Reduced doses of emicizumab achieve good efficacy: Results from a national-wide multicentre real-world study in China.

INTRODUCTION: Reduced doses of emicizumab improve the affordability among patients in developing countries. However, the relationship between variant dose selection and efficacy in the real world of China is still unclear. AIM: This study aimed to investigate the efficacy and safety of emicizumab especially in those on reduced dose regimens in a real-world setting. METHODS: We carried out a multicentre study from 28 hospitals between June 2019 and June 2023 in China and retrospectively analysed the characteristics including demographics, diagnosis, treatment, bleeding episodes, and surgical procedures. RESULTS: In total, 127 patients with haemophilia A, including 42 with inhibitors, were followed for a median duration of 16.0 (IQR: 9.0-30.0) months. Median age at emicizumab initiation was 2.0 (IQR: 1.0-4.0) years. Median (IQR) consumption for loading and maintenance was 12.0 (8.0-12.0) and 4.2 (3.0-6.0) mg/kg/4 weeks, respectively. While on emicizumab, 67 (52.8%) patients had no bleeds, whereas 60 (47.2%) patients had any bleeds, including 26 with treated bleeds. Compared to previous treatments, patients on emicizumab had significantly decreased annualized bleeding rate, annualized joint bleeding rate, target joints and intracerebral haemorrhage. Different dosages had similar efficacy except the proportion of patients with treated spontaneous bleeds and target joints. Adverse events were reported in 12 (9.4%) patients. Postoperative excessive bleeding occurred following two of nine procedures. CONCLUSION: This is the largest study describing patients with HA receiving emicizumab prophylaxis on variant dose regimens in China. We confirmed that nonstandard dose is efficacious and can be considered where full-dose emicizumab is ill affordable.

[Efficacy and safety of anagrelide in treatment of essential thrombocythemia: multicenter, randomized controlled clinical trial].

OBJECTIVE: To evaluate the efficacy and safety of anagrelide in essential thrombocythemia (ET). METHODS: Patients who diagnosed as ET according to the World Health Organization classification were enrolled. Each patient was assigned to take anagrelide hydrochloride capsule or hydroxyurea tablet by random 1∶1 ratio. Dose of anagrelide started at 2 mg/d, then increased gradually and the maximum dose was 10 mg/d until the platelet counts dropped to (100-400) × 109/L, one month later gradually reduced to maintain dose. The dose of hydroxyurea was 1000 mg/d at beginning, then increased gradually, when platelet counts dropped to (100-400)×109/L and kept for one month, reduced to maintain dose as 10 mg·kg⁻¹·d⁻¹. The observation period was 12 weeks. RESULTS: A total of 222 patients were enrolled in seventeen centers (including 113 patients treated with anagrelide and 109 with hydroxyurea). Therapy efficacy can be evaluated in 198 patients (including 97 patients administered with anagrelide and 101 with hydroxyurea). At 12th weeks of therapy, the hematologic remission rate was 87.63% (85/97) in anagrelide group and 88.12% (89/107) in hydroxyurea group, the differences between the two groups were not significant (P=0.173). Treatment with anagrelide lowered the platelet counts by a median of 393 (362-1 339) × 109/L from a median of 827 (562-1657) × 109/L at the beginning of the observation to 400(127-1130)×109/L after 12 weeks (P<0.001), which were similar to the treatment result of hydroxyurea by a median drop of 398 (597-1846)× 109/L (P=0.982). The median time to achieving response of anagrelide group was 7 (3-14) days, superior to that of hydroxyurea for 21 (14-28) significantly (P=0.003). Frequency of anagrelide related adverse events was 65.49 % (74/113), including cardiopalmus (36.28% ), headache (21.24% ), fatigue (14.16% ) and dizzy (11.50% ). CONCLUSION: Anagrelide was effective in patients with ET which had similar hematologic remission rate to hydroxyurea and could take effect more quickly than hydroxyurea. Incidence of adverse events was undifferentiated between anagrelide and hydroxyurea, but anagrelide treatment had tolerable adverse effects and no hematologic toxicity.

[MicroRNA-10a expression in FAB different subtype of acute myeloid leukemia and its relationship with drug resistance].

OBJECTIVE: This study was to investigate the expression of miR-10a in the different FAB subtype of acute myeloid leukemia (AML) and its relationship with drug resistance. METHODS: Forty de novo patients with AML, 16 patients with non-malignant hematologic disease and three AML cell lines HL-60, U937 and HL-60/ADR were enrolled in this study, the MiR-10a expression in bone marrow mononuclear cells of above-mentioned patients and 3 AML cell lines was detected by TaqMan RT-PCR. The correlation of miR-10a with clinicopathological factors of AML patients was analyzed. RESULTS: The miR-10a expression level in HL-60 cell line was higher than that in U937 cell line (P = 0.039). And its expression level in de novo AML patients was higher than that in patients with non-malignant hematologic disease (P < 0.01). FAB-AML-M3 patients exhibited higher expression of miR-10a than that in M1, M2 and M4 (P < 0.05); HL-60/ADR cell line showed higher miR-10a expression than that in HL-60 cell line (P < 0.01) . Except M3, the patients without CR (non-CR) after the first cycle of chemotherapy showed a higher level of miR-10a as compared with CR patients (P < 0.01). CONCLUSION: The high expression of miR-10a may be closely related to over-proliferation of promyelocyte and drug resistance of acute myeloid leukemia cells, except M3.

[Significance of BCL6, MYC, P53 genes abnormalities for the prognosis of diffuse large B-cell lymphoma].

OBJECTIVE: To explore the influence of BCL6, MYC, P53 genes abnormalities can on the prognosis of diffuse large B-cell lymphoma (DLBCL), and to identify independent prognostic factors for DLBCL in order to facilitate clinical prognosis and selection of stratification treatment for the patients. METHODS: Sixty five newly diagnosed DLBCL pathological specimens were collected from 2009 to 2012. Interphase fluorescence in situ hybridization technique (I-FISH) was used to detect the status of BCL6, MYC and P53 genes. Clinical factors were combined with immunohistochemical results for multiple-factor survival analysis. RESULTS: The rates of BCL6 gene rearrangement, P53 gene deletion and MYC rearrangement were 21.5% (14/65), 35.4% (23/65) and 7.7% (5/65), respectively. BCL6 rearrangement group has obviously poorer overall survival (OS)(P< 0.05). COX proportional hazards model analysis showed that gender, BCL6 protein, BCL6 rearrangement, Ki67 index were prognosis factors independent of international prognostic index (IPI). CONCLUSION: BCL6 can influence the prognosis of patients with DLBCL at gene and protein levels and both are independent prognostic factors for DLBCL.

Allogeneic hemopietic stem cell transplants for the treatment of B cell acute lymphocytic leukemia.

OBJECTIVE: Explore the feasibility of allo- hemopietic stem cell transplants in treating patients with B cell acute lymphocytic leukemia. METHODS: Between september 2006 and February 2011, fifteen patients with B cell acute lymphocytic leukemia (ALL) were treated by allo-hemopietic stem cell transplants (HSCT). Stem cell sources were peripheral blood. Six patients were conditioned by busulfan (BU) and cyclophosphamide (CY) and nine patients were conditioned with TBI and cyclophosphamide (CY). Graft versus host disease (GVHD) prophylaxis regimen consisted of cyclosporine A (CSA), methotrex ate (MTX) and mycophenolatemofetil (MMF). RESULTS: Patients received a median of 7.98×108·kg⁻¹ (5.36-12.30×108·kg⁻¹) mononuclear cells (MNC). The median time of ANC>0.5×109/L was day 12 (10-15), and PLT>20.0×109/L was day 13 (11-16). Extensive acute GVHD occurred in 6 (40.0%) patients, and extensive chronic GVHD was recorded in 6 (40.0%) patients. Nine patients were alive after 2.5-65 months follow-up. CONCLUSION: Allogeneic stem cell transplant could be effective in treating patients with B cell acute lymphocytic leukemia.

[Comparison and analysis of SNV results: detected by transcriptome sequencing technology on initial diagnosis and remission stage of a patient with AML-M2].

This study was aimed to further clarify the pathogenesis of acute myeloid leukemia(AML), and to forecast new somatic mutations related with leukemia. The peripheral blood samples on initial diagnosis and remission stage of a patient with partial differentiated AML(AML-M2) were sequenced by high throughput transcriptome sequencing technology. The single nucleotide variation (SNV) which possibly related with pathogenesis of leukemia was screened through comparison of the expressed genes on initial diagnosis and after remission. The results showed that the Reads distributed uniformly in genome and covered completely, detecting most expression genes. by screening the SNV, a total of 29881 mutations were discovered, including 28113 germline mutations and 752 individual mutations. Among them, 11 acquired mutations (P < 0.05) in coding regions were got, including ZRSR1, MLXIP, TLN1, LAP3, HK3. It is concluded that the high throughput sequencing as an unbiased new method can find new tumor-related mutations. MLXIP may be a new molecular marker of AML-M2.

All-trans retinoic acid enhances the effect of 5-aza-2'-deoxycytidine on p16INK4a demethylation, and the two drugs synergistically activate retinoic acid receptor ß gene expression in the human erythroleukemia K562 cell line.

The aim of the current study was to investigate the antineoplastic activities of 5-aza-2'-deoxycytidine (also known as decitabine; DAC) and all-trans retinoic acid (ATRA), administered alone or in combination, in K562 cells in vitro, as well as the effects on the expression of the tumor suppressor genes, p16INK4a (p16) and retinoic acid receptor ß (RAR-ß). Cell growth inhibition, differentiation and apoptosis in K562 cells treated with DAC and/or ATRA were detected. The methylation of the p16 and RAR-ß genes in the K562 cells was detected using the methylation-specific polymerase chain reaction (PCR) method. Quantitative PCR was used for the detection of the mRNA expression of the p16 and RAR-ß genes, and western blot analysis was used to detect protein expression. DAC and ATRA, alone or in combination, had no effect on the growth inhibition, differentiation and apoptosis of the K562 cells. DAC alone induced the demethylation of the p16 gene, and combination of DAC and ATRA demonstrated more evident demethylation of the p16 gene, however, ATRA alone had no effect on methylation. The RAR-ß promoter region was not methylated in the K562 cells. DAC in combination with ATRA appeared to produce a greater activation of the RAR-ß gene, which led to the upregulation of the RAR-ß expression level. ATRA enhanced the effect of DAC on p16 demethylation, and the combination of the two drugs was found to activate RAR-ß expression, which indicated that DAC used in combination with ATRA has clinical potential in the treatment of human erythroleukemia.

RNA-Seq analysis identifies aberrant RNA splicing of TRIP12 in acute myeloid leukemia patients at remission.

Aberrant splicing events play important roles in the pathogenesis of acute myeloid leukemia (AML). To investigate the aberrant splicing events in AML during treatment, we carried out RNA sequencing in peripheral mononuclear cell samples from a patient with complete remission. In addition to the sequencing samples, selected splicing events were confirmed and validated with real-time quantitative RT-PCR in another seven pairs of samples. A total of 4.05 and 3.39 GB clean data of the AML and remission sample were generated, respectively, and 2,223 differentially expressed genes (DEGs) were identified. Integrated with gene expression profiling on T cells from AML patients compared with healthy donors, 82 DEGs were also differentially expressed in AML CD4 T cells and CD8 T cells. Twenty-three alternative splicing events were considered to be confidential, and they were involved in many biological processes, such as RNA processing, cellular macromolecule catabolic process, and DNA binding process. An exon3-skipping event in TRIP12 was detected in patients at remission and further validated in another three independent samples. TRIP12 is an ubiquitin ligase of ARF, which suppresses aberrant cell growth by activating p53 responses. The exon3-skipping isoform of TRIP12 increased significantly after treatment. Our results may provide new understanding of AML, and the confirmed alternative splicing event of TRIP12 may be used as potential target for future investigations.

Combination therapy of rituximab and corticosteroids for patients with refractory chronic immune thrombocytopenic purpura: report of two cases.

Immune thrombocytopenic purpura (ITP) is an autoimmune disorder, for which rituximab has been proven to be an effective treatment. The response rate was reported to be approximately 60% in refractory ITP patients. However, the response time is slower than expected, and the mechanism of action of rituximab in ITP is still unclear. Thus, sometimes, the use of a combination therapy with rituximab according to different patient conditions is necessary. We report two refractory chronic ITP cases. The two patients were administered a low dose of dexamethasone (10 mg, weekly) combined with rituximab and a smaller dose of prednisone (10 mg, daily) as maintenance therapy. Although their peripheral B cells were almost eliminated, no complete reaction was observed. The maintenance therapy with prednisone was helpful in the prevention of bleeding. The patients' responses to rituximab treatment suggest that multiple immunological mechanisms are involved in ITP pathogenesis and that the use of a combination therapy with rituximab according to the different patient conditions is necessary.

[Significance of miR-155, miR-34a and miR-30a expression in diffuse large B-cell lymphoma].

OBJECTIVE: To determine the expressions of miR-155, miR-34a and miR-30a in diffuse large B-cell lymphoma (DLBCL) and to explore their potential correlation with clinicopathological characteristics. METHODS: The expression level of miR-155, miR-34a and miR-30a in 46 DLBCL samples were determined with TaqMan real-time polymerase chain reaction. Interphase fluorescence in situ hybridization (I-FISH) was performed to detect MYC and p53 genes' status, and immunohistochemistry (Envision method) was used to evaluate the expression of CD3, CD10, CD20, BCL-6 and MUM-1 in DLBCL. The DLBCLs were classified into germinal center B cell-like (GCB) and non germinal center B cell-like (non-GCB) subtypes according to Hans' criteria. RESULTS: Compared with normal controls, miR-155 expression level was significantly higher in DLBCL. The expression level of miR-155 in non-GCB type was higher than that in GCB type. It was shown that the patients with MYC rearrangement had lower expression level of miR-155 than the negative controls. Compared with p53 normal group, the expression level of miR-34a was significantly lower in p53 deletion group. It was also shown that the patients with BCL-6 protein expression had lower expression of miR-30a compared with the negative group. CONCLUSION: miR-155 expression level is different in normal controls, DLBCL and patients with subtype DLBCL. It therefore has a diagnosis value for DLBCL. miR-34a is of great prognostic significance. miR-155, miR-34a and miR-30a may be potential therapy targets for DLBCL.

The clinical characteristics and prognostic significance of MN1 gene and MN1-associated microRNA expression in adult patients with de novo acute myeloid leukemia.

This study aimed to determine the clinical characteristics and prognostic significance of the meningioma 1 (MN1) gene and MN1-associated microRNA expression in Chinese adult de novo acute myeloid leukemia (AML) patients. The expression level of MN1, microRNA-20 (miR-20a), and microRNA-181b (miR-181b) in bone marrow mononuclear cells was measured in 158 newly diagnosed AML patients and 20 cases of normal healthy donors by real-time quantitative reverse transcriptase polymerase chain reaction. All AML patients significantly overexpressed MN1 at the level of 0.01983 (P < 0.001) compared with normal controls. High MN1 expression was associated with spleen involvement (P = 0.037), NPM1 wild type (P = 0.001), lower miR-20a expression levels (P = 0.015), and higher miR-181b expression levels (P = 0.035). MiR-20a (P = 0.029) and miR-181b (P = 0.017) overexpressed in the bone marrow cells of patients with certain subtypes of AML compared with healthy donors. High MN1 expressers had lower complete remission (CR) rates and shorter overall survival (OS) within the Southwest Oncology Group classification. In multivariable models, high MN1 expression was associated with worse CR rates (P = 0.01), relapse-free survival (RFS; P = 0.02), and OS (P = 0.02); high miR-20a expression was associated with higher CR rates (P = 0.008) and longer OS (P = 0.04), whereas high miR-181b expression was associated with lower CR rates (P = 0.03), and shorter RFS (P = 0.045) and OS (P = 0.017). High MN1 expression confers worse prognosis in Chinese adult patients with de novo AML. MN1 gene and MN1-associated microRNAs provide clinical prognosis of AML patients and may refine their molecular risk classification.

Identification of circulating microRNAs as potential biomarkers for detecting acute myeloid leukemia.

Acute myeloid leukemia (AML) is the most common acute leukemia in adults. The disease is characterized by various cytogenetic and molecular abnormalities with distinct prognoses and gene expression profiles. Emerging evidence has suggested that circulating microRNAs (miRNAs) could serve as noninvasive biomarkers for cancer detection; however, little is known about circulating miRNA profiles in AML patients. In this study, a genome-wide serum miRNA expression analysis was performed using Solexa sequencing for initial screen, followed by validation with real-time PCR assays. The analysis was conducted on training and verification sets of serum samples from 140 newly diagnosed AML patients and 135 normal adult donors. After a two-phase selection and validation process, 6 miRNAs, miR-10a-5p, miR-93-5p, miR-129-5p, miR-155-5p, miR-181b-5p and miR-320d, were found to have significantly different expression levels in AML compared with control serum samples. Furthermore, unsupervised clustering analysis revealed the remarkable ability of the 6-miRNA profile to differentiate between AML patients and normal controls. The areas under the ROC curve for the selected miRNAs ranged from 0.8129 to 0.9531. More importantly, miR-181b-5p levels in serum were significantly associated with overall survival. These data demonstrated that the expression patterns of circulating miRNAs were systematically altered in AML and miR-181b-5p may serve as a predictor for overall survival in AML patients.

[Correlation of BCL-6, MYC and p53 gene abnormalities with immunological subtypes and prognosis of diffuse large B-cell lymphoma].

OBJECTIVE: To investigate BCL-6, MYC and p53 genes abnormalities in diffuse large B-cell lymphoma (DLBCL) and correlate the result with immunosubtypes and prognosis. METHODS: Interphase fluorescence in situ hybridization (I-FISH) was performed to detect the BCL-6, MYC and p53 genes. Immunohistochemistry (Envision method) was used to measure the expressions of CD3, CD10, CD20, BCL-6, MUM -1, BCL-2 and Ki-67 genes in DLBCL. The patients were classified into germinal center B cell-like (GCB) and non-GCB subtypes according to Hans' algorithm. RESULTS: BCL-6 rearrangement was detected in 10 of 46 DLBCL cases. The presence of gene rearrangement had no correlation with BCL-6 protein expression (P= 0.245). Overall survival (OS, P= 0.138) and progression-free survival (PFS, P= 0.095) were not influenced by BCL-6 rearrangement. All MYC rearrangements were detected in GCB type DLBCL. Deletion of p53 gene was detected in 14 cases and was significantly associated with shorter OS (P= 0.046) and PFS (P= 0.043). CONCLUSION: I-FISH is a rapid, accurate and sensitive method for detecting BCL-6, MYC and p53 abnormalities. No correlation was found between BCL-6 gene rearrangement and BCL-6 protein expression. MYC translocation was more common in GCB type DLBCL compared with non-GCB type ones. Patients with p53 deletion had a poorer prognosis. The p53 gene may provide a useful indicator for the prognosis of DLBCL.

Prognostic power of abnormal cytogenetics for multiple myeloma: a multicenter study in China.

BACKGROUND: Chromosomal abnormalities have been shown to play an important prognostic role in multiple myeloma (MM). Interphase fluorescence in situ hybridization (i-FISH) has been much more effective to identify cytogenetic aberrations in MM than conventional cytogenetic technique (CC). To clearly determine the cytogenetic features of Chinese MM patients and identify their prognostic implications, we designed a multicenter study based on i-FISH including 672 patients from 52 hospitals in China. METHODS: All 672 patients were systematically screened for the following genomic aberrations: del(13q), IgH rearrangement, del(p53) and 1q21 amplifications. RESULTS: The analysis showed that the chromosomal changes were detected in 22.1% patients by CC and in 82.3% patients by i-FISH. The most common abnormalities by CC were chromosome 1 aberrations (48.4%), -13/13q- (37.6%), hyperdiploidy (36.6%), hypodiploidy (30.1%) and IgH rearrangements (23.7%). The most frequent abnormalities by FISH was del(13q), which was found in 60.4% patients, whereas IgH rearrangement, 1q21 amplification and p53 deletions were detected in 57.6%, 49.0% and 34.7% cases, respectively. By statistical analysis, -13/13q- by CC was associated with low level of platelet (P = 0.015), hyperdiploidy was associated with low level of serum albumin (P = 0.028), and IgH rearrangement by FISH was associated with high level of ß2 microglobulin (P = 0.019). Moreover, 1q21 amplification and del(p53) by FISH conferred a high incidence of progressive disease (PD) after initial therapy. Metaphase detection of IgH rearrangements and chromosome 1 aberrations concurrently was associated with a short progression free survival (PFS) (P = 0.036). No significant prognostic implications of other cytogenetic abnormalities were found associated with overall survival and PFS. CONCLUSIONS: Chinese MM patients had similar cytogenetic abnormalities compared with the previous reported studies. However, the prognostic significance of FISH aberrations were not clearly determined and further study is required.

[Clinical study on platelet engraftment by thrombopoietin in patients with hematological malignancies after allogeneic hematopoietic stem cell transplantation].

OBJECTIVE: To evaluate the efficacy and safety of thrombopoietin (TPO) on platelet engraftment in hematological malignancies patients after allogeneic haematopoietic stem cell transplantation (allo-HSCT). METHODS: One hundred and twenty patients were enrolled in a multicenter, open-label, randomized, controlled clinical trial, and were randomized into 4 treatment groups following allo-HSCT. Group A was the control arm without TPO, while group B, C and D were trial arms with received 300 U×kg(-1)×d(-1) of TPO starting from day +1, +4 and +7, respectively. A total of 89 cases were evaluated, of which 22 cases in group A, 23 in group B, 20 in group C and 24 in group D. Efficacy evaluation (the time of platelet engraftment, the number of platelet transfusion) and safety evaluation \[adverse events, routine blood tests, liver and renal function, coagulation function and occurrence of graft-versus-host disease (GVHD)\] were observed. RESULTS: The median platelet engraftment time in experimental groups (groups B, C and D) were on day (13.17 ± 2.89), day (12.15 ± 2.08), day (12.33 ± 1.76), respectively, and that in control group was on day (14.82 ± 5.05). There was statistically significant difference between two groups (P = 0.029), There were no statistically significant difference in the average amount of platelet transfusion, platelet engraftment time, and platelet nadir value among the 3 experimental groups. No significant adverse events were observed in experimental groups. CONCLUSIONS: TPO administration following allo-HSCT for patients with hematologic malignancies appears to shorten platelet engraftment time. TPO given starting from day +7 is effective and safe.

[Influence of 1,25(OH)(2) vitamin D(3) on maturation of human dendritic cells and DC-mediated immune tolerance].

This study was aimed to investigate the effect of 1,25(OH)(2) vitamin D(3) [1,25(OH)(2) Vit D(3)] on the differentiation, maturation and function of human dendritic cells (DC) in vitro and its mechanism. Human peripheral blood mononuclear cells were induced to differentiate to DC in vitro. The DC in test group were cultured with 1,25(OH)(2) Vit D(3) 1 nmol/L for 9 d, while the DC in control group were cultured with the equivalent of absolute alcohol. The expression of co-stimulatory molecules on DC were analyzed by flow cytometry. T cell proliferation induced by DC was assessed by MTT method. The expression of indoleamine 2, 3-dioxygenase (IDO) protein was determined by Western blot. The results showed that compared with the control group, the expression of CD80, CD83 and CD86 on DC in test group was significantly down-regulated (P < 0.05), while the CD1a was up-regulated (P < 0.05). The expression rate of CD80, CD83, CD86, CD1a in test group were (40.43 ± 9.83)%, (20.04 ± 4.73)%, (14.45 ± 5.38)%, (58.48 ± 10.72)% respectively, while in control group were (29.36 ± 13.34)%, (35.91 ± 10.19)%, (27.15 ± 11.64)%, (72.20 ± 12.79)% respectively. Compared with the control group, 1,25(OH)(2) Vit D(3)-treated DC exhibited a markedly reduced ability to stimulate allogenic T cell proliferation and up-regulated IDO protein expression.It is concluded that 1,25(OH)(2) Vit D(3) efficiently inhibits the maturation of DC and DC-mediated T cell proliferation, which may be related to the up-regulation of IDO protein expression.

[Effects of simvastatin on PI3K/AKT signaling pathway in human acute monocytic leukemia cell line SHI-1].

To investigate the effects of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor simvastatin (SV) on proliferation, apoptosis and the PI3K/AKT signaling pathway in human acute monocytic leukemia cell line SHI-1. SHI-1 cells were incubated with different concentrations of SV (5, 10, 15 µmol/L). Otherwise, SHI-1 cells without any treatment were used as control. Cells in different groups were collected at 24, 48 and 72 h after incubation for further detection. MTT method was used to assay the growth inhibition rate and flow cytometry was used to detect the early stage apoptosis ratio. The human PI3K-AKT Signaling Pathway RT(2) Profiler(TM) PCR Array was used to detect the expression of 84 genes involved in PI3K-AKT signaling. The results indicated that the SV inhibited the proliferation and inducted the apoptosis of SHI-1 cells in time- and dose-dependent manners significantly. The growth inhibition rates of SHI-1 cells treated with 15 µmol/L SV for 24, 48 and 72 h were 26.82, 47.09 and 63.92, respectively; and their early stage apoptosis ratios were 5.75, 13.25 and 15.59, respectively. Compared with the control group, expression levels of 39 genes were changed in the group of 15 µmol/L SV at 48 h, among them 26 genes were down-regulated and 13 genes were up-regulated. It is concluded that the SV can inhibit proliferation and induce apoptosis of SHI-1 cells, and the mechanism may be associated with the changes of gene expression level in PI3K-AKT signaling pathway regulated by SV.

[Effects of simvastatin plus all-trans retinoic acid on WT1/hDMP1 gene expression profiles of human promyelocytic leukemia cell line NB4].

OBJECTIVE: To investigate the effects of simvastatin (SV) plus all-trans retinoic acid (ATRA) on the proliferation, differentiation, apoptosis and WT1/hDMP1 gene expression profiles of human promyelocytic leukemia cell line NB4. METHODS: The NB4 cell was incubated with simvastatin and ATRA alone or in combination. And the NB4 cell without any treatment was adopted as a normal control. The cells of different groups were collected at 24, 48 and 72 h post-incubation. Their morphological changes were observed after Wright staining. The method of MTT was employed to assay the growth inhibition rate and flow cytometry was used to detect the early-stage ratios of apoptosis and cell necrosis. Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect the WT1/hDMP1 gene expression levels. RESULTS: The cell inhibition rates increased gradually (F = 7.15, P = 0.000) at 15, 10 and 5 µmol/L SV respectively. And so did the expression levels of CD11b (F = 3.41, P = 0.014) and Annexin-V (F = 43.38, P = 0.000). However the expression levels of WT1 decreased gradually (F = 5.35, P = 0.001) reversely with the elevated levels of hDMP1 (F = 22.61, P = 0.000). Furthermore the NB4 cell exhibited the most significant changes at 15 µmol/L SV. After a 72-hour incubation, the expression levels of CD11b (89.46% ± 9.13%)and hDMP1 (626.9 ± 56.9) in NB4 cells at 15 µmol/L SV plus 0.5 µmol/L ATRA were significantly higher than those with ATRA(71.27% ± 7.27%, P = 0.000 and 421.8 ± 38.3, P = 0.003 in each) and SV alone(62.41% ± 6.37%, P = 0.003 and 241.4 ± 21.9, P = 0.003 in each). A combination of 15 µmol/L SV with 0.5 µmol/L ATRA displayed obvious interactions with the expressions of CD11b and hDMP1 (F = 4.09, P = 0.025 and F = 29.58, P = 0.000 in each). And there was no significant interaction for cell inhibition rates and Annexin-V expression. CONCLUSION: Simvastatin in vitro inhibits the proliferation of NB4 cell, induces its differentiation and promotes its apoptosis. And the lowered expression of WT1 has a dose-dependent correlation with the elevated expression of hDMP1. It indicates that simvastatin has the synergistic in vitro anti-promyelocytic potency.