JIN Formula Inhibits Tumorigenesis Pathways in Human Lung Carcinoma Cells and Tumor Growth in Athymic Nude Mice

cancers by treating early steps in lung carcinogenesis. There is a convergence of basic scienti ﬁ c and clinical ﬁ ndings in lung cancer chemoprevention. Pharmacological interventions also can be used to reverse or arrest the progression of lung carcinogenesis. For this reason, additional clinical trials are needed that emphasize a mechanistic approach in which mechanisms identi ﬁ ed in vitro can be validated in vivo. This study was designed to show the chemopreventive/chemotherapeutic potential of JIN formula against lung cancer. we assessed the efficacy of the JIN formula, which is an ancient herbal formula from classical book JIN KUI YAO LUE (Golden Chamber) for the treatment of lung cancer and explored its probable molecular mechanisms. Initially, employing human lung carcinoma A549 cells, we compared the growth inhibitory effects of JIN formula extracts (JFE ， including E1 to E8). The results showed that E6 and E7 could decrease the viability of A549 cells by the MTT assays. To explore the mechanism of antiproliferative effects of JIN formula against lung cancer, we investigated the involvement of Akt, ERK1/2, JNK1/2, p38 and NF- κ B pathway during the inhibition of tumorigenesis by JFE in A549 and H157 cells. The immunoblot analysis demonstrated that JFE treatment could result in different degree inhibition of above signaling pathways on tumor promotion processes. Oral administration of JIN formula (3%, wt/vol) to athymic nude mice implanted with A549 and H157 cells resulted in a signi ﬁ cant inhibition in tumor growth. In conclusion, the present study demonstrates that human non small cell lung cancer A549 and H157 cells are highly sensitive to JIN formula both in vitro and in vivo experimental models. JIN formula inhibits the development of lung tumorigenesis by modulating the imbalance of

We extracted JIN formula with different solvents, hereafter referred to as JIN formula extracts (JFE). It is hypothesized that JFE may afford chemopreventive as well as chemotherapeutic effects against lung cancer. In the present study, we first demonstrated the antiproliferative effects of JFE (including E1 to E8) in A549 cells. This involves the tumorigenesis network of cells. Next, we determined the chemopreventive potential of JFE on regulation of PI3K/Akt, MAPK, NF-kB pathways in A549 and H157 human lung carcinoma cells. Based on the results of our in vitro data, we next carried out in vivo study in mice. We found that oral administration of a human acceptable dose of JIN formula (3%, wt/vol) to athymic nude mice implanted with A549 and H157 cells resulted in significant inhibition of tumor growth.

Materials
Akt, JNK, and p38 antibodies were obtained from Cell Signaling Technology. The polyclonal antibodies NF-kB/p65, ERK1/2 were procured from Santa Cruz Biotechnology Inc. Antimouse and anti-rabbit secondary antibody horseradish peroxidase (HRP) conjugate was obtained from Amersham Life Science Inc.

Methods 2.2.1 Preparation of JFE
Fresh JIN formula was decocted in distilled water. As shown in Figure 1, different solvents were applied to acquire the following eight extracts: Water extraction concentrate (E1), Ethanol precipitation (E2), Precipitates in ethanol recovery (E3), Ethanol concentration (E4), Cyclohexane extract (E5), Ethyl acetate extract (E6), N-butanol extract (E7) and Water solution (E8). The extracts were condensed and freeze dried. The freeze-dried extracts were stored at 4°C to be used for various treatments.

Cell culture and treatment
The human lung carcinoma A549 and H157 cells were obtained from American Type Culture Collection and cultured in DMEM medium, supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin (P-S) in a 5% CO 2 atmosphere at 37°C. The extracts were dissolved in dimethyl sulfoxide (DMSO) and were used for the treatment of cells. A total of 50-60% confluent cells were treated with the extracts (1-100 µg/ml) for 72 h in complete growth medium.

Cell viability (MTT assay)
The effect of JFE on the viability of cells was determined by 3-[4,5-dimethylthiazol-2-yl]-2,5diphenyl tetrazoliumbromide assay. The cells were plated at 1×10 4 cells/well in 200 µl of complete culture medium containing 1-100 µg/ml concentrations of JFE in 96-well microtiter plates for 72 h. After incubation for specified times at 37°C in a humidified incubator, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazoliumbromide (5 mg/ml in PBS) was added to each well and incubated for 4 h, after which the plate was centrifuged at 1800g for 5 min at 4°C. The absorbance was recorded on a microplate reader at the wavelength of 540 nm. The effect of JFE on growth inhibition was assessed as percent cell viability where DMSO-treated cells were taken as 100% viable. DMSO at the concentrations used was without any effect on cell viability.

Protein extraction and western blotting
Following the treatment of cells as described above, the media was aspirated, the cells were washed with cold PBS (pH 7.4), and ice-cold lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 20 mM NaF, 100 mM Na 3 VO 4 , 0.5% NP-40, 1%Triton X-100, 1 mM PMSF (pH 7.4) with freshly added protease inhibitor cocktail (Protease Inhibitor Cocktail Set III; Calbiochem) over ice for 30 min. The cells were scraped and the lysate was collected in a microfuge tube and passed through needle to break up the cell aggregates. The lysate was cleared by centrifugation at 14 000g for 15 min at 4°C and the supernatant (whole cell lysate) was used or immediately stored at -80°C. For western blotting, 30-50 mg protein was resolved over 8-12% polyacrylamide gels and transferred to a nitrocellulose membrane. The blot was blocked in blocking buffer (5% nonfat dry milk/1% Tween 20; in 20 mM TBS, pH 7.6) for 1 h at room temperature, incubated with appropriate monoclonal or polyclonal primary antibody in blocking buffer for 1.5 hours to overnight at 4°C, followed by incubation with anti-mouse or anti-rabbit secondary antibody HRP conjugate obtained from Amersham Life Science Inc. Densitometric measurements of the band detected by chemiluminescence in western blot analysis were performed using digitalized scientific software program Quantity One.

In vivo tumor xenograft model
Balb/c athymic (nude) mice (male, 6−8 weeks) weighing 21−25 g were purchased from Animal Center, Academy of Military Medical Sciences, (Beijing, China) and were housed under specific pathogen-free conditions according to the guidelines of Animal Care, Nanjing University of Chinese Medicine. The animal room was controlled for temperature (22 ±2°C), light (12 hour light/dark cycle) and humidity (50±10%). All laboratory feed pellets and bedding was autoclaved. The tumor regression model in nude mice has been successfully applied to evaluate antitumor activity. This model was therefore used to evaluate suppression of solid tumor growth in JIN Formula (JIN). A total of 1×10 7 A549, H157 cells in 0.2 ml culture medium were injected subcutaneously into the flank of each mouse using a 26-gauge needle. After 7 days observation, an apparently solid tumor mass was excised from mice inoculated with lung cancer cells. When the tumor volume reached about 50 mm 3 in the nude mice, xenografted tumor model were randomly distributed into normal group, model group, Jin group, after sacrifice of animals with oral administration of JIN formula (3%, wt/vol) for 16 days, tumor inhibiting rate was recorded to show the effects of drugs on tumor growth. Tumor inhibiting rate= (average of tumor weight in model group-average of tumor weight in JIN group) /average of tumor weight in model group ×100%.

Statistical analysis
Results were analyzed using a two-tailed Student's t-test to assess statistical significance and P-values<0.05 were considered significant.

Inhibition of cell growth by JFE in A549 cells
This study was designed to show the chemopreventive/chemotherapeutic potential of JIN formula against lung cancer. Initially in our study, we investigated the antiproliferative effects of JFE treatment on human lung carcinoma A549 cells. Therefore, using A549 cells, we first evaluated the effect of JFE on the growth of these cells by MTT assay. We compared the antiproliferative effects of JFE (including E1 to E8) on A549 cells. As shown in Figure 2, treatment of JFE (1-100 µg/ml) for 72 h was found that E6 and E7 could decrease the viability of A549 cells.

Inhibition of tumorigenesis pathways by JFE in A549 and H157 cells
To explore the mechanism of antiproliferative effects of JIN formula against lung cancer, we investigated the involvement of Akt, ERK1/2, JNK1/2, p38 and NF-κB machinery during the inhibition of tumorigenesis by JFE in A549 and H157 cells. Akt, also known as protein kinase B, which is a serine or threonine kinase, has been identified as an important component of tumorigenesis signaling pathway. The PI3K/Akt promotes cell survival by activating the NF-kB signaling pathway (3). Studies have shown that Akt plays an important role in carcinogenesis. We first investigated the effect of JFE on Akt protein expression in human lung carcinoma cells. In our study, we have demonstrated that the treatment of A549 and H157 cells with JFE (E1-E8) resulted in different degree inhibition of Akt pathways on tumor promotion processes as shown in Figure 3.

Fig. 2. The effects of JFE (including E1 to E8) on A549 cells growth
The MAP kinase superfamily (MAPKs) has been characterized into three groups which include extracellular signal-regulated kinase p44/42 (ERK), JNK/SAPK (c-jun N-terminal kinase/stress activated protein kinase) and p38 MAP kinase. MAPKs, a group of serine/threonine-specific, proline-directed protein kinases are known to modulate transcription factor activities. (4,5). The involvement of the MAPK pathway in tumor proliferation is well documented. Transient activation of ERK is responsible for proliferation and differentiation and has also been shown to be involved in tumor promotion processes (3). Stimulation of JNK/SAPK and p38 can mediate differentiation, inflammatory responses and cell death (6). In the present study, we assessed the effect of JFE on MAPK pathway in A549 and H157 human lung carcinoma cells. The immunoblot analysis demonstrated that the treatment of cells with JFE inhibited ERKl/2, JNK1/2 and p38 proteins, and this inhibition of JNK1/2 and p38 in A549 cells was stronger than in H157 (Figure 3). Several studies have shown that JNK pathway plays a major role in cellular function, such as cell proliferation and transformation, whereas the ERK pathway suppresses apoptosis and enhances cell survival or tumorigenesis (4). ERK1/2 and p38 are also involved in the transcriptional activation of NF-kB (7,8). NF-kB is a sequence specific transcription factor that is known to be involved in the inflammatory and innate immune responses (9,10). NF-kB is sequestered in the cytoplasm in an inactive form through interaction with IkB. Phosphorylation of IkB by IkB kinase (IKK) causes ubiquitination and degradation of IkB, thus releasing NF-kB which then translocates to the nucleus, where it binds to specific kB binding sites in the promoter regions of several genes (11). Studies have shown that NF-kB activation plays an important role in cell survival, by its ability to block or reduce apoptosis (12). In the present study, we further investigated the effect of JFE on the pattern of NF-kB activation and whether treatment with JFE inhibits nuclear translocation of NF-kB/p65 in A549 and H157 cells. As is evident from western blot analysis data and the relative density of bands, we found that JFE (E2, E3, E7) treatment of A549 cells resulted in inhibition of translocation of NF-kB/p65, meanwhile, we demonstrated that NF-kB is activated in H157 human lung carcinoma cells treated with JFE (E2, E3, E7) and is translocated to the nucleus when measured by western blot analysis (Figure 3). The immunoblot analysis demonstrated that JFE (E1-E8) treatment could induce (i) inhibition of AKt, (ii) inhibition of ERK1/2 (p44 and p42), (iii) inhibition of JNK1/2 (p54 and p46), (iv) inhibition of p38 protein and (v) regulation of NF-kB.

Inhibition on the growth of human lung carcinoma A549 and H157 cells by JIN formula in nude mice
JFE inhibits the development of lung tumorigenesis by modulating and inhibiting PI3K/Akt, MAPK, and NF-kB signaling. To establish the relevance of these in vitro findings to in vivo situation, athymic nude mice were implanted with human lung carcinoma A549 and H157 cells. Compared with model group, Jin formula could significantly inhibit volume growth of A549 and H157 xenografted tumors. The inhibiting rate could reach to 28.5% and 25% in A549 and H157 tumor-bearing mice respectively (p＜0.05). We found that the oral administration of JIN formula significantly slowed the progression of A549 and H157 tumor growth in nude mice.

Discussion
Lung cancer as the most common cancer in the world represents a major public health problem. Worldwide it accounts for 1.18 million cancer-related deaths and is the most common cause of cancer death in both men and women (13). Lung carcinogenesis is a chronic and multistep process resulting in malignant lung tumors. This progression from normal to neoplastic pulmonary cells or tissues could be arrested or reversed through pharmacological treatments. These therapeutic interventions should reduce or avoid the clinical consequences of lung cancer by treating early neoplastic lesions before the development of clinically evident signs or symptoms of malignancy. Preclinical, clinical and epidemiological findings relating to different classes of candidate chemopreventive agents provide strong support for lung cancer prevention as a therapeutic strategy (14). Cancer chemoprevention is an attractive approach to reduce lung cancers by treating early steps in lung carcinogenesis. There is a convergence of basic scientific and clinical findings in lung cancer chemoprevention. Pharmacological interventions also can be used to reverse or arrest the progression of lung carcinogenesis. For this reason, additional clinical trials are needed that emphasize a mechanistic approach in which mechanisms identified in vitro can be validated in vivo. This study was designed to show the chemopreventive/chemotherapeutic potential of JIN formula against lung cancer. we assessed the efficacy of the JIN formula, which is an ancient herbal formula from classical book JIN KUI YAO LUE (Golden Chamber) for the treatment of lung cancer and explored its probable molecular mechanisms. Initially, employing human lung carcinoma A549 cells, we compared the growth inhibitory effects of JIN formula extracts (JFE，including E1 to E8). The results showed that E6 and E7 could decrease the viability of A549 cells by the MTT assays. To explore the mechanism of antiproliferative effects of JIN formula against lung cancer, we investigated the involvement of Akt, ERK1/2, JNK1/2, p38 and NF-κB pathway during the inhibition of tumorigenesis by JFE in A549 and H157 cells. The immunoblot analysis demonstrated that JFE treatment could result in different degree inhibition of above signaling pathways on tumor promotion processes. Oral administration of JIN formula (3%, wt/vol) to athymic nude mice implanted with A549 and H157 cells resulted in a significant inhibition in tumor growth.
In conclusion, the present study demonstrates that human non small cell lung cancer A549 and H157 cells are highly sensitive to JIN formula both in vitro and in vivo experimental models. JIN formula inhibits the development of lung tumorigenesis by modulating the imbalance of www.intechopen.com proliferative and apoptotic signaling network. Based on the present study it is tempting to suggest that JIN formula and its effective extracts have strong potential for development as a chemopreventive and possibly as a chemotherapeutic agent against lung cancer.