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BackgroundAlthough the action mechanism of antineoplastic agents is different, oxaliplatin, paclitaxel, or bortezomib as first-line antineoplastic drugs can induce painful neuropathy. In rodents, mechanical allodynia is a common phenotype of painful neuropathy for 3 chemotherapeutics. However, whether there is a common molecular involved in the different chemotherapeutics-induced painful peripheral neuropathy remains unclear.MethodsMechanical allodynia was tested by von Frey hairs following i.p. injection of vehicle, oxaliplatin, paclitaxel, or bortezomib in Sprague-Dawley rats. Reduced representation bisulfite sequencing and methylated DNA immunoprecipitation were used to detect the change of DNA methylation. Western blot, quantitative polymerase chain reaction, chromatin immunoprecipitation, and immunohistochemistry were employed to explore the molecular mechanisms.ResultsIn 3 chemotherapeutic models, oxaliplatin, paclitaxel, or bortezomib accordantly upregulated the expression of transient receptor potential cation channel, subfamily C6 (TRPC6) mRNA and protein without affecting the DNA methylation level of TRPC6 gene in DRG. Inhibition of TRPC6 by using TRPC6 siRNA (i.t., 10 consecutive days) relieved mechanical allodynia significantly following application of chemotherapeutics. Furthermore, the downregulated recruitment of DNA methyltransferase 3 beta (DNMT3b) at paired box protein 6 (PAX6) gene led to the hypomethylation of PAX6 gene and increased PAX6 expression. Finally, the increased PAX6 via binding to the TPRC6 promoter contributes to the TRPC6 increase and mechanical allodynia following chemotherapeutics treatment.ConclusionsThe TRPC6 upregulation through DNMT3b-mediated PAX6 gene hypomethylation participated in mechanical allodynia following application of different chemotherapeutic drugs.

We conducted a phase II, noncomparative, multicenter study to assess the efficacy and safety of allogeneic bone marrow-derived mesenchymal stromal cells (BM-MSCs) expanded in vitro for patients with aplastic anemia (AA) refractory to immunosuppressive therapy. Seventy-four patients from seven centers received allogeneic BM-MSCs at a dose of 1–2 × 106 cells/kg per week for 4 weeks. Responses were assessed at 0.5, 1, 2, 3, 6, 9, and 12 months after the first cells infusion. Patients with response at 1 month continued to receive four infusions. All patients were evaluable. The overall response rate was 28.4% (95% confidence interval, 19%–40%), with 6.8% complete response and 21.6% partial response. The median times to response of leukocytic, erythrocytic, and megakaryocytic linages were 19 (range, 11–29), 17 (range, 12–25), and 31 (range, 26–84) days, respectively. After median follow-up of 17 months, overall survival was 87.8%. Seven patients developed transitory and mild headache and fever, but no other adverse events were observed. Antithymocyte globulin used in previous treatment and no activated infection throughout treatment were predictors for response. Allogeneic BM-MSCs infusion is a feasible and effective treatment option for refractory AA. The trial was registered at www.clinicaltrials.gov as NCT00195624.

Celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, has been demonstrated to exert antitumor activity in a variety of cancer cells. The underlying mechanism involves inhibition of cell cycle progression and induction of apoptosis. Besides, celecoxib has also been found to induce autophagy in some solid tumor cells. The aim of this study was to investigate the effect of celecoxib on cell proliferation in HL-60 human acute leukemia cells and to explore the potential mechanism. HL-60 cells were exposed to various concentrations of celecoxib and cell viability was evaluated by the MTT assay. Apoptosis was analyzed with flow cytometry and the amount of autophagosome was evaluated by LysoTracker probe labelling. The expression of apoptosis- and autophagy-related proteins was assayed by Western blot and LysoSensor probe labelling was used to detect the effect of celecoxib on the lysosomal functions. The results of this study indicated that celecoxib inhibited cell proliferation in a time- and dose-dependent fashion. The flow cytometry analysis showed that celecoxib induced apoptosis at low concentrations and mainly cell necrosis at high concentrations. The Western blot test confirmed the induction of apoptosis by the upregulation of apoptosis-related proteins cleaved caspase-3 and cleaved PARP. Furthermore, this study demonstrated that celecoxib prevented the autophagic flux by inhibiting lysosome function; the fluorescence intensity of the LysoTracker probe and the level of autophagy-related proteins LC3-II and p62 were increased, but the fluorescence intensity of the LysoSensor probe was weakened. These findings show that celecoxib is an autophagy suppresser and has antitumor effects in HL-60 cells by inducing cell apoptosis and necrosis.

Background/Aims: Resistance of leukemia stem cells (LSCs) to chemotherapy in patients with acute myeloid leukemia (AML) causes relapse of disease. Hedgehog (Hh) signaling plays a critical role in the maintenance and differentiation of cancer stem cells. Yet its role in AML remains controversial. The purpose of the present study is to investigate the role of GLI1, the transcriptional activator of Hh signaling, in AML progenitor cells and to explore the anti-AML effects of GLI small-molecule inhibitor GANT61. Methods: The expression of GLI1 mRNA and protein were examined in AML progenitor cells and normal cells. The proliferation, colony formation, apoptosis and differentiation of AML progenitor cells were also analyzed in the presence of GANT61. Results: Kasumi-1 and KG1a cells, containing more CD34+ cells, expressed higher level of GLI1 compared to U937 and NB4 cells with fewer CD34+ cells. Consistently, a positive correlation between the protein levels of GLI1 and CD34 was validated in the bone marrow mononuclear cells (BMMC) of AML patients tested. GANT61 inhibited the proliferation and colony formation in AML cell lines. Importantly, GANT61 induced apoptosis in CD34+ enriched Kasumi-1 and KG1a cells, whereas it induced differentiation in U937 and NB4 cells. Furthermore, GANT61 enhanced the cytotoxicity of cytarabine (Ara-c) in primary CD34+ AML cells, indicating that inhibition of GLI1 could be a promising strategy to enhance chemosensitivity. Conclusions: The present findings suggested that Hh signaling was activated in AML progenitor cells. GLI1 acted as a potential target for AML therapy.

Three new Cd(II) luminescent metal-organic frameworks (LMOFs), namely {[Cd(bib) (bdc)]·DMF·H2O}n (1), {[Cd(bib)(ndc)(H2O)]·DMF}n (2) and {[Cd2(bib)2(bpdc)2]·1.5H2O} (3) (bib ​= ​1,3-bis(1-imidazolyl)benzene, H2bdc ​= ​1,4-benzenedicarboxylic acid, H2ndc ​= ​1,4-naphthalenedicarboxylic acid, H2bpdc ​= ​biphenyl-4,4′-dicarboxylic acid and DMF ​= ​N,N-dimethylformamide), have been synthesized by combining the V-shaped bib ligand and different dicarboxylate ligands under solvothermal conditions. Complex 1 displays a 4-fold interpenetrated 3D framework with sra topology. Complex 2 presents a 2D (4,4) layer structure derived from bib and ndc2− mixed spacers. Complex 3 is a 2-fold interpenetrated array based on 2D (4,4) wave-like coordinated layers. The structural diversity among these complexes can be realized by the abduction of linear dicarboxylate ligands. The results demonstrate that the length of dicarboxylate ligands makes an impact on the structural diversity. Moreover, the complexes 1–3 can be well dispersed in different solvents, which exhibit highly selective and sensitive detection for nitrobenzene (NB) over other solvents by means of fluorescence quenching effect. The work illuminates fine-tuning of structure and in turn sensing properties towards LMOFs based on the reasonable selection of organic ligands.

Background After allogeneic haematopoietic stem cell transplantation (allo-HSCT), Hepatitis B virus reactivation (HBVr) can be observed in patients with previous resolved Hepatitis B virus (HBV) infections. Nephrotic syndrome (NS) is the main clinical manifestation of HBsAg-positive glomerulonephritis. However, the development of HBVr combined with NS after allo-HSCT is uncommon. Case presentation We presented a case of a 47-year-old female with acute myelogenous leukemia who underwent HLA-identical sibling allo-HSCT and achieved leukemia free survival. She had pretransplant serological markers of a resolved HBV infection (HBsAg-negative, anti-HBc and anti-HBs positive). However, she developed HBVr combined with nephrotic syndrome (NS) 16 months after HSCT. Her histological renal lesion was mesangial proliferative glomerulonephritis. IgA+, IgM+, and C1q deposits but not HBV antigens (HBsAg and HBcAg) were identified in her renal biopsy material. Long-term entecavir and immunosuppression resulted in decrease of HBV virus replication, amelioration of proteinuria and stabilisation of renal function. Conclusions Entecavir combined with immunosuppression has efficacy in the treatment of HBVr combined with NS after allo-HSCT, but long course of treatment is needed. Closely monitoring and antiviral prophylaxis might be necessary for allo-HSCT recipients to prevent reactivation of resolved HBV infection and its related complications.

Oxaliplatin-induced chronic painful neuropathy is the most common dose-limiting adverse event that negatively affects cancer patients’ quality of life. However, the underlying molecular mechanisms are still unclear. In the present study, we found that the intraperitoneal administration of oxaliplatin at 4 mg/kg for five consecutive days noticeably upregulated the expression of CXC motif ligand 12 (CXCL12) in the dorsal root ganglion, and the intrathecal injection of an anti-CXCL12 neutralizing antibody or CXCL12 siRNA attenuated the mechanical allodynia and thermal hyperalgesia induced by oxaliplatin. We also found that the signal transducers and transcription activator 3 (STAT3) was activated in the dorsal root ganglion, and inhibition of STAT3 with S3I-201 or the injection of AAV-Cre-GFP into STAT3flox/flox mice prevented the upregulation of CXCL12 expression in the dorsal root ganglion and chronic pain following oxaliplatin administration. Double-label fluorescent immunohistochemistry findings also showed that p-STAT3 was mainly localized in CXCL12-positive cells in the dorsal root ganglion. Furthermore, the results of a chromatin immunoprecipitation assay revealed that p-STAT3 might be essential for oxaliplatin-induced CXCL12 upregulation via binding directly to the specific position of the CXCL12 gene promoter. Finally, we found that cytokine TNF-α and IL-1β increases mediated the STAT3 activation following oxaliplatin treatment. Taken together, these findings suggested that the upregulation of CXCL12 via TNF-α/IL-1β–dependent STAT3 activation contributes to oxaliplatin-induced chronic pain.

Doxycycline, a tetracycline-based antibiotic, has been reported to attenuate melanoma cell migration through inhibiting the focal adhesion kinase (FAK) signaling pathway. However, it remains to be elucidated whether doxycycline exerts this effect on leukemia cell migration. The present study aimed to examine the role of doxycycline in leukemia cell migration. The invasion capacities of the human leukemia cell lines KG1a (acute myelogenous leukemia) and K562 (chronic myelogenous leukemia) were evaluated using Matrigel® matrix-coated Transwell® chamber assays; leukemic cell lines treated with doxycycline (1 µg/ml) or anti-β1-integrin antibodies were added to the upper chamber, while untreated cells were included as controls. Reverse transcription quantitative polymerase chain reaction was performed in order to further understand the influence of doxycycline treatment on the expression of FAK and gelatinases in the KG1a and K562 leukemic cell lines. In addition, FAK protein expression and phosphorylation were determined using western blot analysis in order to investigate the mechanism by which doxycycline inhibited leukemic cell migration. The results revealed that doxycycline treatment significantly attenuated the migration of KG1a and K562 cells, which was demonstrated to be associated with inhibition of the expression and phosphorylation of FAK. In addition, doxycycline treatment inhibited matrix metalloproteinase (MMP)-2 and MMP-9 expression. Furthermore, incubation with blocking anti-β1-integrin antibodies had an analogous inhibitory effect on leukemic cell migration to that of doxycycline. In conclusion, the results of the present study suggested that doxycycline attenuated leukemic cell migration through inhibiting the FAK signaling pathway. Therefore, doxycycline may have potential for use as a novel strategy for the treatment of leukemia.

We conducted a phase II, noncomparative, multicenter study to assess the efficacy and safety of allogeneic bone marrow‐derived mesenchymal stromal cells (BM‐MSCs) expanded in vitro for patients with aplastic anemia (AA) refractory to immunosuppressive therapy. Seventy‐four patients from seven centers received allogeneic BM‐MSCs at a dose of 1–2 × 106 cells/kg per week for 4 weeks. Responses were assessed at 0.5, 1, 2, 3, 6, 9, and 12 months after the first cells infusion. Patients with response at 1 month continued to receive four infusions. All patients were evaluable. The overall response rate was 28.4% (95% confidence interval, 19%–40%), with 6.8% complete response and 21.6% partial response. The median times to response of leukocytic, erythrocytic, and megakaryocytic linages were 19 (range, 11–29), 17 (range, 12–25), and 31 (range, 26–84) days, respectively. After median follow‐up of 17 months, overall survival was 87.8%. Seven patients developed transitory and mild headache and fever, but no other adverse events were observed. Antithymocyte globulin used in previous treatment and no activated infection throughout treatment were predictors for response. Allogeneic BM‐MSCs infusion is a feasible and effective treatment option for refractory AA. The trial was registered at www.clinicaltrials.gov as NCT00195624. Stem Cells Translational Medicine 2017;6:1569–1575

To investigate the effect of valproate treatment on the K562 cell line, a model for chronic myelogenous leukaemia, the growth and survival of the K562 cell line were investigated using the Annexin-V/PI dual staining method, and global profiles of gene expression and alternative splicing in K562 cells were assessed using exon microarrays. A significant increase in cell apoptosis was observed in valproate-exposed K562 cells using flow cytometry. A total of 628 transcripts were identified as being significantly differentially expressed. The number of genes demonstrating increased expression levels was greater than the number of genes demonstrating decreased expression levels (445 genes vs. 183 genes, respectively). The significant enrichment analysis of GO terms for the differentially expressed genes revealed that these genes are involved in many important biological processes such as apoptosis. Six of the genes observed to be differentially expressed that might be involved in apoptosis were selected to undergo qRT-PCR validation. In total, 198 candidates of alternative splicing variants were identified. Among them, three alternative splicing events were selected for validation, and CBLC and TBX1 were confirmed to be alternatively spliced by semi-nested PCR. In conclusion, valproate exposure facilitated cell apoptosis, altered mRNA expression and alternative splicing events in the K562 cell line.

The delivery of adipose-derived stem cells (ADSCs) for promoting tissue repair has become a potential new therapy, while hepatocyte growth factor (HGF) is an important growth factor with angiogenic, antifibrotic, and anti-inflammatory benefits. Therefore, transplantation of ADSCs into acute myocardial infarction (AMI) may improve cardiac function through angiogenesis and anti-fibrosis, and that hHGF may enhance these effects. ADSCs were isolated from human subcutaneous adipose tissue. Lentivirus vector encoding human HGF (lenti-hHGF) was constructed and infected into ADSCs. Results indicated that transplantation of ADSCs led to improvement of left ventricular function, explained partly through their ability to differentiate into endothelial cells, resulting in increased blood flow and decreased fibrosis. Furthermore, hHGF enhanced these effects. This suggests that ADSCs combined with HGF gene transfer may be a useful strategy for the treatment of patients with ischemic heart disease.

To explore the expression and clinical significance of Hedgehog signaling transcription factor Gli1 in acute lymphoblastic leukemia (ALL) patients.The clinical specimens were obtained from 32 newly diagnosed and 6 relapsed ALL patients. Normal bone marrow cells from 15 healthy donors were used as controls. Real-time qPCR and Western blot were applied to detect Gli1 mRNA and protein expression in bone marrow mononuclear cells (BMMNC) of these samples respectively. The relation of Gli1 mRNA levels with clinical parameter was also evaluated.The expression level of Gli1 mRNA in de novo and relapsed ALL patients was significantly higher than that in the normal controls (P0.05). There was no stalistically significant difference of the Gli1 mRNA expression between de novo and relapsed ALL cases (P0.05). In 24 de novo ALL patients with complete remission (CR) after induction chemotherapy, the levels of Gli1 mRNA were significantly reduced as compared with levels before treatment (P0.05). However, in 4 ALL patients without remission, no obvious difference of Gli1 mRNA levels were observed as compared with levels of Gli1 before treatment (P0.05). A positive correlation between the Gli1 mRNA expression level and white blood cell count (WBC) was found in the BMMNC of ALL patients (R = 0.725, P0.05). Similarly, Gli1 protein expression was significantly higher in the de novo and relapsed ALL cases compared with normal controls. The Gli1 protein level was down-regulated when the ALL patients was in CR.The expression of Gli1 mRNA and protein has been found to be high in de novo and relapsed ALL patients, and the change of Gli1 expression maybe relate to therapeutic efficacy and prognosis of ALL patients.

To date, acute myeloid leukemia (AML) shows very poor outcome for conventional chemotherapy. Leukemia stem cells (LSCs) are insensitive to conventional chemotherapeutic drugs and play a central role in the pathogenesis of AML. Failure to effectively ablate these cells may lead to AML relapse following chemotherapy. Phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway is constructively activated in LSCs. This pathway can be inhibited by PI-103, a novel synthesized molecule of the pyridofuropyrimidine class, resulting in the apoptosis of LSCs. Therefore, we investigate the influences of PI-103 in combination with daunorubicin (DNR) on the LSCs. Our data indicate that PI-103 synergistically sensitizes LSCs to DNR-induced cytotoxicity. In addition, the PI-103/DNR co-treatment can induce significant apoptosis in LSCs, but sparing hematopoietic stem cells. The synergistic effect and the LSCs-specific apoptosis mechanism may be associated with the inhibition of PI3K/Akt/mTOR signaling pathway. Our results suggest that PI-103 in combination with DNR may be a potent and less toxic therapy for targeting LSCs and deserve further preclinical and clinical studies in the treatment of AML.

To explore the mechanism underlying antileukaemia effect of sodium valproate, the growth and survival of the K562 cell line were investigated. Global profiles of gene expression in K562 cells exposed to sodium valproate were assessed and validated. The differentially expressed genes identified were further used to query the connectivity map database to retrieve a ranked list of compounds that act on the same intracellular targets as sodium valproate. A significant increase in cell apoptosis and a change in gene expression profile were observed in valproate-exposed K562 cells. The significant enrichment analysis of gene ontology terms for the differentially expressed genes showed that these genes were involved in many important biological processes. Eight differentially expressed genes involved in apoptosis were verified by quantitative real-time PCR. The connectivity map analysis showed gene expression profile in K562 cells exposed to sodium valproate was most similar to that of HDACi and PI3K inhibitors, suggesting that sodium valproate might exert antileukaemic action by inhibiting HDAC as well as inhibiting PI3K pathway. In conclusion, our data might provide clues to elucidate the molecular and therapeutic potential of VPA in leukaemia treatment, and the connectivity map is a useful tool for exploring the molecular mechanism of drug action.

To construct the eukaryotic expression vector of human gene Smac pcDNA3.1-Smac and express it in the lung adenocarcinoma A549 cells.The Smac was amplified from human testis tissue by reverse transcriptase polymerase chain reaction (RT-PCR). Then recombined eukaryotic expression vector pcDNA3.1-Smac was constructed. After the reconbinant plasmid was proved to be constructed correctly by endonucleases digesting and DNA sequencing, we trasfected it into lung adenocarcinama cells A549 through liposome inducing. The expression of Smac in transfectant A549 was detected by RT-PCR and Western blot. And the cell growth inhibition ratio after trasfection was detected by MTT.The amplified fragment by PCR was coincident with the anticipated result, and its sequence was in concordance with that published on GenBank.Therefore, the gene Smac was cloned successfully, and the recombinant plasmid pcDNA3.1-Smac was also constructed successfully. Both on the mRNA level and the protein level, the expression of Smac gene was increased obviously in the transfected A549 detected by RT-PCR and Western blot respectively. The cell growth inhibition ratio in the group transfected pcDNA3.1-Smac was significantly higher compared with the pcDNA3.1 group after 72 hours.The recombinant eukaryotic expression vector pcDNA3.1-Smac was constructed, and it could be obviously expressed in lung adenocarcinoma cells A549. It is also proven that Smac has the function of growth inhibition.

To investigate the effect of Shenqi Fuzheng Injection (SFI) for hemopoietic and immune function reconstruction in patients with hematologic malignancies (HM) after chemotherapy.Eighty HM patients at remission inducing stage of initial treatment were randomly assigned to two groups, the treatment group treated with SFI (250 mL daily) plus chemotherapy and the control group only treated with chemotherapy for 14 days, with same supportive treatments administered to both. Levels of blood routine test, T lymphocyte subsets (CD3+, CD4+, CD4+ /CD8+) and B lymphocyte subsets CD3- CD19+ were determined before and after treatment, and the remission rate was assessed after treatment.The remission rates in the two groups showed no significant difference [90% (36/40) vs 82.5% (33/40), P0.05] statistically. Levels of peripheral leucocyte count and hemoglobin as well as levels of CD3+, CD4+, CD4+ /CD8+; were significantly higher in the treatment group than in the control group (P0.05), but no significant difference was shown between groups in CD3- CD19+ level.SFI can lighten the inhibition of chemotherapy on hemopoietic function of bone marrow, and promote its recovery, enhance the immune function, and improve the quality of life in patients with HM undergoing chemotherapy.