Role Of Autophagy In Apoptosis Research Articles

The role of HIF-1α-mediated autophagy in ionizing radiation-induced testicular injury.

Testis, as a key organ for maintaining male fertility, are extremely sensitive to ionizing radiation (IR). IR-induced testicular dysfunction and infertility are common adverse effects of radiation therapy in patients with pelvic cancer. To study the phenotype and mechanism of IR-induced testicular injury, the mice were irradiated with different radiation doses (0, 2 and 5Gy) below the semi-lethal dose for one month. Our present results showed that testicular weight and the serum testosterone levels significantly decreased with the structural injury of the testis in an IR dose-dependent manner, indicating that IR caused not only the structural damage of the testis, but also the functional damage. Further analysis by TUNEL staining and Western blotting found that IR induced the apoptosis in a dose-dependent manner as indicated by increased expressions of cleaved caspase3, p53 and Bax on Day 15 after IR treatment. Combined with significantly increased oxidative stress, these results indicated that IR-induced testicular damage may be a long-term, progressively aggravated process, accompanied by apoptosis. Given the role of autophagy in apoptosis, the present study also detected and analyzed the localization and expressions of autophagy-related proteins LC-3I/II, beclin1, p62 and Atg12 in testicular cells, and found that LC-3II, beclin1 and Atg12 expressions significantly increased in the testicular cells of mice irradiated with 2Gy and 5Gy, while p62 expression significantly decreased with 5Gy, implying autophagy was involved in the apoptosis of testicular cells induced by IR. Furthermore, the expressions of HIF-1α and BNIP3 were significantly enhanced in the testis cells of mice irradiated with 2Gy and 5Gy, suggesting the potential role of HIF-1α/BNIP3-mediated autophagy in the apoptosis of testicular cells induced by IR. Taken together, our findings demonstrated that HIF-1α/BNIP3-mediated autophagy not only plays a protective effect on the testicular cells of mice irradiated with 2Gy, but also induces the apoptosis of the testicular cells of mice irradiated with 5Gy, indicating the double effects on apoptosis, which will help us further understanding the molecular mechanisms of IR-induced testicular injury, and will facilitate us further studies on testicular radioprotection.

The delay in cell death caused by the induction of autophagy by P2Et extract is essential for the generation of immunogenic signals in melanoma cells.

P2Et extract obtained from the Caesalpinia spinosa plant is abundant in phenolic compounds such as gallic acid and ethyl gallate and can generate signals to activate the immune response by inducing a mechanism known as immunogenic cell death in murine models of breast cancer and melanoma. Immunogenic cell death involves mechanisms such as autophagy, which can be modulated by various natural compounds, including phenolic compounds with a structure similar to those found in P2Et extract. Here, we determine the role of autophagy in apoptosis and the generation of immunogenic signals using murine wild-type B16-F10 melanoma cells and cells with beclin-1 gene knockout. We show that P2Et extract and ethyl gallate induced autophagy, partially protecting tumor cells from death and promoting calreticulin exposure and the release of ATP. Although ethyl gallate showed a mechanism similar to that of P2Et, the induction of apoptosis and immunogenic signals was significantly weaker. In contrast, gallic acid-induced autophagy acted by blocking autophagic flux, which was associated with increased cell death. However, this compound did not induce any of the immunogenic death signals evaluated. Therefore, the complex extract has greater antitumor potential than isolated compounds. Here, we show that inducing autophagic flux with P2Et protects cancer cells from cell death and that this delay in cell death is required for the generation of immunogenic signals.

Valproic Acid Enhanced Apoptosis by Promoting Autophagy Via Akt/mTOR Signaling in Glioma

Glioma is the most common malignant tumor in the central nervous system with a poor median survival. Valproic acid (VPA), a widely used antiepileptic drug, has been found to have antitumor effects on gliomas, but its role still has not been determined. In this study, we investigated VPA-induced apoptotic and autophagic effects on human U251 and SNB19 cells by cell counting kit-8 assay, flow cytometry, terminal deoxynucleotidyl transferase-mediated nick end labeling staining, western blots, and immunofluorescence assay in vitro, and then we further explored the role of autophagy in apoptosis by using the selective antagonist MHY1485. The data showed that VPA inhibited U251 and SNB19 glioma cells viability in a dose-dependent and time-dependent manner and induced apoptosis through the mitochondria-dependent pathway in vitro. In addition, VPA activated the Akt/mTOR pathway by decreasing their protein phosphorylation to promote cellular apoptosis. Surprisingly, the mTOR agonist MHY1485, causing a strong elevation of mTOR activity, partially reduced apoptosis ratio, which supposing that the autophagy of VPA is involved in the regulation of apoptosis. These findings suggest that VPA enhanced apoptosis by promoting autophagy via Akt/mTOR signaling in glioma, which could be further evaluated as a reliable therapy for glioma.

SIRT1-mediated FoxOs pathways protect against apoptosis by promoting autophagy in osteoblast-like MC3T3-E1 cells exposed to sodium fluoride.

Fluorine may result in damage to teeth, bones and other body tissues, and is a serious public health problem. SIRT1 deacetylates FOXOs, which brings about apoptosis and autophagy promotion or suppression. Fluorine may induce cell apoptosis, however, the role of autophagy in apoptosis induced by fluorine is still poorly understood, and the interaction between SIRT1 and FOXOs should be further illustrated. Therefore, this study investigated the mechanisms underlying the NaF- induced apoptosis and autophagy in osteoblast-like MC3T3-E1 cells in vitro through activating or inhibiting SIRT1. Via RT-PCR, western blot, flow cytometry assays, fluorescence and laser confocal microscopy, it was found that NaF induced both cell apoptosis and autophagy. Results also showed that NaF up-regulated SIRT1 expression in a dose-dependent manner. The autophagy of MC3T3-E1 was also up- regulated indirectly whilst apoptosis was significantly attenuated when incubated with the SIRT1 activator SRT1720. When SIRT1 inhibitor Ex-527 was used, the latter effects were reversed. Furthermore, SIRT1 increased deacetylation of FoxO1 and promoted the up-regulation of its target substrate Rab7, as well as increase of Bnip3 which was substrate of FoxO3, and we hypothesize that these pathways may cause an increase in autophagic flux and a reduction in apoptosis. In conclusion, SIRT1-induced autophagy enhancement protects against fluoride-induced apoptosis through autophagy induction in MC3T3-E1 cells, which may be associated with a SIRT1-FoxO1-Rab7 axis and a SIRT1-FoxO3-Binp3 axis. The role of SIRT1 in selecting between cell survival and death provides a potential therapeutic strategy in fluorosis.

Regulatory role of autophagy in globular adiponectin-induced apoptosis in cancer cells.

Adiponectin, an adipokine predominantly secreted from adipose tissue, exhibits diverse biological responses, including metabolism of glucose and lipid, and apoptosis in cancer cells. Recently, adiponectin has been shown to modulate autophagy as well. While emerging evidence has demonstrated that autophagy plays a role in the modulation of proliferation and apoptosis of cancer cells, the role of autophagy in apoptosis of cancer cell caused by adiponectin has not been explored. In the present study, we demonstrated that globular adiponectin (gAcrp) induces both apoptosis and autophagy in human hepatoma cell line (HepG2 cells) and breast cancer cells (MCF-7), as evidenced by increase in caspase-3 activity, Bax, microtubule-associated protein light chain 3-II (LC3 II) protein levels, and autophagosome formation. Interestingly, gene silencing of LC3B, an autophagy marker, significantly enhanced gAcrp-induced apoptosis in both HepG2 and MCF-7 cell lines, whereas induction of autophagy by rapamycin, an mTOR inhibitor, significantly prevented gAcrp-induced apoptosis in hepatoma cells HepG2. Furthermore, modulation of autophagy produced similar effects on gAcrp-induced Bax expression in HepG2 cells. These results implicate that induction of autophagy plays a regulatory role in adiponectin-induced apoptosis of cancer cells, and thus inhibition of autophagy would be a novel promising target to enhance the efficiency of cancer cell apoptosis by adiponectin.

Radiation-induced autophagy contributes to cell death and induces apoptosis partly in malignant glioma cells.

PurposeRadiation-induced autophagy has been shown to play two different roles, in malignant glioma (MG) cells, cytocidal or cytoprotective. However, neither the role of radiation-induced autophagy for cell death nor the existence of autophagy-induced apoptosis, a well-known cell-death pathway after irradiation, has been verified yet.Materials and MethodsWe observed both temporal and dose-dependent response patterns of autophagy and apoptosis to radiation in MG cell lines. Additionally, we investigated the role of autophagy in apoptosis through knockdown of autophagy-related proteins.ResultsAutophagic activity measured by staining of acidic vesicle organelles and Western blotting of LC-3 protein increased in proportion to radiation dose from day 1 to 5 after irradiation. Apoptosis measured by annexin-V staining and Western blotting of cleaved poly(ADP-ribose) polymerase demonstrated relatively late appearance 3 days after irradiation that increased for up to 7 days. Blocking of pan-caspase (Z-VAD-FMK) did not affect apoptosis after irradiation, but silencing of Atg5 effectively reduced radiation-induced autophagy, which decreased apoptosis significantly. Inhibition of autophagy in Atg5 knockdown cells was shown to be beneficial for cell survival. Stable transfection of GFP-LC3 cells was observed after irradiation. Annexin-V was localized in cells bearing GFP-LC3 punctuated spots, indicating autophagy in immunofluorescence. Some of these punctuated GFP-LC3 bearing cells formed conglomerated spots and died in final phase.ConclusionThese findings suggest that autophagy appears earlier than apoptosis after irradiation and that a portion of the apoptotic population that appears later is autophagy-dependent. Thus, autophagy is a pathway to cell death after irradiation of MG cells.

Role of autophagy in insulin-promoting cisplatin-induced apoptosis in EC9706 cells

Objective To investigate the role of autophagy in apoptosis induced by cisplatin and insulin in human esophageal squamous cells.Methods EC9706 cells were treated with 140 mg/L cisplatin,and cell viability was measured by using methyl thiazol tetrazolium (MTT) assay.The time T1 when the number of living cells reached a plateau was 24 h,then the time T2 was found when cell viability reached a new plateau with the addition of 10 mU/ml insulin after 6 h.At T1 and T2 time points,the cells were stained by Annexin V-fluoresceine isothiocyanate (FITC) apoptosis detection kit and monodansylcadaverine (MDC),respectively.Cell fluorescence was observed qualitatively under a fluorescence microscope,and apoptosis and the fluorescence intensity of MDC were detected quantitatively by using flow cytometry.Western blotting was used to investigate autophagy-related Beclin-1 changes that occurred in the course of treatment.Results (1) Apoptosis ratio of T1 plateau cells [(32.6 ± 4.3) ％] was lower than that of T2 plateau cells [(47.5 ± 5.6) ％] (P ＜ 0.05),and the MDC fluorescence intensity at T1 plateau [(104.9 ± 13.2)] was higher than that at T2 plateau [(82.6 ± 10.3)] significantly (P ＜ 0.05).(2) After co-treatment with insulin,the expression of Beclin-1 was significantly down-regulated (P ＜ 0.0l).Conclusion Inhibition of autophagy by insulin enhances the effect of cisplatin-induced apoptosis in esophageal cancer cells.Insulin is likely to be in this way to realize its chemotherapy sensitization effect. Key words: Esophageal squamous cell carcinoma; Autophagy; Apoptosis; Insulin

Dual PI3K/mTOR inhibitor NVP-BEZ235-induced apoptosis of hepatocellular carcinoma cell lines is enhanced by inhibitors of autophagy

Dysregulation of the phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling has been found in several types of human cancer, including hepatocellular carcinoma (HCC). NVP-BEZ235 is a novel, orally bioavailable dual PI3K/mTOR inhibitor that has exhibited promising activity against HCC in preclinical models. Autophagy is a cellular lysosomal degradation pathway essential for the regulation of cell survival and death to maintain homeostasis. This process is negatively regulated by mTOR signaling and often counteracts the efficacy of certain cancer therapeutic agents. In this study, we explored the role of autophagy in apoptosis induced by NVP-BEZ235 in two HCC cell lines, Hep3B and PLC/PRF/5, and identified the mechanism of combinatorial treatment. NVP-BEZ235 was effective in inhibiting the growth of the two HCC cell lines possibly though induction of apoptosis. NVP-BEZ235 also potently increased the expression of LC3-II and decreased the expression of p62, indicating induction of autophagy. When NVP-BEZ235 was used in combination with Atg5 siRNA or the autophagy inhibitor 3-methyladenine (3-MA), enhancement of the inhibitory effects on the growth of HCC cells was detected. In addition, enhanced induction of apoptosis was observed in cells exposed to the combination of NVP-BEZ235 and Atg5 siRNA or 3-MA. Thus, induction of autophagy by NVP-BEZ235 may be a survival mechanism that counteracts its anticancer effects. Based on these data, we suggest a strategy to enhance the anticancer efficacy of BEZ235 by blockade of autophagy. Thus, our study provides a rationale for the clinical development of combinations of NVP-BEZ235 and autophagy inhibitors for the treatment of HCC and other malignancies.

Abstract 4844: Dual effect of Akt on autophagy in pancreatic cancer cells

Abstract Background & Aims: Key steps in autophagic protein degradation are formation of autophagosomes (induction) and their fusion with lysosomes (progression). Akt is a major anti-apoptotic factor in cancer cells; however, its role in autophagy in general, and in pancreatic cancer (PaCa) in particular, is poorly understood. We recently showed that phosphatases PHLPP-I and -II are key negative regulators of Akt in PaCa cells. The present study aimed to elucidate the effects of Akt and PHLPPs on individual steps of autophagy and the role of autophagy in the prosurvival function of Akt in PaCa cells. Methods: We measured apoptosis by DNA fragmentation and caspase-3 activity; autophagy, by conversion of cytosolic LC3-I to autophagic LC3-II (with immunoblot) and LC3 immunofluorescence; autophagy induction, in cells pretreated with NH4Cl to block autophagy progression; autophagosomal fusion with lysosomes, by co-localization of the lysosomal marker LAMP-1 with LC3-II; and efficiency of protein degradation, by level of p62 protein, a specific autophagy target. Results: Akt inactivation by AktVIII inhibitor or siRNA, or by overexpression of PHLPP-I and -II all stimulated autophagic vacuole formation in MiaPaCa-2 and PANC-1 cells. Akt inactivation greatly facilitated LC3-I/LC3-II conversion both in the presence and absence of NH4Cl, indicating increased autophagy induction. At the same time, Akt inactivation inhibited autophagosomal fusion with lysosomes and efficiency of protein degradation, indicating decreased autophagy progression. Thus, Akt has dual effect on autophagy in PaCa cells: it inhibits autophagy induction but promotes autophagosomal fusion with lysosomes and hence autophagy progression. The overall effect of Akt is decreased accumulation of autophagic vacuoles, i.e., inhibition of autophagic death. To study the role of autophagy in apoptosis, we used 3-methyladenine or beclin siRNA to inhibit autophagy induction, and bafilomycin or chloroquine to block the fusion of autophagosomes with lysosomes. All these treatments stimulated apoptosis in PaCa cells, indicating the anti-apoptotic role of autophagy. In cells with inactivated Akt, blocking autophagy induction (e.g., with 3-methyladenine) further greatly stimulated apoptosis. This indicates that the ability of Akt to inhibit autophagy induction greatly diminishes its anti-apoptotic function. Differently, blocking autophagosomal fusion with lysosomes had little effect on apoptosis in cells in which Akt was inactivated, likely because in these cells autophagy progression was already inhibited. Conclusions: Akt inhibition hampers autophagy progression, thus promoting vacuolization and autophagic cell death. At the same time, Akt inhibition greatly stimulates autophagy induction, thus counteracting apoptosis. Therefore, to maximally stimulate apoptosis in PaCa cells we propose to apply Akt inhibitors in combination with inhibitors of autophagy. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4844.

Rottlerin induces autophagy and apoptotic cell death through a PKC-delta-independent pathway in HT1080 human fibrosarcoma cells: The protective role of autophagy in apoptosis

Rottlerin is widely used as a protein kinase C-δ inhibitor. Recently, several reports have shown the possible apoptosis-inducing effect of rottlerin in some cancer cell lines. Here we report that rottlerin induces not only apoptosis but also autophagy via a PKC- δ-independent pathway in HT1080 human fibrosarcoma cells. Rottlerin treatment induced a dose- and time-dependent inhibition of cell growth, and cytoplasmic vacuolations were markedly shown. These vacuoles were identified as acidic autolysosomes by electron microscopy, acidic vesicular organelle (AVO) staining and transfection of green fluorescent protein-LC3. The LC3-II protein level also increased after treatment with rottlerin. Prolonged exposure to rottlerin eventually caused apoptosis via loss of mitochondrial membrane potential and translocation of AIF from mitochondria to the nucleus. However, the activities of caspase-3, -8, and -9 were not changed, and PARP did not show signs of cleavage. Interestingly, the pretreatment of cells with a specific inhibitor of autophagy (3-methyladenine) accelerated rottlerin-induced apoptosis as revealed by an analysis of the subdiploid fraction and TUNEL assay. Nevertheless, the knockdown of PKC-δ by RNA interference neither affected cell growth nor acidic vacuole formation. Similarly, rottlerin-induced cell death was not prevented by PKC-δ overexpression. Taken together, these findings suggest that rottlerin induces early autophagy and late apoptosis in a PKC-δ-independent manner, and the rottlerin-induced early autophagy may act as a survival mechanism against late apoptosis in HT1080 human fibrosarcoma cells.