Seon-Hee Oh

Autophagy Induction by Capsaicin in Malignant Human Breast Cells Is Modulated by p38 and Extracellular Signal-Regulated Mitogen-Activated Protein Kinases and Retards Cell Death by Suppressing Endoplasmic Reticulum Stress-Mediated Apoptosis

In our previous study, we showed that capsaicin induces autophagy in several cell lines. Here, we investigated the molecular mechanisms of capsaicin-induced autophagy in malignant (MCF-7 and MDA-MB-231) and normal (MCF10A) human breast cells. Capsaicin caused nonapoptotic cell cycle arrest of MCF-7 and MDA-MB-231 cells but induced apoptosis in MCF10A cells. In MCF-7 and MDA-MB-231 cells, capsaicin induced endoplasmic reticulum (ER) stress via inositol-requiring 1 and Chop and induced autophagy, as demonstrated by microtubule-associated protein 1 light chain-3 (LC3) conversion. Autophagy blocking by 3-methyladenine (3MA) or bafilomycin A1 (BaF1) activated caspase-4 and -7 and enhanced cell death. In MCF-7 and MDA-MB-231 cells, p38 was activated for more than 48 h by capsaicin treatment, but extracellular signal-regulated kinase (ERK) activation decreased after 12 h, and LC3II levels continuously increased. Furthermore, treatment with 3MA markedly down-regulated capsaicin-induced p38 activation and LC3 conversion, and BaF1 completely down-regulated ERK activation and led to LC3II accumulation. In addition, pharmacological blockade or knockdown of the p38 gene down-regulated Akt activation and LC3II levels but did not affect ERK, and pharmacological blockade or knockdown of the ERK gene up-regulated LC3II induction by capsaicin. Knockdown of inositol-requiring 1 down-regulated p38-Akt signaling. In MCF10A cells, capsaicin did not elicit p38 activation and LC3 conversion and caused the sustained activation of caspase-4. Collectively, capsaicin-induced autophagy is regulated by p38 and ERK; p38 controls autophagy at the sequestration step, whereas ERK controls autophagy at the maturation step, and that autophagy is involved in the retardation of cell death by blocking capsaicin-induced ER stress-mediated apoptosis in MCF-7 and MDA-MB-321 cells.

Endoplasmic Reticulum Stress-Mediated Autophagy/Apoptosis Induced by Capsaicin (8-Methyl-N-vanillyl-6-nonenamide) and Dihydrocapsaicin is Regulated by the Extent of c-Jun NH2-Terminal Kinase/Extracellular Signal-Regulated Kinase Activation in WI38 Lung Epithelial Fibroblast Cells

Endoplasmic reticulum (ER) stress causes cell survival or death, which is dependent on the type of cell and stimulus. Capsaicin (8-methyl-N-vanillyl-6-nonenamide) and its analog, dihydrocapsaicin (DHC), induced caspase-3-independent/-dependent signaling pathways in WI38 lung epithelial fibroblast cells. Here, we describe the molecular mechanisms induced by both chemicals. Exposure to capsaicin or DHC caused induction of p53, p21, and G0/G1 arrest. DHC induced massive cellular vacuolization by dilation of the ER and mitochondria. Classic ER stress inducers elicited the unfolded protein response (UPR) and up-regulation of microtubule-associated protein 1 light chain-3 (LC3) II. DHC induced ER stress by the action of heavy chain-binding protein, IRE1, Chop, eukaryotic initiation factor 2α, and caspase-4 and, to a lesser level, by capsaicin treatment. DHC treatment induced autophagy that was blocked by 3-methyladenine (3MA) and accumulated by bafilomycin A1. Blocking of DHC-induced autophagy by 3MA enhanced apoptotic cell death that was completely inhibited by treatment of cells with benzyl-oxcarbonyl-Val-Ala-Asp-fluoromethyl ketone. Knockdown of Ire1 down-regulated the DHC-induced Chop and LC3II and enhanced caspase-3 activation. DHC induced rapid and high-sustained c-Jun NH2-terminal kinase (JNK)/extracellular signal-regulated kinase (ERK) activation, but capsaicin induced transient activation of JNK/ERK. The JNK inhibitor SP600125 down-regulated the expression of IRE1, Chop, and LC3II induced by DHC, thapsigargin, and MG132 [N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal]. Pharmacological blockade or knockdown of ERK down-regulated LC3II. Capsaicin and DHC induced Akt phosphorylation, and the phosphatidylinositol 3-kinase inhibitors, wortmannin and LY294002 [2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride], induced autophagy via ERK activation. Our results indicate that the differential responses of capsaicin and DHC for cell protection are caused by the extent of the UPR and autophagy that are both regulated by the level of JNK and ERK activation.

Role of autophagy in chemoresistance: Regulation of the ATM-mediated DNA-damage signaling pathway through activation of DNA–PKcs and PARP-1

Capsaicin treatment was previously reported to reduce the sensitivity of breast cancer cells, but not normal MCF10A cells, to apoptosis. The present study shows that autophagy is involved in cellular resistance to genotoxic stress, through DNA repair. Capsaicin treatment of MCF-7 cells induced S-phase arrest and autophagy through the AMPKα-mTOR signaling pathway and the accumulation of p53 in the nucleus and cytosol, including a change in mitochondrial membrane potential. Capsaicin treatment also activated δ-H2AX, ataxia telangiectasia mutated (ATM), DNA-dependent protein kinase catalytic subunit (DNA-PKcs), and poly(ADP-ribose) polymerase (PARP)-1. Genetic or pharmacological disruption of autophagy attenuated capsaicin-induced phospho-ATM and phospho-DNA-PKcs and enhanced apoptotic cell death. ATM inhibitors, including Ku55933 and caffeine, and the genetic or pharmacological inhibition of p53 prevented capsaicin-induced DNA-PKcs phosphorylation and stimulated PARP-1 cleavage, but had no effect on microtubule-associated protein light chain 3 (LC3)-II levels. Ly294002, a DNA-PKcs inhibitor, boosted the capsaicin-induced cleavage of PARP-1. In M059K cells, but not M059J cells, capsaicin induced ATM and DNA-PKcs phosphorylation, p53 accumulation, and the stimulation of LC3II production, all of which were attenuated by knockdown of the autophagy-related gene atg5. Ku55933 attenuated capsaicin-induced phospho-DNA-PKcs, but not LC3II, in M059K cells. In human breast tumors, but not in normal tissues, AMPKα, ATM, DNA-PKcs, and PARP-1 were activated and LC3II was induced. The induction of autophagy by genotoxic stress likely contributes to the sustained survival of breast cancer cells through DNA repair regulated by ATM-mediated activation of DNA-PKcs and PARP-1.

Autophagy involvement in cadmium resistance through induction of multidrug resistance-associated protein and counterbalance of endoplasmic reticulum stress WI38 lung epithelial fibroblast cells

Human multidrug-resistance associated protein (MRP1) is known as a cellular efflux pump of heavy metals and anticancer drugs. In our previous study, MRP was found to have involvement in cell protection against cadmium (Cd) toxicity through apoptosis interruption. The purpose of the present study was to investigate the molecular mechanism of MRP1 in Cd resistance. For this purpose, we developed Cd-resistant cells (RWI38) from WI38 human lung epithelial fibroblast cells, which showed a 4-fold resistance to Cd when compared to WI38 cells. WI38 cells elicited endoplasmic reticulum (ER) stress through RNA-dependent protein kinase-like ER kinase (PERK) and the eukaryotic translation initiation factor 2 alpha (eIF2alpha), Chop, and glucose-regulated protein (Grp78). RWI38 cells responding to Cd did not elicit ER stress or mitochondrial apoptosis, but induced autophagy, as demonstrated by Atg5 induction, LC3 conversion, and formation of GFP-LC3 dots. A pharmacological inhibitor of p38 downregulated Cd-induced Atg5 and LC3II. A pharmacological inhibitor of autophagy or silencing of atg5 dephosphorylated p38 and Akt, and downregulated MRP1 and procaspase-3. However, pharmacological inhibition or silencing of mrp-1 had no affect on Cd-induced phosphorylated p38 and LC3II. These data indicate that Cd induces autophagy in RWI38 cells through a mechanism that involves p38 activation, which is involved in cell protection through counterbalance of ER stress and MRP1 induction.

Capsaicin-induced apoptosis is regulated by endoplasmic reticulum stress- and calpain-mediated mitochondrial cell death pathways

Capsaicin, a pungent compound found in hot chili peppers, induces apoptotic cell death in various cell lines, however, the precise apoptosis signaling pathway is unknown. Here, we investigated capsaicin-induced apoptotic signaling in the human breast cell line MCF10A and found that it involves both endoplasmic reticulum (ER) stress and calpain activation. Capsaicin inhibited growth in a dose-dependent manner and induced apoptotic nuclear changes in MCF10A cells. Capsaicin also induced degradation of tumor suppressor p53; this effect was enhanced by the ER stressor tunicamycin. The proteasome inhibitor MG132 completely blocked capsaicin-induced p53 degradation and enhanced apoptotic cell death. Capsaicin treatment triggered ER stress by increasing levels of IRE1, GADD153/Chop, GRP78/Bip, and activated caspase-4. It led to an increase in cytosolic Ca(2+), calpain activation, loss of the mitochondrial transmembrane potential, release of mitochondrial cytochrome c, and caspase-9 and -7 activation. Furthermore, capsaicin-induced the mitochondrial apoptotic pathway through calpain-mediated Bid translocation to the mitochondria and nuclear translocation of apoptosis-inducing factor (AIF). Capsaicin-induced caspase-9, Bid cleavage, and AIF translocation were blocked by calpeptin, and BAPTA and calpeptin attenuated calpain activation and Bid cleavage. Thus, both ER stress- and mitochondria-mediated death pathways are involved in capsaicin-induced apoptosis.

Proteasome inhibition-induced p38 MAPK/ERK signaling regulates autophagy and apoptosis through the dual phosphorylation of glycogen synthase kinase 3β

Proteasome inhibition is a promising approach for cancer treatment; however, the underlying mechanisms involved have not been fully elucidated. Here, we show that proteasome inhibition-induced p38 mitogen-activated protein kinase regulates autophagy and apoptosis by modulating the phosphorylation status of glycogen synthase kinase 3β (GSK3β) and 70kDa ribosomal S6 kinase (p70S6K). The treatment of MDA-MB-231 cells with MG132 induced endoplasmic reticulum stress through the induction of ATF6a, PERK phosphorylation, and CHOP, and apoptosis through the cleavage of Bax and procaspase-3. MG132 caused the phosphorylation of GSK3β at Ser(9) and, to a lesser extent, Thr(390), the dephosphorylation of p70S6K at Thr(389), and the phosphorylation of p70S6K at Thr(421) and Ser(424). The specific p38 inhibitor SB203080 reduced the p-GSK3β(Ser9) and autophagy through the phosphorylation of p70S6K(Thr389); however, it augmented the levels of p-ERK, p-GSK3β(Thr390), and p-70S6K(Thr421/Ser424) induced by MG132, and increased apoptotic cell death. The GSK inhibitor SB216763, but not lithium, inhibited the MG132-induced phosphorylation of p38, and the downstream signaling pathway was consistent with that in SB203580-treated cells. Taken together, our data show that proteasome inhibition regulates p38/GSK(Ser9)/p70S6K(Thr380) and ERK/GSK3β(Thr390)/p70S6K(Thr421/Ser424) kinase signaling, which is involved in cell survival and cell death.

Selective induction of catalase-mediated autophagy by dihydrocapsaicin in lung cell lines

We reported that dihydrocapsaicin (DHC) induces autophagy in a catalase-regulated manner. In this study, we further examined the role of DHC-induced autophagy in lung cell lines. DHC-induced cytotoxicity was higher in WI38 and H1299 cells than in H460 and A549 cells, and was related to the loss of cell membrane integrity. However, apoptotic cells markedly increased in H460 and A549 cells. In WI38 and H1299 cells, DHC-induced catalase was correlated with a decrease of intracellular reactive oxygen species (ROS) and an increase in the level of LC3II, an autophagy marker, and LC3 conversion was attenuated by the catalase inhibitor 3-amino-1,2,4-triazole (3AT) or by knockdown of the catalase gene. In A549 cells, DHC downregulated catalase, led to ROS accumulation, and blocked LC3 conversion. In H460 cells expressing limited amount of catalase, DHC caused ROS accumulation and blocked LC3 conversion. However, H460 cells overexpressing catalase were able to induce autophagy. In contrast to Earles balanced salt solution and rotenone, H(2)O(2) treatment caused ROS accumulation and did not promote upregulation of catalase and LC3II in lung cell lines. Cytoplasmic vacuolization in WI38 and H1299 cells was blocked by treatment of 3AT and which enhanced caspase-3 activity and LDH release. Suppression of autophagy by 3-methyladenine also enhanced DHC-induced cell death through apoptotic and necrotic cell death. In A549 and H460 cells, treatment of rapamycin attenuated DHC-induced cell death. Collectively, these results suggest that catalase regulates autophagy, which helps protect cells against apoptotic and necrotic cell death.

Serine 9 and Tyrosine 216 Phosphorylation of GSK-3β Differentially Regulates Autophagy in Acquired Cadmium Resistance

Glycogen synthase kinase-3β (GSK-3β) plays an important role in the regulation of apoptosis. To investigate its involvement in acquired cadmium (Cd) resistance, Cd-resistant cells (RH460) were established from H460 lung carcinoma cells. Cd resistance led to interruption of apoptosis and autophagy, as determined by an apoptotic sub-G1 population, procaspase-3 clevage, and LC3-II induction. Cd-induced autophagy preceded apoptosis as determined by 3-methyladenine or zVAD and time-course experiments after Cd treatment. Despite β-catenin accumulation, phospho(p)-Ser/Tyr GSK-3α/β increased in the nucleus until 12h after treatment and then p-Ser partly translocated to the cytoplasm. The GSK-3 inhibitor lithium augmented Cd-induced p-Ser GSK-3α/β, which accumulated in the nucleus and cytoplasm, and increased autophagy. SB216763 inhibited p-Ser/p-Tyr GSK-3α/β and subsequent autophagy. GSK-3β knockdown decreased Cd-induced autophagy. Cd exposure to RH460 cells overexpressed with pcDNA-GSK-3β-HA strongly phosphorylated Ser(9)/Tyr(216) residues and decreased LC3-II. Constitutively active pcDNA-GSK-3β(S9A)-HA overexpression phosphorylated Tyr(216) and decreased LC3-II, suggesting that p-Tyr inhibits autophagy. PI3K inhibitors decreased Cd-induced p-Ser GSK-3αβ and LC3-II, whereas a Ser/Thr phosphatase inhibitor, okadaic acid, hyperphosphorylated Ser residues, which accumulated in the nucleus and cytosol, and enhanced LC3-II. The general tyrosine kinase inhibitor genistein suppressed Cd-induced p-Tyr/p-Ser GSK-3α/β and LC3-II. Mouse lung tissues respond to long-term Cd exposure increased p-Tyr, downregulated LC3-II, and accumulated full-length Bax and procaspase-3. Taken together, this study shows that acquired Cd resistance is regulated by GSK-3β phosphorylation state, but not activation state, and intracellular localization of p-Ser GSK-3 regulates Cd-induced autophagy and apoptosis.

Cadmium adaptation is regulated by multidrug resistance-associated protein-mediated Akt pathway and metallothionein induction

To elucidate the mechanisms involved in adaptation of lung epithelial cells to cadmium (Cd), we established a cell line that exhibits Cd-resistance (RWI38). RWI38 showed ∼5-fold greater Cd-resistance (MTT assays) than WI38 cells, and cross-resistance to Zn and cisplatin. RWI38 cells also demonstrated an upregulated level of multidrug resistance-associated protein (MRP) and metallothionein (MT) (as shown by Western blot analysis and RT-PCR studies). The protein level of MRP decreased after Cd exposure in WI38 cells, but was sustained at high levels in RWI38 cells, leading led to enhanced calcein efflux. Cd induced Akt phosphorylation in RWI38 but not WI38 cells; this was prevented by probenecid or siRNA for MRP, both of which led to enhanced cell death, as demonstrated by capsase-3 activation and decreased cell viability. These results suggest a functional role for MRP in the regulation of the Akt pathway as well in the efflux pumping of drugs, thereby contributing toward the adaptation of cells to Cd toxicity. The findings of this study could be potentially beneficial in the design of therapeutic targets for Cd-induced tumor progression.

Inhibition of autophagy potentiates pemetrexed and simvastatin-induced apoptotic cell death in malignant mesothelioma and non-small cell lung cancer cells

Pemetrexed, a multitarget antifolate used to treat malignant mesothelioma and non-small cell lung cancer (NSCLC), has been shown to stimulate autophagy. In this study, we determined whether autophagy could be induced by pemetrexed and simvastatin cotreatment in malignant mesothelioma and NSCLC cells. Furthermore, we determined whether inhibition of autophagy drives apoptosis in malignant mesothelioma and NSCLC cells. Malignant mesothelioma MSTO-211H and A549 NSCLC cells were treated with pemetrexed and simvastatin alone and in combination to evaluate their effect on autophagy and apoptosis. Cotreatment with pemetrexed and simvastatin induced greater caspase-dependent apoptosis and autophagy than either drug alone in malignant mesothelioma and NSCLC cells. 3-Methyladenine (3-MA), ATG5 siRNA, bafilomycin A, and E64D/pepstatin A enhanced the apoptotic potential of pemetrexed and simvastatin, whereas rapamycin and LY294002 attenuated their induction of caspase-dependent apoptosis. Our data indicate that pemetrexed and simvastatin cotreatment augmented apoptosis and autophagy in malignant mesothelioma and NSCLC cells. Inhibition of pemetrexed and simvastatin-induced autophagy was shown to enhance apoptosis, suggesting that this could be a novel therapeutic strategy against malignant mesothelioma and NSCLC.