Houttuynia cordata Thunb extract modulates G0/G1 arrest and Fas/CD95-mediated death receptor apoptotic cell death in human lung cancer A549 cells

Background Houttuynia cordata Thunb (HCT) is commonly used in Taiwan and other Asian countries as an anti-inflammatory, antibacterial and antiviral herbal medicine. In this study, we investigated the anti-human lung cancer activity and growth inhibition mechanisms of HCT in human lung cancer A549 cells. Results In order to investigate effects of HCT on A549 cells, MTT assay was used to evaluate cell viability. Flow cytometry was employed for cell cycle analysis, DAPI staining, and the Comet assay was used for DNA fragmentation and DNA condensation. Western blot analysis was used to analyze cell cycle and apoptotic related protein levels. HCT induced morphological changes including cell shrinkage and rounding. HCT increased the G0/G1 and Sub-G1 cell (apoptosis) populations and HCT increased DNA fragmentation and DNA condensation as revealed by DAPI staining and the Comet assay. HCT induced activation of caspase-8 and caspase-3. Fas/CD95 protein levels were increased in HCT-treated A549 cells. The G0/G1 phase and apoptotic related protein levels of cyclin D1, cyclin A, CDK 4 and CDK 2 were decreased, and p27, caspase-8 and caspase-3 were increased in A549 cells after HCT treatment. Conclusions The results demonstrated that HCT-induced G0/G1 phase arrest and Fas/CD95-dependent apoptotic cell death in A549 cells


Background
In Taiwan, 26 individuals per 100,000 died from lung cancer each year, based on reports from the "People Health Bureau of Taiwan". Surgery, radiotherapy, and chemo-therapy are used for treating lung cancer patients [1][2][3]. However, those treatments are not satisfactory. Induction of cell cycle arrest and/or apoptosis in lung cancer cells has been considered an influential treatment strategy [4][5][6]. Many researchers have focused on selectively killing cancer cells or reducing cell number through the induction of cell cycle arrest and apoptosis [6,7].

Preparation of HCT
Ethanol extract of Houttuynia cordata Thunb (yield: 6.73% of dry wt.) was obtained by 48 h incubation at room temperature. The ethanol extract was filtered through a 0.45 μm filter (Osmonics, Minnetonka, MN, USA), lyophilized and kept at 4°C. The dried extract was re-solubilized in PBS before use as previously described [32,33].

Cell culture
Human lung cancer A549 cells were obtained from the Bioresource Collection and Research Center (BCRC, Hsinchu, Taiwan), originally from the American Type Culture Collection (ATCC, USA). Cells were maintained in RPMI-1640 containing 100 mL/L FBS with 100,000 U/L penicillin and 100 mg/L streptomycin.

Cell viability
A549 cells were plated onto 96-well plates and incubated with HCT (0, 125, 250 and 500 μg/ml) for 24 and 48 h. MTT was added to each well then incubated for an additional 4 h at 37°C. The blue formazan product was dissolved in 100 μL of DMSO. The plates were read at O. D.570 nm using a spectrophotometric plate reader (Bio-Rad, Tokyo, Japan). The experiments were performed in triplicate (n = 3). Cell viability was calculated as O.D. of drug-treated sample/O.D. of none treated sample × 100% as previously described [32,35].

Cell cycle transition and apoptosis determination
For cell cycle and apoptosis determination, A549 cells were plated onto 24-well plates and incubated with HCT (0, 125, 250 and 500 μg/ml) for 24 h. Cells were fixed gently in 70% ethanol at 4°C and then re-suspended in phosphate-buffered saline (PBS) containing 40 μg/ml PI, 0.1 mg/ml RNase and 0.1% Triton X-100 for 30 min at 37°C. Cell cycle transition and apoptosis were then analyzed by flow cytometry (FACS Calibur™; Becton Dickinson, NJ, USA) as previously described [35].

DAPI staining
A549 cells were plated onto 24-well plates and treated with HCT (0 and 500 μg/ml) for 24 h. After HCT treatment, cells were fixed in 4% paraformaldehyde and then incubated with 1 μg/ml of DAPI staining solution in darkness. The apoptotic cells were observed by fluorescence microscopy (Zeiss, Oberköchen, Germany) as previously described [32,35].

Comet assay
A549 cells were plated onto 24-well plates and incubated with HCT (0 and 500 μg/ml) for 24 h, 1 × 10 4 cells were mixed with 150 μL 0.75% low-melting agarose held at 37°C and layered onto a pre-treated slide with 1.5% regular agarose. After agarose were solidified on a chilled plate, the slides were transferred to the same lysis buffer, held at room temperature for 4 h and stained with propidium iodide as previously described [36].

Western blotting
A549 cells were plated onto T-75 flasks and treated with HCT (0, 125, 250 and 500 μg/ml) for 24 h. Total cell lysates were prepared as previously described. Thirty μg of total protein applied to SDS-PAGE and transferred onto a polyvinylidene fluoride membrane (PVDF; Millipore). After blocking, the blots were incubated with the appropriate dilution of specific monoclonal antibodies for cyclin D1, cyclin A, CDK 4, CDK 2, p27, caspase-8 and caspase-3 (Santa Cruz Biotechnology, USA). Blots were washed and then incubated with horseradish peroxidaseconjugated secondary antibody (Santa Cruz Biotechnology, USA). Protein expressions were detected using enhanced chemiluminescence kits (Amersham, ECL Kits) as previously described [37].

Caspase-8 and caspase-3 activities assays
A549 cells were plated onto T-75 flasks and incubated with HCT (0, 125, 250 and 500 μg/ml) for 24 h. A549 cells were collected in a protein lysis buffer (50 mM Tris-HCl, 1 mM EDTA, 10 mM EGTA, 10 mM digitonin and 2 mM DTT). Cell lysates were centrifuged at 15,000 × g at 4°C and then incubated with caspase-3 and caspase-8 specific substrates (Ac-DEVE-pNA and Ac-IETD-pNA) with reaction buffer in a 96-well plate at 37°C for 1 h. Caspase activity was determined by measuring O.D. 405 nm of the released pN using a spectrophotometric plate reader (Bio-Rad, Tokyo, Japan) as previously described. The experiments were performed in triplicate [37].
Immunostaining assay for Fas/CD95 protein levels Fas/CD95 cell surface antigen expression was measured by flow cytometry. A549 cells were plated onto 24 well and treated with HCT (0, 125, 250 and 500 μg/ml) for 12 h. Cells were collected and rinsed in PBS. Fas/CD95 was analyzed by direct immune-fluorescence staining. FITC-conjugated anti-Fas/CD95 and its FITC-conjugated isotype mAb (BD Biosciences Pharmingen, San Diego, CA, USA) were analyzed using a flow cytometer as previously described [38].

Statistical analysis
The experiments were performed in triplicate (n = 3) and all data were expressed as the mean ± standard error. Student's t-test was used for single variable comparison. For multiple variable comparisons, data were analyzed by oneway ANOVA followed by Dunnett's test. P < 0.05 was considered significant.

Effect of HCT on cell-cycle distribution and apoptosis in A549 cells
To evaluate the effects of HCT on cell-cycle distribution and apoptosis, A549 cells were treated with HCT (0, 125, 250 and 500 μg/ml). Figure 2A and B showed that HCT caused an arrest of cell-cycle transition in the G 0 /G 1 phase, and the proportion of cells in the G 0 /G 1 phase increased markedly in a concentration-dependent manner.   Figure 2B, the cell cycle distribution of G 0 /G 1 by HCT (0, 125, 250 and 500 μg/ml) treatment was 43.48%, 45.06%, 58.59%, and 63.25%, respectively. In addition, A549 cells treated with HCT (0, 125, 250 and 500 μg/ml) showed a significant increase in the sub-G1 apoptotic cell population, and the apoptotic cell population was 4.53%, 13.07%, 18.77% and 31.89%, respectively ( Figure 3C). These results indicated HCT inhibited cell growth.

Effects of HCT on G 0 /G 1 phase-associated protein levels
We investigated the protein levels of the G 0 /G 1 phase. Figure 4 showed that HCT caused an increase in the protein level of p27 and decreased protein levels of CDK4, CDK2, Cyclin D1 and Cyclin A in A549 cells. HCT (0, 125, 250 and 500 μg/ml) treatment increased p27 expression ratio by 1, 1.8, 1.6, and 1.4, respectively.
Among the protein levels of G 0 /G 1 phase, decrease of CDK4 was the most significant one. HCT decreased protein levels of CDK4 by 0%, 20%, 70%, and 90%, respectively.

Effect of HCT on DNA condensation and damage in A549 cells
Effects of HCT on nuclear morphological change were examined by DAPI staining and DNA damage by the Comet assay. As shown in Figure 3A and B, cells exhibited nuclear shrinkage and DNA condensation after a 24 h-incubation with HCT (500 μg/ml) ( Figure 3A). Cells exposed to HCT had an increase in DNA damage as seen in the Comet assay ( Figure 3B), and the length of Comet tail was 2.39 fold increase ( Figure 3D). The results suggest that HCT induces morphological changes and DNA damage in A549 cells.

Effects of HCT on caspase-8 and caspase-3 activities
We determined if HCT would stimulate the caspase-9, caspase-8 and caspase-3 activity in A549 cells. Figure 5, panel A and panel B, show that HCT increased caspase-8 and caspase-3 activities in a concentration-dependent manner but caspase-9 activity was unaffected (data not shown). HCT (125, 250 and 500 μg/ml) treatment increased caspase-8 activity by 51.96%, 124.51%, and 208.82%, respectively; increased caspase-3 activity by 85.29%, 150%, and 256.86%, respectively. Our results showed that HCTinduced apoptosis was mediated through the activation of caspase-8 and caspase-3. Thus, we determined whether the Fas/CD95 (death receptor protein) contributes to HCTinduced apoptosis. Figure 6, panel A showed that Fas/CD95 protein level was increased in a concentration-dependent manner in A549 cells. Figure 6, panel B also showed that caspase-8 and caspase-3 protein levels increased in a concentration-dependent manner when A549 cells were treated with HCT. HCT (125, 250 and 500 μg/ml) treatment increased caspase-8 protein expression by 1.1, 1.6, and 2.1 fold, respectively; increased caspase-3 expression ratio by 2.2, 1.6, and 1.9 fold, respectively. To further verify the involvement of caspase-8 and caspase-3 in HCTinduced apoptosis, cells were pretreated with the caspase-8 inhibitor (z-IETD-fmk) and the caspase-3 inhibitor (z-DEVE-fmk). It can be seen in Figure 7, panels A and B showed that the caspase-3 and caspase-8 inhibitors inhibited the apoptotic effects of HCT.

Discussion
The death receptor apoptotic pathway has previously been proposed as an anti-cancer drug target in human lung cancer [39,40]. Traditional Chinese medicine (TCM) that could stimulate the death receptor apoptotic pathway should have therapeutic potential in human lung cancer treatment [41][42][43]. HCT has been used as TCM in Taiwan for many years [44]. The pharmacological activities of HCT include immuno-regulatory, anti-inflammatory, anti-micro bacterial, anti-viral, and anti-cancer effects [24,25,[27][28][29][30][31][32]. HCT was reported to be active against leukemia, colorectal cancer and HER2/neu-overexpressing breast cancer cells [30][31][32][33][34]. However, the anti-lung cancer effects have not been well-studied. We previously reported that 450 μg/ml of HCT had anti-cancer activity in human primary colorectal cancer cells from patients [33] and HT29 human colon adenocarcinoma cells [32]. In the present study, HCT had anti-lung cancer activity (Figure 1), and this activity was concentration-, and time-dependent. HCT contains quercetin and quercetin 3-β-D-glucoside  (isoquercitrin) which have anti-viral activity [28,45]. Chou SC, et. al. indicated that quercitrin and isoquercitrin isolated from methanolic extracts of Houttuynia cordata showed excellent DPPH radical scavenging activities with IC50 values of 31 μM and 63 μm, respectively [46]. Also, HCT contains chlorogenic acid [47] which has anti-leukemia effects. In this study, we suggested that ethanolic extract of HCT have anti-lung cancer activity.
Previous studies have shown that HCT induces human lymphoblastic leukemic Molt-4 cell death through an endoplasmic reticulum stress pathway [31]. Banjerdpongchai R, et. al. suggested that ethanolic extract of HCT induces human leukemic HL-60 and Molt-4 cell apoptosis through a mitochondrial apoptotic pathway [30].
Evidences showing the link between apoptosis and the cell cycle are compelling. Regulation of cell cycle traverse involves activations of cyclin-dependent kinases (CDKs). CDKs (e.g. CDK1, CDK2, CDK4/6) is paired with the cyclins (e.g. cyclins A, B, E, D 1-3 ) [48]. Activation of CDKs by cyclins leads to phosphorylation of the retinoblastoma protein (pRb) [49] and leads to diminished binding of pRb to the E2F transcription factor, which various genes necessary for cell cycle progression. The activities of CDKs are antagonized by CDK inhibitors. CDK inhibitors directly interact with CDKs and negatively regulate the activity of CDKs. p27 kip1 , belongs to the Cip/Kip (kinase inhibitor protein) family, inhibits most CDKs [50]. Dysregulation of cell cycle is one of the most potent stimuli for apoptosis induction [51]. p27 kip1 [52], cyclin D1 [53], has been shown to influence the apoptotic process. Direct inhibition of CDKs, induction of endogenous CDK inhibitors (e.g. p27 kip1 ) or down-regulation of cyclins required for CDK activation (e.g. cyclin D 1 ) are mechanisms of inhibition of cell cycle progression [48]. Our results showed that HCT increased p27 expression, decreased cyclin D1, cyclin A, CDK 4, and CDK 2 which leaded to cycle G 0 /G 1 arrest. HCT induced apoptosis through caspase8/caspase-3 activation (Figure 5A,B) and up-regulated the protein levels of Fas/CD95 ( Figure 6A), caspase-8 and caspase-3 ( Figure 6B), while HCT did not affect the caspase-9 activity (data not shown) in A549 cells. In addition, HCT induced growth inhibition was significantly attenuated by the specific caspase-3 and caspase-8 inhibitors ( Figure 7A, B). One interpretation of our findings is that HCT induces A549 cell apoptosis and activates caspase-8, and −3 through the Fas/CD95-mediated death receptor apoptotic pathway.

Conclusions
In conclusion, this study demonstrated that HCT has antilung cancer activity by modulating G0/G1 arrest and stimulating the Fas/CD95 protein level, which leads to caspase-8 and caspase-3 activation resulting in the induction of apoptosis in human lung cancer A549 cells (Figure 8).