Mechanisms of autophagy and apoptosis: Recent developments in breast cancer cells

Autophagy,the pathway whereby cell components are degraded by lysosomes,is involved in the cell response to environmental stresses,such as nutrient deprivation,hypoxia or exposition to chemotherapeutic agents.Under these conditions,which are reminiscent of certain phases of tumor development,autophagy either promotes cell survival or induces cell death. This strengthens the possibility that autophagy could be an important target in cancer therapy,as has been proposed.Here,we describe the regulation of survival and death by autophagy and apoptosis,especially in cultured breast cancer cells.In particular,we discuss whether autophagy represents an apoptosis-independent process and/or if they share common pathways. We believe that understanding in detail the molecular mechanisms that underlie the relationships between autophagy and apoptosis in breast cancer cells could improve the available treatments for this disease.     

Growth inhibitory effect of paratocarpin E,a prenylated chalcone isolated from Euphorbia humifusa Wild., by induction of autophagy and apoptosis in human breast cancer cells

Five flavones, including four flavonoids and one prenylated chalcone (paratocarpin E), were isolated from E. humifusa. and their chemical structures were established by spectroscopic analyses. We assessed the efficacy of these compounds against the growth of human breast cancer, leukemic, kidney cancer cell lines. Among them, paratocarpin E showed significant cytotoxicity against these cancer cell lines with an IC₅₀ of 19.6 μM on the growth of MCF-7 cells. Paratocarpin E treatment of MCF-7 cells resulted in typical apoptotic features via increasing expression of activated caspase-8 and -9 and PARP cleavage. Moreover, paratocarpin E altered the expression of Bax and Bcl-2, leading to the release of cytochrome c from the mitochondria into the cytosol, suggesting that the mitochondria-mediated apoptosis was initiated. In addition, paratocarpin E increased the MDC-positive autophagic vacuoles, the ratio of LC3-II/LC3-I protein levels of Beclin-1, but decreased p62 expression, indicating the potent pro-autophagic effects of paratocarpin E in MCF-7 cells. Mechanistically, cell death induced by paratocarpin E is able to induce apoptosis of MCF-7 cells by activating p38 and JNK signaling pathway while inhibiting Erk pathway. Furthermore, paratocarpin E promotes the activation and nuclear translocation of NF-κB, which plays an important role in balancing paratocarpin E-mediated apoptosis and autophagy. The molecular docking study also revealed that paratocarpin E bound to Fas and NF-κB complex. These findings provide initial evidences that paratocarpin E can be used as a potential anti-cancer drug in future for breast cancer therapy.     

Polygonatum odoratum lectin induces apoptosis and autophagy via targeting EGFR-mediated Ras-Raf-MEK-ERK pathway in human MCF-7 breast cancer cells

Polygonatum odoratum lectin (POL), a mannose-binding GNA-related lectin, has been reported to display remarkable anti-proliferative and apoptosis-inducing activities toward a variety of cancer cells; however, the precise molecular mechanisms by which POL induces cancer cell death are still elusive. In the current study, we found that POL could induce both apoptosis and autophagy in human MCF-7 breast cancer cells. Subsequently, we found that POL induced MCF-7 cell apoptosis via the mitochondrial pathway. Additionally, we also found that POL induces MCF-7 cell apoptosis via EGFR-mediated Ras-Raf-MEK-ERK pathway, suggesting that POL may be a potential EGFR inhibitor. Finally, we used proteomics analyses for exploring more possible POL-induced pathways with EGFR, Ras, Raf, MEK and ERK, some of which were consistent with our in silico network prediction. Taken together, these results demonstrate that POL induces MCF-7 cell apoptosis and autophagy via targeting EGFR-mediated Ras-Raf-MEK-ERK signaling pathway, which would provide a new clue for exploiting POL as a potential anti-neoplastic drug for future cancer therapy.     

VMP1 related autophagy and apoptosis in colorectal cancer cells: VMP1 regulates cell death

Vacuole membrane protein 1 (VMP1) is an autophagy-related protein and identified as a key regulator of autophagy in recent years. In pancreatic cell lines, VMP1-dependent autophagy has been linked to positive regulation of apoptosis. However, there are no published reports on the role of VMP1 in autophagy and apoptosis in colorectal cancers. Therefore, to address this gap of knowledge, we decided to interrogate regulation of autophagy and apoptosis by VMP1. We have studied the induction of autophagy by starvation and rapamycin treatment in colorectal cell lines using electron microscopy, immunofluorescence, and immunoblotting. We found that starvation-induced autophagy correlated with an increase in VMP1 expression, that VMP1 interacted with BECLIN1, and that siRNA mediated down-regulation of VMP1-reduced autophagy. Next, we examined the relationship between VMP1-dependent autophagy and apoptosis and found that VMP1 down-regulation sensitizes cells to apoptosis and that agents that induce apoptosis down-regulate VMP1. In conclusion, similar to its reported role in other cell types, VMP1 is an important regulator of autophagy in colorectal cell lines. However, in contrast to its role in pancreatic cell lines, in colorectal cancer cells, VMP1-dependent autophagy appears to be pro-survival rather than pro-cell death.     

Taxol-induced unfolded protein response activation in breast cancer cells exposed to hypoxia: ATF4 activation regulates autophagy and inhibits apoptosis

Understanding the mechanisms responsible for the resistance against chemotherapy-induced cell death is still of great interest since the number of patients with cancer increases and relapse is commonly observed. Indeed, the development of hypoxic regions as well as UPR (unfolded protein response) activation is known to promote cancer cell adaptive responses to the stressful tumor microenvironment and resistance against anticancer therapies. Therefore, the impact of UPR combined to hypoxia on autophagy and apoptosis activation during taxol exposure was investigated in MDA–MB-231 and T47D breast cancer cells. The results showed that taxol rapidly induced UPR activation and that hypoxia modulated taxol-induced UPR activation differently according to the different UPR pathways (PERK, ATF6, and IRE1α). The putative involvement of these signaling pathways in autophagy or in apoptosis regulation in response to taxol exposure was investigated. However, while no link between the activation of these three ER stress sensors and autophagy or apoptosis regulation could be evidenced, results showed that ATF4 activation, which occurs independently of UPR activation, was involved in taxol-induced autophagy completion. In addition, an ATF4-dependent mechanism leading to cancer cell adaptation and resistance against taxol-induced cell death was evidenced. Finally, our results demonstrate that expression of ATF4, in association with hypoxia-induced genes, can be used as a biomarker of a poor prognosis for human breast cancer patients supporting the conclusion that ATF4 might play an important role in adaptation and resistance of breast cancer cells to chemotherapy in hypoxic tumors.     

Inhibition of autophagy enhances effects of PF-04691502 on apoptosis and DNA damage of lung cancer cells

Autophagy modulation has been considered as a potential therapeutic strategy for lung diseases. The PI3K-Akt-mTOR pathway may be one of the main targets for regulation of autophagy. We previously reported that a PI3 K/mTOR dual inhibitor PF-04691502 suppressed hepatoma cells growth in vitro. However, it is still unclear whether PF-04691502 induces autophagy and its roles in DNA damage and cell death in human lung cancer cells. In this study, we investigate the effects of PF-04691502 on the autophagy and its correlation with cell apoptosis and DNA damage in non-small-cell lung cancer (NSCLC) cell lines. PF-04691502 efficiently inhibited the phosphorylation of Akt and showed dose-dependent cytotoxicity in A549 and H1299 cells. PF-04691502 also triggered apoptosis and the cleavage of caspase-3 and PARP. Phosphorylated histone H2AX (γ-H2AX), a hallmark of DNA damage response, was dramatically induced by PF-04691502 treatment. By exposure to PF-04691502, A549 cells acquired a senescent-like phenotype with an increase in the level of β-galactosidase. Furthermore, PF-04691502 enhanced the expression of LC3-II in a concentration-dependent manner. More interestingly, effects of PF-04691502 on toxicity and DNA damage were remarkably increased by co-treatment with an autophagy inhibitor, chloroquine (CQ), in human lung cancer cells. These data suggest that a strategy of blocking autophagy to enhance the activity of PI3 K/mTOR inhibitors warrants further attention in treatment of NSCLC cells.     

Celastrol inhibits gastric cancer growth by induction of apoptosis and autophagy

Recently, the interest in natural products for the treatment of cancer is increasing because they are the pre-screened candidates. In the present study, we demonstrate the therapeutic effect of celastrol, a triterpene extracted from the root bark of Chinese medicine on gastric cancer. The proliferation of AGS and YCC-2 cells were most sensitively decreased in six kinds of gastric cancer cell lines after the treatment with celastrol. Celastrol inhibited the cell migration and increased G1 arrest in cell-cycle populations in both cell lines. The treatment with celastrol significantly induced autophagy and apoptosis and increased the expression of autophagy and apoptosis-related proteins. We also found an increase in phosphorylated AMPK following a decrease in all phosphorylated forms of AKT, mTOR and S6K after the treatment with celastrol. Moreover, gastric tumor burdens were reduced in a dose-dependent manner by celastrol administration in a xenografted mice model. Taken together, celastrol distinctly inhibits the gastric cancer cell proliferation and induces autophagy and apoptosis. [BMB Reports 2014; 47(12): 697-702]     

Synthesis of novel pyrazole carboxamide derivatives and discovery of modulators for apoptosis or autophagy in A549 lung cancer cells

A series of novel 3-aryl-1-arylmethyl-1H-pyrazole-5-carboxamide derivatives 3a–l, were synthesized by the reaction of 3-aryl-1-arylmethyl-1H-pyrazole-5-carbonyl chloride with substituted amine in excellent yields. The compounds 3e–h could suppress A549 lung cancer cell growth. More interestingly, compounds 3e and 3f might inhibit the A549 cell growth by inducing apoptosis; whereas compounds 3g and 3h with fluorine group might inhibit the A549 cell growth by inducing autophagy.     

Dihydroartemisinin potentiates the anticancer effect of cisplatin via mTOR inhibition in cisplatin-resistant ovarian cancer cells: involvement of apoptosis and autophagy

Dihydroartemisinin (DHA) exhibits anticancer activity in tumor cells but its mechanism of action is unclear. Cisplatin (DDP) is currently the best known chemotherapeutic available for ovarian cancer. However, tumors return de novo with acquired resistance over time. Mammalian target of rapamycin (mTOR) is an important kinase that regulates cell apoptosis and autophagy, and its dysregulation has been observed in chemoresistant human cancers. Here, we show that compared with control ovarian cancer cells (SKOV3), mTOR phosphorylation was abnormally activated in cisplatin-resistant ovarian cancer cells (SKOV3/DDP) following cisplatin monotherapy. Treatment with cisplatin combined with DHA could enhance cisplatin-induced proliferation inhibition in SKOV3/DDP cells. This mechanism is at least partially due to DHA deactivation of mTOR kinase and promotion of apoptosis. Although autophagy was also induced by DHA, the reduced cell death was not found by suppressing autophagic flux by Bafilomycin A1 (BAF). Taken together, we conclude that inhibition of cisplatin-induced mTOR activation is one of the main mechanisms by which DHA dramatically promotes its anticancer effect in cisplatin-resistant ovarian cancer cells.     

Blue light emitting diode induces apoptosis in lymphoid cells by stimulating autophagy

The present study was performed to examine the induction of apoptotic cell death and autophagy by blue LED irradiation, and the contribution of autophagy to apoptosis in B cell lymphoma A20 and RAMOS cells exposed to blue LED. Irradiation with blue LED reduced cell viability and induced apoptotic cell death, as indicated by exposure of phosphatidylserine on the plasma outside membrane and fragmentation of DNA. Furthermore, the mitochondrial membrane potential increased, and apoptotic proteins (PARP, caspase 3, Bax, and bcl-2) were observed. In addition, the level of intracellular superoxide anion (O₂⁻) gradually increased. Interestingly the formation of autophagosomes and level of LC3-II were increased in blue LED-irradiated A20 and RAMOS cells, but inhibited after pretreatment with 3-methyladenine (3-MA), widely used as an autophagy inhibitor. Inhibition of the autophagic process by pretreatment with 3-MA blocked blue LED irradiation-induced caspase-3 activation. Moreover, a significant reduction of both the early and late phases of apoptosis after transfection with ATG5 and beclin 1 siRNAs was shown by the annexin V/PI staining, indicating a crucial role of autophagy in blue LED-induced apoptosis in cells. Additionally, the survival rate of mice irradiated with blue LED after injection with A20 cells increased compared to the control group. Our data demonstrate that blue LED irradiation induces apoptosis via the mitochondrial-mediated pathway, in conjunction with autophagy. Further studies are needed to elucidate the precise mechanism of blue LED-induced immune cell death.     

High dose of extracellular ATP switched autophagy to apoptosis in anchorage-dependent and anchorage-independent hepatoma cells

Extracellular adenosine triphosphate (eATP) transduces purinergic signal and plays an important regulatory role in many biological processes, including tumor cell growth and cell death. A large amount of eATP exists in the fast-growing tumor center and inflammatory tumor microenvironment. Tumor cells could acquire anoikis resistance and anchorage independence in tumor microenvironment and further cause metastatic lesion. Whether such a high amount of eATP has any effect on the anchored and non-anchored tumor cells in tumor microenvironment has not been elucidated and is investigated in this study. Our data showed that autophagy helped hepatoma cells to maintain survival under the treatment of no more than 1 mM of eATP. Only when eATP concentration reached a relatively high level (2.5 mM), cell organelle could not be further maintained by autophagy, and apoptosis and cell death occurred. In hepatoma cells under treatment of 2.5 mM of eATP, an AMP-activated protein kinase (AMPK) pathway was dramatically activated while mTOR signaling pathway was suppressed in coordination with apoptosis. Further investigation showed that the AMPK/mTOR axis played a key role in tipping the balance between autophagy-mediated cell survival and apoptosis-induced cell death under the treatment of eATP. This work provides evidence to explain how hepatoma cells escape from eATP-induced cytotoxicity as well as offers an important clue to consider effective manipulation of cancer.     

Sonodynamic therapy induces the interplay between apoptosis and autophagy in K562 cells through ROS

Sonodynamic therapy (SDT) is a relatively new approach in the treatment of various cancers including leukemia cells. The aim of this study is to investigate the occurrence of apoptosis and autophagy after treated by protoporphyrin IX (PpIX)-mediated SDT (PpIX-SDT) on human leukemia K562 cells as well as the relationship between them. Firstly, mitochondrial-dependent apoptosis was observed through morphological observation and biochemical analysis. Meanwhile, SDT was shown to induce autophagy in K562 cells, which caused an increase in EGFP-LC3 puncta cells, a conversion of LC3 II/I, formation of acidic vesicular organelles (AVOs) and co-localization between LC3 and LAMP2 (a lysosome marker). Besides, pretreatment with autophagy inhibitor 3-MA or bafilomycin A1 was shown to provide protection against autophagy and to enhance SDT-induced apoptosis and necrosis, while the apoptosis suppressor z-VAD-fmk failed to affect formation of autophagic vacuoles or partially prevented SDT-induced cytotoxicity, which suggested that SDT-induced autophagy functioned as a survival mechanism. Additionally, this study reported apparent apoptosis and autophagy with dependence on intracellular reactive oxygen species (ROS) production. Preliminary data showed that ROS scavenger N-acetylcysteine (NAC) effectively blocked the SDT induced accumulation of ROS, reversed sono-damage, cell apoptosis and autophagy. Taken together, these data indicate that autophagy may be cytoprotective in our experimental system, and the ROS caused by PpIX-SDT treatment may play an important role in initiating apoptosis and autophagy.     

G15, a GPR30 antagonist,induces apoptosis and autophagy in human oral squamous carcinoma cells

As GPR30 has been implicated in mediating cancer cell proliferation, this study aimed to examine the antitumor effect of the GPR30 antagonist G15 in human oral squamous cell carcinoma (OSCC). G15 induced dose-dependent cytotoxicity, apoptosis and G2/M cell cycle arrest in a panel of OSCC cells. The results showed that G15 could inhibit the growth of the oral cancer cells with IC₅₀ value 11.2 μM for SCC4, 15.6 μM for SCC9, and 7.8 μM for HSC-3, respectively. Flow cytometric analysis and Comet assay indicated that G15 suppressed the viability of SCC4 and HSC-3 cells by inducing apoptosis and G2/M arrest. In addition, G15 down regulated the expression of Akt, cell cycle-related proteins, and mitogen-activated protein kinases, but increased the levels of LC3B-II and the accumulation of autophagosomes. Inhibition of autophagy by chloroquine does not affect the G15-induced apoptosis in SCC4 cells. Mechanistic evidence indicated that the antiproliferative effect was mediated through the downregulation of cdc2, cdc25c and NF-κB expression. Taken together, our findings suggest the potential of G15 in treating OSCC.     

Puerariae radix isoflavones and their metabolites inhibit growth and induce apoptosis in breast cancer cells

Puerariae radix (PR) is a popular natural herb and a traditional food in Asia, which has antithrombotic and anti-allergic properties and stimulates estrogenic activity. In the present study, we investigated the effects of the PR isoflavones puerarin, daidzein, and genistein on the growth of breast cancer cells. Our data revealed that after treatment with PR isoflavones, a dose-dependent inhibition of cell growth occurred in HS578T, MDA-MB-231, and MCF-7 cell lines. Results from cell cycle distribution and apoptosis assays revealed that PR isoflavones induced cell apoptosis through a caspase-3-dependent pathway and mediated cell cycle arrest in the G2/M phase. Furthermore, we observed that the serum metabolites of PR (daidzein sulfates/glucuronides) inhibited proliferation of the breast cancer cells at a 50% cell growth inhibition (GI₅₀) concentration of 2.35 μM. These results indicate that the daidzein constituent of PR can be metabolized to daidzein sulfates or daidzein glucuronides that exhibit anticancer activities. The protein expression levels of the active forms of caspase-9 and Bax in breast cancer cells were significantly increased by treatment with PR metabolites. These metabolites also increased the protein expression levels of p53 and p21. We therefore suggest that PR may act as a chemopreventive and/or chemotherapeutic agent against breast cancer by reducing cell viability and inducing apoptosis.     

Redox signaling: Potential arbitrator of autophagy and apoptosis in therapeutic response

Redox signaling plays important roles in the regulation of cell death and survival in response to cancer therapy. Autophagy and apoptosis are discrete cellular processes mediated by distinct groups of regulatory and executioner molecules, and both are thought to be cellular responses to various stress conditions including oxidative stress, therefore controlling cell fate. Basic levels of reactive oxygen species (ROS) may function as signals to promote cell proliferation and survival, whereas increase of ROS can induce autophagy and apoptosis by damaging cellular components. Growing evidence in recent years argues for ROS that below detrimental levels acting as intracellular signal transducers that regulate autophagy and apoptosis. ROS-regulated autophagy and apoptosis can cross-talk with each other. However, how redox signaling determines different cell fates by regulating autophagy and apoptosis remains unclear. In this review, we will focus on understanding the delicate molecular mechanism by which autophagy and apoptosis are finely orchestrated by redox signaling and discuss how this understanding can be used to develop strategies for the treatment of cancer.     

Formoxanthone C,isolated from Cratoxylum formosum ssp. pruniflorum, reverses anticancer drug resistance by inducing both apoptosis and autophagy in human A549 lung cancer cells

Multidrug resistance (MDR) cancer toward cancer chemotherapy is one of the obstacles in cancer therapy. Therefore, it is of interested to use formoxanthone C (1,3,5,6-tetraoxygenated xanthone; XanX), a natural compound, which showed cytotoxicity against MDR human A549 lung cancer (A549RT-eto). The treatment with XanX induced not only apoptosis- in A549RT-eto cells, but also autophagy-cell death. Inhibition of apoptosis did not block XanX-induced autophagy in A549RT-eto cells. Furthermore, suppression of autophagy by beclin-1 small interfering RNAs (siRNAs) did not interrupt XanX-induced apoptosis, indicating that XanX can separately induce apoptosis and autophagy. Of interest, XanX treatment reduced levels of histone deacetylase 4 (HDAC4) protein overexpressed in A549RT-etocells. The co-treatment with XanX and HDAC4 siRNA accelerated both autophagy and apoptosis more than that by XanX treatment alone, suggesting survival of HDAC4 in A549RT-eto cells. XanX reverses etoposide resistance in A549RT-eto cells by induction of both autophagy and apoptosis, and confers cytotoxicity through down-regulation of HDAC4.     

Neuronal cell death in transmissible spongiform encephalopathies (prion diseases) revisited: from apoptosis to autophagy

Neuronal autophagy, like apoptosis, is one of the mechanisms of the programmed cell death (PCD). In this review, we summarize the presence of autophagic vacuoles in experimentally induced scrapie, Creutzfeldt–Jakob disease and Gerstmann–Sträussler–Scheinker (GSS) syndrome. Initially, a part of the neuronal cytoplasm was sequestrated by concentric arrays of double membranes; the enclosed cytoplasm appeared relatively normal except that its density was often increased. Next, electron density of the central area dramatically increased. The membranes then proliferated within the cytoplasm in a labyrinth-like manner and the area sequestrated by these membranes enlarged into a more complex structure consisting of vacuoles, electron-dense areas and areas of normally-looking cytoplasm connected by convoluted membranes. Of note, autophagic vacuoles form not only in neuronal perikarya but also in neurites and synapses. Finally, a large area of the cytoplasm was transformed into a collection of autophagic vacuoles of different sizes. On a basis of ultrastructural studies, we suggest that autophagy plays a major role in transmissible spongiform encephalopathies (TSEs) and may even participate in a formation of spongiform change.     

Polymethoxylated flavones induce Ca(2+)-mediated apoptosis in breast cancer cells

Flavonoids, polyphenolic phytochemicals which include flavones and isoflavones, are present in the common human diet. It has been suggested that these compounds may exert anticancer activity; however, the mechanisms involved remain unknown. We have recently shown (Sergeev, 2004, Biochem Biophys Res Commun 321: 462-467) that isoflavones can activate the novel apoptotic pathway mediated by cellular Ca(2+). Here, we report that polymethoxyflavones (PMFs) derived from sweet orange (Citrus sinensis L.) inhibit growth of human breast cancer cells via Ca(2+)-dependent apoptotic mechanism. The treatment of MCF-7 breast cancer cells with 5-hydroxy-3,6,7,8,3',4'-hexamethoxyflavone (5-OH-HxMF) and 3'-hydroxy-5,6,7,4'-tetramethoxyflavone (3'-OH-TtMF) induced a sustained increase in concentration of intracellular Ca(2+) ([Ca(2+)](i)) resulting from both depletion of the endoplasmic reticulum Ca(2+) stores and Ca(2+) influx from the extracellular space. This increase in [Ca(2+)](i) was associated with the activation of the Ca(2+)-dependent apoptotic proteases, mu-calpain and caspase-12, as evaluated with the calpain and caspase-12 peptide substrates and antibodies to active (cleaved) forms of the enzymes. Corresponding non-hydroxylated PMFs, 3,5,6,7,8,3',4'-heptamethoxyflavone (HpMF) and 5,6,7,3',4'-pentamethoxyflavone (PtMF), were dramatically less active in inducing Ca(2+)-mediated apoptosis. Our results strongly suggest that the cellular Ca(2+) modulating activity of flavonoids underlies their apoptotic mechanism and that hydroxylation of PMFs is critical for their ability to induce an increase in [Ca(2+)](i) and, thus, activate Ca(2+)-dependent apoptotic proteases.     

Involvement of PKC signal pathways in oridonin-induced autophagy in HeLa cells: A protective mechanism against apoptosis

Our previous studies showed that oridonin could induce both apoptosis and autophagy in HeLa cells, and this autophagy might be a protective mechanism against apoptosis. In this study, the roles of PKC signal pathways in oridonin-induced HeLa cell autophagy and apoptosis were further investigated. We found that inhibition of PKC significantly reduced oridonin-induced autophagy whereas markedly increased apoptosis, while pretreatment with PKC activator caused opposite results. Subsequently, the oridonin-induced autophagy was also suppressed by Raf-1 or JNK inhibition accompanied by the increase of apoptosis, but it was not affected by ERK or p38 inhibition. In addition, oridonin-induced protein levels of Raf-1, JNK and p-JNK were sharply downregulated by PKC inhibitor, and they were enhanced by PKC activator. Taken together, these results demonstrate that PKC enhances oridonin-induced autophagy against apoptosis through regulating its downstream factors Raf-1 and JNK in HeLa cells.