Cyclophilin A inhibits A549 cell oxidative stress and apoptosis by modulating the PI3K/Akt/mTOR signaling pathway

Abstract The excessive and inappropriate production of reactive oxygen species (ROS) can cause oxidative stress and is implicated in the pathogenesis of lung cancer. Cyclophilin A (CypA), a member of the immunophilin family, is secreted in response to ROS. To determine the role of CypA in oxidative stress injury, we investigated the role that CypA plays in human lung carcinoma (A549) cells. Here, we showed the protective effect of human recombinant CypA (hCypA) on hydrogen peroxide (H2O2)-induced oxidative damage in A549 cells, which play crucial roles in lung cancer. Our results demonstrated that hCypA substantially promoted cell viability, superoxide dismutase (SOD), glutathione (GSH), and GSH peroxidase (GSH-Px) activities, and attenuated ROS and malondialdehyde (MDA) production in H2O2-induced A549 cells. Compared with H2O2-induced A549 cells, Caspase-3 activity in hCypA-treated cells was significantly reduced. Using Western blotting, we showed that hCypA facilitated Bcl-2 expression and inhibited Bax, Caspase-3, Caspase-7, and PARP-1 expression. Furthermore, hCypA activates the PI3K/Akt/mTOR pathway in A549 cells in response to H2O2 stimulation. Additionally, peptidyl-prolyl isomerase activity was required for PI3K/Akt activation by CypA. The present study showed that CypA protected A549 cells from H2O2-induced oxidative injury and apoptosis by activating the PI3K/Akt/mTOR pathway. Thus, CypA might be a potential target for lung cancer therapy.


Introduction
Chronic enhanced oxidative stress is a pathogenic feature of most chronic diseases, such as cancer and diabetes, as well as pulmonary, cardiovascular, kidney, and neurodegenerative diseases [1,2]. Oxidative stress usually arise from the excessive accumulation of reactive oxygen species (ROS), which include hydrogen peroxide (H 2 O 2 ), superoxide anions, hydroxyl radicals, and singlet oxygen [3]. Elevated levels of ROS-induced oxidative stress can induce cancer cell death [4][5][6]. To prevent excessive intracellular ROS, cancer cells have been found to maintain a redox balance by increasing their antioxidant potential [7][8][9].
In the present study, we report that CypA inhibits H 2 O 2 -induced oxidative damage and apoptosis in human lung carcinoma A549 cells. Furthermore, we demonstrate that extracellular CypA facilitates Bcl-2 expression and inhibits Bax, Caspase-3, Caspase-7, and PARP-1 expression. Finally, we show that CypA causes activation of the PI3K/Akt/mTOR pathway in A549 cells in response to H 2 O 2 stimulation. Therefore, our present study identifies an important role for CypA in oxidative damage and apoptosis, which makes CypA a new potential target for lung cancer therapy.

Cell viability analysis
Cell viability was measured with a CCK-8 assay. Briefly, A549 cells were seeded in 96-well plates and pretreated with human recombinant CypA (hCypA) at various concentrations (100-1000 ng/ml) and then treated with 200 μmol/l H 2 O 2 . After treatment, 10 μl CCK-8 solution (C0037; Beyotime Institute of Biotechnology, Haimen, China) was added to each well and incubated at 37 • C for 4 h. Then, the optical density of each well was recorded using a microplate reader (Bio-Tek, Winooski, VT, U.S.A.) at 450 nm.

Measurement of the content of LDH and MDA
A549 cells were seeded in 96-well plates and cultured for 24 h. Then, the cells were treated with hCypA at different concentrations ranging from 100 to 1000 ng/ml for 24 h. At the end of treatment, lactate dehydrogenase (LDH) activity and lipid peroxidation in a culture medium were determined using an LDH cytotoxicity assay kit (91963; Sigma-Aldrich, St. Louis, MO, U.S.A.) and malondialdehyde (MDA) ELISA (S0131S; Beyotime Institute of Biotechnology, Haimen, China) according to the manufacturer's instructions.

Detection of Caspase-3 activity
The activity of Caspase-3 was determined using a Caspase-3 Activity Kit (C1115; Beyotime Institute of Biotechnology, Haimen, China). A549 cells with different treatments were lysed and incubated with 2 mM Ac-DEVD-pNA at 37 • C for 4 h. Samples were measured with an ELISA reader at an absorbance rate of 405 nm. The analysis procedure was detailed in the manufacturer's protocol.

Western blot analysis
The A549 cells were lysed. Then, the proteins were quantified using a BCA assay (23225, Thermo Scientific). An equal amount of protein samples was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electro-transferred to 0.22 μm polyvinylidene difluoride membranes (Millipore, HATF09025). The membranes were blocked with 5% BSA and incubated with primary antibodies at 4 • C overnight. The membranes were then incubated with a secondary antibody (Jackson, 1:10,000) for 1 h at room temperature followed by three 10 min washes in TBST. The bands were visualized using an enhanced chemiluminescence (ECL) system. Data within a linear range were quantified using the ImageJ Launcher software (National Institutes of Health).

Statistical analyses
Statistical analyses were performed using Prism 5 software (GraphPad Software, San Diego, CA). Statistics were calculated using Student's t-test. P<0.05 was considered statistically significant.

CypA improved cell viability in H 2 O 2 -stimulated A549 cells
To investigate whether CypA can be induced by H 2 O 2 stimulation, quantitative real-time PCR analysis was done using mRNA extracted from the human lung carcinoma (A549) cell line exposed to H 2 O 2 for the indicated periods of time. The CypA transcripts increased after 3 h incubation and kept increasing up to 24 h under H 2 O 2 -stimulated conditions ( Figure 1A). Next, an immunoblotting analysis was done using the whole cell lysate from A549 exposed to H 2 O 2 for the indicated periods of time. As shown in Figure 1B, a rapid increase in the CypA protein level was observed. To detect the cytotoxicity effect of human recombinant CypA (hCypA) on A549 cells, A549 cells were treated with hCypA at different concentrations ranging from 100 to 1000 ng/ml for 24 h. The LDH cytotoxicity assay showed that hCypA did not exhibit a cytotoxicity effect on A549 cells even at a concentration of 500 ng/ml ( Figure  1C). Therefore, we selected the concentrations of 100, 200, and 500 ng/mL for the following experiments. Then, we used H 2 O 2 to stimulate oxidative injury in A549 cells. A549 cells were pretreated with 500 ng/ml of hCypA for the indicated periods of time and then stimulated with H 2 O 2 for 24 h. As shown in Figure 1D, compared with the control group, the cell viability in the H 2 O 2 stimulation group was markedly reduced. However, treatment with hCypA at 1 h dramatically increased cell viability. Additionally, treatment with hCypA (100, 200, and 500 ng/ml) gave rise to a dose-dependent increase in cell viability ( Figure 1E).

CypA suppressed oxidative stress in H 2 O 2 -stimulated A549 cells
The production of ROS and MDA and the activities of SOD and GSH-Px were examined as markers of oxidative stress, especially by H 2 O 2 . As shown in Figure 2A, intracellular ROS production was remarkably increased in the A549 cells exposed to H 2 O 2 . However, this increased ROS production was attenuated by hCypA in a dose-dependent manner. We also obtained similar results with other lung cancer cell type, such as CaLu-3 stimulated by H 2 O 2 ( Figure 2B). Importantly, ELISA showed that increased hCypA treatment reduced the MDA level and enhanced SOD, GSH-Px, and GSH activities compared with the H 2 O 2 stimulation group ( Figure 2C-F). Previous studies discovered that GSH was depleted by inhibition of GSH synthetase using buthionine sulfoximine (BSO) [32,33]. Whereas hCypA treatment increased GSH activities compared to the BSO stimulation group ( Figure 2G). Collectively, CypA inhibited oxidative stress in H 2 O 2 -stimulated A549 cells.

CypA inhibited cell apoptosis in H 2 O 2 -stimulated A549 cells
We next identified the effect of CypA on Caspase-3 activity in the H 2 O 2 -stimulated A549 cells. As shown in Figure  3A, Caspase-3 activity significantly enhanced the H 2 O 2 -stimulated A549 cells compared with the control cells, while Caspase-3 activity was restored through treatment with hCypA. Furthermore, we observed similar results of Caspase-3 activity in CaLu-3 stimulated by H 2 O 2 ( Figure 3B). Subsequently, Western blot detected the expression levels of apoptosis-related genes. We observed elevated Bcl-2 levels but also a reduction in the expression of Bax, Caspase-7, and Caspase-3 due to hCypA in H 2 O 2 -stimulated A549 compared with the H 2 O 2 stimulation group ( Figure 3C-G). To further confirm that hCypA induces the activation of Caspase-3, the cleavage of PARP was also examined by Western blot. Consistently, hCypA treatment caused the concentration-dependent proteolytic cleavage of PARP-1 ( Figure 3C,H). These data indicated that CypA inhibited cell apoptosis in H 2 O 2 -stimulated A549 and CaLu-3 cells.

CypA induced the PI3K/Akt/mTOR pathway in H 2 O 2 -stimulated A549 cells
The down-regulation of the activation of Caspase-7 and the inactivation of the Bcl-2 family members strongly suggest the involvement of the PI3K/Akt/mTOR pathway. We, therefore, tested the expression of p-PI3K, p-Akt, and p-mTOR in H 2 O 2 -stimulated A549 cells via Western blot analysis. As shown in Figure 4A, exposure to H 2 O 2 resulted in a considerable reduction in p-PI3K, p-Akt, and p-mTOR levels. However, hCypA-pretreated A549 cells presented a dramatic increase in the protein expressions of p-PI3K, p-Akt, and p-mTOR expressions ( Figure 4B-D).

Inhibition of PI3K/Akt reversed the protective effects of CypA on A549 cells
Next, a PI3K/AKT-inhibitor (LY294002) was used to block activation of the PI3K/AKT signaling pathway in A549 cells. The increased cell viability caused by hCypA (500 ng/ml) was inhibited by LY294002 ( Figure 5A). In addition, LY294002 treatment advanced ROS production and Caspase-3 activity compared with the hCypA-pretreated A549 cells ( Figure 5B,C). Collectively, these results clearly indicated that the PI3K/Akt/mTOR pathway mediated the protective effects of hCypA on H 2 O 2 -stimulated A549 cells.

Peptidyl-prolyl isomerase activity is required for CypA-induced PI3K/Akt/mTOR activation
CypA belongs to the immunophilin family, whose members possess peptidyl prolyl cis-trans isomerase (PPIase) activity. CsA is an immunosuppressive drug that powerfully inhibits the PPIase activity of CypA. To investigate whether PPIase activity is required for CypA-induced PI3K/Akt/mTOR activation, hCypA was incubated with CsA and then applied to A549 cells. As shown in Figure 6A, CsA inhibited the hCypA-induced proliferation of A549 cells, while the attenuated ROS production was restored by CsA ( Figure 6B). Moreover, we observed that although a mutant R55A-CypA is 100-fold less active as a PPIase [34], it failed to attenuate ROS production ( Figure 6C). Importantly, CsA significantly inhibited PI3K/Akt/mTOR activation by H 2 O 2 ( Figure 6D-G). Taken together, these results indicate that PPIase activity is involved in PI3K/Akt activation by CypA.

Discussion
Oxidative stress has been postulated to be key a determinant of cancer-related death [35][36][37]. CypA is overexpressed in various cancer types and is associated with tumor invasion, metastasis, and chemoresistance [18,[38][39][40][41][42]. However, the roles of CypA in cancer and in regulating the survival and growth of cancer cells remain obscure. In our study, the mRNA and protein levels of CypA were induced by H 2 O 2 inA549 cells, suggesting that the induction of CypA can be triggered by H 2 O 2 stimulation and that CypA is involved in cellular oxidative stress.
Increasing evidence is demonstrating that CypA has the capacity to enhance endogenous antioxidant enzymatic activities. For instance, overexpressed CypA markedly reduces ROS produced by cyclosporin A [25]. It has also been reported that CypA can be secreted in response to ROS in vascular smooth muscle cells [20]. Thus, it appears that the antioxidant role of CypA is at least partly crucial for the cytoprotection of overexposed CypA against H 2 O 2 treatment. In the present study, we found that CypA significantly improved cell viability of A549 cells, decreased ROS and MDA production, and enhanced SOD, GSH and GSH-Px activities, implying that CypA attenuated oxidative injury in H 2 O 2 -induced A549 cells.
It has been reported that ROS-mediated oxidative stress may lead to apoptosis mediated by the mitochondrial, death receptor, and endoplasmic reticulum pathways [43][44][45][46]. In the mitochondrial pathway, one of the first steps is increasing the permeability of the outer mitochondrial membrane, which is regulated by Bcl-2 family members [47,48]. Thereafter, cytoplasmic Caspase signaling is activated, which contributes to the execution of apoptosis [3,49]. Our results showed that CypA treatment caused an increase in Bcl-2 and a decrease in Bax, Caspase-7, Caspase-3, as well as PARP-1. These findings suggest that CypA can protect A549 cells from H 2 O 2 -induced oxidative injury and apoptosis.
In many cancers, including lung carcinoma, the PI3K/AKT/mTOR pathway is hyperactive, thus blocking apoptosis through the regulation of downstream signaling molecules, such as inhibiting the activation of Caspase-7, as well inactivating Bcl-2 family members [50,51]. Emerging evidence has demonstrated that activation of the PI3K/Akt signaling pathway protects A549 cells from oxidative stress and apoptosis [52][53][54]. Intriguingly, a previous study reported that extracellular CypA promotes platelet adhesion via cluster of differentiation 147 (CD147)-mediated PI3K/Akt-signaling [55]. In the present study, we demonstrated that CypA caused a dramatic activation of the PI3K/Akt/mTOR pathway in A549 cells in response to H 2 O 2 stimulation. Moreover, the inhibition of PI3K/Akt/mTOR blocked the protective effects of CypA on H 2 O 2 -induced oxidative injury and apoptosis in A549 cells, which suggests that the cytoprotective effects of A549 are mediated by the PI3K/Akt/mTOR signaling pathway. We also found that the cytoprotection of CypA might be dependent on its PPIase activity because the CsA, which is a well-known inhibitor of PPIase activity in CypA, also aggravates ROS generation and inhibits activation of the PI3K/Akt/mTOR pathway. Consistent with our proposal, CsA has been reported to have chemotherapeutic effects in various cancer cells, including non-small cell lung cancer [56,57]. Hence, the molecular mechanism of CypA as an antioxidant should be investigated in future studies.
In conclusion, our present study demonstrate that CypA has protective effects on H 2 O 2 -induced oxidative injury and apoptosis in A549 cells. On the one hand, these protective effects may be mediated by activation of the PI3K/Akt/mTOR signaling pathway. On the other hand, the cytoprotective role of CypA seems to depend upon its PPIase activity. Hence, the PPIase activity of CypA may be a potent chemotherapeutic target for cancer therapy. Our results indicate that CypA might be a potential therapeutic strategy against solid tumors.

Data Availability
All data included in the present study are available from the corresponding author on reasonable request.