Apoptosis In Human Lung Cancer Research Articles

Potential anticancer activity of chemically characterized extract of Olea europaea (Olive) leaves

Olea europaea Linn. (Olive) is considered as essential component of Mediterranean diets. Olive leaves, fruits, and oil are traditionally known for several health benefits including diabetes, cardiac complications, cancer, etc. The objective of the present study is to determine the anticancer potential of chemically characterized O. europaea extract in MTT assay and EB/AO double staining method using Human lung cancer cell lines (A549). The chemical constituents present in the ethyl acetate extract of O. europaea leaves were characterized by GC-MS and its cytotoxic activity was assessed by MTT assay and EB/AO double staining method. The GC-MS analysis identified 63 chemical constituents, and neophytadiene (21.80%), zingiberenol (12.36%), and allohimachalol (5.49%) was found as major chemical constituents in ethyl acetate extract of O. europaea leaves. O. europaea produces a time and dose-dependent inhibition of cell proliferation of A549 cell lines. The cell viability of A549 cell lines after 24 hrs treatment with O. europaea ranged from 97.96±3.44 to 18.95±2.14 % for a concentration range of 0.5-500 μg/mL, respectively, with IC50 value of 21.91±1.8 μg/mL. EB/AO double staining shows significant apoptosis in early and late apoptotic, and necrotic cells with increased volume and showed uneven orange-red fluorescence at their periphery. The study outcome shows that O. europaea extract significantly inhibited cell proliferation and apoptosis in human lung cancer (A549) cell lines, and it also explores the chemical composition of O. europaea leaves extract.
 Keywords: Apoptosis, A549 cell lines, Cancer, GC-MS, MTT assay, Olea europea, Olive.
 

Delivery of Therapeutic miR-148b Mimic via Poly(β Amino Ester) Polyplexes for Post-transcriptional Gene Regulation and Apoptosis of A549 Cells.

In this study, we utilized selectively modified, biodegradable polymer-based polyplexes to deliver custom, exogenous miR-148b mimics to induce apoptosis in human lung cancer (A549) cells. The gene regulatory effects of the payload miRNA mimics (miR-148b-3p) were first evaluated through bioinformatic analyses to uncover specific gene targets involved in critical carcinogenic pathways. Hyperbranched poly(β amino ester) polyplexes (hPBAE) loaded with custom miR-148b mimics were then developed for targeted therapy. When evaluated in vitro, these hPBAE-based polyplexes sustained high intracellular uptake, low cytotoxicity, and efficient escape from endosomes to deliver functionally intact miRNA mimics to the cytosol. High-resolution confocal microscopy revealed successful intracellular uptake, cell viability was assessed through qualitative fluorescence microscopy and fluorescence-based DNA quantification, and successful cytosolic delivery of intact miRNA mimics was evaluated using real-time polymerase chain reaction (RT-PCR) to demonstrate target gene knockdown. The hPBAE-miRNA mimic polyplexes were shown to induce apoptosis among A549 cells through direct modulation of intracellular protein expression, targeting multiple potential carcinogenic pathways at the gene level. These results indicated that spatially controlled miR-148b mimic delivery can promote efficient cancer cell death in vitro and may lead to an enhanced therapeutic design for in vivo application.

VLX1570 regulates the proliferation and apoptosis of human lung cancer cells through modulating ER stress and the AKT pathway.

The ubiquitin‐proteasome system (UPS) possesses unique functions in tumorigenesis and has great potential for targeting tumours. The effectiveness of inhibitors targeting UPS in solid tumours remains to be studied. We screened a library of inhibitors targeting the ubiquitination system and found the highly potent, low‐concentration inhibitor molecule VLX1570 that specifically killed lung cancer cells. VLX1570 is an inhibitor of deubiquitinating enzyme activity and has also shown potential for preclinical cancer treatment. We found that VLX1570 significantly inhibited lung cancer cells proliferation and colony formation. VLX1570 induced a G2/M cell cycle arrest by downregulating CDK1 and CyclinB1. Moreover, VLX1570 significantly promoted the mitochondrial‐associated apoptosis. Mechanistically speaking, VLX1570 activated the PERK/IRE1/ATF6 pathway and induced ER stress in lung cancer cell lines. The inhibition of ER stress by tauroursodeoxycholic acid sodium or 4‐phenylbutyric acid enhanced VLX1570‐induced apoptosis. In addition, VLX1570 treatment led to the inactivation of Akt signalling and inhibited the proliferation of lung cancer cells by downregulating the Akt pathway. Moreover, combined treatment with VLX1570 and Afatinib or Gefitinib induced synergistic anti‐lung cancer activity. Our present study demonstrated a novel therapy using VLX1570, which inhibited the proliferation and increased apoptosis in human lung cancer.

VDAC upregulation and αTAT1‑mediated α‑tubulin acetylation contribute to tanespimycin‑induced apoptosis in Calu‑1 cells.

Voltage‑dependent anion channel 1 (VDAC1) functions as a porin in the mitochondrial outer membrane (MOM) and plays important roles in mitochondria‑mediated cell apoptosis. VDAC1 interacts with a variety of proteins, such as Bcl‑2 family proteins, hexose kinase (HK), adenine nucleotide translocase (ANT) and α‑tubulin. However, the association between VDAC1 and α‑tubulin, particularly between VDAC1 and acetylated α‑tubulin (Ac‑α‑tubulin), in apoptosis remains unclear. The present study revealed that the heat shock protein 90 inhibitor, tanespimycin, induced VDAC1 upregulation and α‑tubulin acetylation during Calu‑1 cell apoptosis in human lung cancer. Hsp90 mediated the expression level of VDAC1, and the acetylation of α‑tubulin was enhanced in an α‑tubulin acetyltransferase 1 (αTAT1)‑dependent manner following an increase in VDAC1 expression. Docetaxel, as an inhibitor of microtubules, augmented the expression of Ac‑α‑tubulin, VDAC1 and Bax induced by tanespimycin and increased the degree of caspase activation. Immunoprecipitation (IP) experiments revealed that Ac‑α‑tubulin, α‑tubulin and VDAC1 were co‑precipitated in the IP complex, in which α‑tubulin expression was decreased and VDAC1 proteins were oligomerized, and that the p‑AKT/glycogen synthase kinase 3β (GSK3β) signalling pathway mediated the opening of VDAC1. Therefore, it can be asserted that the acetylation of α‑tubulin and VDAC1 upregulation or oligomerization induced by tanespimycin may lead to mitochondrial permeability and consequently induce the apoptosis of lung cancer cells. These findings provide evidence for the use of a combination of drugs that target VDAC1 and tubulin to induce tumour cell apoptosis.

Elephantopus mollis Kunth extracts induce antiproliferation and apoptosis in human lung cancer and myeloid leukemia cells

Ethnopharmacological relevanceElephantopus mollis Kunth (EM), which belongs to Asteraceae family, has been used as a folk medicine with diverse therapeutic properties. Previous studies reported that crude extracts of this plant could inhibit several cancer cell lines, including breast carcinoma MCF-7, liver carcinoma HepG2, colorectal carcinoma DLD-1, lung carcinoma NCI–H23, etc. AimIn this study, the anticancer activity and associated molecular mechanism of EM which is distributed in Vietnam were investigated. Materials and methodsThe cytotoxicity of various EM extracts was evaluated on different cell lines by MTT assay. In addition, the effects of EM extracts on cell growth, cell morphology, nuclear morphology, caspase-3 activation, and mRNA expression levels of apoptosis-related genes were also examined. ResultsOur results demonstrated that ethyl acetate extract (EM-EA) caused proliferative inhibition and apoptotic induction towards A549 lung cancer cells (IC50 = 18.66 μg/ml, SI = 5.8) and HL60 leukemia cells (IC50 = 7.45 μg/ml, SI = 14.5) while petroleum ether extract (EM-PE) showed high toxicity to HL60 cell line (IC50 = 11.14 μg/ml, SI = 6.7). Notably, Raji lymphoma cells were also affected by these extracts (IC50 < 20 μg/ml, SI > 4), which has not been reported yet. Furthermore, mechanisms of EM extracts were elucidated. The significant downregulation of PCNA mRNA level induced by EM-EA/PE extracts contributed to the cell-growth restraint. EM-EA extract might activate apoptosis in A549 cells through both extrinsic and intrinsic signaling pathways by causing a 1.55-fold increase in BID, 3.65-fold increase in BAK and 3.11-fold decrease in BCL-2 expression level. Meanwhile, with EM-EA-extract treatment, HL60 cells might encounter P53-dependent apoptotic deaths. ConclusionsThe combination of antiproliferation and apoptosis activation contributed to the high efficacy of EM extracts. These findings not only proved the anticancer potential of EM but also provided further insights into the mechanisms of EM extracts.

Triptolide-induced apoptosis in non-small cell lung cancer via a novel miR204-5p/Caveolin-1/Akt-mediated pathway.

Lung cancer is one of the most prevalent malignancies world-wide with non-small cell lung cancer (NSCLC) comprising nearly 80% of all cases. Unfortunately, many lung cancer patients are diagnosed at advanced stages of the disease with an associated poor prognosis. Recently, the Chinese herb root extract Triptolide/Minnelide (TL) has shown significant promise as a therapeutic agent for NSCLC treatment both in vitro and in vivo. The aim of this study was to investigate the underlying mechanism(s) of action regarding TL-induced cytotoxicity in NSCLC. We demonstrate that triptolide treatment of A549 and H460 NSCLC cells decreases Caveolin-1 (CAV-1) mRNA/protein expression, resulting in activation of the Akt/Bcl-2-mediated mitochondrial apoptosis pathway. CAV-1 down-regulation was triggered by Micro-RNA 204-5p (miR204-5p) up-regulation and could be significantly blocked by pre-treatment with both Sirt-1/Sirt-3 specific siRNA and SIRT-1/SIRT-3 enzyme inhibitors, EX-527 and nicotinamide. Overall, our results provide evidence for a novel mechanism by which TL exerts its cytotoxic effects on NSCLC via CAV-1 down-regulation. Furthermore, these findings demonstrate a pivotal role for TL induction of the Akt/Bax pathway in apoptosis of human lung cancer.

The novel microRNA hsa-miR-CHA1 regulates cell proliferation and apoptosis in human lung cancer by targeting XIAP

ObjectivesMicroRNAs have critical roles in cancer development by regulating the expression of oncogenes or tumor suppressor genes. We identified and characterized a novel miRNA, miR-CHA1, in human lung cancer cells. The aim of this study was to investigate its novel function in human lung cancer by targeting XIAP. Material and methodsNovel miRNA cloning, Real-time qRT-PCR, western blotting, dual luciferase assay, miRNA transfection, proliferation and apoptosis assay were carried on human lung cancer cell line A549. Fifteen paired NSCLC tissues and noncancerous lung tissues were collected. In vivo xenograft assay was performed. ResultsExpression of miR-CHA1 was downregulated in human lung cancer cell lines and tissues compared with normal cells and tissues. We identified a putative target gene, XIAP, whose expression was regulated by miR-CHA1 overexpression. XIAP is an inhibitor of apoptosis that represses the activation of caspase 3 and 9. XIAP mRNA and protein levels were directly suppressed by miR-CHA1. XIAP has an important role in carcinogenesis, and previous studies suggest that it may regulate cell survival and proliferation by its anti-apoptotic ability. ConclusionTaken together, miR-CHA1 inhibited cell proliferation and induced apoptosis in vitro and in vivo by targeting XIAP. These data can be applied to identify novel therapeutic targets for lung cancer therapy.

Suppression of CEP55 reduces cell viability and induces apoptosis in human lung cancer.

Centrosomal protein55(CEP55), identified as a centrosome‑associated protein, has been reported to be involved in human malignancies. However, its biological function in human lung cancer remains largely unknown. In the present study, we firstly analyzed the expression of CEP55 in 20pairs of lung cancer and matched non‑tumor tissues using quantitative RT‑PCR analysis and found that CEP55 mRNA was significantly increased in lung cancer tissues compared with that in matched tumor‑adjacent tissues. Then we performed a loss‑of‑function assay using lung cancer cell lines A549 and 95D. Functionally, knockdown of CEP55 markedly inhibited cell viability and proliferation ability as determined by MTT and colony formation assays. Moreover, CEP55‑silenced cells were obviously arrested in the G0/G1 phase and presented significant cell apoptosis as determined using flow cytometric analysis. Mechanistically, western blot analysis further revealed that knockdown of CEP55 decreased the expression of CDK4, p21 and Bcl‑2, while it increased the expression of pro‑apoptotic protein, Bad, caspase‑3 and PARP in 95D cells. In conclusion, our data highlight the crucial role of CEP55 in promoting lung cancer cell proliferation invitro and its inhibition may be a novel therapeutic strategy for lung cancer.

Abstract 4523: Decreased H2B monoubiquitination is associated with enhanced proliferation and resistance to apoptosis in human lung cancer

Abstract Monoubiquitination of histone H2B (H2Bub1) is an important chromatin modification that plays multiple roles in transcription, mRNA processing, DNA repair, cell differentiation, and maintenance of stemness. Recent studies have indicated that dysregulation of H2Bub1 and the resulting alterations of global gene expression are key contributors in cellular transformation and metastasis. However, the importance of H2B ubiquitination in lung cancer is unknown. Here, by using immunohistochemical analysis (IHC), we have demonstrated that the protein level of H2Bub1 is frequently decreased in a cohort of human lung cancer tissues in comparison to paired adjacent normal tissues. We also found that decrease in H2Bub1 is associated with increased cellular proliferation, as measured by Ki67, and is associated with decreased overall survival. Furthermore, using RNA-seq and transcriptional profiling analysis, we identified that reduction of H2Bub1 via attenuating the expression of histone H2B E3 ubiquitin ligase ring finger protein 20 (RNF20) leads to significant changes of global gene expression, modulating multiple malignancy-promoting genes, including c-Met, E2F2, and BMF. Signaling pathways including p53, DNA replication, DNA repair, and cellular proliferation, and regulation of apoptosis in lung cancer cells are also altered. Additionally, we have further shown that decrease of H2Bub1 by RNF20 knockdown significantly promotes growth, migration/invasion, and strongly enhances resistance to cytotoxic chemotherapy-induced apoptosis in lung cancer cells. Our findings suggest that dysregulation of H2Bub1 signaling may play an important role in growth and drug resistance of lung cancers, and may potentially serve as a prognostic biomarker and therapeutic target for lung cancer patients. Citation Format: Keqiang Zhang, Jinhui Wang, Xiwei Wu, Theresa A. Reno, Tommy R. Tong, Jae Kim, Dan Raz. Decreased H2B monoubiquitination is associated with enhanced proliferation and resistance to apoptosis in human lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4523.

Apoptosis Induction by MEK Inhibition in Human Lung Cancer Cells Is Mediated by Bim

AZD6244 (ARRY-142886) is an inhibitor of MEK1/2 and can inhibit cell proliferation or induce apoptosis in a cell-type dependent manner. The precise molecular mechanism of AZD6244-induced apoptosis is not clear. To investigate mechanisms of AZD6244 induced apoptosis in human lung cancer, we determined the molecular changes of two subgroups of human lung cancer cell lines that are either sensitive or resistant to AZD6244 treatment. We found that AZD6244 elicited a large increase of Bim proteins and a smaller increase of PUMA and NOXA proteins, and induced cell death in sensitive lung cancer cell lines, but had no effect on other Bcl-2 related proteins in those cell lines. Knockdown of Bim by siRNA greatly increased the IC50 and reduced apoptosis for AZD6244 treated cells. We also found that levels of endogenous p-Thr32-FOXO3a and p-Ser253-FOXO3a were lower in AZD6244-sensitive cells than in AZD6244-resistant cells. In the sensitive cells, AZD6244 induced FOXO3a nuclear translocation required for Bim activation. Moreover, the silencing of FOXO3a by siRNA abrogated AZD6244-induced cell apoptosis. In addition, we found that transfection of constitutively active AKT up-regulated p-Thr32-FOXO3a and p-Ser253-FOXO3a expression and inhibited AZD6244-induced Bim expression in sensitive cells. These results show that Bim plays an important role in AZD6244-induced apoptosis in lung cancer cells and that the PI3K/AKT/FOXO3a pathway is involved in Bim regulation and susceptibility of lung cancer cells to AZD6244. These results have implications in the development of strategies to overcome resistance to MEK inhibitors.

Peroxisome proliferator-activated receptor gamma and early folliculogenesis during an acute hyperandrogenism condition

Acute hyperandrogenism decreases serum P levels and induces early apoptosis of antral follicles by a mechanism mediated by the peroxisome proliferator-activated receptor gamma system and independent of the steroidogenic acute regulator protein.

The eIF4E/eIF4G Interaction Inhibitor 4EGI-1 Augments TRAIL-Mediated Apoptosis through c-FLIP Down-regulation and DR5 Induction Independent of Inhibition of Cap-Dependent Protein Translation

The small molecule 4EGI-1 was identified as an inhibitor of cap-dependent translation initiation owing to its disruption of the eIF4E/eIF4G association through binding to eIF4E. 4EGI-1 exhibits growth-inhibitory and apoptosis-inducing activity in cancer cells; thus, we were interested in its therapeutic efficacy in human lung cancer cells. 4EGI-1, as a single agent, inhibited the growth and induced apoptosis of human lung cancer cells.When combined with the death ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), enhanced apoptosis-induced activity was observed. As expected, 4EGI-1 inhibited eIF4E/eIF4G interaction and reduced the levels of cyclin D1 and hypoxia-inducing factor-1α (HIF-1α), both of which are regulated by a cap-dependent translation mechanism. Moreover, 4EGI-1 induced CCAAT/enhancer-binding protein homologous protein-dependent DR5 expression and ubiquitin/proteasome- mediated degradation of cellular FLICE-inhibitory protein (c-FLIP). Small interfering RNA-mediated blockade of DR5 induction or enforced expression of c-FLIP abrogated 4EGI-1's ability to enhance TRAIL-induced apoptosis, indicating that both DR5 induction and c-FLIP down-regulation contribute to enhancement of TRAIL-induced apoptosis by 4EGI-1. However, inhibition of eIF4E/eIF4G interaction by knockdown of eIF4E effectively reduced the levels of cyclin D1 and HIF-1α but failed to induce DR5 expression, downregulate c-FLIP levels, or augment TRAIL-induced apoptosis. These results collectively suggest that 4EGI-1 augments TRAIL-induced apoptosis through induction of DR5 and down-regulation of c-FLIP, independent of inhibition of cap-dependent protein translation.

Plasma Membrane Alterations and Cytoskeletal Changes in Apoptosis

During apoptosis, one of the first membrane changes that can be detected is exposure of phosphatidylserine residues at the outer plasma membrane leaflet, while early apoptosis is also accompanied by changes in the cytoskeletal organization. In this study we investigated the relationship between these two phenomena during olomoucine- and roscovitin-induced apoptosis in human lung cancer and neuroblastoma cell lines. Loss of membrane asymmetry was detected by biotin-labeled or FITC-labeled annexin V binding to negatively charged phosphatidylserine, while cytoskeletal components were visualized by immunocytochemistry. The apoptotic, annexin V-positive, cells were analyzed by flow cytometry, confocal scanning laser microscopy, and Western blotting. We report that cytokeratin and vimentin aggregation in early apoptosis occurs simultaneously with phosphatidylserine exposure and chromatin condensation. In contrast to these intermediate filament proteins, which were disassembled and proteolytically cleaved in early apoptosis, microfilaments and microtubuli were not proteolytically degraded but were found to be present as aggregated filaments in the apoptotic bodies. We also show that loss of membrane asymmetry and cytokeratin aggregation are independent processes, sinceN-ethylmaleimide-induced phosphatidylserine exposure does not cause cytokeratin disassembly. Vice versa, phorbol 12-myristate 13-acetate-induced cytokeratin filament aggregation does not result in phosphatidylserine exposure.