Iwona Ciechomska

Inhibition of Akt kinase signalling and activation of Forkhead are indispensable for upregulation of FasL expression in apoptosis of glioma cells

Activation of Akt signalling pathway is frequently found in glioma cells and may contribute to their resistance to undergo apoptosis in response to conventional therapies. We found that cyclosporin A (CsA) induces apoptosis of C6 glioma cells, which is associated with transcriptional activation of fasL. In the present paper, we investigated an involvement of Akt signalling in the regulation of FasL expression in CsA-induced apoptosis. We demonstrated that the level of active Akt decreases significantly after CsA treatment, which results in the decrease of Forkhead phosphorylation and its translocation to the nucleus. It correlated with an increase of binding to the Forkhead-responsive element FHRE from the FasL promoter, as demonstrated by gel-shift assays. Although treatment with LY294002, a specific inhibitor of PI3 K, decreased the phosphorylation of Akt and increased Fkhr translocation to the nucleus, these events were not sufficient to induce FasL expression and apoptosis of C6 glioma cells. Interference with Akt/Forkhead signalling by membrane-targeted Akt or removal of the FKHR-binding sites from the FasL promoter significantly abolished its activation. These results indicate that downregulation of Akt signalling and activation of Forkhead is a prerequisite for the induction of FasL promoter. It may be clinically important for pharmacological intervention in gliomas.

Endoplasmic reticulum stress triggers autophagy in malignant glioma cells undergoing cyclosporine A-induced cell death

Autophagy is a conserved, self-digestion process that is activated in response to nutrient limitation but acting also as an alternative death mechanism under certain conditions. It is accompanied by the progressive formation of vesicle structures from autophagosomes to autophagolysosomes orchestrated by autophagy effectors (Atg proteins) and modulators (that is, mTOR—mammalian target of rapamycin as a negative regulator). Malignant gliomas are highly resistant to current therapies that induce apoptosis, thus induction of the alternative cell death is an attractive strategy. We demonstrate that cyclosporine A (CsA, an immunophilin/calcineurin inhibitor) induces cell death with some apoptotic features but also accompanied by the appearance of numerous cytoplasmic vacuoles, immunostained for endoplasmic reticulum (ER) and autophagy markers. The induction of ER stress in glioma cells by CsA was evidenced by detection of unfolded protein response activation (phosphorylation of PERK, accumulation of IRE1α) and accumulation of ER stress-associated proteins (BIP and CHOP). Formation of the acidic vesicular organelles, increase of autophagic vacuoles, GFP-LC3 punctation (microtubule-associated protein light chain 3) and LC3-II accumulation upon CsA treatment confirmed activation of autophagy. Decrease of phosphorylation of 4E-BP1, p70S6K1 and its downstream target S6 ribosomal protein demonstrate inhibition of mTOR signaling by CsA. Salubrinal and silencing of PERK and IRE1α partially blocked CsA-induced accumulation of LC3-II. It suggests that ER stress precedes CsA-induced autophagy. Surprisingly, silencing of autophagy effectors ULK1, Atg5 or Atg7 increased the level of active caspases 3, 7 and PARP degradation in CsA-treated cells. Our results demonstrate that CsA induces both apoptosis and autophagy in malignant glioma cells via induction of ER stress and inhibition of mTOR/p70S6K1 pathway, however autophagy is cytoprotective in this context.

Curcumin induces permanent growth arrest of human colon cancer cells: Link between senescence and autophagy

Curcumin, a natural polyphenol derived from the rhizome of Curcuma longa, is a potent anticancer agent, which restricts tumor cell growth both in vitro and in vivo. Thus far curcumin was shown to induce death of cancer cells. This study reports the induction of cellular senescence of human colon cancer cells HCT116 upon curcumin treatment. The SA-β-galactosidase activation was observed both in p53+/+ and p53-/- cells, however the latter ones were less sensitive to the prosenescent activity of curcumin. Upregulation of p53 and p21 proteins was observed in p53+/+ HCT116, while p53-independent induction of p21 was noticed in p53-/- HCT116. Moreover, the senescence of HCT116 cells was accompanied by autophagy, that was confirmed by electron microscopy observations of autophagosomes in the curcumin-treated cells as well as LC3-II expression, punctue staining of LC3 and increased content of acidic vacuoles. Inhibition of autophagy, due to the diminished expression of ATG5 by RNAi decreased the number of senescent cells induced by curcumin, but did not lead to increased cell death. Altogether, we demonstrated a new antitumor activity of curcumin leading to cancer cell senescence and revealed the presence of a functional link between senescence and autophagy in curcumin-treated cells.

Contents of myosin heavy chains in denervated slow and fast rat leg muscles

The total content of myosin heavy chain (MHC) and individual MHC isoforms were studied in 14-day denervated rat leg muscles: the slow-twitch (soleus) and fast-twitch (extensor digitorum longus and gastrocnemius) by biochemical methods. The weight of the denervated muscles decreased by about 50%, as compared to the control muscles. In all denervated muscles the total content of MHCs decreased, more so in the slow than in the fast muscles. We have observed that the proportion among the MHC isoforms changed: while MHC-1 and MHC-2B decreased, MHC-2A and MHC-2X increased. Taking into account muscle atrophy, the loss of MHC total content and the shift in pattern of MHC isoforms, the total net changes of the particular MHC isoforms were evaluated. It was found that the muscle content of each of the MHCs decreased after denervation, but their tissue concentration changed variously. The concentration of the MHC-1 and MHC-2B decreased in all denervated muscles, but that of the MHC-2A and MHC-2X changed variously, depending on the muscle. The concentration of MHC-2A decreased in the soleus and increased in the fast muscles, whereas the concentration of the MHC-2X changed inversely. In the denervated soleus a considerable amount of MHC-2X was expressed, while in the contralateral muscles this isoform was undetectable or appeared at trace levels.

Non-Autophagic GFP-LC3 Puncta Induced by Saponin and Other Detergents

Expression of GFP-LC3 is now in widespread use to visualize autophagy in cultured cells. Recently, Kuma et al. (Autophagy 2007; 3:323-8) highlighted some complications using GFP-LC3, demonstrating that punctate dots containing GFP-LC3 do not always represent autophagic structures. We report here that GFP-LC3 can also rapidly aggregate into autophagosome look-alike structures when cells are permeabilized with saponin before cell fixation. Treatment with saponin reduced diffuse cytosolic and nuclear GFP-LC3 but caused an increase in the number and intensity of fluorescent puncta per cell regardless of whether the cells were induced to undergo autophagy. Saponin also induced GFP-LC3 puncta in Atg5-/- MEF transfected with GFP-LC3, where no LC3-II is produced, demonstrating that the puncta are autophagosome-independent. The increase in GFP-LC3 puncta was not matched by an increase in endogenous LC3-II or GFP-LC3-II detected by immunoblotting when protein samples were normalized to cell number. A qualitatively similar effect was observed when cells were treated with other detergents commonly used for membrane permeabilization, such as CHAPS, Triton X-100 or digitonin. We also noted that tubulin could not be used to normalize for protein loading on blots after applying saponin as it was selectively extracted from untreated cells but not from cells treated with vinblastine. When using mild detergents to remove background fluorescence, we recommend using a membrane-associated protein such as ATP synthase β for normalization. Thus, detergents used prior to fixation may precipitate GFP-LC3 aggregation into structures that appear autophagosomal and so should be used with caution.

Loss of dystrophin and some dystrophin-associated proteins with concomitant signs of apoptosis in rat leg muscle overworked in extension.

Abstract This study investigated the basis for the high severity of damage to skeletal muscle due to eccentric exercise, i.e., to muscles generating force while lengthened. Fast and slow rat leg muscles maintained in an extended position were examined after 2–24 h of continuous stimulation. The treatment caused the injury to some regions of both muscles. Within the better preserved parts of the muscles, i.e., those without signs of necrotic processes, dystrophin, spectrin, and some of the dystrophin-associated proteins (β-dystroglycan, α-sarcoglycan, and γ-sarcoglycan) disappeared from sarcolemma of many fibers. The reduction or loss of dystrophin from the sarcolemma was more evident than that of other proteins examined, with sarcoglycans apparently being the most preserved. Several muscle fibers devoid of dystrophin contained apoptotic nuclei. Simultaneously, Bax, Bcl-2 and caspase-3 proteins appeared in many fibers. Our results indicate that a normal muscle overworking in an extended position undergoes the loss of several membrane skeletal proteins because of the excessive stress to the membrane cytoskeleton, which can lead to fiber death by either apoptosis or necrosis. This experimental model may represent a good model for mimicking the pathogenetic events in several muscular dystrophies.

Cyclosporine A and its non‐immunosuppressive derivative NIM811 induce apoptosis of malignant melanoma cells in in vitro and in vivo studies

Advanced melanoma is a highly malignant tumor with an increasing incidence that has a poor prognosis due to resistance to common therapeutic strategies. We have demonstrated previously that cyclosporine A (CsA) induces apoptosis of rat glioma cells, reactive astrocytes, and fibroblasts. In our present study, we investigated effects of CsA and its nonimmunosuppressive derivative NIM811 on survival of human and murine melanoma cells. We demonstrated that CsA and NIM811 affect survival of human and murine melanoma cells and induce morphological changes, alterations in nuclear morphology and an internucleosomal DNA fragmentation, consistent with an apoptotic type of death. Western blot analysis showed an activation of caspases 9, 7, 3 and PARP cleavage detectable at 24 hr after exposure of human melanoma cells to the drugs. CsA and NIM811 induced a significant increase in subG1 population of murine B16F10 melanoma cells indicative of apoptotic DNA fragmentation. Studies in murine model of melanoma showed that NIM811, but not CsA, retards tumor progression and significantly decreases tumor volume after intratumoral application. Our findings indicate that CsA and its derivatives may be new candidates for the treatment of melanoma patients.

BIX01294, an inhibitor of histone methyltransferase, induces autophagy-dependent differentiation of glioma stem-like cells

Glioblastoma (GBM) contains rare glioma stem-like cells (GSCs) with capacities of self-renewal, multi-lineage differentiation, and resistance to conventional therapy. Drug-induced differentiation of GSCs is recognized as a promising approach of anti-glioma therapy. Accumulating evidence suggests that unique properties of stem cells depend on autophagy. Here we demonstrate that BIX01294, an inhibitor of a G9a histone methyltransferase (introducing H3K9me2 and H3K27me3 repressive marks) triggers autophagy in human glioma cells. Pharmacological or genetic inhibition of autophagy decreased LC3-II accumulation and GFP-LC3 punctation in BIX01294-treated cells. GSCs-enriched spheres originating from glioma cells and GBM patient-derived cultures express lower levels of autophagy related (ATG) genes than the parental glioma cell cultures. Typical differentiation inducers that upregulate neuronal and astrocytic markers in sphere cultures, increase the level of ATG mRNAs. G9a binds to the promoters of autophagy (LC3B, WIPI1) and differentiation-related (GFAP, TUBB3) genes in GSCs. Higher H3K4me3 (an activation mark) and lower H3K9me2 (the repressive mark) levels at the promoters of studied genes were detected in serum-differentiated cells than in sphere cultures. BIX01294 treatment upregulates the expression of autophagy and differentiation-related genes in GSCs. Pharmacological inhibition of autophagy decreases GFAP and TUBB3 expression in BIX01294-treated GSCs suggesting that BIX01294-induced differentiation of GSCs is autophagy-dependent.

Why doesn’t Beclin 1, a BH3-only protein, suppress the anti-apoptotic function of Bcl-2?

Beclin 1 binds to Bcl-2 through its BH3 domain and this interaction inhibits starvation-induced autophagy. However, we have found that when Beclin 1 binds thus to Bcl-2, it fails to inhibit Bcl-2-mediated protection against four different inducers of apoptosis. In this punctum, we discuss possible reasons why Beclin 1 fails to behave like other BH3-only proteins and induce apoptosis.

Molecular Links Between Autophagy and Apoptosis

Macroautophagy (herein referred to as autophagy) contributes to the control of life and death throughout the animal and plant kingdoms. Bilateral links have been found between apoptosis and autophagy where inducers of apoptosis also induce autophagy and vice versa. In some cases, autophagy delays the onset of apoptosis and thus prolongs life although it may also promote apoptosis and other forms of cell death. It is thus of great biological and medical interest to understand the molecular connections between these two pathways, and try to utilize-or block-them selectively to aid induction of cell death (e.g., cancer cells) or inhibit death (e.g., in degenerative disorders). This chapter describes methods for studying apoptotic induction of autophagy and its effects on cell function. We also discuss potential pitfalls. Although cell lines are used as model systems, the substances and methods described here can be applied to primary cells and tissues.