Download PectaSol-C Modified Citrus Pectin Induces Apoptosis and

PectaSol-C Modified Citrus Pectin
Induces Apoptosis and Inhibition
of Proliferation in Human and
Mouse Androgen-Dependent and Independent Prostate Cancer Cells
Integrative Cancer Therapies
9(2) 197­–203
© The Author(s) 2010
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DOI: 10.1177/1534735410369672
http://ict.sagepub.com
Jun Yan, PhD,1 and Aaron Katz, MD1
Abstract
Aim: To demonstrate the efficacy of PectaSol-C modified citrus pectin (MCP) on prostate cancer in vitro. Method: Cytotoxicity
analysis of PectaSol-C was performed by MTT assay, as were parallel studies with the former brand version of MCP
called PectaSol. Apoptosis and inhibition of cell growth were investigated by Western blotting. Results: Androgen-dependent
and -independent human prostate cancer cell lines (LNCaP and PC3, respectively), androgen-dependent and -independent
murine prostate cancer cell lines (CASP2.1 and CASP1.1, respectively), as well as noncancerous human benign prostate
hyperplasia BPH-1 cell line, were used in the study. MTT assay revealed that 1.0% PectaSol exerted cytotoxicity on LNCaP,
PC3, CASP2.1, CASP1.1, and BPH-1 cells for 4-day treatment by 48.0% ± 2.1%, 54.4% ± 0.3%, 15.4% ± 0.8%, 46.1% ± 1.7%,
and 27.4% ± 1.6%, respectively; whereas 1.0% PectaSol-C showed cytotoxity by 52.2% ± 1.8%, 48.2% ± 2.9%, 23.0% ± 2.6%,
49.0% ± 1.3%, and 26.8% ± 2.6%, respectively. Western blotting further confirmed that both MCPs inhibit MAP kinase
activation, increase the expression level of its downstream target Bim, a pro-apoptotic protein, and induce the cleavage
of Caspase-3 in PC3 and CASP1.1 prostate cancer cells. Conclusion: PectaSol MCP and PectaSol-C MCP can inhibit cell
proliferation and apoptosis in prostate cancer cell lines. Our data suggested that 1.0% PectaSol-C can be used for further
chemopreventive and chemotherapeutic analysis in vivo.
Keywords
prostate cancer, modified citrus pectin, Caspase-3, apoptosis, MAPK
Introduction
Dietary carbohydrates have been suggested to have various
health-promoting properties.1-9 Pectin is a complex carbohydrate-soluble fiber. Although pectins are not digestible
by humans, modified citrus pectin (MCP) is altered by
decreasing its molecular weight and thus believed to increase
its absorbability into the blood circulation. The results of
in vivo animal and human clinical studies with oral
administration of MCP, which are discussed below, suggest
absorption into the circulatory system. The MCPs used in
this study are composed of short slightly branched carbohydrate chains derived from the soluble albedo fraction of
citrus fruit peels. MCP polysaccharides are derived from
commercial pectin isolated from citrus fruits, which have a
molecular weight of 100 000 to 200 000 Da. Modification
via pH, temperature, and/or enzymatic degradation produces MCP that is much lower in molecular weight and
less structurally complex, compared with the original citrus
pectin. As a result, MCP dissolves readily in water and is
believed to be better absorbed and utilized by the body than
ordinary, long-chain pectin. In the United States, MCP is
registered as a dietary supplement.
MCP is relatively rich in galactose and antagonizes a
binding protein galectin-3 (Gal-3), which results in suppression of cancer metastasis.7,10 It has been shown that
MCP can increase prostate-specific antigen (PSA) doubling
time in 7 of 10 men (with biochemical recurrence of prostate cancer following local therapy) after taking MCP for
12 months, compared with before taking MCP.11 Moreover,
oral intake of MCP can reduce lung metastases of rat prostate cancer cells in the Copenhagen rat.12 These studies are
1
Columbia University, New York, NY, USA
Corresponding Author:
Jun Yan, Department of Urology, Columbia University, 1130 St. Nicholas
Avenue, New York, NY 10032, USA
Email: [email protected]
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Integrative Cancer Therapies 9(2)
indicative that MCP can act as a potent agent for the treatment of cancer.
The shorter polysaccharide units afford this type of MCP
with its ability to access and bind tightly to galactose-binding
lectins (galectins) on the surface of certain types of cancer
cells. For metastasis to occur, cancerous cells must first
clump together; galectins on their surface membranes are
thought to be responsible for much of this metastatic potential. Galactose-rich MCP has a binding affinity for galectins
on the surface of cancer cells, resulting in an inhibition, or
blocking, of cancer cell aggregation, adhesion, and metastasis.13 MCP acts as a ligand for galectin-3, which plays a
major role in tumor formation and progression.14-16 It has
been recently shown by using a combination of fluorescence
microscopy, flow cytometry, and atomic force microscopy,
for the first time, specific binding of a pectin galactan to the
recombinant form of human galectin-3.17 MCP is thought to
render galectin-3 incapable of binding its receptors that
would result in angiogenesis.10 Moreover, MCP also shows
antimetastatic effects on cancer cells in vitro or in vivo.12,18-20
The first published human clinical trial to use MCP11 demonstrated its effect on PSA doubling time. A more recent
clinical trial that used MCP showed a significant improvement of quality of life and stabilization of disease for
patients with advanced solid tumors.21 One patient in the
latter study, who had advanced and hormonal resistant prostate cancer, had a 50% decrease in PSA with significant
improvement in his quality of life after taking 15 g of MCP
per day. Most of the patients had improvements in their life
quality as reflected in the Karnofsky scores.21
Apart from therapeutic roles against prostate cancer,
MCP has an immunomodulation effect.9 It can also remove
toxic metals from the body.22,23 It is also effective against
various types of cancer, such as melanoma, colon, and breast
cancer.18,20,24,25 The primary purpose of this study was to
confirm the anticancer efficacy of MCP, using the MCPs
included in previous cancer clinical trials.11,21 MCP is not a
carefully defined term as a dietary supplement because there
are multiple MCPs on the market that are not well characterized molecularly, and are not the same molecular weight
range or low esterification as the MCP marketed under the
PectaSol name. Of note, the MCPs studied here are the only
MCPs that have been used in human clinical trials.11,21-23
High-performance size exclusion chromatography
(HPSEC) analysis of the 2 MCPs has previously determined the average molecular weight of PectaSol-C MCP to
be between 5000 and 10 000 Da, lower than that of the PectaSol MCP, which was determined to be between 12 000
and 16 000 Da. The average amount of monogalacturonic
acid content of PectaSol-C MCP was determined to be 5%
compared with 19% for PectaSol MCP. The percentage of
monogalacturonic acid represents the degradation of the
MCP structure to single carbohydrate units. The lower
molecular weight PectaSol-C MCP may have better bioavailability than higher molecular weight PectaSol MCP
in vivo. In this study, we examined the anticancer effects
of PectaSol-C MCP and PectaSol MCP on androgendependent and -independent prostate cancer cells. This study
was performed to demonstrate and establish the efficacy of
the new preparation of PectaSol-C MCP before further
chemopreventive and chemotherapeutic analysis in vivo.
Materials and Methods
Reagents
PectaSol MCP and PectaSol-C MCP (EcoNugenics, Santa
Rosa, CA) were added to medium immediately before
treatment. Penicillin, streptomycin, RPMI 1640 medium,
D-MEM medium, and fetal bovine serum (FBS) were
obtained from Invitrogen (Carlsbad, CA). Anti-pERK1/2,
total ERK1/2, Bim, Caspase-3 antibodies were from Cell
Signaling (Danvers, MA).
Cell Culture
LNCaP and PC3 cells were obtained from the American
Type Culture Collection (Manassas, VA) and maintained in
RPMI 1640 supplemented with 10% heat-inactivated FBS
and 1% antibiotic antimycotic (Invitrogen, Carlsbad, CA).
Two mouse prostate cancer cell lines, CASP2.1 and CASP1.1
were derived from the tumors of Nkx3.1;Pten;p27 compound mutant mice.26 Noncancerous benign prostate
hyperplasia BPH-1 cell line is kindly provided by Dr Simon
W. Hayward.27 These cells were kept in our laboratory. All
the cells were cultured at 37°C in a humidified atmosphere
of 95% air and 5% CO2.
Cell Viability Assay
Cells were seeded at 4 × 103 cells/well in 96-well plates overnight before treatment with MCPs. MTT solution (10 mL;
5 mg/mL in phosphate-buffered saline) was added to each
well of the plates and incubated for 3 hours at 37°C. MTT
lysis buffer was then added to dissolve the formazan. The
optical density was measured at 570 nm using a mQuant
Microplate Spectrophotometer (Biotek, Winooski, VT).
The percentage of viable cells was calculated as the relative
optical density compared to the control.
Western Blotting Assay
Cells were lyzed in RIPA buffer containing a protease
inhibitor cocktail (Roche Diagnostics, Indianapolis, IN)
and phosphotase inhibitor (Sigma, St Louis, MO). Cell
lysates (20 mg) were resolved on sodium dodecyl
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Yan and Katz
Figure 1. Photographs of prostate cancer cell lines (CASP1.1 and CASP2.1) taken after 48 hours treatment with 0.1%, 1.0% PectaSol
and PectaSol-C, as well as vehicle treatment (scale bar, 200 mm).
sulfate–polyacrylamide gel electrophoresis, and transferred
onto a PVDF membrane. After blotting in 5% non-fat dry
milk in Tris-buffered saline containing 0.1% Tween 20
(TBST), the membranes were incubated with primary antibodies at 1:1000 dilutions in TBST overnight at 4°C. The
blots were washed and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 hour,
and finally detected by ECL reagent (GE Healthcare,
Buckinghamshire, UK).
Results
Growth Inhibitory Effects of PectaSol MCP
and PectaSol-C MCP on Prostate Cancer Cells
We examined the anticancer effects of the MCPs in vitro,
with the long-term goal for preclinical study in future.
Because we will use nkx3.1;pten compound mutant mice
for future preclinical study, we chose 2 mouse prostate cancer
cell lines, CASP1.1 and CASP2.1, with the losses of nkx3.1
and pten alleles.26 Meanwhile, we also included 2 human
prostate cancer cell lines (LNCaP and PC3), as well as
1 noncancerous benign prostate hyperplasia (BPH-1) cells
for cytotoxicity study.
At first, we treated androgen-independent CASP1.1 and
androgen-dependent CASP2.1 prostate cancer cell lines
with the MCPs. Figure 1 shows that treatment with 1.0% of
both MCPs for 48 hours induced changes in cellular morphologies (such as cell shrinkage and round-up) in CASP1.1
cells. CASP2.1 cells also turn to be rounded up under both
of the treatments; however, the percentage is less than that in
CASP1.1. The morphologic changes were dependent on
dosage of MCPs. To investigate the cytotoxic effects of
MCPs, we performed an MTT assay on 4 different prostate
cancer cell lines. Two of them are androgen dependent
(CASP2.1 and LNCaP) and the other 2 are androgen independent (CASP1.1 and PC3). The results in Figure 2 indicate
that both MCPs can suppress cell proliferation in a dosedependent manner. Consistently, the data from MTT assay
demonstrated that 1.0% of PectaSol MCP exerts cytotoxicity on CASP1.1 cells (46.1% ± 1.7%) more effectively than
on CASP2.1 cells (15.4% ± 0.8%); whereas cytotoxicity of
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Figure 2. Effects of PectaSol and PectaSol-C on cell viability on 2 androgen-dependent prostate cancer cell lines (LNCaP and
CASP 2.1) and 2 androgen-independent prostate cancer cell lines (PC3 and CASP 1.1), as well as 1 noncancerous human benign
prostate hyperplasia BPH-1 cells
The cells were exposed to modified citrus pectins at the indicated dosages. The cytotoxicity was determined by MTT assay.Vehicle-treated cells
were arbitrarily set as 0% of cytotoxicity. Error bars and all data were expressed as mean ± standard error in triplicate. *P < .05, compared with the
no-treatment group.
following 1.0% PectaSol-C MCP treatment was greater in
CASP1.1 cells (49.0% ± 1.3%) than in CASP2.1 cells
(23.0% ± 2.6%). Similarly, both PectaSol and PectaSol-C
showed cytotoxic effects on 2 human prostate cancer cells,
LNCaP (48.0% ± 2.1%, 52.2% ± 1.8%) and PC3 cells
(54.4% ± 0.3%, 48.2% ± 2.9%), respectively. Although
1.0% of PectaSol and 1.0% of PectaSol-C MCPs also
showed cytotoxicity on human BPH-1 cells (27.4% ± 1.6%
and 26.8% ± 2.6%), respectively, the cytotoxic effects are
significantly lower (all P value <.05) than human prostate
cancer cells (LNCaP and PC3). These data indicate that both
MCPs have anticancer properties with PectaSol-C MCP
showing increased cytotoxicity in most of the cell lines.
Cytotoxic Effects of PectaSol MCP and PectaSol-C
MCP on PC3 and CASP1.1 Prostate Cancer
Cells by Suppression of MAPK signaling and
Induction of the Cleavage of Caspase-3
To confirm the cytotoxicity of the MCPs on prostate cancer
cells, we performed Western blotting assay on PC3 cells, as
well as CASP1.1 cells. It revealed that both MCPs can suppress mitogen-activated protein kinase (MAPK) signaling
through reducing phosphorylation forms of ERK1/2
(Figure 3). Bim, a pro-apoptotic member of the Bcl-2 protein family member, was reported to be targeted by ERK1/2
through phosphorylation and eventually leading to
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Figure 3. Involvement of MAPK signal pathway and Caspase-3 in PC3 (A) and CASP1.1 (B) prostate cancer cell growth inhibition and
apoptosis by PectaSol and PectaSol-C at indicated concentrations
Western blotting analysis of phosphorylated ERK1/2 at theronine 202 and tyrosine 204, Bim and Caspase-3 in PC3 and CASP1.1 cells. Cells were
treated with modified citrus pectins at the indicated dose. An amount of 20 mg proteins were loaded for the assay. Total ERK1/2 was used as a loading
control. FL, full length; cCaspase-3, cleaved Caspase-3.
degradation.28,29 We observed an increase of Bim protein
levels in both cell lines, after treatment of MCPs, with the
inactivation of ERK1/2. Consistently, we also detected the
cleavage of pro-Caspase-3 after treatment of MCPs in both
PC3 and CASP1.1 cell lines. These results indicate that
both MCPs induce cytotoxic effects through the inhibition
of prosurvival MAPK signaling and the induction of apoptosis by the activation of Caspase-3.
Discussion
In this study, we showed that both MCPs can induce
cytotoxicity in both androgen-dependent and -independent
prostate cancer cells in vitro. The anticancer effects of both
MCPs are very similar in most of cancer cells we tested.
MAPK signal pathway was impaired by the treatment
of MCPs. The lower average molecular weight and monogalacturonic acid content of PectaSol-C MCP may be
important for the absorbance and distribution in the body.
MCP has shown to have positive impact clinical benefit
for patients with advanced solid tumors.21 For prostate
cancer patients, MCP may lengthen the PSA doubling time
in those with recurrent prostate cancer.11 Our studies show
that both PectaSol MCP and PectaSol-C MCP showed
cytotoxicity on human and mouse prostate cancer cells.
Although we observed some cytotoxicity in the BPH-1 cell
line, there appears to be selectivity toward the cancerous
cell lines. For mechanistic study, both MCPs can induce
apoptosis in androgen-independent PC3 and CASP1.1
prostate cancer cells. The cleavage of Caspase-3 suggests
that the induction of apoptosis by the MCPs may be caspase-3 dependent. Moreover, these MCPs can strongly
suppress the MAPK signaling pathway, which is related to
prostate cancer cell proliferation and survival,30 manifested
by the reduction of phosphorylation of ERK1/2. Of note,
the reduction of ERK1/2 by MCPs correlates with the
increase of Bim protein levels in both cell lines, indicating
that the effect is not cell line dependent. Bim is a BH3-only
protein, promoting apoptosis through binding to prosurvival Bcl-2 protiens, thereby releasing pro-apoptotic Bax or
Bak proteins and eventually leading to apoptosis.31 ERK1/2
has recently shown to phosphorylate Bim to facilitate its
proteasome-dependent degradation.28,29 Therefore, these
MCPs can inhibit cell growth through the inhibition of the
prosurvival MAPK pathway to induce apoptosis.
MCP was shown to inhibit cell adhesion of breast cancer
cells to human umbilical vein endothelial cells (HUVECs),
through binding to and suppressing the activity of galectin-3.10
A notable feature of galectin-3 is its implication in neoplastic
transformation and cancer progression.14 Anti-galectin-3 antibody inhibited adhesion of cancer cells and liver metastasis by
adenocarcinoma cell lines.32 These studies suggest the potential for carbohydrate-mediated cancer therapy.
This study shows that PectaSol-C, a promising MCP
with average lower molecular weight, has similar anticancer effects to previous MCP with average slightly higher
molecular weight. Although this study is not conclusive, the
data shown here strongly suggested that PectaSol-C may be
a promising anticancer agent for prostate cancer cells and
further in vivo study is warranted.
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In summary, our results demonstrated that both MCPs
can efficiently reduce cell viability in vitro, independent of
their androgen dependency. These MCPs can induce cell
growth inhibition and apoptosis partially through the inhibition of MAPK signal pathway and activation of Caspase-3.
These data support the role of these specific dietary carbohydrate compounds as chemopreventive and/or therapeutic
agents, and further clinical and basic science data is warranted in this area.
Acknowledgments
The authors would like to thank Dr Isaac Eliaz for his development of PectaSol and PectaSol-C and his contribution to this study
as well as Dr Geovanni Espinosa, Dr Arland Hotchkiss, and Barry
Wilk for their contributions in helping with this study and the
preparation of this manuscript. We would also like to thank Dr
Cory Abate-Shen and Dr Simon W. Hayward for the kind gifts of
CASP cell lines, BPH-1 cell line, and support.
Declaration of Conflicting Interests
The author(s) declared no conflicts of interest with respect to the
authorship and/or publication of this article.
Funding
The author(s) disclosed receipt of the following financial support
for the research and/or authorship of this article:
This work was supported by a sponsored research grant from
EcoNugenics, Inc, Santa Rosa, CA.
References
1. Anderson JW. Dietary fiber, complex carbohydrate and coronary artery disease. Can J Cardiol. 1995;11:55G-62G.
2. Anderson JW, Jones AE, Riddell-Mason S. Ten different
dietary fibers have significantly different effects on serum and
liver lipids of cholesterol-fed rats. J Nutr. 1994;124:78-83.
3. Barondes SH, Cooper DN, Gitt MA, Leffler H. Galectins.
Structure and function of a large family of animal lectins. J Biol
Chem. 1994;269:20807-20810.
4. Bingham SA, Day NE, Luben R, et al. Dietary fiber in food
and protection against colorectal cancer in the European Prospective Investigation into Cancer and Nutrition (EPIC): an
observational study. Lancet. 2003;361:1496-1501.
5. Chen CH, Sheu MT, Chen TF, et al. Suppression of endotoxininduced proinflammatory responses by citrus pectin through
blocking LPS signalling pathways. Biochem Pharmacol. 2006;
72:1001-1009.
6. Nangia-Makker P, Conklin J, Hogan V, Raz A. Carbohydratebinding proteins in cancer, and their ligands as therapeutic
agents. Trends Mol Med. 2002;8:187-192.
7. Kidd P. A new approach to metastasis cancer prevention:
modified citrus pectin (MCP), a unique pectin that blocks cell
surface lectins. Altern Med Rev. 1996;1;4-10.
8. Olano-Martin E, Rimbach GH, Gibson GR, Rastall RA.
Pectin and pectic-oligosaccharides induce apoptosis in in
vivo human colonic adenocarcinoma cells. Anticancer Res.
2003;23:341-346.
9. Wong CK, Leung KN, Fung KP. Immunomodulatory and antitumor polysaccharides from medicinal plants. J Int Med Res.
1994;22:299-312.
10. Nangia-Makker P, HoganV, Honjo Y, et al. Inhibition of
human cancer cell growth and metastasis in nude mice by oral
intake of modified citrus pectin. J Natl Cancer Inst. 2002;24:
1854-1862.
11. Guess BW, Scholz MC, Strum SB, Lam RY, Johnson HJ,
Jennrich RI. Modified citrus pectin (MCP) increases the
prostate-specific antigen doubling time in men with prostate
cancer: a phase II pilot study. Prostate Cancer Prostatic Dis.
2003;6:301-304.
12. Pienta KJ, Naik H, Akhtar A, et al. Inhibition of spontaneous
metastasis in a rat prostate cancer model by oral administration of modified citrus pectin. J Natl Cancer Inst. 1995;87:
348-353.
13. Glinsky VV, Raz A. Modified citrus pectin anti-metastatic
properties: one bullet multiple targets. Carbohydr Res. 2009;
344:1788-1791.
14. Zou J, Glinsky VV, Landon LA, Matthews L, Deutscher SL.
Peptides specific to the galectin-3 carbohydrate recognition
domain inhibit metastasis-associated cancer cell adhesion.
Carcinogenesis. 2005;26:309-318.
15. Johnson KD, Glinskii OV, Mossine VV, et al. Galectin-3 as a
potential therapeutic target in tumors arising from malignant
endothelia. Neoplasia. 2007;9:662-670.
16. Nangia-Makker P, Honjo Y, Sarvis R, et al. Galectin-3 induces
endothelial cell morphogenesis and angiogensis. Am J Pathol.
2000;156:899-909.
17. Gunning AP, Bongaerts RJ, Morris VJ. Recognition of galactan components of pectin by galectin-3. FASEB J. 2009;23:
415-424.
18. Platt D, Raz A. Modulation of the lung colonization of
B16-F1 melanoma cells by citrus pectin. J Natl Cancer Inst.
1992;84;438-442.
19. Glinsky VV, Huflejt M, Glinsky G, Deutscher S, Quinn T.
Effects of Thomsen-Friendenreich antigen-specific peptide
p-30 on b-galactoside-mediated homotypic aggregation and
adhesion to the endothelium of MDA-MB-435 human breast
carcinoma cells. Cancer Res. 2000;60:2584-2588.
20. Liu HY, Huang ZL, Yang GH, Lu WQ, Yu NR. Inhibitory effect of modified citrus pectin on liver metastases in a
mouse colon cancer model. World J Gastroenterol. 2008;14:
7386-7391.
21. Azemar M, Hildenbrand B, Haering B, Heim ME, Unger C.
Clinical benefit in patients with advanced solid tumors treated
with modified citrus pectin: a prospective pilot study. Clin
Med: Oncol. 2007;I:73-80.
Downloaded from http://ict.sagepub.com by Barry Wilk on June 29, 2010
203
Yan and Katz
22. Eliaz I, Hotchkiss A, Fishman M. Rode D. The effect of modified citrus pectin on urinary excretion of toxic elements. Phytother Res. 2006;20:859-864.
23. Zhao ZY, Liang L, Fan X, et al. The role of modified citrus
pectin as an effective chelator of lead in children hospitalized
with toxic lead levels. Altern Ther Health Med. 2008;14:34-38.
24. Glinskii OV, Huxley VH, Glinsky GV, Pienta KJ, Raz A,
Glinsky VV. Mechanical entrapment is insufficient and intercellular adhesion is essential for metastatic cell arrest in distant organs. Neoplasia. 2005;7:522-527.
25. Inohara H, Raz A. Effects of natural complex carbohydrate
(citrus pectin) on murine melanoma cell properties related to
galectin-3 functions. Glycoconj J. 1994;11:527-532.
26. Gao H, Ouyang X, Banach-Petrosky W, et al. A critical role of
p27kip1 gene dosage in a mouse model of prostate carcinogenesis. Proc Natl Acad Sci U S A. 2004;101:17204-17209.
27. Hayward SW, Dahiya R, Cunha GR, Bartek J, Deshpande N,
Narayan P. Establishment and characterization of an immortalized but non-transformed human prostate epithelial cell
line: BPH-1. In Vitro Cell Dev Biol Anim. 1995;31:14-24.
28. Ley R, Balmanno K, Hadfield K, Weston C, Cook SJ.
Activation of the ERK1/2 signaling pathway promotes
phosphorylation and proteasome-dependent degradation
of the BH3-only protein, Bim. J Biol Chem. 2003;278:
18811-18816.
29. Luciano F, Jacquel A, Colosetti P, et al. Phosphorylation of
Bim-EL by Erk1/2 on serine 69 promotes its degradation via
the proteasome pathway and regulates its proapoptotic function. Oncogene. 2003;22:6785-6793.
30. Kinkade CW, Castillo-Martin M, Puzio-Kuter A, et al. Targeting AKT/mTOR and ERK MAPK signaling inhibits hormonerefractory prostate cancer in a preclinical mouse model. J Clin
Invest. 2008;118:305-364.
31. Ewings KE, Wiggins CM, Cook SJ. Bim and the pro-survival
Bcl-2 proteins. Cell Cycle. 2007;18:2236-2240.
32. Khaldoyanidi SK, Glinsky VV, Sikora L, et al. MDA-MB-435
human breast carcinoma cell homo- and heterotypic adhesion under flow conditions is mediated in part by ThomsenFriedenreich antigengalectin-3 interactions. J Biol Chem.
2003:278:4127-4134.
Downloaded from http://ict.sagepub.com by Barry Wilk on June 29, 2010