E2F1 downregulation by arsenic trioxide in lung adenocarcinoma

Abstract

Lung cancer is one of the most common cancers worldwide. Arsenic trioxide (ATO) has been approved by the US Food and Drug Administration for the treatment of acute promyelocytic leukemia. Nonetheless preliminary data have suggested potential activity of ATO in solid tumors including lung cancer. This study aimed to examine the underlying mechanisms of ATO in the treatment of lung adenocarcinoma. Using a panel of 7 lung adenocarcinoma cell lines, the effects of ATO treatment on cell viability, expression of E2F1 and its downstream targets, phosphatidylserine externalization, mitochondrial membrane depolarization and alteration of apoptotic/anti-apoptotic factors were studied. Tumor growth inhibition in vivo was investigated using a nude mouse xenograft model. ATO decreased cell viability with clinically achievable concentrations (8 µM) in all cell lines investigated. This was accompanied by reduced expression of E2F1, cyclin A2, skp2, c-myc, thymidine kinase and ribonucleotide reductase M1, while p-c-Jun was upregulated. Cell viability was significantly decreased with E2F1 knockdown. Treatment with ATO resulted in phosphatidylserine externalization in H23 cells and mitochondrial membrane depolarization in all cell lines, associated with truncation of Bid, downregulation of Bcl-2, upregulation of Bax and Bak, caspase-9 and -3 activation and PARP cleavage. Using the H358 xenograft model, the tumor growth was suppressed in the ATO treatment group during 8 days of treatment, associated with downregulation of E2F1 and upregulation of truncated Bid and cleaved caspase-3. In conclusion, ATO has potent in vitro and in vivo activity in lung adenocarcinoma, partially mediated through E2F1 downregulation and apoptosis.

Tumour growth suppressive effect of arsenic trioxide in squamous cell lung carcinoma

“…A previous clinical study demonstrated that patients with higher tumoral RRM1 expression exhibited a lower survival rate when treated with gemcitabine-based therapy (28). ATO downregulated RRM1 expression in lung adenocarcinoma, which may cause inhibition of DNA synthesis (11).…”

“…Phosphatidylserine externalization has been observed in H841 SCLC cells (9) and H23 lung adenocarcinoma cells (10). Mitochondrial membrane depolarization is another indicator of apoptosis; it has been demonstrated that ATO can induce mitochondrial membrane depolarization in lung cancer cells (11). DNA damage is typically sensed at the G 1 /S checkpoint or G 2 /M checkpoint, leading to either DNA repair or cell apoptosis.…”

“…High tumoral TYMS expression has been associated with poor clinical outcomes (25). In addition, ATO has been demonstrated to downregulate TYMS protein and mRNA expression, leading to tumour growth inhibition in lung adenocarcinoma cell lines (11).…”

“…JC-1 (Sigma-Aldrich; Merck KGaA) staining was performed as previously described (11). Cells were treated for 72 h with ATO (1.25, 2.5 and 5 µM) and untreated cells served as a control.…”

“…Western blot analysis was performed as previously reported (11). Cells (1x10 7 ) were collected and lysed for 1 h on ice with RIPA buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na 2 EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na 3 VO 4 and 1 µg/ml leupeptin) containing a protease inhibitor cocktail.…”