Effects of a Sublethal and Transient Stress of the Endoplasmic Reticulum on the Mitochondrial Population.

Endoplasmic reticulum (ER) and mitochondria are not discrete intracellular organelles but establish close physical and functional interactions involved in several biological processes including mitochondrial bioenergetics, calcium homeostasis, lipid synthesis, and the regulation of apoptotic cell death pathways. As many cell types might face a transient and sublethal ER stress during their lifetime, it is thus likely that the adaptive UPR response might affect the mitochondrial population. The aim of this work was to study the putative effects of a non-lethal and transient endoplasmic reticulum stress on the mitochondrial population in HepG2 cells. The results show that thapsigargin and brefeldin A, used to induce a transient and sublethal ER stress, rapidly lead to the fragmentation of the mitochondrial network associated with a decrease in mitochondrial membrane potential, O2 (•-) production and less efficient respiration. These changes in mitochondrial function are transient and preceded by the phosphorylation of JNK. Inhibition of JNK activation by SP600125 prevents the decrease in O2 (•-) production and the mitochondrial network fragmentation observed in cells exposed to the ER stress but has no impact on the reduction of the mitochondrial membrane potential. In conclusion, our data show that a non-lethal and transient ER stress triggers a rapid activation of JNK without inducing apoptosis, leading to the fragmentation of the mitochondrial network and a reduction of O2 (•-) production. J. Cell. Physiol. 231: 1913-1931, 2016. © 2015 Wiley Periodicals, Inc.

miRNA-196b inhibits cell proliferation and induces apoptosis in HepG2 cells by targeting IGF2BP1.

BACKGROUND: Tumor hypoxia is one of the features of tumor microenvironment that contributes to chemoresistance. miRNAs have recently been shown to play important roles in tumorigenesis and drug resistance. Moreover, hypoxia also regulates the expression of a series of miRNAs. However, the interaction between chemoresistance, hypoxia and miRNAs has not been explored yet. The aim of this study is to understand the mechanisms activated/inhibited by miRNAs under hypoxia that induce resistance to chemotherapy-induced apoptosis. METHODS: TaqMan low-density array was used to identify changes in miRNA expression when cells were exposed to etoposide under hypoxia or normoxia. The effects of miR-196b overexpression on apoptosis and cell proliferation were studied in HepG2 cells. miR-196b target mRNAs were identified by proteomic analysis, luciferase activity assay, RT-qPCR and western blot analysis. RESULTS: Results showed that hypoxia down-regulated miR-196b expression that was induced by etoposide. miR-196b overexpression increased the etoposide-induced apoptosis and reversed the protection of cell death observed under hypoxia. By a proteomic approach combined with bioinformatics analyses, we identified IGF2BP1 as a potential target of miR-196b. Indeed, miR-196b overexpression decreased IGF2BP1 RNA expression and protein level. The IGF2BP1 down-regulation by either miR-196b or IGF2BP1 siRNA led to an increase in apoptosis and a decrease in cell viability and proliferation in normal culture conditions. However, IGF2BP1 silencing did not modify the chemoresistance induced by hypoxia, probably because it is not the only target of miR-196b involved in the regulation of apoptosis. CONCLUSIONS: In conclusion, for the first time, we identified IGF2BP1 as a direct and functional target of miR-196b and showed that miR-196b overexpression reverses the chemoresistance induced by hypoxia. These results emphasize that the chemoresistance induced by hypoxia is a complex mechanism.

Differential effect of hypoxia on etoposide-induced DNA damage response and p53 regulation in different cell types.

Among the main causes of cancer cell resistance to chemotherapy are p53 mutation and hypoxic tumor microenvironment. However, the effect of hypoxia can be very different from one cell type to the other. We studied the effect of hypoxia on the etoposide-induced cell death in two cancer cell lines, HepG2 and A549 cells. Hypoxia decreased etoposide-induced apoptosis in HepG2 cells but not in A549 cells. Here, we evidenced two pathways, known to play important roles in cancer cell resistance, that are differently affected by hypoxia in these two cell types. First, in HepG2 cells, hypoxia decreased p53 protein level and activity by acting post-transcriptionally and independently of HIF-1. The results suggest an effect of hypoxia on p53 translation. On the other hand, in A549 cells, no effect of hypoxia was observed on p53 level. Secondly, hypoxia decreased DNA damage response in HepG2 cells while this was not the case in A549 cells. Indeed, a decrease in the phosphorylation level of CHK2 and H2AX with a decrease in ATM activity was observed. Importantly, these results evidenced that hypoxia can prevent cancer cell apoptosis by acting at different levels in the cell and that these effects are strongly cell-type dependent.

A critical role for heme synthesis and succinate in the regulation of pluripotent states transitions.

Using embryonic stem cells (ESCs) in regenerative medicine or in disease modeling requires a complete understanding of these cells. Two main distinct developmental states of ESCs have been stabilized in vitro, a naïve pre-implantation stage and a primed post-implantation stage. Based on two recently published CRISPR-Cas9 knockout functional screens, we show here that the exit of the naïve state is impaired upon heme biosynthesis pathway blockade, linked in mESCs to the incapacity to activate MAPK- and TGFß-dependent signaling pathways after succinate accumulation. In addition, heme synthesis inhibition promotes the acquisition of 2 cell-like cells in a heme-independent manner caused by a mitochondrial succinate accumulation and leakage out of the cell. We further demonstrate that extracellular succinate acts as a paracrine/autocrine signal, able to trigger the 2C-like reprogramming through the activation of its plasma membrane receptor, SUCNR1. Overall, this study unveils a new mechanism underlying the maintenance of pluripotency under the control of heme synthesis.

Characterization of the Proteins Secreted by Equine Muscle-Derived Mesenchymal Stem Cells Exposed to Cartilage Explants in Osteoarthritis Model.

BACKGROUND: Osteoarthritis (OA) is a highly prevalent joint degenerative disease for which therapeutic treatments are limited or invasive. Cell therapy based on mesenchymal stem/stromal cells (MSCs) is therefore seen as a promising approach for this disease, in both human and horses. As the regenerative potential of MSCs is mainly conferred by paracrine function, the goal of this study was to characterize the secreted proteins of muscle-derived MSCs (mdMSCs) in an in vitro model of OA to evaluate the putative clinical interest of mdMSCs as cell therapy for joint diseases like osteoarthritis. METHODS: An equine osteoarthritis model composed of cartilage explants exposed to pro-inflammatory cytokines was first developed. Then, the effects of mdMSC co-culture on cartilage explant were studied by measuring the glycosaminoglycan release and the NO2- production. To identify the underlying molecular actors, stable isotope-labeling by amino acids in cell culture based secreted protein analyses were conducted, in the presence of serum. The relative abundance of highly sequenced proteins was finally confirmed by western blot. RESULTS: Co-culture with muscle-derived MSCs decreases the cytokine-induced glycosaminoglycan release by cartilage explants, suggesting a protecting effect of mdMSCs. Among the 52 equine proteins sequenced in the co-culture conditioned medium, the abundance of decorin and matrix metalloproteinase 3 was significantly modified, as confirmed by western blot analyses. CONCLUSIONS: These results suggest that muscle-derived MSCs could reduce the catabolic effect of TNFα and IL-1ß on cartilage explant by decreasing the secretion and activity of matrix metalloproteinase 3 and increasing the decorin secretion. mdMSCs capacity to reduce the catabolic consequences of cartilage exposure to pro-inflammatory cytokines. These effects can be explained by mdMSC-secreted bioactive such as TIMP-1 and decorin, known as an inhibitor of MMP3 and an anti-inflammatory protein, respectively.

Senescence Induced by UVB in Keratinocytes Impairs Amino Acids Balance.

Skin is one of the most exposed organs to external stress. Namely, UV rays are the most harmful stress that could induce important damage leading to skin aging and cancers. At the cellular level, senescence is observed in several skin cell types and contributes to skin aging. However, the origin of skin senescent cells is still unclear but is probably related to exposure to stresses. In this work, we developed an in vitro model of UVB-induced premature senescence in normal human epidermal keratinocytes. UVB-induced senescent keratinocytes display a common senescent phenotype resulting in an irreversible cell cycle arrest, an increase in the proportion of senescence-associated ß-galactosidase‒positive cells, unrepaired DNA damage, and a long-term DNA damage response activation. Moreover, UVB-induced senescent keratinocytes secrete senescence-associated secretory phenotype factors that influence cutaneous squamous cell carcinoma cell migration. Finally, a global transcriptomic study highlighted that senescent keratinocytes present a decrease in the expression of several amino acid transporters, which is associated with reduced intracellular levels of glycine, alanine, and leucine. Interestingly, the chemical inhibition of the glycine transporter SLC6A9/Glyt1 triggers senescence features.

Taking Advantage of the Senescence-Promoting Effect of Olaparib after X-ray and Proton Irradiation Using the Senolytic Drug, ABT-263.

Radiotherapy (RT) is a key component of cancer treatment. Although improvements have been made over the years, radioresistance remains a challenge. For this reason, a better understanding of cell fates in response to RT could improve therapeutic options to enhance cell death and reduce adverse effects. Here, we showed that combining RT (photons and protons) to noncytotoxic concentration of PARP inhibitor, Olaparib, induced a cell line-dependent senescence-like phenotype. The senescent cells were characterized by morphological changes, an increase in p21 mRNA expression as well as an increase in senescence-associated ß-galactosidase activity. We demonstrated that these senescent cells could be specifically targeted by Navitoclax (ABT-263), a Bcl-2 family inhibitor. This senolytic drug led to significant cell death when combined with RT and Olaparib, while limited cytotoxicity was observed when used alone. These results demonstrate that a combination of RT with PARP inhibition and senolytics could be a promising therapeutic approach for cancer patients.

Mitochondrial Protein Cox7b Is a Metabolic Sensor Driving Brain-Specific Metastasis of Human Breast Cancer Cells.

Distant metastases are detrimental for cancer patients, but the increasingly early detection of tumors offers a chance for metastasis prevention. Importantly, cancers do not metastasize randomly: depending on the type of cancer, metastatic progenitor cells have a predilection for well-defined organs. This has been theorized by Stephen Paget, who proposed the "seed-and-soil hypothesis", according to which metastatic colonization occurs only when the needs of a given metastatic progenitor cell (the seed) match with the resources provided by a given organ (the soil). Here, we propose to explore the seed-and-soil hypothesis in the context of cancer metabolism, thus hypothesizing that metastatic progenitor cells must be capable of detecting the availability of metabolic resources in order to home in a secondary organ. If true, it would imply the existence of metabolic sensors. Using human triple-negative MDA-MB-231 breast cancer cells and two independent brain-seeking variants as models, we report that cyclooxygenase 7b (Cox7b), a structural component of Complex IV of the mitochondrial electron transport chain, belongs to a probably larger family of proteins responsible for breast cancer brain tropism in mice. For metastasis prevention therapy, this proof-of-principle study opens a quest for the identification of therapeutically targetable metabolic sensors that drive cancer organotropism.

MitoQ Inhibits Human Breast Cancer Cell Migration, Invasion and Clonogenicity.

To successfully generate distant metastases, metastatic progenitor cells must simultaneously possess mesenchymal characteristics, resist to anoïkis, migrate and invade directionally, resist to redox and shear stresses in the systemic circulation, and possess stem cell characteristics. These cells primarily originate from metabolically hostile areas of the primary tumor, where oxygen and nutrient deprivation, together with metabolic waste accumulation, exert a strong selection pressure promoting evasion. Here, we followed the hypothesis according to which metastasis as a whole implies the existence of metabolic sensors. Among others, mitochondria are singled out as a major source of superoxide that supports the metastatic phenotype. Molecularly, stressed cancer cells increase mitochondrial superoxide production, which activates the transforming growth factor-ß pathway through src directly within mitochondria, ultimately activating focal adhesion kinase Pyk2. The existence of mitochondria-targeted antioxidants constitutes an opportunity to interfere with the metastatic process. Here, using aggressive triple-negative and HER2-positive human breast cancer cell lines as models, we report that MitoQ inhibits all the metastatic traits that we tested in vitro. Compared to other mitochondria-targeted antioxidants, MitoQ already successfully passed Phase I safety clinical trials, which provides an important incentive for future preclinical and clinical evaluations of this drug for the prevention of breast cancer metastasis.

Pillar[5]arene-Based Polycationic Glyco[2]rotaxanes Designed as Pseudomonas aeruginosa Antibiofilm Agents.

Pseudomonas aeruginosa (P.A.) is a human pathogen belonging to the top priorities for the discovery of new therapeutic solutions. Its propensity to generate biofilms strongly complicates the treatments required to cure P.A. infections. Herein, we describe the synthesis of a series of novel rotaxanes composed of a central galactosylated pillar[5]arene, a tetrafucosylated dendron, and a tetraguanidinium subunit. Besides the high affinity of the final glycorotaxanes for the two P.A. lectins LecA and LecB, potent inhibition levels of biofilm growth were evidenced, showing that their three subunits work synergistically. An antibiofilm assay using a double ΔlecAΔlecB mutant compared to the wild type demonstrated that the antibiofilm activity of the best glycorotaxane is lectin-mediated. Such antibiofilm potency had rarely been reached in the literature. Importantly, none of the final rotaxanes was bactericidal, showing that their antibiofilm activity does not depend on bacteria killing, which is a rare feature for antibiofilm agents.

HDAC2 and 7 down-regulation induces senescence in dermal fibroblasts.

Originally simply reported to be in a stable and irreversible growth arrest in vitro, senescent cells are now clearly associated with normal and pathological ageing in vivo. They are characterized by several biomarkers and changes in gene expression that may depend on epigenetic factors, such as histone acetylation, involving a balance between histone acetyltransferases (HATs) and histone deacetylases (HDACs). In this study, we investigate the expression and the role of HDACs on the senescent phenotype of dermal fibroblasts. We report that during replicative senescence, most canonical HDACs are less expressed. Moreover, treatment with SAHA, a histone deacetylase inhibitor (HDACi) also known as Vorinostat, or the specific downregulation of HDAC2 or HDAC7 by siRNA, induces the appearance of senescence biomarkers of dermal fibroblasts. Conversely, the ectopic re-expression of HDAC7 by lentiviral transduction in pre-senescent dermal fibroblasts extends their proliferative lifespan. These results demonstrate that HDACs expression can modulate the senescent phenotype, highlighting their pharmaceutical interest in the context of healthy ageing.

Optimization of label-free nano LC-MS/MS analysis of the placental proteome.

The placenta can be regarded as a mirror of the events to which the fetus is exposed during development. The placental proteome has been studied with several methodologies differing in sample handling, protein extraction, and processing. We optimized a protocol to analyze the placental proteome by means of label-free nano-LC-MS/MS mass spectrometry with regard to sample treatment, protein extraction, and protein digestion, in order to obtain a high protein concentration for identification of a specific protein signature according to the conditions studied. We recommend mechanical tissue disruption, blood removal prior to protein extraction, and FASP-based or in-gel digestion.

Characterization of the role of TMEM45A in cancer cell sensitivity to cisplatin.

TMEM45A is a transmembrane protein involved in tumor progression and cancer resistance to chemotherapeutic agents in hypoxic condition. It is correlated to a low breast cancer patient overall survival. However, little is known about this protein, in particular the mechanisms by which TMEM45A modulates cancer cell chemosensitivity. In this work, the messenger RNA expression of TMEM45A was assessed in head and neck squamous cell carcinoma (HNSCC) and renal cell carcinoma (RCC) biopsies. TMEM45A was upregulated in patients diagnosed for head and neck or renal cancer. Then, the implication of this protein in cisplatin sensitivity was explored in SQD9 and RCC4 + pVHL cells. TMEM45A inactivation decreased cell proliferation and modulated cell responses to cisplatin. Indeed, TMEM45A inactivation increased the sensitivity of SQD9 cells to cisplatin, whereas it rendered RCC4 + pVHL cells resistant to this anticancer agent. Through RNA-sequencing analysis, we identified several deregulated pathways that indicated that the impact on cisplatin sensitivity may be associated to the inhibition of DNA damage repair and to UPR pathway activation. This study demonstrated, for the first time, an anti or a pro-apoptotic role of this protein depending on the cancer type and highlighted the role of TMEM45A in modulating patient responses to treatment.

Appropriate Sequence for Afatinib and Cisplatin Combination Improves Anticancer Activity in Head and Neck Squamous Cell Carcinoma.

Despite a better understanding in head and neck tumors pathogenesis as well as improvements in radiotherapy and surgery, locally advanced head and neck squamous cell carcinoma (HNSCC) remains of poor prognosis. One promising target is the epidermal growth factor receptor (EGFR), which is overexpressed in the majority of HNSCC and is associated to tumor progression and resistance to treatment. However, in several clinical trials, the combination of EGFR inhibitors with chemotherapy and/or radiotherapy generates moderate results. In this study, we investigated the anti-tumor activity of afatinib, an irreversible pan-EGFR inhibitor, combined to cisplatin in different schedules of exposure. For that, we used two human EGFR wild-type HNSCC cell lines and we evaluated the cytotoxicity of the two drugs combined in different sequences. The efficiency of each strategy was assessed by evaluating the effects on cell cycle distribution, DNA damage, cell death and downstream pathways of ErbB family receptors. We demonstrated that cisplatin treatment followed by afatinib exposure displayed more cytotoxic effects than the opposite timing or than simultaneous association. This higher anticancer activity is probably due to afatinib-induced cell cycle arrest, which prevents the repair of cisplatin-induced DNA damage and promotes cell death by various mechanisms including apoptosis. These data suggest the importance of an appropriate timing administration between an EGFR inhibitor and a conventional chemotherapy in order to obtain the best clinical benefit for patients with a head and neck cancer.

Identification of a cytotoxic molecule in heat-modified citrus pectin.

Modified forms of citrus pectin possess anticancer properties. However, their mechanism of action and the structural features involved remain unclear. Here, we showed that citrus pectin modified by heat treatment displayed cytotoxic effects in cancer cells. A fractionation approach was used aiming to identify active molecules. Dialysis and ethanol precipitation followed by HPLC analysis evidenced that most of the activity was related to molecules with molecular weight corresponding to low degree of polymerization oligogalacturonic acid. Heat-treatment of galacturonic acid also generated cytotoxic molecules. Furthermore, heat-modified galacturonic acid and heat-fragmented pectin contained the same molecule that induced cell death when isolated by HPLC separation. Mass spectrometry analyses revealed that 4,5-dihydroxy-2-cyclopenten-1-one was one cytotoxic molecule present in heat-treated pectin. Finally, we synthesized the enantiopure (4R,5R)-4,5-dihydroxy-2-cyclopenten-1-one and demonstrated that this molecule was cytotoxic and induced a similar pattern of apoptotic-like features than heat-modified pectin.

Heat-modified citrus pectin induces apoptosis-like cell death and autophagy in HepG2 and A549 cancer cells.

Cancer is still one of the leading causes of death worldwide, and finding new treatments remains a major challenge. Previous studies showed that modified forms of pectin, a complex polysaccharide present in the primary plant cell wall, possess anticancer properties. Nevertheless, the mechanism of action of modified pectin and the pathways involved are unclear. Here, we show that citrus pectin modified by heat treatment induced cell death in HepG2 and A549 cells. The induced cell death differs from classical apoptosis because no DNA cleavage was observed. In addition, Z-VAD-fmk, a pan-caspase inhibitor, did not influence the observed cell death in HepG2 cells but appeared to be partly protective in A549 cells, indicating that heat-modified citrus pectin might induce caspase-independent cell death. An increase in the abundance of the phosphatidylethanolamine-conjugated Light Chain 3 (LC3) protein and a decrease in p62 protein abundance were observed in both cell types when incubated in the presence of heat-modified citrus pectin. These results indicate the activation of autophagy. To our knowledge, this is the first time that autophagy has been revealed in cells incubated in the presence of a modified form of pectin. This autophagy activation appears to be protective, at least for A549 cells, because its inhibition with 3-methyladenine increased the observed modified pectin-induced cytotoxicity. This study confirms the potential of modified pectin to improve chemotherapeutic cancer treatments.

Taxol-induced unfolded protein response activation in breast cancer cells exposed to hypoxia: ATF4 activation regulates autophagy and inhibits apoptosis.

Understanding the mechanisms responsible for the resistance against chemotherapy-induced cell death is still of great interest since the number of patients with cancer increases and relapse is commonly observed. Indeed, the development of hypoxic regions as well as UPR (unfolded protein response) activation is known to promote cancer cell adaptive responses to the stressful tumor microenvironment and resistance against anticancer therapies. Therefore, the impact of UPR combined to hypoxia on autophagy and apoptosis activation during taxol exposure was investigated in MDA-MB-231 and T47D breast cancer cells. The results showed that taxol rapidly induced UPR activation and that hypoxia modulated taxol-induced UPR activation differently according to the different UPR pathways (PERK, ATF6, and IRE1α). The putative involvement of these signaling pathways in autophagy or in apoptosis regulation in response to taxol exposure was investigated. However, while no link between the activation of these three ER stress sensors and autophagy or apoptosis regulation could be evidenced, results showed that ATF4 activation, which occurs independently of UPR activation, was involved in taxol-induced autophagy completion. In addition, an ATF4-dependent mechanism leading to cancer cell adaptation and resistance against taxol-induced cell death was evidenced. Finally, our results demonstrate that expression of ATF4, in association with hypoxia-induced genes, can be used as a biomarker of a poor prognosis for human breast cancer patients supporting the conclusion that ATF4 might play an important role in adaptation and resistance of breast cancer cells to chemotherapy in hypoxic tumors.

Hypoxia-induced modulation of apoptosis and BCL-2 family proteins in different cancer cell types.

Hypoxia plays an important role in the resistance of tumour cells to chemotherapy. However, the exact mechanisms underlying this process are not well understood. Moreover, according to the cell lines, hypoxia differently influences cell death. The study of the effects of hypoxia on the apoptosis induced by 5 chemotherapeutic drugs in 7 cancer cell types showed that hypoxia generally inhibited the drug-induced apoptosis. In most cases, the effect of hypoxia was the same for all the drugs in one cell type. The expression profile of 93 genes involved in apoptosis as well as the protein level of BCL-2 family proteins were then investigated. In HepG2 cells that are strongly protected against cell death by hypoxia, hypoxia decreased the abundance of nearly all the pro-apoptotic BCL-2 family proteins while none of them are decreased in A549 cells that are not protected against cell death by hypoxia. In HepG2 cells, hypoxia decreased NOXA and BAD abundance and modified the electrophoretic mobility of BIM(EL). BIM and NOXA are important mediators of etoposide-induced cell death in HepG2 cells and the hypoxia-induced modification of these proteins abundance or post-translational modifications partly account for chemoresistance. Finally, the modulation of the abundance and/or of the post-translational modifications of most proteins of the BCL-2 family by hypoxia involves p53-dependent and -independent pathways and is cell type-dependent. A better understanding of these cell-to-cell variations is crucial in order to overcome hypoxia-induced resistance and to ameliorate cancer therapy.

Copper(II) oxide nanoparticles penetrate into HepG2 cells, exert cytotoxicity via oxidative stress and induce pro-inflammatory response.

The potential toxic effects of two types of copper(II) oxide (CuO) nanoparticles (NPs) with different specific surface areas, different shapes (rod or spheric), different sizes as raw materials and similar hydrodynamic diameter in suspension were studied on human hepatocarcinoma HepG2 cells. Both CuO NPs were shown to be able to enter into HepG2 cells and induce cellular toxicity by generating reactive oxygen species. CuO NPs increased the abundance of several transcripts coding for pro-inflammatory interleukins and chemokines. Transcriptomic data, siRNA knockdown and DNA binding activities suggested that Nrf2, NF-κB and AP-1 were implicated in the response of HepG2 cells to CuO NPs. CuO NP incubation also induced activation of MAPK pathways, ERKs and JNK/SAPK, playing a major role in the activation of AP-1. In addition, cytotoxicity, inflammatory and antioxidative responses and activation of intracellular transduction pathways induced by rod-shaped CuO NPs were more important than spherical CuO NPs. Measurement of Cu(2+) released in cell culture medium suggested that Cu(2+) cations released from CuO NPs were involved only to a small extent in the toxicity induced by these NPs on HepG2 cells.