Objective To study the antitumor activity of extract from Salvia plebeia and investigate whether the extract induce apoptosis of K562 cells. Methods The aqueous, petroleum ether, dichloromethane (CH2Cl2), ethyl acetate, and butanol extracts were prepared from the aerial parts of 5. plebeia. Taking fluorouracil as reference, the cytotoxic activities of these extracts on HeLa, A549, SGC-7901, HCT-116, K562, LoVo, DU-145, and HepG2 cells were evaluated. To clarify the apoptosis of K562 cells induced by CH2Cl2 extract, the methods of Hoechst 33258 staining, flow cytometry assay, and DNA ladder assay were investigated. Results The CH2Cl2 extract showed the most potent cytotoxic effect against K562 cells, with an IC50 < 15 μg/mL for 3 d treatment. The characteristic apoptotic symptoms such as DNA fragmentation and chromatin condensation were also observed in the K562 cells. Conclusion The CH2Cl2 extract from S. plebeia may inhibit the cancer cell proliferation by inducing cell apoptosis.

OBJECTIVE: To study the correlation of hematomorphology, bone marrow cytogenetics and clinical biochemical parameters with the prognosis of non-Hodgkin's lymphoma with bone marrow invasion. METHODS: Morphological analysis of bone marrow cells was performed by routine bone marrow puncture.Chromosome samples were prepared by short-term bone marrow culture. Karyotype analysis was carried out by R-banding in 28 patients. P53 gene was detected by fluorescence in situ hybridization (FISH). Serum lactate dehydrogenase (LDH) of all patients was determined and compared. RESULTS: In all patients, bone marrow morphology showed invasion of lymphoma. Chromosome analysis revealed abnormal karyotypes in 19 cases, which yielded an incidence of 67.85%. The proportion of lymphoma cells in bone marrow among those with an abnormal karyotype was much higher than those with a normal karyotype (60.2% vs. 33.5%, P<0.05). FISH assay showed that 9 (32.14%) patients had P53 gene deletion. And the deletion was much more common among those with an abnormal karyotype (42.11% vs. 11.11%, P<0.05). The serum LDH level in patients with an abnormal karyotype was significantly higher compared with whose with a normal karyotype (1464.37 U/L vs. 294.33 U/L, P<0.05). CONCLUSION Patients with abnormal karyotypes have a higher rate of P53 gene deletion, and their LDH level is significantly higher than those with a normal karyotype, which predicted a relatively poor prognosis.
Adult ; Bone Marrow ; Child ; Chromosome Aberrations ; Humans ; In Situ Hybridization, Fluorescence ; Karyotyping ; Lymphoma, Non-Hodgkin

Objective To investigate the mechanism of matrine in inhibition of proliferation the proliferation of human chronic myeloid leukemia (CML) K562 cells via MEK-ERK signaling pathway. Methods Western blot was used to detect the expression of MEK1, ERK1/2, Shc and SHP2 (the signal effect molecules of MEK-ERK pathway) in K562 cells. The transcription and translation of bcr-abl and target protein (bcl-xL, Cyclin D1, c-myc and p27) were detected by RT-PCR and Western blot. Results Matrine was able to significantly inhibit the phosphorylation of MEK1, ERK1/2, Shc and SHP2 in K562 cells and suppress the protein and mRNA expression of bcr-abl. Moreover, the expressions of bcl-xL, Cyclin D1 and c-myc were down-regulated significantly, while the expression level of p27 (a negative regulator of cell cycle progression) was increased markedly after matrine treatment. Conclusions Suppression of the growth of human CML K562 cells is related to the inhibition of bcr-abl-mediated MEK-ERK pathway activity. The down-regulation of phosphorylated proteins or protein kinases activity in signaling pathways might be an important molecular mechanism in control the activity of MEK-ERK pathway.

OBJECTIVE: To characterize the mutational profile of patients with core-binding factor acute myeloid leukemia (CBF-AML). METHODS: A total of 81 acute myeloid leukemia patients were recruited, which included 36 cases of CBF-AML and 45 cases of cytogenetically normal acute myeloid leukemia (CN-AML) . Mutations of FLT3-ITD, FLT3-TKD, NPM1, c-KIT, NRAS, KRAS, TET2, IDH1/2, RUNX1, DNMT3A, GATA2, ASjXL1, TP53, PTPN11, JAK2V617F, SETBP1 and CEBPA genes were simultaneously detected by DNA-based PCR and Sanger sequencing. RESULTS: Over all, mutations were detected in 68 patients (83.9%), with the most common ones including double CEBPA mutations (n=17), followed by NPM1 (n=15), c-KIT (n=11), NRAS (n=10), TET2 (n=9), FLT3-TKD (n=9), FLT3-ITD (n=8), IDH1 (n=7), RUNX1 (n=7), KRAS (n=7), DNMT3A (n=6), IDH2 (n=4), and GATA2 (n=4) mutations. AML1-ETO and CBFβ-MYH11 fusions were present in 21 and 15 patients, respectively. Coexistence of ≥2 mutations was more common in CN-AML comparing with CBF-AML. The mutation rate of NPM1, FLT3-ITD, DNMT3A, IDH1 and CEBPA double mutations were higher in patients with CN-AML. NRAS, c-KIT and KRAS mutations were identified more frequently in patients with CBF-AML (P<0.05). Based on the function, aberration of genes involved in DNA methylation, NPM1 proteins and transcription predominated in CN-AML, while tyrosine kinase receptor signaling and RAS pathways have predominated in CBF-AML. CONCLUSION The genomic landscape of CBF-AML patients has differed from that of CN-AML patients. Synergy of fusion genes with particular mutations may impact the clinical phenotype and prognosis of patients.
Core Binding Factors ; genetics ; DNA Mutational Analysis ; Humans ; Leukemia, Myeloid, Acute ; genetics ; Mutation ; Prognosis

OBJECTIVE: To characterize the molecular genetics of 81 patients with acute monocytic leukemia (AML). METHODS: Fluorescence in situ hybridization (FISH) was employed to detect MLL gene rearrangements. Combined mutations of 17 genes were detected by DNA-based PCR and Sanger sequencing. RESULTS: Sixty seven patients were found to harbor at least one mutation. The most commonly mutated gene was NPM1 (n=18), which was followed by FLT3-ITD (n=16), NRAS (n=16), DNMT3A (n=15), TET2 (n=12), RUNX1 (n=11) and KRAS (n=9). Based on the functions of mutated genes, the most frequently involved genes were those involved in DNA methylation (38.27%), tyrosine kinase receptor signaling (32.1%), transcription regulation (28.4%), and RAS pathway (24.7%). Single gene mutation predominated in patient with cytogenetic abnormalities, while coexistence of 2 mutations have predominated in patient with normal cytogenetic findings. Stratified by cytogenetic findings, patients with single gene mutations (intermediate-risk group) had significantly higher complete remission (CR) rates than those with ≥2 gene mutations (unfavorable-risk group) (91.7% vs. 57.6% , 87.5% vs. 25.0%, P =0.0319, 0.0117, respectively). CONCLUSION Over 80% of AML patients were found to harbor at least one mutation. Their clinical phenotype and prognosis may be impacted by the synergy of MLL gene rearrangement and multiple mutations. For patients under the same risk stratification, the number of mutations is reversely correlated with the CR rate.
Cytogenetics ; Humans ; In Situ Hybridization, Fluorescence ; Leukemia, Monocytic, Acute ; Leukemia, Myeloid, Acute ; Mutation ; Prognosis ; fms-Like Tyrosine Kinase 3

OBJECTIVE: To delineate the clinical and molecular characteristics of a patient with myeloid neoplasm and co-existence of t(7;11)(p15;p15) and t(5;12)(q33;p13) translocations. METHODS: Clinical data of the patient was collected. Conventional karyotyping, reverse transcriptase (RT)-PCR and next generation sequencing (NGS) were carried out to delineate its genetic features. RESULTS: The patient has featured recurrent rash, fatigue, loss of appetite and splenomegaly. Laboratory test suggested hyperleukocytosis of FAB-M2-subtype. Neither eosinophilia nor basophilia was presented. NUP98/HOXA9 and ETV6/PDGFRB fusion genes were detected by RT-PCR. NGS and DNA-PCR showed the co-existence of WT1 p.C423Y, KRAS p.G12D and DNMT3A p.R882C mutations. The patient achieved morphological remission after imatinib plus coventional chemotherapy (standard IAC regimen). However, the disease has relapsed shortly after. Treatment was switched to HHT-Ara-C-Acla regimen, no hematological response was observed. The ETV6/PDGFRB fusion gene was undetectable in bone marrow sample, though strong expression of NUP98/HOXA9 was detectable throughout the whole course. CONCLUSION Acute myeloid leukemia in association with the co-existence of NUP98/HOXA9 and ETV6/PDGFRB fusion genes have unique clinical and genetic features. Imatinib seems to have no impact on the overall survival in such cases.
Chromosomes, Human ; Humans ; Karyotyping ; Leukemia, Myeloid, Acute ; Myeloproliferative Disorders ; Oncogene Proteins, Fusion ; Translocation, Genetic

Objective:To explore the coexisting gene mutations of FLT3-ITD mutation and its association with partial clinical parameters in acute myeloid leukemia (AML).Methods:The clinical data of 236 newly diagnosed AML outpatients and hospitalized patients of Changzhou No.2 People's Hospital and the Second People's Hospital of Wuxi between December 2012 and August 2019 were retrospectively analyzed. Genome DNA-polymerase chain reaction (PCR) combined with Sanger sequencing was used to detect FLT3-ITD mutations, and 51 tumor target gene mutations in patients with FLT3-ITD mutations were detected by using high-throughput DNA sequencing combined with Sanger sequencing.Results:Among 236 AML patients, FLT3-ITD mutations were found in 71 cases (30.1%). About 97.2% (69/71) patients with FLT3-ITD mutations were accompanied by additional mutations, of which 19 patients harbored double coexisting genes mutations, 24 patients harbored 3 coexisting genes mutations and 26 patients harbored ≥ 4 coexisting genes mutations. The most common coexisting genes mutations were NPM1 (55 cases, 77.5%), followed by DNMT3A (36 cases, 50.7%), TET2 (9 cases, 12.7%), CEBPA (5 cases, 7.0%), IDH1 (4 cases, 5.6%) and NRAS (4 cases, 5.6%). In FLT3-ITD mutation group, the hemoglobin level of patients with DNMT3A mutation type was lower than that of those with DNMT3A wild type ( t = -2.37, P = 0.020); the hemoglobin level of patients with NPM1 mutation type was higher than that of those with NPM1 wild type ( t = 2.04, P = 0.045). The platelet in patients with 3 mutations and ≥ 4 mutations was higher than that in those with double mutations ( χ2 = 7.72, P = 0.021). After chemotherapy in 71 patients, the curative effect of 66 cases was evaluable, and the white blood count of 18 patients who did not reach complete remission was higher than that of 48 patients who reached complete remission ( Z = -2.74, P = 0.006). Conclusions:Most FLT3-ITD mutated patients with AML commonly show coexisting gene mutations, and the mutation types of coexisting genes are correlated with the clinical features of patients.

Objective The inhibitory effect of the PARP inhibitor olaparib on human acute myeloid leukemia HL-60 cells was studied. Methods The HL-60 cells in logarithmic growth phase were treated with different concentrations(1. 25,2. 5,5 and 10 μmol/L) of olaparib for different time. The CCK-8 assay was used to detect the inhibitory effect of olaparib on HL-60 cells. The apoptotic level of HL-60 cells was detected by Annexin-V/PI double staining method,and the expression of related signal proteins ( PARP-1 and caspase-3)in HL-60 cells was detected by Western blot. Results HL-60 cells were inhibited by olaparib at dif-ferent concentrations(1. 25,2. 5,5 and 10 μmol/L) for 48 h,and the inhibition rate gradually increased with the prolongation of the action time;at the same time,the apoptotic rate was increased in HL-60 cells after olaparib treatment for 48 h,showing a dose-de-pendent manner;the PARP activity was inhibited and caspase-3 was activated in HL-60 cells treated with olaparib. Conclusion The PARP inhibitor olaparib not only inhibits proliferation of HL-60 cells,but it also promotes apoptosis of HL-60 cells by inhibi-ting PARP activity and activating caspase-3.

Objective: To explore the effects and possible mechanisms of the novel pan-FGFR inhibitor BGJ398 on KG-1 cells in vitro. Methods: Effects of BGJ398 on cells proliferation were detected by CCK-8, the apoptosis was assessed by Annexin V-FITC. Reverse transcriptionquantitative polymerase chain reaction (q-PCR) analysis was used to detect the expression of apoptosis-related genes B cell lymphoma-2 (Bcl-2) and caspase-3. Western blotting analysis was performed to explore the proteins expression levels of Bcl-2, caspase-3 and the expression of p-AKT, p-S6K, p-ERK and FGFR1. Results: BGJ398 effectively inhibited cell proliferation by dose-dependent manners. BGJ398(1.4 µmol/L) induced apoptosis of KG-1 cells by 36.4%, compared with 4.5% in the control group(P<0.001). Treatment with BGJ398 at 1.4 µmol/L led to significant increases in the expression levels of caspase-3, and decreases in the expression of Bcl-2 (P<0.005). In accordance with these results, Western blot analysis further confirmed the increased expression of Bcl-2 protein along with elevated caspase-3 activity. In addition, BGJ398 markedly down-regulated FGFR1OP2-FGFR1 fusion protein, p-AKT and p-S6K expression, but not p-ERK expression. Conclusion Novel pan-FGFR inhibitor BGJ398 substantially suppressed KG-1 cell growth and induced apoptosis by inhibiting the expression of FGFR1, p-AKT, p-S6K and regulating apoptosis-related proteins.

OBJECTIVETodelineate the clinical and genetic features of a patient with myeloproliferative neoplasm (MPN) in association with PDGFRA and EVI1 genes rearrangements.

METHODSClinical data of the patient was collected. Conventional cytogenetics, fluorescence in situ hybridization (FISH) and nested PCR were carried out for the patient.

RESULTSThe patient has featured recurrent rash, joint pain, and intermittent fever. Laboratory tests showed hyperleukocytosis and marked eosinophilia. Physical examination revealed splenomegaly. His karyotype was 46,XY,t(3;5)(q26;q15)[6]/46,XY[10]. FISH assay showed that both PDGFRA and EVI1 genes were rearranged. Molecular studies of the mRNA suggested that there was a in-frame fusion between exon 12 of the PDGFRA gene and exon 9 of the FIP1L1 gene. Imatinib was initiated at a dosage of 200 mg, and after 10 months, the signal of the FIP1L1-PDGFRA fusion gene was undetectable in bone marrow sample. However, the expression of EVI1 mRNA was stable, with no significant difference found between the patient and 10 healthy controls.

CONCLUSIONMPN in association with PDGFRA and EVI1 genes rearrangements have unique clinical and genetic features. Genetic testing is helpful for early diagnosis. Imatinib may be effective for the treatment.

Antineoplastic Agents ; therapeutic use ; Base Sequence ; Chromosome Banding ; Chromosomes, Human, Pair 3 ; genetics ; Chromosomes, Human, Pair 5 ; genetics ; DNA-Binding Proteins ; genetics ; Gene Rearrangement ; Humans ; Imatinib Mesylate ; therapeutic use ; In Situ Hybridization, Fluorescence ; Karyotyping ; MDS1 and EVI1 Complex Locus Protein ; Male ; Myeloproliferative Disorders ; drug therapy ; genetics ; Proto-Oncogenes ; genetics ; Receptor, Platelet-Derived Growth Factor alpha ; genetics ; Transcription Factors ; genetics ; Translocation, Genetic ; Treatment Outcome ; Young Adult