phenanthrenes and Precursor-Cell-Lymphoblastic-Leukemia-Lymphoma

Cryptotanshinone, a well-known diterpene quinone from a widely used traditional Chinese herb named Salvia miltiorrhiza, has been reported for its therapeutical potentials on diverse activities. In this study, pharmacological effects of cryptotanshinone on acute lymphoblastic leukemia cells were investigated. IC50 values of 5.0 and 4.8 were obtained in CEM/ADR5000 and CCRF-CEM. Microarray-based mRNA expression revealed that cryptotanshinone regulated genes associated with cell cycle, DNA damage, reactive oxygen species (ROS), NFκB signaling and cellular movement. The involvement of these pathways in the mode of action of cryptotanshinone was subsequently validated by additional independent in vitro studies. Cryptotanshinone stimulated ROS generation and induced DNA damage. It arrested cells in G2/M phase of the cell cycle and induced apoptosis as measured by annexin V-FITC-conjugating fluorescence. The induction of the intrinsic apoptotic pathway by cryptotanshinone was proved by loss of mitochondrial membrane potential and increased cleavage of caspase 3/7, caspase 9 and poly ADP ribose polymerase (PARP). DNA-binding motif analysis of the microarray-retrieved deregulated genes in the promoter region revealed NFκB as potential transcription factor involved in cryptotanshinone's mode of action. Molecular docking and Western blotting provided supportive evidence, suggesting that cryptotanshinone binds to IKK-β and inhibits the translocation of p65 from the cytosol to the nucleus. In addition, cryptotanshinone inhibited cellular movement as shown by a fibronectin-based cellular adhesion assay, indicating that this compound exerts anti-invasive features. In conclusion, cryptotanshinone exerts profound cytotoxicity, which is caused by multispecific modes of actions, including G2/M arrest, apoptosis and inhibition of cellular movement. The inhibitory activities of this compound may be explained by inhibition of NFκB, which orchestrates all these mechanisms.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Apoptosis Regulatory Proteins; Cell Adhesion; Cell Cycle Proteins; Cell Line, Tumor; Cell Movement; Cell Survival; DNA Damage; Dose-Response Relationship, Drug; G2 Phase Cell Cycle Checkpoints; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Inhibitory Concentration 50; Membrane Potential, Mitochondrial; Molecular Docking Simulation; NF-kappa B; Oligonucleotide Array Sequence Analysis; Phenanthrenes; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Reactive Oxygen Species; Signal Transduction

Unfolded protein responses (UPR) determine cell fate and are recognized as anticancer targets. In a previous research, we reported that cryptotanshinone (CPT) exerted cytotoxic effects toward acute lymphoblastic leukemia cells through mitochondria-mediated apoptosis.. In the present study, we further investigated the role of UPR in CPT-induced cytotoxicity on acute lymphoblastic leukemia cells by applying tools of pharmacogenomics and bioinformatics.. Gene expression profiling was performed by mRNA microarray hybridization. Potential transcription factor binding motifs were identified in the promoter regions of the deregulated genes by Cistrome software. Molecular docking on eIF-4A and PI3K was performed to investigate the inhibitory activity of CPT on translation initiation.. CPT regulated genes related to UPR and eIF2 signaling pathways. The DNA-Damage-Inducible Transcript 3 (DDIT3) gene, which is activated as consequence of UPR malfunction during apoptosis, was induced and validated by in vitro experiments. Transcription factor binding motif analysis of the microarrary-retrieved deregulated genes in the promoter region emphasized the relevance of transcription factors, such as ATF2, ATF4 and XBP1, regulating UPR and cell apoptosis. Molecular docking suggested inhibitory effects of CPT by binding to eIF-4A and PI3K providing evidence for a role of CPT's in the disruption of protein synthesis.. CPT triggered UPR and inhibited protein synthesis via eIF-mediated translation initiation, potentially supporting CPT-induced cytotoxic effects toward acute leukemia cells.

Topics: Apoptosis; Cell Line, Tumor; Computational Biology; Eukaryotic Initiation Factor-2; Eukaryotic Initiation Factor-4A; Eukaryotic Initiation Factors; Humans; Molecular Docking Simulation; Pharmacogenetics; Phenanthrenes; Phosphatidylinositol 3-Kinases; Precursor Cell Lymphoblastic Leukemia-Lymphoma; RNA, Messenger; Signal Transduction; Transcription Factor CHOP; Transcription Factors; Unfolded Protein Response

Chemoresistance represents a major challenge for treatment of acute lymphoblastic leukemia (ALL). Thus, new drugs to overcome chemoresistance in ALL are urgently needed. To this end, we established a cytarabine (araC)-resistant ALL cell line (NALM-6/R), which interestingly displayed cross-resistance towards doxorubicin (ADM). Here we report that low dose of triptolide (TPL), a natural product used for treating inflammatory diseases such as arthritis, could reverse araC and ADM resistance and in NALM-6/R cells as well as primary cells from patients with relapsed or refractory (R/R) ALL, reflected by inhibition of cell proliferation and induction of apoptosis in vitro, and repression of tumor growth in vivo in a mouse xenograft model. Mechanistically, these events were associated with impaired mitochondrial membrane potential and increased reactive oxygen species (ROS) production. Co-treatment with TPL and araC or ADM upregulated pro-apoptotic caspase-9 protein, inhibited checkpoint kinase 1 (Chk1) and 2 (Chk2) phosphorylation, and induced γH2A.X (a DNA damage marker). Notably, the combination regimen of TPL and conventional chemotherapeutics also rapidly diminished tumor burden in a patient with R/R ALL. Together, these findings provide preclinical evidence for repurposing use of TPL in combination with chemotherapeutic agents to treat R/R ALL as an alternative salvage regimen.

Topics: Adolescent; Adult; Aged; Animals; Antimetabolites, Antineoplastic; Antineoplastic Agents, Alkylating; Apoptosis; Caspase 9; Cell Line, Tumor; Cell Proliferation; Checkpoint Kinase 1; Checkpoint Kinase 2; Cytarabine; Diterpenes; DNA Damage; Dose-Response Relationship, Drug; Doxorubicin; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Epoxy Compounds; Female; Histones; Humans; Inhibitory Concentration 50; Male; Membrane Potential, Mitochondrial; Mice, Inbred NOD; Mice, SCID; Middle Aged; Oxidative Stress; Phenanthrenes; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Reactive Oxygen Species; Signal Transduction; Tumor Cells, Cultured; Xenograft Model Antitumor Assays; Young Adult

Miltirone (1) is a diterpene quinone extracted from a well-known Chinese traditional herb (Salvia miltiorrhiza). We investigated the cytotoxic effects of miltirone toward sensitive and multidrug-resistant acute lymphoblastic leukemia cell lines. Miltirone inhibited multidrug-resistant P-glycoprotein (P-gp)-overexpressing CEM/ADR5000 cells better than drug-sensitive CCRF-CEM wild-type cells, a phenomenon termed collateral sensitivity. Flow cytometric analyses revealed that miltirone induced G2/M arrest and apoptosis. Furthermore, miltirone stimulated reactive oxygen species (ROS) generation and mitochondrial membrane potential (MMP) disruption, which in turn induced DNA damage and activation of caspases and poly ADP-ribose polymerase (PARP). Downregulation of CCNB1 (cyclin B1) and CDC2 mRNA and upregulation of CDKN1A (p21) mRNA were in accord with miltirone-induced G2/M arrest. Moreover, miltirone decreased cell adherence to fibronectin. Molecular docking revealed that miltirone bound to the ATP-binding site of IKK-β. In conclusion, miltirone was collateral sensitive in multidrug-resistant P-gp-overexpressing cells, induced G2/M arrest, and triggered apoptosis via ROS-generated breakdown of MMP and DNA damage. Therefore, miltirone may be a promising candidate for cancer chemotherapy.

Topics: Apoptosis; ATP Binding Cassette Transporter, Subfamily B; Caspases; Cyclin-Dependent Kinase Inhibitor p21; G2 Phase Cell Cycle Checkpoints; Humans; Membrane Potential, Mitochondrial; Molecular Structure; Phenanthrenes; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Reactive Oxygen Species; Salvia miltiorrhiza

Triptolide, a natural product derived from the Chinese plant Tripterygium wilfordii, is reported to exhibit antitumor effects in a broad range of cancers. The antitumor activity of triptolide is associated with its biologic activities, as it inhibits various proproliferative or antiapoptotic factors that are dominantly expressed in given types of cancer cells. Herein, we show that triptolide induced apoptosis in a subgroup of acute lymphoblastic leukemia (ALL) cells overexpressing the MDM2 oncoprotein by inhibiting MDM2 expression. More specifically, we found that triptolide inhibited MDM2 at the transcriptional level by suppressing its mRNA synthesis. This MDM2 inhibition led in turn to increased levels of p53 protein; however, p53 functionality was not activated due to the fact that triptolide-treated cells lacked induction of p21 and PUMA as well as in G(1) cell-cycle arrest. Triptolide-mediated downregulation of MDM2 increased inhibition of X-linked inhibitor of apoptosis protein (XIAP), its translational target, in a manner distinct from reactions to cellular stress and DNA-damaging agent ionizing radiation that induce XIAP due to p53-activated MDM2. These results suggest that increased inhibition of XIAP due to downregulation of MDM2 may play a critical role in triptolide-induced apoptosis in MDM2-overexpressing cancers.

Topics: Antineoplastic Agents, Alkylating; Apoptosis; Child; Diterpenes; Epoxy Compounds; Humans; Phenanthrenes; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Promoter Regions, Genetic; Proto-Oncogene Proteins c-mdm2; Signal Transduction; Transfection; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Anticancer therapies that target single signal transduction pathways often fail to prevent proliferation of cancer cells because of overlapping functions and cross-talk between different signaling pathways. Recent research has identified that balanced multi-component therapies might be more efficacious than highly specific single component therapies in certain cases. Ideally, synergistic combinations can provide 1) increased efficacy of the therapeutic effect 2) reduced toxicity as a result of decreased dosage providing equivalent or increased efficacy 3) the avoidance or delayed onset of drug resistance. Therefore, the interest in combinatorial drug discovery based on systems-oriented approaches has been increasing steadily in recent years.. Here we describe the development of Combinatorial Drug Assembler (CDA), a genomics and bioinformatics system, whereby using gene expression profiling, multiple signaling pathways are targeted for combinatorial drug discovery. CDA performs expression pattern matching of signaling pathway components to compare genes expressed in an input cell line (or patient sample data), with expression patterns in cell lines treated with different small molecules. Then it detects best pattern matching combinatorial drug pairs across the input gene set-related signaling pathways to detect where gene expression patterns overlap and those predicted drug pairs could likely be applied as combination therapy. We carried out in vitro validations on non-small cell lung cancer cells and triple-negative breast cancer (TNBC) cells. We found two combinatorial drug pairs that showed synergistic effect on lung cancer cells. Furthermore, we also observed that halofantrine and vinblastine were synergistic on TNBC cells.. CDA provides a new way for rational drug combination. Together with phExplorer, CDA also provides functional insights into combinatorial drugs. CDA is freely available at http://cda.i-pharm.org.

Topics: Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Combinatorial Chemistry Techniques; Computational Biology; Drug Discovery; Drug Resistance; Female; Gene Expression Profiling; Gene Expression Regulation; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Models, Statistical; Phenanthrenes; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Signal Transduction; Transcription, Genetic; Vinblastine

Drug-induced cell death can occur as a result of DNA damage, which in turn may lead to the reduction of bcl-2 expression and activation of caspase-3 expression. In the present study, we investigated the effect of armepavines and atherosperminine on the cell survival rate and expression of bcl-2 and caspase-3 in CCRF-CEM cells. Our data have revealed that armepavine oxalate reduced the survival rate of CCRF-CEM cells in a dose- and time-dependent manner by MTT assay. However, no significant effects of armepavine MeI and atherosperminine N-oxide on the survival rate of the CCRF-CEM cell were observed. Armepavine oxalate-induced cell death was considered to be apoptotic on the basis of observed formation of the DNA ladder and the typical apoptotic morphological change by Hoechst 33258 staining. The expression of bcl-2 protein in CCRF-CEM cells treated with 30 microM armepavine oxalate was significantly decreased in western blotting analysis. In contrast, the expression of active caspase-3 in the cells was increased by armepavine oxalate in a dose-dependent manner. These findings indicate the involvement of bcl-2 and caspase-3 in the apoptotic process of CCRF-CEM cells induced by armepavine oxalate. The increased expression of active caspase-3 as well as decreased expression of bcl-2 support the assumption the armepavine oxalate-treated cells may be capable to complete the entire apoptotic process ending in cell fragmentation.

Topics: Alkaloids; Apoptosis; Benzylisoquinolines; Caspase 3; Caspases; Cell Line, Tumor; Cell Survival; DNA Fragmentation; Dose-Response Relationship, Drug; Humans; Oxalates; Phenanthrenes; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Proto-Oncogene Proteins c-bcl-2

We investigated the early events involved in loss of mitochondrial membrane potential (DeltaPsi(mt)) leading to apoptosis in cells derived from patients with acute lymphocytic leukemia after exposure to phenolic antioxidants. Using the nitric oxide binding dye diaminofluorescein-FM diacetate, we found that intracellular nitric oxide (NO) levels increased significantly within 4h after exposure to the antioxidants curcumin, carnosol, and quercetin. Inhibition of nitric oxide synthetase (NOS) activity with mercaptoethylguanidine increased the percentage of leukemia cells with depolarized mitochondria membranes after antioxidant treatment. These data suggest that NO production in the leukemia-derived cells may be a protective response to maintain DeltaPsi(mt) after antioxidant exposure and inhibition of NOS increases the disruption of mitochondrial homeostasis induced by the antioxidants.

Topics: Abietanes; Antioxidants; Curcumin; Enzyme Inhibitors; Guanidines; Humans; Intracellular Membranes; Membrane Potentials; Mitochondria; Nitric Oxide; Nitric Oxide Synthase; Phenanthrenes; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Quercetin; Tumor Cells, Cultured