The International Journal of Biochemistry & Cell Biology 38 (2006) 21022113

External Qi of Yan Xin Qigong differentially regulates the Akt and 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
Institute of Chongqing Traditional Chinese Medicine, Chongqing, PR China b New Medical Science Research Institute, New York, NY 10107, USA c Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA d Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA e Childrens Hospital, Harvard Medical School, Boston, MA 02115, USA Received 14 February 2006; received in revised form 21 May 2006; accepted 2 June 2006 Available online 27 June 2006
a

Abstract Long-term clinical observations and ongoing studies have shown signicant antitumor effect of external Qi of Yan Xin Qigong which originated from traditional Chinese medicine. In order to understand the molecular and cellular mechanisms underlying the antitumor effect of external Qi of Yan Xin Qigong, we have examined its cytotoxic effect on BxPC3 pancreatic cancer cells and its effect on the Akt and extracellular signal-regulated kinase pathways. We found that external Qi of Yan Xin Qigong dramatically inhibited basal phosphorylation levels of Akt and extracellular signal-regulated kinases, epidermal growth factor-mediated phosphorylation of extracellular signal-regulated kinases, and phosphatidylinositol 3-kinase activity. External Qi of Yan Xin Qigong also inhibited constitutive and inducible activities of nuclear factor-kappa B, a target of the Akt and epidermal growth factor receptor pathways. Furthermore, a single 5 min exposure of BxPC3 cells to external Qi of Yan Xin Qigong induced apoptosis, accompanied by 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 of broblasts with external Qi of Yan Xin Qigong induced transient activation of extracellular signal-regulated kinases and Akt, and caused no cytotoxic effect. These ndings suggest that external Qi of Yan Xin Qigong may differentially regulate these survival pathways 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 uoride; TCM, traditional Chinese 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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1. Introduction The concept External Qi (of Qigong) refers to the technology and ability of the Qi deployment therapy and health preservation of traditional Chinese medicine (TCM) and has been described in classic literatures of TCM and in Chinese medical textbooks (Yan et al., 2004). External Qi therapy of Chinese medicine has long been one of the medical practices in China and is under management by the Chinese health authorities. Multiple studies have shown that external Qi can be emitted by highly talented/trained Qigong practitioners, while ordinary people are unable to deploy external Qi of therapeutic, physical or chemical effects (Lu, 1997; Yan et al., 2004). Qi is considered as the basic element of human vital energy in TCM. The underlying theory of TCM is fully based on balancing Qi according to the theory of Yin-Yang, an approach that also takes into account the biological rhythms, and the chronotherapeutic principles is integrated into external Qi therapy of TCM (Seki et al., 2005). Long-term clinical observations and ongoing studies have shown that patients with cancer and other medical conditions have received signicant benecial effects from the exposure to external Qi of Yan Xin Qigong (YXQ) which originated from TCM, and in some cases conditions of cancer patients have even been dramatically improved (Fong, 1997; Ming, 1988; Wang & Zhu, 1997; Zhang, Zhao, & Zhang, 1997). External Qi of YXQ can also help patients improve or avoid side effects associated with radio- and chemotherapy (Fong, 1997; Ming, 1988; Wang & Zhu, 1997; Zhang et al., 1997). In order to understand the molecular basis underlying these effects, numerous laboratory studies have been conducted in the past 20 years (e.g. Li et al., 1990; Lu, 1997; Yan, Fong, Jiang, et al., 2002; Yan, Fong, Wolf, Wolf, & Cao, 2001; Yan, Fong, Wolf, et al., 2002; Yan, Li, Liu, et al., 1988; Yan, Li, Yang, & Lu, 1988; Yan, Li, Yu, Li, & Lu, 1988; Yan et al., 1999; Yan et al., 2004; Yan, Zhao, Yin, & Lu, 1988; Yan, Zheng, Zou, & Lu, 1988; Yan, Xia, Shen, & Traynor-Kaplan, 2002). These studies have demonstrated that external Qi of YXQ is able to alter molecular structure and properties of experimental samples in multiple disciplines (Lu, 1997; Yan et al., 1999; Yan, Lu, et al., 2002). In particular, external Qi of YXQ has been shown to inuence the molecular structure and function of DNA, RNA and protein molecules (Li et al., 1990; Lu, 1997; Yan, Zheng, et al., 1988), enhance or repress phosphatidylinositol 3-kinase (PI3K) activity in vitro and in vivo (Yan, Xia, et al., 2002; Yan et al., 2004). Furthermore, it is also able to modulate gene expression, signal transduction, cell survival and apop-

tosis (Yan et al., 2001; Yan, Fong, Jiang, et al., 2002; Yan, Fong, Wolf, et al., 2002; Yan, Lu, et al., 2002; Yan et al., 2004). Carcinoma of the pancreas is the fth leading cause of cancer-related deaths in Western countries, with an overall 1-year survival rate of 12% and 5-year survival rate of 35% (Greenlee, Hill-Harmon, Murray, & Thun, 2001). Resistance to chemotherapy is a major cause of treatment failure and poor prognosis in pancreatic cancer (Shi, Liu, Kleeff, Friess, & Buchler, 2002). Multiple genetic and epigenetic changes occur at very high frequencies 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; Shi et al., 2002). The majority of pancreatic cancers overexpress epidermal growth factor receptor (EGFR). EGFR and its downstream signaling pathways, Ras-Raf-MEKERK axis, play important roles in the development of pancreatic cancer (Boucher et al., 2000; Feng et al., 2002; Matsuda et al., 2002; Murphy et al., 2001). The PI3K/Akt pathway is also important for survival, proliferation and resistance to apoptosis of pancreatic cancer cells (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 nuclear factor-kappa B (NF- B) activity is important in establishing chemoresistance of BxPC3 human pancreatic cancer cells (Arlt et al., 2003). These pathways have been targets for the development of therapeutic drugs for pancreatic cancer (Dhar et al., 2005; Lockhart, Rothenberg, & Berlin, 2005; Xiong, 2004). In order to get insight into the molecular and cellular basis of the clinical benet of external Qi of YXQ, we have investigated its effect on these pathways in BxPC3 cells and broblasts. We found that external Qi of YXQ inhibited Akt and ERK1/2 pathways in BxPC3 human pancreatic cancer cells, while it induced transient activation of these pathways in broblasts. Furthermore, external Qi of YXQ exhibited potent cytotoxic effects on BxPC3 cells, but not on broblasts. 2. Materials and methods 2.1. Cell culture BxPC3 cells and human broblast cells were maintained in RPMI 1640 and DMEM containing 10% fetal bovine serum (FBS), 100 g/ml penicillin G and 0.25% streptomycin at 37 C and 5% CO2 . RPMI 1640 medium was 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 essentially as previously described (Yan, Zheng, et al., 1988; Yan et al., 2004). Briey, cells cultured to near conuency were transferred to the treatment room, treated by external Qi of YXQ for 5 min and then returned to the incubator. To determine the effect of external Qi of YXQ on Akt and ERK1/2 phosphorylation, cells were harvested and analyzed by Western blot 10 min after the treatment. To examine the effect of external Qi of YXQ on the EGF-mediated ERK1/2 activation, BxPC3 cells were serum-starved for 48 h before they were exposed to external Qi of YXQ. Ten minutes after the exposure to external Qi of YXQ the cells were stimulated with EGF (100 ng/ml) for 20 min and harvested for Western blot analysis. To investigate the effect of external Qi of YXQ on the Akt and ERK pathways in broblasts, the cells were subjected to 24 h of serum starvation, treated by external Qi of YXQ, and harvested at 0.5, 1, 12 and 24 h after the treatment. 2.3. Cytotoxic assay For cytotoxicity assessment of external Qi of YXQ, cells were seeded into 96-well plates at (12) 104 cells per well one day before the treatment. Cells were treated by external Qi of YXQ for 5 min and cell viability was determined using Trypan Blue exclusion assay 3, 6 and 24 h after the treatment. In some experiments, cells were exposed to external Qi of YXQ for three times, 5 min each time with a 25 min interval (i.e. the cells were returned to and kept in the incubator for 25 min between treatments). Cell viability assays were performed 10 min after the third exposure to external Qi of YXQ, using MTS [3-(4,5dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)2-(4-sulfophenyl)-2H-tetrazolium, inner salt] assay and LDH (lactic dehydrogenase) assay. A CellTiter 96Aqueous One Solution Cell Proliferation Assay kit (Promega, Madison, WI) was used in the MTS assay and run according to the manufacturer. Briey, 20 l of One Solution Reagent containing MTS were added to the cells. After incubation at 37 C and 5% CO2 for 24 h, the plates were read at 490 nm with a plate reader. Data were calculated as percentage of the control cells after subtracting the background OD (optical density) of the culture medium. LDH activity was measured using a CytoTox-ONE Homogeneous Membrane Integrity Assay kit according to the protocol provided by the manufacturer (Promega, Madison, WI). Briey, 50 l of cell culture medium were mixed with

50 l of CytoTox-ONE Reagent, and incubated at room temperature for 10 min. The reaction was stopped with 25 l of Stop Solution. Fluorescence was recorded with an excitation wavelength of 544 nm and an emission wavelength of 590 nm. Maximum LDH release was obtained by adding Lysis solution (1:50, v/v) to the cell culture. Data were calculated as a percentage of maximum LDH release after subtracting the background from the culture medium. 2.4. Electrophoresis mobility shift assay (EMSA) Nuclear extract was prepared from BxPC3 cells as described by Arlt et al. (2003). Briey, cells were incubated in hypotonic HEPES/HCl buffer (10 mM HEPES, pH 7.6, 50 mM KCl, 0.1 mM PMSF, 0.5 ng/ml aprotinin, 0.1 mM dithiothreitol) for 20 min on ice. Nuclei were collected by centrifugation at 10,000 rpm for 5 min and washed once with the hypotonic buffer. A nuclear fraction was prepared from the nuclei by extraction with high-salt buffer (50 mM HEPES, pH 7.9, 0.5 M NaCl, 1 mM MgCl2 , 0.1 mM PMSF, 0.5 g/ml aprotinin, 0.1 mM dithiothreitol) for 15 min on ice followed by centrifugation at 10,000 rpm for 15 min. EMS A was performed with 32 P-labelled synthetic NF- B oligonucleotide (Promega, Madison, WI) according to the protocol provided by the manufacturer. 2.5. Western blot analysis Cells were lysed in RIPA buffer (50 mM TrisCl, pH 7.4; 150 mM NaCl, 0.5% sodium deoxycholate, 1% NP40, 0.1% SDS, 1 mM EDTA) supplemented with 2 mM Na3 VO4 and 1 MiniComplete (protease inhibitors from Roche, Minneapolis, MN). Cell lysates were subjected to electrophoresis on SDS polyacrylamide gels (420%). Proteins were transferred to nitrocellulose membranes (Millipore, Bedford, MA), the membranes were probed with antibodies against Akt, ERK1/2, phospho-Akt, phospho-ERK1/2, caspases 3 and 9 (Cell Signaling, Beverly, MA), caspase 8 and poly(ADPribose) polymerase (PARP) (Santa Cruz Biotechnology, Santa Cruz, CA), or -actin (Sigma, St. Louis, MO), as indicated. 2.6. PI3 kinase assay Cells were lysed in lysis buffer containing 20 mM TrisCl, pH 7.4, 10 mM EDTA, 100 mM NaCl, 1% (octylphenoxy)polyethoxyethanol, 1 mM Na3 VO4 , 50 mM NaF, and 1 Minicomplete protease inhibitors (Roche, Minneapolis, MN). PI3K activity assays were

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performed directly on total cell lysates in 50 l of the reaction mixture containing 0.2 mg/ml PI-4,5-P2 , 50 M ATP, 0.2 Ci [ -32 P]ATP, 5 mM MgCl2 , and 10 mM HEPES buffer (pH 7.5) as previously described (Yan et al., 2004). After incubation for 15 min at room temperature the reactions were stopped by the addition of 100 l of 1N HCl followed by 200 l of chloroformmethanol (1:1, v/v). Lipids were extracted and resolved on oxalatecoated silica gel 60 thin layer chromatography (TLC) plates with a solvent system of 2-propanol/2 M acetic acid (65:35, v/v). TLC plates were exposed to X-ray lm and radioactive lipids were scraped off the plates and quantied by liquid scintillation counting. 2.7. Cell cycle analysis Cells grown in normal medium were subjected to YXQ external Qi treatment for 5 min and harvested by trypsinization 12 h post the treatment. The cells were xed with 70% ethanol and treated with RNase A (100 ng/ml) at 37 C for 30 min. DNA was stained with propidium iodide (40 g/ml). Samples were analyzed by ow cytometry using a FACSVantage ow cytometer (BD Bioscience, San Jose, CA). The fraction of cells in the sub-G1 peak was considered apoptotic. 2.8. DNA fragmentation analysis Cells were harvested by trypsinization and resuspended in lysis buffer (20 mM TrisCl, pH 8.0, 1% SDS, 25 mM EDTA, and 1 mg/ml proteinase K). After incubation overnight at 50 C, ribonuclease A was added to 100 g/ml and incubated for an additional 2 h at 37 C. The chromosomal DNA was extracted with phenol/chloroform, precipitated with 0.3 M sodium acetate and 2.5 volumes ethanol, and analyzed by agarose gel electrophoresis. DNA was stained with ethidium bromide (0.5 ng/ml) and visualized under ultraviolet light. 2.9. Statistic analysis Results are presented as mean S.D. The signicance of differences in means was determined using the two-tailed Students t-test. p < 0.05 was considered signicant. 3. Results 3.1. External Qi of YXQ inhibits Akt and ERK1/2 phosphorylation in BxPC3 cells The activation of both Akt kinase and MAP kinase ERK plays important roles in growth and chemoresis-

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 examined the effect of external Qi of YXQ on the activation of Akt and ERK1/2 in BxPC3 cells. Activation was analyzed by immunoblotting using antibodies recognizing phospho-Akt and phospho-ERK1/2, respectively. Under normal growth conditions, BxPC3 cells showed moderate levels of Akt and ERK1/2 phosphorylation (Fig. 1A, lane 1). The phosphorylation levels of both Akt 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 treatment (Fig. 1C). Consistent with this observation, PI3K activities were also dramatically inhibited by external Qi of YXQ as the formation of PI-3,4,5-P3, the product of PI3K, decreased signicantly after the treatment (Fig. 1D). We further investigated the effect of external Qi of YXQ on the ERK1/2 activation mediated by EGF. BxPC3 cells were subjected to 48 h serum starvation and then incubated with EGF for 20 min. The phosphorylation levels of ERK1/2 increased 3.5-fold after EGF treatment (Fig. 1E, lane 2 and F). However, pretreatment of BxPC3 cells by external Qi of YXQ abrogated the EGF-mediated ERK1/2 activation (Fig. 1E, lane 3 and F). These ndings indicate that external Qi of YXQ can profoundly interfere with the Akt and ERK pathways in BxPC3 cells. 3.2. External Qi of YXQ suppresses NF-B activity in BxPC3 cells It has been shown that constitutive NF- B activity is important for chemoresistance of BxPC3 cells (Arlt et al., 2003), and that NF- B can be activated through Akt and EGFR pathways (Ozes et al., 1999; Zhang et al., 2005). Therefore, we examined the effect of external Qi of YXQ on NF- B activity in BxPC3 cells using EMS A. Constitutive NF- B activity was detected in BxPC3 cells under normal growth conditions (Fig. 2A, lane 1), and decreased by 75% after the treatment by external Qi of YXQ (Fig. 2A, lane 2 and B). NF- B activity increased 2.2-fold after the incubation of BxPC3 cells with TNFfor 30 min (Fig. 2C, lane 2 and D). This induction was abolished by pretreatment of BxPC3 cells with external Qi of YXQ (Fig. 2C, lane 3 and D). 3.3. External Qi of YXQ activates Akt and ERK1/2 in broblasts Our previous studies have shown that external Qi of YXQ protected neurons from H2 O2 -induced apopto-

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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 Qi of 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 min after the treatment. A representative Western blot (A) and graph of mean S.D. (B) of phosphorylation from three to four independent experiments are 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 Qi of 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 of external 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 and used in PI3K activity assay. PI-3,4,5-P3, the PI3K product, was separated from other lipids by thin layer chromatography (TLC). A representative TLC (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 by external 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. A representative Western blot (E) and graph of mean S.D. of phosphorylation (F) from three independent experiments are shown (* p < 0.01 vs. cells treated with EGF only).

sis, indicating that external Qi of YXQ has a protective effect on normal cells (Yan et al., 2004). The activation of the Akt and ERK pathways is known to protect cells 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 broblasts. The cells were subjected to 24 h of serum starvation before they were treated by external Qi of YXQ. As shown in Fig. 3, external Qi of YXQ activated Akt and ERK1/2 in a

time-dependent manner. A signicant increase in Akt and ERK1/2 phosphorylation was detected as early as 0.5 h post the treatment. The highest levels of phosphoAkt and phospho-ERK1/2 were observed at 1 h post the treatment. Thereafter Akt and ERK1/2 phosphorylation levels gradually declined. Interestingly, PI3K activities in broblasts remained unchanged after the treatment by external Qi of YXQ (Fig. 3C). These results suggest that external Qi of YXQ may have induced transient activation of Akt in a PI3K-independent manner in broblasts.

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Fig. 2. Effect of external Qi of YXQ on NF- B activity in BxPC3 cells. (A and B) Effect of external Qi of YXQ on constitutive NFB activity in BxPC3 cells. Cells were used as control or treated by external Qi of YXQ for 5 min and harvested 10 min after the treatment. NF- B activity was determined by EMSA. A representative gel (A) and graph of mean S.D. of NF- B activity (B) from three independent experiments are shown (* p 0.01 vs. control cells). (C and D) Effect of external Qi of YXQ on the TNF- -induced NF- B activation. Cells were or were not treated by external Qi of YXQ for 5 min. Ten minutes later the cells were treated with TNF- for 30 min and harvested for EMSA. A representative EMSA gel (C) and graph of mean S.D. of NF- B activity (D) from three independent experiments are shown (* p 0.01 vs. cells treated with TNF- only).

3.4. Differential cytotoxic effects of external Qi of YXQ on BxPC3 cells and broblasts The inhibition of the Akt and ERK pathways has been shown to cause cytotoxic effect on cells (Bondar et al., 2002; Boucher et al., 2000; Cantly, 2002; Perugini et al., 2000). Therefore we investigated the effect of external Qi of YXQ on BxPC3 cell viability. Treatment of BxPC3 cells by external Qi of YXQ for 5 min caused a time-dependent decrease in cell viability (Fig. 4A). Complete loss of BxPC3 cell viability was observed 24 h post the treatment. In order to determine if external Qi of YXQ induced apoptotic death in BxPC3 cells, cells were stained with PI and analyzed on a ow cytometer for formation of a sub-G1 peak that is considered apoptotic. The percentage of cells in the sub-G1 population was 1.2% in control cells, but increased to 31.6% after the treatment, with concomitant decreases of cell populations in the G1 and G2 phases of the cell cycle (Fig. 4B). Furthermore, external Qi of YXQ induced DNA fragmentation (Fig. 4C), cleavage of procaspases 3, 8 and 9, and cleavage of PARP (Fig. 4D). These results indicate that external Qi of YXQ induced apoptosis in BxPC3 cells. In contrast, the viability and cell cycle distribution of broblasts was not signicantly affected by external Qi of YXQ (Fig. 4A and B). Consistent with these results,

Fig. 3. Effect of external Qi of YXQ on Akt and ERK1/2 phosphorylation and PI3K activity in broblasts. (A and B) Akt and ERK1/2 phophorylation levels. Serum-starved broblasts were exposed to external Qi of YXQ for 5 min and harvested at different time points as indicated and ERK1/2 phosphorylation was analyzed by Western blot. A representative immunoblot (A) and graph of mean S.D. of fold induction of phosphorylation (B) from three independent experiments are shown (* p 0.05 vs. control cells). Lane C on the immunoblot and column C on the bar chart denote control cells. (C) PI3K activity. Serum-starved broblasts were treated with external Qi of YXQ for 5 min and harvested 30 min after the treatment. Whole cell lysates were prepared and used in PI3K activity assays. A representative TLC (left panel) and mean S.D. of percent PI3K activity (right panel) from six independent experiments are shown. The slight difference in PI3K activities between the control and treated groups was statistically insignicant.

DNA fragmentation, cleavage of caspases 3, 8 and 9 or cleavage of PARP was not detected in broblasts treated with external Qi of YXQ (Fig. 4C and D). In order to investigate the role of inhibition of Akt and/or ERK1/2

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Fig. 4. Differential cytotoxic effects of external Qi of YXQ on BxPC3 cells and broblasts. Cells were grown in normal medium and treated by external 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 calculated as 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 percent viability from three independent experiments. (B) Cell cycle distribution of BxPC3 cells and broblasts. Control cells (left panel) and cells at 12 h after treatment with external Qi of YXQ (right panel) were stained with PI and analyzed by ow cytometry. Also shown are the relative percentages of cells in the G1, S, G2 and sub-G1 phases. (C) DNA fragmentation analysis of BxPC3 cells and broblasts. Lanes 1 and 3, DNA isolated from control broblasts and BxPC3 cells, respectively; lanes 2 and 4, DNA isolated from broblasts and BxPC3 cells at 16 h post treatment with external Qi of YXQ, respectively. (D) Western blot analysis of caspases 3, 8 and 9 and PARP in broblasts (left panel) and BxPC3 cells (right panel). Lane 1, 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) BxPC3 cells 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.

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Fig. 5. Prolonged exposure to external Qi of YXQ caused lysis of BxPC3 cells, but not broblasts. Cells were sequentially treated by external Qi of YXQ for three times over 65 min (5 min each time with 25 min interval between consecutive treatments). Cell viability was determined using MTS assay (A) and LDH release assay (B) 10 min after the third exposure to external Qi of YXQ. Results are presented as mean S.D. of percent viability (n = 3, * p 0.01 vs. control cells). (C) Microscopic examination of cells. a and c, control BxPC3 cells and broblasts, respectively; b and d, BxPC3 cells and broblasts treated by external Qi of YXQ, respectively.

phosphorylation in growth and apoptosis, BxPC3 cells were treated with PI3K specic inhibitor LY294002, ERK inhibitor PD098059, or in combination for 24 h and cell viability and cell cycle distribution were determined. Treatment with LY294002, PD098059, and in combination inhibited cell growth by 40%, 32% and 50%, respectively (Fig. 4E), and increased the percentage of the sub-G1 population by 8%, 6% and 20%, respectively (data not shown). Cleavage of pro-casapse 3 was detected in BxPC3 cells treated with LY294002, and to a lesser extent, with PD098059 (Fig. 4F). These results indicate that inhibition of Akt and ERK1/2 phosphorylation plays a role in growth inhibition and induction of apoptosis of BxPC3 cells. Interestingly, when BxPC3 cells were treated by external Qi of YXQ for three times over a period of 65 min,

a dramatic reduction in cell viability was detected as early as 10 min after the completion of the treatment (Fig. 5A). In concordance with the rapid decline in cell viability, LDH activity released into the medium rapidly increased to the levels comparable to the maximum LDH release (Fig. 5B). Microscopic examinations revealed that BxPC3 cells treated in this way had been lysed and essentially no intact cells were observed (Fig. 5C). In contrast, the viability, LDH release and morphology of broblasts were unaffected by the same treatment (Fig. 5C). 4. Discussion The activation of both the Akt and ERK pathways promotes 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 development of pancreatic tumors and chemoresistance of pancreatic cancer 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 et al., 2000; Yip-Schneider et al., 2003). The inhibition of these pathways may potentially be benecial for cancer patients and these pathways are candidate targets for chemotherapy of pancreatic cancer. Various inhibitors and neutralizing antibodies of these pathways have been developed and are in clinical trials (Dhar et al., 2005; Lockhart et al., 2005; Xiong, 2004). However, the Akt and ERK pathways are also critical for multiple physiological processes in normal cells and inhibition of these pathways may thus be toxic to the patients (Fang & Richardson, 2005; Nicholson & Anderson, 2002). The ability to use inhibitors of these pathways will depend on their ability to alter tumor progression relative to their toxicity to normal cellular functions. In this report we show that external Qi of YXQ has opposite effects on the Akt and ERK pathways in BxPC3 pancreatic cancer cells versus broblasts; while it inhibits basal Akt and ERK1/2 activity and the EGF-induced ERK activation in BxPC3 cells, it transiently activates these kinases in broblasts. This suggests that external Qi of YXQ may have cytotoxic effects on cancer cells while protecting normal cells. Indeed, we demonstrate in this report by various methods that external Qi of YXQ has potent cytotoxic effects on BxPC3 pancreatic cancer cells, but not broblasts. Furthermore, external Qi of YXQ was also highly cytotoxic to various human cancer cell lines including breast, prostate, colon cancer and leukemia cell lines, but not human umbilical vein endothelial cells (HUVEC) or peripheral blood monocytes (PBMC) (Yan, Fong, Jiang, et al., 2002; manuscript in preparation). We have also demonstrated that external Qi of YXQ protected neurons from H2 O2 -induced apoptosis (Yan et al., 2004). The observations that external Qi of YXQ helps cancer patients to minimize or avoid side effects associated with conventional chemo- and radiotherapy also support the notion of a protective effect for external Qi of YXQ on normal cells (Fong, 1997; Ming, 1988; Wang & Zhu, 1997; Zhang et al., 1997). The mechanisms by which external Qi of YXQ exhibits 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 be able to inuence the structure and properties of proteins and has been successfully used to promote crystallization

of a Fab protein (Lu, 1997; Yan et al., 1999). Recently, we have shown that external Qi of YXQ is able to enhance PI3K activity in neurons (Yan et al., 2004), and increase or repress PI3K activity of a highly enriched PI3K preparation (Yan, Xia, et al., 2002). We showed in this report that external Qi of YXQ dramatically inhibited Akt activation with concomitant inhibition of PI3K activity in BxPC3 cells, while it stimulated Akt phosphorylation in broblasts without elevating PI3K activity. These ndings suggest that external Qi of YXQ may modulate Akt activation through PI3K dependent and independent mechanisms (Cantly, 2002; Nicholson & Anderson, 2002; Song, Ouyang, & Bao, 2005). A single exposure of BxPC3 cells to external Qi of YXQ induced apoptosis as it caused a dramatic increase in the sub-G1 population, DNA fragmentation and cleavage of caspases and PARP. Two major pathways of caspase activation are known: the cell surface death receptor pathway and the mitochondria-initiated pathway (Budihardjo, Oliver, Lutter, Luo, & Wang, 1999). In the death receptor pathway, activation of caspase 8 is the critical event that transmits the death signal. In the mitochondria-initiated pathway, caspase 9 is activated and will then cleave and activate downstream caspases such caspases 3, 6 and 7. Thus, the cleavage of caspases 8 and 9 in BxPC3 cells induced by external Qi of YXQ suggests that both apoptotic pathways were initiated after the treatment. Inhibition of ERK pathway by PD098059 has been shown to activate both apoptotic pathways in pancreatic cancer MIA PaCa-2 cells (Boucher et al., 2000). In this report we show that inhibition of Akt and ERK phosphorylation by LY294002 and PD098059, respectively, attenuated BxPC3 cell growth and caused a small but signicant increase in apoptosis of BxPC3 cells. Similar effects of LY294002 on BxPC3 cell growth and apoptosis have also been reported (Perugini et al., 2000). Thus, it is reasonable to assume that inhibition of PI3K/Akt and ERK pathways by external Qi of YXQ was at least in part responsible for the induction of apoptosis in BxPC3 cells. Further studies using negative mutants or constantly active forms of Akt and ERK are required to dissect their role in the initiation of these two apoptotic pathways by external Qi of YXQ, and elucidate additional mechanisms involved in induction of apoptosis of BxPC3 cells by external Qi of YXQ. Interestingly, prolonged exposure to external Qi of YXQ caused rapid lysis of BxPC3 cells, probably through non-apoptotic mechanism(s). The precise mechanisms underlying this cytolytic effect of external Qi of YXQ remains to be investigated. Previously, we have observed that external Qi of YXQ could dramatically

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inuence the structure and properties of liposomes, an articial model for bio-membrane studies (Yan, Zhao, et al., 1988). These ndings suggest that prolonged treatment by external Qi of YXQ might have caused profound damage to the structure and function of the BxPC3 cell membrane, resulting in rapid osmotic lysis. Earlier studies have used physical signal detectors to verify the existence of external Qi and have sometimes detected signals of electricity, magnetism and sound in external Qi (Chen, 2004). However, these physical aspects are most likely secondary or side effects of external Qi and have not revealed the primary nature of external Qi (Chen, 2004; Lu, 1997). Furthermore, while ultrasound, low-frequency pulsating electromagnetic elds (LF-PEMF) and low-level direct currents induce apoptotic and/or necrotic death of cancer cells including leukemia cell lines (K-562, U-937 and HL60), they also have signicant cytotoxic effects on normal cells including broblasts and PBMC (Ashush et al., 2000; Feigl, Volklein, Iro, Ell, & Schneider, 1996; Lejbkowicz, Zwiran, & Salzberg, 1993; Radeva & Berg, 2004; Tachibana, Uchida, Hisano, & Morioka, 1997; Tang et al., 2005; Yen et al., 1999). In contrast, external Qi of YXQ has no cytotoxic effect on normal cells including broblasts, PBMC and HUVEC, while it completely kills cancer cells such as leukemia cell lines K-562, U-937 and HL-60 (Yan, Fong, Jiang, et al., 2002; manuscript in preparation). Therefore, it is unlikely that these physical aspects, if exist, could be responsible for the cytotoxic effects of external Qi of YXQ on cancer cells. The activation of NF- B is mediated by two kinases, I B and IKK (Arlt et al., 2001, 2002; Dong et al., 2002). In response to various stimuli, I B is phosphorylated by IKK and degraded in the cytoplasm, leading to nuclear translocation of NF- B and activation of its target genes. Akt has been shown to be required for TNF- -mediated NF- B activation by phosphorylation of IKK and subsequent degradation of I B (Ozes et al., 1999). The inhibition of Akt may thus contribute to the suppression of the TNF- -induced NF- B activity in BxPC3 cells by external Qi of YXQ. Constitutive NF- B activation has been observed in many types of solid tumors and has been shown to contribute to survival and resistance of cancer cells to apoptosis induced by various agents (Arlt et al., 2001, 2002, 2003; Nicholson & Anderson, 2002). It has been shown that the blockade of NF- B activation increases the sensitivity of malignant cells to the apoptotic effects of anticancer drugs and radiation (Arlt et al., 2001; Dong et al., 2002). These ndings suggest that external Qi of YXQ may also sensitize cancer cells to chemotherapy through inhibiting NF- B activity.

Resistance to chemotherapy is a major cause of treatment 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 pathways in BxPC3 cells by external Qi of YXQ is noteworthy as these pathways play important roles in pancreatic cancer cell growth and drug resistance (Boucher et al., 2000; Perugini et al., 2000). Furthermore, BxPC3 cells are resistant to apoptosis mediated by gemcitabine, which has become the standard chemotherapy for locally advanced and metastatic adenocarcinoma of pancreas (Richards, 2005), and by the Fas pathway (Elnemr et al., 2001). However, BxPC3 cells are highly sensitive to external Qi of YXQ. Taken together, our ndings suggest that external Qi of YXQ could potentially be an effective approach for therapy of pancreatic carcinomas. Acknowledgement This work was supported in part by Yan Xin Foundation. References
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