Sensitization of K562 Leukemia Cells to Doxorubicin by the Viscum album Extract

Toxicity of conventional chemotherapeutics highlights the requirement for complementary or alternative medicines that would reduce side effects and improve their anticancer effectiveness. European mistletoe (Viscum album) has long been used as a complementary and alternative medicine supporting cancer therapy. The aim of this study was to investigate synergistic antitumor action of V. album extract and doxorubicin during co‐treatment of chemoresistant chronic myelogenic leukemia K562 cells. Combined treatment of leukemia cells led to inhibitory synergism at sub‐apoptotic doxorubicin concentrations and multifold reduction of cytotoxic effects in healthy control cells. Prolonged co‐treatment was associated with reduced G2/M accumulation and increased expression of early and late apoptotic markers. Our data indicate that V. album extract increases antileukemic effectiveness of doxorubicin against resistant K562 cells by preventing G2/M arrest and inducing apoptosis. Copyright © 2015 John Wiley & Sons, Ltd.     

小檗碱增强K562/DOX细胞对多柔比星敏感性的研究

 目的 探讨小檗碱增强耐药K562/DOX细胞对化疗药多柔比星的敏感性的作用。方法 四甲基偶氮唑蓝法检测小檗碱的细胞毒性及其对多柔比星抗肿瘤活性的增强作用;高内涵活细胞成像系统检测无毒剂量小檗碱作用后,多柔比星在K562/DOX细胞内的蓄积量;PI/Hoechst33342双染法检测小檗碱对多柔比星诱导的K562/DOX细胞凋亡的影响;罗丹明123蓄积实验检测小檗碱对P-糖蛋白外排功能的影响。结果 1 μmol·L^-1为小檗碱的无毒剂量,在此无毒剂量下,小檗碱使多柔比星对K562/DOX细胞的IC₅₀降低了1.5倍;1 μmol·L^-1小檗碱可使多柔比星在K562/DOX细胞内的蓄积量增加,增强多柔比星诱导的K562/DOX细胞凋亡, 增加K562/DOX细胞内罗丹明123的蓄积量,从而抑制P-糖蛋白的外排功能。结论 小檗碱可通过抑制K562/DOX细胞膜上P-糖蛋白的外排功能,增加K562/DOX细胞内多柔比星浓度,促进多柔比星对耐药细胞的诱导凋亡作用,逆转K562/DOX细胞的多药耐药性。     

In Vitro and In Silico Evaluation of NF‐κB Targeted Costunolide Action on Estrogen Receptor‐Negative Breast Cancer Cells—A Comparison with Normal Breast Cells

Costunolide, a sesquiterpene lactone is a plant‐derived secondary metabolite found to be present in most of the pharmacologically active herbs, being the cause for their medicinal values. The present study aims to evaluate the cytotoxic effect of costunolide isolated from Costus speciosus rhizome extract on MDA‐MB‐231 cells and explore its targeted action in comparison with its action on the normal breast cells (MCF 10A). The effect of costunolide on cell viability of the cells was assessed by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide viability assay. The targeted action of the compound was analyzed comparing the effectiveness of the compound to alter the protein expression levels of NF‐κB subunits in the normal and the cancer cells using western blotting analysis. In silico studies were performed to predict the targeted interaction of costunolide with the NF‐κB subunit proteins. Costunolide inhibited the cell viability of MDA‐MB‐231 cells in a dose‐dependent manner leaving no significant change in the viability of the normal breast cells. The over expressed NF‐κB subunits – p65, 52 and 100 in the cancer cells were found to be downregulated when treated with costunolide at an effective dose of 20 and 40 μM costunolide. In silico results provided stable interactions between costunolide and the target proteins, supporting the in vitro results in addition. Thus, costunolide derived from C. speciosus plant source elevates a fresh conviction for its use in breast cancer therapy for its cytotoxic efficacy and non‐toxic nature. Copyright © 2014 John Wiley & Sons, Ltd.     

Cytotoxic Impact of Costunolide Isolated from Costus speciosus on Breast Cancer via Differential Regulation of Cell Cycle—An In‐vitro and In‐silico Approach

Costunolide, a sesquiterpene lactone, is a biologically active molecule found in most of the medicinally valuable plants. The present study aims to evaluate the anticancer property of costunolide isolated from Costus speciosus against breast cancer cell lines (MCF‐7 and MDA‐MB‐231). Costunolide effectively reduced the viability of both MCF‐7 and MDA‐MB‐231 cell lines at an IC₅₀ value of 40 μM. Flow cytometric analysis revealed costunolide mediated cell cycle arrest at G2/M phase in both the cell types. Western blotting results confirmed the alterations in the expression of cell cycle regulators (cyclin D1, D3, CDK‐4, CDK‐6, p18 INK4c, p21 CIP1/Waf‐1 and p27 KIP1) and apoptosis inducers (caspase‐3 and caspase‐9) upon costunolide treatment in comparison with their expressions in normal breast cell line (MCF‐10A). Costunolide mediated downregulation of positive cell cycle regulators and upregulation of negative cell cycle regulators were related to the induction of apoptosis in cancer cells. The above results were validated with in‐silico results that predicted stable interactions between costunolide and cancer targets. Thus costunolide effectively induced breast cancer cell apoptosis targeting cell cycle regulation, and the compound can be used as an effective herbal therapeutic molecule to treat breast cancer with further explorations. Copyright © 2015 John Wiley & Sons, Ltd.     

Paris saponin VII induces cell cycle arrest and apoptosis by regulating Akt/MAPK pathway and inhibition of P‐glycoprotein in K562/ADR cells

Paris saponinVII (PSVII) is a steroidal saponin isolated from the roots and rhizomes of Trillium tschonoskii Maxim. We found that PSVII could inhibit the growth of adriamycin‐resistant human leukemia cells (K562/ADR) in a dose‐dependent manner. Furthermore, the molecular mechanism underlying the cytotoxicity and downregulation of P‐glycoprotein (P‐gp) expression by PSVII was clarified. PSVII significantly suppressed cell proliferation by cell cycle arrest in the G0/G1 phase, which was associated with an obvious decrease in cyclin B1/D1 and CDK2/4/6 protein expression. Moreover, PSVII could attenuate mitochondrial membrane potential, increase the expression of apoptosis‐related proteins, such as Bax and cytochrome c, and decrease the protein expression levels of Bcl‐2, caspase‐9, caspase‐3, PARP‐1, and p‐Akt. We also found that JNK, ERK1/2, and p38 were regulated by PSVII in K562/ADR cells. And further studies indicated that the decrease in the reactive oxygen species level inhibited intrinsic P‐gp expression. Therefore, PSVII‐induced apoptosis in K562/ADR cells was associated with Akt/MAPK and the inhibition of P‐gp. In addition, PSVII induced a robust autophagy in K562/ADR cells as demonstrated by the degradation of LC3‐I. These results provide a biochemical basis for possible clinical applications of PSVII in the treatment of leukemia.     

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。     

复方浙贝药物血清影响K562/A02细胞积聚外排功能和细胞凋亡研究

目的 观察复方浙贝药物血清对K562/A02细胞积聚外排功能和细胞凋亡的影响。方法 Wistar 雄性大鼠40只, 分为空白对照组（血清组）、含阿霉素（ADR）血清组及含复方浙贝颗粒高［8 g/（kg·d）］、中［4 g/（kg·d）］、低［2 g/（kg·d）］剂量血清组。用流式细胞仪定量检测不同时间药物血清干预后的K562/A02细胞内ADR和罗丹明123（Rh123）浓度;Annexin V/PI双染法检测K562/A02细胞早期凋亡率。结果 复方浙贝药物血清不同剂量组均能减缓K562/A02细胞内ADR和Rh123荧光下降速度,其中高剂量组作用最明显,中剂量组次之,低剂量组较弱;K562/A02细胞早期凋亡率依次为：中剂量组24.60％、高剂量组11.21％、低剂量组5.71％、空白组1.40％。结论 复方浙贝颗粒体外能够抑制K562/A02细胞对药物的外排功能,并能够诱导细胞凋亡。     

吴茱萸碱逆转K562/Adr细胞多药耐药的实验研究

目的 探讨吴茱萸碱(evodiamine,EVO)对白血病K562细胞及其耐药株K562/Adr的增殖、细胞周期及多药耐药性(MDR)的影响。方法用细胞增殖毒性检测试剂盒(CCK-8)检测EVO和/或柔红霉素(DNR)对细胞增殖的影响,并计算耐药指数(RI)和逆转倍数(RF);流式细胞仪检测EVO和/或DNR对K562及K562/Adr细胞周期的影响;流式细胞仪检测K562及K562/Adr细胞内DNR的荧光强度;定量PCR检测K562及K562/Adr细胞中MDR1基因的表达;Western blotting检测K562及K562/Adr细胞中MDR1、BCRP蛋白的表达。结果EVO、DNR作用于K562和K562/Adr细胞后,细胞增殖受到抑制,且呈剂量和时间依赖性;与K562细胞相比,K562/Adr细胞对DNR的RI为30.54,K562/Adr细胞对EVO的RI为19.09。当EVO(0.125 gmol/L)与不同浓度DNR联合作用后,能使DNR对K562/Adr细胞的IC_(50)明显下降,DNR+EVO对K562/Adr细胞的RF为12.07;EVO、DNR单独或联合作用于K562及K562/Adr细胞后,能够使K562/Adr细胞BCRP、MDR1蛋白及mRNA表达水平均明显下降。结论EVO能有效逆转白血病K562/Adr细胞对DNR的耐药现象,而这种作用可能与EVO通过减少细胞膜上多药耐药蛋白MDR1的表达有关。     

Cariporide降低K562/DOX细胞株表达P-糖蛋白而逆转耐药的影响

 目的 探讨cariporide对耐药细胞株K562/DOX中P-糖蛋白（P-glycoprotein,P-gp）的影响,为抗白血病多药耐药（multidrug resistance, MDR）提供新方法。方法 应用cariporide对细胞进行酸化,应用激光共聚焦显微镜测定野生型细胞系K562及耐药细胞株K562/DOX细胞内pH值及细胞酸化对K562和K562/DOX细胞内阿霉素累积的影响。采用MTT法观察细胞酸化对细胞活力的影响。应用流式细胞术检测细胞酸化对K562/DOX细胞中P-gp功能的影响。采用实时定量RT-PCR技术检测MDR1基因在mRNA表达水平的变化。结果 Cariporide处理3 h对K562及K562/DOX细胞的活力影响较小。在K562/DOX细胞中,P-gp的外排药物能力随细胞内pH值的降低而减弱,cariporide明显增加了细胞对罗丹明123（rhodaminel 123,Rh123）和阿霉素的累积。细胞酸化还在mRNA水平抑制了K562/DOX细胞中P-gp的表达。结论 Cariporide能够抑制K562/DOX耐药细胞株中MDR1基因表达和P-gp的功能。     

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。     

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。     

梁金菇多糖诱导K562细胞凋亡的研究

目的 :探讨梁金菇多糖对K562细胞凋亡的影响及其机制。方法 :应用集落形成实验观察梁金菇多糖对K562细胞增殖的影响 ,细胞形态学、DNA凝胶电泳等检测细胞凋亡 ,逆转录 聚合酶链反应 (RT-PCR)检测凋亡相关基因FasmRNA的表达。结果 ;梁金菇多糖能明显抑制K562细胞的增殖 ,促进细胞凋亡 ,细胞凋亡率的增加呈剂量依赖性。Fas基因的表达与K562细胞凋亡呈正相关。结论 :梁金菇多糖具有抑制K562细胞增殖、诱导其凋亡的作用 ,Fas基因表达的上调可能是梁金菇多糖诱导细胞凋亡的机制之一。     

欧白芷素对K562/A02细胞阿霉素耐药的逆转作用(英文)

目的:探讨欧白芷素对耐药细胞株K562/A02中P-糖蛋白(P-glycoprotein,P-gp)的影响,为抗白血病多药耐药(multi-drug resistance,MDR)提供新方法。方法:采用MTT法观察阿霉素对细胞活力的影响。应用流式细胞术检测欧白芷素对K562和K562/A02细胞内阿霉素累积和细胞中P-gp功能的影响。采用实时定量RT-PCR技术检测MDR1基因在mRNA表达水平的变化。结果:欧白芷素对耐药细胞株K562/A02有显著的逆转耐药活性,最大逆转倍数为7.36。在K562/A02细胞中,欧白芷素明显增加阿霉素的累积,增加了罗丹明123(rhodaminel123,Rh123)蓄积,抑制了Rh123的外排,同时欧白芷素还在mRNA水平抑制了K562/A02细胞中P-gp的表达。结论:欧白芷素能够抑制K562/A02耐药细胞株中MDR1基因表达和P-gp的功能。     

灵芪胶囊含药血清对K562白血病细胞的诱导凋亡作用

[目的]观察灵芪胶囊含药血清在体外对K562白血病细胞凋亡的影响.[方法]采用血清药理学方法制备灵芪胶囊含药血清,以不同浓度的含药血清处理体外培养的K562白血病细胞,采用流式细胞仪(FCM)及DNA片段凝胶电泳观察和检测细胞凋亡情况.[结果]不同浓度灵芪胶囊含药血清能够诱导K562细胞的凋亡,且凋亡率随着剂量的增加而增大,与空白对照组比较有显著性差异(P<0.01);DNA琼脂糖凝胶电泳谱可见典型的DNA梯形带形成.[结论]灵芪胶囊含药血清具有诱导细胞凋亡的作用,其作用强度与时间-浓度呈正相关.灵芪胶囊含药血清对K562细胞发生细胞毒作用可能与诱导K562细胞凋亡有关.     

Bioactivity‐guided Isolation of Cytotoxic Sesquiterpene Lactones of Gochnatia polymorpha ssp. floccosa

Phytochemical study of Gochnatia polymorpha (Less) Cabr. ssp. floccosa Cabr. trunk bark, guided by antiproliferative assays on 10 human cancer cell lines and the VERO cell line, yielded six known compounds identified as the triterpene bauerenyl acetate, the guaianolide 11α,13‐dihydrozaluzanin C and the dimeric guaianolides 10‐desoxygochnatiolide A, gochnatiolide A, 8‐hydroxi‐10‐desoxygochnatiolide A and 8‐hydroxigochnatiolide A. Extracts, fractions of extracts and isolated compounds were tested in vitro against a panel of human cancer cell lines, including U251 (glioma, CNS), UACC‐62 (melanoma), MCF‐7 (breast), NCI‐ADR/RES (drug‐resistant ovarian), 786.0 (kidney), NCI‐H460 (lung, no small cells), PC‐3 (prostate), OVCAR‐3 (ovarian), HT‐29 (colon), K562 (leukemia) and against the VERO no cancer cell line. Bauerenyl acetate was inactive, while 11α,13‐dihydrozaluzanin C showed weak activity against UACC62 and the VERO cell line. The most active compounds were 10‐desoxygochnatiolide A and gochnatiolide A, which inhibited the growth of kidney, melanoma, ovarian‐resistant and glioma cell lines with values of TGI (total growth inhibition) varying 0.21–1.09 µg/mL. This is the first report about cytotoxic activity of dimeric lactones against cell lines. Copyright © 2011 John Wiley & Sons, Ltd.     

Brassinin Induces Apoptosis in PC‐3 Human Prostate Cancer Cells through the Suppression of PI3K/Akt/mTOR/S6K1 Signaling Cascades

The oncogenic PI3K/Akt/mammalian target of rapamycin (mTOR) signaling axis and its downstream effector, the ribosomal protein S6 kinase 1 (S6K1) play a key role in mediating cell survival in various tumor cells. Here, we investigated the effects of brassinin (BSN), a phytoalexin first identified as a constituent of cabbage, on the PI3K/Akt/mTOR/S6K1 activation, cellular proliferation, and apoptosis in PC‐3 human prostate cancer. BSN exerted a significant dose‐dependent cytotoxicity and reduced constitutive phosphorylation of Akt against androgen‐independent PC‐3 cells as compared to androgen‐dependent LNCaP cells. Moreover, knockdown of androgen receptor (AR) by small interfering RNA enhanced the potential effect of BSN on induction of apoptosis in LNCaP cells. BSN clearly suppressed the constitutive activation of PI3K/Akt/mTOR/S6K1 signaling cascade, which correlated with the induction of apoptosis as characterized by accumulation of cells in subG1 phase, positive Annexin V binding, TUNEL staining, loss of mitochondrial membrane potential, down‐regulation of antiapoptotic and proliferative proteins, activation of caspase‐3, and cleavage of PARP. Additionally, BSN could block broad‐spectrum inhibition of PI3K/Akt/mTOR/S6K1 axes, and aberrant Akt activation by pcDNA3‐myr‐HA‐Akt1 plasmid could not prevent the observed suppressive effect of BSN on constitutive mTOR activation. Finally, overexpression of Bcl‐2 also attenuated BSN‐mediated apoptosis in PC‐3 cells. Taken together, our findings suggest that BSN can interfere with multiple signaling cascades involved in tumorigenesis and might be provided as a potential therapeutic candidate for both the prevention and treatment of prostate cancer. Copyright © 2013 John Wiley & Sons, Ltd.     

四物合剂对人红白血病细胞株K562/ADM多药耐药性的逆转作用研究

目的 :观察四物合剂对人红白血病细胞株K5 62 /ADM多药耐药性的逆转作用。方法 :以维拉帕米为阳性对照 ,MTT法观察耐药细胞株K5 62 /ADM的耐药倍数及四物合剂的逆转倍数 ;采用反相高效液相色谱法 (RP-HPLC)检测细胞内的ADM浓度 ;免疫荧光法测定细胞膜P-糖蛋白 (Pgp)表达。结果 :四物合剂和ADM合用时 ,对K562/ADM耐药性的逆转倍数及细胞内的ADM含量比ADM单独使用时明显增高 (P<0.01);但对K562/ADM细胞膜Pgp表达的影响差异不显著 (P>0.05)。结论 :四物合剂在无毒性剂量时能逆转细胞株K562/ADM对ADM的耐药性 ,但对细胞膜Pgp表达影响不大 ,其逆转作用可能与降低Pgp药物外排作用、增加细胞内药物浓度有关。