| Research Article |
Open Access |
|
| Free Base Lysine Increases Survival and Reduces Metastasis in Prostate
Cancer Model |
| Arig Ibrahim-Hashim1*, Jonathan W. Wojtkowiak1, Maria de Lourdes Coelho Ribeiro1, Veronica Estrella1, Kate M. Bailey1, Heather H.
Cornnell1, Robert A. Gatenby2 and Robert J Gillies1,2 |
| 1Department of Imaging, H. Lee Moffitt Cancer Center Tampa, FL 33612 |
| 2Department of Radiology, H. Lee Moffitt Cancer Center Tampa, FL 33612 |
| *Corresponding author: |
Arig Ibrahim-Hashim
Department of Radiology
H. Lee
Moffitt Cancer Center and Research Institute
12902 Magnolia Dr, Tampa, FL,
33612, USA
E-mail: arig.ibrahimhashim@moffitt.org |
|
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| Received November 03, 2011; Accepted November 17, 2011; Published
November 19, 2011 |
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| Citation: Ibrahim-Hashim A, Wojtkowiak JW, de Lourdes Coelho Ribeiro M, Estrella
V, Bailey KM, et al. (2011) Free Base Lysine Increases Survival and Reduces
Metastasis in Prostate Cancer Model. J Cancer Sci Ther S1. doi:10.4172/1948-
5956.S1-004 |
| |
| Copyright: © 2011 Ibrahim-Hashim A, et al. This is an open-access article
distributed under the terms of the Creative Commons Attribution License, which
permits unrestricted use, distribution, and reproduction in any medium, provided
the original author and source are credited. |
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| Abstract |
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| Background: Malignant tumor cells typically metabolize glucose anaerobically to lactic acid even under normal
oxygen tension, a phenomenon called aerobic glycolysis or the Warburg effect. This results in increased acid
production and the acidification of the extracellular microenvironment in solid tumors. H+ ions tend to flow along
concentration gradients into peritumoral normal tissue causing extracellular matrix degradation and increased
tumor cell motility thus promoting invasion and metastasis. We have shown that reducing this acidity with sodium
bicarbonate buffer decreases the metastatic fitness of circulating tumor cells in prostate cancer and other cancer
models. Mathematical models of the tumor-host dynamics predicted that buffers with a pka around 7 will be more
effective in reducing intra- and peri-tumoral acidosis and, thus, and possibly more effective in inhibiting tumor
metastasis than sodium bicarbonate which has a pKa around 6. Here we test this prediction the efficacy of free
base lysine; a non-bicarbonate / non-volatile buffer with a higher pKa (~10), on prostate tumor metastases model. |
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| Methods: Oxygen consumption and acid production rate of PC3M prostate cancer cells and normal prostate
cells were determined using the Seahorse Extracellular Flux (XF-96) analyzer. In vivo effect of 200 mM lysine
started four days prior to inoculation on inhibition of metastasis was examined in PC3M-LUC-C6 prostate cancer
model using SCID mice. Metastases were followed by bioluminescence imaging. |
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| Results: PC3M prostate cancer cells are highly acidic in comparison to a normal prostate cell line indicating
that reduction of intra- and perit-tumoral acidosis should inhibit metastases formation. In vivo administration of 200
mM free base lysine increased survival and reduced metastasis. |
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| Conclusion: PC3M prostate cancer cells are highly glycolytic and produce large amounts of acid when
compared to normal prostate cells. Administration of non-volatile buffer decreased growth of metastases and
improved survival indicating acidity plays a significant role in growth and invasion in-vivo. |
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| Keywords |
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| Tumor Acidosis; PC3M; Prostate Cancer; Oxygen
consumption rate; Extracellular acidification rate; Buffers; Free base
Lysine |
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| Introduction |
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| Tumor cells are highly glycolytic even in the presence of oxygen
and hence produce free protons (H+) at a higher rate than normal cells,
a phenomenon known as the Warburg effect [1]. As a consequence,
the microenvionment of solid tumors is acidic and significantly affects
tumor growth and invasion. Low extracellular pH leads to increased release of
Cathepsin B and other proteolytic enzymes that result in degradation
of the extracellular matrix (ECM) [2,3]. Interestingly tumor cells are
relatively resistant to acidic pHe most likely due to mutations of the
p53 tumor suppressor gene or other components of the apoptotic
pathway [4]. These observations have led to the acid-mediated invasion
hypothesis which proposes that H+ flow along concentrations gradients
from the tumor into peritumoral normal tissue causing normal cell
death and ECM degradation. Cancer cells, which are acid-adapted, are
then able to invade into the damaged adjacent normal tissue. Acidic
pHe has been shown to pre-dispose cancers to increased invasive and
metastatic phenotypes in animal models. Exposure of tumor cells to
acidic growth conditions prior to intravascular injection substantially
increases their ability to metastasize [5]. Our previous studies have
shown that neutralizing tumor acidity with oral sodium bicarbonate
can lead to a reduction in spontaneous and experimental metastasis in
different animal models including prostate [6]. The data also showed
that bicarbonate inhibits extravasation and/or colonization, and not the rate of intravasation, as the level of circulating tumor cells was
unchanged. Bicarbonate therapy does not alter the pH of blood and
healthy tissues, which can be explained by steady-state physiological
reaction-diffusion modeling [7]. These mathematical models indicated
that equal and perhaps improved anti tumor effects could be obtained
by non-volatile buffers with a pka closer to physiologic (i.e. pKa around
7 compared to the bicarbonate pka of 6). This was confirmed by
observations that imidazoles (IEPA) is equally effective in raising the
pH and reducing metastases [8]. Here we investigate the potential role
of lysine, a freely available amino acid with a pKa of 10, in buffering
tumors and reducing metastases. We hypothesize that lysine will
alkalize the stomach, and through the alkalinization of the stomach lumen will increase the availability of bicarbonate in blood, in what is
known as "alkaline tide". |
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| A prostate cancer model was chosen because preliminary
observation, using fluorescence microscopy in dorsal window
chambers, demonstrated that prostate cancer (PC3) xenografts export
acid into the surrounding normal tissue [9]. Here we investigate acid
production by PC3M cells and normal prostate cells in-vitro. After
demonstrating increased glycolysis and acid production we examined
the effect of lysine buffer on growth of metastases in vivo. |
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| Materials and Methods |
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| Cell culture |
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| Experiments were performed using PC3M cells (-Luc6 clone)
obtained from Xenogen Caliber (Hopkinton, MA) and Normal Human
Primary Prostate Epithelial Cells obtained form ATCC (Manassas, VA
). PC3M cells were cultured using MEM/EBSS media, supplemented with
10% Fetal Bovine Serum, 1% Penicillin Streptomycin, 1% nonessential
amino acids, 1% sodium pyruvate and 1% MEM vitamins. Normal
prostate cells were grown in phenol red-free Prostate Epithelial Cell
Basal Medium supplemented with L-Glutamine: 6 mM , Extract P:
0.4% ,Epinephrine: 1.0 μM , rh TGF-α: 0.5 ng/mL , Hydrocortisone:
100 ng/mL , rh Insulin: 5 μg/mL , Apo-transferrin: 5 μg/mL . Cells were
maintained in 37°C and 5% CO2. |
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| Oxygen consumption and extracellular acidification measurements |
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| Real-time basal oxygen consumption (OCR) and extracellular
acidification rates (ECAR) for PC3M and normal human primary
prostate epithelial cells (PCS) were determined using the Seahorse
Extracellular Flux (XF-96) analyzer (Seahorse Bioscience, Chicopee,
MA). The XF-96 measures the concentration of oxygen and free
protons in the medium above a monolayer of cells in real-time. Cells
seeded in a XF microplate were cultured for 2 hours in the presence or
absence of 2 g/L D-glucose prior to OCR and ECAR measurements.
Protein concentration was determined for each well using a standard
BCA protein assay. OCR and ECAR values are normalized to mg/
protein and are plotted as the mean +/- standard deviation. |
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| Glycolysis stress test |
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| Glycolysis and glycolytic capacity were determined for PC3M
and PCS cells using the Seahorse Extracellular Flux (XF-96) analyzer.
Cells were cultured for 2 hours in the absence of glucose. Three
sequential injections of D-glucose (2 g/L), oligomycin (1 μM), and
2-Deoxyglucose (100 mM) provided extracellular acdification (ECAR)
associated with glycolysis, the maxiumum glycolytic capacity, and
non-glycolytic ECAR. Glycolysis was defined as ECAR following the
addition of D-glucose and maximum glycolytic capacity was defined as
ECAR following the addition oligomycin. ECAR following treatment
with 2-Deoxyglucose is associated with non-glycolytic activity. |
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| Animals housing and diet |
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| All animals were maintained in accordance with IACUC standards
of care in pathogen free rooms, in the USF Vivarium on site at the
Moffitt Cancer Research Center. All imaging was performed within
the facility. 4-6 week old male beige SCID mice (Harlan, Madison,
WI) were placed in two cohorts, which were allowed to drink either
tap water or 200 mM free base lysine. Lysine (free base from Sigma
Aldrich, St. Louis MO) dissolved in tap water at a concentration of 200
mM was started four days prior to injection. Water consumption was
recorded biweekly by weighing the water bottles. Animal weights were
measured and recorded twice weekly. |
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| Cell preparation for injections: Cells were trypsinized, then
rinsed once with sterile phosphate buffered saline (PBS), prepared for
injection at a concentration of 5 x 106 cells in 200 μL PBS. The cells were
injected intravenously. Injection was confirmed by bioluminescent
imaging immediately following cell injection. |
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| Bioluminescent imaging |
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| Animals were anesthetized with isoflurane and injected
intraperitoneally with 10 µl per g body weight of sterile d-luciferin
substrate prepared in PBS at 15 mg/ml. Five minutes after the mice
were transferred to the thermoregulated, dark chamber of the In
Vivo Imaging System (IVIS 200), a photographic image was acquired
followed by overlaying of the bioluminescent image. Bioluminescent
images were acquired by measuring photons emitted from luciferaseexpressing
cells and transmitted through the tissue. Images were
analyzed using the LIVINGIMAGE V. 3.2 software. |
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| Histology |
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| At necropsy, ex vivo bioluminescence images were obtained of
the Lung/Heart for each animal. Tissues were
processed, embedded in paraffin, and 4 - 5 μm slices of the tissues were
obtained. Slides were stained with hematoxylin and eosin (H&E) stain,
and were graded by a pathologist (MB) for presence of tumor tissue.
Histology slides were scanned using the Aperio™ (Vista, CA) ScanScope XT with a 20x/0.8NA objective lens (200x) at a rate of 2 minutes per
slide via Basler tri-linear-array. |
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| Data processing |
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| Microsoft Excel and Graphpad Prism were used for data processing,
and to calculate statistical significance. |
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| Results |
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| We determined the metabolic profile of PC3M and PCS cells
using the XF analyzer to test the hypothesis that prostate cancer cells
have acquired a glycolytic phenotype and would potentially benefit
from buffer therapy (Figure 1A). Both cell lines were glucose starved
for approximately 2 hours to limit extracellular acidification (ECAR)
coupled to glycolysis. PC3M ECAR was approximately two fold greater
than PCS cells under glucose starved conditions implying elevated
acidification by cancer cells independent of glycolysis. Glyolytic
ECAR was measured immediately following the addition of glucose.
Acidification of the extracellular space in PC3M cultures occurred at a
much greater rate than normal prostate cell cultures. Complete cellular
glycolytic capacity was determined by treating cultures with oligomycin,
an inhibitor of ATP synthase. Cells treated with oligomycin will reduce mitochondrial respiration and maximize glycolytic ATP production.
Reserved glycolytic capacity was observed in PC3M cultures and not
PCS cells further suggesting that prostate cancer cells have acquired a
glycolytic phenotype to satisfy ATP energetic demands. The addition
of 2-deoxyglucose (2-DG), an inhibitor of the first step of glycolysis,
was used to confirm that the ECAR measured was a result of glycolytic
metabolism. ECAR was restored to non-glycolytic levels in both cell
lines following 2-DG treatment. Basal measurements of mitochondrial
respiration (OCR) and glycolysis (ECAR) were measured in the
presence and absence of glucose to study if PC3M acid production was
a consequence of high glucose metabolism. ECAR increased three fold,
while OCR decreased three fold in PC3M cells after administration of
glucose (Figure 1B,C) indicating that acid production of PC3M cells is
critically dependent on glycolysis. |
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|
Figure 1: The glycolytic activity and maximum glycolytic capacity was
determined for PC3M and PCS cells in real-time using the Seahorse extracellular
flux analyzer A) A series of extracellular acidification rates (ECAR)
were calculated for cells glucose starved for two hours and subsequently treated
with 2 g/L D-glucose (a), 1 µM oligomycin (b), and 100 mM 2-Deoxyglucose
(2-DG) (c). ECAR following the addition of glucose defines glycolysis and
ECAR following oligomycin represents maximum glycolytic capacity. ECAR
prior to the addition of glucose and following treatment with 2-DG represents
acidification associated with non-glycolytic activity. The data represent the
mean ± standard deviation. B-C) Basal oxygen consumption rates (OCR) and
extracellular acidification rates (ECAR) were determined for PC3M and PCS
cells either in the presence or absence of 2 g/L D-gluocose. The data represent
the mean ± standard deviation. A two-tailed Student's t-test was used to
calculate statistical significance: ** p < 0.001 and *** p < 0.0001. |
|
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| High acid production of cancer cells supports rapid, invasive and
metastatic growth. This growth can be reduced by buffers. To examine
this we used 200 mM free base lysine buffer. Animals can tolerate the
lysine with no observed reduction on their weight or change in their
behavior (Figure 2). Two separate experiments were conducted; the
first experiment consisted of intravenous injections of PC3M cells
(n=10 mice for each group) followed by treatment with or without
lysine for seven weeks. The groups that were treated with lysine showed a significant decrease in metastasis (p< 0.04) compared to tap water
with all control animals showing metastases and only two showing
metastases from the lysine group (Figure 3). The second experiment was
a longitudinal survival study. Animals treated with lysine for six weeks
lived significantly (p<.002) longer (Figure 4A) than animals on tap
water. The bioluminescence imaging (Figure 4B) showed an increase
in the tumor burden in all tap animals (two had to be euthanized
at 5 weeks), while only two of the six animals on lysine showed any
significant tumor burden (Figure 4C). Bioluminescence images from
the tap water group showed signal emanating from all areas of the
animal, and necropsy indicated that the tap cohort showed evidence
of metastatic disease, with lymph, jaw and backbone metastases in
addition to lung. Conversely, only one third of the lysine animals developed any measurable tumor burden, and these metastases were
located mainly in the inguinal lymph nodes, and the lungs. |
| |
|
Figure 2: Average animals weight and water consumption A) Name and
Chemical Structure of lysine. B) Average water consumption over time in the
two cohorts (tap and lysine) indicating no difference in the amount of water
consumption between the treatments. C) Average mouse weight over time
between cohorts of mice drinking either tap water (Tap) or tap water buffered
with 200mM free base lysine, showing no significant difference in weight gain
or loss between the two treatments. |
|
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|
Figure 3: Effect of lysine on PC3M experimental metastasis A) Bioluminescence
images of representative mice from the tap versus lysine groups at
the indicated time points after venous injection of luciferin expressing PC3M
cancer cells to induce experimental metastases. B) Ex-vivo Bioluminescence
images of representative mice from the tap versus lysine groups at the end
time point confirming the in vivo images. C) Mean tumor bioluminescence in
each group after induction of experimental metastases, indicating significantly
fewer metastases in the lysine cohort than in the tap (p<0.04) note log scale). |
|
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|
Figure 4: Effect of lysine on survival, and tumor burden A) Kaplan Meyer
Survival Curve illustrating that the animals treated with lysine survived significantly
(p<.002) longer than the tap cohort. B) Representative bioluminescence
images demonstrating little tumor burden present in the lysine animal as compared
to the tap animal. C) The lifetime average tumor burden of two cohorts,
showing that all tap animals had to be euthanized after 6 weeks,while only two
of the six lysine animals developed measureable metastases. |
|
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| Histology of lungs collected from animals that were sacrificed at
the same time (7 weeks) demonstrates that animals treated with lysine
(Figure 5A) still had healthy lung tissue, where as the tap animal
(Figure 5B) consisted of poorly differentiated lung tissue, indicating
metastases. Moreover, histology from lung samples taken from the
animals which were allowed to live until they demonstrated signs of
severe discomfort show similar results; with the lysine lung appearing
more normal (Figure 5C) with healthy tissue and the tap group having
poorly differentiated lung tissue (Figure 5D). |
|
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|
Figure 5: Histological images for treated and non treated mice. A) Representative
images from mice at 7 weeks , showing a healthy lung on the lysine
treated mouse B) and poorly differentiated lung on the control mouse. C-D)
Representative images from the longitudinal study, showing a mouse sacrificed
at 16 weeks old on lysine treated and 7 weeks on the control mouse. All
images are at 200x magnification and scale bar is 100μm. |
|
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| Discussion |
| |
| Cancers typically maintain a highly acidic microenvironment.
In part this is due to regions of insufficient perfusion leading to
inadequate oxygen supply (hypoxia), which requires upregulation of
glycolysis to maintain ATP levels. In addition, it has been commonly
observed since Warburg that many tumors use glycolytic metabolism
even in the presence of normal oxygen. This is commonly described as
aerobic glycolysis or the Warburg effect. Because it is less efficient in
ATP production, glycolysis requires increased glucose flux resulting
in increased lactic acid production and extracellular acidosis [10].
Although the glycolytic phenotype is thought to be a near-universal
phenomenon in cancer cells, it has not been specifically described in
PC3M. Therefore, we first investigated the metabolic state of PC3M
cells in comparison to a normal prostate cell line (PCS) in vitro. Real
time basal metabolic measurements of oxygen consumption and
proton production were significantly higher in PC3M cells suggesting
that prostate cancer cells are more metabolically active than normal
prostate cells. In support of this observation, PC3M cells were found
to have a greater glycolytic capacity in the presence of glucose and
following inhibition of mitochondrial ATP production implying that
PC3M cells have the ability to depend on glycolysis much more so than
PCS cells to meet energetic demands. |
| |
| Based on the in vitro results, we examine the potential role of a
systemic buffer, lysine, in inhibiting the development of metastases from the PC3M cell in vivo. We found, consistent with our predictions,
that lysine substantially inhibits development of metastases and
prolongs survival. |
| |
| The precise mechanism by which lysine acts to inhibit metastases
cannot be specifically answered in our study. pH plays an important role
in almost all steps of metastasis [11]. Metastases are the cause of 90%
of human cancer deaths [12] and metastatic disease in prostate cancer
is uniformly fatal. Metastasis is a multistep process that is defined as
the spread of cells from a primary tumor to a distant secondary organ
or site. The metastatic process involves multiple interactions between
the tumor cells and their microenvironments [13]. Tumor cells locally
invade through the basement membrane into the lymphatic and
blood vasculature followed by extravasation into secondary organs
[14]. Numerous explanations and mechanisms could potentially
contribute to the effect of buffers on tumor metastasis. Acid-mediated
invasion can occur via destruction of the extracellular matrix, which is
promoted by proteases and glycosidases. Metalloproteinases (MMP-2
and MMP-9) are believed to be critical for invasion and extravasation
[15]. MMPs are a family of proteolytic enzymes that degrade the
extracellular matrix and junctional proteins and further increase
endothelial permeability [16]. Low pH up regulates angiogenic factors
such as vascular endothelial growth factor (VEGEF) and interlukin 8
(IL-8) stimulating neovascularization and promoting metastasis. Thus
it is possible that lysine can reverse acidosis and consequently decrease
proteolytic enzyme activity and or anigiogenesis which will lead to the
inhibition of extravasations and colonization of circulating tumor cells
decreasing successful metastases. |
| |
| The clinical application of these results will require additional
investigation. A practical concern is that the amount of buffer necessary
to match the dose applied to the mice is in the range of 15 to 30 grams
per day. Compliance would likely be limited by the large amount and
possible related GI toxicity. An alternative may focus on perturbation
of proton pumps, which export protons from across the cytoplasmic
membrane and acidify the intracellular compartments. These pumps
play a pivotal role in the regulation of cell pH in normal cells and, to
a much greater extent, in tumour cells [17]. Inhibiting these pumps
can be an alternative approach to alleviate extracellular tumor acidity
although this may be attenuated by systemic effects that, for example,
might decrease buffering capacity and, therefore, paradoxically
increase tumoral acid concentrations. Thus, this approach may be
useful for tumor control [18] but it is likely that the complex tumor and
systemic dynamics will require extensive experimental investigation
and mathematical modeling to optimize treatment strategies. |
| |
| In summary our findings suggest that manipulation of the
extracellular acidosis of prostate tumor with Lysine buffer reduces
metastatic disease and prolongs survival which supports the hypothesis
that non-volatile buffers with pKa > 7 should be more effective in
buffering extra cellular acidity. This suggests new potential therapeutic
strategies in treatment of prostate cancer. |
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| Acknowledgements |
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| This work was supported by NIH Grant CA 077575. |
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| References |
| |
- Warburg O (1956) On the origin of cancer cells. Science 123: 309-314.
- Montcourrier P, Mangeat PH, Valembois C, Salazar G, Sahuquet A, et al.
(1994) Characterization of very acidic phagosomes in breast cancer cells and
their association with invasion. J Cell Sci 107: 2381-2391.
- Cuvier C, Jang A, Hill RP (1997) Exposure to hypoxia, glucose starvation and
acidosis: effect on invasive capacity of murine tumor cells and correlation with
cathepsin (L + B) secretion. Clin Exp Metastasis 15: 19-25.
- Ohtsubo T, Wang X, Takahashi A, Ohnishi K, Saito H, et al. (1997) p53-
dependent induction of WAF1 by a low-pH culture condition in human
glioblastoma cells. Cancer Res 57: 3910-3913.
- Rofstad EK, Mathiesen B, Kindem K, Galappathi K (2006) Acidic extracellular
pH promotes experimental metastasis of human melanoma cells in athymic
nude mice. Cancer Res 66: 6699-6707.
- Robey IF, Baggett BK, Kirkpatrick ND, Roe DJ, Dosescu J, et al. (2009) Bicarbonate increases tumor pH and inhibits spontaneous metastases. Cancer
Res 69: 2260-2268.
- Silva AS, Yunes JA, Gillies RJ, Gatenby RA (2009) The potential role of
systemic buffers in reducing intratumoral extracellular pH and acid-mediated
invasion. Cancer Res 69: 2677-2684.
- Ibrahim Hashim A, Cornnell HH, Coelho Ribeiro Mde L, Abrahams D,
Cunningham J, et al. (2011) Reduction of metastasis using a non-volatile
buffer. Clin Exp Metastasis 28: 841-849.
- Gatenby RA, Gawlinski ET, Gmitro AF, Kaylor B, Gillies RJ (2006) Acidmediated
tumor invasion: a multidisciplinary study. Cancer Res 66: 5216-5223.
- Vaupel P, Kallinowski F, Okunieff P (1989) Blood flow, oxygen and nutrient
supply, and metabolic microenvironment of human tumors: a review. Cancer
Res 49: 6449-6465.
- Hashim AI, Zhang X, Wojtkowiak JW, Martinez GV, Gillies RJ (2011) Imaging
pH and metastasis. NMR in biomedicine 24: 582-591.
- Weigelt B, Peterse JL, van 't Veer LJ (2005) Breast cancer metastasis: markers
and models. Nat Rev Cancer 5: 591-602.
- Fidler IJ (2003) The pathogenesis of cancer metastasis: the 'seed and soil'
hypothesis revisited. Nat Rev Cancer 3: 453-458.
- Ruoslahti E (1996) How cancer spreads. Sci Am 275: 72-77.
- Nguyen JH, Yamamoto S, Steers J, et al. (2006) Matrix metalloproteinase-9
contributes to brain extravasation and edema in fulminant hepatic failure mice.
J Hepatol 44: 1105-1114.
- Alexander JS, Elrod JW (2002) Extracellular matrix, junctional integrity and
matrix metalloproteinase interactions in endothelial permeability regulation. J
Anat 200: 561-574.
- Martinez-Zaguilan R, Lynch RM, Martinez GM, Gillies RJ (1993) Vacuolar-type
H(+)-ATPases are functionally expressed in plasma membranes of human
tumor cells. The American journal of physiology 265: C1015-C1029.
- Luciani F, Spada M, De Milito A, Molinari A, Rivoltini L, et al. (2004) Effect of
proton pump inhibitor pretreatment on resistance of solid tumors to cytotoxic
drugs. J Natl Cancer Inst 96: 1702-1713.
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