Graveoline Isolated from Ethanolic Extract of Ruta graveolens Triggers Apoptosis and Autophagy in Skin Melanoma Cells: A Novel Apoptosis‐Independent Autophagic Signaling Pathway

Anti‐cancer drugs generally kill cancer cells by apoptosis but fail to do so when they become resistant and escape apoptosis signals. But these resistant cells can still be killed by autophagy. Therefore, drugs having both apoptotic and autophagic abilities are solicited in effective cancer management. In search of such a drug, we examined the efficacy of graveoline, a bioactive compound isolated from Ruta graveolens on skin melanoma A375 cells through the use of specific signaling cascades and their inhibitors. Cytotoxicity of graveoline was tested by conducting MTT assay. Induction of autophagy and apoptosis was checked. Expression of related proteins and their localization were studied by conducting immunoblot assay and through confocal microscopy, respectively. We found graveoline‐induced Beclin‐1 associated autophagy in A375 cells and 3‐methyladenine, an inhibitor of autophagy did not affect apoptosis. Conversely, caspase inhibitor that blocked apoptosis did not affect autophagic cell death, suggesting thereby that these two were independent events. Use of reactive oxygen species (ROS) scavengers inhibited cell death, but blocking autophagy did not affect graveoline‐induced ROS generation, suggesting that ROS generation ensued autophagy. Thus, graveoline‐induced both apoptotic and autophagic cell death in skin melanoma cells, a desirable quality in effective anti‐cancer drug design. Copyright © 2013 John Wiley & Sons, Ltd.     

Cytotoxic and apoptosis-inducing activities of triterpene acids from Poria cocos

Six lanostane-type triterpene acids (1a-6a), isolated from Poria cocos , and their methyl ester (1b-6b) and hydroxy derivatives (1c-6c) were prepared. Upon evaluation of the cytotoxic activity of these compounds against leukemia (HL60), lung (A549), melanoma (CRL1579), ovary (NIH:OVCAR-3), breast (SK-BR-3), prostate (DU145), stomach (AZ521), and pancreas (PANC-1) cancer cell lines, 11 compounds (5a, 6a, 2b-5b, 1c, and 3c-6c) exhibited activity with single-digit micromolar IC(50) values against one or more cell lines. Poricotriol A (1c), a hydroxy derivative of poricoic acid A (1a), exhibited potent cytotoxicities against six cell lines with IC(50) values of 1.2-5.5 μM. Poricotriol A induced typical apoptotic cell death in HL60 and A549 cells on evaluation of the apoptosis-inducing activity by flow cytometric analysis. Western blot analysis in HL60 cells showed that poricotriol A activated caspases-3, -8, and -9, while increasing the ratio of Bax/Bcl-2. This suggested that poricotriol A induced apoptosis via both mitochondrial and death receptor pathways in HL60. On the other hand, poricotriol A did not activate caspases-3, -8, and -9, but induced translocation of apoptosis-inducing factor (AIF) from mitochondria and increased the ratio of Bax/Bcl-2 in A549. This suggested that poricotriol A induced apoptosis via the mitochondrial pathway mostly by translocation of AIF, independent from the caspase pathway in A549. Furthermore, poricotriol A was shown to possess high selective toxicity in lung cancer cells since it exhibited only weak cytotoxicity against a normal lung cell line (WI-38).     

Drug‐like dietary vanilloids induce anticancer activity through proliferation inhibition and regulation of bcl‐related apoptotic proteins

In this study, a series of 20 structurally similar vanilloids (Vn) were tested for their antiproliferative effects against 12 human cancer cells: human breast (MCF‐7 and MDA‐MB‐231), cervical (HeLa), ovarian (Caov‐3), lung (A549), liver (HepG2), colorectal (HT‐29 and HCT116), nasopharyngeal (CNE‐1 and HK‐1), and leukemic (K562 and CEM‐SS) cancer cells. Among all the tested vanilloids, Vn16 (6‐shogaol) exhibited the most potent cytotoxic effects against human colorectal cancer cells (HT‐29). The apoptotic induction effects exhibited by Vn16 on HT‐29 cells were confirmed using dual staining fluorescence microscopy and enzyme‐linked immunosorbent assay. The effects of Vn16 on regulation of 43 apoptotic‐related markers were determined in HT‐29. The results suggested that 8 apoptotic markers (caspase 8, BAD, BAX, second mitochondrial‐derived activator, caspase 3, survivin, bcl‐2, and cIAP‐2) were either upregulated or downregulated. These results further support the chemopreventive properties of foods that contain vanilloids.     

Haeamtang induces apoptosis of colon cancer HT-29 cells through activation of caspase-3

The effect of Haeamtang (HAT) on the colon cancer HT-29 cells was investigated in this study. A water extract of HAT significantly decreased the number of HT-29 cells in a dose-and time-dependent manner as determined by a MTT assay. Flow cytometry results revealed a dose- and time-dependent increase of dead cells in HT-29 cells treated with HAT extract. The anticancer activity of the H AT extract is attributed to apoptosis induced in HT-29 cells, which was demonstrated by increased caspase-3 activity and poly-ADP-ribose polymerase fragmentation. A selective caspase inhibitor, z-VAD-fmk, inhibited the HAT-induced cell death. Taken together, these results demonstrate that HAT extract induces apoptosis in HT-29 cells.     

Ethyl acetate extract of Patrinia scabiosaefolia downregulates anti-apoptotic Bcl-2/Bcl-X(L) expression, and induces apoptosis in human breast carcinoma MCF-7 cells independent of caspase-9 activation

Patrinia scabiosaefolia Fisch. is a Chinese medicinal herb used traditionally for treating intestinal carbuncle. Although Patrinia scabiosaefolia has also been suggested for cancer therapy, there has not been any scientific evidence supporting this application. In this study, a panel of human cancer cells, including breast carcinoma MCF-7; hepatocellular carcinoma HepG2; skin melanoma A375; lung carcinoma A549 and prostate adenocarcinoma PC-3, were treated in vitro with ethyl acetate extract of Patrinia scabiosaefolia (EAE-PS) for 48 h. Results from MTT study showed that MCF-7 was the most responsive (IC50 = 112.3 microg/ml) while PC-3 was the most resistant (IC50 = 348.7 microg/ml) one to cell growth inhibition. DNA flow cytometry demonstrated that EAE-PS induced apoptosis in the resistant MCF-7 cells by 14.5-fold of the control level after 36 h of treatment. Immunoblot studies further illustrated that although EAE-PS downregulated the anti-apoptotic Bcl-2/Bcl-X(L) expression in breast cancer cells, the induced apoptosis could not be prevented by the caspase-9 inhibitor (Z-LEHD-FMK). All these results suggest that EAE-PS retards MCF-7 cell growth by activating the caspase-independent mitochondrial cell death pathway. Results from this study support future research and development of the bioactive ingredients from Patrinia scabiosaefolia as anticancer agents, especially against those apoptosis-resistant cancers with deregulated Bcl-2/Bcl-X(L) expression.     

1,7‐Bis(4‐hydroxyphenyl)‐4‐hepten‐3‐one from Betula platyphylla induces apoptosis by suppressing autophagy flux and activating the p38 pathway in lung cancer cells

Betula platyphylla (BP) is frequently administered in the treatment of various human diseases, including cancers. This study was undertaken to investigate the pharmacological function of the active components in BP and the underlying mechanism of its chemotherapeutic effects in human lung cancer cells. We observed that BP extracts and 1,7‐bis(4‐hydroxyphenyl)‐4‐hepten‐3‐one (BE1), one of the components of BP, effectively decreased the cell viability of several lung cancer cell lines. BE1‐treated cells exhibited apoptosis induction and cell cycle arrest at the G2/M phase. Further examination demonstrated that BE1 treatment resulted in suppression of autophagy, as evidenced by increased protein expression levels of both LC3 II and p62/SQSTM1. Interestingly, the pharmacological induction of autophagy with rapamycin remarkably reduced the BE1‐induced apoptosis, indicating that apoptosis induced by BE1 was associated with autophagy inhibition. Our data also demonstrated that BE1 exposure activated the p38 pathway resulting in regulation of the pro‐apoptotic activity. Taken together, we believe that BE1 is a potential anticancer agent for human lung cancer, which exerts its effect by enhancing apoptosis via regulating autophagy and the p38 pathway.     

厚朴酚通过自噬途径促进肺癌细胞死亡

目的:研究厚朴酚诱导人非小细胞性肺癌H460细胞的死亡作用并探讨其抗癌作用机制。方法:采用MTT法检测厚朴酚对H460细胞的生长抑制作用,TdT介导dUTP缺口末端标记法(TdT-medi-ated dUTP Nick-End Labeling,TUNEL)法和乳酸脱氢酶(L-Lactate Dehydrogenase,LDH)活力检测法检测细胞凋亡及坏死率,monodansyl-cadaverine(MDC)荧光染色法标记检测细胞自噬情况。结果:厚朴酚可时间依赖性抑制H460肺癌细胞的生长,荧光显微镜下观察厚朴酚作用24h后的细胞出现大量空泡;乳酸脱氢酶活力检测,厚朴酚处理48h后细胞坏死;TUNEL法检测发现,高浓度处理组的细胞很少发生凋亡。结论:厚朴酚主要通过细胞自噬途径而非凋亡途径诱导H460肺癌细胞的死亡。     

钩吻总碱对人结肠癌细胞增殖、凋亡的影响及其机制

目的:探讨钩吻总生物碱提取物(钩吻总碱)促进人结肠癌HT-29细胞凋亡的机制。方法:钩吻总碱体外干预HT-29细胞24 h后,用噻唑蓝(MTT)比色法评估其对细胞增殖的影响,4,6-二脒基-2-苯基吲哚(DAPI)染色法及流式细胞术(Annexin V-FITC/PI)观察HT-29细胞的凋亡情况,逆转录聚合酶链反应(RT-PCR)观察HT-29细胞B型淋巴细胞瘤-2(Bcl-2)相关X蛋白(Bax),Bcl-2 mRNA表达情况,利用紫外分光光度法检测含半胱氨酸的天冬氨酸水解酶-3(Caspase-3)表达量的变化。结果:钩吻总碱可抑制HT-29细胞增殖,200 mg·L~(-1)钩吻总碱干预24 h后,对HT-29细胞增殖的平均抑制率达54.17%。DAPI染色及流式细胞术(Annexin V-FITC/PI)观察到钩吻总碱可促进HT-29细胞凋亡。RT-PCR观察到钩吻总碱能降低HT-29细胞Bcl-2 mRNA表达(P0.05),对Bax mRNA表达无明显影响,提高了Bax/Bcl-2 mRNA表达(P0.05)。利用紫外分光光度法检测到随着钩吻总碱浓度的升高,HT-29细胞的Caspase-3表达呈上升趋势(P0.05)。结论:钩吻总碱能有效地抑制HT-29细胞的增殖,促进HT-29细胞的凋亡,其机制可能与通过Bax/Bcl-2途径诱导细胞凋亡相关。     

Anti‐lung Cancer Effects of Polyphyllin VI and VII Potentially Correlate with Apoptosis In Vitro and In Vivo

Polyphyllin VI (PVI) and polyphyllin VII (PVII) derived from Paris polyphylla possess anti‐cancer activities. However, the mechanisms for the anti‐lung cancer effects of PVI and PVII remain poorly understood. In this study, PVI and PVII exhibited inhibitory effects on the proliferation of A549 and NCI‐H1299 cells. PVI and PVII induced G2/M cell cycle arrest and triggered apoptosis. PVI and PVII upregulated the tumor suppressor protein p53 and downregulated cyclin B1. The two treatments significantly increased the expression levels of death receptor 3, death receptor 5, Fas, cleaved PARP, and cleaved caspase‐3. Furthermore, PVI and PVII significantly inhibited the growth of A549 cells in vivo. The tumor inhibitory rates of PVI were 25.74%, 34.62%, and 40.43% at 2, 3, and 4 mg/kg, respectively, and those of PVII were 25.63%, 41.71%, and 40.41% at 1, 2, and 3 mg/kg, respectively. Finally, PVI and PVII regulated the expression of proteins related to the apoptotic pathway in A549 xenografts. In summary, PVI and PVII exhibited strong inhibitory effects on lung cancer cell growth in vitro and in vivo by inducing G2/M cell cycle arrest and triggering apoptosis. Copyright © 2015 John Wiley & Sons, Ltd.     

Fucoidan from Undaria pinnatifida induces apoptosis in A549 human lung carcinoma cells

Fucoidan, a sulfated polysaccharide, has various biological activities, such as anticancer, antiangiogenic and antiinflammatory effects; however, the mechanisms of action of fucoidan on anticancer activity have not been fully elucidated. The anticancer effects of fucoidan from Undaria pinnatifida on A549 human lung carcinoma cells were examined. Treatment of A549 cells with fucoidan resulted in potent antiproliferative activity. Also, some typical apoptotic characteristics, such as chromatin condensation and an increase in the population of sub-G1 hypodiploid cells, were observed. With respect to the mechanism underlying the induction of apoptosis, fucoidan reduced Bcl-2 expression, but the expression of Bax was increased in a dose-dependent manner compared with the controls. Furthermore, fucoidan induced caspase-9 activation, but decreased the level of procaspase-3. Cleavage of poly-ADP-ribose polymerase (PARP), a vital substrate of effector caspase, was found. The study further investigated the role of the MAPK and PI3K/Akt pathways with respect to the apoptotic effect of fucoidan, and showed that fucoidan activates ERK1/2 in A549 cells. Unlike ERK1/2, however, treatment with fucoidan resulted in the down-regulation of phospho-p38 expression. In addition, fucoidan resulted in the down-regulation of phospho-PI3K/Akt. Together, these results indicate that fucoidan induces apoptosis of A549 human lung cancer cells through down-regulation of p38, PI3K/Akt, and the activation of the ERK1/2 MAPK pathway.     

Induction of apoptosis by a synergistic lignan composition from Cedrus deodara in human cancer cells

AP9‐cd, a synergistic lignan mixture from Cedrus deodara (Pinaceae) consisting of (?)‐wikstromal, (?)‐matairesinol and dibenzyl butyrolactol, depicted cytotoxic effects against numerous human cancer cell lines reported previously. The aim of this study was to investigate the mechanism of cell death in human cancer cells. The viability, morphological and ultrastructural changes in Molt‐4 cells were investigated. Using the trypan blue exclusion assay, we demonstrated that AP9‐cd significantly reduced the viability of Molt‐4 cells in a time‐ and dose‐dependent manner. Apoptotic assays using light microscopy revealed that this agent induced Molt‐4 cell apoptosis at varied concentrations. The treatment causes a loss in cell viability by activating the apoptotic process as identified by light and electron microscopy. The morphological changes of intracellular organelles in Molt‐4 cells treated with 30 µg/ml of AP9‐cd revealed the disruption of mitochondrial cristae. Other features included the vacuolization, chromatin condensation and formation of micronuclei. Surface ultrastructural studies of four different tumor cell lines (Molt‐4, HL‐60, PC‐3 and A‐549) treated with AP9‐cd depicted loss of surface projections, condensation and formation of apoptotic bodies. AP9‐cd treatment to transgenic fruit fly, Drosophila, carrying human adenomatous polpyposis coli (hAPC) gene enhanced eye phenotypes and therefore may inhibit Wnt/Wg pathway which is important in the aetiology of a number of human cancers. Copyright © 2008 John Wiley & Sons, Ltd.     

Alboatisins A-C, ent-atisene diterpenoids from Isodon albopilosus

Three biogenetically interesting ent-kaurane-derived metabolites, alboatisins A-C (1-3), featuring a C-13-oxygenated ent-atisane skeleton, have been isolated from the aerial parts of Isodon albopilosus. Their structures were determined on the basis of spectroscopic evidence. Compound 2 exhibited cytotoxicity against A549 and HT-29 human cancer cells, with IC50 values of 8.3 and 7.9 microM, respectively.     

HBV X蛋白对阿霉素诱导的肝癌细胞凋亡的影响

目的：探讨乙型肝炎病毒X蛋白（hepatitis BvirusX protein,HBVX）对阿霉素诱导的肝癌细胞凋亡及p53、PTEN表达的影响。方法：用阿霉素（2.5g/mL）分别处理HepG及稳定表达GFP、GFP-HBVX融合蛋白的细胞系HepG/GFP、22HepG/GFP-HBVX,处理后不同时间在显微镜下观察细胞形态变化,流式细胞术检测细胞凋亡。结果：流式细胞术检测显2示阿霉素处理后36h,HepG/GFP-HBVX细胞凋亡率为3.94%,明显低于HepG（59.03%）、HepG/GFP细胞（61.38%）222（P〈0.05）,而与未处理对照组细胞（2.12%、2.78%、2.55%）无明显差别（P〉0.05）。结论：HBVX蛋白能够抑制阿霉素诱导的细胞凋亡。     

Aqueous extract of clove inhibits tumor growth by inducing autophagy through AMPK/ULK pathway

Cloves (Syzygium aromaticum), a traditional Chinese medicinal herb, displays broad biological activity. In the present study, the aqueous extract of clove (AEC) was prepared, and its anticancer affects were studied. 3‐(4,5‐Dimethylthiazol‐2‐yl)‐5‐(3‐carboxymethoxyphenyl)‐2‐(4‐sulfophenyl)‐2H‐tetra‐zolium (MTS) analysis revealed that AEC was able to inhibit cancer cell growth in vitro on several cancer cell lines; the IC₅₀ is around 150 μg/ml for human pancreatic ASPC‐1 and human colon HT‐29 cancer cells. Treatment of the cancer cells with AEC also diminished the colony formation significantly in both human pancreatic ASPC‐1 cancer cells and human colon HT‐29 cancer cells. In vivo study revealed that AEC inhibited the tumor growth significantly in HT‐29 xenograft mice model. Transmission electron microscope, flow cytometry assay, and fluorescence microscope analysis confirmed that AEC is capable of inducing cell autophagy. Further study showed that AMPK/ULK pathway plays an important role in AEC‐induced autophagy in cancer cells. Analysis of AEC components was performed by liquid chromatograph mass spectrometer approach, and more than nine constitutes were identified in AEC fraction. The study provides evidence that AEC has potential to be developed as a novel anticancer agent or as an adjuvant in cancer chemotherapy.     

ERK1/2在黄芪多糖促进HepG2细胞凋亡中的作用

目的:阐明黄芪多糖与ERK1/2通路在HepG2细胞凋亡中的作用.方法:实验分为4组:空白对照组(control);B组,20 mg?L-1黄芪多糖3个剂量处理组(AP,20,40,80 mg?L-1黄芪多糖处理组( AP,40,80 mg?L-1),药物处理24 h后检测各指标.应用Western blot,MTT,TUNEL,线粒体膜电势检测等方法检测黄芪多糖对HepG2细胞生存凋亡及细胞中ERK1/2表达量的影响.结果:MTT与TUNEL表明黄芪多糖对HepG2细胞的凋亡具有促进作用,增加HepG2细胞的caspase-3的活性,降低(HepG2)细胞线粒体的膜电位,降低Bcl-2表达;黄芪多糖可以降低ERK1/2的表达,表明黄芪多糖的凋亡促进作用可能通过ERK1/2途径.结论:黄芪多糖可以通过抑制ERK1/2信号通路促进HepG2细胞的凋亡.     

虎皮楠生物碱daphnioldhanin E诱导HepG2细胞类凋亡研究

目的研究虎皮楠生物碱daphnioldhanin E诱导人肝癌HepG2细胞死亡的机制。方法 HepG2细胞用daphnioldhanin E(10、30μg/mL)处理后,瑞姬氏染色法观察细胞形态变化,DNA琼脂糖凝胶电泳检测细胞凋亡情况,MTT法检测细胞凋亡抑制剂Z-VAD-FMK对daphnioldhanin E抗癌活性的影响,分光光度法检测caspase-3酶活性的变化。结果瑞姬氏染色显示,HepG2细胞经daphnioldhanin E处理48 h后,细胞体积增大,细胞核清晰可见,周围出现大量的胞浆空泡,但细胞膜仍保持完整;DNA琼脂糖凝胶电泳结果可见大量50～200 bp的片段;MTT法检测结果显示Z-VAD-FMK不能阻断daphnioldhanin E对HepG2细胞增殖的抑制作用;分光光度法测定显示,daphnioldhanin E给药组与对照组HepG2细胞caspase-3酶活性无明显变化,表明daphnioldhanin E对HepG2细胞的杀伤作用与caspase-3激活通路无关。结论Daphnioldhanin E可能通过类凋亡方式明显诱导HepG2细胞程序性死亡,该过程不依赖于caspase-3途径。     

Antiproliferative properties of polyketides isolated from Virola sebifera leaves

An activity-guided fractionation of Virola sebifera Aubl. methylene chloride-soluble fraction provided novel 3,5-dihydro-2-(1'-oxo-3'-hexadecenyl)-2-cyclohexen-1-one (3), two known lignans (1, 2) and dehydro hexadecanoyl resorcinol (4). Isolation and purification were conducted with the application of column chromatography and structures were assigned by spetral analysis (1D and 2D NMR, HREIMS). Compounds 1-4 were evaluated for cytotoxic activities against human tumour cell lines UACC62 (melanoma), MCF-7 (breast), NCI 460 (lung, non-small cells), OVCAR03 (ovarian), PC-03 (prostate), HT-29 (colon), 786-0 (renal) and NCI-ADR (breast expressing phenotype multiple drugs resistance) in vitro. The new polyketide (3) showed selectivity against human OVCAR03 and NCI-ADR cell lines, ranging from 2 to 4 microg/mL.     

人参皂苷Rh_2通过激活p38诱导K562细胞自噬凋亡的实验研究

为了研究人参皂苷Rh2对人白血病细胞K562的凋亡作用,并从自噬角度来探讨其机制。实验采用CCK-8方法检测人参皂苷单体Rh2对人白血病K562细胞增殖抑制作用;流式细胞术检测细胞凋亡;Hoechest染色观察细胞核染色质的形态;吖啶橙和MDC染色观察Rh2对细胞自噬的影响;Western blot和RT-PCR检测Rh2对白血病细胞自噬重要基因的表达的影响;运用自噬抑制剂(3-MA)研究自噬对细胞增殖和凋亡的影响。CCK-8显示人参皂苷Rh2在低浓度时能有效抑制白血病细胞增殖,且其抑制作用具有浓度和时间依赖性;FCM和Hoechest显示Rh2能增加细胞的凋亡和染色质呈凋亡形态改变;吖啶橙和MDC染色发现Rh2组细胞的绿色荧光增强,并出现大量酸性自噬小泡;Western blot和RT-PCR结果发现Rh2能上调Beclin-1,LC3A,LC3B,激活型Caspase-3和p-p38的表达;运用细胞自噬剂(3-MA)会削弱Rh2对K562的增殖抑制和促凋亡作用。人参皂苷Rh2可能通过激活磷酸化的p38,诱导细胞自噬途径,从而抑制K562细胞增殖和促进凋亡作用。     

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.