An inhibition of p62/SQSTM1 caused autophagic cell death of several human carcinoma cells

p62/SQSTM1 (p62) is a multifunctional protein implicated in several signal transduction pathways and selectively degraded by autophagy, a process for lysosomal degradation of both protein and organelle. p62 was also recently reported to be overexpressed in various malignancies and its inhibition to suppress carcinoma cell proliferation. However, its correlation with autophagy in carcinoma cells has remained largely unknown. Therefore, in this study, we examined the effects of p62 inhibition on the regulation of autophagy and cell survival in p62‐positive carcinoma cells. p62‐silencing dramatically suppressed cell proliferation and induced autophagy in p62 expressing PC9 and A549 cells. Electron microscopical analysis revealed the formation of autophagosomes with multilayer membranes caused by p62‐silencing. p62 silencing‐mediated reduced cell viability was restored by both genomic and pharmacological inhibition of autophagy but not that of apoptosis. These findings were also detected in several types of carcinoma cell lines including adenocarcinomas and squamous cell carcinomas. Results of our present study revealed that an inhibition of p62 resulted in the formation of mis‐regulated autophagosomes with multilayer membranes and an autophagic cell death, and p62 can therefore be an attractive target for the development of anti‐neoplastic agents.     

p53-regulated autophagy is controlled by glycolysis and determines cell fate

The tumor suppressor p53 regulates downstream targets that determine cell fate. Canonical p53 functions include inducing apoptosis, growth arrest, and senescence. Non-canonical p53 functions include its ability to promote or inhibit autophagy and its ability to regulate metabolism. The extent to which autophagy and/or metabolic regulation determines cell fate by p53 is unclear. To address this, we compared cells resistant or sensitive to apoptosis by the p53 activator Nutlin-3a. In resistant cells, glycolysis was maintained upon Nutlin-3a treatment, and activated p53 promoted prosurvival autophagy. In contrast, in apoptosis sensitive cells activated p53 increased superoxide levels and inhibited glycolysis through repression of glycolytic pathway genes. Glycolysis inhibition and increased superoxide inhibited autophagy by repressing ATG genes essential for autophagic vesicle maturation. Inhibiting glycolysis increased superoxide and blocked autophagy in apoptosis-resistant cells, causing p62-dependent caspase-8 activation. Finally, treatment with 2-DG or the autophagy inhibitors chloroquine or bafilomycin A1 sensitized resistant cells to Nutlin-3a-induced apoptosis. Together, these findings reveal novel links between glycolysis and autophagy that determine apoptosis-sensitivity in response to p53. Specifically, the findings indicate 1) that glycolysis plays an essential role in autophagy by limiting superoxide levels and maintaining expression of ATG genes required for autophagic vesicle maturation, 2) that p53 can promote or inhibit autophagy depending on the status of glycolysis, and 3) that inhibiting protective autophagy can expand the breadth of cells susceptible to Nutlin-3a induced apoptosis.     

The cell death response to the ROS inducer, cobalt chloride, in neuroblastoma cell lines according to p53 status

Cobalt chloride (CoCl2), a hypoxia-mimetic agent, induces reactive oxygen species (ROS) generation, leading to cell death. Divergent data have been reported concerning p53 implication in this apoptotic mechanism. In this study, we studied cobalt-induced cell death in neuroblastoma cell lines carrying wild-type (WT) p53 ( SHSY5Y) and a mutated DNA-binding domain p53 [SKNBE(2c)]. CoCl2 induced an upregulation of p53, p21 and PUMA expression in WT cells but not in SKNBE(2c). In SHSY5Y cells, p53 serine-15 phosphorylation appeared early (6 h) in the mitochondria, and also in the nucleus after 12 h. In contrast, in SKNBE(2c) cells, the slight nuclear signal disappeared with CoCl2 treatment. In SHSY5Y cells, a mitochondrial pathway dependent on caspases [collapse of mitochondrial transmembrane potential (?Ψmt), caspase 3 and 9 activation], was activated in a time-dependent manner. SKNBE(2c) cells exhibited a delay in the cell death executive phase linked to a caspase-independent pathway, involving apoptosis inducing factor nuclear translocation, but also an autophagic process attested by LC3-II expression and cathepsin-B activation. The downregulation of p53 in SHSY5Y cells by siRNA induced a cell death pathway related to the one observed in SKNBE(2c) cells. Finally, CoCl2 induced time-dependent canonical p53 mitochondrial apoptosis in the WT p53 cell line, and caspase-independent cell death in cells with a mutated or KO p53.     

Dasatinib induces autophagic cell death in human ovarian cancer

BACKGROUND:

Dasatinib, an inhibitor of Src/Abl family kinases, can inhibit tumor growth of several solid tumors. However, the effect and mechanism of action of dasatinib in human ovarian cancer cells remains unknown.

METHODS:

Dasatinib‐induced autophagy was determined by acridine orange staining, punctate localization of GFP‐LC3, LC3 protein blotting, and electron microscopy. Significance of beclin 1, AKT, and Bcl‐2 in dasatinib‐induced autophagy and growth inhibition was assayed by small interfering RNA (siRNA) silencing and/or overexpression of the gene of interest.

RESULTS:

Dasatinib inhibited cell growth by inducing little apoptosis, but substantial autophagy in SKOv3 and HEY ovarian cancer cells. In vivo studies showed dasatinib inhibited tumor growth and induced both autophagy and apoptosis in a HEY xenograft model. Knockdown of beclin 1 and Atg12 expression with their respective siRNAs diminished dasatinib‐induced autophagy, whereas knockdown of p27Kip1 with specific siRNAs did not. Small hairpin RNA knockdown of beclin 1 expression reduced dasatinib‐induced autophagy and growth inhibition. Dasatinib reduced the phosphorylation of AKT, mTOR, p70S6K, and S6 kinase expression. Constitutive expression of AKT1 and AKT2 inhibited dasatinib‐induced autophagy in both HEY and SKOv3 cells. Dasatinib also reduced Bcl‐2 expression and activity. Overexpression of Bcl‐2 partially prevented dasatinib‐induced autophagy.

CONCLUSIONS:

Dasatinib induces autophagic cell death in ovarian cancer that partially depends on beclin 1, AKT, and Bcl‐2. These results may have implications for clinical use of dasatinib. Cancer 2010. © 2010 American Cancer Society.     

The induction of the p53 tumor suppressor protein bridges the apoptotic and autophagic signaling pathways to regulate cell death in prostate cancer cells

The p53 tumor suppressor protein plays a crucial role in influencing cell fate decisions in response to cellular stress. As p53 elicits cell cycle arrest, senescence or apoptosis, the integrity of the p53 pathway is considered a key determinant of anti-tumor responses. p53 can also promote autophagy, however the role of p53-dependent autophagy in chemosensitivity is poorly understood. VMY-1-103 (VMY), a dansylated analog of purvalanol B, displays rapid and potent anti-tumor activities, however the pathways by which VMY works are not fully defined. Using established prostate cancer cell lines and novel conditionally reprogrammed cells (CRCs) derived from prostate cancer patients; we have defined the mechanisms of VMY-induced prostate cancer cell death. Herein, we show that the cytotoxic effects of VMY required a p53-dependent induction of autophagy, and that inhibition of autophagy abrogated VMY-induced cell death. Cancer cell lines harboring p53 missense mutations evaded VMY toxicity and treatment with a small molecule compound that restores p53 activity re-established VMY-induced cell death. The elucidation of the molecular mechanisms governing VMY-dependent cell death in cell lines, and importantly in CRCs, provides the rationale for clinical studies of VMY, alone or in combination with p53 reactivating compounds, in human prostate cancer.     

Autophagic cell death induced by 5-FU in Bax or PUMA deficient human colon cancer cell

Autophagy is a membrane process that results in the transporting of cellular contents to lysosomes for degradation. Autophagic cell death is another way of programed cell death called type II PCD, which has complicated connection with apoptosis, both of these two types of cell death play an important role in tumor development. In this study, we investigated chemotherapeutic agent induced cell death pathway in wild type (WT), Bax^−/− and PUMA^−/− HCT116 cells. Bax or PUMA deficient cells had similar chemosensitivity to WT cells but were defective in undergoing apoptosis. The results of electron microscopy and GFP–LC3 localization assay showed that autophagy was induced in Bax or PUMA deficient cells but not in WT cells. mTOR activity was decreased in Bax or PUMA deficient cells which further indicated the up-regulation of autophagy. Inhibition of autophagy by 3-Methyladenine (3-MA) decreased the cell death in Bax or PUMA deficient cells. Taken together, these results suggest that autophagic cell death can be used as an alternative cell death pathway in apoptosis defective cells and may bring a new target for cancer therapy.     

Lapatinib induces autophagic cell death and inhibits growth of human hepatocellular carcinoma

Lapatinib, an orally adminstered small-molecule tyrosine kinase inhibitor targeting epidermal growth factor receptors (EGFR) and Her2/Neu, has been widely accepted in the treatment of breast cancer. In this study, we found that lapatinib induced cytotoxicity in human hepatoma Huh7, HepG2 and HA22T cells. For the mode of cell death, we found lapatinib induced a higher percent of dead cells and a lower percent of hypodiploid cells, suggesting non-apoptotic cell death in lapatinib-treated hepatoma cells. Moreover, lapatinib-induced autophagy in hepatoma cells was confirmed by the detection of autophagic LC3-II conversion, the up-regulation of autophagy-related proteins, and the down-regulation of p62 by immunoblotting. Autophagic cell death was demonstrated by images of punctuated LC3 patterns, a higher percent of acridine orange positive cells, as well as a partial rescue of cell death by autophagy inhibitor 3-methyladenine or chloroquine. We also found massive vacuoles in lapatinib-treated hepatoma cells by electronic microscopy. In addition, the shRNA of knocked-down autophagy-related proteins rescued the hepatoma cells from lapatinib-induced growth inhibition. We also demonstrated a reduction of tumorigenesis by lapatinib in vivo. In conclusion, lapatinib induced autophagic cell death and the growth of human hepatoma cells. Our study provides potential cancer therapies by using lapatinib as a treatment for hepatoma.     

mTORC2/mTORC1抑制剂OSI-027诱导骨髓增生异常综合征转化急性髓系白血病细胞发生自噬性死亡

目的:研究mTORC2/mTORC1抑制剂OSI-027对骨髓增生异常综合征转化急性髓系白血病(myel-odysplastic syndrome/acute myeloid leukemia,MDS/AML)细胞SKM-1增殖的影响,并从凋亡和自噬两方面研究其作用机制.方法:采用不同浓度的雷帕霉素和OSI-027分别...     

Ras-mediated activation of ERK by cisplatin induces cell death independently of p53 in osteosarcoma and neuroblastoma cell lines

Activation of the mitogen-activated protein kinases ERK1/2 by the chemotherapeutic agent cisplatin has been shown to result in either survival or cell death. The downstream mediators of these opposing effects are unknown, as are the upstream signaling molecules. Activation of ERK is required for accumulation and phosphorylation of p53 following cisplatin treatment. We studied the role of ERK activation after cisplatin treatment under p53-negative and p53-positive conditions using a tetracycline-dependent expression vector in Saos-2 osteosarcoma cells. Dose-dependent activation of ERK first occurred 3-6 h after a 2-h cisplatin incubation and declined after 12-24 h in several tumor cell lines. Incubation of cell lines with the MEK1 inhibitors PD98059 or UO126 after, but not during, cisplatin treatment completely inhibited cisplatin-induced activation of ERK. The activation of ERK by cisplatin was inhibited by transient transfection with dominant-negative Ras-N17 in Saos-2 cells. Treatment of cells with PD98059 or UO126 after cisplatin incubation or inhibition of signaling through ERK by tetracycline-regulated expression of dominant-inhibitory ERK enhanced resistance to cisplatin in p53-negative osteosarcoma cells and reduced cisplatin-induced apoptosis. P53 was stabilized and phosphorylated in a MEK1-dependent manner after cisplatin incubation in Kelly neuroblastoma cells. Inhibition of signaling through ERK increased cell survival after cisplatin treatment in these cells as well. Expression of functional p53 did not change the proapoptotic effects of ERK activation in response to cisplatin in Saos-2 cells. Our results suggest that cisplatin-induced activation of ERK is mediated by Ras. ERK activation increased cisplatin-induced cell death independently of p53 in osteosarcoma and neuroblastoma cell lines.     

Inhibition of autophagy overcomes glucocorticoid resistance in lymphoid malignant cells

Glucocorticoid (GC) resistance remains a major obstacle to successful treatment of lymphoid malignancies. Till now, the precise mechanism of GC resistance remains unclear. In the present study, dexamethasone (Dex) inhibited cell proliferation, arrested cell cycle in G0/G1-phase, and induced apoptosis in Dex-sensitive acute lymphoblastic leukemia cells. However, Dex failed to cause cell death in Dex-resistant lymphoid malignant cells. Intriguingly, we found that autophagy was induced by Dex in resistant cells, as indicated by autophagosomes formation, LC3-I to LC3-II conversion, p62 degradation, and formation of acidic autophagic vacuoles. Moreover, the results showed that Dex reduced the activity of mTOR pathway, as determined by decreased phosphorylation levels of mTOR, Akt, P70S6K and 4E-BP1 in resistant cells. Inhibition of autophagy by either chloroquine (CQ) or 3-methyladenine (3-MA) overcame Dex-resistance in lymphoid malignant cells by increasing apoptotic cell death in vitro. Consistently, inhibition of autophagy by stably knockdown of Beclin1 sensitized Dex-resistant lymphoid malignant cells to induction of apoptosis in vivo. Thus, inhibition of autophagy has the potential to improve lymphoid malignancy treatment by overcoming GC resistance.     

MiR-15a and miR-16 induce autophagy and enhance chemosensitivity of Camptothecin

It has been reported that persistent or excessive autophagy promotes cancer cell death during chemotherapy, either by enhancing the induction of apoptosis or mediating autophagic cell death. Here, we show that miR-15a and miR-16 are potent inducers of autophagy. Rictor, a component of mTORC2 complex, is directly targeted by miR-15a/16. Overexpression of miR-15a/16 or depletion of endogenous Rictor attenuates the phosphorylation of mTORC1 and p70S6K, inhibits cell proliferation and G1/S cell cycle transition in human cervical carcinoma HeLa cells. Moreover, miR-15a/16 dramatically enhances anticancer drug camptothecin (CPT)-induced autophagy and apoptotic cell death in HeLa cells. Collectively, these data demonstrate that miR-15a/16 induced autophagy contribute partly to their inhibition of cell proliferation and enhanced chemotherapeutic efficacy of CPT.     

Renal cancer-selective Englerin A induces multiple mechanisms of cell death and autophagy

Renal cell carcinoma (RCC), the most common malignancy of the kidney, is refractory to standard therapy and has an incidence that continues to rise. Screening of plant extracts in search of new agents to treat RCC resulted in the discovery of englerin A (EA), a natural product exhibiting potent selective cytotoxicity against renal cancer cells. Despite the establishment of synthetic routes to the synthesis of EA, very little is known about its mechanism of action. The results of the current study demonstrate for the first time that EA induces apoptosis in A498 renal cancer cells in addition to necrosis. The induction of apoptosis by EA required at least 24 h and was caspase independent. In addition, EA induced increased levels of autophagic vesicles in A498 cells which could be inhibited by nonessential amino acids (NEAA), known inhibitors of autophagy. Interestingly, inhibition of autophagy by NEAA did not diminish cell death suggesting that autophagy is not a cell death mechanism and likely represents a cell survival mechanism which ultimately fails. Apart from cell death, our results demonstrated that cells treated with EA accumulated in the G₂ phase of the cell cycle indicating a block in G₂/M transition. Moreover, our results determined that EA inhibited the activation of both AKT and ERK, kinases which are activated in cancer and implicated in unrestricted cell proliferation and induction of autophagy. The phosphorylation status of the cellular energy sensor, AMPK, appeared unaffected by EA. The high renal cancer selectivity of EA combined with its ability to induce multiple mechanisms of cell death while inhibiting pathways driving cell proliferation, suggest that EA is a highly unique agent with great potential as a therapeutic lead for the treatment of RCC.     

MDM-2 antagonists induce p53-dependent cell cycle arrest but not cell death in renal cancer cell lines

Renal cell cancers (RCC) are notoriously resistant to chemotherapy and radiotherapy. While mutations of the p53 tumor suppressor gene frequently contribute to therapy resistance in other epithelial cancers, p53 mutations are relatively rare in RCC. To date, there is conflicting evidence as to whether p53 signaling and function are otherwise proficient or defective in tumors with wild-type p53. In this study, we assayed p53 function in a series of RCC cell lines and normal proximal epithelial tubule cells using two different MDM-2 antagonists, Nutlin-3a and MI-219. Most cell lines with wild-type p53 responded to MDM-2 antagonists as evidenced by induction of p53 and its target gene p21. RCC cell lines treated with MDM-2 antagonists consistently accumulated in the G2/M phase of the cell cycle and this event was associated with inhibition of proliferation in RCC cell lines but not in normal proximal epithelial tubule cells. MDM-2 antagonists did not induce significant cell death in RCC cell lines, even with induction of p53-dependent pro-apoptotic genes. In contrast, MDM-2 antagonists caused significant cell death in LNCaP prostate adenocarcinoma cells. RCC cell lines with reduced p53, either by mutation or through ectopic expression of p53 shRNA, demonstrated enhanced sensitivity to cell death following sequential treatment with DNA damage and G2/M checkpoint abrogation. Our results suggest that wild-type p53 RCC cell lines are proficient in p53-dependent cell cycle arrest but defective in p53-dependent cell death.     

hTERT: a novel endogenous inhibitor of the mitochondrial cell death pathway

hTERT is the catalytic subunit of the telomerase and is hence required for telomerase maintenance activity and cancer cell immortalization. Here, we show that acute hTERT depletion has no adverse effects on the viability or proliferation of cervical and colon carcinoma cell lines, as evaluated within 72 h after transfection with hTERT-specific small interfering RNAs (siRNAs). Within the same time frame, hTERT depletion facilitated the induction of apoptotic cell death by cisplatin, etoposide, mitomycin C and reactive oxygen species, yet failed to sensitize cells to death induction via the CD95 death receptor. Experiments performed with p53 knockout cells or chemical p53 inhibitors revealed that p53 was not involved in the chemosensitizing effect of hTERT knockdown. However, the proapoptotic Bcl-2 family protein Bax was involved in cell death induction by hTERT siRNAs. Depletion of hTERT facilitated the conformational activation of Bax induced by genotoxic agents. Moreover, Bax knockout abolished the chemosensitizing effect of hTERT siRNAs. Inhibition of mitochondrial membrane permeabilization by overexpression of Bcl-2 or expression of the cytomegalovirus-encoded protein vMIA (viral mitochondrial inhibitor of apoptosis), which acts as a specific Bax inhibitor, prevented the induction of cell death by the combination of hTERT depletion and chemotherapeutic agents. Altogether, our data indicate that hTERT inhibition may constitute a promising strategy for facilitating the induction of the mitochondrial pathway of apoptosis.     

蟾蜍灵诱导人胃癌SGC7901细胞自噬与凋亡的研究

目的：探讨自噬在蟾蜍灵（bufalin）诱导人胃癌SGC7901细胞死亡中的作用.方法：不同浓度的bu-falin处理人胃癌SGC7901细胞,采用MTT法检测bufalin对SGC7901细胞增殖的抑制作用.透射电镜观察给药后SGC7901细胞的自噬现象;流式细胞术检测细胞凋亡.Western blot检测自噬标志LC3蛋白表达.结果：Bufalin对胃癌SGC7901细胞生长有显著的抑制作用,且此作用呈明显的时间-剂量依赖性.Bufalin给药后可诱导SGC7901细胞发生自噬;并且在bufalin给药前用氯喹阻断自噬明显提高了bufalin对SGC7901的细胞毒性（P＜0.05）.结论：Bufalin能明显抑制SGC7901细胞的生长,并且诱导其发生保护性自噬.Bufalin和自噬抑制剂的联合应用可能为胃癌治疗提供新的策略.     

Potentiation of kinesin spindle protein inhibitor-induced cell death by modulation of mitochondrial and death receptor apoptotic pathways

Targeting the mitotic motor kinesin kinesin spindle protein (KSP) is a new strategy for cancer therapy. We have examined the molecular events induced by KSP inhibition and explored possible mechanisms of resistance and sensitization of tumor cells to KSP inhibitors. We found that KSP inhibition induced cell death primarily via activation of the mitochondrial death pathway. In HeLa cells, inhibition of KSP by small-molecule inhibitor monastrol resulted in mitotic arrest and rapid caspase activation. BclXL phosphorylation and loss of mitochondrial membrane potential was detected before significant caspase activation, which was required to trigger the subsequent apoptotic pathway. In A549 cells, however, KSP inhibition did not induce mitochondrial damage, significant caspase activity, or cell death. A549 cells aberrantly exited mitosis, following a prolonged drug-induced arrest, and arrested in a G(1)-like state with 4N DNA content in a p53-dependent manner. Overexpression of BclXL provided a protective mechanism, and its depletion rescued the apoptotic response to monastrol. In addition, Fas receptor was up-regulated in A549 cells in response to monastrol. Treatment with Fas receptor agonists sensitized the cells to monastrol-induced cell death, following exit from mitosis. Thus, activation of the death receptor pathway offered another mechanism to enhance KSP inhibitor-induced apoptosis. This study has elucidated cellular responses induced by KSP inhibitors, and the results provide insights for a more effective cancer treatment with these agents.     

5-ALA mediated photodynamic therapy induces autophagic cell death via AMP-activated protein kinase

Photodynamic therapy (PDT) has been developed as an anticancer treatment, which is based on the tumor-specific accumulation of a photosensitizer that induces cell death after irradiation of light with a specific wavelength. Depending on the subcellular localization of the photosensitizer, PDT could trigger various signal transduction cascades and induce cell death such as apoptosis, autophagy, and necrosis. In this study, we report that both AMP-activated protein kinase (AMPK) and mitogen-activated protein kinase (MAPK) signaling cascades are activated following 5-aminolevulinic acid (ALA)-mediated PDT in both PC12 and CL1-0 cells. Although the activities of caspase-9 and -3 are elevated, the caspase inhibitor zVAD-fmk did not protect cells against ALA-PDT-induced cell death. Instead, autophagic cell death was found in PC12 and CL1-0 cells treated with ALA-PDT. Most importantly, we report here for the first time that it is the activation of AMPK, but not MAPKs that plays a crucial role in mediating autophagic cell death induced by ALA-PDT. This novel observation indicates that the AMPK pathway play an important role in ALA-PDT-induced autophagy.