Rational combinations of siRNAs targeting Plk1 with breast cancer drugs

Commonly used drugs for the treatment of breast cancer patients like paclitaxel and Herceptin often show severe side effects or induce resistance in clinical settings. Thus, we analysed a combination of Plk1 (polo-like kinase 1)-specific small interfering RNAs (siRNAs), a powerful tool to induce 'mitotic catastrophe' in cancer cells, together with these drugs to identify conditions for enhanced drug sensitivity. After transfection, the antineoplastic agents were added and cell proliferation, apoptosis and cell cycle distribution in breast cancer cells (MCF-7, SK-BR-3, MDA-MB-435 and BT-474) and in primary human mammary epithelial cells were determined. Downregulation of cellular Plk1 levels led to an elevated percentage of cells in G(2)/M phase. The percentage of apoptotic nuclei in MCF-7, MDA-MB-435, SK-BR-3 and BT-474 cells was clearly increased after incubation with Plk1-specific siRNAs and paclitaxel. Interestingly, the caspase pathway was activated after treatment with Plk1-specific siRNAs and paclitaxel or Herceptin. Treatment of breast cancer cells with siRNAs targeting Plk1 improved the sensitivity toward paclitaxel and Herceptin in a synergistic manner. In all experiments, very low concentrations across a wide range of clinically relevant concentrations were sufficient to induce an antiproliferative effect. The combination of Plk1-specific siRNAs with modern breast cancer drugs seems to represent rational combinations to be tested in preclinical trials.     

Enhanced sensitization to taxol-induced apoptosis by herceptin pretreatment in ErbB2-overexpressing breast cancer cells

The recombinant humanized anti-ErbB2/HER2 monoclonal antibody Herceptin (Trastuzumab) has been shown to significantly enhance the tumoricidaleffects of antitumor drugs such as paclitaxel (Taxol) in patients with ErbB2-overexpressing breast cancers. Here, we investigated the molecular mechanisms by which Herceptin enhances the antitumor effects of Taxol. Because activation of p34(Cdc2) is required for Taxol-induced apoptosis and because overexpression of ErbB2 blocks Taxol-induced apoptosis by inhibiting p34(Cdc2) activation, we studied the effect of Herceptin treatment on p34(Cdc2) kinase activation and apoptosis in Taxol-treated human breast carcinoma cell lines MDA-MB-435, SKBr3, MDA-MB-453, and 435.eB, which is an ErbB2 transfectant of MDA-MB-435. Herceptin treatment down-regulated ErbB2, reduced the inhibitory phosphorylation of Cdc2 on Tyr-15, and down-regulated the expression of p21(Cip1), a Cdc2 inhibitor. Herceptin plus Taxol treatment led to higher levels of p34(Cdc2) kinase activity and apoptosis in ErbB2-overexpressing breast cancer cells, which is likely attributable to inhibition of Cdc2-Tyr-15 phosphorylation and p21(Cip1) expression. Because significant dephosphorylation of Cdc2-Tyr-15 and down-regulation of p21(Cip1) occur at least 24 h after Herceptin treatment, we investigated whether 24 h Herceptin pretreatment will render ErbB2-overexpressing breast cancer cells more sensitive to Taxol-induced apoptosis compared with the simultaneous treatment of Herceptin plus Taxol. Indeed, Herceptin pretreatment increased Taxol-induced apoptosis and cytotoxicity in vitro and more effectively inhibited the growth of tumor xenografts with enhanced in vivo apoptosis. Thus, Herceptin treatment of ErbB2-overexpressing cells can inhibit ErbB2-mediated Cdc2-Tyr-15 phosphorylation and p21(Cip1) up-regulation, which allows effective p34(Cdc2) activation and induction of apoptosis upon Taxol treatment. Herceptin pretreatment renders ErbB2-overexpressing breast cancers more susceptible to Taxol-induced cell death, which may have important clinical therapeutic implications.     

Vitamin E succinate (VES) induces Fas sensitivity in human breast cancer cells: role for Mr 43,000 Fas in VES-triggered apoptosis

Fas (CD95/APO-1) is an important mediator of apoptosis. We show that Fas-resistant MCF-7, MDA-MB-231, and MDA-MB-435 human breast cancer cells become responsive to anti-Fas (CD95) agonistic antibody-triggered apoptosis after pretreatment or cotreatment with vitamin E succinate (VES; RRR-alpha-tocopheryl succinate). In contrast, no enhancement of anti-Fas agonistic antibody-triggered apoptosis was observed following VES pretreatment or cotreatment with Fas-sensitive primary cultures of human mammary epithelial cells, immortalized MCF-10A cells, or T47D human breast cancer cells. Although VES is itself a potent apoptotic triggering agent, the 6-h pretreatment procedure for Fas sensitization did not initiate VES-mediated apoptosis. The combination of VES plus anti-Fas in pretreatment protocols was synergistic, inducing 2.8-, 3.0-, and 6.3-fold enhanced apoptosis in Fas-resistant MCF-7, MDA-MB-231, and MDA-MB-435 cells, respectively. Likewise, cotreatment of Fas-resistant MCF-7, MDA-MB-231, and MDA-MB-435 cells with VES plus anti-Fas enhanced apoptosis 1.9-, 2.0-, and 2.6-fold, respectively. Functional knockout of Fas-mediated signaling with either Fas-neutralizing antibody (MCF-7-, MDA-MB-231-, and MDA-MB-435-treated cells) or Fas antisense oligomers (MDA-MB-435-treated cells only), reduced VES-triggered apoptosis by approximately 50%. Analyses of whole cell extracts from Fas-sensitive cells revealed high constitutive expression of Mr 43,000 Fas, whereas Fas-resistant cells expressed low levels that were confined to the cytosolic fraction. VES treatment of the Fas-resistant cells caused a depletion of cytosolic Mr 43,000 Fas with a concomitant increase in Mr 43,000 membrane Fas. These data show that VES can convert Fas-resistant human breast cancer cells to a Fas-sensitive phenotype, perhaps by translocation of cytosolic Mr 43,000 Fas to the membrane and show that VES-mediated apoptosis involves Mr 43,000 Fas signaling.     

Paclitaxel induces apoptosis via protein kinase A- and p38 mitogen-activated protein-dependent inhibition of the Na+/H+ exchanger (NHE) NHE isoform 1 in human breast cancer cells.

PURPOSE: The molecular signal components essential to paclitaxel-dependent apoptosis in breast cancers are potential targets for combined therapy. However, the signal mechanisms underlying paclitaxel action still need to be better defined. EXPERIMENTAL DESIGN: In a breast cancer cell line, pharmacological agents and transient transfection with dominant interfering and constitutive active mutants were used to identify the signal transduction module involved in the regulation of paclitaxel-induced apoptosis and to evaluate its potential as a therapeutic target. RESULTS: In MDA-MB-435 cells, paclitaxel treatment stimulated the activity of both protein kinase A and p38, and inhibited the activity of the Na(+)/H(+) exchanger isoform 1 (NHE1) with similar IC(50) concentrations as for its activation of apoptosis. Activation and inhibition experiments demonstrated that protein kinase A and p38 participate sequentially upstream of the NHE1 in regulating the paclitaxel-induced apoptotic pathway. Importantly, concurrent specific inhibition of the NHE1 with paclitaxel treatment resulted in a synergistic induction of apoptosis and a reduction in the paclitaxel IC(50) for apoptosis. This sensitization of paclitaxel apoptotic action by specific inhibition of NHE1 was verified in breast cancer cell lines with different paclitaxel sensitivity. CONCLUSIONS: We have, for the first time, identified NHE1 as an essential component of paclitaxel-induced apoptosis in breast cancer cells and, importantly, identified that simultaneous inhibition of the NHE1 results in a synergistic potentiation of low-dose paclitaxel apoptotic action. As specific NHE1 inhibitors have finished Phase II/Phase III clinical trials for myocardial protection, there is the possibility for a rapid biological translation of this novel therapeutic strategy to a clinical setting.     

Downregulation of Plk1 Expression By Receptor-Mediated Uptake of Antisense Oligonucleotide-Loaded Nanoparticles

Human serum albumin (HSA) nanoparticles represent a promising tool for targeted drug delivery to tumor cells. The coupling of the antibody trastuzumab to nanoparticles uses the capability of human epidermal growth factor receptor 2 (HER2)-positive cells to incorporate agents linked to HER2. In our present study, we developed targeted nanoparticles loaded with antisense oligonucleotides (ASOs) against polo-like kinase 1 (Plk1). We evaluated the receptor-mediated uptake into HER2-positive and -negative breast cancer and murine cell lines. We performed quantitative real-time PCR and Western blot analyses to monitor the impact on Plk1 expression in HER2-positive breast cancer cells. Antibody-conjugated nanoparticles showed a specific targeting to HER2-overexpressing cells with cellular uptake by receptor-mediated endocytosis and a release into HER2-positive BT-474 cells. We observed a significant reduction of Plk1 mRNA and protein expression and increased activation of Caspase 3/7. Thus, this is the first report about ASO-loaded HSA nanoparticles, where an impact on gene expression could be observed. The data provide the basis for the further development of carrier systems for Plk1-specific ASOs to reduce off-target effects evoked by systemically administered ASOs and to achieve a better penetration into primary and metastatic target cells. Treatment of tumors using trastuzumab-conjugated ASO-loaded HSA nanoparticles could be a promising approach to reach this goal.     

Contribution of reactive oxygen species and caspase-3 to apoptosis and attenuated ICAM-1 expression by paclitaxel-treated MDA-MB-435 breast carcinoma cells

Paclitaxel is a microtubule-stabilizing and apoptosis-inducing drug that is commonly used to treat metastatic breast cancer, although the mechanism of paclitaxel-induced apoptosis remains incompletely understood. Furthermore, adhesion molecule expression is attenuated on mouse mastocytoma and human leukemia cells that survive short-term culture in the presence of paclitaxel. In the present study we show that MDA-MB-435 human breast carcinoma cells that survived culture for 72 h in the presence of submaximal cytotoxic concentrations of paclitaxel (0.02 and 0.01 microg/ml) showed decreased expression of the adhesion molecule ICAM-1. Paclitaxel treatment of MDA-MB-435 cells was associated with the generation of reactive oxygen species (ROS), dissipation of mitochondrial transmembrane potential, and the activation of caspase-3. The antioxidant glutathione protected MDA-MB-435 cells from paclitaxel-induced cytotoxicity and reduced ICAM-1 expression. In addition, a selective inhibitor of caspase-3 (Z-DEVD-FMK), as well as a pan-caspase inhibitor (Z-VAD-FMK), partially prevented the decrease in ICAM-1 expression observed following paclitaxel treatment, but did not protect against paclitaxel-induced cytotoxicity. We conclude that the paclitaxel-induced reduction in ICAM-1 expression by MDA-MB-435 breast carcinoma cells is both ROS- and caspase-dependent, whereas paclitaxel-induced cytotoxicity is ROS-dependent and does not involve caspases. Decreased ICAM-1 expression by breast carcinoma cells that survive paclitaxel treatment may negatively impact on cytotoxic lymphocyte-mediated destruction of paclitaxel-resistant breast cancer cells in the context of chemo-immunotherapy or chemo-adoptive immunotherapy.     

Downregulation of human polo-like kinase activity by antisense oligonucleotides induces growth inhibition in cancer cells

A central role for polo-like kinases (PLK) in regulating several stages of mitotic progression has been born out in several species. Overexpression of PLK1 is observed in the majority of hitherto analysed human tumors. PLK1 overexpression is a negative prognostic factor in patients suffering from non-small cell lung cancer, head and neck tumors, esophageal carcinomas and melanomas. In order to define the role of PLK1 for mitotic progression of human cells and for neoplastic cell growth, phosphorothioate antisense oligonucleotides (ASOs) were tested to selectively downregulate PLK1 expression in MDA-MB-435 (breast cancer), HeLa S3 (cervical carcinoma) and A549 (non-small cell lung cancer) cells. ASOs were identified which suppress PLK1 mRNA and protein in a dose-dependent and sequence-specific manner. This approach also led to reduced PLK1 serine/threonine kinase activity. Downregulation of cellular PLK1 levels in cancer cells altered cell cycle progression moderately with an elevated percentage (20-30%) of cells in G(2)/M. Furthermore, cells with reduced PLK1 protein gained a rounded phenotype with multiple centrosomes. Moreover, ASO treatment resulted in potent antiproliferative effects in cell culture. Considerable antitumor activity was observed in vivo against A549 cells. This study suggests that antisense inhibitors targeted against PLK1 at well tolerated doses may be considered as a cancer therapeutic agent.     

In vitro exposure to paclitaxel modulates integrin expression by human T lymphocytes and inhibits T cell adhesion to breast carcinoma cells

Paclitaxel (Taxol) is a drug that is commonly used in the treatment of metastatic breast cancer. In this study, we investigated the effect of prior exposure to submaximal cytotoxic concentrations (EC(25) and EC(50)) of paclitaxel on the subsequent ability of human Jurkat T lymphocytes to adhere to monolayers of MDA-MB-435 human breast carcinoma cells. Jurkat T cells that survived culture for 24 h in the presence of paclitaxel (0.1 or 3 micro g/ml) exhibited a diminished ability to adhere to MDA-MB-435 breast cancer cell monolayers. Flow cytometric analysis of paclitaxel-treated Jurkat T cells revealed reduced surface expression of alphaL, alpha4, alpha5, and beta7 integrins, but normal beta1 integrin expression. These data suggest that paclitaxel interferes with the expression of alphaLbeta2 (LFA-1), alpha4beta1 (VLA-4), alpha5beta1 (VLA-5), and alpha4beta7 (LPAM-1) adhesion molecules by surviving T cells. Blocking experiments with antibodies against alphaL, alpha4, alpha5, beta1, and beta7 integrins revealed that adhesion of Jurkat T cells to MDA-MB-435 breast cancer cell monolayers was dependent upon alphaL, alpha4, and beta7 but not alpha5 or beta1 integrins. We conclude that prior exposure to paclitaxel caused a reduction in Jurkat T cell adhesion to MDA-MB-435 breast carcinoma cells by preventing optimal alphaLbeta2 and alpha4beta7 interactions with their corresponding ligands on tumor cells. A similar effect by paclitaxel on T lymphocytes of breast cancer patients may compromise cell-mediated anti-tumor immune responses.     

Bcl-2/bcl-xL bispecific antisense treatment sensitizes breast carcinoma cells to doxorubicin,paclitaxel and cyclophosphamide

Overexpression of the anti-apoptotic proteins bcl-2 and bcl-xL is implicated in breast cancer development, tumor progression and drug resistance. Here we describe the use of the bcl-2/bcl-xL bispecific antisense oligonucleotide 4625 to sensitize breast carcinoma cells to anti-cancer drugs routinely used in breast cancer therapy. MCF7 cells were treated with oligonucleotide 4625, doxorubicin, paclitaxel or cyclophosphamide alone, or with combinations of oligonucleotide and the anti-cancer drugs. As measured in cell viability assays, treatment with the various combinations reduced the number of viable MCF7 cells more effectively than treatment with the single drugs alone. Treatment with a sequence control oligonucleotide did not affect cell viability. All combination treatments induced apoptosis as demonstrated by the appearance of massive nuclear condensation in a high proportion of the cells. To further characterize the interaction between 4625 and doxorubicin, paclitaxel or cyclophosphamide, the median-effect method was used. In MCF7 cells all combinations resulted in potent synergistic effects over a broad range of toxicity with combination indices ranging from 0.8 to 0.1. Similarly, strong synergistic interactions between oligonucleotide 4625 and the anti-cancer drugs were also observed in cultures of the breast carcinoma cell line MDA-MB-231. Our data suggest the use of 4625 as a potent adjuvant in breast cancer chemotherapy.     

Synergism between the anticancer actions of 2-methoxyestradiol and microtubule-disrupting agents in human breast cancer

2-Methoxyestradiol (2-MeO-E(2)), a well-known nonpolar endogenous metabolite of 17beta-estradiol, has strong antiproliferative, apoptotic, and antiangiogenic actions in vitro and in vivo at pharmacologic concentrations. We determined in the present study whether 2-MeO-E(2) can enhance the anticancer actions of paclitaxel or vinorelbine (two commonly used microtubule-disrupting agents) in several human breast cancer cell lines, including the estrogen receptor-positive MCF-7 and T-47D cells and the receptor-negative MDA-MB-435s and MDA-MB-231 cells. 2-MeO-E(2) in combination with paclitaxel or vinorelbine exhibited a synergistic anticancer effect in these human breast cancer cells in vitro, and this synergistic effect was more pronounced when each of the drugs was used at relatively low concentrations. Additional experiments using female athymic BALB/c nu/nu mice showed that p.o. administration of 2-MeO-E(2) at 30 mg/kg body weight, once a week for 6 weeks, markedly enhanced the activity of paclitaxel or vinorelbine against the growth of the estrogen receptor-negative MDA-MB-231 human breast cancer xenografts in these animals. By contrast, combination of 2-MeO-E(2) with 5-fluorouracil only had a partial additive effect against the growth of these cell lines in culture, and no synergistic effect was observed. Interestingly, when doxorubicin was used in combination with 2-MeO-E(2), the antiproliferative effect of 2-MeO-E(2) was somewhat antagonized by doxorubicin when it was present at high concentrations. Our results showed that 2-MeO-E(2) at nontoxic or subtoxic doses selectively enhanced the effects of certain microtubule-disrupting agents (such as paclitaxel and vinorelbine) against the growth of the receptor-negative human breast cancer cells in culture and also in athymic nude mice.     

Lactoferricin-induced apoptosis in estrogen-nonresponsive MDA-MB-435 breast cancer cells is enhanced by C6 ceramide or tamoxifen

Bovine lactoferricin (LfcinB) is a cationic peptide that selectively induces caspase-dependent apoptosis in human leukemia and carcinoma cell lines. Ceramide is a second messenger in apoptosis signaling that has been shown to increase the cytotoxicity of various anti-cancer drugs. In this study, we determined whether manipulation of intracellular ceramide levels enhanced LfcinB-induced apoptosis of estrogen-nonresponsive MDA-MB-435 breast carcinoma cells. LfcinB caused DNA fragmentation and morphological changes consistent with apoptosis in MDA-MB-435 breast cancer cell cultures, but did not affect the viability of untransformed mammary epithelial cells. MDA-MB-435 breast cancer cells also exhibited DNA fragmentation and morphological changes consistent with apoptosis following exposure to the cell-permeable ceramide analog C6. An additive increase in DNA fragmentation was observed when both LfcinB and C6 ceramide were added to MDA-MB-435 breast cancer cell cultures. A greater than additive increase in DNA fragmentation was seen when LfcinB was used in combination with tamoxifen, which prevents the metabolism of endogenous ceramide to glucosylceramide by glucosylceramide synthase, as well as blocking estrogen receptor signaling. However, a selective inhibitor of glucosylceramide synthase,1-phenyl-2-palmitoylamino-3-morpholino-1-propanol, failed to further increase DNA fragmentation by LfcinB, suggesting that tamoxifen enhanced LfcinB-induced apoptosis in breast cancer cells via a mechanism that did not involve glucosylceramide synthase inhibition. We conclude that combination therapy with LfcinB and tamoxifen warrants further investigation for possible use in the treatment of breast cancer.     

反义硫代寡核苷酸增强细胞毒药物抗乳腺癌活性的体外研究

目的研究以HER2mRNA为靶点的反义硫代脱氧寡核苷酸(SODNs)HA6722单用及与紫杉醇合用时对HER2过表达乳腺癌细胞株MDAMB453体外增殖的抑制作用。方法选择HER2过表达的MDAMB453细胞与HER2低表达的MDAMB231细胞,MTT法观察HA6722单用及与紫杉醇合用时对两种肿瘤细胞增殖的影响;以末端转移酶介导的dUTP切口末端标记法(TUNEL)检测细胞凋亡。结果HA6722及紫杉醇单用均可以剂量依赖方式抑制MDAMB453细胞的体外增殖,IC50值分别为47.6±7.9nmol·L1(n=3,mean±s)和19.4±4.1nmol·L-1(n=3,mean±s)。加用低剂量的HA6722可增强紫杉醇对MDAMB453细胞的抑制作用,IC50值由合用前的19.4±4.1nmol·L-1降至13.6±5.2nmol·L-1(n=3,P<0.05)。联合应用时,在IC50浓度下二者之间的联合指数(CI)为0.81±0.43(n=3),其对MDAMB453细胞的相互作用表现为正性协同作用。结论反义寡核苷酸HA6722与细胞毒药物紫杉醇合用对HER2过表达乳腺癌细胞的体外增殖抑制具有协同作用...     

Induction of apoptosis and inhibition of c-erbB-2 in MDA-MB-435 cells by genistein.

Breast cancer is the most common cancer and second leading cause of cancer related deaths in women in the United States. Genistein is a protein tyrosine kinase inhibitor and prominent isoflavonoid in soy products and has been proposed as the agent responsible for lowering the rate of breast cancer in Asian women. We have previously shown that genistein inhibits the growth of MDA-MB-231 breast cancer cells, regulates the expression of apoptosis-related genes, and induces apoptosis through a p53-independent pathway. In this study, we investigated these effects of genistein in the breast cancer cell line MDA-MB-435 and 435.eB cells that were established by transfecting c-erbB-2 cDNA into MDA-MB-435. We also investigated the effect of genistein on matrix metalloproteinase (MMP) secretion previously shown to be effected by erbB-2 transfection. Genistein was found to inhibit MDA-MB-435 and 435.eB cell growth. Induction of apoptosis was also observed in these cell lines when treated with genistein, as measured by DNA laddering, poly(ADP-ribose) polymerase (PARP) cleavage, and flow cytometric analysis. We also found an up-regulation of Bax and p21WAF1 expression and down-regulation of Bcl-2 and c-erbB-2 in genistein-treated cells. Gelatin zymography showed that genistein inhibits the secretion of MMP in the breast cancer cells. From these results, we conclude that genistein inhibits the growth of MDA-MB-435 breast cancer cells, induces apoptosis, regulates the expression of genes, and may inhibit invasion and metastasis of breast cancer cells. These findings suggest that genistein may be a potentially effective chemopreventive or therapeutic agent against breast cancer.     

Inhibitory effects of the combination of HER-2 antisense oligonucleotide and chemotherapeutic agents used for the treatment of human breast cancer.

Poor response to chemotherapy in patients with breast cancer is often associated with overexpression of HER-2/neu. Interference with HER-2 mRNA translation by means of antisense oligonucleotides might improve the efficacy of chemotherapy. To test this hypothesis, eight breast cancer cell lines and a normal human fibroblast cell line were examined for their level of HER-2 expression, their sensitivity to phosphorothioate antisense oligonucleotides (AS HER-2 ODN), and to various chemotherapeutic agents, and the combination of the two. No correlation was found between the intrinsic HER-2 level and either the sensitivity to a particular chemotherapeutic agent alone, or the amount of growth inhibition observed with a specific AS HER-2 ODN concentration. Although sequence specificity and extent of AS HER-2 ODN inhibition of HER-2 synthesis were somewhat higher in the HER-2 overexpressing MDA-MB-453 and SK-BR-3 cells, we found that antisense treatment significantly sensitized all of the breast cancer cells, even MDA-MB-231 and MDA-MB-435 cells, with approximately basal levels of HER-2, to various chemotherapeutic agents. In addition, the combination of AS HER-2 ODN and taxol was shown to synergistically induce apoptosis in MDA-MB-435. These results demonstrate that overexpression of HER-2 would not be a prerequisite for the effective use of AS HER-2 ODN as a combination treatment modality for breast cancer and suggest that the use of AS HER-2 ODN, as part of a combination treatment modality, need not be limited to breast tumors that display elevated levels of HER-2.     

A new superinvasive in vitro phenotype induced by selection of human breast carcinoma cells with the chemotherapeutic drugs paclitaxel and doxorubicin

Doxorubicin- and paclitaxel-selected variants of an in vitro invasive clonal population of the human breast cancer cell line, MDA-MB-435S, were established by pulse selection, and exhibited a novel 'superinvasive' phenotype. This phenotype is characterised by an ability to relocate to another surface following invasion through matrigel and membrane pores, by decreased adhesion to extracellular matrix proteins and by increased motility. This may represent an in vitro model of a step in the metastatic process occurring subsequent to invasion. The paclitaxel-resistant variants, MDA-MB-435S-F/Taxol-10p and MDA-MB-435S-F/Taxol-10p4p were resistant to paclitaxel, vincristine and docetaxel, but not to doxorubicin, carboplatin, etoposide or 5-fluorouracil. The doxorubicin-selected variants MDA-MB-435S-F/Adr-10p and MDA-MB-435S-F/Adr-10p10p, in contrast, exhibited only small increases in resistance to doxorubicin, although they were slightly resistant to VP-16 and docetaxel, and exhibited increased sensitivity to paclitaxel, carboplatin and 5-fluorouracil.     

Integrin signaling inhibits paclitaxel-induced apoptosis in breast cancer cells

Inherent or acquired drug resistance is one of the major problems in chemotherapy. The mechanisms by which cancer cells survive and escape the cytotoxic effects of chemotherapeutic agents are essentially unknown. In the present study, we demonstrate that in the MDA-MB-231 and MDA-MB-435 breast cancer cells, ligation of beta1 integrins by their extracellular matrix ligands inhibits significantly apoptosis induced by paclitaxel and vincristine, two microtubule-directed chemotherapeutic agents that are widely used in the therapy of breast cancer. We show that beta1 integrin signaling inhibits drug-induced apoptosis by inhibiting the release of cytochrome c from the mitochondria in response to drug treatment. Further, integrin-mediated protection from drug-induced apoptosis and inhibition of cytochrome c release are dependent on the activation of the PI 3-kinase/Akt pathway. Our results identify beta1 integrin signaling as an important survival pathway in drug-induced apoptosis in breast cancer cells and suggest that activation of this pathway may contribute to the generation of drug resistance.     

Paclitaxel sensitivity of breast cancer cells requires efficient mitotic arrest and disruption of Bcl-xL/Bak interaction

Taxanes are being used for the treatment of breast cancer. However, cancer cells frequently develop resistance to these drugs with the subsequent recurrence of the tumor. MDA-MB-231 and T-47D breast cancer cell lines were used to assess the effect of paclitaxel treatment on apoptosis and cell cycle, the possible mechanisms of paclitaxel resistance as well as the enhancement of paclitaxel-induced apoptosis based on its combination with phenylethyl isothiocyanate (PEITC). T-47D cells undergo apoptosis in response to paclitaxel treatment. The induction of apoptosis was associated with a robust mitotic arrest and the disruption of Bcl-xL/Bak interaction. By contrary, MDA-MB-231 cells were insensitive to paclitaxel-induced apoptosis and this was associated with a high percentage of cells that slip out of paclitaxel-imposed mitotic arrest and also with the maintenance of Bcl-xL/Bak interaction. The sequential treatment of MDA-MB-231 cells with PEITC followed by paclitaxel inhibited the slippage induced by paclitaxel and increased the apoptosis induction achieved with any of the drugs alone. In breast cancer tissues, high Bcl-xL expression was correlated with a shorter time of disease-free survival in patients treated with a chemotherapeutic regimen that contains paclitaxel, in a statistically significant way. Thus, resistance to paclitaxel in MDA-MB-231 cells is related to the inability to disrupt the Bcl-xL/Bak interaction and increased slippage. In this context, the combination of a drug that induces a strong mitotic arrest, such as paclitaxel, with another that inhibits slippage, such as PEITC, translates into increased apoptotic induction.     

槲皮素对人乳腺癌细胞MDA-MB-435S增殖及侵袭力的影响

 目的 探讨槲皮素对人乳腺癌细胞MDA-MB-435S 增殖及侵袭能力的影响。 方法 人乳腺癌细胞MDA-MB-435S以12.5、25、50、100、200μM 终浓度的槲皮素处理后,台盼蓝拒染法检测细胞的增殖状况、流式细胞术法检测细胞的凋亡率、Boyden小室法检测细胞的侵袭能力。 结果 人乳腺癌细胞MDA-M-435S经槲皮素处理后,其体外增殖及侵袭能力明显下降的同时凋亡率明显上升,且与药物的剂量呈正相关,当槲皮素终浓度达到200μM时,MDA-MB-435S细胞的生长及侵袭能力几乎完全受到抑制。 结论 槲皮素在体外剂量依赖性的抑制人乳腺癌细胞MDA-MB-435S的增殖及侵袭能力并能诱导其凋亡,为槲皮素用于乳腺癌的预防和治疗提供了部分依据。     

Riluzole synergizes with paclitaxel to inhibit cell growth and induce apoptosis in triple-negative breast cancer

Purpose

One in eight women will develop breast cancer, 15–20% of whom will have triple-negative breast cancer (TNBC), an aggressive breast cancer with no current targeted therapy. We have demonstrated that riluzole, an FDA-approved drug for treating amyotrophic lateral sclerosis, inhibits growth of TNBC. In this study, we explore potential synergism between riluzole and paclitaxel, a chemotherapeutic agent commonly used to treat TNBC, in regulating TNBC proliferation, cell cycle arrest, and apoptosis.

Methods

TNBC cells were treated with paclitaxel and/or riluzole and synergistic effects on cell proliferation were quantified via MTT assay and CompuSyn analysis. Apoptosis was observed morphologically and by measuring cleaved PARP/caspase three products. Microarray analysis was performed using MDA-MB-231 cells to examine cell cycle genes regulated by riluzole and any enhanced effects on paclitaxel-mediated cell cycle arrest, determined by FACS analysis. These results were confirmed in vivo using a MDA-MB-231 xenograft model.

Results

Strong enhanced or synergistic effects of riluzole on paclitaxel regulation of cell cycle progression and apoptosis was demonstrated in all TNBC cells tested as well as in the xenograft model. The MDA-MB-231, SUM149, and SUM229 cells, which are resistant to paclitaxel treatment, demonstrated the strongest synergistic or enhanced effect. Key protein kinases were shown to be upregulated in this study by riluzole as well as downstream cell cycle genes regulated by these kinases.

Conclusions

All TNBC cells tested responded synergistically to riluzole and paclitaxel strongly suggesting the usefulness of this combinatorial treatment strategy in TNBC, especially for patients whose tumors are relatively resistant to paclitaxel.

     

Mechanisms of the synergistic interaction between the bisphosphonate zoledronic acid and the chemotherapy agent paclitaxel in breast cancer cells in vitro.

Breast cancer patients often receive both paclitaxel and zoledronic acid as part of their treatment, and these drugs are reported to have synergistic effects on the induction of apoptosis of breast cancer cells in vitro. We have found that the synergistic interaction is drug sequence dependent, with maximal levels of apoptosis achieved when cells are treated with paclitaxel followed by zoledronic acid, as opposed to the reverse sequence or simultaneous treatment. The synergistic interaction persists at clinically relevant concentrations and incubation periods. We report that the sequential treatment is associated with cell cycle changes and depends on breast cancer cell characteristics, with hormone independence, mutated p53 status and presence of BRCA1 gene being associated with higher levels of apoptosis. Finally, we have found that the synergistic induction of apoptosis is via zoledronic acid-mediated inhibition of the mevalonate pathway.