| Background and ObjectivesUse of the conventional cancer chemotherapy is seriously limited in tumor cells exhibiting pre-existing or acquired resistance, which is caused by changes in the level or activity of drug transporters and proteins that affect drug actions and/or metabolism. Chemo-sensitization has been a major focus of cancer research. Different groups have been investigating the activity of short-chain cell permeable ceramide(C6) to augment the anti-tumor activity of conventional chemotherapeutic agents. C6 ceramide dramatically enhances the activity of multiple anti-cancer drugs, including paclitaxel, doxorubicin and histone deacetylase inhibitors(HDACi). The molecular mechanisms underlying this sensitization remain to be inconclusive.Vincristine is a commonly used anti-cancer chemo-drug. It is a cell-cycle-specific drug that binds to tubulin, inhibiting assembly of microtubule structures and arresting mitosis in metaphase in cancer cells undergoing mitosis. It has been widely used in various types of chemotherapy regimens to treat patients with cancer, although the response rates vary among different cancers, and cancer cells trend to develop resistance after initial uses. The molecular basis for induction cancer cell death by vincristine, however, remains unclear. Our previous study has shown that activation of AMP-activated protein kinase(AMPK) might be the key signaling hub to mediate its activity in vitro. Our group and others have been focusing on the inhibitory ability of AMPK against cancer cells. Sustained AMPK activation is shown to induce cancer cell death and growth inhibition through regulating multiple downstream signal targets, including in-activating mTOR complex 1(mTORC1), phosphorylating p53, activating autophagy, among many others.Here we show that C6 ceramide dramatically enhances vincristine-induced activity in multiple established human cancer cell lines both in vivo and in vitro. At the molecular level, we propose that AMPK-dependent p53 activation might be the key signaling-hub to mediate the sensitization effect.Methods and materialsHuman colon cancer HCT-116 cells, human pancreatic cancer PANC-1 cells and human ovarian cancer A2780 cells, all purchased from Shanghai Institute of Biological Science(Shanghai, China), 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) and/or Vincristine, and cultured for indicated time, Cell apoptosis was analyzed by Histone-DNA ELISA assay,“Clonogenicity” assay and Annexin V FACS assay. MTT assay and trypan blue staining were utilized to test cell survival. Lactate dehydrogenase(LDH) release assay of cell necrosis. mitochondria isolated by mitochondria immunoprecipitation, measured mitochondrial membrane potential(MMP) by JC-10 dye, Western blot detected total and phosphorylated AMPKα, ACC, P53, CYP-D, Bcl- 2, HIF-1α, S6K1 signaling pathway changings; and used inhibitors of programmed cell death and apoptosis inhibitor to further validate C6 ceramide and vincristine-induced cell death. Establishing CYP-D-shRNA, p53-sh RNA, AMPKα1-shRNA and AMPK-α1 dominant negative(DN) mutant(DN-AMPK-α1) cDNA stably transfected tumor cells, and using the above-described method further validate the changing of signaling pathway. Using the above-described method to verify vitro activity of C6 ceramide liposome. In order to validate the liposome C6 enhance the anti-tumor mechanism of ceramide and vincristine co-administration, nude model was established and grouped by different durg treatment, mice survival rate and tumor size were recorded, meanwhile AMPKα expression was detected by immunohistochemistry.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.Results1. C6 ceramide dramatically enhances vincristine-induced cytotoxicity in multiple established human cancer cell linesCancer cell viability was examined through MTT assay, and results showed that vincristine(0.1-10 μM) alone only moderately inhibited HCT-116 colon cancer cell survival, co-administration with C6 ceramide(10 μg/m L) dramatically sensitized vincristine’s activity, resulting in substantial viability reduction. The effect of C6 ceramide was dose-dependent, and 2.5-10.0 μg/mL of C6 ceramide was able to significantly sensitize the activity of vincristine, while C6 ceramide by itself had almost no effect on HCT-116 cell survival. Trypan blue staining assay and clonogenicity assay results showed that C6 ceramide and vincristine co-administration resulted in massive cell death, and the combined efficiency was significantly higher than either agent alone. The synergistic effect was also observed in two other human cancer cell lines: A2780 ovarian cancer cells and PANC-1 pancreatic cancer cells. Together, these results show that C6 ceramide dramatically sensitizes vincristine’s activity in vitro.2. C6 ceramide facilitates vincristine-induced necrosis and apoptosisThe histone DNA-ELISA assay and Annexin V FACS assay were performed to test cell apoptosis, and results demonstrated that treatment of vincristine(1 μM) alone only induced minimal apoptosis in HCT-116 cells, which was significantly augmented by C6 ceramide(10 μg/m L) co-treatment. The sensitization effect by C6 ceramide on cell apoptosis was almost abolished by the broad caspase inhibitor z-VAD-fmk. vincristine-induced HCT-116 cell necrosis, detected by increased LDH release, was also enhanced by C6 ceramide co-treatment, and cell necrosis was expectably blocked by the necrosis inhibitor necrostatin-1(Nec-1). Importantly, C6 ceramide plus vincristine-induced cytotoxicity against HCT-116 cells was patricianly inhibited by z-VAD-fmk or Nec-1 alone, but was almost blocked by the two combination. Note that Nec-1 was more efficient than z-VAD-fmk in alleviating the cytotoxicity. Similar results were also seen in A2780 ovarian cancer cells and PANC-1 pancreatic cancer cells(Data not shown). Based on these data, we suggest that both cell necrosis and apoptosis participate in C6 ceramide plus vincristine-induced cytotoxicity, and necrosis appears playing a more dominant role.3. C6 ceramide and vincristine synergistically activates AMPK-dependent mTORC1 inhibition as well as cell apoptosis and necrosisWestern blot results demonstrated that, in HCT-116 cells and A2780 cells, C6 ceramide and vincristine synergistically activated AMPK pathway(AMPK/ACC phosphorylations), more efficiently than either agent alone. Activation of mTORC1, detected by phosphorylation of S6K1(Thr-389), and expressions of mTORC1-dependent genes, including Bcl-2 and HIF-1α, were significantly downregulated by the co-administration, which was more potently than each single treatment.Inhibition of AMPK activation, either by genetic silencing(shRNA knockdown) or by introducing a dominant negative(DN) mutation, restored S6K1 phosphorylation and Bcl-2/HIF-1α expressions in co-stimulated cells. Importantly, C6 ceramide plus vincristine-induced viability decease, cell apoptosis and cell necrosis were all inhibited by AMPK silencing or mutation in HCT-116 cells. On the other hand, AMPK activator AICAR mimicked the actions on signalings by the co-administration, and promoted cancer HCT-116 cell apoptosis and necrosis. Thus, C6 ceramide and vincristine co-administration induces a profound AMPK activation, causing mTORC1 in-activation, Bcl-2/HIF-1α downregulation as well as cell apoptosis and necrosis.4. C6 ceramide and vincristine synergistically induces AMPK-dependent p53 activation, its mitochondrial translocation and Cyp-D complexationC6 ceramide or vincristine alone induced moderate p53 activation(phosphorylation and upregulation) in HCT-116 cells. Combination of the two induced a further increased p53 activation. Notably, inhibition of AMPK by shRNA depletion or mutation suppressed p53 activation by the co-treatment. Western blot assay examining mitochondrial proteins showed that C6 ceramide plus vincristine led to p53 translocation to mitochondria, note that both p53 and phosphorylated-p53 travelled to mitochondria. Further, mito-IP assay results showed that translocated p53 formed a complex with Cyp-D, such complexation was inhibited by depletion of AMPK or Cyp-D. Further, input assay showed that p53 translocation by co-administration was inhibited by AMPK depletion, but not by Cyp-D knockdown. Reversely, AMPK activator AICAR induced p53 activation, causing it mitochondrial translocation as well as Cyp-D complexation. we tested MMP in HCT-116 cells after stimulation through JC-10 dye, and results showed that C6 ceramide and vincristine synergistically decreased MMP, more potent than either agent alone(Figure 4F). Depletion of AMPK or Cyp-D by targeted shRNA inhibited co-administration-induced MMP reduction.5. Mitochondrial protein Cyp-D is required for C6 ceramide and vincristine co-administration-induced necrosis, but not apoptosiscyclosporin A(CsA) as well as Cyp-D sh RNA knockdown almost blocked co-administration-induced cell necrosis, whiling leaving cell apoptosis non-affected. As a result, C6 ceramide plus vincristine-induced viability decrease was alleviated by Cyp-D inhibition. Interestingly, p53 depletion by shRNA not only suppressed co-administration-induced necrosis, but also inhibited cell apoptosis.6. Liposomal C6 ceramide efficiently sensitizes vincristine’s activity in vitro and in vivoWe were able to synthesize a liposomal C6 ceramide based on the previous protocol. In vitro study results showed that this liposomal C6 ceramide enhanced vincristine-induced viability decrease and apoptosis in both HCT-116 cells and A2780 cells, with higher efficiency than non-liposomal C6 ceramide. Liposomal C6 ceramide containing 1 μg/m L of C6 ceramide significantly increased vincristine’s in vitro activity, while same concentration of non-liposomal C6 ceramide(1 μg/m L) failed to achieve the sensitization effect. Note that control liposome containing no C6 ceramide failed to affect vincristine’s activity in cultured HCT-116 cells or A2780 cells. In vivo, HCT-116 or A2780 xenograft growth was dramatically inhibited in mice administrated with liposomal C6 ceramide plus vincristine. HCT-116 or A2780 in vivo growth was only slightly suppressed by liposomal C6 ceramide or by vincristine as a single treatment. In consistent with results of non-liposomal C6 ceramide, IHC assay results showed that liposomal C6 ceramide and vincristine co-administration(48 hours) in vivo also induced a significant AMPK phosphorylation/activation in tumor xenograft. Control liposome containing no C6 ceramide had no effect on xenograft growth alone or in combination with vincristine. We failed to detect any obvious deleterious effects by the drugs tested in experimental mice. Mice body weight was also not affected by the regimens used above.ConclusionsC6 dramatically sensitizes vincristine-induced cancer cell apoptosis and necrosisC6 and vincristine synergistically activates AMPK-p53, mediating apoptosis/necrosisAMPK activation by co-treatment causes mTOR inhibition and Bcl-2/HIF-1α degradationAMPK-dependent p53-Cyp-D mitochondrial complexation mediates necrosis by co-treatmentA liposomal C6 ceramide sensitizes vincristine-induced activity in vitro and in vivo... |
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