A THESIS SUBMITTED TO THE GRADUATE DIVISION OF THE · evidence support the conclusion that...
Transcript of A THESIS SUBMITTED TO THE GRADUATE DIVISION OF THE · evidence support the conclusion that...
UNIVERSITY OF HAWAI'I LIBRARY
CANCER CHEMOPREVENTION BY WATER SOLUBLE ASTAXANTHIN
DERIVATIVES
A THESIS SUBMITTED TO THE GRADUATE DIVISION OF THEUNIVERSITY OF HAWAtI IN PARTIAL FULFILLMENT OF THE
REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE
IN
BIOMEDICAL SCIENCES(CELL AND MOLECULAR BIOLOGY)
MAY 2004
ByLauraM. Hix
Thesis Committee:
Tom Humphreys, Co-ChairpersonJohn Bertram, Co-Chairperson
Pratibha Nerurkar
TABLE OF CONTENTS
List of Figures , .IV
Chapter 1: Introduction 1
Chapter 2: Materials and Methods 7
2.1. Materials 72.1.1. Compounds 72.1.2. Cel/lines and culture conditions 9
2.2. Methods 92.2.1. Analysis ofCx43 expression 92.2.2. Immunofluorescence 102.2.3. Gapjunctional communication assay 102.2.4. MCA-induced neoplastic transformation assay 112.2.5. Statistical analysis 12
Chapter 3: Results 13
3.1. Formulation and Solubility Studies 133.2. Molecular Analysis ofCx43 , 153.3. Cellular Analysis ofCx43 203.4. Induction of Gap Junctional Communication 223.5. Inhibition of MCA-induced Neoplastic Transformation 23
Chapter 4: Discussion 26
Appendix 31
References 32
III
LIST OF FIGURES
Figure Page
I. Structures of the disodium salt astaxanthin derivatives 8
2. UV/vis absorption spectra ofracemic dAST and AST 14
3. Western blot demonstrating Cx43 protein expression in lOTI/2 cells treated with
two different formulations of racemic dAST 15
4. Western blot demonstrating induction ofCx43 protein expression in IOT1I2 cells
treated with dAST derivatives 17
5. Western blot demonstrating induction ofCx43 protein expression in IOTII2 cells
treated with pAST, AST and CTX 19
6. Immunofluorescence demonstrating dAST increases assembly of Cx43
immunoreactive junctional plaques .21
7. Micrographs demonstrating dAST enhances functional GJIC in lOTI/2 cells .23
8. Dose-dependent effects ofpAST and AST on MCA-induced neoplastic
transformation 25
IV
CHAPTER 1. INTRODUCTION
It is now well established that the major contributing factor to cancer risk in
Western societies is lifestyle rather than genetics. Current epidemiological data suggests
that approximately 70% of the estimated 1.37 million cancer cases in 2004 will be linked
to preventable lifestyle factors such as tobacco use, alcohol, diet, infections (including
sexually transmitted diseases), occupational exposures, pollution, sunlight exposure (UV
radiation), and stress (Cancer prevention and early detection, American Cancer Society,
2004); [1]. Despite considerable improvements in the detection and treatment of cancer,
it is estimated that 563,700 Americans will die ofcancer in the year 2004 (Cancer facts
and figures, American Cancer Society, 2004). Cancer chemoprevention, defined as the
reduction in cancer incidence through the use of pharmaceuticals, vitamins, minerals and
other chemicals, has emerged as a powerful strategy in the fight against cancer. Potential
targets for chemoprevention include the general population, subgroups at risk due to
lifestyle or other environmental factors, individuals with precancerous lesions, and cancer
survivors at risk for secondary tumors.
A growing body of epidemiological evidence has linked increased intake and/or
blood levels of several dietary carotenoids, plant pigments found in green, yellow and
orange fruits and vegetables, to decreased risk of cancer at many anatomic sites. This
activity appears to be independent of the pro-vitamin A activity of these carotenoids [2].
Studies in experimental animal and cell culture models ofcarcinogenesis have confirmed
the cancer chemopreventative activity of many dietary carotenoids. In these systems,
activity does not correlate with the pro-vitamin A properties of these compounds, nor
does it correlate with their ability to act as lipid-phase antioxidants [3].
In the C3H/lOTl/2 mouse embryo fibroblast (lOTl/2) cell system developed in
the lab of the late Charles Heidelberger, cells undergo neoplastic transformation in
response to many chemical and physical carcinogens in a dose-dependent manner [4].
This cell system has been shown to effectively mimic the initiation and transformation
events of tumor formation in whole animals, and represents one of the most widely
characterized in vitro models for carcinogenesis [5]. Studies conducted in the 10Tl/2 cell
system revealed that carotenoids active in inhibiting neoplastic transformation
upregulated gap junctional intercellular communication (GJlC) in direct relationship to
their activity as chemopreventative agents [3].
GJIC permits the transfer of ions and small hydrophilic molecules <1 kiloDalton
(kDa) in size by passive diffusion through aqueous channels (connexons) that span the
plasma membranes of adjoining cells. Individual connexons are comprised of proteins
known as connexins. Communicating cells typically form several thousand connexons
that assemble into plaques. A family of approximately 20 connexin members is
expressed in mammals. These connexins exhibit developmental- and differentiation
specific expression, allowing the formation of communicating compartments between
compatible family members. There is growing evidence that these compartments are
vital to normal growth and development.
Interest in gap junctions and carcinogenesis stems from several independent lines
of evidence. Following the discovery ofjunctional communication in cultured normal
cells, it was soon discovered that neoplastically-transformed cells were deficient in GJlC.
Moreover, inhibition ofGJIC was a very early event after activation ofthe oncogene src
[6]. Human and animal tumors of many pathologic types were found to be deficient in
2
GJIC, either as a consequence of decreased connexin expression or faulty assembly into
the plasma membrane. Restoration ofjunctional communication between transformed
cells and growth-inhibited normal cells resulted in growth arrest of the transformed cells
in direct proportion to the extent of heterologous cell coupling [7]. Tumor promoters
agents that accelerate the process of carcinogenesis-were found to inhibit GJIC, while
cancer preventive agents such as retinoids and carotenoids-agents that delay
carcinogenesis-had the opposite effect. .Finally, transfection of molecular expression
constructs into human and animal neoplastic cells, utilizing either constitutive or
inducible promoters, demonstrated that connexin re-expression in these cells decreased
their neoplastic potential in in vitro and in vivo assays. Collectively, these lines of
evidence support the conclusion that connexins can function as tumor-suppressor genes
[8].
Studies of human and animal cells have demonstrated that connexin 43 (Cx43),
the most widely expressed connexin in human and animal tissues, is upregulated at the
message and protein level by chemopreventive retinoids and carotenoids. The ability of
carotenoids to regulate Cx43 expression appears to be independent of their conversion to
retinoids. This is exemplified by compounds such as lycopene, a straight-chain
hydrocarbon that is not cleaved to vitamin A in mammals, yet possesses this regulatory
ability. It is unclear how much the parent compound contributes to the observed increase
in Cx43 expression; for example two oxidation products of lycopene are active in this
respect [9;10]. Moreover, the potential conversion of carotenoids to
retinoids--chemopreventive agents which are highly potent inducers ofCx43 and GJIC
[11;12]-eannot be ignored. Indeed, there is evidence that conversion of canthaxanthin
3
to 4-oxo-retinoic acid, an active retinoid, may be in part responsible for increased Cx43
expression in lOTl/2 cells [13], although we found no evidence of this conversion in the
same system [14].
In the 10Tl/2 system, upregulated GJIC as a consequence of increased Cx43
expression is highly correlated with the ability of carotenoids to prevent neoplastic
transformation of these cells [15;16]. Due to these multiple lines ofevidence linking
GJIC with inhibition of carcinogenesis and decreased neoplastic potential of established
transformed cells, Bertram and others have proposed that GJIC allows for the
transmission of growth controlling signals between normal and transformed cells [17]. In
this model, the ability of carotenoids and retinoids to inhibit tumor progression is a result
of enhanced GJIC between carcinogen-initiated cells and surrounding growth-controlled
normal cells. By extension, the ability to upregulate GJIC may be an important indicator
of chemopreventative potential.
Astaxanthin, found predominantly as a dietary source in shrimp, lobster and
salmon, has been associated with reduced risk of diseases such as age-related macular
degeneration and ischemic diseases, effects attributed to its potent antioxidant activity
[18]. In addition, the antioxidant activity of astaxanthin has been reported to be 10 times
stronger than that of other carotenoids, namely, zeaxanthin, lutein, canthaxanthin, and ~
carotene [19;20]. In experimental animal studies astaxanthin has been shown to be
capable of inhibiting chemically induced oral and bladder carcinogenesis [21 ;22]; its
usefulness as an immunomodulating agent in experimental cancer studies is also well
documented (reviewed [23;24]). Based on these findings, astaxanthin has significant
cancer chemopreventative potential.
4
One problem confronting researchers investigating the effects of carotenoids such
as astaxanthin in cell culture and/or whole animals has been the delivery of these highly
lipophilic molecules to target cells. For cell culture studies, the Bertram lab developed
the use of tetrahydrofuran (THF) as a delivery solvent, the use of which creates a highly
bioavailable and non-toxic pseudo-solution of carotenoids in cell culture medium [25].
However, caution must be taken to protect THF from oxidation to toxic species, and this
method is unsuitable for use in whole animal studies. Delivery of carotenoids to
experimental animals or humans is usually achieved by delivery of carotenoids as a
suspension in oil, frequently as a micro-dispersed emulsion. Unfortunately,
bioavailability is usually low in animals (e.g. rodents) and can be variable in humans.
To circumvent these problems of drug delivery Hawaii Biotech, Inc. has
synthesized a set of novel carotenoid derivatives, disodium salt disuccinate (dAST) and
disodium salt diphosphate (PAST) derivatives of synthetic astaxanthin (3,3'-dihydroxy-p,
p-carotene-4,4'-dione), in all-trans (all-E) form. Synthetic astaxanthin can be
commercially obtained most economically as the statistical mixture of stereoisomers
[optically active (38,3'8) and (3R,3'R) and optically inactive (3R,3'S; meso) in a 1:1:2
ratio]; the statistical mixture of stereoisomers is known as (3RS,3 ,RS)-astaxanthin or
"racemic"astaxanthin (Buckton Scott, India). The individual stereoisomers are also
available commercially from Hoffman-LaRoche (Switzerland) and BASF (Germany).
The racemic mixture of the novel derivative, as well as purified stereoisomeric forms of
the derivative, were successfully synthesized and tested in the current study. The
derivatives exhibit several unique characteristics that increase their utility for use in the
cancer chemoprevention setting. They are water soluble (critical micelle concentration =
5
0.3 mg/mL) and water dispersible, with the maximum aqueous dispersibility greater than
8 mg/mL (approximately 10 mM), allowing them to be introduced into cell culture
without a co-solvent and delivered parenterally to experimental animals [27;28];
(Lockwood, unpublished results). Additionally, the intact synthetic derivatives retain
antioxidant activity prior to enzymatic cleavage to mono-succinates and non-esterified,
free astaxanthin [29]. Derivatives that enhance astaxanthin's water solubility and
bioavailability should prove invaluable in assessing the carotenoids' abilities in vitro and
in vivo.
This thesis evaluates the ability of the novel derivatives delivered in several
aqueous formulations to upregulate Cx43 protein expression, induce functional GllC, and
inhibit carcinogen-induced neoplastic transformation in the lOTI/2 cell culture system
described above. The compounds were found to induce expression of functional Cx43
protein, significantly increase GllC and significantly inhibit MeA-induced neoplastic
transformation with enhanced ability over the parent carotenoid astaxanthin. These
results indicate that the major hurdle ofdelivering hydrophobic carotenoids to biological
tissues in model systems has been overcome, and that evaluation of these highly
bioavailable astaxanthin derivatives in in vivo models of cancer chemoprevention should
be pursued.
6
CHAPTER 2. MATERIALS AND METHODS
2.1 Materials
2.1.1. Compounds
The racemic disodium salt diphosphate derivative ofastaxanthin (racemic pAST; >
90% purity by HPLC) was synthesized from commercial astaxanthin and its structure
verified. It was prepared in a fonnulation of20% EtOH and sterile water (0.2% final
EtOH cone.) to minimize supramolecular aggregation. The racemic disodium salt
disuccinate derivative ofastaxanthin (racemic dAST; > 90% purity by HPLC) was
synthesized from commercial astaxanthin and its structure verified. It was prepared in a
fonnulation of33% EtOH and sterile water (0.33% final EtOH cone.). Non-esterified,
all-E synthetic astaxanthin (AST) (> 96% purity by HPLC; Sigma, St. Louis, MO) was
added to tetrahydrofuran (THF). Synthetic canthaxanthin (CTX) (CaroteNatur,
Switzerland) was added to THF. The synthetic retinoid TTNPB [p-«E)-2-(5,6,7,8
Tetrahydro-5,5,8,8-tetramethyl-2-napthyl) propenyl>benzoic acid] (Hoffman-LaRoche,
Nutley, NJ) and retinol acetate (Sigma, St. Louis, MO) were prepared in acetone (Sigma,
St. Louis, MO). TTNPB, canthaxanthin and astaxanthin concentrations were confinned
by comparing their UV absorption and their published extinction coefficients. The
chemical structures of the three stereoisomers are shown in Fig. IA. These are:
(3R,3'R)-, (3S,3'S)-, and (3R,3'S; meso)-astaxanthin disuccinate, disodium salt. The
structure of the all-E-astaxanthin diphosphate, disodium salt is shown in Figure. IB. Due
to the sensitivity ofcarotenoids to light, heat and oxygen, special precautions were taken
7
throughout the study. All compounds were stored under nitrogen at -70° C and care was
taken to ensure minimal exposure to direct sunlight or UV light.
A
3R,3'R disodium salt disuccinateastaxanthin derivative
o
3S.3'S disodium salt disuccinateastaxanthin derivative
o3R,3'S disodium salt disuceinateastaxanlltin derivative
BRacemic disodium diphosphateastaxanthin derivative
oNaO-~...,.,
NaO' U
o
oONa
O~P/-ONaIIo
Fig. 1. (A) Structures of the three stereoisomers of the disodium salt disuccinate astaxanthin derivatives
(clAST) evaluated in the current study (shown as the all-E geometric isomers). The racemic mixture of
stereoisomers, referred to as ''racemic clAST" in the text, contains (38,3'S)-astaxanthin disuccinate,
disodium salt; (3R,3'R)-astaxanthin disuccinate, disodium salt; and (3R,3'S; meso)-astaxanthin disuccinate,
disodium salt in a 1: 1:2 ratio. (B) Structure of the racemic disodium salt diphosphate astaxanthin derivative
8
(pAST), containing (38,3'S)-astaxanthin diphosphate, disodium salt; (3R,3'R)-astaxanthin diphosphate,
disodium salt; and (3R,3'S; meso)-astaxanthin diphosphate, disodium salt in a 1:1:2 ratio. Racemic pAST,
racemic dAST and all individual dAST stereoisomers (38,3'8 dAST, meso dAST, and 3R,3'R dAST) were
separated to > 90% purity by HPLC.
2.1.2. Cell lines and culture conditions
Mouse embryonic fibroblast lOTl/2 cells were cultured in Eagle's basal medium
with Earle's salts supplemented with 4% fetal calf serum (Atlanta Biologicals, Norcross,
GA), 25 J.lg/mL gentamicin sulfate (Sigma, St. Louis, MO), and passaged by
trypsin/EDTA. Cells were incubated at 37° C in 5% C02 as described previously [1;2],
and confluent cells were treated on the 7th day after seeding, unless otherwise indicated in
methods.
2.2. Methods
2.2.1. Analysis ofCx43 expression
Expression of Cx43 protein in murine fibroblasts was assessed by immuno
(Western) blotting essentially as described [1;2]. Briefly, lOTl/2 cells were treated with
the novel astaxanthin derivatives and/or retinoids at confluence on the 7th day after
seeding in 100 mm dishes (Fisher Scientific, Pittsburgh, PA). Cells were harvested after
4 days and total protein content was measured using the Protein Assay Reagent kit
(Pierce Chemical Co., Rockford, IL) according to the manufacturer's instructions. Cell
lysates containing 50 J.lg of protein were analyzed by Western blotting using the NuPage
Western blotting kit (Invitrogen, Carlsbad, CA). Cx43 was detected using a rabbit
polyclonal antibody (Zymed, San Francisco, CA) raised against a synthetic polypeptide
9
corresponding to the C-terminal domain common to mouse, human and rat Cx43. Cx43
immunoreactive bands were visualized by chemiluminescence using an anti-rabbit HRP
conjugated secondary antibody (Pierce Chemical Co., Rockford, IL). Images were
obtained by exposure to X-ray film as previously described [1 ;2] or obtained with a
cooled CCD camera, and quantitative densitometry was performed (Bio-Rad, Richmond,
CA). Equal protein loading of the lanes was confirmed by staining with Coomassie blue
protein (Sigma, S1. Louis, MO) and digital image analysis.
2.2.2. Immunofluorescence
Expression and assembly ofCx43 into plaques was assessed by
immunofluorescence staining essentially as described [3]. Briefly, confluent cultures of
10Tl/2 cells were grown on Permanox plastic 4-chamber slides (Nalge Nunc
International, Naperville, IL) and treated for 4 days with (1) racemic dAST dissolved in
33% EtOH; (2) TTNPB at 1 x 10-8 M in acetone (0.1 % final acetone concentration in
assay) as a positive control; or (3) 33% EtOH (0.33% EtOH final concentration) as a
solvent-only control. Cells were fixed with - 20°C methanol overnight, washed in buffer,
blocked in 1% bovine serum albumin (Sigma, S1. Louis, MO) in PBS, incubated with the
rabbit anti-Cx43 antibody, and finally detected with Alexa568 conjugated anti-rabbit
secondary antibody (Molecular Probes, Eugene, OR). Slides were illuminated with 568
nm light and images were acquired at a wavelength of 600 nm using a Zeiss Axioplan
microscope and a Roper Scientific cooled CCD camera.
10
2.2.3. Gap junctional communication assay
Junctional permeability was assayed by microinjection of the fluorescent dye Lucifer
Yellow CH (10% in 0.1 M LiCI) (Sigma, St. Louis, MO) into confluent cells as described
previously [4]. Confluent cultures of lOTl/2 cells were treated for 4 days with: (1)
racemic dAST at lO-5 M in 33% EtOH; (2) TTNPB in acetone at lO-8 M as positive
control; or (3) 33% EtOH as solvent-only control as described above. Single cells were
injected with the fluorescent dye and digital images were taken under UV illumination
after 2 minutes. The number of fluorescent cells adjacent to the injected cell was later
determined by digital image analysis, and this number was used as an index ofjunctional
communication, as described previously [4].
2.2.4. MeA-induced neoplastic transformation assay
In this experiment, the novel derivatives were assessed for their ability to prevent
carcinogenic neoplastic transformation in the C3H/1 OT1I2 cell assay developed in the
Bertram lab [4;5]. Using the protocols previously described, cells were initiated with the
potent carcinogen methylcholanthrene (MCA, Sigma, St. Louis, MO) in acetone at a final
concentration of 5 f,lg/mL, and media was replaced 24 hours later. Cells were treated
with either the novel carotenoid derivative (pAST) in 20% EtOH (0.2% final EtOH
concentration) or the parent carotenoid astaxanthin (AST) in THF (0.1 % final THF
concentration) beginning 7 days after removal of carcinogen and continuing to the end of
the four-week culture period [6]. The ability to inhibit neoplastic transformation was
assessed at concentrations of lO-5, lO-6, lO-7 and 10-8M. Negative controls include MCA
treated cells followed by media only treatments, Acetone-treated cells followed by media
11
only treatments, and MCA-treated cells followed by treatment with THF as a solvent
control. A total of 24 dishes/treatment has previously been shown to yield an expected 50
transformed foci in MCA-only controls and 10 foci in canthaxanthin 10-5 M treated cells,
and allows statistical significance to be met over most of the dose range [7].
2.2.5 Statistical Analysis
Statistical analyses were conducted with the NCSS 2001 and PASS 2002
statistical software (J. Hintze, Number Croneher Statistical Systems, Kaysville, UT). All
tests were conducted at the a = 0.05 level.
12
CHAPTER 3. RESULTS
3.1. Formulation and Solubility Studies
In pure aqueous formulation, racemic dAST and the purified stereoisomeric dAST
derivatives demonstrate supramolecular assembly, a form of 3-dimensional aggregation
that can limit solubility and bioavailability [27;31]. The aggregation is of the so called
H- or "card-pack" type, and may be disrupted into a completely monomeric solution with
the addition of a less polar solvent (such as EtOH) [32]. We observed precipitation of red
solid onto the cellular monolayer several days after introduction of dAST from purely
aqueous stock solutions, without effect on the cellular viability (data not shown). This
phenomenon has also been observed after introduction of non-esterified, free astaxanthin
in EtOH to cell culture systems [Lockwood, unpublished results]. To enhance both
solubility and bioavailability, several EtOH/water formulations were tested for
aggregation with UVIvis spectroscopy. The "solubility" of the derivatives was
significantly enhanced by the use of 1:1 (50% EtOH) and 1:2 (33% EtOH) EtOH/water
formulations. These formulations have been previously demonstrated to maintain the
carotenoid derivatives in monomeric form [26]. The addition of EtOH at 50% final
concentration caused the expected red-shifted, hyperchromic changes in the UVIvis
absorption spectrum of the racemic dAST derivative, from 423 nm (card-pack aggregate)
to 484 nm (Amax of the non-esterified, free astaxanthin chromophore measured in
acetone), due to the disaggregation of the compound to molecular monomers (Figs. 2A
and 2B) [33]. Therefore, ethanolic formulations were utilized in subsequent analyses in
the current study. Non-esterified, synthetic all-E astaxanthin exhibited a lack of
13
solubility in both the water and EtOH/water fonnulations as assessed
spectrophotometrically (Figs. 2C and 2D), demonstrating the significant enhancement of
water solubility achieved by the derivatives over the parent carotenoid.
A 1.0 C 1.0
c c0 .~';:. 0.5 Q, D.S'" ..i <:l..
.Q .c-< '(
0.0 I65fj 200 6SO
WIl'v"len:tb (11m) WavtleDtth (om)
1.0B D 1.0
-I c~
',ca. 0.5 e- t.S..j 0
ill•< ~
0.1) «..0200 65ft 200 6S0
Waveleagth (nm) 'W'.vtl~llth(nrn)
Fig, 2, UVivis absorption spectra ofracemic dAST dissolved in water, 2A, compared to a solution in 50%
EtOH, 2B (all solutions at 10-5 M racemic dAST). The Amax in 50% EtOH (484 nm) is red-shifted and
hyperchromic relative to the aggregated state in water (423 nm), expected spectral changes for carotenoids
which demonstrate supramolecular assembly in aqueous solution, Panel2C: lack ofUV/vis absorption of
non-esterified, synthetic all-E astaxanthin in water, or as shown in 2D, in 50% EtOH, illustrating the dramatic
increase in aqueous solubility achieved with the disodium disuccinate derivatization ofnon-esterified, free
astaxanthin.
Studies conducted using the disodium diphosphate derivative (pAST) yielded
similar disaggregation effects with the use of 20% EtOH. This allowed the compound to
be directly dissolved in media without precipitation for up to 24 hours, upon which
14
phosphatases present in the media and serwn presumably resulted in cleavage of the
diphosphates to the parent moiety and subsequent precipitation in solution.
3.2. Molecular Analysis of Cx43
Initial treatments of the lOT1/2 cells were performed with the goal of comparing the
relative biological efficacy of the racemic dAST in both water and in 50% EtOH to
enhance connexin 43 expression. Cells were treated with racemic dAST in water at 10-5,
10-6, and 10-7 M, as well as in a 50% EtOH formulation at 10-5 M (0.5% final EtOH
concentration in assay). 50% EtOH was used as a solvent-only control. Racemic dAST
caused induction of Cx43 in comparison with solvent-only treated controls in a dose-
dependent manner when dissolved in sterile water (Fig. 3). Additionally, induction levels
were higher with the EtOH formulation at 10-5 M than for the formulations in sterile
water alone, demonstrating enhanced biological efficacy using EtOH as a co-solvent, as
suggested by the physico-chemical studies described in Section 3.1 above. Racemic
dAST in pure aqueous formulation was able to upregulate Cx43 expression with
equivalent or greater potency than that previously observed for other carotenoids in
organic vehicle [15;16]. Importantly, this activity was achieved via direct delivery in
sterile water alone, eliminating the absolute need for introduction of a co-solvent into cell
culture.
A
2 4 5
15
B
Fold Induction
4.00 ...---------------------------.......
3.S03.00
2.S0
2.00
1.S0
1.00O.SO0.00
dAST 10-SH20
dAST 10-6H20
dAST 10-7H20
dAST 10-SEtOH
EtOH
Fig. 3. Western blot demonstrating Cx43 protein expression in IOTl/2 cells treated for four days with two
different formulations ofracemic dAST. (A) Lanes 1-3: racemic dAST in water at IO-s, 10-6, and 10-7 M,
respectively; Lane 4: racemic dAST at 10-s M in 50% EtOH; Lane 5: 50% EtOH solvent-only control. (B)
Relative Cx43 induction levels by racemic dAST obtained by digital analysis ofdata shown in Panel A;
Cx43 protein levels in 50% EtOH solvent control set to 1.0. The upper immunoreactive bands in A are
believed to represent phosphorylated forms ofthe protein assembled into gap junctions; lower bands
unphosphorylated proteins [34]. Figure is representative of three separate replicates. Immunoblot stained
with Coomassie blue and confirmed by densitometry for equal protein loading and transfer.
Racemic dAST and the purified stereoisomers 38,3'8 dAST, 3R,3'S (meso) dAST,
and 3R,3'R dAST were also added to cell cultures in a formulation of33% EtOH at I x
10-5 M. The potent chemopreventive retinoids TTNPB at 10-8 M in acetone (0.1 % fmal
acetone concentration in assay) and retinol acetate ("RetAC") at 10-5 M in acetone (0.1 %
final acetone concentration in assay) were included as positive controls. All compounds
induced increased expression ofCx43 in comparison to cell cultures treated with 33%
16
EtOH as a solvent-only control (Fia. 4). TreatIneaM with racemic clAST induced the
highest level ofCx43 ofall novel derivatives tested (::::3-fold). As previously observed
with unmodified carotenoids, connexin 43 induction by the astaxanthin derivatives (::::3-
fold or less) was several-fold lower than induction levels observed with the retinoids (up
to 9-fold induction). Interestingly, in cells treated with the novel astaxanthin derivatives,
the majority of the Cx43 protein was located in the higher molecular weight regions of the
gel, whereas the majority ofconnexin 43 iDduced by the retinoids remained in the lower
regions (Fig. 4A). These higher molecular weight isoforms are believed to represent
phosphorylated forms of the protein assembled into gap junctions, whereas the lower
blinds are comprised ofnon-phosphorylated, unassembled proteins [39].
A1 2 7
-mlUI . \", GIlD,
[.'4~ '. ", . ,,~:t.
B
fold Inductieft
10.009.008.007.008.005.004.003.002.001.000.00
Media RetAC TTNPB Rac R,R S,S Meso
17
Fig.4. Western blot demonstrating induction ofCx43 protein expression in 10Tl/2 cells treated for four
days by novel astaxanthin derivatives delivered in 33% EtOH, versus positive and negative controls. (A)
Lane 1: 33% EtOH (final EtOH concentration in assay 0.3%) as solvent-only control. Lane 2: TTNPB at
10-8 M in acetone (final acetone concentration in assay 0.1%) as positive control. Lane 3: Retinol acetate
("RetAC") at 10-5 M in acetone (final acetone concentration in assay 0.1 %) as positive control. Lane 4:
racemic clAST at 10-5 M; Lane 5: 3R,3'R clAST at 10-5 M; Lane 6: 38,3'8 clAST at 10-5 M; Lane 7: meso
clAST at 10-5 M. (B) Relative induction levels ofCx43 expression versus solvent-only control, as in Fig.
3B. Figure is representative ofthree separate replicates. Racemic clAST at 10-5 M in 33% EtOH
demonstrates greatest induction ofCx43 protein levels (> 3-fold), similar to that obtained with racemic
dAST at 10-5 M in 50% EtOH vehicle (Fig. 3B). Figure is representative of three separate replicates.
Immunoblot stained with Coomassie blue and confirmed by densitometry for equal protein loading and
transfer.
Separate treatments of the 10TI12 cells were also performed with the goal of
comparing the relative biological efficacy of the racemic pAST in a 20% EtOH
formulation to enhance connexin 43 expression as compared to the parent carotenoid
astaxanthin, the related xanthophyll canthaxanthin and the retinoid control. Cells were
treated with racemic pAST in 20% EtOH (0.2% final EtOH cone.), AST in THF (0.1 %
final THF cone.), and CTX in THF (0.1 % final THF cone.) at 10-5, 10-6
, and 10-7 M.
TTNPB in acetone at 10.8 M was used as a positive control and media without compound
was used as a negative control. Racemic pAST caused induction of Cx43 in comparison
with non-treated controls in a dose-dependent manner at concentrations of 10-6 and 10-7
M, although Cx43 induction was not observed at 10-5 M (Fig. 5). Cx43 induction by the
astaxanthin derivatives (::::3.5-fold or less) was several-fold lower than induction levels
observed with the retinoid (up to 12-fold induction). CTX exhibited potent induction of
Cx43 in comparison with non-treated controls in a dose-dependent manner, with strong
18
induction observod at 10-5 M (::::7-fold). NeitberpAST nor AST at 10-5 M demonstrated
sipificant Cx43 induction. This finding was corroborated by Lucifer dye transfer
studies, where treatment ofcells with pAST and AST at 10-5 M demonstrated a lack of
induced gap junctional communication (data not shown). CTX induced greater
expression 0 Cx43 at 10-6 M (:::4.5-fold as compared to pAS at 10-6 M (::::3.5-fold)
and comparable induction at 10.7 M (::::2-fold). AST exhibited very weak induction at 10
5 and 10-6 M (::::2-fold or less), and no induction at 10-7 M.
A1 2 3 .. 5 8 7 8 9 10 11
B..--- .. __ .. ~43kD
Fold Induction
14.00 -....--------------------------,
12.00
10.00
8.00
6.00
4.00
2.00
0.00
/ ....~ ....rJ> ....f:1~ ....f:1+, ....t? ....~ ~+, ....J:' ....f:1~ ~.v¢~v¢',,~-!"/q$'
19
Fig. 5. Western blot demonstrating induction ofCx43 protein expression in IOTl/2 cells treated for four
days by novel astaxanthin derivatives, versus the related carotenoids astaxanthin and canthaxanthin and
positive and negative controls. (A) Lane 1: TTNPB at 10'8 M in acetone (final acetone concentration in
assay 0.1 %) as positive control. Lane 2: CTX at 10-5 M in THF (fmal THF concentration in assay 0.1 %).
Lane 3: : CTX at 10-6 M in THF (final THF concentration in assay 0.1%). Lane 4: CTX at 10-7 M in THF
(fmal THF concentration in assay 0.1%). Lane 5: AST at 10-5 M in THF (final THF concentration in
assay 0.1%). Lane 6: AST at 10,6 M in THF (final THF concentration in assay 0.1 %). Lane 7: AST at
10-7 M in THF (final THF concentration in assay 0.1 %). Lane 8: Racemic pAST at 10-5 Min 20% EtOH
(final EtOH concentration in assay 0.2%). Lane 9: Racemic pAST at 10-6M in 20% EtOH (final EtOH
concentration in assay 0.2%). Lane 10: Racemic pAST at 1O-7 M in 20% EtOH (final EtOH concentration
in assay 0.2%). Lane 11: Non-treated sample as negative control. (8) Relative induction levels ofCx43
expression versus non-treated control. Figure is representative of two separate replicates. Racemic pAST
at 10-6 M in 20% EtOH demonstrates greater induction ofCx43 protein levels «;::::3.5-fold) over the parent
AST carotenoid at 10-6 M (;::::2-fold), although less than the related carotenoid CTX at 10-6 M (;::::4.5-fold).
Figure is representative of two separate replicates. Immunoblot stained with Coomassie blue and
confirmed by densitometry for equal protein loading and transfer.
3.3. Cellular Analysis of Cx43
Racemic dAST increased assembly of immunoreactive Cx43 into plaques in regions
of the cell membrane in direct contact with adjacent cells. Such localization is consistent
with formation of functional gap junctions. Cells treated with TTNPB at 10.8 M in
acetone (0.1% final acetone concentration in assay) also exhibited more extensive
immunoreactive plaques than untreated cells. In solvent-only treated cultures, such
immunoreactive plaques were infrequent, and were smaller than those detected in cells
treated with racemic dAST or with TTNPB as a positive control. The frequency of these
plaques and their size is consistent with the functional differences in gap junctional
20
penneability as detected by the Lucifer Yellow dye transfer experiments (described
below), and with the degree of induction of Cx43 as detected in the immunoblot
experiments described in Section 3.2 above. Representative photomicrographs are shown
in Fig. 6.
E F
Fig. 6. Racemic dAST increases assembly ofCx43 immunoreactive junctional plaques. Confluent
cultures of lOT1/2 cells were treated for 4 days as in Figs. 3-5, and immunostained with a Cx43 antibody.
Panels: (A) racemic dAST at 10.5 M in 33% EtOH; (B) 33% EtOH as solvent-only control; (C) TTNPB
at 10-8 M in acetone (final acetone concentration in assay 0.1 %). Panels D, E, F: digital analysis of
micrographs in Panels A, B, and C, respectively, demonstrating pixels above a fixed set threshold positive
for fluorescent intensity. Yellow arrows: immunoreactive junctional plaques; red arrows: position of cell
nuclei. Note the greater number and intensity ofjunctional immunoreactive plaques in the cultures treated
with racemic dAST in comparison with solvent-only treated controls. The junctional plaques shown in
21
Panels B and E represent infrequent plaques seen in controls; most cells in these cultures were negative for
Cx43 staining, in contrast to the extensive immunoreactivity in racemic dAST- and lTNPB-treated cells.
3.4. Induction of Gap Junctional Communication
To assess the functional capacity of these junctional plaques for direct intercellular
communication, Lucifer Yellow dye microinjection studies were performed to assess the
ability of racemic clAST to enhance dye transfer among IOTI/2 cells. As discussed
above, this ability has been previously highly correlated with the ability of carotenoids to
inhibit carcinogen-induced neoplastic transformation [15;16]. Racemic clAST in 33%
EtOH formulation at a concentration of I x 10-5 M effectively increased the extent of
junctional communication over that seen in solvent-only treated controls. Of 22
microinjected treated cells, IS (56%) were functionally coupled by gap junctions in
contrast to only 3 out of II (27%) solvent-only treated control cells. These differences
were statistically significant (P < 0.03; Fisher's Exact test). Representative
photomicrographs are shown in Fig. 7.
A
D
B
E
22
c
F
Fig. 7. Racemic dAST enhances functional GJIC in 1OTl/2 cells as shown by transfer of microinjection
dye. Confluent cultures were treated for 4 days as described in Fig. 6, then assayed for the ability to transfer
the fluorescent dye Lucifer Yellow. Arrows indicate the cell injected with Lucifer Yellow. Panels: (A)
racemic dAST at 10-5 M in 33% EtOH; (B) 33% EtOH solvent-only control; (C) TTNPB at 10-8 M in
acetone (final acetone concentration in assay 0.1%). Panels D, E, F: digital analysis ofmicrographs in
Panels A, B, and C, respectively, demonstrating pixels above a set threshold positive for Lucifer Yellow
fluorescence. Because cell nuclei have the most volume, they accumulate the most Lucifer Yellow and
exhibit the greatest fluorescence. Racemic dAST-treated cells demonstrate significantly increased
functional coupling (56%) over solvent-only treated controls (27%); P < 0.03, Fisher's Exact test.
3.5. Inhibition of MeA-induced Neoplastic Transformation
The Bertram lab has previously reported that carotenoids such as canthaxanthin
and ~-carotene act as potent inhibitors of transformation when added in the postinitiation
phase of carcinogenesis [36]. Here we examine the ability of the pAST derivative and its
parent carotenoid moiety astaxanthin to similarly inhibit carcinogen-induced neoplastic
transformation in a dose-dependent manner. As per Bertram lab protocols, compounds
were added in all cases 7 days after the removal of the carcinogen so as not to potentially
interfere with the production of carcinogen-initiated cells. Treatments were continued at
weekly intervals after re-feeding with fresh media 24 hours after MCA treatment.
Results are presented as the average number of foci per dish (Fig. 8). Cells treated with
pAST and AST at 10-5 M demonstrated highly significant inhibition of transformation (P
< 0.0001), however visualization of the cell monolayers for both treatments by
miscroscopy revealed regions of incomplete monolayer formation. This is most likely
due to cell toxicity at high concentrations, and such toxicity acts to inhibit transformation.
23
This may also account for their lack ofCx43 protein expression and induced GJIC at
these concentrations. This effect was not observed at the lower concentrations. At 10-6
M pAST demonstrated 100% inhibition of neoplastic transfonnation, and at 10-7 M pAST
exhibited significant inhibition of transformation (P > 0.04). At 10-8 M pAST did not
demonstrate significant inhibition. AST at concentrations of 10-6 M, 10-7 M, and 10-8 M
did not significantly demonstrate inhibition of transfonnation, which corroborates earlier
findings of a significant lack of Cx43 expression and induction of GJIC for all doses of
AST.
2.50
2.00
s::.fI) 1.50C:::CJ
1.000LL
0.50
0.00 -.---~-
Fig. 8. Dose-dependent effects of pAST and AST on MCA-induced neoplastic transfonnation. Results arepresented as average number of foci per dish. As a negative control, cells were treated with acetone andfollowed by media-alone treatments as per protocol. Positive controls were treated with MCA andfollowed by either media-alone or THF as a solvent control. Statistical significance was calculated bystudent's paired t-test.
24
CHAPTER 4. DISCUSSION
The accumulated body of evidence demonstrates that tumor cells exhibit a lack of
communication with surrounding normal cells. This is either a consequence of lack of
connexin gene expression, or a loss of functional gap junctional communication due to
faulty trafficking or assembly into the plasma membrane [37]. Unlike other tumor
suppressor genes, inactivating mutations of connexins are apparently rare and lack of
expression in tumors appears a consequence of increased DNA methylation of the
connexin promoter [38]. While fully established neoplastic cells can be expected to be
non-responsive to carotenoid or retinoid treatments, pre-neoplastic cells may retain some
responsiveness. Indeed, in the lOTl/2 cell system, isolated carcinogen-initiated cells
were shown to be less junctionally coupled than normal cells, yet able to respond to
retinoids with increased GJIC [39]. In contrast, once transformation has occurred,
retinoids--and one would expect carotenoids as well--are not able to induce junctional
communication between these and surrounding normal cells, nor suppress their ability to
proliferate and form transformed foci [11]. A similar situation may exist clinically. The
Bertram lab has shown that in cervical dysplasia and leukoplakia ofthe oral cavity, Cx43
expression is strongly downregulated in these pre-neoplastic lesions induced by HPV
infection and tobacco exposure respectively, in contrast to its strong expression in normal
epithelium [40;41]. In the cervix, retinoic acid, and in the oral cavity retinoic acid and f3-
carotene, have separately been shown to have chemopreventative activity. In this context
it is of interest that retinoic acid only suppresses oral tumors after a delay of six months
to one year. This suggests a lack of effect on initially latent, fully transformed cells and
25
instead an inhibition of progression of pre-neoplastic cells [42;43]. In the Bertram model
of gap junctional communication and proliferation control, the restoration of OJIC by
retinoids or carotenoids would lead to suppression of aberrant proliferation and inhibition
of the development of cells bearing multiple mutations leading to full carcinogenic
potential.
Unlikecarotenoids, the use of retinoids as chemopreventative agents is limited by
toxicity. ~-carotene, a pro-vitamin A carotenoid whose consumption in foods and
presence in serum strongly correlates in epidemiological studies with decreased lung
cancer risk in smokers, has been evaluated most thoroughly. In two interventional
studies of individuals at high risk of lung cancer as a consequence of tobacco and/or
asbestos exposure, supplemental ~-carotene at levels approximately 10-fold higher than
found in a "healthy" diet, actually increased lung cancer rates over placebo controls
[42;43]. However, in a third study oflow-risk physicians, few of whom were smokers, ~
carotene did not influence lung cancer rates [44]. In a fourth study, also in current and
former smokers, ~-carotene tended to decrease cancers of the oral cavity but increase
those ofthe lung [45]. This suggests a direct interaction between tobacco smoke and~
carotene. This was confirmed in animal studies as in which ~-carotene or its breakdown
products were shown to enhance smoke-induced pathological changes and to interfere
with retinoic acid signaling [32]. Such interactions appear unlikely with non-pro-vitamin
A carotenoids; a conclusion confirmed in a follow-up study. Here lycopene replaced~
carotene which resulted in protection against smoke-induced lung pathology without
associated toxicity [46].
Carotenoids may act as effective antioxidants at low oxygen tension and low
26
physiologic concentration, in comparison to other chain-breaking antioxidants such as
vitamin E. Certain carotenoids may be pro-oxidant under high oxygen tension and high
physiologic concentration environments (e.g. p-carotene) [47;48]. Therefore,
supplementation of p-carotene at high levels could add an additional oxidative stress
insult (particularly in the lung) to these individuals. In contrast, astaxanthin has been
described as a "class three" antioxidant; that is an excellent antioxidant that effectively
quenches excited molecular states as well as ground state radicals, without evidence of
pro-oxidant activity [48]. Ifpro-vitamin A activity, retinoid toxicity, and pro-oxidant
behavior are subsequently definitively implicated in the increased lung cancer rates in
supplemented human populations, then the astaxanthin derivatives described here may be
particularly well suited for clinical cancer chemoprevention. The influence on GJIC
demonstrated in the current study suggests that novel astaxanthin derivatives with
increased bioavailability may be especially useful chemopreventative agents.
Lycopene, a non-pro-vitamin A carotenoid found in tomatoes, may also have both
therapeutic and preventive properties. Epidemiological evidence suggests that tomato
consumption is protective against prostate cancer [49]. When administered as a
supplement to men scheduled for radical prostatectomy after diagnosis of prostate cancer,
a dose of 30 mg/day for approximately three weeks apparently decreased indices of
neoplasia in excised tumors and increased expression of connexin 43 [50]. When
lycopene was administered in conjunction with a-tocopherol to patients at high-risk of
liver cancer as a consequence of infection with hepatitis C, this combination reduced the
incidence of hepatocellular carcinoma after a delay of approximately one year [51].
Carotenoids have been shown to enhance antibody production, increase T-helper
27
cell numbers, T- and B-cell proliferation and even to increase the cytotoxicity ofnatural
killer cells [52]. Experimental evidence on tumor immunity suggests that ~-carotene's
suppressive effects on tumor growth may be attributed to enhanced production of tumor
specific antigens [53]. Astaxanthin has also demonstrated the ability to inhibit tumor
formation in the livers of mice through enhanced T cell-mediated immune response
[54;55]. While enhanced immune function may playa role during tumorigenesis in the
whole animal, cell culture studies demonstrating inhibition of cancer during the
promotion phase ofcarcinogenesis suggest that it is unlikely to be the central mechanism
ofaction. Rather, the current body ofevidence suggests that induction of Cx43
expression and increase in GJIC is the major mechanism by which initiated cells prevent
transformation. In the current study, astaxanthin did not significantly induce Cx43
expression, increase GJIC, or prevent carcinogen-induced neoplastic transformation, in
contrast to the novel compounds derived from astaxanthin. This apparent discrepancy
may be attributed to differences in uptake or cleavage of the derivatives as compared to
the parent astaxanthin moiety. Further studies need to be performed to elucidate the
mechanism behind this discrepancy. It is noteworthy that the diphosphate derivative was
demonstrated to have enhanced biological efficacy over the parent carotenoid despite the
precipitation of the compound in solution 24 hours post treatment.
The availability of a water-soluble, highly bioavailable derivative of astaxanthin
should facilitate the evaluation of its cancer chemopreventative properties in
experimental animals, and if successful, will simplify its evaluation as a
chemopreventative agent in the clinic. The demonstration that novel astaxanthin
derivatives are able to upregulate the expression ofCx43, increase the size and number of
28
Cx43 immunoreactive gap junctional plaques, functionally increase GIIC and inhibit
carcinogen-induced neoplastic transformation with enhanced activity over the parent
carotenoid suggests that these derivatives will have potent chemopreventive properties in
vivo. In view of this potential, further cell culture and animal studies utilizing these
derivatives are warranted.
29
APPENDIX
Hix, L.M., Lockwood, S.P., and Bertram, J.S. (2004) Upregulation of Connexin 43Protein Expression and Increased Gap Junctional Communication by Water SolubleDisodium Disuccinate Astaxanthin Derivatives. Cancer Letters, in press.
30
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