulation enhanced angiogenesis by increasing PIM1 expression and activating the AKT/mTOR signaling pathway in HUVECs under high glucose and low serum conditions.

Metastatic brain tumors are regarded as the most advanced stage of certain types of cancer; however, chemotherapy has played a limited role in the treatment of brain metastases. Here, we established murine models of brain metastasis using cell lines derived from human brain metastatic tumors, and aimed to explore the antitumor efficacy of perifosine, an orally active allosteric Akt inhibitor. We evaluated the effectiveness of perifosine by using it as a single agent in ectopic and orthotopic models created by injecting the DU 145 and NCI-H1915 cell lines into mice. Initially, the injected cells formed distant multifocal lesions in the brains of NCI-H1915 mice, making surgical resection impractical in clinical settings. We determined that perifosine could distribute into the brain and remain localized in that region for a long period. Perifosine significantly prolonged the survival of DU 145 and NCI-H1915 orthotopic brain tumor mice; additionally, complete tumor regression was observed in the NCI-H1915 model. Perifosine also elicited much stronger antitumor responses against subcutaneous NCI-H1915 growth; a similar trend of sensitivity to perifosine was also observed in the orthotopic models. Moreover, the degree of suppression of NCI-H1915 tumor growth was associated with long-term exposure to a high level of perifosine at the tumor site and the resultant blockage of the PI3K/Akt signaling pathway, a decrease in tumor cell proliferation, and increased apoptosis. The results presented here provide a promising approach for the future treatment of patients with metastatic brain cancers and emphasize the importance of enriching a patient population that has a higher probability of responding to perifosine.

Simple Summary The Microtubule-associated protein Tau is expressed in different cancers; however, its role and prognostic value are still debated. In the present work, we evaluated the role of Tau in glioblastoma by down-regulating its expression in glioblastoma cells. We showed that Tau: (1) is required for tumor progression in nude mice; (2) is necessary for glioblastoma 3D cell organization, growth, and migration; and (3) regulates the PI3K/AKT signaling pathway. Abstract The Microtubule-Associated Protein Tau is expressed in several cancers, including low-grade gliomas and glioblastomas. We have previously shown that Tau is crucial for the 2D motility of several glioblastoma cell lines, including U87-MG cells. Using an RNA interference (shRNA), we tested if Tau contributed to glioblastoma in vivo tumorigenicity and analyzed its function in a 3D model of multicellular spheroids (MCS). Tau depletion significantly increased median mouse survival in an orthotopic glioblastoma xenograft model. This was accompanied by the inhibition of MCS growth and cell evasion, as well as decreased MCS compactness, implying N-cadherin mislocalization. Intracellular Signaling Array analysis revealed a defective activation of the PI3K/AKT pathway in Tau-depleted cells. Such a defect in PI3K/AKT signaling was responsible for reduced MCS growth and cell evasion, as demonstrated by the inhibition of the pathway in control MCS using LY294002 or Perifosine, which did not significantly affect Tau-depleted MCS. Finally, analysis of the glioblastoma TCGA dataset showed a positive correlation between the amount of phosphorylated Akt-Ser473 and the expression of MAPT RNA encoding Tau, underlining the relevance of our findings in glioblastoma disease. We suggest a role for Tau in glioblastoma by controlling 3D cell organization and functions via the PI3K/AKT signaling axis.

Background Radiation-induced lung injury (RILI) is considered one of the most common complications of thoracic radiation. Recent studies have focused on stem cell properties to obtain ideal therapeutic effects, and Sox9 has been reported to be involved in stem cell induction and differentiation. However, whether Sox9-expressing cells play a role in radiation repair and regeneration remains unknown. Methods We successfully obtained Sox9CreER, RosatdTomato and RosaDTA mice and identified Sox9-expressing cells through lineage tracing. Then, we evaluated the effects of the ablation of Sox9-expressing cells in vivo. Furthermore, we investigated the underlying mechanism of Sox9-expressing cells during lung regeneration via an online single-cell RNA-seq dataset. Results In our study, we demonstrated that Sox9-expressing cells promote the regeneration of lung tissues and that ablation of Sox9-expressing cells leads to severe phenotypes after radiation damage. In addition, analysis of an online scRNA-Seq dataset revealed that the PI3K/AKT pathway is enriched in Sox9-expressing cells during lung epithelium regeneration. Finally, the AKT inhibitor perifosine suppressed the regenerative effects of Sox9-expressing cells and the AKT pathway agonist promotes proliferation and differentiation. Conclusions Taken together, the findings of our study suggest that Sox9-expressing cells may serve as a therapeutic target in lung tissue after RILI.

BackgroundChemotherapy resistance is the major cause of failure in neuroblastoma (NB) treatment. ATXN3 has been linked to various types of cancer and neurodegenerative diseases; however, its roles in NB have not been established. The aim of our study was to explore the role of ATXN3 in the cell death induced by AKT inhibitor (perifosine or MK-2206) or chemotherapy drugs (etoposide or cisplatin) in NB cells.MethodsThe expressions of ATXN3 and BCL-2 family members were detected by Western blot. Cell survival was evaluated by CCK8, cell confluence was measured by IncuCyte, and apoptosis was detected by flow cytometry. AS and BE2 were treated with AKT inhibitors or chemotherapeutics, respectively.ResultsDownregulation of ATXN3 did not block, but significantly increased the perifosine/MK-2206-induced cell death. Among the BCL-2 family members, the expression of pro-apoptotic protein BIM and anti-proapoptotic protein Bcl-xl expression increased significantly when ATXN3 was down-regulated. Downregulation of BIM protected NB cells from the combination of perifosine/MK-2206 and ATXN3 downregulation. Downregulation of ATXN3 did not increase, but decrease the sensitivity of NB cells to etoposide/cisplatin, and knockdown of Bcl-xl attenuated this decrease in sensitivity.ConclusionDownregulation of ATXN3 enhanced AKT inhibitors (perifosine or MK-2206) induced cell death by BIM, but decreased the cell death induced by chemotherapeutic drugs (etoposide or cisplatin) via Bcl-xl. The expression of ATXN3 may be an indicator in selecting different treatment regimen.

Background/aim The P13K/Akt signaling pathway is a growth-regulating cellular pathway that is constitutively activated in a variety of human cancers. In previous studies, we reported that a solenopsin analog, compound B (MU-06-SC-608-7), shows inhibitory effects on Akt phosphorylation at a key activation site, as well as on proliferation of tumorigenic cells at sub-micromolar concentrations. The purpose of this study was to evaluate the effect of compound B on downstream effectors of Akt kinase, phosphorylation of Akt at a second activation site, Akt kinase activity in vitro, tumorigenic cell viability and other signaling pathways. Materials and methods Western blot analyses were performed using WBras1 epithelial and H2009 human carcinoma cells and cell viability assays were performed on H2009 cells. In vitro Akt kinase assays were performed using a commercially available kit. Results Compound B decreased the phosphorylation of Akt at the Thr308 activation site and key downstream effectors of Akt kinase, but did not directly inhibit Akt kinase. Substantial decreases in cell viability were observed at concentrations above 5 μM. No effect was seen on ERK or JNK pathways. Conclusion The results earmark this compound for further studies as a potential targeted cancer therapy.

PURPOSE: Perifosine (PRF) is an oral alkylphospholipid with antineoplastic effects and reasonable tolerability. It inhibits signaling through the PI3/AKT axis and other cascades of biologic importance in glioblastoma, and has promising pre-clinical activity in vitro and in vivo. Therefore, we conducted a phase II open-label single-arm clinical trial of perifosine for patients with recurrent glioblastoma (GBM). METHODS: We planned to accrue up to 30 adults with recurrent GBM with a minimum Karnofsky Performance Status of 50 following radiotherapy but without other restrictions on the number or types of prior therapy. Concurrent p450 stimulating hepatic enzyme inducing anticonvulsants were prohibited. Patients were treated with a loading dose of 600mg PRF (in 4 divided doses on day 1) followed by 100mg daily until either disease progression or intolerable toxicity. The primary endpoint was the 6-month progression free survival (PFS6) rate, with at least 20% considered promising. Accrual was continuous but if 0 of the first 12 patients with GBM reached PFS6, then further accrual would terminate for futility. Patients with other high grade gliomas were accrued concurrently to an exploratory cohort. RESULTS: Treatment was generally well tolerated; gastrointestinal toxicities were the most common side effects, although none resulted in treatment discontinuation. However, there was limited to no efficacy in GBM (n=16): the PFS6 rate was 0%, median PFS was 1.58 months [95%CI: (1.08, 1.84)], median overall survival was 3.68 months [95%CI: (2.50, 7.79)], with no radiographic responses. There was a confirmed partial response in one patient with anaplastic astrocytoma (n=14). CONCLUSIONS: PRF is tolerable but ineffective as monotherapy for GBM. Preclinical data suggests synergistic effects of PRF in combination with other approaches, and further study is ongoing.

Hypoxia-induced chemoresistance is a major obstacle in the development of effective cancer therapy. In our study, the reversal abilities of NADPH oxidase 4 (NOX4) silence on hypoxia resistance and the potential mechanism were investigated. Our data showed that the expression of NOX4 was upregulated in human neuroblastoma cells SH-SY5Y under hypoxia condition time dependently. Knockdown of NOX4 expression by siRNA inhibited glycolysis induced by hypoxia through decreasing the expression of glycolysis-related proteins (HIF-1α, LDHA, and PDK1), decreasing glucose uptake, lactate production, and ROS production, while increasing mitochondria membrane potential. Moreover, NOX4 silence inhibited cell growth under hypoxia condition through suppressing cell proliferation and proliferation-related proteins (Ki-67 and PCNA) compared with the hypoxia 24 h + siRNA NC group. Further, Western blot experiments exhibited that NOX4 siRNA could downregulate the rate of p-Akt/Akt. Treatment with PI3K/Akt signaling activator IGF-1 blocked, while treatment with Akt inhibitor perifosine enhanced the inhibitory effect of si-NOX4 on glycolysis and cell growth. In summary, knockdown of NOX4 had the ability of reversing hypoxia resistance, and the major mechanism is considered to be the inhibition of glycolysis and cell growth via the PI3K/Akt signaling pathway. Therefore, NOX4 could be a novel target against hypoxia resistance in neuroblastoma.

Lipid rafts are ordered membrane domains, which provide an environment for the proteins participating in signal transduction. Perifosine is an alkylphospholipid (APL) that inhibits the AKT pathway, cytotoxic to neoplastic cells. We have shown that the lipid raft adaptor protein NTAL is a target of APLs in leukemic cells. Using human mantle cell lymphoma (MCL) Granta-519 cell line we showed here that perifosine decreased NTAL in lipid raft fractions reducing AKT phosphorylation before apoptosis. We also showed that the NTAL-knockdown by shRNA induced a state of reduced AKT activation. Experimental NTAL-knockdown in NSG mouse MCL xenografts reduced AKT activity, increased the basal apoptotic rate by 3-fold (n = 8) and decreased tumor weight by 2.7-fold (n = 5), indicating that NTAL participates in tumor growth. NTAL protein was detected by western blotting in circulating cells of 7 of 8 MCL patients in the leukemic phase, suggesting involvement in the progression of the disease.

Background: Perifosine, is a third generation alkylphospholipid analog which has promising anti-tumor efficacy in clinical trials of refractory/recurrent neuroblastoma (NB). However, perifosine’s mechanism of action remains unclear. Previously, we have shown that perifosine changes global proteome and acetylome profiles in NB. Methods: To obtain a more comprehensive understanding of the perifosine mechanism, we performed a quantitative assessment of the lysine ubiquitylome in SK-N-AS NB cells using SILAC labeling, affinity enrichment and high-resolution liquid chromatography combined with mass spectrometry analysis. To analyse the data of ubiquitylome, we performed enrichment analysis with gene ontology (GO), the Encyclopedia of Genes and Genomes (KEGG) pathway, ubiquitylated lysine motif, protein complex and protein domain. Protein-protein interaction was conducted to explore the crosstalk between ubiquitylome and previous global proteome/acetylome. Co-immunoprecipitation and western blotting were used to validate the results of the ubiquitylome analysis. Results: Altogether, 3,935 sites and 1,658 proteins were quantified. These quantified ubiquitylated proteins participated in various cellular processes such as binding, catalytic activity, biological regulation, metabolic process and signaling pathways involving non-homologous end-joining, steroid biosynthesis and Ras signaling pathway. Ubiquitylome and proteome presented negative connection. We identified 607 sites which were modified with both ubiquitination and acetylation. We selected 14 proteins carrying differentially quantified lysine ubiquitination and acetylation sites at the threshold of 1.5 folds as potential targets. These proteins were enriched in activities associated with ribosome, cell cycle and metabolism. Conclusions: Our study extends our understanding of the spectrum of novel targets that are differentially ubiquitinated after perifosine treatment of NB tumor cells.

BackgroundGlioma has one of the highest mortality rates of all tumors of the nervous system and commonly used treatments almost always fail to achieve tumor control. Low-dose carbon-ion radiation can effectively target cancer and tumor cells, but the mechanisms of growth inhibition induced by heavy-ion radiation via the PI3K/Akt signaling pathway are unknown, and inhibition by heavy-ion radiation is minor in C6 cells.MethodsCarbon-ion radiation was used to investigate the effects of heavy-ion radiation on C6 cells, and suppression of Akt was performed using perifosine. MTT assays were used to investigate optimal perifosine treatment concentrations. Clone formation assays were used to investigate the growth inhibition effects of carbon-ion radiation and the effects of radiation with Akt inhibition. Lactate dehydrogenase release, superoxide dismutase activity, and malondialdehyde content were assessed to investigate oxidative stress levels. Expression levels of proteins in the PI3K/Akt/p53 signaling pathway were assessed via western blotting.ResultsThe 10% maximum inhibitory concentration of perifosine was 19.95 μM. In clone formation assays there was no significant inhibition of cell growth after treatment with heavy-ion irradiation, whereas perifosine enhanced inhibition. Heavy-ion radiation induced lactate dehydrogenase release, increased the level of malondialdehyde, and reduced superoxide dismutase activity. Akt inhibition promoted these processes. Heavy-ion radiation treatment downregulated Akt expression, and upregulated B-cell lymphoma-2 (Bcl-2) expression. p53 and Bcl-2 expression were significantly upregulated, and Bcl-2-associated X protein (Bax) expression was downregulated. The expression profiles of pAkt, Bcl-2, and Bax were reversed by perifosine treatment. Caspase 3 expression was upregulated in all radiation groups.ConclusionsThe growth inhibition effects of low-dose heavy-ion irradiation were not substantial in C6 cells, and Akt inhibition induced by perifosine enhanced the growth inhibition effects via proliferation inhibition, apoptosis, and oxidative stress. Akt inhibition enhanced the effects of heavy-ion radiation, and the PI3K/Akt/p53 signaling pathway may be a critical component involved in the process.

Colorectal cancer (CRC) is a disease with constantly increasing incidence and high mortality. The treatment efficacy could be curtailed by drug resistance resulting from poor drug penetration into tumor tissue and the tumor-specific microenvironment, such as hypoxia and acidosis. Furthermore, CRC tumors can be exposed to different pH depending on the position in the intestinal tract. CRC tumors often share upregulation of the Akt signaling pathway. In this study, we investigated the role of external pH in control of cytotoxicity of perifosine, the Akt signaling pathway inhibitor, to CRC cells using 2D and 3D tumor models. In 3D settings, we employed an innovative strategy for simultaneous detection of spatial drug distribution and biological markers of proliferation/apoptosis using a combination of mass spectrometry imaging and immunohistochemistry. In 3D conditions, low and heterogeneous penetration of perifosine into the inner parts of the spheroids was observed. The depth of penetration depended on the treatment duration but not on the external pH. However, pH alteration in the tumor microenvironment affected the distribution of proliferation- and apoptosis-specific markers in the perifosine-treated spheroid. Accurate co-registration of perifosine distribution and biological response in the same spheroid section revealed dynamic changes in apoptotic and proliferative markers occurring not only in the perifosine-exposed cells, but also in the perifosine-free regions. Cytotoxicity of perifosine to both 2D and 3D cultures decreased in an acidic environment below pH 6.7. External pH affects cytotoxicity of the other Akt inhibitor, MK-2206, in a similar way. Our innovative approach for accurate determination of drug efficiency in 3D tumor tissue revealed that cytotoxicity of Akt inhibitors to CRC cells is strongly dependent on pH of the tumor microenvironment. Therefore, the effect of pH should be considered during the design and pre-clinical/clinical testing of the Akt-targeted cancer therapy.

International audience; Alkylphospholipids (APLs) have elicited great interest as antitumor agents due to their unique mode of action on cell membranes. However, their clinical applications have been limited so far by high hemolytic activity. Recently, cationic prodrugs of erufosine, a most promising APL, have been shown to mediate efficient intracellular gene delivery, while preserving the antiproliferative properties of the parent APL. Here, cationic prodrugs of the two APLs that are currently used in the clinic, miltefosine, and perifosine, are investigated and compared to the erufosine prodrugs. Their synthesis, stability, gene delivery and self-assembly properties, and hemolytic activity are discussed in detail. Finally, the potential of the pro-miltefosine and pro-perifosine compounds M E12 and P E12 in combined antitumor therapy is demonstrated using pUNO1-hTRAIL, a plasmid DNA encoding TRAIL, a member of the TNF superfamily. With these pro-APL compounds, we provide a proof of concept for a new promising strategy for cancer therapy combining gene therapy and APL-based chemotherapy.

Background: Survivin, a member of the inhibitor of apoptosis protein family, has been associated with protection from cell apoptosis and regulation of mitosis. Phosphorylated-Survivin at Ser81 was reported to provide cytoprotection against tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) in L929 cells by inducing a backloop activation of phosphatidylinositol 3-kinase (PI3K). Therefore Akt as a possible substrate of PI3K was investigated. Methods : L929 cells were pretreated with/without 50 μM LY294002 or 10 μM Perifosine, and infected with viral particle of Survivin, anti sense of Survivin, Ser81Ala mutated Survivin or vector only. Cells were then harvested, lysed and subjected to immunoblot assay to detect Akt, phosphorylated Akt (Ser473), mammalian target of rapamycin (mTOR), phosphorylated-mTOR (Ser2448). Results: Survivin induced Akt and mTOR phosphorylations in a viral particle concentration dependent manner. Pretreatment of LY294002 or Perifosine prior to Survivin infection, attenuated Akt or mTOR phosphorylations, respectively. Low Akt or mTOR phosphorylations were observed when L929 cells were infected with Ser81Ala mutated Survivin. Conclusion: Ser81 phosphorylation site of Survivin played an important role in activating Survivin/PKA/PI3K/Akt/mTOR signaling pathway. Keywords: survivin, Ser81, Akt, mTOR, LY294002, perifosine

Background Tamoxifen is widely administered for patients with estrogen receptor-positive breast cancer. Despite many patients benefiting from Tamoxifen as an effective anti-hormonal agent in adjuvant therapy, a noticeable number of patients tend to develop resistance. Objective The aim of this study was to shed light upon the molecular mechanisms associated with Tamoxifen resistance which can help improve current treatment strategies available for stimulating responsiveness and combating resistance. Methods Relevant articles were obtained from PubMed and google scholar, nearly all dated from 2010 to 2017. Articles were screened to select the ones meeting the objective. The molecular interactions in the resistant network were extracted from the appropriate articles. Results The mechanisms of developing Tamoxifen resistance were briefly outlined. Overactivation of Receptor Tyrosine Kinases (RTKs) pathways, commonly known as alternative growth cascades, is one of the main players in acquired cancer cell stemness, which can induce unrestricted proliferation in the presence of Tamoxifen. There are seven recent patents including 6291496B1 as an anti-HER2, 8143226B2 as an inhibitor of RTK phosphorylation, 9062308B2 as an anti-HOXB7, Lapatinib functioning as an anti-EGFR/HER2, Everolimus as an inhibitor of mTOR, Exemestane as an aromatase inhibitor and Perifosine as an AKT inhibitor. Conclusion Altogether, it seems that tumor cells express a stemness phenotype which tends to override anti-hormonal adjuvant therapies. Since RTKs are overactivated and overexpressed in such cells, specialized targeted therapies suppressing RTKs would be a novel and effective way in restoring Tamoxifen sensitivity in resistant breast cancer tumor cells.

Telomeres protect chromosomal ends from deterioration and have been shown to be susceptible to shortening by reactive oxygen species (ROS)-induced damage. ROS levels increase during the progression from early to advanced hepatocellular carcinoma (HCC). An independent study found that the telomeres in most HCC tissues lengthened during carcinogenic advancement. Activated telomerase has been hypothesized to elongate telomeres during the progression of malignant HCC, but it remains unclear which signaling pathway is necessary for telomerase activation in HCC. Here, we showed using cell lines derived from human HCC that H2O2, which is a major component of ROS in living organisms, elongates telomeres by increasing telomerase activity via AKT activation. The AKT inhibitor perifosine decreased telomere length, cellular viability, and H2O2-mediated migration and invasion capacity in HCC cells while also inhibiting AKT activation, telomere maintenance, and tumor growth in nude mice. Advanced HCC tissues showed a positive correlation among ROS levels, pAKT levels, and telomere length. Furthermore, patients with HCC tumors that have high ROS levels and long telomeres displayed poorer survival rates. These data demonstrate the significant utilities of ROS levels, pAKT levels, and telomere length for predicting a poor prognosis in patients with HCC. Taken together, AKT activation could be essential for telomere maintenance in advanced HCC tumors as well as being an important contributor to malignant HCC progression. Conclusion: We showed that H2O2 contributes to telomere elongation via AKT activation in advanced HCC, suggesting that an AKT inhibitor such as perifosine may be useful for treating patients with malignant HCC. This article is protected by copyright. All rights reserved.

Malignant pleural mesothelioma (MPM), an asbestos‐related occupational disease, is an aggressive and incurable tumor of the thoracic cavity. Despite recent advances in MPM treatment, overall survival of patients with MPM is very low. Recent studies have implicated that PI3K/Akt signaling is involved in MPM cell survival and development. To investigate the effects of Akt inhibitors on MPM cell survival, we examined the effects of nine selective Akt inhibitors, namely, afuresertib, Akti‐1/2, AZD5363, GSK690693, ipatasertib, MK‐2206, perifosine, PHT‐427, and TIC10, on six MPM cell lines, namely, ACC‐MESO‐4, Y‐MESO‐8A, MSTO‐211H, NCI‐H28, NCI‐H290, and NCI‐H2052, and a normal mesothelial cell line MeT‐5A. Comparison of IC 50 values of the Akt inhibitors showed that afuresertib, an ATP‐competitive specific Akt inhibitor, exerted tumor‐specific effects on MPM cells. Afuresertib significantly increased caspase‐3 and caspase‐7 activities and apoptotic cell number among ACC‐MESO‐4 and MSTO‐211H cells. Moreover, afuresertib strongly arrested the cell cycle in the G1 phase. Western blotting analysis showed that afuresertib increased the expression of p21WAF 1/ CIP 1 and decreased the phosphorylation of Akt substrates, including GSK‐3β and FOXO family proteins. These results suggest that afuresertib‐induced p21 expression promotes G1 phase arrest by inducing FOXO activity. Furthermore, afuresertib significantly enhanced cisplatin‐induced cytotoxicity. Interestingly, results of gene set enrichment analysis showed that afuresertib modulated the expression E2F1 and MYC, which are associated with fibroblast core serum response. Together, these results suggest that afuresertib is a useful anticancer drug for treating patients with MPM.

Tripchlorolide (T4) has been shown to induce A549 lung cancer cell death predominantly by activating an autophagy pathway. However, the underlying mechanism remains unclear. Herein, we demonstrated that compared with T4 treatment alone, pretreatment with wortmannin (an inhibitor of phosphatidylinositol 3-kinase), perifosine (an inhibitor of AKT) or rapamycin (an inhibitor of mTOR) combined with a subsequent T4 treatment significantly impaired the cell viability of A549 and A549/DDP lung cancer cells. We found that either treatment scheme markedly reduced the activity of P13K and AKT. Expression of LC3II increased in parallel to the increase of the T4 concentration in both A549 and A549/DDP cells and was repressed by overexpression of AKT. The expression levels of PI3-K, PI3-P, AKT, TSC2, mTOR, p70S6K and 4E-BP1 were minimally affected by the wortmannin, perifosine, or rapamycin plus T4 treatments, but their phosphorylated products were greatly affected in A549 lung cancer cells and slightly affected in A549/DDP lung cancer cells. These results indicate that T4 induces autophagy in lung cancer cells by inhibiting the PI3K/AKT/mTOR signaling pathway. We further found that T4 decreased expression of MDR1 and improved cisplatin sensitivity of A549/DDP cells. Altogether, these results have meaningful implications for tumor therapy in the future.

Bevacizumab (BVZ) as an antiangiogenesis therapy leads to a transient therapeutic efficacy in high-grade glioma. However, the proapoptotic potential of BVZ has not been well elucidated, yet. There is also a tumor resistance to BVZ that is linked to post-treatment metalloproteinases and AKT activities. Herein, the association between therapeutic efficacy and putative proapoptotic activity of low-dose BVZ either alone or in combination with a specific inhibitor of AKT called perifosine (PRF), in a glioma model was investigated. BALB/c mice bearing C6 glioma tumor were treated with BVZ and PRF either alone or combined for 13 days (n = 11/group). At the end of treatments, apoptosis, proliferation and vascular density, in the xenografts (3/group) were detected by TUNEL staining, Ki67 and CD31 markers, respectively. Relative levels of cleaved-caspase3, phospho-AKT (Ser473) and matrix metalloproteinase2 (MMP2) were measured using western blotting. PRF and BVZ separately slowed down tumor growth along with the cell apoptosis induction associated with a profound increase in caspase3 activity through an AKT inhibition-related pathway for PRF but not BVZ. Unlike PRF, BVZ significantly increased the intratumor MMP2 and phospho-AKT (Ser473) levels coupled with the slight antiproliferative and significant antivascular effects. Co-administration of PRF and BVZ versus monotherapies potentiated the proapoptotic effects and reversed the BVZ-induced upregulation of phospho-AKT (Ser473) and MMP2 levels in C6 xenografts, leading to the optimal antiproliferative activity and tumor growth regression and longer survival. In conclusion, BVZ plus PRF renders a paramount proapoptotic effect, leading to a major therapeutic efficacy and might be a new substitute for GBM therapy in the clinic.

Background/Aims: The alkylphospholipid perifosine is used for the treatment of malignancy. The substance is effective by triggering suicidal tumor cell death or apoptosis. Side effects of perifosine include anemia. At least in theory, perifosine-induced anemia could result from stimulation of suicidal erythrocyte death or eryptosis. Hallmarks of eryptosis are cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Cellular mechanisms participating in the orchestration of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress, increase of ceramide abundance, as well as activation of staurosporine sensitive protein kinase C and/or of SB203580 sensitive p38 kinase. The present study explored, whether perifosine induces eryptosis and, if so, whether its effect involves and/or requires Ca2+ entry, oxidative stress, ceramide and kinase activation. Methods: Flow cytometry was employed to quantify phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, reactive oxygen species (ROS) abundance from DCFDA dependent fluorescence, and ceramide abundance utilizing specific antibodies. Hemolysis was estimated from hemoglobin concentration in the supernatant. Results: A 24 hours exposure of human erythrocytes to perifosine (2.5 µg/ml) significantly increased the percentage of annexin-V-binding cells, significantly decreased average forward scatter, significantly increased the percentage of shrunken erythrocytes, and significantly decreased the percentage of swollen erythrocytes. Perifosine significantly increased the percentage of hemolytic erythrocytes. Perifosine significantly increased Fluo3-fluorescence, but decreased DCFDA fluorescence and ceramide abundance. The effect of perifosine on annexin-V-binding was significantly blunted by removal of extracellular Ca2+ and by addition of staurosporine (1 µM), but not by addition of SB203580 (2 µM). Conclusions: Perifosine triggers eryptosis, an effect at least in part due to Ca2+ entry and activation of staurosporine sensitive kinases.

Diffuse intrinsic pontine glioma (DIPG) is a devastating disease with an extremely poor prognosis. Recent studies have shown that platelet-derived growth factor receptor (PDGFR) and its downstream effector pathway, PI3K/AKT/mTOR, are frequently amplified in DIPG, and potential therapies targeting this pathway have emerged. However, the addition of targeted single agents has not been found to improve clinical outcomes in DIPG, and targeting this pathway alone has produced insufficient clinical responses in multiple malignancies investigated, including lung, endometrial, and bladder cancers. Acquired resistance also seems inevitable. Activation of the Ras/Raf/MEK/ERK pathway, which shares many nodes of cross talk with the PI3K/AKT pathway, has been implicated in the development of resistance. In the present study, perifosine, a PI3K/AKT pathway inhibitor, and trametinib, a MEK inhibitor, were combined, and their therapeutic efficacy on DIPG cells was assessed. Growth delay assays were performed with each drug individually or in combination. Here, we show that dual inhibition of PI3K/AKT and MEK/ERK pathways synergistically reduced cell viability. We also reveal that trametinib induced AKT phosphorylation in DIPG cells that could not be effectively attenuated by the addition of perifosine, likely due to the activation of other compensatory mechanisms. The synergistic reduction in cell viability was through the pronounced induction of apoptosis, with some effect from cell cycle arrest. We conclude that the concurrent inhibition of the PI3K/AKT and MEK/ERK pathways may be a potential therapeutic strategy for DIPG.

Physiological shear stress has been demonstrated to serve an atheroprotective function by stimulating endothelial nitric oxide synthase (eNOS) multi‑site phosphorylation. Low shear stress (LSS) serves an atheroprone role by increasing endothelial cell apoptosis and inflammation. The present study assessed whether LSS inhibited nitric oxide (NO) production in human umbilical vein endothelial cells by modulating eNOS phosphorylation and potential signaling pathways. A parallel flow chamber imposed with 2 dyn/cm2 shear stress on endothelial cells was used. Western blotting and 4,5‑diaminofluorescein diacetate were used to analyze the protein expression levels and NO production. LSS activated eNOS‑Ser1177 and eNOS‑Thr495, but inhibited eNOS‑Ser633. NO production was decreased after a transient increase at 5 min. LSS‑stimulated phosphorylation of eNOS‑Ser1177 and ‑Thr495 were suppressed by the Akt inhibitor, perifosine, and extracellular signal regulated kinases1/2 (ERK1/2) inhibitor, PD98059, respectively. Additionally, the phosphorylation of eNOS‑Ser633 inhibited by LSS was restored by the protein kinase A activator, 8‑Bromo‑cAMP. PD98059 completely inhibited the LSS‑induced downregulation of NO production. NO downregulation in response to LSS was intensified by perifosine and was partly inhibited by 8‑Bromo‑cAMP. These results indicated that LSS‑induced activation of ERK1/2/eNOS‑Thr495 serves a major role in inhibiting endothelial NO synthase, which may explain the proinflammatory and proatherosclerotic properties of LSS.

// Weidong Liu 1, * , Li Mao 2, * , Feng Ji 1 , Fengli Chen 3 , Shouguo Wang 1 , Yue Xie 1 1 Department of Orthopedics, Huai’an First People’s Hospital, Nanjing Medical University, Huai’an, China 2 Department of Endocrinology, Huai’an First People’s Hospital, Nanjing Medical University, Huai’an, China 3 Clinical Laboratory, Huai’an First People’s Hospital, Nanjing Medical University, Huai’an, China * Co-first authors Correspondence to: Feng Ji, email: huaiaifengjidr@163.com Keywords: icariside II, dexamethasone, EGFR-Akt, Nrf2 signaling, oxidative stress Received: November 07, 2016 Accepted: November 21, 2016 Published: December 01, 2016 ABSTRACT The potential effect of icariside II on dexamethasone-induced osteoblast cell damages was evaluated here. In MC3T3-E1 osteoblastic cells and the primary murine osteoblasts, co-treatment with icariside II dramatically attenuated dexamethasone- induced cell death and apoptosis. Icariside II activated Akt signaling, which is required for its actions in osteoblasts. Akt inhibitors (LY294002, perifosine and MK-2206) almost abolished icariside II-induced osteoblast cytoprotection against dexamethasone. Further studies showed that icariside II activated Nrf2 signaling, downstream of Akt, to inhibit dexamethasone-induced reactive oxygen species (ROS) production in MC3T3-E1 cells and primary osteoblasts. On the other hand, Nrf2 shRNA knockdown inhibited icariside II-induced anti-dexamethasone cytoprotection in MC3T3-E1 cells. Finally, we showed that icariside II induced heparin-binding EGF (HB-EGF) production and EGFR trans-activation in MC3T3-E1 cells. EGFR inhibition, via anti-HB-EGF antibody, EGFR inhibitor AG1478 or EGFR shRNA knockdown, almost blocked icariside II-induced Akt-Nrf2 activation in MC3T3-E1 cells. Collectively, we conclude that icariside II activates EGFR-Akt-Nrf2 signaling and protects osteoblasts from dexamethasone. Icariside II might have translational value for the treatment of dexamethasone-associated osteoporosis/osteonecrosis.

Background Pulmonary arterial hypertension (PAH) is a severe pulmonary vascular disease characterized by unbalanced proliferation and apoptosis of pulmonary arterial smooth muscle cells (PASMCs). Prohibitin 1 (PHB1) is known for its significant anti-proliferative activity. However, the role of PHB1 in PASMCs and PAH have not been elucidated. Methods Monocrotaline (MCT 60 mg/kg) was used to build a PAH model in SD rats. Right ventricular systolic pressure (RVSP) and right ventricle (RV) hypertrophy were measured. Morphology of pulmonary vessels was observed by Hematoxylin-Eosin (HE) staining. Expression of PHB1 in pulmonary arteries and PASMCs was determinated by immunoblot and immunofluorescence. Cell proliferation was detected by CCK8 and EDU when PASMCs were stimulated by PDGF-BB (20 ng/mL). Furthermore, siRNA for PHB1 and Akt inhibitor were conducted to investigate the mechanism behind the role of PHB1 and AKT signaling pathway in PASMCs proliferation and apoptosis. Results The protein expression of PHB1 in PAH rats lung tissue was significantly up-regulated accompanied by elevated RVSP and enhanced RV hypertrophy. Immunohistochemistry showed that PHB1 was mainly localized in the pulmonary vascular smooth muscle layer. PDGF-BB significantly up-regulated the expression of PHB1 in rat primary PASMCs in a time- and dose-dependent manner. After PHB1 knock down, PASMCs proliferation was significantly suppressed while apoptosis was significantly recovered. Meanwhile the level of proliferating cell nuclear antigen (PCNA) and P-Akt were significantly down-regulated. Perifosine (Akt inhibitor) also significantly inhibit proliferation of PASMCs. Conclusion PHB1 contributes to pulmonary vascular remodeling by accelerating proliferation of PASMCs which involves AKT phosphorylation.

Triple-negative breast cancer (TNBC) treatment remains a great challenge for clinical practice and novel therapeutic strategies are urgently needed. UCHL3 is a deubiquitinase that is overexpressed in TNBC and correlates with poor prognosis. UCHL3 deubiquitinates RAD51 thereby promoting the recruitment of RAD51 to DNA damage sites and augmenting DNA repair. Therefore, UCHL3 overexpression can render cancer cells resistant to DNA damage inducing chemo and radiotherapy, and targeting UCHL3 can sensitize TNBC to radiation and chemotherapy. However, small molecule inhibitors of UCHL3 are yet to be identified. Here we report that perifosine, a previously reported Akt inhibitor, can inhibit UCHL3 in vitro and in vivo. We found low dose (50 nM) perifosine inhibited UCHL3 deubiquitination activity without affecting Akt activity. Furthermore, perifosine enhanced Olaparib-induced growth inhibition in TNBC cells. Mechanistically, perifosine induced RAD51 ubiquitination and blocked the RAD51-BRCA2 interaction, which in turn decreased ionizing radiation-induced foci (IRIF) of Rad51 and, thereby, homologous recombination (HR)-mediated DNA double strand break repair. In addition, combination of perifosine and Olaparib showed synergistic antitumor activity in vivo in TNBC xenograft model. Thus, our present study provides a novel therapeutic approach to optimize PARP inhibitor treatment efficiency.

Developing novel therapeutic agents against chondrosarcoma is important. SF2523 is a PI3K-Akt-mTOR and bromodomain-containing protein 4 (BRD4) dual inhibitor. Its activity in human chondrosarcoma cells is tested. Our results show that SF2523 potently inhibited survival, proliferation and migration, and induced apoptosis activation in SW1353 cells and primary human chondrosarcoma cells. The dual inhibitor was yet non-cytotoxic to the primary human osteoblasts and OB-6 osteoblastic cells. SF2523 blocked Akt-mTOR activation and downregulated BRD4-regulated genes (Bcl-2 and c-Myc) in chondrosarcoma cells. It was more efficient in killing chondrosarcoma cells than other established PI3K-Akt-mTOR and BRD4 inhibitors, including JQ1, perifosine and OSI-027. In vivo, intraperitoneal injection of SF2523 (30 mg/kg) potently inhibited subcutaneous SW1353 xenograft tumor growth in severe combined immunodeficient mice. Akt-mTOR inhibition as well as Bcl-2 and c-Myc downregulation were detected in SF2523-treated SW1353 tumor tissues. In conclusion, targeting PI3K-Akt-mTOR and BRD4 by SF2523 potently inhibited chondrosarcoma cell growth in vitro and in vivo.

Endometrial cancer is the most common cancer of the female reproductive system. Perifosine has shown anti-tumor activity in a variety of cancers by inhibition of AKT phosphorylation. The active metabolite of vitamin D (1,25(OH)2D3) has anti-proliferative and pro-apoptotic properties and is recognized as an agent with the great potential for cancer intervention. Combination treatment with specific agents has been widely used as targeted therapy for cancer. In this study, we aimed to investigate the anti-apoptotic effect of varying concentrations of perifosine and vitamin D on endometrial cancer cell line (HEC1A). The expression levels of P53, BCL2 and BAX were assessed in cultured HEC1A cells at 10 and 30 μM concentrations of perifosine, 50 and 200 nM of vitamin D and combination groups using qRT PCR. Apoptosis evaluation in HEC-1A cells was performed using flow cytometry after 48 and 72 h of treatment with perifosine, vitamin D and combinations of both. According to the obtained data, treatment with different doses of perifosine and vitamin D significantly induced apoptosis in a time and dose-dependent manner in HEC-1A cells. The mRNA expression levels of P53, BAX, BCL2 in HEC-1A cells were found to be significant in the experimental groups as compared with the control. Perifosine displayed substantial anti-tumor activity in HEC-1A. These effects were increased with vitamin D. Therefore, perifosine and vitamin D combinations should be further evaluated in clinical studies in endometrial cancer.

Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) mutation in colon cancer contributes to the poor prognosis of the disease and chemoresistance of tumors. New therapies are needed; however, the lack of knowledge of the mechanism of chemoresistance has hindered progress. In this study, we investigated the mechanism of the reduced sensitivity of colon cancer cells to 5-fluorouracil (5-FU) and oxaliplatin (L-OHP), and the effects of perifosine, an Akt inhibitor that enhances the cytotoxicity of 5-FU and L-OHP in colon cancer cells harboring the PIK3CA mutation. The use of 5-FU or L-OHP alone or in combination induced significant death of Caco-2 cells (PIK3CA wild type), but only weakly decreased the viability of DLD-1 and SW948 cells harboring the PIK3CA mutation. The use of 5-FU and L-OHP, either alone or in combination, strongly suppressed Akt activation, Survivin, Bcl-2, and Bcl-xL expression, and enhanced Puma, phospho-p53, and p53 expression in Caco-2 cells than in DLD-1 cells. In addition, perifosine enhanced the cytotoxicity of the 5-FU and L-OHP combination, inhibited Akt activation and the expression of Survivin, Bcl-2, and Bcl-xL, and increased the expression of Puma, phospho-p53, and p53 in DLD-1 cells. These results indicate that PIK3CA mutation contributes to reduced sensitivity to 5-FU and L-OHP via Akt activation in colon cancer cells. Perifosine increases the efficacy of 5-FU and L-OHP by suppressing Akt activation. Thus, the use of an Akt inhibitor in combination with 5-FU and L-OHP may be beneficial in colon cancer with cells harboring the PIK3CA mutation.

Hepatocellular carcinoma is highly resistant to chemotherapy. Research data supported that cancer stem cells (CSC) may be responsible for the chemoresistance and strategies that suppress CSCs stemness could also inhibit the drug resistance. In this study, we found that nuclear receptor binding protein 2 (NRBP2) expression was downregulated in the CD133+ hepatocellular carcinoma CSCs. Most adjacent noncancerous liver tissue analyzed expressed higher level of NRBP2 compared with cancerous tissue in hepatocellular carcinoma patients, and high NRBP2 expression indicated a better prognosis. Real-time PCR results showed that NRBP2 negatively correlated with stemness-related genes, including Oct3/4, Nanog, Notch1, Ep300, and CD133 mRNA expression. High NRBP2 expression in hepatocellular carcinoma cells downregulated CK19 protein expression, inhibited tumorsphere formation, and tumorigenesis ability, indicating that high NRBP2 expression restrains the hepatocellular carcinoma cell stemness. Overexpression of NRBP2 reduced the IC50 of sorafenib in hepatocellular carcinoma cells, and NRBP2 expression was negatively correlated with hepatocellular carcinoma cell resistance to the chemotherapy agents, including cisplatin and the Akt signaling inhibitor perifosine. Coimmunoprecipitation results showed that NRBP2 could bind with Annexin A2 (ANXA2) and inhibit ANXA2 expression. Coexpression of ANXA2 restored the chemoresistant ability in NRBP2-overexpressing hepatocellular carcinoma cells. Further analysis showed that NRBP2 downregulated Akt and its downstream signaling target Bad phosphorylation level. ANXA2 coexpression partially restored the Akt phosphorylation. Analysis of the expression of Bcl2 family proteins showed that NRBP2 may increase hepatocellular carcinoma cell chemosensitivity by regulating expression of survival proteins involved in the Akt and Bcl2 pathway. These results suggest that NRBP2 plays an important role in the tumor progression and chemotherapeutic resistance of hepatocellular carcinoma. Cancer Res; 76(23); 7059–71. ©2016 AACR.

Clinical trials are in progress on AZD5363, an inhibitor of protein kinase B (AKT), to assess its effects on the phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway. Cells treated with AKT inhibitors have been reported to activate alternative pathways in order to escape growth inhibition. AZD5363-sensitized Hs578T breast cancer cells displayed reduced levels of phosphorylated glycogen synthase kinase 3 beta (pGSK3β). Interestingly, in AZD5363-treated cells, the level of phosphorylated (activated) AKT (pAKT) increased. Since pAKT positively correlates with cancer growth and survival, we aimed to identify conditions that could reduce AZD5363-induction of pAKT. We examined whether AZD5363 induction of pAKT could be reduced by co-treatment with inhibitors of the PI3K/AKT/mTOR pathway (LY294002, MK-2206, wortmannin, perifosine, rapamycin, everolimus, and temsirolimus). We observed that co-treatment of LY294002 or MK-2206 with AZD5363 reduced the level of pAKT. Since MK-2206 is clinically used, we propose that co-treatment using MK-2206 with AZD5363 would prove beneficial in blocking the AZD5363-induced pAKT signaling pathway. Our findings contribute to the development of AZD5363-based sensitization therapies for patients with cancer.

Brain-derived neurotrophic factor (BDNF) and its tyrosine kinase receptor TrkB have been reported to be associated with poor prognosis in neuroblastoma (NB) patients. Our previous studies indicated that BDNF activation of TrkB induces chemo-resistance through activation of phosphoinositide-3-kinase (PI3K)/Akt pathway. In this study, we investigated the role of BDNF/TrkB on metastasis in NB. A tetracycline-regulated TrkB-expressing NB cell line (TB3) was used. Scratch wound healing assay, Boyden chamber migration, and invasion assays were performed to study the migration and invasion of TB3 cells. A tumor xenograft model using SCID-Beige mice was utilized to detect the metastasis of NB tumors in vivo. Inhibitors of PI3K, MAPK, Akt, and mTOR were used. Western blotting was performed to study the expressions of P-Akt, P-Erk, and P-mTOR. Our results showed that in TrkB-expressing NB cells, BDNF treatment significantly increased gap closing (P

Patients with basal cell nevus syndrome (BCNS), also known as Gorlin syndrome, develop numerous basal cell carcinomas (BCC) due to germline mutations in the tumor suppressor PTCH1 and aberrant activation of Hedgehog (Hh) signaling. Therapies targeted at components of the Hh pathway, including the smoothened (SMO) inhibitor vismodegib, can ablate these tumors clinically, but tumors recur upon drug discontinuation. Using SKH1-Ptch1+/− as a model that closely mimics the spontaneous and accelerated growth pattern of BCCs in patients with BCNS, we show that AKT1, a serine/threonine protein kinase, is intrinsically activated in keratinocytes derived from the skin of newborn Ptch1+/− mice in the absence of carcinogenic stimuli. Introducing Akt1 haplodeficiency in Ptch1+/− mice (Akt1+/− Ptch1+/−) significantly abrogated BCC growth. Similarly, pharmacological inhibition of AKT with perifosine, an alkyl phospholipid AKT inhibitor, diminished the growth of spontaneous and UV-induced BCCs. Our data demonstrate an obligatory role for AKT1 in BCC growth, and targeting AKT may help reduce BCC tumor burden in BCNS patients. Cancer Prev Res; 9(10); 794–802. ©2016 AACR.

AKT plays a pivotal role in driving the malignant phenotype of many cancers, including high-risk neuroblastoma (HR-NB). AKT signaling, however, is active in normal tissues, raising concern about excessive toxicity from its suppression. The oral AKT inhibitor perifosine showed tolerable toxicity in adults and in our phase I trial in children with solid tumors (clinicaltrials.gov NCT00776867). We now report on the HR-NB experience. HR-NB patients received perifosine 50-75mg/m2/day after a loading dose of 100-200mg/m2 on day 1, and continued on study until progressive disease. The 27 HR-NB patients included three treated for primary refractory disease and 24 with disease resistant to salvage therapy after 1-5 (median 2) relapses; only one had MYCN-amplified HR-NB. Pharmacokinetic studies showed μM concentrations consistent with cytotoxic levels in preclinical models. Nine patients (all MYCN-non-amplified) remained progression-free through 43+ to 74+ (median 54+) months from study entry, including the sole patient to show a complete response and eight patients who had persistence of abnormal 123I-metaiodobenzylguanidine skeletal uptake but never developed progressive disease. Toxicity was negligible in all 27 patients, even with the prolonged treatment (11-to-62 months, median 38) in the nine long-term progression-free survivors. The clinical findings 1) confirm the safety of therapeutic serum levels of an AKT inhibitor in children; 2) support perifosine for MYCN-non-amplified HR-NB as monotherapy after completion of standard treatment or combined with other agents (based on preclinical studies) to maximize antitumor effects; and 3) highlight the welcome possibility that refractory or relapsed MYCN-non-amplified HR-NB is potentially curable. This article is protected by copyright. All rights reserved.

Histone deacetylase (HDAC) overactivity in colorectal cancer (CRC) promotes cancer progression. In the current study, we showed that 4SC-202, a novel class I HDAC inhibitor (HDACi), potently inhibited survival and proliferation of primary human colon cancer cells and established CRC lines (HT-29, HCT-116, HT-15, and DLD-1). Yet, the same 4SC-202 treatment was non-cytotoxic to colon epithelial cells where HDAC-1/-2 expressions were extremely low. 4SC-202 provoked apoptosis activation in CRC cells, while caspase inhibitors (z-VAD-CHO and z-DVED-CHO) significantly alleviated 4SC-202-exerted cytotoxicity in CRC cells. Meanwhile, 4SC-202 induced dramatic G2-M arrest in CRC cells. Further studies showed that AKT activation might be an important resistance factor of 4SC-202. 4SC-202-induced cytotoxicity was dramatically potentiated with serum starvation, AKT inhibition (by perifosine or MK-2206), or AKT1-shRNA knockdown in CRC cells. On the other hand, exogenous expression of constitutively active AKT1 (CA-AKT1) decreased the sensitivity by 4SC-202 in HT-29 cells. Notably, 4SC-202, at a low concentration, enhanced oxaliplatin-induced in vitro anti-CRC activity. In vivo, we showed that oral gavage of 4SC-202 inhibited HT-29 xenograft growth in nude mice, and when combined with oxaliplatin, its activity was further strengthened. Together, these pre-clinical results indicate that 4SC-202 may be further investigated as a valuable anti-CRC agent/chemo-adjuvant.

Renal cell carcinoma (RCC) is the most lethal urologic malignancy; however, the molecular events supporting RCC carcinogenesis and progression remain poorly understood. In this study, based on the analysis of gene expression profile data from human clear cell RCC (ccRCC) and the corresponding normal tissues, we discovered that Hedgehog (HH) pathway component genes GLI1 and GLI2 were significantly elevated in ccRCC. Survival analysis of a large cohort of ccRCC samples demonstrated that the expression of GLI1 and GLI2 was negatively correlated with patient overall survival. Clinical sample-based VHL mutation and cell model-based VHL manipulation studies all indicated that the activation of GLI1 and GLI2 was not affected by VHL status. Further signaling pathway dissections demonstrated that GLI1 and GLI2 were activated by the phosphoinositide 3-kinase (PI3K)/AKT pathway, but not mediated by the canonical HH/SMO/GLI signaling. Up-regulation of GLI1 and GLI2 promoted RCC proliferation and clonogenic ability, whereas, a combination of GLIs inhibitor Gant61 and AKT inhibitor Perifosine synergistically suppressed RCC growth and induced apoptosis in vitro and in vivo. Therefore, this study identifies that GLI1 and GLI2 are critical for RCC carcinogenesis, and also provides an alternative therapeutic strategy for RCC.

The role of Hedgehog (HH)/ glioma-associated oncogene homolog 1 (GLI1) pathway has been implicated in a variety of cancer entities, and the targeted pathway inhibition mediated by GLI1 is of therapeutic relevance. However, its oncogenicity and cross-talks with other cancer pathways including PI3K/Akt/NFκB, which modulates the HH/GLI1 signal strength, have rarely been explored in colorectal adenocarcinoma. We assessed the expression of GLI1 and its relationship with other cancer stemness genes, cell cycle markers, epithelial-mesenchymal transition (EMT), PI3K/Akt/NFκB signaling pathway genes, and HIF1α in 100 paraffin-embedded colorectal adenocarcinoma tissue samples using immunohistochemistry. We further addressed the effect of GLI1 on EMT, cell cycle, and its putative interaction with the PI3K/Akt/NFκB cascade in colorectal adenocarcinoma cell lines. The expression of GLI1 in colorectal adenocarcinoma tissues was found to correlate with the clinical stages, and distant metastasis. Moreover, GLI1 was found to be an independent predictor of poor overall survival and disease-free survival in colorectal adenocarcinoma. GLI1-expressing cancer cells also expressed their representative cancer stem-like cell (CSC) markers (SOX9 and CD133), as well as HIF1α. GLI1 expression was also strongly linked to EMT-related and PI3K/Akt/NFκB signaling genes. Downregulation of GLI1 by inhibitor treatment in colorectal adenocarcinoma cell lines resulted in reduced expression of CSC markers, cell clonogenicity, S-phase subpopulations, as well as the migration and invasion ability. Importantly, Akt inhibitor Perifosine significantly inhibited the expression of pAkt and GLI1 in colorectal adenocarcinoma cells. Combination of GLI1 inhibitor GANT61 and NFκB p65 inhibitor QZN exhibited much higher inhibition compared to using any of them individually on colorectal adenocarcinoma cells. We suggested that GLI1 may be a novel stem cell marker, and cancer stemness was activated via PI3K/Akt/NFκB pathway. In addition, co-targeting GLI1 and PI3K/Akt/NFκB signaling simultaneously might provide an alternative therapeutic strategy for colorectal adenocarcinoma patients.

The incidence of intrahepatic cholangiocarcinoma (ICC) is progressively increasing worldwide, and its prognosis remains poor. Accumulating evidence has demonstrated that tumor necrosis factor receptor-associated factor 4 (TRAF4), an adaptor protein, is involved in the carcinogenesis and progression of several tumor types. However, the function of TRAF4 in predicting prognosis, and mediating migration and invasion of ICC remains to be elucidated. In the present study, immunohistochemistry, western blotting and reverse transcription-quantitative polymerase chain reaction assays were used to determine that the expression of TRAF4 at the mRNA and protein levels in ICC tissues was significantly higher compared with that in non‑tumor tissues. The overexpression of TRAF4 was positively correlated with poor differentiation, regional lymphatic metastasis, and high tumor‑node-metastasis staging. Inhibiting the expression of TRAF4 using small interfering RNA decreased the migration and invasion of ICC cells in vitro. In addition, the AKT inhibitor perifosine eliminated the effect of TRAF4 on the invasion and migration of ICC cells in vitro. Clinically, the overexpression of TRAF4 was correlated with shorter overall survival rate and elevated recurrence rate in patients with ICC. Furthermore, patients with ICC with a high expression of TRAF4 and lymphatic metastasis were closely associated with a poorer prognosis compared with the other groups. Multivariate analysis indicated that the overexpression of TRAF4 was an independent prognostic indicator for patients with ICC. It was identified that a high level of TRAF4 facilitated the invasiveness of ICC cells via the activation of AKT signaling. The overexpression of TRAF4 may be a prognostic biomarker and candidate therapeutic target for patients with ICC.

Most tumors circumvent telomere-length imposed replicative limits through expression of telomerase, the reverse transcriptase that maintains telomere length. Substantial evidence that AKT activity is required for telomerase activity exists, indicating that AKT inhibitors may also function as telomerase inhibitors. This possibility has not been investigated in a clinical context despite many clinical trials evaluating AKT inhibitors. We tested if Perifosine, an AKT inhibitor in clinical trials, inhibits telomerase activity and telomere maintenance in tissue culture and orthotopic xenograft models as well as in purified CLL samples from a phase II Perifosine clinical trial. We demonstrate that Perifosine inhibits telomerase activity and induces telomere shortening in a wide variety of cell lines in vitro, though there is substantial heterogeneity in long-term responses to Perifosine between cell lines. Perifosine did reduce primary breast cancer orthotopic xenograft tumor size, but did not impact metastatic burden in a statistically significant manner. However, Perifosine reduced telomerase activity in four of six CLL patients evaluated. Two of the patients were treated for four to six months and shortening of the shortest telomeres occurred in both patients' cells. These results indicate that it may be possible to repurpose Perifosine or other AKT pathway inhibitors as a novel approach to targeting telomerase.

The aim of this study was to determine the role of AKT as a therapeutic target in ovarian clear cell carcinoma (CCC), an aggressive, chemoresistant histologic subtype of ovarian cancer. AKT activation was assessed by immunohistochemistry (IHC) using human tissue microarrays of primary ovarian cancers, composed of both CCC and serous adenocarcinoma (SAC). The growth-inhibitory effect of AKT-specific targeting by the small-molecule inhibitor, perifosine, was examined using ovarian CCC cell lines in vitro and in vivo. Finally, the activity of perifosine was examined using in CCC-derived tumors that had acquired resistance to anti-VEGF or chemotherapeutics such as bevacizumab or cisplatin, respectively. Interestingly, AKT was frequently activated both in early-stage and advanced-stage CCCs. Treatment of CCC cells with perifosine attenuated the activity of AKT–mTORC1 signaling, inhibited proliferation, and induced apoptosis. The effect of perifosine was more profound under conditions of high AKT activity compared with low AKT activity. Increased AKT activation and enhanced sensitivity to perifosine were observed in the context of cisplatin-resistant CCC. Treatment with perifosine concurrently with cisplatin significantly enhanced the antitumor effect of cisplatin. Moreover, perifosine showed significant antitumor activity in CCC-derived tumors that had acquired resistance to bevacizumab or cisplatin. Collectively, these data reveal that AKT is frequently activated in ovarian CCCs and is a promising therapeutic target in aggressive forms of ovarian cancer. Implications: AKT-targeted therapy has value in a first-line setting as well as a second-line treatment for recurrent disease developing after platinum-based chemotherapy or bevacizumab treatment. Mol Cancer Res; 13(4); 795–806. ©2014 AACR.

Central nervous system tumors are histologically and biologically heterogeneous. Standard treatment for malignant tumors includes surgery, radiation and chemotherapy, yet surgical resection is not always an option and chemotherapeutic agents have limited benefit. Recent investigations have focused on molecularly targeted therapies aimed at critical tumorigenic pathways. Several tumor types, including high-grade gliomas and pediatric pontine gliomas, exhibit Akt activation. Perifosine, an orally bioavailable, synthetic alkylphospholipid and potent Akt inhibitor, has demonstrated activity in some preclinical models, but absent activity in a genetically engineered mouse model of pontine glioma. We evaluated the plasma and cerebrospinal fluid pharmacokinetics of orally administered perifosine in a non-human primate model to evaluate CNS penetration.Perifosine was administered orally to three adult rhesus monkeys as a single dose of 7.0 mg/kg perifosine. Serial paired plasma and CSF samples were collected for up to 64 days. Perifosine was quantified with a validated HPLC/tandem mass spectrometry assay. Pharmacokinetic parameters were estimated using non-compartmental methods. CSF penetration was calculated from the areas under the concentration-time curves.Peak plasma concentrations (C max) ranged from 11.7-19.3 µM, and remained1 µM for28 days. Time to C max (T max) was 19 h. The median (range) AUCPl was 3148 (2502-4705) µM/h, with a median (range) terminal half-life (t 1/2) of 193 (170-221) h. Plasma clearance was 494 (329-637) mL/h/kg. Peak CSF concentrations were 4.1-10.1 nM (T max 64-235 h). CSF AUCs and t 1/2 were 6358 (2266-7568) nM/h and 277 (146-350) h, respectively. Perifosine concentrations in the CSF remained over nM for35 days. The mean CSF penetration was 0.16 %.CNS penetration of perifosine after systemic administration is poor. However, levels were measurable in both plasma and CSF for an extended time (2 months) after a single oral dose.

Radiation-induced gastrointestinal (GI) syndrome currently has no effective prophylactic or therapeutic treatment. Previous studies and our data have demonstrated the important role of p53 in acute radiation-induced GI syndrome in mice. Many cytokines, such as tumor necrosis factor-α and fibroblast growth factor (bFGF), have been found to protect against radiation-induced intestinal injury, although the underlying mechanisms remain to be identified. Here, we report blockage of p53 through a protein kinase B (Akt) pathway in intestinal progenitor/stem cells or crypt cells as a novel molecular mechanism of growth factor-mediated intestinal radioprotection. Treatment with platelet-derived growth factor (PDGF-BB) or bFGF activated Akt phosphorylation in the intestinal crypt, lessened intestinal crypt p53 expression, decreased radiation-induced apoptosis in mouse intestinal progenitor/stem cell marker leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5)-positive cells by an average of 50%, and increased the survival rate of mice with abdominal radiation by 3 days in average. Conversely, the Akt inhibitor perifosine obstructed growth factor-simulated Akt phosphorylation while promoting radiation-induced p53 expression in intestinal crypts. Importantly, reduced Akt phosphorylation and elevated p53 expression due to the Akt inhibitor perifosine impaired intestinal progenitor/stem cells radioprotection provided by PDGF-BB and bFGF. Consistently, PDGF-BB and bFGF both upregulated Akt activation, suppressed radiation-induced p53 expression, and abrogated radiation-induced apoptosis in IEC-6 cells, although p53 overexpression in IEC-6 cells partially counteracted the radioprotection of PDGF-BB and bFGF. Our data suggest that intestinal crypt radioprotection by PDGF-BB and bFGF is dependent on regulation of Akt/p53 signaling.

Fragile histidine triad (FHIT) loss by the two-hit mechanism of loss of heterozygosity and promoter hypermethylation commonly occurrs in non-small cell lung cancer (NSCLC) and may confer cisplatin resistance in NSCLC cells. However, the underlying mechanisms of FHIT loss in cisplatin resistance and the response to cisplatin-based chemotherapy in NSCLC patients have not yet been reported. In the present study, inhibition concentration of 50% cell viability induced by cisplatin (IC50) and soft agar growth and invasion capability were increased and decreased in FHIT-knockdown and -overexpressing cells, respectively. Mechanistically, Slug transcription is upregulated by AKT/NF-κB activation due to FHIT loss and, in turn, Slug suppresses PUMA expression; this decrease of PUMA by FHIT loss is responsible for cisplatin resistance. In addition, cisplatin resistance due to FHIT loss can be conquered by AKT inhibitor-perifosine in xenograft tumors. Among NSCLC patients, low FHIT, high p-AKT, high Slug and low PUMA were correlated with shorter overall survival, relapse-free survival and poorer response to cisplatin-based chemotherapy. Therefore, the AKT inhibitor perifosine might potentially overcome the resistance to cisplatin-based chemotherapy in NSCLC patients with low-FHIT tumors, and consequently improve the outcome.

Acquisition of multidrug resistance (MDR) is a common cause of treatment failure during chemotherapy for dogs with lymphoma (lymphosarcoma). Overexpression of P-glycoprotein (P-gp), encoded by the ABCB1 gene, is associated with MDR. Perifosine, an Akt inhibitor, downregulates the expression of P-gp. In this study, the antitumour effect of perifosine and its ability to modulate ABCB1 expression were examined in four canine lymphoid tumour cell lines (GL-1, CLBL-1, UL-1 and Ema). GL-1 and CLBL-1 were inherently negative for P-gp, while UL-1 and Ema were inherently positive for P-gp. GL-1 and UL-1 were sensitive to perifosine, whereas CLBL-1 and Ema were resistant. The amount of ABCB1 mRNA significantly decreased after treatment with perifosine in UL-1, associated with activation of the c-Jun NH2-terminal kinase (JNK) pathway, but such an effect was not observed in Ema. In UL-1, perifosine decreased the efflux of rhodamine 123 dye and reduced the 50% inhibitory concentration of vincristine, but such effects were not observed in Ema. Perifosine had an antitumour effect in 2/4 canine lymphoid tumour cell lines. In 1/4 cell lines, perifosine downregulated ABCB1 gene expression through activation of the JNK pathway and increased sensitivity to vincristine.

Alkylphospholipid (APL) analogues are promising candidates in the search for treatments for cancer. In contrast to standard chemotherapeutic drugs, these lipophilic agents target the cell membrane without interacting directly with DNA. A variety of mechanisms have been suggested to explain the actions of these compounds, which can induce apoptosis and/or cell growth arrest. In this review, we focus on recent advances in our understanding of the actions of clinically-relevant APLs, such as hexadecylphosphocholine (HePC), edelfosine, erucylphosphocholine (ErPC) and perifosine on the human hepatoma HepG2 cell line, which is commonly used for lipid metabolism studies with a special emphasis on cholesterol metabolism. One consistent finding is that HePC and other APLs cause a reduction in the biosynthesis of phosphatidylcholine (PC) by inhibiting the rate-limiting enzyme CTP:phosphocholine cytidylyltransferase (CT). Our research group has been at the forefront in demonstrating that exposure to APLs affects cholesterol homeostasis in mammalian cells. Treatment with HePC, for example, causes a marked enhancement in cholesterol synthesis, which has been related to an impairment in the arrival of cholesterol at the endoplasmic reticulum (ER). In a similar way to HePC, edelfosine, ErPC and perifosine increase the de novo synthesis and uptake of cholesterol and also inhibit the arrival of plasma-membrane cholesterol at the ER, which induces a significant cholesterogenic response in these cells, involving an increase in gene expression and higher levels of several proteins related to the biosynthetic pathway and receptor-mediated uptake of cholesterol. It is generally accepted nowadays that the maintenance of a tightly controlled free-cholesterol/PC ratio is crucial to optimum cell behaviour and that alterations to this ratio may lead to necrosis and/or apoptosis. Our results have considerable bearing on this idea because an increase in cholesterol biosynthesis associated with a decrease in the synthesis of choline-containing phospholipids and cholesterol esterification leads to a modification in the free-cholesterol/PC ratio in cells exposed to APLs. It is well accepted that cholesterol is critical for the formation of lipid rafts and therefore drugs that alter cell cholesterol content should modify the properties of these membrane domains and consequently the signal-transduction pathways, which depends upon lipid-raft integrity. Results on the whole show that APLs share a common active mechanism consisting of disrupting PC and sphingomyelin (SM) biosyntheses and cholesterol homeostasis, all of which leads to a disturbance in the native membrane structure, thus affecting signaling processes vital to cell survival and growth.

Our improved understanding of the molecular processes that determine cellular sensitivity to ionizing radiation has accelerated the identification of new targets for intervention. Indeed, novel agents have become available for combined clinical use to overcome radioresistance and increase the therapeutic ratio of radiotherapy. Synthetic alkyl-phospholipid analogs (APLs), such as edelfosine, ilmofosine, miltefosine, perifosine and erucylphosphocholine, are a novel class of anti-tumor agents that target cell membranes to induce growth arrest and apoptosis. In addition, APLs strongly enhance the cytotoxic effect of radiation in preclinical models making these compounds attractive candidates as clinical radiosensitizers. In this review, we will discuss mechanisms of action underlying the rationale to combine APLs with radiotherapy and highlight the clinical perspective of this novel combined modality treatment.

Mantle cell lymphoma (MCL) is a neoplasm classified as a B-cell malignancy, that accounts for approximately 3 to 8% of Non-Hodgkin’s lymphoma (NHL) cases diagnosed annually. MCL is difficult to treat and seldom considered cured. The pathobiology of MCL is complex and includes alteration in the cell cycle, abnormalities in the DNA damage response, and constitutive activation of key antiapoptotic pathways including phosphatidyl-inositol 3-kinase (PI3K)/Akt and nuclear factor-kB. This has promoted the identification of new targeted treatments and new agents that have shown promising efficacy for future MCL therapies. The phosphatidylinositol 3-kinase (PI3K) mammalian target of rapamicin (mTOR) pathway mediates proliferation, survival, and drug resistance in lymphoma cells. NVP-BEZ235 (BEZ235) is a new, orally bioavailable inhibitor of PI3K and mTOR and a representative of a new class of anti-tumour agents.In this study, we analysed the in vitro inhibitory effects of NVP-BEZ235 on mantle lymphoma cell lines (GRANTA-519 and JeKo-1) and its effects in combination with enzastaurin, everolimus and perifosine. Our data suggest that in mantle lymphoma cell lines, BEZ235 in combination with enzastaurin elicits its antitumour effect better than combined with perifosine and everolimus. Our data reveal that the drug combination targets phosphorylation of PI3K/Akt/mTOR pathways and induces both intrinsic and extrinsic apoptosis pathways. Furthermore, inhibition of Bcl-2 anti-apoptosis family members may, in part, explain the efficacy of signalling blockade in lymphoma cells and suggests an additional therapeutic targeting strategy. Therefore, these preclinical data support the potential use of BEZ235 in patients with mantle lymphoma, and in particular provide rationale for combination with enzastaurin.