Methods

Cell culture

The human lung adenocarcinoma cell lines (A549) was obtained from the American Type Culture Collection. The cells were maintained in Dulbecco's modified Eagle's medium/Nutrient Mixture Ham's F12 (DMEM/F12) medium which was supplemented with 10% heat-inactivated FCS, 2mM-glutamine, penicillin (100 U/ml) and streptomycin (100 ng/ml) at 37°C with 5% CO₂.

MTT assay

Cell viability was determined by MTT assay which are laboratory tests and standard colorimetric assays (an assay which measures changes in color) for measuring the activity of enzymes that reduce MTT to formazan. Yellow MTT(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide is reduced to purple formazan in living cells (Fig. 2). After treatment with honokiol for 2 days, cultures were washed with PBS. MTT (0.5 mg/ml) was then added to each well and the mixture was incubated for 2 h at 37 °C.

Culture medium was then replaced with an equal volume of DMSO to

dissolve formazan crystals[71]. After the mixture was shaken at room temperature for 10 min, absorbance of each well was determined at 550 nm using a microplate reader (Bio-Tek, Winooski, VT). Each clone was plated in triplicate in each experiment, and each experiment was repeated at least three times.

Fig. 2 Yellow MTT(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide is reduced to purple formazan

Quantification of apoptosis by flow cytometry

Apoptosis was assessed using annexin V, a protein that binds to phosphatidylserine (PS) residues which are exposed on the cell surface of apoptotic cells, as previously described. Cells were treated with vehicle or honokiol for indicated time intervals. After treatment, cells were washed twice with PBS (pH 7.4), and resuspended in staining buffer containing 1 μg/ml Propidium iodine (PI) and 0.025 μg/ml annexin V-FITC.

Double-labeling was performed at room temperature for 10 min in the dark before the flow cytometric analysis[71, 72]. Cells were immediately analyzed using FACScan and the Cellquest program (Becton Dickinson).

Quantitative assessment of apoptotic cells was also assessed by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick endlabeling (TUNEL) method, which examines DNA-strand breaks during apoptosis by using BD ApoAlert™ DNA Fragmentation Assay Kit.

Briefly, cells were incubated with honokiol for the indicated times. The cells were trypsinized, fixed with 4% paraformaldehyde, and permeabilized with 0.1% Triton-X-100 in 0.1% sodiumcitrate. After being washed, the cells were incubated with the reaction mixture for 60 min at 37 °C. The stained cells were then analyzed with flow cytometer[71, 73].

Western blot analysis

The cellular lysates were prepared as described previously. Proteins were resolved on SDS-PAGE and transferred to Immobilon polyvinyldifluoride (PVDF) membranes. The blots were blocked with 4%

BSA for 1 h at room temperature and then probed with rabbit anti-human antibodies against Bax, Bcl-2 (1:1000) for 1 h at room temperature. After three washes, the blots were subsequently incubated with a donkey anti-rabbit peroxidase conjugated secondary antibody (1:1000) for 1 h at room temperature. The blots were visualized by enhanced chemiluminescence using Kodak X-OMAT LS film (Eastman Kodak, Rochester, NY).

Caspase activity

The assay is based on the ability of the active enzyme to cleave the Ac-DEVD-pNA (for caspase-3). The cell lysates were prepared and incubated with specific caspase-3 antibodies. Immunocomplexes were incubated with peptide substrate in assay buffer (100 mMNaCl, 50 mM 4-(2-hydroxyethyl)-1-piperazine-ethanesulphonic acid (HEPES), 10mM dithiothreitol, 1mM EDTA, 10% glycerol, 0.1%

3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate(CHAPS), pH 7.4) for 2 h at 37 °C. The release of p-nitroaniline was monitored at 405 nm.

Results are represented as the percent change of the activity compared to the untreated control.

In vivo tumor xenograft study

Male nude mice [6 weeks old; BALB/cA-nu (nu/nu)] were purchased from the National Science Council Animal Center (Taipei, Taiwan) and maintained in pathogen-free conditions. A549 cells were injected subcutaneously into the flanks of these nude mice (1 ×10⁶ cells in 200μl) (Fig. 3A,B), and tumors were allowed to develop for ~14 days until they reached a size of approximately 100 mm³, when treatment was initiated. The mice were treated with vehicle, intraperitoneal injection 1.5 mg/kg Honokiol every day for 21 days (6 mice/group). The volume of the implanted tumor in dorsal side of mice was measured once a week with a caliper (Fig. 3C,D), using the formula V = (LW²) π/6: where V, volume (mm³); L, biggest diameter (mm); W, smallest diameter (mm). Xenografts that were smaller than pretreatment size were defined as tumor reduction[9, 10]. Three weeks after the beginning of the treatment, the xenografts were removed, and the specimens were processed for pathological analyses[8]. All protocols complied with institutionalguidelines and were approved by Animal Care Committee of China Medical Taiwan University.

Fig. 3 A549 cells were injected subcutaneously into the flanks of these nude mice [A,B]. The volume of the implanted tumor in dorsal side of mice was measured once a week with a caliper.[C,D]

Meridian Selection

The acupuncture points used were the bilateral Zusanli (ST 36) acupoints, located on the calf of the anterior-tibia myo-muscle near the knee, according to the manual designed by Huang’s research.

Non-acupoints were selected in the contralateral side of the upper limbs. It was demonstrated that the calcium ion concentration was significantly higher in the acupoint area than that in the non-acupoint and non-meridian locations. Thus, ion-selected EA electrodes were introduced for further detection of calcium ion concentration in acupoints in rats to ensure an

accurate location of acupoints and nonacupoints for the study. The immunomodulatory effect of Zusanli (ST 36) acupoint was reported in many studies. For example, it could increase IL-2 level and NK cell activity and decreased level of IL-6 and IL-10 in the acupuncture group of cancer patient after 10 days of treatment[8, 57]; moxibustion on this point increase the white blood counts of the patients with late stage cancer; increase lymphocyte subgroups CD3⁺ and CD4⁺ and increased lymphocyte proliferation rate[10].

Operation of ST 36 electrical-acupuncture in mice

The mice anesthetized by FORANE (isoflurane, USP), a nonflammable liquid administered by vaporizing, were placed on the wooden operating table. A 0.5-inch No. 36 EA needle (Han’s Instrument, Taiwan) was stabbed into the connective tissues perpendicularly 5 mm deep at the bilateral Zusanli (ST 36) acupoints. The needle in the right knee was connected to the positive lead, and the needle in the left knee was connected to the negative lead (Fig. 4). An electrical stimulus (2 Hz pulses) was delivered for 30 minutes, followed by 90 minutes of recovery, 1.5 mg/kg honokiol were injected intra-peritoneal in combined (EA+HK) group.

Fig. 4 The mice anesthetized by FORANE. A 0.5-inch No. 36 EA needle was stabbed into the connective tissues perpendicularly 5 mm deep at the bilateral Zusanli (ST 36) acupoints. The needle in the left knee was connected to the positive lead, and the needle in the left knee was connected to the negative lead

Electro-acupuncture protocol

 Instrument：stainless steel needle : 0.20 mm*13 mm (36#)

 Acupoint：susanli acupoint (ST36) and non-acupoint (near ST-36) connected to the positive (anode) and the negative (cathode) wicks of a stimulator (mode Trio 300, Ito Co., Ltd., Japan)

 Deepness：The depth of insertion from 2 to 4 mm according to the

 Electro-acupuncture parameter：

 Pulse : square biphasic wave, positive and negative

 Width : 50 micro-sec

 Frequncy: 2 Hz

 Intensities : 0.8 to 1 mA to produce a visual muscle contraction

 The electrical stimulation was applied to the animals for 15 minutes every day

Grouping

Mice were injected subcutaneously with A549 cells. After the tumors reached 100mm³ in size, electroacupuncture group (EA), Honokiol group (HK), electroacupuncture conbined with Honokiol group (EA+HK) and vehicle (CON) was administered daily for 3 weeks( Fig. 5).

39

40

Flow Chart Flow Chart

Day 0: grouping & record body weight Day 1 : A549 cells injecting subcutaneously Day 14: 1. record tumor size & body weight

2. start electro-acupuncture Day 21: record tumor size & body weight Day 28: record tumor size & body weight Day 35 : 1. record tumor size & body weight

2. BLI Procedure and Image Analysis Day 36 : sacrifice & dissect tumor sample

EAHK

Fig. 5 Electro-acupuncture grouping and experiment flow chart

BLI Procedure and Image Analysis

Each mouse were anesthetized by FORANE. To generate the bioluminescence reaction catalyzed by Renilla luciferase, we injected its substrate, coelenterazine (20 mg in 100 mL of a 10% methanol/ 90%

phosphate-buffered saline [pH 7.4] solution), intraperitoneal injection.

Immediately afterward, we placed the animal supine in an IVIS system (Xenogen Inc.), which consists of a cooled charge coupled- device camera (2105C) mounted on a light tight chamber. The photograph and biolumicent image were superimposed using Living Image software (version 2.20;

Xenogen Inc.) (Fig. 6)

Fig. 6 IVIS system

Pathological analysis

For hematoxylin-eosin (H&E) staining the xenografts were fixed in 10%

neutral buffered formalin and embedded in paraffin. We performed the following procedures to complete H&E staining:

1. Deparafinize and hydrate slides to distilled water.

2. Stain in Harris’Hematoxylin for 4 minutes.

3. Rinse in tap water for 1 minute.

4. Clarify in 1% hydrochloric acid solution for 1 minute.

5. Rinse in tap water for 1 minute.

6. Blue in 0.5% ammonium hydroxide solution for 1 minute.

7. Rinse in tap water for 1 minute.

8. Dip in 95% alcohol three times.

9. Dip in Eosin four times.

10. Dehydrate in 95% alcohol, absolute alcohol and clear in xylene, two

changes each.

11. Mount with synthetic resin.

The sections were then observed, and representative images were taken under a microscope (Olympus, Tokyo, Japan).

Statistics

The values given are means ± S.E.M. Statistical analysis between two samples was performed using Student’s t test. Statistical comparisons of more than two groups were performed using one-way analysis of variance (ANOVA) with Bonferroni’s post hoc test. In all cases, P < 0.05 was considered as significant.