Stimulation of ALK by the growth factor midkine renders glioma cells resistant to autophagy-mediated cell death

Δ⁹-tetrahydrocannabinol (THC), the main active component of marijuana, promotes cancer cell death via autophagy stimulation. We find that activation of the tyrosine kinase receptor ALK by its ligand midkine interferes with the signaling mechanism by which THC promotes autophagy-mediated glioma cell death.     

Stimulation of ALK by the growth factor midkine renders glioma cells resistant to autophagy-mediated cell death

Δ?-tetrahydrocannabinol (THC), the main active component of marijuana, promotes cancer cell death via autophagy stimulation. We find that activation of the tyrosine kinase receptor ALK by its ligand midkine interferes with the signaling mechanism by which THC promotes autophagy-mediated glioma cell death.     

Simian Virus 40 Small T Antigen Activates AMPK and Triggers Autophagy To Protect Cancer Cells from Nutrient Deprivation

As tumors grow larger, they often experience an insufficient supply of oxygen and nutrients. Hence, cancer cells must develop mechanisms to overcome these stresses. Using an in vitro transformation model where the presence of the simian virus 40 (SV40) small T (ST) antigen has been shown to be critical for tumorigenic transformation, we investigated whether the ST antigen has a role to play in regulating the energy homeostasis of cancer cells. We find that cells expressing the SV40 ST antigen (+ST cells) are more resistant to glucose deprivation-induced cell death than cells lacking the SV40 ST antigen (−ST cells). Mechanistically, we find that the ST antigen mediates this effect by activating a nutrient-sensing kinase, AMP-activated protein kinase (AMPK). The basal level of active, phosphorylated AMPK was higher in +ST cells than in −ST cells, and these levels increased further in response to glucose deprivation. Additionally, inhibition of AMPK in +ST cells increased the rate of cell death, while activation of AMPK in −ST cells decreased the rate of cell death, under conditions of glucose deprivation. We further show that AMPK mediates its effects, at least in part, by inhibiting mTOR (mammalian target of rapamycin), thereby shutting down protein translation. Finally, we show that +ST cells exhibit a higher percentage of autophagy than −ST cells upon glucose deprivation. Thus, we demonstrate a novel role for the SV40 ST antigen in cancers, where it functions to maintain energy homeostasis during glucose deprivation by activating AMPK, inhibiting mTOR, and inducing autophagy as an alternate energy source.The localization of most mammalian cells within a 100- to 150-μm distance from blood vessels ensures a continuous supply of oxygen and nutrients, a prerequisite for cell survival. However, tumors often grow beyond this limit, thereby experiencing oxygen and nutrient deprivation (28). Tumors overcome this barrier by initiating neoangiogenesis, a process that supplies new blood vessels (44). However, before neoangiogenesis can set in, incipient tumors must survive the stresses of nutrient deprivation. Therefore, an understanding of the molecular mechanisms that regulate cancer cell survival under conditions of nutrient deprivation is fundamental in cancer biology. Additionally, targeting the ability of cancer cells to survive under nutrient-deprived conditions can be exploited for designing novel cancer therapeutics.Glucose is the major source of energy for mammalian cells. Several types of cancer cells exhibit marked resistance to cell death upon glucose deprivation (22). In this study we have attempted to delineate the mechanisms that allow cancer cells to survive under conditions of glucose deprivation by using human foreskin fibroblasts that have been transformed by the serial introduction of the simian virus 40 (SV40) early region (coding for the large T [LT] and small T [ST] antigens), the catalytic subunit of human telomerase (hTERT), and an oncogenic allele of H-Ras (H-Ras V12) (referred to below as +ST cells) (32). In this model, human cells lacking the ST antigen but expressing the rest of these genetic elements (referred to below as −ST cells) are nontumorigenic (16, 32), highlighting the importance of the ST antigen in human cell transformation. However, little is known about the specific cellular functions moderated by the ST antigen that aid in transformation (3).Since glucose is the major source of energy for mammalian cells, and cancer cells experience glucose deprivation when they are beyond the diffusion limit, we investigated whether the ST antigen has any role to play under conditions of glucose deprivation. We report here a novel link between the ST antigen and AMP-activated protein kinase (AMPK) activation that enables cancer cell survival under glucose deprivation via inhibition of protein synthesis and activation of autophagy as an alternate energy source.     

Role of Pin1 in UVA-induced cell proliferation and malignant transformation in epidermal cells

The hepatitis B virus X protein (HBx) has been implicated in the development of hepatocellular carcinoma (HCC) associated with chronic infection. As a multifunctional protein, HBx regulates numerous cellular pathways, including autophagy. Although autophagy has been shown to participate in viral DNA replication and envelopment, it remains unclear whether HBx-activated autophagy affects host cell death, which is relevant to both viral pathogenicity and the development of HCC. Here, we showed that enforced expression of HBx can inhibit starvation-induced cell death in hepatic (L02 and Chang) or hepatoma (HepG2 and BEL-7404) cell lines. Starvation-induced cell death was greatly increased in HBX-expressing cell lines treated either with the autophagy inhibitor 3-methyladenine (3-MA) or with an siRNA directed against an autophagy gene, beclin 1. In contrast, treatment of cells with the apoptosis inhibitor Z-Vad-fmk significantly reduced cell death. Our results demonstrate that HBx-mediated cell survival during starvation is dependent on autophagy. We then further investigated the mechanisms of cell death inhibition by HBx. We found that HBx inhibited the activation of caspase-3, an execution caspase, blocked the release of mitochondrial apoptogenic factors, such as cytochrome c and apoptosis-inducing factor (AIF), and inhibited the activation of caspase-9 during starvation. These results demonstrate that HBx reduces cell death through inhibition of mitochondrial apoptotic pathways. Moreover, increased cell viability was also observed in HepG2.2.15 cells that replicate HBV and in cells transfected with HBV genomic DNA. Our findings demonstrate that HBx promotes cell survival during nutrient deprivation through inhibition of apoptosis and activation of autophagy. This highlights an important potential role of autophagy in HBV-infected hepatocytes growing under nutrient-deficient conditions.     

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.     

TRB3 links ER stress to autophagy in cannabinoid antitumoral action

Δ⁹-tetrahydrocannabinol (THC), the main active component of marijuana, is being investigated as a potential anti-tumoral agent. We find that THC stimulates an endoplasmic reticulum (ER) stress-related signaling pathway, which activates autophagy via inhibition of the Akt/mTORC1 axis. We also show that autophagy is upstream of apoptosis in cannabinoid-induced cancer cell death and that activation of this pathway is necessary for the anti-tumoral action of cannabinoids in vivo.     

ROS-induced DNA damage and PARP-1 are required for optimal induction of starvation-induced autophagy

In response to nutrient stress, cells start an autophagy program that can lead to adaptation or death. The mechanisms underlying the signaling from starvation to the initiation of autophagy are not fully understood. In the current study we show that the absence or inactivation of PARP-1 strongly delays starvation-induced autophagy. We have found that DNA damage is an early event of starvation-induced autophagy as measured by γ-H2AX accumulation and comet assay, with PARP-1 knockout cells displaying a reduction in both parameters. During starvation, ROS-induced DNA damage activates PARP-1, leading to ATP depletion (an early event after nutrient deprivation). The absence of PARP-1 blunted AMPK activation and prevented the complete loss of mTOR activity, leading to a delay in autophagy. PARP-1 depletion favors apoptosis in starved cells, suggesting a pro-survival role of autophagy and PARP-1 activation after nutrient deprivation. In vivo results show that neonates of PARP-1 mutant mice subjected to acute starvation, also display deficient liver autophagy, implying a physiological role for PARP-1 in starvation-induced autophagy. Thus, the PARP signaling pathway is a key regulator of the initial steps of autophagy commitment following starvation.     

The pseudokinase tribbles homologue-3 plays a crucial role in cannabinoid anticancer action

Δ⁹-Tetrahydrocannabinol (THC), the major active ingredient of marijuana, and other cannabinoids inhibit tumor growth in animal models of cancer. This effect relies, at least in part, on the up-regulation of several endoplasmic reticulum stress-related proteins including the pseudokinase tribbles homologue-3 (TRIB3), which leads in turn to the inhibition of the AKT/mTORC1 axis and the subsequent stimulation of autophagy-mediated apoptosis in tumor cells. Here, we took advantage of the use of cells derived from Trib3-deficient mice to investigate the precise mechanisms by which TRIB3 regulates the anti-cancer action of THC. Our data show that RasV¹²/E1A-transformed embryonic fibroblasts derived from Trib3-deficient mice are resistant to THC-induced cell death. We also show that genetic inactivation of this protein abolishes the ability of THC to inhibit the phosphorylation of AKT and several of its downstream targets, including those involved in the regulation of the AKT/mammalian target of rapamycin complex 1 (mTORC1) axis. Our data support the idea that THC-induced TRIB3 up-regulation inhibits AKT phosphorylation by regulating the accessibility of AKT to its upstream activatory kinase (the mammalian target of rapamycin complex 2; mTORC2). Finally, we found that tumors generated by inoculation of Trib3-deficient cells in nude mice are resistant to THC anticancer action. Altogether, the observations presented here strongly support that TRIB3 plays a crucial role on THC anti-neoplastic activity. This article is part of a Special Issue entitled Lipid Metabolism in Cancer.     

Aiduqing formula suppresses breast cancer metastasis via inhibiting CXCL1-mediated autophagy

BackgroundMetastasis is the most common lethal cause of breast cancer-related death. Recent studies have implied that autophagy is closely implicated in cancer metastasis. Therefore, it is of great significance to explore autophagy-related molecular targets involved in breast cancer metastasis and to develop therapeutic drugs.PurposeThis study was designed to investigate the anti-metastatic effects and autophagy regulatory mechanisms of Aiduqing (ADQ) formula on breast cancer.Study Design/MethodsMultiple cellular and molecular experiments were conducted to investigate the inhibitory effects of ADQ formula on autophagy and metastasis of breast cancer cells in vitro. Meanwhile, autophagic activator/inhibitor as well as CXCL1 overexpression or interference plasmids were used to investigate the underlying mechanisms of ADQ formula in modulating autophagy-mediated metastasis. Furthermore, the zebrafish xenotransplantation model and mouse xenografts were applied to validate the inhibitory effect of ADQ formula on autophagy-mediated metastasis in breast cancer in vivo.ResultsADQ formula significantly inhibited the proliferation, migration, invasion and autophagy but induced apoptosis of high-metastatic breast cancer cells in vitro. Similar results were also observed in starvation-induced breast cancer cells which exhibited elevated metastatic ability and autophagy activity. Mechanism investigations further approved that either CXCL1 overexpression or autophagic activator rapamycin can significantly abrogated the anti-metastatic effects of ADQ formula, suggesting that CXCL1-mediated autophagy may be the crucial pathway of ADQ formula in suppressing breast cancer metastasis. More importantly, ADQ formula suppressed breast cancer growth, autophagy, and metastasis in both the zebrafish xenotransplantation model and the mouse xenografts.ConclusionOur study not only revealed the novel function of CXCL1 in mediating autophagy-mediated metastasis but also suggested ADQ formula as a candidate drug for the treatment of metastatic breast cancer.     

Impaired autophagy due to constitutive mTOR activation sensitizes TSC2-null cells to cell death under stress

It has been well documented that cells deficient in either TSC1 or TSC2 are highly sensitive to various cell death stimuli. In this study, we utilized the TSC2^-/- mouse embryonic fibroblasts (MEFs) to study the involvement of autophagy in the enhanced susceptibility of TSC2-null cells to cell death. We first confirmed that both TSC1-null and TSC2-null MEFs are more sensitive to apoptosis in response to amino acid starvation (EBSS) and hypoxia. Second, we found that both the basal and inducible autophagy in TSC2^-/- MEFs is impaired, mainly due to constitutive activation of mTORC1. Third, suppression of autophagy by chloroquine and Atg7 knockdown sensitizes TSC2^+/+ cells, but not TSC2^-/- cells, to EBSS-induced cell death. Conversely, the inhibition of mTORC1 by raptor knockdown and rapamycin activates autophagy and subsequently rescues TSC2^-/- cells. Finally, in starved cells, nutrient supplementations (insulin-like growth factor-1 (IGF-1) and leucine) enhanced cell death in TSC2^-/- cells, but reduced cell death in TSC2^+/+ cells. Taken together, these data indicate that constitutive activation of mTORC1 in TSC2^-/- cells leads to suppression of autophagy and enhanced susceptibility to stress-mediated cell death. Our findings thus provide new insights into the complex relationships among mTOR, autophagy and cell death, and support the possible autophagy-targeted intervention strategies for the treatment of TSC-related pathologies.     

Plant autophagy puts the brakes on cell death by controlling salicylic acid signaling

It has long been recognized that autophagy in plants is important for nutrient recycling and plays a critical role in the ability of plants to adapt to environmental extremes such as nutrient deprivation. Recent reverse genetic studies, however, hint at other roles for autophagy, showing that autophagy defects in higher plants result in early senescence and excessive immunity-related programmed cell death (PCD), irrespective of nutrient conditions. Until now, the mechanisms by which cells die in the absence of autophagy were unclear. In our study, using biochemical, pharmacological and genetic approaches, we reveal that excessive salicylic acid (SA) signaling is a major factor in autophagy-defective plant-dependent cell death and that the SA signal can induce autophagy. These findings suggest a novel physiological function for plant autophagy that operates via a negative feedback loop to modulate proper SA signaling.     

Research Article: Involvement of autophagy induction in penta-1,2,3,4,6-O-galloyl-β-D-glucose-induced senescence-like growth arrest in human cancer cells

Growing evidence has demonstrated that autophagy plays important and paradoxical roles in carcinogenesis, while senescence is considered to be a crucial tumor-suppressor mechanism in cancer prevention and treatment. In the present study we demonstrated that both autophagy and senescence were induced in response to penta-1,2,3,4,6-O-galloyl-β-D-glucose (PGG), a chemopreventive polyphonolic compound, in multiple types of cancer cells. Analysis of these 2 events over the experimental time course indicated that autophagy and senescence occurred in parallel early in the process and dissociated later. The long-term culture study suggested that a subpopulation of senescent cells may have the capacity to reenter the cell cycle. Inhibition of autophagy by either a chemical inhibitor or RNA interference led to a significant reduction of PGG-induced senescence, followed by induction of apoptosis. These results suggested that autophagy promoted senescence induction by PGG and that PGG might exert its anticancer activity through autophagy-mediated senescence. For the first time, these findings uncovered the relationships among autophagy, senescence, and apoptosis induced by PGG. In addition, we identified that unfolded protein response signaling played a pivotal role in the autophagy-mediated senescence phenotype. Furthermore, our data showed that activation of MAPK8/9/10 (mitogen-activated protein kinase 8/9/10/c-Jun N-terminal kinases) was an essential upstream signal for PGG-induced autophagy. Finally, the key in vitro results were validated in vivo in a xenograft mouse model of human HepG2 liver cancer. Our findings provided novel insights into understanding the mechanisms and functions of PGG-induced autophagy and senescence in human cancer cells.     

The life span-prolonging effect of Sirtuin-1 is mediated by autophagy

The life span of various model organisms can be extended by caloric restriction as well as by autophagy-inducing pharmacological agents. Life span-prolonging effects have also been observed in yeast cells, nematodes and flies upon the overexpression of the deacetylase Sirtuin-1. Intrigued by these observations and by the established link between caloric restriction and Sirtuin-1 activation, we decided to investigate the putative implication of Sirtuin-1 in the response of human cancer cells and Caenorhabditis elegans to multiple triggers of autophagy. Our data indicate that the activation of Sirtuin-1 (by the pharmacological agent resveratrol and/or genetic means) per se ignites autophagy, and that Sirtuin-1 is required for the autophagic response to nutrient deprivation, in both human and nematode cells, but not for autophagy triggered by downstream signals such as the inhibition of mTOR or p53. Since the life span-extending effects of Sirtuin-1 activators are lost in autophagy-deficient C. elegans, our results suggest that caloric restriction and resveratrol extend longevity, at least in experimental settings, by activating autophagy.     

OsATG7 is required for autophagy-dependent lipid metabolism in rice postmeiotic anther development

In flowering plants, the tapetum, the innermost layer of the anther, provides both nutrient and lipid components to developing microspores, pollen grains, and the pollen coat. Though the programmed cell death of the tapetum is one of the most critical and sensitive steps for fertility and is affected by various environmental stresses, its regulatory mechanisms remain mostly unknown. Here we show that autophagy is required for the metabolic regulation and nutrient supply in anthers and that autophagic degradation within tapetum cells is essential for postmeiotic anther development in rice. Autophagosome-like structures and several vacuole-enclosed lipid bodies were observed in postmeiotic tapetum cells specifically at the uninucleate stage during pollen development, which were completely abolished in a retrotransposon-insertional OsATG7 (autophagy-related 7)-knockout mutant defective in autophagy, suggesting that autophagy is induced in tapetum cells. Surprisingly, the mutant showed complete sporophytic male sterility, failed to accumulate lipidic and starch components in pollen grains at the flowering stage, showed reduced pollen germination activity, and had limited anther dehiscence. Lipidomic analyses suggested impairment of editing of phosphatidylcholines and lipid desaturation in the mutant during pollen maturation. These results indicate a critical involvement of autophagy in a reproductive developmental process of rice, and shed light on the novel autophagy-mediated regulation of lipid metabolism in eukaryotic cells.     

OsATG7 is required for autophagy-dependent lipid metabolism in rice postmeiotic anther development

In flowering plants, the tapetum, the innermost layer of the anther, provides both nutrient and lipid components to developing microspores, pollen grains, and the pollen coat. Though the programmed cell death of the tapetum is one of the most critical and sensitive steps for fertility and is affected by various environmental stresses, its regulatory mechanisms remain mostly unknown. Here we show that autophagy is required for the metabolic regulation and nutrient supply in anthers and that autophagic degradation within tapetum cells is essential for postmeiotic anther development in rice. Autophagosome-like structures and several vacuole-enclosed lipid bodies were observed in postmeiotic tapetum cells specifically at the uninucleate stage during pollen development, which were completely abolished in a retrotransposon-insertional OsATG7 (autophagy-related 7)-knockout mutant defective in autophagy, suggesting that autophagy is induced in tapetum cells. Surprisingly, the mutant showed complete sporophytic male sterility, failed to accumulate lipidic and starch components in pollen grains at the flowering stage, showed reduced pollen germination activity, and had limited anther dehiscence. Lipidomic analyses suggested impairment of editing of phosphatidylcholines and lipid desaturation in the mutant during pollen maturation. These results indicate a critical involvement of autophagy in a reproductive developmental process of rice, and shed light on the novel autophagy-mediated regulation of lipid metabolism in eukaryotic cells.     

Autophagy in hypoxia protects cancer cells against apoptosis induced by nutrient deprivation through a beclin1‐dependent way in hepatocellular carcinoma

Oxygen deficiency and nutrient deprivation widely exists in solid tumors because of the poor blood supply. However, cancer cells can survive this adverse condition and proliferate continuously to develop. To figure out the way to survive, we investigated the role of autophagy in the microenvironment in hepatocellular carcinoma. In order to simulate the tumor microenvironment more veritably, cells were cultured in oxygen‐nutrient‐deprived condition following a hypoxia preconditioning. As a result, cell death under hypoxia plus nutrient deprivation was much less than that under nutrient deprivation only. And the decreased cell death mainly attributed to the decreased apoptosis. GFP‐LC3 and electron microscopy analysis showed that autophagy was significantly activated in the period of hypoxia preconditioning. However, autophagic inhibitor—3‐MA significantly abrogated the apoptosis reduction in hypoxia, which implied the involvement of autophagy in protection of hepatocellular carcinoma cells against apoptosis induced by starvation. Furthermore, Beclin 1 was proved to play an important role in this process. siRNA targeting Beclin 1 was transfected into hepatocellular carcinoma cells. And both data from western blot detecting the expression of LC3‐II and transmission microscopy observing the accumulation of autophagosomes showed that autophagy was inhibited obviously as a result of Beclin 1 knockdown. Besides, the decreased apoptosis of starved cells under hypoxia was reversed. Taken together, these results suggest that autophagy activated by hypoxia mediates the tolerance of hepatocellular carcinoma cells to nutrient deprivation, and this tolerance is dependent on the activity of Beclin 1. J. Cell. Biochem. 112: 3406–3420, 2011. © 2011 Wiley Periodicals, Inc.     

Autophagy Constitutes a Protective Mechanism against Ethanol Toxicity in Mouse Astrocytes and Neurons

Ethanol induces brain damage and neurodegeneration by triggering inflammatory processes in glial cells through activation of Toll-like receptor 4 (TLR4) signaling. Recent evidence indicates the role of protein degradation pathways in neurodegeneration and alcoholic liver disease, but how these processes affect the brain remains elusive. We have demonstrated that chronic ethanol consumption impairs proteolytic pathways in mouse brain, and the immune response mediated by TLR4 receptors participates in these dysfunctions. We evaluate the in vitro effects of an acute ethanol dose on the autophagy-lysosome pathway (ALP) on WT and TLR4^-/- mouse astrocytes and neurons in primary culture, and how these changes affect cell survival. Our results show that ethanol induces overexpression of several autophagy markers (ATG12, LC3-II, CTSB), and increases the number of lysosomes in WT astrocytes, effects accompanied by a basification of lysosomal pH and by lowered phosphorylation levels of autophagy inhibitor mTOR, along with activation of complexes beclin-1 and ULK1. Notably, we found only minor changes between control and ethanol-treated TLR4^-/- mouse astroglial cells. Ethanol also triggers the expression of the inflammatory mediators iNOS and COX-2, but induces astroglial death only slightly. Blocking autophagy by using specific inhibitors increases both inflammation and cell death. Conversely, in neurons, ethanol down-regulates the autophagy pathway and triggers cell death, which is partially recovered by using autophagy enhancers. These results support the protective role of the ALP against ethanol-induced astroglial cell damage in a TLR4-dependent manner, and provide new insight into the mechanisms that underlie ethanol-induced brain damage and are neuronal sensitive to the ethanol effects.     

Hypoxia signals autophagy in tumor cells via AMPK activity, independent of HIF-1, BNIP3, and BNIP3L

Macroautophagy (called autophagy hereafter) is a catabolic process activated by various types of stress, most notably by nutrient deprivation. The autophagic degradation of intracellular macromolecules provides metabolic support for the cell; however, this physiological process can also initiate a form of cell death (type 2 programmed cell death). Here we report that oxygen deprivation can activate the autophagic pathway in human cancer cell lines. We observed that hypoxia induced distinct cellular changes characteristic of autophagy such as an increase in cytoplasmic acidic vesicles, and processing and cellular localization of microtubule-associated protein-1 light chain 3. Oxygen deprivation-induced autophagy did not require nutrient deprivation, hypoxia-inducible factor-1 (HIF-1) activity, or expression of the HIF-1 target gene BNIP3 (Bcl-2 adenovirus E1a nineteen kilodalton interacting protein 3) or BNIP3L (BNIP3 like protein). Hypoxia-induced autophagy involved the activity of 5'-AMP-activated protein kinase (AMPK). Finally, we determined that cells lacking the autophagy gene ATG5 were unable to activate the autophagic machinery in hypoxia, had decreased oxygen consumption and increased glucose uptake under hypoxia, had increased survival in hypoxic environments, and exhibited accelerated growth as xenografted tumors. Together, these findings suggest that the autophagic degradation of cellular macromolecules contributes to the energetic balance governed by AMPK, and that suppression of autophagy in transformed cells can increase both resistance to hypoxic stress and tumorigenicity.     

Coupling endoplasmic reticulum stress to the cell death program in mouse melanoma cells: effect of curcumin

The microenvironment of cancerous cells includes endoplasmic reticulum (ER) stress the resistance to which is required for the survival and growth of tumors. Acute ER stress triggers the induction of a family of ER stress proteins that promotes survival and/or growth of the cancer cells, and also confers resistance to radiation and chemotherapy. Prolonged or severe ER stress, however, may ultimately overwhelm the cellular protective mechanisms, triggering cell death through specific programmed cell death (pcd) pathways. Thus, downregulation of the protective stress proteins may offer a new therapeutic approach to cancer treatment. In this regard, recent reports have demonstrated the roles of the phytochemical curcumin in the inhibition of proteasomal activity and triggering the accumulation of cytosolic Ca²⁺ by inhibiting the Ca²⁺-ATPase pump, both of which enhance ER stress. Using a mouse melanoma cell line, we investigated the possibility that curcumin may trigger ER stress leading to programmed cell death. Our studies demonstrate that curcumin triggers ER stress and the activation of specific cell death pathways that feature caspase cleavage and activation, p23 cleavage, and downregulation of the anti-apoptotic Mcl-1 protein.