Achilleas Mitrakas

Autophagosome Proteins LC3A, LC3B and LC3C Have Distinct Subcellular Distribution Kinetics and Expression in Cancer Cell Lines

LC3s (MAP1-LC3A, B and C) are structural proteins of autophagosomal membranes, widely used as biomarkers of autophagy. Whether these three LC3 proteins have a similar biological role in autophagy remains obscure. We examine in parallel the subcellular expression patterns of the three LC3 proteins in a panel of human cancer cell lines, as well as in normal MRC5 fibroblasts and HUVEC, using confocal microscopy and western blot analysis of cell fractions. In the cytoplasm, there was a minimal co-localization between LC3A, B and C staining, suggesting that the relevant autophagosomes are formed by only one out of the three LC3 proteins. LC3A showed a perinuclear and nuclear localization, while LC3B was equally distributed throughout the cytoplasm and localized in the nucleolar regions. LC3C was located in the cytoplasm and strongly in the nuclei (excluding nucleoli), where it extensively co-localized with the LC3A and the Beclin-1 autophagy initiating protein. Beclin 1 is known to contain a nuclear trafficking signal. Blocking nuclear export function by Leptomycin B resulted in nuclear accumulation of all LC3 and Beclin-1 proteins, while Ivermectin that blocks nuclear import showed reduction of accumulation, but not in all cell lines. Since endogenous LC3 proteins are used as major markers of autophagy in clinical studies and cell lines, it is essential to check the specificity of the antibodies used, as the kinetics of these molecules are not identical and may have distinct biological roles. The distinct subcellular expression patterns of LC3s provide a basis for further studies.

Therapeutic interactions of autophagy with radiation and temozolomide in glioblastoma: evidence and issues to resolve

Glioblastoma is a unique model of non-metastasising disease that kills the vast majority of patients through local growth, despite surgery and local irradiation. Glioblastoma cells are resistant to apoptotic stimuli, and their death occurs through autophagy. This review aims to critically present our knowledge regarding the autophagic response of glioblastoma cells to radiation and temozolomide (TMZ) and to delineate eventual research directions to follow, in the quest of improving the curability of this incurable, as yet, disease. Radiation and TMZ interfere with the autophagic machinery, but whether cell response is driven to autophagy flux acceleration or blockage is disputable and may depend on both cell individuality and radiotherapy fractionation or TMZ schedules. Potent agents that block autophagy at an early phase of initiation or at a late phase of autolysosomal fusion are available aside to agents that induce functional autophagy, or even demethylating agents that may unblock the function of autophagy-initiating genes in a subset of tumours. All these create a maze, which if properly investigated can open new insights for the application of novel radio- and chemosensitising policies, exploiting the autophagic pathways that glioblastomas use to escape death.

EVALUATION OF THE ALAMARBLUE ASSAY FOR ADHERENT CELL IRRADIATION EXPERIMENTS

The AlamarBlue assay is based on fluorometric detection of metabolic mitochondrial activity of cells. In this study, we determined the methodology for application of the assay to radiation response experiments in 96-well plates. AlamarBlue was added and its reduction measured 7 hours later. Selection of the initial number of plated cells was important so that the number of proliferating cells remains lower than the critical number that produced full AlamarBlue reduction (plateau phase) at the time points of measurements. Culture medium was replaced twice a week to avoid suppression of viability due to nutrient competition and metabolic waste accumulation. There was no need to replace culture medium before adding AlamarBlue. Cell proliferation continued after irradiation and the suppression effect on cell viability was most evident on day 8. At this time point, by comparing measurements from irradiated vs. non-irradiated cells, for various dose levels, a viability dose response curve was plotted. Immediately after the 8th day (nadir), cells started to re-grow at a rate inversely related to the radiation dose. By comparing measurements at the time point of nadir vs. a convenient subsequent time point, re-growth dose response abilities were plotted, simulating clonogenic assays.

Fever-Range Hyperthermia vs. Hypothermia Effect on Cancer Cell Viability, Proliferation and HSP90 Expression

Purpose The current study examines the effect of fever-range hyperthermia and mild hypothermia on human cancer cells focusing on cell viability, proliferation and HSP90 expression. Materials and Methods A549 and H1299 lung carcinoma, MCF7 breast adenocarcinoma, U87MG and T98G glioblastoma, DU145 and PC3 prostate carcinoma and MRC5 normal fetal lung fibroblasts cell lines were studied. After 3-day exposure to 34°C, 37°C and 40°C, cell viability was determined. Cell proliferation (ki67 index), apoptosis (Caspase 9) and HSP90 expression was studied by confocal microscopy. Results Viability/proliferation experiments demonstrated that MRC5 fibroblasts were extremely sensitive to hyperthermia, while they were the most resistant to hypothermia. T98G and A549 were thermo-tolerant, the remaining being thermo-sensitive to a varying degree. Nonetheless, as a universal effect, hypothermia reduced viability/proliferation in all cell lines. Hyperthermia sharply induced Caspase 9 in the U87MG most thermo-sensitive cell line. In T98G and A549 thermo-tolerant cell lines, the levels of Caspase 9 declined. Moreover, hyperthermia strongly induced the HSP90 levels in T98G, whilst a sharp decrease was recorded in the thermo-sensitive PC3 and U87MG cell lines. Hyperthermia sensitized thermo-sensitive cancer cell lines to cisplatin and temozolomide, whilst its sensitizing effect was diminished in thermo-tolerant cell lines. Conclusions The existence of thermo-tolerant and thermo-sensitive cancer cell lines was confirmed, which further encourages research to classify human tumor thermic predilection for patient stratification in clinical trials. Of interest, mild hypothermia had a universal suppressing effect on cancer cell proliferation, further supporting the radio-sensitization hypothesis through reduction of oxygen and metabolic demands.

Repression of the autophagic response sensitises lung cancer cells to radiation and chemotherapy

Background:The cellular autophagic response to radiation is complex. Various cells and tissues respond differentially to radiation, depending on both the dose of exposure and the time post irradiation. In the current study, we determined the autophagosomal and lysosomal response to radiation in lung cancer cell lines by evaluating the expression of the associated proteins, as well as the effect of relevant gene silencing in radio and chemosensitisation. Furthermore, tumour sensitisation was evaluated in in vivo autophagic gene silencing model after irradiation.Methods:A549 and H1299 cell lines were utilised as in vitro cancer models. Both cell lines were transfected with various small-interfering RNAs, silencing auto-lysosomal genes, and irradiated with 4 Gy. Cell growth response was evaluated with AlamarBlue assay. Western blot and confocal microscopy were utilised for the characterisation of the auto-lysosomal flux. Also, the H1299 cell line was stable transfected with small-hairpin RNA of the MAP1LC3A gene, and the tumour radiosensitisation in Athymic Nude-Foxn1nu was evaluated.Results:Following exposure to 4 Gy of radiation, A549 cells exhibited a significant induction of the autophagic flux, which was not supported by transcriptional activation of auto-lysosomal genes (LC3A, LC3B, p62, TFEB and LAMP2a), resulting in aggresome accumulation. Recovery of transcriptional activity and autophagy efficacy occurred 7 days post irradiation. Alternatively, H1299 cells, a relatively radio-resistant cell line, sharply responded with an early (at 2 days) transcriptional activation of auto-lysosomal genes that sustained an effective autophagosomal flux, resulting in adequate aggresome clearance. Subsequently, we tested the silencing of four genes (LC3A, LC3B, TFEB and LAMP2a), confirming a significant radiosensitisation and chemosensitisation to various chemotherapeutic agents, including cisplatin and taxanes. In mouse xenografts, exposure to radiation significantly reduced tumour growth (P<0.001), which was exacerbated among shLC3A-H1299 transfected tumours.Conclusions:The ability of lung cancer cells to survive after irradiation at 4 Gy depends on their ability to sustain a functional autophagic flux. Abrogation of such ability results in increased radiosensitivity and susceptibility to various chemotherapy agents. Selective inhibitors of cancer cell autophagic function may prove important for the eradication of lung cancer.

Effect of mitochondrial metabolism-interfering agents on cancer cell mitochondrial function and radio/chemosensitivity.

Abnormal mitochondrial function is common in cancer cells and activates metabolic pathways suppressed in normal tissues. Experimental and clinical studies suggest that mitochondria might serve as targets for novel anticancer therapies. We investigated whether mitochondrial metabolism-interfering agents (MMIAs) available currently in clinical practice affect cancer cell mitochondrial metabolism and synergize with chemotherapy and radiotherapy. Two cancer cell lines A549 (lung cancer) and DU145 (prostate cancer) were treated with a variety of MMIAs (metformin, nimodipine, memantine, oxytetracycline, amiodarone, and sodium azide) and their response was assessed using a resazurin reduction method and confocal microscopy. Focusing on amiodarone and metformin, we investigated their potential sensitizing effect on cancer cells when treated with ionizing radiation, cisplatin, and docetaxel. Resazurin reduction was increased by metformin and decreased by amiodarone at nontoxic concentrations. Amiodarone induced mitochondrial swelling, whereas metformin exerted no apparent effect on their morphology. Amiodarone and metformin exerted a weak radiosensitization effect on A549, whereas a synergetic activity with cisplatin and docetaxel was evident in both cell lines. It can be concluded that amiodarone and metformin, being well-established drugs in clinical practice, constitute two potential drugs for further experimental and clinical evaluation as cancer cell sensitizers to chemotherapy and radiotherapy.

Metabolic cooperation between co-cultured lung cancer cells and lung fibroblasts

Cooperation of cancer cells with stromal cells, such as cancer-associated fibroblasts (CAFs), has been revealed as a mechanism sustaining cancer cell survival and growth. In the current study, we focus on the metabolic interactions of MRC5 lung fibroblasts with lung cancer cells (A549 and H1299) using co-culture experiments and studying changes of the metabolic protein expression profile and of their growth and migration abilities. Using western blotting, confocal microscopy and RT-PCR, we observed that in co-cultures MRC5 respond by upregulating pyruvate dehydrogenase (PDH) and the monocarboxylate transporter MCT1. In contrast, cancer cells increase the expression of glucose transporters (GLUT1), LDH5, PDH kinase and the levels of phosphorylated/inactivated pPDH. H1299 cells growing in the same culture medium with fibroblasts exhibit a ‘metastasis-like’ phenomenon by forming nests within the fibroblast area. LDH5 and pPDH were drastically upregulated in these nests. The growth rate of both MRC5 and cancer cells increased in co-cultures. Suppression of LDHA or PDK1 in cancer cells abrogates the stimulatory signal from cancer cells to fibroblasts. Incubation of MRC5 fibroblasts with lactate resulted in an increase of LDHB and of PDH expression. Silencing of PDH gene in fibroblasts, or silencing of PDK1 or LDHA gene in tumor cells, impedes cancer cell’s migration ability. Overall, a metabolic cooperation between lung cancer cells and fibroblasts has been confirmed in the context of direct Warburg effect, thus the fibroblasts reinforce aerobic metabolism to support the intensified anaerobic glycolytic pathways exploited by cancer cells.

Trachycladines and Analogues: Synthesis and Evaluation of Anticancer Activity

The synthesis of four new analogues of marine nucleoside trachycladine A was accomplished by direct regio‐ and stereoselective Vorbrüggen glycosylations of 2,6‐dichloropurine and 2‐chloropurine with a d‐ribose‐derived chiron. Naturally occurring trachycladines A and B and a series of analogues were examined for their cytotoxic activity against a number of cancer cell lines (glioblastoma, lung, and cervical cancer). Parent trachycladine A and two analogues (the diacetate of the 2,6‐dichloropurine derivative and N‐cyclopropyl trachycladine A) resulted in a significant decrease in cell viability, with the latter exhibiting a stronger effect. The same compounds enhanced the cytotoxic effect of docetaxel in lung cancer cell lines, whereas additional experiments revealed that their mode of action relies on mitotic catastrophe rather than DNA damage. Moreover, their activity as autophagic flux blockers was postulated.

Differential effect of hypoxia and acidity on lung cancer cell and fibroblast metabolism

This study examined the metabolic response of lung cancer cells and normal lung fibroblasts to hypoxia and acidity. GLUT1 and HXKII mRNA/protein expression was up-regulated under hypoxia in the MRC5 fibroblasts and in the A549 and H1299 lung cancer cell lines, indicating intensified glucose absorption and glycolysis. Under hypoxia, the LDHA mRNA and LDH5 protein levels increased in the cancer cells but not in the fibroblasts. Acidity suppressed the above-mentioned hypoxia effect. PDH-kinase-1 (PDK1 mRNA and protein) and inactive phosphorylated-PDH protein levels were induced under hypoxia in the cancer cells, whereas these were reduced in the MRC5 lung fibroblasts. In human tissue sections, the prevalent expression patterns supported the contrasting metabolic behavior of cancer cells vs. tumor fibroblasts. The monocarboxylate/lactate transporter 1 (MCT1) was up-regulated in all the cell lines under hypoxic conditions, but it was suppressed under acidic conditions. The mitochondrial DNA (mtDNA) content per cell decreased significantly in the A549 cancer cell line under hypoxia, but it increased in the MRC5 fibroblasts. Taking into account these findings, we suggest that, under hypoxia, cancer cells intensify the anaerobic direction in glycolysis, while normal fibroblasts prefer to seek energy by intensifying the aerobic use of the available oxygen.

Exploring the Molecular Determinants of Tumor-Stroma Interaction in Non-small Cell Lung Cancer Through the Utilization of RNA-seq Data from Lung Biopsies

Lung cancer is responsible for most fatalities amongst cancers worldwide. Within the frames of the Metaboli-Ca project, in order to gain deeper insight regarding the molecular crosstalk taking place between the tumor cells and the surrounding stroma, we analyze with the help of next generation sequencing (NGS) techniques, specimens from lung section biopsies derived from non-small cell lung cancer (NSCLC) patients. Specifically, we exploit RNA sequencing data for the derivation of differentially expressed genes in cancer cells compared to the adjacent tumor associated stroma and perform functional analysis to uncover the underlying molecular mechanisms involved in tumor progression and cancer cell survival. We advanced a pipeline for quantitative analysis, using various state-of-the-art tools, and combined different statistical methodologies to investigate the outcomes from a broader point of view. On the basis of the results of this analysis we see a clear effect on signaling and repair mechanisms, allowing the referred development of cancer and tumor cell sustainability.