Well-differentiated Cell Line Research Articles

ESR Essentials: staging and restaging with FDG-PET/CT in oncology-practice recommendations by the European Society for Hybrid, Molecular and Translational Imaging.

Positron emission tomography (PET) stands as the paramount clinical molecular imaging modality, especially in oncology. Unlike conventional anatomical-morphological imaging methods such as computed tomography (CT) and magnetic resonance imaging (MRI), PET provides detailed visualizations of internal activity at the molecular and cellular levels. 18-fluorine-fluorodeoxyglucose ([18F]FDG)-PET combined with contrast-enhanced CT (ceCT) significantly improves the detection of various cancers. Appropriate patient selection is crucial, and physicians should carefully assess the appropriateness of [18F]FDG-PET/CT based on specific clinical criteria and evidence. Due to its high diagnostic accuracy, [18F]FDG-PET/CT is indispensable for evaluating the extent of disease, staging, and restaging known malignancies, and assessing the response to therapy. PET/CT imaging offers significant advantages in patient management, particularly by identifying occult metastases that might otherwise go undetected. This can help prevent unnecessary surgeries, allowing many patients to be redirected to systemic chemotherapy instead. However, it is important to note that the gold standard for surgical planning remains CT and/or MRI, depending on the body region. These imaging modalities, with or without associated angiography, provide superior contrast and spatial resolution, essential for detailed surgical preparation and planning. [18F]FDG-PET/CT has a central role in the precise and early diagnosis of cancer, contributing significantly to personalized treatment plans. However, it has limitations, including non-tumor-specific uptake and the potential to inaccurately capture the metabolic activity of certain tumor types due to low uptake in some well-differentiated tumor cell lines. Therefore, it should be utilized in clinical scenarios where it offers crucial diagnostic insights not readily available with other imaging modalities. KEY POINTS: Use [18F]FDG-PET/CT selectively based on clinical appropriateness criteria and existing evidence to optimize resource utilization and minimize patient exposure. Employ [18F]FDG-PET/CT in treatment planning and monitoring, particularly for assessing chemotherapy or radiotherapy response in FDG-avid lymphoma and solid tumors. When available, [18F]FDG-PET/CT can be integrated with other diagnostic tools, such as MRI, to enhance overall diagnostic accuracy.

Abstract 5870: T-cell factor-4 variants regulate PD-L1 expression in liver cancer cells

Abstract Background: We previously identified 14 isoforms of T-cell factor (TCF)-4, a key transcriptional factor in the Wnt signaling pathway, from human hepatocellular carcinoma (HCC) cell lines (Exp Cell Res 2011). The TCF-4J and K pair has been characterized based on the presence (K) or absence (J) of a conserved SxxSS motif at the head of exon 9. TCF-4J-overexpressing HCC cells (J cells) exhibited high tumorigenic potential in contrast to TCF-4K-overexpressing cells (K cells) (PLoS ONE 2012); however, resistance to anti-cancer agents was more prominent in K cells than J cells. Another analysis demonstrated that loss of exon 4 increased resistance to chemotherapeutic agents and induced epithelial-mesenchymal transition (EMT) (Liver Int 2013). EMT and the expression of programmed cell death 1-ligand 1 (PD-L1) is closely associated with each other, and our preliminary study revealed that EMT was regulated by TCF-4 variants, suggesting possible involvement of the variants in the regulation of PD-L1 expression. In general, PD-L1 is known to be upregulated by hypoxia-inducible factor-1α (HIF-1α) under hypoxic conditions. Therefore, if the TCF-4 variants regulate PD-L1 expression under normoxia, it would be a unique mechanism bridging the Wnt signaling pathway and the tumor immune microenvironment (TIME). Aim: To assess whether or not the TCF-4 variants regulate the expression of PD-L1 in cancer cells under normoxia. Methods: HAK-1A, a well-differentiated HCC cell line (Hepatology 1993), was used in this study. TCF-4K mutants (S269A, S272A, and S273A) were prepared with the conversion of serine (S) in the SxxSS motif to alanine (A) by site-directed mutagenesis. HAK-1A-derived stable clones overexpressing TCF-4J, K, and K-mutants (S269A, S272A, and S273A cells, respectively) were established. Empty vector-transfected mock cells (EV cells) were used as a control. Western blot analysis and real-time RT-PCR were employed to evaluate protein and mRNA expression levels, respectively. Results: J cells and K cells expressed PD-L1 mRNA 2-fold and 4-fold of that in the EV cells, respectively. Of note, the PD-L1 expression mRNA was enhanced up to 11-fold in S269A cells, 11-fold in S272A cells, and 17-fold in S273A cells. The increased mRNA expressions were verified in protein levels. Conclusion: PD-L1 expression was regulated by TCF-4 variants in liver cancer cells under normoxic conditions. The finding suggests that the Wnt signaling pathway fine-tunes the TIME by regulating the expression levels of PD-L1 in a HIF-1α-independent manner. Citation Format: Hironori Koga, Yasuko Imamura, Toshimitsu Tanaka, Hideki Iwamoto, Toru Nakamura, Atsutaka Masuda, Takahiko Sakaue, Hiroyuki Suzuki, Hirohisa Yano, Takumi Kawaguchi. T-cell factor-4 variants regulate PD-L1 expression in liver cancer cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5870.

EphA4 signaling is involved in the phenotype of well-differentiated oral squamous cell arcinoma with decreased tumor immunity

Metronomic chemotherapy is defined as a high-frequency low-dose schedule of chemotherapy drug administration. Although metronomic chemotherapy is widely used, the mechanisms underlying resistance to metronomic chemotherapy remain unclear. Therefore, we herein conducted a single institutional phase I/II trial to assess the efficacy and safety of metronomic chemotherapy with bleomycin plus S-1, an oral 5-FU prodrug, in the neoadjuvant setting for patients with oral squamous cell carcinoma (OSCC). The response rate of well-differentiated OSCC to metronomic chemotherapy was significantly lower. We investigated differences in molecular profiles between poorly or moderately differentiated head and neck squamous cell carcinoma (HNSCC) and well-differentiated HNSCC from patients with HNSCC TCGA data. EphA4 expression positively correlated with histological differentiation. An upstream regulator analysis correlated with EphA4 expression identified pathways associated with decreased mTORC1 signaling and T cell activation, including TCR, CD3, CD28, and CD40LG. An EphA4 blocking peptide (KYL) induced mTOR activation in well-differentiated OSCC cell lines. Plasmacytoid dendritic cell and CD8+ T cell numbers were higher in the microenvironment of poorly or moderately differentiated HNSCC than in that of well-differentiated HNSCC. Well-differentiated HNSCC had the characteristics of “cold tumors” (immune-excluded tumors). Moreover, KYL used with chemotherapeutic drugs synergistically increased cancer cell death. Well-differentiated OSCC is depleted of immune cells, which may be partly explained by the receptor tyrosine kinase EphA4.

Nickel chloride induces anticancer biological responses in hepatocellular carcinoma cell lines.

Nickel has long been known to have a toxic effect in humans and has been defined as a human carcinogen. However, recent studies have suggested that nickel chloride (NiCl2) may also possess anticancer properties. The liver is one of the target organs for nickel, and thus, the present study aims to evaluate the effect of NiCl2 on anticancer biological responses in hepatocellular carcinoma (HCC) cell lines. Both HuH-7, a well-differentiated HCC cell line, and Mahlavu cell line, a poorly differentiated HCC cell line, were exposed to NiCl2. It was determined that NiCl2 decreased cell viability in both cell lines in a dose- and time-dependent manner. Nickel chloride exposure at IC50 doses were observed to suppress the ability of HCC cells to produce colonies and also induce apoptosis of HCC cells by increasing Cleaved Caspase-3 protein levels. It was found that NiCl2 exposure affected cellular morphology, increased the LC3-II protein levels, and induced autophagy in parallel to increased apoptosis in HCC cells. It was also observed that NiCl2 suppressed cell migration, decreased the size and viability of HCC tumor spheroids generated in 3D cell cultures, and disrupted the spheroid structure of the tumor cells depending on E-cadherin expression levels. Furthermore, it was observed that all anticancer biological responses induced by NiCl2 occurred independently of the AKT signaling pathway. In conclusion, our results suggested that NiCl2 induced anticancer biological responses in HCC cell lines. Moreover, this study provided important new molecular and cellular biological basic data about the action mechanisms of NiCl2 in HCC.

Circular RNAs hsa_circ_0001438 and hsa_circ_0000417 are downregulated and upregulated, respectively, in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most predominant type of liver cancer and is frequently fatal. Alpha-fetoprotein, alpha-fetoprotein-L3, and protein induced by vitamin K absence or antagonist-II are used as biomarkers to diagnose HCC. However, these biomarkers are not highly specific, especially for early-stage HCC diagnosis; therefore, more specific biomarkers are needed. Recently, circular RNA (circRNA) biomarkers have been used to diagnose several intractable diseases. In this study, we sought to identify circRNA biomarkers for the specific diagnosis of HCC. To this end, we compared the expression levels of circRNAs in primary HCC and normal tissues using publicly available RNA-seq data. Our analysis revealed that the expression levels of eight circRNAs were altered in primary HCC tissues compared with normal tissues. To confirm our findings, we examined the expression levels of selected circRNAs in HCC cell lines and normal hepatocytes. The expression level of hsa_circ_0001438, a circRNA that was downregulated in primary HCC, was lower in poorly and well-differentiated HCC cell lines than in normal hepatocytes. By contrast, the expression level of hsa_circ_0000417, which was increased in primary HCC, was strongly upregulated in a well-differentiated HCC cell line compared with normal hepatocytes. Thus, hsa_circ_0001438 and hsa_circ_0000417 might be potential biomarkers for the specific diagnosis of HCC. The experimental strategy described here, using publicly available RNA-seq data, is a useful and cost-effective method of identifying circRNA biomarkers.

Activation of nano-photosensitizers by Y-90 microspheres to enhance oxidative stress and cell death in hepatocellular carcinoma

While radioembolization with yttrium-90 (Y-90) microspheres is a promising treatment for hepatocellular carcinoma (HCC), lower responses in advanced and high-grade tumors present an urgent need to augment its tumoricidal efficacy. The purpose of this study was to determine whether clinically used Y-90 microspheres activate light-responsive nano-photosensitizers to enhance hepatocellular carcinoma (HCC) cell oxidative stress and cytotoxicity over Y-90 alone in vitro. Singlet oxygen and hydroxyl radical production was enhanced when Y-90 microspheres were in the presence of several nano-photosensitizers compared to either alone in cell-free conditions. Both the SNU-387 and HepG2 human HCC cells demonstrated significantly lower viability when treated with low activity Y-90 microspheres (0.1–0.2 MBq/0.2 mL) and a nano-photosensitizer consisting of both titanium dioxide (TiO2) and titanocene (TC) labelled with transferrin (TiO2-Tf-TC) compared to Y-90 microspheres alone or untreated cells. Cellular oxidative stress and cell death demonstrated a linear dependence on Y-90 at higher activities (up to 0.75 MBq/0.2 mL), but was significantly more accentuated in the presence of increasing TiO2-Tf-TC concentrations in the poorly differentiated SNU-387 HCC cell line (p < 0.0001 and p = 0.0002 respectively) but not the well-differentiated HepG2 cell line. Addition of TiO2-Tf-TC to normal human hepatocyte THLE-2 cells did not increase cellular oxidative stress or cell death in the presence of Y-90. The enhanced tumoricidal activity of nano-photosensitizers with Y-90 microspheres is a potentially promising adjunctive treatment strategy for certain patient subsets. Applications in clinically relevant in vivo HCC models are underway.

Extracellular ATP Induced S-Phase Cell Cycle Arrest via P2Y Receptor-Activated ERK Signaling in Poorly Differentiated Oral Squamous Cell Carcinoma SAS Cells.

Extracellular ATP in the tumor microenvironment exhibits either pro- or antitumor effect via interaction with P2Y receptors, but the intracellular signaling and functional roles of P2Y receptors in oral squamous cell carcinoma (OSCC) are unclear. We aimed to study the effect of ATP on OSCC cell lines and the potential mechanisms involved. Through GEPIA dataset analysis, high expression levels of mRNA encoding P2Y receptors, the ATP-induced G protein-coupled receptors, were associated with better overall patient survival in head and neck squamous cell carcinoma. qPCR analysis showed that the poorly differentiated OSCC SAS cell line, had higher P2RY1 expression level compared to the well-differentiated H103 and H376 cell lines. Western blotting and flow cytometry analyses revealed that ATP phosphorylated ERK and elevated intracellular calcium signaling in all tested cell lines. A significant S-phase cell cycle arrest was observed in SAS, and preincubation with the MEK inhibitor PD0325901 reversed the ATP-induced S-phase arrest. We further demonstrated that ATP induced a slight reduction in cell count and colony formation yet significant apoptosis in SAS. Overall, we postulate that the ATP-induced S-phase arrest effect in SAS cells may be regulated through P2Y receptor-mediated ERK signaling, thus suggesting a potential antitumor effect of ATP via interaction with its distinct profile of P2Y receptors.

LncRNA BC200 Promotes Esophageal Squamous Cell Cancer Migration and Invasion and Can Regulate ATF4 Expression.

Background: The main reason for esophageal squamous cell carcinoma (ESCC) treatment failure is metastasis. Little is known about the mechanisms involved in the metastasis of ESCC, and there is a lack of effective therapeutic targets. In our previous study, we found that patients with high levels of BC200 tended to have poor prognoses.Methods: First, we applied qRT-PCR to detect the expression level of BC200 in normal esophageal squamous epithelial cells and ESCC cells with different degrees of differentiation ability. Then, we changed BC200 expression by transfecting constructed lentiviruses that included BC200 shRNA (LV-BC200-shRNA, KD), negative control (CON053, NC), or BC200 gene (LV-BC200, BC200) to create BC200-deficient cell models in KYSE410 and KYSE70 cells and BC200 overexpression cell models in EC9706 cells and verified the transfection effect by qRT-PCR. Then, we examined cell migration by wound healing assay, invasion by Transwell assay, and proliferation by MTT assay and examined the metastasis ability in a xenograft mouse model. Gene expression profiling was performed to screen a panel of mRNAs following inhibition of BC200 expression. We then used ingenuity pathway analysis (IPA) to analyze the functions of the changed molecules and their interactions. The results from the microarray were validated by qRT-PCR and Western blotting.Results: In this study, we found that the expression of BC200 in poorly differentiated cell lines was significantly higher than that in well-differentiated cell lines. BC200 can significantly promote the migration and invasion but not the proliferation ability of ESCC cells in vitro and BC200 shRNA can significantly suppress tumor metastasis in vivo. Our genome-wide expression profile chip showed 406 differentially expressed genes, with 91 upregulated genes and 315 downregulated genes. The upstream regulator analysis showed that ATF4 was predicted to be strongly inhibited and 21 genes were consistently inhibited by this gene. Our qRT-PCR and Western blotting data also identified the reduced expression of ATF4 and some selected downstream genes, such as SNAIL2, GADD45A, and PSAT1, as a consequence of downregulating BC200 expression in ESCC.Conclusion: Our data showed that BC200 promoted the metastasis of ESCC cells and could regulate the expression of ATF4 and its downstream genes.

Chloroquine induces apoptosis in pancreatic neuroendocrine neoplasms via endoplasmic reticulum stress.

Although pancreatic neuroendocrine neoplasms (PanNENs) are generally indolent, patients with distant metastasis have a dismal prognosis. Recently, the autophagy inhibitor chloroquine (CQ) has been shown to suppress the tumour growth of PanNENs, but the detailed mechanisms have not been elucidated. Furthermore, these results were obtained from poorly differentiated cell lines rather than well-differentiated cell lines, which is the most prevalent type in this tumour. To explore the mechanism and efficacy of CQ on PanNENs, we applied CQ to cell lines and evaluated the resulting apoptosis and endoplasmic reticulum (ER) stress. CQ treatment induced ER stress, and an unfolded protein response was activated through the PERK-eIF2α-ATF4 pathway, resulting in the expression of the pro-apoptotic protein C/EBP homologous protein (CHOP), which reflects ER-stress-mediated apoptotic cell death. Furthermore, hydroxychloroquine (HCQ) was effective in Men1 heterozygous-deficient (Men1+/ΔN3-8) mice, a mouse PanNEN model that is considered to correspond to human low-grade PanNEN. HCQ administration decreased tumour size in Men1+/ΔN3-8 mice. In the HCQ group, histological analyses revealed that proliferative activity was unchanged, but apoptosis was accelerated, accompanied by CHOP expression. These results suggest that autophagy inhibition by CQ/HCQ could be used for the treatment of PanNEN, including the well-differentiated type.

Decreased miR-200b-3p in cancer cells leads to angiogenesis in HCC by enhancing endothelial ERG expression

Transcription factor ERG (erythroblast transformation-specific (ETS)-related gene) is essential in endothelial differentiation and angiogenesis, in which microRNA (miR)-200b-3p targeting site is expected by miRNA target prediction database. miR-200b is known decreased in hepatocellular carcinoma (HCC), however, the functional relation between ERG and miR-200b-3p, originating from pre-miR-200b, in HCC angiogenesis remains unclear. We investigated whether hepatocyte-derived miR-200b-3p governs angiogenesis in HCC by targeting endothelial ERG. Levels of miR-200b-3p in HCC tissues were significantly lower than those in adjacent non-HCC tissues. Poorly differentiated HCC cell line expressed lower level of miR-200b-3p compared to well-differentiated HCC cell lines. The numbers of ERG-positive endothelial cells were higher in HCC tissues than in adjacent non-HCC tissues. There was a negative correlation between the number of ERG-positive cells and miR-200b-3p expression in HCC tissues. Culture supernatants of HCC cell lines with miR-200b-3p-overexpression reduced cell migration, proliferation and tube forming capacity in endothelial cells relative to the control, while those with miR-200b-3p-inhibition augmented the responses. Exosomes isolated from HCC culture supernatants with miR-200b-3p overexpression suppressed endothelial ERG expression. These results suggest that exosomal miR-200b-3p from hepatocytes suppresses endothelial ERG expression, and decreased miR-200b-3p in cancer cells promotes angiogenesis in HCC tissues by enhancing endothelial ERG expression.

Cell Survival Is Regulated via SOX9/BCL2L1 Axis in HCT-116 Colorectal Cancer Cell Line.

Colorectal cancer (CRC) is one of the most frequent types of malignancies and one of the major causes of cancer-related death worldwide. Sex-determining region Y (SRY)-box 9 protein (SOX9) is a member of the SOX family of transcription factors which are involved in the regulation of differentiation and development. Recently, several reports suggest an important role of SOX9 in tumorigenesis since its overexpression correlates with tumor progression and poor outcome in several types of cancer; however, its role in CRC is not clear until now. Therefore, in this work, we searched for novel SOX9-regulated genes involved in cell survival of CRC. We silenced SOX9 in the poorly differentiated HCT-116 cell line, using a specific siRNA, to identify differential expressed genes by DNA microarrays and analyzed the role or candidate genes in apoptosis and autophagy. Transcriptome analysis showed that diverse cellular pathways, associated with CRC carcinogenesis such as Wnt/β-catenin, MAPK, TGF-β, and mTOR, were modulated after SOX9 silencing. Interestingly, we found that SOX9 silencing promotes downregulation of BCL2L1 and overexpression of CASP3, proteins related to apoptosis, which was further confirmed in SW-480, a moderated-differentiated cell line, but not in HT-29, well-differentiated cell line. Moreover, inhibition of BCL2L1 by ABT-737 (BH3 mimetic) in SOX9-silenced HCT-116 cells resulted in an increased apoptosis percentage. However, downregulation of BCL2L1 was not enough to induce autophagy. This is the first report, suggesting that cell survival in poorly and moderated-differentiated CRC cells lines is regulated by SOX9/BCL2L1 axis, but not in well-differentiated cell lines.

Epstein-Barr virus induces morphological and molecular changes in thyroid neoplastic cells.

Although the evolution of differentiated thyroid cancer (DTC) is usually indolent, some tumors grow fast, metastasize, and may be fatal. Viruses have been associated with many human tumors, especially the Epstein-Barr virus (EBV), which shows a high viral load in DTC. In order to evaluate the ability of the virus to cause morphological and molecular changes in neoplastic thyroid cell lines TPC-1, BCPAP, and 8505C, a viral adaptation was performed for the analysis of EBV cytopathic effect (CPE), viral kinetics and gene expression analysis of oncogenes KRAS, NRAS, HRAS, and TP53. Comparison of inoculated cells with non-inoculated control cells showed that all tumor cell lines were permissive to the virus. The virus caused CPE in the TPC-1 and 8505C, but not in BCPAP cells. Viral kinetic was similar in both BCPAP and 8505C with a point of eclipse at 24 h post infection. TPC-1 cell line displayed a decreasing growth curve, with highest viral load right after inoculation, which decreased over time. There was hyperexpression of TP53 and NRAS in BCPAP cell (p = 0.012 and p = 0.0344, respectively). The 8505C cell line presented NRAS hyperexpression (p = 0.0255), but lower TP53 expression (p = 0.0274). We concluded that neoplastic thyroid cell lines are permissive to EBV that the virus presents different viral kinetic patterns in different cell lines and produces a CPE on both well-differentiated and undifferentiated thyroid cell lines. We also demonstrated that EBV interferes in oncogene expression in thyroid neoplastic cell lines, suggesting that these effects could be related to different tumor progression patterns.

CSF1-associated decrease in endometrial macrophages may contribute to Asherman's syndrome.

Asherman's syndrome (AS) is characterized by endometrial fibrosis leading to intrauterine adhesions and symptoms like hypomenorrhea, infertility, and recurrent pregnancy loss. Macrophages are key regulators of inflammation, tissue repair, regeneration, and fibrosis. However, the role of macrophages in AS remains unclear. Endometrial biopsies of AS patients and controls were collected during the late proliferating phase of menstrual cycle. Fibrosis and proliferation markers were detected by Masson's trichrome staining and immunohistochemistry. Macrophages were examined by immunostaining and flow cytometry. The expression levels of CCL2, CSF1, CSF1R, and GM-CSF were detected by quantitative real-time polymerase chain reaction (q-PCR) and immunohistochemistry. A well-differentiated endometrial cell line Ishikawa (IK) was used for in vitro studies. Macrophages differentiating from THP-1 monocytic cells were polarized by IL-4/IL-13. Their culture supernatants (M(IL-4/13)-S) were applied to H2 O2 or bleomycin-damaged IK cells. In AS patients, endometrial stroma was replaced by fibrous tissue and cell proliferation was reduced. Macrophages in endometrial tissue were mainly alternative activated macrophages and their number was significantly decreased in AS patients. The CSF1 expression level was reduced in AS patients. M(IL-4/13)-S promoted the growth and migration of IK cells and inhibited H2 O2 -induced apoptosis. M(IL-4/13)-S protected IK cells from bleomycin-induced fibrosis. Macrophages are critical cells involved in the process of endometrial repair and fibrosis. The decreased amount of endometrial macrophages may be attributed to the reduced expression level of CSF1. Manipulation of macrophage activation/function may provide a novel therapeutic target for AS.

Modulatory effect of Prosopis juliflora leaves on hepatic fibrogenic and fibrolytic alterations induced in rats by thioacetamide

This study investigated the antifibrotic effect of Prosopis juliflora leaves crude methanolic extract (PJEL) against thioacetamide (TAA)-induced liver fibrosis. The phytochemical analysis of PJEL was performed via HPLC/MS in association with evaluating its free radical scavenging and cytotoxic activities. The antifibrotic activity of PJEL was assessed by dividing Wistar rats into 8 groups: normal control, PJEL1-administered rats (2 mg/ Kg b.w.), PJEL2-administered rats (4 mg/ Kg b.w.), PJEL3-administered rats (8 mg/Kg b.w.), TAA-induced hepatic fibrosis, TTA + PJEL1, TAA + PJEL2, and TAA + PJEL3. Results indicated that PJEL crude methanolic extract is rich in polyphenolic compounds and alkaloids. PJEL exerted free radical scavenging activity with IC50 of 123.5 μg/mL and cytotoxic activity against a well-differentiated hepatocellular cell line (IC50 = 11.1 μg/mL). PJEL at a dose of 4 mg/Kg b.w. ameliorated serum ALT activity and improved serum albumin level and hepatic hydroxyproline content in association with a reduction in the fibrosis stage. PJEL elevated hepatic tumor necrosis factor-α and interleukin-6 contents with less necrosis grade. PJEL post-therapy ameliorated the relative expression of Bcl-2, Col1A1, Mmp-9, and Mmp-2 genes in liver. ConclusionPJEL possesses a good therapeutic activity against TAA-induced liver fibrosis via enhancing extracellular matrix removal and stimulating hepatic regeneration to decrease hepatic necrosis.

SAT-572 Morphological and Molecular Alterations in Thyroid Neoplastic Cells Infected by Epstein-Barr Virus May Be Associated to Tumor Evolution

ABSTRACT Title: Morphological and molecular alterations in thyroid neoplastic cells infected by Epstein-Barr virus may be associated to tumor evolution. The Epstein-Barr Virus (EBV) is an ubiquitous human herpes virus and more than 90% of adults, worldwide, have evidence of infection. Since the virus is able to cause a latent and persistent infection, interfering in the metabolism of host cells and triggering a tumorigenic process, it has been considered a human carcinogen by the International Agency for Research on Cancer. EBV has been held responsible for 2% of the malignancies, and is associated to nasopharyngeal carcinoma, gastric cancer, Burkitt’s and Hodgkin’s lymphoma. More recently, our group reported the presence of high EBV viral load in malignant thyroid tumors. Thyroid cancer prevalence has been raising all over the world but its typical evolution is indolent and tumors may even regress. In fact, current guidelines advise small low risk tumors observation without immediate surgical intervention. Looking for the putative clinical role of EBV infection in the evolution of thyroid tumors, we aimed to evaluate the ability of the virus to cause morphological and molecular changes in neoplastic thyroid cell lines TPC-1, BCPAP and 8505C. Viral adaptation for the analysis of the cytopathic effect of the neoplastic thyroid cell lines was performed, followed by viral kinetics of 48 hours for viral load and genes expression analysis of KRAS, NRAS, HRAS and TP53. Comparison of the inoculated cells with non-inoculated control cells showed that all tumor cell lines were permissive to the virus; however, the virus caused cytopathic effect, altering the cell morphology only in the TPC-1 and 8505C. There was hyperexpression of KRAS and NRAS in all cell lines whereas the virus was associated to an initial increase of TP53 that further decreased its expression. We conclude that neoplastic thyroid cell lines are permissive to EBV and that the virus is capable of causing cytopathic effect in both well-differentiated and undifferentiated thyroid cell lines. We also demonstrated that EBV interferes in gene expression in thyroid neoplastic cell lines producing an increase in the expression of the oncogenes KRAS, NRAS and TP53 followed by the loss of expression of the tumor suppressor gene TP53. We suggest that these effects could be related to tumor progression. Keywords: Humam Herpesvirus 4. EBV. Thyroid Cancer. Viral Load. RAS expression.

SUN-042 Loss of the 18-Kda Translocator Protein Tspo Destabilizes Cholesterol Homeostasis in a Hepatic Steatosis Cell Model

The 18-kDa translocator protein TSPO is a conserved ubiquitous high affinity cholesterol binding protein localized in the outer mitochondrial membrane. It plays a critical role in the segregation and transport of free cholesterol (FC) from the outer to the inner mitochondrial membrane in steroidogenic cells. Numerous studies have shown the presence of TSPO in liver. Imaging studies in humans, using specific TSPO ligands, showed that TSPO is highly expressed and accurately mirrors the histological picture of non-alcoholic fatty liver disease (NAFLD)/nonalcoholic steatohepatitis (NASH). The function of TSPO in simple steatosis and NASH is unknown. Wild type Huh7 (WT) is a well-differentiated human hepatocyte-derived carcinoma cell line, which contains TSPO in mitochondria. In the present study, we generated a stable TSPO knockdown (TSPO KD) in the Huh7 cell line. To mimic hepatic steatosis in vitro, we treated WT and TSPO KD cells with either acetylated LDL (AcLDL), to deliver cholesterol into the cells, or AcLDL plus 58035, an Acetyl-CoA Acetyltransferase (ACAT) and thus cholesterol esterification inhibitor, to induce the accumulation of FC. AcLDL treatment significantly increased FC levels in WT cells; this effect was more pronounced in TSPO KD cells. In contrast, treatment of the cells with AcLDL and 58035 resulted in reduced FC levels likely due to the damage induced by FC accumulation that led to reduced cell proliferation. Considering that TSPO binds FC with high affinity, we investigated the underlying mechanism/link between TSPO KD and FC accumulation by examining the conversion between FC and cholesterol ester (CE) upon AcLDL treatment. The results obtained showed that knocking down TSPO expression reduced ACAT2 (transforming FC to CE) levels, but not cholesterol ester hydrolase (CEH) converting CE to FC, expression. TSPO was found to directly interact with ACAT2. Under these conditions, the accumulation of FC led to the appearance of lipid droplets (LD) of increased size in TSPO KD compared to WT cells, suggesting that TSPO plays a role in LD formation and/or dynamics in liver cells. GRP78, IRE1A, and PERK endoplasmic reticulum stress markers were also elevated in TSPO KD cells following AcLDL treatment while the mitochondrial membrane potential decreased compared to WT cells. Moreover, increased numbers of lysosomes and autophagosomes were observed in TSPO KD cells, an effect further potentiated by the treatment with AcLDL plus 58035. This finding was validated by the up regulation of the LAMP1 and LC3B, lysosome and autophagic structure markers, respectively. Taken together, these data suggest that under cholesterol treatment, TSPO loss would trigger ER stress, mitochondrial dysfunction, and lipid droplet accumulation via FC accumulation, and ultimately cause intracellular protein and organelle degradation, hallmarks of hepatic steatosis.

Targeting EphA4 abrogates intrinsic resistance to chemotherapy in well-differentiated cervical cancer cell line

Alkylating reagent chemotherapy for human cancers is not curative, and relapse occurs due to the continued presence of tumor cells, referred to as minimal residual disease (MRD). The survival of MRD cells after chemotherapy, a phenomenon referred to as intrinsic resistance, depends on reactive oxygen species. Well-differentiated regions of the tumor are intrinsically resistant to chemotherapy. Receptor tyrosine kinase erythropoietin-producing human hepatocellular receptor A4 (EphA4) protein is highly expressed in the well-differentiated tumor-derived cervical cancer cell line Caski, but not in poorly differentiated tumor-derived cervical cancer cell lines such as HeLa or SiHa. Here, we report that reactive oxygen species produced by cisplatin exposure induce tyrosine phosphorylation of EphA4. After observing that EphA4 is activated by cisplatin, we rationalized a combination chemotherapy that induces well-differentiated cervical cancer death. Pharmacological inhibition of EphA4 increased cisplatin-induced cell death in Caski cells. Moreover, we observed increased expression levels of the senescence marker cyclin-dependent kinase inhibitor 2A (p16) in the absence of EphA4 kinase function after stimulation of Caski cells with cisplatin exposure. Mechanistically, cisplatin induces chemotherapy resistance of Caski cells by upregulating Lyn, a Src family kinase (SFK) that interacts with EphA4, through a pathway involving reactive oxygen species. Thus, the reactive oxygen species–SFK–EphA4 axis presents new potential drug targets for chemotherapy resistance.

Plexin C1 Marks Liver Cancer Cells with Epithelial Phenotype and Is Overexpressed in Hepatocellular Carcinoma.

Background and Aims Hepatocellular carcinoma is an aggressive malignancy of the liver and is ranked as the sixth most common cancer worldwide. There is still room for novel markers to improve the diagnosis and monitoring of HCC. Our observations in cancer databases that PLXNC1 is upregulated in HCC led us to investigate the expression profile of Plexin C1 mRNA and protein in HCC cell lines and tissues. Methods A recombinant protein encompassing part of the extracellular domain of Plexin C1 was used as an antigen for monoclonal antibody development. Transcript and protein levels of Plexin C1 in HCC cell lines were determined by RT-qPCR and Western blotting, respectively. In vivo evaluation of Plexin C1 expression in HCC tissues was accomplished by immunohistochemistry studies in tissue microarrays. Results A monoclonal antibody, clone PE4, specific to Plexin C1, was generated. In silico and in vitro analyses revealed a Plexin C1-based clustering of well-differentiated HCC cell lines. Staining of HCC and nontumoral liver tissues with PE4 showed a membrane-localized overexpression of Plexin C1 in tumors (p=0.0118). In addition, this expression was correlated with the histological grades of HCC cases. Conclusions Plexin C1 distinguishes HCC cells of epithelial characteristics from those with the mesenchymal phenotype. Compared to the nontumoral liver, HCC tissues significantly overexpress Plexin C1. The newly generated PE4 antibody can be evaluated in larger HCC cohorts and might be exploited for the examination of Plexin C1 expression pattern in other epithelial malignancies.

Liver cancer cell lines distinctly mimic the metabolic gene expression pattern of the corresponding human tumours

BackgroundAlthough metabolism is profoundly altered in human liver cancer, the extent to which experimental models, e.g. cell lines, mimic those alterations is unresolved. Here, we aimed to determine the resemblance of hepatocellular carcinoma (HCC) cell lines to human liver tumours, specifically in the expression of deregulated metabolic targets in clinical tissue samples.MethodsWe compared the overall gene expression profile of poorly-differentiated (HLE, HLF, SNU-449) to well-differentiated (HUH7, HEPG2, HEP3B) HCC cell lines in three publicly available microarray datasets. Three thousand and eighty-five differentially expressed genes in ≥2 datasets (P < 0.05) were used for pathway enrichment and gene ontology (GO) analyses. Further, we compared the topmost gene expression, pathways, and GO from poorly differentiated cell lines to the pattern from four human HCC datasets (623 tumour tissues). In well- versus poorly differentiated cell lines, and in representative models HLE and HUH7 cells, we specifically assessed the expression pattern of 634 consistently deregulated metabolic genes in human HCC. These data were complemented by quantitative PCR, proteomics, metabolomics and assessment of response to thirteen metabolism-targeting compounds in HLE versus HUH7 cells.ResultsWe found that poorly-differentiated HCC cells display upregulated MAPK/RAS/NFkB signaling, focal adhesion, and downregulated complement/coagulation cascade, PPAR-signaling, among pathway alterations seen in clinical tumour datasets. In HLE cells, 148 downregulated metabolic genes in liver tumours also showed low gene/protein expression – notably in fatty acid β-oxidation (e.g. ACAA1/2, ACADSB, HADH), urea cycle (e.g. CPS1, ARG1, ASL), molecule transport (e.g. SLC2A2, SLC7A1, SLC25A15/20), and amino acid metabolism (e.g. PHGDH, PSAT1, GOT1, GLUD1). In contrast, HUH7 cells showed a higher expression of 98 metabolic targets upregulated in tumours (e.g. HK2, PKM, PSPH, GLUL, ASNS, and fatty acid synthesis enzymes ACLY, FASN). Metabolomics revealed that the genomic portrait of HLE cells co-exist with profound reliance on glutamine to fuel tricarboxylic acid cycle, whereas HUH7 cells use both glucose and glutamine. Targeting glutamine pathway selectively suppressed the proliferation of HLE cells.ConclusionsWe report a yet unappreciated distinct expression pattern of clinically-relevant metabolic genes in HCC cell lines, which could enable the identification and therapeutic targeting of metabolic vulnerabilities at various liver cancer stages.

Method for Biomonitoring DNA Adducts in Exfoliated Urinary Cells by Mass Spectrometry.

Tobacco smoking contributes to about 50% of the bladder-cancer (BC) cases in the United States. Some aromatic amines in tobacco smoke are bladder carcinogens; however, other causal agents of BC are uncertain. Exfoliated urinary cells (EUCs) are a promising noninvasive biospecimen to screen for DNA adducts of chemicals that damage the bladder genome, although the analysis of DNA adducts in EUCs is technically challenging because of the low number of EUCs and limiting quantity of cellular DNA. Moreover, EUCs and their DNA adducts must remain viable during the time of collection and storage of urine to develop robust screening methods. We employed RT4 cells, a well-differentiated transitional epithelial bladder cell line, as a cell-model system in urine to investigate cell viability and the chemical stability of DNA adducts of two prototypical bladder carcinogens: 4-aminobiphenyl (4-ABP), an aromatic amine found in tobacco smoke, and aristolochic acid I (AA-I), a nitrophenanthrene found in Aristolochia herbaceous plants used for medicinal purposes worldwide. The cell viability of RT4 cells pretreated with 4-ABP or AA-I in urine exceeded 80%, and the major DNA adducts of 4-ABP and AA-I, quantified by liquid chromatography-mass spectrometry, were stable for 24 h. Thereafter, we successfully screened EUCs of mice treated with AA-I to measure DNA adducts of AA-I, which were still detected 25 days following treatment with the carcinogen. EUCs are promising biospecimens that can be employed for the screening of DNA adducts of environmental and dietary genotoxicants that may contribute to the development of BC.