Autophagy In Lung Cancer Cells Research Articles

Benzyl isothiocyanate induces protective autophagy in human lung cancer cells through an endoplasmic reticulum stress-mediated mechanism.

Isothiocyanates, such as allyl isothiocya¬nate (AITC), benzyl isothiocyanate (BITC), phenethyl isothio¬cyanate (PEITC) and sulforaphane (SFN), are natural compounds abundant in cruciferous vegetables, which have substantial chemopreventive activities against various human malignancies. However, the mechanisms underlying the inhibition of tumor cell growth by isothiocyanates are not fully understood. Since autophagy has dual functions in cancer, in the present study we investigated the effects of BITC on autophagy induction in human lung cancer cells in vitro and in vivo. BITC (1-100 μmol/L) dose-dependently inhibited the growth of 3 different human lung cancer cell lines A549 (adenocarcinoma), H661 (large cell carcinoma) and SK-MES-1 (squamous cell carcinoma) with IC50 values of 30.7±0.14, 15.9±0.22 and 23.4±0.11 μmol/L, respectively. BITC (10-40 μmol/L) induced autophagy in the lung cancer cells, evidenced by the formation of acidic vesicular organelles (AVOs), the accumulation of LC3-II, the punctate pattern of LC3, and the expression of Atg5. Pretreatment with the autophagy inhibitor 3-MA (5 mmol/L) significantly enhanced the BITC-caused growth inhibition in the lung cancer cells. Furthermore, BITC (20-40 μmol/L) activated ER stress, as shown by the increased cytosolic Ca2+ level and the phosphorylation of the ER stress marker proteins PERK and eIF2α in the lung cancer cells. Pretreatment with the ER stress inhibitor 4-PBA (5 mmol/L) attenuated the autophagy induction and potentiated the BITC-induced cell growth inhibition. In nude mice bearing A549 xenografts, administration of BITC (100 mg·kg-1·d-1, ip) for 8 weeks markedly suppressed the lung tumor growth, and significantly enhanced both autophagy and ER stress in the tumor tissues. Our results demonstrate that BITC inhibits human lung cancer cell growth in vitro and in vivo. In addition, BITC induces autophagy in the lung cancer cells, which protects the cancer cells against the inhibitory action of BITC; the autophagy induction is mediated by the ER stress response.

Synthesis and biological evaluation of matrine derivatives containing benzo-α-pyrone structure as potent anti-lung cancer agents.

Matrine, an active component of root extracts from Sophora flavescens Ait, is the main chemical ingredient of Fufang Kushen injection which was approved by Chinese FDA (CFDA) in 1995 as an anticancer drug to treat non-small cell lung cancer and liver cancer in combination with other anticancer drugs. Owning to its druggable potential, matrine is considered as an ideal lead compound for modification. We delineate herein the synthesis and anticancer effects of 17 matrine derivatives bearing benzo-α-pyrone structures. The results of cell viability assays indicated that most of the target compounds showed improved anticancer effects. Further studies showed that compound 5i could potently inhibit lung cancer cell proliferation in vitro and in vivo with no obvious side effects. Moreover, compound 5i could induce G1 cell cycle arrest and autophagy in lung cancer cells through up-regulating P27, down-regulating CDK4 and cyclinD1 and attenuating PI3K/Akt/mTOR pathway. Suppression of autophagy attenuated 5i induced proliferation inhibition. Collectively, our results infer that matrine derivative 5i bears therapeutic potentials for lung cancer.

Acid-induced autophagy protects human lung cancer cells from apoptosis by activating ER stress

An acidic tumor microenvironment exists widely in solid tumors. However, the detailed mechanism of cell survival under acidic stress remains unclear. The aim of this study is to clarify whether acid-induced autophagy exists and to determine the function and mechanism of autophagy in lung cancer cells. We have found that acute low pH stimulated autophagy by increasing LC3-positive punctate vesicles, increasing LC3 II expression levels and reducing p62 protein levels. Additionally, autophagy was inhibited by the addition of Baf or knockdown of Beclin 1, and cell apoptosis was increased markedly. In mouse tumors, the expression of cleaved caspase3 and p62 was enhanced by oral treatment with sodium bicarbonate, which can raise the intratumoral pH. Furthermore, the protein levels of ER stress markers, including p-PERK, p-eIF2α, CHOP, XBP-1s and GRP78, were also increased in response to acidic pH. The antioxidant NAC, which reduces ROS accumulation, alleviated acid-mediated ER stress and autophagy, and knocking down GRP78 reduced autophagy activation under acidic conditions, which suggests that autophagy was induced by acidic pH through ER stress. Taken together, these results indicate that the acidic microenvironment in non-small cell lung cancer cells promotes autophagy by increasing ROS-ER stress, which serves as a survival adaption in this setting.

Crizotinib induces autophagy through inhibition of the STAT3 pathway in multiple lung cancer cell lines.

Autophagy is an evolutionarily conserved survival pathway in eukaryote and is frequently upregulated in cancer cells after chemotherapy or targeted therapy. Thus induction of autophagy has emerged as a drug resistance mechanism. In this study, we found that crizotinib induced a high level of autophagy in lung cancer cells through inhibition of STAT3. Ectopic expression of wild-type or constitutive activated STAT3 significantly suppressed the effect of crizotinib on autophagy. Interestingly, crizotinib-mediated inhibition of STAT3 is in a step-wise manner. Firstly it inhibited cytoplasmic STAT3, which leads to the phosphorylation of EIF2A, then inhibited nuclear STAT3, which leads to the downregulation of BCL-2. Cell death induced by crizotinib was greatly enhanced after the inhibition of autophagy by the pharmacological inhibitors or shRNAs against Beclin-1. Moreover, the autophagy inhibitor HCQ significantly augmented the anti-tumor effect of crizotinib in a mouse xenograft model. In conclusion, crizotinib can induce cytoprotective autophagy by suppression of STAT3 in lung cancer cells. Thus, autophagy inhibition represents a promising approach to improve the efficacy of crizotinib in the treatment of targeted lung cancer patients.

Abstract 1014: Dihydrotanshione I induces caspase-independent cell death and autophagy in lung cancer cells

Abstract In the present study the antitumor mechanism of dihydrotanshinone I (DHT) from Salvia miltiorrhiza Bunge was elucidated in lung cancer cells. DHT significantly exerted cytotoxicity and increased the apoptosis by Annexin V assay in NSCLC (A549 and H460 cells) and lung mesothelioma cells (H28, H2452, MSTO). Furthermore, DHT reduced clonogenic ability in A549 cells. Interestingly, DHT increased PARP cleavage, but no activation of caspase-9 and caspase-3, although caspase-8 and, 3 were partially activated. FACS analysis revealed that the pan-caspase inhibitor z-VAD-fmk increased apoptosis induced by DHT. Furthermore, MTT assay showed that z-VAD-fmk treatment did not block cell cytotoxicity induced by DHT. In contrast, DHT induced the features of autophagy, including formation of autophagosomes by immunofluorescence and an increase of LC3II level after DHT exposure. Treatment of 3MA, an inhibitor of autophagy, blocked the expression level of LC3II induced by DHT and induced cytoprotective autophagy in A549 cells. Taken together, our data suggest that DHT induces caspase-independent cell death and autophagy in lung cancer cells as a potent agent for lung cancer treatment. Citation Format: Sung-Hoon Kim, Gunho Won, Myoung Seok Jeong, Sang Wook Yoon, Jihyun Lee, Bonglee Kim, Eun Jung Sohn. Dihydrotanshione I induces caspase-independent cell death and autophagy in lung cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1014. doi:10.1158/1538-7445.AM2015-1014

MiR-144 Inhibits Proliferation and Induces Apoptosis and Autophagy in Lung Cancer Cells by Targeting TIGAR

Background: MiRNAs are noncoding RNAs of 20-24 nucleotides that function as post-transcriptional negative regulators of gene expression. MiRNA genes are usually transcribed by RNA polymerase II in the nucleus. Their initial products are pre-miRNAs which have cap sequences and polyA tails. The p53-induced glycolysis and apoptosis regulator (TIGAR) was discovered through microarray analysis of gene expression following activation of p53. However, little is known about the effect of miR-144 on cell proliferation and apoptosis and how it interacts with TIGAR. Methods: We performed real-time PCR, western blotting, CCK8, colony formation, tumor growth, flow cytometry, Caspase3/7 activity, Hoechst 33342 staining, MDC staining of autophagic cells and luciferase reporter assays to detect the influence of miR-144 to lung cancer cells. Results: miR-144 targeted TIGAR, inhibited proliferation, enhanced apoptosis, and increased autophagy in A549 and H460 cells. Conclusions: Our study improves our understanding of the mechanisms underlying lung cancer pathogenesis and may promote the development of novel targeted therapies.

Phosphorylation‐Regulated Degradation of the Tumor‐Suppressor Form of PED by Chaperone‐Mediated Autophagy in Lung Cancer Cells

PED/PEA-15 is a death effector domain (DED) family member with a variety of effects on cell growth and metabolism. To get further insight into the role of PED in cancer, we aimed to find new PED interactors. Using tandem affinity purification, we identified HSC70 (Heat Shock Cognate Protein of 70 kDa)-which, among other processes, is involved in chaperone-mediated autophagy (CMA)-as a PED-interacting protein. We found that PED has two CMA-like motifs (i.e., KFERQ), one of which is located within a phosphorylation site, and demonstrate that PED is a bona fide CMA substrate and the first example in which phosphorylation modifies the ability of HSC70 to access KFERQ-like motifs and target the protein for lysosomal degradation. Phosphorylation of PED switches its function from tumor suppression to tumor promotion, and we show that HSC70 preferentially targets the unphosphorylated form of PED to CMA. Therefore, we propose that the up-regulated CMA activity characteristic of most types of cancer cell enhances oncogenesis by shifting the balance of PED function toward tumor promotion. This mechanism is consistent with the notion of a therapeutic potential for targeting CMA in cancer, as inhibition of this autophagic pathway may help restore a physiological ratio of PED forms.

Autophagy facilitates TLR4- and TLR3-triggered migration and invasion of lung cancer cells through the promotion of TRAF6 ubiquitination

Autophagy contributes to the pathogenesis of cancer, whereas toll-like receptors (TLRs) also play an important role in cancer development and immune escape. However, little is known about the potential interaction between TLR signaling and autophagy in cancer cells. Here we show that autophagy induced by TLR4 or TLR3 activation enhances various cytokine productions through promoting TRAF6 (TNF receptor-associated factor 6, E3 ubiquitin protein ligase) ubiquitination and thus facilitates migration and invasion of lung cancer cells. Stimulation of TLR4 and TLR3 with lipopolysaccharide (LPS) and polyinosinic-polycytidylic acid [poly(I:C)] respectively triggered autophagy in lung cancer cells. This was mediated by the adaptor protein, toll-like receptor adaptor molecule 1 (TICAM1/TRIF), and was required for TLR4- and TLR3-induced increases in the production of IL6, CCL2/MCP-1 [chemokine (C-C motif) ligand 2], CCL20/MIP-3α [chemokine (C-C motif) ligand 20], VEGFA (vascular endothelial growth factor A), and MMP2 [matrix metallopeptidase 2 (gelatinase A, 72 kDa gelatinase, 72 kDa type IV collagenase)]. These cytokines appeared to be necessary for enhanced migration and invasion of lung cancer cells upon TLR activation. Remarkably, inhibition of autophagy by chemical or genetic approaches blocked TLR4- or TLR3-induced Lys63 (K63)-linked ubiquitination of TRAF6 that was essential for activation of MAPK and NFKB (nuclear factor of kappa light polypeptide gene enhancer in B-cells) pathways, both of which were involved in the increased production of the cytokines. Collectively, these results identify induction of autophagy by TLR4 and TLR3 as an important mechanism that drives lung cancer progression, and indicate that inhibition of autophagy may be a useful strategy in the treatment of lung cancer.

Autophagy induction by low-dose cisplatin: The role of p53 in autophagy

The majority of chemotherapy treatments for lung cancer use cisplatin; however, its use is limited as it has several side-effects. Autophagy (or type II cell death) is an important mechanism by which programmed cell death occurs. The purpose of this study was to determine whether low-dose cisplatin treatment induces autophagy in lung cancer cells. We also examined whether autophagy inhibition results in p53-mediated apoptosis. NCI-H460 (wild-type p53) and NCI-H1299 (null-type p53) cells were treated with 5 or 20 µM cisplatin for 12, 24 or 48 h. An MTT assay was performed to measure the cell viability following cisplatin treatment. To detect cisplatin-induced autophagy, cell morphology (autophagic vacuole) and LC3 localization were examined. The outcome of autophagy inhibition was determined using 3-methyladenine (3-MA) to detect Annexin V (+), propidium iodide (PI) (-) and acridine orange (+) cells by FACS analysis. To determine whether cisplatin induced autophagy, we examined the role of p53 as a cell survival regulator in autophagy. Low-doses of cisplatin (5 µM) induced cell death and this was augmented by 3-MA in both cell lines. Autophagic vacuoles and cytoplasmic LC3 formation was more evident in H460 cells than in H1299 cells. The induction of autophagy by low-dose cisplatin was increased by 2-fold in H460 compared to H1299 cells. However, the tests for apoptosis showed no difference between the 2 cell lines. Following 3-MA pretreatment, cisplatin-induced autophagy was found to be markedly reduced (a 3-fold reduction) in wild-type p53 compared to null-type p53 cells. However, cisplatin-induced apoptosis increased in wild-type p53 compared to null-type p53 cells. Autophagy induction and apoptotic shift after autophagy inhibition may be mediated by p53 activation in lung cancer cells treated with low-dose cisplatin.

Cisplatin-induced downregulation of SOX1 increases drug resistance by activating autophagy in non-small cell lung cancer cell

SOX1 was aberrant methylated in hepatocellular cancer and non-small cell lung cancer (NSCLC). Long-term cisplatin exposure promotes methylation of SOX1 in ovarian cancer cell, suggesting that SOX1 may be involved in cisplatin resistance. Our aim was to test the hypothesis that cisplatin resistance is associated with alteration of SOX1 expression in NSCLC. Expression of levels of SOX1 was examined using RT-PCR in cisplatin resistance cells and parental cells. The level of SOX1 mRNA in cisplatin resistance cells was markedly reduced when compared to parental cells. Promoter methylation of SOX1 was induced in cisplatin resistance cells. We also found that SOX1 silencing enhanced the cisplatin-mediated autophagy in NSCLC. This study shows that inactivation of SOX1 by promoter hypermethylation, at least in part, is responsible for cisplatin resistance in human NSCLC.

Neferine from Nelumbo nucifera induces autophagy through the inhibition of PI3K/Akt/mTOR pathway and ROS hyper generation in A549 cells

Previously we have reported that neferine from the medicinal plant Nelumbo nucifera, inhibited cancer cell proliferation by inducing apoptosis. The present study was focused on the action mechanism of neferine in inducing autophagy in lung cancer cells. Neferine markedly inhibited A549 cell proliferation in a dose dependent manner. Acidic vesicular accumulation was observed in neferine treated cells as an indication of autophagy. Neferine could induce the conversion of LC3B-I to LC3B-II without affecting the expression levels of PI3KCIII and Beclin1. It has been observed that neferine mediated autophagy is dependent on inhibition of PI3K/Akt/mTOR signaling by neferine. Neferine treatment could also lead to the ROS hypergeneration and depletion of cellular antioxidant, GSH. The results demonstrate that neferine-induced autophagy is mediated through ROS hypergeneration and mTOR inhibition. Taken together, the present study unveils a novel mechanism of action of neferine on lung cancer cells in the induction of autophagy.

Gambogenic acid induces G1 arrest via GSK3β-dependent cyclin D1 degradation and triggers autophagy in lung cancer cells

Cyclin D1, an oncogenic G1 cyclin which can be induced by environmental carcinogens and whose over-expression may cause dysplasia and carcinoma, has been shown to be a target for cancer chemoprevention and therapy. In this study, we investigated the effects and underlying mechanisms of action of a polyprenylated xanthone, gambogenic acid (GEA) on gefitinib-sensitive and -resistant lung cancer cells. We found that GEA inhibited proliferation, caused G1 arrest and repressed colony-forming activity of lung cancer cells. GEA induced degradation of cyclin D1 via the proteasome pathway, and triggered dephosphorylation of GSK3β which was required for cyclin D1 turnover, because GSK3β inactivation by its inhibitor or specific siRNA markedly attenuated GEA-caused cyclin D1 catabolism. GEA induced autophagy of lung cancer cells, possibly due to activation of GSK3β and inactivation of AKT/mTOR signal pathway. These results indicate that GEA is a cyclin D1 inhibitor and a GSK3β activator which may have chemopreventive and therapeutic potential for lung cancer.

Chloroquine enhances the cytotoxicity of topotecan by inhibiting autophagy in lung cancer cells

Although the anti-malaria drug chloroquine (CQ) has been shown to enhance chemotherapy and radiation sensitivity in clinical trials, the potential mechanisms underlying this enhancement are still unclear. Here, we examined the relevant mechanisms by which the multipotent CQ enhanced the cytotoxicity of topotecan (TPT). The lung cancer cell line A549 was treated with TPT alone or TPT combined with CQ at non-cytotoxic concentrations. Cell viability was assessed using the MTT assay. The percentage of apoptotic cells and the presence of a side population of cells were both determined by flow Cytometry. Autophagy and the expression of Bcl-2 family proteins were examined by Western blotting. The accumulation of YFP-LC3 dots and the formation of acidic vesicular organelles were examined by confocal microscopy. CQ sensitized A549 cells to TPT and enhanced TPT-induced apoptosis in a Bcl-2 family protein-independent fashion. CQ inhibited TPT-induced autophagy, which modified the cytotoxicity of TPT. However, CQ failed to modify the transfer of TPT across the cytoplasmic membrane and did not increase lysosomal permeability. This study showed that CQ at non-cytotoxic concentrations potentiated the cytotoxicity of TPT by interfering with autophagy, implying that CQ has significant potential as a chemotherapeutic enhancer.

Knockdown of integrin β4‐induced autophagic cell death associated with P53 in A549 lung adenocarcinoma cells

Integrin beta4 is a tissue-specific protein, but its role in autophagy of lung adenocarcinoma cells is not clear. In this study, we used microtubule-associated protein 1 light chain 3 processing and acridine orange staining to reveal that knockdown of integrin beta4 by its specific siRNA induced autophagic cell death in A549 lung cancer cells. Next, we investigated the effects of siRNA-mediated downregulation of integrin beta4 on cell death and the level of p53. The proportion of dead cells and level of p53 were significantly increased. Inhibition of autophagy by the inhibitor 3-methyladenine attenuated the cell death induced by integrin beta4 knockdown. To further understand the relationship between p53 and integrin beta4 in autophagic cell death, we inhibited the expression of integrin beta4 by its specific siRNA in p53-mutated H322 lung cancer cells. Knockdown of integrin beta4 could not induce autophagic cell death in H322 cells. The data suggest that integrin beta4 is implicated in and associated with p53 in autophagy of lung cancer cells.