Apigetrin inhibits cytotoxicity and dysregulation of ACE2, IL1α and TGFβ expression induced by recombinant spike protein of SARS-CoV-2

SARS-CoV-2 virus is a novel coronavirus that was first identified in Wuhan, China in December 2019 and has caused an ongoing global health crisis. It has been a worldwide focus in research to understand viral pathogenesis and discover effective therapies. SARS-CoV-2 belongs to the same genus as the viruses responsible for Severe Acute Respiratory Syndrome (SARS) and the Middle East Respiratory Syndrome (MERS). Spike protein (SP) on SARS-CoV-2 plays a key role in the pathogenesis of SARS-CoV-2. The virus enters human cells via the binding of SP to the angiotensin-converting enzyme 2 (ACE2) on human cells. The binding of SP inhibits ACE2 function by reducing formation of angiotensin-(1-7), a compound that has inhibitory effects on inflammation. In addition, SARS-CoV-2 induces excessive proinflammatory cytokine production through various other signaling pathways such as the NFκB and NLRP3 inflammasome pathways. Previous evidence showed that apigenin (APG), a plant phenolic compound, can bind to SP. However, whether or not apigetrin (APT), the glucoside conjugate of APG, can protect human cells against cell injury caused by SARS-CoV-2 is still unknown. Studies have shown that SARS-CoV-2 induced dysregulation of host cell ACE2 expression is one of the major pathophysiological factors of COVID-19 infection. Our current study demonstrated that recombinant SP significantly reduced ACE2 level in human neuronal cells in a dose dependent manner using ELISA assay. Interestingly, APT reversed the SP induced ACE2 downregulation in these cells. In this study, the effect of SP on cell proliferation and immune regulation was also investigated. Using MTT and LDH assays, I discovered that SP had a cytotoxic effect on these cells and significantly inhibited cell proliferation. This cytotoxic effect was mitigated by adding APT treatment. Furthermore, APT reduced SP induced cytokine production such as IL1α and TGFβ . In sum, my study demonstrated that APT inhibited SARS-CoV-2 SP induced dysregulation of human cells and reduced its cytotoxic effects on cells. APT significantly upregulated ACE2 expression and inhibited the production of cytokines IL1α and TGFβ in the cells treated with SP. My study indicated that APT has potential to be a novel therapy for COVID-19 infection. More experiments to further elucidate molecular mechanisms of how APT modulates pathological effect of SP with different in vitro models including other human cell lines and in vivo animal models are currently being performed.

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Norepinephrine induces calcium spikes and proinflammatory actions in human hepatic stellate cells

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00537.2005 ◽

2006 ◽

Vol 291 (5) ◽

pp. G877-G884 ◽

Cited By ~ 38

Author(s):

Pau Sancho-Bru ◽

Ramón Bataller ◽

Jordi Colmenero ◽

Xavier Gasull ◽

Montserrat Moreno ◽

...

Keyword(s):

Cell Proliferation ◽

Portal Hypertension ◽

Liver Fibrosis ◽

Hepatic Stellate Cells ◽

Molecular Mechanisms ◽

Nuclear Translocation ◽

Stellate Cells ◽

Luciferase Reporter ◽

Dependent Manner ◽

Cell Contraction

Catecholamines participate in the pathogenesis of portal hypertension and liver fibrosis through α1-adrenoceptors. However, the underlying cellular and molecular mechanisms are largely unknown. Here, we investigated the effects of norepinephrine (NE) on human hepatic stellate cells (HSC), which exert vasoactive, inflammatory, and fibrogenic actions in the injured liver. Adrenoceptor expression was assessed in human HSC by RT-PCR and immunocytochemistry. Intracellular Ca2+ concentration ([Ca2+]i) was studied in fura-2-loaded cells. Cell contraction was studied by assessing wrinkle formation and myosin light chain II (MLC II) phosphorylation. Cell proliferation and collagen-α1(I) expression were assessed by [3H]thymidine incorporation and quantitative PCR, respectively. NF-κB activation was assessed by luciferase reporter gene and p65 nuclear translocation. Chemokine secretion was assessed by ELISA. Normal human livers expressed α1A-adrenoceptors, which were markedly upregulated in livers with advanced fibrosis. Activated human HSC expressed α1A-adrenoceptors. NE induced multiple rapid [Ca2+]i oscillations (Ca2+ spikes). Prazosin (α1-blocker) completely prevented NE-induced Ca2+ spikes, whereas propranolol (nonspecific β-blocker) partially attenuated this effect. NE caused phosphorylation of MLC II and cell contraction. In contrast, NE did not affect cell proliferation or collagen-α1(I) expression. Importantly, NE stimulated the secretion of inflammatory chemokines (RANTES and interleukin-8) in a dose-dependent manner. Prazosin blocked NE-induced chemokine secretion. NE stimulated NF-κB activation. BAY 11-7082, a specific NF-κB inhibitor, blocked NE-induced chemokine secretion. We conclude that NE stimulates NF-κB and induces cell contraction and proinflammatory effects in human HSC. Catecholamines may participate in the pathogenesis of portal hypertension and liver fibrosis by targeting HSC.

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Screening of FDA-Approved Drug Library Identifies Adefovir Dipivoxil as Highly Potent Inhibitor of T Cell Proliferation

Frontiers in Immunology ◽

10.3389/fimmu.2020.616570 ◽

2021 ◽

Vol 11 ◽

Author(s):

Linda Voss ◽

Karina Guttek ◽

Annika Reddig ◽

Annegret Reinhold ◽

Martin Voss ◽

...

Keyword(s):

Cell Proliferation ◽

T Cells ◽

T Cell ◽

Autoimmune Diseases ◽

Adefovir Dipivoxil ◽

Cytokine Production ◽

T Cell Proliferation ◽

Potential Candidate ◽

Dependent Manner ◽

Approved Drugs

Repositioning of approved drugs for identifying new therapeutic purposes is an alternative, time and cost saving strategy to classical drug development. Here, we screened a library of 786 FDA-approved drugs to find compounds, which can potentially be repurposed for treatment of T cell-mediated autoimmune diseases. Investigating the effect of these diverse substances on mitogen-stimulated proliferation of both, freshly stimulated and pre-activated (48 h) peripheral blood mononuclear cells (PBMCs), we discovered Adefovir Dipivoxil (ADV) as very potent compound, which inhibits T cell proliferation in a nanomolar range. We further analyzed the influence of ADV on proliferation, activation, cytokine production, viability and apoptosis of freshly stimulated as well as pre-activated human T cells stimulated with anti-CD3/CD28 antibodies. We observed that ADV was capable of suppressing the proliferation in both T cell stimulation systems in a dose-dependent manner (50% inhibition [IC50]: 63.12 and 364.8 nM for freshly stimulated T cells and pre-activated T cells, respectively). Moreover, the drug impaired T cell activation and inhibited Th1 (IFN-γ), Th2 (IL-5), and Th17 (IL-17) cytokine production dose-dependently. Furthermore, ADV treatment induced DNA double-strand breaks (γH2AX foci expression), which led to an increase of p53-phospho-Ser15 expression. In response to DNA damage p21 and PUMA are transactivated by p53. Subsequently, this caused cell cycle arrest at G0/G1 phase and activation of the intrinsic apoptosis pathway. Our results indicate that ADV could be a new potential candidate for treatment of T cell-mediated autoimmune diseases. Prospective studies should be performed to verify this possible therapeutic application of ADV for such disorders.

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IcarisideII facilitates the differentiation of ADSCs to SCs via let-7i/STAT3 axis to preserve erectile function

Biological Research ◽

10.1186/s40659-019-0262-3 ◽

2019 ◽

Vol 52 (1) ◽

Author(s):

Pingyu Ge ◽

Yinxue Guo ◽

Jun Shen

Keyword(s):

Cell Proliferation ◽

Erectile Function ◽

Molecular Mechanisms ◽

Therapeutic Option ◽

Luciferase Reporter ◽

Dependent Manner ◽

Regulatory Relationship ◽

Rat Models ◽

Protein Levels ◽

Dose Dependent

Abstract Background IcarisideII (ICAII) could promote the differentiation of adipose tissue-derived stem cells (ADSCs) to Schwann cells (SCs), leading to improvement of erectile function (EF) and providing a realistic therapeutic option for the treatment of erectile dysfunction (ED). However, the underlying molecular mechanisms of ADSCs and ICAII in this process remain largely unclear. Methods ADSCs were treated with different concentrations of ICAII. Cell proliferation was determined by MTT assay. qRT-PCR and western blot were performed to detect expressions of SCs markers, signal transducer and activator of transcription-3 (STAT3), and microRNA-let-7i (let-7i). Luciferase reporter assay was conducted to verify the regulatory relationship between let-7i and STAT3. The detection of intracavernosal pressure (ICP) and the ratio of ICP/mean arterial pressure (MAP) were used to evaluate the EF in bilateral cavernous nerve injury (BCNI) rat models. Results ICAII promoted cell proliferation of ADSCs in a dose-dependent manner. The mRNA and protein levels of SCs markers were increased by ICAII treatment in a dose-dependent manner in ADSCs. Moreover, let-7i was significantly decreased in ICAII-treated ADSCs and upregulation of let-7i attenuated ICAII-induced promotion of SCs markers. In addition, STAT3 was a direct target of let-7i and upregulated in ICAII-treated ADSCs. Interestingly, overexpression of STAT3 abated the let-7i-mediated inhibition effect on differentiation of ADSCs to SCs and rescued the ICAII-mediated promotion effect on it. Besides, combination treatment of ADSCs and ICAII preserved the EF of BCNI rat models, which was undermined by let-7i overexpression. Conclusion ICAII was effective for preserving EF by promoting the differentiation of ADSCs to SCs via modulating let-7i/STAT3 pathway.

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Molecular Mechanisms of Antitumor Activity of the Selective Inhibitor of Nuclear Export (SINE) CRM1 Antagonist KPT-185 in Mantle Cell Lymphoma.

Blood ◽

10.1182/blood.v120.21.2438.2438 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2438-2438

Author(s):

Yoko Tabe ◽

Kensuke Kojima ◽

Linhua Jin ◽

Takashi Miida ◽

Sharon Shacham ◽

...

Keyword(s):

Gene Expression ◽

Cell Proliferation ◽

Cyclin D1 ◽

Signaling Pathways ◽

Nuclear Export ◽

Molecular Mechanisms ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Dependent Manner ◽

Antitumor Effects

Abstract Abstract 2438 CRM1, a member of the importin b super family of nuclear transport receptors, functions as a major nuclear export factor by shuttling transcription factors including p53, p21, I-kB, and FOXO3a from nucleus to cytoplasm, thereby preventing their activity. CRM1 is also involved in the transport of rRNA and a certain subset of mRNAs including Cyclin D1. Upregulated CRM1 expression has been reported to correlate with poor prognosis in various hematopoietic malignancies. MCL is a subtype of B-cell lymphoma which is frequently resistant to standard chemotherapy. The t(11,14)(q13;32) translocation of MCL juxtaposes the cyclin D1 gene, and constitutively overexpressed cyclin D1 is believed to be associated with oncogenesis. Additional genetic events such as mutation/overexpression of TP53 have been reported as adverse prognostic indicators. TP53 mutations are rare in typical MCL, although about 30% of aggressive blastoid MCL have mt-TP53. Because of the multiple signaling pathways that are dysregulated in MCL, a novel strategy aimed at restoring multiple anti-oncogenetic pathways, especially targeting p53-independent signaling pathways, is of considerable interest. In this study, we investigated the antitumor effects and molecular mechanisms of the SINE CRM1 antagonist KPT-185 (Karyopharm Therapeutics) in 4 MCL cells with known TP53 mutation status (wt-TP53: JVM2, Z138; mt-TP53: MINO, Jeko-1). Treatment with KPT-185 resulted in reduction of cell proliferation in a concentration-dependent manner without significant differences between wt- and mt-TP53 cells (IC50 at 72hrs by trypan blue exclusion method; 35nM for Z138, 92 nM for JVM2, 96 nM for MINO, 103 nM for Jeko-1). KPT-185 exhibited limited pro- apoptotic activity in the tested MCL cells except Z138 (ED50 at 48hrs by Annexin V positivity; 62 nM for Z138, 910 nM for JVM2, 665 nM for MINO, 618 nM for Jeko-1). We then investigated KPT-185-induced TP53 target gene expression changes (24 genes) by TaqMan low density arrays (TLDA) (Applied Biosystems). In wt-TP53 JVM2 and Z138 cells, KPT-185 (100nM for Z138, MINO, and 500nM for JVM2, Jeko-1) upregulated classical p53 targets such as p21 and MDM2 mRNA (>2.0 fold), while there was no increase in mt-TP53 MINO and Jeko-1 cells. Of note, in both wt- and mt-TP53 cells, KPT-185 upregulated gene expression of PUMA which is a target of FOXO3a, p73 and p53 (3.3 fold for JVM2, 2.5 fold for Z138, 3.3 fold for MINO, 4.8 fold for Jeko-1). Recently, CRM1 has been reported to positively modulate the nuclear export of Cyclin D1 mRNA in a eIF4E-dependent manner. We therefore examined Cyclin D1 protein levels by western blot analysis, and observed significantly high baseline expression of Cyclin D1 in Z-138 cells which are highly sensitive to KPT-185, as compared to less sensitive MCL cells. KPT-185 treatment decreased Cyclin D1 expression in a dose-dependent manner (50nM and 100nM) after 12hrs of treatment accompanied by p21 induction and decreased p-Rb. These findings demonstrate that KPT-185 successfully inhibits CRM1 activity in MCL resulting in inhibition of Cyclin D1 and cell proliferation, and in the p-53-independent upregulation of pro-apoptotic PUMA. In conclusion, CRM1 inhibition by KPT-185 results in cell growth inhibition and in moderate cell death in a TP-53 independent manner. Results also suggest that the sensitivity to KPT-185 in MCL may be dependent on Cyclin D1 expression. Therefore, KPT-185may be an effective agent for the treatment of MCL. Disclosures: Shacham: Karyopharm Therapeutics: Employment. Kauffman:Karyopharm Therapeutics Inc: Employment.

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Thymoquinone induces cell proliferation inhibition and apoptosis in acute myeloid leukemia cells: role of apoptosis-related WT1 and BCL2 genes

European Journal of Cell Science ◽

10.34154/2019-ejcs-0101-02-09/euraass ◽

2019 ◽

pp. 02-09

Author(s):

Mishary G. Musalli ◽

Mohammed A. Hassan ◽

Ryan A. Sheikh ◽

Abdulaziz A. Kalantan ◽

Majed A. Halwani ◽

...

Keyword(s):

Cell Proliferation ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Molecular Mechanisms ◽

Leukemia Cells ◽

Hl60 Cell ◽

Dependent Manner ◽

Cell Proliferation Inhibition ◽

Acute Myeloid Leukemia Cells ◽

Acute Myeloid

Acute myeloid leukemia (AML) is an aggressive and heterogeneous disease characterized by an abnormal proliferation and impaired differentiation of the myeloid precursor cells. The outcome for most AML patients remains poor with high relapse rates and chemotherapy remains the first line treatment for AML. The Wilms tumor wt1 and the anti-apoptotic BCL2 genes are upregulated in AML and are known to be involved in apoptosis inhibition. In the present study we evaluated the molecular mechanisms underlie the anti-proliferative and pro-apoptotic activities exerted by thymoquinone (TQ), the major biologically active compound of the black seed oil on acute myeloid leukemia (AML) cell line-HL60. Cell proliferation was determined by WST-1 assay and apoptosis rate was assessed by flow cytometry using annexin-V/7AAD staining. The expression of target genes was analyzed by real-time RT–PCR analysis. TQ significantly reduced HL60 cell viability and induced apoptosis in a dose and time-dependent manner. In order to decipher the molecular mechanisms underlie the anti-cancer activities induced by TQ in AML cells, we investigated its effect on the expression of WT1 and BCL2 genes. TQ significantly decreased the expression of WT1 and BCL2 genes in a dose and time-dependent manner. In summary, these findings suggest that TQ induces cell proliferation inhibition and apoptosis in acute myeloid leukemia cells most likely through targeting the apoptosis-related WT1 and BCL2 genes and also suggest that TQ could be a promising strategy for AML therapy.

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SYT8 promotes pancreatic cancer progression via the TNNI2/ERRα/SIRT1 signaling pathway

Cell Death Discovery ◽

10.1038/s41420-021-00779-4 ◽

2021 ◽

Vol 7 (1) ◽

Author(s):

Zhiping Fu ◽

Xing Liang ◽

Ligang Shi ◽

Liang Tang ◽

Danlei Chen ◽

...

Keyword(s):

Pancreatic Cancer ◽

Cell Proliferation ◽

Cancer Progression ◽

Tumor Metastasis ◽

Molecular Mechanisms ◽

Hormone Secretion ◽

Migration And Invasion ◽

Dependent Manner ◽

Orphan Nuclear Receptor

AbstractPancreatic cancer is a highly lethal malignancy due to failures of early detection and high metastasis in patients. While certain genetic mutations in tumors are associated with severity, the molecular mechanisms responsible for cancer progression are still poorly understood. Synaptotagmin-8 (SYT8) is a membrane protein that regulates hormone secretion and neurotransmission, and its expression is positively regulated by the promoter of the insulin gene in pancreatic islet cells. In this study, we identified a previously unknown role of SYT8 in altering tumor characteristics in pancreatic cancer. SYT8 levels were upregulated in patient tumors and contributed towards increased cell proliferation, migration, and invasion in vitro and in vivo. Increased SYT8 expression also promoted tumor metastasis in an in vivo tumor metastasis model. Furthermore, we showed that SYT8-mediated increase in tumorigenicity was regulated by SIRT1, a protein deacetylase previously known to alter cell metabolism in pancreatic lesions. SIRT1 expression was altered by orphan nuclear receptor ERRα and troponin-1 (TNNI2), resulting in cell proliferation and migration in an SYT8-dependent manner. Together, we identified SYT8 to be a central regulator of tumor progression involving signaling via the SIRT1, ERRα, and TNNI2 axis. This knowledge may provide the basis for the development of therapeutic strategies to restrict tumor metastasis in pancreatic cancer.

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3BDO Inhibits proliferation, epithelial–mesenchymal transition (EMT) and stemness via suppressing survivin in human glioblastoma cells

10.21203/rs.3.rs-179269/v1 ◽

2021 ◽

Author(s):

zhaotao wang ◽

yongping Li ◽

minyi liu ◽

danmin chen ◽

yunxiang ji ◽

...

Keyword(s):

Cell Proliferation ◽

Mouse Model ◽

Molecular Mechanisms ◽

Migration And Invasion ◽

Therapeutic Effects ◽

Dependent Manner ◽

Mesenchymal Transition ◽

Xenograft Mouse Model

Abstract BackgroundGlioblastoma (GBM) is a tumor of the central nervous system carries an extremely poor prognosis. Unfortunately, it also is the most frequently encountered tumor in this region. These tumors arise from glioblastoma stem cells (GSCs), which are glioma cells that are known to possess high degrees of stemness. GBM invades through the process of EMT, which features loss of cell differentiation and polarity. Survivin is a type of apoptotic inhibitor that has been characterized in several malignancies such as glioma. Normal tissues rarely express survivin. On the other hand, 3-benzyl-5-((2-nitrophenoxy) methyl) dihydrofuran-2(3H)-one (3BDO) represents an autophagy inhibitor and activates the mTOR pathway. It has been reported that 3BDO shows anti-cancer activities in lung carcinoma. However, the effects of 3BDO on GBM reminds unknown. Therefore, the purpose of this study was to explore the role and molecular mechanisms that 3BDO mediates in GBM.MethodCCK-8 experiments and clone formation assay were performed to detect the cell proliferation. Transwell assay was conducted to examined cell migration and invasion. Western blotting and immunofluorescence staining was used to analyze protein expression levels. Xenograft mouse model was used to evaluate the effect of 3BDO in vivo.ResultsWe found that 3BDO inhibited U87 and U251 cell proliferation in a dose-dependent manner. Additonally, 3BDO decreased the sphere formation and stemness markers (sox2, nestin and CD133) in GSCs. 3BDO also inhibited migration, invasion and suppressed EMT markers (N-cadherin, vimentin and snail) in GBM cells. Moreover, we found that 3BDO downregulated survivin expression of survivin both in GBM cells (U87, U251) and GSCs. Furthermore, overexpression of survivin reduced the therapeutic effects of 3BDO on GBM cell EMT, invasion, migration and proliferation, as well as decreased stemness in GSCs. Finally, we demonstrated that 3BDO inhibited tumor growth in a tumor xenograft mouse model constructed using U87 cells. Similar to the in vitro findings, 3BDO diminished suvivin expression, stemness and levels of EMT makers in vivo.Conclusionsour results demonstrated that 3BDO repressed GBM via downregulating survivin-mediated stemness and EMT both in vitro and in vivo.

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MSTMP, a Stilbene Derivative, Protects SH-SY5Y Cells Against Oxidative Stress

Canadian Journal of Neurological Sciences / Journal Canadien des Sciences Neurologiques ◽

10.1017/s0317167100017340 ◽

2014 ◽

Vol 41 (3) ◽

pp. 382-388

Author(s):

Wei-Guo Liu ◽

Jun-Yi Zhao ◽

Hao Zhang ◽

Xin-Yong Liu ◽

Xiu-Li Guo

Keyword(s):

Nitric Oxide ◽

Cell Viability ◽

Molecular Mechanisms ◽

Caspase 3 ◽

Cell Injury ◽

Neuroblastoma Cell ◽

Dependent Manner ◽

Caspase 9 ◽

Sod Activity ◽

Stilbene Derivative

Objective:The protective effects of a novel stilbene derivative, (E)-2-(3,4,5-trimethoxystyryl)-3,5,6-trimethylpyrazine (MSTMP), on hydrogen peroxide (H2O2)-induced human derived neuroblastoma cell (SH-SY5Y) damage and its molecular mechanisms were investigated.Methods:SH-SY5Y cells were exposed to 200 μmol·L−1 H2O2 for 12 h. The effect of MSTMP on cell viability and apoptosis was assessed by 3-(4,5-dimethyl- thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and flow cytometry method. The activities of lactate dehydrogenase (LDH), superoxide dismutase (SOD) and nitric oxide synthetase (NOS) and the content of malondialdehyde (MDA), reduced glutathione (GSH) and nitric oxide (NO) in cells were determined by commercial kits. The expressions of pro-apoptotic factor caspase-3, caspase-9 and inducible NOS (iNOS) were detected by Western blotting. Intracellular formation of reactive oxygen species (ROS) was assessed using 6-carboxy-2',7'-dichlorofluorescin diacetate (DCFH-DA) fluorescent probe.Results:MSTMP increased the SH-SY5Y cell viability by inhibition of cell apoptosis induced by H2O2. These effects were accompanied by an increase of SOD activity, GSH level, and a decrease of MDA content. Moreover, MSTMP showed stronger effects on inhibition of LDH leakage, apoptotic cells, intracellular ROS level and the expression of caspase-3 and caspase-9 than TMP. Furthermore, MSTMP induced a decrease of NO level and the activity of iNOS, tNOS in a time-dependent manner.Conclusions:MSTMP prevents H2O2-induced cell injury through anti-oxidation and anti-apoptosis via ROS-NO pathway.

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MiR-137 Targets the 3′ Untranslated Region of MSH2: Potential Implications in Lynch Syndrome-Related Colorectal Cancer

Cancers ◽

10.3390/cancers13184662 ◽

2021 ◽

Vol 13 (18) ◽

pp. 4662

Author(s):

Raffaella Liccardo ◽

Raffaele Sessa ◽

Silvia Trombetti ◽

Marina De Rosa ◽

Paola Izzo ◽

...

Keyword(s):

Colorectal Cancer ◽

Cell Proliferation ◽

Lynch Syndrome ◽

Molecular Mechanisms ◽

Genome Instability ◽

Cell Cycle Control ◽

Site Directed Mutagenesis ◽

Dependent Manner ◽

Colon Cells ◽

Sw480 Cells

Mismatch Repair (MMR) gene dysregulation plays a fundamental role in Lynch Syndrome (LS) pathogenesis, a form of hereditary colorectal cancer. Loss or overexpression of key MMR genes leads to genome instability and tumorigenesis; however, the mechanisms controlling MMR gene expression are unknown. One such gene, MSH2, exerts an important role, not only in MMR, but also in cell proliferation, apoptosis, and cell cycle control. In this study, we explored the functions and underlying molecular mechanisms of increased MSH2 expression related to a c.*226A>G variant in the 3′untranslated (UTR) region of MSH2 that had been previously identified in a subject clinically suspected of LS. Bioinformatics identified a putative binding site for miR-137 in this region. To verify miRNA targeting specificity, we performed luciferase gene reporter assays using a MSH2 3′UTR psiCHECK-2 vector in human SW480 cells over-expressing miR-137, which showed a drastic reduction in luciferase activity (p > 0.0001). This effect was abolished by site-directed mutagenesis of the putative miR-137 seed site. Moreover, in these cells we observed that miR-137 levels were inversely correlated with MSH2 expression levels. These results were confirmed by results in normal and tumoral tissues from the patient carrying the 3′UTR c.*226A>G variant in MSH2. Finally, miR-137 overexpression in SW480 cells significantly suppressed cell proliferation in a time- and dose-dependent manner (p < 0.0001), supporting a role for MSH2 in apoptosis and cell proliferation processes. Our findings suggest miR-137 helps control MSH2 expression via its 3′UTR and that dysregulation of this mechanism appears to promote tumorigenesis in colon cells.

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Chiaurinib Selectively Inhibits Colorectal Cancer with KRAS Wild-Type by Modulation of ROS through Activating the p53 Signaling Pathway

10.21203/rs.3.rs-46779/v1 ◽

2020 ◽

Author(s):

Haofan Yin ◽

Jineye Xie ◽

Ping Jiang ◽

Xi Jiang ◽

Deyu Duan ◽

...

Keyword(s):

Colorectal Cancer ◽

Cell Proliferation ◽

Signaling Pathway ◽

Molecular Mechanisms ◽

Tunel Staining ◽

Dependent Manner ◽

Ros Production ◽

Wild Type ◽

P53 Signaling ◽

P53 Signaling Pathway

Abstract Background: Colorectal cancer (CRC) is one of the top three most deadly cancers despite using chemotherapy based on oxaliplatin or irinotecan combined with targeted therapy. Chiaurinib has recently been identified to be a promising anticancer candidate with impressive efficacy and safety. However, the role and molecular mechanisms of Chiaurinib in the treatment of CRC remain to be elucidated.Methods: Cell proliferation and apoptosis were detected by CCK-8, EDU staining, Colony formation assay, TUNEL staining and flow cytometric analysis. ROS production was confirmed by Mito-SOX and DCF-DA fluorescence. RNA-Seq and GSEA analysis were used to explore the mechanisms of the effect of Chiaurinib in KRAS wild-type CRC cells.Results: Our study shows that Chiaurinib inhibits cell proliferation and induces apoptosis in KRAS wild-type CRC cells in a dose- and time-dependent manner, but not mutation ones. Meanwhile, Chiaurinib increases ROS production in KRAS wild-type CRC cells. Moreover, Chiaurinib selectively suppresses KRAS wild-type CRC cells growth in vivo. Mechanistically, Chiaurinib inhibits KRAS wild-type CRC cells by triggering ROS production via activating the p53 signaling pathway. Further, KRAS mutation CRC cells are resistant to Chiaurinib by increasing Nrf2 to stably elevate the basal antioxidant program and thereby lower intracellular ROS induced by Chiaurinib.Conclusions: Taken together, we reveal that Chiauranib induces p53 upregulation, resulting in ROS accumulation, thus inhibiting cell proliferation and inducing apoptosis in KRAS wild-type CRC cells. Our findings provide the rationale for further clinical evaluation of Chiaurinib as a therapeutic agent in treating KRAS wild-type CRC.

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