The International Journal of Biochemistry & Cell Biology 38 (2006) 2102–2113

External Qi of Yan Xin Qigong differentially regulates the Aktand extracellular signal-regulated kinase pathways and is

cytotoxic to cancer cells but not to normal cells

Xin Yan a,b,∗, Hua Shen b, Hongjian Jiang c, Chengsheng Zhang d,Dan Hu d, Jun Wang b, Xinqi Wu e

a Institute of Chongqing Traditional Chinese Medicine, Chongqing, PR Chinab New Medical Science Research Institute, New York, NY 10107, USA

c Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USAd Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA

e Children’s Hospital, Harvard Medical School, Boston, MA 02115, USA

Received 14 February 2006; received in revised form 21 May 2006; accepted 2 June 2006Available online 27 June 2006

Abstract

Long-term clinical observations and ongoing studies have shown significant antitumor effect of external Qi of Yan Xin Qigongwhich originated from traditional Chinese medicine. In order to understand the molecular and cellular mechanisms underlying theantitumor effect of external Qi of Yan Xin Qigong, we have examined its cytotoxic effect on BxPC3 pancreatic cancer cells and itseffect on the Akt and extracellular signal-regulated kinase pathways. We found that external Qi of Yan Xin Qigong dramaticallyinhibited basal phosphorylation levels of Akt and extracellular signal-regulated kinases, epidermal growth factor-mediated phos-phorylation of extracellular signal-regulated kinases, and phosphatidylinositol 3-kinase activity. External Qi of Yan Xin Qigong alsoinhibited constitutive and inducible activities of nuclear factor-kappa B, a target of the Akt and epidermal growth factor receptorpathways. Furthermore, a single 5 min exposure of BxPC3 cells to external Qi of Yan Xin Qigong induced apoptosis, accompaniedby a dramatic increase of the sub-G1 cell population, DNA fragmentation, and cleavage of caspases 3, 8 and 9, and poly(ADP-ribose)polymerase. Prolonged treatment with external Qi of Yan Xin Qigong caused rapid lysis of BxPC3 cells. In contrast, treatment offibroblasts with external Qi of Yan Xin Qigong induced transient activation of extracellular signal-regulated kinases and Akt, andcaused no cytotoxic effect. These findings suggest that external Qi of Yan Xin Qigong may differentially regulate these survivalpathways in cancer versus normal cells and exert cytotoxic effects preferentially on cancer cells, and that it could potentially be a

valuable approach for therapy of pancreatic carcinomas.© 2006 Elsevier Ltd. All rights reserved.

Keywords: Akt; ERK1/2; External Qi; Yan Xin Qigong; Pancreatic cancer

Abbreviations: EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; EMSA, electrophoresis mobility shift assay; ERK,extracellular signal-regulated kinase; FBS, fetal bovine serum; IkB, inhibitor of NF-�B; IKK, I�B kinase; LDH, lactic dehydrogenase; MTS,[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; inner salt]; NF-�B, nuclear factor-kappa B; PARP,poly(ADP-ribose) polymerase; PI, propidium iodide; PI3K, phosphatidylinositol 3-kinase; PMSF, phenylmethylsulfonyl fluoride; TCM, traditionalChinese medicine; TLC, thin layer chromatography; TNF-�, tumor necrotic factor �; YXQ, Yan Xin Qigong

∗ Corresponding author. Tel.: +1 617 325 7784; fax: +1 617 325 7784.E-mail address: smkj2006@yahoo.com (X. Yan).

1357-2725/$ – see front matter © 2006 Elsevier Ltd. All rights reserved.doi:10.1016/j.biocel.2006.06.002

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. Introduction

The concept External Qi (of Qigong) refers to theechnology and ability of the “Qi deployment” therapynd health preservation of traditional Chinese medicineTCM) and has been described in classic literatures ofCM and in Chinese medical textbooks (Yan et al.,004). External Qi therapy of Chinese medicine has longeen one of the medical practices in China and is underanagement by the Chinese health authorities. Multi-

le studies have shown that external Qi can be emittedy highly talented/trained Qigong practitioners, whilerdinary people are unable to deploy external Qi of ther-peutic, physical or chemical effects (Lu, 1997; Yan etl., 2004). “Qi” is considered as the basic element ofuman vital energy in TCM. The underlying theory ofCM is fully based on balancing Qi according to the

heory of Yin-Yang, an approach that also takes intoccount the biological rhythms, and the chronothera-eutic principles is integrated into external Qi therapyf TCM (Seki et al., 2005). Long-term clinical obser-ations and ongoing studies have shown that patientsith cancer and other medical conditions have received

ignificant beneficial effects from the exposure to exter-al Qi of Yan Xin Qigong (YXQ) which originatedrom TCM, and in some cases conditions of canceratients have even been dramatically improved (Fong,997; Ming, 1988; Wang & Zhu, 1997; Zhang, Zhao, &hang, 1997). External Qi of YXQ can also help patients

mprove or avoid side effects associated with radio- andhemotherapy (Fong, 1997; Ming, 1988; Wang & Zhu,997; Zhang et al., 1997). In order to understand theolecular basis underlying these effects, numerous lab-

ratory studies have been conducted in the past 20 yearse.g. Li et al., 1990; Lu, 1997; Yan, Fong, Jiang, et al.,002; Yan, Fong, Wolf, Wolf, & Cao, 2001; Yan, Fong,olf, et al., 2002; Yan, Li, Liu, et al., 1988; Yan, Li,ang, & Lu, 1988; Yan, Li, Yu, Li, & Lu, 1988; Yant al., 1999; Yan et al., 2004; Yan, Zhao, Yin, & Lu,988; Yan, Zheng, Zou, & Lu, 1988; Yan, Xia, Shen,

Traynor-Kaplan, 2002). These studies have demon-trated that external Qi of YXQ is able to alter moleculartructure and properties of experimental samples in mul-iple disciplines (Lu, 1997; Yan et al., 1999; Yan, Lu, etl., 2002). In particular, external Qi of YXQ has beenhown to influence the molecular structure and func-ion of DNA, RNA and protein molecules (Li et al.,990; Lu, 1997; Yan, Zheng, et al., 1988), enhance or

epress phosphatidylinositol 3-kinase (PI3K) activity initro and in vivo (Yan, Xia, et al., 2002; Yan et al.,004). Furthermore, it is also able to modulate genexpression, signal transduction, cell survival and apop-

istry & Cell Biology 38 (2006) 2102–2113 2103

tosis (Yan et al., 2001; Yan, Fong, Jiang, et al., 2002;Yan, Fong, Wolf, et al., 2002; Yan, Lu, et al., 2002; Yanet al., 2004).

Carcinoma of the pancreas is the fifth leading causeof cancer-related deaths in Western countries, with anoverall 1-year survival rate of ∼12% and 5-year survivalrate of 3–5% (Greenlee, Hill-Harmon, Murray, & Thun,2001). Resistance to chemotherapy is a major causeof treatment failure and poor prognosis in pancreaticcancer (Shi, Liu, Kleeff, Friess, & Buchler, 2002). Mul-tiple genetic and epigenetic changes occur at very highfrequencies in pancreatic tumors (Arlt et al., 2001, 2002;Dong et al., 2002; Garcea, Neal, Pattenden, Steward, &Berry, 2005; Kalthoff et al., 1993; Moore et al., 2001; Shiet al., 2002). The majority of pancreatic cancers overex-press epidermal growth factor receptor (EGFR). EGFRand its downstream signaling pathways, Ras-Raf-MEK-ERK axis, play important roles in the development ofpancreatic cancer (Boucher et al., 2000; Feng et al.,2002; Matsuda et al., 2002; Murphy et al., 2001). ThePI3K/Akt pathway is also important for survival, prolif-eration and resistance to apoptosis of pancreatic cancercells (Asano et al., 2004; Bondar, Sweeney-Gotsch,Andreeff, Mills, & McConkey, 2002; Ng, Tsao, Chow,& Hedley, 2000; Perugini, McDade, Vittimberga, &Callery, 2000; Yip-Schneider, Wiesenauer, & Schmidt,2003). It has also been shown that constitutive nuclearfactor-kappa B (NF-�B) activity is important in estab-lishing chemoresistance of BxPC3 human pancreaticcancer cells (Arlt et al., 2003). These pathways have beentargets for the development of therapeutic drugs for pan-creatic cancer (Dhar et al., 2005; Lockhart, Rothenberg,& Berlin, 2005; Xiong, 2004). In order to get insight intothe molecular and cellular basis of the clinical benefit ofexternal Qi of YXQ, we have investigated its effect onthese pathways in BxPC3 cells and fibroblasts. We foundthat external Qi of YXQ inhibited Akt and ERK1/2pathways in BxPC3 human pancreatic cancer cells,while it induced transient activation of these pathways infibroblasts. Furthermore, external Qi of YXQ exhibitedpotent cytotoxic effects on BxPC3 cells, but not on fibro-blasts.

2. Materials and methods

2.1. Cell culture

BxPC3 cells and human fibroblast cells were main-

tained in RPMI 1640 and DMEM containing 10% fetalbovine serum (FBS), 100 �g/ml penicillin G and 0.25%streptomycin at 37 ◦C and 5% CO2. RPMI 1640 mediumwas also supplemented with 2 mM glutamine.

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2.2. Treatment of cells by external Qi of YXQ

Cells were treated by external Qi of YXQ essentiallyas previously described (Yan, Zheng, et al., 1988; Yan etal., 2004). Briefly, cells cultured to near confluency weretransferred to the treatment room, treated by externalQi of YXQ for 5 min and then returned to the incu-bator. To determine the effect of external Qi of YXQon Akt and ERK1/2 phosphorylation, cells were har-vested and analyzed by Western blot 10 min after thetreatment. To examine the effect of external Qi of YXQon the EGF-mediated ERK1/2 activation, BxPC3 cellswere serum-starved for 48 h before they were exposedto external Qi of YXQ. Ten minutes after the exposureto external Qi of YXQ the cells were stimulated withEGF (100 ng/ml) for 20 min and harvested for Westernblot analysis. To investigate the effect of external Qi ofYXQ on the Akt and ERK pathways in fibroblasts, thecells were subjected to 24 h of serum starvation, treatedby external Qi of YXQ, and harvested at 0.5, 1, 12 and24 h after the treatment.

2.3. Cytotoxic assay

For cytotoxicity assessment of external Qi of YXQ,cells were seeded into 96-well plates at (1–2) × 104

cells per well one day before the treatment. Cellswere treated by external Qi of YXQ for 5 min andcell viability was determined using Trypan Blueexclusion assay 3, 6 and 24 h after the treatment. Insome experiments, cells were exposed to external Qiof YXQ for three times, 5 min each time with a 25 mininterval (i.e. the cells were returned to and kept inthe incubator for 25 min between treatments). Cellviability assays were performed 10 min after the thirdexposure to external Qi of YXQ, using MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] assayand LDH (lactic dehydrogenase) assay. A CellTiter96Aqueous One Solution Cell Proliferation Assaykit (Promega, Madison, WI) was used in the MTSassay and run according to the manufacturer. Briefly,20 �l of One Solution Reagent containing MTS wereadded to the cells. After incubation at 37 ◦C and 5%CO2 for 2–4 h, the plates were read at 490 nm witha plate reader. Data were calculated as percentage ofthe control cells after subtracting the background OD(optical density) of the culture medium. LDH activity

was measured using a CytoTox-ONE HomogeneousMembrane Integrity Assay kit according to the protocolprovided by the manufacturer (Promega, Madison, WI).Briefly, 50 �l of cell culture medium were mixed with

istry & Cell Biology 38 (2006) 2102–2113

50 �l of CytoTox-ONE Reagent, and incubated at roomtemperature for 10 min. The reaction was stopped with25 �l of Stop Solution. Fluorescence was recorded withan excitation wavelength of 544 nm and an emissionwavelength of 590 nm. Maximum LDH release wasobtained by adding Lysis solution (1:50, v/v) to thecell culture. Data were calculated as a percentage ofmaximum LDH release after subtracting the backgroundfrom the culture medium.

2.4. Electrophoresis mobility shift assay (EMSA)

Nuclear extract was prepared from BxPC3 cells asdescribed by Arlt et al. (2003). Briefly, cells were incu-bated in hypotonic HEPES/HCl buffer (10 mM HEPES,pH 7.6, 50 mM KCl, 0.1 mM PMSF, 0.5 ng/ml apro-tinin, 0.1 mM dithiothreitol) for 20 min on ice. Nucleiwere collected by centrifugation at 10,000 rpm for 5 minand washed once with the hypotonic buffer. A nuclearfraction was prepared from the nuclei by extractionwith high-salt buffer (50 mM HEPES, pH 7.9, 0.5 MNaCl, 1 mM MgCl2, 0.1 mM PMSF, 0.5 �g/ml apro-tinin, 0.1 mM dithiothreitol) for 15 min on ice followedby centrifugation at 10,000 rpm for 15 min. EMS A wasperformed with 32P-labelled synthetic NF-�B oligonu-cleotide (Promega, Madison, WI) according to the pro-tocol provided by the manufacturer.

2.5. Western blot analysis

Cells were lysed in RIPA buffer (50 mM Tris–Cl, pH7.4; 150 mM NaCl, 0.5% sodium deoxycholate, 1% NP-40, 0.1% SDS, 1 mM EDTA) supplemented with 2 mMNa3VO4 and 1× MiniComplete (protease inhibitorsfrom Roche, Minneapolis, MN). Cell lysates were sub-jected to electrophoresis on SDS polyacrylamide gels(4–20%). Proteins were transferred to nitrocellulosemembranes (Millipore, Bedford, MA), the membraneswere probed with antibodies against Akt, ERK1/2,phospho-Akt, phospho-ERK1/2, caspases 3 and 9 (CellSignaling, Beverly, MA), caspase 8 and poly(ADP-ribose) polymerase (PARP) (Santa Cruz Biotechnology,Santa Cruz, CA), or �-actin (Sigma, St. Louis, MO), asindicated.

2.6. PI3 kinase assay

Cells were lysed in lysis buffer containing 20 mM

Tris–Cl, pH 7.4, 10 mM EDTA, 100 mM NaCl,1% (octylphenoxy)polyethoxyethanol, 1 mM Na3VO4,50 mM NaF, and 1× Minicomplete protease inhibitors(Roche, Minneapolis, MN). PI3K activity assays were

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erformed directly on total cell lysates in 50 �l of theeaction mixture containing 0.2 mg/ml PI-4,5-P2, 50 �MTP, 0.2 �Ci [�-32P]ATP, 5 mM MgCl2, and 10 mMEPES buffer (pH 7.5) as previously described (Yan et

l., 2004). After incubation for 15 min at room tempera-ure the reactions were stopped by the addition of 100 �lf 1N HCl followed by 200 �l of chloroform–methanol1:1, v/v). Lipids were extracted and resolved on oxalate-oated silica gel 60 thin layer chromatography (TLC)lates with a solvent system of 2-propanol/2 M aceticcid (65:35, v/v). TLC plates were exposed to X-raylm and radioactive lipids were scraped off the platesnd quantified by liquid scintillation counting.

.7. Cell cycle analysis

Cells grown in normal medium were subjected toXQ external Qi treatment for 5 min and harvested by

rypsinization 12 h post the treatment. The cells werexed with 70% ethanol and treated with RNase A100 ng/ml) at 37 ◦C for 30 min. DNA was stained withropidium iodide (40 �g/ml). Samples were analyzed byow cytometry using a FACSVantage flow cytometerBD Bioscience, San Jose, CA). The fraction of cells inhe sub-G1 peak was considered apoptotic.

.8. DNA fragmentation analysis

Cells were harvested by trypsinization and resus-ended in lysis buffer (20 mM Tris–Cl, pH 8.0, 1% SDS,5 mM EDTA, and 1 mg/ml proteinase K). After incu-ation overnight at 50 ◦C, ribonuclease A was addedo 100 �g/ml and incubated for an additional 2 h at7 ◦C. The chromosomal DNA was extracted with phe-ol/chloroform, precipitated with 0.3 M sodium acetatend 2.5 volumes ethanol, and analyzed by agarose gellectrophoresis. DNA was stained with ethidium bro-ide (0.5 ng/ml) and visualized under ultraviolet light.

.9. Statistic analysis

Results are presented as mean ± S.D. The signifi-ance of differences in means was determined using thewo-tailed Student’s t-test. p < 0.05 was considered sig-ificant.

. Results

.1. External Qi of YXQ inhibits Akt and ERK1/2

hosphorylation in BxPC3 cells

The activation of both Akt kinase and MAP kinaseRK plays important roles in growth and chemoresis-

istry & Cell Biology 38 (2006) 2102–2113 2105

tance of pancreatic cancer cells (Asano et al., 2004;Bondar et al., 2002; Ng et al., 2000; Perugini et al.,2000; Yip-Schneider et al., 2003). Therefore, we exam-ined the effect of external Qi of YXQ on the activationof Akt and ERK1/2 in BxPC3 cells. Activation wasanalyzed by immunoblotting using antibodies recog-nizing phospho-Akt and phospho-ERK1/2, respectively.Under normal growth conditions, BxPC3 cells showedmoderate levels of Akt and ERK1/2 phosphorylation(Fig. 1A, lane 1). The phosphorylation levels of bothAkt and ERK1/2 in BxPC3 cells were reduced by ∼80%after the treatment by external Qi of YXQ (Fig. 1A,lane 2 and B), and remained low 16 h post the treat-ment (Fig. 1C). Consistent with this observation, PI3Kactivities were also dramatically inhibited by externalQi of YXQ as the formation of PI-3,4,5-P3, the prod-uct of PI3K, decreased significantly after the treatment(Fig. 1D). We further investigated the effect of externalQi of YXQ on the ERK1/2 activation mediated by EGF.BxPC3 cells were subjected to 48 h serum starvation andthen incubated with EGF for 20 min. The phosphoryla-tion levels of ERK1/2 increased ∼3.5-fold after EGFtreatment (Fig. 1E, lane 2 and F). However, pretreat-ment of BxPC3 cells by external Qi of YXQ abrogatedthe EGF-mediated ERK1/2 activation (Fig. 1E, lane 3and F). These findings indicate that external Qi of YXQcan profoundly interfere with the Akt and ERK pathwaysin BxPC3 cells.

3.2. External Qi of YXQ suppresses NF-κB activityin BxPC3 cells

It has been shown that constitutive NF-�B activity isimportant for chemoresistance of BxPC3 cells (Arlt etal., 2003), and that NF-�B can be activated through Aktand EGFR pathways (Ozes et al., 1999; Zhang et al.,2005). Therefore, we examined the effect of external Qiof YXQ on NF-�B activity in BxPC3 cells using EMS A.Constitutive NF-�B activity was detected in BxPC3 cellsunder normal growth conditions (Fig. 2A, lane 1), anddecreased by ∼75% after the treatment by external Qi ofYXQ (Fig. 2A, lane 2 and B). NF-�B activity increased2.2-fold after the incubation of BxPC3 cells with TNF-�for 30 min (Fig. 2C, lane 2 and D). This induction wasabolished by pretreatment of BxPC3 cells with externalQi of YXQ (Fig. 2C, lane 3 and D).

3.3. External Qi of YXQ activates Akt and ERK1/2

in fibroblasts

Our previous studies have shown that external Qiof YXQ protected neurons from H2O2-induced apopto-

2106 X. Yan et al. / The International Journal of Biochemistry & Cell Biology 38 (2006) 2102–2113

Fig. 1. Effect of external Qi of YXQ on Akt and ERK1/2 phosphorylation and PI3 kinase activity in BxPC3 cells. (A and B) Effect of external Qiof YXQ on basal Akt and ERK1/2 activities. Cells were treated by external Qi of YXQ for 5 min and harvested for Western blot analysis 10 minafter the treatment. A representative Western blot (A) and graph of mean ± S.D. (B) of phosphorylation from three to four independent experimentsare shown (*p ≤ 0.01 vs. control cells). (C) Time course of Akt and ERK1/2 phosphorylation in BxPC3 cells after 5 min treatment of external Qiof YXQ. Cells were lysed 0.5, 1 and 16 h post treatment and phosphorylation levels of Akt and ERK1/2 were tested by Western blot. (D) Effect ofexternal Qi of YXQ on PI3 kinase activity. Cells were treated by external Qi of YXQ for 5 min and 10 min later whole cell lysates were prepared andused in PI3K activity assay. PI-3,4,5-P3, the PI3K product, was separated from other lipids by thin layer chromatography (TLC). A representativeTLC (left panel) and mean ± S.D. of PI3K activity (%) (right panel) from three independent experiments are shown (*p < 0.01 vs. control). (E and F)Effect of external Qi of YXQ on the EGF-mediated ERK1/2 phosphorylation. BxPC3 cells serum starved for 48 h were used as control or treated byexternal Qi of YXQ for 5 min. Ten minutes later the cells were stimulated with EGF for an additional 20 min and harvested for Western blot analysis.

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A representative Western blot (E) and graph of mean ± S.D. of phospcells treated with EGF only).

sis, indicating that external Qi of YXQ has a protectiveeffect on normal cells (Yan et al., 2004). The activa-tion of the Akt and ERK pathways is known to protectcells from apoptosis and necrosis (Boonstra et al., 1995;Bruns et al., 2000; Cantly, 2002; Chang et al., 2003;Datta, Brunet, & Geenberg, 1999; Yao & Cooper, 1995).Thus, we examined the effect of external Qi of YXQ on

Akt and ERK1/2 kinases in fibroblasts. The cells weresubjected to 24 h of serum starvation before they weretreated by external Qi of YXQ. As shown in Fig. 3,external Qi of YXQ activated Akt and ERK1/2 in a

ion (F) from three independent experiments are shown (*p < 0.01 vs.

time-dependent manner. A significant increase in Aktand ERK1/2 phosphorylation was detected as early as0.5 h post the treatment. The highest levels of phospho-Akt and phospho-ERK1/2 were observed at 1 h post thetreatment. Thereafter Akt and ERK1/2 phosphorylationlevels gradually declined. Interestingly, PI3K activitiesin fibroblasts remained unchanged after the treatment

by external Qi of YXQ (Fig. 3C). These results sug-gest that external Qi of YXQ may have induced tran-sient activation of Akt in a PI3K-independent manner infibroblasts.

X. Yan et al. / The International Journal of Biochemistry & Cell Biology 38 (2006) 2102–2113 2107

Fig. 2. Effect of external Qi of YXQ on NF-�B activity in BxPC3cells. (A and B) Effect of external Qi of YXQ on constitutive NF-�B activity in BxPC3 cells. Cells were used as control or treated byexternal Qi of YXQ for 5 min and harvested 10 min after the treatment.NF-�B activity was determined by EMSA. A representative gel (A) andgraph of mean ± S.D. of NF-�B activity (B) from three independentexperiments are shown (*p ≤ 0.01 vs. control cells). (C and D) Effectof external Qi of YXQ on the TNF-�-induced NF-�B activation. Cellswere or were not treated by external Qi of YXQ for 5 min. Ten minuteslater the cells were treated with TNF-� for 30 min and harvested forEN(

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Fig. 3. Effect of external Qi of YXQ on Akt and ERK1/2 phosphory-lation and PI3K activity in fibroblasts. (A and B) Akt and ERK1/2phophorylation levels. Serum-starved fibroblasts were exposed toexternal Qi of YXQ for 5 min and harvested at different time points asindicated and ERK1/2 phosphorylation was analyzed by Western blot.A representative immunoblot (A) and graph of mean ± S.D. of foldinduction of phosphorylation (B) from three independent experimentsare shown (*p ≤ 0.05 vs. control cells). Lane C on the immunoblotand column C on the bar chart denote control cells. (C) PI3K activ-ity. Serum-starved fibroblasts were treated with external Qi of YXQfor 5 min and harvested 30 min after the treatment. Whole cell lysateswere prepared and used in PI3K activity assays. A representative TLC(left panel) and mean ± S.D. of percent PI3K activity (right panel)

DNA fragmentation, cleavage of caspases 3, 8 and 9 or

MSA. A representative EMSA gel (C) and graph of mean ± S.D. ofF-�B activity (D) from three independent experiments are shown

*p ≤ 0.01 vs. cells treated with TNF-� only).

.4. Differential cytotoxic effects of external Qi ofXQ on BxPC3 cells and fibroblasts

The inhibition of the Akt and ERK pathways has beenhown to cause cytotoxic effect on cells (Bondar et al.,002; Boucher et al., 2000; Cantly, 2002; Perugini etl., 2000). Therefore we investigated the effect of exter-al Qi of YXQ on BxPC3 cell viability. Treatment ofxPC3 cells by external Qi of YXQ for 5 min causedtime-dependent decrease in cell viability (Fig. 4A).omplete loss of BxPC3 cell viability was observed 24 host the treatment. In order to determine if external Qif YXQ induced apoptotic death in BxPC3 cells, cellsere stained with PI and analyzed on a flow cytometer

or formation of a sub-G1 peak that is considered apop-otic. The percentage of cells in the sub-G1 populationas 1.2% in control cells, but increased to 31.6% after

he treatment, with concomitant decreases of cell popula-ions in the G1 and G2 phases of the cell cycle (Fig. 4B).urthermore, external Qi of YXQ induced DNA frag-entation (Fig. 4C), cleavage of procaspases 3, 8 and 9,

nd cleavage of PARP (Fig. 4D). These results indicatehat external Qi of YXQ induced apoptosis in BxPC3

ells. In contrast, the viability and cell cycle distributionf fibroblasts was not significantly affected by externali of YXQ (Fig. 4A and B). Consistent with these results,

from six independent experiments are shown. The slight difference inPI3K activities between the control and treated groups was statisticallyinsignificant.

cleavage of PARP was not detected in fibroblasts treatedwith external Qi of YXQ (Fig. 4C and D). In order toinvestigate the role of inhibition of Akt and/or ERK1/2

2108 X. Yan et al. / The International Journal of Biochemistry & Cell Biology 38 (2006) 2102–2113

Fig. 4. Differential cytotoxic effects of external Qi of YXQ on BxPC3 cells and fibroblasts. Cells were grown in normal medium and treated byexternal Qi of YXQ for 5 min. (A) Cell viability was determined 3, 6 and 24 h after the treatment using Trypan blue exclusion assay and calculatedas a percentage of the control cells (at time 0 h) that were not exposed to external Qi of YXQ. Results are presented as mean ± S.D. of percentviability from three independent experiments. (B) Cell cycle distribution of BxPC3 cells and fibroblasts. Control cells (left panel) and cells at 12 hafter treatment with external Qi of YXQ (right panel) were stained with PI and analyzed by flow cytometry. Also shown are the relative percentagesof cells in the G1, S, G2 and sub-G1 phases. (C) DNA fragmentation analysis of BxPC3 cells and fibroblasts. Lanes 1 and 3, DNA isolated fromcontrol fibroblasts and BxPC3 cells, respectively; lanes 2 and 4, DNA isolated from fibroblasts and BxPC3 cells at 16 h post treatment with externalQi of YXQ, respectively. (D) Western blot analysis of caspases 3, 8 and 9 and PARP in fibroblasts (left panel) and BxPC3 cells (right panel). Lane1, untreated control cells; lanes 2 and 3, cells treated by external Qi of YXQ and incubated for additional 6 and 18 h, respectively. (E and F) BxPC3cells grown in normal medium were treated with 50 �M of LY294002 (a PI3K inhibitor), PD098059 (an ERK inhibitor), or combined for 24 h.Cell viability (E) was determined using MTS assay and is presented as mean ± S.D. of percent viability from three to six independent experiments(*p < 0.01 vs. control cells). Cleavage of caspase 3 (F) was determined by Western blot analysis.

X. Yan et al. / The International Journal of Biochemistry & Cell Biology 38 (2006) 2102–2113 2109

Fig. 5. Prolonged exposure to external Qi of YXQ caused lysis of BxPC3 cells, but not fibroblasts. Cells were sequentially treated by external Qiof YXQ for three times over 65 min (5 min each time with 25 min interval between consecutive treatments). Cell viability was determined usingM sure tov ion of cd ively.

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TS assay (A) and LDH release assay (B) 10 min after the third expoiability (n = 3, *p ≤ 0.01 vs. control cells). (C) Microscopic examinat, BxPC3 cells and fibroblasts treated by external Qi of YXQ, respect

hosphorylation in growth and apoptosis, BxPC3 cellsere treated with PI3K specific inhibitor LY294002,RK inhibitor PD098059, or in combination for 24 h andell viability and cell cycle distribution were determined.reatment with LY294002, PD098059, and in combi-ation inhibited cell growth by 40%, 32% and 50%,espectively (Fig. 4E), and increased the percentage ofhe sub-G1 population by 8%, 6% and 20%, respectivelydata not shown). Cleavage of pro-casapse 3 was detectedn BxPC3 cells treated with LY294002, and to a lesserxtent, with PD098059 (Fig. 4F). These results indicatehat inhibition of Akt and ERK1/2 phosphorylation plays

role in growth inhibition and induction of apoptosis ofxPC3 cells.

Interestingly, when BxPC3 cells were treated by ext-rnal Qi of YXQ for three times over a period of 65 min,

external Qi of YXQ. Results are presented as mean ± S.D. of percentells. a and c, control BxPC3 cells and fibroblasts, respectively; b and

a dramatic reduction in cell viability was detected asearly as 10 min after the completion of the treatment(Fig. 5A). In concordance with the rapid decline in cellviability, LDH activity released into the medium rapidlyincreased to the levels comparable to the maximum LDHrelease (Fig. 5B). Microscopic examinations revealedthat BxPC3 cells treated in this way had been lysedand essentially no intact cells were observed (Fig. 5C).In contrast, the viability, LDH release and morphologyof fibroblasts were unaffected by the same treatment(Fig. 5C).

4. Discussion

The activation of both the Akt and ERK pathways pro-motes cell survival and protects cells against apoptosis

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and necrosis (Boonstra et al., 1995; Bruns et al., 2000;Chang et al., 2003; Datta et al., 1999; Proskuryakov,Konoplyannikov, & Gabai, 2003; Yao & Cooper, 1995).These pathways play important roles in the developmentof pancreatic tumors and chemoresistance of pancreaticcancer cells (Asano et al., 2004; Bondar et al., 2002;Boucher et al., 2000; Feng et al., 2002; Matsuda et al.,2002; Murphy et al., 2001; Ng et al., 2000; Perugini etal., 2000; Yip-Schneider et al., 2003). The inhibition ofthese pathways may potentially be beneficial for can-cer patients and these pathways are candidate targets forchemotherapy of pancreatic cancer. Various inhibitorsand neutralizing antibodies of these pathways have beendeveloped and are in clinical trials (Dhar et al., 2005;Lockhart et al., 2005; Xiong, 2004). However, the Aktand ERK pathways are also critical for multiple physio-logical processes in normal cells and inhibition of thesepathways may thus be toxic to the patients (Fang &Richardson, 2005; Nicholson & Anderson, 2002). Theability to use inhibitors of these pathways will depend ontheir ability to alter tumor progression relative to theirtoxicity to normal cellular functions. In this report weshow that external Qi of YXQ has opposite effects onthe Akt and ERK pathways in BxPC3 pancreatic can-cer cells versus fibroblasts; while it inhibits basal Aktand ERK1/2 activity and the EGF-induced ERK activa-tion in BxPC3 cells, it transiently activates these kinasesin fibroblasts. This suggests that external Qi of YXQmay have cytotoxic effects on cancer cells while protect-ing normal cells. Indeed, we demonstrate in this reportby various methods that external Qi of YXQ has potentcytotoxic effects on BxPC3 pancreatic cancer cells, butnot fibroblasts. Furthermore, external Qi of YXQ wasalso highly cytotoxic to various human cancer cell linesincluding breast, prostate, colon cancer and leukemiacell lines, but not human umbilical vein endothelial cells(HUVEC) or peripheral blood monocytes (PBMC) (Yan,Fong, Jiang, et al., 2002; manuscript in preparation). Wehave also demonstrated that external Qi of YXQ pro-tected neurons from H2O2-induced apoptosis (Yan et al.,2004). The observations that external Qi of YXQ helpscancer patients to minimize or avoid side effects asso-ciated with conventional chemo- and radiotherapy alsosupport the notion of a protective effect for external Qiof YXQ on normal cells (Fong, 1997; Ming, 1988; Wang& Zhu, 1997; Zhang et al., 1997).

The mechanisms by which external Qi of YXQexhibits differential effect on the Akt and ERK pathways

in cancer versus normal cells remain to be investigated.Nonetheless, external Qi of YXQ has been shown to beable to influence the structure and properties of proteinsand has been successfully used to promote crystallization

istry & Cell Biology 38 (2006) 2102–2113

of a Fab protein (Lu, 1997; Yan et al., 1999). Recently, wehave shown that external Qi of YXQ is able to enhancePI3K activity in neurons (Yan et al., 2004), and increaseor repress PI3K activity of a highly enriched PI3K prepa-ration (Yan, Xia, et al., 2002). We showed in this reportthat external Qi of YXQ dramatically inhibited Akt acti-vation with concomitant inhibition of PI3K activity inBxPC3 cells, while it stimulated Akt phosphorylation infibroblasts without elevating PI3K activity. These find-ings suggest that external Qi of YXQ may modulateAkt activation through PI3K dependent and indepen-dent mechanisms (Cantly, 2002; Nicholson & Anderson,2002; Song, Ouyang, & Bao, 2005).

A single exposure of BxPC3 cells to external Qi ofYXQ induced apoptosis as it caused a dramatic increasein the sub-G1 population, DNA fragmentation and cleav-age of caspases and PARP. Two major pathways ofcaspase activation are known: the cell surface deathreceptor pathway and the mitochondria-initiated path-way (Budihardjo, Oliver, Lutter, Luo, & Wang, 1999).In the death receptor pathway, activation of caspase 8 isthe critical event that transmits the death signal. In themitochondria-initiated pathway, caspase 9 is activatedand will then cleave and activate downstream caspasessuch caspases 3, 6 and 7. Thus, the cleavage of cas-pases 8 and 9 in BxPC3 cells induced by external Qiof YXQ suggests that both apoptotic pathways were ini-tiated after the treatment. Inhibition of ERK pathwayby PD098059 has been shown to activate both apop-totic pathways in pancreatic cancer MIA PaCa-2 cells(Boucher et al., 2000). In this report we show that inhibi-tion of Akt and ERK phosphorylation by LY294002 andPD098059, respectively, attenuated BxPC3 cell growthand caused a small but significant increase in apopto-sis of BxPC3 cells. Similar effects of LY294002 onBxPC3 cell growth and apoptosis have also been reported(Perugini et al., 2000). Thus, it is reasonable to assumethat inhibition of PI3K/Akt and ERK pathways by exter-nal Qi of YXQ was at least in part responsible for theinduction of apoptosis in BxPC3 cells. Further studiesusing negative mutants or constantly active forms of Aktand ERK are required to dissect their role in the initia-tion of these two apoptotic pathways by external Qi ofYXQ, and elucidate additional mechanisms involved ininduction of apoptosis of BxPC3 cells by external Qiof YXQ.

Interestingly, prolonged exposure to external Qi ofYXQ caused rapid lysis of BxPC3 cells, probably

through non-apoptotic mechanism(s). The precise mech-anisms underlying this cytolytic effect of external Qi ofYXQ remains to be investigated. Previously, we haveobserved that external Qi of YXQ could dramatically

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nfluence the structure and properties of liposomes, anrtificial model for bio-membrane studies (Yan, Zhao,t al., 1988). These findings suggest that prolongedreatment by external Qi of YXQ might have causedrofound damage to the structure and function of thexPC3 cell membrane, resulting in rapid osmotic lysis.arlier studies have used physical signal detectors toerify the existence of external Qi and have sometimesetected signals of electricity, magnetism and soundn external Qi (Chen, 2004). However, these physicalspects are most likely secondary or side effects ofxternal Qi and have not revealed the primary naturef external Qi (Chen, 2004; Lu, 1997). Furthermore,hile ultrasound, low-frequency pulsating electromag-etic fields (LF-PEMF) and low-level direct currentsnduce apoptotic and/or necrotic death of cancer cellsncluding leukemia cell lines (K-562, U-937 and HL-0), they also have significant cytotoxic effects on nor-al cells including fibroblasts and PBMC (Ashush et

l., 2000; Feigl, Volklein, Iro, Ell, & Schneider, 1996;ejbkowicz, Zwiran, & Salzberg, 1993; Radeva & Berg,004; Tachibana, Uchida, Hisano, & Morioka, 1997;ang et al., 2005; Yen et al., 1999). In contrast, exter-al Qi of YXQ has no cytotoxic effect on normal cellsncluding fibroblasts, PBMC and HUVEC, while it com-letely kills cancer cells such as leukemia cell lines-562, U-937 and HL-60 (Yan, Fong, Jiang, et al., 2002;anuscript in preparation). Therefore, it is unlikely that

hese physical aspects, if exist, could be responsible forhe cytotoxic effects of external Qi of YXQ on cancerells.

The activation of NF-�B is mediated by two kinases,�B and IKK (Arlt et al., 2001, 2002; Dong et al., 2002).n response to various stimuli, I�B is phosphorylated byKK and degraded in the cytoplasm, leading to nuclearranslocation of NF-�B and activation of its target genes.kt has been shown to be required for TNF-�-mediatedF-�B activation by phosphorylation of IKK and sub-

equent degradation of I�B (Ozes et al., 1999). Thenhibition of Akt may thus contribute to the suppressionf the TNF-�-induced NF-�B activity in BxPC3 cellsy external Qi of YXQ. Constitutive NF-�B activationas been observed in many types of solid tumors and haseen shown to contribute to survival and resistance ofancer cells to apoptosis induced by various agents (Arltt al., 2001, 2002, 2003; Nicholson & Anderson, 2002).t has been shown that the blockade of NF-�B activationncreases the sensitivity of malignant cells to the apop-

otic effects of anticancer drugs and radiation (Arlt etl., 2001; Dong et al., 2002). These findings suggest thatxternal Qi of YXQ may also sensitize cancer cells tohemotherapy through inhibiting NF-�B activity.

istry & Cell Biology 38 (2006) 2102–2113 2111

Resistance to chemotherapy is a major cause of treat-ment failure and poor prognosis in pancreatic cancer(Dhar et al., 2005; Lockhart et al., 2005; Xiong, 2004).The inhibition of Akt, ERK1/2 and NF-�B pathwaysin BxPC3 cells by external Qi of YXQ is notewor-thy as these pathways play important roles in pancre-atic cancer cell growth and drug resistance (Boucher etal., 2000; Perugini et al., 2000). Furthermore, BxPC3cells are resistant to apoptosis mediated by gemcitabine,which has become the standard chemotherapy for locallyadvanced and metastatic adenocarcinoma of pancreas(Richards, 2005), and by the Fas pathway (Elnemr etal., 2001). However, BxPC3 cells are highly sensitive toexternal Qi of YXQ. Taken together, our findings suggestthat external Qi of YXQ could potentially be an effectiveapproach for therapy of pancreatic carcinomas.

Acknowledgement

This work was supported in part by Yan Xin Founda-tion.

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