• 1 •Chin J Integr Med

Lung cancer is one of the most common and devastating cancers worldwide, and is a major cause of cancer-associated death. The Akt signaling pathway plays an important role in the development of lung cancer, and activation of Akt signaling pathway can inhibit apoptosis provoked by multiple stimuli in parallel with promoting cell cycle progression, thus contributing to the survival and proliferation of cancer cells. Together with its involvement in angiogenesis, this pathway has an important role in the aggressiveness and metastasis of tumors.

Xiaoji Decoction (消积饮, XJD), an oral liquid of Chinese medicine developed by Dr. LIU Wei-sheng of Guangdong Provincial Hospital of Traditional Chinese Medicine, is composed of Coriolus Versicolor , Psoralea Corylifolia L., Hedyotis Diffusa, Astragalus Mongholicus, Scorpion, Centipede and Rhubarb. XJD has shown promising clinical effi cacy for patients with lung cancer since it was approved for use as a medical treatment in 1993.(1-3) Many experiments have proven that XJD can suppress the proliferation and metastasis of lung cancer cell in Lewis mice, prolong the survival of tumor-bearing mice by downregulating cyclinD1 expression, arrest the tumor cells at G0/G1 and prevent tumor cells entering S phase for DNA

replication.(4-6) In addition, XJD can downregulate the expression of PCNA, survivin protein and BCL-2 gene, induce apoptosis, and reduce tumor metastatic potential.(6,7)

In this study, we observed the effect of XJD in the human lung cancer A549 cells and explored the mechanism by which XJD inhibit A549 cells proliferation.

METHODS

Experimental AnimalsEighty male Wistar rats, weighing 180±20 g,

were purchased from the Center of Experiment Animal of Sun Yat-sen University [SCXK (Yue) 2011-0015, Guangzhou, China]. The animal experimental protocol

ORIGINAL ARTICLE

Xiaoji Decoction (消积饮) Inhibited Cell Proliferation and Induced Apoptosis through Akt Signaling Pathway in

Human Lung Cancer A549 Cells

CHAI Xiao-shu (柴小姝)1, ZHANG Xiao-xuan (张晓轩)2, and WU Wan-yin (吴万垠)1

©The Chinese Journal of Integrated Traditional and Western Medicine Press and Springer-Verlag Berlin Heidelberg 2014Supported by Canadian Terry Fox Run Foundation for Cancer Research1. Department of Oncology, Guangdong Provincial Hospital of Traditional Chinese Medicine, Guangzhou, (510120), China; 2. The Second Clinical College, Guangzhou University of Chinese Medicine, Guangzhou, (510045), ChinaCorrespondence to: Dr. CHAI Xiao-shu, Tel: 86-20-39318281, E-mail: Chaixiaoshu@126.comDOI: 10.1007/s11655-014-1772-4

ABSTRACTABSTRACT ObjectiveObjective: To investigate the inhibitive effect and the underlying mechanism of Xiaoji Decoction (消: To investigate the inhibitive effect and the underlying mechanism of Xiaoji Decoction (消

积饮, XJD) in human lung cancer A549 cells. 积饮, XJD) in human lung cancer A549 cells. MethodsMethods: A549 cells in logarithmic proliferation were cultivated in : A549 cells in logarithmic proliferation were cultivated in

RPMI-1640 containing 10% low, medium or high dosages of XJD serum. The inhibitive effect of XJD in A549 cell RPMI-1640 containing 10% low, medium or high dosages of XJD serum. The inhibitive effect of XJD in A549 cell

proliferation was assessed by methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. The pro-apoptotic effect proliferation was assessed by methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. The pro-apoptotic effect

of XJD in A549 cells was observed by fl uorescence microscope via Hoechst 33258 staining. The role of the Akt of XJD in A549 cells was observed by fl uorescence microscope via Hoechst 33258 staining. The role of the Akt

signaling pathway was observed by examining the presence of p-Akt protein by Western blot and the mRNA signaling pathway was observed by examining the presence of p-Akt protein by Western blot and the mRNA

expression of downstream proteins such as Bcl-2/Bcl-XL-associated death promoter (BAD) and caspase-9 by expression of downstream proteins such as Bcl-2/Bcl-XL-associated death promoter (BAD) and caspase-9 by

real time polymerase chain reaction. real time polymerase chain reaction. ResultsResults: MTT assay revealed that XJD could inhibit A549 proliferation in : MTT assay revealed that XJD could inhibit A549 proliferation in

a dose- and time-dependent manner. Hoechst 33258 staining showed that XJD induced the typical nuclear a dose- and time-dependent manner. Hoechst 33258 staining showed that XJD induced the typical nuclear

apoptotic morphology after XJD treatment. Moreover, XJD could reduce the phosphorylation of Akt and increase apoptotic morphology after XJD treatment. Moreover, XJD could reduce the phosphorylation of Akt and increase

the mRNA expression of BAD and caspase-9. the mRNA expression of BAD and caspase-9. ConclusionsConclusions: XJD can inhibit the proliferation of A549 cells in a : XJD can inhibit the proliferation of A549 cells in a

dose- and time-dependent manner through signaling Akt pathway via up-regulating the expression of BAD and dose- and time-dependent manner through signaling Akt pathway via up-regulating the expression of BAD and

caspase-9. XJD may provide a novel therapeutic model for lung cancer and deserve further study.caspase-9. XJD may provide a novel therapeutic model for lung cancer and deserve further study.

KEYWORDSKEYWORDS Xiaoji Decoction, A549 cells, proliferation inhibition, apoptosis induction, Akt signaling pathway Xiaoji Decoction, A549 cells, proliferation inhibition, apoptosis induction, Akt signaling pathway

• 2 • Chin J Integr Med

was reviewed and approved by the Institutional Animal Care and Use Committee of Guangzhou University of Chinese Medicine. All experimental procedures performed were conducted in accordance with institutional guidelines for the care and use of laboratory animals in research.

Serum PreparationsXJD was composed of Coriolus Versicolor ,

Psoralea Corylifolia L., Hedyotis Diffusa, Astragalus Mongholicus, Scorpion, Centipede and Rhubarb. XJD (100 mL/bottle, per 1 mL equivalent to 1.5 g raw herb) was purchased from Guangdong Provincial Hospital of Traditional Chinese Medicine. Rats were randomly divided into 4 groups: XJD high dosage group, medium dosage group, low dosage group and control group (n=20 per group). Based on the clinical dosage of XJD (50 mL orally administered twice a day), the high, medium and low dosages (4.5 mL gastric infusion twice a day) for rats were 10 times, 5 times and 2.5 times the daily adult dosage, respectively. Medicine was given through intragastric administration while equivalent volumes of normal saline were given for the control group. After 7 days administration, the rats were anesthetized with 10% chloral hydrate solution at 1 h after the last feed, and then sacrificed by a rapid exsanguination via the abdominal aorta. Blood was drawn into sterile test tubes and centrifuged at 3,000 r/min for 15 min. The serum was removed, denatured at 56 ℃ for 30 min, filtered through a 0.22 μm filter to remove bacteria, and stored at –70 ℃.

Instruments and ReagentsVICTORTM X5 automated micro plated reader

was from PerkinElmer (Waltham, USA). Fluorescence microscope was from Olympus (Tokyo, Japan). SYBR Green PCR Master Mix was from Toyobo (Shanghai, China). ABI Prism 7300 sequence detection system was from Ambion (Austin TX, USA). Methylthiazolyldiphenyl-tetrazolium bromide (MTT), Hoechst 33258 and dimethylsulfoxide (DMSO) were purchased from Sigma (St. Louis, USA). Lysis buffer and enhanced chemiluminesense (ECL) reagents were from Beyotime (Nantong, China). Trizol was brought from Invitrogen Life Technologies (Carlsbad, CA, USA). The primary antibody against phospho-Akt (clone No. Ser473, serial No. 193H12), Akt (ser) and horseradish peroxidase (HRP)-labeled secondary antibody was from Santa Cruz Biotechnology (CA, USA).

Cell Lines and CulturesThe human lung cancer A549 cel ls were

originally purchased from the Cell Bank of the Chinese Academy of Science (Beijing, China). A549 cells were cultured in RPMI-1640 medium (Hyclone, USA) supplemented with 10% fetal bovine serum (Hyclone, serial No. NRE 0007, USA) and 100 U/mL penicillin and 100 μg/mL streptomycin. The cells were incubated at 37 ℃ in a humidifi ed atmosphere with 5% CO2. Then, A549 cells in the logarithmic growth phase were collected for the following experiments.

Cell Proliferation Assay A549 cells were seeded in a 96-well culture plate

at a density of approximately 5×104 cell/mL overnight. Then, the medium containing 5% control serum, low, medium or high dosages of XJD serum were incubated. After 24, 48 and 72 h, MTT was added at 5.0 mg/mL to each well. After further incubation for 4 h at 37 ℃, 150 μL DMSO was loaded to each well to dissolve the crystals of the viable cells. The optical density (OD) value was read at 490 nm using an automated micro plated reader. Proliferation inhibition rate = [(OD of control group – OD of experimental group)/OD of control group] × 100%.

Hoechst 33258 StainingA549 cells treated with XJD for 48 h were

co l lec ted , then washed tw ice w i th i ce -co ld phospharate buffer solution (PBS) and fi xed with 4% paraformaldehyde for 30 min. After washed twice with ice-cold PBS again and staining with Hoechst 33258 (5 μg/mL) for 30 min, A549 cells were observed by a fluorescence microscope and nuclear morphological changes were recorded.

Real-Time Polymerase Chain ReactionAfter collecting A549 cells treated for 48 h, total

RNA was extracted using Trizol and then the real-time polymerase chain reaction (RT-PCR) was performed using SYBR Green PCR Master Mix. After primer

Table 1. RT-PCR Primer Design

Gene Primer sequrence

qh-BAD Forward: 5'-TGACGAGTTTGTGGACTCCT-3'

Reverse: 5'-GCATAGGCCTGAGGGAAGTA-3'

qh-Caspase-9 Forward: 5'-TCCAGGAAGGTTTGAGGACC-3'

Reverse: 5'-CCCTTTCACCGAAACAGCAT-3'

18SrRNA Forward: 5'-CCTGGATACCGCAGCTAGGA-3'

Reverse: 5'-GCGGCGCAATACGAATGCCC-3'

• 3 •Chin J Integr Med

design (Table 1), PCR was conducted by ABI Prism 7300 sequence detection system.

Protein Extraction and Western Blot AnalysisProteins expressions in A549 cells of different

groups were measured by Western blot. Cells were harvested and washed twice with ice-cold PBS before lysis buffer was added. The lysates were ultracentrifuged at 12,000×g for 30 min and the supernatants were collected for protein assay. Aliquots of proteins were separated on 10% SDS-polyacrylamide gel and transferred onto nitrocellulose (NC) membranes. After the membranes were blocked with 5% nonfat milk, the NC membranes were incubated with primary antibodies (1:1000) overnight and HRP-labeled antibody (1:2000) for 2 h. Finally, the proteins were detected with ECL reagents.

Statistical AnalysisAll data analyses were performed in SPSS

15.0 using one-way analysis of variance (ANOVA). Independent samples t-test was used for analyzing inter-group differences. P<0.05 was considered to represent a signifi cant difference.

RESULTS

Effect of XJD on A549 Cells ProliferationVarying dosages of XJD significantly inhibited

the proliferation of A549 lung cancer cells compared with the control group (P<0.05 or P<0.01, Figure 1), and no difference was observed between the medium and high dose group.

nuclear blebbing and DNA fragmentation. Moreover, the number of apoptotic cells was increased and the cell density was decreased in a dose-dependent manner while cells in the control group had clear shape and intact cell membranes (Figure 2), demonstrating that higher dosages of XJD were more effective in proliferation inhibition of A549 cells. These changes are in accordance with the characteristics of apoptosis.

Figure 1. Proliferation Inhibition of XJD in A549 CellsNotes: P<0.05, P<0.01, compared with the control group

Effect of XJD on Nuclear Morphological Changes in A549 Cells

After adding varying dosages of XJD to A549 cells for 48 h, A549 cells showed bright fluorescent

Figure 2. Effect of XJD on Nuclear Morphological Changes in A549 Cells (Hoechst 33258 staining, 200×)

Notes: A: control group; B: low dosage group; C: medium dosage group; D: high dosage group

A B

DC

Effect of XJD on BAD and Caspase-9 mRNA Expression

Compared with the control group, BAD and caspase-9 mRNA expressions were slightly increased in the low and medium dosage group after 48 h XJD treatment while the high dosage group resulted in a significantly higher △△Ct value (P<0.01, Figure 3). There were no signifi cant differences among the control group, low dosage group and medium dosage group.

Effect of XJD on Expression of Akt PhosphorylationWestern b lot analys is revealed that the

expressions of Akt phosphorylation were decreased in a dose-dependent manner after A549 cells treated with XJD for 48 h, which was significantly different compared with the control group (P<0.01, Figure 4).

DISCUSSION

Lung cancer, one of the most common malignant tumors worldwide, is a major cause of cancer-related death.(8) In China, Chinese medicines are received by almost all tumor patients and becoming a promising method in cancer treatment. Dr. LIU Wei-sheng, a Guangdong Provincial famous doctor, basing on his

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30 years of clinical experience, developed a Chinese medicine decoction named "XJD", which targets the etiology and pathogenesis such as deficiency,

toxin, phlegm and blood stasis. Since XJD has being a clinical application for over a decade, some clinical researches found that XJD may extend the survival time and improve the quality of life of lung cancer patients.(1,2) Experiments showed that XJD can inhibit the growth and metastasis of Lewis mice lung cancer in Lewis mice and extend survival time. The mechanism may be related to the reduction of cyclinD1 in Lewis mice lung cancer cells, halting the cell cycle at G0/G1, leading the cells unable to enter phase S for DNA replication ultimately. In addition, XJD may downregulate PCNA, survivin and BCL-2 gene expression in tumor cells to induce apoptosis, and stabilize the lesion and reduce metastasis.(4-7)

Apoptosis is a complex multi-step process, caspase cascade and Bcl-2 fami ly members are the key mediators in the apoptotic signaling transduction pathway.(9) Apoptotic factors such as caspase-9 are initiators and effectors of apoptosis, and activated Akt can phosphorylate and thus inactivate caspase-9 at Ser196, therefore, inhibiting its apoptotic effects.(10) Activated Akt, an important regulatory factor in multiple apoptosis process, can resist apoptosis through numerous mechanisms and effect on transcription factor families in the control of cell survival. Recently, others have observed that activated Akt can induce or suppress downstream target proteins such as BAD, caspase-9, nuclear factor-κB, GSK-3β and FKHR through phosphorylation, and thereby regulate proliferation, differentiation, apoptosis and metastasis. (10-14) Otherwise, Akt is an important regulator of BAD and phosphorylated BAD on Ser136, which makes BAD dissociate from the Bcl-2/Bcl-XL complex and lose the pro-apoptotic function.(15-17)

In our exper iments , we noted that XJD significantly inhibited cell proliferation of A549 in a time- and dose-dependent manner identifi ed by MTT assay. We observed that the typical apoptotic cells were increased examined by a fl uorescent microscope at different doses of XJD treatments. It was interesting to unders tand whether BAD and caspase-9 contributed to apoptosis in this study. Thereby, the expressions of BAD and caspase-9 were evaluated by RT-PCR. Results showed dramatic significant decrease of BAD and caspase-9 after XJD treatments for 48 h. To further assess the Akt signaling pathway that XJD inhibited A549 cells proliferation via induction

Figure 3. Effect of XJD on the mRNA Expression of BAD and Caspase-9 in A549 Cells

Note: P<0.01, compared with the control group

Figure 4. Effect of XJD on Akt Phosphorylation in A549 Cells

Notes: A: (1) control group; (2) low dosage group; (3); medium dosage group; (4) high dosage group. B: P<0.01, compared with the control group

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• 5 •Chin J Integr Med

of apoptosis, we focused on the expression of Akt. We found that phosphoxylation of p-Akt was decreased in a dose-dependent manner after treated with XJD. Our evidences suggested that XJD induced apoptosis in A549 cells was referred to Akt signaling pathway via activation of BAD and caspase-9.

Our experiments discovered that XJD may inhibit the proliferation and induce the apoptosis of A549 cells through inhibiting the p-Akt activation. This mechanism may be associated with blocking the activation of downstream apoptotic factors. Thus, further investigations are required in the future.

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(Received June 16, 2011)Edited by WANG Wei-xia