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

Abstract

Lung squamous cell carcinoma (SCC) is the second most common subtype of non-small cell lung carcinoma. The anticancer effects of arsenic trioxide (ATO) in lung adenocarcinoma and small-cell lung cancer have previously been reported; however its effects in SCC remain unclear. An MTT assay and western blot analysis were performed to determine cell viability and protein expression, respectively, in the SK-MES-1 and SW900 SCC cell lines following treatment with ATO. Phosphatidylserine externalization, mitochondrial membrane depolarization and cell cycle distribution were studied using flow cytometry and the in vivo effects of ATO on tumour growth were investigated with a xenograft model. The results demonstrated that SK-MES-1 and SW900 SCC cells were sensitive to clinically relevant concentrations of ATO. ATO induced apoptosis, mitochondrial membrane depolarization and G₂/M arrest. In addition, treatment with ATO resulted in the downregulation of X-linked inhibitor of apoptosis, B-cell lymphoma-2 (Bcl-2), E2F transcription factor 1 (E2F1), thymidylate synthase and ribonucleotide reductase M1 in addition to the upregulation of Bcl-2 antagonist/killer protein, cleaved poly ADP-ribose polymerase and cleaved caspase 3 in a cell-line specific manner. In the SW900 xenograft model, tumour growth was inhibited by ATO with the formation of apoptotic bodies and downregulation of Bcl-2 and E2F1. In conclusion, ATO suppresses the growth of SCC in vitro and in vivo.

Keywords: apoptosis; arsenic trioxide; squamous cell lung carcinoma; xenograft.

Figure 1.

ATO reduced cell viability, induced phosphatidylserine externalization and mitochondrial membrane depolarization in SK-MES-1 cells. (A) SK-MES-1 and SW900 cells were sensitive to ATO treatment with IC₅₀ values of 2.5 and 5 µM respectively. H520 and H2170 cells were relative resistance to ATO. (B) ATO increased the percentage of cells undergoing apoptosis in SK-MES-1 cells in a dose-dependent manner. (C) ATO increased the proportion of cells undergoing mitochondrial membrane depolarization in SK-MES-1 cells in a dose-dependent manner. Results were measured in triplicate experiments. *P<0.05, compared with the control group. ATO, arsenic trioxide; IC₅₀, half-maximal inhibitory concentration.

Figure 2.

Alteration of protein expression following ATO treatment. ATO induced (A) XIAP downregulation (SW900), (B) Bcl-2 suppression (SK-MES-1), (C) Bak upregulation (SW900), (D) CC3 elevation (SK-MES-1 and SW900), (E) cleaved PARP upregulation (SK-MES-1), (F) E2F1 suppression (SK-MES-1 and SW900), (G) RRM1 downregulation (SK-MES-1) and (H) TYMS suppression (SK-MES-1). Results were measured in triplicate experiments. *P<0.05, **P<0.01 and ***P<0.001 compared with the control group. XIAP, X-linked inhibitor of apoptosis; Bcl-2, apoptosis regulator Bcl-2; Bak, Bcl-2 antagonist/killer protein; CC3, cleaved caspase 3; PARP, poly ADP-ribose polymerase; RRM1, ribonucleotide reductase M1; TYMS, thymidylate synthase.

Figure 3.

ATO suppressed squamous cell lung carcinoma tumour growth in vivo. (A) ATO (7.5 mg/kg) suppressed tumour growth in SW900 xenograft compared with the control group. (B) The expression of Bcl-2 and E2F1 was downregulated in the 7.5 mg/kg ATO treatment group compared with the control group. (C) Detection of apoptotic bodies (indicated by arrows) in ATO treatment groups using hematoxylin and eosin staining. *P<0.05, **P<0.01 compared with the control.