ACSL5, a prognostic factor in acute myeloid leukemia, modulates the activity of Wnt/β-catenin signaling by palmitoylation modification / 医学前沿

Acyl-CoA synthetase long chain family member 5 (ACSL5), is a member of the acyl-CoA synthetases (ACSs) family that activates long chain fatty acids by catalyzing the synthesis of fatty acyl-CoAs. The dysregulation of ACSL5 has been reported in some cancers, such as glioma and colon cancers. However, little is known about the role of ACSL5 in acute myeloid leukemia (AML). We found that the expression of ACSL5 was higher in bone marrow cells from AML patients compared with that from healthy donors. ACSL5 level could serve as an independent prognostic predictor of the overall survival of AML patients. In AML cells, the ACSL5 knockdown inhibited cell growth both in vitro and in vivo. Mechanistically, the knockdown of ACSL5 suppressed the activation of the Wnt/β-catenin pathway by suppressing the palmitoylation modification of Wnt3a. Additionally, triacsin c, a pan-ACS family inhibitor, inhibited cell growth and robustly induced cell apoptosis when combined with ABT-199, the FDA approved BCL-2 inhibitor for AML therapy. Our results indicate that ACSL5 is a potential prognosis marker for AML and a promising pharmacological target for the treatment of molecularly stratified AML.

Effects of ABT-737 on the growth and angiogenesis of ovarian cancer cells in the co-culture of TAMs and SKOV3 cells / 中国临床药理学与治疗学

AIM: To explore the effect of Bcl-2 small molecule inhibitor ABT-737 on the growth and angiogenesis mimicry of SKOV3 cells in a co-culture system of Tumour-associated macrophages (TAMs) and human ovarian cancer cells SKOV3. METHODS: PMA and IL-4 was used to induce THP-1 cells into TAMs cells in vitro; MTT method was used to detect the cell survival rate of SKOV3 cells after 24 hours of treatment with different concentrations of ABT-737 culture medium; a co-culture system of SKOV3 cells and TAMs cells was established; the experimental groups were divided into control group, SKOV3+ABT-737 group (containing 5.0 μmol/L ABT-737 cultured cells), TAMs+SKOV3 group (SKOV3 cells co-cultured with TAMs cells), TAMs+SKOV3+ABT-737 group (SKOV3 cells Co-cultured with TAMs cells, and added ABT-737 containing 5.0 μmol/L), cells after 24 h was collected, MTT method was used to detect cell survival rate, EdU staining for cell proliferation, ranswell chamber experiment for cell migration and invasion, Flowcytometry for cell apoptosis, the vascular mimicry experiment for the ability of cells to form blood vessels, Western blot for the expression of vascular endothelial growth factor (VEGF), matrix metalloproteinase-2 (MMP-2) and MMP-9 in cells. RESULTS: THP-1 cells were successfully induced for TAMs cells; the survival rate of SKOV3 cells decreased under the action of ABT-737 (P<0.01); compared with the control group, the survival rate of SKOV3 cells in the SKOV3+ABT-737 group decreased, the number of EdU-labeled positive cells decreased, the number of cell migration and invasion also decreased, the rate of apoptosis increased, and the duct branches decreased, The protein expression of VEGF, MMP-2, MMP-9 decreased (P<0.01); Compared with the TAMs+SKOV3 group, the cell survival rate of the TAMs+SKOV3+ABT-737 group decreased, the number of EdU-labeled positive cells and the number of cell migration and invasion also decreased, the apoptosis rate increased, and the duct branches decreased. At the same time, the protein expression of VEGF, MMP-2, MMP-9 decreased (P<0.01). CONCLUSION: ABT-737 can inhibit SKOV3 cell proliferation, metastasis, apoptosis and angiogenesis in a co-culture system, and affect tumor progression.

Targeted therapy with a selective BCL-2 inhibitor in older patients with acute myeloid leukemia

ABSTRACT Background: Older patients with acute myeloid leukemia are particularly difficult to treat, as they have a high risk of comorbidities, poor performance status and less tolerability to chemotherapy, as well as a more aggressive disease biology, responsible for the resistance to treatment. There is a need to explore novel therapeutic agents that are more effective and tolerable. Venetoclax, a BCL-2 inhibitor is a promising agent, as BCL-2 overexpression is present in 84% of acute myeloid leukemia patients at diagnosis and 95% of patients at relapse and has been associated with leukemia cell survival, chemotherapy resistance and poor prognosis. Objective: To review the available data about venetoclax in acute myeloid leukemia and how it can influence the treatment in older patients. Methods: Using the Pubmed database, we selected 29 articles published within the last 15 years, considering preclinical and clinical trials and review studies that combined venetoclax with acute myeloid leukemia. Results: Venetoclax has demonstrated promising results in preclinical and clinical trials, especially in patients with poor prognosis and the IDH mutation, with an excellent side-effect profile. However, resistance seems to develop rapidly with venetoclax monotherapy, because of antiapoptotic escape mechanisms. Conclusions: While the results with the use of venetoclax seem encouraging, it is not likely that targeting a single pathway will result in long-term disease control. The solution includes the use of combined therapy to block resistance mechanisms and enhance apoptosis, by reducing MCL-1, increasing BIM or inhibiting the complex IV in the mitochondria.

Expression of activator of basal transcription 1 in gastric cancer tissue and its clinical significance / 吉林大学学报(医学版)

Objective: To investigate the expression of activator of basal transcription 1 (ABT1) protein in gastric cancer tissue and its relationships with the clinical parameters and prognosis of gastric cancer patients, and to clarify the role of ABT1 in the occurrence and development of gastric cancer. Methods: A total of 100 cases of cancer tissue of the gastric cancer patients and 80 pairs of adjacent tissue were selected. The expressions of ABT1 in cancer tissue and adjacent tissue were detected by immunohistochemistry and the proportion of stained cells and the degree of staining in the immunohistochemistry results were analyzed using semi-quantitative analysis. The relationships between the semi-quantitative analysis results and the clinical parameters of gastric cancer patients were statistically analyzed. Kaplan-Meier method was used to analyze the correlation between the ABT1 protein expression level and the survival of gastric cancer patients. Results: ABTl-positive staining was observed in the nucleus and cytoplasm of gastric cancer tissue and adjacent gastric tissue. The expression level of ABT1 in gastric cancer tissue was lower than that in adjacent tissue (P= 0.021). The ABT1 protein expression level in gastric cancer tissue was significantly negatively correlated with the pathological grade (r=-0.224, P = 0. 026). The Kaplan-Meier analysis results of the survival curve showed that the high expression of ABT1 was associated with good prognosis in the gastric cancer patients (HR=1. 483, P<.0. 01). The survival rate of gastric cancer patients with high ABT1 expression was significantly higher than that of the patients with low ABT1 expression (HR = 2.411, P=0. 0272). Conclusion: The expression of ABT1 in gastric cancer tissue is lower, indicating that ABT1 can be used one of the markers of good prognosis of gastric cancer.

Expression of activator of basal transcription 1 in gastric cancer tissue and its clinical significance / 吉林大学学报(医学版)

Objective:To investigate the expression of activator of basal transcription 1 (ABT1) protein in gastric cancer tissue and its relationships with the clinical parameters and prognosis of gastric cancer patients, and to clarify the role of ABT1in the occurrence and development of gastric cancer.Methods:A total of 100cases of cancer tissue of the gastric cancer patients and 80pairs of adjacent tissue were selected.The expressions of ABT1in cancer tissue and adjacent tissue were detected by immunohistochemistry and the proportion of stained cells and the degree of staining in the immunohistochemistry results were analyzed using semi-quantitative analysis.The relationships between the semi-quantitative analysis results and the clinical parameters of gastric cancer patients were statistically analyzed.Kaplan-Meier method was used to analyze the correlation between the ABT1 protein expression level and the survival of gastric cancer patients.Results:ABT1-positive staining was observed in the nucleus and cytoplasm of gastric cancer tissue and adjacent gastric tissue.The expression level of ABT1in gastric cancer tissue was lower than that in adjacent tissue (P=0.021) .The ABT1protein expression level in gastric cancer tissue was significantly negatively correlated with the pathological grade (r=-0.224, P=0.026) .The Kaplan-Meier analysis results of the survival curve showed that the high expression of ABT1was associated with good prognosis in the gastric cancer patients (HR=1.483, P＜0.01) .The survival rate of gastric cancer patients with high ABT1expression was significantly higher than that of the patients with low ABT1expression (HR=2.411, P=0.0272) .Conclusion:The expression of ABT1in gastric cancer tissue is lower, indicating that ABT1can be used one of the markers of good prognosis of gastric cancer.

Abt-737 enhances apoptosis of gastric cancer cells induced by MCL-1 small-molecule inhibitor UMI-77 / 中国药理学通报

Aim: To investigate the promoting effect of Bcl-2/Bcl-xL inhibitor ABT-737 on apoptosis of gastric cancer cells induced by small molecule Mcl-1 inhibitor UMI-77, and to explore its possible mechanism. Methods: The response of gastric cancer MGC-803 and HGC-27 cells to different concentrations of UMI-77 was detected by MTS assay. In the UMI-77-resistant cell lines, the effect of treatment with UMI-77/ABT-737 alone or in combination on cell viability was detected by MTS assay. The apoptotic rate and the changes of the mitochondrial membrane potential were analyzed by flow cytometry. The cleavage of caspase-9, caspase-3 and PARP-1, as well as the expression level of Bcl-2 family members and IAP proteins, were determined by Western blot. Results: Compared with MGC-803 cells, HGC-27 cells were resistant to UMI-77. Treatment with ABT-737 alone in HGC-27 cells also induced minimal level of cell death. While treatment with both agents induced much greater decreased cell viability. All the dead cells were positive for Annexin V and mitochondrial membrane potential collapsed. Caspase-9, caspase-3 and its substrate PARP-1 were cleaved. All of these proved that the sensitization effect was achieved by activating the mitochondrial apoptotic pathway. Protein levels of XIAP, cIAP1 and cIAP2 decreased after treatment with UMI-77 plus ABT-737. It also resulted in the increase of NOXA and Bcl-2 along with the decline of PUMA and Mcl-1. Conclusions: The combination of UMI-77 and ABT-737 could significantly increase the sensitivity of gastric cancer cells to the Mcl-1 small molecule inhibitor UMI-77.

Mechanism of gemcitabine enhances ABT-199 induced apoptosis in Philadelphia chromosome-positive acute lymphoblastic leukemia:a preliminary study / 白血病·淋巴瘤

Objective To investigate the effects of gemcitabine and ABT-199 on proliferation inhibition and apoptosis induction of Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL) cell line SUP-B15, and to explore its synergistic mechanism. Methods SUP-B15 cells in logarithmic growth phase were treated with gemcitabine (0.025 and 0.050 μmol/L), ABT-199 (0, 0.5, 1.0, 2.0, 4.0, 8.0 μmol/L) or two drugs for 24 h. Cell proliferation was detected by CCK-8 method, apoptosis was detected by flow cytometry (FCM), mitochondrial membrane potential was detected by JC-1 method, and expression of mitochondrial apoptosis pathway-related protein was analyzed by Western blot. Results The 50 ％ inhibitory concentration (IC50) of SBT-B15 cells treated with ABT-199 for 24 h was (4.13±0.89) μmol/L. However, gemcitabine (0.025, 0.050 μmol/L) significantly enhanced the inhibitory effect of ABT-199 on proliferation of SUP-B15 cells, the IC50 values were (2.23 ±0.73) and (1.15 ±0.45) μmol/L, respectively. The results of FCM assay showed that compared with the monotherapy group [(7.33±1.54)％], 0.025 umol/L gemcitabine combined with ABT-199 (1.0 and 2.0 μmol/L) acted on SUP-B15 cells for 24 h, the proportions of apoptotic cells were (32.42±1.45) ％and (44.33±1.86) ％, the difference was statistically significant (F=70.78, P<0.001);compared with the monotherapy group [(9.60 ±2.76) ％], 0.05 μmol/L gemcitabine combined with ABT-199 (1.0 and 2.0 μmol/L) acted on SUP-B15 cells for 24 h, the proportion of apoptotic cells increased to (47.63 ± 3.81) ％ and (58.73 ±4.33) ％, respectively, and the difference was statistically significant (F= 79.21, P<0.001). The JC-1 experiment showed that treated with ABT-199 and gemcitabine for 12 h, the percentage of depolarizing cell was significantly higher than that in single agent group, and the difference was statistical significant (P<0.001). Western blot showed that the anti-apoptotic proteins bcl-2, bcl-xL and Mcl-1 decreased after treated by gemcitabine combined with ABT-199 for 12 h. Conclusion Gemcitabine could enhance the proliferation inhibition and induce apoptosis of Ph+ALL cells by ABT-199, and its mechanism may be related to down-regulation of anti-apoptosis-related proteins.

ABT-737 ameliorates docetaxel resistance in triple negative breast cancer cell line

PURPOSE: This study aimed to validate the synergistic effect of ABT-737 on docetaxel using MDA-MB-231, a triple negative breast cancer (TNBC) cell line overexpressing B-cell lymphoma-2 (Bcl-2). METHODS: Western blot analysis was performed to assess expression levels of Bcl-2 family proteins and caspase-related molecules. Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell cycle distribution was determined by flow cytometry analysis. Benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (z-VAD-fmk) was used for pretreatment to assess the role of caspases. RESULTS: Cell viability of MDA-MB-231 after combination treatment with ABT-737 and docetaxel was significantly lower than that after docetaxel or ABT-737 monotherapy based on MTT assay (both P < 0.001), with a combination index of 0.41. The proportion of sub-G1 population after combination treatment was significantly higher than that after docetaxel or ABT-737 monotherapy (P = 0.001, P = 0.003, respectively). Pretreatment with z-VAD-fmk completely restored cell viability of MDA-MB-231 from apoptotic cell death induced by combination therapy (P = 0.001). Although pro-caspase-8 or Bid did not show significant change in expression level, pro-casepase-9 showed significantly decreased expression after combination treatment. Cleaved caspase-3 showed increased expression while poly (ADP-ribose) polymerase cleavage was induced after combination treatment. However, hypoxia-inducible factor 1-alpha and aldehyde dehydrogenase 1 totally lost their expression after combination treatment. CONCLUSION: Combination of ABT-737 with docetaxel elicits synergistic therapeutic effect on MDA-MB-231, a TNBC cell line overexpressing Bcl-2, mainly by activating the intrinsic pathway of apoptosis. Therefore, adjunct of ABT-737 to docetaxel might be a new therapeutic option to overcome docetaxel resistance of TNBCs overexpressing Bcl-2.

ABT-263 induced A431 cell apoptosis and its effect on AKT downstream signaling pathway / 中国职业医学

OBJECTIVE: To investigate the effect of ABT-263,an anti-apoptotic protein inhibitor,on human cutaneous squamous cell carcinoma A431 cells,and to explore its molecular mechanisms. METHODS: i) Total protein was extracted from human immortalized epidermal cells( Ha Ca T cells) and A431 cells in logarithmic growth phase. The protein expression of B-cell lymphoma-2( BCL-2) and BCL2-like 1( BCL-XL) was detected by Western blotting. ii) The A431 cells were treated with ABT-263( inhibitor group) and dimethyl sulfoxide( control group) at a concentration of 50 μmol/L for 4 and 9 hours. The morphological changes of the cells were examined by transmission electron microscopy. iii) The A431 cells were treated with 0,10,25,40,and 50 μmol/L of ABT-263 for 24 hours,and the cell viability was determined by CCK-8 assay. iv) The A431 cells were treated with different doses of ABT-263,and the expression of cleaved Caspase-3, cleaved poly( ADP-ribose) polymerase-1( PARP-1), phosphorylated protein kinase B [p AKT(ser473)],phosphorylated glycogen synthase kinase-3β(p GSK3β) and phosphorylated histone H2 AX(γH2 AX) was detected by Western blot. RESULTS: The relative expression of BCL-2 and BCL-XL in A431 cells were higher than those in Ha Ca T cells( P < 0. 01). Transmission electron microscopy results showed that A431 cells in inhibitor group gradually changed from normal morphology to apoptotic morphology,showing loss of microvilli,increased nuclear chromatin density and aggregation around the nuclear membrane,and nuclear fragmentation. The cell viability of A431 cells in 10,25,40 and 50 μmol/L groups were lower than those in control group( P < 0. 05). The relative expression of cleaved Caspase-3 and cleaved PARP-1 in A431 cells in 10,30 and 50 μmol/L groups were higher than those in control group( P < 0. 05).The relative expression of p AKT( ser473) and p GSK3β in A431 cells in 10,25,40 and 50 μmol/L groups were lower than those of the control group( P < 0. 05) and γH2 AX protein expression was higher than that of the control group( P <0. 05). A431 cell viability and p GSK3β protein expression decreased with the increase of inhibitor dosage( P < 0. 01).The relative expression of cleaved Caspase-3 and γH2 AX protein increased with the increase of inhibitor dosage( P <0. 01),showing dose-effect relationship. CONCLUSION: ABT-263 can induce apoptosis of A431 cells through mitochondria pathway and induce the inactivation of AKT/GSK3β pathway,which can promote the apoptosis of A431 cells with a doseeffect relationship.

Effects of emodin in Polygonum multiflorum on liver cytotoxicity and CYP450 isoenzymes expression in L02 cells / 中国药理学通报

Aim To study the effect of emodin in Po-lygonum multiflorum on the expression of CYP450 isoenzymes in L02 hepatocytes and explore its mecha-nism of cytotoxicity. Methods L02 cells were treated with different concentrations of emodin. Cell viability was examined by MTS assay kit, and cell membrane injury was examined by detecting the release rate of lactate dehydrogenase( LDH) . The expression of cyto-chrome P450 mRNA was detected by real time PCR. Results The result of MTS assay showed that L02 cells viability was significantly reduced following expo-sure to emodin in a concentration and time dependent manner. The LDH release rate of L02 cells significant-ly increased after exposure to emodin for 48 h com-pared with the control group. On the mRNA level, compared with the control group,emodin had inductive effects on mRNA of each CYP450 enzyme, while had significant inductive effects on mRNA of CYP1 A1 and CYP1 B1 in a concentration and time dependent man-ner. Conclusion Emodin in Polygonum multiflorum may generate significant liver injury in L02 cells and has inductive effects on CYP450 enzyme activity.

Effect of dopamine D4 receptor agonist ABT-724 on behaviors in rat model of attention-deficit hyperactivity disorder / 中华行为医学与脑科学杂志

Objective To investigate the effects of a highly selective dopamine D4 receptor (ABT-724)on behaviors in Spontaneously hypertensive rats (SHR),a rat model of attention-deficit hyperactivity disorder (ADHD).Methods After intervention of methylphenidate(5mg/kg) and three different doses of ABT-724(0.04mg/kg,0.16mg/kg,0.64mg/kg),behaviors of SHR were verified by open-field test,Morris water maze and Làt maze.Results Number of square crossing after intervention of methylphenidate and ABT-724 in SHR( 70.67 ± 8.59,76.50 ± 10.75,79.17 ± 10.44,65.67 ± 20.62) was less than the saline control group( 130.33 ± 1 1.40) (P＜0.05).During Morris water maze,SHRs(52.50 ± 4.04,52.17 ± 2.99,61 ± 8.15,53.83 ± 9.87 ) after intervention of both had a better spatial memory ability than control group(38.83 ±7.17) (P＜0.05).In Làt maze,number of rearing after intervention of both in SHRs(57.17 ± 5.67,60.83 ± 8.28,55.17 ± 9.45,65.33 ± 9.50 ) was less than saline control group(78.00 ± 13.84) (P＜0.05).Conclusion ABT-724 could improve behaviors of spontaneous locomotor activity,cognitive ability,non-selective attention in SHR.

Effect of ABT-594 a Selective Nicotinic Agonist, on Voiding Function in Spinal Cord Injury Rat

PURPOSE: In this study we demonstrate effect of selective nicotinic agonist, ABT-594 on voiding in spinal cord injury rat. MATERIALS AND METHODS: Spinal cord injury rat was made by complete resection of the spinal cord(T8~9) and checked cystometry three weeks after injury. In female Sprague-Dawley awake rats, an intravesical catheter was inserted through the bladder dome and intravenous catheter was inserted to right jugular vein. After the surgery, cystometry was performed by infusing saline into the bladder(0.08 ml/min). Cumulative doses of ABT-594 (0.01, 0.1, 1, 10 ug/kg) were injected intravenously in spinal cord injury rat at 1 hour intervals. RESULTS: All dose(0.01~10 ug/kg) of ABT-594 did not affect the intercontraction interval(ICI), maximal voiding pressure(MVP), and pressure threshold(PT), and from voiding contraction to non-voiding contraction(from VC to NVC), voiding volume(VV), residual volume(RV). But moderate dose(1 ug) of ABT-594 significantly decreased number of non-voiding contraction(55.6+/-13.7% of control)(p<0.05). CONCLUSION: These result suggest that the nicotinic agonist ABT-594 might in part affect voiding function through stimulation of serotonergic nerve to lumbosacral level in spinal cord injury rat.