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Tubulin-binding agents such as taxol, vincristine or vinblastine are well-established drugs in clinical treatment of metastatic cancer. However, because of their highly complex chemical structures, the synthesis and hence the supply issues are still quite challenging. Here we set on stage pretubulysin, a chemically accessible precursor of tubulysin that was identified as a potent microtubule-binding agent produced by myxobacteria. Although much simpler in chemical structure, pretubulysin abrogates proliferation and long-term survival as well as anchorage-independent growth, and also induces anoikis and apoptosis in invasive tumor cells equally potent to tubulysin. Moreover, pretubulysin posseses in vivo efficacy shown in a chicken chorioallantoic membrane (CAM) model with T24 bladder tumor cells, in a mouse xenograft model using MDA-MB-231 mammary cancer cells and finally in a model of lung metastasis induced by 4T1 mouse breast cancer cells. Pretubulysin induces cell death via the intrinsic apoptosis pathway by abrogating the expression of pivotal antiapoptotic proteins, namely Mcl-1 and Bcl-xL, and shows distinct chemosensitizing properties in combination with TRAIL in two- and three-dimensional cell culture models. Unraveling the underlying signaling pathways provides novel information: pretubulysin induces proteasomal degradation of Mcl-1 by activation of mitogen-activated protein kinase (especially JNK (c-Jun N-terminal kinase)) and phosphorylation of Mcl-1, which is then targeted by the SCF(Fbw7) E3 ubiquitin ligase complex for ubiquitination and degradation. In sum, we designate the microtubule-destabilizing compound pretubulysin as a highly promising novel agent for mono treatment and combinatory treatment of invasive cancer.
Smac (second mitochondria-derived activator of caspase) mimetics are considered as promising anticancer therapeutics and used to induce apoptosis by antagonizing inhibitor of apoptosis proteins, which are often abundantly expressed in cancer cells. Here, we identify interferon regulatory factor 1 (IRF1) as a novel critical regulator of Smac mimetic BV6-induced apoptosis and proinflammatory cytokine secretion with impact on the immune response. IRF1 knockdown rescues cells from BV6-induced apoptosis and attenuates BV6-stimulated upregulation of tumor necrosis factor-α (TNFα), indicating that IRF1 mediates BV6-triggered cell death, at least in part, by inducing TNFα. This notion is supported by data showing that exogenous supply of TNFα restores BV6-induced cell death in IRF-knockdown cells. Interestingly, IRF1 selectively controls the induction of nuclear factor-κB (NF-κB) target genes, as IRF1 depletion attenuates BV6-stimulated upregulation of TNFα and interleukin-8 (IL-8) but not p100 and RelB. Concomitant knockdown of IRF1 and p65 cooperate to inhibit BV6-induced cell death, implying a cooperative interaction of IRF1 and NF-κB. In addition, IRF1 silencing hampers TNFα induction by TNFα itself as an another prototypical NF-κB stimulus. Importantly, IRF1 depletion impedes BV6-stimulated secretion of additional proinflammatory cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-8, IL-6 and monocyte chemoattractant protein-1, and migration of primary monocytes to BV6-treated tumor cells. In conclusion, this identification of IRF1 as a dual regulator of BV6-induced apoptosis and inflammatory cytokine secretion provides novel insights into determinants of sensitivity towards Smac mimetic and possible implications of Smac mimetic treatment on tumor microenvironment and immune response.
Hypoxia enhances the antiglioma cytotoxicity of b10, a glycosylated derivative of betulinic acid
(2014)
B10 is a glycosylated derivative of betulinic acid with promising activity against glioma cells. Lysosomal cell death pathways appear to be essential for its cytotoxicity. We investigated the influence of hypoxia, nutrient deprivation and current standard therapies on B10 cytotoxicity. The human glioma cell lines LN-308 and LNT-229 were exposed to B10 alone or together with irradiation, temozolomide, nutrient deprivation or hypoxia. Cell growth and viability were evaluated by crystal violet staining, clonogenicity assays, propidium iodide uptake and LDH release assays. Cell death was examined using an inhibitor of lysosomal acidification (bafilomycin A1), a cathepsin inhibitor (CA074-Me) and a short-hairpin RNA targeting cathepsin B. Hypoxia substantially enhanced B10-induced cell death. This effect was sensitive to bafilomycin A1 and thus dependent on hypoxia-induced lysosomal acidification. Cathepsin B appeared to mediate cell death because either the inhibitor CA074-Me or cathepsin B gene silencing rescued glioma cells from B10 toxicity under hypoxia. B10 is a novel antitumor agent with substantially enhanced cytotoxicity under hypoxia conferred by increased lysosomal cell death pathway activation. Given the importance of hypoxia for therapy resistance, malignant progression, and as a result of antiangiogenic therapies, B10 might be a promising strategy for hypoxic tumors like malignant glioma.