Benzyl isothiocyanate targets mitochondrial respiratory chain to trigger reactive oxygen species-dependent apoptosis in human breast cancer cells

Benzyl isothiocyanate (BITC), a dietary cancer chemopreventive agent, causes apoptosis in MDA-MB-231 and MCF-7 human breast cancer cells, but the mechanism of cell death is not fully understood. We now demonstrate that the BITC-induced apoptosis in human breast cancer cells is initiated by reactive oxygen species (ROS) due to inhibition of complex III of the mitochondrial respiratory chain. The BITC-induced ROS production and apoptosis were significantly inhibited by overexpression of catalase and Cu,Zn-superoxide dismutase and pharmacological inhibition of the mitochondrial respiratory chain. The mitochondrial DNA-deficient Rho-0 variant of MDA-MB-231 cells was nearly completely resistant to BITC-mediated ROS generation and apoptosis. The Rho-0 MDA-MB-231 cells also resisted BITC-mediated mitochondrial translocation (activation) of Bax. Biochemical assays revealed inhibition of complex III activity in BITC-treated MDA-MB-231 cells as early as at 1 h of treatment. The BITC treatment caused activation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), which function upstream of Bax activation in apoptotic response to various stimuli. Pharmacological inhibition of both JNK and p38 MAPK conferred partial yet significant protection against BITC-induced apoptosis. Activation of JNK and p38 MAPK resulting from BITC exposure was abolished by overexpression of catalase. The BITC-mediated conformational change of Bax was markedly suppressed by ectopic expression of catalytically inactive mutant of JNK kinase 2 (JNKK2(AA)). Interestingly, a normal human mammary epithelial cell line was resistant to BITC-mediated ROS generation, JNK/p38 MAPK activation, and apoptosis. In conclusion, the present study indicates that the BITC-induced apoptosis in human breast cancer cells is initiated by mitochondria-derived ROS.     

Mitochondria of highly metastatic breast cancer cell line MDA-MB-231 exhibits increased autophagic properties

Autophagy is a cellular housekeeping process that removes damaged or unwanted cellular components and recycles them to build new constituents. It is essential for tumor growth under adverse environment. Mitochondria play an important role in the formation of autophagosome and its subsequent docking and fusion with lysosome. To understand the contribution of mitochondria to the regulation of homeostatic autophagy in cancer cells, we used the transmitochondrial cytoplasmic hybrid (cybrid) model. Cybrid system allowed us to compare mitochondria from different cell types including highly metastatic breast cancer cell line MDA-MB-231 (c231), less metastatic breast cancer cell lines: MDA-MB-436 (c436) and MDA-MB-468 (c468), as well as non-cancerous mammary epithelial cell MCF-10A (c10A) in a defined nuclear background. The c231 exhibited lower LC3-II levels but higher ratio of LC3-II/LC3-I than c436, c468 and c10A. In addition, c231 displayed more punctate LC3-positive cells and had lower levels of sequestosome 1 (p62/SQSTM1) than other cybrids. These suggested that mitochondria could contribute to the increased autophagy and autophagic flux in metastatic cancer. This increased autophagy was found to be non-selective autophagy instead of selective mitophagy since LC3 puncta in c231 did not co-localize with mitochondria labeled by Mitotracker red or Tomm 20. The promotion of mitochondrial permeability transition (MPT) in c231 also contributed to increased autophagy. Block of MPT by the inhibition of low-conductance stage of MPT pores resulted in a decrease of LC3 puncta in c231. These results suggested that mitochondria from highly metastatic breast cancer cell line MDA-MB-231 can promote homeostatic autophagy of cancer through opening low-conductance MPT pores.     

Molecular iodine induces caspase-independent apoptosis in human breast carcinoma cells involving the mitochondria-mediated pathway

Molecular iodine (I2) is known to inhibit the induction and promotion of N-methyl-n-nitrosourea-induced mammary carcinogenesis, to regress 7,12-dimethylbenz(a)anthracene-induced breast tumors in rat, and has also been shown to have beneficial effects in fibrocystic human breast disease. Cytotoxicity of iodine on cultured human breast cancer cell lines, namely MCF-7, MDA-MB-231, MDA-MB-453, ZR-75-1, and T-47D, is reported in this communication. Iodine induced apoptosis in all of the cell lines tested, except MDA-MB-231, shown by sub-G1 peak analysis using flow cytometry. Iodine inhibited proliferation of normal human peripheral blood mononuclear cells; however, it did not induce apoptosis in these cells. The iodine-induced apoptotic mechanism was studied in MCF-7 cells. DNA fragmentation analysis confirmed internucleosomal DNA degradation. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling established that iodine induced apoptosis in a time- and dose-dependent manner in MCF-7 cells. Iodine-induced apoptosis was independent of caspases. Iodine dissipated mitochondrial membrane potential, exhibited antioxidant activity, and caused depletion in total cellular thiol content. Western blot results showed a decrease in Bcl-2 and up-regulation of Bax. Immunofluorescence studies confirmed the activation and mitochondrial membrane localization of Bax. Ectopic Bcl-2 overexpression did not rescue iodine-induced cell death. Iodine treatment induces the translocation of apoptosis-inducing factor from mitochondria to the nucleus, and treatment of N-acetyl-L-cysteine prior to iodine exposure restored basal thiol content, ROS levels, and completely inhibited nuclear translocation of apoptosis-inducing factor and subsequently cell death, indicating that thiol depletion may play an important role in iodine-induced cell death. These results demonstrate that iodine treatment activates a caspase-independent and mitochondria-mediated apoptotic pathway.     

Estrogen down-regulates uncoupling proteins and increases oxidative stress in breast cancer

Oxidative stress has been postulated as one of the mechanisms underlying the estrogen carcinogenic effect in breast cancer. Estrogens are known to increase mitochondrial-derived reactive oxygen species (ROS) by an unknown mechanism. Given that uncoupling proteins (UCPs) are key regulators of mitochondrial energy efficiency and ROS production, our aim was to check the presence and activity of UCPs in estrogen receptor (ER)-positive and ER-negative breast cancer cells and tumors, as well as their relation to oxidative stress. Estrogen (1 nM) induced higher oxidative stress in the ER-positive MCF-7 cell line, showing increased mitochondrial membrane potential, H₂O₂ levels, and DNA and protein damage compared to ER-negative MDA-MB-231 cells. All isoforms of uncoupling proteins were highly expressed in ER-positive breast cancer cells and tumors compared to negative ones. ROS production in mitochondria isolated from MCF-7 was increased by inhibition of UCPs with GDP, but not in mitochondria from MDA-MB-231. Estrogen treatment decreased uncoupling protein and catalase levels in MCF-7 and decreased GDP-dependent ROS production in isolated mitochondria. On the whole, these results suggest that estrogens, through an ER-dependent mechanism, may increase mitochondrial ROS production by repressing uncoupling proteins, which offers a new perspective on the understanding of why estrogens are a risk factor for breast cancer.     

The cytotoxic nature of Acanthopanax sessiliflorus stem bark extracts in human breast cancer cells

Acanthopanax sessiliflorus, a small woody shrub has traditionally been referred to have anticancer activity, but it has not been scientifically explored so far. Therefore, to investigate the anticancer effects of A. sessiliflorus stem bark extracts (ASSBE), MDA-MB-231 and MCF-7 human breast cancer cells were treated with one of its bioactive fractions, n-hexane (ASSBE-nHF). Cytotoxicity (24 h) was determined by MTT assay and antiproliferative effect was assessed by counting cell numbers after 72 h treatment using hemocytometer. The role of ASSBE-nHF on apoptosis was analysed by annexin V-FITC/PI, Hoechst 33342 staining, DNA fragmentation pattern and immunoblotting of apoptosis markers. For the assay of enhanced production of ROS and mitochondrial membrane depolarization, specific stains such as DCFH-DA and JC-1 were used, respectively. To understand the mode of action of ASSBE-nHF on MCF-7 cells, cells were pre-treated with antioxidant, n-acetylcysteine. The hexane fraction of ASSBE showed maximum activity towards human breast cancer cells compared to other two fractions at a minimal concentration of 50 μg/ml. The annexin V-FITC/PI, Hoechst 33342 staining, DNA fragmentation and immunoblotting assays showed that ASSBE-nHF induces non-apoptotic cell death in MCF-7 and MDA-MB-231 cells. ASSBE-nHF significantly increased the production of ROS and decreased the mitochondrial membrane potential (MMP) in MCF-7 cells. Similarly, it decreased the MMP in MDA-MB-231 cells, but had no effect on ROS production. Further, the cytotoxic effect of ASSBE-nHF in MCF-7 cells was not significantly reversed even in the presence of n-acetylcysteine, an antioxidant. These findings revealed that ASSBE-nHF induces non-apoptotic cell death via mitochondria associated with both ROS dependent and independent pathways in human breast cancer cells.     

桑葚花色苷提取物对乳腺癌细胞凋亡及线粒体膜电位的影响

目的:观察桑葚花色苷提取物对人乳腺癌细胞株MDA-MB-453、MDA-MB-231和MCF-7细胞凋亡及线粒体膜电位的影响.方法:利用超声辅助乙醇萃取法提取桑葚花色苷,pH示差法测定提取物花色苷总含量,以50、100和150 mg/mL桑葚花色苷提取物作用三种乳腺癌细胞MDA-MB-231、MDA-MB-453和MCF-7 24h,采用Annexin V/PI双染流式细胞分析法检测细胞凋亡水平变化,JC-1探针染色激光共聚焦扫描显微镜观察MDA-MB-453细胞线粒体膜电位水平变化.结果:凋亡分析结果表明,桑葚花色苷提取物作用后三种乳腺癌细胞凋亡率均升高,显示出促凋亡效应,且具有剂量-效应关系,100和150 mg/mL组凋亡率显著升高(P＜0.05).激光共聚焦扫描显微镜检测结果显示,桑葚花色苷提取物作用24h,可使MDA-MB-453细胞线粒体膜电位显著下降,表现为红色/绿色荧光的比值显著降低(P＜0.05).结论:桑葚花色苷提取物可显著降低乳腺癌细胞线粒体膜电位,并促发细胞凋亡.     

Withaferin A-induced apoptosis in human breast cancer cells is mediated by reactive oxygen species

Withaferin A (WA), a promising anticancer constituent of Ayurvedic medicinal plant Withania somnifera, inhibits growth of MDA-MB-231 and MCF-7 human breast cancer cells in culture and MDA-MB-231 xenografts in vivo in association with apoptosis induction, but the mechanism of cell death is not fully understood. We now demonstrate, for the first time, that WA-induced apoptosis is mediated by reactive oxygen species (ROS) production due to inhibition of mitochondrial respiration. WA treatment caused ROS production in MDA-MB-231 and MCF-7 cells, but not in a normal human mammary epithelial cell line (HMEC). The HMEC was also resistant to WA-induced apoptosis. WA-mediated ROS production as well as apoptotic histone-associated DNA fragment release into the cytosol was significantly attenuated by ectopic expression of Cu,Zn-superoxide dismutase in both MDA-MB-231 and MCF-7 cells. ROS production resulting from WA exposure was accompanied by inhibition of oxidative phosphorylation and inhibition of complex III activity. Mitochondrial DNA-deficient Rho-0 variants of MDA-MB-231 and MCF-7 cells were resistant to WA-induced ROS production, collapse of mitochondrial membrane potential, and apoptosis compared with respective wild-type cells. WA treatment resulted in activation of Bax and Bak in MDA-MB-231 and MCF-7 cells, and SV40 immortalized embryonic fibroblasts derived from Bax and Bak double knockout mouse were significantly more resistant to WA-induced apoptosis compared with fibroblasts derived from wild-type mouse. In conclusion, the present study provides novel insight into the molecular circuitry of WA-induced apoptosis involving ROS production and activation of Bax/Bak.     

Induction of Fas-mediated extrinsic apoptosis, p21WAF1-related G2/M cell cycle arrest and ROS generation by costunolide in estrogen receptor-negative breast cancer cells, MDA-MB-231

Costunolide (C(15)H(20)O(2)) is a sesquiterpene lactone that was isolated from many herbal medicines and it has diverse effects according to previous reports. However, the anti-cancer effects and the mechanism of actions are still unknown in breast cancer. In this study, we first observed that costunolide inhibits cell growth in a dose-and time-dependent manner. To examine the mechanism by which costunolide inhibits cell growth, we checked the effect of costunolide on apoptosis and the cell cycle. Costunolide induced apoptosis through the extrinsic pathway, including the activation of Fas, caspase-8, caspase-3, and degradation of PARP. However, did not have the same effect on the intrinsic pathway as revealed by analysis of mitochondrial membrane potential (Δψm) with JC-1 dye and expression of Bcl2 and Bax proteins level. Furthermore, costunolide induced cell cycle arrest in the G2/M phase via decrease in Cdc2, cyclin B1 and increase in p21WAF1 expression, independent of p53 pathway in p53-mutant MDA-MB-231 cells and increases Cdc2-p21WAF1 binding. In addition, costunolide had a slight induced effect on ROS generation. Among the mechanisms of p21WAF1 induction examined, costunolide-induced increase in p21WAF1 expression was related with protein stability and ROS generation. Through this study we confirm that costunolide induces G2/M cell cycle arrest and apoptotic cell death via extrinsic pathway in MDA-MB-231 cells suggesting that it could be a promising anticancer drug especially for ER-negative breast cancer.     

Gain-of-function mutant p53-R280K mediates survival of breast cancer cells

Missense mutations in TP53 resulting in the expression of p53-R175H, p53-R273H, or p53-R280K are frequently detected in human breast cancer. Currently, the role of mutant p53-R280K in breast cancer is relatively unknown, and therefore, the present study analyzed the function of mutant p53-R280K in breast cancer cell growth. To this end, we used small interfering RNA to study the role of mutant p53-R280K in MDA-MB-231 cells, which endogenously express the mutant protein. We found that curcumin induced apoptosis in MDA-MB-231 cells and downregulated mutant p53-R280K. We also observed that knockdown of mutant p53 by small interfering RNA induced apoptosis in MDA-MB-231 cells. Curcumin-induced apoptosis was further enhanced by the overexpression of wild-type p53, but was decreased by mutant p53-R280K overexpression. Our findings indicate that mutant p53-R280K has an important role in mediating the survival of triple-negative breast cancer MDA-MB-231 cells. Furthermore, this study suggests mutant p53-R280K could be used as a therapeutic target for breast cancer cells harboring this TP53 missense mutation.     

Verrucarin A, a protein synthesis inhibitor, induces growth inhibition and apoptosis in breast cancer cell lines MDA-MB-231 and T47D

Verrucarin A (VA), a protein synthesis inhibitor, derived from the pathogen fungus Myrothecium verrucaria, inhibits growth of leukemia cell lines and activates caspases and apoptosis and inflammatory signaling in macrophages. We have investigated VA-induced growth inhibition in breast cancer cells MDA-MB-231 and T47D and, particularly, the mechanism of VA-induced apoptosis. VA treatment brought about apoptotic cell death in a dose- and time-dependent manner which was associated with chromatin condensation, cell shrinkage, nuclear fragmentation and intracellular ROS production. Mitochondrial membrane depolarization, activation of caspase-3, down-regulation of Bcl-2 expression and up-regulation of Bax and p53 expression were observed. VA thus affects the viability of both the breast cancer cells by triggering ROS-mediated intrinsic mechanism of apoptosis.     

Different effects of genistein on molecular markers related to apoptosis in two phenotypically dissimilar breast cancer cell lines

The association between consumption of genistein-containing soybean products and lower risk of breast cancer suggests a cancer chemopreventive role for genistein. Consistent with this suggestion, exposing cultured human breast cancer cells to genistein inhibits cell proliferation, although this is not completely understood. To better understand how genistein works, the ability of genistein to induce apoptosis was compared in phenotypically dissimilar MCF-7 and MDA-MB-231 human breast cancer cells that express the wild-type and mutant p53 gene, respectively. After 6 days of incubation with 50 microM genistein, MCF-7 but not MDA-MB-231 cells, showed morphological signs of apoptosis. Marginal proteolytic cleavage of poly-(ADP-ribose)-polymerase and significant DNA fragmentation were also detected in MCF-7 cells. In elucidating these findings, it was determined that after 2 days of incubation with genistein, MCF-7 but not MDA-MB-231 cells, had significantly higher levels of p53. Accordingly, the expression of certain proteins modulated by p53 was studied next. Levels of p21 increased in both of the genistein-treated cell lines, suggesting that p21 gene expression was activated but in a p53-independent manner, whereas no significant changes in levels of the pro-apoptotic protein, Bax, were found. In MCF-7 cells, levels of the anti-apoptotic protein, Bcl-2, decreased slightly at 18-24 h but then increased considerably after 48 h. Hence, the Bax:Bcl-2 ratio initially increased but later decreased. These data suggest that at the genistein concentration tested, MCF-7 cells in contrast to MDA-MB-231 cells were sensitive to the induction of apoptosis by genistein, but Bax and Bcl-2 did not play clear roles.     

Identification and structural characterization of a new pro-apoptotic cyclic octapeptide cyclosaplin from somatic seedlings of Santalum album L.

Small cyclic peptides exhibiting potent biological activity have great potential for anticancer therapy. An antiproliferative cyclic octapeptide, cyclosaplin was purified from somatic seedlings of Santalum album L. (sandalwood) using gel filtration and RP-HPLC separation process. The molecular mass of purified peptide was found to be 858 Da and the sequence was determined by MALDI-ToF-PSD-MS as ‘RLGDGCTR’ (cyclic). The cytotoxic activity of the peptide was tested against human breast cancer (MDA-MB-231) cell line in a dose and time-dependent manner. The purified peptide exhibited significant antiproliferative activity with an IC₅₀ 2.06 μg/mL. In a mechanistic approach, apoptosis was observed in differential microscopic studies for peptide treated MDA-MB-231 cells, which was further confirmed by mitochondrial membrane potential, DNA fragmentation assay, cell cycle analysis and caspase 3 activities. The modeling and docking experiments revealed strong affinity (kcal/mol) of peptide toward EGFR and procaspase 3. The co-localization studies revealed that the peptide sensitizes MDA-MB-231 cells by possibly binding to EGFR and induces apoptosis. This unique cyclic octapeptide revealed to be a favorable candidate for development of anticancer agents.     

Chetomin induces apoptosis in human triple-negative breast cancer cells by promoting calcium overload and mitochondrial dysfunction

Human triple-negative breast cancer (TNBC) is poorly diagnosed and unresponsive to conventional hormone therapy. Chetomin (CHET), a fungal metabolite synthesized by Chaetomium cochliodes, has been reported as a promising anticancer and antiangiogenic agent but the complete molecular mechanism of its anticancer potential remains to be elucidated. In our study, we explored the anti-neoplastic action of CHET on TNBC cells. Cytotoxicity studies were performed in human TNBC cells viz. MDA-MB-231 and MDA-MB-468 cells by Sulforhodamine B assay. It exhibited antiproliferative response and induced apoptosis in both the cell types. Cell cycle analysis revealed that it increases the sub G0/G1 phase cell population. Modulation of mitochondrial membrane potential, activation of caspase 3/7 and a remarkable increase in the expression of cleaved PARP and increased chromatin condensation was observed after CHET treatment in MDA-MB-231 and MDA-MB-468 cells. Additionally, an elevated level of intracellular Ca²⁺ played an important role in CHET mediated cell death response. Calcium overload in mitochondria led to release of cytochrome c which in turn triggered caspase-3 mediated cell death. Inhibition of calcium signalling using BAPTA-AM reduced apoptosis confirming the involvement of calcium signalling in CHET induced cell death. Chetomin also inhibited PI3K/mTOR cell survival pathway in human TNBC cells. The overall findings suggest that Chetomin inhibited the growth of human TNBC cells by caspase-dependent apoptosis and modulation of PI3K/mTOR signalling and could be used as a novel chemotherapeutic agent for the treatment of human TNBC in future.     

Cytotoxic effect of sanguiin H-6 on MCF-7 and MDA-MB-231 human breast carcinoma cells

Sanguiin H-6 is a dimer of casuarictin linked by a bond between the gallic acid residue and one of the hexahydroxydiphenic acid units. It is an effective compound extracted from Rubus coreanus. It has an anticancer effect against several human cancer cells; however, its effect on breast cancer cells has not been clearly demonstrated. Thus, we aimed to investigate the anticancer effect and mechanism of action of sanguiin H-6 against two human breast carcinoma cell lines (MCF-7 and MDA-MB-231). We found that sanguiin H-6 significantly reduced cell viability in a concentration-dependent manner. It also increased the rates at which MCF-7 and MDA-MB-231 cells underwent apoptosis. Furthermore, sanguiin H-6 induced the cleavage of caspase-8, caspase-3, and poly(ADP-ribose) polymerase, which resulted in apoptosis. However, cleavage of caspase-9 was only detectable in MCF-7 cells. In addition, sanguiin H-6 increased the ratio of Bax to Bcl-2 in both MCF-7 and MDA-MB-231 cells. These findings suggest that sanguiin H-6 is a potent therapeutic agent against breast cancer cells. In addition, it exerts its anticancer effect in an estrogen-receptor-independent manner.     

New Castanospermine Glycoside Analogues Inhibit Breast Cancer Cell Proliferation and Induce Apoptosis without Affecting Normal Cells

sp²-Iminosugar-type castanospermine analogues have been shown to exhibit anti-tumor activity. However, their effects on cell proliferation and apoptosis and the molecular mechanism at play are not fully understood. Here, we investigated the effect of two representatives, namely the pseudo-S- and C-octyl glycoside 2-oxa-3-oxocastanospermine derivatives SO-OCS and CO-OCS, on MCF-7 and MDA-MB-231 breast cancer and MCF-10A mammary normal cell lines. We found that SO-OCS and CO-OCS inhibited breast cancer cell viability in a concentration- and time-dependent manner. This effect is specific to breast cancer cells as both molecules had no impact on normal MCF-10A cell proliferation. Both drugs induced a cell cycle arrest. CO-OCS arrested cell cycle at G1 and G2/M in MCF-7 and MDA-MB-231cells respectively. In MCF-7 cells, the G1 arrest is associated with a reduction of CDK4 (cyclin-dependent kinase 4), cyclin D1 and cyclin E expression, pRb phosphorylation, and an overexpression of p21^Waf1/Cip1. In MDA-MB-231 cells, CO-OCS reduced CDK1 but not cyclin B1 expression. SO-OCS accumulated cells in G2/M in both cell lines and this blockade was accompanied by a decrease of CDK1, but not cyclin B1 expression. Furthermore, both drugs induced apoptosis as demonstrated by the increased percentage of annexin V positive cells and Bax/Bcl-2 ratio. Interestingly, in normal MCF-10A cells the two drugs failed to modify cell proliferation, cell cycle progression, cyclins, or CDKs expression. These results demonstrate that the effect of CO-OCS and SO-OCS is triggered by both cell cycle arrest and apoptosis, suggesting that these castanospermine analogues may constitute potential anti-cancer agents against breast cancer.     

Down-Regulation of NDUFB9 Promotes Breast Cancer Cell Proliferation,Metastasis by Mediating Mitochondrial Metabolism

Despite advances in basic and clinical research, metastasis remains the leading cause of death in breast cancer patients. Genetic abnormalities in mitochondria, including mutations affecting complex I and oxidative phosphorylation, are found in breast cancers and might facilitate metastasis. Genes encoding complex I components have significant breast cancer prognostic value. In this study, we used quantitative proteomic analyses to compare a highly metastatic cancer cell line and a parental breast cancer cell line; and observed that NDUFB9, an accessory subunit of the mitochondrial membrane respiratory chain NADH dehydrogenase (complex I), was down-regulated in highly metastatic breast cancer cells. Furthermore, we demonstrated that loss of NDUFB9 promotes MDA-MB-231 cells proliferation, migration, and invasion because of elevated levels of mtROS, disturbance of the NAD⁺/NADH balance, and depletion of mtDNA. We also showed that, the Akt/mTOR/p70S6K signaling pathway and EMT might be involved in this mechanism. Thus, our findings contribute novel data to support the hypothesis that misregulation of mitochondrial complex I NADH dehydrogenase activity can profoundly enhance the aggressiveness of human breast cancer cells, suggesting that complex I deficiency is a potential and important biomarker for further basic research or clinical application.     

Different anticancer effects of Saxifragifolin A on estrogen receptor-positive and estrogen receptor-negative breast cancer cells

BackgroundBreast cancer is the leading cause of cancer-related death among women worldwide. For treating breast cancer, numerous natural products have been considered as chemotherapeutic drugs.Hypothesis/purposeThe present study aims to investigate the apoptotic effect of Saxifragifolin A (Saxi A) isolated from Androsace umbellata in two different human breast cancer cells which are ER-positive MCF-7 cells and ER-negative MDA-MB-231 cells, and examine the molecular basis for its anticancer actions.Study designThe inhibitory effects of Saxi A on cell survival were examined in MCF-7 cells and MDA-MB-231 cells in vitro.MethodsMTT assays, Annexin V/PI staining analysis, ROS production assay, Hoechst33342 staining and Western blot analysis were performed.ResultsOur results showed that MDA-MB-231 cells were more sensitive to Saxi A-induced apoptosis than MCF-7 cells. Saxi A induced apoptosis in MDA-MB-231 cells through ROS-mediated and caspase-dependent pathways, whereas treatment with Saxi A induced apoptosis in MCF-7 cells in a caspase-independent manner. In spite of Saxi A-induced activation of MAPKs in both breast cancer cell lines, only p38 MAPK and JNK mediated Saxi A-induced apoptosis. In addition, cell survival of shERα-transfected MCF-7 cells was decreased, while MDA-MB-231 cells that overexpress ERα remained viable.ConclusionSaxi A inhibits cell survival in MCF-7 cells and MDA-MB-231 cells through different regulatory pathway, and ERα status appears to be important for regulating Saxi A-induced apoptosis in breast cancer cells. Thus, Saxi A may have a potential therapeutic use for treating breast cancer.     

Anticancer effect of tert-butyl-2(4,5-dihydrogen-4,4,5,5-tetramethyl-3-O-1H-imidazole-3-cationic-1-oxyl-2)-pyrrolidine-1-carboxylic ester on human hepatoma HepG2 cell line

Tert-butyl-2(4,5-dihydrogen-4,4,5,5-tetramethyl-3-O-1H-imidazole-3-cationic-1-oxyl-2)-pyrrolidine-1-carboxylic ester (L-NNP) is a stable nitroxyl nitroxide radical, which have displayed cytotoxicity on human breast cancer MCF-7 and MDA-MB-231 cell lines. In the present study, we investigated the selective cytotoxicity of L-NNP on isogenetic human hepatoma HepG2 and normal L-02 cell lines. Cell growth inhibition, intracellular reactive oxygen species production, the mitochondrial membrane potential loss, malondialdehyde generation and glutathione levels were analyzed. The expression of Bax, Bcl-2 and NF-κBp65 proteins was also examined. The anticancer activity was evaluated in a HepG2 cell xenograft nude mice model. The results showed that 10, 20, 40 μg/ml L-NNP exposure for 48 h caused 52%, 82% and 91% cell growth inhibition of HepG2 cells, compared with 5%, 10% and 15% that of L-02 cells (p < 0.01). Concentrations of 10, 20, 40 μg/ml L-NNP induced cell death by increasing the generation of intracellular reactive oxygen species and MDA, by depolarizing the mitochondrial membrane potential, and by decreasing intracellular GSH levels in HepG2 cells. Western blot assay showed that Bax, Bcl-2 and NF-κBp65 might be implicated in L-NNP-induced selective HepG2 cell death. L-NNP was also found to inhibit HepG2 hepatoma growth and extend the life span of nude mice model (p < 0.01). The pretreatment and co-treatment of 10 mM N-acetyl-cysteine alleviated L-NNP exposure induced intracellular reactive oxygen species increase and cell growth inhibition demonstrated that L-NNP exhibited neoplasm-selective cytotoxicity and pro-apoptotic activities via reactive oxygen species mediated oxidative damage in HepG2 cells. It might be promising for developing a new class of anticancer agent for liver cancer.     

Suppression of growth of highly-metastatic human breast cancer cells by norcantharidin and its mechanisms of action

The effects of norcantharidin (NCTD) on the growth of highly-metastatic human breast cancer cells were investigated by in vitro and ex vivo assays. Our results indicated that norcantharidin inhibited the in vitro growth of human breast cancer MDA-MB-231 cell line in dose- and time-dependent manners after the cancer cells were treated with norcantharidin at the concentrations of 6, 30 and 60 μmol/L for 24, 48 and 72 h. Moreover, the sera from the NCTD-treated rabbits after intravenous injection of NCTD at 15 and 30 min significantly suppressed the growth of the cancer cells ex vivo. The analyses by Hoechst 33258 staining and flow cytometry showed that the typical apoptotic morphological changes appeared and cell cycles arrested at G2/M phase in MDA-MB-231 cells after the cells were treated for 48 h with NCTD. In addition, NCTD down-regulated the expressions of anti-apoptotic protein Bcl-2 and up-regulated the expressions of pro-apoptotic protein Bax, eventually leading to the reduction of Bcl-2/Bax ratio in MDA-MB-231 cells. Furthermore, NCTD at concentrations of 6, 30 and 60 μmol/L dose-dependently reduced the phosphorylation of Akt and NF-κB expression in the breast cancer cell line. Induction of apoptosis and cell cycle arrest as well as reduction of Bcl-2/Bax ratio by NCTD may be the important mechanisms of action of NCTD suppressing the growth of MDA-MB-231 cells, which are associated with inhibition of the Akt and NF-κB signaling. Our findings suggest that norcantharidin may have a wide therapeutic and/or adjuvant therapeutic application in the treatment of human breast cancer.