CDKN2A promoter methylation enhances self-renewal of glioblastoma stem cells and confers resistance to carmustine.

BACKGROUND: Glioblastoma, a highly aggressive form of brain cancer, poses significant challenges due to its resistance to therapy and high recurrence rates. This study aimed to investigate the expression and functional implications of CDKN2A, a key tumor suppressor gene, in glioblastoma cells, building upon the existing background of knowledge in this field. METHOD: Quantitative reverse transcription PCR (qRT-PCR) analysis was performed to evaluate CDKN2A expression in U87 glioblastoma cells compared to normal human astrocytes (NHA). CDKN2A expression levels were manipulated using small interfering RNA (siRNA) and CDKN2A overexpression vector. Cell viability assays and carmustine sensitivity tests were conducted to assess the impact of CDKN2A modulation on glioblastoma cell viability and drug response. Sphere formation assays and western blot analysis were performed to investigate the role of CDKN2A in glioblastoma stem cell (GSC) self-renewal and pluripotency marker expression. Additionally, methylation-specific PCR (MSP) assays and demethylation treatment were employed to elucidate the mechanism of CDKN2A downregulation in U87 cells. RESULT: CDKN2A expression was significantly reduced in glioblastoma cells compared to NHA. CDKN2A overexpression resulted in decreased cell viability and enhanced sensitivity to carmustine treatment. CDKN2A inhibition promoted self-renewal capacity and increased pluripotency marker expression in U87 cells. CDKN2A upregulation led to elevated protein levels of p16INK4a, p14ARF, P53, and P21, which are involved in cell cycle regulation. CDKN2A downregulation in U87 cells was associated with high promoter methylation, which was reversed by treatment with a demethylating agent. CONCLUSION: Our findings demonstrate that CDKN2A downregulation in glioblastoma cells is associated with decreased cell viability, enhanced drug resistance, increased self-renewal capacity, and altered expression of pluripotency markers. The observed CDKN2A expression changes are mediated by promoter methylation. These results highlight the potential role of CDKN2A as a therapeutic target and prognostic marker in glioblastoma.

Functionalized Carbon Nano-Onions as a Smart Drug Delivery System for the Poorly Soluble Drug Carmustine for the Management of Glioblastoma.

The drug delivery system for transporting anticancer agents to targeted tissues in the body is a challenging issue. In search of a suitable biocompatible carrier having controlled and sustained drug release properties of poorly soluble drugs, carbon nano-onions (CNOs) were loaded with an anticancer drug, bis-chloroethyl nitrosourea (BCNU/carmustine). CNOs being autofluorescent, drug-loaded functionalized CNOs (f-CNO-BCNU) can be detected in vivo. Transmission electron microscopy (TEM) and differential light scattering (DLS) techniques were used to analyze the sizes of these f-CNOs. The molecular study revealed that the f-CNO-BCNU readily and noncovalently binds with the folate receptors present on the cancer cell surface in excess. Computer modeling and molecular dynamics simulation followed by binding free energy calculation shows f-CNOs have -29.9 kcal/mol binding free energy, and it noncovalently binds the receptor FRα using loop dynamics of three essential loops present in the protein along with polar stabilization interactions provided by Asp55 and Glu86 residues present in the active site. The f-CNO effectively decreased cancer cell viability with a low IC50 value (the concentration that led to 50% killing of the cells). The cell-based Franz diffusion assay was performed to study the drug release profile. The f-CNO-BCNUs also decreased the mitochondrial membrane potential of U87 cells, increased reactive oxygen species release, and caused a loss of mitochondrial membrane integrity. The f-CNOs also increased the percentage of apoptotic cells observed by the Annexin V assay. Based on observed results, it can be concluded that the f-CNO-BCNU efficiently targets the cancer cells, enhances the bioavailability of carmustine, and can be used as a smart chemotherapeutic agent. This strategy offers better patient compliance and greater bioavailability of the drug.

Development and in vitro evaluation of carmustine delivery platform: A hypoxia-sensitive anti-drug resistant nanomicelle with BBB penetrating ability.

Glioma is extremely difficult to be completely excised by surgery due to its invasive nature. Thus, chemotherapy still is the mainstay in the treatment of glioma after surgery. However, the natural blood-brain barrier (BBB) greatly restricts the penetration of chemotherapeutic agents into the central nervous system. As a front-line anti-glioma agent in clinical, carmustine (BCNU) exerts antitumor effect by inducing DNA damage at the O6 position of guanine. However, the therapeutic effect of BCNU was largely decreased because of the drug resistance mediated by O6-alkylguanine-DNA alkyltransferase (AGT) and insufficient local drug concentrations. To overcome these obstacles, we synthesized a BCNU-loaded hypoxia-responsive nano-micelle with BBB penetrating capacity and AGT inhibitory activity, named as T80-HA-AZO-BG/BCNU NPs. In this nano-system, Tween 80 (T80) serves as a functional coating on the surface of the micelle, promoting transportation across the BBB. Hyaluronic acid (HA) with active tumor-targeting capability was linked with the hydrophobic O6-benzylguanine (BG) analog via a hypoxia-sensitive azo bond. Under hypoxic tumor microenvironment, the azo bond selectively breaks to release O6-BG as AGT inhibitor and BCNU as DNA alkylating agent. The synthesized T80-HA-AZO-BG/BCNU NPs showed good stability, favorable biocompatibility and hypoxia-responsive drug-releasing ability. T80 modification improved the transportation of the micelle across an in vitro BBB model. Moreover, T80-HA-AZO-BG/BCNU NPs exhibited significantly enhanced cytotoxicity against glioma cell lines with high AGT expression compared with traditional combined medication of BCNU plus O6-BG. We expect that the tumor-targeting nano-micelle designed for chloroethylnitrosourea will provide new tools for the development of effective glioma therapy.

A multi-functional hypoxia/esterase dual stimulus responsive and hyaluronic acid-based nanomicelle for targeting delivery of chloroethylnitrosouea.

Carmustine (BCNU), a vital type of chloroethylnitrosourea (CENU), inhibits tumor cells growth by inducing DNA damage at O6 position of guanine and eventually forming dG-dC interstrand cross-links (ICLs). However, the clinical application of BCNU is hindered to some extent by the absence of tumor selectivity, poor stability and O6-alkylguanine-DNA alkyltransferase (AGT) mediated drug resistance. In recent years, tumor microenvironment has been widely utilized for advanced drug delivery. In the light of the features of tumor microenvironment, we constructed a multifunctional hypoxia/esterase-degradable nanomicelle with AGT inhibitory activity named HACB NPs for tumor-targeting BCNU delivery and tumor sensitization. HACB NPs was self-assembled from hyaluronic acid azobenzene AGT inhibitor conjugates, in which O6-BG analog acted as an AGT inhibitor, azobenzene acted as a hypoxia-responsive linker and carboxylate ester bond acted as both an esterase-sensitive switch and a connector with hyaluronic acid (HA). The obtained HACB NPs possessed good stability, favorable biosafety and hypoxia/esterase-responsive drug-releasing ability. BCNU-loaded HACB/BCNU NPs exhibited superior cytotoxicity and apoptosis-inducing ability toward the human uterine cervix carcinoma HeLa cells compared with traditional combined medication of BCNU plus O6-BG. In vivo studies further demonstrated that after a selective accumulation in the tumor site, the micelles could respond to hypoxic tumor tissue for rapid drug release to an effective therapeutic dosage. Thus, this multifunctional stimulus-responsive nanocarrier could be a new promising strategy to enhance the anticancer efficacy and reduce the side effects of BCNU and other CENUs.

Hypoxia and CD44 receptors dual-targeted nano-micelles with AGT-inhibitory activity for the targeting delivery of carmustine.

Carmustine (BCNU) is a typical chemotherapy used for treatment of cerebroma and other solid tumors, which exerts antitumor effect by inducing DNA damage at O6 position of guanine. However, the clinical application of BCNU was extremely limited due to the drug resistance mainly mediated by O6-alkylguanine-DNA alkyltransferase (AGT) and absence of tumor-targeting ability. To overcome these limitations, we developed a hypoxia-responsive nanomicelle with AGT inhibitory activity, which was successfully loaded with BCNU. In this nano-system, hyaluronic acid (HA) acts as an active tumor-targeting ligand to bind the overexpressing CD44 receptors on the surface of tumor cells. An azo bond selectively breaks in hypoxic tumor microenvironment to release O6-benzylguanine (BG) as AGT inhibitor and BCNU as DNA alkylating agent. The obtained HA-AZO-BG NPs with shell core structure had an average particle size of 176.98 ± 11.19 nm and exhibited good stability. Meanwhile, HA-AZO-BG NPs possessed a hypoxia-responsive drug release profile. After immobilizing BCNU into HA-AZO-BG NPs, the obtained HA-AZO-BG/BCNU NPs exhibited obvious hypoxia-selectivity and superior cytotoxicity in T98G, A549, MCF-7 and SMMC-7721 cells with IC50 at 189.0, 183.2, 90.1 and 100.1 µm, respectively, under hypoxic condition. Near-infrared imaging in HeLa tumor xenograft models showed that HA-AZO-BG/DiR NPs could effectively accumulate in tumor site at 4 h of post-injection, suggesting its good tumor-targetability. In addition, in vivo anti-tumor efficacy and toxicity evaluation indicated that HA-AZO-BG/BCNU NPs was more effective and less harmful compared to the other groups. After treatment, the tumor weight of HA-AZO-BG/BCNU NPs group was 58.46 % and 63.33 % of the control group and BCNU group, respectively. Overall, HA-AZO-BG/BCNU NPs was expected to be a promising candidate for targeted delivery of BCNU and elimination of chemoresistance.

CRISPR/Cas9-induced knockout reveals the role of ABCB1 in the response to temozolomide, carmustine and lomustine in glioblastoma multiforme.

Glioblastoma multiforme (GBM) is the most common malignant brain tumor with limited therapeutic options. Besides surgery, chemotherapy using temozolomide, carmustine or lomustine is the main pillar of therapy. However, therapy success is limited and prognosis still is very poor. One restraining factor is drug resistance caused by drug transporters of the ATP-binding cassette family, e.g. ABCB1 and ABCG2, located at the blood-brain barrier and on tumor cells. The active efflux of xenobiotics including drugs, e.g. temozolomide, leads to low intracellular drug concentrations and subsequently insufficient anti-tumor effects. Nevertheless, the role of efflux transporters in GBM is controversially discussed. In the present study, we analyzed the role of ABCB1 and ABCG2 in GBM cells showing that ABCB1, but marginally ABCG2, is relevant. Applying a CRISPR/Cas9-derived ABCB1 knockout, the response to temozolomide was significantly augmented demonstrated by decreased cell number (p < 0.001) and proliferation rate (p = 0.04), while apoptosis was increased (p = 0.04). For carmustine, a decrease of cells in G1-phase was detected pointing to cell cycle arrest in the ABCB1 knockout (p = 0.006). For lomustine, however, loss of ABCB1 did not alter the response to the treatment. Overall, this study shows that ABCB1 is involved in the active transport of temozolomide out of the tumor cells diminishing the response to temozolomide. Interestingly, loss of ABCB1 also affected the response to the lipophilic drug carmustine. These findings show that ABCB1 is not only relevant at the blood-brain barrier, but also in the tumor cells diminishing success of chemotherapy.

Protective role of Codiaeum variegatum against genotoxicity induced by carmustine in somatic and germ cells of male mice.

BACKGROUND: Carmustine (Cr) is an important chemotherapeutic drug, widely used in the treatment of brain tumors. Herein, the protective role of Codiaeum variegatum leaves ethyl acetate fraction was determined against genotoxicity of Cr. The technique HPLC-qTOF-MS/MS was used to identify the constituents in C. variegatum. MATERIALS: 90 male mice were used to evaluate micronuclei (MPCEs) in bone marrow, chromosomal aberration (CAs) in bone marrow and mouse spermatocytes, sperm abnormalities, and gene expression (qRT-PCR). The following groups were included, I: Negative control (ethanol 30%), II: Positive control (i.p injected once with 30 mg/kg Cr), III: Control orally treated with C. variegatum at 500 mg/kg, four days. IV-VI: treated with 100, 300, and 500 mg/kg of the plant (4 days) plus a single dose of Cr. RESULTS: In bone marrow, Cr induced significant increase in MPCEs and CAs by 3 and 7-folds respectively over the control. Cr also induced a significant percentage of CAs in spermatocytes in meiosis in the form of univalent (X-Y and autosomal univalent) and also a significant percentage of morphological sperm abnormalities was recorded. A large number of coiled tail abnormalities were detected indicating the effect of Cr in sperm motility. Cr induced an overexpression of p53 gene. C. variegatum mitigated all deleterious genotoxic effects of Cr. Chemical analysis showed that flavones (35.21%) and phenolic acids (17.62%) constitute the main components. CONCLUSIONS: The results indicated that Cr is genotoxic in both somatic and germ cells. The active components in C. variegatum together participate in the obtained protective role.

2-Deoxy-D-glucose increases the sensitivity of glioblastoma cells to BCNU through the regulation of glycolysis, ROS and ERS pathways: In vitro and in vivo validation.

Chloroethylnitrosoureas (CENUs) exert antitumor activity via producing dG-dC interstrand crosslinks (ICLs). However, tumor resistance make it necessary to find novel strategies to improve the therapeutic effect of CENUs. 2-Deoxy-D-glucose (2-DG) is a well-known glycolytic inhibitor, which can reprogram tumor energy metabolism closely related to tumor resistance. Here, we investigated the chemosensitization effect of 2-DG on l,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) against glioblastoma cells and the underlying mechanisms. We found that 2-DG significantly increased the inhibitory effects of BCNU on tumor cells compared with BCNU alone, while 2-DG showed no obvious enhancing effect on the BCNU-induced cytotoxicity for normal HaCaT and HA1800 cells. Proliferation, migration and invasion determinations presented the same trend as survival on tumor cells. 2-DG plus BCNU increased the energy deficiency through a more effective inhibition of glycolytic pathway. Notably, the combination of 2-DG and BCNU aggravated oxidative stress in glioblastoma cells, along with a significant decrease in glutathione (GSH) levels, and an increase in intracellular reactive oxygen species (ROS). Subsequently, we demonstrated that the combination treatment led to increased apoptosis via activating mitochondria and endoplasmic reticulum stress (ERS) related apoptosis pathways. Finally, we found that the dG-dC level was significantly increased after 2-DG pretreatment compared to BCNU alone by HPLC-ESI-MS/MS analysis. Finally, in vivo, 2-DG plus BCNU significantly suppressed tumor growth with lower side effects compared with BCNU alone in tumor-bearing mice. In summary, we proposed that 2-DG may have potential to increase the sensitivity of glioblastoma cells to BCNU by regulating glycolysis, ROS and ERS pathways in clinical setting.

Bendamustine-EAM versus R-BEAM after high-dose cytarabine-based induction in newly diagnosed patients with mantle cell lymphoma, a LYSA retrospective study.

Cytarabine-based immuno-chemotherapy followed by autologous stem cell transplantation (ASCT) consolidation is standard of care for fit patients with Mantle Cell Lymphoma (MCL). BEAM (Carmustine, Etoposide, Aracytine, Melphalan) is among the most frequently used conditioning regimen. Studies comparing BEAM with Bendamustine-EAM (BeEAM) have suggested that patients treated with BeEAM have a better progression-free survival (PFS). We performed a cross-study analysis to better evaluate BeEAM. Thirty-five patients from a retrospective study who received R-DHAP/BeEAM were compared to 245 patients from the LyMa trial (NCT00921414) who all received R-DHAP followed by R-BEAM. PFS and Overall Survival (OS) were estimated using Kaplan-Meier methods. At 2 years there was no difference between R-BEAM and BeEAM in either PFS (84.9% versus 87.9%; p = 0.95) or OS (91.8% versus 94.2%; p = 0.30). Analyses were repeated on a propensity score to reduce biases. Each patient from the BeEAM cohort (n = 30) was matched to three patients from the R-BEAM cohort (n = 90) for age, sex, MIPI score, pre-transplant status disease and rituximab maintenance (RM). PFS and OS at 2 years remained similar between R-BEAM and BeEAM with more renal toxicity in BeEAM group. MCL patients who received R-DHAP induction before ASCT have similar outcome after R-BEAM or BeEAM conditioning regimen.

Covalent inhibition of P. falciparum ferredoxin-NADP+ reductase: Exploring alternative strategies for the development of new antimalarial drugs.

The protozoan Plasmodium falciparum is the main aetiological agent of tropical malaria. Characteristic of the phylum is the presence of a plastid-like organelle which hosts several homologs of plant proteins, including a ferredoxin (PfFd) and its NADPH-dependent reductase (PfFNR). The PfFNR/PfFd redox system is essential for the parasite, while mammals share no homologous proteins, making the enzyme an attractive target for novel and much needed antimalarial drugs. Based on previous findings, three chemically reactive residues important for PfFNR activity were identified: namely, the active-site Cys99, responsible for hydride transfer; Cys284, whose oxidation leads to an inactive dimeric form of the protein; and His286, which is involved in NADPH binding. These amino acid residues were probed by several residue-specific reagents and the two cysteines were shown to be promising targets for covalent inhibition. The quantitative and qualitative description of the reactivity of few compounds, including a repurposed drug, set the bases for the development of more potent and specific antimalarial leads.

Effect of Superparamagnetic DMSO@γ-Fe2O3 Combined with Carmustine on Cervical Cancer.

This study aimed to investigate the effects of DMSO@γ-Fe2O3 nanomagnetic fluid thermotherapy combined with the chemotherapy drug carmustine on cervical cancer cells under a certain intensity of alternating magnetic field. And the role of Mir-590-3P in the development and progression of cervical cancer. The optimal thermotherapy concentration of γ-Fe2O3 nanomaterials on cervical cancer cells was determined by in vitro heating. In addition, the MTT colorimetric method was used to evaluate the toxic effect of γ-Fe2O3 magnetic nanoparticles on cervical cancer cells, and the optimal therapeutic concentration of carbachol on cervical cancer cells was optimized (0.015 g · L-1). The cervical cancer cells were divided into control, γ-Fe2O3 hyperthermia, chemotherapy, and DMSO@γ-Fe2O3 combined chemotherapy groups. After 2 h exposure to hypothermic conditions, flow cytometry was used to assess cell apoptosis for each group. The heating effect of the γ-Fe2O3 magnetic nanomaterials was apparent. When the concentration of γ-Fe2O3 was ≥6 g· L-1, the temperature rise above 41 °C. γ-Fe2O3 is non-toxic to cervical cancer cells and has good biocompatibility. Taking the drug concentration of IC25 as the working concentration of this study, the working concentration of carmustine was 0.015 g · L-1. Both the 41 °C heat treatment and chemotherapy alone had a killing effect on glioma and cervical cancer cells (P < 0.05). Additionally, the combined inhibitory effect of DMSO@γ-Fe2O3 nanomagnetic fluid thermotherapy and drugs at this temperature was significantly stronger than that of thermotherapy and chemotherapy alone (P < 0.05). For the control, gamma-Fe2O3 hyperthermia, chemotherapy, and DMSO@γ-Fe2O3 combined chemotherapy groups, the apoptosis rates of the cervical cancer cells were 1.4%, 18.6%, 24.12%, and 38.97%, respectively. DMSO@γ-Fe2O3 nanomagnetic fluid thermotherapy combined with the chemotherapeutic drug carmustine exerted a noticeable toxic effect on the cervical cancer cells, and DMSO@γ-Fe2O3 significantly enhanced the killing effect of carmustine on cervical cancer cells.

MGMT Epigenetics: The Influence of Gene Body Methylation and Other Insights Derived from Integrated Methylomic, Transcriptomic, and Chromatin Analyses in Various Cancer Types.

BACKGROUND: MGMT (O6-methylguanine-DNA methyltransferase) is primarily responsible for limiting the activity of some widely used chemotherapeutic agents, including temozolomide (TMZ) and carmustine (BCNU). The gene encoding this protein is epigenetically regulated, and assessment of methylation at its promoter region is used to predict glioma patients' response to TMZ. METHODS: In this report, we employed a bioinformatic approach to elucidate MGMT's epigenetic regulation. Integrated for the analysis were genome-wide methylation and transcription datasets for > 8,600 human tissue (representing 31 distinct cancer types) and 500 human cancer cell line samples. Also crucial to the interpretation of results were publicly available data from the ENCODE Project: tracks for histone modifications (via ChIP-seq) and DNase I hypersensitivity (via DNaseseq), as well as methylation and transcription data for representative cell lines (HeLa-S3, HMEC, K562). RESULTS AND DISCUSSION: We were able to validate (perhaps more comprehensively) the contrasting influences of CpG methylation at promoter region and at gene body on MGMT transcription. While the MGMT promoter is populated by CpG sites whose methylation levels displayed high negative correlation (R) with MGMT mRNA counts, the gene body harbors CpG sites exhibiting high positive R values. The promoter CpG sites with very high negative R's across cancer types include cg12981137, cg12434587, and cg00618725. Among the notable gene body CpG sites (high positive R's across cancer types) are cg00198994 (Intron 1), cg04473030 (Intron 2), and cg07367735 (Intron 4). For certain cancer types, such as melanoma, gene body methylation appears to be a better predictor of MGMT transcription (compared to promoter methylation). In general, the CpG methylation v. MGMT expression R values are higher in cell lines relative to tissues. Also, these correlations are noticeably more prominent in certain cancer types such as colorectal, adrenocortical, esophageal, skin, and head and neck cancers, as well as glioblastoma. As expected, hypomethylation at the promoter region is associated with more open chromatin, and enrichment of histone marks H3K4m1, H3K4m2, H3K4m3, and H3K9ac. CONCLUSION: Overall, our analysis illustrated the contrasting influence of promoter and gene body methylation on MGMT expression. These observations may help improve diagnostic assays for MGMT.

FKBP51 acts as a biomarker of early metastasis and is related to carmustine sensitivity in human glioma cells.

OBJECTIVE: Given that FK506 binding protein 51 (FKBP51) is upregulated in multiple cancers, we designed the present study to characterize its role as well as underlying regulatory mechanisms in glioma in the presence and absence of the chemotherapeutic carmustine (BCNU). MATERIALS AND METHODS: Through lentiviral overexpression and shRNA knockdown of FKBP51, we examined the effects on BT325 glioma cell proliferation, migration and invasion using quantitative reverse transcription PCR (qRT-PCR), CCK-8 assay, flow cytometry, and transwell assay. RESULTS: The upregulation of FKBP51 resulted in significantly decreased BT325 cell proliferation and cell viability, cell cycle arrest, reduced BCNU chemosensitivity and AKT pathway inactivation. However, FKBP51-overexpressed BT325 cells showed enhanced migration and invasion, which was supported by corresponding increase in phosphorylated IKKα (p-IKKα), MMP-2, and MMP-9 levels, as well as increased NF-κB p65 nuclear translocation. By contrast, FKBP51-suppressed BT325 cells showed excessive proliferation and BCNU resistance due to increased p-AKT activation and attenuated migration and invasion. CONCLUSIONS: We demonstrated that the effects of FKBP51 on BT325 glioma cell proliferation, migration, invasion and BCNU chemosensitization are modulated via the AKT and NF-κB pathways. Furthermore, our findings suggest the potential of FKBP51 as a prognostic glioma biomarker and an indicator of patient response to chemotherapy.

microRNA-181a mediates the chemo-sensitivity of glioblastoma to carmustine and regulates cell proliferation, migration, and apoptosis.

Glioblastoma multiform (GBM) as the most frequent and lethal brain tumor is defined by aggressive invasiveness and considerable resistance to chemotherapy. The molecular mechanisms underlying GBM tumorigenesis still needs to be further investigated. Considering that, the current study was aimed to investigate the function of miR-181a in human glioblastoma cells in combination with carmustine. U373 cell line with the low expression levels of miR-181a was selected for functional investigations. MTT assay was used to determine cell viability and Annexin V/PI and DAPI staining were employed to evaluate apoptosis induction. Also, cell migration and cell cycle progression were investigated using wound healing test and flow cytometry, respectively. qRT-PCR was used for the quantification of gene expression. MTT assay results revealed that miR-181a replacement increased the sensitivity of U373 cells to low doses of carmustine. Moreover, miR-181a was shown to increase the sub G1 cell cycle arrest and apoptosis induction by carmustine via regulating the expression of related genes including caspase-9, Bcl-2, and SIRT1. Furthermore, this miRNA combined with carmustine suppressed cell migration via downregulation of MMP-2 and Bach1 and reduced the clonogenic ability of U373 cells. Additionally, miR-181a-mediated downregulation of AKT1 implied that this miRNA could inhibit cell proliferation by modulating PI3K/AKT signaling pathway. In conclusion, the findings of this study suggest that miR-181a replacement, regarding its tumor-suppressive effects and sensitization of glioblastoma cells to carmustine, could be considered as a potential therapeutic strategy to improve the efficiency of glioblastoma chemotherapy.

3-Bromopyruvate regulates the status of glycolysis and BCNU sensitivity in human hepatocellular carcinoma cells.

Chloroethylnitrosoureas (CENUs) are bifunctional antitumor alkylating agents, which exert their antitumor activity through inducing the formation of dG-dC interstrand crosslinks (ICLs) within DNA double strand. However, the complex process of tumor biology enables tumor cells to escape the killing triggered by CENUs, as for instance with the detoxifying activity of O6-methylguanine DNA methyltransferase (MGMT) to accomplish DNA damage repair. Considering the fact that most tumor cells highly depend on aerobic glycolysis to provide energy for survival even in the presence of oxygen (Warburg effect), inhibition of aerobic glycolysis may be an attractive strategy to overcome the resistance and improve the chemotherapeutic effects of CENUs. Especially, 3-bromopyruvate (3-BrPA), a small molecule alkylating agent, has been emerged as an effective glycolytic inhibitor (energy blocker) in cancer treatment. In view of its tumor specificity and inhibition on cellular multiple targets, it is likely to reduce the chemoresistance when chemotherapeutic drugs are combined with 3-BrPA. In this study, we investigated the effects of 3-BrPA on the chemosensitivity of two human hepatocellular carcinoma (HCC) cell lines to the cytotoxic effects of l,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and the underlying molecular mechanism. The sensitivity of SMMC-7721 and HepG2 cells to BCNU was significantly increased by 2 h pretreatment with micromolar dosage of 3-BrPA. Moreover, 3-BrPA decreased the cellular ATP and GSH levels, and extracellular lactate excreted by tumor cells, and the effects were more effective when 3-BrPA was combined with BCNU. Cellular hexokinase-II (HK-II) activity was also reduced after exposure to the treatment of 3-BrPA plus BCNU. Based on the above results, the effects of 3-BrPA on the formation of dG-dC ICLs induced by BCNU was investigated by stable isotope dilution high-performance liquid chromatography electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). The results indicated that BCNU produced higher levels of dG-dC ICLs in SMMC-7721 and HepG2 cells pretreated with 3-BrPA compared to that without 3-BrPA pretreatment. Notably, in MGMT-deficient HepG2 cells, the levels of dG-dC ICLs were significantly higher than MGMT-proficient SMMC-7721 cells. In general, these findings revealed that 3-BrPA, as an effective glycolytic inhibitor, may be considered as a potential clinical chemosensitizer to optimize the therapeutic index of CENUs.

90Y-Daclizumab (Anti-CD25), High-Dose Carmustine, Etoposide, Cytarabine, and Melphalan Chemotherapy and Autologous Hematopoietic Stem Cell Transplant Yielded Sustained Complete Remissions in 4 Patients with Recurrent Hodgkin's Lymphoma.

Background: Despite advances in therapy of Hodgkin's lymphoma (HL), a proportion of patients will not respond or relapse. The authors had previously identified CD25, IL-2Rα, as a target for systemic radioimmunotherapy of HL since most normal cells do not express CD25, but it is expressed by a minority of Hodgkin/Reed-Sternberg (HRS) cells and most Tregs rosetting around HRS cells. Study Design and Treatment: This was a single institution, nonrandomized, open-label phase I/II trial of radiolabeled 90Y-daclizumab, an anti-CD25 monoclonal antibody, BEAM (carmustine, etoposide, cytarabine, and melphalan) conditioning treatment followed by autologous hematopoietic stem cell transplant (ASCT). Four patients with refractory and relapsed HL were treated in this trial with 3 patients receiving a single dose of 564.6-574.6 MBq 90Y-daclizumab and the fourth patient receiving two doses of 580.9-566.1 MBq 90Y-daclizumab followed by high-dose chemotherapy and ASCT. Results: All 4 evaluable patients treated with 90Y-daclizumab obtained complete responses (CRs) that are ongoing 4.5-7 years following their stem cell transplant. The spectrum and severity of adverse events were mild and more importantly none of the patients, including several with multiple therapies before this treatment, developed the myelodysplastic syndrome. Discussion: Targeting by daclizumab was not directed primarily at tumor cells, but rather the nonmalignant CD25-expressing T cells adjacent to the HRS cells and 90Y-daclizumab provided strong enough ß emissions to kill CD25-negative tumor cells at a distance by a crossfire effect. Furthermore, the strong ß irradiation killed normal cells in the tumor microenvironment. Conclusions: 90Y-daclizumab (anti-CD25), high-dose BEAM chemotherapy and ASCT was well tolerated and yielded sustained complete remissions in all 4 patients with recurrent HL patients who completed their treatment. Significance: Despite advances, a proportion of patients with HL will not have a CR to their initial treatment, and some with CRs will relapse. They demonstrated that the addition of 90Y-daclizumab into the preconditioning regimen for refractory and relapsed HL patients with high-dose BEAM chemotherapy and ASCT provided sustained CRs in the 4 patients studied. Two of these patients were highly refractory to multiple prior treatments with bulky disease at entry into this study, including 1 patient who never entered a remission and had failed 6 different therapeutic regimens. Despite the small number of patients treated in this study, the sustained clinical benefit in these patients indicates a highly effective treatment. The daclizumab was directed primarily not at HRS cells themselves but toward nonmalignant T cells rosetting around malignant cells. 90Y provided strong ß emissions that killed antigen nonexpressing tumor cells at a distance by a crossfire effect. Furthermore, the strong ß radiation killed normal cells in the tumor microenvironment that nurtured the malignant cells in the lymphomatous mass. The present study supports expanded analysis of 90Y-daclizumab as part of the regimen of ASCT in patients with refractory and relapsed HL.

Glycolytic inhibition by 3-bromopyruvate increases the cytotoxic effects of chloroethylnitrosoureas to human glioma cells and the DNA interstrand cross-links formation.

DNA interstrand cross-links (ICLs) are essential for the antitumor activity of chloroethylnitrosoureas (CENUs). Commonly, CENUs resistance is mainly considered to be associated with O6-methylguanine-DNA methyltransferase (MGMT) within tumors. Bypassing the MGMT-mediated resistance, to our knowledge, herein, we first utilized a novel glycolytic inhibitor, 3-bromopyruvate (3-BrPA), to increase the cytotoxic effects of l,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) to human glioma cells based on the hypothesis that blocking energy metabolism renders tumor cells more sensitive to chemotherapy. We found 3-BrPA significantly increased the cell killing by BCNU in human glioma SF763 and SF126 cell lines. Significantly decreased levels of extracellular lactate, cellular ATP and glutathione (GSH) were observed after 3-BrPA treatment, and the effects were more remarkable with 3-BrPA in combination with BCNU. Considering that the role of ATP and GSH in drug efflux, DNA damage repair and drug inactivation, we determined the effect of 3-BrPA on the formation of dG-dC ICLs induced by BCNU using stable isotope dilution high-performance liquid chromatography electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). As expected, the levels of lethal dG-dC ICLs induced by BCNU were obviously enhanced after 3-BrPA pretreatment. Based on these results, 3-BrPA and related glycolytic inhibitors may be promising to enhance the cell killing effect and reverse the clinical chemoresistance of CENUs and related antitumor agents.

A novel strategy to the formulation of carmustine and bioactive nanoparticles co-loaded PLGA biocomposite spheres for targeting drug delivery to glioma treatment and nursing care.

Gliomas are the most common brain tumors in humans. Different chemotherapeutics are available to treat gliomas. However, they are costly and pose numerous side effects. The development of nanocomposite based on chemotherapeutic drug and metallic nanoparticle loaded with polymer could be highly useful against glioma. In this study, carmustine loaded with gold NPs and linked with PLGA-PSPE was produced as a bio-nanocomposite and its efficacy in treating glioma and burn wound were investigated. The synthesized biocomposite was characterized by biophysical techniques. It was observed that the synthesized composite was hexagonal and crystalline nature. TGA analysis showed that the particle had good combustible property. Interestingly, the Cm-Au-PLGA-PSPE composite had exhibited remarkable anti-tumor property against U251 human glioma. The flow cytometry showed a greater increase in the apoptosis rate (62.31%) of glioma cells exposed to the bio-nanocomposite. In addition, a greater reduction in the viability of U251 cells was recorded following treatment with Cm-Au-PLGA-PSPE composite. Quick healing of the heart, liver, spleen, lung and kidney tissue wounds in mouse was noticed with Cm-Au-PLGA-PSPE composite treatment. This study concludes that the newly produced Cm-Au-PLGA-PSPE composite would be a promising alternative in treating human gliomas and associated wounds with increased biomedical applications.

Microcolony Size Distribution Assay Enables High-Throughput Cell Survival Quantitation.

Cell survival is a critical and ubiquitous endpoint in biology. The broadly accepted colony formation assay (CFA) directly measures a cell's ability to divide; however, it takes weeks to perform and is incompatible with high-throughput screening (HTS) technologies. Here, we describe the MicroColonyChip, which exploits microwell array technology to create an array of colonies. Unlike the CFA, where visible colonies are counted by eye, using fluorescence microscopy, microcolonies can be analyzed in days rather than weeks. Using automated analysis of microcolony size distributions, the MicroColonyChip achieves comparable sensitivity to the CFA (and greater sensitivity than the 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide [XTT] assay). Compared to CellTiter-Glo, the MicroColonyChip is as sensitive and also robust to artifacts caused by differences in initial cell seeding density. We demonstrate efficacy via studies of radiosensitivity and chemosensitivity and show that the approach is amenable to multiplexing. We conclude that the MicroColonyChip is a rapid and automated alternative for cell survival quantitation.

BEAM versus BUCYVP16 Conditioning before Autologous Hematopoietic Stem Cell Transplant in Patients with Hodgkin Lymphoma.

High-dose chemotherapy followed by autologous hematopoietic stem cell transplant (AHSCT) is a standard of care for patients with relapsed Hodgkin lymphoma. Different conditioning regimens before AHSCT have been used, with the 2 most common being BEAM (carmustine, etoposide, cytarabine, and melphalan) and BUCYVP16 (busulfan, cyclophosphamide, and etoposide). We retrospectively compared the outcomes of patients treated with BEAM (n = 128) or BUCYVP16 (n = 105) followed by AHSCT. After a median follow-up of 4.2 years for BEAM and 3.8 for BUCYVP16 from AHSCT, the 5-year cumulative incidence of relapse was 29% with BEAM compared with 56% with BUCYVP16 (P < .001). Median progression free survival (PFS) and overall survival (OS) were not reached with BEAM and were 2.0 and 7.8 years with BUCYVP16, respectively. Improved PFS (P < .001) and OS (P = .001) were observed with BEAM for patients who needed transplant within 24 months from diagnosis and for patients not in complete remission (non-CR; P = .001 and P < .001, respectively) at AHSCT. In this large retrospective comparison the use of BEAM conditioning before AHSCT resulted in a statistically significant improved PFS and OS and lower relapse compared with BUCYVP16. This supports the use of BEAM as a frontline conditioning regimen before AHSCT for early relapsed and non-CR Hodgkin lymphoma.