Novel thiosemicarbazides induced apoptosis in human MCF-7 breast cancer cells via JNK signaling

Abstract

In this study, novel thiosemicarbazides and 1,3,4-oxadiazoles were synthesized and evaluated for their anticancer effects on human MCF-7 breast cancer cell lines. Among the synthesized derivatives studied, compound 2-(3-(4-chlorophenyl)-3-hydroxybutanoyl)-N-phenylhydrazinecarbothioamide 4c showed the highest cytotoxicity against MCF-7 breast cancer cells as it reduced cell viability to approximately 15% compared to approximately 25% in normal breast epithelial cells. Therefore, we focused on 4c for further investigations. Our data showed that 4c induced apoptosis in MCF-7 cells which was further confirmed by TUNEL assay. Western blotting analysis showed that compound 4c up-regulated the pro-survival proteins Bax, Bad and ERK1/2, while it down-regulated anti-apoptotic proteins Bcl-2, Akt and STAT-3. Additionally, 4c induced phosphorylation of SAPK/JNK in MCF-7 cells. Pretreatment of MCF-7 cells with 10?µM of JNK inhibitor significantly reduced 4c-induced apoptosis. Molecular docking results suggested that compound 4c showed a binding pattern close to the pattern observed in the structure of the lead fragment bound to JNK1. Collectively, the data of current study suggested that the thiosemicarbazide 4c might trigger apoptosis in human MCF-7 cells by targeting JNK signaling.     

Tehranolide inhibits proliferation of MCF-7 human breast cancer cells by inducing G0/G1 arrest and apoptosis

Tehranolide, a novel natural sesquiterpene lactone with an endoperoxide group, bears a structural similarity to artemisinin and has been shown to inhibit cell growth. However, the underlying mechanisms of these activities remain obscure. The purpose of this study was to investigate the fundamental mechanisms by which tehranolide inhibits growth in MCF-7 cells. Cell growth was determined by using the MTT viability assay and counting cells. Apoptosis and cell-cycle progression were evaluated by means of Hoechst 33258 staining, flow cytometry with annexin-V/propidium iodide double staining, and ROS formation. The protein expression of Bax and Bcl-2 was demonstrated by Western blotting. Moreover, to determine the molecular mechanism whereby tehranolide mediates G0/G1 arrest, the expression of PI3K, p-PI3K, Akt, p-Akt, p27kip1, cyclin D1, and CDK4 was monitored. Cell proliferation was significantly inhibited by tehranolide in a dose- and time-dependent manner. This compound inhibited cell proliferation and induced G0/G1 arrest through the PI3K/Akt/cyclin D1 pathway. It also induced apoptosis and an increase in ROS. In addition, an increase in cytochrome c and Bax, as well as a decrease in Bcl-2, was observed. Moreover, blocking the CD95 receptor with an anti-CD95 antibody (ZB4) had no effect on tehranolide-mediated apoptosis. This study has yielded promising results, which show for the first time that tehranolide does inhibit the growth of cancer cells. The selective inhibition of cancer cell growth, the apoptosis induction via the mitochondrial pathway, and the G0/G1 arrest by modulating the PI3K/AKT signaling pathway and downregulating cyclin D1, which leads to the release of p27kip1 and the association of this inhibitor with the cyclin E/CDK2 complex, ultimately preventing cell-cycle progression from G1 to S phase, all serve to provide support for further studies of tehranolide as a possible anticancer drug in the clinical treatment of cancer.     

Rottlerin inhibits the nuclear factor kappaB/cyclin-D1 cascade in MCF-7 breast cancer cells

In the course of a project aimed to clarify the molecular mechanisms by which phorbol 12-myristate 13-acetate (PMA)-activated forms of protein kinase C (PKC) promote growth arrest in an MCF-7 cell line, we found that the PKCdelta inhibitor Rottlerin was able by itself to block cell proliferation. In the current study, we investigated further the antiproliferative response to Rottlerin. Western blotting analysis of cytoplasmic/nuclear extracts showed that the drug did not prevent either extracellular signal-regulated kinase (ERK) activation by PMA or Akt phosphorylation, but did interfere with the NFkappaB activation process (both basal and PMA-stimulated), by lowering the levels of phospho-IkappaBalpha and preventing p65 nuclear migration. The growth arrest evoked by Rottlerin was not mediated by cell-cycle inhibitors p21 and p27 but was accompanied by a dramatic fall in the cyclin-D1 protein, the levels of which were not altered by the pan-PKC inhibitor GF 109203X, thus excluding a PKC-mediated mechanism in the Rottlerin effect. The parallel drop in cyclin-D1 mRNA suggested a down-regulation of the gene caused by the inhibition of nuclear factor-kappa B (NFkappaB), which occurs via a PKC-, Akt-, ERK- and mitochondrial uncoupling-independent mechanism. We provide preliminary evidence that the interference on the NFkappaB activation process likely occurs at the level of calcium/calmodulin-dependent protein kinase II (CaMKII), a known Rottlerin target. Indeed the drug prevented calcium-induced CaMKII autophosphorylation which, in turn, led to decreased NFkappaB activation.     

Activation of a cysteine protease in MCF-7 and T47D breast cancer cells during beta-lapachone-mediated apoptosis

beta-Lapachone (beta-lap) effectively killed MCF-7 and T47D cell lines via apoptosis in a cell-cycle-independent manner. However, the mechanism by which this compound activated downstream proteolytic execution processes were studied. At low concentrations, beta-lap activated the caspase-mediated pathway, similar to the topoisomerase I poison, topotecan; apoptotic reactions caused by both agents at these doses were inhibited by zVAD-fmk. However at higher doses of beta-lap, a novel non-caspase-mediated "atypical" cleavage of PARP (i.e., an approximately 60-kDa cleavage fragment) was observed. Atypical PARP cleavage directly correlated with apoptosis in MCF-7 cells and was inhibited by the global cysteine protease inhibitors iodoacetamide and N-ethylmaleimide. This cleavage was insensitive to inhibitors of caspases, granzyme B, cathepsins B and L, trypsin, and chymotrypsin-like proteases. The protease responsible appears to be calcium-dependent and the concomitant cleavage of PARP and p53 was consistent with a beta-lap-mediated activation of calpain. beta-Lap exposure also stimulated the cleavage of lamin B, a putative caspase 6 substrate. Reexpression of procaspase-3 into caspase-3-null MCF-7 cells did not affect this atypical PARP proteolytic pathway. These findings demonstrate that beta-lap kills cells through the cell-cycle-independent activation of a noncaspase proteolytic pathway.     

Tamoxifen induces TGF-β1 activity and apoptosis of human MCF-7 breast cancer cells in vitro

We report here that the antiestrogen tamoxifen (TAM) induces cell death in human breast cancer cell line MCF-7. We assessed the type of cell death induced by TAM in this breast cancer cell line on the basis of morphological and biochemical characteristics. Dying cells showed morphological characteristics of apoptosis, such as chromatin condensation and nuclear disintegration. DNA isolated from these cells revealed a pattern of distinctive DNA bands on agarose gel. The DNA fragmentation in MCF-7 cells induced by TAM could also be detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin end labeling. Northern blot hybridization revealed a substantial increase in the amounts of TRPM-2 and TGF-β1 mRNAs in MCF-7 cells after treatment with TAM. In contrast, the mRNA level of the estrogen-induced pS2 gene was strongly suppressed. The biological activity of TGF-β was increased at least fourfold in the media from MCF-7 cells treated with TAM. The results presented in this study suggest that TAM induces apoptosis of MCF-7 cells and it may be mediated by the secretion of active TGF-β. © 1996 Wiley-Liss, Inc.     

抑制PI3K信号通路促进TRAIL诱导人乳腺癌细胞MCF-7凋亡

肿瘤坏死因子相关凋亡诱导配体（tumor necrosis factor-related apoptosis-inducing ligand, TRAIL）对癌细胞有独特的细胞毒性作用,而对正常细胞没有影响. 但乳腺癌细胞耐受TRAIL诱导凋亡.本研究探索磷脂酰肌醇-3激酶（phosphatidylinositol 3-kinase,PI3K）信号通路对人乳腺癌MCF-7细胞耐受TRAIL的影响. 采用MTT法、显微照相以及DAPI染色观察TRAIL对MCF-7细胞生长的抑制作用以及诱导细胞凋亡状况;流式细胞分析细胞凋亡的情况;激光共聚焦显微镜观察多聚ADP核糖多聚酶-1（poly(ADP-ribose) polymerase -1,PARP-1）的迁移和定位;Western印迹分析死亡受体、caspase-3/8、磷酸化的AKT［pAKT(Ser473)］、Src和PARP-1等蛋白质表达. 结果显示,小剂量TRAIL（< 80 nmol/L）和Ly294002（< 40μmol/L）对MCF-7细胞生长没有显著的抑制作用,但是大剂量TRAIL（160 nmol/L）和Ly294002（80 μmol/L）则能抑制MCF-7细胞生长;低剂量Ly294002协同TRAIL抑制MCF-7细胞生长,并诱导细胞凋亡;Ly294002和TRAIL共同作用能促进PARP-1从胞浆进入细胞核;蛋白质表达分析显示,MCF-7细胞均表达死亡受体DR4、DR5、诱骗受体DcR1和DcR2、以及caspase-8,但是不表达caspase-3;Ly294002和TRAIL共同作用也能抑制pAKT(Ser473)和Src的表达,并且导致PARP-1断裂. 本研究结果提示,抑制PI3K信号可增加MCF-7细胞对TRAIL诱导的敏感性;MCF-7细胞通过PI3K/AKT途径促进Src的表达耐受TRAIL的细胞毒性作用Ly294002联合TRAIL是一种新的药物组合方式治疗乳腺癌.     

Insulin-like growth factor I triggers nuclear accumulation of cyclin D1 in MCF-7S breast cancer cells

Stimulation of the breast cancer-derived MCF-7S cell line with insulin-like growth factor I (IGF-I; 20 ng/ml) leads to enhanced expression of cyclin D1, hyperphosphorylation of pRb, DNA synthesis, and cell division. 17beta-Estradiol (E(2); 10(-9) m) is not able to stimulate proliferation of MCF-7S cells, although addition of E(2) to serum-starved cells does result in induction of cyclin D1. However, in combination with submitogenic amounts of IGF-I (2 ng/ml), E(2) induces cell proliferation. We have previously shown that the synergistic action of E(2) and IGF-I emanates from the ability of both hormones to induce cyclin D1 expression and that IGF-I action is required to induce activity of the cyclin D1-CDK4 complex, which triggers cell cycle progression. Here, we show that IGF-I (but not E(2)) is able to induce nuclear accumulation of cyclin D1 by a phosphatidylinositol 3-kinase-dependent mechanism. Nuclear accumulation of cyclin D1 and cell cycle progression were also observed when LiCl, a known inhibitor of GSK3beta, was added to E(2)-stimulated cells. Thus, inhibition of GSK3beta activity appears to trigger nuclear accumulation of cyclin D1 and cell cycle progression. This notion was confirmed by overexpression of constitutively active GSK3beta, which blocks IGF-I-induced nuclear accumulation of cyclin D1 as well as S phase transition.     

Polygonatum odoratum lectin induces apoptosis and autophagy via targeting EGFR-mediated Ras-Raf-MEK-ERK pathway in human MCF-7 breast cancer cells

Polygonatum odoratum lectin (POL), a mannose-binding GNA-related lectin, has been reported to display remarkable anti-proliferative and apoptosis-inducing activities toward a variety of cancer cells; however, the precise molecular mechanisms by which POL induces cancer cell death are still elusive. In the current study, we found that POL could induce both apoptosis and autophagy in human MCF-7 breast cancer cells. Subsequently, we found that POL induced MCF-7 cell apoptosis via the mitochondrial pathway. Additionally, we also found that POL induces MCF-7 cell apoptosis via EGFR-mediated Ras-Raf-MEK-ERK pathway, suggesting that POL may be a potential EGFR inhibitor. Finally, we used proteomics analyses for exploring more possible POL-induced pathways with EGFR, Ras, Raf, MEK and ERK, some of which were consistent with our in silico network prediction. Taken together, these results demonstrate that POL induces MCF-7 cell apoptosis and autophagy via targeting EGFR-mediated Ras-Raf-MEK-ERK signaling pathway, which would provide a new clue for exploiting POL as a potential anti-neoplastic drug for future cancer therapy.     

Acacetin-induced apoptosis of human breast cancer MCF-7 cells involves caspase cascade, mitochondria-mediated death signaling and SAPK/JNK1/2-c-Jun activation

The mechanism of acacetin-induced apoptosis of human breast cancer MCF-7 cells was investigated. Acacetin caused 50% growth inhibition (IC50) of MCF-7 cells at 26.4% 0.7% M over 24 h in the MTT assay. Apoptosis was characterized by DNA fragmentation and an increase of sub-G1 cells and involved activation of caspase-7 and PARP (poly-ADP-ribose polymerase). Maximum caspase 7 activity was observed with 100 microM acacetin for 24 h. Caspase 8 and 9 activation cascades mediated the activation of caspase 7. Acacetin caused a reduction of Bcl-2 expression leading to an increase of the Bax:Bcl-2 ratio. It also caused a loss of mitochondrial membrane potential that induced release of cytochrome c and apoptosis inducing factor (AIF) into the cytoplasm, enhancing ROS generation and subsequently resulting in apoptosis. Pretreatment of cells with N-acetylcysteine (NAC) reduced ROS generation and cell growth inhibition, and pretreatment with NAC or a caspase 8 inhibitor (Z-IETD-FMK) inhibited the acacetin-induced loss of mitochondrial membrane potential and release of cytochrome c and AIF. Stress-activated protein kinase/c-Jun NH4-terminal kinase 1/2 (SAPK/ JNK1/2) and c-Jun were activated by acacetin but extracellular-regulated kinase 1/2 (Erk1/2) nor p38 mitogen-activated protein kinase (MAPK) were not. Our results show that acacetin-induced apoptosis of MCF-7 cells is mediated by caspase activation cascades, ROS generation, mitochondria-mediated cell death signaling and the SAPK/JNK1/2-c-Jun signaling pathway, activated by acacetin-induced ROS generation.     

Role of mitochondria and caspases in vitamin D-mediated apoptosis of MCF-7 breast cancer cells

Vitamin D(3) compounds are currently in clinical trials for human breast cancer and offer an alternative approach to anti-hormonal therapies for this disease. 1alpha,25-Dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)), the active form of vitamin D(3), induces apoptosis in breast cancer cells and tumors, but the underlying mechanisms are poorly characterized. In these studies, we focused on the role of caspase activation and mitochondrial disruption in 1alpha,25(OH)(2)D(3)-mediated apoptosis in breast cancer cells (MCF-7) in vitro. The effect of 1alpha,25(OH)(2)D(3) on MCF-7 cells was compared with that of tumor necrosis factor alpha, which induces apoptosis via a caspase-dependent pathway. Our major findings are that 1alpha,25(OH)(2)D(3) induces apoptosis in MCF-7 cells by disruption of mitochondrial function, which is associated with Bax translocation to mitochondria, cytochrome c release, and production of reactive oxygen species. Moreover, we show that Bax translocation and mitochondrial disruption do not occur after 1alpha,25(OH)(2)D(3) treatment of a MCF-7 cell clone selected for resistance to 1alpha,25(OH)(2)D(3)-mediated apoptosis. These mitochondrial effects of 1alpha,25(OH)(2)D(3) do not require caspase activation, since they are not blocked by the cell-permeable caspase inhibitor z-Val-Ala-Asp-fluoromethylketone. Although caspase inhibition blocks 1alpha,25(OH)(2)D(3)-mediated events downstream of mitochondria such as poly(ADP-ribose) polymerase cleavage, external display of phosphatidylserine, and DNA fragmentation, MCF-7 cells still execute apoptosis in the presence of z-Val-Ala-Asp-fluoromethylketone, indicating that the commitment to 1alpha,25(OH)(2)D(3)-mediated cell death is caspase-independent.     

Autophagy-dependent EIF2AK3 activation compromises ursolic acid-induced apoptosis through upregulation of MCL1 in MCF-7 human breast cancer cells

Ursolic acid (UA) is a pentacyclic triterpenoid with promising cancer chemopreventive properties. A better understanding of the mechanisms underlying anticancer activity of UA is needed for further development as a clinically useful chemopreventive agent. Here, we found that both endoplasmic reticulum (ER) stress and autophagy were induced by UA in MCF-7 human breast cancer cells. Surprisingly, ER stress was identified as an effect rather than a cause of UA-induced autophagy. Autophagy-dependent ER stress protected the cells from UA-induced apoptosis through EIF2AK3-mediated upregulation of MCL1. Activation of MAPK1/3 but not inhibition of MTOR pathway contributed to UA-induced cytoprotective autophagy in MCF-7 cells. Our findings uncovered a novel cellular mechanism involved in the anticancer activity of UA, and also provided a useful model to study biological significance and mechanisms of autophagy-mediated ER stress.     

BRCA1 negatively regulates formation of autophagic vacuoles in MCF-7 breast cancer cells

In recent years, the function of different tumour suppressors in the regulation of macroautophagy has been studied. We show here that BRCA1, unlike other tumour suppressors, negatively regulates formation of autophagosomes and lysosomal mass under conditions of both basal and enhanced autophagy. In MCF-7 breast cancer cells, increased formation of autophagic vacuoles after inactivation of BRCA1 by siRNAs is associated with an increase in reactive oxygen species, such as superoxide anion and hydrogen peroxide. This allows one to propose an antioxidant function for BRCA1 and suggests that dysfunctional mitochondria and the generated reactive oxygen species excess could explain the increased macroautophagy observed in the absence of BRCA1. In addition, a quick decrease in BRCA1 levels occurs when MCF-7 cells are switched to a nutrient-poor environment that stimulates macroautophagy and that is also reminiscent of certain phases of tumour growth. Inhibition of BRCA1 synthesis has an important role in this reduction, while there are almost no changes in BRCA1 degradation by lysosomes and proteasomes. Therefore, BRCA1 produces macroautophagy inhibition by reducing the formation of autophagic vacuoles, and this, together with the other results presented here, shows new functional aspects of BRCA1 that could help to clarify the role of autophagy in cancer development.     

trans-Resveratrol induces apoptosis in human breast cancer cells MCF-7 by the activation of MAP kinases pathways

Polyphenols represent a large class of plant-derived molecules with a general chemical structure that act as potent free radical scavengers. They have long been recognized to possess several therapeutic activities ranging from anti-thrombotic to antioxidant. Moreover, the capability of polyphenols to act as reducing or oxidizing molecules depends on the presence of environmental metals and on the concentrations used. In this work we demonstrated that the stilbene trans-resveratrol was able to commit human breast cancer MCF-7 cells to apoptosis. Mainly, we evidenced a pivotal role of the mitochondria in this phenomenon as cytochrome c release into the cytosol was found after the treatment. We further showed that trans-resveratrol was able to affect cellular redox state. In particular, it induced an early production of ROS and lipid oxidation, and only later compromised the GSH/GSSG ratio. This mode of action was mirrored by a temporally different activation of JNK and p38(MAPK), with the former rapidly induced and the latter weakly activated at long intervals. The results obtained demonstrate a pro-apoptotic activity for trans-resveratrol, and suggest a preferential activation of different classes of MAP kinases in response to different oxidative stimuli (ROS versus GSH/GSSG alteration).     

Combinatory effects of phytoestrogens and 17beta-estradiol on proliferation and apoptosis in MCF-7 breast cancer cells

Phytoestrogens have been described to be weak estrogens, SERMs or exhibit antiestrogenic properties. However, information about their activity in presence of estrogens is limited. Therefore, we have analysed the dose dependent combinatory activity of the phytoestrogens genistein (Gen), daidzein (Dai) and coumestrol (Cou), and 17ß-estradiol (E2) on cell proliferation and apoptosis induction in human MCF-7 breast cancer cells. Neither additive nor antagonistic effects on proliferation could be observed, but in contrast all phytoestrogens possessed the ability to inhibit apoptosis in the presence of 17ß-estradiol.

In summary, our in vitro results demonstrate that Gen does not exhibit any antiestrogenic properties. The additive growth stimulatory effects of Gen, Dai and Cou in the presence of E2 are not the result of a stimulation of proliferation; these phytoestrogens, at least in MCF-7 cells, could be characterised as inhibitors of apoptosis.     

RhoBTB2 (DBC2) functions as tumor suppressor via inhibiting proliferation, preventing colony formation and inducing apoptosis in breast cancer cells

RhoBTB2 was isolated recently as a tumor suppressor gene from sporadic breast cancer. Although RhoBTB2 was found to be frequently lost in breast cancer and a variety of cancers, its antitumor effect, however, remains unclear. In this study, we constructed a recombinant expression vector pEGFP-N1-RhoBTB2 and transfected it into RhoBTB2-negative breast tumor cell line T-47D. Stable transformanted cells were identified by fluorescence microscope, RT-PCR and Western blot. Cell viability was measured by MTT assay. Colony forming efficiency of breast tumor cells was detected by colony formation assay. Morphological change of apoptotic cells was observed by hematoxylin-eosin staining. Apoptotic ratio was determined by flow cytometry. Cell invasion and migration ability assay were performed using transwell system. Overexpression of RhoBTB2 in breast tumor cells significantly inhibited the proliferation and colony formation of tumor cells. In addition, RhoBTB2 also elevated the apoptotic ratio and caused typical changes of apoptotic morphology in breast tumor cells of RhoBTB2 overexpression. But RhoBTB2 did not influence the invasion and migration ability of breast tumor cells. Therefore, RhoBTB2 is an important tumor suppressor gene related with breast cancer and may play antitumor roles by inhibiting proliferation, preventing colony formation and promoting the apoptosis of tumor cells. However, the precise mechanism behind the antitumor effects of RhoBTB2 needs to be investigated further.