Antitumor Activity of TRAIL-Mu3 Protein in vitro and in vivo and the Mechanisms

YANG Ting, ZHU Ai-jing, WANG Jing-song, JIN Zhao

Abstract

TRAIL-Mu3 was obtained by mutating the N-terminus of human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene to an eight continuous arginine sequence. The present study was designed to explore the antitumor effect of this soluble mutant protein and the underlying mechanisms.  Methods  The inhibitory effect of TRAIL-Mu3 on the proliferation of lung cancer cell lines NCI-H460, A549, NCI-H1299 and calu-1 was tested by CCK8 assay. The apoptotic rates of A549 and NCI-H460 treated by TRAIL-Mu3 were detected by flow cytometer (FCM). The expressions of apoptosis related proteins death receptor (DR) 4, DR5, Caspase-3, Caspase-8 and X-linked inhibitor of apoptosis protein (XIAP) were detected by Western blot. Moreover, a subcutaneous xenograft tumor mouse model of NCI-H460 was established and treated with TRAIL-Mu3 daily or every other day or three times a week. The expressions of DR4, DR5, Caspase-3, Caspase-8 and XIAP were detected by immunohistochemical staining.  Results  The in vitro study demonstrated that as compared to the TRAIL, the TRAIL-Mu3 was more toxic and pro-apoptotic by up-regulation of the expression and activity of DR4, Caspase-3 and Caspase-8. Also, the animal study showed a similar antitumor effect between treatment with TRAIL-Mu3 every other day and three time a week, which was better than daily use. All treatments significantly suppressed the growth of xenograft tumor, increased the expression or activity of DR4 and Caspase-3, and down-regulated the expression of XIAP (P<0.05).  Conclusion  TRAIL-Mu3 could improve antitumor activity in vivo and in vitro through elevating DR4 expression, activating Caspase-3/-8, and inhibiting XIAP activation.

 

 Keywords: TRAIL, Mutant, Arginine, Lung cancer, Apoptosis

 

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References


WILEY S R， SCHOOLEY K， SMOLAK P J， et al. Identification and characterization of a new member of the TNF family that induces apoptosis. Immunity，1995，3(6): 673–682.

PITTI R M， MARSTERS S A， RUPPERT S， et al. Induction of apoptosis by Apo-2 ligand, a new member of the tumor necrosis factor cytokine family. J Biol Chem，1996，271(22): 12687–12690.

STUCKEY D W， SHAH K. TRAIL on trial: preclinical advances in cancer therapy. Trends Mol Med，2013，19(11): 685–694.

KANTARI C， WALCZAK H. Caspase-8 and bid: caught in the act between death receptors and mitochondria. Biochim Biophys Acta，2011，1813(4): 558–563.

SANTOS C A， SOUZA A P. Solubilization， folding， and purification of a recombinant peptidoglycan-associated lipoprotein (PAL) expressed in Escherichia coli. Curr Protoc Protein Sci， 2018， 92(1): e53[2020-03-14].https://doi.org/10.1002/cpps.53.

UPADHYAY V， SINGH A， JHA D， et al. Recovery of bioactive protein from bacterial inclusion bodies using trifluoroethanol as solubilization agent. Microb Cell Fact， 2016， 15: 100[2020-03-14]. https://micro bialcellfactories.biomedcentral.com/articles/10.1186/s12934-016-0504-9. doi: 10.1186/s12934-016-0504–9.

WAJANT H. Molecular mode of action of TRAIL receptor agonists-common principles and their translational exploitation. Cancers (Basel)， 2019， 11(7). pii: E954[2020-03-14]. https://doi.org/10.3390/cancers11070954.

ZHU H， YAN J， XU Q， et al. TRAIL mutant membrane penetrating peptide alike (TMPPA) TRAIL-MU3 enhances the antitumor effects of TRAIL in vitro and in vivo. Mol Med Rep，2017，16(6): 9607–9612.

HUANG M， ZHU H， YI C， et al. A novel TRAIL mutant-TRAIL-MU3 enhances the antitumor effects by the increased affinity and the up-expression of DR5 in pancreatic cancer. Cancer Chemother Pharmacol， 2018，82(5): 829–838.

LOCKSLEY R M， KILLEEN N， LENARDO M J. The TNF and TNF receptor superfamilies: integrating mammalian biology. Cell，2001， 104(4): 487–501.

GUIMARAES P P G， GAGLIONE S， SEWASTIANIK T， et al. Nanoparticles for immune cytokine TRAIL-based cancer therapy. ACS Nano，2018，12(2): 912–931.

BERG D， LEHNE M， MEULLER N， et al. Enforced covalent trimerization increases the activity of the TNF ligand family members TRAIL and CD95L. Cell Death Differ，2007，14(12): 2021–2034.

CARDOSO A L， BERGER M D， KOUTSANDREAS T， et al. Non-apoptotic TRAIL function modulates NK cell activity during viral infection. EMBO Rep， 2020， 21(1): e48789[2020-03-14]. https://doi.org/10.15252/embr.201948789.

VON KARSTEDT S， WALCZAK H. An unexpected turn of fortune: targeting TRAIL-Rs in KRAS-driven cancer. Cell Death Discov， 2020， 6: 14[2020-03-14]. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC 7078304/. doi: 10.1038/s41420-020-0249–4.

HYMOWITZ S G， O'CONNELL M P， ULTSCH M H， et al. A unique zinc-binding site revealed by a high-resolution X-ray structure of homotrimeric Apo2L/TRAIL. Biochemistry，2000，39(4): 633–640.

KELLEY R F， TOTPAL K， LINDSTROM S H， et al. Receptor-selective mutants of apoptosis-inducing ligand 2/tumor necrosis factor-related apoptosis-inducing ligand reveal a greater contribution of death receptor (DR) 5 than DR4 to apoptosis signaling. J Biol Chem，2005，280(3): 2205–2212.

REIS C R， VAN DER SLOOT A M， NATONI A， et al. Rapid and efficient cancer cell killing mediated by high-affinity death receptor homotrimerizing TRAIL variants. Cell Death Dis， 2010， 1: e83[2020-03- 14]. https://www.nature.com/articles/cddis201061. doi: 10.1038/cddis.2010.61.

YAGOLOVICH A V， ARTYKOV A A， DOLGIKH D A， et al. A new efficient method for production of recombinant antitumor cytokine TRAIL and its receptor-selective variant DR5-B. Biochemistry (Mosc)， 2019，86(6): 627–636.

BOSMAN M C， REIS C R， SCHURINGA J J， et al. Decreased affinity of recombinant human tumor necrosis factor-related apoptosis-inducing ligand (rhTRAIL) D269H/E195R to osteoprotegerin (OPG) overcomes TRAIL resistance mediated by the bone microenvironment. J Biol Chem， 2014，289(2): 1071–1078.

YU R， ALBARENQUE S M， COOL R H， et al. DR4 specific TRAIL variants are more efficacious than wild-type TRAIL in pancreatic cancer. Cancer Biol Ther，2014，15(12): 1658–1666.

MEIJER A， KRUYT F A， VAN DER ZEE A G， et al. Nutlin-3 preferentially sensitises wild-type p53-expressing cancer cells to DR5-selective TRAIL over rhTRAIL. Br J Cancer，2013，109(10): 2685–2695.

SZEGEZDI E， REIS C R， VAN DER SLOOT A M， et al. Targeting AML through DR4 with a novel variant of rhTRAIL. J Cell Mol Med，2011，


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