Cells were incubated with DMSO (0.1%) or each compound for 16 h. drugs. Of note, the CdCl2 exposure increased the levels of the Notch1 intracellular domain and of the downstream Notch1 target genes Snail and Slug. Strikingly, siRNA-mediated Notch1 silencing partially suppressed the CdCl2-induced EMT, stress fiber formation, high cell motility, and antitumor drug resistance. In addition, we found that prolonged CdCl2 exposure induced reduction of E-cadherin in BEAS-2B human bronchial epithelial cells and antitumor drug resistance in H1975 human tumor-derived non-small-cell lung cancer cells depending on Notch1 signaling. Moreover, Notch1, HIF-1, and IGF-1R/Akt/ERK/S6K1 activated each other to induce EMT in the CdCl2-exposed A549 cells. These results suggest that Notch1, along with HIF-1 and IGF-1R/Akt/ERK/S6K1 signaling pathways, promotes malignant progression stimulated by prolonged cadmium exposure in this lung adenocarcinoma model. < 0.01, significant difference between the samples. Notch1 is involved in prolonged cadmium exposure-induced EMT, stress fiber formation, and high cell motility in A549 cells Although Notch3 is highly activated in A549 cells (25), its mRNA expression decreased in cells with prolonged CdCl2 exposure (supplemental Fig. S3gene (Notch1-1 and Notch1-2) (Fig. 2gene (Snail-1 and Snail-2) suppressed cadmium-induced reduction of E-cadherin expression (Fig. 2and < 0.01, significant difference between the samples. Notch1 is involved in prolonged cadmium exposure-induced antitumor drug resistance in A549 cells We determined the viability of prolonged CdCl2-exposed A549 cells treated with cisplatin, gemcitabine, and etoposide, antitumor drugs Rabbit Polyclonal to Glucokinase Regulator commonly used in lung cancer chemotherapy, using trypan blue exclusion assays. Prolonged CdCl2 exposure resulted in lower cell death induced by treatment with cisplatin (Fig. 3and and < 0.05; **, < 0.01, significant difference between the samples. HIF-1 regulates Notch1 activity in prolonged cadmium-exposed A549 cells The transcription factor HIF-1 is an important trigger and modulator of EMT and activates Notch1 signaling through a multistep process (28,C32). Therefore, we examined whether HIF-1 is involved in Notch1 activation and induces EMT in prolonged CdCl2-exposed A549 cells. HIF-1 protein levels increased in cells exposed to 5C20 m CdCl2 for 10 weeks (Fig. 4gene (HIF-1-1 and HIF-1-2) suppressed Notch1-ICD but not Notch1-NTM in prolonged CdCl2-exposed cells (Fig. 4< 0.05; **, < 0.01, significant difference between the samples. HIF-1 transcriptional activity is not required for the activation of Notch1 signaling in prolonged cadmium-exposed A549 cells It has been reported that HIF-1 increases the expression of Jagged2 and anterior pharynx-defective 1 (APH-1), a component of the -secretase complex, through the binding to their promoters (29, 30), resulting in the activation of Notch1 signaling. To investigate whether the transcriptional activity of HIF-1 is required for Notch1 activation in prolonged CdCl2-exposed cells, we depleted the expression of arylhydrocarbon receptor nuclear translocator (ARNT), Indomethacin (Indocid, Indocin) an HIF-1 binding partner for DNA binding (28). Transfection with siRNAs targeted against the human gene (ARNT-1 and ARNT-2) markedly suppressed ARNT expression (Fig. 5and < 0.05; **, < 0.01, significant difference between the samples (and and and and and < 0.01, significant difference between the Indomethacin (Indocid, Indocin) samples. Discussion A549 cells are a human lung adenocarcinoma cell line with properties of type II alveolar epithelial cells (42). We found that exposure to CdCl2 for more than 8 weeks enhanced the proliferative ability of A549 cells. The transcription factor Nrf2 has been reported to be one of the key factors that induce a high proliferative ability in cadmium-transformed BEAS-2B cells (9), and its downstream target, heme oxygenase-1, is involved in the suppression of cadmium toxicity in kidney and pulmonary cells (43, 44). Consistent with these findings, knockdown of Nrf2 (supplemental Fig. S9, model of prolonged cadmium-exposed lung epithelial cells. We found that prolonged CdCl2 exposure induced EMT, stress fiber formation, high cell motility, and antitumor drug resistance in A549 cells. In concordance with the findings that chronic cadmium exposure induces EMT-like characteristics in HPL-1D human peripheral lung epithelial cells (45), cadmium-induced malignant progression was also clearly observed in our model using A549 cells. Furthermore, consistent with our previous findings in HK-2 human renal proximal epithelial cells treated Indomethacin (Indocid, Indocin) with CdCl2 (22), an increase in the levels of Notch1-ICD and its downstream targets, Snail and Slug, was found in prolonged CdCl2-exposed A549 cells. Notch1 knockdown partially suppressed prolonged CdCl2-induced EMT, stress fiber formation, high cell motility, and antitumor drug resistance. In addition, we also found that prolonged CdCl2 exposure induced reduction of E-cadherin in BEAS-2B cells and antitumor drug resistance in H1975 cells depending on Notch1 signaling. These findings demonstrate for the first time, to our knowledge, that Notch1 signaling is involved in cadmium-induced malignant progression in lung cancer cells, including A549 cells. Because these findings remained in the prolonged CdCl2-exposed A549 cells after removal of CdCl2 from culture medium for 10 weeks, cadmium-induced malignant progression via the Notch1 pathway may be maintained (supplemental Fig..