Berberine exerts anti-tumor activity in diffuse large B-cell lymphoma by modulating c-myc/CD47 axis.

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma (NHL) with high clinical heterogeneity and poor prognosis. Immune escape mediated by CD47 overexpression contributes to the limited efficacy of rituximab, an anti-CD20 antibody, which indicates a target to improve the efficacy of DLBCL treatment. Here, we validated berberine, a natural compound, as a suppressor of CD47 and revealed the involved mechanism and biological function in DLBCL. Berberine downregulated the expression of CD47 in DLBCL at the transcriptional level by suppressing c-myc expression. Berberine-induced CD47 inhibition enhanced the phagocytosis of macrophages, thereby eliminating DLBCL cells in vitro and in vivo. Interestingly, berberine enhanced the efficiency of anti-CD47 antibody and rituximab-mediated phagocytosis. Moreover, a novel prognostic model based on the combination of CD47 and CD68, a biomarker of macrophages, was established in DLBCL. Our results highlighted for the first time that berberine could restore macrophage function in the tumor microenvironment, enhance rituximab-mediated phagocytosis and promote anti-CD47 antibody function via suppressing CD47 expression, which revealed a new anti-tumor mechanism of berberine and provided novel insights into the rituximab-based immunochemotherapy and CD47-targeted immunotherapy in DLBCL.

Directly targeting c-Myc contributes to the anti-multiple myeloma effect of anlotinib.

Despite the significant advances in the treatment of multiple myeloma (MM), this disease is still considered incurable because of relapse and chemotherapy resistance, underscoring the need to seek novel therapies with different mechanisms. Anlotinib, a novel multi-targeted tyrosine kinase inhibitor (TKI), has exhibited encouraging antitumor activity in several preclinical and clinical trials, but its effect on MM has not been studied yet. In this study, we found that anlotinib exhibits encouraging cytotoxicity in MM cells, overcomes the protective effect of the bone marrow microenvironment and suppresses tumor growth in the MM mouse xenograft model. We further examined the underlying molecular mechanism and found that anlotinib provokes cell cycle arrest, induces apoptosis and inhibits multiple signaling pathways. Importantly, we identify c-Myc as a novel direct target of anlotinib. The enhanced ubiquitin proteasomal degradation of c-Myc contributes to the cell apoptosis induced by anlotinib. In addition, anlotinib also displays strong cytotoxicity against bortezomib-resistant MM cells. Our study demonstrates the extraordinary anti-MM effect of anlotinib both in vitro and in vivo, which provides solid evidence and a promising rationale for future clinical application of anlotinib in the treatment of human MM.

L20, a Calothrixin B analog, induces intrinsic apoptosis on HEL cells through ROS/γ-H2AX/p38 MAPK pathway.

Erythroleukemia is a malignant disease in the blood system. Quinones consists of a class of antitumor agents. Calothrixin B is a carbazole-1,4-quinone alkaloid isolated from Calothrix cyanobacteria with a unique indolo[3,2-j] phenanthridine framework. This study aimed to investigate the anti-leukemic effect of the new Calothrixin B derivative, L20, and to dig up the underlying mechanisms. Cytotoxicity analysis of L20 has revealed that it shows significant IC50 concentrations in HEL cells at low doses (1.10 ± 0.05 µM) than in K562, and KG-1a (5.46 ± 3.09, and 1.82 ± 1.08 µM respectively). The study even revealed that the L20 could induce a dose and time-dependent cellular death in HEL cells. The L20 increased expression of phospho-γ-H2A.X and phospho-p38 in HEL cells, causing DNA damage and nuclear alterations due to the G2/M phase cell cycle arrest. The HEL cells even lost the mitochondrial membrane potential (MMP) and resulted in the release of reactive oxygen species (ROS). Additionally, L20 inhibited the proliferation of HEL cells by inducing apoptosis through the mitochondrial pathway, depending on the caspase family. The study even established this may be due to the upregulation of the p-P38MAPK and downregulation of p-ERK. Pretreatment with P38/ERK inhibitors, SB203580, and U0126, decreased L20-induced apoptosis. These findings indicated that L20 induced mitochondrial mediated-apoptosis and G2/M arrest through DNA damage and modulation of p38 MAPK pathways. Thus, the study suggests L20, a chemical analog of Calothrixin B, as a novel chemotherapeutic agent against erythroleukemia.

Tissue-specific transcriptional regulation of seven heavy metal stress-responsive miRNAs and their putative targets in nickel indicator castor bean (R. communis L.) plants.

R.communis L. has high capability to accumulate nickel which is a trace nutrient for higher plants and also an environmental contaminant causes toxicity related symptoms at higher concentrations. MicroRNAs (miRNAs) are known to be important modulators of responses against heavy metal stress for detoxification of the metal. In this study, we experimentally measured and validated the transcript levels of the seven heavy metal stress response-related miRNAs and the expression levels of target genes in both leaf and root tissues of R. communis L. subjected to three different concentrations of nickel stress via qRT-PCR quantification. The results demonstrated differential regulations of heavy metal stress-responsive miRNAs and their putative targets in both tissues in same stress treatments. This dynamic regulation suggest that regulatory processes differ between the tissues under nickel stress. Our data suggest that, miR838 was the most responsive to the Ni2+ stress. miR398 target gene Cu-Zn/SOD was found to be up-regulated in both root and leaf tissues. The relations between TCP and expression levels of miR159 and miR319 were also found statistically significant exclusive to leaf tissues. In leaf tissue, changes in miR395 level and its putative target genes, sulphate transporter and sulphate adenyltransferase gene were found in relation whereas, only expression level of sulphate transporter represented a statistically significant relation in root tissue. The sharp decrease in transcript levels of 2r3 myb gene at lower nickel dose suggest to investigate the role of r2r3 myb and the all MYB family members in primary and secondary metabolisms against nickel stress.

Convenient method for the synthesis of a flexible cyclic polyamide for selective targeting of c-myb G-quadruplex DNA.

A convenient efficient method for synthesis of a flexible cyclic polyamide (cß, 1) was developed through cyclodimerization. Electrospray ionization mass spectrometry and nuclear magnetic resonance results showed that 1 selectively binds to the c-myb G-quadruplex with high affinity, and there was no binding with the ILPR, bcl-2, and c-kit G-quadruplexes. This is the first time that a flexible cyclic polyamide was found to have high selectivity for the c-myb G-quadruplex.

Role of c-Myb in the survival of pre B-cell acute lymphoblastic leukemia and leukemogenesis.

Acute lymphoblastic leukemia (ALL) is the most common cancer in children. The current treatment protocol for ALL involves an intense chemotherapy regimen yielding cure rates of nearly 80%. However, new therapies need to be designed not only to increase the survival rate but also to combat the risk of severe therapy associated toxicities including secondary malignancies, growth problems, organ damage, and infertility. The c-Myb proto-oncogene is highly expressed in immature hematopoietic cells. In this study, we demonstrate that loss of c-Myb itself decreased the viability of these leukemic cells. Additionally, the inhibition of c-Myb caused a decrease in cell proliferation, significantly increased the number of cells in G(0) /G(1) phase of the cell cycle, increased the sensitivity of pre-B-ALL cells to cytotoxic agents in vitro, and significantly delayed disease onset in a mouse model of leukemia. Furthermore, we demonstrate that Bcl-2 is a target of c-Myb in pre-B-ALL cells. Our results identify c-Myb as a potential therapeutic target in pre-B-ALL and suggest that suppression of c-Myb levels or activity, in combination with currently used therapies and/or dose reduction, may lead to a decrease in toxicity and an increase in patient survival rates. Because c-Myb is aberrantly expressed in several other malignancies, targeting c-Myb will have broad clinical applications.

Telomestatin impairs glioma stem cell survival and growth through the disruption of telomeric G-quadruplex and inhibition of the proto-oncogene, c-Myb.

PURPOSE: Glioma stem cells (GSC) are a critical therapeutic target of glioblastoma multiforme (GBM). EXPERIMENTAL DESIGN: The effects of a G-quadruplex ligand, telomestatin, were evaluated using patient-derived GSCs, non-stem tumor cells (non-GSC), and normal fetal neural precursors in vitro and in vivo. The molecular targets of telomestatin were determined by immunofluorescence in situ hybridization (iFISH) and cDNA microarray. The data were then validated by in vitro and in vivo functional assays, as well as by immunohistochemistry against 90 clinical samples. RESULTS: Telomestatin impaired the maintenance of GSC stem cell state by inducing apoptosis in vitro and in vivo. The migration potential of GSCs was also impaired by telomestatin treatment. In contrast, both normal neural precursors and non-GSCs were relatively resistant to telomestatin. Treatment of GSC-derived mouse intracranial tumors reduced tumor sizes in vivo without a noticeable cell death in normal brains. iFISH revealed both telomeric and non-telomeric DNA damage by telomestatin in GSCs but not in non-GSCs. cDNA microarray identified a proto-oncogene, c-Myb, as a novel molecular target of telomestatin in GSCs, and pharmacodynamic analysis in telomestatin-treated tumor-bearing mouse brains showed a reduction of c-Myb in tumors in vivo. Knockdown of c-Myb phenocopied telomestatin-treated GSCs both in vitro and in vivo, and restoring c-Myb by overexpression partially rescued the phenotype. Finally, c-Myb expression was markedly elevated in surgical specimens of GBMs compared with normal tissues. CONCLUSIONS: These data indicate that telomestatin potently eradicates GSCs through telomere disruption and c-Myb inhibition, and this study suggests a novel GSC-directed therapeutic strategy for GBMs.

c-erbB2 and c-myb induce mouse oocyte maturation involving activation of maturation promoting factor.

Proto-oncogenes are involved in cell growth, proliferation, and differentiation. In the present study, we investigated the roles and mediating pathways of proto-oncogenes c-erbB(2) and c-myb in mouse oocyte maturation by RT-PCR, real-time quantitative PCR, western blot, and recombinant proto-oncogene protein microinjection. Results showed that both c-erbB(2) and c-myb antisense oligodeoxynucleotides (c-erbB(2) ASODN and c-myb ASODN) inhibited germinal vesicle breakdown and the first polar body extrusion in a dose-dependent manner. However, microinjection of recombinant c-erbB(2) or c-myb protein into germinal vesicle stage oocytes stimulated oocyte meiotic maturation. In addition, the expression of c-erbB(2) and c-myb mRNA was detected in oocytes; and c-erbB(2) ASODN and c-myb ASODN inhibited c-erbB(2) mRNA and c-myb mRNA expression, respectively. Maturation promoting factor (MPF) inhibitor roscovitine did not affect the expression of c-erbB(2) mRNA and c-myb mRNA, but blocked the effects of recombinant c-erbB(2) and c-myb protein-induced oocyte maturation. Further, cyclin B1 protein expression in oocytes was remarkably inhibited by c-erbB(2) ASODN, c-myb ASODN, and roscovitine. Nonsense tat ODN had no effect on the expression of c-erbB(2), c-myb, and cyclin B1. These results suggest that c-erbB(2) and c-myb may induce oocyte maturation through mediating a pathway involving the activation of MPF.

The effects of 1,4-benzoquinone on c-Myb and topoisomerase II in K-562 cells.

Exposure to benzene, a ubiquitous environmental pollutant, has been linked to leukemia, although the mechanism of benzene-initiated leukemogenesis remains unclear. Benzene can be bioactivated to toxic metabolites such as 1,4 benzoquinone (BQ), which can alter signaling pathways and affect chromosomal integrity. BQ has been shown to increase the activity of c-Myb, which is an important transcription factor involved in hematopoiesis, cell proliferation, and cell differentiation. The c-Myb protein has also been shown to increase topoisomerase IIalpha (Topo IIalpha) promoter activity specifically in cell lines with hematopoietic origin. Topo IIalpha is a critical nuclear enzyme that removes torsional strain by cleaving, untangling and religating double-stranded DNA. Since Topo IIalpha mediates DNA strand breaks, aberrant Topo IIalpha activity or increased protein levels may increase the formation of DNA strand breaks, leaving the cell susceptible to mutational events. We hypothesized that BQ can increase c-Myb activity, which in turn increases Topo IIalpha promoter activity resulting in increased DNA strand breaks. Using luciferase reporter assays in K-562 cells we demonstrated that BQ (25 and 37microM) exposure caused an increase in c-Myb activity after 24h. Contradictory to previous findings, overexpression of exogenous c-Myb or a polypeptide consisting of c-Myb's DNA binding domain (DBD), which competitively inhibits the binding of endogenous c-Myb to DNA, did not affect Topo IIalpha promoter activity. However, BQ (37microM for 24h) exposure caused a significant increase in Topo IIalpha promoter activity, which could be blocked by the overexpression of the DBD polypeptide, suggesting that BQ exposure increases Topo IIalpha promoter activity through the c-Myb signaling pathway.

Transcriptional repression of c-Myb and GATA-2 is involved in the biologic effects of C/EBPalpha in p210BCR/ABL-expressing cells.

Ectopic C/EBPalpha expression in p210(BCR/ABL)-expressing hematopoietic cells induces granulocytic differentiation, inhibits proliferation, and suppresses leukemogenesis. To assess the underlying mechanisms, C/EBPalpha targets were identified by microarray analyses. Upon C/EBPalpha activation, expression of c-Myb and GATA-2 was repressed in 32D-BCR/ABL, K562, and chronic myelogenous leukemia (CML) blast crisis (BC) primary cells but only c-Myb levels decreased slightly in CD34(+) normal progenitors. The role of these 2 genes for the effects of C/EBPalpha was assessed by perturbing their expression in K562 cells. Ectopic c-Myb expression blocked the proliferation inhibition- and differentiation-inducing effects of C/EBPalpha, whereas c-Myb siRNA treatment enhanced C/EBPalpha-mediated proliferation inhibition and induced changes in gene expression indicative of monocytic differentiation. Ectopic GATA-2 expression suppressed the proliferation inhibitory effect of C/EBPalpha but blocked in part the effect on differentiation; GATA-2 siRNA treatment had no effects on C/EBPalpha induction of differentiation but inhibited proliferation of K562 cells, alone or upon C/EBPalpha activation. In summary, the effects of C/EBPalpha in p210(BCR/ABL)-expressing cells depend, in part, on transcriptional repression of c-Myb and GATA-2. Since perturbation of c-Myb and GATA-2 expression has nonidentical consequences for proliferation and differentiation of K562 cells, the effects of C/EBPalpha appear to involve dif-ferent transcription-regulated targets.

The effects of benzene and the metabolites phenol and catechol on c-Myb and Pim-1 signaling in HD3 cells.

Exposure to the environmental toxicant benzene has been proposed to lead to leukemogenesis. The transcription factor c-Myb plays a role in blood cell differentiation and can be regulated by the serine-threonine kinase Pim-1. Overexpressed versions of c-Myb and Pim-1 are believed to play a key role in the development of a wide variety of leukemias and tumors. In our study, we evaluated the effects of benzene and the metabolites catechol and phenol on c-Myb signaling to investigate our hypothesis that benzene exerts its toxicity by interfering with this pathway. To evaluate this hypothesis, HD3 chicken erythroblast cells were transiently transfected with a c-Myb responsive luciferase reporter plasmid and then exposed to benzene, catechol, or phenol (0-300 microM) for 1-24 h before nonproprietary dual luciferase activities were measured. Our results demonstrated that catechol exposure caused a time- and concentration-dependent increase in c-Myb activity with significance occurring at 100 and 300 microM after 24 h of exposure, which was independent of increased Pim-1 protein, but dependent on increased c-Myb phosphorylation. Benzene and phenol exposure resulted in small but significant decreases in c-Myb activity that were not dose- and time-dependent, nor was increased Pim-1 protein involved. These results are consistent with other studies, which suggest metabolite differences in benzene-mediated toxicity. More importantly, this study supports the hypothesis that benzene may mediate its toxicity through metabolite-mediated alterations in the c-Myb signaling pathway.

Induction of transcription factor c-myb expression in reactive astrocytes following intracerebroventricular kainic acid injection in mouse hippocampus.

The transcription factor c-myb is known to play an important role in the regulation of cellular proliferation and differentiation. Recently, the constitutive and aberrant expression of c-myb in the normal and Cu/Zn SOD mutant mouse brain was reported. However, the expression of c-myb in the process of reactive gliosis is not known yet. Here we report the delayed and protracted induction of c-myb in the brain of mice following kainic acid (KA) induced seizure. Our western blot analysis revealed that the amount of c-myb was dramatically increased in the brain 3 days after KA treatment. The induction of c-myb was sustained for more than 7 days after KA treatment. The c-myb immunoreactivity (IR) was restricted to neurons of the hippocampus in control mice. Three days after KA treatment, a strong c-myb IR was found in reactive astrocytes in the whole areas of the CA3 region. Thereafter, c-myb IR astrocytes were gradually concentrated around the CA3 region undergoing selective neuronal loss. A few c-myb IR astrocytes were continuously persisted in the CA3 region 14 days after KA treatment. These findings suggest a role of c-myb signal pathway in reactive gliosis in mice with KA induced seizure.

Trichostatin A suppresses transformation by the v-myb oncogene in BM2 cells.

BM2 cells are chicken monoblasts transformed by the v-myb oncogene of avian myeloblastosis virus. The constitutively high v-myb expression interferes with the terminal differentiation of BM2 cells, but these cells can be induced to differentiate into macrophage-like cells by phorbol esters. Histone acetylation plays an important role in regulation of transcription and is particularly relevant to the regulation and pathology of hematopoiesis. In the present study, we examined the contribution of elevated histone acetylation to the differentiation of BM2 cells. Inhibition of the activity of endogenous histone deacetylases by trichostatin A (TSA) resulted in histone hyperacetylation causing cell cycle arrest and differentiation of BM2 cells into macrophage polykaryons. TSA did not affect the level of v-Myb protein in BM2 cells, but it downregulated its transcription activation capability. This suggests that chromatin remodeling can be significantly engaged in regulation of proliferation and differentiation of leukemic cells.

Targeting c-Myb expression in human disease.

c-Myb is a transcription factor employed in the haematopoietic system and gastrointestinal tract to regulate the exquisite balance between cell division, differentiation and survival. In its absence, these tissues either fail to form, or show aberrant biology. Mice lacking a functional c-myb gene die in utero by day 15 of development. When inappropriately expressed, as is common in leukaemia and epithelial cancers of the breast, colon and gastro-oesophagus, c-Myb appears to activate gene targets of key importance to cancer progression and metastasis. These genes include cyclooxygenase-2 (COX-2), Bcl-2, BclX(L) and c-Myc, which influence diverse processes such as angiogenesis, proliferation and apoptosis. The clinical potential for blocking c-Myb expression in malignancies is based upon strong preclinical data and some trial-based evidence. The modest clinical experience to date has been with haematopoietic malignancies, but other disease classes may be amenable to similar interventions. The frontline agents to achieve this are nuclease-resistant oligodeoxynucleotides (ODNs), which are proving to be acceptable therapeutic reagents in terms of tolerable toxicities and delivery. Nevertheless, further effort must be focused on improving their efficacy, eliminating non-specific toxicity and optimising delivery. Optimisation issues aside, it would appear that anti-c-Myb therapies will be used with most success when combined with other agents, some of which will be established cytotoxic and differentiation-inducing drugs. This review will explore the future strategic use of ODNs in vivo, focusing on a wide spectrum of diseases, including several beyond the haematopoietic malignancies, in which c-Myb appears to play a role.

Activation of the nuclear transcription factor kappaB (NFkappaB) and differential gene expression in U87 glioma cells after exposure to the cytoprotector amifostine.

PURPOSE: Amifostine has been approved as a therapy to decrease the incidence of moderate-to-severe xerostomia in patients undergoing postoperative radiation treatment for head-and-neck cancer. As a reducing agent capable of participating in intracellular reductive/oxidative processes, it has the potential to affect redox-sensitive transcription factors and gene expression. Amifostine's active free thiol WR-1065 was investigated to determine its effect on nuclear transcription factor kappaB (NFkappaB) activation and subsequent gene expression in U87 glioma cells. METHODS AND MATERIALS: The human glioma cell line U87 was grown to confluency and then exposed to WR-1065 at a concentration of 40 microM for times ranging from 30 min to 24 h. Changes in cell cycle were monitored by flow cytometry. The effect of WR-1065 on NFkappaB activation was determined by a gel shift assay. Changes in gene expression as a function of time of exposure to WR-1065 were determined by Northern blot and the Atlas Human cDNA Expression Array (Clontech, Palo Alto, CA). Changes in gene expression using the Atlas Array were verified by reverse transcriptase-polymerase chain reaction (RT-PCR) with gene-specific primers. RESULTS: Exposure of U87 cells to 40 microM WR-1065 resulted in a marked activation of NFkappaB between 30 min and 1 h after treatment. Expression of MnSOD, an NFkappaB-responsive gene, was enhanced by over 2-fold after 16 h of treatment and remained elevated at 24 h. During this period of time, no changes in cell cycle distribution were observed. To assess changes in the expression levels of NFkappaB-responsive genes as a function of WR-1065 exposure, cDNA arrays containing 49 genes identified as having DNA-binding motifs for NFkappaB were used. Only five genes were found to be significantly affected at 1, 4, and/or 16 h of treatment. GST-3 and c-myc were repressed up to 2- and 4-fold, respectively. The expression levels of IL-2Ra, RANTES, and c-myb, in contrast, were enhanced up to 14-, 3-, and 2-fold, respectively. The remaining genes having NFkappaB-responsive elements in their promoter regions were either not expressed (20 genes) or were not affected (24 genes) by exposure to WR-1065. CONCLUSIONS: The redox-sensitive transcription factor NFkappaB can be activated in U87 glioma cells by the active thiol form of the cytoprotector amifostine. Activation of NFkappaB by the antioxidant WR-1065 is accompanied by a reduced expression of the oncogene c-myc and an enhanced expression of the antioxidant gene MnSOD, a gene whose expression in tumor cells is relatively low, but when overexpressed has been correlated with a suppression of the malignant phenotype. Activation of NFkappaB by WR-1065, however, results in selective rather than global changes in the expression of genes containing NFkappaB-responsive elements.

Oligodeoxynucleotide-mediated inhibition of c-myb gene expression in autografted bone marrow: a pilot study.

Antisense oligodeoxynucleotide (ODN) drugs might be more effective if their delivery was optimized and they were targeted to short-lived proteins encoded by messenger RNA (mRNA) species with equally short half-lives. To test this hypothesis, an ODN targeted to the c-myb proto-oncogene was developed and used to purge marrow autografts administered to allograft-ineligible chronic myelogenous leukemia patients. CD34(+) marrow cells were purged with ODN for either 24 (n = 19) or 72 (n = 5) hours. After purging, Myb mRNA levels declined substantially in approximately 50% of patients. Analysis of bcr/abl expression in long-term culture-initiating cells suggested that purging had been accomplished at a primitive cell level in more than 50% of patients and was ODN dependent. Day-100 cytogenetics were evaluated in surviving patients who engrafted without infusion of unmanipulated "backup" marrow (n = 14). Whereas all patients were approximately 100% Philadelphia chromosome-positive (Ph(+)) before transplantation, 2 patients had complete cytogenetic remissions; 3 patients had fewer than 33% Ph(+) metaphases; and 8 remained 100% Ph(+). One patient's marrow yielded no metaphases, but fluorescent in situ hybridization evaluation approximately 18 months after transplantation revealed approximately 45% bcr/abl(+) cells, suggesting that 6 of 14 patients had originally obtained a major cytogenetic response. Conclusions regarding clinical efficacy of ODN marrow purging cannot be drawn from this small pilot study. Nevertheless, these results lead to the speculation that enhanced delivery of ODN, targeted to critical proteins of short half-life, might lead to the development of more effective nucleic acid drugs and the enhanced clinical utility of these compounds in the future.