Arkadiusz Orzechowski

Molecular basis of parthenolide-dependent proapoptotic activity in cancer cells

This review outlines the molecular events that accompany the anti-tumor action of parthenolide (PN). Parthenolide (PN) is naturally derived compound, isolated from plant Tanacetum parthenium. PN has been previously shown to withdraw cells from cell cycle or to promote cell differentiation, and finally to induce programmed cell death. Recent advances in molecular biology indicate that this sesquiterpene lactone might evoke the above-mentioned effects by indirect action on genes. PN was shown to inhibit NF-kappaB- and STATs-mediated antiapoptotic gene transcription. On one hand, the proapoptotic activity of PN includes stimulation of intrinsic apoptotic pathway with the higher level of intracellular ROS and modifications of Bcl-2 family proteins (conformational changes of Bak and Bax, Bid cleavage). On the other hand, PN amplifies the apoptotic signal through the sensitization of cancer cells to extrinsic apoptosis, induced by TNF-alpha. These effects are specific to tumor cells. Unique properties of PN make this agent a promising metabolic inhibitor to retard tumorigenesis and to suppress tumor growth.

Calcium Homeostasis and ER Stress in Control of Autophagy in Cancer Cells

Autophagy is a basic catabolic process, serving as an internal engine during responses to various cellular stresses. As regards cancer, autophagy may play a tumor suppressive role by preserving cellular integrity during tumor development and by possible contribution to cell death. However, autophagy may also exert oncogenic effects by promoting tumor cell survival and preventing cell death, for example, upon anticancer treatment. The major factors influencing autophagy are Ca2+ homeostasis perturbation and starvation. Several Ca2+ channels like voltage-gated T- and L-type channels, IP3 receptors, or CRAC are involved in autophagy regulation. Glucose transporters, mainly from GLUT family, which are often upregulated in cancer, are also prominent targets for autophagy induction. Signals from both Ca2+ perturbations and glucose transport blockage might be integrated at UPR and ER stress activation. Molecular pathways such as IRE 1-JNK-Bcl-2, PERK-eIF2α-ATF4, or ATF6-XBP 1-ATG are related to autophagy induced through ER stress. Moreover ER molecular chaperones such as GRP78/BiP and transcription factors like CHOP participate in regulation of ER stress-mediated autophagy. Autophagy modulation might be promising in anticancer therapies; however, it is a context-dependent matter whether inhibition or activation of autophagy leads to tumor cell death.

Mitofusin 2 (Mfn2): a key player in insulin-dependent myogenesis in vitro

We have previously shown that mitochondrial activity increases in response to insulin in differentiating muscle cells. Moreover, the protein kinase kinase/extracellular-signal-regulated kinase (MAPKK/ERK-MEK) inhibitor PD98059 accelerates insulin-mediated myogenesis, whereas the phosphatidylinositol 3-kinase (PI3-K) inhibitor LY294002 or blockade of mitochondrial respiration abrogates insulin-mediated myogenesis. Our present study focuses on the mitochondrial transmembrane protein, hyperplasia suppressor gene/mitofusin2 (HSG/Mfn2), which regulates both mitochondrial fusion (as demonstrated by perinuclear mitochondria clustering) and insulin-dependent myogenesis in vitro. Increased mitochondrial length and interconnectivity are not observed after the inhibition of PI3-K activity with LY294002. Insulin induces Mfn2 and subunits I and IV of cytochrome-c oxidase (MTCOI and NCOIV) in L6 myoblasts. Inhibition of the MEK-dependent signalling pathway elevates the Mfn-2 protein level. The molecular mechanism of this phenomenon is unknown, although immunoprecipitation studies indicate that, during insulin-mediated myogenesis, Ras protein (an upstream activator of the MAPK/ERK1/2 cascade) interacts with HSG/Mfn2 in muscle cells. Interaction of Ras with Mfn2 continues unless insulin is present and is reduced after PD98059 co-treatment indicating that insulin-mediated myogenesis is increased by the inhibition of MEK, most probably by the lack of mitogenic signals opposing muscle differentiation. We conclude that insulin-mediated myogenesis depends on PI3-K activity, which stimulates mitochondrial activity and the extensive fusion of mitochondria. We further suggest that insulin stimulates the expression of Mfn2 protein, which in turn binds to Ras and inhibits the MEK-dependent signalling pathway. At the same time, the PI3-K-dependent signalling pathway is boosted, mitochondrial respiration increases and the rate of myogenesis is accelerated.

Not only insulin stimulates mitochondriogenesis in muscle cells, but mitochondria are also essential for insulin-mediated myogenesis

Abstract.  Viability and myogenesis from C2C12 muscle cells and L6 rat myoblasts were dose‐dependently stimulated by insulin. The metabolic inhibitors of phosphatidyl‐inositol‐3‐kinase (PI‐3K, LY294002) and of MAPKK/ERK kinase (MEK, PD98059) differently affected insulin‐stimulated myogenesis of the cells. After LY294002 and PD98059 treatment, viability deteriorated and apparently an additive effect of both metabolic inhibitors was observed, irrespective of the method of measurement (neutral red or MTT assay). These inhibitors were antagonistic in myogenesis. Our results confirm that insulin regulates cell viability by at least two distinct pathways, namely by PI‐3K‐ and MEK‐dependent signalling cascades. Both pathways are agonistic in cell viability, whereas PI‐3K rather than MEK supports insulin‐mediated myogenicity. Accordingly, inhibition of insulin action by LY294002, but not PD98059, was accompanied with a reduced level of Ser473‐phosphorylated Akt with additional loss of myogenin protein. Besides, repression of insulin signalling by either PI‐3K or MEK inhibitor diminished expression of selected subunits of the mitochondrial oxidative phosphorylation enzymes (OXPHOS). In turn, insulin raised and accelerated protein expression of subunits I and IV of mitochondrial cytochrome‐c oxidase (COX). In addition, the level of myogenin, the molecular marker of terminal and general muscle differentiation indices decreased if selected OXPHOS enzymes were individually blocked by rotenone, myxothiazol or oligomycin. Summing up, our results pointed to mitochondria as an essential organelle for insulin‐dependent myogenesis. Insulin positively affects mitochondrial function by induction of OXPHOS enzymes, which provide energy indispensable for the anabolic effect of insulin.

Comparison of metabolic effects of EGF, TGF-α, and TGF-β1 in primary culture of fetal bovine myoblasts and rat L6 myoblasts

Abstract 1. 1. Comparative studies of EGF, TGF-α, and TGF-βl action on the synthesis of DNA and cellular proteins in rat L6 myogenic cells and fetal bovine myoblasts demonstrated considerable differences between particular growth factors, dependent on dose and target cells. 2. 2. Among examined growth factors only EGF exerted mitostimulatory action, more pronounced at lower concentrations. EGF, progressively with dose, stimulated protein synthesis much more effectively in fetal bovine myoblasts than in L6 cells. 3. 3. The dynamics of stimulation of protein synthesis by TGF-α was greater than by EGF in both examined types of cell cultures. 4. 4. The maximal response of fetal bovine myoblasts to TGF-α in a concentration of 100 ng/ml reached 370%, whereas EGF in a 10 times higher concentration stimulated protein synthesis only to 123% of control. 5. 5. In contrast to EGF, TGF-α significantly inhibits DNA synthesis. Inhibition of the mitogenic response with simultaneous stimulation of protein synthesis by TGF-α may indicate changes toward cell differentiation. 6. 6. TGF-β 1 in smallest concentration inhibits both DNA and protein synthesis. The suppressive action of TGF-β 1 was more distinct in fetal bovine myoblasts than in the L6 cell line. 7. 7. Increasing concentrations of TGF-β l diminished its inhibitory effect, even leading to stimulation of protein synthesis at higher doses in L6 myoblasts.

Sodium butyrate sensitizes human colon adenocarcinoma COLO 205 cells to both intrinsic and TNF-α-dependent extrinsic apoptosis

Overexpression of cFLIP protein seems to be critical in the antiapoptotic mechanism of immune escape of human COLO 205 colon adenocarcinoma cells. Actually, cFLIP appears to inhibit the death receptor ligand-mediated cell death. Application of the metabolic inhibitor sodium butyrate (NaBt), short-chain volatile fatty acid, sensitized COLO 205 cells to TNF-α-mediated apoptosis. Western-blot analysis revealed that the susceptibility of human COLO 205 cells to apoptogenic stimuli resulted from time-dependent reduction in cFLIP and simultaneous up-regulation of TNF-R1 protein levels. Additionally, the combined TNF-α and NaBt treatment caused cleavage of Bid and caspase-9 activation, as well as cytochrome c release from mitochondria. Thus, the evidence of this study indicates that NaBt facilitates the death receptor signal evoked by TNF-α. Moreover, NaBt alone initiated intrinsic apoptosis, that in turn was abolished by intracellular BCL-2 delivery. It confirms the involvement of mitochondria in the proapoptotic activity of NaBt. The activation of mitochondrial pathway was substantiated by up-regulated expression of BAK with concomitant reduction of antiapoptotic BCL-xL, XIAP and survivin proteins. These findings suggest that NaBt could represent a good candidate for the new therapeutic strategy aimed to improve chemo- and immunotherapy of colon cancer.

Preconditioning with millimolar concentrations of Vitamin C or N-acetylcysteine protects L6 muscle cells insulin-stimulated viability and DNA synthesis under oxidative stress

The effect of reactive oxygen/nitrogen species (ROS/RNS)(hydrogen peroxide -- H(2)O(2), superoxide anion radical O(2)*- and hydroxyl radical *OH -- the reaction products of hypoxanthine/xanthine oxidase system), nitric oxide (NO* from sodium nitroprusside -- SNP), and peroxynitrite (ONOO(-) from 3-morpholinosydnonimine -- SIN-1) on insulin mitogenic effect was studied in L6 muscle cells after one day pretreatment with/or without antioxidants. ROS/RNS inhibited insulin-induced mitogenicity (DNA synthesis). Insulin (0.1 microM), however, markedly improved mitogenicity in the muscle cells treated with increased concentrations (0.1, 0.5, 1 mM) of donors of H(2)O(2), O(2)*-, *OH, ONOO(-) and NO*. Cell viability assessed by morphological criteria was also monitored. Massive apoptosis was induced by 1 mM of donors of H(2)O(2) and ONOO(-), while NO* additionally induced necrotic cell death. Taken together, these results have shown that ROS/RNS provide a good explanation for the developing resistance to the growth promoting activity of insulin in myoblasts under conditions of oxidative or nitrosative stress. Cell viability showed that neither donor induced cell death when given below 0.5 mM. In order to confirm the deleterious effects of ROS/RNS prior to the subsequent treatment with ROS/RNS plus insulin one day pretreatment with selected antioxidants (sodium ascorbate - ASC (0.01, 0.1, 1 mM), or N-acetylcysteine - NAC (0.1, 1, 10 mM) was carried out. Surprisingly, at a low dose (micromolar) antioxidants did not abrogate and even worsened the concentration-dependent effects of ROS/RNS. In contrast, pretreatment with millimolar dose of ASC or NAC maintained an elevated mitogenicity in response to insulin irrespective of the ROS/RNS donor type used.

Delineation of signalling pathway leading to antioxidant-dependent inhibition of dexamethasone-mediated muscle cell death.

The molecular mechanism of the cell death-promoting effect of dexamethasone (Dex) was studied during myogenesis (10 days) in L6 muscle cells by making use of several indices such as cell viability (protein synthesis, mitochondrial respiration), mortality (DNA fragmentation, chromatin condensation, structural modifications) and immunocytochemical studies [hydrogen peroxide, m-calpain (calpain 2)]. Dex initially (2 nM) stimulated protein synthesis (P < 0.001), but a further increase (20 nM) did not stimulate, whereas a higher dose (200 nM) inhibited formation of cellular proteins (P < 0.001). The latter, apparently, resulted from impaired cell viability (P < 0.001). From the day 4, structural changes featuring cell death were observed. Antioxidants [sodium ascorbate (ASC), catalase (CAT) or N-acetyl-L-cysteine (NAC)] as well as the inhibition of transcription and translation by actinomycin D abrogated Dex-induced cell death (P < 0.001). Using a fluorescent probe (DCFH-DA) we directly corroborated the working hypothesis of the mediating role of H2O2 in the reduction of cell viability by the excess of glucocorticoids. We also found that tPKC, PLCγ, PLA2 were required to induce Dex-dependent cell death since inactivation of tPKC by H7 completely abolished the cytotoxic effect of Dex, while the blockade of PLCγ and PLA2 by U 73122 partially abolished the effect. Cell death was triggered by Ca2+ influx necessary to activate m-calpain since it was reversed by the calcium chelator EGTA or m-calpain inhibitor ALLN but not EDTA nor ALLM. However, cell viability impaired by Ca2+ ionophore A 23187 (P < 0.001) was neither reversed by EGTA, nor EDTA, nor caspase-3 blocker – Ac DEVD CHO, nor ALLN, nor antioxidants – ASC, NAC, CAT. Specific caspase-3 inhibitor Ac DEVD CHO also did not rescue cells from Dex-induced cell death (P < 0.001), in contrast to m-calpain inhibitor – ALLN. Taken together, these findings suggest that reactive oxygen species inhibit protein synthesis and amplify m-calpain-dependent proteolysis. The events that led to the death of L6 muscle cells most likely resulted from Dex-mediated repression of antioxidative defences on the genomic level.

Involvement of polyamines in epidermal growth factor (EGF), transforming growth factor (TGF)-α and -β1 action on culture of L6 and fetal bovine myoblasts

Abstract 1. 1. α-Difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase significantly abolished stimulation of protein synthesis evoked by EGF, TGF-α or -β 1 in L6 and fetal bovine myoblasts. 2. 2. The participation of polyamines in early events evoked by growth factors was shown by a significant stimulation of ornithine decarboxylase and Sdenosylmethionine decarboxylase activity as well as increased concentration of spermidine and spermine in L6 cells exposed to TGF-α and EGF. 3. 3. TGF-β 1 at a high concentration (1 ng/ml) increased protein synthesis in L6 myoblasts but inhibited it in fetal bovine myoblasts. Metabolic effects of TGF-β 1 in L6 cells was associated with an enhancement of decarboxylase activities, however there were no significant changes in cellular polyamine concentrations. Presented data suggest that polyamines are involved in the signal transduction pathway of EGF, TGF-α, and -β 1 in L6 and fetal bovine myoblasts.

Antiapoptotic action of prolactin is associated with up-regulation of Bcl-2 and down-regulation of Bax in HC11 mouse mammary epithelial cells.

The effect of prolactin on apoptosis and the expression of bcl-2 and bax in HC11 mouse mammary epithelial cells were investigated. Flow cytometric analysis of Bcl-2 level (FITC-conjugated monoclonal anti-Bcl-2 antibody and FITC-conjugated monoclonal anti-IgG1 antibody as a negative control), number of apoptotic cells and cell cycle phases (DNA stained with DAPI) was performed. Bax transcript was measured using the RT-PCR method with GAPDH serving as a reference gene. Administration of prolactin (5lg/ml) in the presence of insulin stimulated differentiation of mammary epithelial cells, which manifested in stopping cells at G0/G1 phase, cell swelling and increase of cell number with enhanced protein content. Moreover, prolactin highly significantly reduced the extent of apoptosis of HC11 cells during 48 h of incubation. Nevertheless, the apoptotic cell number rose with increased time length of cell culture, probably due to the resulting high cell density and EGF withdrawal from t he incubation medium. The antiapoptotic effect of prolactin was associated with up-regulation of bcl-2 expression, shown as an increase in cell numbers expressing this protooncogene and elevated Bcl-2 content in these cells. A negative relationship (r=−0.87, p≤0.001) between the number of apoptotic cells and those expressing bcl-2 was also found. Prolactin administration lowered Bax transcript by 68.8% and 70.7% after 3 and 6h, respectively. In conclusion, the results presented indicate that stimulation of bcl-2 expression with simultaneous suppression of bax may be key events in the mechanism of antiapoptotic action of prolactin in HC11 mammary epithelial cells.