Polyphyllin VII, a novel moesin inhibitor, suppresses cell growth and overcomes bortezomib resistance in multiple myeloma.

Multiple myeloma is a plasma cell malignancy, accounting for approximately 1% of new cancer cases. It is the second most common hematological malignancy. Novel clinical agents such as the proteasome inhibitor-bortezomib, have shown improved survival rates in recent decades. However, multiple myeloma remains incurable, as most patients eventually relapse and become refractory to current treatments. Therefore, there is an urgent need for developing new regimens to overcome the bortezomib resistance. Here, we screened a library of 2370 bioactives and found that polyphyllin VII selectively suppressed multiple myeloma cell growth in vitro and in vivo. We identified moesin, one of the critical regulators of the Wnt/ß-catenin pathway, as a target of polyphyllin VII by drug affinity responsive target stability assay and cellular thermal shift assay. Polyphyllin VII binds to moesin and induces its degradation via the ubiquitin-proteasome pathway, thereby impairing the Wnt/ß-catenin pathway activity and leading to a reduction in the side population cells to overcome bortezomib resistance. Our study identified polyphyllin VII as a promising compound and moesin as a potential diagnostic and therapeutic target for treating multiple myeloma.

Albendazole inhibits NF-κB signaling pathway to overcome tumor stemness and bortezomib resistance in multiple myeloma.

Multiple myeloma (MM) is incurable and the second most common hematologic malignancy in plasma cells. Multiple myeloma stem cell-like cells (MMSCs), a rare population of MM cells, are believed to be the major cause of drug resistance and high recurrence rates in patients with MM. Therefore, developing novel strategies to eradicate MMSCs may favor myeloma treatment. In this study, based on the drug repositioning strategy, we found that albendazole (ABZ), a broad-spectrum antiparasitic drug, selectively suppresses the proliferation of multiple myeloma cells in vitro and in vivo and decreases number of aldehyde dehydrogenase (ALDH)-positive MMSCs in MM. Furthermore, RNA-seq of MM cells after ABZ treatment revealed that inhibition of the nuclear factor kappa-B (NF-κB) pathway is a key mediator of ABZ against MM. Moreover, we demonstrated that ABZ can resensitize cells resistant to bortezomib and overcome MMSCs-induced bortezomib resistance by decreasing ALDH1+ MMSCs numbers. Our findings provide preclinical evidence for utilizing the previously known pharmacologically active drug albendazole for the treatment of multiple myeloma.

Targeting lactate dehydrogenase A (LDHA) exerts antileukemic effects on T-cell acute lymphoblastic leukemia.

BACKGROUND: T-cell acute lymphoblastic leukemia (T-ALL) is an uncommon and aggressive subtype of acute lymphoblastic leukemia (ALL). In the serum of T-ALL patients, the activity of lactate dehydrogenase A (LDHA) is increased. We proposed that targeting LDHA may be a potential strategy to improve T-ALL outcomes. The current study was conducted to investigate the antileukemic effect of LDHA gene-targeting treatment on T-ALL and the underlying molecular mechanism. METHODS: Primary T-ALL cell lines Jurkat and DU528 were treated with the LDH inhibitor oxamate. MTT, colony formation, apoptosis, and cell cycle assays were performed to investigate the effects of oxamate on T-ALL cells. Quantitative real-time PCR (qPCR) and Western blotting analyses were applied to determine the related signaling pathways. A mitochondrial reactive oxygen species (ROS) assay was performed to evaluate ROS production after T-ALL cells were treated with oxamate. A T-ALL transgenic zebrafish model with LDHA gene knockdown was established using CRISPR/Cas9 gene-editing technology, and then TUNEL, Western blotting, and T-ALL tumor progression analyses were conducted to investigate the effects of LDHA gene knockdown on T-ALL transgenic zebrafish. RESULTS: Oxamate significantly inhibited proliferation and induced apoptosis of Jurkat and DU528 cells. It also arrested Jurkat and DU528 cells in G0/G1 phase and stimulated ROS production (all P < 0.001). Blocking LDHA significantly decreased the gene and protein expression of c-Myc, as well as the levels of phosphorylated serine/threonine kinase (AKT) and glycogen synthase kinase 3 beta (GSK-3ß) in the phosphatidylinositol 3'-kinase (PI3K) signaling pathway. LDHA gene knockdown delayed disease progression and down-regulated c-Myc mRNA and protein expression in T-ALL transgenic zebrafish. CONCLUSION: Targeting LDHA exerted an antileukemic effect on T-ALL, representing a potential strategy for T-ALL treatment.

Deregulation of mitochondrial ATPsyn-ß in acute myeloid leukemia cells and with increased drug resistance.

The mechanisms underlying the development of multidrug resistance in acute myeloid leukemia are not fully understood. Here we analyzed the expressions of mitochondrial ATPsyn-ß in adriamycin-resistant cell line HL-60/ADM and its parental cell line HL-60. Meanwhile we compared the differences of mitochondrial ATPsyn-ß expression and ATP synthase activity in 110 acute myeloid leukemia (AML, non-M3) patients between relapsed/refractory and those in remission. Our results showed that down-regulation of ATPsyn-ß expression by siRNA in HL-60 cells increased cell viability and apoptotic resistance to adriamycin, while up-regulation of mitochondrial ATPsyn-ß in HL-60/ADM cells enhanced cell sensitivity to adriamycin and promoted apoptosis. Mitochondrial ATPsyn-ß expression and ATP synthase activity in relapsed/refractory acute myeloid leukemia patients were downregulated. This downregulated ATPsyn-ß expression exhibited a positive correlation with the response to adriamycin of primary cells. A lower expression of ATPsyn-ß in newly diagnosed or relapsed/refractory patients was associated with a shorter first remission duration or overall survival. Our findings show mitochondrial ATPsyn-ß plays an important role in the mechanism of multidrug resistance in AML thus may present both a new marker for prognosis assessment and a new target for reversing drug resistance.

Fms-like tyrosine kinase (FLT) 3 and FLT3 internal tandem duplication in different types of adult leukemia: analysis of 147 patients.

AIM: To assess the expression level of fms-like tyrosine kinase 3 (FLT3), the incidence of FLT3/internal tandem duplications (ITD) mutation, and prognostic value of FLT3 changes in different types of adult leukemia. METHODS: Bone marrow mononuclear cells were isolated from 147 adult patients with leukemia. Reverse transcriptase polymerase chain reaction (PCR) was used to screen FLT3/ITD mutation and quantitative PCR was performed to evaluate the expression of the FLT3 transcript. Flow cytometry was used for detection of FLT3 receptor protein expression on bone marrow mononuclear cells. Pearson correlation analysis was performed to estimate the significance of FLT3. RESULTS: FLT3 expression was higher in acute myeloid leukemia and B-acute lymphoid leukemia than in T-acute lymphoid leukemia (P=0.006, P=0.001) and chronic myelogenous leukemia (P<0.001). In chronic myelogenous leukemia, FLT3 expression in blast transformation phase was higher than in acceleration phase (P=0.023). Surface expression of FLT3 protein was correlated with high percentage of bone marrow blasts and with FLT3 mRNA expression (r=0.366, P<0.001) in acute leukemia. FLT3/ITDs in the juxtamembrane domain were found in 25% of patients with acute myeloid leukemia and 7% of patients with acute lymphoid leukemia. FLT3/ITD positive sequences had 36, 42, and 57 nucleotides. FLT3/ITD mutation was associated with a higher white blood cell count, higher marrow blast percentage, and elevated serum lactate dehydrogenase (P=0.045, P=0.014, P<0.001, respectively) and not associated with a higher FLT3 mRNA and FLT3 protein expression, and lower complete remission (P=0.091, P=0.060, P=0.270, respectively). CONCLUSION: FLT3 expression levels differed in different types of adult leukemia. Overexpression of FLT3 and presence of a positive FLT3/ITD mutation in acute leukemia were associated with unfavorable clinical characteristics and poor prognosis.

Dup-697, a specific COX-2 inhibitor, suppresses growth and induces apoptosis on K562 leukemia cells by cell-cycle arrest and caspase-8 activation.

This investigation was designed to assess the effect of DuP-697 on growth and apoptosis in a human chronic myeloid leukemia (CML) cell line (K562 cells) and primary CML cells from CML patient bone marrow. DuP-697 significantly suppressed K562 cells and primary CML cells growth and induced apoptosis in a concentration-dependent manner and the growth-inhibiting effect was independent on Philadelphia chromosome. The IC50 of DuP-697 at 36 h was 31.7 muM. It arrested G1-S phase transmit on cell cycle and its apoptosis activity was partially abrogated by pretreating K562 cells with Z-IETD-fmk, a specific inhibitor of caspase-8. This study suggested that Dup-697 suppresses growth and induces apoptosis on K562 leukemia cells by cell-cycle arrest and caspase-8 activation.

[Cytotoxic activities of Celecoxib on leukemic cells and the synergistic effects of Celecoxib with Imatinib thereupon].

OBJECTIVE: To explore the effects of Celecoxib on cell growth, early apoptosis, PRb and P27(Kip) protein expression of human leukemic cells of the line K562 and to determine its synergistic effects in combination of Imatinib. METHODS: K562 cel1s were incubated with Celecoxib of the concentrations of 0, 10, 20, 40, 80, and 160 micromol/L for 36 hours, the cell vitality was determined by MTT assay, the early apoptosis was examined by flow cytometry (FC). The percentage of annexin V positive was detected by FC so as to evaluate the early apoptosis. The expression of PRb and P27(Kip) protein was detected by Western blotting. Another K562 cells were incubated with Celecoxib of the concentration of 40 micromol/L, Imatinib of the concentration of 0.2 micromol/L, or Celecoxib 40 micromol/L + Imatinib 0.2 micromol/L for 36 hours. Then the cell vitality and early apoptosis of the cells were detected so as to evaluate the synergistic effects of the combination of Celecoxib and Imatinib. RESULTS: Celecoxib inhibited the K562 cells vitality significantly in a dose-dependent manner. The apoptotic rate of K562 cells was elevated with the increasing Celecoxib concentration. Celecoxib down-regulated the expression of PRb protein and up-regulated the expression of P27(Kip) protein in the K562 cells. The combination of Celecoxib and Imatinib exhibited evidently synergistic effects in terms of anti-proliferation on K562 cells. CONCLUSION: Celecoxib inhibits the proliferation and induces apoptosis on leukemic cells in a dose-dependent fashion. The anti-proliferation effect of Celecoxib may be associated with the down-regulation of PRb expression and up-regulation of P27(Kip) expression. Combination of Celecoxib and Imatinib has obviously synergistic effects in terms of anti-proliferation on K562 cells.

[Analysis of angiogenesis related proteins and its implication in type-2 hereditary hemorrhagic telangiectasia].

OBJECTIVE: To detect the level of transforming growth factor-beta1 (TGF-beta1), TGF-beta2, vascular endothelial growth factor (VEGF) and platelet-derived growth factor receptor-alpha (PDGFRalpha) in plasma and peripheral blood leukocytes in a hereditary hemorrhagic telangiectasia type 2 (HHT-2) family, and explore the implication of angiogenesis related proteins in HHT-2 pathogenesis. METHODS: The diagnosis of the HHT-2 patient was based on clinical features and further confirmed by determining a C1231T mutation of activin receptor-like kinase 1 (ALK1) gene. Five other new members in this family were evaluated with ALK1 gene screening and clinical manifestation. Plasma level of TGF-beta1, TGF-beta2 or VEGF was measured by ELISA, and the expression of PDGFRalpha,TGF-beta1, and VEGF in peripheral blood leukocytes by flow cytometry combined with direct or indirect immunofluorescence. RESULTS: No C1231T mutation was detected in exon 8 of ALK1 gene in the 5 new members. Plasma TGF-beta1 and TGF-beta2 concentration in 3 affected HHT case was (16 954 +/- 3 709) ng/L and (11 548 +/- 2 611) ng/L, respectively, compared with that of normal control, the difference was not significant (P > 0.05). VEGF concentration in the 3 HHT patients, 6 unaffected family members and 6 normal controls was (179.2 +/- 22.0) microg/L, (149.8 +/- 22.7) microg/L and (132.9 +/- 21.0) microg/ L, respectively. Plasma VEGF level in HHT patients was significantly higher than that in normal subjects (P < 0.025). Peripheral leukocyte PDGFRalpha and VEGF in HHT patients and unaffected family members were markedly higher than that of normal control (P < 0.05 and P < 0.02), while TGF-beta1 distribution was similar in HHT patients and normal subjects. CONCLUSION: Compared with normal controls there is no difference in plasma TGF-beta1 concentration on peripheral leukocytes of HHT patients. Plasma VEGF concentration or leukocytes VEGF expression in HHT is significantly higher than that of normal subjects. Leukocytes PDGFRalpha expression in HHT is significantly higher than that of normal control. These changes may be associated with a compensable mechanism in HHT.

[Acute monocytic leukemia after orthotopic liver transplantation: clinical features, molecular genetics, and significance thereof].

OBJECTIVE: To study the clinical features and molecular genetics of acute monocytic leukemia (AML) after orthotopic liver transplantation and significance thereof. METHODS: The clinical manifestations, laboratory findings, development, diagnosis, treatment, and prognosis of the first case of AML after orthotopic liver transplantation in the world, a Chinese, male, aged 46, were observed. RT-PCR was used to analyze the mRNA expression of FLT3, Pim-1, and Hsp-70 in the bone marrow mononuclear cells (BMMCs) of the patient. Nucleotide sequence analysis was used to detect the mutation of FLT3-ITD. Flow cytometry was used to examine the protein expression of C-kit, a stem cell factor receptor, and platelet derived growth factor receptor-alpha (PDGFRalpha). RESULTS: (1) Three months after the orthotopic liver transplantation the patient manifested symptoms and signs of anemia and thrombocytopenia. Laboratory tests found increase of white blood cells and myeloblasts with Auer's rods. Cytogenetic analysis showed a genotype of 46XY/47XY with an extra chromosome 8. Bone marrow examination revealed increased promonocytes. The diagnosis of chronic myelomonocytic leukemia was made. Five months after the liver transplantation the disease developed to AML. The patient underwent combined chemotherapy (HA or DA regimens) for 5 courses and showed a partial remission both hematologically and in bone marrow examination at first, however, became resistant to all chemotherapeutic agents. RT-PCR showed absence of wild type FLT3 allele. At last the patient died of infection. (2) A FLT3-ITD mutation of "insertion" type was identified in the BMMCs. The Pim-1 mRNA was weakly expressed and the expression of Hsp-70 mRNA was upregulated in the BMMCs. (3) The protein expression of C-kit and that of PDGFRalpha were both upregulated in the BMMCs as showed by flow cytometry. CONCLUSION: Orthotopic liver transplantation may be complicated with acute leukemia heterogeneous in clinical features and hematology. Certain defects in cytogenetics/molecular genetics may attribute to unfavorable prognosis.