The Antitumor Effect of the DNA Polymerase Alpha Inhibitor ST1926 in Glioblastoma: A Proteomics Approach.

Glioblastoma Multiforme (GBM) is the most aggressive form of malignant brain tumor. The median survival rate does not exceed two years, indicating an imminent need to develop novel therapies. The atypical adamantyl retinoid ST1926 induces apoptosis and growth inhibition in different cancer types. We have shown that ST1926 is an inhibitor of the catalytic subunit of DNA polymerase alpha (POLA1), which is involved in initiating DNA synthesis in eukaryotic cells. POLA1 levels are elevated in GBM versus normal brain tissues. Therefore, we studied the antitumor effects of ST1926 in several human GBM cell lines. We further explored the global protein expression profiles in GBM cell lines using liquid chromatography coupled with tandem mass spectrometry to identify new targets of ST1926. Low sub-micromolar concentrations of ST1926 potently decreased cell viability, induced cell damage and apoptosis, and reduced POLA1 protein levels in GBM cells. The proteomics profiles revealed 197 proteins significantly differentially altered upon ST1926 treatment of GBM cells involved in various cellular processes. We explored the differential gene and protein expression of significantly altered proteins in GBM compared to normal brain tissues.

Development and Challenges of Synthetic Retinoid Formulations in Cancer.

Retinoids represent a class of chemical compounds derived from or structurally and functionally related to vitamin A. Retinoids play crucial roles in regulating a range of crucial biological processes spanning embryonic development to adult life. These include regulation of cell proliferation, differentiation, and cell death. Due to their promising characteristics, retinoids emerged as potent anti-cancer agents, and their effects were validated in vitro and in vivo preclinical models of several solid and hematological malignancies. However, their clinical translation remained limited due to poor water solubility, photosensitivity, short half-life, and toxicity. The development of retinoid delivery formulations was extensively studied to overcome these limitations. This review will summarize some preclinical and commercial synthetic retinoids in cancer and discuss their different delivery systems.

Multi-approach LC-MS methods for the characterization of species-specific attributes of monoclonal antibodies from plants.

In this work, an analytical platform based on the use of chromatography and mass spectrometry (MS), has been applied to the characterization of Rituximab (RTX) obtained from two plant expression systems (rice and tobacco) in comparison to the mammalian cell-derived reference monoclonal antibody (mAb). Different chromatographic approaches, hyphenated to high resolution MS (HRMS), were applied to RTX structural investigation both at middle- and peptide level. In particular, cation exchange chromatography (CEX), size exclusion chromatography (SEC), reversed phase (RPLC) and hydrophilic interaction liquid chromatographic (HILIC) methods were developed and applied on intact mAbs, IdeS-, and trypsin digests in order to address critical attributes such as primary structure, glycan composition, species-related heterogeneity, glycosylation degree, charge variants, aggregation tendency and enzymatic stability. All the collected data highlight the features and criticalities of each production approach. Production in rice results in a heterogeneous but stable product over time, suggesting the absence of proteases in seeds; while tobacco expression system leads to more homogeneous glycosylation, but protein stability seems to be a critical issue probably due to the presence of proteases. This analytical strategy represents a robust support to scientists in the selection and optimization of the best plant expression system to produce recombinant humanized mAbs.

Antitumor activity of novel POLA1-HDAC11 dual inhibitors.

Hybrid molecules targeting simultaneously DNA polymerase α (POLA1) and histone deacetylases (HDACs) were designed and synthesized to exploit a potential synergy of action. Among a library of screened molecules, MIR002 and GEM144 showed antiproliferative activity at nanomolar concentrations on a panel of human solid and haematological cancer cell lines. In vitro functional assays confirmed that these molecules inhibited POLA1 primer extension activity, as well as HDAC11. Molecular docking studies also supported these findings. Mechanistically, MIR002 and GEM144 induced acetylation of p53, activation of p21, G1/S cell cycle arrest, and apoptosis. Oral administration of these inhibitors confirmed their antitumor activity in in vivo models. In human non-small cancer cell (H460) xenografted in nude mice MIR002 at 50 mg/kg, Bid (qd × 5 × 3w) inhibited tumor growth (TGI = 61%). More interestingly, in POLA1 inhibitor resistant cells (H460-R9A), the in vivo combination of MIR002 with cisplatin showed an additive antitumor effect with complete disappearance of tumor masses in two animals at the end of the treatment. Moreover, in two human orthotopic malignant pleural mesothelioma xenografts (MM473 and MM487), oral treatments with MIR002 and GEM144 confirmed their significant antitumor activity (TGI = 72-77%). Consistently with recent results that have shown an inverse correlation between POLA1 expression and type I interferon levels, MIR002 significantly upregulated interferon-α in immunocompetent mice.

Investigation of the Complexes Formed between PARP1 Inhibitors and PARP1 G-Quadruplex at the Gene Promoter Region.

DNA repair inhibitors are one of the latest additions to cancer chemotherapy. In general, chemotherapy produces DNA damage but tumoral cells may become resistant if enzymes involved in DNA repair are overexpressed and are able to reverse DNA damage. One of the most successful drugs based on modulating DNA repair are the poly(ADP-ribose) polymerase 1 (PARP1) inhibitors. Several PARP1 inhibitors have been recently developed and approved for clinical treatments. We envisaged that PARP inhibition could be potentiated by simultaneously modulating the expression of PARP 1 and the enzyme activity, by a two-pronged strategy. A noncanonical G-quadruplex-forming sequence within the PARP1 promoter has been recently identified. In this study, we explored the potential binding of clinically approved PARP1 inhibitors to the G-quadruplex structure found at the gene promoter region. The results obtained by NMR, CD, and fluorescence titration confirmed by molecular modeling demonstrated that two out the four PARP1 inhibitors studied are capable of forming defined complexes with the PARP1 G-quadruplex. These results open the possibility of exploring the development of better G-quadruplex binders that, in turn, may also inhibit the enzyme.

New Antimicrobials Based on the Adarotene Scaffold with Activity against Multi-Drug Resistant Staphylococcus aureus and Vancomycin-Resistant Enterococcus.

The global increase in infections by multi-drug resistant (MDR) pathogens is severely impacting our ability to successfully treat common infections. Herein, we report the antibacterial activity against S. aureus and E. faecalis (including some MDR strains) of a panel of adarotene-related synthetic retinoids. In many cases, these compounds showed, together with favorable MICs, a detectable bactericidal effect. We found that the pattern of substitution on adarotene could be modulated to obtain selectivity for antibacterial over the known anticancer activity of these compounds. NMR experiments allowed us to define the interaction between adarotene and a model of microorganism membrane. Biological assessment confirmed that the scaffold of adarotene is promising for further developments of non-toxic antimicrobials active on MDR strains.

Restoration of ceramide de novo synthesis by the synthetic retinoid ST1926 as it induces adult T-cell leukemia cell death.

Ceramide (Cer) is a bioactive cellular lipid with compartmentalized and tightly regulated levels. Distinct metabolic pathways lead to the generation of Cer species with distinguishable roles in oncogenesis. Deregulation of Cer pathways has emerged as an important mechanism for acquired chemotherapeutic resistance. Adult T-cell leukemia (ATL) cells are defective in Cer synthesis. ATL is an aggressive neoplasm that develops following infection with human T-cell lymphotropic virus-1 (HTLV-1) where the viral oncogene Tax contributes to the pathogenesis of the disease. ATL cells, resistant to all-trans-retinoic acid, are sensitive to pharmacologically achievable concentrations of the synthetic retinoid ST1926. We studied the effects of ST1926 on Cer pathways in ATL cells. ST1926 treatment resulted in early Tax oncoprotein degradation in HTLV-1-treated cells. ST1926 induced cell death and a dose- and time-dependent accumulation of Cer in malignant T cells. The kinetics and degree of Cer production showed an early response upon ST1926 treatment. ST1926 enhanced de novo Cer synthesis via activation of ceramide synthase CerS(s) without inhibiting dihydroceramide desaturase, thereby accumulating Cer rather than the less bioactive dihydroceramide. Using labeling experiments with the unnatural 17-carbon sphinganine and measuring the generated Cer species, we showed that ST1926 preferentially induces the activities of a distinct set of CerS(s). We detected a delay in cell death response and interruption of Cer generation in response to ST1926 in Molt-4 cells overexpressing Bcl-2. These results highlight the potential role of ST1926 in inducing Cer levels, thus lowering the threshold for cell death in ATL cells.

Novel adamantyl retinoid-related molecules with POLA1 inhibitory activity.

Atypical retinoids (AR) or retinoid-related molecules (RRMs) represent a promising class of antitumor compounds. Among AR, E-3-(3'-adamantan-1-yl-4'-hydroxybiphenyl-4-yl)acrylic acid (adarotene), has been extensively investigated. In the present work we report the results of our efforts to develop new adarotene-related atypical retinoids endowed also with POLA1 inhibitory activity. The effects of the synthesized compounds on cell growth were determined on a panel of human and hematological cancer cell lines. The most promising compounds showed antitumor activity against several tumor histotypes and increased cytotoxic activity against an adarotene-resistant cell line, compared to the parent molecule. The antitumor activity of a selected compound was evaluated on HT-29 human colon carcinoma and human mesothelioma (MM487) xenografts. Particularly significant was the in vivo activity of the compound as a single agent compared to adarotene and cisplatin, against pleural mesothelioma MM487. No reduction of mice body weight was observed, thus suggesting a higher tolerability with respect to the parent compound adarotene.

Calcium Regulates HCC Proliferation as well as EGFR Recycling/Degradation and Could Be a New Therapeutic Target in HCC.

Calcium is the most abundant element in the human body. Its role is essential in physiological and biochemical processes such as signal transduction from outside to inside the cell between the cells of an organ, as well as the release of neurotransmitters from neurons, muscle contraction, fertilization, bone building, and blood clotting. As a result, intra- and extracellular calcium levels are tightly regulated by the body. The liver is the most specialized organ of the body, as its functions, carried out by hepatocytes, are strongly governed by calcium ions. In this work, we analyze the role of calcium in human hepatoma (HCC) cell lines harboring a wild type form of the Epidermal Growth Factor Receptor (EGFR), particularly its role in proliferation and in EGFR downmodulation. Our results highlight that calcium is involved in the proliferative capability of HCC cells, as its subtraction is responsible for EGFR degradation by proteasome machinery and, as a consequence, for EGFR intracellular signaling downregulation. However, calcium-regulated EGFR signaling is cell line-dependent. In cells responding weakly to the epidermal growth factor (EGF), calcium seems to have an opposite effect on EGFR internalization/degradation mechanisms. These results suggest that besides EGFR, calcium could be a new therapeutic target in HCC.

Combined Treatment with Doxorubicin and Rapamycin Is Effective against In Vitro and In Vivo Models of Human Glioblastoma.

Despite numerous clinical trials, glioblastoma (GBM) remains a tumor that is difficult to treat. The aim of this study was to investigate the potential of a new pharmacological approach, combining doxorubicin (Dox) and rapamycin (Rapa), in in vitro and in vivo GBM models. Cytotoxic and anti-proliferative effects of Rapa plus Dox treatments were analyzed in GBM cell lines. The in vivo effectiveness of these treatments was investigated in an orthotopic xenograft mice model of GBM. In vitro results demonstrated that prolonged exposure to Rapa sensitize GBM cells to Dox treatments. In vivo results demonstrated that Rapa (5 mg/kg) plus Dox (5 mg/kg) determined the major tumor growth inhibition (-97.29% vs. control) but results in greater toxicity. The combination Rapa plus Dox (2.5 mg/kg) showed a tumor inhibition like Rapa plus Dox (5 mg/kg) with a toxicity comparable to Rapa alone. Thus, this study demonstrated the efficacy of this pharmacological approach, providing the rationale for a clinical application of this combinational therapy in "poor-responder" GBM patients.

The synthetic retinoid ST1926 attenuates prostate cancer growth and potentially targets prostate cancer stem-like cells.

Retinoids are vitamin A derivatives that regulate crucial biological processes such as cellular proliferation, apoptosis, and differentiation. The use of natural retinoids in cancer therapy is limited due to their toxicity and the acquired resistance by cancer cells. Therefore, synthetic retinoids were developed, such as the atypical adamantyl retinoid ST1926 that provides enhanced bioavailability and reduced toxicity. We have assessed the in vitro and in vivo antitumor properties and mechanism of action of ST1926 in targeting cancer stem-like cells population of human prostate cancer (PCa) cell lines, DU145 and PC3, and mouse PCa cell lines, PLum-AD and PLum-AI. We demonstrated that ST1926 substantially reduced proliferation of PCa cells and induced cell cycle arrest, p53-independent apoptosis, and early DNA damage. It also decreased migration and invasion of PCa cells and significantly reduced prostate spheres formation ability in vitro denoting sufficient eradication of the self-renewal ability of the highly androgen-resistant cancer stem cells. Importantly, ST1926 potently inhibited PCa tumor growth and progression in vivo. Our results highlight the potential of ST1926 in PCa therapy and warrant its clinical development.

Aldoxorubicin and Temozolomide combination in a xenograft mice model of human glioblastoma.

Glioblastoma Multiforme (GBM) is still an incurable disease. The front-line Temozolomide (TMZ)-based therapy suffers from poor efficacy, underlining the need of new therapies. Preclinically, Aldoxorubicin (Aldox), a novel prodrug of Doxorubicin (Dox), has been successfully tested against GBM, encouraging the study of its association with other agents. For the first time, we evaluated the effectiveness of Aldox combined to TMZ in preclinical models of GBM. Our in vitro results demonstrated that the anti-glioma effect of Aldox was more marked than TMZ and their combination increased the killing effect of the anthracycline in TMZ-resistant GBM cells. Moreover, unlike Dox, Aldox was able to accumulate in P-glycoprotein (P-gp)-overexpressed cells due to a negative regulation of the P-gp function. We also compared efficacy and safety of weekly administrations of Aldox (16 mg/kg), with or without TMZ (0.9 mg/kg, daily injections), in the U87 xenograft mouse model. Aldox therapy induced a moderate tumor volume inhibition (TVI) and an increased survival rate (+12.5% vs vehicle). On the other hand, when combined to TMZ, Aldox caused a significant TVI (P=0.0175 vs vehicle) and delayed the mortality during the experimental period, although TVI and endpoint survival percentage (+37.5% vs vehicle) were not significantly different from TMZ alone. Our preliminary data showed that Aldox exerts anti-glioma effects in vitro and in vivo. It also enhances its antitumor activity when combined with TMZ, resulting in a superior efficacy compared to the single agents, without adverse side effects.

Hybrid topoisomerase I and HDAC inhibitors as dual action anticancer agents.

Recent studies have shown that HDAC inhibitors act synergistically with camptothecin derivatives in combination therapies. To exploit this synergy, new hybrid molecules targeting simultaneously topoisomerase I and HDAC were designed. In particular, a selected multivalent agent containing a camptothecin and a SAHA-like template showed a broad spectrum of antiproliferative activity, with IC50 values in the nanomolar range. Preliminary in vivo results indicated a strong antitumor activity on human mesothelioma primary cell line MM473 orthotopically xenografted in CD-1 nude mice and very high tolerability.

Mechanism of action of the atypical retinoid ST1926 in colorectal cancer: DNA damage and DNA polymerase α.

Despite advances in therapeutic strategies, colorectal cancer (CRC) remains the third cause of cancer-related deaths with a relatively low survival rate. Resistance to standard chemotherapy represents a major hurdle in disease management; therefore, developing new therapeutic agents demands a thorough understanding of their mechanisms of action. One of these compounds is ST1926, an adamantyl retinoid that has shown potent antitumor activities in several human cancer models. Here, we show that ST1926 selectively suppressed the proliferation of CRC cells while sparing normal counterparts, and significantly reduced tumor volume in a xenograft cancer mouse model. Next, we investigated the effects of ST1926 in CRC cells and observed early DNA damage, S-phase arrest, dissipation of mitochondrial membrane potential, and apoptosis induction, in a p53 and p21-independent manner. To address the underlying mechanism of resistance to ST1926, we generated ST1926-resistant HCT116 cells and sequenced DNA polymerase α (POLA1), which was reported to be a direct target to the drug's parent molecule, CD437. We identified similar mutations in POLA1 that conferred resistance to ST1926 and CD437. These mutations were absent in 5-fluorouracil-resistant HCT116 cells, clearly validating the specificity of these mutations to the lack of DNA damage and acquired resistance to ST1926. ST1926 also inhibited POLA1 activity and reduced its protein expression levels. Further, in silico analysis of normal and malignant tissue expression data demonstrated that POLA1 levels are elevated in CRC cells and tissues compared to normal counterparts as well as to other cancer types. Our findings highlight previously uncharacterized mechanisms of action of ST1926 in CRC and suggest that elevated POLA1 expression is a pertinent molecular feature and an attractive target in CRC.

Antitumor activity of the synthetic retinoid ST1926 on primary effusion lymphoma in vitro and in vivo models.

Primary effusion lymphoma (PEL) is a rare B-cell neoplasm, associated with Kaposi sarcoma-associated herpes virus/human herpes virus-8 (KSHV/HHV-8), arising as malignant effusions in body cavities. PEL cells do not harbor conventional genetic cancer mutations; however, their oncogenesis is mainly attributed to HHV-8 latent genes. Treatment strategies are inefficient resulting in poor prognosis of PEL patients, stressing the need for new effective therapy. ST1926 is a synthetic retinoid with favorable antitumor properties and no cross-resistance with the natural retinoid, all-trans retinoic acid. ST1926 has shown potent apoptotic activities on a variety of solid tumors and hematologic malignancies in in vitro and in vivo models. In the present study we elucidated the antitumor activities and underlying molecular mechanism of ST1926 using in vitro, ex vivo, and in vivo PEL preclinical models. ST1926, at sub­micromolar concentrations, displayed potent antiproliferative effects on PEL cell lines and malignant ascites. Furthermore, ST1926 treatment of PEL cells and ascites resulted in their accumulation in the sub-G1 region, S phase cell cycle arrest, early DNA damage, PARP cleavage and p53 activation including the upregulation of its target genes p21 and Bax. However, ST1926 did not significantly modulate HHV-8 latent viral transcripts. Importantly, ST1926 delayed formation of ascites and enhanced survival of PEL mice. These results highlight the therapeutic potential of ST1926 in combination with drugs that target HHV-8 in PEL patients.

Camptothecin-psammaplin A hybrids as topoisomerase I and HDAC dual-action inhibitors.

Recent studies have demonstrated enhanced anticancer effects of combination therapy consisting of camptothecin derivatives and HDAC inhibitors. To exploit this synergy in a single active compound, we designed new dual-acting multivalent molecules simultaneously targeting topoisomerase I and HDAC. In particular, a selected compound containing a camptothecin and the psammaplin A scaffold showed a broad spectrum of antiproliferative activity, with IC50 values in the nanomolar range. Preliminary in vivo results indicated a strong antitumor activity on human mesothelioma primary cell line MM473 orthotopically xenografted in CD-1 nude mice and very high tolerability.

Tumor response of temozolomide in combination with morphine in a xenograft model of human glioblastoma.

Despite multimodal treatments comprising, radiation therapy (RT) and chemotherapy with temozolomide (TMZ), the prognosis of glioblastoma multiforme (GBM) remains dismal and consolidated therapy yields a median survival of 14.6 months. Blood Brain Barrier (BBB) mediated chemoresistance and high dose related toxicity make necessary the development of new therapeutic approach to sensitize GBM to TMZ. The aim of the present study was to investigate the potential of the treatment morphine plus TMZ metronmic doses (1,77 and 0,9 mg/kg) in GBM therapy. The effect of morphine, on tumor cell growth and P-glycoprothein (P-gp) activity, was investigate in in vitro models. The results demonstrated that GBM cells growth is not influenced by morphine treatment and, for the first time, we show that morphine is an inhibitor of the activity of P-gp efflux transporter who is markedly expressed on BBB. In vivo, response to the treatments TMZ plus morphine was investigated in an orthotopic nude mice model of GBM. Animals treated with TMZ metronomic doses showed a significant tumor growth inhibition compared to untreated mice and association with morphine appears to improve TMZ efficacy. Moreover, the combination of morphine with lower dose of TMZ result in a cytostatic effect on tumor growth over the period of the pharmacological treatments. In conclusion this novel approach could be a successful strategy to overcome chemoresistance and side effects TMZ mediated, reducing drug dosage and improving long term response, in GBM therapy.

Antitumor Effect of the Atypical Retinoid ST1926 in Acute Myeloid Leukemia and Nanoparticle Formulation Prolongs Lifespan and Reduces Tumor Burden of Xenograft Mice.

Acute myeloid leukemia (AML) is one of the most frequent types of blood malignancies. It is a complex disorder of undifferentiated hematopoietic progenitor cells. The majority of patients generally respond to intensive therapy. Nevertheless, relapse is the major cause of death in AML, warranting the need for novel treatment strategies. Retinoids have demonstrated potent differentiation and growth regulatory effects in normal, transformed, and hematopoietic progenitor cells. All-trans retinoic acid (ATRA) is the paradigm of treatment in acute promyelocytic leukemia, an AML subtype. The majority of AML subtypes are, however, resistant to ATRA. Multiple synthetic retinoids such as ST1926 recently emerged as potent anticancer agents to overcome such resistance. Despite its lack of toxicity, ST1926 clinical development was restricted due to its limited bioavailability and rapid excretion. Here, we investigate the preclinical efficacy of ST1926 and polymer-stabilized ST1926 nanoparticles (ST1926-NP) in AML models. We show that sub-µmol/L concentrations of ST1926 potently and selectively inhibited the growth of ATRA-resistant AML cell lines and primary blasts. ST1926 induced-growth arrest was due to early DNA damage and massive apoptosis in AML cells. To enhance the drug's bioavailability, ST1926-NP were developed using Flash NanoPrecipitation, and displayed comparable anti-growth activities to the naked drug in AML cells. In a murine AML xenograft model, ST1926 and ST1926-NP significantly prolonged survival and reduced tumor burden. Strikingly, in vivo ST1926-NP antitumor effects were achieved at four fold lower concentrations than the naked drug. These results highlight the promising use of ST1926 in AML therapy and encourage its further development. Mol Cancer Ther; 16(10); 2047-57. ©2017 AACR.

Antitumor activities of the synthetic retinoid ST1926 in two-dimensional and three-dimensional human breast cancer models.

Despite recent advances in chemotherapy, aggressive and metastatic breast cancers remain refractory to targeted therapy and the development of novel drugs is urgently needed. Retinoids are crucial regulators of cellular proliferation, differentiation, and cell death, and have shown potent chemotherapeutic and chemopreventive properties. The major drawback of the use of all-trans retinoic acid (ATRA) in cancer therapy is disease relapse. Therefore, synthetic retinoids, specifically ST1926, have emerged as potent anticancer agents. Given the importance of the microenvironment in modulating the response of cancer cells to chemotherapeutic drugs, we investigated the antitumor activities of ST1926 in two-dimensional (2D) and different three-dimensional (3D) human breast cancer models and compared them with ATRA. We have shown that in 2D cell culture models, ATRA-resistant MCF-7 and MDA-MB-231 cells were sensitive to ST1926 at submicromolar concentrations that spared the 'normal-like' breast epithelial cells. ST1926 induced apoptosis and S-phase arrest, caused DNA damage, and downregulated the Wnt/ß-catenin pathway in breast cancer cells in 2D and 3D cell culture models. ST1926-mediated growth inhibition was independent of the retinoid receptor-signaling pathway. Long-term treatments with low submicromolar ST1926 concentrations reduced the anchorage-independent growth and decreased the sphere-forming ability of breast cancer progenitor cells in the sphere formation assay. Furthermore, ST1926 potently induced cell death of breast cancer cells under 3D conditions and spared the lumen-forming ability of normal-like breast epithelial cells. In tested 3D models, ATRA had minimal effects on the growth of breast cancer cells compared with ST1926. In summary, our results highlight the therapeutic potential of ST1926 in breast cancer and warrant its further clinical development.

Morphine modulates doxorubicin uptake and improves efficacy of chemotherapy in an intracranial xenograft model of human glioblastoma.

Morphine may alter the permeability of Blood-Brain Barrier (BBB), enhancing the access of molecules normally unable to cross it, as Doxorubicin (Dox). In addition, morphine seems to mediate the uptake of Dox into the brain by its reduced efflux mediated by P-glycoprotein (P-gp). We evaluated the antitumor efficacy of Dox plus morphine treatment by an orthotopic glioblastoma xenograft model. Foxn1 mice were injected with U87MG-luc cells in the left lobe of the brain and treated with Dox (5 mg/kg and 2.5 mg/kg, weekly) with or without morphine pretreatment (10 mg/kg, weekly). Bioluminescence imaging (BLI) was used to monitoring tumor growth and response to therapy. Additionally, we investigated the role of morphine on the uptake of Dox by MDCKII cells transfected with human MDR1 gene encoding for P-gp. The data demonstrate that only Dox 5 mg/kg determined a significant tumor regression while the lower dose (2.5 mg/kg) was not effective. However, if combined with morphine, the group treated with Dox 2.5 mg/kg showed a decreasing tumor growth. The average BLI for Dox 2.5 mg/kg plus morphine was 5 fold lower than Dox 2.5 mg/kg alone (P=0.0053) and 8 fold lower than vehicle (P=0.0004). Additionally, Dox increased in MDCKII-P-gp transfected cells only in the presence of morphine with a significantly higher level comparing control group (3.84) vs Dox plus morphine group (12.29, P<0.05). Our results indicate that Dox alone and in combination with morphine appear to be effective in controlling the growth of glioblastoma in a xenograft mouse model.