phenanthrenes and Leukemia--Myeloid--Acute

In this work, a series of water-soluble triptolide prodrugs were synthesized, and their triptolide release rate, pharmacokinetic characteristics and anti-tumor effect were measured. We found that inserting glycolic acid as a linker between triptolide and the cyclic amino acid accelerated the release of triptolide from prodrugs into the plasma while preserving its safety. Among them, prodrug TP-P1 was significantly better than Minnelide (the only water-soluble triptolide prodrug in clinical trials) in terms of release rate in plasma and synthetic yield. In mouse models of human acute myeloid leukemia (AML), TP-P1 was effective in reducing xenograft tumors at dose levels as low as 25 μg/kg, and eliminating tumors at dose 100 μg/kg. Furthermore, TP-P1 could significantly enhance the efficacy of FLT3 inhibitors in the treatment of AML. These experimental results showed the potential of TP-P1 as water-soluble prodrugs of triptolide.

Topics: Animals; Diterpenes; Epoxy Compounds; Humans; Leukemia, Myeloid, Acute; Mice; Phenanthrenes; Prodrugs; Water

Leukemia stem cells (LSCs) are considered to be the root of relapse for acute myeloid leukemia (AML). Conventional chemotherapeutic drugs fail to eliminate LSCs. Therefore, new therapeutic strategies eliminating LSCs are urgently needed. Our results showed that low-dose Triptolide (TPL) enhanced the anti-AML activity of Idarubicin (IDA) in vitro against LSC-like cells (CD34 + CD38- KG1αand CD34 + CD38- kasumi-1 cells) and CD34+ primary AML cells, while sparing normal cells. Inspiringly, the combination treatment with low-dose TPL and IDA was also effective against CD34 + blasts from AML patients with FLT3-ITD mutation, which is an unfavorable risk factor for AML patients. Moreover, the combination of TPL and IDA induced a remarkable suppression of human leukemia growth in a xenograft mouse model. Mechanistically, the enhanced effect of low dose TPL on IDA against LSCs was attributed to inhibiting DNA damage repair response. Thus, our study may provide a theoretical basis to facilitate the development of a novel LSCs-targeting strategy for AML.Graphical abstract.

Topics: Animals; Diterpenes; DNA Damage; DNA Repair; Drug Synergism; Epoxy Compounds; Humans; Idarubicin; Leukemia, Myeloid, Acute; Mice; Neoplastic Stem Cells; Phenanthrenes

The bromodomain and extra-terminal (BET) domain inhibitor JQ1 exerts potent anticancer activity in various cancer cells. However, the resistance to BET inhibitors in leukemia stem cells limits its implication in acute myeloid leukemia (AML). High concentration of triptolide (TPL) presents anticancer activities but with adverse effects. Here, we investigated whether the combination of low-dose TPL with JQ1 could help to circumvent the dilemma of drug resistance and side effect in treating AML. AML cell lines, primary cells from 10 AML patients with different status, as well as AML mice model were subjected to different treatments and apoptotic related protein expression were evaluated. Data showed that low-dose TPL combined with JQ1 effectively killed AML cell lines and primary cells from AML patients without exerting significantly greater lethal activity against normal cells. Mechanism study revealed that low-dose TPL combined with JQ1 triggered reactive oxygen species production and induced mitochondrial-mediated apoptosis in AML cells, in which the inhibition of RNA polymerase II to downregulate c-Myc was mainly responsible for the enhanced activity of TPL in combination with JQ1. In vivo study presented that cotreatment with low-dose TPL and JQ1 significantly reduced tumor burden of the NOD/SCID mice engrafted with MOLM-13 cells. In conclusion, low-dose TPL enhanced the antitumor effect of JQ1 on AML without increasing the side effects, supporting a potential option for AML treatment.

Topics: Adult; Animals; Antineoplastic Agents, Alkylating; Apoptosis; Azepines; Biomarkers, Tumor; Cell Proliferation; Diterpenes; Drug Resistance, Neoplasm; Epoxy Compounds; Female; Gene Expression Regulation, Enzymologic; Humans; In Vitro Techniques; Leukemia, Myeloid, Acute; Male; Mice; Mice, Inbred BALB C; Mice, Inbred NOD; Mice, Nude; Mice, SCID; Middle Aged; Neoplastic Stem Cells; Phenanthrenes; Prognosis; Reactive Oxygen Species; RNA Polymerase II; Signal Transduction; Triazoles; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Bcl-2 inhibitors display an effective activity in acute myeloid leukemia (AML), but its clinical efficacy as a monotherapy was limited in part owing to failure to target other antiapoptotic Bcl-2 family proteins, such as Mcl-1. In this context, the combination strategy may be a promising approach to overcome this barrier. Here, we report the preclinical efficacy of a novel strategy combining ABT-199 with triptolide (TPL), a natural product extracted from a traditional Chinese medicine, in AML. Combination treatment exhibited markedly increased cytotoxicity in leukemic cells irrespective of p53 status while largely sparing normal cells of the hematopoietic lineage. Moreover, co-administration of ABT-199 with TPL dramatically suppressed leukemia progression as well as prolonged animal survival in a xenograft AML model. The potentiated effect of ABT-199 and TPL against AML was associated with activation of the mitochondrum-related intrinsic apoptotic pathway through a mechanism reciprocally modulating Bcl-2 family proteins. In this case, TPL not only downregulated Mcl-1 but also upregulated proapoptotic BH3-only proteins, thereby overcoming the resistance toward ABT-199. Conversely, ABT-199 abrogated Bcl-2-mediated cytoprotection against TPL. Together, these findings suggest that the regimen combining TPL and ABT-199 might be active against AML by inducing robust apoptosis through reciprocal regulation of anti- and proapoptotic Bcl-2 family proteins, therefore providing a strong rationale for the clinical investigation of this combination regimen for the treatment of AML.

Topics: Adolescent; Adult; Aged; Animals; Apoptosis; Blast Crisis; Bridged Bicyclo Compounds, Heterocyclic; Cell Line, Tumor; Child; Diterpenes; Drug Synergism; Epoxy Compounds; Female; Humans; Leukemia, Myeloid, Acute; Male; Mice, Inbred BALB C; Mice, Nude; Middle Aged; Mitochondria; Phenanthrenes; Proto-Oncogene Proteins c-bcl-2; Sulfonamides; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays

To investigate the effects of inhibiting proliferation and inducing apoptosis of low-dose triptolide and sorafenib alone or in combination on FLT3-ITD. The MV4-11 cells were treated with low dose triptolide(IC. The treatment of MV4-11 cells with low dose triptolide and sorafenib alone and in combination for 48 hours could inhibit cell proliferation and induce cell apoptosis, moreover the inhibitory rate and apoptotic rate of MV4-11 cells in drug-combination group both were higher than those in single drug group. The mRNA expression and protein expression of FLT3,STAT5 signaling pathway in drug combination group were significantly lower than those in single drug group.. Low-dose triptolide combined with sorafenib can synergistically inhibit the proliferation and induce the apoptosis of MV4-11 cells, which may be related with the inhibition of FLT3 and STAT5 pathway.. 低剂量雷公藤内酯醇和索拉非尼单药及其联用对AML细胞株MV411及其STAT5信号通路的作用.. 探讨低剂量雷公藤内酯醇、索拉非尼单药及二者联合对FLT3-ITD突变阳性人急性髓系白血病细胞株 MV4-11 细胞的抑制增殖和诱导凋亡的作用，以及对STAT5通路的影响.. 采用低剂量雷公藤内酯醇 (IC. 雷公藤内酯醇、索拉非尼单药及二者联合用药48 h对MV4-11细胞株有抑制增殖和诱导凋亡的作用，联合用药组抑制率及凋亡率均高于单药组，联合用药组FLT3、STAT5通路基因及蛋白的表达显著低于单药组.. 低剂量雷公藤内酯醇联合索拉非尼可协同抑制MV4-11细胞的增殖及诱导其凋亡，其机制可能与抑制FLT3及STAT5 通路有关.

Topics: Apoptosis; Cell Line, Tumor; Diterpenes; Epoxy Compounds; fms-Like Tyrosine Kinase 3; Humans; Leukemia, Myeloid, Acute; Phenanthrenes; Sorafenib; STAT5 Transcription Factor

There is an urgent need for novel and effective treatment options for acute myeloid leukemia (AML). Triptolide, a diterpenoid tri-epoxide compound isolated from the herb Tripterygium wilfordii and its water-soluble pro-drug-Minnelide have shown promising anti-cancer activity. A recent clinical trial for patients with solid tumors confirmed the safety and efficacy at biologically equivalent doses of 0.2 mg/kg/day and lower.. Cell viability of multiple AML cell lines as well as patient apheresis samples were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) based assay. Apoptosis was evaluated by estimating the amount of cleaved caspase. AML cell line (THP1-Luc) was implanted in immunocompromised mice and treated with indicated doses of Minnelide. Leukemic burden before and after treatment was evaluated by imaging in an In Vivo Imaging System (IVIS).. In the current study, we show that Minnelide, at doses below maximum tolerated dose (MTD) demonstrates leukemic clearance of both primary AML blasts and luciferase expressing THP-1 cells in mice. In vitro, multiple primary AML apheresis samples and AML cell lines (THP-1, KG1, Kasumi-1, HL-60) were sensitive to triptolide mediated cell death and apoptosis in low doses. Treatment with triptolide led to a significant decrease in the colony forming ability of AML cell lines as well as in the expression of stem cell markers. Additionally, it resulted in the cell cycle arrest in the G1/S phase with significant downregulation of c-Myc, a major transcriptional regulator mediating cancer cell growth and stemness.. Our results suggest that Minnelide, with confirmed safety and activity in the clinic, exerts a potent anti-leukemic effect in multiple models of AML at doses easily achievable in patients.

Topics: Animals; Apoptosis; Biomarkers, Tumor; Cell Cycle Checkpoints; Cell Line, Tumor; Disease Models, Animal; Disease Progression; Diterpenes; Down-Regulation; Drug Evaluation, Preclinical; Epoxy Compounds; Humans; Leukemia, Myeloid, Acute; Mice; Neoplastic Stem Cells; Organophosphates; Phenanthrenes; Proto-Oncogene Proteins c-myc; Tumor Burden; Tumor Stem Cell Assay

Identifying and targeting oncogenic fusion genes have revolutionized the treatment of leukemia, such as PML-RARα fusion gene in acute promyelocytic leukemia. Here we identified an intrachromosomal fusion gene located on chromosome 19q.13 between UBA2 and WTIP gene in a case of acute myeloid leukemia. The UBA2-WTIP fusion gene contains the N-terminal E1_enzyme_family, VAE_Ubl domains of UBA2, and the C-terminal LIM domains of WTIP. The UBA2-WTIP fusion was detected by reverse transcriptase polymerase chain reaction and Sanger sequencing in 19 of 56 acute myeloid leukemia samples (33.9%). Ectopic expression of the UBA2-WTIP fusion in human acute myeloid leukemia KG-1a cells showed enhanced cell proliferation both in vitro and in vivo. The UBA2-WTIP fusion induced phosphorylation of STAT3, STAT5 and ERK1/2, and abrogates WTIP-mediated mammalian processing body formation. Finally, triptolide displayed selective cytotoxicity against KG-1a cells harboring the UBA2-WTIP fusion. Collectively, our findings suggest that the UBA2-WTIP fusion is an oncogenic fusion gene, as well as a promising therapeutic target for the treatment of acute myeloid leukemia.

Topics: Animals; Antineoplastic Agents, Alkylating; Apoptosis; Carrier Proteins; Cell Line, Tumor; Cell Proliferation; Cell Survival; Co-Repressor Proteins; Cytoskeletal Proteins; Diterpenes; Epoxy Compounds; Gene Expression Regulation, Leukemic; Humans; Leukemia, Myeloid, Acute; Leukocytes; Mice; Mice, Inbred NOD; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Oncogene Proteins, Fusion; Phenanthrenes; Protein Domains; RNA, Messenger; STAT3 Transcription Factor; STAT5 Transcription Factor; Survival Analysis; Ubiquitin-Activating Enzymes; Xenograft Model Antitumor Assays

Leukemia stem cells (LSCs) are considered to be the main reason for relapse and are also regarded as a major hurdle for the success of acute myeloid leukemia chemotherapy. Thus, new drugs targeting LSCs are urgently needed. Triptolide (TPL) is cytotoxic to LSCs. Low dose of TPL enhances the cytotoxicity of idarubicin (IDA) in LSCs. In this study, the ability of TPL to induce apoptosis in leukemic stem cell (LSC)-like cells derived from acute myeloid leukemia cell line KG1a was investigated. LSC-like cells sorted from KG1a were subjected to cell cycle analysis and different treatments, and then followed by in vitro methyl thiazole tetrazolium bromide cytotoxicity assay. The effects of different drug combinations on cell viability, intracellular reactive-oxygen species (ROS) activity, colony-forming ability and apoptotic status were also examined. Combination index-isobologram analysis indicates a synergistic effect between TPL and IDA, which inhibits the colony-forming ability of LSC-like cells and induces their apoptosis. We further investigated the expression of Nrf2, HIF-1α and their downstream target genes. LSC-like cells treated with both TPL and IDA have increased levels of ROS, decreased expression of Nrf2 and HIF-1α pathways. Our findings indicate that the synergistic cytotoxicity of TPL and IDA in LSCs-like cells may attribute to both induction of ROS and inhibition of the Nrf2 and HIF-1α pathways.

Topics: Apoptosis; Blotting, Western; Cell Cycle; Cell Line; Cell Survival; Diterpenes; Epoxy Compounds; Flow Cytometry; Humans; Idarubicin; Immunophenotyping; Leukemia, Myeloid, Acute; Neoplastic Stem Cells; NF-E2-Related Factor 2; Phenanthrenes; Reactive Oxygen Species; Real-Time Polymerase Chain Reaction

Triptolide, isolated from the herb Tripterygium wilfordii, has been shown to potently induce apoptosis in various malignant cells by inhibiting RNA synthesis and nuclear factor-κB activity. Previously, we showed that triptolide promotes apoptosis in acute myeloid leukemia (AML) cells via the mitochondria-mediated pathway, in part, by decreasing levels of the anti-apoptotic proteins XIAP and Mcl-1. MRx102 is a triptolide derivative, currently in preclinical development. Here we show that MRx102 potently promoted apoptosis in AML cell lines, with EC(50) values of 14.5±0.6 nM and 37.0±0.9 nM at 48 h for OCI-AML3 and MV4-11 cells, respectively. MRx102, at low nanomolar concentrations, also induced apoptosis in bulk, CD34(+) progenitor, and more importantly, CD34(+)CD38(-) stem/progenitor cells from AML patients, even when they were protected by coculture with bone marrow derived mesenchymal stromal cells. MRx102 decreased XIAP and Mcl-1 protein levels and inhibited RNA synthesis in OCI-AML3 cells. In vivo, MRx102 greatly decreased leukemia burden and increased survival time in non-obese diabetic/severe combined immunodeficiency mice harboring Ba/F3-ITD cells. Collectively, we demonstrated that MRx102 has potent antileukemic activity both in vitro and in vivo, has the potential to eliminate AML stem/progenitor cells and overcome microenvironmental protection of leukemic cells, and warrants clinical investigation.

Topics: Animals; Antineoplastic Agents, Alkylating; Apoptosis; Bone Marrow; Cell Line, Tumor; Disease Models, Animal; Female; Humans; Leukemia, Myeloid, Acute; Male; Mice; Mice, Inbred NOD; Mice, SCID; Myeloid Cell Leukemia Sequence 1 Protein; Neoplastic Stem Cells; Phenanthrenes; Proto-Oncogene Proteins c-bcl-2; Transcription, Genetic; Tumor Microenvironment; X-Linked Inhibitor of Apoptosis Protein; Xenograft Model Antitumor Assays

Triptolide is a compound isolated from the traditional Chinese medicinal herb Tripterygium wilfordii that shows potent anti-tumor activities, but its effects on acute myeloid leukemia with t(8;21) remain unclear. Here we report that triptolide inhibits cell proliferation and induces apoptosis in a dose- and time-dependent manner of t(8;21)-bearing Kasumi-1, SKNO-1 and CD34+ cells harvested from bone marrow samples of patients with t(8;21) leukemia. We show that triptolide triggers cleavage of the resultant AML1-ETO fusion protein of t(8;21), and causes downregulation of C-KIT followed by inhibition of JAK-STAT signaling. Triptolide downregulates p65 and inhibits the DNA-binding activity of NF-κB. Our data indicate that triptolide might be an effective agent for t(8;21) leukemia.

Topics: Antineoplastic Agents, Alkylating; Apoptosis; Blotting, Western; Cell Proliferation; Cells, Cultured; Chromosomes, Human, Pair 21; Chromosomes, Human, Pair 8; Core Binding Factor Alpha 2 Subunit; Diterpenes; Epoxy Compounds; Humans; Janus Kinases; Leukemia, Myeloid, Acute; Oncogene Proteins, Fusion; Phenanthrenes; Proto-Oncogene Proteins c-kit; Reverse Transcriptase Polymerase Chain Reaction; RUNX1 Translocation Partner 1 Protein; Signal Transduction; STAT Transcription Factors

Triptolide has shown antitumor activity in a broad range of solid tumors and on leukemic cells in vitro.. The THP1 cell line and primary acute myeloid leukemia (AML) cells were cultured with triptolide alone or in association with AraC or idarubicin in increasing concentrations. Apoptosis was measured by flow cytometry using DiOC6(3) for the cell line and fluorescein isothiocyanateAnnexin-V and CD45 labeling for fresh blast cells. Protein expression was measured by Western blot. Cell cycle distribution of apoptotic cells was measured by flow cytometry.. A synergistic effect was observed when triptolide was added to idarubicin or to AraC to induce apoptosis of THP-1 leukemic cells. The triptolide/AraC association was also investigated in vitro on primary blast cells from 25 AML patients. This combination induced significantly higher percentages of apoptosis vs treatment with each drug separately (p<0.005). The IkappaB and X-linked inhibitor of apoptosis protein contents, which were altered by triptolide in idarubicin-treated cells, were not modified in AraC-treated cells. The association of AraC with triptolide increased the number of cells blocked in the S phase and most underwent apoptosis.. These results suggest that, by modifying the cell cycle kinetics, AraC sensitizes AML cells to apoptosis induced by low concentration triptolide. The in vitro proapoptotic effect of triptolide associated with the antiproliferative activity of AraC warrants further clinical investigation for treatment of AML patients, especially elderly patients for whom low-dose AraC treatment could be improved by the addition of triptolide.

Topics: Annexin A5; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Cell Line, Tumor; Cytarabine; Diterpenes; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Drug Synergism; Epoxy Compounds; Humans; I-kappa B Proteins; Idarubicin; Leukemia, Myeloid, Acute; Leukocyte Common Antigens; Phenanthrenes; X-Linked Inhibitor of Apoptosis Protein

Acute myeloid leukemia (AML) cells are relatively resistant to tumor necrosis factor alpha-related apoptosis-inducing ligand (TRAIL). We previously reported that triptolide, a potent anticancer agent from a Chinese herb, decreases XIAP in leukemic cells. We evaluated the combination of triptolide and TRAIL and found synergistic promotion of apoptosis in AML cells. XIAP-overexpressing U937 cells (U937XIAP) were more resistant to TRAIL than U937neo cells, and inhibition of XIAP with the small-molecule inhibitor 1396-11 enhanced TRAIL-induced apoptosis, implying XIAP as a resistance factor in AML. Furthermore, triptolide increased DR5 levels in OCI-AML3, while the DR5 increase was blunted in p53-knockdown OCI-AML3 and p53-mutated U937 cells, confirming a role for p53 in the regulation of DR5. In support of this finding, disruption of MDM2-p53 binding with subsequent increase in p53 levels by nutlin3a increased DR5 levels and sensitized OCI-AML3 cells to TRAIL. The combination of 1396-11 plus nutlin3a plus TRAIL was more effective than either the 1396-11 and TRAIL or nutlin3a and TRAIL combinations in OCI-AML3 cells, further supporting the role of triptolide as a sensitizer to TRAIL-induced apoptosis in part by independent modulation of XIAP expression and p53 signaling. Thus, the combination of triptolide and TRAIL may provide a novel strategy for treating AML by overcoming critical mechanisms of apoptosis resistance.

Topics: Aniline Compounds; Antineoplastic Agents, Alkylating; Apoptosis; Diterpenes; Drug Synergism; Epoxy Compounds; Female; Humans; Imidazoles; Leukemia, Myeloid, Acute; Male; Phenanthrenes; Piperazines; Proto-Oncogene Proteins c-mdm2; Receptors, TNF-Related Apoptosis-Inducing Ligand; Signal Transduction; TNF-Related Apoptosis-Inducing Ligand; Tumor Suppressor Protein p53; U937 Cells; X-Linked Inhibitor of Apoptosis Protein

NK314 is a novel synthetic benzo[c]phenanthridine alkaloid that has recently entered clinical trials as an antitumor compound, based on impressive activities in preclinical models. The present investigations were directed at determining the mechanism of action of this agent. NK314 induced significant G(2) cell cycle arrest in several cell lines, independent of p53 status, suggesting the existence of a common mechanism of checkpoint activation. The Chk1-Cdc25C-Cdk1 G(2) checkpoint pathway was activated in response to 100 nmol/L NK314 in ML-1 human acute myeloid leukemia cells. This was associated with the phosphorylation of the histone variant H2AX, an action that was predominant in the G(2) population, suggesting that double-strand DNA breaks caused cells to activate the checkpoint pathway. Double-strand DNA breaks were visualized as chromosomal aberrations when the G(2) checkpoint was abrogated by 7-hydroxystaurosporine. In vitro assays showed that NK314 inhibited the ability of topoisomerase IIalpha to relax supercoiled DNA and trapped topoisomerase IIalpha in its cleavage complex intermediate. CEM/VM1 cells, which are resistant to etoposide due to mutations in topoisomerase IIalpha, were cross-resistant to NK314. However, CEM/C2 cells, which are resistant to camptothecin due to mutations in topoisomerase I, retained sensitivity. These findings support the conclusion that the major mechanism of NK314 is to inhibit topoisomerase IIalpha, an action that leads to the generation of double-strand DNA breaks, which activate the G(2) DNA damage checkpoint pathway.

Topics: Antigens, Neoplasm; Antineoplastic Agents; Cell Line, Tumor; Chromosome Aberrations; Clinical Trials as Topic; DNA Topoisomerases, Type II; DNA-Binding Proteins; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Enzyme Inhibitors; G2 Phase; Histones; Humans; Leukemia, Myeloid, Acute; Phenanthrenes; Phenanthridines; Phosphorylation; Staurosporine; Topoisomerase II Inhibitors