Costunolide enhances sensitivity of K562/ADR chronic myeloid leukemia cells to doxorubicin through PI3K/Akt pathway

Costunolide, a sesquiterpene lactone, a small molecular monomer extracted from Inula helenium, has been reported to possess antiproliferative effects on several cancer cell lines. The current study was designed to evaluate the effect of costunolide on sensitivity of K562/ADR chronic myeloid leukemia cells to doxorubicin. The antiproliferative effect of costunolide was assessed by CCK‐8 assay. Flow cytometry and Western blot were used to examine the mechanisms of antileukemia action. Costunolide dramatically enhanced doxorubicin‐induced antiproliferative activity against K562/ADR cells through inhibition of PI3K/Akt pathway, activation of caspases 3, cleavage of poly (ADP‐ribose) polymerase, and downregulation of p‐glycoprotein expression. These results demonstrate that costunolide may be a potent therapeutic agent against CML.     

复方三根制剂对MDR细胞株K562/ADR和K562/VCR逆转作用的研究

目的：研究复方中药——复方三根制剂对多药耐药(MDR)细胞株的逆转作用。方法：应用MTT比色法，观察复方三根制剂对MDR细胞的逆转作用。运用流式细胞仪(FCM)，测定其对耐药细胞积聚和外排阿霉素(ADR)的影响，以及对耐药株细胞表达P=gp的影响。结果：复方三根制剂可部分恢复K562／ADR和K562／VCR耐药细胞对ADR的敏感性，而对VCR抗药性的逆转作用不明显；可增加K562／ADR和K562／VCR耐药细胞内ADR的积聚，并对外排ADR有一定影响；可部分下调p-gp的表达。结论：复方三根制剂可部分逆转耐药细胞的抗药性。     

Growth Inhibition Effects of Isoalantolactone on K562/A02 Cells: Caspase‐dependent Apoptotic Pathways,S Phase Arrest,and Downregulation of Bcr/Abl

Isoalantolactone, a sesquiterpene lactone, is the active component of Inula helenium (Compositae). It has been reported that isoalantolactone has the capacity to inhibit tumor cell growth through induction of apoptosis. The purposes of this study were to evaluate the effects of isoalantolactone on the human erythroleukemia drug‐resistant cell line K562/A02 and to provide evidence of its function as a potent therapeutic agent in patients with chronic myelogenous leukemia with the Bcr/Abl phenotype. Our results showed that isoalantolactone significantly inhibited K562/A02 cell growth by downregulating Bcr/Abl expression. Isoalantolactone also induced apoptosis via increase generation of reactive oxygen species, modulation of the protein levels of Bcl‐2 family members, caspase activation, poly ADP‐ribose polymerase (PARP) cleavage, and release of cytochrome c. We also observed that isoalantolactone inhibited proliferation by inducing cell cycle arrest in the S phase. Taken together, all these findings support that growth inhibition effects of isoalantolactone on K562/A02 cells may be mediated through caspase‐dependent apoptotic pathways, S phase arrest, and downregulation of Bcr/Abl. Copyright © 2014 John Wiley & Sons, Ltd.     

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??OBJECTIVE To study the anti-leukemia activities and mechanisms of bergenin derivative D-23. METHODS CCK-8 method was applied to investigate anti-tumor activities of D-23. Flow cytometry and immunofluorescence assay were used to observe the effects of D-23 on the apoptosis and autophagy in K562 and Jurkat human leukemia cell lines. Western blot analysis was used to investigate the mechanisms that compound D-23 induced tumor cell apoptosis and autophagy. RESULTS Bergenin derivative D-23 could significantly inhibit the proliferation of K562 and Jurkat cells by inducing cell apoptosis and autophagy. The mitochondrial membrane potential was decreased,protein kinase B(Akt)and heat shock protein 70 (Hsp70) were inhibited,and the expressions of apoptotic related proteins caspase 3 and caspase 9 were activated. In addition, mammalian target of rapamycin (P-mTOR)(Ser 2448 and Ser 2481)protein was inhibited. CONCLUSION Bergenin derivative D-23 shows obvious anti-leukemia activities by inducing cell apoptosis and autophagy. The apoptosis may be associated with the reduction of mitochondrial membrane potential and activation of caspase pathway. The autophagy may be related to the inhibition of Akt/mTOR signaling pathway.     

Poncirin downregulates ATP‐binding cassette transporters to enhance cisplatin sensitivity in cisplatin‐resistant osteosarcoma cells

Poncirin, a flavanone glycoside with bitter taste extracted from dried immature fruit of Poncirus trifoliate, exhibits multiple biological activities including anti‐tumor activity. Our study aimed to determine the effect and potential mechanism of poncirin on cisplatin resistance in osteosarcoma (OS) cells. CCK‐8, flow cytometry analysis, and caspase‐3/7 activity assays were used to evaluate cisplatin sensitivity. The expression changes of multidrug resistance 1 (MDR1), multidrug resistance‐associated protein (MRP1), breast cancer resistance protein (BCRP), and phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt) pathway‐related proteins were detected by RT‐qPCR or western blot analyses. Results showed that poncirin exposure enhanced cisplatin sensitivity, promoted apoptosis, and increased caspase‐3/7 activity in cisplatin‐resistant OS cells. Poncirin decreased the expression levels of MDR1, MRP1, and BCRP, and inhibited the PI3K/Akt signaling in OS cells. Rescue experiments suggested that activation of the PI3K/Akt signaling by 740Y‐P abolished poncirin‐induced expression reduction of MDR1, MRP1, and BCRP, and attenuated the facilitative effects of poncirin on cisplatin sensitivity and apoptosis in cisplatin‐resistant OS cells. In summary, poncirin suppressed cisplatin resistance in cisplatin‐resistant OS cells by downregulating the expression of MDR1, MRP1, and BCRP through inhibiting the PI3K/Akt pathway.     

小檗胺对多药耐药K562/Adr细胞作用的研究

目的研究小檗胺诱导人白血病K562／Adr细胞凋亡及逆转多药耐药的作用及机理。方法采用MTT法测IC50值,流式细胞仪Annexin V FITC-PI法检测细胞凋亡发生率,PI染色法检测凋亡峰及细胞周期,同时以FCM检测Caspase-3、P-GP蛋白表达及细胞内药物积聚能力,RT-PCR法检测mdr-1基因表达。结果小檗胺能抑制人白血病K562／Adr细胞生长且呈剂量依赖关系,并能诱导细胞凋亡,使Caspase-3蛋白表达及细胞药物外排能力增加,同时降低mdr-1基因mRNA和蛋白表达水平。结论小檗胺能激活Caspase-3以诱导人白血病K562／Adr细胞凋亡,同时能通过降低mdr-1表达逆转多药耐药。     

Caffeoylserotonin Protects Human Keratinocyte HaCaT Cells against H2O2‐Induced Oxidative Stress and Apoptosis through Upregulation of HO‐1 Expression via Activation of the PI3K/Akt/Nrf2 Pathway

Caffeoylserotonin (CaS) has strong radical scavenging activity as well as antioxidant activities, protecting cells from lipid peroxidation, intracellular reactive oxygen species generation, DNA damage, and cell death. The molecular mechanism by which CaS protects against oxidative stress is not well understood. Here, we analyzed the cytoprotective activity of CaS in hydrogen peroxide (H₂O₂)‐treated keratinocyte HaCaT cells. H₂O₂ induced apoptosis in the cells through activation of pro‐apoptotic p21, Bax, and caspase‐3. Pretreatment with CaS inhibited apoptotic gene expression and activated the anti‐apoptotic gene, Bcl‐xL. Although CaS did not directly affect heme oxygenase‐1 (HO‐1) expression, pretreatment with CaS augmented HO‐1 expression through an increase in NF‐E2‐related factor (Nrf2) stability and stimulation of Nrf2 translocation to the nucleus upon H₂O₂ exposure. H₂O₂ also induced the phosphorylation and subsequent activation of ERK, p38 MAPK, and Akt. Analysis using specific inhibitors of p38 MAPK and Akt demonstrated that only Akt activation was involved in HO‐1 and Nrf2 expressions. In addition, PI3K and PKC inhibitors suppressed HO‐1/Nrf2 expression and Akt phosphorylation. These results demonstrate that CaS protects against oxidative stress‐induced keratinocyte cell death in part through the activation of Nrf2‐mediated HO‐1 induction via the PI3K/Akt and/or PKC pathways, but not MAPK signaling. Copyright © 2013 John Wiley & Sons, Ltd.     

Decursin in Angelica gigas Nakai (AGN) Enhances Doxorubicin Chemosensitivity in NCI/ADR‐RES Ovarian Cancer Cells via Inhibition of P‐glycoprotein Expression

Angelica gigas Nakai (AGN, Korean Dang‐gui) is traditionally used for the treatment of various diseases including cancer. Here, we investigated multidrug‐resistant phenotype‐reversal activities of AGN and its compounds (decursin, ferulic acid, and nodakenin) in doxorubicin‐resistant NCI/ADR‐RES ovarian cancer cells. Our results showed that a combination of doxorubicin with either AGN or decursin inhibited a proliferation of NCI/ADR‐RES cells. These combinations increased the number of cells at sub‐G1 phase when cells were stained with Annexin V‐fluorescein isothiocyanate. We also found that these combinations activated caspase‐9, caspase‐8, and caspase‐3 and increased cleaved PARP level. Moreover, an inhibition of P‐glycoprotein expression by either AGN or decursin resulted in a reduction of its activity in NCI/ADR‐RES cells. Therefore, our data demonstrate that decursin in AGN inhibits doxorubicin‐resistant ovarian cancer cell proliferation and induces apoptosis in the presence of doxorubicin via blocking P‐glycoprotein expression. Therefore, AGN would be a potentially novel treatment option for multidrug‐resistant tumors by sensitizing to anticancer agents. Copyright © 2016 John Wiley & Sons, Ltd.     

Chebulinic Acid Isolated From the Fruits of Terminalia chebula Specifically Induces Apoptosis in Acute Myeloid Leukemia Cells

Chebulinic acid, an ellagitannin found in the fruits of Terminalia chebula, has been extensively used in traditional Indian system of medicine. It has shown to have various biological activities including antitumor activity. The present study aims to investigate the cytotoxic potential of chebulinic acid in human myeloid leukemia cells. Interestingly, chebulinic acid caused apoptosis of acute promyelocytic leukemia HL‐60 and NB4 cells but not K562 cells. In vitro antitumor effects of chebulinic acid were investigated by using various acute myeloid leukemia cell lines. Chebulinic acid treatment to HL‐60 and NB4 cells induced caspase activation, cleavage of poly(ADP‐ribose) polymerase, DNA fragmentation, chromatin condensation, and changes in the mitochondrial membrane permeability. Additionally, inhibition of caspase activation drastically reduced the chebulinic acid‐induced apoptosis of acute promyelocytic leukemia cells. Our data also demonstrate that chebulinic acid‐induced apoptosis in HL‐60 and NB4 cells involves activation of extracellular signal‐regulated kinases, which, when inhibited with ERK inhibitor PD98059, mitigates the chebulinic acid‐induced apoptosis. Taken together, our findings exhibit the selective potentiation of chebulinic acid‐induced apoptosis in acute promyelocytic leukemia cells. Copyright © 2017 John Wiley & Sons, Ltd.     

Apigenin as an anticancer agent

Apigenin is an edible plant‐derived flavonoid that has been reported as an anticancer agent in several experimental and biological studies. It exhibits cell growth arrest and apoptosis in different types of tumors such as breast, lung, liver, skin, blood, colon, prostate, pancreatic, cervical, oral, and stomach, by modulating several signaling pathways. Apigenin induces apoptosis by the activation of extrinsic caspase‐dependent pathway by upregulating the mRNA expressions of caspase‐3, caspase‐8, and TNF‐α. It induces intrinsic apoptosis pathway as evidenced by the induction of cytochrome c, Bax, and caspase‐3, while caspase‐8, TNF‐α, and B‐cell lymphoma 2 levels remained unchanged in human prostate cancer PC‐3 cells. Apigenin treatment leads to significant downregulation of matrix metallopeptidases‐2, ?9, Snail, and Slug, suppressing invasion. The expressions of NF‐κB p105/p50, PI3K, Akt, and the phosphorylation of p‐Akt decreases after treatment with apigenin. However, apigenin‐mediated treatment significantly reduces pluripotency marker Oct3/4 protein expression which might be associated with the downregulation of PI3K/Akt/NF‐κB signaling.     

Harmine Hydrochloride Triggers G2 Phase Arrest and Apoptosis in MGC‐803 Cells and SMMC‐7721 Cells by Upregulating p21, Activating Caspase‐8/Bid,and Downregulating ERK/Bad Pathway

This study aimed to investigate the effects of harmine hydrochloride (HMH) on digestive tumor cells in vitro and its molecular mechanism. MTT assays showed that HMH inhibited the proliferation of some human cancer cell lines and had no obvious inhibitory effects on human LO2 cells. Flow cytometry assays showed that HMH trigged G2 phase arrest in MGC‐803 cells and SMMC‐7721 cells, while the expression of cyclin A, cyclin B, p21, Myt1, and p‐cdc2 (Tyr15) was upregulated. Flow cytometry assays also showed that the percentages of apoptotic cells were increased, the mitochondrial transmembrane potential (ΔΨm) decreased, and the cleavage of caspase‐9, caspase‐3, and poly (Adenosine diphosphate ribose) polymerase (PARP) were observed, the expression of Bad increased, phospho‐Bad (S112) decreased, pro‐caspase‐8 was cleaved, and Bid (22 kDa) was cleaved. The expression of p‐ERK decreased in both cells. In conclusion, these results demonstrated that HMH upregulates the expression of p21, activates Myt1 and inhibits cdc2 by phospho‐cdc2 (Y15), and triggers G2 phase arrest in both MGC‐803 cells and SMMC‐7721 cells. It can also activate the mitochondria‐related cell apoptosis pathway through the caspase‐8/Bid pathway, inhibiting the ERK/Bad pathway and promoting apoptosis in both of these two cell types. Copyright © 2015 John Wiley & Sons, Ltd.     

升血颗粒含药血清对人白血病K562/ADR细胞多药耐药的逆转作用

目的:观察升血颗粒(PG)含药血清对人白血病K562/ADR细胞多药耐药性的逆转作用,并探讨其逆转机制。方法:采用MTT法测定细胞的药敏性及耐药逆转性,应用流式细胞仪检测非细胞毒性的含药血清处理后K562/ADR细胞内阿霉素(ADM)的浓度。结果:低、中、高剂量含药血清对K562/ADR细胞无明显细胞毒性。低、中、高剂量含药血清作用后,ADM对K562/ADR细胞的半数抑制浓度(I C50)显著下降,逆转倍数分别为1.34、1.71、1.82倍;ADM外渗试验显示,低、中、高剂量含药血清处理后K562/ADR细胞内ADM浓度显著增加,增加倍数分别为1.41、1.67、2.04倍。结论:升血颗粒含药血清对人白血病K562/ADR细胞耐药性有一定的逆转作用。     

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??OBJECTIVE To investigate the effects of interferon-??(IFN-??) and all-trans retinoic acid(ATRA) on multidrug resistance reversal effect and mechanism of human leukemia K562/ADM cells. METHODS The cytotoxicity and reversal times of IFN-?? and ATRA were detected by CCK-8 method. Apoptosis rate and cell cycle were detected by flow cytometry. PI3K, Akt and Bad mRNA were detected by RT-PCR method. Western blot method was used to detect the expression of PI3K, AKt, P-AKt and Bad protein.RESULTS The drug resistance of K562/ADM cells to adriamycin(ADM) was 54 times. ADM, respectively, with IFN-??, ATRA or combined application, the drug resistance of K562/ADM cells to ADM was 1.24, 2.34 and 8.14, respectively. The apoptosis rate of K562/ADM cells was significantly increased by using ADM 4 mg??L^-1alone or in combination with IFN-?? 2.5??10⁶ U??L^-1, ATRA 7.5 ??mol??L^-1, and the cell cycle was blocked in G₀/G₁ phase. PI3K mRNA and protein expression were significantly lowered, Akt mRNA and protein has no obvious change, Bad mRNA and protein expression are raised, phosphorylated Akt protein expression decreased, the expression is more obvious when the two drug combination. CONCLUSION IFN-?? and ATRA can reverse the multidrug resistance of K562/ADM cells, its mechanism may be the inhibition of the PI3K/Akt pathway.     

丹参酮ⅡA诱导白血病细胞凋亡过程中端粒酶活性的改变

目的观察丹参酮Ⅱ_A(TanⅡ_A)对HL60,K562细胞端粒酶活性的抑制作用和对凋亡相关基因的影响,探讨丹参酮Ⅱ_A对造血细胞的作用机理。方法以HL60,K562为靶细胞,应用细胞培养技术,流式细胞术,透射电镜观察TanⅡ_A对HL60,K562细胞的作用,利用PCRTRAP方法检测TanⅡ_A处理前后HL60,K562细胞端粒酶活性的改变。结果经05μg·mL^-1TanⅡ_A作用6d后,HL60,K562细胞生长明显受到抑制,生长抑制率分别为756%和563%。经丹参酮诱导后,HL60,K562细胞发生凋亡,出现亚二倍体峰;同时显著下调HL60及K562细胞的cmyc,bcl2基因表达,上调cfos基因表达。HL60,K562细胞在TanⅡ_A作用后,端粒酶活性受到抑制,端粒酶活性抑制率分别为308%,508%。结论TanⅡ_A可明显抑制HL60和K562细胞的增殖和细胞端粒酶活性,并诱导细胞凋亡。     

PESV干预K562细胞PI3K/Akt 信号通路凋亡调控的研究

[目的]探讨PESV对K562细胞PI3K/Akt信号蛋白及凋亡调控因子Bcl-2和Bad表达的影响。[方法]将体外培养K562细胞,经PESV处理不同时间后,流式细胞术检测细胞凋亡率,Western blot检测PI3K及p-Akt蛋白水平变化,实时荧光定量RT-PCR检测Bcl-2、Bad mRNA水平变化。[结果]与对照组相比,PESV处理后K562细胞凋亡率显著增加,PI3K及p-Akt表达降低,抗凋亡相关基因Bcl-2 mRNA表达降低,促凋亡基因Bad mRNA表达增加。[结论]PESV可能通过降低PI3K、Akt信号蛋白表达,调节Bcl-2和Bad表达,抑制K562细胞增殖,促进其凋亡。     

人参皂苷Rg1诱导人白血病K562细胞株衰老的实验研究

目的:观察人参皂苷Rg1(Rg1)诱导人白血病K562细胞株衰老的作用及其机制。方法:MTT比色法检测Rg1对K562细胞增殖的影响,筛选最佳作用浓度及时间(20μmol.L-1,48 h)。流式细胞术检测Rg1对细胞周期的影响;SA-β-Gal染色检测细胞阳性染色百分率;RT-PCR法检测衰老相关基因p16,p53,p21,Rb的表达;电镜观察细胞衰老超微形态学改变。结果:Rg1在体外能明显抑制K562细胞增殖,使细胞阻滞于G2/M期;SA-β-Gal染色阳性细胞百分率显著增高(P<0.05);细胞衰老相关基因的表达上调(P<0.05);超微结构观察显示细胞增大,异染色质凝集、碎裂,线粒体体积增大,溶酶体体积增大、数目增多等衰老形态学变化。结论:Rg1能诱导K562细胞衰老,p53-p21-Rb,p16-Rb信号转导通路在其衰老调控中起重要作用。     

雷公藤红素对白血病细胞Akt信号通路的影响及在细胞凋亡中的作用

目的 研究雷公藤红素（celastrol)对人类白?∠赴闗562细胞Akt信号通路的影响及其作用。     

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??OBJECTIVE To investigate the inhibitory activity, induced differentiation-inducing activity and apoptosis-inducingactivity of hydroxyl morpholine(QDML-01) on chronic myelocytic leukemia cells line K562. METHODS The cell growth curve was drawn based on cell counting method. The IC₅₀ value of QDML-01 and positive control medicine to K562 cells were evaluated by methyl thiazolyl tetrazolium (MTT) assay method. Double soft agar assay method was carried out to study the ability of cell proliferation to determine efficacy of phamacognosy. The pathomorphism was analyed by the Wright-Giemsa staining method. The mechanism of cell apoptosis from morphology and gene level were investigated, by AO-EB double-staining method and DNA breakage test. The effect of QDML-01 on K562 cells from the protein level was determined by Western-blot. RESULTS The growth curves showed the K562 cells had strong cell vitality. They came into logarithmic phase on the third generation. The MTT assay RESULTS showed that the IC₅₀ values of QDML-01 and imatinib to K562 cells were 5.81 and 596.88 nmol??L^-1. Double soft agar colony formation test showed that clone formed at 21 d and the inhibitory rate of QDML-01 was 81.7%.It indicated that K562 cells were sensitive to QDML-01.Morphology test result showed that QDML-01 induced K562 cells to normal cells. The RESULTS of AO-EB double-staining method showed that QDML-01 induced the apoptosis of K562 cells. The study of DNA breakage test indicated that QDML-01 can induce the apoptosis of K562 cells to produce DNA banding with step-like. Western-blot analysis result suggested that QDML-01 can downregulated the expression of P210^bcr/abl protein. CONCLUSION QDML-01 has the inhibitory activity on chronic myelocytic leukemia cells line K562 by promoting the apoptosis of K562 cells and inducing differentiation to normal cells.     

甲异靛诱导K562细胞凋亡及对传导信号STAT5表达影响的研究

目的 探讨甲异靛诱导K562原代细胞凋亡及对传导信号分子STAT5表达影响的作用.方法 K562细胞甲异靛处理12h、24 h、48 h后,采用流式细胞术检测细胞凋亡及细胞周期等相关指标,Western blot检测STAT5的改变.结果 ①甲异靛作用K562细胞12h、24 h、48 h凋亡率分别为12.5±0.69,19.4±1.07和42.5±3.22,与对照组相同时间段比较,差异均有统计学意义（P＜0.05）.②甲异靛可使K562细胞阻滞于S期,S期细胞比例增加至（70.48±6.34）,G0/G1期、G2/M期细胞减少.③在甲异靛作用12 h后至48 h,STAT5蛋白含量逐渐减少.结论 甲异靛促进K562细胞凋亡,可能与STAT5蛋白含量表达减少有关.