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Investigation of the effects of the toll-like receptor 4 pathway on immune checkpoint vista in pancreatic cancer

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Abstract

Pancreatic ductal adenocarcinoma (PDAC) is one of the most common malignant tumors of the pancreas. Preclinical studies show that it evades the immune system with immune checkpoints and promotes tumor development. V-domain Ig suppressor of T cell activation (VISTA) is a new immune-check point from the B7 family and is highly expressed in cancer cells. Overexpression of toll like receptor 4 (TLR4) in pancreatic adenocarcinoma is associated with induced tumorigenesis, tumor growth, resistancy to chemotherapy. Naloxone is an opioid and inhibits TLR4-ligand association. In this study, we investigated the relation of TLR4 and downstream pathways with immune-check point VISTA in pancreatic cancer proliferation. We initially collected pancreatic cancer-related datasets using the GEPIA2 and UALCAN databases. Based on this data obtained the effect of various concentrations and incubation times of naloxone were used on PANC-1 cells proliferation. A combination of naloxone and VISTA-siRNA were applied, and the effect of both naloxone and combined treatment on TLR4, Interleukin 1 receptor associated kinase 4 (IRAK4) and VISTA gene expression were analyzed in pancreatic cancer cells. As a result of analysis with Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR), gene expression levels of TLR4, IRAK4 and VISTA were significantly suppressed and cell proliferation was significantly reduced. We found that administration of naloxone and VISTA-siRNA in combination with PDAC cells suppressed signaling. Therefore, we considered that the relationship between VISTA and TLR4 signaling pathways and the other possible associated signal molecules may be an important marker in determining the response of immune checkpoint inhibitors in cancer treatment.

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Data availability

All data generated or analyzed during this study are included in this published article. Raw data is available under reasonable request.

Abbreviations

ANOVA:

Analysis of variance

cDNA:

Complementary DNA

CO2 :

Carbon dioxide

DAMPs:

Damage-associated molecular pattern

DMEM:

Dulbecco's modified eagle's medium

DNA:

Deoxyribonucleic acid

FBS:

Fetal bovine serum

GAPDH:

Glyceraldehyde-3-phosphate dehydrogenase

GEPIA2:

Gene expression profiling interactive analysis 2

GTEx:

Genotype-tissue expression

HepG2:

Hepatocellular carcinoma

HUVEC:

Human umbilical vein endothelial cells

IC50:

50% Inhibitory concentration

IL-6:

Interleukin 6

IRAK4:

Interleukin-1 receptor-associated kinase 4

LPS:

Lipopolysaccharides

MD2:

Myeloid differentiation factor 2

MTT:

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

Myd88:

Myeloid differentiation factor 88

NF-κB:

Nuclear factor kappa B; nm, nanometer; nM, nanomolar

NO:

Nitric oxide

NOS:

Nitric oxide synthase

NSCLC:

Non-small cell lung cancer

NT-siRNA:

Non target small interfering RNA

PANC-1:

Pancreatic cancer cells-1

PD-1:

Programmed cell death protein 1

PDAC:

Pancreatic ductal adenocarcinoma

PD-L1:

Programmed cell death-1 ligand1

qRT-PCR:

Quantitative real-time polymerase chain reaction

RNA:

Ribonucleic acid

RNAi:

RNA interference

siRNA:

Small interfering RNA

TCGA:

The cancer genome atlas

TLR4:

Toll like receptor 4

VISTA:

V-domain Ig suppressor of T cell activation

VSIR:

V-set immunoregulatory receptor

μL:

Microliter

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Acknowledgements

We acknowledge the GEPIA2 and UALCAN databases for free use.

Funding

This study was funded by Necmettin Erbakan University Scientific Research Projects Coordination Unit, Project number 191315002.

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Authors and Affiliations

  1. Department of Molecular Biology and Genetics, Faculty of Science, Bartın University, Bartın, Turkey

    Kubra Sena Bas Topcu

  2. Department of Molecular Biology and Genetics, Faculty of Science, Necmettin Erbakan University, Konya, Turkey

    Emine Nedime Korucu

  3. Department of Medical Biochemistry, Faculty of Medicine, Selcuk University, Konya, Turkey

    Esma Menevse

  4. Department of Medical Genetics, Faculty of Medicine, Selcuk University, Konya, Turkey

    Nadir Kocak

  5. Department of Medical Genetics, Faculty of Medicine, KTO Karatay University, Konya, Turkey

    Tugce Duran

Authors
  1. Kubra Sena Bas Topcu
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  2. Emine Nedime Korucu
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  3. Esma Menevse
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  4. Nadir Kocak
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  5. Tugce Duran
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Contributions

Emine Nedime Korucu conceptualized the Project. Emine Nedime Korucu, Nadir Kocak and Kubra Sena Bas Topcu designed experiments. Emine Nedime Korucu, Kubra Sena Bas Topcu and Tugce Duran performed experiments. Esma Menevse analyzed data. Kubra Sena Bas Topcu wrote the original manuscript draft. Esma Menevse wrote a revised manuscript draft. Emine Nedime Korucu supervised and administered the project.

Corresponding author

Correspondence to Emine Nedime Korucu.

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Ethical approval

This study were approved by the Pharmaceutical and Non-Medical Research Ethical Committee of Necmettin Erbakan University, Meram Faculty of Medicine (Ethics number: 2019/2070).

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The authors declare no conflict of interest.

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Topcu, K.S.B., Korucu, E.N., Menevse, E. et al. Investigation of the effects of the toll-like receptor 4 pathway on immune checkpoint vista in pancreatic cancer. Invest New Drugs 40, 519–528 (2022). https://doi.org/10.1007/s10637-021-01209-z

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  • DOI: https://doi.org/10.1007/s10637-021-01209-z

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