Selective and effective targeting of chronic myeloid leukemia stem cells by topoisomerase II inhibitor etoposide in combination with imatinib mesylate in vitro.

Imatinib mesylate (IM) and other BCR-ABL tyrosine kinase inhibitors (TKIs) have improved chronic myeloid leukemia (CML) patient survival markedly but fail to eradicate quiescent CML leukemia stem cells (LSCs). Thus, strategies targeting LSCs are required to induce long-term remission and achieve cure. Here, we investigated the ability of topoisomerase II (Top II) inhibitor etoposide (Eto) to target CML LSCs. Treatment with Eto combined with IM markedly induced apoptosis in primitive CML CD34+ CD38- stem cells resistant to eradication by IM alone, but not in normal hematopoietic stem cells, CML and normal mature CD34- cells, and other leukemia and lymphoma cell lines. The interaction of IM and Eto significantly inhibited phosphorylation of PDK1, AKT, GSK3, S6, and ERK proteins; increased the expression of pro-apoptotic gene Bax; and decreased the expression of anti-apoptotic gene c-Myc in CML CD34+ cells. Top II inhibitors treatment represents an attractive approach for targeting LSCs in CML patients undergoing TKIs monotherapy.

Targeting miR-21 sensitizes Ph+ ALL Sup-b15 cells to imatinib-induced apoptosis through upregulation of PTEN.

Philadelphia chromosome positive (Ph+) acute lymphoblastic leukemia (ALL) cells are insensitive to BCR-ABL tyrosine kinase inhibitor imatinib, the underlying mechanisms remain largely unknown. Here, we showed that imatinib treatment induced significant upregulation of miR-21 and downregulation of PTEN in Ph+ ALL cell line Sup-b15. Transient inhibition of miR-21 resulted in increased apoptosis, PTEN upregulation and AKT dephosphorylation, whereas ectopic overexpression of miR-21 further conferred imatinib resistance. Furthermore, knockdown of PTEN protected the cells from imatinib-induced apoptosis achieved by inhibition of miR-21. Additionally, PI3K inhibitors also notably enhanced the effects of imatinib on Sup-b15 cells and primary Ph+ ALL cells similar to miR-21 inhibitor. Therefore, miR-21 contributes to imatinib resistance in Ph+ ALL cells and antagonizing miR-21 demonstrates therapeutic potential by sensitizing the malignancy to imatinib therapy.

Predicting adverse pregnancy outcomes of pregnant mothers with syphilis based on a logistic regression model: a retrospective study.

Objective: Maternal syphilis could cause serious consequences. The aim of this study was to identify risk factors for maternal syphilis in order to predict an individual's risk of developing adverse pregnancy outcomes (APOs). Methods: A retrospective study was conducted on 768 pregnant women with syphilis. A questionnaire was completed and data analyzed. The data was divided into a training set and a testing set. Using logistic regression to establish predictive models in the training set, and its predictive performance was evaluated in the testing set. The probability of APOs occurrence is presented through a nomogram. Results: Compared with the APOs group, pregnant women in the non-APOs group participated in a longer treatment course. Course, time of the first antenatal care, gestation week at syphilis diagnosis, and gestation age at delivery in weeks were independent predictors of APOs, and they were used to establish the nomogram. Conclusions: Our study investigated the impact of various characteristics of syphilis pregnant women on pregnancy outcomes and established a prediction model of APOs in Suzhou. The incidence of APOs can be reduced by controlling for these risk factors.

[Effect of Etoposide on Elimination of Chronic Myeloid Leukemia Stem Cells by Imatinib in Vivo].

OBJECTIVE: To investigate the effect of etoposide (ETO) on elimination of chronic myeloid leukemia (CML) stem cells by imatinib mesylate(IM) in vivo. METHODS: SCL-tTA/BCR-ABL mice were used as CML animal model. Flow cytometry was used to assess the effect of ETO alone or in combination with IM on the number of leukemia stem cell （LSC） in bone marrow and spleen, and peripheral blood neutrophils in CML mice and normal control FVB mice. RESULTS: The results showed that in CML mice, the number and proportion of LSC in bone marrow and the proportion of neutrophils in peripheral blood decreased significantly after ETO and IM combined treatment, and the degree of decrease was more significant than that of both alone. While in wild type FVB mice, the combination of ETO and IM showed no significant effect on the number and proportion of LSK cells in bone marrow and the proportion of neutrophils in spleen. CONCLUSION: ETO can selectively enhance elimination of CML LSC by IM in vivo.

[Heterologous expression of attacin gene from Musca domestica in Pichia pastoris].

OBJECTIVE: To construct recombinant expression plasmid with an antibacterial peptide (attacin) mature peptide gene from Musca domestica and express an antibacterial peptide attacin in Pichia pastoris. METHODS: The coding region of attacin mature peptide was cloned into the expression vector pPIC9K. The recombinant plasmid was linearized by digestion with Sac I and transformed into P. pastoris GS115 by electroporation. The insert clones containing multiple copies were selected with geneticin, pha, enotype and PCR. The expression of P. pastoris GS115/pPIC9K-attacin were induced with methanol. The supernatant of the expressed protein was analyzed by SDS-PAGE and Western blotting. The anti-bacterial activity of expression product was analyzed by agarose diffusion assay. RESULTS: The expression plasmid was constructed and transformed into P. pastoris GS115. SDS-PAGE and Western blotting analysis indicated that the recombinant containing recombinant plasmid pPIC9K-attacin expressed a Mr 22000 protein. The expression product inhibited the growth of E. coli K12D31. CONCLUSION: The attacin gene from Musca domestica has been expressed in P. pastoris.

Silencing of miR-21 sensitizes CML CD34+ stem/progenitor cells to imatinib-induced apoptosis by blocking PI3K/AKT pathway.

BCR-ABL tyrosine kinase inhibitor imatinib fails to eradicate leukemia stem cells (LSCs), the underlying mechanisms maintaining CML LSCs remain poorly understood. Here, we showed that transient inhibition of miR-21 by antagomiR-21 markedly increased imatinib-induced apoptosis in CML, but not normal CD34+ stem/progenitor cells. Furthermore, PI3K inhibitors also significantly sensitized CML CD34+ cells to imatinib-induced apoptosis. MiR-21 or PI3K inhibitor in combination with imatinib treatment significantly decreased AKT phosphorylation and c-Myc expression than either agent did alone, but did not affect Bim and Bcl-6 expresssion. These findings indicate that miR-21 is required for maintaining the imatinib-resistant phenotype of CML CD34+ cells through PI3K/AKT signaling pathway, thus providing the basis for a promising therapeutic approach to eliminate CML LSCs.

Scientific evaluation of the subchronic toxicity of Musca domestica larvae extracts in Sprague Dawley rats.

Musca domestica larvae extracts (MDLE) is a potential drug used to treat lipopolysaccharide-induced atherosclerosis pro-inflammatory responses. The purpose of the study was to evaluate the safety of MDLE via a 13-week repeated dose subchronic toxicity test in rats. Both male and female Sprague Dawley rats were divided into four groups, eight animals each from the control and high-dose group (33.0 g/kg) were allocated into recovery groups. The four groups of rats were administrated with MDLE (0, 13.2, 22.0, 33.0 g/kg) in the diet for 13weeks respectively. During the experimental period, the rats were observed for symptoms and signs of gross toxicity daily, food consumption and body weight were measured weekly. Urinalysis, thrombotest, blood biochemical and hematological analyses were performed regularly; Expression of peroxide dismutase gene in liver was quantified and a histopathological examination was also performed. There were no MDLE-induced abnormalities in any of the groups during or after the 13 weeks except the relative weight of liver of high-dose group and middle-dose group was significantly higher than that of control group in male rats (P<0.05). The results indicate a no observed adverse effect level for MDLE is 13.2 and 33.0 g/kg bw/day in male and female rats, respectively.

Curcumin induces FasL-related apoptosis through p38 activation in human hepatocellular carcinoma Huh7 cells.

AIM: The aim of this study is to explore the underlying molecular mechanism of curcumin-induced apoptosis in human hepatocellular carcinoma (HCC) Huh7 cells. MAIN METHODS: Fas and FasL mRNA expression was analyzed by reverse transcription PCR. Western blot was applied to detect the protein expression of Bcl-2 family members, MAPK family members, c-Jun, c-Fos, ATF-2, caspase-3, PARP, TNF receptor family members and the respective ligands. Apoptotic cells were assayed with annexin V/PI double staining and flow cytometry. KEY FINDINGS: Curcumin treatment resulted in a fast and significant increase of Fas and Fas ligand (FasL) along with activation of caspase-3 and cleavage of PARP in Huh7 cells. Inhibition of caspase-3 activity by the specific inhibitor Z-DEVD-FMK rescued Huh7 cells from curcumin-induced apoptosis. Neutralization of FasL significantly protected the cells from curcumin-induced caspase-3 activation and apoptosis in a dose-dependent manner. Moreover, p38 was rapidly activated in response to curcumin, and inactivation of p38 by pharmacologic inhibitor SB203580 dramatically suppressed curcumin-induced FasL expression and apoptosis. SIGNIFICANCE: Our results demonstrated that curcumin induces apoptosis through p38-denpendent up-regulation of FasL in Huh7 cells.