The Interplay between Autophagy and Apoptosis Induced by One Synthetic Curcumin Derivative Hydrazinobenzoylcurcumin in A549 Lung Cancer Cells

The two important cell autonomic responses, autophagy, and apoptosis, play critical roles in cellular homeostasis and survival. By studying of the synthetic curcumin derivative hydrazinobenzoylcurcumin (HBC), we revealed that it could induce autophagy in nonsmall lung cancer cells (A549). Here, we use the Hoechst 33342 staining, Annexin V/propyliodide double dyeing and Western blotting analysis of PARP protein to demonstrate that HBC could also induce apoptosis in A549 cells. Apoptosis inhibitor (Z‐VAD‐FMK, 10 μM) treatment helps to promote the cells survival. Moreover, inhibition of apoptosis‐promoted HBC‐induced autophagy of A549 cells by morphological detection and Western blotting analysis (vice versa). These data indicate that there exist some interconnections between the autophagy and apoptosis induced by HBC. The following work will be carried out to characterize the specific regulation processes between the two cell pathways in A549 cells.     

Role of autophagy in inhibiting the proliferation of A549 cells by type III interferon

Interferons (IFNs) have anti‐viral and anti‐tumour effects. Type III interferon, as a member of the recently discovered interferon family, has been proved to inhibit tumour proliferation and promote the apoptosis of various tumour cells. However, whether type III IFN could inhibit the proliferation of lung cancer was not clear. In this study, we found that interferon λ (IFN λ) could inhibit the proliferation of A549 cells and induce autophagy and apoptosis of A549 cells. IFN λ could promote the expression of autophagy gene Beclin1 and interfere the expression of autophagy gene Beclin1 with small interfering RNA, thus inhibiting the effect of type III interferon on anti‐proliferation and promoting apoptosis of lung cancer cell. These results suggested that IFN λ could inhibit the proliferation of A549 cells by activating autophagy pathway, and IFN λ might be one of the potential therapeutic drugs for lung cancer.     

Licarin A induces cell death by activation of autophagy and apoptosis in non-small cell lung cancer cells

Lung cancer has a relatively poor prognosis with a low survival rate and drugs that target other cell death mechanism like autophagy may help improving current therapeutic strategy. This study investigated the anti-proliferative effect of Licarin A (LCA) from Myristica fragrans in non-small cell lung cancer cell lines—A549, NCI-H23, NCI-H520 and NCI-H460. LCA inhibited proliferation of all the four cell lines in a dose and time dependent manner with minimum IC50 of 20.03?±?3.12, 22.19?±?1.37 µM in NCI-H23 and A549 cells respectively. Hence NCI-H23 and A549 cells were used to assess the ability LCA to induce autophagy and apoptosis. LCA treatment caused G1 arrest, increase in Beclin 1, LC3II levels and degradation of p62 indicating activation of autophagy in both NCI-H23 and A549 cells. In addition, LCA mediated apoptotic cell death was confirmed by MMP loss, increased ROS, cleaved PARP and decreased pro-caspase3. To understand the role of LCA induced autophagy and its association with apoptosis, cells were analysed following treatment with a late autophagy inhibitor-chloroquine and also after Beclin 1 siRNA transfection. Data indicated that inhibition of autophagy resulted in reduced anti-proliferative as well as pro-apoptotic ability of LCA. These findings confirmed that LCA brought about autophagy dependent apoptosis in non-small cell lung cancer cells and hence it may serve as a potential drug candidate for non-small cell lung cancer therapy.     

Paraptosis accompanied by autophagy and apoptosis was induced by celastrol, a natural compound with influence on proteasome, ER stress and Hsp90

In the present study, we found that celastrol, a natural compound with well-known apoptosis-inducing effect, could also induce paraptosis-like cytoplasmic vacuolization in cancer cell lines including HeLa cells, A549 cells and PC-3 cells derived from cervix, lung and prostate, respectively. Further study using HeLa cells indicated that the vacuoles induced by celastrol might be derived from dilation of endoplasmic reticulum. And, in celastrol-treated cells, markers of autophagy such as transformation of microtubule-associated protein 1 light chain 3 (LC3)I to LC3II and LC3 punctates formation were identified. Interestingly, autophagy inhibitors could not interrupt but enhance the induction of cytoplasmic vacuolization. Furthermore, MAPK pathways were activated by celastrol and inhibitors of MEK and p38 pathways could prevent the formation of cytoplasmic vacuolization. Celastrol treatment also induced G2/M cell cycle arrest and apoptosis in HeLa cells. In conclusion, celastrol induced a kind of paraptosis accompanied by autophagy and apoptosis in cancer cells. The coincidence of apoptosis and autophagy together with paraptosis might contribute to the unique characteristics of paraptosis in celastrol-treated cells such as the dependence of paraptosis on MAPK pathways and dynamic change of LC3 proteins. Both paraptosis and apoptosis could contribute to the cell death induced by celastrol while autophagy might serve as a kind of survival mechanism. The potency of celastrol to induce paraptosis, apoptosis and autophagy at the same dose might be related to its capability to affect a variety of pathways including proteasome, ER stress and Hsp90.     

Afatinib Decreases P‐Glycoprotein Expression to Promote Adriamycin Toxicity of A549T Cells

We investigated the reversal effect of afatinib (AFT) on activity of adriamycin (ADR) in A549T cells and clarified the related molecular mechanisms. A549T cells overexpressing P‐glycoprotein (P‐gp) were resistant to anticancer drug ADR. AFT significantly increased the antitumor activity of ADR in A549T cells. AFT increased the intracellular concentration of ADR by inhibiting the function and expression of P‐gp at mRNA and protein levels in A549T cells. Additionally, the reversal effect of AFT on P‐gp mediated multidrug resistance (MDR) might be related to the inhibition of PI3K/Akt pathway. Cotreatment with AFT and ADR could enhance ADR‐induced apoptosis and autophagy in A549T cells. Meanwhile, the co‐treatment significantly induced cell apoptosis and autophagy accompanied by increased expression of cleaved caspase‐3, PARP, LC3B‐II, and beclin 1. Apoptosis inhibitors had no significant effect on cell activity, while autophagy inhibitors decreased cell viability, suggesting that autophagy may be a self protective mechanism of cell survival in the absence of chemotherapy drugs. Interestingly, when combined with AFT and ADR, inhibition of apoptosis and/or autophagy could enhance cell viability. These results indicated that in addition to inhibit P‐gp, ADR‐induced apoptosis, and autophagy promoted by AFT contributed to the antiproliferation effect of combined AFT and ADR on A549T cells. These findings provide evidence that AFT combined ADR may achieve a better therapeutic effect to lung cancer in clinic. J. Cell. Biochem. 119: 414–423, 2018. © 2017 Wiley Periodicals, Inc.     

AKT/mTOR and c-Jun N-terminal kinase signaling pathways are required for chrysotile asbestos-induced autophagy

Chrysotile asbestos is closely associated with excess mortality from pulmonary diseases such as lung cancer, mesothelioma, and asbestosis. Although multiple mechanisms in which chrysotile asbestos fibers induce pulmonary disease have been identified, the role of autophagy in human lung epithelial cells has not been examined. In this study, we evaluated whether chrysotile asbestos induces autophagy in A549 human lung epithelial cells and then analyzed the possible underlying molecular mechanism. Chrysotile asbestos induced autophagy in A549 cells based on a series of biochemical and microscopic autophagy markers. We observed that asbestos increased expression of A549 cell microtubule-associated protein 1 light chain 3 (LC3-II), an autophagy marker, in conjunction with dephosphorylation of phospho-AKT, phospho-mTOR, and phospho-p70S6K. Notably, AKT1/AKT2 double-knockout murine embryonic fibroblasts (MEFs) had negligible asbestos-induced LC3-II expression, supporting a crucial role for AKT signaling. Chrysotile asbestos also led to the phosphorylation/activation of Jun N-terminal kinase (JNK) and p38 MAPK. Pharmacologic inhibition of JNK, but not p38 MAPK, dramatically inhibited the protein expression of LC3-II. Moreover, JNK2^−/− MEFs but not JNK1^−/− MEFs blocked LC3-II levels induced by chrysotile asbestos. In addition, N-acetylcysteine, an antioxidant, attenuated chrysotile asbestos-induced dephosphorylation of P-AKT and completely abolished phosphorylation/activation of JNK. Finally, we demonstrated that chrysotile asbestos-induced apoptosis was not affected by the presence of the autophagy inhibitor 3-methyladenine or autophagy-related gene 5 siRNA, indicating that the chrysotile asbestos-induced autophagy may be adaptive rather than prosurvival. Our findings demonstrate that AKT/mTOR and JNK2 signaling pathways are required for chrysotile asbestos-induced autophagy. These data provide a mechanistic basis for possible future clinical applications targeting these signaling pathways in the management of asbestos-induced lung disease.     

Radiosensitizing effects of curcumin alone or combined with GLUT1 siRNA on laryngeal carcinoma cells through AMPK pathway-induced autophagy

In this study, we investigated the ability of curcumin alone or in combination with GLUT1 siRNA to radiosensitize laryngeal carcinoma (LC) through the induction of autophagy. Protein levels in tumour tissues and LC cells were measured by immunohistochemistry and Western blotting. In vitro, cell proliferation, colony formation assays, cell death and autophagy were detected. A nude mouse xenograft model was established through the injection of Tu212 cells. We found that GLUT1 was highly expressed and negatively associated with autophagy-related proteins in LC and that curcumin suppressed radiation-mediated GLUT1 overexpression in Tu212 cells. Treatment with curcumin, GLUT1 siRNA, or the combination of the two promoted autophagy. Inhibition of autophagy using 6-amino-3-methypourine (3-MA) promoted apoptosis after irradiation or treatment of cells with curcumin and GLUT1 siRNA. 3-MA inhibited curcumin and GLUT1 siRNA-mediated non-apoptotic programmed cell death. The combination of curcumin, GLUT1 siRNA and 3-MA provided the strongest sensitization in vivo. We also found that autophagy induction after curcumin or GLUT1 siRNA treatment implicated in the AMP-activated protein kinase-mTOR-serine/threonine-protein kinase-Beclin1 signalling pathway. Irradiation primarily caused apoptosis, and when combined with curcumin and GLUT1 siRNA treatment, the increased radiosensitivity of LC occurred through the concurrent induction of apoptosis and autophagy.     

Cardiac glycosides induce autophagy in human non-small cell lung cancer cells through regulation of dual signaling pathways

Na(+)/K(+)-ATPase targeted cancer therapy has attracted increasing interests of oncologists in lung cancer field. Although multiple anti-cancer mechanisms of cardiac glycosides as Na(+)/K(+)-ATPase inhibitors are revealed, the role of autophagy and related molecular signaling pathway for the class of compounds in human non-small cell lung cancer (NSCLC) cells has not been systematically examined. We herein investigated the anti-cancer effects of two representative cardiac glycosides, digoxin and ouabain, in A549 and H460 cell lines. Both agents caused significant growth inhibition at nanomolar level. The cardiac glycosides were found to induce moderate G(2)/M arrest but not apoptosis at IC(50) level in the NSCLC cell lines. Moreover, autophagy was markedly induced by both agents, as evidenced by the time- and dose-dependent increase of LC3-II, up-regulation of Atg5 and Beclin1, as well as by the observations through acridine orange staining, transmission electron microscopy and quantification of GFP-LC3 fluorescence. Importantly, AMP-activated protein kinase (AMPK) pathway was activated, resulting in mammalian target of rapamycin (mTOR) deactivation during autophagy induction. Moreover, extracellular-signal-regulated kinase 1/2 (ERK1/2) activation was simultaneously found to be involved in the autophagy regulation. Co-treatment with respective inhibitors or siRNAs could either block the autophagic phenotypes and signals, or significantly increase the cellular viability, indicating the drugs-induced autophagy plays tumor-suppressing role. This work provides first evidence showing that the cardiac glycosides induce autophagy in human NSCLC cells through regulation of both mTOR and ERK1/2 signaling pathways. The autophagy may at least partially account for the growth inhibitory effects of the compounds in human NSCLC cells.     

Role of p38 MAPK in enhanced human cancer cells killing by the combination of aspirin and ABT‐737

Regular use of aspirin after diagnosis is associated with longer survival among patients with mutated‐PIK3CA colorectal cancer, but not among patients with wild‐type PIK3CA cancer. In this study, we showed that clinically achievable concentrations of aspirin and ABT‐737 in combination could induce a synergistic growth arrest in several human PIK3CA wild‐type cancer cells. In addition, our results also demonstrated that long‐term combination treatment with aspirin and ABT‐737 could synergistically induce apoptosis both in A549 and H1299 cells. In the meanwhile, short‐term aspirin plus ABT‐737 combination treatment induced a greater autophagic response than did either drug alone and the combination‐induced autophagy switched from a cytoprotective signal to a death‐promoting signal. Furthermore, we showed that p38 acted as a switch between two different types of cell death (autophagy and apoptosis) induced by aspirin plus ABT‐737. Moreover, the increased anti‐cancer efficacy of aspirin combined with ABT‐737 was further validated in a human lung cancer A549 xenograft model. We hope that this synergy may contribute to failure of aspirin cancer therapy and ultimately lead to efficacious regimens for cancer therapy.     

Organic anion transporters and PI3K–AKT–mTOR pathway mediate the synergistic anticancer effect of pemetrexed and rhein

The aim of this study was to explore whether rhein could enhance the effects of pemetrexed (PTX) on the therapy of non-small-cell lung cancer (NSCLC) and to clarify the associated molecular mechanism. Our study shows that rhein in combination with PTX could obviously increase the systemic exposure of PTX in rats, which would be mediated by the inhibition of organic anion transporters (OATs). Furthermore, the toxicity of PTX was significantly raised by rhein in A549 cells in a concentration-dependent manner. Concomitant administration of rhein and PTX-induced cell apoptosis compared with PTX alone in flow cytometry assays, which was further validated by the protein expressions of the apoptotic markers B-cell lymphoma-2/Bcl-2-associated x (Bcl-2/Bax) and Cleaved-Caspase3 (Cl-Caspase3). Meanwhile, the results of monodansylcadaverine (MDC) dyeing experiments showed that PTX-induced autophagy could be enhanced by combination therapy with rhein in A549 cells. Western blot analysis indicated that the synergistic effect of rhein on PTX-mediated autophagy may be interrelated to PI3K–AKT–mTOR pathway inhibition and to the enhancement of p-AMPK and light chain 3-II (LC3-II) protein levels. From these findings, it could be surmised that rhein enhanced the antitumor activity of PTX through influencing autophagy and apoptosis by modulating the PI3K–AKT–mTOR pathway and Bcl-2 family of proteins in A549 cells. Our findings demonstrated that the potential application of rhein as a candidate drug in combination with PTX is promising for treatment of the human lung cancer.     

Mesenchymal stem cells promote cell invasion and migration and autophagy‐induced epithelial‐mesenchymal transition in A549 lung adenocarcinoma cells

Mesenchymal stem cells (MSCs) are recruited into the tumour microenvironment and promote tumour growth and metastasis. Tumour microenvironment‐induced autophagy is considered to suppress primary tumour formation by impairing migration and invasion. Whether these recruited MSCs regulate tumour autophagy and whether autophagy affects tumour growth are controversial. Our data showed that MSCs promote autophagy activation, reactive oxygen species production, and epithelial‐mesenchymal transition (EMT) as well as increased migration and invasion in A549 cells. Decreased expression of E‐cadherin and increased expression of vimentin and Snail were observed in A549 cells cocultured with MSCs. Conversely, MSC coculture‐mediated autophagy positively promoted tumour EMT. Autophagy inhibition suppressed MSC coculture‐mediated EMT and reduced A549 cell migration and invasion slightly. Furthermore, the migratory and invasive abilities of A549 cells were additional increased when autophagy was further enhanced by rapamycin treatment. Taken together, this work suggests that microenvironments containing MSCs can promote autophagy activation for enhancing EMT; MSCs also increase the migratory and invasive abilities of A549 lung adenocarcinoma cells. Mesenchymal stem cell‐containing microenvironments and MSC‐induced autophagy signalling may be potential targets for blocking lung cancer cell migration and invasion.     

Discovery of a new autophagy inducer for A549 lung cancer cells

Biological activities of a series of fluorescent compounds against human lung cancer cell line A549 were investigated. The results showed that (E)-1,3,3-trimethyl-2-(4-(piperidin-1-yl)styryl)-3H-indol-1-ium iodide (8) and (E)-2-(5,5-dimethyl-3-(4-(piperazin-1-yl)styryl)cyclohex-2-en-1-ylidene) malononitrile (11) could inhibit the growth of A549 cancer cells in a dose and time-dependent manner. Furthermore, compound 8 could trigger autophagy and apoptosis, but not obviously induce necrosis under the stimulatory condition. Therefore, 8 can be used as autophagy activator to investigate the regulatory mechanism of autophagy and may offer a new candidate for the treatment of lung cancer.     

Type I interferons induce autophagy in certain human cancer cell lines

Autophagy is an evolutionarily conserved cellular recycling mechanism that occurs at a basal level in all cells. It can be further induced by various stimuli including starvation, hypoxia, and treatment with cytokines such as IFNG/IFNγ and TGFB/TGFβ. Type I IFNs are proteins that induce an antiviral state in cells. They also have antiproliferative, proapoptotic and immunomodulatory activities. We investigated whether type I IFN can also induce autophagy in multiple human cell lines. We found that treatment with IFNA2c/IFNα2c and IFNB/IFNβ induces autophagy by 24 h in Daudi B cells, as indicated by an increase of autophagy markers MAP1LC3-II, ATG12–ATG5 complexes, and a decrease of SQSTM1 expression. An increase of MAP1LC3-II was also detected 48 h post-IFNA2c treatment in HeLa S3, MDA-MB-231, T98G and A549 cell lines. The presence of autophagosomes in selected cell lines exposed to type I IFN was confirmed by electron microscopy analysis. Increased expression of autophagy markers correlated with inhibition of MTORC1 in Daudi cells, as well as inhibition of cancer cell proliferation and changes in cell cycle progression. Concomitant blockade of either MTOR or PI3K-AKT signaling in Daudi and T98G cells treated with IFNA2c increased the level of MAP1LC3-II, indicating that the PI3K-AKT-MTORC1 signaling pathway may modulate IFN-induced autophagy in these cells. Taken together, our findings demonstrated a novel function of type I IFN as an inducer of autophagy in multiple cell lines.     

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.     

Production of CCL20 from lung cancer cells induces the cell migration and proliferation through PI3K pathway

Tumour inflammatory microenvironment is considered to play a role in the sensitivity of tumour cells to therapies and prognosis of patients with lung cancer. The expression of CCL20, one of the critical chemoattractants responsible for inflammation cells recruitment, has been shown overexpressed in variety of tumours. This study aimed at investigating potential mechanisms of CCL20 function and production in human non‐small cell lung cancer (NSCLC). Expression of CCL20 gene and protein in lung tissues of patients with NSCLC and NSCLC cells (A549) were determined. The interleukin (IL)‐1β‐induced signal pathways in A549 and the effect of CCL20‐induced A549 cell migration and proliferation were determined using migration assays and cell‐alive monitoring system. Mechanisms of signal pathways involved in the migration of CCL20 were also studied. We initially found that NSCLC tumour tissues markedly overexpressed CCL20 in comparison with normal lung samples. In addition, IL‐1β could directly promote CCL20 production in lung cancer cells, which was inhibited by extracellular signal‐regulated kinase (ERK)1/2 inhibitor, p38 mitogen‐activated protein kinase (p38 MARP) inhibitor or PI3K inhibitors. CCL20 promoted lung cancer cells migration and proliferation in an autocrine manner via activation of ERK1/2‐MAPK and PI3K pathways. Our data indicated that IL‐1β could stimulate CCL20 production from lung cancer cells through the activation of MAPKs and PI3K signal pathways, and the auto‐secretion of CCL20 could promote lung cancer cell migration and proliferation through the activation of ERK and PI3K signal pathways. Our results may provide a novel evidence that CCL20 could be a new therapeutic target for lung cancer.     

MicroRNA‐144‐3p suppressed TGF‐β1‐induced lung cancer cell invasion and adhesion by regulating the Src–Akt–Erk pathway

Lung cancer remains a leading cause to cancer‐related death worldwide. The anti‐cancer ability of microRNA‐144‐3p has been reported in many cancer types. This study focused on the mechanisms underlying miR‐144‐3p in inhibiting lung cancer. The expression levels of miR‐144‐3p and steroid receptor coactivator (Src) in different lung cancer cell lines and those in bronchial epithelial cells (16HBE) were compared. miR‐144‐3p mimic and siSrc were transfected into A549 cells. Under the conditions of transforming growth factor‐β1 (TGF‐β1). Small interfering transfection or TGF‐β1 treatment, cell invasive and adhesive abilities were analyzed by Transwell and cell adhesion assays. miR‐144‐3p inhibitor and siSrc were co‐transfected into A549 cells and the changes in cell invasion and adhesion were detected. The activation of Src–protein kinase B–extracellular‐regulated protein kinases (Src–Akt–Erk) pathway was determined using Western blot. The downregulated miR‐144‐3p and upregulated Src were generally detected in lung cancer cell lines and were the most significant genes in A549 cells. Both miR‐144‐3p overexpression and Src inhibition could obviously inhibit the invasion and adhesion abilities of A549 cells in the presence or absence of the effects of TGF‐β1. The inhibition of Src could block the promotive effects of miR‐144‐3p inhibitor and TGF‐β1 on cell invasion and adhesion. Furthermore, we found that miR‐144‐3p could negatively regulate the phosphorylation levels of Akt and Erk. Our data indicated the essential role of Src in the mechanisms underlying TGF‐β1‐induced cell invasion and adhesion of lung cancer, and that miR‐144‐3p could effectively suppress TGF‐β1‐induced aggressive lung cancer cells by regulating Src expression.     

Defects in Mitochondrial Fission Protein Dynamin-Related Protein 1 Are Linked to Apoptotic Resistance and Autophagy in a Lung Cancer Model

Evasion of apoptosis is implicated in almost all aspects of cancer progression, as well as treatment resistance. In this study, resistance to apoptosis was identified in tumorigenic lung epithelial (A549) cells as a consequence of defects in mitochondrial and autophagic function. Mitochondrial function is determined in part by mitochondrial morphology, a process regulated by mitochondrial dynamics whereby the joining of two mitochondria, fusion, inhibits apoptosis while fission, the division of a mitochondrion, initiates apoptosis. Mitochondrial morphology of A549 cells displayed an elongated phenotype–mimicking cells deficient in mitochondrial fission protein, Dynamin-related protein 1 (Drp1). A549 cells had impaired Drp1 mitochondrial recruitment and decreased Drp1-dependent fission. Cytochrome c release and caspase-3 and PARP cleavage were impaired both basally and with apoptotic stimuli in A549 cells. Increased mitochondrial mass was observed in A549 cells, suggesting defects in mitophagy (mitochondrial selective autophagy). A549 cells had decreased LC3-II lipidation and lysosomal inhibition suggesting defects in autophagy occur upstream of lysosomal degradation. Immunostaining indicated mitochondrial localized LC3 punctae in A549 cells increased after mitochondrial uncoupling or with a combination of mitochondrial depolarization and ectopic Drp1 expression. Increased inhibition of apoptosis in A549 cells is correlated with impeded mitochondrial fission and mitophagy. We suggest mitochondrial fission defects contribute to apoptotic resistance in A549 cells.     

Mimulone-Induced Autophagy through p53-Mediated AMPK/mTOR Pathway Increases Caspase-Mediated Apoptotic Cell Death in A549 Human Lung Cancer Cells

Anticancer properties and mechanisms of mimulone (MML), C-geranylflavonoid isolated from the Paulownia tomentosa fruits, were firstly elucidated in this study. MML prevented cell proliferation in a dose- and time-dependent way and triggered apoptosis through the extrinsic pathway in A549 human lung adenocarcinoma cells. Furthermore, MML-treated cells displayed autophagic features, such as the formation of autophagic vacuoles, a primary morphological feature of autophagy, and the accumulation of microtubule-associated protein 1 light chain 3 (LC3) puncta, another typical maker of autophagy, as determined by FITC-conjugated immunostaining and monodansylcadaverine (MDC) staining, respectively. The expression levels of LC3-I and LC3-II, specific markers of autophagy, were also augmented by MML treatment. Autophagy inhibition by 3-methyladenine (3-MA), pharmacological autophagy inhibitor, and shRNA knockdown of Beclin-1 reduced apoptotic cell death induced by MML. Autophagic flux was not significantly affected by MML treatment and lysosomal inhibitor, chloroquine (CQ) suppressed MML-induced autophagy and apoptosis. MML-induced autophagy was promoted by decreases in p53 and p-mTOR levels and increase of p-AMPK. Moreover, inhibition of p53 transactivation by pifithrin-α (PFT-α) and knockdown of p53 enhanced induction of autophagy and finally promoted apoptotic cell death. Overall, the results demonstrate that autophagy contributes to the cytotoxicity of MML in cancer cells harboring wild-type p53. This study strongly suggests that MML is a potential candidate for an anticancer agent targeting both autophagy and apoptotic cell death in human lung cancer. Moreover, co-treatment of MML and p53 inhibitor would be more effective in human lung cancer therapy.     

Curcumin reverses cis-platin resistance and promotes human lung adenocarcinoma A549/DDP cell apoptosis through HIF-1α and caspase-3 mechanisms

Curcumin, a yellow pigment derived from Curcuma longa Linn, has been favored by the Eastern as dietary ingredients for centuries. During the past decade, extensive investigations have revealed curcumin sensitized various chemotherapeutic agents in human breast, colon, pancreas, gastric, liver, brain and hematological malignant disorders in vivo and in vitro. Several pathways and specific targets including NF-κB, STAT3, COX-2, Akt and multidrug resistant protein have been identified to facilitate curcumin as a chemosensitizer. Recent studies suggest HIF-1α participated in the development of drug resistance in cancer cells and targeting HIF-1α either by RNAi or siRNA successfully overcame chemotherapeutic resistance. To investigate the mechanism basis of curcumin as a chemosensitizer in lung cancer, we examined curcumin's effects on HIF-1α in cis-platin (DDP) sensitive A549 and resistant A549/DDP cell lines by RT-PCR and Western blot. HIF-1α in A549/DDP cells was found to be overexpressed at both mRNA and protein levels together with a poor response to DDP. Results from transient transfection and flow cytometry showed the HIF-1α abnormality contributed to DDP resistance in A549/DDP lung cancer cells. Combined curcumin and DDP treatment markedly inhibited A549/DDP cells proliferation, reversed DDP resistance and triggered apoptotic death by promoting HIF-1α degradation and activating caspase-3, respectively. Expression of HIF-1α-dependent P-gp also seemed to decrease as response to curcumin in a dose-dependent manner. Our findings shed light on drug resistant reversing effect of curcumin in lung cancer cells by inhibiting HIF-1α expression and activating caspase-3.     

A triazole-conjugated benzoxazone induces reactive oxygen species and promotes autophagic apoptosis in human lung cancer cells

Numerous approaches suggested that compounds with conjugated triazole moieties or benzoxazone pharmacores are effective to antagonize proliferation of human tumors. The current study reported that a synthetic triazole-conjugated benzoxazone, 4-((5-benzyl-1H-1,2,3-triazol-3-yl)-methyl)-7-methoxy-2H-benzo[b][1,4]-oxazin-3(4H)-one (BTO), inhibited growth rates of human non-small cell lung cancer cells. The cytotoxicity can be enhanced with increasing drug concentrations. More evidence supported that the induced reactive oxygen species lead to ultimate apoptotic cell death by recruiting autophagy. The mechanistic pathway as elucidated involved tumor suppressor p53 activation and LC3-1 conversion followed by PARP and procaspase-3 cleavage. Autophagy inhibition reverted apoptotic death and restored cell viabilities. BTO suppressed the development of A549 cell xenograft tumors by activating autophagy and apoptosis simultaneously. As an efficient tumor growth inhibitor with relatively small molecular weight, BTO is a viable addition to the existing list of lung cancer treatment.