Epigallocatechin‐3‐gallate enhances differentiation of acute promyelocytic leukemia cells via inhibition of PML‐RARα and HDAC1

The use of all‐trans retinoic acid (ATRA) has dramatically improved the treatment and survival rate of patients with acute promyelocytic leukemia (APL). However, toxicity and resistance to this drug are major problems in the treatment of APL with ATRA. Earlier studies have suggested that the green tea polyphenol epigallocatechin gallate (EGCG) induces cell death in hematopoietic neoplasms without adversely affecting normal cells. In the present study, the potential therapeutic effect of EGCG in APL and the underlying molecular mechanisms were investigated. EGCG (100 μM) significantly inhibited proliferation and induced apoptosis in HL‐60 and NB4 cells. This effect was associated with decreased expressions of multidrug resistance proteins ABCB1, and ABCC1, whereas the expressions of pro‐apoptotic genes CASP3, CASP8, p21, and Bax/Bcl‐2 ratio were significantly increased. EGCG, at 25 μM concentration, induced differentiation of leukemic cells towards granulocytic pattern in a similar manner to that observed for ATRA (1 μM). Furthermore, EGCG suppressed the expression of clinical marker PML/RARα in NB4 cells and reduced the expression of HDAC1 in leukemic cells. In conclusion, the results suggested that EGCG can be considered as a potential treatment for APL.     

清开灵注射液及其有效成分诱导人急性早幼粒白血病细胞凋亡的研究

目的：研究清开灵注射液及其有效成分治疗急性白血病的作用机理。方法：采用体外培养技术、MTT法、细胞形态学、DNA凝胶电泳及流式细胞检测技术，对清开灵及其有效成分诱导的人急性早幼粒白血症（HL－60）细胞凋亡进行分析。结果：清开灵及其有效成分黄芩甙、猪去氧胆酸对HL－60细胞均有很强的细胞毒作用，牛黄胆酸的作用次之；前三者在体外作用6h即可诱导细胞凋亡，流式细胞仪检测出现典型凋亡峰；牛黄胆酸无诱导HL－60细胞凋亡作用。结论：清开灵注射液及其有效成分可在体外诱导白血病细胞凋亡，可能是其治疗急性白血病的作用机理之一。     

Antiproliferative activity and apoptosis induction by trijuganone C isolated from the root of Salvia miltiorrhiza Bunge (Danshen)

In this study, we found that the hexane fraction of Danshen, the dried root of Salvia miltiorrhiza (Lamiaceae), exerted antiproliferative effects on human leukemia cells. Phytochemical investigation of the hexane fraction achieved the isolation of the tanshinone diterpenes: dihydrotanshinone I ( 1 ), trijuganone C ( 2 ), trijuganone B ( 3 ), cryptotanshinone ( 4 ), tanshinone IIA ( 5 ), and tanshinone I ( 6 ). Compound 2 showed significant antiproliferative activities against human leukemia cells HL‐60, Jurkat, and U937. The antiproliferative activities of 2 against human cancer and normal cells indicated that 2 exhibited potent antiproliferative activities with IC₅₀ values less than 10 μM against HL‐60 and Jurkat cells as well as on the colon cancer cells DLD‐1, COLO 205, and Caco‐2. Compound 2 induced chromatin condensation, DNA fragmentation, activation of caspase‐3, ‐8, and ‐9, and the cleavage of poly (ADP‐ribose) polymerase (PARP) in HL‐60 cells. Moreover, 2 activated Bid and Bax, leading to the loss of mitochondrial membrane potential, and 2 induced the cytochrome c release from mitochondria into cytosol. In contrast, Bcl‐2 and Bcl‐xL were unaffected by 2 . These results suggest that 2 exerts antiproliferative effects via apoptosis induction mediated by mitochondrial dysfunction and caspase activation. Compound 2 may serve as a candidate of potential chemotherapeutic agent for human leukemia.     

蓬子菜总黄酮诱导急性早幼粒细胞白血病NB4细胞株凋亡的研究

目的:探讨蓬子菜总黄酮(FGVL)抗急性早幼粒细胞白血病NB4细胞株的作用,为以后临床治疗早幼粒细胞白血病提供一个新的依据。方法:以急性早幼粒细胞白血病细胞株NB4为研究对象,实验分为3个实验组(FGVL质量浓度依次为50,100,200 mg·L~(-1))和空白组(相同体积的培养液),通过台盼蓝染色观察FGVL对NB4细胞生长的影响;噻唑蓝法(MTT)检测FGVL对细胞增殖的抑制作用;吖啶橙/溴乙锭(AO/EB)荧光染色观察NB4细胞凋亡的形态学变化;DNA琼脂糖凝胶电泳检测细胞凋亡;逆转录-聚合酶链式反应(RT-PCR)检测凋亡相关基因B细胞淋巴瘤/白血病-2(Bcl-2),Bcl-2相关X蛋白(Bax)mRNA的表达。结果:与空白组比较,FGVL(50,100,200 mg·L-1)能抑制NB4细胞的增殖(P0.01);24 h细胞形态学可见明显的细胞凋亡特征以及DNA琼脂糖凝胶电泳显示典型的DNA"梯形"凋亡条带;RT-PCR实验表明,与空白组比较,FGVL下调了抑制细胞凋亡基因Bcl-2 mRNA的表达,并上调促凋亡基因Bax mRNA的表达(P0.01),其作用与剂量呈正相关。结论:FGVL抗急性早幼粒细胞白血病NB4细胞的增殖并诱导细胞凋亡,其作用机制与调节Bcl-2/Bax表达有关,可为急性早幼粒细胞白血病临床化疗用药提供一个新的可能。     

β‐Eudesmol Induces JNK‐Dependent Apoptosis through the Mitochondrial Pathway in HL60 Cells

β‐eudesmol, a natural sesquiterpenol present in a variety of Chinese herbs, is known to inhibit the proliferation of human tumor cells. However, the molecular mechanisms of the effect of β‐eudesmol on human tumor cells are unknown. In the present study, we report the cytotoxic effect of β‐eudesmol on the human leukemia HL60 cells and its molecular mechanisms. The cytotoxic effect of β‐eudesmol on HL60 cells was associated with apoptosis, which was characterized by the presence of DNA fragmentation. β‐eudesmol‐induced apoptosis was accompanied by cleavage of caspase‐3, caspase‐9, and poly (ADP‐ribose) polymerase; downregulation of Bcl‐2 expression; release of cytochrome c from mitochondria; and decrease in mitochondrial membrane potential (MMP). Activation of c‐Jun N‐terminal kinases (JNK) mitogen‐activated protein kinases was observed in β‐eudesmol‐treated HL60 cells, and the inhibitor of JNK blocked the β‐eudesmol‐induced apoptosis, downregulation of Bcl‐2, and the loss of MMP. These data suggest that β‐eudesmol induces apoptosis in HL60 cells via the mitochondrial apoptotic pathway, which is controlled through JNK signaling. Copyright © 2012 John Wiley & Sons, Ltd.     

应用基因芯片研究雄黄对NB4细胞的作用

目的 :研究雄黄在诱导急性早幼粒细胞白血病细胞株NB4分化及凋亡过程中基因表达谱的改变。方法 :应用包含 1003条人类基因的cDNA表达谱芯片 ,检测雄黄作用于NB4细胞前后基因表达的调控。结果 :NB4细胞在雄黄作用后 12h ,9条基因上调 ,37条基因下调 ;2条参与蛋白酶体降解途径的基因显著上调 ,多条与细胞信号传导、RNA加工及蛋白质合成相关的基因下调。结论 :PSMC2 ,PSMD1及ITGB1基因的表达改变可能与NB4细胞的分化和凋亡有密切关系。     

低剂量雄黄诱导NB4细胞凋亡的研究

目的：探讨复方黄黛片中主要成分雄黄治疗急性早幼粒细胞白血病的作用机理。方法：应用台盘蓝排染法、流式细胞仪、DNA电泳、免疫印迹等多项方法,体外研究雄黄对NB4细胞的促凋亡作用。结果：雄黄时NB4细胞的生长有抑制作用,并有促凋亡效应,对细胞周期的影响表现为G2/M期的阻滞,药物处理的早期细胞凋亡,未见伴有bcl-2的下调。结论：雄黄作用的早期主要以细胞凋亡为主,并且可能通过其他途径诱导细胞凋亡。     

Curcumin and tetrahydrocurcumin induce cell death in Ara‐C‐resistant acute myeloid leukemia

Most anticancer agents induce cancer cell death; however, multidrug‐resistant cancers often lead to treatment failure. The effective use of curcumin as an anticancer agent has been demonstrated in clinical trials. Tetrahydrocurcumin, a major curcumin metabolite, exhibits pharmacological activities similar to those of curcumin. Curcumin induces cell death mainly through the apoptosis pathway, and tetrahydrocurcumin induces cell death mainly via an autophagy pathway in HL60 cells. Here, we investigated whether curcumin and tetrahydrocurcumin can induce apoptosis‐ and autophagy‐mediated cell deaths in Ara‐C‐resistant cancer cells, respectively. The results demonstrated that curcumin and tetrahydrocurcumin induced cell death by apoptosis and autophagy, respectively, in Ara‐C‐resistant HL60 cells. Thus, curcumin and tetrahydrocurcumin have potential applications in the treatment of acute myeloid leukemia with Ara‐C resistance.     

Antitumour effects of selected plant polyphenols,gallic acid and ellagic acid,on sensitive and multidrug‐resistant leukaemia HL60 cells

The aim of this study was to examine the antitumour effects of plant phenolic acids, gallic acid (GA) and ellagic acid (EA), on human promyelocytic leukaemia sensitive HL60 cell line and its resistant sublines exhibiting two MDR phenotypes: HL60/VINC (overexpressing P‐glycoprotein) and HL60/MX2 (characterized by the presence of mutated α isoform of topoisomerase II). Both studied compounds exerted comparable cytotoxic activities towards sensitive HL60 cells and their MDR counterparts. It was also found that GA and EA modulated the cellular level of reactive oxygen species in a dose‐dependent and time‐dependent manner. Furthermore, it was demonstrated that GA (IC₉₀) and EA (IC₅₀ and IC₉₀) significantly increased the percentage of sub‐G1 subpopulation of all studied leukaemia cells causing oligonucleosomal DNA fragmentation. Both compounds used at IC₉₀ triggered mainly the apoptotic death of these cells. However, GA had no effect on the activity of caspase‐3 as well as caspase‐8 in sensitive HL60 cells and their MDR counterparts. In contrast, EA provoked a significant activation of these caspases in all studied leukaemia cells. It was also found that lysosomes were not involved in triggering programmed death of sensitive HL60 and MDR cells by GA and EA.     

人参皂甙诱导HL-60细胞凋亡的研究

目的：观察人参皂甙（GS）是否具有诱导髓性白血病HL－60细胞株的凋亡作用。方法：取不同浓度的GS处理HL－60细胞，观察GS所致的细胞形态学的变化；流式细胞术分析细胞DNH含量的改变，并进行DNA片段分析（DNA Ladder);用Annexin V-FITC试验法分析细胞凋亡百分率。结果：人参皂甙能够抑制HL－60细胞生长，在一定剂量和时间范围内可引起细胞凋亡。结论：提示人参皂甙能特异性地诱导HL－60细胞凋亡，可为临床应用人参皂甙作为化疗药物的辅助剂治疗白血病提供实验依据。     

(-)-Epigallocatechin-3-O-gallate induces nonapoptotic cell death in leukemia cells independent of the 67 kDa laminin receptor

The 67 kDa laminin receptor (67 LR) mediates (-)-epigallocatechin-3-O-gallate (1; EGCG)-67 LR direct action only at physiological concentrations. The relevancy of biological effects of 1 at physiological concentrations to 67 LR was investigated in myeloid and lymphoid leukemia cells using flow cytometric analysis. It was shown that physiological concentrations of 1 suppressed the cell growth of HL60 myeloid leukemia cells and Raji lymphoid leukemic cells independent of 67 LR expression. Moreover, there was no discernible change in the levels of intracellular reactive oxygen species, characteristics of apoptosis such as phosphatidylserine translocation and activated caspase-3. The activity of 1 at physiological concentrations does not depend on direct 67 LR-mediated actions, and this compound induces necrosis-like death of promyelocytic leukemia and non-Hodgkin's lymphoma cells.     

全蝎对人白血病HL-60细胞作用的研究

[目的]观察全蝎提取液对急性早幼粒细胞株(HL-60)细胞及相关基因的影响。[方法]全蝎诱导HL-60细胞凋亡的形态学观察及流式细胞仪分析；全蝎对HL-60细胞周期分布的影响；免疫组化法观察全蝎对HL一60细胞凋亡调控基因表达的影响。[结果]全蝎可以明显促进HL-60细胞的程序化死亡，并在一定范围内呈正比关系，最高浓度(10mg／mL)时，凋亡细胞比例明显增加，达21．44%。中、小剂量药物浓度时，细胞凋亡比例略有增加，分别为12．56％和9．47％，对照组仅7．36％。同时可显著提高P53因的表达水平(与对照组相比，P＜O．01)和降低bcl-2基因的表达。[结论]全蝎提取液对HL-60细胞凋亡及相关基因的表达具有益影响。     

Acacetin‐induced cell apoptosis in head and neck squamous cell carcinoma cells: Evidence for the role of muscarinic M3 receptor

Aacacetin, a plant flavone has shown antitumor efficacy recently. However, its associated mechanisms are poorly known. We hypothesized that the muscarinic M3 receptor (M₃R), which is highly expressed in some cancer tissue, is related to the antitumor effect of acacetin in head and neck squamous cell carcinoma (HNSCC) cells. Our results showed that 12.5‐ to 200‐μM acacetin inhibited cell viability in dose‐ and time‐dependent manners in HNSCC cells, but a relative higher concentration was needed for oral adenoid cystic carcinoma cells. M₃R expression level was higher in HNSCC cells than that in adenoid cystic carcinoma cells. Flow cytometry and electron microscopy confirmed acacetin‐induced cell apoptosis in 22B cells, a HNSCC cell line. Acacetin promoted mitochondrial cytochrome c release and caspase 9, 3 processing. Knocking down of M₃R expression by specific siRNA significantly prevented the acacetin‐induced cell viability damage, cell apoptosis, and caspase 3 activation. Besides, M₃R was also involved in acacetin‐induced elevation of reactive oxygen species and intracellular calcium ([Ca²⁺]_i). These data indicate that acacetin‐induced cell apoptosis in HNSCC cells may through M₃R related calcium signaling and caspase 3 activation. Acacetin is a potent natural antitumor reagent especially for the tumor cells, which highly expressed M₃R.     

Corosolic Acid Triggers Mitochondria and Caspase‐dependent Apoptotic Cell Death in Osteosarcoma MG‐63 Cells

The response of osteosarcoma MG‐63 cells to corosolic acid treatment has been investigated. The results showed that corosolic acid significantly inhibited cell viability in both a dose and a time dependent manner. It was found that corosolic acid increased the Bax/Bcl‐2 ratio by up‐regulating Bax expression, disrupted mitochondrial membrane potential and triggered the release of cytochrome c from mitochondria into the cytoplasm. Corosolic acid treatment triggered the activation of caspase‐8, 9 and 3. The apoptosis was obviously inhibited by pretreatment with a general caspase inhibitor, z‐VAD‐FMK. Moreover, pretreatment of CsA, a cyclophilin D ligand that inhibits mitochondria potential uncoupling, prevented the activation of caspase‐9 and caspase‐3, but not caspase‐8, and the apoptosis of MG‐63 cells, triggered by corosolic acid. All these results indicated that corosolic acid‐induced apoptosis was associated with the activation of caspases via a mitochondrial pathway. Copyright © 2011 John Wiley & Sons, Ltd.     

构建雄黄诱导维甲酸耐药急性早幼粒细胞白血病NB4-R1细胞凋亡的蛋白质双向电泳图谱

目的 构建雄黄（四硫化四砷, tetra-arsenic tetra-sulfide, As4S4）诱导急性早幼粒细胞白?。ˋPL)维甲酸耐药细胞株NB4-R1细胞凋亡前后的差异蛋白质组表达谱。     

雄黄对NB4和MR2细胞组织因子表达的影响

目的 :探讨雄黄对NB4细胞 (维甲酸敏感的急性早幼粒细胞白血病细胞株 )和MR2 细胞 (维甲酸耐药的急性早幼粒细胞白血病细胞株 )促凝活性 (PCA)和组织因子 (TF)表达的影响。方法 :用 300μg·L^-1雄黄与NB₄和MR₂ 细胞共同孵育 ,分别用复钙时间、ELISA方法、半定量RT PCR检测未处理及雄黄处理6 ,12 ,24 ,48,72h后NB₄和MR₂ 细胞的促凝活性、TF抗原表达及TFmRNA转录的变化。同时检测未处理的HL 6 0 ,K562细胞的PCA ,TF抗原表达作为对照。结果 :NB₄和MR₂细胞的PCA和TF抗原水平明显高于HL 60和K562细胞。雄黄呈时间依赖方式下调NB₄和MR₂ 细胞的促凝活性 ,TF抗原的表达及TFmRNA的转录。结论 :下调NB₄和MR₂ 细胞TF的表达并降低其PCA可能是雄黄改善APL患者弥散性血管内凝血 (DIC)早期出血症状的主要机制之一。     

六神丸诱导NB4细胞凋亡的实验研究

目的 :观察六神丸诱导 NB4白血病细胞凋亡的作用。方法 :应用 TUNEL法和 Annexin V/ PI双染法检测不同浓度和不同时间段六神丸诱导 NB4细胞的凋亡率。结果 :TU NEL法表明凋亡率与剂量呈正相关性 ,10 0μg/ ml六神丸凋亡率最高。 Annexin V / PI双染法表明 5 0μg/ ml六神丸作用 32 h凋亡率最高。结论 :六神丸可诱导白血病细胞凋亡。     

The ethyl acetate extract of Phellinus linteus grown on germinated brown rice induces G0/G1 cell cycle arrest and apoptosis in human colon carcinoma HT29 cells

It is well known that Phellinus linteus has a variety of biological functions, such as antitumor and immunomodulating activities. In our previous studies, we developed a P. linteus grown on germinated brown rice (PBR) and found that organic solvent extracts of PBR possessed immunomodulating activity to regulate a balance of cytokine network in mice. The components of PBR are ergosterol peroxide, γ‐aminobutyric acid (GABA) and B‐glucan. In this study, we demonstrate that an organic solvent extract of P. linteus grown on PBR induced apoptotic cell death through the induction of G₀/G₁ arrest of cell cycle and the apoptosis via DNA fragmentation in human colon carcinoma HT‐29 cells. Cell death induced by the extract of P. linteus grown on PBR was shown to be associated with the upregulation of p21^CIP1/WAF1, the downregulation of cyclin D1, anti‐apoptotic protein, Bcl‐2, the release of cytochrome c, and the activation of caspase‐9, caspase‐3 and caspase‐8. This study suggests that the ethyl acetate extract of P. linteus grown on PBR induces apoptosis accompanied by cell cycle arrest at G₀/G₁ phase and regulates apoptosis‐regulatory proteins, which may be applicable to anticancer therapy. Copyright © 2009 John Wiley & Sons, Ltd.     

Auraptene Induces Apoptosis via Myeloid Cell Leukemia 1‐Mediated Activation of Caspases in PC3 and DU145 Prostate Cancer Cells

Although auraptene, a prenyloxy coumarin from Citrus species, was known to have anti‐oxidant, anti‐bacterial, antiinflammatory, and anti‐tumor activities, the underlying anti‐tumor mechanism of auraptene in prostate cancers is not fully understood to date. Thus, in the present study, we have investigated the anti‐tumor mechanism of auraptene mainly in PC3 and DU145 prostate cancer cells, because auraptene suppressed the viability of androgen‐independent PC3 and DU145 prostate cancer cells better than androgen‐sensitive LNCaP cells. Also, auraptene notably increased sub‐G1 cell population and terminal deoxynucleotidyl transferase dUTP nick end labeling‐positive cells as features of apoptosis in two prostate cancer cells compared with untreated control. Consistently, auraptene cleaved poly(ADP‐ribose) polymerase, activated caspase‐9 and caspase‐3, suppressed the expression of anti‐apoptotic proteins, including Bcl‐2 and myeloid cell leukemia 1 (Mcl‐1), and also activated pro‐apoptotic protein Bax in both prostate cancer cells. However, Mcl‐1 overexpression reversed the apoptotic effect of auraptene to increase sub‐G1 population and induce caspase‐9/3 in both prostate cancer cells. Taken together, the results support scientific evidences that auraptene induces apoptosis in PC3 and DU145 prostate cancer cells via Mcl‐1‐mediated activation of caspases as a potent chemopreventive agent for prostate cancer prevention and treatment. Copyright © 2017 John Wiley & Sons, Ltd.