Cancer Chemoprevention By Water Soluble Astaxanthin Derivatives

Hix, Lauram.
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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 of cancer 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 of carcinogenesis 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 (GJIC) in direct relationship to their activity as chemopreventative agents [3]. GJIC permits the transfer of ions and small hydrophilic molecules <1 kilo Dalton (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 of junctional communication in cultured normal cells, it was soon discovered that neoplastically-transformed cells were deficient in GJIC. Moreover, inhibition of GJIC was a very early event after activation of the oncogene src [6]. Human and animal tumors of many pathologic types were found to be deficient in GJIC, either as a consequence of decreased connexin expression or faulty assembly into the plasma membrane. Restoration of junctional 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]-cannot be ignored. Indeed, there is evidence that conversion of canthaxanthin 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 of evidence 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. 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 = 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 GJIC, 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 GJIC and significantly inhibit MCA-induced neoplastic transformation with enhanced ability over the parent carotenoid astaxanthin. These results indicate that the major hurdle of delivering 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.
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Theses for the degree of Master of Science (University of Hawaii at Manoa). Biomedical Sciences (Cell and Molecular Biology) ; no. 3856
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