Afatinib Reduces STAT6 Signaling of Host ARPE-19 Cells Infected with <i>Toxoplasma gondii</i>

Abstract

Specific gene expressions of host cells by spontaneous STAT6 phosphorylation are major strategy for the survival of intracellular Toxoplasma gondii against parasiticidal events through STAT1 phosphorylation by infection provoked IFN-γ. We determined the effects of small molecules of tyrosine kinase inhibitors (TKIs) on the growth of T. gondii and on the relationship with STAT1 and STAT6 phosphorylation in ARPE-19 cells. We counted the number of T. gondii RH tachyzoites per parasitophorous vacuolar membrane (PVM) after treatment with TKIs at 12-hr intervals for 72 hr. The change of STAT6 phosphorylation was assessed via western blot and immunofluorescence assay. Among the tested TKIs, Afatinib (pan ErbB/EGFR inhibitor, 5 µM) inhibited 98.0% of the growth of T. gondii, which was comparable to pyrimethamine (5 µM) at 96.9% and followed by Erlotinib (ErbB1/EGFR inhibitor, 20 µM) at 33.8% and Sunitinib (PDGFR or c-Kit inhibitor, 10 µM) at 21.3%. In the early stage of the infection (2, 4, and 8 hr after T. gondii challenge), Afatinib inhibited the phosphorylation of STAT6 in western blot and immunofluorescence assay. Both JAK1 and JAK3, the upper hierarchical kinases of cytokine signaling, were strongly phosphorylated at 2 hr and then disappeared entirely after 4 hr. Some TKIs, especially the EGFR inhibitors, might play an important role in the inhibition of intracellular replication of T. gondii through the inhibition of the direct phosphorylation of STAT6 by T. gondii.

A Human Proteome Array Approach to Identifying Key Host Proteins Targeted by Toxoplasma Kinase ROP18

“…where S 0 is the total number of unique substrates in the database; K i is the number of substrates of kinase i; n is the number of ROP18 substrates detected in the database; and m is the number of common targets between ROP18 and kinase i. In Vitro Infection-Cells were infected by tachyzoites of RH strain, as described previously (35) with slight modifications. Briefly, cells were plated in 12-well plates 24 h before infection.…”

Suppressors for Human Epidermal Growth Factor Receptor 2/4 (HER2/4): A New Family of Anti-Toxoplasmic Agents in ARPE-19 Cells

“…ROP2 family of rhoptry proteins (ROPs) has a very important role in creating the parasitophorous vacuole membrane (PVM) within the host cells during this process. Some of these ROPs, and especially ROP16, have kinase domains in their C-terminal halves, which may function in signal transduction across the PVM as a protein kinase (PK) to maintain the host cell-parasite relationship and may be candidate targets for new drugs [ 4 ].…”

“…Numerous TKs have been identified as oncogenes in various tumors, so a strict regulation of their catalytic activity is an absolute requirement. They have also been implicated in various diseases such as diabetic retinopathy, atherosclerosis, psoriasis [ 7 ], and infections [ 4 ].…”

“…These differences of T. gondii PKs in structure and function from mammalian PKs can be used as drug targets. The functions of some of these PKs have been revealed and their inhibitors have been found, which look promising as novel drugs against toxoplasmosis [ 4 , 10 – 15 ].…”

“…PKs, and also T. gondii PKs, can be inhibited by so called ‘small molecule kinase inhibitors’ which have been developed for the chemotherapy of various types of cancer [ 16 ]. A tyrosine kinase inhibitor (TKI) is such a pharmaceutical drug that inhibits TKs [ 4 ], and numerous TKIs developed recently for the chemotherapy of cancers have expanded their efficacy for the treatment of infectious diseases [ 17 , 18 ]. In the present study we evaluated the effects of TKIs on the growth inhibition of intracellular T. gondii inside ARPE-19 cells, and tried to determine their mechanisms.…”