WO2008008333A2

WO2008008333A2 - Bioassays for quality control of nutriceuticals that inhibit, upregulate or otherwise modulate translation initiation

Info

Publication number: WO2008008333A2
Application number: PCT/US2007/015721
Authority: WO
Inventors: José A. HALPERIN; Huseyin Aktas
Original assignee: President And Fellows Of Harvard College
Priority date: 2006-07-11
Filing date: 2007-07-09
Publication date: 2008-01-17
Also published as: CL2007002011A1; WO2008008333A3

Abstract

The present invention relates to bioassays for detecting the ability of one sample of a food substance, nutritional supplement, therapeutic agent or disease preventive agent relative to that of a second sample of such a substance, supplement or agent to inhibit, upregulate or otherwise modulate translation initiation, and thereby demonstrate a disease curative or preventive effect in a human or animal that consumes a such substance, supplement or agent or to whom a such substance, supplement or agent is administered.

Description

BIOASSAYS FOR QUALITY CONTROL OF NUTRICEUTICALS THAT INHIBIT, UPREGULATE OR OTHERWISE MODULATE TRANSLATION INITIATION

FIELD OF THE INVENTION

[01] The present invention relates to bioassays for detecting the ability of one sample of a food substance, nutritional supplement, therapeutic agent or disease preventive agent relative to that of a second sample of such a substance, supplement or agent to inhibit, upregulate or otherwise modulate translation initiation, and thereby demonstrate a disease curative or preventive effect in a human or animal that consumes a such substance, supplement or agent or to whom a such substance, supplement or agent is administered.

BACKGROUND OF THE INVENTION

[02] Translation, the mRNA-directed synthesis of proteins, occurs in three distinct steps: initiation, elongation and termination. Translation initiation is a complex process in which the two ribosomal subunits and methionyl tRNA (mtRNA) assemble on a properly aligned mRNA to commence chain elongation at the AUG initiation codon. The established scanning mechanism for initiation involves the formation of a ternary complex among eukaryotic initiation factor 2 (eIF2), GTP and met-tRNA. The ternary complex recruits the 4OS ribosomal subunit to form the 43 S pre-initiation complex. This complex recruits mRNA in cooperation with other initiation factors such as eIF4E, which recognizes the 7-methyl-guanidine cap (m-⁷GTP cap) in an mRNA molecule and forms the 48S pre-initiation complex. Cap recognition facilitates the 43 S complex entry at the 5' end of a capped mRNA. Subsequently, this complex migrates linearly until it reaches the first AUG codon, where a 60S ribosomal subunit joins the complex, and the first peptide bond is formed (Pain (1996) Eur. J. Biochem. 236:747-771).

[03] Translation initiation plays a critical role in the regulation of cell growth and malignant transformation because expression of most oncogenic and cell growth regulatory proteins is translationally regulated (Flynn et al. (1996) Cancer Surv. 27:293; Sonenberg et al. (1998) Curr. Opin. Cell Biol. 10:268). For this reason, translation initiation is a tightly regulated cellular process. Many examples demonstrate that disregulation of translation initiation contributes to the genesis and progression of cancer (Donze et al. (1995) Embo J. 14: 3828; Rosenwald (1996) Bioessays 18: 243-50. (1996); De Benedetti et al. (2004) Oncogene 23: 3189-99 (2004); and Rosenwald (2004) Oncogene 23:3230). Conversely, inhibition of translation initiation reverts transformed phenotypes (Jiang et al. (2003) Cancer Cell Int. 3:2; Graff et al. (1995) Int. J. Cancer 60:255). The eIF2 GTPMet-tRNAi complex is a critical site in the regulation of translation initiation that is "targeted" by novel anti-cancer agents generally known as inhibitors of translation initiation.

[04] Following the recruitment of the 60S ribosomal subunit at the AUG initiator codon, the GTP bound to eIF2 in the ternary complex is hydrolyzed to GDP. This GDP must be exchanged for another molecule of GTP in order to regenerate the ternary complex and initiate a new round of translation. This GDP-GTP exchange is catalyzed by the guanine nucleotide exchange factor eIF2B, and is inhibited when eIF2α is phosphorylated. Phosphorylated elF2α has a much higher affinity for and inhibits the function of eIF2B because when bound to phosphorylated eIF2α, eIF2B cannot catalyze the GDP-GTP exchange (Pain (1996) Eur. J. Biochem. 236:747). In other words, phosphorylated eIF2α is an inhibitor of eIF2B. Because the stoichiometric ratio of eIF2B and eIF2α in the cytosol is quite low, i.e., molecules of eIF2α are far more abundant than molecules of eIF2B, even partial phosphorylation of eIF2α is sufficient to decrease the availability of eIF2B necessary to re-cycle the eIF2.GDP into the functional eIF2.GTP form. The net result of even partial phosphorylation of eIF2α is a reduction in the availability of the ternary complex necessary to initiate a new round of translation (Brostrom et el. (1989) J Biol. Chem. 264:1644; Srivastava et al. (1995) J Biol. Chem. 270: 16619).

[05] eIF2α is phosphorylated on its serine 51 residue by eIF2α protein kinases including PKR (protein kinase R) and PERK (PKR-like protein kinase). eIF2α kinases are activated by signals from a "stressed" endoplasmic reticulum triggering a cascade of events generally termed the endoplasmic reticulum (ER) stress response. Most proteins synthesized in the cytoplasm are translocated to the ER for folding and post- translational modifications. Increased protein synthesis that overwhelms the ER's capacity for folding or other disturbances that prevent protein folding or transport, induce ER stress (Harding et al. (2000) MoI. Cell 5:897; Kaufman (1999) Genes Dev. 13:1211). Another ER stressor is the partial depletion of intracellular Ca⁺* stores, probably because Ca⁺* stored in the ER contributes to protein folding, although the exact mechanism by which reduction of ER calcium triggers the ER stress response is not clearly understood. It is well established and supported by an extensive body of experimental evidence that partial depletion of Ca^+4" stores rapidly induces the ER stress response, and activates eTF2α kinases phosphorylating eIF2α and reducing the rate of translation initiation (Brostrom et al. (1989) J. Biol. Chem. 264:1644; Aktas et al. (1998) Proc. Natl. Acad. ScL U.S.A. 95:8280).

[06] Phosphorylation of eIF2α limits the availability of the ternary complex and preferentially affects the translation of mRNAs coding for oncogenic proteins such as the Gl cyclins or c-myc but not or much less the translation of housekeeping proteins such as β-actin or ubiquitin (Clemens et al. (1999) Int. J. Biochem. Cell Biol. 31 :1). Paradoxically, a subset of mRNAs is translated more efficiently when the ternary complex is scarce than when it is abundant (Aktas et al. (2004) Journal of Nutrition). These include the mRNA encoding for the transcription factor ATF-4, which transcriptionally up-regulates many of the ER stress response genes such as pro- apoptotic C/EBP-homologous protein (CHOP) or the ER chaperone binding protein (BiP) (Harding et al. (2000) MoI. Cell 6:1099). An isoform of the BRCAl mRNA belongs to this subset of mRNAs that are more efficiently translated when the ternary complex is scarce. The n-3 polyunsaturated fatty acid eicosapentaenoic acid (EPA) up-regulates CHOP and BiP in cancer cells and in tumors excised from either animal cancer models or human patients. EPA also increases the translation of BRCAl mRNAb in breast cancer cell lines.

SUMMARY OF THE INVENTION

[07] Regulation of translation initiation plays a critical role in the control of cell proliferation, apoptosis and cancer because expression of growth regulatory, pro- apoptotic, oncogenic, and tumor suppressor proteins is tightly regulated at the level of translation and is highly dependent on the activity of translation initiation factors. In many human cancers, some degree of disregulation of translation initiation favors the expression of oncogenic over "housekeeping" proteins and contributes to the initiation and maintenance of the malignant phenotypes. Without intending to be bound by theory, inhibitors of translation initiation such as eicosapentaenoic acid (EPA), the primary n-3 polyunsaturated fatty acid (PUFA) of marine fish oils, restore translational control to reduce the expression of oncogenic proteins, and favor the expression of pro-apoptotic proteins and tumor-suppressor proteins, thereby suppressing malignant phenotypes. Accordingly, dietary supplements such as marine fish oils that contain translation initiation inhibitors, represent attractive commercial products for treatment and/or prevention of cancer and/or proliferative diseases in which abnormal cell proliferation is a characteristic pathological abnormality. Such dietary supplements can also act as translation initiation upregulators, and represent attractive commercial products for treatment and/or prevention of metabolic diseases such as obesity and diabetes.

[08] Dietary supplements with therapeutic/preventive effects for human diseases represent a fast growing, multibillion dollar industry worldwide. However, a major unresolved problem in this industry is the lack of quality control of products that are extracted from natural sources to assure a specific biological activity and potency, a homogeneity in biological activity among different preparations extracted/produced from the same plant/animal source, and a comparable potency among the preparations extracted from the same plant or animal species but originating from different geographical regions and/or industry sources.

[09] Inhibitors, upregulators or other modulators of translation initiation have broad- spectrum anti-cancer, anti-cell proliferation effects as well as broad-spectrum effects on energy balance. Nutriceuticals containing inhibitors, upregulators or other modulators of translation initiation, including but not limited to fish oil preparations, could be used for prevention of human diseases characterized by abnormal cell proliferation, including cancer. However, the current absence of bioassays to determine the biological activity of nutriceuticals of this kind makes it impossible to control their quality, potency and/or homogeneity among different brands or among batches of the same brand. [10] For example, marine fish oils are rich in and constitute and excellent source of n-3 PUFAs such as EPA. Without intending to be bound by theory, the same fish produced in aquaculture farms contain low levels of n-3 PUFAs because they are fed with vegetables rich in n-6 PUFAs. For this reason, fish oils from aquaculture farm fish are low in n-3 PUFAs.

[11] Accordingly, the present invention provides translation initiation-specific bioassays that can be used to quantitatively assess the biological activity of compounds, e.g., nutriceuticals, that contain inhibitors, upregulators or modulators of translation initiation. The translation initiation-specific assays provided herein assess the quality (e.g., biological activity), potency and batch homogeneity of nutriceuticals that contain products, e.g., endogenous products, that act as inhibitors, upregulators or other modulators of translation initiation.

[12] In one embodiment, the present invention provides a method for performing quality control of a composition having a translation initiation inhibition, upregulation or other modulation activity including the steps of detecting the translation initiation inhibition, upregulation or other modulation activity of the composition, and comparing the translation initiation inhibition, upregulation or other modulation activity of the composition to a standard.

[13] In another embodiment, the present invention provides a method of determining potency of a composition as a translation initiation inhibitor, upregulator or modulator including the steps of detecting the translation initiation inhibition, upregulation or other modulation activity of the composition, and comparing the translation initiation inhibition, upregulation or other modulation activity of the composition to a standard to determine the potency of the composition as a translation initiation inhibitor.

[14] In another embodiment, the present invention provides a method of determining batch homogeneity of a plurality of individual compositions comprising the steps of detecting translation initiation inhibition, upregulation or other modulation activity of at least one of the individual compositions, and comparing the translation initiation inhibition, upregulation or other modulation activity of the at least one of the individual compositions to a standard to determine batch homogeneity. [15] In certain aspects of the invention, translation initiation inhibition, upregulation or other modulation activity is between about 50% and about 150% of the activity of the standard, between about 80% and about 120% of the activity of the standard, between about 90% and about 110% of the activity of the standard, or between about 95% and about 105% of the activity of the standard.

[16] In certain aspects of the invention, the translation initiation inhibition, upregulation or other modulation activity is detected using a bioassay such as an ATF-4 ternary complex formation assay, an S51-eIF2α phosphorylation assay, a CHOP expression assay, a BiP expression assay, or a BRCAl expression assay.

[17] In still other aspects of the invention, the standard comprises an omega-3 fatty acid, such as eicosapentaenoic acid. The standard may be derived from marine fish oil or flax seed oil.

[18] In still another embodiment the present invention provides a method of producing a standard of translation initiation inhibition, upregulation or other modulation comprising obtaining a composition having a translation initiation inhibitor, upregulator or modulator activity, and quantitating the level of translation initiation inhibitor, upregulator or modulator activity using a bioassay such as an ATF-4 ternary complex formation assay, an S51-eIF2α phosphorylation assay, a CHOP expression assay, a BiP expression assay or a BRCAl expression assay.

BRIEF DESCRIPTION OF THE DRAWINGS

[19] The foregoing and other features and advantages of the present invention will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings in which:

[20] Figures JA-JD illustrate eIF2α phosphorylation in primary tumors from prostate biopsies and prostatectomy samples from prostate cancer patients. Representative photographs of a prostate biopsy (left, A and C) and a radical prostatectomy specimen (right, B and D) from two prostate cancer patients treated with 15 grams of corn oil (top, A and B) or 15 grams of fish oil (bottom, C and D) for five weeks between the diagnostic prostate biopsy and surgical prostatectomy are shown. [21] Figures 2A-2C depict an ATF-4 ternary complex assay. Figure 2A shows a schematic of a dual luciferase vector. Figure 2B graphically depicts an increase in the firefly luciferase (F) / renilla luciferase (R) ratio in stable KLN-luciferase (KLN-luc) cells in the presence of increasing EPA (all concentrations are in micromoles). Figure 2C graphically depicts the F/R ratio in KLN-luc tumor-bearing mice treated orally with fish oil (shaded bar) or corn oil (unshaded bar) for two days. Dual luciferase activity was measured in tumor lysates.

[22] Figure 3 depicts phosphorylation of eIF2α, suppression of cyclin Dl, and an increase in BiP expression in KLN tumors in the presence of fish oil. Mice bearing KLN tumors were treated with EPA (2.5 g / kg / day) for seven days. Tumors were excised and stained with specific antibodies. A, B, C depict treatment with corn oil; D₃ E, F depict treatment with fish oil. eIF2α phosphorylation is shown in A and D; cyclin Dl expression is shown in B and E; BiP expression is shown in C and F.

[23] Figure 4 graphically depicts a CLT-induced increase of firefly luciferase expression when its mRNA contained the 5' UTR of BRCAl mRNAb (gray) but not the 5' UTR of BRCAl mRNAa (black).

DETAILED DESCRIPTION OF THE INVENTION

[24] Embodiments of the present invention are based on the discovery of bioassays that may be used to quantitate the ability of a compound, e.g., a nutriceutical, to inhibit, upregulate or otherwise modulate translation initiation. Bioassays of the present invention include, but are not limited to: ATF-4 ternary complex formation assays; S51-eIF2α phosphorylation assays; CHOP expression assays; BiP expression assays; and BRCAl expression assays. Each of these bioassays is discussed further herein.

[25] The term "nutriceutical," as used herein, is a combination of "nutritional" and "pharmaceutical," and refers to an ingestible substance that has one or more beneficial effects on an organism such as a human. The term nutriceutical can also refer to one or more compounds which are present in an ingestible substance. Ingestible substances include, but not limited to dietary supplements, foods, beverages and the like. [26] Nutriceuticals of the present invention include oils derived from fish such as cold water fish, warm water fish, fresh water fish, salt water fish, brackish water fish, wild fish, farm-raised fish and the like, and preparations of fatty acids such as those containing omega-3 fatty acids.

[27] The term "omega-3 fatty acid," as used herein, refers to polyunsaturated fatty acids such as those found in oil from oily fish such as mackerel, salmon, sardines and the like, or vegetable sources such as the seeds of chia, perilla, flax, walnut, purslane, ligonberry, seabuckthorn, hemp, and the like, and fruits from plants such as the acai palm. Omega-3 fatty acids include, but are not limited to, α-linoleic acid (ALA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and the like.

[28J Certain aspects of the invention are directed to methods of determining the potency of a composition to inhibit, upregulate or modulate translation initiation. The term "potency," as used herein, is intended to include, but is not limited to, the effectiveness of a compound, e.g., a nutriceutical, to inhibit, upregulate or otherwise modulate translation initiation. The potency of a composition can be defined as the ability of the composition to inhibit, upregulate or otherwise modulate translation initiation relative to a standard.

[29] The present invention provides assays in which the translation inhibition, upregulation or modulation activity of a composition is compared to a standard using one or more of the bioassays described herein. A composition may have an activity level that is 0.001%, 0.01%, 0.1%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1 10%, 1 15%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 195%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950%, 1000% or greater than the activity of the standard.

[30] In certain aspects, a composition may have an activity level that is between about 1% and 200%, between about 5% and 195%, between about 10% and 190%, between about 20% and 180%, between about 30% and 170%, between about 40% and 160%, between about 50% and 150%, between about 60% and 140%, between about 65% and 135%, between about 70% and 130%, between about 75% and 125%, between about 80% and 120%, between about 85% and 115%, between about 90% and 110%, between about 91% and 109%, between about 92% and 108%, between about 93% and 107%, between about 94% and 106%, between about 95% and 105%, between about 96% and 104%, between about 97% and 103%, between about 98% and 102%, or between about 99% and 101%, of the activity of the standard.

[31] A nutriceutical or composition including a nutriceutical of the present invention may be diluted or concentrated to decrease or increase its translation inhibition, upregulation or modulation activity relative to the Standard, respectively.

[32] The present invention also provides assays in which batch homogeneity of compositions is determined by comparing the relative activity of two or more (e.g., 10, 100, 1000, 10,000 1,000,000 or more) compositions using one or more of the bioassays described herein. As used herein, the term "batch homogeneity" is intended to refer, but is not limited to, the relative translation initiation inhibition upregulation or modulation activity of two or more compositions in a batch. As used herein, the term "batch" refers, but is not limited to, a group of two or more compositions. A batch includes compositions prepared together or compositions from two or more sources (e.g., geographical, plant, animal, commercial, and/or synthetic sources).

[33] A standard of the present invention is a compound or composition having a translation initiation inhibition, upregulation or modulation activity as determined by one or more of the bioassays described herein. Standards may be obtained from a variety of sources such as the sources of omega-3 fatty acids described herein. Standards may be synthesized in the laboratory or obtained from commercial sources. A standard may be diluted or concentrated to decrease or increase its translation inhibition, upregulation or modulation activity, respectively.

[34] In at least certain examples, the nutriceutical s disclosed herein can be used in the treatment of disorders associated with aberrant cellular proliferation such as cellular proliferative disorders, (e.g., cancer). Treatment of cellular proliferative disorders is intended to include inhibition of proliferation including rapid proliferation. As used herein, the term "cellular proliferative disorder" includes disorders characterized by undesirable or inappropriate proliferation of one or more subset(s) of cells in a multicellular organism. The term "cancer" refers to various types of malignant neoplasms, most of which can invade surrounding tissues, and may metastasize to different sites (see, for example, PDR Medical Dictionary 1st edition (1995)). The terms "neoplasm" and "tumor" refer to an abnormal tissue that grows by cellular proliferation more rapidly than normal and continues to grow after the stimuli that initiated proliferation is removed (see, for example, PDR Medical Dictionary 1st edition (1995)). Such abnormal tissue shows partial or complete lack of structural organization and functional coordination with the normal tissue which may be either benign (i.e., benign tumor) or malignant (i.e., malignant tumor).

|35] The language "treatment of cellular proliferative disorders" is intended to include the prevention of the growth of neoplasms in a subject or a reduction in the growth of preexisting neoplasms in a subject. The inhibition also can be the inhibition of the metastasis of a neoplasm from one site to another. Examples of the types of neoplasms intended to be encompassed by the present invention include but are not limited to those neoplasms associated with cancers of the breast, skin, bone, prostate, ovaries, uterus, cervix, liver, lung, brain, larynx, gallbladder, pancreas, rectum, parathyroid, thyroid, adrenal gland, immune system, neural tissue, head and neck, colon, stomach, bronchi, and/or kidneys.

[36] Cellular proliferative disorders can. further include disorders associated with hyperproliferation of vascular smooth muscle cells such as proliferative cardiovascular disorders, e.g., atherosclerosis and restinosis. Cellular proliferation disorders can also include disorders such as proliferative skin disorders, e.g., X-linked ichthyosis, psoriasis, atopic dermatitis, allergic contact dermatitis, epidermolytic hyperkeratosis, and seborrheic dermatitis. Cellular proliferative disorders can further include disorders such as autosomal dominant polycystic kidney disease (ADPKD), mastocystosis, and cellular proliferation disorders caused by infectious agents such as viruses.

[37] In at least certain examples, the nutriceuticals disclosed herein can be used in the treatment of disorders associated with energy balance such as metabolic disorders including, but not limited to, diabetes, obesity, glycogen storage diseases, lipid storage disorders, mitochondrial diseases and the like (See also the worldwide website: emedicine.com/ped/GENETICS_AND_METABOLIC_DISEASE.htm, incorporated herein by reference in its entirety for all purposes). In certain aspects, the nutriceuticals disclosed herein modulate weight gain by interacting with the 5' UTR of the leptin receptor.

[38] This invention is further illustrated by the following examples, which should not be construed as limiting. The contents of all references, patents and published patent applications cited throughout this application are hereby incorporated by reference.

EXAMPLE I Human Cancer

An ti -cancer properties of EPA are mediated by eIF2α phosphorylation and inhibition of translation initiation

[39] It was discovered that the n-3 polyunsaturated fatty acid (n-3 PUFA) EPA, exerts anticancer activity in cancer cell lines and animal models of experimental cancer. By staining with phospho-specific anti S51P-eIF2α human cancer cell lines, tumors excised from mice treated EPA or marine fish oils, and prostate cancer samples from patients fed fish oil for 5 weeks prior to surgery, it was demonstrated that phosphorylation of eIF2α was induced in vitro and in vivo (Figures 1, 3 and 4).

The Concept of Tarfiet Accreditation

[40] "Target accreditation," as used herein, is the demonstration that mechanism-specific anti-cancer agents affect in vivo the same target they affect in vitro. Target accreditation is considered critical in modern cancer therapy. The most powerful accreditation strategy is to mutate a putative target in a manner that ablates its response to the test compound, rendering cancers with this mutation resistant to treatment. This can only be achieved in animal models and is part of modern development strategies for target-specific anti-cancer drugs.

[41] Another powerful tool for target accreditation is the identification of biomarkers as reporters of target-specific activity of the compound in vivo, ideally in humans. However, very few if any anti-cancer drugs currently in use in the treatment of human cancers met the accreditation criterion in humans, i.e., the demonstration that a given "target-specific drug" does indeed interact with its putative target in the human cancers against which the drug is used.

Target Accreditation of EPA in Human Cancer

[42] Using phospho-S51-specifc anti-eIF2α antibodies, it was determined that oral administration of a dietary marine fish oil supplement rich in EPA for five weeks prior to surgery induced phosphorylation of eIF2α in tumors from prostate cancer patients (Figures IA- ID).

[43] Although target accreditation is critical for the successful development of a target- specific anti-cancer drug, very few anti-cancer agents can be "accredited" in humans. Taking advantage of the fact that EPA is a food supplement, an Institutional Review Board (IRB) approved preliminary study was conducted in patients diagnosed with prostate cancer by prostate biopsy who later underwent radical prostatectomy at Brigham and Women's Hospital. Two patients received 15 grams distilled purified fish oil (OMEGARX™, 75% EPA, 25% DHA, referred to hereafter as EPA) and two received 15 grams (equicaloric) of the n-6 polyunsaturated fatty acid (PUFA) corn oil, daily for five weeks prior to surgery.

[44] Levels of S51-P-eIF2α and total eIF2α were compared between the biopsy and the prostatectomy specimens of the subjects. In the prostatectomy specimens of the patients that received EPA prior to radical prostatectomy, there was a remarkable increase in phosphorylation of eIF2α as compared with the biopsy sample, although there was no change in the levels of total eIF2α that was not observed in the corn oil group. Both patients had similar results. Figures IA- IB and 1C- ID each depict representative results from a single patient.

[45] The specificity of the staining for phosphorylated eIF2α was determined by Western blot analysis of cells treated with or without thapsogargin (Tg). Tg is a specific inhibitor the calcium ATPase of the ER (SERCA- ATPase) and induces Ca++ release and phosphorylation of eIF2α. It is a well accepted "positive" control for detection of eIF2α phosphorylation. EXAMPLE II The ATF-4 Ternary Complex Assay

[46] To demonstrate that EPA inhibited translation initiation by reducing the availability of the ternary complex, a cell-based reporter assay was developed in which the expression of the reporter depended on the availability of the ternary complex.

Background

[47] Very recently it has been recognized that the availability of the ternary complex not only determines the overall rate of translation initiation but also differentially affects the translation of specific mRNAs (Harding, 2000 #4141 ; Dever, 2002 #8359). When the ternary complex is scarce, mRNA translation is generally down-regulated. Paradoxically, translation of some mRNAs such as ATF-4 mRNA is significantly more efficient under conditions that limit the availability of the ternary complex because their 5¹ UTRs contain multiple uORF (Vattem, 2004 #9162; Harding, 2000 #4141). This novel finding was taken advantage of to develop a ternary complex specific assay.

Assay Development

[48] A bi-directional eukaryotic expression vector was constructed that contained on one side the renilla luciferase (R) and on the other side the firefly luciferase (F) ORFs, both flanked by a 90 nucleotide 5' UTR and the human β-globin polyadenylation signal. Recombinant rabbit monoclonal anti-P-S51 -eIF2α recognized P-S51-eIF2α as a single band in cell extracts from human prostate cancer cells treated with Tg, a specific inhibitor of SERCA ATPase that releases ER-Ca²⁺ and induces phosphorylation of eIF2α. Expression of the respective reporters was detected by a dual luciferase assay and the results were expressed as the F/R ratio. A dual reporter assay was developed to internally control for potential effects of compounds on transcription, mRNA and protein stability, translation elongation and termination, which are similar for both reporters.

[49] To render expression of firefly luciferase dependent on availability of the ternary complex, the 267 bp 5' UTR of mouse ATF-4 was inserted in front of its ORF, this plasmid was transfected into KLN cancer cells, and stable cell lines (KLN-luc) were established. Using these cell lines it was confirmed that the 5' UTR of ATF-4 rendered expression of the reporter gene inversely dependent on the availability of the ternary complex. Consistently, treatment with EPA resulted in a dramatic dose- dependent increase of the F/R ratio due to an increased expression of firefly luciferase and decreased expression of renilla luciferase (Figure 2B). This novel in vitro assay can be utilized for quality control of dietary supplements containing compounds that deplete the ternary complex.

The ternary complex assay in vivo

[50] To determine whether EPA similarly depleted the ternary complex in vivo, KLN-luc cells were injected intradermally into mice to establish tumors. The tumor-bearing animals were then treated with orally EPA with orally for two days, the tumors were excised and homogenized, and the luciferase activity was measured with the dual luciferase assay. The results indicated that treatment of mice with EPA increased the F/R ratio (Figure 2C), an indication that 1) EPA depleted the ternary complex in vivo as it does in vitro, and 2) the assay could be utilized for quality control of dietary supplements containing compounds that deplete the ternary complex in vivo.

EXAMPLE III Phosphorylation of eIF2α

[51] EPA and other inhibitors of translation initiation induce phosphorylation of eIF2α in vivo and in vitro. Phosphorylation of eIF2α detected with anti-P-51 S-specific antibodies is another assay that will be used for the quality of dietary supplements/nutriceuticals. Phosphorylation of eIF2α was detected in human breast cancer MCF-7 cells treated with 25, 50 or 100 μM EPA dissolved in DMSO. Accordingly, phosphorylation of eIF2α will be tested in cultured cells treated with an appropriate preparation of given nutriceutical.

[52] Phosphorylation of eIF2α was determined by Western blot analysis of xenograft KLN cell carcinomas excised from mice fed diets rich in either com or fish oil for five days prior to excision using anti-P-S51-eIF2α-specific antibodies. Phosphorylated eIF2α was detected in the same tumors immunostained with anti-P-51 S-specific antibodies. Accordingly, phosphorylation of eIF2α will also be tested in xenograft tumors excised from mice treated with a diet supplement containing compounds that induce phosphorylation of eIF2α.

EXAMPLE IV Up-Regulation of CHOP and BiP Expression

[53] EPA was determined experimentally to increase the expression of BiP and CHOP in different human cancer cell lines and in xenograft tumors excised from mice fed a fish oil diet (Figure 3). 25 μM EPA up-regulated BiP and CHOP expression in breast cancer MCF-7 cells and in prostate cancer DU 145 cells. B-actin was used as a loading control.

EXAMPLE V Expression of BRCAl

[54] The tumor suppressor BRCAl is a major breast and ovarian cancer susceptibility gene. BRCAl germ line mutations contribute to only 3% of all breast cancers in Caucasians, and somatic mutations are very rare in sporadic breast cancer (Khoo, 1999 #8704) and ovarian cancer (Merajver, 1995 #8709).

[55] The 5¹ UTR region of the BRCAl gene contains two alternative first exons, Ia and Ib, resulting in two BRCAl transcripts with different 5' UTR but the same open reading frame for BRCAl protein. It was discovered that the structure of the 5' UTR of BRCAl mRNAb contained three upstream open reading frames (uORFs) in a manner that was remarkably similar to the 5' UTR of ATF-4. Based on this finding it was postulated that the 5' UTR of mRNAb may reduce its translational efficacy when the ternary complex is abundant but enhance it when the complex is restricted.

[56] To test this hypothesis, MCF-7 breast cancer cells, known to have very low levels of BRCAl protein, were exposed to EPA. EPA was determined by Western blot to induce a significant up-regulation of BRCAl in a dose-dependent manner (75 or 150 μM EPA) in the human breast cancer cell lines MCF7 and HME. BRCAl -specific antibodies were used to detect BRCAl protein, and laminin was used as a loading control. Dose-dependent up-regulation of BRCAl was observed in MCF-7 breast cancer cells was observed for EPA (25 or 50 μM EPA) and CLT (5 or 10 μM CLT). CLT also increased the expression of firefly luciferase when its mRNA contained the 5' UTR of BRCAl mRNAb, but not of mRNAa (Figure 4).

[57] Mice bearing MCF-7 human breast cancer cell tumors were fed diets prepared with either corn or fish oil as the source of fatty acids (MCF-7 cells were xenografted into the mammary fat pad of female nude mice, and allowed to grow for two weeks. The mice were then fed identical diets in which either corn or fish oil was the source of fatty acids.). The tumors were then processed for expression of BRCAl . The results indicated that the fish oil diet induced a significant increase in the expression level of BRCAl in the tumors, while the corn oil diet had no effect.

Claims

What is claimed:

1. A method of performing quality control for a composition having a translation initiation inhibition activity comprising the steps of: detecting the translation initiation inhibition activity of the composition; and comparing the translation initiation inhibition activity of the composition to a standard, wherein the translation initiation inhibition activity of the composition is between about 50% and about 150% of the activity of the standard.

2. The method of claim 1, wherein the translation initiation inhibition activity is between about 80% and about 120% of the activity of the standard.

3. The method of claim 1, wherein the translation initiation inhibition activity is between about 90% and about 1 10% of the activity of the standard.

4. The method of claim 1, wherein the translation initiation inhibition activity is between about 95% and about 105% of the activity of the standard.

5. The method of claim 1 , wherein the translation initiation inhibition activity is detected using a bioassay selected from the group consisting of: ATF-4 ternary complex formation assay, S51-eIF2α phosphorylation assay, CHOP expression assay, BiP expression assay, and BRCAl expression assay.

6. The method of claim 1, wherein the standard comprises an omega-3 fatty acid.

7. The method of claim 6, wherein the omega-3 fatty acid is eicosapentaenoic acid.

8. The method of claim 1, wherein the standard is derived from marine fish oil or flax seed oil.

9. A method of determining batch homogeneity of a plurality of individual compositions comprising the steps of: detecting translation initiation inhibition activity at least one of the individual compositions; and comparing the translation initiation inhibition activity of the at least one of the individual compositions to an activity of a standard to determine a translation initiation inhibition activity, wherein the translation initiation inhibition activity is between about 50% and about 150% of the activity of the standard.

10. The method of claim 9, wherein the relative translation initiation inhibition activity is between about 80% and about 120%.

1 1. The method of claim 9, wherein the relative translation initiation inhibition activity is between about 90% and about 110%.

12. The method of claim 9, wherein the relative translation initiation inhibition activity is between about 95% and about 1 15%.

13. The method of claim 9, wherein the translation initiation inhibition activity is detected using a bioassay selected from the group consisting of: ATF-4 ternary complex formation assay, S51-eIF2α phosphorylation assay, CHOP expression assay, BiP expression assay, and BRCAl expression assay.

14. The method of claim 9, wherein the standard comprises an omega-3 fatty acid.

15. The method of claim 14, wherein the omega-3 fatty acid is eicosapentaenoic acid.

16. The method of claim 9, wherein the standard is derived from marine fish oil or flax seed oil.

17. A method of determining potency of a composition as a translation initiation inhibitor comprising the steps of: detecting the translation initiation inhibition activity of the composition; and comparing the translation initiation inhibition activity of the composition to a standard to determine the potency of the composition as a translation initiation inhibitor.

18. The method of claim 17, wherein the translation initiation inhibition activity of the composition is between about 50% and about 150% of the activity of the standard.

19. The method of claim 17, wherein the translation initiation inhibition activity is between about 80% and about 120% of the activity of the standard.

20. The method of claim 17, wherein the translation initiation inhibition activity is between about 90% and about 110% of the activity of the standard.

21. The method of claim 17, wherein the translation initiation inhibition activity is between about 95% and about 105% of the activity of the standard.

22. The method of claim 17, wherein the translation initiation inhibition activity is detected using a bioassay selected from the group consisting of: ATF-4 ternary complex formation assay, S51-eIF2α phosphorylation assay, CHOP expression assay, BiP expression assay, and BRCAl expression assay.

23. The method of claim 17, wherein the standard comprises an omega-3 fatty acid.

24. The method of claim 23, wherein the omega-3 fatty acid is eicosapentaenoic acid.

25. The method of claim 17, wherein the standard is derived from marine fish oil or flax seed oil.

26. A method of producing a standard of translation initiation inhibition comprising: obtaining a composition having a translation initiation inhibitor activity; and quantitating the level of translation initiation inhibitor activity using a bioassay selected from the group consisting of: ATF-4 ternary complex formation assay, S51-eIF2α phosphorylation assay, CHOP expression assay, BiP expression assay, and BRCAl expression assay.

27. The method of claim 26, wherein the standard comprises an omega-3 fatty acid.

28. The method of claim 27, wherein the omega-3 fatty acid is eicosapentaenoic acid.

29. The method of claim 26, wherein the standard is derived from marine fish oil or flax seed oil.