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  • br Considering the significant impact of AAMP on

    2020-08-12


    Considering the significant impact of AAMP on ERK1/2 activation and the interaction between AAMP and EGFR, we wondered if EGFR is an intermediate protein in AAMP-ERK1/2 regulation. We measured
    A
    siCTRL
    siAAMP-1
    siAAMP-2
    -Icotinib
    PI
    +Icotinib
    AnnexinV-FITC
    B
    C
    siCTRL
    AAMP
    CASP3
    ACTB
    Fig. 6. AAMP downregulation elevated the sensitivity to icotinib in H1975 cells. (A) H1975 UNC-1999 were transfected with AAMP siRNAs for 24 h and then treated with icotinib [20 μM] for 24 h. Flow cytometry was used to measure apoptosis rate (n = 4). (B) H1975 cells were treated with icotinib [20 μM] for 24 h after being transfected with AAMP siRNAs for 24 h. The cleavage of caspase 8 and caspase 3 was detected by Western blot. (C) H1975 cells were reseeded in 96-well plate after AAMP downregulating. Cells were fixed separately after icotinib [20 μM] treated for 0, 24, 48 and 72 h. SRB assay was performed to detect survival rate (n = 5).
    phosphorylation level of ERK1/2 after AAMP knockdown and EGFR overexpression in the same cells. Western blot data show ERK1/2 phosphorylation was repressed after AAMP silencing. Interestingly, EGFR overexpression recovered p-ERK1/2 level after AAMP silencing (Fig. 5D). These data demonstrate EGFR overexpression rescues the reduction of ERK1/2 phosphorylation caused by AAMP inhibition, in-dicating AAMP activates ERK1/2 through EGFR activation.
    3.5. AAMP silencing elevated sensitivity to icotinib in H1975 cells
    H1975 cells were confirmed as a first-generation EGFR-TKIs re-sistance cell lines with two EGFR activating mutations (L858R and T790M). Thus, we wonder whether AAMP suppression elevates sensi-tivity to icotinib in H1975 cells. AAMP siRNA transfection was perform in H1975 cells for 24 h. Apoptosis was detected by western blot and flow cytometry after icotinib treatment at 20 μM for 24 h. Results show that combination between AAMP inhibition and icotinib increased the percentage of apoptosis cells (Fig. 6A) and cleaved forms of caspase-8 and caspase-3 (Fig. 6B). Furthermore, the percentage of survival cells was decreased in a time-dependent manner with icotinib [20 μM]/24 h treatment when AAMP was silenced (Fig. 6C). These data suggest AAMP downregulation enhanced sensitivity to icotinib in H1975 cells 
    with TKIs resistance EGFR mutations.
    3.6. EGFR has minimum influence on AAMP expression
    Since AAMP plays an important role in EGFR activation, we wonder whether EGFR exert an influence on AAMP. As AAMP is an adaptor protein, and no enzymatic activity has been found yet. Thus, we de-tected the level of AAMP after EGFR knockdown in EGFR wild type and mutant cell lines respectively. The results showed that EGFR repression had minimum effect on AAMP expression (Fig. S4A and B). In addition, we transfected EGFR single mutation (L858R) and double mutation (L858R/T790M) plasmids into NSCLC cells to test the protein level of AAMP. Similarly, no big difference in AAMP levels was found (Fig. S4C). These results indicate AAMP regulates EGFR activity, while EGFR has minimum effect on AAMP level.
    3.7. AAMP inhibition promoted apoptosis caused by doxorubicin in NSCLC
    Our previous studies have found that doxorubicin induces EGFR downregulation in dose-dependent and time-dependent manners in NSCLC cells (Fig. 7A). Besides, evidence above revealed that suppres-sion of AAMP impaired proliferation and survival through EGFR
    A
    B
    siCTRL
    Doxorubicin[µM]/24h
    EGFR
    ACTB
    AAMP
    Cleaved
    CASP8
    EGFR
    ACTB
    CASP3
    ACTB
    C
    AAMP
    PARP-1 Cleaved
    CASP3
    ACTB
    Fig. 7. AAMP reduced the sensitivity to doxorubicin in NSCLC cells. (A) H1792 cells were treated with doxorubicin at 0, 0.5, 1, 2, 4 μM for 24 h. A549 cells were treated with doxorubicin at 1 μM for 0, 12, 24 and 48 h. The level of EGFR was measured by WB. (B) AAMP downregulation and doxorubicin [1 μM]/24 h treatment were performed in H1792 and Calu-1 cells. WB was used to detect the cleavage of caspase 8 and caspase 3. (C) H460 and A549 cells were treated with doxorubicin [1 μM] for 24 h after being transfected with pcDNA3.1-AAMP. The cleavage of caspase 8 and caspase 3 were measured by WB. (D) AAMP downregulation and upregulation were performed in Calu-1 and A549 cells. Apoptosis rate was detected by flow cytometry after doxorubicin [1 μM]/24 h treatment (n = 4).
    activation inhibiting in NSCLC. To test the specific role of AAMP in apoptosis caused by doxorubicin in cancer cells, we treated H1792 and Calu-1 cells with doxorubicin for 24 h after AAMP siRNA transfection. The data show that AAMP downregulation enhanced cleavage of cas-pase-8 and caspase-3 caused by doxorubicin significantly (Fig. 7B), while the cleavage in H460 and A549 cells overexpressing AAMP was declined (Fig. 7C). We achieved similar results using flow cytometry (Fig. 7D). These results collectively demonstrate that the combination of doxorubicin with AAMP inhibition potently induces apoptosis in NSCLC cells.