| IntroductionThe metastatic breast cancer, which is seen in 30% to 40% of all patients with breast cancer, is one of the most aggressive human malignancies with an extremely low 5-year overall survival. It is characterized by a rapid disease progression without specific symptoms, commonly precluding early diagnosis and curative treatment. These patients commonly present in an advanced stage with only a minority being amenable to surgical intervention. Due to its high recurrence rate, chemotherapy is an essential adjunct for the metastatic breast cancer treatment. The taxane-based therapy is now the standard treatment for the metastatic breast cancer, especially for patients in whom anthracyclines have failed. Clinical studies have revealed that single-agent docetaxel treatment significantly improved overall survival, superior time to disease progression and response rates, as compared with a mitomycin plus vinblastine combination therapy. Further, although paclitaxel(Taxol) and docetaxel are both approved for the metastatic breast cancer treatment, docetaxel showed a better response in terms of overall survival and time to disease progression. Recently, it has been reviewed that taxane-based combination regimens could further improve tumor response and time to disease progression verse single-agent taxane chemotherapy. For example, docetaxel-capecitabine was superior to docetaxel in efficiency, although non-hematologic toxicities were also higher. Further, study by Albain et al., showed that gemcitabine plus paclitaxel regimen had a better efficacy than paclitaxel mono-therapy. In the current study, by using in vitro cellular models, we were set to test the potential anti-breast cancer effect of a short-chain cell permeable ceramide(C6) in combination with docetaxel.Ceramides, found in high concentration within cell membranes, are a family of lipid molecules to act as structural elements. Ceramides are also active signaling molecules for apoptosis. Numerous studies have shown a strong association between ceramides production and apoptosis in tumor cells, and processes that could enhance the production of intracellular ceramides will likely provide a favorable pro-apoptotic outcome. The cell–permeable short chain ceramides(i.e. C2 or C6) have shown activity against a variety of cancer cell lines including melanoma and soft tissue sarcoma, Jurkat leukemia, head and neck squamous cancer.A major focus in our laboratory is to examine the potential of C6 ceramide to augment the anti-tumor effects of known chemotherapeutic agents. Our previous studies have shown that C6 ceramide could be useful as an adjunct to conventional chemotherapeutics(i.e. Taxol). The molecular mechanisms involved in this synergistic effect continue to be largely elusive.Methods and materialsHuman breast cancer MCF-7 cells, MDA-231 cell all purchased from Shanghai Institute of Biological Science(Shanghai, China)and primary cultures of metastatic breast cancer tissues from patients with advantage stage diseases were obtained at the time of surgery.All cells were maintained in RPMI/DMEM medium(Sigma, St. Louis, MO), with a 10% FBS(Sigma, St. Louis, MO), penicillin/streptomycin(1:100, Sigma), and in a CO2 incubator at 37 oC. Cells were treated with indicated ceramide(C6, Alabaster, AB, CN: 860506P) and/or docetaxel, and cultured for indicated time, MTT assay and trypan blue staining were utilized to test cell survival.Cell apoptosis was analyzed by Annexin V FACS assay. Western blot detected total and phosphorylated AMPKα, ACC signaling pathway changings; and used inhibitors of apoptosis inhibitor to further validate C6 ceramide and docetaxel-induced cell death. Establishing CYP-D-sh RNA, AMPKα1-sh RNA and AMPK-α1 dominant negative(DN) mutant(DN-AMPK-α1) c DNA stably transfected tumor cells, and using the indicated method further validate the changing of signaling pathway. To test whether m PTP rupture is associated with C6 ceramide plus docetaxel-induced cancer cell death, we examined the MPP in C6 plus docetaxel-treated breast cancer cells. The mitochondrial membrane potential(MMP) was measured by JC-10 dye and the cellular ROS level was measured by a DCFH-DA fluorescent dye(Molecular Probes/Invitrogen, Shanghai, China). Western blot detected cytosol cytochrome C, caspase-3 cleavage,Her-1/2,Akt/Erk signaling changings. And used inhibitors of Cyp-D(sanglifehrin A, Sigma St. Louis, MO), inhibitors of JNK(SP-600125, JNK Inhibitor V, Calbiochem,San Diego, CA)as well as the ROS scavenger N-acetyl-L-cysteine(NAC)( Sigma St. Louis, MO)further validate C6 ceramide and docetaxel-induced cell death. Establishing CYP-D-sh RNA stably transfected tumor cells, Cyp-D over-expressing MDA-231 cells and using the indicated method further validate the changing of signaling pathway.Statistical analysis-In each experiment, a minimum of three wells/dishes were used. Each experiment was repeated a minimum of three times, with similar results obtained each time. Data were presented as mean ± standard deviation(SD). Statistic was analyzed by one-way ANOVA followed by a Scheffe’ and Tukey Test by SPSS 15.0 software(SPSS Inc., Chicago, IL, USA). Significance was chosen as P < 0.05.Results3.1 C6 ceramide dramatically enhances docetaxel-induced cytotoxicity in cultured breast cancer cellsAs demonstrated, either docetaxel(DTX, 1.0 μg/m L) or C6 ceramide(C6, 10 μg/m L) alone had moderate effect on MDA-231 cancer cell death, whereas combination of these two agents induced a dramatic cell death and growth inhibition, with over 90% dead cell was seen 72 hrs after treatment. Similarly, in primary cultured human breast cancer cells, C6 ceramide significantly enhanced docetaxel-induced cytotoxicity in primary cells.3.2 C6 ceramide significantly augments docetaxel-induced breast cancer cell apoptosisIn cultured MDA-231 or MCF-7 cells, C6 ceramide or docetaxel as a single agent only induced moderate cell apoptosis; However, a combination of the two caused dramatic increase of Annexin V positive cells(apoptosis). Cell apoptosis after the co-administration was also confirmed by Western-blots detecting apoptosis-associated proteins. For example, PARP was downregulated after co-administration in MCF-7 and MDA-231 cells, while cleaved-caspase-9 was upregulated.Significantly, z VADfmk, the general caspase inhibitor, almost blocked C6+docetaxel-induced viability reduction in both MDA-231 and MCF-7 cells. In primary cultured human breast cancer cells, z VADfmk dramatically inhibited cell death by the co-administration. Thus, apoptosis mediates C6 ceramide plus docetaxel-induced breast cancer cell death.3.3 C6 ceramide facilitates docetaxel-induced growth inhibition in cultured breast cancer cellsC6 ceramide and docetaxel as a single agent moderately inhibited growth of MDA-231 and MCF-7 cells, as the number of large colonies decreased after the corresponding single treatment. Significantly, combination of C6 and docetaxel caused a profound inhibition of cell growth, with the number of large colonies dropped sharply after combination treatment in both cell lines. Meanwhile, the expression of cyclin D1 was also largely inhibited by the co-administration.Thus, C6 ceramide facilitates docetaxel-induced growth inhibition while inhibiting docetaxel-induced G2 M arrest in cultured breast cancer cells.3.4 C6 ceramide plus docetaxel co-administration inhibits the mammalian target of rapamycin complex 1(m TORC1) through activating AMPKwe tested AMPK activation in C6 ceramide and/or docetaxeltreated breast cancer cells.In both MDA-231 and MCF-7 cells, C6 ceramide and docetaxel synergistically induced AMPK activation, which was demonstrated by phosphorylation of AMPKα(Thr 172) and its downstream ACC(Ser 79). The expression of regular AMPK and ACC was not changed by the co-treatment. Significantly, AMPK silencing by sh RNA knockdown inhibited C6+docetaxel-induced cytotoxicity in MDA-231 cells. Meanwhile, in HEK-293 cells, C6 plus docetaxel-caused cytotoxicity was suppressed by AMPK dominant negative(DN, T172A) mutation. These results suggested that activation of AMPK is important for cancer cell death by the co-administration. Next, we tested m TORC1 activation in MDA-231 cells treated with C6+docetaxel. As demonstrated, S6 phosphorylation, an indicator of m TORC1 activation, was largely inhibited by C6+docetaxel in MDA-231 cells. Significantly, AMPK sh RNA knockdown almost abolished S6 inhibition by the co-administration, suggesting that AMPK activation is required for m TORC1 inhibition by the co-treatment. Based on these results, we suggest that C6 ceramide plus docetaxel activates AMPK to inhibit m TORC1 and cell survival in breast cancer cells.3.5 JNK activation is involved in C6 ceramide plus docetaxel co-administration-induced cytotoxicity in MDA-231 cellsC6 ceramide plus docetaxel co-administration induced a synergistic and sustained JNK activation in MDA-231 cells cells. JNK activation by the co-administration was more potent than either agent alone. To test whether JNK activation was involved in the cytotoxicity of the co-administration, MDA-231 cells were pre-treated with two different JNK inhibitors including SP 600125 and JNK inhibitor V(JNKi V), and results showed that both inhibitors suppressed co-treatment-induced viability reduction, cell death and apoptosis in MDA-231 cells. Thus, JNK activation is involved in C6 ceramide plus docetaxel-mediated cytotoxicity in MDA-231 cells.3.6 The m PTP opening and subsequent ROS production are important mediators for C6 ceramide plus docetaxel-induced JNK/AMPK activation and cytotoxicityTo test whether m PTP rupture is associated with C6 ceramide plus docetaxel-induced cancer cell death, we examined the MPP in C6 plus docetaxel-treated breast cancer cells. The results demonstrated that MPP was significantly decreased after the co-administration in MDA-231 cells. Further, the ROS production, cytosol cytochrome C release and caspase-3 cleavage were also increased, Similar results were also seen in MCF-7 cells. Significantly, sanglifehrin A(Sf A), the m PTP inhibitor as well as the ROS scavenger N-acetyl-L-cysteine(NAC) largely inhibited C6+docetaxel-induced MDA-231 cell death, further, cytochrome C release and caspase-3 cleavage were also inhibited. Based on these data, we suggest that m PTP opening and subsequent ROS production are important mediators for co-administration-induced cytotoxicity in cultured breast cancer cells. In supporting of this hypothesis, we found that inhibition of Cyp-D, a key component of m PTP, by si RNA or its inhibitor Cs A,significantly alleviated cell death caused by the co-administration. While, Cyp-D over-expressing MDA-231 cells were significantly more sensitive to C6+docetaxel. Since ROS are known activators of AMPK [39, 40] and JNK [41], we tested whether ROS were required for AMPK and JNK activation by the co-administration. As demonstrated, the ROS scavengers NAC inhibited AMPK and JNK phosphorylation induced by the co-administration. On the other hand, hydrogen peroxide(H2O2, a ROS) activated AMPK and JNK in MDA-231 cells. Importantly, m PTP inhibitor Sf A pre-treatment similarly inhibited co-administration induced AMPK/JNK activation in MDA-231 cells. Note that Sf A or NAC alone had no effect on AMPK/JNK activation in MDA-231 cells.Thus, we proposed that m PTP opening and ROS production mediates AMPK/JNK activation and cell death induced by the co-administration.3.7 C6 ceramide plus docetaxel co-administration induces HER-1/2 degradation and Akt/Erk inhibition in MDA-231 cellsIn MDA-231 cells, C6 ceramide and docetaxel stimulation caused a synergistic degradation of HER-1/2, as well as inhibition of Akt and Erk1/2 signalings. Note that HER-1/2 degradation started 6-12 hrs after the co-administration, so did the p-Akt and p-Erk, indicating that Akt/Erk inhibition by the co-administration might be due to HER-1/2 degradation. As a matter of fact, AG1478, the HER inhibitor also inhibited Akt and Erk phosphorylation in both cell lines. Meanwhile, MDA-231/MCF-7 cell viability was also inhibited when AG-1478 was present 。 Thus, C6 ceramide plus docetaxel co-administration induces HER-1/2 degradation and Akt/Erk inhibition.Conclusion1. C6 ceramide dramatically enhances docetaxel-induced cytotoxicity in cultured breast cancer cells2. C6 ceramide significantly augments docetaxel-induced breast cancer cell apoptosis3. C6 ceramide facilitates docetaxel-induced growth inhibition in cultured breast cancer cells4. C6 ceramide plus docetaxel co-administration inhibits the mammalian target of rapamycin complex 1(m TORC1) through activating AMPK5. JNK activation is involved in C6 ceramide plus docetaxel co-administration-induced cytotoxicity in cultured breast cancer cells6. The m PTP opening and subsequent ROS production are important mediators for C6 ceramide plus docetaxel-induced JNK/AMPK activation and cytotoxicity7. C6 ceramide plus docetaxel co-administration induces HER-1/2 degradation and Akt/Erk inhibition in breast cancer cells... |
PDF Full Text Request