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• W2035242519 abstract "Autophagy is being increasingly implicated in both cell survival and death. However, the intricate relationships between drug-induced autophagy and apoptosis remain elusive. Here we demonstrate that a tubulin-binding noscapine analog, (R)-9-bromo-5-((S)-4,5-dimethoxy-1,3-dihydroisobenzofuran-1-yl)-4-methoxy-6-methyl-5,6,7,8-tetrahydro-[1,3]-di-oxolo[4,5-g]isoquinoline (Red-Br-nos), exerts a novel autophagic response followed by apoptotic cell death in human prostate cancer PC-3 cells. Red-Br-nos-induced autophagy was an early event detectable within 12 h that displayed a wide array of characteristic features including double membranous vacuoles with entrapped organelles, acidic vesicular organelles, and increased expression of LC3-II and beclin-1. Red-Br-nos-triggered release of reactive oxygen species (ROS) and attenuation of ROS by tiron, a ROS scavenger, reduced the sub-G1 population suggesting ROS-dependent apoptosis. Abrogation of ROS also reduced autophagy indicating that ROS triggers autophagy. Pharmacological and genetic approaches to inhibit autophagy uncovered the protective role of Red-Br-nos-induced autophagy in PC-3 cells. Direct effects of the drug on mitochondria viz. disruption of normal cristae architecture and dissipation of mitochondrial transmembrane potential revealed a functional link between ROS generation, autophagy, and apoptosis induction. This is the first report to demonstrate the protective role of ROS-mediated autophagy and induction of caspase-independent ROS-dependent apoptosis in PC-3 cells by Red-Br-nos, a member of the noscapinoid family of microtubule-modulating anticancer agents. Autophagy is being increasingly implicated in both cell survival and death. However, the intricate relationships between drug-induced autophagy and apoptosis remain elusive. Here we demonstrate that a tubulin-binding noscapine analog, (R)-9-bromo-5-((S)-4,5-dimethoxy-1,3-dihydroisobenzofuran-1-yl)-4-methoxy-6-methyl-5,6,7,8-tetrahydro-[1,3]-di-oxolo[4,5-g]isoquinoline (Red-Br-nos), exerts a novel autophagic response followed by apoptotic cell death in human prostate cancer PC-3 cells. Red-Br-nos-induced autophagy was an early event detectable within 12 h that displayed a wide array of characteristic features including double membranous vacuoles with entrapped organelles, acidic vesicular organelles, and increased expression of LC3-II and beclin-1. Red-Br-nos-triggered release of reactive oxygen species (ROS) and attenuation of ROS by tiron, a ROS scavenger, reduced the sub-G1 population suggesting ROS-dependent apoptosis. Abrogation of ROS also reduced autophagy indicating that ROS triggers autophagy. Pharmacological and genetic approaches to inhibit autophagy uncovered the protective role of Red-Br-nos-induced autophagy in PC-3 cells. Direct effects of the drug on mitochondria viz. disruption of normal cristae architecture and dissipation of mitochondrial transmembrane potential revealed a functional link between ROS generation, autophagy, and apoptosis induction. This is the first report to demonstrate the protective role of ROS-mediated autophagy and induction of caspase-independent ROS-dependent apoptosis in PC-3 cells by Red-Br-nos, a member of the noscapinoid family of microtubule-modulating anticancer agents. IntroductionChemotherapy remains the mainstay of treatment for both early stage as well as metastatic tumors. The ultimate goal of successful chemotherapy is to strategically induce robust apoptosis with simultaneous suppression of survival signaling circuitries in cancer cells with minimal toxicity to normal cells. Various chemotherapeutic agents induce cell death by halting cell cycle progression, enhancing expression of pro-apoptotic molecules whereas down-regulating survival molecules that impede apoptosis. However, beyond the provocation of death-inducing signals, chemotherapeutic drugs unfavorably up-regulate apoptosis-inhibitory molecules such as survivin and XIAP (1Peng X.H. Karna P. O'Regan R.M. Liu X. Naithani R. Moriarty R.M. Wood W.C. Lee H.Y. Yang L. Mol. Pharmacol. 2007; 71: 101-111Crossref PubMed Scopus (70) Google Scholar), and trigger various stress-response-activated pathways that promote cell survival and adaptation (2Yu L. Wan F. Dutta S. Welsh S. Liu Z. Freundt E. Baehrecke E.H. Lenardo M. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 4952-4957Crossref PubMed Scopus (581) Google Scholar). One such stress-response mechanism is autophagy, a well conserved ancient mechanism of cellular self-destruction that mediates survival (3Levine B. Cell. 2005; 120: 159-162Abstract Full Text Full Text PDF PubMed Scopus (686) Google Scholar, 4Levine B. Yuan J. J. Clin. Invest. 2005; 115: 2679-2688Crossref PubMed Scopus (1465) Google Scholar, 5Levine B. Klionsky D.J. Dev. Cell. 2004; 6: 463-477Abstract Full Text Full Text PDF PubMed Scopus (3140) Google Scholar). Regulation of autophagy is highly complex with inputs from the cellular environment that promote cell survival through the phosphatidylinositol 3-OH kinase (PI3K) 2The abbreviations used are: PI3Kphosphatidylinositol 3-OH kinaseRed-Br-nos(R)-9-bromo-5-((S)-4,5-dimethoxy-1,3-dihydro-isobenzofuran-1-yl)-4-methoxy-6-methyl-5,6,7,8-tetrahydro-[1,3]di-oxolo[4,5-g]isoquinolineAVOacidic vesicular organellesAOacridine orangeDCFDA2′,7′-dichlorofluorescein diacetate3-MA3-methyladenineDHEdihydroethidiumJC-15,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolyl carbocyanine iodideZ-VAD-fmkbenzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethyl ketoneROSreactive oxygen speciessiRNAsmall interfering RNADMSOdimethyl sulfoxidePBSphosphate-buffered salineGFPgreen fluorescent proteinEndo-Gendonuclease GLC3light chain 3. /Akt/mammalian target of rapamycin pathway, members of the Bcl2 family, and p53 (6Pattingre S. Levine B. Cancer Res. 2006; 66: 2885-2888Crossref PubMed Scopus (248) Google Scholar, 7Pattingre S. Tassa A. Qu X. Garuti R. Liang X.H. Mizushima N. Packer M. Schneider M.D. Levine B. Cell. 2005; 122: 927-939Abstract Full Text Full Text PDF PubMed Scopus (2896) Google Scholar, 8Levine B. Abrams J. Nat. Cell. Biol. 2008; 10: 637-639Crossref PubMed Scopus (180) Google Scholar, 9Rosenbluth J.M. Pietenpol J.A. Autophagy. 2009; 5: 114-116Crossref PubMed Scopus (71) Google Scholar). Although autophagy has recently gained much attention for its paradoxical roles in cell survival and cell death, the true identity of autophagy lies in its adaptive cellular homeostatic and housekeeping mechanisms (10Levine B. Kroemer G. Cell Death Differ. 2009; 16: 1-2Crossref PubMed Scopus (152) Google Scholar). Induction of autophagy thus constitutes a protective mechanism that renders tumor cells resistant to death (11Kroemer G. Levine B. Nat. Rev. Mol. Cell Biol. 2008; 9: 1004-1010Crossref PubMed Scopus (1140) Google Scholar).Drug-induced autophagy is being increasingly implicated in modulating cell death responses. When considering treatment options, it is conceivable that autophagy could limit the effects of cytotoxic anticancer drugs through its ability to clear damaged organelles and proteins. It may also help cancer cells to survive chemotherapy-induced stress. In these scenarios, inhibiting autophagy would be beneficial to treatment outcome. Equally plausible, however, is the notion that drug-induced autophagy might contribute to tumor cell demise and in this case inhibition of autophagy would lead to enhanced tumor growth. Several anticancer agents that induce autophagy include tamoxifen, arsenic trioxide, temozolomide, HDAC inhibitors, etoposide, vitamin D analogs, and tubulin-binding drugs (paclitaxel and 2-methoxyestradiol) (12Høyer-Hansen M. Bastholm L. Mathiasen I.S. Elling F. Jäättelä M. Cell Death Differ. 2005; 12: 1297-1309Crossref PubMed Scopus (237) Google Scholar, 13Lee S.B. Tong S.Y. Kim J.J. Um S.J. Park J.S. DNA Cell Biol. 2007; 26: 713-720Crossref PubMed Scopus (34) Google Scholar, 14Shao Y. Gao Z. Marks P.A. Jiang X. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 18030-18035Crossref PubMed Scopus (532) Google Scholar, 15Kanzawa T. Germano I.M. Komata T. Ito H. Kondo Y. Kondo S. Cell Death Differ. 2004; 11: 448-457Crossref PubMed Scopus (831) Google Scholar, 16Kanzawa T. Kondo Y. Ito H. Kondo S. Germano I. Cancer Res. 2003; 63: 2103-2108PubMed Google Scholar, 17Bursch W. Ellinger A. Kienzl H. Török L. Pandey S. Sikorska M. Walker R. Hermann R.S. Carcinogenesis. 1996; 17: 1595-1607Crossref PubMed Scopus (460) Google Scholar). In these examples, autophagy has been shown to be pro-survival or death inducing in a context-dependent manner. Thus, manipulation of autophagy in favor of death induction in cancer cells would be contingent on context, such as cell-type, nature, and duration of stress etc.Ongoing efforts in our laboratory are actively focused on expansion of a novel class of anticancer agents, noscapinoids, based upon the parent noscapine (18Aneja R. Lopus M. Zhou J. Vangapandu S.N. Ghaleb A. Yao J. Nettles J.H. Zhou B. Gupta M. Panda D. Chandra R. Joshi H.C. Cancer Res. 2006; 66: 3782-3791Crossref PubMed Scopus (53) Google Scholar, 19Aneja R. Zhou J. Vangapandu S.N. Zhou B. Chandra R. Joshi H.C. Blood. 2006; 107: 2486-2492Crossref PubMed Scopus (65) Google Scholar, 20Aneja R. Liu M. Yates C. Gao J. Dong X. Zhou B. Vangapandu S.N. Zhou J. Joshi H.C. Cancer Res. 2008; 68: 1495-1503Crossref PubMed Scopus (29) Google Scholar, 21Aneja R. Zhou J. Zhou B. Chandra R. Joshi H.C. Mol. Cancer Ther. 2006; 5: 2366-2377Crossref PubMed Scopus (49) Google Scholar). Unlike conventional tubulin-binding agents, these novel molecules are weak affinity tubulin binders, and subtly attenuate microtubule dynamics without appreciably perturbing the steady state monomer/polymer ratio of tubulin (22Zhou J. Gupta K. Aggarwal S. Aneja R. Chandra R. Panda D. Joshi H.C. Mol. Pharmacol. 2003; 63: 799-807Crossref PubMed Scopus (147) Google Scholar, 23Zhou J. Panda D. Landen J.W. Wilson L. Joshi H.C. J. Biol. Chem. 2002; 277: 17200-17208Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). This unique property perhaps underlies the “kinder and gentler” nature of noscapine and its analogs and represents a unique edge over conventional anti-tubulin agents (24Heidemann S. Blood. 2006; 107: 2216-2217Crossref Scopus (9) Google Scholar). A novel noscapinoid, (R)-9-bromo-5-((S)-4,5-dimethoxy-1,3-dihydro-isobenzofuran-1-yl)-4-methoxy-6-methyl-5,6,7,8-tetrahydro-[1,3]di-oxolo[4,5-g]isoquinoline (hereon referred to as Red-Br-nos) has been shown to be significantly more potent than the founding molecule, noscapine (22Zhou J. Gupta K. Aggarwal S. Aneja R. Chandra R. Panda D. Joshi H.C. Mol. Pharmacol. 2003; 63: 799-807Crossref PubMed Scopus (147) Google Scholar). Previous reports demonstrate that Red-Br-nos arrests cell cycle at the G2/M phase and induces apoptosis in ovarian cancer cells in vitro and in vivo without detectable toxicity (25Zhou J. Liu M. Luthra R. Jones J. Aneja R. Chandra R. Tekmal R.R. Joshi H.C. Cancer Chemother. Pharmacol. 2005; 55: 461-465Crossref PubMed Scopus (35) Google Scholar, 26Zhou J. Liu M. Aneja R. Chandra R. Joshi H.C. Biochem. Pharmacol. 2004; 68: 2435-2441Crossref PubMed Scopus (30) Google Scholar). The identity of key determinants that arbitrate life and death decisions upon Red-Br-nos exposure and contribute to the final outcome of cell death are still unknown.The present study reports a novel protective autophagic response in PC-3 human prostate cancer cells upon Red-Br-nos treatment. The induction of autophagy preceded the onset of apoptosis. Both opposing responses, autophagy and apoptosis, were ROS triggered. Attenuation of ROS reduced autophagic protection and enhanced apoptosis induction, suggesting that ROS production was upstream of these two events. Red-Br-nos directly affected the mitochondria that generates ROS to trigger a protective autophagic response that perhaps finally sets the stage for apoptosis in the presence of overwhelming damage. This is the first report to identify the induction of autophagy by a novel noscapine family member, as a protective defense mechanism against apoptotic cell death.RESULTSRed-Br-nos Induces Robust Autophagy in Prostate Cancer CellsFormation of Double Membranous Autophagosomes in Red-Br-nos-treated PC-3 CellsSeveral members of the noscapinoid family (EM011, EM015) activate a mitochondrially mediated intrinsic apoptotic pathway to induce cell death in lymphoma and breast cancer cells (18Aneja R. Lopus M. Zhou J. Vangapandu S.N. Ghaleb A. Yao J. Nettles J.H. Zhou B. Gupta M. Panda D. Chandra R. Joshi H.C. Cancer Res. 2006; 66: 3782-3791Crossref PubMed Scopus (53) Google Scholar, 19Aneja R. Zhou J. Vangapandu S.N. Zhou B. Chandra R. Joshi H.C. Blood. 2006; 107: 2486-2492Crossref PubMed Scopus (65) Google Scholar, 20Aneja R. Liu M. Yates C. Gao J. Dong X. Zhou B. Vangapandu S.N. Zhou J. Joshi H.C. Cancer Res. 2008; 68: 1495-1503Crossref PubMed Scopus (29) Google Scholar, 21Aneja R. Zhou J. Zhou B. Chandra R. Joshi H.C. Mol. Cancer Ther. 2006; 5: 2366-2377Crossref PubMed Scopus (49) Google Scholar). Because mitochondrial damage has been widely implicated in the induction of autophagy, we asked if Red-Br-nos can also induce autophagy. Classically, electron microscopy has been considered as the gold standard to demonstrate autophagosomes in cells (28Kondo Y. Kondo S. Autophagy. 2006; 2: 85-90Crossref PubMed Scopus (279) Google Scholar). Thus, the ultrastructure of control and 25 μm Red-Br-nos-treated PC-3 cells was first examined using transmission electron microscopy. The choice of drug concentration (25 μm) was based upon dose response (supplemental Fig. S1) and time course (supplemental Fig. S2) flow cytometric experiments that determined the sub-G1 population that is indicative of apoptosis. As can be seen in Fig. 1A, PC-3 cells treated with Red-Br-nos revealed the appearance of large double-membranous cytoplasmic vacuoles (black arrowheads) as early as 12 h that progressively accumulated upon increasing the drug exposure time. These vacuoles resembled autophagosomes and several of them showed entrapped intracellular organelles such as mitochondria or endoplasmic reticulum, and digested residual material (Fig. 1A, black arrows). On the contrary, control PC-3 cells displayed large nuclei with finely dispersed chromatin material surrounded by cytoplasm with a normal complement of healthy looking mitochondria at all time points (12, 24, and 48 h) studied. After 48 h of drug exposure, cells with fragmented nuclei were observable indicating induction of apoptosis (data not shown). These results indicated that Red-Br-nos treatment caused formation of autophagosome-like structures and this cellular response preceded the onset of apoptosis in PC-3 cells.Recruitment of LC3-II into Autophagolysosomes and Up-regulation of Beclin-1 ExpressionMore recently, the microtubule-associated protein-1 LC3 has been used as a marker of autophagy. LC3 exists in two forms, an 18-kDa cytosolic protein (LC3-I) and a processed 16-kDa form (LC3-II) that is membrane-bound and increased during autophagy by conversion from LC3-I (28Kondo Y. Kondo S. Autophagy. 2006; 2: 85-90Crossref PubMed Scopus (279) Google Scholar). To gain insights into the mechanism of Red-Br-nos-induced autophagy, we examined drug effects on LC3, the mammalian homolog of the yeast autophagy protein Apg8/Aut7p (29Kabeya Y. Mizushima N. Ueno T. Yamamoto A. Kirisako T. Noda T. Kominami E. Ohsumi Y. Yoshimori T. EMBO J. 2000; 19: 5720-5728Crossref PubMed Scopus (5363) Google Scholar, 30Kirisako T. Baba M. Ishihara N. Miyazawa K. Ohsumi M. Yoshimori T. Noda T. Ohsumi Y. J. Cell Biol. 1999; 147: 435-446Crossref PubMed Scopus (708) Google Scholar). Red-Br-nos treatment caused processing of full-length LC3-I (18 kDa) to LC3-II (16 kDa) as evident by immunoblotting data using lysates from PC-3 cells treated with either DMSO (control) or 25 μm Red-Br-nos for the indicated time points (Fig. 1B, i). An immunoreactive band corresponding to processed LC3-II (16 kDa) was present with a weak intensity (3 and 6 h) in drug-treated lysates. However, at 12 h, LC3-II levels were increased by ∼6-fold compared with controls, suggesting induction of LC3-II, the form that is recruited to autophagosomes (Fig 1B, i). The autophagic response was not restricted to the lower drug concentration of 25 μm. Higher dose levels of Red-Br-nos (50 and 100 μm) also showed induction of autophagy as seen by increased LC3-II expression at 24 h post-treatment (supplemental Fig. S3).Next, we confirmed autophagy by quantifying GFP-LC3-tagged compartments by fluorescence microscopy. To this end, transient transfectants of PC-3 cells expressing GFP-LC3 were generated. Although there was a diffused localization of GFP-LC3 in control cells (Fig. 1B, ii), treatment of cells with Red-Br-nos for 24 h produced a punctate pattern for GFP-LC3 fluorescence, indicating recruitment of LC3 to autophagosomes during Red-Br-nos-induced autophagy (Fig. 1B, ii). GFP-LC3 compartments at 24 h post-treatment were quantified by counting GFP-LC3 dots in control and Red-Br-nos-treated cells (Fig. 1B, iii). Our results show that there was a significant increase in the number of GFP-LC3 dots by ∼80% in 24-h drug-treated cells compared with controls (Fig. 1B, ii). This clearly suggested increased induction of autophagy over time following Red-Br-nos treatment.An essential autophagy effector, beclin-1, is a central player in autophagosome formation (31Liang X.H. Jackson S. Seaman M. Brown K. Kempkes B. Hibshoosh H. Levine B. Nature. 1999; 402: 672-676Crossref PubMed Scopus (2700) Google Scholar). Immunoblotting data showed a time-dependent increase of beclin-1 expression upon drug exposure suggesting autophagic induction (Fig. 1C, i). Quantitation of Western data using ImageJ revealed a ∼3-fold increase in beclin-1 expression at 12 h compared with controls. There was an increase of beclin-1 positive cells by ∼3.7-fold at 24 h post-treatment as revealed by quantitation of at least 200 cells from random fields (Fig. 1C, iii).Red-Br-nos Causes Formation of AVOsYet another characteristic feature of cells engaged in autophagy is the formation of AVOs following treatment with different stimuli (32Daido S. Kanzawa T. Yamamoto A. Takeuchi H. Kondo Y. Kondo S. Cancer Res. 2004; 64: 4286-4293Crossref PubMed Scopus (353) Google Scholar, 33Paglin S. Hollister T. Delohery T. Hackett N. McMahill M. Sphicas E. Domingo D. Yahalom J. Cancer Res. 2001; 61: 439-444PubMed Google Scholar). Thus, we visualized the effect of Red-Br-nos treatment on formation of AVOs in PC-3 cells using fluorescence microscopy upon staining with the lysosomotropic agent AO (Fig. 2A). Essentially, AO is a weak base that traverses freely across biological membranes in an uncharged state characterized by green fluorescence. Its protonated form accumulates as aggregates in acidic compartments characterized by red fluorescence. As is visually evident in Fig. 2A, control cells primarily displayed green fluorescence with minimal red fluorescence, indicating a lack of AVOs. On the other hand, drug-treated PC-3 cells showed a ∼3.2-fold increase in red fluorescent AVOs at 24 h post-treatment compared with controls (Fig. 2B). We also quantitated AO fluorescence using flow cytometry (Fig. 2C). Histogram profiles show the mean fluorescence intensity of unstained cells (no AO, blue profile), control cells (AO, green profile), and drug-treated cells (profile), and drug-treated cells (AO, red profile) (Fig. 2C). There was an increase in red fluorescence intensity upon drug treatment indicating an enhancement of AVOs. Fig. 2D is a bar graph quantitation showing a ∼78% increase in red fluorescent cells upon drug treatment for 24 h compared with controls. These results provided further evidence to conclude that Red-Br-nos treatment induced autophagy in PC-3 cells.FIGURE 2Panel A, immunofluorescence microscopy of acridine orange-stained PC-3 cells treated for 24 h with DMSO (control) or 25 μm Red-Br-nos. Increase in number of cells with AO accumulating acidic vesicular organelles (orange-red fluorescence) in Red-Br-nos-treated cells was evident. Panel B, quantitation of cells with AVOs from 6 to 8 random image fields totaling ∼200 cells and reported as mean ± S.D. (p < 0.05). Panel C, histogram profiles of control and Red-Br-nos-treated cells that were read flow cytometrically. Blue profile shows unstained cells without AO as negative controls. Green profile depicts control cells that were stained with AO and the red profile shows drug-treated cells with increased AO fluorescence, indicative of numerous red AVOs. Panel D, bar graph representation of the quantitation of the mean fluorescence intensity in control and drug-treated PC-3 cells. Columns, mean ± S.D. (p < 0.05, compared with controls).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Red-Br-nos Triggers ROS GenerationSeveral reports provide strong evidence for the involvement of ROS in the induction of autophagy as well as apoptosis (2Yu L. Wan F. Dutta S. Welsh S. Liu Z. Freundt E. Baehrecke E.H. Lenardo M. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 4952-4957Crossref PubMed Scopus (581) Google Scholar, 34Xu Y. Kim S.O. Li Y. Han J. J. Biol. Chem. 2006; 281: 19179-19187Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). To test whether Red-Br-nos induced ROS production, we stained cells using DCFDA and read them flow cytometrically (Fig. 3A, i). DCFDA is cell permeable, cleaved by nonspecific cellular esterases, and reacts mainly with H2O2 and other peroxides to yield fluorescent DCF (35Cathcart R. Schwiers E. Ames B.N. Anal. Biochem. 1983; 134: 111-116Crossref PubMed Scopus (723) Google Scholar, 36Vanden Hoek T.L. Li C. Shao Z. Schumacker P.T. Becker L.B. J. Mol. Cell Cardiol. 1997; 29: 2571-2583Abstract Full Text PDF PubMed Scopus (331) Google Scholar). Red-Br-nos-treated PC-3 cells (red profile) exhibited a statistically significant increase in DCF mean fluorescence intensity by ∼1.8-fold compared with controls (blue profile) (Fig. 3A, i and ii). Microscopic inspection of DCFDA-stained drug-treated cells showed a significant increase in intensity of DCF staining compared with controls (Fig. 3A, iii). The increase in DCF fluorescence was also measured fluorimetrically using excitation and emission wavelengths at 485 and 535 nm, respectively. As shown in Fig. 3A, iv, drug treatment resulted in a ∼28% increase of fluorescence signal compared with controls, at 24 h post-treatment.FIGURE 3Red-Br-nos-triggered ROS generation in PC-3 cells. Panel A, i, histogram profiles of control and drug-treated cells that were read flow cytometrically upon DCFDA staining (an indicator of ROS generation). Panel ii, bar graph representation of the quantitation of the increase in the mean fluorescence intensity (DCF-positive cells) in PC-3 cultures treated with DMSO (control) or Red-Br-nos for 24 h. Columns, mean ± S.D. (*, p < 0.05, compared with controls). Panel iii, microscopic visualization of DCF fluorescence in control and drug-treated PC-3 cells. Panel iv, fluorimetric data showing an increase in DCF fluorescence upon drug treatment compared with controls. Panel B, i, flow cytometric histogram profiles of control (blue profile) and drug-treated (red profile) cells upon DHE staining (another indicator of ROS generation). Panel ii, bar graph representation of the quantitation of the increase in the mean fluorescence intensity (EtBr-positive cells) in PC-3 cultures treated with DMSO (control) or Red-Br-nos for 24 h. Panel iii, microscopic visualization of EtBr (EB) fluorescence in control and drug-treated PC-3 cells. Panel iv, fluorimetric data showing an increase in EtBr fluorescence upon drug treatment compared with controls. Panel C, i, attenuation of ROS by tiron reduced the autophagic response as seen by a decrease in the number of red acidic compartments (AVOs), a characteristic feature of autophagic vacuoles. Panel ii, abrogation of ROS by tiron also decreased beclin-1 expression levels upon Red-Br-nos treatment for 24 h, suggesting a clear involvement of ROS in induction of autophagy. Panel iii, ROS plays a role in inducing Red-Br-nos-induced apoptosis. Attenuating ROS levels by tiron reduced the sub-G1 population as shown in the three-dimensional disposition of cell-cycle profiles. Panel iv, bar graph representation of the quantitation of the sub-G1 population upon drug treatment in the presence or absence of tiron. Panel v shows an immunoblot analysis for total and cleaved caspase-3 at the noted time points. β-Actin was used as a loading control. Panel vi is a bar graph representation for caspase-3/7 activity measured at 24 h upon treatment with vehicle control, Red-Br-nos and docetaxel. Columns, mean ± S.D. (*, p < 0.05, compared with controls).View Large Image Figure ViewerDownload Hi-res image Download (PPT)The results observed using DCF fluorescence were next confirmed with another ROS probe, DHE that yields ethidium bromide (EtBr) upon DHE oxidation and accumulates as red fluorescence in the nucleus (36Vanden Hoek T.L. Li C. Shao Z. Schumacker P.T. Becker L.B. J. Mol. Cell Cardiol. 1997; 29: 2571-2583Abstract Full Text PDF PubMed Scopus (331) Google Scholar). Flow cytometry data showed a ∼1.6-fold increase of mean fluorescence intensity of EtBr upon drug treatment (red profile) compared with controls (blue profile) (Fig. 3B, i and ii). Indeed, upon drug treatment, nuclear staining with ethidium bromide was evident microscopically, indicating that PC-3 cells accumulated peroxides (Fig. 3A, iii). Fluorimetric data also showed an enhanced fluorescence emission at 580 nm correlating with a ∼27% increase of EtBr-stained cells at 24 h post-treatment compared with controls (Fig. 3B, iv).Red-Br-nos-induced ROS Triggers AutophagyROS serve as signaling molecules in many cellular processes including growth, differentiation, and apoptosis (37Scherz-Shouval R. Shvets E. Fass E. Shorer H. Gil L. Elazar Z. EMBO J. 2007; 26: 1749-1760Crossref PubMed Scopus (1624) Google Scholar, 38Azad M.B. Chen Y. Gibson S.B. Antioxid. Redox Signal. 2009; 11: 777-790Crossref PubMed Scopus (583) Google Scholar, 39Wu W.S. Cancer Metastasis Rev. 2006; 25: 695-705Crossref PubMed Scopus (616) Google Scholar). ROS specifically, H2O2, and superoxide anion O2̇̄ have been reported to induce autophagy (37Scherz-Shouval R. Shvets E. Fass E. Shorer H. Gil L. Elazar Z. EMBO J. 2007; 26: 1749-1760Crossref PubMed Scopus (1624) Google Scholar, 39Wu W.S. Cancer Metastasis Rev. 2006; 25: 695-705Crossref PubMed Scopus (616) Google Scholar, 40Scherz-Shouval R. Elazar Z. Trends Cell Biol. 2007; 17: 422-427Abstract Full Text Full Text PDF PubMed Scopus (754) Google Scholar). Having identified that Red-Br-nos induced generation of ROS, we next asked if ROS triggered autophagy in PC-3 cells. To investigate a functional link between ROS production and autophagy induction, we used tiron, a ROS scavenger, and examined its effect on AVO formation and beclin-1 expression over the Red-Br-nos treatment time. Attenuation of ROS levels by tiron significantly decreased the number of AVOs upon Red-Br-nos treatment (Fig. 3C, i). This was in contrast to Red-Br-nos treatment alone that caused a significant induction of cells with AVOs (Fig. 2A, i). In addition, treatment with tiron in the absence or presence of Red-Br-nos did not induce ROS production as evident by DCF staining (supplemental Fig. S4). Furthermore, there was an absence of drug-induced beclin-1 expression upon drug treatment in the presence of tiron as seen by immunoblotting methods (Fig. 3C, ii). Taken together, these data suggested ROS-mediated induction of autophagy in PC-3 cells.Red-Br-nos Induces ROS-dependent Apoptosis That Is Caspase-independentSeveral tubulin-binding drugs recruit mitochondrially mediated ROS signaling to induce apoptotic cell death (41Mizumachi T. Suzuki S. Naito A. Carcel-Trullols J. Evans T.T. Spring P.M. Oridate N. Furuta Y. Fukuda S. Higuchi M. Oncogene. 2008; 27: 831-838Crossref PubMed Scopus (52) Google Scholar). To investigate the involvement of ROS in Red-Br-nos-induced cell death, we monitored the drug-induced sub-G1 population (an indicator of cell death) in the absence or presence of tiron using flow cytometry. Fig. 3C, iii, is a three-dimensional representation of cell-cycle profiles of PC-3 cells that were treated with drug in the absence or presence of tiron. Quantitation data showed that there was a decline in the drug-induced sub-G1 population by ∼2.7-fold when ROS was inhibited by tiron, suggesting that ROS played a crucial role in induction of cell death (Fig. 3C, iv). Because caspase activation is a hallmark of classical apoptosis, cleaved caspase-3 expression was examined by immunoblotting (Fig. 3C, v). There was an absence of cleaved caspase-3 expression upon drug treatment over time (Fig. 3C, v). Furthermore, we also measured caspase-3/7 activity at 24 h of drug treatment (Fig. 3C, vi). Docetaxel was included as a positive control. We found that there were no changes in caspase-3/7 activity patterns in drug-treated cells compared with controls (Fig. 3C, vi). However, 2 nm docetaxel increased caspase-3 activity (Fig. 3C, vi). In addition, there was an absence of activated caspase-3, caspase-7, caspase-8, caspase-9, and caspase-2 upon a 48-h Red-Br-nos treatment (supplemental Fig. S5). Docetaxel treatment, however, showed an increase of activated caspase-3, caspase-7, caspase-8, and caspase-9 compared with controls (supplemental Fig. S5). Drug treatment in the presence of the pan-caspase inhibitor (Z-VAD-fmk) showed no significant" @default.
• W2035242519 created "2016-06-24" @default.
• W2035242519 creator A5019846100 @default.
• W2035242519 creator A5051568194 @default.
• W2035242519 creator A5063670409 @default.
• W2035242519 creator A5069985048 @default.
• W2035242519 creator A5077311826 @default.
• W2035242519 creator A5077988118 @default.
• W2035242519 date "2010-06-01" @default.
• W2035242519 modified "2023-10-03" @default.
• W2035242519 title "Induction of Reactive Oxygen Species-mediated Autophagy by a Novel Microtubule-modulating Agent" @default.
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