[Establishment of a Patient-Derived T-Cell Acute Lymphoblastic Leukemia Xenograft Model in Novel Immunodeficient NCG Mice].

OBJECTIVE: The leukemia cells from patients with T-cell acute lymphoblastic leukemia (T-ALL) were inoculated into NCG mice to establish a stable human T-ALL leukemia animal model. METHODS: Leukemia cells from bone marrow of newly diagnosed T-ALL patients were isolated, and the leukemia cells were inoculated into NCG mice via tail vein. The proportion of hCD45 positive cells in peripheral blood of the mice was detected regularly by flow cytometry, and the infiltration of leukemia cells in bone marrow, liver, spleen and other organs of the mice was detected by pathology and immunohistochemistry. After the first generation mice model was successfully established, the spleen cells from the first generation mice were inoculated into the second generation mice, and after the second generation mice model was successfully established, the spleen cells from the second generation mice were further inoculated into the third generation mice, and the growth of leukemia cells in peripheral blood of the mice in each group was monitored by regular flow cytometry to evaluate the stability of this T-ALL leukemia animal model. RESULTS: On the 10th day after inoculation, hCD45+ leukemia cells could be successfully detected in the peripheral blood of the first generation mice, and the proportion of these cells was gradually increased. On average, the mice appeared listless 6 or 7 weeks after inoculation, and a large number of T lymphocyte leukemia cells were found in the peripheral blood and bone marrow smear of the mice. The spleen of the mice was obviously enlarged, and immunohistochemical examination showed that hCD3+ leukemia cells infiltrated into bone marrow, liver and spleen extensively. The second and third generation mice could stably develop leukemia, and the average survival time was 4-5 weeks. CONCLUSION: Inoculating leukemia cells from bone marrow of patients with T-ALL into NCG mice via tail vein can successfully construct a patient-derived tumor xenografts (PDTX) model.

OGG1 contributes to hepatocellular carcinoma by promoting cell cycle-related protein expression and enhancing DNA oxidative damage repair in tumor cells.

BACKGROUND: This study aimed to analyze the expression of 8-oxoguanine DNA glycosylase (OGG1) in patients with hepatocellular carcinoma (HCC) and its effect on prognosis by bioinformatics techniques and to determine its possible carcinogenic mechanism through data mining. METHODS: The difference in OGG1 expression between healthy people and HCC patients was searched and analyzed by TCGA and GEO databases, and the effect of OGG1 on prognosis was judged by survival analysis. Meanwhile, the possible molecular mechanism of OGG1 in the tumorigenesis and development of HCC was explored by GO analysis, KEGG analysis, immune infiltration analysis, protein-protein interaction network, promoter methylation analysis, and so forth. Quantitative polymerase chain reaction (qPCR) was used to examine the gene expression in 36 pairs of HCC tissues and adjacent tissues. RESULTS: The expression of OGG1 in HCC patients was higher than that in healthy people, and the overexpression of OGG1 might stimulate cell proliferation by increasing the activity of cell cycle-related proteins. CONCLUSION: The alteration of OGG1 was significantly correlated with the tumorigenesis and development of HCC. OGG1 is expected to be a new biomarker for evaluating the prognosis of HCC and a new target for the treatment of HCC.

[Effect of Celastrol Based on IRAK4/ERK/p38 Signaling Pathway on Proliferation and Apoptosis of Multiple Myeloma Cells].

OBJECTIVE: To investigate the effect of celastrol on the proliferation and apoptosis of human multiple myeloma (MM) cell lines, reveal the relationship between IRAK4/ERK/p38 signaling pathway and celastrol regulating the proliferation and apoptosis of H929 and ARP-1 cells, and explore whether celastrol combined with bortezomib has synergistic effect. METHODS: CCK-8 method was used to detect the viability of MM cell lines H929 and ARP-1 treated by different concentrations of celastrol, bortezomib, and their combination, and the synergistic effect was determined by Kim's formula. The apoptosis rate of H929 cells and necrosis rate of ARP-1 were detected by Annexin V/PI method. The expression of key proteins and apoptosis proteins in IRAK4/ERK/p38 signaling pathway were detected by Western blot. RESULTS: Celastrol could significantly inhibit the proliferation of H929 and ARP-1 cells (r=0.9018, r=0.9244) and induce apoptosis in a time-dependent manner. Compared with the control group, celastrol could significantly up-regulate the expression of PARP and cleaved caspase-3 while down-regulate the expression of p-IRAK4, p-ERK, and p-p38 in H929 and ARP-1 cells. Celastrol and bortezomib alone inhibited the proliferation of H929 and ARP-1 cells. Compared with celastrol and bortezomib alone, their combination had lower cell survival rate and higher apoptosis rate (P<0.05). CONCLUSION: Celastrol can inhibit the proliferation and promote the apoptosis of H929 and ARP-1 cells, which may be related to inhibiting the phosphorylation of IRAK4 and blocking the activation of IRAK4/ERK/p38 signaling pathway. Celastrol combined with bortezomib has synergistic effect, which can more effectively inhibit the proliferation and induce apoptosis of H929 and ARP-1 cells.

[Analysis of Serum Metabonomics in Patients with Multiple Myeloma Based on Liquid Chromatography-Mass Spectrometry].

OBJECTIVE: To observe the changes of serum metabolites in patients with multiple myeloma (MM) by metabonomics, and explore the potential biomarkers for diagnosis, prognosis, and progression of MM. METHODS: Serum samples were collected from 26 patients with MM and 50 healthy controls. The data detected by liquid chromatography-mass spectrometry (LC-MS) was input into SIMCA-14.0 software for multivariate statistical analysis. Principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), and orthogonal partial least squares discriminant analysis (OPLS-DA) were used to analyze the changes of metabolites. RESULTS: The metabolic change of uric acid and trans-vaccenic acid in serum samples of MM patients was 9.39 times and 2.77 times of these in healthy people, respectively, which were significantly higher than those of healthy people, and the difference was statistically significant(P<0.01). CONCLUSION: Uric acid and trans-vaccenic acid are expected to be important metabolic indicators for the diagnosis, prognosis, and efficacy evaluation of MM, thus providing some clues for the pathogenesis of MM.

[Clinical Prognostic Factors Analysis of Initially Treated AML Children with t(8;21)/RUNX1-RUNX1T1].

OBJECTIVE: To investigate the clinical prognostic factors of initially-treated AML children with t(8;21)/RUNX1-RUNX1T1+. METHODS: Clinical data of 41 initially-treated AML children with t(8;21)/RUNX1-RUNX1T1+ in our hospital in period from January 2009 to January 2017 were retrospectively analyzed. The baseline clinical characteristics, cumulative recurrence, event-free survival (EFS) and overall survival (OS) were recorded, and the influencing factors of prognosis were evaluated by χ2 test and Cox regression model. RESULTS: The complete remission (CR) rates in the first course and the second course of induction chemotherapy were respectively 82.93% (34/41) and 97.56% (40/41). The median EFS time and OS time were 30 months and 31 months respectively. The EFS rate and OS rate of children with CR after the first treatment course were significantly higher than those of children without CR (P＜0.05). The EFS rate of male children was significantly higher than that of female children (P＜0.05). The OS rate of children < 10 years old was significantly higher than that of children≥10 years old (P＜0.05). The expression level of RUNX1-RUNX1T1 gene after the second induction remission was the influencing factor of cumulative recurrence rate, EFS rate and OS rate in children (P＜0.05). Multivariate analysis by Cox regression model showed that the decreased levels of RUNX1-RUNX1T1 gene expression < 3 log after the second induction remission was the independent risk factor for EFS rate and OS rate in children (P＜0.05). The cumulative recurrence rate of children with RUNX1-RUNX1T1 gene expression increase for＞1 log after decreased 3 log was significantly higher than that of children with≤1 log (P＜0.05). CONCLUSION: Iuithally-treated AML children with t(8;21)/RUNX1-RUNX1T1+ show the fine clinical prognosis after standard chemotherapy. The expression level of RUNX1-RUNX1T1 gene should be closely relates with the recurrence and long-term survival of AML children.

[Modified Shengma Biejia Decoction Combined with CAG Program for Elderly Acute Myeloid Leuke- mia Patients with Yin Deficiency Toxin Stasis Syndrome].

OBJECTIVE: To observe the efficacy and safety of modified Shengma Biejia Decoction (MSBD) combined with CAG program in treating elderly acute myeloid leukemia (AML) patients with yin deficiency toxin stasis syndrome (YDTSS). METHODS: Totally 46 elderly AML patients were assigned to the treatment group (24 cases; treated with MSBD + CAG) and the control group (22 cases; treated with CAG + placebos of Chinese medicine) according to random digit table. The therapeutic course of CM placebo or MSBD was 21 days. The clinical efficacy and adverse reactions were observed. Meanwhile, physical state (ECOG Score), transfusion dependency, and TCM syndrome score were compared before and after treatment. RESULTS: (1) The complete remission rate was 54% (13/24) and the objective response rate (ORR) was 71% (17/24) in the treatment group, obviously higher than those of the control group [36% (8/22); 54% (13/24)], with statistical difference (P = 0.036, 0.042). When comparing the efficacy based on risk level, the moderate and poor ORR was 71% (10/14) and 67% (6/9) in the treatment group, and 57% (8/14) and 33% (2/6) in the control group, with statistical difference between the two groups (P = 0.048; P = 0.010). (2) Compared with before treatment in the same group, the ECOG score significantly decreased, the average infusion time of red cells and platelets were markedly prolonged in the treatment group after treatment (P < 0.05). ECOG score, the average infusion time of red cells and platelets were significantly better in the treatment group than in the control group after treatment (P < 0.05). (3) Compared with before treatment in the same group, scores of fever, hemorrhage, and bone pain were markedly reduced in the control group (P < 0.05); scores of fever, fatigue, hemorrhage, dry mouth, and bone pain were markedly reduced in the treatment group (P < 0.05). Better effect in relief of fever, fatigue, hemorrhage, dry mouth, and so on was obtained in the treatment group than in the control group (P < 0.05). (4) In aspect of hematotoxicity, the incidence of neutropenia, anemia, thrombocytopenia was obviously lower in the treatment group than in the control group [29.2% (7/24) vs 54.5% (12/22); 16.7% (4/ 24) vs 45.5% (10/22); 33.3% (8/24) vs 63.6% (14/22); P < 0.05]. The incidence of fatigue and anorexia was obviously lower in the treatment group than in the control group [37.5% (9/24) vs 63.6% (14/22), 37.5% (9/24) vs 81.8% (18/22); P < 0.05]. CONCLUSION: MSBD combined with CAG program in treating elderly AML patients with YDTSS, with efficacy enhancing toxicity reducing effect, had distinct advantages in improving physical condition and clinical symptoms, and reducing transfusion dependency.

[Apoptosis of Kasumi-1 cells induced by puerariae radix flavones and its molecular mechanism].

OBJECTIVE: To explore the effects and the molecular mechanism of puerariae radix flavones (PRF) on acute myeloid leukemia cell line Kasumi-1 cells in vitro. METHODS: Kasumi-1 cells treated by PRF for 48 hours were observed with Wright's and Hoechst 33258 dying. The apoptotic cells were analyzed by flow cytometry with AnnexinV/PI staining. The expression levels of bcl-2, Bim and Caspase-3/-8/-9 protein were assayed by Western blot and the AML1-ETO fusion gene was detected by real-time polymerase chain reaction. RESULTS: PRF could induce Kasumi-1 cells to apoptosis effectively. The proportion of apoptotic cells in 50, 200 and 500 µg/ml PRF treatment groups were (14.1 ± 0.8)%, (17.7 ± 1.3)% and (32.4 ± 1.4)%, respectively, and significantly higher than that of control \[(7.8 ± 0.7)%\]. The relative expression levels of the anti-apoptotic Bcl-2 protein were 0.85 ± 0.05, 0.62 ± 0.07 and 0.43 ± 0.05; the apoptotic Bim protein were 0.21 ± 0.06, 0.39 ± 0.04 and 0.75 ± 0.05; the caspase-3 and caspase-9 were 0.92 ± 0.04, 1.21 ± 0.07, 1.33 ± 0.04 and 0.35 ± 0.05, 0.53 ± 0.03, 0.69 ± 0.07, respectively. Compared to the blank control group, all these changes were significant (P < 0.01). Nevertheless, nearly no changes could be observed on the expression level of AML1-ETO fusion gene and caspase-8 protein. CONCLUSION: Apoptosis of Kasumi-1 cells induced by PRF might correlate to the down-regulation of Bcl-2 protein expression and the activation of caspase-3 and caspase-8 protein in the cells. It seemed that all these effects had no relationship with the AML1-ETO fusion gene.

Combining nilotinib and imatinib improves the outcome of imatinib-resistant blast phase CML.

Imatinib resistance is an important hurdle in the treatment of chronic myeloid leukemia (CML), and CML patients with this drug resistance are often given a dismal prognosis. In this case report, an imatinib-refractory blast phase CML patient was treated with a combination of imatinib and nilotinib. A complete hematologic response was achieved within 3 months, the drug combination was well tolerated, and there was a relatively long bone-marrow complete remission. These results suggest that combining imatinib and nilotinib treatment may improve the outcome of imatinib-resistant CML patients in the blast phase. We hypothesize regarding the possible mechanism for the effectiveness of the drug combination by reviewing the recent literature.

A case report of a 33-year chronic phase survivor of chronic myeloid leukemia.

Here, we report a Philadelphia chromosome-positive chronic myeloid leukemia case with the longest chronic phase and overall survival to our knowledge ever reported in the medical literature. During the 33-year chronic phase, he was asymptomatic without any treatment and had normal blood cell values. BCR-ABL silencing might be referred to the uncommon long-term survivor.

Advanced glycation end-products induce heparanase expression in endothelial cells by the receptor for advanced glycation end products and through activation of the FOXO4 transcription factor.

As an endo-ß (1-4)-D: -glucuronidase, heparanase can specifically cleave carbohydrate chains of heparan sulfate (HS) and has been implicated in development of endothelial cells dsyfunction. The advanced glycation end products (AGEs) play a pivotal role in the pathology of diabetic complications. In the present study, we investigated the effect of AGE-bovine serum albumin (AGE-BSA) on heparanase expression in human microvascular endothelial cells (HMVECs) and the underlying molecular mechanisms. The results indicated that in vitro direct exposure of HMVECs to AGE-BSA (300, 1000, and 3000 µg/ml) could increase heparanase mRNA and protein expression in a dose and time-dependent manner. The effect of 1000 µg/ml AGE-BSA could be abolished by neutralization with antibody of the receptor for advanced glycation end products (RAGE). Moreover, pretreatment with inhibitors of nuclear factor-κB (NF-κB) or PI3-kinase did not affect heparanase expression induced by AGE-BSA. Nevertheless, small interference RNA (siRNA) for transcriptional factor FOXO4 could reduce the increase of heparanase expression in HMVECs induced by 1000 µg/ml AGE-BSA. These results suggest that AGEs could induce heparanase expression in HMVECs by RAGE and predominantly through activation of the FOXO4 transcription factor.

[Clinical and biological features in refractory anemia with ringed sideroblasts with fluctuant platelet counts].

The objective of this study was to explore the differences between refractory anemia with ringed sideroblast (RARS) and RARS associated with marked thrombocytosis (RARS-T) in the clinical, biological features and prognosis. The morphological changes of cells were observed by bone marrow smear and biopsy. Immunologic phenotype was analyzed by flow cytometry, and chromosome was examined by conventional chromosomal analysis. JAK2 V617F and MPL W515L mutations were screened by allele-specific polymerase chain reaction (AS-PCR) and sequence analysis. The results showed that this case was clinically diagnosed as RARS with thrombophilia, the level of serum potassium was positively related with platelet counts. When platelets increased, the clusters of atypical giant platelets and megakaryocytes were observed in peripheral blood and bone marrow examined by bone marrow smear and bone marrow biopsy respectively, JAK2 V617F and MPL W515L mutations were negative. It is concluded that RARS may transform into RARS-T accompanied with megakaryocyte proliferation, large atypical platelets and negative JAK2 V617F. Preventing thrombophilia and monitoring relative gene mutations are necessary when atypical giant platelets and fluctuant platelet counts occurred in process of RARS with tendency to RARS-T.

[Inhibitory effect of flavonoids of puerarin on proliferation of different human acute myeloid leukemia cell lines in vitro].

This study was aimed to investigate the inhibitory effect of flavonoids of puerarin (PR) in different concentrations on proliferation of 4 kinds of acute myeloid leukemia (AML) cell lines (Kasumi-1, HL-60, NB4 and U937), and to explore its possible mechanism. The MTT method was used to detected the inhibitory effect of PR on proliferation of AML cell lines. The flow cytometry was adopted to determine the change of cell cycle in vitro. The results showed that a certain concentration of PR could inhibit the proliferation of these 4 cell lines effectively in time-and dose-dependent manners, and the intensity of inhibition on 4 kinds of AML cell lines was from high to low as follows: NB4>Kasumi-1>U937>HL-60. Meanwhile, PR could also change cycle process, cell proportion in G1/G0 phase decreased, cells in S phase increased and Sub-diploid peak also appeared. It is concluded that PR can selectively inhibit the proliferation of 4 AML cell lines and block cell cycle process, especially for NB4 cells.

[Apoptosis of NB4 cells induced by flavonoids of puerarin in vitro].

This study was aimed to investigate the effects of flavonoids of puerarin (PR) on apoptosis of acute promyelocytic leukemia (APL) cell line NB4 cells and its mechanism. The NB4 were treated with PR in vitro, the MTT assay was used to detect the inhibitory effect of PR on cell proliferation. The apoptosis of NB4 cells were detected by flow cytometry labelled with Annexin V/PI. The expressions of pml/rar alpha, bcl-2 and survivin were detected by real time reverse transcription-polymerase chain reaction (real time RT-PCR), the expressions of JNK, p38 MAPK, FasL, caspase 3, caspase 8 were detected by Western blot. The results showed that with the increasing of PR concentrations, the apoptosis rates of NB4 cells were gradually elevated. Simultaneously, the mRNA expression of pml/rar alpha, bcl-2 and survivin decreased, while the protein expression of JNK, FasL, caspase 3 and caspase 8 increased, which presented the positive correlation to PR concentrations. When PR combined with arsenic trioxide (ATO), the expression levels of above mentioned mRNA and protein decreased or increased more significantly. It is concluded that PR can effectively induce the apoptosis of NB4 cells. PR combined with ATO displays synergistic effect. It may be triggered by the activation of JNK signal pathway.

[Chronic myeloid leukemia onset with marked thrombocythemia].

This study was aimed to investigate the clinical, pathological and biological features of a special case of chronic myeloid leukemia (CML) with marked thrombocythemic onset. The morphological changes of cells were analyzed by using bone marrow smear and biopsy; Ph chromosome, a specific marker of CML, was assayed by conventional chromosomal analysis and fluorescence in situ hybridization, bcr/abl fusion gene was detected by reverse transcription-polymerase chain reaction. The results indicated that CML mimicked essential thrombocythemia (ET) at presentation was relatively rare and might be misdiagnosed as ET, bone marrow smear and biopsy revealed, marked thrombocytosis and moderate leukocytosis; RT-PCR, FISH and conventional chromosomal analysis demonstrated the existence of Ph chromosome and bcr/abl fusion gene. This special CML could progress into accelerated phase or blast crisis. The megakaryocytes in Ph+ ET were smaller than normal ones and had typically hypolobulated round nuclei. Patients diagnosed as Ph+ ET might progress into CML and showed a high tendency to myelofibrosis and blastic transformation. It is concluded that the value of routine cytogenetical and molecular biological analysis in diagnosis for potential CML cases, which mimicked ET as in this presentation, is very distinctive, and the importance is magnified by the recent availability of imatinib, a specific inhibitor of the bcr/abl tyrosine kinase produced by the Philadelphia chromosome. Every case of "ET" should be tested for the Philadelphia chromosome and bcr/abl transcript.