Effect Of EATP And NLRP3Inflammasome On Hepatocellular Carcinoma

AimsAdenosine triphosphate (ATP) is a vital molecule used by living organisms as a universal source of energy required to drive the cogwheels of intracellular biochemical reactions necessary for growth and development. In response to tissue damage and cellular stress, intracellular ATP is actively released into the extracellular environment and acts as an important signaling molecule. Extracellular ATP (eATP) exerts many functions in physiological and pathological conditions and its effect on cells is mediated by ionotropic (P2X) and metabotropic (P2Y) receptors. eATP could be hydrolyzed by ectonucleotidases, thus its concentration is maintained at very Low level in physiological conditions. However, in some pathological conditions, such as tumor microenvironment and fast growing tumor center where are less vascularized or have lots of cell death, a large amount of intracellular ATP is released from damaged cells, and the concentration of eATP in pericellular space is maintained at a relatively high level. Whether such a high concentration of eATP has any beneficial or detrimental effect on hepatoma cells in tumor microenvironment remains elusive.It is well known that when epithelial cells are deprived of anchorage, they undergo detachment-induced apoptosis, also known as anoikis. However, many cancer cells acquire anoikis resistance and maintain survival without adhering strongly to the substratum. Anchorage independent cells obtain more opportunities to escape from immune surveillance and traditional cancer therapy, maintain survival in the circulation and lodge to a secondary place as metastatic lesions. The high concentration of extracellular ATP in local tumor microenvironment is a harsh stress and the first challenge that detached hepatoma cells encounter. Whether anchorage independent hepatoma cells could escape from this stress and acquire more opportunities to maintain survival than anchorage dependent cells is not known and is of great significance to be extensively clarified, and is investigated in this study.Our data showed that autophagy helped hepatoma cells to maintain survival under the treatment of no more than1mM of eATP. Only when eATP concentration reached to a relatively high level (2.5mM), cell organelle could not be further maintained by autophagy, apoptosis and cell death occurred. In hepatoma cells under treatment of2.5mM of eATP, AMPK pathway was dramatically activated while mTOR signaling pathway was suppressed in coordination with apoptosis. Further investigation showed that AMPK/mTOR axis played a key role in tipping the balance between autophagy-mediated cell survival and apoptosis-induced cell death under the treatment of eATP. This work provides evidence to explain how hepatoma cells escape from eATP-induced cytotoxicity as well as offering an important clue to consider effective manipulation of cancer.Materials and Methods1. Establishment of anchorage-dependent and independent hepatoma modelHuman BEL7402, SMMC7721and HepG2hepatoma cells were maintained in RPMI1640medium supplemented with10%FBS.6-well plates and96-well plates were coated with1.5mL and0.1mL of poly-HEMA solution, respectively. Coated plates were allowed to air dry under sterile conditions in a laminar flow hood. Before use, poly-HEMA-coated dishes were washed with PBS for3times. Hepatoma cells grown on normal plates and poly-HEMA-coated plates were established as anchorage-dependent and independent models, respectively.2. Effect of eATP on anchorage dependent and independent hepatoma cells2.1Anchorage-dependent and independent BEL7402cells were treated with different concentrations of ATP (0,0.05mM,0.1mM,0.5mM,1mM,2.5mM) and cultured for 24h, followed by morphology investigation.2.2Anchorage-dependent and independent BEL7402, SMMC7721and HepG2cells were treated with different concentrations of ATP (0,0.05mM,0.1mM,0.5mM,1mM,2.5mM) and cultured for24h. Effect of eATP on cell viabilities was detected by CCK-8.2.3Anchorage-dependent and independent BEL7402cells were treated with different concentrations of ATP (0,0.05mM,0.1mM,0.5mM,1mM,2.5mM) and cultured for24h. Western blot was performed to analyze the expression and activation of apoptosis and autophagy-related proteins.2.4Anchorage-dependent and independent BEL7402cells were treated with high dose of eATP (2.5mM) at different time points, and the apoptotic Caspase cascade, autophagy marker LC3-Ⅱ and the intracellular signaling pathway were detected by western blot.2.5BEL7402hepatoma cells were pretreated with autopahgy inhibitor3-MA before treatment with1mM of eATP. Immunofluorescence and CCK-8assay were performed to determine the effect of1mM of eATP treatment after autophagy was blocked.3. Detection of type of cell death induced by high dose of eATP3.1Anchorage-dependent and independent BEL7402cells were treated with BzATP for24h, and western blot was performed to confirm the effect of eATP.3.2Anchorage-dependent and independent BEL7402cells were pre-treated with apyrase for30min, before2.5mM of eATP was added, and the apoptotic Caspase cascade, autophagy marker LC3-Ⅱ and the intracellular signaling pathway were detected by western blot.4. Verification of high dose of eATP-mediated apoptosis induction and autophgy inhibition4.1Anchorage-dependent and independent BEL7402cells were pre-treated with KN-62for30min to block P2X7receptor, and then treated with2.5mM of eATP for24h. The apoptotic Caspase cascade, autophagy marker LC3-Ⅱ and the intracellular signaling pathway were detected by western blot. 4.2Anchorage-dependent and independent BEL7402cells were pre-treated with compound C, which was the AMPK inhibitor, and then treated with2.5mM of eATP for24h. AMPK and mTOR signaling pathway were detected by western blot.Results1. High dose of eATP induced apoptosis and inhibited autophagy in both anchorage-dependent and independent hepatoma cells1.1Morphology was observed by microscopeAfter cells were treated with2.5mM of eATP and cultured for24h, morphology of both of the anchorage dependent and independent hapatoma cells changed significantly, which indicated a dramatically cytotoxicity induced by eATP.1.2Cell viability was detected by CCK-8When hepatoma cells were treated with high concentration of eATP, significantly decreased cell viability was detected in both types of cells.1.3Caspase-3was activated and LC3-Ⅱ was decreased under treatment of high dose of eATPBEL7402hepatoma cells were treated by eATP during or after the formation of anchorage dependent or independent model, respectively. Both of apoptosis and autophagy markers were analyzed by western blot. When eATP concentration was no more than lmM, there is rather low Caspase-3and Caspase-9activation. When the eATP concentration reached to2.5mM, both of Caspase-3and Caspase-9were dramatically activated while the LC3-Ⅱ level was dramatically decreased.1.42.5mM of eATP mediated time-dependent Caspase-3activation and LC3-II inhibitionAnchorage-dependent and independent BEL7402cells were treated with high dose of eATP (2.5mM) at different time points. Caspase-3activation and autophagy marker LC3were analyzed by western blot. In both of the anchorage dependent and independent model, Caspase-3was effectively activated by2.5mM of eATP in a time-dependent manner while LC3-Ⅱ level was significantly decreased in accordance with the increased level of Caspase-3activation. 1.5Inhibition of autophagy significantly increased apoptosis induced by lmM of eATPBEL7402hepatoma cells were pretreated with autopahgy inhibitor3-MA before treatment with1mM of eATP. CCK-8and immunofluorescence were applied and showed that inhibition of autophagy significantly increased the apoptosis rate induced by1mM of eATP, a concentration that failed to induce significant apoptosis while working alone.2. Further confirmation of eATP-induced cytotoxicity in hepatoma cells2.1Effect of eATP on hepatoma cells was further confirmed by non-hydrolysable ATP analogue, BzATPAnchorage-dependent and independent BEL7402cells were treated with a non-hydrolysable form of eATP, BzATP.Under the treatment of2.5mM of BzATP, AMPK pathway was activated while mTOR pathway was significantly inhibited in accordance with Caspase3activation and autophagy inhibition.2.2Effect of eATP on hepatoma cells was reversed by degradation of eATPAnchorage dependent and independent BEL7402cells were pretreated with soluble NTPDase, apyrase for0.5h before treatment with2.5mM of eATP for24h. CCK-8and western blot analysis showed that degradation of eATP inhibited high dose of eATP-induced purinergic signaling, and abrogated its apoptosis-inducing effect.3. Further clarification molecular mechanism of eATP-induced cytotoxicity3.1Blockade of eATP-induced purinergic signaling abrogated its cytotoxic effectAnchorage dependent and independent BEL7402cells were pretreated with a potent P2X7receptor antagonist, KN-62for0.5h before treatment with2.5mM of eATP for24h. CCK-8and western blot analysis showed that blockade of P2X7receptor inhibited high dose of eATP-induced purinergic signaling, and abrogated its apoptosis-inducing effect.3.2High dose of eATP-induced cytotoxicity was mediated through AMPK/mTOR pathway We used compound C, an AMPK inhibitor to block AMPK signaling and analyzed activation of mTOR pathway. Our data indicated that AMPK pathway activation was responsible for mTOR pathway inhibition, which further confirmed that AMPK/mTOR pathway was responsible for, high dose of eATP-induced effect on anchorage dependent and independent hepatoma cells.Conclusions1. eATP induced cell death of anchorage dependent and independent hepatoma cellsthrough tipping the balance between apoptosis and autophagy.2. AMPK/mTOR signaling pathway is responsible for eATP-induced cytotoxicity in hepatoma cells3. Autophagy inhibitor in conjugation with eATP achieved efficient anti-tumor effect at lower concentrations of eATP, which opened a new avenue for the efficient manipulation of cancer.Innovations and Significances1. Anchorage-dependent and independent hepatoma models are established, and the effect of eATP on both cell types is investigated for the first time to indicate whether eATP plays the same role in different stages of HCC progression.2. It is the first time to show that eATP could mediate the balance between apoptosis and autophagy in hepatoma cells.3. It is the first time to show that AMPK/mTOR signaling pathway is involved to regulate the eATP-induced cytotoxicity in hepatoma cells. AimsThe nucleotide-binding domain, leucine rich family (NLR), pyrin containing3(NLRP3) inflammasome, has been shown to orchestrate multiple innate and adaptive immune responses, however, little is known about its role in cancer. This study is to investigate roles of NLRP3inflammasome in the progression of hepatocellular carcinoma (HCC), a typical inflammation-induced cancer. Three cohorts of patients were investigated in this study for the expression of NLRP3inflammasome components in HCC cells. Our data showed that expression of all the NLRP3inflammasome components was either completely lost or significantly down-regulated in human HCC, and loss of NLRP3inflammasome components was significantly correlated with advanced stages and poor pathological differentiation. Reconstitution of NLRP3inflammasome in HCC cells had antitumor effects and significantly reversed malignant behaviors of these cancer cells. Our data offered basis for elucidation of pathogenesis of HCC and potential anti-cancer strategy by reconstitution of NLRP3inflammasome in HCC patients.Methods1. Detection of NLRP3inflammasome expression in HCC and corresponding peritumoral liver tissues.1.1Two TMAs, including128HCC patients with HCC tissues and the corresponding non-cancer tissue cores were used for IHC analysis of NLRP3, ASC, Caspase-1and IL-1β expression.1.2To further confirm the down-regulated expression of NLRP3inflammasome components in HCC tissues, immunoreactivity in cancer and non-cancer TMA staining was analyzed by SPSS software. Statistical analysis of the immunohistochemical score of NLRP3, ASC, Caspase-1and IL-1β expression was performed to determine whether the four molecules were correlated with each other. Then, to further elucidate the potential link between NLRP3components expression and clinic pathological features, correlation analysis was applied.1.3Validation of the expression tendency of NLRP3inflammasome components was further performed by RT-PCR, real-time-PCR and western blot analysis to detect mRNA and protein level of NLRP3inflammasome components in fresh HCC tissues and corresponding non-cancer liver tissues from24HCC patients.2. Reconstitution of NLRP3inflammasome in HCC cells2.1NLRP3inflammasome reconstitution was performed by transfecting SMMC7721and HepG2cells with eukaryotic expression plasmids, including15ng of pcDNA3-myc-NLRP3,5ng of pcDNA3-myc-ASC,5ng of pcDNA3-myc-pro-Caspase-1and150ng of pRC/cMV-proIL-1β. Successful inflammasome reconstitution was confirmed by mRNA and protein assay of NLRP3inflammasome components by RT-PCR and western blot.2.2Effect of NLRP3inflammasome on the proliferation and colony formation capability ofHCC cellsSMMC7721and HepG2cells were co-transfected with NLRP3inflammasome components. Cell viabilities and the colony formation capability were analyzed by CCK-8kit, LDH Cytotoxicity Assay Kit and the colony formation assay.2.3Effect of NLRP3inflammasome on migration capability of HCC cells.SMMC7721and HepG2cells were co-transfected with NLRP3inflammasome components. Cell migration capability was studied by wound healing assay and transwell migratory assay.3. Effect of NLRP3inflammasome on the downstream signaling pathwayThe NLRP3inflammasome was over-expressed in SMMC7721and HepG2cells by co-transfection of NLRP3inflammasome components. Activation of the signaling transduction pathways, including mTOR and ERK1/2pathways were detected by western blot.Results1. Expression of NLRP3inflammasome components in HCC tissues was down-regulated1.1The IHC assay suggested that expression of NLRP3inflammasome-related proteins-NLRP3, ASC, Caspase-1and IL-1β was dramatically decreased in HCC tissues compared to that of corresponding non-cancer liver tissues.1.2Immunoreactivity in cancer and non-cancer TMA staining was assessed byx2-test, and the statistical data showed significant down-regulation of NLRP3inflammasome components, compared with that of the pair-matched non-cancer tissues. Association between NLRP3inflammasome components was performed using a two sided Spearman’s rank correlation test, and the data showed that expression of the four proteins had statistically positive correlations with one another. In addition, association between protein expression and clinicopathological parameters were assessed by the Spearman’s rank correlation test, which indicated that patients with weaker immunoreactivity of the NLRP3inflammasome components were prone to have poorer differentiation and higher TNM stages.1.3Total RNA and proteins were respectively extracted from HCC tissues and the corresponding non-cancer liver tissues from24HCC patients. RT-PCR, real-time PCR and western blot analysis revealed that both of the mRNA and protein level of NLRP3, ASC, Caspase1and IL-1β were significantly decreased in HCC tissues compared with those of non-cancer liver tissues2. Reconstitution of NLRP3inflammasome reversed malignant behavior of HCC cells 2.1The SMMC7721and HepG2cells were co-transfected with plasmids encoding NLRP3, ASC, pro-caspasel, and pro-IL-1β. Expression of each component of NLRP3inflammasome was confirmed by RT-PCR and immunoblotting.2.2Reconstitution of NLRP3inflammasome inhibited HCC cell proliferation and colony formationThe NLRP3inflammasome was reconstructed in SMMC7721and HepG2cells. CCK-8assay, LDH release assay and colony formation assay revealed that over-expression of NLRP3inflammasome in HepG2and SMMC7721cells inhibited HCC cell proliferation and colony formation.2.3Reconstitution of NLRP3inflammasome inhibited the migratory ability of HCC cells.The NLRP3inflammasome was over-expressed in SMMC7721and HepG2cells by co-transfection of NLRP3inflammasome components. To further verify the effect of NLRP3inflammasome on the migratory ability of tumor cells, we performed transwell migratory assay and wound healing assay in HCC cells, which revealed over-expression of NLRP3inflammasome molecular platform significantly decreased the migratory ability of HCC cells.3. Reconstitution of NLRP3inflammasome inhibited mTOR signaling pathway and activated ERK1/2pathwayThe NLRP3inflammasome was reconstituted in SMMC7721and HepG2cells. Western blot analysis revealed that over-expression the NLRP3inflammasome significantly decreased the p-mTOR and p-S6K1level, which suggested mTOR signaling pathway was down-regulated by NLRP3inflammasome. In contrast, p-ERK1/2level was significantly increased, which indicated that NLRP3inflammasome could up-regulate ERK1/2signaling pathway.Conclusions1. IHC, western blot and real-time PCR results confirmed that expression of all of the NLRP3inflammasome components was significantly down-regulated at both of the mRNA and protein level in the liver cancer cells compared with that of their adjacent non-cancer hepatocytes.2. Expression of NLRP3inflammasome components was negatively correlated with the HCC progression.3. NLRP3inflammasome was reconstituted successfully, and its reconstitution inhibited the proliferation and migration of hepatoma cells. Thus, the malignant behavior of hepatoma cells was reversed.4. mTOR and ERK1/2signaling pathway were regulated by NLRP3inflammasome by NLRP3inflmmasome.Innovations and significances1. Most NLRP3inflammasome studies focus on innate immunity, and roles of NLRP3inflammasome remain elusive. In our study, expression and function of NLRP3inflammasome in hepatama cells are studied for the first time. It reveals that NLRP3inflammasome plays an important role in HCC progression, which provides a new approach for elucidation and final manipulation of HCC.2. NLRP3inflammasome is down-regulated in HCC tissues, and its reconstitution reverses the malignant behavior of HCC cells. This report reveals relationship between NLRP3inflammasome deficiency and HCC progression.