WO2005070403A1

WO2005070403A1 - Anti-estrogens and methods of use

Info

Publication number: WO2005070403A1
Application number: PCT/US2005/002111
Authority: WO
Inventors: Brian W. Loggie; Zafar Nawaz
Original assignee: Creighton University
Priority date: 2004-01-23
Filing date: 2005-01-24
Publication date: 2005-08-04

Abstract

The present invention is directed novel methods of using anti-estrogens such as tamoxifen and raloxifene, including, for instance, decreasing mucin production by a cell, and decreasing progression of, for instance, a disease wherein mucin production is prognositc of increased morbidity and/or mortality.

Description

ANTI-ESTROGENS AND METHODS OF USE

CONTINUING APPLICATION DATA

This application claims the benefit of U.S. Provisional Application Serial No. 60/538,740, filed January 23, 2004, which is incorporated by reference herein.

BACKGROUND

Mucins are high-molecular weight glycoproteins with a high content of clustered oligosaccharides O-linked to tandem repeat peptides rich in serine, threonine and proline. There are two structurally and functionally distinct classes of mucins: secreted gel-forming mucins (for instance, mucin-2, mucin-5AC, mucin-5B, and mucin-6) and transmembrane mucins (for instance, mucin- 1, mucin-3A, mucin-3B, mucin-4, mucin- 12, mucin- 17). Under normal physiological conditions, mucins are known to play a protective role for the adjoining epithelial tissues. Mucins may be involved in the renewal and differentiation of the epithelium, as well as in modulation of cell adhesion and in cell signaling. In general, mucins follow a defined spatial and temporal pattern of expression throughout the development of an organism. However, recent studies have demonstrated the association of a deregulated expression of mucins with various types of malignancies. In pancreatic adenocarcinoma, tumors and tumor cell lines can overex press mucin- 1 (Yonezawa et al., Pathol Int., 47:813-830 (1997), Satoh et al., Int J Cancer, 88:507-518 (2000)). Mucin- 1 is increased in expression in human colon cancers, which correlates with a worse progression (Nakomori et al., Gastroenterol., 106:353-361 (1994)), and has been linked to aggressive tumors in human breast carcinoma. Mucin-2 may play a role in the metastasis of mucinous colon cancer (Schwartz et al., Int. J. Cancer, 52:60-65 (1992), Sternberg et al., Gastroenterol., 1 16:363-371 (1999 ), Bresalier et al., J. Clin. Invest., 87:1037-1045 (1991)) although mucin-2 down versus up-regulation in non-mucinous colorectal cancer is still debatable (Chang et al., Gastroenterol., 107: 160- 172 (1994); Weiss et al, 1994, and Chu et al., Am. J. Clin. Pathol.,

121(6):884-92 (2004)). Mucins and mucin binding proteins in colorectal cancer have been recently reviewed by Byrd (Byrd et al., Cancer and Metastasis Reviews 23:77-99 (2004)). Pseudomyxoma peritonei is characterized by pathologic overproduction of mucins, including mucin- 2, which accumulates as an insoluble extracellular jelly (Bechtold et al., Abdom. Imaging, 26:406-410 (2001), Jackson et al., Mod. Pathol., 14:664-671 (2001), and O'Connell et al., Am. J. Pathol., 161: 551-564 (2002). The accumulation of mucin is a significant cause of the disease's morbidity and mortality.

SUMMARY OF THE INVENTION

The present invention represents an advance in treating diseases, such as diseases having deregulated expression of a mucin. The present invention provides methods for decreasing mucin production by a cell. The methods include contacting a mucin producing cell with an effective amount of an anti- estrogen, such as tamoxifen, raloxifene, or a combination thereof, wherein mucin production by the cell is decreased. In some aspects, the cell is a cancer cell, such as a lung cancer cell, a colon cancer cell, an appendix cancer cell, a pancreas cancer cell, or a neoplastic cell, for instance a neoplastic cell obtained from a subject with pseudomyxoma peritonei. In some aspects, the cell is not a breast cancer cell. The cell may be in vivo, for instance, present in a human. The present invention also provides methods for decreasing disease progression. The methods include administering to a subject an effective amount of an anti-estrogen, wherein the subject has or is at risk of having a disease wherein mucin production is prognositc of increased morbidity, mortality, or the combination thereof. The anti-estrogen can be, for instance, tamoxifen, raloxifene, or a combination thereof, and can be combined with other compounds useful to decrease disease progression. The disease can be, for instance, lung cancer, colon cancer, appendix cancer, pancreas cancer, or pseudomyxoma peritonei. In some aspects, the cell is not a breast cancer cell. Administration may result in a decrease in mucin production by a cell associated with the disease. Examples of mucin include mucin- 1, mucin-2, mucin-5AC, or the combination thereof. The present invention further provides methods for treating a symptom of a disease, such as cancer. The methods include administering to a subject having a disease an effective amount of an anti-estrogen, wherein a symptom of the disease is decreased or stabilized, and wherein the disease is a disease where mucin production is prognositc of increased morbidity, mortality, or the combination thereof. Examples of disease include, for instance, lung cancer, colon cancer, appendix cancer, pancreas cancer, or pseudomyxoma peritonei. In some aspects, the cell is not a breast cancer cell. The anti-estrogen can be, for instance, tamoxifen, raloxifene, or a combination thereof, and can be combined with other compounds useful to decrease disease progression. Administration may result in a decrease in mucin production by a cell associated with the disease. Examples of mucin include mucin- 1, mucin-2, mucin-5AC, or the combination thereof. The terms "comprises" and variations thereof do not have a limiting meaning where these terms appear in the description and claims. Unless otherwise specified, "a," "an," "the," and "at least one" are used interchangeably and mean one or more than one.

BRIEF DESCRIPTION OF THE FIGURES Figure 1. MUC2 protein expression after treatment with either estrogen or both estrogen and Tamoxifen in HT-29 cell line. Control, MUC2 protein expression in control cells; E2, MUC2 protein expression in cells that were treated with estrogen; E2+T, MUC2 protein expression in cells co-treated with estrogen and Tamoxifen DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION The present invention relates to methods for using anti-estrogens. As used herein, "anti-estrogen" refers to a compound that blocks the effect of estrogen on a cell. While not intending to be limiting, an anti-estrogen may block the effect of estrogen on a cell by, for instance, competitively blocking the binding of estrogen to its receptor, by blocking binding of receptor to DNA (for instance, to an estrogen response element), by blocking the release of negative regulators from DNA, by blocking the association of positive regulators to the receptor, or a combination thereof. Whether a compound is an anti-estrogen can be evaluated in terms of inhibition of estradiol-induced activity of target genes in target tissue or cells. Examples of target genes are genes having an estrogen response element present in the promoter, and examples of cells include estrogen-dependent cell lines (see, for instance, Nawaz et al., Cancer Res., 59:372-376 (1999)). Typically, a compound is considered an anti-estrogen if it inhibits estradiol-induced activity of target gene in target cells or tissues by at least 30%, preferably by at least about 45%, more preferably by at least about 75%, and most preferably by at least about 90%. An anti-estrogen may be nonsteroidal or steroidal. Examples of nonsteriodal anti-estrogens include, for example, tamoxifen, raloxifene, clomiphene, and derivatives thereof (see Day et al., J. Med. Chem., 34:842-851 Day (1991)). Examples of steroidal anti- estrogens include, for instance, ICI 164,384 (Wakeling et al. J. Steroid Biochem. 31 :645-653 (1988)), ICI 182,780 (Wakeling et al., Cancer Res. 51 :3867-3873 (1991), Wakeling et al., J. Steroid Biochem. Molec. Biol. 37:771-774 (1990)), and RU 58668 (Van de Velde et al., Ann. N.Y. Acad. Sci. 761: 164-175 (1995), Van de Velde et al., Pathol. Biol 42:30 (1994), Nique et al., Drugs Future 20:362-366 (1995)). In one aspect, the present invention is directed to methods for decreasing mucin production by a cell. The method includes contacting a mucin producing cell with an effective amount of an anti-estrogen. As used herein, an "effective amount" of an anti-estrogen as provided herein is a sufficient amount of the anti- estrogen to provide the desired effect. As discussed herein, the exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease being treated, and the particular anti-estrogenic agent and mode of administration, and the like. Thus, it is not possible to specify an exact "effective amount." However, an appropriate "effective" amount in any individual case may be determined by one of ordinary skill in the art using only routine experimentation. The cell may be ex vivo or in vivo. As used herein, the term "ex vivo" refers to a cell that has been removed, for instance, isolated, from the body of a subject. Ex vivo cells include, for instance, primary cells (e.g., cells that have recently been removed from a subject and are capable of limited growth or maintenance in tissue culture medium), and cultured cells (e.g., cells that are capable of extended growth or maintenance in tissue culture medium). As used herein, the term "in vivo" refers to a cell that is within the body of a subject. An in vivo cell may be a cell present in an organ. The cell is preferably a mammalian cell, such as, for instance, mouse, rat, livestock (e.g., pig, horse, cow, goat, sheep), or primate (e.g., monkey, human), preferably, human.

Preferably, mucin production by the cell is decreased by at least 30%, preferably by at least about 45%, more preferably by at least about 75%, and most preferably by at least about 90%. With ex vivo cells, the anti-estrogen is typically introduced by adding the anti-estrogen directly to the medium. When the cells are in vivo, the anti- estrogen can be introduced systemically, for instance, orally or by intravenous injection. Preferably, when the cells are in vivo the anti-estrogen is introduced systemically, preferably orally. The mucin can be one of the mucins expressed by epithelial cells. Examples of mucin include the polypeptides encoded by the currently identified mucin-encoding open reading frames. Such mucins are referred to in the art as mucin- 1, mucin-2, mucin-3, and so on. Preferred examples of mucins are mucin- 1, mucin-2, and mucin-5AC. Mucin- 1 refers to a polypeptide having a molecular weight of greater than approximately 250 kDa as determined by sodium dodecyl sulfate

SDS-polyacrylamide gel electrophoresis, and bound by an antibody that specifically binds to a human mucin- 1, such as the polypeptide disclosed at Genbank accession number AY463543. Such antibodies are commercially obtainable from, for instance, BD Biosciences (San Jose, CA), and Zymed Laboratories, (San Fransico, CA). Mucin-2 refers to a polypeptide having a molecular weight of greater than approximately 250 kDa as determined by sodium dodecyl sulfate SDS-polyacrylamide gel electrophoresis, and bound by an antibody that specifically binds to a human mucin-2, such as the polypeptide disclosed at Genbank accession number L21998. Such antibodies are commercially obtainable from, for instance, BD Biosciences (San Jose, CA), and Zymed Laboratories, (San Fransico, CA). Mucin-5AC refers to a polypeptide having a molecular weight of greater than approximately 250 kDa as determined by sodium dodecyl sulfate SDS-polyacrylamide gel electrophoresis, and bound by an antibody that specifically binds to a human mucin-5AC, such as the polypeptide disclosed at Genbank accession number AJ001403. Such antibodies are commercially obtainable from, for instance, Zymed Laboratories (San Francisco, CA). As used herein, an antibody that can "specifically bind" a polypeptide is an antibody that interacts only with the epitope of the antigen that induced the synthesis of the antibody, or interacts with a structurally related epitope. An antibody that "specifically binds" to an epitope will, under the appropriate conditions, interact with the epitope even in the presence of a diversity of potential binding targets. Antibody to mucins can also be made using routine methods known in the art. See, for example, Antibodies: A Laboratory Manual, Harlow and Lane, eds., Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York, 1988). Whether a cell expresses a mucin, preferably mucin-1, mucin-2, mucin- 5AC, or a combination thereof, can be determined using methods that are routine and known in the art including, for instance, Western immunoblot, ELISA, immunoprecipitation, or immunohistochemistry. Western immunoblot, immunoprecipitation, or the combination thereof are generally used with ex vivo cells, and immunohistochemistry is generally used with in vivo cells. Whether in vivo cells produce mucin to result in the accumulation of extracellular mucin can be determined by, for instance, immunohistochemistry, computed tomographic (CT) scan (Jacquet et al., J. Am. Coll. Surg., 181, 530-538 (1995)), by surgery, or a combination thereof. Examples of cultured cells expressing a mucin are those obtained from tissue, preferably cancerous tissue, such as breast, colon, lung, appendix, or pancreas. Examples of cultured cells expressing mucin-1 include HT-29 (ATCC number HTB-38), HCT-116 (ATCC number CCL-247), LS174T, and MCF-7 (ATCC number HTB-22). Examples of cultured cells expressing mucin-2 include HT-29 (ATCC number HTB-38), LS174T, and HCT-116 (ATCC number CCL-247). Examples of cultured cells expressing mucin-5 AC include HT-29 (ATCC number HTB-38). Primary cells that produce a mucin, such as mucin-1, mucin-2, mucin-5AC, or the combination thereof, can be obtained from tissues of patients with a disease having the characteristic of extracellular mucin accumulation (for instance, pseudomyxoma peritonei), or a cancer (for instance, colon cancer, breast cancer, lung cancer, appendix cancer, or pancreas cancer). With respect to pseudomyxoma peritonei, the primary cell can be, for instance, a neoplastic cell from a benign, malignant or so-called intermediate tumour associated with pseudomyxoma peritonei. In some aspects of the present invention, the cancer does not include colon cancer, breast cancer, lung cancer, appendix cancer, pancreas cancer, or a combination thereof. Other cells can also be modified to express one or more of the mucins by introducing into a cell a vector having a polynucleotide encoding the mucin. The present invention is further directed to methods for treating certain diseases in a subject. The subject is a mammal, for instance, mouse, rat, livestock (e.g., pig, horse, cow, goat, sheep), or primate (e.g., monkey, human), preferably, human. As used herein, the term "disease" refers to any deviation from or interruption of the normal structure or function of a part, organ, or system, or combination thereof, of a subject that is manifested by a characteristic symptom or set of symptoms. In some aspects, diseases include estrogen-dependent diseases, for instance, diseases that are estrogen induced or estrogen stimulated. In other aspect, the diseases include estrogen-independent diseases. Diseases include those having abnormal mucin production, typically mucin overproduction. Diseases also include those where mucin production is prognositc of increased morbidity, mortality, or the combination thereof. Examples of such diseases include breast cancer, colon cancer, lung cancer, pseudomyxoma peritonei, appendix cancer, and pancreas cancer. Typically, whether a subject has a disease, and whether a subject is responding to treatment, is determined by evaluation of symptoms associated with the disease. As used herein, the term "symptom" refers to objective evidence of a disease present in a subject. Symptoms associated with diseases referred to herein and the evaluation of such symptoms are routine and known in the art. Examples of symptoms of cancers include, for instance, the presence and size of tumors, and the presence and amount of biomarkers. Examples of symptoms of pseudomyxoma peritonei include, for instance, accumulation of extracellular mucin (which can lead to large tumor masses within the belly, and cause bowel blockage or lead to massive abdominal distention resulting in pain and difficulty breathing) and amount of biomarkers. Biomarkers are compounds, typically polypeptides, present in a subject and indicative of the presence and/or progression of cancer. Examples of biomarkers include carcinoembryonic antigen (CEA), carbohydrate antigen 19.9 (CA19.9), and cancer antigen 125 (CA125). Treatment of a disease can be prophylactic or, alternatively, can be initiated after the development of a disease. Treatment that is prophylactic, for instance, initiated before a subject manifests symptoms of a disease, is referred to herein as treatment of a subject that is "at risk" of developing a disease. An example of a subject that is at risk of developing a disease is a person having a risk factor that is associated with the disease. Risk factors include genetic markers. Examples of genetic markers indicating a subject has a predisposition to develop certain cancers such as breast cancer include alterations in the BRAC1 and/or BRAC2 genes. Treatment can be performed before, during, or after the occurrence of the diseases described herein. Treatment may result in reduction in severity of symptoms, reduction in disease progression (e.g., symptoms remain stable, and do not increase or decrease in severity), elimination of symptoms and/or underlying cause of the symptoms, or prevention of the occurrence of symptoms and/or their underlying cause. The anti-estrogens useful in the present invention may be formulated into compositions including one or more anti-estrogen and a pharmaceutically acceptable carrier. As used herein "pharmaceutically acceptable carrier" includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Additional compounds, for instance, a chemotherapeutic or radiotherapeutic compound, can also be incorporated into the compositions. Suitable chemotherapeutic and radiotherapeutic agents are known to the art. A composition may be prepared by methods well known in the art of pharmacy. In general, a composition can be formulated to be compatible with its intended route of administration. Examples of routes of administration include perfusion and parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions can include the following components: a sterile diluent such as water for administration, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates; electrolytes, such as sodium ion, chloride ion, potassium ion, calcium ion, and magnesium ion, and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. A composition can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Compositions can include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). A composition is typically sterile and, when suitable for injectable use, should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin. Sterile solutions can be prepared by incorporating the active compound (i.e., an anti-estrogen) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring. For administration by inhalation, the active compounds are delivered in the form of an aerosol spray from a pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art. The active compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery. The active compounds may be prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Such formulations can be prepared using standard techniques. The materials can also be obtained commercially from, for instance, Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art. Toxicity and therapeutic efficacy of such active compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD₅₀ (the dose lethal to

50% of the population) and the ED₅₀ (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD₅₀ ED₅₀. Compounds which exhibit high therapeutic indices are preferred. The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For a compound used in the methods of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC₅₀ (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography. . The compositions can be administered one or more times per day to one or more times per week, including once every other day. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Generally, however, dosage will be in the range of approximately 0.01 mg/kg/day to 10.0 mg/kg/day, more preferably in the range of about 1.0 mg/kg/day to 5.0 mg/kg/day.

The present invention is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein. EXAMPLES Example 1 Decreasing production of mucin-2

The aim of this study was to evaluate the effect of anti-estrogens on mucin-2 production in cell lines.

Methods The ability of the anti-estrogens Tamoxifen and Raloxifene to block mucin-2 expression in vitro was analyzed in the colon cancer cell lines HT-29 (ATCC accession number HTB-38) and HCT-116 (ATCC accession number CCL-247). The ability of estrogen to stimulate estrogen production was also analyzed using the two cell lines. The expression of mucin-2 was analyzed by immunohistochemistry and Western blot analyses. Tamoxifen and estrogen were obtained from CalBiochem (San Diego, CA). Raloxifene was obtained from Sigma (St.Louis, MO). For immunohistochemistry analysis, cells were grown in chamber slides in hormone stripped serum (Gibco-BRL, Gaithersburg, MD) for 24 hours. Following this, the cells were treated with either 10^"6 M estrogen, 10^"9 M anti -estrogen, or both, for 24 hours. The expression of MUC2 was analyzed using anti-mucin-2 antibody (BD Biosciences, San Jose, CA) and a biotin conjugated secondary antibody (Vector Laboratories,

Burlingame, CA). Positive signal for mucin-2 was a brown staining of the cytoplasm (nuclear blue staining was seen as the result of counter staining by hematoxylin). For western blot analysis, HT-29 and HCT- 1 16 cell lines were grown in hormone stripped serum for 24 hours and then cells were treated with either 10^"6 M estrogen, 10^"9 M anti-estrogen, or both, for 24 hours. Equal amounts of proteins were loaded on 4-20% gradient gels, transferred to a membrane, and then analyzed using the anti-mucin-2 antibody. The expression of mucin-2 was analyzed by Western blot analysis with quantification by NIH image scan program 1.62. The NIH image scan program is publicly available through the NIH Image FTP site, rsbweb.nih.gov.

Results mucin-2 expression in different colon cancer cell lines was evaluated by Western blot analysis and immunohistochemical analysis. The data showed that the colon cancer cell lines used in this study, HT- 29 and HCT-116, express mucin-2. The effects of Estrogen and anti-estrogen (Raloxifene) on the expression of mucin-2 in HCT-1 16 cell line. HCT- 1 16 cells were treated with either estrogen alone or estrogen and Raloxifene together and mucin-2 expression was analyzed by immunohistochemistry. Estrogen enhanced the expression of mucin-2 protein. Raloxifene reduced the expression of mucin-2. Mucin-2 protein expression after treatment with either estrogen or both estrogen and anti-estrogen (Tamoxifen) in HT-29 cell line. The cells were treated with either estrogen alone or estrogen in combination with Tamoxifen. Mucin-2 protein levels were determined by using Western blot analysis (Fig. 1). Figure 1 shows results with the HT-29 cells, and similar results were observed with HCT-119 cells. Lane C represents control cells while Lane E2 represents cells that were treated with estrogen. Lane E2+T represents cells that were co-treated with estrogen and Tamoxifen. Actin expression was used as a loading control (left). Quantitative analysis (right) shows significant increases in mucin-2 expression after estrogen exposure and marked reduction of mucin-2 expression following treatment with Tamoxifen. In conclusion, Estrogen enhanced the expression of mucin-2 in colon cancer cell lines, and the anti-estrogens Tamoxifen and Raloxifene blocked expression of mucin-2 in the presence of estrogen. Example 2

Effect of Tamoxifen on symptoms associated with pseudomyxoma peritonei

Patient 1 was a 46 year old woman with a form of pseudomyxoma peritonei (peritoneal mucinous carcinomatosis with intermediate features - PMCAI) characterized by the extensive accumulation of mucin-2 in the belly which leads to progressive abdominal distention and associated symptoms. She had failed surgery and prior several chemotherapy regimens including: oxaliplatin/irenotecan, cisplatin/capecitabine/celecoxib, and thalidomide. The patient presented with persistent symptomatic ascites and progressive disease when first seen by us. An oral treatment combination of celecoxib and capecitabine were continued but with rising seen in three tumor markers. The tumor markers were CEA (carcinoembryonic antigen), CA19.9 (carbohydrate antigen 19.9), and CA125 (cancer antigen 125). The patient was begun on Tamoxifen 20 mg per day orally. The tumor markers peaked 2 months later and then steadily declined over the next 6 months to less than 50% of peak values. The only change in therapy had been the addition of tamoxifen. There were no side effects and the patient's clinical status was stable for that period. Following that, markers began to rise and the patient underwent surgical debulking about 10 months after tamoxifen treatment was started. Patient 2 was a 52 year old man with a form of pseudomyxoma peritonei (disseminated peritoneal mucinosis - DP AM) characterized by the extensive accumulation of mucin-2 in the belly. He had had a prior attempt at surgical debulking and presented to us with disease progression. He had received and failed prior chemotherapy with the combination systemic chemotherapy carboplatin/gemzar/capecitabine.

CEA tumor marker rose from 20 at the time he first presented to us to 48 approximately 6 months later on celecoxib/capecitabine. Tamoxifen was started at 20 mg per day orally. CEA tumor marker fell to 22 approximately two months after treatment was begun and was 23 approximately 12 months later. During this period, there were no side effects and the patient had clinically stable disease. The addition of tamoxifen was the only clinical change during this period. More recently, the patient has begun to show signs of disease progression and will undergo debulking surgery for disease progression and symptoms. In summary, these two patients with previously refractory disease have shown clinical responses to tamoxifen therapy. These responses of the patients are particularly remarkable since both patients had failed systemic chemotherapy.

The complete disclosure of all patents, patent applications, and publications, and electronically available material (including, for instance, nucleotide sequence submissions in, e.g., GenBank and RefSeq, and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) cited herein are incorporated by reference. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims. All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

Claims

What is claimed is:

1. A method for decreasing mucin production by a cell comprising: contacting a mucin producing cell with an effective amount of an anti-estrogen, wherein mucin production by the cell is decreased.

2. The method of claim 1 wherein the cell is in vivo.

3. The method of claim 2 wherein the cell is present in a human.

4. The method of claim 3 wherein the human has a symptom of pseudomyxoma peritonei.

5. The method of claim 4 wherein the mucin producing cell is a neoplastic cell.

6. The method of claim 1 wherein the cell is a cancer cell, with the proviso that the cell is not a breast cancer cell.

7. The method of claim 6 wherein the cancer cell is a lung cancer cell, a colon cancer cell, an appendix cancer cell, a pancreas cancer cell, or a neoplastic cell obtained from a subject with pseudomyxoma peritonei.

8. The method of claim 1 wherein the anti-estrogen is tamoxifen, raloxifene, or a combination thereof.

9. The method of claim 1 wherein the mucin is mucin-1, mucin-2, mucin- 5AC, or a combination thereof.

10. A method for decreasing disease progression comprising: administering to a subject an effective amount of an anti -estrogen, wherein the subject has or is at risk of having a disease wherein mucin production is prognositc of increased morbidity, mortality, or the combination thereof, with the proviso that the disease is not breast cancer.

1 1. The method of claim 10 wherein the disease is lung cancer, colon cancer, appendix cancer, pancreas cancer, or pseudomyxoma peritonei.

12. The method of claim 10 wherein the anti-estrogen is tamoxifen, raloxifene, or a combination thereof.

13. The method of claim 10 wherein the administering results in a decrease in mucin production by a cell associated with the disease.

14. The method of claim 13 wherein the mucin produced is mucin-1, mucin- 2, mucin-5AC, or the combination thereof.

15. A method for treating a symptom of a disease comprising: administering to a subject having a disease an effective amount of an anti-estrogen, wherein a symptom of the disease is decreased or stabilized, and wherein the disease is a disease where mucin production is prognositc of increased morbidity, mortality, or the combination thereof, with the proviso that the disease is not breast cancer.

16. The method of claim 15 wherein the disease is lung cancer, colon cancer, appendix cancer, pancreas cancer, or pseudomyxoma peritonei.

17. The method of claim 15 wherein the anti-estrogen is tamoxifen, raloxifene, or a combination thereof.

18. The method of claim 15 wherein the administering results in a decrease in mucin production by a cell associated with the disease.

19. The method of claim 18 wherein the mucin produced is mucin-1, mucin- 2, mucin-5AC, or the combination thereof.

20. A method for treating cancer comprising: administering to a subject having a cancer an effective amount of an anti -estrogen, wherein a symptom of the cancer is decreased or stabilized, with the proviso that the cancer is not breast cancer.