ALBERT

Description: Crystal Growth & Design DOI: 10.1021/cg301286w

Description: The performance of the regional water environment integrated governance is affected by many factors. This study took place in Henan Province, China, as the research area, and constructed an index system through the comprehensive consideration of three target layers based on the Ecological-Social-Economic (ESE) framework. Due to advantages such as strong objectivity and operability, the improved entropy-weight technique for the order of preference by similarity to the ideal solution (TOPSIS) model can greatly overcome subjective human interference and render the evaluation results more reliable. Therefore, it was introduced to evaluate the water environment integrated governance in Henan from 2007 to 2016. By applying the obstacle degree model, the obstacle factors were then diagnosed. The results of this study show that the overall performance of the integrated governance was generally improved in Henan from 2007 to 2016. Performance levels of the three target layers exhibited different trends, of which the social and economic benefits presented a linear increase year by year, but the ecological benefits presented a fluctuating downward trend. The obstacle on the Henan water environment integrated governance mainly comes from the ecological and economic benefits index. Therefore, a series of countermeasures have been proposed as a means of improving the governance performance in Henan.

Description: Background The emergence of Imatinib has brought a new era for the treatment of chronic myeloid leukemia. However, resistant to Imatinib might lead to the treatment failure and death of patients. Thus, finding a drug which could enhance the Imatinib-induced apoptosis in vitro could provide the experimental base for the treatment of chronic myeloid leukemia. K562 cell line is a common cell line used in the study of chronic myeloid leukemia while K562/G cell line which is resistant to Imatinib is derived from K562 cell line. Aim This study aims to explore whether low-dose Triptolide(TPL) could enhance the Imatinib-induced apoptosis in K562/G cells and related mechanism. Methods K562/G cells were subjected to different treatments and thereafter MTT assay, flow cytometry and Western blot or RT-PCR were used to determine IC50, apoptotic status and expression of Nrf2, HIF-1α and their target genes. Results Triptolide is highly cytotoxic to K562/G cells in a concentration-dependent manner. To determine the combination effect of TPL and anticancer agents, K562/G cells were exposed to Imatinib(50μM) with or without TPL (25nM) for 24h. The apoptotic cells were determined by PI/Annexin V staining and flow cytometric analysis. Apoptotic ratio of cells treated by Imatinib together with TPL is significantly increased compared to cells treated by Imatinib alone (24.78±1.12 vs. 77.52±7.75, P

Description: Background Disulfiram(DS), an old drug clinically used for alcoholism, was reported to have antitumor effects, recent studies have found that Copper(Cu) can significantly enhance the DS-induced cell death in vitro in a variety of tumor cells. Our previous studies also demonstrated that disulfiram/copper (DS/Cu) couldtarget human leukemia cell lines(like KG1α,Molt4) through the activation of JNK, in vitro. However, there is few report about the ability of DS/Cu in killing cancer cells in vivo. Aims This study aims to explore the effect of DS/Cu on acute myeloid leukemia cell line KG1αin vivo and clarify the underlining mechanism. Methods 6-8 week old female NOD/SCID mice were sublethally irradiated with 2Gy X-ray the day before transplantation, followed by intravenous injection of KG1α cells (1×107 cells) suspended in 0.2 mL of PBS. 5 weeks after transplantation mice were randomly divided into three treatment groups: vehicle (0.9% saline), a combination of DS and Cu daily for 2 weeks, Ara-C alone twice before killing. Mice were sacrificed after 2 weeks treatment with tissues of spleen, liver, bone marrow being observed using histopathology method to detect the invasion of leukemia. The DS/Cu-induced p-c-jun activation was also examined by western blot using tissues of spleen, liver, bone marrow. Statistical analysis was carried out with one-way ANOVA to assess statistical significance (*p 〈 0.05). Results 4 weeks after transplantation, mice were dispirited with low appetite, down-bent gait, wrinkled fur, slow move, just like suffered from leukemia. What’s more, immature blasts like morphology similar to KG1α were found in the peripheral blood of the mice(11%±3.41). All the mice were sacrificed after 2 weeks treatment, mice in control group were observed with slightly larger spleen and liver with the morphology of invasion of leukemia such as a granular appearance than the other two groups. Histopathology examination showed that leukemia cells infiltrate liver, spleen and bone marrow, and the immunohistochemistry examination found that the leukemia cells in spleen, liver and bone marrow expressed human specific antigen CD45 with the highest expression level in the control group. Moreover, solid tumor could be observed in the peritoneal cavity of two mice in the control group with expression of human specific antigen CD45detected by immunohistochemistry examination. Western blot in this study showed DS/Cu complex induced phosphorylation of c-Jun expression in the spleen, liver and bone marrow. Conclusion DS/Cu complex could effectively target the acute myeloid leukemia cells in the acute leukemia NOD/SCID mice while inhibiting the invasion of leukemia to some extent, and the activation of JNK might play a functional role in DS/Cu mediated antileukemic effects. Disclosures: No relevant conflicts of interest to declare.

Description: Background Relapse has been a major hurdle for the success of acute myeloid leukemia chemotherapy, whereas leukemia stem cells (LSCs) have been considered to be responsible for relapse recently. Thus new drug targeting LSCs is urgently needed. Triptolide (TPL), a diterpenoid triepoxide derived from Tripterygium wilfordii, has been used in Traditional Chinese Medicine (TCM) for centuries. Recently, it has been reported that TPL has the potential of depleting quiescent CD34+ primitive CML progenitor cells. Moreover, normal CD34+ hemopoietic stem cells are less sensitive than AML blasts to TPL, suggesting the seclectivity of TPL in targeting LSCs. Aims In this study, the ability of triptolide to induce apoptosis in leukemia stem and progenitor cells was investigated with the underlying mechanism being explored. Methods Leukemia stem and progenitor cells were sorted from KG1a cells using flow cytometry with cell cycle being analyzed and then were subjected to different treatments and thereafter MTT assay, flow cytometry and Western blot or RT-PCR, intracellular ROS measurement colony forming assay were used to determine IC50, apoptotic status and expression of Nrf2, HIF-1α and their target genes, ROS level and colony forming ability. Results TPL is highly cytotoxic to leukemia stem and progenitor cells with IC20 of 5.0±0.81nM and IC50 of 20.48±1.6nM after 72h exposure. Leukemia stem and progenitor cells were also exposed to series concentrations of IDA with or without TPL(IC20F5.0nM). TPL significantly enhanced cytotoxicity of IDA to LSCs (IC50-IDA: 285.20±13.7 nM vs. IC50-IDA+TPL: 27.01±0.73 nM, P

Description: Abstract 4088 Backgrounds Acute myeloid leukemia(AML) is a hierarchical disease initiating from a rare population of cells known as leukemia stem cells (LSCs), which are typically enriched in CD34+CD38- cells and presumed responsible for the relapse and refractory of AML. Moreover, current regimens may not effectively discriminate between normal and malignant cells. For this reason, it is important to identify therapies that can specifically target the LSC population without affecting normal cells. Disulfiram (DS) is an anti-alcoholism drug that has recently been indicated to show cytotoxic to multiple cancers including acute myeloid leukemia (AML) and the antineoplastic activity was enhanced in the present of copper (Cu). In the present study, we investigated the effect of DS/Cu on LSCs and further explored its mechanism. Methods and Results CD34+CD38- leukemia stem cell (LSC) enriched subpopulations were sorted from both KG1a cell lines and primary AML bone marrow or peripheral blood mononuclear cells (n=6) by fluoresce-activated cell sorting (FACS) analysis. Using MTT cell proliferation assay and Annexin-V/PI staining assay, We demonstrated that DS/Cu inhibited proliferation and induced apoptosis in CD34+CD38−KG1a cells (IC50= 0.788± 0.451 μM at 24h). With the increasing concentrations of DS (DS=0.05, 0.5, 5, 50μM), the apoptotic proportion increased from 7.2% to 89.5% at 24h. Apoptosis was also observed in CD34+CD38- primary AML cells and the exposure to DS/Cu (DS=0.01, 0.1, 1μM;Cu=0.5μM clearly inhibited the growth of AML-colony-forming units (CFUs) for both CD34+CD38-LSC enriched subpopulations (AML-CFUs decreased from 34.2% to 0% in KG1a cells), but was relatively sparing to normal hematopoietic progenitors. Further more, using flow cytometric analysis, western blot and RT-PCR, we identified that the change in redox status and redox-dependent signaling events play a crucial role in DS/Cu-induced apoptosis. We showed that DS/Cu(DS= 0.625,1.25,2.5,5μM, Cu=1μM) increased reactive oxygen species (ROS) and activated its downstream apoptosis-related SAPK/JNK pathway in association with blockade translocation of Nrf2 and expression of Nrf2-regulated genes in CD34+CD38−KG1a cells. Notably, blockade of ROS by glutathione precursor N-acetylcysteine (NAC)(10mM) strongly diminished DS/Cu mediated lethality and restored Nrf2 nuclear translocation and blocked JNK activation. Additionally, consistent with the ROS accumulation, we also seen that translocation of RelA/p65 and the expression of NF-κb-related gene, associated with abnormal apoptotic response of LSCs, were significantly inhibited by DS/Cu. Conclusion Taken together, we concluded that DS/Cu might selectively eradicate LSCs by induction of oxidatibe stress and blockade the NF-κb pathway and offers a potential therapeutic option in AML. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4869 Disulfiram (DS), an antialcoholism drug, demonstrates strong antitumor activity in a copper (Cu)-dependent manner. Our previous study showed that it is highly cytotoxic in doxorubicin resistant leukemia cells and enhances cytotoxicity of doxorubicin. DS/Cu induces reactive oxidative stress (ROS) which activates stress related signaling pathway (c-Jun-amino-terminal kinase, JNK). Cancer cells possess higher ROS activity and some antiapoptotic factors. Therefore cancer cells may be effectively targeted by simultaneous inducing ROS and inhibiting antiapoptotic factors. Our study investigated the cytotoxicity of DS/Cu complex in acute lymphoblastic leukemia and Burkitt's lymphoma cell lines. MTT assay showed that at a low concentration (1μM) of Cu2+, DS induces cytotoxicity to Molt4 and Raji cells with IC50s of 0.435±0.109μM and 0.085±0.015μM respectively. The morphology and Annexin-V/PI flow cytometric analysis indicated that DSF/Cu induced apoptosis of Molt4 and Raji cells. The apoptotic proportion of Molt4 cells increased from 17.75% to 79.5% when exposed to increasing concentrations of DS (0.125, 0.25, 0.5, 1μM) for 24h. The DS/Cu induced apoptosis is also time-dependent. The apoptotic proportion of Raji cells increased from 18.89±5.86% to 81.03±7.91% when exposed to DS (3.3μM) and Cu (1μM) for 6, 12 and 24h. Nrf2 is a key antioxidant factor. Western blot indicated that Nrf2 nuclear translocation was changed in a time-dependent manner after cells being treated by DS/Cu. Nrf2 expression was increased when Raji cells were treated for less than 12h and decreased after 18h or 24h treatment (Figure 1). Inhibition of Nrf2 could also be seen in Molt4 cells after 24h treatment. QT-PCR showed that DS/Cu down-regulated Nrf2 gene expression in a concentration dependent manner. ROS levels are closely related to Nrf2. Flow cytometric analysis showed that DS/Cu induced ROS generation. Western blot manifested that DS/Cu complex induced phosphorylation of JNK expression and inhibited P65 activity (Figure 2). N-acetyl-L-cysteine (NAC), an antioxidant, can partially attenuate DS/Cu complex-induced apoptosis, restore Nrf2 nuclear translocation, reactivate P65 activity and block JNK activation (Figure 3). In conclusion, our study showed that DS/Cu induces apoptosis in vitro and shows anticancer activity in vivo to lymphoid malignancy cell lines. We also demonstrated that ROS played a key role in DS/Cu- induced apoptosis. ROS can partially inhibit P65 activity and activate JNK while having bi phase regulation of Nrf2 expression. The initially increased Nrf2 expression decreases after 18h and 24h treatment. Therefore the DS/Cu induced ROS may be higher than that antioxidant factors could protect and thus the Nrf2-mediated cellular survival mechanism was disabled through down regulation of Nrf2 to allow initiation of death process. Figure 1. Bi phase regulation (right) and decrease (left) of Nrf2 expression in Molt4 (left) and Raji (right) cells Figure 1. Bi phase regulation (right) and decrease (left) of Nrf2 expression in Molt4 (left) and Raji (right) cells Figure 2. Inhibition of P65 activity and activation of JNK by DS/Cu in Molt4 (left) and Raji (right) cells Figure 2. Inhibition of P65 activity and activation of JNK by DS/Cu in Molt4 (left) and Raji (right) cells Figure 3. Partially restoration of Nrf2 nucelar translocation, block of inhibition of P65 activity and activation of JNK in Molt4 (A) and Raji (B) cells Figure 3. Partially restoration of Nrf2 nucelar translocation, block of inhibition of P65 activity and activation of JNK in Molt4 (A) and Raji (B) cells Disclosures: No relevant conflicts of interest to declare.

Description: Background Relapse and treatment-related complication have been the hurdle for the cure of acute myeloid leukemia patients. Finding a new drug-sensitizer could significantly prolong the survival time of patients. Triptolide (TPL) has been shown to enhance the drug-sensitivity of a variety of cancer cells when used in a low-concentration. HL60/DOX is a kind of acute myeloid leukemia cell line which is resistant to a commonly used drug Doxorubicin. This kind of cell line is widely used in the study of drug-sensitization. Aim This study aims to investigate whether low-dose TPL could enhance the drug-sensitivity of resistant acute myeloid leukemia cell line HL60/DOX to Doxorubicin and related mechanism. Method HL60/DOX cells were subjected to different treatments and thereafter MTT assay, flow cytometry and Western blot or RT-PCR were used to determine IC50, apoptotic status and expression of Nrf2, HIF-1α and their target genes. Results HL60/DOX cells were exposed to increasing concentrations of Doxorubicin with TPL at a constant IC20 concentration (14nM) for 48h. In comparison with anticancer agent alone, TPL enhanced the cytotoxicity of Doxorubicin (IC50:14.36±2.23 vs. 7.9±0.33μM, 1.82 fold; P=0.008) to HL60/DOX. Results of combination index showed that TPL and anti-cancer agents had synergistic effects when fraction affected was below 60%. Flow cytometry analysis also showed that apoptoticratio of cells treated by Doxorubicin together with TPL was significantly increased compared to cell streated by Doxorubicin alone (19.55±1.70%vs. 72.62±4.83%, P