Induction Of Lysosomal Membrane Permeabilization Research Articles

(-)-Oleocanthal rapidly and selectively induces cancer cell death via lysosomal membrane permeabilization

(-)-Oleocanthal (OC), a phenolic compound present in extra-virgin olive oil (EVOO), has been implicated in the health benefits associated with diets rich in EVOO. We investigated the effect of OC on human cancer cell lines in culture and found that OC induced cell death in all cancer cells examined as rapidly as 30 minutes after treatment in the absence of serum. OC treatment of non-transformed cells suppressed their proliferation but did not cause cell death. OC induced both primary necrotic and apoptotic cell death via induction of lysosomal membrane permeabilization (LMP). We provide evidence that OC promotes LMP by inhibiting acid sphingomyelinase (ASM) activity, which destabilizes the interaction between proteins required for lysosomal membrane stability. The data presented here indicate that cancer cells, which tend to have fragile lysosomal membranes compared to non-cancerous cells, are susceptible to cell death induced by lysosomotropic agents. Therefore, targeting lysosomal membrane stability represents a novel approach for the induction of cancer-specific cell death.

The acidic transformed nano-VO2 causes macrophage cell death by the induction of lysosomal membrane permeabilization and Ca2+ efflux

Because of its outstanding thermochromic characteristics and metal-insulator transition (MIT) property, nano-vanadium dioxide (abbreviated as nano-VO2 or nVO2) has been applied widely in electrical/optical devices and design of intelligent window. However, the biological effect of nVO2 is not well understood, especially when affected by environmental factors or living organisms. For VO2 is an amphoteric oxide, we simulated pH's influence to nVO2’s physicochemical properties by exposure nVO2 in water of different pH values. We found that nVO2 transformed to a new product after exposure in acidic water for two weeks, as revealed by physicochemical characterization such as SEM, TEM, XRD, and DLS. This transformation product formed in acidic water was referred as (acidic) transformed nVO2). Both pristine/untransformed and transformed nVO2 displayed no obvious toxicity to common epithelial cells; however, the acidic transformed nVO2 rapidly induced macrophage cell death. Further investigation demonstrated that transformed nVO2 caused macrophage apoptosis by the induction of Ca2+ efflux and the following mitochondrial membrane permeabilization (MMP) process. And a more detailed time course study indicated that transformed nVO2 caused lysosomal membrane permeabilization (LMP) at the earlier stage, indicating LMP could be chosen as an earlier and sensitive end point for nanotoxicological study. We conclude that although nVO2 displays no acute toxicity, its acidic transformation product induces macrophage apoptosis by the induction of LMP and Ca2+ efflux. This report suggests that the interplay with environmental factors or living organisms can results in physicochemical transformation of nanomaterials and the ensuing distinctive biological effects.

WIN induces apoptotic cell death in human colon cancer cells through a block of autophagic flux dependent on PPARγ down-regulation

Cannabinoids have been reported to possess anti-tumorigenic activity in cancer models although their mechanism of action is not well understood. Here, we show that the synthetic cannabinoid WIN55,212-2 (WIN)-induced apoptosis in colon cancer cell lines is accompanied by endoplasmic reticulum stress induction. The formation of acidic vacuoles and the increase in LC3-II protein indicated the involvement of autophagic process which seemed to play a pro-survival role against the cytotoxic effects of the drug. However, the enhanced lysosomal membrane permeabilization (LMP) blocked the autophagic flux after the formation of autophagosomes as demonstrated by the accumulation of p62 and LC3, two markers of autophagic degradation. Data also provided evidence for a role for nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) in cannabinoid signalling. PPARγ expression, at both protein and mRNA levels, was significantly down-regulated after WIN treatment and its inhibition, either by specific antagonists or by down-regulation via gene silencing, induced effects on cell viability as well as on ER stress and autophagic markers similar to those obtained in the presence of WIN. Moreover, the observation that the increase in p62 level and the induction of LMP were also modified by PPARγ antagonists seemed to indicate that PPARγ down-regulation was crucial to determinate the block of autophagic flux, thus confirming the critical role of PPARγ in WIN action. In conclusion, at our knowledge, our results are the first to show that the reduction of PPARγ levels contributes to WIN-induced colon carcinoma cell death by blocking the pro-survival autophagic response of cells.

Combating apoptosis and multidrug resistant cancers by targeting lysosomes

Acquired therapy resistance is one of the prime obstacles for successful cancer treatment. Partial resistance is often acquired already during an early face of tumor development when genetic changes causing defects in classical caspase-dependent apoptosis pathway provide transformed cells with a growth advantage by protecting them against various apoptosis inducing stimuli including transforming oncogenes themselves and host immune system. Apoptosis defective cells are further selected during tumor progression and finally by apoptosis inducing treatments. Another form of resistance, multidrug resistance, arises during cancer treatment when cancer cells with effective efflux of cytotoxic agents escape the therapy. Remarkably, induction of lysosomal membrane permeabilization has recently emerged as an effective way to kill apoptosis resistant cancer cells and some lysosome targeting drugs can also re-sensitize multidrug resistant cells to classical chemotherapy. In this review, we highlight recent data on lysosomal cell death pathways and their implications for the future treatment of apoptosis defective and multidrug resistant aggressive tumors.

Zinc(II) ion mediates tamoxifen-induced autophagy and cell death in MCF-7 breast cancer cell line

Treatment of MCF-7 cells with tamoxifen induced vacuole formation and cell death. Levels of the autophagy marker, microtubule-associated protein light chain 3 (LC3)-II also increased, and GFP-LC3 accumulated in and around vacuoles in MCF-7 cells exposed to tamoxifen, indicating that autophagy is involved in tamoxifen-induced changes. Live-cell confocal microscopy with FluoZin-3 staining and transmission electron microscopy with autometallographic staining revealed that labile zinc(II) ion (Zn(2+)) accumulated in most acidic LC3(+) autophagic vacuoles (AVs). Chelation of Zn(2+) with N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN) blocked the increase in phospho-Erk and LC3-II levels, and attenuated AV formation and cell death. Conversely, the addition of ZnCl(2) markedly potentiated tamoxifen-induced extracellular signal-regulated kinase (Erk) activation, autophagy and cell death, indicating that Zn(2+) has an important role in these events. Tamoxifen-induced death was accompanied by increased oxidative stress and lysosomal membrane permeabilization (LMP) represented as release of lysosomal cathepsins into cytosol. Treatment with the antioxidant N-acetyl-L-cysteine (NAC) blunted the increase in Zn(2+) levels and reduced LC3-II conversion, cathepsin D release and cell death induced by tamoxifen. And cathepsin inhibitors attenuated cell death, indicating that LMP contributes to tamoxifen-induced cell death. Moreover, TPEN blocked tamoxifen-induced cathepsin D release and increase in oxidative stress. The present results indicate that Zn(2+) contributes to tamoxifen-induced autophagic cell death via increase in oxidative stress and induction of LMP.

Regulation of Neuron Death in Neonatal Hypoxia‐Ischemia

Hypoxia ischemia (HI) is a major cause of neonatal brain injury and often results in long‐term neurological disability. We have shown that the pro‐apoptotic molecules such as Bax critically regulate neuronal injury following HI. Alterations in the autophagy‐lysosome pathway (ALP) are known to regulate neuron death following neonatal HI and our goal is to delineate mechanisms by which ALP regulates neuron death following neonatal HI. Postnatal day 7 B57BL6/J mice underwent unilateral carotid ligation followed by 45 min exposure to 8% oxygen, and were euthanized 3‐48h later. Brain samples were processed either for IHC or western blot analysis. Within 12h after neonatal HI, intense cytosolic CD immunoreactivity was observed in the ipsilateral hippocampus and peaked at 24‐48h, suggesting induction of lysosomal membrane permeabilization. Increased CD levels were confirmed by western blot analysis. Cytosolic CD co‐localized with NeuN and cleaved caspase‐3 but not p53 in the ipsilateral hippocampus. Bax deficiency attenuated cleaved caspase‐3 immunoreactivity but this attenuation was incomplete, suggesting an additional role for the extrinsic apoptotic pathway in neonatal HI. These results suggest the existence of multiple neuron death pathways following neonatal HI and studies are ongoing to determine if these pathways are regulated through alterations in the ALP.

467 POSTER Induction of lysosomal membrane permeabilization by compounds that induce p53-independent apoptosis

467 POSTER Induction of lysosomal membrane permeabilization by compounds that induce p53-independent apoptosis

Cardenolide-Induced Lysosomal Membrane Permeabilization Demonstrates Therapeutic Benefits in Experimental Human Non-Small Cell Lung Cancers

Non-small cell lung cancers (NSCLCs) are the leading cause of cancer deaths in most developed countries. Targeting heat shock protein 70 (Hsp70) expression and function, together with the induction of lysosomal membrane permeabilization (LMP), could overcome the multiple anti-cell death mechanisms evidenced in NSCLCs that are responsible for the failure of currently used chemotherapeutic drugs. Because cardenolides bind to the sodium pump, they affect multiple signaling pathways and thus have a number of marked effects on tumor cell behavior. The aim of the present study was to characterize in vitro and in vivo the antitumor effects of a new cardenolide (UNBS1450) on experimental human NSCLCs. UNBS1450 is a potent source of in vivo antitumor activity in the case of paclitaxeland oxaliplatin-resistant subcutaneous human NCIH727 and orthotopic A549 xenografts in nude mice. In vitro UNBS1450-mediated antitumor activity results from the induction of nonapoptotic cell death. UNBS1450 mediates the decrease of Hsp70 at both mRNA and protein levels, and this is at least partly due to UNBS1450-induced downregulation of NFAT5/ TonEBP (a factor responsible for the transcriptional control of Hsp70). These effects were paralleled by the induction of LMP, as evidenced by acridine orange staining and immunofluorescence analysis for cathepsin B accumulation.

Bypassing p53

Many antineoplastic drugs elicit DNA damage, thereby activating p53-dependent apoptotic pathways. The existence of p53 mutations in many cancers thus likely contributes to drug resistance. Erdal et al . exposed p53 wild-type and p53 −/− cultured HCT116 colon carcinoma cell lines to each of 879 compounds (the National Cancer Institute mechanistic diversity set, known to cause growth inhibition through different mechanisms) in an effort to identify drugs that elicited apoptosis independently of p53. Of these, 175 compounds elicited apoptosis [assessed with an enzyme-linked immunosorbent assay (ELISA) for caspase-cleaved cytokeratin-18] at a concentration of 5 μM or less. Further analysis of 15 compounds that induced apoptosis with little or no dependence on p53 expression indicated that Bax was required for apoptosis with all 15 and that 11 of them activated caspase-3 (assessed by flow cytometry) in enucleated cells, suggesting a cytoplasmic target. Vital staining with Acridine Orange indicated that seven of the drugs with cytoplasmic targets caused permeabilization of lysosomal membranes; moreover, these seven compounds stimulated translocation of the proteases cathepsin B and cathepsin D from lysosomes to cytoplasm. Pepstatin A, a cathepsin D inhibitor, partially inhibited apoptosis in response to these seven compounds. The authors concluded that these drugs stimulated a p53-independent pathway to apoptosis that involved lysosomal permeabilization and that drugs using such a mechanism might be useful in cancer therapy. H. Erdal, M. Berndtsson, J. Castro, U. Brunk, M. C. Shoshan, S. Linder, Induction of lysosomal membrane permeabilization by compounds that activate p53-independent apoptosis. Proc. Natl. Acad. Sci. U.S.A. 102 , 192-197 (2005). [Abstract] [Full Text]

Induction of lysosomal membrane permeabilization by compounds that activate p53-independent apoptosis.

The p53 protein activates cellular death programs through multiple pathways. Because the high frequency of p53 mutations in human tumors is believed to contribute to resistance to commonly used chemotherapeutic agents, it is important to identify drugs that induce p53-independent cell death and to define the mechanisms of action of such drugs. Here we screened a drug library (the National Cancer Institute mechanistic set; 879 compounds with diverse mechanisms of actions) and identified 175 compounds that induced caspase cleavage of cytokeratin-18 in cultured HCT116 colon cancer cells at <5 microM. Interestingly, whereas most compounds elicited a stronger apoptotic response in cells with functional p53, significant apoptosis was observed also in p53-null cells. A subset of 15 compounds showing weak or no dependence on p53 for induction of apoptosis was examined in detail. Of these compounds, 11 were capable of activating caspase-3 in enucleated cells. Seven such compounds with nonnuclear targets were found to induce lysosomal membrane permeabilization (LMP). Translocation of the lysosomal proteases cathepsin B and cathepsin D into the cytosol was observed after treatment with these drugs, and apoptosis was inhibited by pepstatin A, an inhibitor of cathepsin D. Apoptosis depended on Bax, suggesting that LMP induced a mitochondrial apoptotic pathway. We conclude that a large number of potential anticancer drugs induce p53-independent apoptosis and that LMP is a mediator of many such responses.