ATP-Binding Pocket-Targeted Suppression of Src and Syk by Luteolin Contributes to Its Anti-Inflammatory Action

Luteolin is a flavonoid identifiedas a major anti-inflammatory componentofArtemisia asiatica. Numerous reports have demonstrated the ability of luteolin to suppress inflammation in a variety of inflammatory conditions. However, its exact anti-inflammatory mechanism has not been fully elucidated. In the present study, the anti-inflammatory mode of action in activated macrophages of luteolin fromArtemisia asiaticawas examined by employing immunoblotting analysis, a luciferase reporter gene assay, enzyme assays, and an overexpression strategy. Luteolin dose-dependently inhibited the secretion of nitric oxide (NO) and prostaglandin E2(PGE2) and diminished the levels of mRNA transcripts of inducible NO synthase (iNOS), tumor necrosis factor- (TNF-)α, and cyclooxygenase-2 (COX-2) in lipopolysaccharide- (LPS-) and pam3CSK-treated macrophage-like RAW264.7 cells without displaying cytotoxicity. Luteolin displayed potent NO-inhibitory activity and also suppressed the nuclear translocation of NF-κB (p65 and p50) via blockade of Src and Syk, but not other mitogen-activated kinases. Overexpression of wild type Src and point mutants thereof, and molecular modelling studies, suggest that the ATP-binding pocket may be the luteolin-binding site in Src. These results strongly suggest that luteolin may exert its anti-inflammatory action by suppressing the NF-κB signaling cascade via blockade of ATP binding in Src and Syk.

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Albuminuria induces a proinflammatory and profibrotic response in cortical collecting ducts via the 24p3 receptor

AJP Renal Physiology ◽

10.1152/ajprenal.00006.2013 ◽

2013 ◽

Vol 305 (7) ◽

pp. F1053-F1063 ◽

Cited By ~ 30

Author(s):

Eva Dizin ◽

Udo Hasler ◽

Stellor Nlandu-Khodo ◽

Marc Fila ◽

Isabelle Roth ◽

...

Keyword(s):

Transforming Growth Factor ◽

Nuclear Translocation ◽

Collecting Duct ◽

Transforming Growth Factor Β ◽

Luciferase Reporter ◽

Lipocalin 2 ◽

Luciferase Reporter Gene ◽

Puromycin Aminonucleoside ◽

Gene Assay ◽

Albumin Endocytosis

Albuminuria is strongly associated with progressive kidney tubulo-interstitial damage and chronic kidney disease (CKD) progression. In proteinuric nephropathies, albumin reabsorption by the proximal tubule is saturated and the distal nephron is exposed to high concentrations of luminal albumin that may produce adverse effects. Since proximal tubular cells exposed to albuminuria exhibit a proinflammatory and profibrotic response, we assessed the effect of albuminuria in the collecting duct (CD). With the use of kidney sections and isolated cortical CDs (CCDs) from puromycin-aminonucleoside-induced nephrotic rats (PAN rats) exhibiting proteinuria, immunofluorescence microscopy revealed internalized albumin in CD cells. In these proteinuric rats, increased expression levels of cytokines and profibrotic signaling markers were detected in isolated CCDs and bands of inflammatory fibrosis could be observed around CDs. Albumin endocytosis was confirmed by FITC-albumin uptake in cultured murine CCD (mCCDcl1) cells. Exposure of mCCDcl1 cells to albumin induced NF-κB activation as assessed by luciferase reporter gene assay, nuclear translocation of NF-κB p65 subunit, and increased NF-κB target gene expression. Moreover, albuminuria-like condition results in transforming growth factor-β1 (TGF-β1) overexpression and the upregulation of profibrotic signaling markers such as Snail or vimentin via an autocrine mechanism. In mCCDcl1 cells, neutrophil gelatinase-associated lipocalin (NGAL)/lipocalin-2/24p3 receptor (24p3R) mediates albumin endocytosis as well as activation of NF-κB and TGF-β1 signaling pathways. Therefore, CD may play a key role in initiation and/or progression of inflammation and fibrosis in response to proteinuria.

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AP-1/IRF-3 Targeted Anti-Inflammatory Activity of Andrographolide Isolated fromAndrographis paniculata

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2013/210736 ◽

2013 ◽

Vol 2013 ◽

pp. 1-16 ◽

Cited By ~ 29

Author(s):

Ting Shen ◽

Woo Seok Yang ◽

Young-Su Yi ◽

Gi-Ho Sung ◽

Man Hee Rhee ◽

...

Keyword(s):

Molecular Mechanisms ◽

Inflammatory Responses ◽

Peritoneal Macrophages ◽

Luciferase Reporter ◽

Prostaglandin E ◽

Dependent Manner ◽

Molecular Signaling ◽

Luciferase Reporter Gene ◽

Necrosis Factor Alpha ◽

Anti Inflammatory

Andrographolide (AG) is an abundant component of plants of the genusAndrographisand has a number of beneficial properties including neuroprotective, anticancer, anti-inflammatory, and antidiabetic effects. Despite numerous pharmacological studies, the precise mechanism of AG is still ambiguous. Thus, in the present study, we investigated the molecular mechanisms of AG and its target proteins as they pertain to anti-inflammatory responses. AG suppressed the production of nitric oxide (NO) and prostaglandin E2(PGE2), as well as the mRNA abundance of inducible NO synthase (iNOS), tumor necrosis factor-alpha (TNF-α), cyclooxygenase (COX)-2, and interferon-beta (IFN-β) in a dose-dependent manner in both lipopolysaccharide- (LPS-) activated RAW264.7 cells and peritoneal macrophages. AG also substantially ameliorated the symptoms of LPS-induced hepatitis and EtOH/HCl-induced gastritis in mice. Based on the results of luciferase reporter gene assays, kinase assays, and measurement of nuclear levels of transcription factors, the anti-inflammatory effects of AG were found to be clearly mediated by inhibition of both (1) extracellular signal-regulated kinase (ERK)/activator protein (AP)-1 and (2) IκB kinaseε(IKKε)/interferon regulatory factor (IRF)-3 pathways. In conclusion, we detected a novel molecular signaling pathway by which AG can suppress inflammatory responses. Thus, AG is a promising anti-inflammatory drug with two pharmacological targets.

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Comparison of Variability and Sensitivity between Nuclear Translocation and Luciferase Reporter Gene Assays

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057108328016 ◽

2008 ◽

Vol 14 (1) ◽

pp. 59-65 ◽

Cited By ~ 4

Author(s):

Vincent Unterreiner ◽

Yvonne Ibig-Rehm ◽

Marjo Simonen ◽

Hanspeter Gubler ◽

Daniela Gabriel

Keyword(s):

Reporter Gene ◽

Fluorescent Protein ◽

Nuclear Translocation ◽

Mean Shift ◽

Luciferase Reporter ◽

Luciferase Reporter Gene ◽

Forkhead Transcription Factor ◽

Primary Screening ◽

Gene Assay ◽

Subcellular Imaging

High-content screening (HCS), a technology based on subcellular imaging by automated microscopy and sophisticated image analysis, has emerged as an important platform in small-molecule screening for early drug discovery. To validate a subcellular imaging assay for primary screening campaigns, an HCS assay was compared with a non—image-based readout in terms of variability and sensitivity. A study was performed monitoring the accumulation of the forkhead transcription factor of the O subfamily (FOXO3a) coupled with green fluorescent protein in the nucleus of human osteosarcoma (U-2 OS) cells. In addition, the transcription of a luciferase gene coupled with a FOXO3a-responsive promoter was monitored. This report demonstrates that both assay formats show good reproducibility in primary and concentration response screening despite differences in statistical assay quality. In primary screening, the correlation of compound activity between the 2 assays was low, in contrast to the good correlation of the IC50 values of confirmed compounds. Furthermore, the high-content imaging assay showed a mean shift of 2.63-fold in IC50 values compared with the reporter gene assay. No chemical scaffold was specifically found with 1 of the technologies only, however these results validate the HCS technology against established assays for screening of new molecular entities. ( Journal of Biomolecular Screening 2009:59-65)

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Phosphatidylinositide 3-Kinase Contributes to the Anti-Inflammatory Effect of Abutilon crispum L. Medik Methanol Extract

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2018/1935902 ◽

2018 ◽

Vol 2018 ◽

pp. 1-10 ◽

Cited By ~ 5

Author(s):

Stephanie Triseptya Hunto ◽

Kon Kuk Shin ◽

Han Gyung Kim ◽

Sang Hee Park ◽

Junsang Oh ◽

...

Keyword(s):

Nitric Oxide ◽

Methanol Extract ◽

Luciferase Reporter ◽

Luciferase Reporter Gene ◽

Promoter Regions ◽

Luciferase Reporter Gene Assay ◽

Traditional Remedy ◽

Gene Assay ◽

Anti Inflammatory ◽

Inflammatory Effect

Abutilon crispum L. Medik, better known as bladdermallow, is used as a traditional remedy in India, for its anti-inflammatory effect due to its high content of flavonoids. However, research about its anti-inflammatory effect at the molecular level has not been performed. In this study, we aimed to investigate the mechanism of Abutilon crispum methanol extract (Ac-ME) in inhibiting the inflammatory response by conducting several experiments including cellular and molecular assays. Ac-ME inhibited the production of nitric oxide (NO) in RAW264.7 cells during treatment of LPS and Pam3CSK4 without exhibiting cytotoxicity. Ac-ME also suppressed the mRNA expression of inducible nitric oxide (iNOS) and proinflammatory cytokines such as interleukin (IL)-1β and IL-6. Moreover, Ac-ME was shown to inhibit the NF-κB pathway, according to the luciferase reporter gene assay performed with a NF-κB-Luc construct containing NF-κB-binding promoter regions under MyD88 and TRIF overexpression conditions, and immunoblotting analysis by determining the phospho-form levels of IκBα, IKKα/β, and p85, a regulatory domain of phosphatidylinositide 3-kinase (PI3K). Finally, we observed that the level of phospho-p85 induced by the overexpression of spleen tyrosine kinase (Syk) and Src was decreased by Ac-ME at 200 μg/ml. Therefore, these results suggest that Ac-ME has an anti-inflammatory effect by targeting PI3K in the NF-κB signaling pathway.

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IRAK1/4-Targeted Anti-Inflammatory Action of Caffeic Acid

Mediators of Inflammation ◽

10.1155/2013/518183 ◽

2013 ◽

Vol 2013 ◽

pp. 1-12 ◽

Cited By ~ 23

Author(s):

Woo Seok Yang ◽

Deok Jeong ◽

Young-Su Yi ◽

Jae Gwang Park ◽

Hyohyun Seo ◽

...

Keyword(s):

Caffeic Acid ◽

Nuclear Translocation ◽

Biological Activities ◽

Kinase Assay ◽

Raw264.7 Cells ◽

Prostaglandin E ◽

Mrna Levels ◽

Action Mechanism ◽

Anti Inflammatory ◽

Inflammatory Action

Caffeic acid (CA) is a phenolic compound that is frequently present in fruits, grains, and dietary supplements. Although CA has been reported to display various biological activities such as anti-inflammatory, anti-cancer, anti-viral, and anti-oxidative effects, the action mechanism of CA is not yet fully elucidated. In this study, the anti-inflammatory action mechanism of CA was examined in lipopolysaccharide (LPS) treated macrophages (RAW264.7 cells) and HCl/EtOH-induced gastritis. CA was found to diminish nitric oxide (NO) and prostaglandin E2(PGE2) production in LPS-stimulated RAW264.7 cells. Additionally, mRNA levels of tumor necrosis factor (TNF)-α, cyclooxygenase (COX)-2, and inducible NO synthase (iNOS) were downregulated by CA. CA also strongly suppressed the nuclear translocation of AP-1 family proteins and the related upstream signaling cascade composed of interleukin-1 receptor-associated kinase 1 (IRAK1), IRAK4, TGF-β-activated kinase 1 (TAK1), mitogen-activated protein kinase kinase 4/7 (MKK4/7), and c-Jun N-terminal kinase (JNK). In a direct kinase assay, CA was revealed to directly inhibit IRAK1 and IRAK4. CA also ameliorated HCl/EtOH-induced gastric symptoms via the suppression of JNK, IRAK1, and IRAK4. Therefore, our data strongly suggest that CA acts as an anti-inflammatory drug by directly suppressing IRAK1 and IRAK4.

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Anti-Inflammatory Effects of Licania macrocarpa Cuatrec Methanol Extract Target Src- and TAK1-Mediated Pathways

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2019/4873870 ◽

2019 ◽

Vol 2019 ◽

pp. 1-13 ◽

Cited By ~ 1

Author(s):

Kon Kuk Shin ◽

Jae Gwang Park ◽

Yo Han Hong ◽

Nur Aziz ◽

Sang Hee Park ◽

...

Keyword(s):

Mrna Expression ◽

Methanol Extract ◽

Tracking System ◽

Signal Pathway ◽

Luciferase Reporter ◽

No Production ◽

Luciferase Reporter Gene ◽

Gene Assay ◽

Cox 2 ◽

Anti Inflammatory

It is very important for the corresponding author to have a linked ORCID (Open Researcher and Contributor ID) account on MTS. To register a linked ORCID account, please go to the Account Update page (http://mts.hindawi.com/update/) in our Manuscript Tracking System and after you have logged in click on the ORCID link at the top of the page. This link will take you to the ORCID website where you will be able to create an account for yourself. Once you have done so, your new ORCID will be saved in our Manuscript Tracking System automatically."?>In this study, we investigated the anti-inflammatory effects of Licania macrocarpa Cuatrec methanol extract (Lm-ME) in vitro and in vivo and found pharmacological target proteins of Lm-ME in TLR4-mediated inflammatory signaling. This extract reduced NO production and mRNA expression of inflammatory cytokines such as iNOS, COX-2, IL-6, and IL-1β. In the NF-κB- and AP-1-mediated luciferase reporter gene assay, transcription factor activities decreased under cotransfection with MyD88 or TRIF. Phosphorylated protein levels of Src, PI3K, IKKα/β, and IκBα as well as p50 and p65 in the NF-κB signal pathway were downregulated, and phosphorylation of TAK1, MEK1/2, MKK4/7, and MKK3/6 as well as ERK, JNK, and p38 was decreased in the AP-1 signal pathway. Through overexpression of HA-Src and HA-TAK1, respectively, Lm-ME inhibited autophosphorylation of overexpressed proteins and thereby activated fewer downstream signaling molecules. Lm-ME also attenuated stomach ulcers in an HCl/EtOH-induced acute gastritis model mice, and COX-2 mRNA expression and phosphorylated TAK1 levels in gastric tissues were diminished. The flavonoids kaempferol and quercetin were identified in the HPLC analysis of Lm-ME; both are actively anti-inflammatory. Therefore, these results suggest that Lm-ME can be used for anti-inflammatory remedy by targeting Src and TAK1.

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BRUCE silencing leads to axonal dystrophy by repressing autophagosome-lysosome fusion in Alzheimer’s disease

Translational Psychiatry ◽

10.1038/s41398-021-01427-2 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Lu Zhang ◽

Yu Fang ◽

Xinyu Zhao ◽

Yake Zheng ◽

Yunqing Ma ◽

...

Keyword(s):

Alzheimer’S Disease ◽

Alzheimer's Disease ◽

Nuclear Translocation ◽

Nicotinamide Adenine Dinucleotide Phosphate ◽

Axon Growth ◽

Luciferase Reporter ◽

Axonal Dystrophy ◽

Luciferase Reporter Gene ◽

Ht22 Cells ◽

Gene Assay

AbstractAxonal dystrophy is a swollen and tortuous neuronal process that contributes to synaptic alterations occurring in Alzheimer’s disease (AD). Previous study identified that brain-derived neurotrophic factor (BDNF) binds to tropomyosin-related kinase B (TrkB) at the axon terminal and then the signal is propagated along the axon to the cell body and affects neuronal function through retrograde transport. Therefore, this study was designed to identify a microRNA (miRNA) that alters related components of the transport machinery to affect BDNF retrograde signaling deficits in AD. Hippocampus tissues were isolated from APP/PS1 transgenic (AD-model) mice and C57BL/6J wild-type mice and subject to nicotinamide adenine dinucleotide phosphate and immunohistochemical staining. Autophagosome-lysosome fusion and nuclear translocation of BDNF was detected using immunofluorescence in HT22 cells. The interaction among miR-204, BIR repeat containing ubiquitin-conjugating enzyme (BRUCE) and Syntaxin 17 (STX17) was investigated using dual luciferase reporter gene assay and co-immunoprecipitation assay. The expression of relevant genes and proteins were determined by RT-qPCR and Western blot analysis. Knockdown of STX17 or BRUCE inhibited autophagosome–lysosome fusion and impacted axon growth in HT22 cells. STX17 immunoprecipitating with BRUCE and co-localization of them demonstrated BRUCE interacted with STX17. BRUCE was the target of miR-204, and partial loss of miR-204 by inhibitor promoted autophagosome–lysosome fusion to prevent axon dystrophy and accumulated BDNF nuclear translocation to rescue BDNF/TrkB signaling deficits in HT22 cells. The overall results demonstrated that inhibition of miR-204 prevents axonal dystrophy by blocking BRUCE interaction with STX17, which unraveled potential novel therapeutic targets for delaying AD.

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Benzisothiazolone Derivatives Exhibit Cytotoxicity in Hodgkin’s Lymphoma Cells through NF-κB Inhibition and are Synergistic with Doxorubicin and Etoposide

Anti-Cancer Agents in Medicinal Chemistry ◽

10.2174/1871520620666200213103513 ◽

2020 ◽

Vol 20 (6) ◽

pp. 715-723

Author(s):

Natarajan Nandakumar ◽

Pushparathinam Gopinath ◽

Jacob Gopas ◽

Kannoth M. Muraleedharan

Keyword(s):

Synergistic Effect ◽

Hodgkin’S Lymphoma ◽

A549 Cells ◽

Hodgkin's Lymphoma ◽

Luciferase Reporter ◽

Luciferase Reporter Gene ◽

Lymphoma Cells ◽

Nuclear Extracts ◽

Gene Assay

Background: The authors investigated the NF-κB inhibitory role of three Benzisothiazolone (BIT) derivatives (1, 2 and 3) in Hodgkin’s Lymphoma cells (L428) which constitutively express activated NF-κB. All three compounds showed dose-dependent NF-κB inhibition (78.3, 70.7 and 34.6%) in the luciferase reporter gene assay and were found cytotoxic at IC50 values of 3.3μg/ml, 4.35μg/ml and 13.8μg/ml, respectively by the XTT assay. BIT 1and BIT 2 (but not BIT 3) suppressed both NF-κB subunits p50 and p65 in cytoplasmic and nuclear extracts in a concentration-dependent manner. Furthermore, BIT 1 showed a moderate synergistic effect with the standard chemotherapy drugs etoposide and doxorubicin, whereas BIT 2 and 3 showed a moderate additive effect to antagonistic effect. Cisplatin exhibited an antagonist effect on all the compounds tested under various concentrations, except in the case of 1.56μg/ml of BIT 3 with 0.156μg/ml of cisplatin. The compounds also inhibited the migration of adherent human lung adenocarcinoma cells (A549) in vitro. We conclude that especially BIT 1 and BIT 2 have in vitro anti-inflammatory and anti-cancer activities, which can be further investigated for future potential therapeutic use. Methods: Inspired by the electrophilic sulfur in Nuphar alkaloids, monomeric and dimeric benzisothiazolones were synthesized from dithiodibenzoic acid and their NF-κB inhibitory role was explored. NF-κB inhibition and cytotoxicity of the synthesized derivatives were studied using luciferase reporter gene assay and XTTassay. Immunocytochemistry studies were performed using L428 cells. Cell migration assay was conducted using the A549 cell line. L428 cells were used to conduct combination studies and the results were plotted using CompuSyn software. Results: Benzisothiazolone derivatives exhibited cytotoxicity in Hodgkin’s Lymphoma cells through NF-κB inhibition. Potent compounds showed suppression of both NF-κB subunits p50 and p65 in a concentrationdependent manner, both in cytoplasmic and nuclear extracts. Combination studies suggest that benzisothiazolone derivatives possess a synergistic effect with etoposide and doxorubicin. Furthermore, the compounds also inhibited the migration of A549 cells. Conclusion: Benzisothiazolones bearing one or two electrophilic sulfur atoms as part of the heterocyclic framework exhibited cytotoxicity in Hodgkin’s Lymphoma cells through NF-κB inhibition. In addition, these derivatives also exhibited a synergistic effect with etoposide and doxorubicin along with the ability to inhibit the migration of A549 cells. Our study suggests that BIT-based new chemical entities could lead to potential anticancer agents.

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Sevoflurane inhibits proliferation, invasion, but enhances apoptosis of lung cancer cells by Wnt/β-catenin signaling via regulating lncRNA PCAT6/ miR-326 axis

Open Life Sciences ◽

10.1515/biol-2020-0017 ◽

2020 ◽

Vol 15 (1) ◽

pp. 159-172

Author(s):

Guoning Su ◽

Zhibing Yan ◽

Min Deng

Keyword(s):

Lung Cancer ◽

Cancer Cells ◽

Volatile Anesthetic ◽

Lung Cancer Cells ◽

Luciferase Reporter ◽

Dependent Manner ◽

Luciferase Reporter Gene ◽

Western Blot Assay ◽

Gene Assay ◽

Cancer Tissues

AbstractSevoflurane was frequently used as a volatile anesthetic in cancer surgery. However, the potential mechanism of sevoflurane on lung cancer remains largely unclear. In this study, lung cancer cell lines (H446 and H1975) were treated by various concentrations of sevoflurane. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assessment and colony formation assay were performed to detect the cell viability and proliferation, separately. Also, transwell assay or flow cytometry assay was applied as well to evaluate the invasive ability or apoptosis in lung cancer cells, respectively. Western blot assay was employed to detect the protein levels of β-catenin and Wnt5a. Moreover, quantitative real-time polymerase chain reaction (qRT-PCR) was used to examine the expression level of prostate cancer-associated transcript 6 (PCAT6) and miR-326 in lung cancer tissues and cells. The target interaction between miR-326 and PCAT6 or Wnt5a was predicted by bioinformatics analysis and verified by the dual-luciferase reporter gene assay. Sevoflurane inhibited the abilities on viability, proliferation, invasion, and activation of Wnt/β-catenin signaling, but promoted apoptosis of H446 and H1975 cells in a dose-dependent manner. The expression of PCAT6 was increased in lung cancer tissues and cells, except for that of miR-326. Besides, sevoflurane could lead to expressed limitation of PCAT6 or improvement of miR-326. This process presented a stepwise manner. Up-regulation of PCAT6 restored the suppression of sevoflurane on abilities of proliferation, invasion, rather than apoptosis, and re-activated the Wnt5a/β-catenin signaling in cells. Moreover, the putative binding sites between miR-326 and PCTA6 or Wnt5a were predicted by starBase v2.0 software online. PCAT6 suppressing effects on cells could be reversed by pre-treatment with miR-326 vector. The promotion of Wnt5a inverted effects led from miR-326 or sevoflurane. Our study indicated that sevoflurane inhibited the proliferation, and invasion, but enhanced the apoptosis in lung cancer cells by regulating the lncRNA PCAT6/miR-326/Wnt5a/β-catenin axis.

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Impact of RARα and miR-138 on retinoblastoma etoposide resistance

Tumor Biology ◽

10.3233/tub-200072 ◽

2021 ◽

Vol 43 (1) ◽

pp. 11-26

Author(s):

Maike Busch ◽

Natalia Miroschnikov ◽

Jaroslaw Thomas Dankert ◽

Marc Wiesehöfer ◽

Klaus Metz ◽

...

Keyword(s):

Cell Viability ◽

Cell Lines ◽

Cancer Progression ◽

Treated Patient ◽

Luciferase Reporter ◽

Luciferase Reporter Gene ◽

Gene Assay ◽

The Impact

BACKGROUND: Retinoblastoma (RB) is the most common childhood eye cancer. Chemotherapeutic drugs such as etoposide used in RB treatment often cause massive side effects and acquired drug resistances. Dysregulated genes and miRNAs have a large impact on cancer progression and development of chemotherapy resistances. OBJECTIVE: This study was designed to investigate the involvement of retinoic acid receptor alpha (RARα) in RB progression and chemoresistance as well as the impact of miR-138, a potential RARα regulating miRNA. METHODS: RARα and miR-138 expression in etoposide resistant RB cell lines and chemotherapy treated patient tumors compared to non-treated tumors was revealed by Real-Time PCR. Overexpression approaches were performed to analyze the effects of RARα on RB cell viability, apoptosis, proliferation and tumorigenesis. Besides, we addressed the effect of miR-138 overexpression on RB cell chemotherapy resistance. RESULTS: A binding between miR-138 and RARα was shown by dual luciferase reporter gene assay. The study presented revealed that RARα is downregulated in etoposide resistant RB cells, while miR-138 is endogenously upregulated. Opposing RARα and miR-138 expression levels were detectable in chemotherapy pre-treated compared to non-treated RB tumor specimen. Overexpression of RARα increases apoptosis levels and reduces tumor cell growth of aggressive etoposide resistant RB cells in vitro and in vivo. Overexpression of miR-138 in chemo-sensitive RB cell lines partly enhances cell viability after etoposide treatment. CONCLUSIONS: Our findings show that RARα acts as a tumor suppressor in retinoblastoma and is downregulated upon etoposide resistance in RB cells. Thus, RARα may contribute to the development and progression of RB chemo-resistance.

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