Curcumin can induce p53-independent apoptosis. However, the underlying mechanism remains to be defined. Here, we show that curcumin-induced apoptosis in a panel of tumor cells with mutant p53. ...Curcumin rapidly induced activation of the mitogen-activated protein kinases (MAPKs) including extracellular signal-regulated kinase 1/2 (Erk1/2) and c-Jun N-terminal kinase (JNK). Inhibition of JNK (with SP600125) or Erk1/2 (with U0126) partially prevented curcumin-induced cell death in the cells. Similarly, expression of dominant negative c-Jun or downregulation of Erk1/2 in part attenuated curcumin-induced cell death. It appears that curcumin-induced activation of MAPKs and apoptosis was due to induction of reactive oxygen species (ROS), as pretreatment with N-acetyl-L-cysteine, a ROS scavenger, blocked these events. Furthermore, we found that curcumin-induced activation of MAPK pathways was related to inhibition of the serine/threonine protein phosphatases 2A (PP2A) and 5 (PP5). Overexpression of PP2A or PP5 partially prevented curcumin-induced activation of JNK and Erk1/2 phosphorylation as well as cell death. The results suggest that curcumin induction of ROS activates MAPKs, at least partially by inhibiting PP2A and PP5, thereby leading to p53-independent apoptosis in tumor cells.
Dihydroartemisinin (DHA) is a derivative of artemisinin, a natural product isolated from the plant, Artemisia annua. It is also the active metabolite of first-generation artemisinin derivatives ...(artemisinin, artesunate, and artemether). Artemisinins have been clinically used to treat malaria for decades. Recent studies have demonstrated that artemisinin and its derivatives possess anticancer activity. This study was set to investigate the anticancer mechanism of DHA. Using rhabdomyosarcoma (Rh30 and RD) cells as an experimental model, we found that DHA potently inhibited proliferation in the tumor cells, by arresting cells in G1/G0 and G2/M phases, which was linked to down regulation of cyclin D1, CDK4 and CDC25A, as well as CDK1 and CDC25C expression, respectively. We also observed that DHA induced caspase-mediated apoptosis in the cells, which was related to downregulation of survivin, Mcl-1 and Bcl2 and upregulation of BAD. As the mammalian target of rapamycin (mTOR) is a master kinase that regulates cell proliferation and survival, we further assessed whether DHA inhibits mTOR signaling. We found that DHA inhibited mTORC1, but not mTORC2, in the tumor cells. This is supported by our findings that 1) DHA inhibited mTORC1-mediated phosphorylation of p70 S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1), in a concentration- and time-dependent manner; 2) DHA enhanced the binding of 4E-BP1 to eIF4E, by the m7-GTP pull-down assay; and 3) DHA did not obviously affect mTORC2-mediated phosphorylation of Akt. Next, we focused on identifying the molecular mechanism(s) by which DHA inhibits mTORC1 signaling. We found that DHA neither affected phosphorylation of IGF-1 receptor β subunit or p85 of phosphatidylinositide 3-kinase, two positive regulators of mTORC1, nor phosphorylation of phosphatase and tensin homolog (PTEN), a negative regulator of mTORC1. However, DHA enhanced the level of ROS (reactive oxygen species), lowered the intracellular ATP, and activated AMPK, a negative regulator of mTORC1 activity. A ROS scavenger, N-acetyl-L-cysteine, attenuated DHA-mediated anti-proliferation of Rh30 cells. Moreover, expression of a dominant-negative form of AMPK or downregulation of TSC2 (another negative regulator of mTORC1) attenuated DHA inhibition of mTORC1 in Rh30 cells. DHA did not induced phosphorylation of raptor, but hindered raptor from associating with mTOR. Although DHA inhibited the mTORC1 kinase activity, by an in vitro mTOR kinase assay using recombinant 4E-BP1 as a substrate, we failed to detect significant binding of 3H-DHA to mTOR. Taken together, our results indicate that DHA inhibits mTORC1 at least in part through AMPK-TSC pathway, and in part by dissociating raptor from mTOR. More studies are required to address how DHA activates AMPK and disrupts mTORC1 formation.
Mammalian target of rapamycin (mTOR) controls lymphangiogenesis. However, the underlying mechanism is not clear. Here we show that rapamycin suppressed insulin-like growth factor 1 (IGF-1)- or fetal ...bovine serum (FBS)-stimulated lymphatic endothelial cell (LEC) tube formation, an
in vitro
model of lymphangiogenesis. Expression of a rapamycin-resistant and kinase-active mTOR (S2035T, mTOR-T), but not a rapamycin-resistant and kinase-dead mTOR (S2035T/D2357E, mTOR-TE), conferred resistance to rapamycin inhibition of LEC tube formation, suggesting that rapamycin inhibition of LEC tube formation is mTOR kinase activity dependent. Also, rapamycin inhibited proliferation and motility in the LECs. Furthermore, we found that rapamycin inhibited protein expression of VEGF receptor 3 (VEGFR-3) by inhibiting protein synthesis and promoting protein degradation of VEGFR-3 in the cells. Down-regulation of VEGFR-3 mimicked the effect of rapamycin, inhibiting IGF-1- or FBS-stimulated tube formation, whereas over-expression of VEGFR-3 conferred high resistance to rapamycin inhibition of LEC tube formation. The results indicate that rapamycin inhibits LEC tube formation at least in part by downregulating VEGFR-3 protein expression.
Background
In view of the limited success of available treatment modalities for a wide array of cancer, alternative and complementary therapeutic strategies need to be developed. Virotherapy ...employing conditionally replicative adenoviruses (CRAds) represents a promising targeted intervention relevant to a wide array of neoplastic diseases. Critical to the realization of an acceptable therapeutic index using virotherapy in clinical trials is the achievement of oncolytic replication in tumor cells, while avoiding non-specific replication in normal tissues. In this report, we exploited cancer-specific control of mRNA translation initiation in order to achieve enhanced replicative specificity of CRAd virotherapy agents. Heretofore, the achievement of replicative specificity of CRAd agents has been accomplished either by viral genome deletions or incorporation of tumor selective promoters. In contrast, control of mRNA translation has not been exploited for the design of tumor specific replicating viruses to date. We show herein, the utility of a novel approach that combines both transcriptional and translational regulation strategies for the key goal of replicative specificity.
Methods
We describe the construction of a CRAd with cancer specific gene transcriptional control using the CXCR4 gene promoter (TSP) and cancer specific mRNA translational control using a 5′-untranslated region (5′-UTR) element from the FGF-2 (Fibroblast Growth Factor-2) mRNA.
Results
Both in vitro and in vivo studies demonstrated that our CRAd agent retains anti-tumor potency. Importantly, assessment of replicative specificity using stringent tumor and non-tumor tissue slice systems demonstrated significant improvement in tumor selectivity.
Conclusions
Our study addresses a conceptually new paradigm: dual targeting of transgene expression to cancer cells using both transcriptional and mRNA translational control. Our novel approach addresses the key issue of replicative specificity and can potentially be generalized to a wide array of tumor types, whereby tumor selective patterns of gene expression and mRNA translational control can be exploited.
We previously showed that introduction of transporter associated with antigen processing (TAP) 1 into TAP-negative CMT.64, a major histocompatibility complex class I (MHC-I) down-regulated mouse lung ...carcinoma cell line, enhanced T-cell immunity against TAP-deficient tumour cells. Here, we have addressed two questions: (1) whether such immunity can be further augmented by co-expression of TAP1 with B7.1 or H-2Kb genes, and (2) which T-cell priming mechanism (tumour direct priming or dendritic cell cross-priming) plays the major role in inducing an immune response against TAP-deficient tumours. We introduced the B7.1 or H-2Kb gene into TAP1-expressing CMT.64 cells and determined which gene co-expressed with TAP1 was able to provide greater protective immunity against TAP-deficient tumour cells. Our results show that immunization of mice with B7.1 and TAP1 co-expressing but not H-2Kb and TAP1 co-expressing CMT.64 cells dramatically augments T-cell-mediated immunity, as shown by an increase in survival of mice inoculated with live CMT.64 cells. In addition, our results suggest that induction of T-cell-mediated immunity against TAP-deficient tumour cells could be mainly through tumour direct priming rather than dendritic cell cross-priming as they show that T cells generated by tumour cell-lysate-loaded dendritic cells recognized TAP-deficient tumour cells much less than TAP-proficient tumour cells. These data suggest that direct priming by TAP1 and B7.1 co-expressing tumour cells is potentially a major mechanism to facilitate immune responses against TAP-deficient tumour cells.
A number of groups have developed libraries of siRNAs to identify genes through functional genomics. While these studies have validated the approach of making functional RNAi libraries to understand ...fundamental cellular mechanisms, they require information and knowledge of existing sequences since the RNAi sequences are generated synthetically. An alternative strategy would be to create an RNAi library from cDNA. Unfortunately, the complexity of such a library of siRNAs would make screening difficult. To reduce the complexity, longer dsRNAs could be used; however, concerns of induction of the interferon response and off-target effects of long dsRNAs have prevented their use. As a first step in creating such libraries, long dsRNA was expressed in mammalian cells. The 250 nt dsRNAs were capable of efficiently silencing a luciferase reporter gene that was stably transfected in MDA-MB-231 cells without inducing the interferon response or off-target effects any more than reported for siRNAs. In addition, a long dsRNA expressed in the same cell line was capable of silencing endogenous c-met expression and inhibited cell migration, whereas the dsRNA against luciferase had no effect on c-met or cell migration. The studies suggest that large dsRNA libraries are feasible and that functional selection of genes will be possible.
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