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. 2021 Aug 19;11:711043. doi: 10.3389/fonc.2021.711043

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Copyright © 2021 Booth, West, Moore, Von Hoff and Dent

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Resistance to ERBB1 inhibitors is associated with a reduced ability to form autophagosomes. (A) H1975 [wild-type sensitive and afatinib-resistant (AR)] were transfected to express LC3-GFP-RFP and subsequently treated with vehicle, pemetrexed (500 nM), GZ17-6.02 (2 μM curcumin final), or the drugs in combination for 4 h and 8 h. The number of intense staining GFP+ and RFP+ punctae was determined randomly in at least 50 cells, and the mean number of punctae per cell was determined (n = 3 ± SD). ^# p < 0.05 greater than GZ17-6.02 value; ^¶ p < 0.05 greater than the corresponding values after 4 h; ~p < 0.05 less than the corresponding values in wild-type sensitive cells. (B) Afatinib-resistant H1975 cells were transfected with siRNA molecules to knock down protein levels or with plasmids to express activated forms of mTOR or STAT3 and then subsequently treated with vehicle or [pemetrexed (500 nM) + GZ17-6.02 (2 μM curcumin final)] in combination for 4 h and 8 h. The number of intense staining GFP+ and RFP+ punctae were determined randomly in at least 50 cells, and the mean number of punctae per cell was determined (n = 3 ± SD). ^¶ p < 0.05 greater than the corresponding values after 4 h; *p < 0.05 less than the corresponding values in siSCR/CMV-transfected cells. (C) Osimertinib-resistant H1975 cells were transfected with siRNA molecules to knock down protein levels or with plasmids to express activated forms of mTOR or STAT3 and then subsequently treated with vehicle or [pemetrexed (500 nM) + GZ17-6.02 (2 μM curcumin final)] in combination for 4 h and 8 h. The number of intense staining GFP+ and RFP+ punctae was determined randomly in at least 50 cells and the mean number of punctae per cell determined (n = 3 ± SD). ^¶ p < 0.05 greater than the corresponding values after 4 h; ~~p < 0.05 less than the corresponding values in afatinib-resistant H1975 cells; *p < 0.05 less than the corresponding values in siSCR/CMV-transfected cells.