Calpeptin Induces Apoptosis in A549 Non-Small Cell Lung Cancer Cells

Lung cancer is a leading cause of cancer-related death worldwide, and most are non-small cell lung cancers (NSCLC). Since the overall survival remains very poor for NSCLC patients with advanced-stage disease, the development of novel treatments is needed. Previous studies reported a relationship between calpain and tumorigenesis. In this study, we examined the apoptotic effects of calpeptin (Cal), a calpain inhibitor, on A549 NSCLC cells. We assessed whether Cal induced apoptosis in A549 cells. Cal induced apoptosis in A549 cells and also activated p38MAPK. These results suggest a possible clinical use of Cal for the treatment of NSCLC.


Introduction
Lung cancer is a leading cause of cancer-related death worldwide and is categorized into two histological subtypes; small cell lung cancer and non-small cell lung cancer (NSCLC). Furthermore, NSCLC are mainly adenocarcinomas. Although several treatments, such as platinum doublet chemotherapy and molecular target therapy, are clinically used for the treatment of NSCLC, the overall survival of NSCLC patients with advanced-stage disease or metastatic lesions remains very poor [1,2]. Therefore, the development of novel treatments for NCSLC is needed.
Calpain is a calcium-dependent intracellular cysteine protease, and the mammalian calpain family comprises of fteen gene products. Classical calpains, such as calpain-1 and calpain-2, are ubiquitously expressed, whereas others are expressed in speci c tissues such as the skeletal muscle and gastrointestinal tract. Calpain-1 and calpain-2 are also referred to as µ-calpain and m-calpain, respectively, because they require speci c calcium concentrations (in µM and mM amounts, respectively) for their activation. Calpain plays important roles in various cellular processes that include cell growth and cellular signaling [3]. Previous studies reported a relationship between calpain and tumorigenesis [4] in cancers such as schwannomas [5], colon cancer [6] and renal cell carcinoma [7].
We previously reported that calpeptin (Cal), an inhibitor of calpain-1 and calpain-2, prevents A549 NSCLC cell proliferation [8]. However, the relationship between calpain and the apoptosis of lung cancer cells have not yet been studied in detail. Therefore, we assessed whether calpeptin (Cal), an inhibitor of calpain-1 and calpain-2, exert apoptosis-inducing effects in A549 NSCLC cells. Cell apoptosis assay A549 cells were cultured with or without 100 nM Cal for 72 hours. After treatment, cytoplasmic extracts were prepared using the Nuclear Extract Kit (Active Motif, Carlsbad, CA), and protein concentrations in the cytoplasmic extracts were measured as previously described [9,10]. Cell apoptosis was evaluated using Cell Death Detection ELISA PLUS (Roche, Mannheim, Germany) according to the manufacturer's instructions.

Measurement of p38MAPK
A549 cells were cultured with or without 100 nM Cal for 1 hour. After treatment, cytoplasmic extracts were prepared and protein concentrations in the extracts were measured as previously described [9,10].

Statistical analysis
The results are shown as the mean ± SD. Statistical analyses were performed using the Bonferroni-Dunn multiple comparisons test.

Cal-induced apoptosis effects in A549 cells
We examined the effects of Cal on apoptosis in A549 cells. Apoptosis in A549 cells was induced by treatment of Cal after 72 h of culture (p<0.01) (Fig. 1A).

P38mapk Is Involved In Cal-induced Apoptosis In A549 Cells
In order to understand the mechanism underlying the induced effect of Cal on apoptosis in A549 cells, we examined whether any kinases are required for this process. We showed that the levels of cytoplasmic p38MAPK in cells were activated in the presence of Cal after 1 h of cultivation (p< 0.01, Fig. 1B). Cellular levels of cytoplasmic ERK1/2 were not affected by Cal (data not shown).

Discussion
In this study, we investigated the relationship between Cal and apoptosis in A549 cells and found that Cal induced apoptosis in these cells. We then attempted to elucidate the mechanism underlying the induction of A549 cell apoptosis by Cal.
The MAPK signaling pathway plays an important role in major cellular processes including proliferation and apoptosis. MAPKs consist of kinases such as p38MAPK and ERK1/2 [11,12], and the former is involved in inducing apoptosis [13]. Kong et al. reported that the activation of p38MAPK and suppression of ERK1/2 plays a central role in the apoptosis of diffuse large B cell lymphoma cells [14]. Furthermore, Ye et al. showed the induction of apoptosis by p38MAPK activation in A549 NSCLC cells [15]. In this study, we examined the relationship between Cal and MAPKs and showed that the levels of cytoplasmic p38MAPK, but not ERK1/2, in A549 cells were activated in the presence of Cal.
Idiopathic pulmonary brosis (IPF) occasionally occurs in patients with lung cancer (largely NSCLC) because of smoking history or complication such as emphysema [16][17][18]; however, the mechanism by which it occurs remains mostly unknown. Chemotherapy or radiation therapy are effective for NSCLC, however these therapies occasionally induce pulmonary brosis and so these are not selectable for NSCLC complicated with pulmonary brosis.
We previously reported that Cal prevented bleomycin-induced pulmonary brosis in mice [19]. Cal also decreases the expression of IL-6, TGF-β 1 , angiopoietin-1, and collagen type Iα1 mRNA in mouse lung tissues. In vitro studies showed that Cal decreased the production of IL-6, TGF-β 1 , and angiopoietin-1 as well as the synthesis of collagen by lung broblasts. Furthermore, Cal decreased IL-6-dependent proliferation and angiopoietin-1-dependent migration of lung broblasts. These may be possible mechanisms by which Cal suppresses pulmonary brosis.
We previously reported that Cal prevents A549 NSCLC cell proliferation [8]. Moreover, we showed that Cal induced apoptosis in A549 cells in this study, suggesting its clinical application in patients with NSCLC complicated with IPF.
In summary, we demonstrated the apoptosis-inducing effects of Cal in A549 cells which may have been due to the activation of p38MAPK. Although the precise cellular mechanism underlying the Cal-induced apoptosis of A549 cell is not fully understood, our results may lead to the development of novel strategies for the treatment of NSCLC.