Human Non-small Cell Lung Carcinoma Research Articles

Targeting the SREBP-1/Hsa-Mir-497/SCAP/FASN Oncometabolic Axis Inhibits the Cancer Stem-like and Chemoresistant Phenotype of Non-Small Cell Lung Carcinoma Cells.

Background: Lung cancer remains a leading cause of cancer-related death, with an annual global mortality rate of 18.4%. Despite advances in diagnostic and therapeutic technologies, non–small cell lung carcinoma (NSCLC) continues to be characterized by a poor prognosis. This may be associated with the enrichment of cancer stem cells (CSCs) and the development of chemoresistance—a double-edged challenge that continues to impede the improvement of long-term outcomes. Metabolic reprogramming is a new hallmark of cancer. Sterol regulatory element-binding proteins (SREBPs) play crucial regulatory roles in the synthesis and uptake of cholesterol, fatty acids, and phospholipids. Recent evidence has demonstrated that SREBP-1 is upregulated in several cancer types. However, its role in lung cancer remains unclear. Objective: This study investigated the role of SREBP-1 in NSCLC biology, progression, and therapeutic response and explored the therapeutic exploitability of SREBP-1 and SREBP-1-dependent oncometabolic signaling and miRNA epigenetic regulation. Methods: We analyzed SREBP-1 levels and biological functions in clinical samples and the human NSCLC cell lines H441 and A549 through shRNA-based knock down of SREBP function, cisplatin-resistant clone generation, immunohistochemical staining of clinical samples, and cell viability, sphere-formation, Western blot, and quantitative PCR assays. We conducted in-silico analysis of miRNA expression in NSCLC samples by using the Gene Expression Omnibus (GSE102286) database. Results: We demonstrated that SREBP-1 and SCAP are highly expressed in NSCLC and are positively correlated with the aggressive phenotypes of NSCLC cells. In addition, downregulation of the expression of tumor-suppressing hsa-miR-497-5p, which predictively targets SREBP-1, was observed. We also demonstrated that SREBP-1/SCAP/FASN lipogenic signaling plays a key role in CSCs-like and chemoresistant NSCLC phenotypes, especially because the fatostatin or shRNA targeting of SREBP-1 significantly suppressed the viability, cisplatin resistance, and cancer stemness of NSCLC cells and because treatment induced the expression of hsa-miR-497. Conclusion: Targeting the SREBP-1/hsa-miR-497 signaling axis is a potentially effective anticancer therapeutic strategy for NSCLC.

Abstract 1632: An orthotopic model for luciferase-labeled human lung epithelial carcinoma with response to standard of care treatment

Abstract The human lung epithelial carcinoma A549 cell line was first developed in 1972 after culturing pulmonary carcinoma tissue from a 58-year-old Caucasian male. Similar to human non-small cell lung carcinoma (NSCLC), this cell line carries a KRAS mutation making an attractive model for drug discovery/oncology research applications. NSCLC tumors grow slower and less aggressively than small cell lung cancers, yet the 5-year survival rate for NSCLC is 25%. Thus, more relevant preclinical models are still needed to evaluate the potential therapeutic activities of agents that can be used in the clinic. Orthotopic implantation of luciferase-labeled cancer cell lines facilitate the evaluation of disease progression using bioluminescence in vivo imaging. Here we report the development of a novel model of orthotopically implanted Luciferase-labeled A549-luc cells, its tumor growth kinetics and response to standard of care therapy. To establish the orthotopic model, we implanted cells by direct injection into the left lung lobe through a small incision on the lateral thorax between the 6th and 7th rib. Disease progression was evaluated by testing cell implants at multiple cell inoculums and following each group for tumor growth using an IVIS Spectrum CT. In line with the observed slow growth of NSCLC, A549 tumor growth kinetics revealed long latency with a median survival of 69.5-days post-implant. Mean flux values correlated with the number of cells inoculated and clinical observations of respiratory distress. Gross necropsies provided visual evidence of masses in the left lung lobe and thoracic cavity. Following the A549-luc orthotopic model development, we sought to evaluate the survival benefit in response to standard of care agents Paclitaxel and the tyrosine kinase inhibitors Erlotinib and Afatinib. Paclitaxel monotherapy was the most active agent in a 101-day study. A single treatment cycle showed robust decrease tumor burden as determined via imaging during the first few weeks following treatment, but disease progression was apparent despite the overall improved survival over the vehicle control group. To establish durable responses, we evaluated the efficacy of Paclitaxel administered for two dosing cycles. Palbociclib, a cyclin-dependent kinase 4/6 inhibitor that has been recently identified as potential target in KRAS-mutant (NSCLC) was also evaluated. Our preliminary results show significant activity for both Palbociclib and Paclitaxel therapies in the A549-luc lung orthotopic xenograft model. This lung orthotopic model provides a sensible alternative that recapitulates the difficulties of treating NCSL tumors in addition to establishing a more clinically relevant model to evaluate cancer therapies compared to standard subcutaneous implanted models. Histopathological analysis of Ki67, Cyclin D1, and CDK4 will be presented from lungs of treated and control groups. Citation Format: Elizabeth Rainbolt, Andrew Wong, Chassidy Hall, Patrick Wood, Paula L. Miliani De Marval. An orthotopic model for luciferase-labeled human lung epithelial carcinoma with response to standard of care treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1632.

AGE/RAGE axis regulates reversible transition to quiescent states of ALK-rearranged NSCLC and pancreatic cancer cells in monolayer cultures

Cancer recurrence due to tumor cell quiescence after therapy and long-term remission is associated with cancer-related death. Previous studies have used cell models that are unable to return to a proliferative state; thus, the transition between quiescent and proliferative states is not well understood. Here, we report monolayer cancer cell models wherein the human non-small cell lung carcinoma cell line H2228 and pancreatic cancer cell line AsPC-1 can be reversibly induced to a quiescent state under hypoxic and serum-starved (HSS) conditions. Transcriptome and metabolome dual-omics profiles of these cells were compared with those of the human lung adenocarcinoma cell line A549, which was unable to enter a quiescent state under HSS conditions. The quiescence-inducible cells had substantially lower intracellular pyruvate and ATP levels in the quiescent state than in the proliferative state, and their response to sudden demand for energy was dramatically reduced. Furthermore, in quiescence-inducible cells, the transition between quiescent and proliferative states of these cells was regulated by the balance between the proliferation-promoting Ras and Rap1 signaling and the suppressive AGE/RAGE signaling. These cell models elucidate the transition between quiescent and proliferative states, allowing the development of drug-screening systems for quiescent tumor cells.

Glaucocalyxin A induces apoptosis of non-small cell lung carcinoma cells by inhibiting the PI3K/Akt/GSK3β pathway.

Lung cancer is one of the fastest growing malignancies in morbidity and mortality, and current therapies are in general not sufficiently effective for this deadly disease. This study characterizes the anticancer effects of glaucocalyxin A (GLA) and explores the underlying mechanisms using human non-small cell lung carcinoma (NSCLC) cells. First, our data showed that GLA suppressed the viability of cancer cells, whereas no effect was observed in the normal bronchial epithelial cell BEAS-2B cells. Second, GLA inhibited colony formation, induced apoptosis of cancer cells. Third, GLA downregulated the expression of B-cell lymphoma-2 (Bcl-2) protein; upregulated the expression of Bcl2-associated X protein (Bax), and strengthened cleavage of caspase 3 and polyadenyl diphosphate ribose polymerase (PARP). Fourth, GLA also diminished mitochondrial membrane potential and inhibited phosphatidylinositol 3-kinase (PI3K)/Akt/ glycogen synthase kinase-3β (GSK3β) pathway. In addition, injection of GLA (20 mg/kg) every 2 days significantly inhibited A549 xenograft tumour growth, accompanied by increased apoptosis and decreased proliferation. Together, our study provides evidence that the anticancer effect of GLA in NSCLC is mediated by inducing apoptosis through inhibiting PI3K/Akt/GSK3β pathway and suggests that GLA may be used as a promising natural medicine for NSCLC therapy.

Evodiamine suppresses the progression of non-small cell lung carcinoma via endoplasmic reticulum stress-mediated apoptosis pathway in vivo and in vitro.

BackgroundEvodiamine (EVO) is one of the major components isolated from Evodia rutaecarpa (Juss.). Recent studies have shown that EVO has an anti-cancer effect. However, the pharmacological mechanism by which EVO impacts cancer is still poorly understood.ObjectivesThis study focused on asking the anti-cancer effect of EVO in human non-small cell lung carcinoma (NSCLC), and in particular to investigate whether EVO acts via modulating the endoplasmic reticulum stress (ERS)-mediated apoptosis pathway.Materials and MethodsA Lewis lung carcinoma (LLC) tumor-bearing mouse model was treated with low-dose EVO (5 mg/kg) and high-dose EVO (10 mg/kg) intraperitoneally for 14 d. The effects of EVO on tumor growth, apoptosis, and ERS were assessed. In addition, NSCLC A549 and LLC cells were treated with EVO in vitro. The effects of EVO on cell proliferation, apoptosis, and ERS were investigated. Finally, 4-phenylbutyric acid (4-PBA), an ERS inhibitor, was used to validate whether EVO induced apoptosis of NSCLC cells by modulating ERS.ResultsEVO treatment significantly inhibited tumor growth in LLC tumor-bearing mice. H&E staining indicated that EVO treatment reduced the number of tumor cells and the nucleo-plasmic ratio. Immunostaining showed that EVO treatment significantly decreased the expression of Ki-67. TUNEL staining revealed that EVO induced apoptosis in the tumor. Likewise, EVO treatment up-regulated the expression of apoptosis-related genes and proteins and increased activation of the ERS pathway in the tumor. Additionally, EVO inhibited cell proliferation and increased cell apoptotic rates in A549 and LLC cells. EVO also increased the expression levels of genes and proteins associated with ERS-mediated apoptosis pathway in vitro. The effects of EVO on apoptosis were abolished by 4-PBA treatment.ConclusionsOur study demonstrated that EVO suppresses the progression of NSCLC by modulating the ERS-mediated apoptosis pathway.

Aloe emodin 3-O-glucoside inhibits cell growth and migration and induces apoptosis of non-small-cell lung cancer cells via suppressing MEK/ERK and Akt signalling pathways

AimsNon-small-cell lung cancer (NSCLC) is the most frequent type of lung cancer with a high mortality rate. Glycosylation of phenolic compounds may increase water-solubility and pharmacological activities and reduce the toxicity of aglycones. This study aimed to evaluate and compare the anticancer effect of aloe emodin 3-O-glucoside (AE3G) and its aglycone, aloe emodin (AE), against NSCLC. Main methodA human adenocarcinoma cell line (A549) and other human non-small cell lung carcinoma cell lines (NCI-H460 cells and NCI-H1299 cells) and BALB/c nu/nu xenograft mice harbouring A549 cells were used as the NSCLC models. Inhibition of cell migration, disruption of mitochondrial membrane potential (MMP), DNA fragmentation, and expression levels of apoptotic proteins were measured by western blot, wound healing assay, JC-1 staining, or TUNEL staining. Histopathological changes in tumour tissues were observed by H&E and TUNEL staining. ResultsWith no significant cytotoxicity against noncancerous cells (Vero cells), AE3G (5–50 μM) significantly and more effectively inhibited the growth, attachment, migration, Bcl-2 expression, and activation of MEK/ERK and Akt signalling proteins and induced cytochrome c release and Bax expression in A549 cells than AE. AE3G also significantly decreased the growth of other NSCLC cells, NCI-H460 cells and NCI-H1299 cells. AE3G suppressed the mRNA expression of matrix metalloproteinases, MMP2 and MMP9, and augmented the collapse of the mitochondrial MMP, cleavage of caspases (caspase 9, 8, and 3) and PARP, and DNA fragmentation. Intraperitoneal injection of AE3G (13 and 26 mg/kg/day) reduced the tumour volume and weight and induced apoptotic cell death in tumour tissues of xenograft NSCLC mice. SignificanceThe present study demonstrated that AE3G significantly and more effectively diminished human NSCLC cell growth and migration by triggering mitochondria-dependent intrinsic apoptosis than AE, providing AE3G as a new potent candidate to prevent or treat human NSCLC.

Green tea-derived theabrownin suppresses human non-small cell lung carcinoma in xenograft model through activation of not only p53 signaling but also MAPK/JNK signaling pathway

Ethnopharmacological relevanceAccording to the theory and practice of traditional Chinese medicine (TCM), the pathogenesis of lung carcinoma is associated with many syndromes, such as "sputum stasis", "cough", "lung fever", "lung toxin", and "hemoptysis", which should be removed for therapeutic purpose. Tea is not only a world-wide beverage, but also a TCM herb, possessing activities against the above syndromes. Recently, green tea extract exerted inhibitory effects on a variety of tumor cells. As a pigment active substance of green tea, theabrownin (TB) has been found to inhibit many cancer cells. Aim of the studyThis study focused on the efficacy and mechanism of TB on non-small cell lung cancer (NSCLC) cell lines. The in vivo efficacy of TB on p53-deficient NSCLC (H1299) cells and p53-wild type NSCLC (A549) cells NSCLC cells were determined, and its mechanism of action was explored. Materials and methodsIn vivo, two lung cancer cell lines, H1299 (p53-deficient) and A549 (p53-wild type) were selected to establish xenograft models of larval zebrafish, respectively. For in vitro experiments, wound healing assay, DAPI staining, TUNEL assay, immunofluorescence assay, and flow cytometry were conducted in these two cell lines. RNA sequencing (RNAseq), real time PCR (qPCR) and Western blot (WB) were performed for the mechanism study. ResultsThe in vivo results showed that TB significantly inhibited the H1299 and the A549 xenograft tumor growth in larval zebrafish (dosage ranged from 2.13 to 21.3 μg/ml). Wound healing assay results showed that TB suppressed the migration of H1299 cells. DAPI staining, TUNEL assay, and immunofluorescence assay results showed that TB inhibited the growth of H1299 cells by inducing apoptosis. RNAseq, qPCR and WB data showed that TB significantly up-regulated the MAPK/JNK pathway-related proteins (ASK-1, JNK and c-JUN) through phosphorylation activation, accompanying with down-regulation of the epithelial–mesenchymal transition (EMT)-associated genes (N-CADHERIN, SLUG, FIBROWNECTIN and ZEB1) and anti-apoptotic molecules (BCL-2), and up-regulation of the metastasis-related gene HSPA6 and the pro-apoptotic molecules (BIM, BAX, PARP, c-PARP, γ-H2A.X, c-CASP3, c-CASP8, c-CASP9, DDIT3 and DUSP8). ConclusionThis study determined the in vivo efficacy of green tea-derived TB on p53-deficient NSCLC (H1299) cells and p53-wild type NSCLC (A549) cells and clarified its p53-independent mechanism mediated by the activation of MAPK/JNK signaling pathway.

The interaction of CD4+ helper T cells with dendritic cells shapes the tumor microenvironment and immune checkpoint blockade response.

Despite their key regulatory role and therapeutic potency, the molecular signatures of interactions between T cells and antigen-presenting myeloid cells within the tumor microenvironment remain poorly characterized. Here, we systematically characterize these interactions using RNA sequencing of physically interacting cells (PIC-seq) and find that CD4+PD-1+CXCL13+ T cells are a major interacting hub with antigen-presenting cells in the tumor microenvironment of human non-small cell lung carcinoma. We define this clonally expanded, tumor-specific and conserved T-cell subset as T-helper tumor (Tht) cells. Reconstitution of Tht cells in vitro and in an ovalbumin-specific αβ TCR CD4+ T-cell mouse model, shows that the Tht program is primed in tumor-draining lymph nodes by dendritic cells presenting tumor antigens, and that their function is important for harnessing the antitumor response of anti-PD-1 treatment. Our molecular and functional findings support the modulation of Tht-dendritic cell interaction checkpoints as a major interventional strategy in immunotherapy.

CircPTN promotes angiogenesis via the MiR-595/LYRM5 signaling pathway in non-small cell lung cancer.

BackgroundNon-small cell lung carcinoma (NSCLC) is a highly malignant tumor with a poor prognosis worldwide. Some studies have demonstrated that circular pleiotrophin (circPTN) plays critical roles in tumorigenesis and tumor development. However, little is known about the role of circPTN in NSCLC.MethodsThe circPTN expression in human NSCLC tissues was measured via quantitative real-time polymerase chain reaction (qRT-PCR). The function and potential mechanisms of circPTN in NSCLC angiogenesis were also investigated. We aimed to explore the function and potential mechanisms and clinical significance of circPTN in NSCLC.ResultsWe first found that circPTN was markedly upregulated in NSCLC tissues. A higher circPTN level was closely associated with angiogenesis and significantly shorter overall survival in patients with NSCLC. We then found that circPTN promoted angiogenesis in NSCLC. More importantly, we found that circPTN facilitated angiogenesis by regulating the expression of LYRM5 in NSCLC. Mechanistically, LYRM5 could be a direct target of microRNA-595 (miR-595). Additionally, we demonstrated that circPTN upregulated LYRM5 expression by sponging miR-595, which promoted NSCLC angiogenesis in NSCLC.ConclusionsWe found that circPTN serves as a competing endogenous ribonucleic acid that promotes angiogenesis via the miR-595/LYRM5 signaling pathway in NSCLC. Targeting circPTN might be a promising new therapeutic strategy for NSCLC.

ING2-WTAP is a potential therapeutic target in non-small cell lung cancer

Non-small-cell lung carcinoma (NSCLC) is the most common kind of lung cancer, which is also the largest cause of cancer-linked deaths globally. A lack of effective prognostic biomarkers contributes to the poor prognosis of NSCLC. For better patient outcomes, NSCLC diagnosing, prognostic, and predictive biomarkers are critically needed. A component of the chromatin-modulatory complex that dampens gene expression, ING2 (inhibitor of growth family member 2), has been linked to cellular mechanisms that enhance tumor repression. Nevertheless, its role in human NSCLC is unclear. Herein, we observed that ING2 downregulation in NSCLC tissues correlates with poor NSCLC prognosis. Functional analysis illustrated that ING2 dampened growth, infiltration along with metastasis and blocked apoptosis of NSCLC cells in vitro, as well as in vivo. The mechanism might be implicated in the EMT (epithelial-mesenchymal transition) process. Mechanistically, we found that ING2 silencing suppresses the expressions of Wilms tumor 1-associated protein (WTAP) and that WTAP expression negatively correlates with ING2 expression. Moreover, rescue experiments illustrated that WTAP overexpression partially counteracts the effect of ING2 silencing on proliferation. Finally, we found that elevated WTAP levels correlate with poor NSCLC prognosis. These results highlight the prognostic and therapeutic potential of ING2-WTAP in NSCLC.

Study on a Ru(III) complex containing picolinate with potent inhibition effect against melanoma cell line

A ruthenium(III) complex formulated as [Ru(pyc)3]·H2O (where pyc is pyridine-2-carboxylate) was prepared through a one-pot reaction of pyridine-2,6-dicarboxylic acid and anhydrous ruthenium(III) chloride. Here, hydrothermal conditions led to decarboxylation from pyridine-2,6-dicarboxylic acid into pyridine-2-carboxylate. The compound was identified by spectroscopic methods and its crystal structure was determined by single-crystal X-ray diffraction. The cytotoxicity of the compounds was evaluated by MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) assay against three cancer cell lines containing a human melanoma (A375), a human non-small cell lung carcinoma (H1299), a human colon adenocarcinoma (HT29), and also one normal cell human foreskin fibroblast (HFF). A strong and selective inhibitory effect of the complex (IC50 = 6.50 μM, viability inhibition = 67.12%) was exhibited toward A375 cells. The apoptosis through a mitochondrial pathway was suggested via reactive oxygen species (ROS) production and mitochondria membrane potential (MMP) collapse. The cytostatic effect of the complex in the A375 3 D spheroid model was depicted.

Transcriptomics reveals in vivo efficacy of PARP inhibitor combinatorial synergy with platinum-based chemotherapy in human non-small cell lung carcinoma models.

Inhibitors of poly(ADP)-ribose polymerase (PARP) exploit defective DNA repair pathways existing in several forms of cancer, such as those with BRCA mutations, and have proven clinical efficacy as chemosensitizers. However, platinum-based chemopotentiation by PARP inhibitors (PARPi), particularly for non-small cell lung cancer (NSCLC), has only been confirmed in a few preclinical models and the molecular mechanisms that drive PARPi combinatorial synergy with chemotherapeutics remains poorly defined. To better understand these mechanisms, we characterized cisplatin and veliparib efficacy in A549 and Calu6 NSCLC in vivo tumor xenograft models and observed combinatorial synergy in the Calu6 model. Transcriptome-wide analysis of xenografts revealed several differentially expressed genes (DEGs) between untreated and cisplatin + veliparib-treated groups, which were unique from genes identified in either of the single-agent treatment arms. Particularly at 10- and 21-days post-treatment, these DEGs were enriched within pathways involved in DNA damage repair, cell cycle regulation, and senescence. Furthermore, TGF-β- and integrin-related pathways were enriched in the combination treatment arm, while pathways involved in cholesterol metabolism were identified at earlier time points in both the combination and cisplatin-only groups. These data advance the biological underpinnings of PARPi combined with platinum-based chemotherapy and provides additional insight into the diverse sensitivity of NSCLC models.

Effects of Methadone on the Toll-like Receptor 4 Expression in Human Non-Small Cell Lung Carcinoma A549 Cell Line Using In-silico and In vitro Techniques.

In this study, the effects of methadone and naloxone on the expression of toll-like receptor 4 (TLR4) gene have been evaluated in human non-small cell lung carcinoma A549 cell line migration using in-silico and in vitro techniques. Lung cancer A549 cell cultures were stimulated for 24 h with methadone (5, 10, and 20 μM) and naloxone (20 and 40 μM) concentrations. The level of TLR4 expression was determined by the quantitative real-time polymerase chain reaction. Migration of the A549 cells was investigated after a 4-h incubation period with methadone using the Boyden Chamber assay. Migration rate of the A549 cells treated with 5 (P < 0.05) and 20 (P < 0.01) μM methadone was, respectively, increased and decreased with 20 μM naloxone (P < 0.05). Furthermore, the TLR4 expression was enhanced with 5 (P < 0.05) and 20 (P < 0.01) μM methadone and decreased with 20 (P < 0.05) and 40 μM naloxone (P < 0.01). In addition, in silico docking analysis revealed docking of methadone to MD-2 and TLR4. According to the present DATA, methadone affects the TLR4 expression. It may however cause adverse consequences by increasing the TLR4 expression. Therefore, the useful analgesic properties of methadone should be separated from the unwanted TLR4-mediated side effects.

Spherical Invasion Assay: A Novel Method to Measure Invasion of Cancer Cells.

The invasion of tumor cells into the neighboring blood vessels and lymph nodes is a vital step for distant metastasis. Traditionally, the invasive activity of growth factors (or the anti-invasive activity of drugs) is measured with the Boyden chamber assay. However, this assay has a few disadvantages like poor physiological relevance of transwell inserts and an inability to control chemokine gradients. The Boyden chamber assay is one of the most prevalent methods to measure the invasion of cancer cells. It would be advantageous to develop another assay that could validate the results of the Boyden chamber assay. With this in mind, our laboratory developed the spherical invasion assay (SIA) to measure the pro-invasive activity of human cancer cells. The SIA also circumvents some of the drawbacks of the Boyden chamber assay. The present manuscript measures the anti-invasive activity of the Src kinase inhibitor PP2 in A549 human non-small cell lung carcinoma (NSCLC) cells using the SIA. The SIA protocol is comprised of two steps. In the first step, A549 human NSCLC cells (treated or not with PP2) were mixed with Matrigel and seeded in the middle of an eight-well chamber slide. After 24 h, a second layer of Matrigel was overlaid over the first layer. Over the course of the next 24 h, the A549 cells invade from the primary to the secondary Matrigel layers. Subsequently, the cells are visualized by phase-contrast microscopy and the images obtained are quantified using ImageJ to calculate the anti-invasive activity of PP2 in A549 cells. The results of the SIA correlate well with Boyden chamber assays. The SIA may be adapted for multiple experimental designs, such as drug screening (to combat invasion and metastasis), measuring the pro-invasive activity of growth factors, and elucidating the signaling pathways underlying the pro-invasive/anti-invasive activity of biological modifiers. Graphic abstract: Diagrammatic illustration of the spherical invasion assay ( Hurley et al., 2017 ) . A. The first layer is comprised of human cancer cells mixed in a 1:1 suspension with Phenol Red containing Matrigel (represented as LAYER 1 in the figure). After 24 h, the cancer cells grow and extend up to the boundary of this first layer. B. A second layer of 1:1 solution Phenol Red-free Matrigel, in Phenol Red-free RPMI (represented as LAYER 2 in the figure) is added on top of the first Matrigel spot. The cells are incubated for 24 h at 37°C. C. Over these 24 h, the cancer cells invade from the primary layer into the secondary Matrigel layer. The chamber slides are observed by phase-contrast microscopy. D. A representative photograph of the images obtained by the SIA is shown. The black arrow indicates the cancer cells invading into the second layer of Matrigel. The dotted line represents the interface between the two layers. The distance to which the cells have traveled (into the secondary Matrigel layer) is measured at ten sites (for each photograph) in a randomized double-blind fashion by three independent observers, using NIH ImageJ Version 1.47. This process is repeated for three separate photographic fields per sample.

Efekat metformina na vijabilitet i mitohondrijalni status tumorske i netumorske ćelijske linije

Introduction: Metformin is a drug of choice in the therapy of type II Diabetes mellitus. There is a growing evidence of metformin's antitumor activity, but the suggested mechanisms of such activity are still not fully elucidated. Aim: To investigate the effect of therapeutic doses of metformin on viability and mitochondrial status of human non-small cell lung carcinoma (NCI-H460) and human immortalized lung fibroblasts (MRC-5) cell lines. Material and methods: Acid phosphatase and Crystal Violet assays were used for the determination of NCI-H460 and MRC-5 cell viability after the treatment with metformin (10-60 µM) for 1-7 days. Mitochondrial membrane potential, production of reactive oxygen species and superoxide anion, as well as mitochondrial mass were measured using flow cytometry after the treatment of the cells for 3, 24 and 120 h, followed by staining with appropriate fluorochromes: JC-1, DHR, DHE and Mitotracker Red. Results: Metformin did not change the viability of both NCI-H460 and MRC-5 cells in all investigated time-points and all used concentrations. Depolarization of mitochondrial membrane was observed 3 h post-treatment in MRC-5 cells. Prolonged treatment (120 h) increased superoxide anion production and mitochondrial mass in NCI-H460 cells. No significant changes in production of reactive oxygen species were observed in both cells lines after short or extended exposure to metformin. Conclusion: : Therapeutic concentrations of metformin do not influence the viability of NCI-H460 and MRC-5 cells, but induce mitochondrial depolarization after short-term exposure in lung fibroblasts and increase production of superoxide anion and mitochondrial mass in lung carcinoma cells after prolonged treatment.

Fish Oil, Plant Polyphenols, and Their Combinations Have No Tumor Growth Promoting Effects on Human Lung and Colon Carcinoma Xenograft Mice

The goal of this study was to evaluate if combinations of ingredients with known anti-cachexia benefits (Fish oil-FO with either curcumin or Green tea extract-GTE), have adverse effects on tumor growth, using human carcinoma xenograft mice models. FO (EPA/DHA 360 mg/kg bw), GTE (90 mg/kg bw), and curcumin (180 mg/kg bw) were administered orally, alone or in combination, to nude mice bearing either A549 human non-small cell lung carcinoma or SW620 human colon carcinoma tumors. Bodyweight, tumor growth, survival time, and other clinical endpoints were assessed. The ingredients either alone or in combinations were well tolerated in both lung and colon tumor-bearing mice. There were no significant group differences between individual or combination treatments for tumor growth (A549 or SW620) as measured by the median time in days to endpoint of tumor volume (TTE). TTE results indicate that these ingredients (alone or combinations) did not adversely impact tumor growth. No significant differences in body weights or survival were observed between controls and treatment groups indicating no adverse health effects of the ingredients. In conclusion, FO, GTE or curcumin administered as monotherapies and in combination were well tolerated and displayed no adverse effects on tumor growth in mouse xenograft models of lung and colon cancer.

Cold Plasma Irradiation Regulates Inflammation and Oxidative Stress in Human Bronchial Epithelial Cells and Human Non-Small Cell Lung Carcinoma.

Atmospheric pressure cold plasma has shown multiple biological effects of anti-bacteria and anti-cancer. In this study, the effect of atmospheric pressure cold plasma on respiratory inflammation and oxidant stress is explored. Tunicamycin was used to stimulate human bronchial epithelial cells (HBECs) and A549 cells for inflammatory response and oxidative stress, followed by atmospheric pressure cold plasma treatment. For HBECs and A549 cells, atmospheric pressure cold plasma was able to alleviate tunicamycin-induced cell proliferation inhibition, inflammation and oxidant stress, and enhance nuclear factor-erythroid-2-related factor 2 (NRF2) pathway activation. Moreover, NRF2/ARE (anti-oxidant response elements) pathway was involved in the regulation of atmospheric pressure cold plasma on tunicamycin-induced oxidative stress. These results suggest the positive effect of atmospheric pressure cold plasma on inflammation and oxidant stress of respiratory system, indicating the therapeutic potential of atmospheric pressure cold plasma for respiratory diseases.

Transthyretin Suppressed Tumor Progression in Nonsmall Cell Lung Cancer by Inactivating MAPK/ERK Pathway.

Background: Lung malignancy is a main source of disease passing all throughout the planet, whereas the transthyretin (TTR) is a specific biomarker for clinical diagnosis. However, its role in lung malignancy stays to be obscure. Materials and Methods: In the current examination, the authors made an endeavor to research impact of abnormal expression of TTR on nonsmall cell lung carcinoma (NSCLC) by overexpression or knockdown of TTR. To further explore the instruments' fundamental mechanism part of TTR in NSCLC, several signal pathways were searched and verified. To confirm the effect of TTR overexpression on tumors, in vivo experiments were conducted. Result: It was found that upregulated TTR clearly stifled cell proliferation, migration, invasion, and expanded apoptosis. Significant suppression of phosphor-extracellular signal-regulated kinase (ERK) was observed in TTR-treated NSCLC cells, implying that TTR was important for cellular progress by regulating mitogen-activated protein kinase/ERK signaling pathway. In in vivo experiment, overexpression of TTR promoted cell apoptosis and inhibited tumor growth. Conclusion: Overall, the results suggest that TTR has a potential antitumor effect in human NSCLC progression, which provides theoretical basis for the diagnosis and treatment of NSCLC. Above all, further understanding of TTR was useful for clinical care. Clinical Trial Registration Number: 2016-08.

New Therapeutic Strategy for Overcoming Multidrug Resistance in Cancer Cells with Pyrazolo[3,4-d]pyrimidine Tyrosine Kinase Inhibitors.

Simple SummaryP-glycoprotein (P-gp) is an ATP-binding cassette transporter whose overexpression in cancer cells is one of the main causes of multidrug resistance (MDR). Tyrosine kinase inhibitors (TKIs) have been reported to interact with ABC transporters and in some cases, increase the susceptibility of cancer cells to chemotherapy. We investigated the potential of novel TKI pyrazolo[3,4-d] pyrimidines and their prodrugs to inhibit P-gp in two MDR cancer cell lines with P-gp overexpression. The tested compounds were able to suppress P-gp by inhibiting its ATPase activity. Interestingly, prodrugs displayed a stronger potential to modulate P-gp and showed higher interaction energies in the docking simulations compared to their parent drugs. Furthermore, prodrugs showed significant potential to inhibit P-gp activity even in prolonged treatment and therefore to enhance the efficacy of doxorubicin and paclitaxel in MDR cancer cells. All of these characteristics imply that the new TKIs could be considered a valuable strategy for combating resistant cancers, especially in combination with other chemotherapeutics.Tyrosine kinase inhibitors (TKIs) often interact with the multidrug resistant (MDR) phenotype of cancer cells. In some cases, TKIs increase the susceptibility of MDR cancer cells to chemotherapy. As the overexpression of membrane transporter P-glycoprotein (P-gp) is the most common alteration in MDR cancer cells, we investigated the effects of TKI pyrazolo[3,4-d]pyrimidines on P-gp inhibition in two cellular models comprising sensitive and corresponding MDR cancer cells (human non-small cell lung carcinoma and colorectal adenocarcinoma). Tested TKIs showed collateral sensitivity by inducing stronger inhibition of MDR cancer cell line viability. Moreover, TKIs directly interacted with P-gp and inhibited its ATPase activity. Their potential P-gp binding site was proposed by molecular docking simulations. TKIs reversed resistance to doxorubicin and paclitaxel in a concentration-dependent manner. The expression studies excluded the indirect effect of TKIs on P-gp through regulation of its expression. A kinetics study showed that TKIs decreased P-gp activity and this effect was sustained for seven days in both MDR models. Therefore, pyrazolo[3,4-d]pyrimidines with potential for reversing P-gp-mediated MDR even in prolonged treatments can be considered a new therapeutic strategy for overcoming cancer MDR.

MicroRNA-324-5p-CUEDC2 Axis Mediates Gain-of-Function Mutant p53-Driven Cancer Stemness.

Regulation of cancer stemness has recently emerged as a new gain-of-function (GOF) property of mutant p53. In this study, we identify miR-324-5p as a critical epigenetic regulator of cancer stemness and demonstrate its role in mediating GOF-mutant p53-driven stemness phenotypes. We report that miR-324-5p is upregulated in human cancer cell lines and non-small cell lung carcinoma (NSCLC) tumors carrying TP53 GOF mutations. Mechanistically, we show that GOF mutant p53 upregulates miR-324-5p expression via c-Myc, an oncogenic transcription factor in cancer cells. Our experimental results suggest that miR-324-5p-induced CSC phenotypes stem from the downregulation of CUEDC2, a downstream target gene of miR-324-5p. Accordingly, CUEDC2 complementation diminishes elevated CSC marker expression in miR-324-5p-overexpressing cancer cells. We further demonstrate that mutant p53 cancer cells maintain a low level of CUEDC2 that is rescued upon miR-324-5p inhibition. Importantly, we identify CUEDC2 downregulation as a novel characteristic feature of TP53-mutated human cancers. We show that activation of NF-κB due to downregulation of CUEDC2 by miR-324-5p imparts stemness in GOF mutant p53 cancer cells. Finally, we provide evidence that TP53 mutations coupled with high miR-324-5p expression predict poor prognosis in patients with lung adenocarcinoma. Thus, our study delineates an altered miR-324-5p-CUEDC2-NF-κB pathway as a novel regulator of GOF mutant p53-driven cancer stemness. IMPLICATIONS: Our findings implicate miRNA-324-5p as a novel epigenetic modifier of human cancer stemness.