RIP1 potentiates BPDE-induced transformation in human bronchial epithelial cells through catalase-mediated suppression of excessive reactive oxygen species

Abstract

Cell survival signaling is important for the malignant phenotypes of cancer cells. Although the role of receptor-interacting protein 1 (RIP1) in cell survival signaling is well documented, whether RIP1 is directly involved in cancer development has never been studied. In this report, we found that RIP1 expression is substantially increased in human non-small cell lung cancer and mouse lung tumor tissues. RIP1 expression was remarkably increased in cigarette smoke-exposed mouse lung. In human bronchial epithelial cells (HBECs), RIP1 was significantly induced by cigarette smoke extract or benzo[a]pyrene diol epoxide (BPDE), the active form of the tobacco-specific carcinogen benzo(a)pyrene. In RIP1 knockdown HBECs, BPDE-induced cytotoxicity was significantly increased, which was associated with induction of cellular reactive oxygen species (ROS) and activation of mitogen-activated protein kinases (MAPKs), including c-jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) and p38. Scavenging ROS suppressed BPDE-induced MAPK activation and inhibiting ROS or MAPKs substantially blocked BPDE-induced cytotoxicity, suggesting ROS-mediated MAPK activation is involved in BPDE-induced cell death. The ROS-reducing enzyme catalase is destabilized in an ERK- and JNK-dependent manner in RIP1 knockdown HBECs and application of catalase effectively blocked BPDE-induced ROS accumulation and cytotoxicity. Importantly, BPDE-induced transformation of HBECs was significantly reduced when RIP1 expression was suppressed. Altogether, these results strongly suggest an oncogenic role for RIP1, which promotes malignant transformation through protecting DNA-damaged cells against carcinogen-induced cytotoxicity associated with excessive ROS production.

Fig. 1.

Increased RIP1 expression in human lung cancer tissues, and cigarette carcinogen induces RIP1 expression in HBECs. (A), RIP1 expression was detected in a human non-small cell lung cancer tissue array by immunohistostaining. Representative images from normal lung, adenocarcinoma and squamous cell carcinoma are shown. (B) The summary of tumors with increased RIP1 expression. (C) RIP1 was detected in mouse tumors and matched lung tissues. The intensity of the individual bands was quantified by densitometry and normalized to the corresponding input control (β-actin) bands. Relative RIP1 expression was calculated with the respective lung tissues taken as 1. (D) RIP1 in lung tissues from six cigarette smoke and six control fresh air mice. Relative RIP1 expression levels shown were calculated as in (C). (E) HBEC-13 and HBEC-2 cells were treated with BPDE (0.2 μM) or CSE (10 μg/ml total particulate material) for indicated time points, RIP1 expression was detected by western blot. β-Actin was used as an input control.

Fig. 2.

RIP1 knockdown sensitizes HBECs to BPDE-induced cell cytotoxicity. (A and B) Cells were treated with increasing concentrations of BPDE for 36h. Cell death was detected by LDH release assay. Data shown are mean ± SD. **P < 0.05. Knockdown of RIP1 was confirmed by western blot. β-Actin was used as an input control.

Fig. 3.

RIP1 suppresses activation of MAPKs induced by BPDE. (A and B) RIP1 stable knockdown and control (negative shRNA transfected) cells were treated with BPDE (0.4 μM) for indicated times. The indicated proteins were detected by western blot. β-Actin was used as input control. (C and D) Cells were pretreated with indicated inhibitors (SP600125, 10 μM; SB203580, 10 μM; U0126, 10 μM) for 45min and then treated with BPDE (0.4 μM) for another 36h. Cell death was measured by LDH release assay. Data shown are mean ± SD. **P < 0.05. (E) Cells were pretreated with indicated inhibitors (SP600125, 10 μM; SB203580, 10 μM; U0126, 10 μM) for 45min, then treated with BPDE (0.4 μM) for an additional 4 h. The expression of indicated proteins was detected by western blot. β-Actin was used as the input control.

Fig. 4.

BPDE-induced intracellular ROS accumulation contributes to potentiated MAPK activation in RIP1 knockdown cells. (A and B) Cells were treated with BPDE (0.4 μM) for 2 h and incubated with 5-(and-6)-chloromethyl-2′,7′-dichlorodihydro fluorescein diacetate, acetyl ester (5 μM) for 30 min before being collected for ROS detection. Data shown are the mean ± SD. **P < 0.05. (C and D) Cells were pretreated with indicated ROS scavengers (BHA, 100 μM; NAC, 3mM) for 45min, then treated with BPDE (0.4 μM) for an additional 36h. Cytotoxicity was detected by LDH release assay. Data shown are the mean ± SD. **P < 0.05. (E) Cells were treated with BHA (100 μM) or NAC (3mM) for 45min followed by BPDE (0.4 μM) for another 4 h. The indicated proteins were detected by western blot. β-Actin was detected as the input control.

Fig. 5.

Reduced catalase expression and activity in RIP1 knockdown cells are involved in BPDE-induced cytotoxicity. (A) Catalase and manganese superoxide dismutase expression was detected by western blot. β-Actin was detected as the input control. (B) Catalase activity was detected in the indicated cells. Data shown are the average of triplicates and mean ± SD. **P < 0.05. (C) Cells were treated with exogenous catalase (250U/ml) and BPDE (0.4 μM) or remained untreated for 36h. Cell cytotoxicity was detected by LDH release assay. Data shown are the mean ± SD. **P < 0.05. (D) The cells were treated with catalase (250U/ml) and BPDE (0.4 μM) for 2 h. ROS was detected with fluorescence plate reader. Data shown are the mean ± SD. **P < 0.05.

Fig. 6.

JNK and ERK mediate catalase degradation in RIP1 knockdown cells. (A) Cells were treated with indicated inhibitors (U0126, 10 μM; SP600125, 10 μM; CSB203580, 10 μM) for 16h. The expression of catalase was detected by western blot. β-Actin was detected as the input control. (B) Left, cells were treated with cycloheximide (10 μg/ml) for the indicated times. Catalase and β-actin were detected by western blot. Right, quantification of the results in Left. The intensity of the individual bands was quantified by densitometry (NIH Image 1.62) and normalized to the corresponding input control (β-actin) bands. (C) HBEC-2 cells were treated with the proteasome inhibitor MG132 (10 μM) or lysosome inhibitor chloroquine (20 μM) for 16h. Catalase was detected by western blot. β-Actin was detected as the input control.

Fig. 7.

RIP1 knockdown suppresses BPDE-induced transformation in HBEC-13 cells. (A) Cells (1 × 10⁴) were seeded in 6-well plates and treated with BPDE (0.2 μM) every 2 days for 1 week or remained untreated, then seeded in soft agar and incubated for 2 weeks. Representative images are shown. (B) Quantitative representation of colony formation in soft agar. Bars show the averages of colony numbers of six randomly selected fields. Data shown are mean ± SD. **P < 0.01. (C) A model of RIP1 in BPDE-induced lung carcinogenesis. RIP1 expression is increased by cigarette smoke carcinogens, which stabilizes catalase, resulting in suppression of ROS accumulation and MAPK activation-mediated cytotoxicity in DNA-damaged bronchial epithelial cells. This process facilitates cell survival and contributes to malignant transformation.