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The International Journal of Biochemistry & Cell Biology xxx (2006) xxx–xxx
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
a
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 Children’s Hospital, Harvard Medical School, Boston, MA 02115, USA
Received 14 February 2006; received in revised form 21 May 2006; accepted 2 June 2006
Abstract
Long-term clinical observations and ongoing studies have shown significant 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
fibroblasts with external Qi of Yan Xin Qigong induced transient activation of extracellular signal-regulated kinases and Akt, and
caused no cytotoxic effect. These findings 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 fluoride; 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
Please cite this article as: Xin Yan et al., External Qi of Yan Xin Qigong differentially regulates the Akt and extracellular signalregulated kinase pathways and is cytotoxic to cancer cells but not to normal cells, The International Journal of Biochemistry &
Cell Biology (2006), 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
significant beneficial 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 influence 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 fifth leading cause
of cancer-related deaths in Western countries, with an
overall 1-year survival rate of ∼12% and 5-year survival
rate of 3–5% (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 benefit of
external Qi of YXQ, we have investigated its effect on
these pathways in BxPC3 cells and fibroblasts. 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
fibroblasts. Furthermore, external Qi of YXQ exhibited
potent cytotoxic effects on BxPC3 cells, but not on fibroblasts.
2. Materials and methods
2.1. Cell culture
BxPC3 cells and human fibroblast 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.
Please cite this article as: Xin Yan et al., External Qi of Yan Xin Qigong differentially regulates the Akt and extracellular signalregulated kinase pathways and is cytotoxic to cancer cells but not to normal cells, The International Journal of Biochemistry &
Cell Biology (2006), doi:10.1016/j.biocel.2006.06.002.
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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). Briefly, cells cultured to near confluency 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 fibroblasts, 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 (1–2) × 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. Briefly,
20 ␮l of One Solution Reagent containing MTS were
added to the cells. After incubation at 37 ◦ C and 5%
CO2 for 2–4 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).
Briefly, 50 ␮l of cell culture medium were mixed with
3
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). Briefly, 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 Tris–Cl, 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
(4–20%). 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
Tris–Cl, 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
Please cite this article as: Xin Yan et al., External Qi of Yan Xin Qigong differentially regulates the Akt and extracellular signalregulated kinase pathways and is cytotoxic to cancer cells but not to normal cells, The International Journal of Biochemistry &
Cell Biology (2006), doi:10.1016/j.biocel.2006.06.002.
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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 chloroform–methanol
(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
film and radioactive lipids were scraped off the plates
and quantified 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
fixed 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
flow cytometry using a FACSVantage flow 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 Tris–Cl, 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.
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 significantly 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 findings 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-κB activity
in BxPC3 cells
3. Results
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 TNF-␣
for 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).
3.1. External Qi of YXQ inhibits Akt and ERK1/2
phosphorylation in BxPC3 cells
3.3. External Qi of YXQ activates Akt and ERK1/2
in fibroblasts
The activation of both Akt kinase and MAP kinase
ERK plays important roles in growth and chemoresis-
Our previous studies have shown that external Qi
of YXQ protected neurons from H2 O2 -induced apopto-
2.9. Statistic analysis
Results are presented as mean ± S.D. The significance of differences in means was determined using the
two-tailed Student’s t-test. p < 0.05 was considered significant.
Please cite this article as: Xin Yan et al., External Qi of Yan Xin Qigong differentially regulates the Akt and extracellular signalregulated kinase pathways and is cytotoxic to cancer cells but not to normal cells, The International Journal of Biochemistry &
Cell Biology (2006), doi:10.1016/j.biocel.2006.06.002.
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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 fibroblasts. 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 significant 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 fibroblasts 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
fibroblasts.
Please cite this article as: Xin Yan et al., External Qi of Yan Xin Qigong differentially regulates the Akt and extracellular signalregulated kinase pathways and is cytotoxic to cancer cells but not to normal cells, The International Journal of Biochemistry &
Cell Biology (2006), doi:10.1016/j.biocel.2006.06.002.
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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 NF␬B 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 fibroblasts
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 flow 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 fibroblasts was not significantly 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 fibroblasts. (A and B) Akt and ERK1/2
phophorylation levels. Serum-starved fibroblasts 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 fibroblasts 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
insignificant.
DNA fragmentation, cleavage of caspases 3, 8 and 9 or
cleavage of PARP was not detected in fibroblasts treated
with external Qi of YXQ (Fig. 4C and D). In order to
investigate the role of inhibition of Akt and/or ERK1/2
Please cite this article as: Xin Yan et al., External Qi of Yan Xin Qigong differentially regulates the Akt and extracellular signalregulated kinase pathways and is cytotoxic to cancer cells but not to normal cells, The International Journal of Biochemistry &
Cell Biology (2006), doi:10.1016/j.biocel.2006.06.002.
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Fig. 4. Differential cytotoxic effects of external Qi of YXQ on BxPC3 cells and fibroblasts. 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 fibroblasts. 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 flow 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 fibroblasts. Lanes 1 and 3, DNA isolated from
control fibroblasts and BxPC3 cells, respectively; lanes 2 and 4, DNA isolated from fibroblasts 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 fibroblasts (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.
Please cite this article as: Xin Yan et al., External Qi of Yan Xin Qigong differentially regulates the Akt and extracellular signalregulated kinase pathways and is cytotoxic to cancer cells but not to normal cells, The International Journal of Biochemistry &
Cell Biology (2006), doi:10.1016/j.biocel.2006.06.002.
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Fig. 5. Prolonged exposure to external Qi of YXQ caused lysis of BxPC3 cells, but not fibroblasts. 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 fibroblasts, respectively; b and
d, BxPC3 cells and fibroblasts treated by external Qi of YXQ, respectively.
phosphorylation in growth and apoptosis, BxPC3 cells
were treated with PI3K specific 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 fibroblasts 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
Please cite this article as: Xin Yan et al., External Qi of Yan Xin Qigong differentially regulates the Akt and extracellular signalregulated kinase pathways and is cytotoxic to cancer cells but not to normal cells, The International Journal of Biochemistry &
Cell Biology (2006), doi:10.1016/j.biocel.2006.06.002.
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X. Yan et al. / The International Journal of Biochemistry & Cell Biology xxx (2006) xxx–xxx
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 beneficial 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 fibroblasts; while it inhibits basal Akt
and ERK1/2 activity and the EGF-induced ERK activation in BxPC3 cells, it transiently activates these kinases
in fibroblasts. 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 fibroblasts. 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 influence the structure and properties of proteins
and has been successfully used to promote crystallization
9
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
fibroblasts without elevating PI3K activity. These findings 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 significant 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
Please cite this article as: Xin Yan et al., External Qi of Yan Xin Qigong differentially regulates the Akt and extracellular signalregulated kinase pathways and is cytotoxic to cancer cells but not to normal cells, The International Journal of Biochemistry &
Cell Biology (2006), doi:10.1016/j.biocel.2006.06.002.
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influence the structure and properties of liposomes, an
artificial model for bio-membrane studies (Yan, Zhao,
et al., 1988). These findings 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 fields (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 significant cytotoxic effects on normal cells including fibroblasts 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 fibroblasts, 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 findings 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 findings 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.
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