Radix Tetrastigma Hemsleyani Flavone Induces Apoptosis in Human Lung Carcinoma A 549 Cells by Modulating the MAPK Pathway

Lung cancer is the leading cause of cancer deaths throughout the world. Approximately 80-85% of all lung cancers are classified as non-small cell lung cancer (NSCLC), Current therapies include active surveillance, surgery, radiation therapy, chemotherapy and immunotherapy. Chemotherapy still plays an important role in this setting, however, these drugs are highly toxic with a low survival profile (De et al., 2006). Therefore, it is imperative to search for chemotherapeutic agents with high effectiveness and low adverse reactions. In the past few decades, traditional Chinese herbal medicine has begun to attain great popularity for disease prevention as well as being used as complementary medicines for a variety of diseases worldwide. Many scientists have focused on the potential roles of extracts of traditional herbs as alternative and complementary medications for cancer treatment. Herb extracts are often administered together with conventional cancer therapies to increase survival rates and enhance the quality of patient life (Li et al., 2009; Messina et al., 2011; Liu et al., 2012).


Introduction
Lung cancer is the leading cause of cancer deaths throughout the world.Approximately 80-85% of all lung cancers are classified as non-small cell lung cancer (NSCLC), Current therapies include active surveillance, surgery, radiation therapy, chemotherapy and immunotherapy.Chemotherapy still plays an important role in this setting, however, these drugs are highly toxic with a low survival profile (De et al., 2006).Therefore, it is imperative to search for chemotherapeutic agents with high effectiveness and low adverse reactions.In the past few decades, traditional Chinese herbal medicine has begun to attain great popularity for disease prevention as well as being used as complementary medicines for a variety of diseases worldwide.Many scientists have focused on the potential roles of extracts of traditional herbs as alternative and complementary medications for cancer treatment.Herb extracts are often administered together with conventional cancer therapies to increase survival rates and enhance the quality of patient life (Li et al., 2009;Messina et al., 2011;Liu et al., 2012).

Radix Tetrastigma Hemsleyani Flavone Induces Apoptosis in Human Lung Carcinoma A549 Cells by Modulating the MAPK Pathway
Liang-Rui Zhong 1 , Xian Chen 2 , Ke-Min Wei 3 * Radix Tetrastigma Hemsleyani (RTH) is a traditional Chinese herb endowing with detoxification, antiinflammation, antivirus and anticancer bioactivities.Flavone is the major composition extracted from the Chinese herb RTH, which has now been shown that Radix Tetrastigma Hemsleyani Flavone (RTHF) induces growth inhibition and apoptosis in various cancer cells including the human liver carcinoma cell line HepG2, gastric carcinoma cell line SGC-7901 and leukemic cell line K562 (Ding et al., 2005;Feng et al., 2006;Xu et al., 2010).It also can inhibit tumor growth (Ni et al., 2008;Li et al., 2012).However, the mechanism is not well described.This study investigated how RTHF affects cell growth and apoptosis of A549 human NSCLC cells.The anticancer mechanism of RTHF was also elucidated by analyzing apoptosis in A549 cells through activation of caspases and Bcl-2 family proteins and MAPKs pathways.

Materials and reagents
Radix Tetrastigma Hemsleyani was purchased from

Cell line and culture
Human lung cancer A549 cells obtained from Zhejiang Province Academy of medical science, China and cultured in RPMI 1640 medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) and 100 IU/ ml penicillin, 100 mg/ml streptomycin, incubated in 5% CO 2 incubator at 37℃.Cells were passaged every 2-3 days and used at about 80-90% of the confluence.

MTT assay
The cell proliferation was measured by MTT method.A549 cells were seeded in 96-well plates at a density of 5×10 3 cells/well, treated with various concentrations of RTHF (0, 0.5, 1, 5 and 10 mg/mL).After incubation for 24 h, 48 h or 72 h, discard medium and replaced with fresh medium (180 μl/well).20 μl of MTT solution (5 mg/ml) added to each well and the plates were incubated for additional 4 h at 37℃.Then, discard the medium and 150 μl of DMSO were added to each well.The absorbance was read at 570 nm as test wavelengths using a microplate reader (Thermo Scientific, USA).Cell viability was calculated as percentage of viable cells in total population.

Cell morphological changes
Cells were treated with RTHF (0, 1, 5 and 10 mg/mL) for 48 h, then morphological changes of the cells were observed under a inverted microscope (Olympus IX71, Japan), recorded with a CCD camera (Olympus DP72, Japan).

Nuclear staining with Hoechst 33258
A549 cells were seeded in 6-well plates, and incubated with various concentrations of RTHF (0, 1, 5 and 10 mg/ mL)for 48 h.Cells were fixed and stained with DNA fluorochrome Hoechst 33258 for 20 min.After washed with PBS, the morphological features of apoptosis (including cellular nucleus shrinkage, chromatin condensation, intense fluorescence and nuclear fragmentation) were monitored by fluorescence microscopy (Olympus IX71, Japan), recorded with a CCD camera (Olympus DP72, Japan).

Flow cytometry analysis of apoptosis
A549 cells were cultured in 6-well plates, incubated overnight, and then exposed to different concentrations of RTHF (0, 1, 5 and 10 mg/mL)for 48 h.cells were harvested by trypsin and washed with PBS.The cells were resuspended in binding buffer and then stained sequentially with Annexin V-FITC and PI as manufacture's instructions.The apoptotic cells were analyzed using a flow cytometry (BD FACSCalibur).

Western blot analysis
After treatments, A549 cells were harvested, and homogenized in 200 μl RIPA lysis buffer.then were extracted and the protein concentration was determined by Lowry method.Protein lysates (40 μg) from each sample were subjected to SDS-PAGE on 10% acrylamide gel and the separated proteins transferred to a PVDF membrane.After transfer, the membranes were blocked with 5% non-fat dry milk in TBS for 1 h at room temperature, then incubated with primary antibodies overnight at 4°C followed by secondary horseradish peroxidase-labeled antibody (1:2000).The bound antibodies were visualized using the ECL blotting detection system.

Statistical analysis
The data were expressed as means ± SD and were analyzed by one-way analysis of variance (ANOVA) by using SPSS software (version 13.0).P<0.05 was considered statistically significant.

RTHF induced cell morphological changes of A549 cells
A549 cell morphological changes were observed under a inverted microscope.Cells were treated with various concentrations of RTHF (0, 1, 5 and 10 mg/mL) for 48 h.As compared to control group, treated group showed a  significant decrease in adherent cells which accompanied an increase in floating cells in culture medium.This study also revealed that the RTHF treated A549 cells acquired a round and shrunken shape significantly in contrast to normal polyclonal structure of normal cells (Figure 2).

RTHF induced apoptosis of A549 cells
Apoptotic cell is characterized by major features like DNA fragmentation and loss of plasma membrane integrity.Morphologic changes in the nucleus representing cellular apoptosis were assessed by staining with the membrane-permeable DNA binding dye Hoechst 33258.As showed in Figure 3(A), A549 cells were treated with different concentrations of RTHF (0, 1, 5 and 10 mg/mL) for 48 h, normal cells exhibited regular and round shaped nuclei with a pallid blue, whereas apoptotic cells were characterized by the condensation and the fragmentation of nuclei with bright fluorescence.To further investigate RTHF-induced apoptotic effect, Flow cytometry analysis was used to quantify the apoptotic A549 cells were double stained with Annexin V-FITC and PI after treatment with different concentrations of RTHF (0, 1, 5 and 10 mg/mL) for 48 h.The proportion of apoptotic cells was increased from 3.45 ± 2.61% to 66.80 ± 0.98% (Figure 3B).The results revealed that RTHF induced apoptosis in A549 cells in a dose-dependent manner (p<0.01).

Effect of RTHF on expressions of Bcl-2/Bax and caspase family proteins
To further examine the possible mechanism of RTHFinduced apoptosis, we tested the protein expressions of Bcl-2, Bax, pro-caspase3/9, cle-caspase3/9, PARP.Western blotting analysis revealed that the Bax expression was obviously increased, whereas the Bcl-2 expression was decreased.In addition, there was significantly decrease in the pro-caspase3/9, pro-PARP expression and increase in the cle-caspase3/9, cle-PARP expression as compared to control (Figure 4A).These results indicated that RTHF up-regulation the Bax/Bcl-2 ratio and cle-caspase3/9, cle-PARP expression in a dose-dependent manner.

Effects of RTHF on MAPKs pathway
In order to better elucidate the molecular basis of RTHF induced apoptosis, we tested the expression of p-p38, p-ERK, p-JNK.The results revealed that RTHF increased the protein levels of p-p38, and decreased p-ERK via a dose-dependent manner, meanwhile little change of p-JNK (Figure 4B).

Discussion
Herbal medicines have been proven to be a major source of novel agents with various pharmaceutical activities (Mishra et al., 2011;De et al., 2012;Mondal et al., 2012).Natural products have provided a rich source of drugs for many diseases, including cancer.Radix Tetrastigma Hemsleyani (RTH) is a traditional Chinese herbal medicine have long been used in China in medical practice widely.Flavone is the major composition extracted from RTH (RTHF) have the functions of improving immune response, anti-inflammation and anticancer.In this study, we used RTHF to interfere in the growth of lung carcinoma A549 cell lines in vitro.The results showed that RTHF inhibited the proliferation of A549 cells in a dose-and time-dependent manner, meanwhile, the apoptotic rates of RTHF group showed statistically significance compared to control group.
Apoptosis is an important regulator in developmental processes, maintenance of homeostasis and elimination of the damaged cells.Generally, apoptosis pathways includes signals through the death receptor (extrinsic) or the mitochondria (intrinsic) pathway (Denault et al., 2004), The intrinsic pathway of apoptosis is regulated by the Bcl-2 family proteins.A balance between pro-apoptotic (BAX, BID, BAK or BAD) and anti-apoptotic (Bcl-XL, Bcl-2, BCLW or MCL1) proteins of the Bcl-2 family controls the mitochondrial apoptosis pathway (Zinkel et al., 2006).Many anticancer agents induced apoptosis by targeting the proteins of Bcl-2 family and the ratio of Bax/Bcl-2 played a critical role in apoptosis of cells (Gupta et al., 2002;Emi et al., 2005).The caspase family is an executioner of apoptosis, in which caspase-3 is the key point (Heimlich et al., 2004).Mitochondrial proteins, such as cytochrome c, which bind with Apaf-1 and procaspase-9 in a dATPdependent manner to form the apoptosome (Mignotte et al., 1998).The apoptosome can induce activation of caspase-9, thereby initiating apoptotic caspase cascades (Hengartner., 2000;Sun et al., 2004).Sequential activation of caspase-9 and caspase-3 in the intrinsic pathway results in PARP cleavage, leading to apoptosis (Shi, 2002).Thus caspases have been shown to be activated during apoptosis in many cells and play critical roles in both initiation and execution of apoptosis (Liu et al., 2009).In this study, we examined the effects of RTHF on expression of Bax, Bcl-2 and caspases proteins using Western blot analysis, and found that RTHF increased Bax, cle-caspase-3/-9 and cle-PARP expression and decreased Bcl-2, pro-caspase-3/-9 and pro-PARP expression, leading to up-regulate the ratio of Bax/Bcl-2 and inducing caspase-dependent apoptosis.This might be one of the molecular mechanisms in which RTHF induces apoptosis in A549 cells.
MAP kinase cascades consist of three core protein kinases such as ERK1/2, p38, and JNK pathways (Pearson et al., 2001;Raman et al., 2007).They are activated by phosphorylation induced by various mitogens and cell stressors, including osmotic stress, heat shock and proinflammatory cytokines.MAPK signaling pathways can induce cell proliferation, differentiation and apoptosis (Johnson et al., 2002).The expression of ERK is known as a prominent feature in many human cancers including nonsmall cell lung cancer (Lee et al., 2002).ERK can antagonize apoptosis by up-regulating pro-apoptotic Bcl-2 proteins such as Bax (Kim et al., 2005).The p38 and JNK MAPK pathways are activated by a variety of cell stressors, including ultraviolet light (UV), radiation, cytotoxic drugs, and cytokines such as tumor necrosis factor alpha and interleukin, and related to the occurrence, development and apoptosis of tumor cells.
Thus, to further understand the molecular mechanism of RTHF, the potential involvement of MAPK pathway in RTHF induced apoptosis was investigated by western blotting.This study showed that ERK and p38 phosphorylation in A549 cells was found enhanced in response to RTHF treatment, which plays an important role in apoptotic signaling through regulating the activities of Bcl-2 family proteins and mediating caspase activation (Tournier et al., 2000).
In conclusion, this study demonstrates that RTHF significantly inhibited proliferation and induced apoptosis in human lung cancer A549 cells, which is associated with activating of MAPKs pathways, then increased Bax/Bcl-2 ratio and caspases proteins expression.Therefore, RTHF may as a promisingly effective chemopreventive agent against lung adenocarcinoma.

Figure 1 .Figure 3 .
Figure 1.Proliferative Inhibition Effect of RTHF on A549 cells.Cell survival rates were measured using MTT assay.The cells were treated with various concentrations of RTHF for 24 h,48 h and 72 h.All data represent the mean values ±SD of at least three independent experiments.*p<0.01 vs. control group