Diallyl trisulfide-induced prostate cancer cell death is associated with Akt/PKB dephosphorylation mediated by P-p66shc

PURPOSE: P66Shc, an isoform of adaptor proteins, is known to mediate various signals including those leading to apoptosis or cell proliferation. Previously, we have shown that diallyl trisulfide (DATS)-induced prostate cancer cell death was mediated by increased ROS formation. In this study, we investigated the role of p66Shc protein and its serine 36 phosphorylation in DATS induced decrease in prostate cancer cell viability (PC-3). METHODS: PC-3 prostate cancer cells were used in this study. Stable cell lines expressing p66ShcS36A or an empty vector have been obtained. Cell viability, concentration of ROS, changes in P-p66Shc and P-Akt and DNA damage were determined. RESULTS: We observed that DATS treatment increased p66Shc phosphorylation at serine 36. Importantly, the phosphorylation was abolished by JNK inhibitor SP600125. Cells expressing plasmid-encoded variant of p66ShcS36A showed much higher resistance to DATS-induced cells death. In addition to that, we observed that DATS-induced ROS formation was completely abolished in cells expressing the p66ShcS36A variant. Interestingly, SP600125 proved to prevent DATS-induced Akt inactivation. In order to confirm that the observed effect is related to phosphorylation of p66Shc, we performed experiments on a stable cell line expressing p66ShcS36A. In such cells, DATS-induced Akt dephosphorylation was significantly reduced. On the other hand, hydrogen peroxide induced Akt activation in PC-3 cells, which was abrogated in cells expressing p66ShcS36A. CONCLUSIONS: Our results uncover a novel signaling pathway with p66Shc being indispensable for DATS-induced inactivation of Akt due to hypophosphorylation

NADH-generating substrates reduce peroxyl radical toxicity in RL-34 cells

There is general agreement that oxidative stress may induce apoptotic and necrotic cell death. Recently it has been shown that NADH can be considered an important antioxidant as it reacts with peroxyl and alkoxyl radicals under in vitro conditions. Therefore, in the present study we hypothesized that an increase in intracellular NADH using specific substrates will protect RL-34 cells against cytotoxicity of 2’-azobis (2-amidinopropane) dihydrochloride (AAPH), which is a peroxyl radical generating compound. Cells treated for 24 hours with 6.0 mM AAPH were severely damaged: mitochondria were vacuolated, and the level of free radicals significantly increased. Both apoptotic and necrotic cells were detected (11.1% and 11.4%, respectively) even after 5 hours of treatment. Pretreatment of the cells with substrates which increase the intracellular level of NADH, such as lactate, beta-hydroxybutyrate, and ethanol, distinctly inhibited AAPH-induced reactive oxygen species (ROS) formation and cell death. On the other hand, acetoacetate (AcA), which decrease the intracellular level of NADH, had opposite effects. Interestingly, NADH-generating substrates augment, while AcA reduced superoxide radical formation induced by AAPH. These results may suggest that although NADH generating substrates may exert some deleterious effects within a cell by inducing reductive stress, they diminish alkoxyl or peroxyl radical cytotoxicity. The protection is associated with a decrease in ROS formation measured by dichlorofluorescein, but with an increase in superoxide radical formation

A role for accessory genes rI.-1 and rI.1 in the regulation of lysis inhibition by bacteriophage T4

Lysis inhibition (LIN) is a known feature of the T-even family of bacteriophages. Despite its historical role in the development of modern molecular genetics, many aspects of this phenomenon remain mostly unexplained. The key element of LIN is an interaction between two phage-encoded proteins, the T holin and the RI antiholin. This interaction is stabilized by RIII. In this report, we demonstrate the results of genetic experiments which suggest a synergistic action of two accessory proteins of bacteriophage T4, RI.-1, and RI.1 with RIII in the regulation of LIN

Benefits and risks of the hormetic effects of dietary isothiocyanates on cancer prevention

The isothiocyanate (ITC) sulforaphane (SFN) was shown at low levels (1-5 µM) to promote cell proliferation to 120-143% of the controls in a number of human cell lines, whilst at high levels (10-40 µM) it inhibited such cell proliferation. Similar dose responses were observed for cell migration, i.e. SFN at 2.5 µM increased cell migration in bladder cancer T24 cells to 128% whilst high levels inhibited cell migration. This hormetic action was also found in an angiogenesis assay where SFN at 2.5 µM promoted endothelial tube formation (118% of the control), whereas at 10-20 µM it caused significant inhibition. The precise mechanism by which SFN influences promotion of cell growth and migration is not known, but probably involves activation of autophagy since an autophagy inhibitor, 3-methyladenine, abolished the effect of SFN on cell migration. Moreover, low doses of SFN offered a protective effect against free-radical mediated cell death, an effect that was enhanced by co-treatment with selenium. These results suggest that SFN may either prevent or promote tumour cell growth depending on the dose and the nature of the target cells. In normal cells, the promotion of cell growth may be of benefit, but in transformed or cancer cells it may be an undesirable risk factor. In summary, ITCs have a biphasic effect on cell growth and migration. The benefits and risks of ITCs are not only determined by the doses, but are affected by interactions with Se and the measured endpoint

The C-terminal domain of the Escherichia coli RNA polymerase α subunit plays a role in the CI-dependent activation of the bacteriophage λ pM promoter

The bacteriophage λ pM promoter is required for maintenance of the λ prophage in Escherichia coli, as it facilitates transcription of the cI gene, encoding the λ repressor (CI). CI levels are maintained through a transcriptional feedback mechanism whereby CI can serve as an activator or a repressor of pM. CI activates pM through cooperative binding to the OR1 and OR2 sites within the OR operator, with the OR2-bound CI dimer making contact with domain 4 of the RNA polymerase σ subunit (σ4). Here we demonstrate that the 261 and 287 determinants of the C-terminal domain of the RNA polymerase α subunit (αCTD), as well as the DNA-binding determinant, are important for CI-dependent activation of pM. We also show that the location of αCTD at the pM promoter changes in the presence of CI. Thus, in the absence of CI, one αCTD is located on the DNA at position −44 relative to the transcription start site, whereas in the presence of CI, αCTD is located at position −54, between the CI-binding sites at OR1 and OR2. These results suggest that contacts between CI and both αCTD and σ are required for efficient CI-dependent activation of pM

The Isoxazole Derivative of Usnic Acid Induces an ER Stress Response in Breast Cancer Cells that Leads to Paraptosis-like Cell Death

Derivatives of usnic acid (UA), a secondary metabolite from lichens, were synthesized to improve its anticancer activity and selectivity. Recently we reported the synthesis and activity of an UA isoxazole derivative, named 2b, against cancer cells of different origins. Herein, the molecular mechanisms underlying its activity and efficacy in vivo were tested. The viability of breast cancer or normal cells has been tested using an MTT assay. Cell and organelle morphology was analyzed using light, electron and fluorescence microscopy. Gene expression was evaluated by RNAseq and protein levels were evaluated by Western blotting. In vivo anticancer activity was evaluated in a mice xenograft model. We found that 2b induced massive vacuolization which originated from the endoplasmic reticulum (ER). ER stress markers were upregulated both at the mRNA and protein levels. ER stress was caused by the release of Ca2+ ions from the ER by IP3R channels which was mediated, at least partly, by phospholipase C (PLC)-synthetized 1,4,5-inositol triphosphate (IP3). ER stress led to cell death with features of apoptosis and paraptosis. When applied to nude mice with xenografted breast cancer cells, 2b stopped tumour growth. In mice treated with 2b, vacuoli-zation was observed in tumour cells, but not in other organs. This study shows that the antiprolif-erative activity of 2b relates to the induction of ER stress in cancer, not in healthy, cells and it leads to breast cancer cell death in vitro and in vivo

Erratum: Synthesis of Usnic Acid Derivatives and Evaluation of Their Antiproliferative Activity against Cancer Cells (Journal of Natural Products (2019) 82:7 (1768-1778) DOI: 10.1021/acs.jnatprod.8b00980)

The (+)-enantiomer of compound 1 was incorrectly assigned as the S-configuration where it is in fact the Rconfiguration. As a consequence, compounds 2b, 3b, 3d, 3f, 3h, 3j, 3l, and 3n are also the R-configuration. Page 1769: The table within Scheme 2 should be substituted with the following:. The compound names within the Supporting Information (pp 2-6) need to be altered as follows: • (R)-8-Acetyl-5,7-dihydroxy-3,4a,6-trimethyl-1-phenyl- 1,4a-dihydro-4Hbenzofuro[3,2-f ]indazol-4-one (3d) • (R)-8-Acetyl-5,7-dihydroxy-1-(4-methoxyphenyl)- 3,4a,6-trimethyl-1,4a-dihydro4H-benzofuro[3,2-f ]- indazol-4-one (3f) • (R)-8-Acetyl-1-(4-fluorophenyl)-5,7-dihydroxy-3,4a,6- trimethyl-1,4a-dihydro-4Hbenzofuro[3,2-f ]indazol-4- one (3h). • (R)-8-Acetyl-1-(3,4-dichlorophenyl)-5,7-dihydroxy- 3,4a,6-trimethyl-1,4a-dihydro4H-benzofuro[3,2-f ]- indazol-4-one (3j) (Table Presented). • (R)-8-Acetyl-1-(4-chlorophenyl)-5,7-dihydroxy-3,4a,6- trimethyl-1,4a-dihydro4H-benzofuro[3,2-f ]indazol-4- one (3n) Accordingly, the structures of compounds 2b, 3b, 3d, 3f, 3h, 3j, 3l, and 3n in the Supporting Information have been revised. Additionally, the Abstract is revised as follows: Moreover, they induced massive cytoplasmic vacuolization, which was associated with elevated dynamin-dependent endocytosis, a process that has not been reported for usnic acid and indicates a novel mechanism of action of its synthetic derivatives. The authors sincerely apologize for any inconvenience caused by these errors

Activation of Nrf2 signaling augments Vesicular Stomatitis Virus Oncolysis via Autophagy-Driven Suppression of Antiviral Immunity

Oncolytic viruses (OVs) offer a promising therapeutic approach to treat multiple types of cancer. In this study, we show that the manipulation of the anti-oxidant network via transcription factor Nrf2 augments vesicular stomatitis virus Δ51 (VSVΔ51) replication and sensitizes cancer cells to viral oncolysis. Activation of Nrf2 signaling by the antioxidant compound sulforaphane (SFN) leads to enhanced VSVΔ51 spread in OV-resistant cancer cells and improves the therapeutic outcome in different murine syngeneic and xenograft tumor models. Furthermore, chemoresistant A549 lung cancer cells that display a constitutive dominant hyperactivation of Nrf2 signaling are particularly vulnerable to VSVΔ51 oncolysis. Mechanistically, enhanced Nrf2 signaling stimulates viral replication in cancer cells and disrupts the type I IFN response via increased autophagy. This study reveals a previously unappreciated role for Nrf2 in the regulation of autophagy and the innate antiviral response that complements the therapeutic potential of VSV-directed oncolysis against multiple types of OV-resistant or chemoresistant cancer

Regulation of the switch from early to late bacteriophage lambda DNA replication

There are two modes of bacteriophage k DNA replication following infection of its host, Escherichia coli. Early after infection, replication occurs according to the theta (teta or circle-to-circle) mode, and is later switched to the sigma (sigma or rolling-circle) mode. It is not known how this switch, occurring at a specific time in the infection cycle, is regulated. Here it is demonstrated that in wildtype cells the replication starting from orik proceeds both bidirectionally and unidirectionally, whereas in bacteria devoid of a functional DnaA protein, replication from orik is predominantly unidirectional. The regulation of directionality of replication from orik is mediated by positive control of lambda pR promoter activity by DnaA, since the mode of replication of an artificial lambda replicon bearing the ptet promoter instead of pR was found to be independent of DnaA function. These findings and results of density-shift experiments suggest that in dnaA mutants infected with lambda, phage DNA replication proceeds predominantly according to the unidirectional teta mechanism and is switched early after infection to the sigma mode. It is proposed that in wild-type E. coli cells infected with lambda, phage DNA replication proceeds according to a bidirectional teta mechanism early after infection due to efficient transcriptional activation of ori lambda, stimulated by the host DnaA protein. After a few rounds of this type of replication, the resulting increased copy number of teta genomic DNA may cause a depletion of free DnaA protein because of its interaction with the multiple DnaA-binding sites in lambda DNA. It is proposed that this may lead to inefficient transcriptional activation of orik resulting in unidirectional teta replication followed by sigma type replication