WO2011133983A2 - Reishi polysaccharide-based compositions and methods for treatment of cancer - Google Patents

Abstract

The present invention relates to methods and compositions for treating cancers with extracts of Ganoderma lucidum (Reishi or Ling-Zhi). Methods for treating tumor progression and metastasis by modulating epithelial-mesenchymal transition (EMT) are provided. Methods for reducing migration and invasion of cancer cells are provided. Alkaline extracts of Ganoderma lucidum comprising immune-modulating activity for modulating EMT and inhibiting progression of cancers are provided.

Description

REISHI POLYS AC CHARIDE-BASED COMPOSITIONS AND METHODS FOR

TREATMENT OF CANCER

INVENTORS:

Chi-Huey Wong, Hsien-Yeh Hsu, Shih-Ting Weng, Wei-Ting Wang, Tseng-Rong Tu, Chia-Feng Li, Wen-Cheng Lin and Eugene Fan

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This patent application claims the benefit of U.S. Provisional Patent Application Serial No. 61/327,635, filed April 23, 2010, entitled "Reishi polysaccharide-based compositions and methods for treatment of cancer" the contents of which are incorporated herein in their entirety by reference.

TECHNICAL FIELD OF THE INVENTION

[0002] The present invention relates to methods and compositions for treating cancers. In particular, the invention relates to methods for treating tumor progression and metastasis by modulating EMT. More particularly, the invention relates to extracts of Ganoderma lucidum (Reishi or Ling-Zhi) for modulating EMT and inhibiting progression of cancers, including lung cancer and breast cancer.

BACKGROUND OF THE INVENTION

[0003] Epithelial cell cancers, for example, prostate cancer, breast cancer, colon cancer, lung cancer, pancreatic cancer, ovarian cancer, cancer of the spleen, testicular cancer, cancer of the thymus, etc., are diseases characterized by abnormal, accelerated growth of epithelial cells. This accelerated growth initially causes a tumor to form. Eventually, metastasis to different organ sites can also occur. Although progress has been made in the diagnosis and treatment of various cancers, these diseases still result in significant mortality.

[0004] Lung cancer is a leading cause of cancer mortality. In the United States, 106,374 men and 90,080 women were diagnosed with lung cancer, and 89,243 men and 69,356 women died from lung cancer in 2006. (U.S. Cancer Statistics Working Group. United States Cancer Statistics: 1999— 2006 Incidence and Mortality Web-based Report. Atlanta (GA): Department of Health and Human Services, Centers for Disease Control and Prevention, and National Cancer Institute; 2010.)

[0005] Lung cancer is broadly divided into small-cell-lung-cancer (SCLC, comprising 20% of lung cancer), and non-small-cell lung cancer (NSCLC, comprising 80% of lung cancers). The adenocarcinoma subtype of NSCLC represents the most common histological type of lung cancer. Most patients with advanced NSCLC present with metastatic disease and have a median survival after diagnosis of 4-5 months and a 1-year survival of less than 10%. (Sharma SV, Bell DW, Settleman J, & Haber DA (2007) Epidermal growth factor receptor mutations in lung cancer. Nat Rev Cancer l(3): l69-m.)

[0006] Worldwide, breast cancer comprises 10.4% of all cancer incidences among women, making it the second most common type of non-skin cancer (after lung cancer) and the fifth most common cause of cancer death. There are many different types of breast cancer, with different stages (spread), aggressiveness, and genetic makeup; survival varies greatly depending on those factors. In 2009, in the United States there were 192,370 (female) and 1,910 (male) new cases, and 40, 170 (female) and 440 (male) deaths from breast cancer. One of the potential therapeutic strategies for the treatment of cancer is the inhibition of metastasis.

[0007] Ganoderma lucidum (Reishi or Ling-Zhi), a saprophytic fungus, has been recommended in traditional Chinese medicine used as a dietary supplement (Wang Y-Y, et al. (2002) Studies on the immuno-modulating and antitumor activities of Ganoderma lucidum (Reishi) polysaccharides: functional and proteomic analyses of a fucose-containing glycoprotein fraction responsible for the activities. Bioorganic & Medicinal Chemistry 10(4): 1057-1062). A protein Ling Zhi-8 in G.

lucidum LZ-8 has positive effects on systemic anaphylaxis, and has been used for the treatment of liver cancer and preventing diabetes. (Kino K. et al, J. Biol. Chem. 1989; 264(1): 472-8).

[0008] Previous studies have demonstrated that a glucan extract of Reishi-derived polysaccharides (EORP), as glycoprotein fractions containing either a polysaccharide backbone with -l,3-linkages or a polymannose backbone with - 1 ,4-linkages, exerts multi-immunological functions and antitumor activity (Chen H-S, et al. (2004) Studies on the immuno-modulating and anti-tumor activities of Ganoderma lucidum (Reishi) polysaccharides. Bioorganic & Medicinal Chemistry 12(21):5595- 5601; Hsu H-Y, et al. (2004) Extract of Reishi Polysaccharides Induces Cytokine Expression via TLR4-modulated Protein Kinase Signaling Pathways. J Immunol 173(10):5989-5999). For example, polysaccharides from Reishi inhibited tumor growth and induced expression of a variety of inflammatory cytokines. Therefore, the polysaccharide compounds may be an anti-tumor agent based on the ability to enhance the host's defense system (Cao Q-Z & Lin Z-B (2006) Ganoderma lucidum polysaccharides peptide inhibits the growth of vascular endothelial cell and the induction of VEGF in human lung cancer cell. Life Sciences 78(13): 1457-1463). Although Reishi have been found to inhibit transcription factors NF-κΒ and AP-1, and decrease secretion of VEGF and TGF- βΐ (Stanley G, Harvey K, Slivova V, Jiang J, & Sliva D (2005) Ganoderma lucidum suppresses angiogenesis through the inhibition of secretion of VEGF and TGF-Bl from prostate cancer cells. Biochemical and Biophysical Research Communications 330(l):46-52). However, molecular mechanisms for the anti-metastasis function of EORP are far from clear.

[0009] TGF-β plays dual roles in cancer biology, i.e., tumor suppression and tumor promotion. TGF-β inhibits the proliferation of epithelial, endothelial and haematopoietic cell lineages as a tumor suppressor. During tumor development processes, the tumors are resistant to TGF-β- mediated growth inhibition and over-express TGF-β. At this stage, TGF-β promotes tumor growth and metastasis (Dumont N & Arteaga CL (2003) Targeting the TGFB signaling network in human neoplasia. Cancer Cell 3(6):531-536; Siegel PM & Massague J (2003) Cytostatic and apoptotic actions of TGF-B in homeostasis and cancer. Nat Rev Cancer 3(1 1):807-820; Wakefield LM & Roberts AB (2002) TGF-B signaling: positive and negative effects on tumorigenesis. Current Opinion in Genetics & Development 12(l):22-29). As pre-malignant lesion progresses, the role of TGF-β changes from tumor suppressor to tumor promoter. Early epithelial tumors convert to invasive, metastatic tumors by TGF-β induced epithelial mesenchymal transition (EMT) (Thiery JP (2002) Epithelial-mesenchymal transitions in tumour progression. Nat Rev Cancer 2(6):442-454). The cells can intravasate into lymph or blood vessels, allowing their passive transport to distant organs (Id.).

[0010] Epithelial-mesenchymal transition (EMT) is a critical phenotypic alteration of cancer cells that triggers invasion and metastasis. Snail, a zinc-finger transcription factor, is known to be involved in EMT. It has been shown to attenuate cell cycle and offer a survival advantage to tumor cells during the extavasation, dissemination and intravasation cycle (Vega S, et al. (2004) Snail blocks the cell cycle and confers resistance to cell death. Genes & Development 18(10): 1131- 1 143).

[0011] EMT-associated cellular transitions and cellular interactions occur in non-small cell lung cancer (NSCLC), colorectal cancer (CRC), and pancreatic carcinoma. During EMT, epithelial cells down-regulate their intercellular adhesion, lose the apical-basal polarity, and undergo

morphological changes from a monolayer of cuboidal-shape cells to dispersed, spindle-shaped fibroblast-like cells. The expression of differentiation markers switches from cell-cell junction proteins such as E-cadherin to mesenchymal markers including N-cadherin and fibronectin.

Furthermore, the stationary cells convert to migratory cells capable of invasion through extracellular matrix. EMT is importance for carcinogenesis and metastasis (Janda E, et al. (2002) Ras and TGF-B cooperatively regulate epithelial cell plasticity and metastasis: dissection of Ras signaling pathways. J Cell Biol 156(2):299-313). SUMMARY OF THE INVENTION

[0012] There is a need for improved methods for treatment of solid tumors such as lung carcinoma with agents that modulate epithelial-mesenchymal transition (EMT).

[0013] The present disclosure relates to the effects of Reishi F3 fraction on the progression and metastasis of lung adenocarcinoma. A Reishi F3 fraction reverses TGF-βΙ induced EMT in A549 human NSCLC cell line in vitro. In addition, the Reishi F3 fraction suppresses expression of experimental metastasis of lung and liver in LLC-bearing mice model in vivo. The disclosure reveals that Reishi F3 fraction can inhibit EMT and metastasis related to solid tumors such as NSCLC.

[0014] The invention relates to a pharmaceutical composition comprising: one or more sub- fractions of an extract of Ganoderma lucidum (Reishi), wherein the one or more sub-fractions (Reishi F3 Fraction) are present in an amount sufficient to (a) modulate epithelial-mesenchymal transition (EMT), or (b) reduce migration and invasion of non-small cell lung cancer (NSCLC) cells, or both; and optionally, a pharmaceutically acceptable excipient.

[0015] In some aspects the one or more sub-fractions are obtainable by: homogenizing tissue of Ganoderma lucidum; extracting the homogenized plant tissue with aqueous alkaline solution to form a crude extract; neutralizing the crude extract to a pH of about 7.2±0.2 and filtering to obtain a clear Reishi Crude Extract; and subjecting the clear Reishi Crude Extract to tangential flow filtration to remove low molecular weight components and obtain a purified Reishi F3 Fraction.

[0016] In some embodiments, the Reishi F3 Fraction displays an HPLC profile according to Figure 6. In some embodiments, Reishi F3 Fraction comprises a polysaccharide or glycopeptide comprising terminal fucose residues.

[0017] Also disclosed are methods for preventing, treating or reducing a cancer-associated event in a cell, the methods comprising: contacting the cell with a composition comprising a Reishi F3 fraction, wherein the Reishi F3 fraction has a HPLC profile according the Figure 6, and further wherein the composition comprises an amount of Reishi F3 fraction sufficient for (a) modulating epithelial-mesenchymal transition (EMT), or (b) reducing migration and invasion of lung cancer cells, or both. In some embodiments, the cell is a mammalian cell. In some embodiments, the mammalian cell is in a human.

[0018] In some aspects, the cancer cell comprises neuroblastoma, melanoma, non-Hodgkin's lymphoma, Epstein-Barr related lymphoma, Hodgkin's lymphoma, retinoblastoma, small cell lung cancer, brain tumors, leukemia, epidermoid carcinoma, prostate cancer, renal cell carcinoma, transitional cell carcinoma, breast cancer, ovarian cancer, lung cancer colon cancer, liver cancer, stomach cancer, and other gastrointestinal cancers.

[0019] In some aspects, the cancer cell comprises a solid tumor selected from non-small cell lung cancer (NSCLC), pancreatic cancer, colorectal cancer, breast cancer and hepatocellular cancer.

[0020] In some aspects, modulating epithelial-mesenchymal transition (EMT) comprises reduction of one or more symptoms selected from: loss of epithelial-cell markers; loss of cell polarity and cell-junction proteins by epithelial tumor cells; and acquisition of protein mesenchymal-cell markers.

[0021] In some aspects, modulating epithelial-mesenchymal transition (EMT) comprises depletion of EMT with one or more effects selected from: conversion of fibroblastic to epithelial morphology; up-regulation of major epithelial cell markers expression; and down-regulation of mesenchymal cell markers expression.

[0022] In some aspects, the EMT is induced by EMT-related signaling pathways driven via receptors such as platelet derived growth factor receptor (PDGFR); fibroblast growth factor receptor (FGFR); cMET; TGFBR; IGF-1R; and kinases selected from PI3K, AKT and mTOR.

[0023] In some embodiments, the EMT is induced by TGF-βΙ. In some embodiments, the reduction of EMT comprises suppression of TGF-β Ι -mediated signal transduction comprising one or more effects selected from (i) reduction of TGF-βΙ production, (ii) down-regulation of TGF-β receptor II expression, (iii) decrease of Smad2/3 phosphorylation, and (iv) decrease of Snail protein expression.

[0024] In some embodiments, the epithelial-cell markers are selected from E-cadherin and γ- catenin. In some embodiments, the protein mesenchymal-cell markers are selected from vimentin, fibronectin, and N-cadherin.

[0025] In some aspects, reducing migration and invasion of lung cancer cells comprises an anti- metastatic effect. In some embodiments, the lung cancer cells are non-small cell lung cancer (NSCLC) cells. In some embodiments, the anti-metastatic effect comprises reducing a

Mesenchymal Epithelial Transition (MET) and thereby reducing proliferation and growth of epithelial tumor cells at sites distal from the primary tumor.

[0026] In some embodiments, the Reishi F3 fraction is administered in conjunction with a therapeutically effective amount of a chemotherapeutic agent selected from cisplatin, doxorubicin, Taxol, daunorubicin, mitomycin, actinomycin D, bleomycin, VP 16, tumor necrosis factor, vincristine, vinblastine, carmustine, melphalan, cyclophosphamide, chlorambucil, bisulfan, lomustine, penicillin, erythromycin, amoxicillin, cefazolin, imipenem, aztreonam, sulbactam, linezolid, gentamicin, sulfamethoxazole, vancomycin, ciprofloxacin, fusidic acid, trimethoprim, metronidazole, clindamycin, mupirocin, amphotericin B, rifampin, fluconazoleor, or a combination thereof.

[0027] In some embodiments, the Reishi F3 fraction is administered in conjunction with a therapeutically effective amount of czs-diamminedichloroplatinum (II) (CDDP or cisplatin). In some embodiments, the cisplatin is administered intraperitoneally. In some embodiments, administration of Reishi F3 fraction with cisplatin reduces distant metastases by one or more of (a) reducing lung tumor multiplicity, and (b) reducing tumor volume, wherein the tumor multiplicity or volume is reduced by an amount greater than that achieved by administration of cisplatin alone.

[0028] In some aspects, the Reishi F3 fraction is administered in conjunction with a an anti-cancer therapy selected from a drug, a hormone, a gene therapy composition, a radionuclide, a

nutraceutical, a surgical procedure, a radiation procedure or a combination thereof. In some embodiments, the Reishi F3 fraction is administered prior to or following the anti-cancer therapy.

[0029] In some embodiments, the Reishi F3 fraction is administered orally, intravenously, subcutaneous ly, intramuscularly, intraperitoneally, intranasally or trans dermally, concurrently or sequentially.

[0030] These and other aspects will become apparent from the following description of the preferred embodiment taken in conjunction with the following drawings, although variations and modifications therein may be affected without departing from the spirit and scope of the novel concepts of the disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, the inventions of which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0032] FIG. 1 shows that A549 cells undergo EMT in response to Reishi F3 fraction (shown as "EORP") preparation. (1A) Untreated A549 cells (l x l0⁵cells/ml) show epithelial morphology. B- D, A549 cells were incubated with TGF-βΙ (1, 2 and 5 ng/ml) for 72 h. E-H, Cells treated with 120 μg/ml Reishi F3 fraction containing various concentration of TGF-βΙ (0, 1, 2 and 5 ng/ml) for 72 h (magnification x200). (IB) A549 cells (l l0⁵cells/ml) were incubated with 5 ng/ml of TGF-βΙ for 24 h. 24 h later, stimulated with Reishi F3 fraction (120 μg/ml) for 24 hours. Total cell lysates were collected, and EMT markers were detected by western blot. (1C) A549 cells (2x l0⁵cells/total) were treated with Reishi F3 fraction (120 μg/ml) for 24 h after cultured with or without TGF-βΙ (5 ng/ml) for 24h. After treatment, cells were seeded to the Transwell system. Cell migration was measured by Transwell system with polycarbonate filters (pore size, 8 μιη). Migration ability of A549 cells were quantified by counting the number of cells that migrated to the underside of the porous polycarbonate membrane under microscopy.

[0033] Figure 2 shows that Reishi F3 fraction (shown as EORP) preparation reduces TGF l signaling pathway in A549 cells. (2A) A549 cells were cultured in the medium with Reishi F3 fraction or absence for 6 and 24 h. Cultured media were collected for measurement of TGF- β 1 determined using ELISA. Data represent mean± S.D. * Significant differences between control and Reishi F3 fraction treated A549 cells, p<0.05. A549 cells were treated with or without Reishi F3 fraction for 3h after cultured with or without TGF-βΙ for 24 h. A549 cells were treated with or without TGF-βΙ (5 ng/ml) for 24 h, then replace fresh medium. Next, A549 cells treated with Reishi F3 fraction (120 μg/ml) for 3h. Expression of (2B) phosphorylation Smad2/3, (2C) Snail and Slug were detected by western blot.

[0034] Figure 3 shows the Reishi F3 fraction (shown as EORP) preparation inhibited migration, invasion and colony formation of A549 and LLC-1 cells. (3A) LLC-1 cells (4 x 10⁴cells / 200μ1) were treated with Reishi F3 fraction (120 μg/ml) for 18h. Cell migration and cell invasion were measured by Transwell system with polycarbonate filters (pore size, 8 μιη). Migration and invasion ability of cells were quantified by counting the number of cells that invaded to the underside of the porous polycarbonate membrane under microscopy. (3B) LLC-1 cells were plated in 6-well plates with 0.33% agar on a 0.5% agar under layer containing DMEM containing 10% FBS. Cells were cultured for 16 days and colonies were counted. Data shown are mean+ SD of triplicate samples of a representative experiment, similar result were obtained in three independent experiments. * p<0.05 and **P<0.01 vs. control.

[0035] Figure 4 shows that Reishi F3 fraction (shown as EORP) preparation combined with CDDP inhibited tumor metastasis. LLC-1 cells were implanted into mice via tail vein injection (n=8 in each group). After 7 days, mice were treated with CDDP (0.5 mg/g) i.p. injection at the same time treated with Reishi F3 fraction (0.075 mg/g) i.p. injection at intervals of 2 days or daily oral administration with Reishi F3 fraction (0.075mg/g) for 4 weeks. (4A) Left panel is lung tumor multiplicities; right panel is tumor volume (mm³) of lung lesion. * p<0.05, **p<0.01 and ***P<0.001 vs. control or CDDP. (4B) Upper panel of lung; medium panel of HE stained lung sections: x 20 magnification; lower panel of HE stained lung sections: x 400 magnification. (4C) Upper panel of HE stained liver sections: x 100 magnification; lower panel of HE stained liver sections: x 400 magnification.

[0036] FIG. 5 shows the HPLC pattern of Reishi (Ganoderma lucidum) Crude Extract by using a size exclusion column (Tosoh TSK-GEL G5000PW).

[0037] FIG. 6 shows the HPLC pattern of the purified Reishi F3 Fraction by using a size exclusion column (Tosoh TSK-GEL G5000PW).

DETAILED DESCRIPTION OF THE INVENTION

[0038] The terms used in this specification generally have their ordinary meanings in the art, within the context of the invention, and in the specific context where each term is used. Certain terms that are used to describe the invention are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner regarding the description of the invention. For convenience, certain terms may be highlighted, for example using italics and/or quotation marks. The use of highlighting has no influence on the scope and meaning of a term; the scope and meaning of a term is the same, in the same context, whether or not it is highlighted. It will be appreciated that same thing can be said in more than one way. Consequently, alternative language and synonyms may be used for any one or more of the terms discussed herein, nor is any special significance to be placed upon whether or not a term is elaborated or discussed herein. Synonyms for certain terms are provided. A recital of one or more synonyms does not exclude the use of other synonyms. The use of examples anywhere in this specification including examples of any terms discussed herein is illustrative only, and in no way limits the scope and meaning of the invention or of any exemplified term. Likewise, the invention is not limited to various embodiments given in this specification.

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the case of conflict, the present document, including definitions will control.

[0040] "Tumor" is used herein, for purposes of the specification and claims, to mean solid nonlymphoid primary tumor of ductal epithelial cell origin, including, but not limited to, tumors originating in the breast, prostate, colon, lung, pancreas, liver, stomach, bladder, or reproductive tract (cervix, ovaries, endometrium etc.), brain, and bone marrow; melanoma; or lymphoma.

[0041] "Inhibiting tumor growth" is used herein, for purposes of the specification and claims, to mean one or more of slowing the growth of the tumor, halting growth of the tumor, causing reduction or regression of the tumor, inhibiting tumor invasion, causing tumor cell death, and causing reduction or regression of metastases.

[0042] "Prevention of tumor development" is used herein, for purposes of the specification and claims, to mean inhibiting growth of the tumor; and more specifically, causing tumor cell death in preventing tumor mass formation.

[0043] An "anti-cancer" agent is capable of negatively affecting cancer in a subject, for example, by killing cancer cells, inducing apoptosis in cancer cells, reducing the growth rate of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing the blood supply to a tumor or cancer cells, promoting an immune response against cancer cells or a tumor, preventing or inhibiting the progression of cancer, or increasing the life span of a subject with cancer. More generally, these compositions and methods would be provided in a combined amount effective to kill or inhibit proliferation of the cell. This process may involve contacting the cells with an anticancer agent and multiple factor(s) at the same time. This may be achieved by contacting the cell with a single composition or pharmacological formulation that includes both agents, or by contacting the cell with two distinct compositions or formulations, at the same time, wherein one composition includes, for instance, an expression construct and the other includes the second agent(s), such as a radionuclide or anticancer drug.

[0044] The F3 extract of Reishi polysaccharides exerts antitumor activities. The disclosure reveals the function of Reishi F3 composition in modulating epithelial-mesenchymal transition (EMT), and reducing of migration and invasion in non-small cell lung cancer (NSCLC).

[0045] Reishi F3 fraction compositions of the present disclosure, reverses EMT in A549, a human NSCLC cell line. Pre-incubation of A549 cells with TGF-βΙ followed by Reishi F3 composition treatment resulted in the depletion of TGF-βΙ -induced EMT, including (i) conversion of fibroblastic to epithelial morphology, (ii) up-regulation of major epithelial cell markers expression: E-cadherin and γ-catenin, (iii) down-regulation of mesenchymal cell expression markers: vimentin and N-cadherin.

[0046] Without being bound by theory, a suggested mechanism for altering EMT can be based on the observations that the Reishi F3 fraction suppresses TGF-βΙ -mediated signal transductions via (i) reduction of TGF-βΙ production, (ii) down-regulation of TGF-β receptor II expression, (iii) decrease of Smad2/3 phosphorylation, or (iv) decrease of Snail protein expression.

[0047] Further, using murine Lewis lung carcinoma (LLC) in vitro, the Reishi F3 fraction reduces cell migration, cell invasion and colony formation. Further therapeutic uses of the Reishi F3 fraction for NSCLC, are based on the effects of oral feeding of and injection demonstration of the Reishi F3 fraction on metastasis in LLC -xenograft mice. Specifically, LLC-1 cells were implanted into mice via tail vein injection. After 7 days, mice were treated with CDDP (0.5 mg/g) i.p.

injection at the same time treated with EORP (0.075 mg/g) i.p. injection at intervals of 2 days or daily oral administration with EORP (0.075mg/g) for 4 weeks. Intraperitoneal injection of the mice tail vein LLC-xenografts with the Reishi F3 fraction had statistically and significantly lower lung cancer metastasis than that of control mice. Moreover, the marker and size of metastatic nodules on lung was also significantly reduced by the treatment of combination the Reishi F3 fraction and cisplatin. The addition of the Reishi F3 fraction to cisplatin augmented the suppression of metastasis to lung and liver. Our current results demonstrate that the Reishi F3 fraction plays key roles in defining phenotypes associated with EMT and its reverse process as well as in the inhibition of metastasis activity of lung cancer in animal model.

Epithelial Mesenchymal Transition (EMT) and Cancer

[0048] A loss of epithelial-cell markers and gain of mesenchymal-cell markers has been observed in patient tumor samples, particularly at the leading edge or invasive front of solid tumors such as non-small cell lung cancer (NSCLC), pancreatic, colorectal, and hepatocellular cancers. Such changes in phenotypic epithelial-like and mesenchymal-like cellular markers have been associated with the degree of tumor progression. The loss of epithelial-cell markers (e.g. E-cadherin, γ- catenin, others) is associated with disease progression and metastatic potential of a tumor. It has become evident that cancer cells can dedifferentiate through activation of specific biological pathways associated with EMT, thereby gaining the ability to migrate and invade. Hence, what has been observed experimentally regarding EMT and normal organ development is also thought to apply in the progression of solid tumors— transcriptional reprogramming processes whereby epithelial tumor cells lose cell polarity and cell-junction proteins and at the same time acquire protein mesenchymal-cell markers (e.g. vimentin, fibronectin, others) and signal transduction activities associated with mesenchymal cells facilitating migration and survival in an anchorage- independent environment, and ultimately metastasis at distal sites.

[0049] Mesenchymal-like tumor cells gain migratory capacity at the expense of proliferative potential. As in normal EMT, pathological EMT in tumor cells results from a transcriptional reprogramming of the cell leading to its transition into mesenchymal-like cellular phenotype, promoted by EMT-related signaling pathways driven, in part, through abnormal survival signals via receptors such as platelet derived growth factor receptor (PDGFR); fibroblast growth factor receptor (FGFR); cMET; TGFBR; IGF-1R; and regulatory kinases such as PI3K, AKT and mTOR. Cellular changes resulting in a more mesenchymal-like state driven by pathological EMT in cancer are thought to play a major role in disease progression has been associated with poor prognosis.

[0050] The reverse, Mesenchymal Epithelial Transition (MET), whereby mesenchymal tumor cells change to become more epithelial-like in phenotype, is thought to be required to regenerate a proliferative state and form macrometastases resembling the primary tumor at distant sites. It has been hypothesized that MET facilitates proliferation and growth of epithelial tumor cells at sites distal from the primary tumor.

Reishi F3 fraction reverses TGF-pi-induced EMT in human A549 NSCLC

[0051] It reported that A549 cells were changed from cuboidal to an elongated spindle shape in response to TGF-βΙ, and showed decreased expression of epithelial markers and enhanced expression of mesenchymal markers (Kasai H, Allen J, Mason R, Kamimura T, & Zhang Z (2005) TGF-betal induces human alveolar epithelial to mesenchymal cell transition (EMT). Respiratory Research 6(1):56). TGF-βΙ -treated cells for 72h show a decrease in cell-cell contacts and turn from round into spindle-like mesenchymal phenotype (Figure 1A, compare panel a with panel b-d). Flat, epithelial shape of A549 cells grown in normal medium was not changed by treatment with Reishi F3 fraction (Figure 1A, panel a, and e). Therefore, we estimated the optimum concentrations and time required for TGF-βΙ to initiate EMT in A549 were incubated with up to 5 ng/ml of TGF-βΙ for 24 h. Mesenchymal markers, including vimentin and N-cadherin, were up-regulated by TGF-βΙ treatment, whereas Epithelial markers, including E-cadherin and γ-catenin, were down regulated (Figure IB, compare lane 3 with lane 1). To investigate the phenotype of TGF-βΙ -treated cells co- treated with Reishi F3 fraction for 72h. Reishi F3 fraction treated with A549 abolished TGF-βΙ- induced EMT, as shown by the changes in cell morphology (Figure 1A, compare panel b-d with panel f-h), and EMT markers, including E-cadherin, γ-catenin, N-cadherin and vimentin (Figure IB, compare lane 3 with lane 4). EMT marker of A549 cells grown in normal medium was not changed by treatment with Reishi F3 fraction (Figure IB, compare lane 1 with lane 2). Because enhancement of cell motility is a well-known phenotypic change associated with EMT, cell motility change was analyzed by migration assay. To test the effect of Reishi F3 fraction on TGF-βΙ induced A549 cells migration. The results revealed that Reishi F3 fraction suppressed TGF-βΙ induced A549 cells migration to the underside of the porous polycarbonate membrane (Fig. 1C). Reishi F3 fraction reversed the features of TGF-βΙ -induced EMT in A549 cells. Reishi F3 fraction inhibits TGF-pi-induced signaling in A549 relevant to EMT

[0052] Because cancer cells often increase their production of active TGF-βΙ, which not only triggers EMT and allows the cells to become invasive (Derynck R, Akhurst RJ, & Balmain A (2001) TGF-beta signaling in tumor suppression and cancer progression. Nat Genet 29(2): 117- 129), we investigated the TGF-βΙ production after treatment of Reishi F3 fraction in A549 cells and its association with EMT. The result indicated that Reishi F3 fraction could suppress TGF-βΙ production in A549 cells at 6h or 24h (Fig. 2A). To explore the mechanism by which Reishi F3 fraction suppresses TGF-βΙ -induced EMT, we measured the effect of Reishi F3 fraction on early TGF-βΙ -induced signaling events in A549 cells. The major TGF-βΙ -induced signaling pathway is Smad signaling. Since the phosphorylation of Smad2/3 is needed for ΤϋΡβ-Ι -induced epithelial- mesenchymal transition. Therefore, we investigated the effect of Reishi F3 fraction on TGF-βΙ- induced phosphorylation of Smad2/3. We found Reishi F3 fraction treatment reduces TGF-βΙ- induced phosphorylation of Smad2/3 at 3 h (Fig. 2B, compare lane 3 with lane 4). Snail and Slug are known to regulate the E-cadherin transcription. Both transcription factors are induced in response to TGF-βΙ in cells that undergo TGF-βΙ induced EMT (Cano A, et al. (2000). The transcription factor Snail controls epithelial-mesenchymal transitions (EMTs) by repressing E- cadherin expression. Nat Cell Biol 2(2):76-83). To define the mechanisms through which Reishi F3 fraction reverses TGF-βΙ -induced EMT, the protein levels of Snail and Slug were investigated by western blot. When treated with Reishi F3 fraction, which blocks the TGF-βΙ pathway downstream of the receptor, in A549 cells, Snail was decreased. However, Slug was not changed with Reishi F3 fraction (Fig. 2C, compare lane 3 with lane 4).

Reishi F3 fraction suppresses invasion and colony formation of LLC

[0053] Since it was found that Reishi F3 fraction reverses TGF-βΙ -induced EMT in vitro, the anti- metastasis effect of Reishi F3 fraction in vivo were investigated in LLC-bearing mice model. Firstly, we examined the anti-metastasis effect of Reishi F3 fraction on cell migration, invasion, and colony formation of LLC cells. At the concentration of Reishi F3 fraction (120 μg/ml), LLC cell invasion was significantly decreased to 33.8% compared to the untreated controls (**, p<0.01) (Fig. 3 A). In addition, the effect of Reishi F3 fraction (120 μg/ml) was a significant decrease to 40% in colony formation compared with control wells (*, p<0.05) (Fig. 3B). Therefore, Reishi F3 fraction decreases cell invasion and colony formation in LLC. Inhibitory effect of Reishi F3 fraction in combination with cisplatin on LLC-bearing mice

[0054] To investigate the anti- metastatic capacity of Reishi F3 fraction in mice model in vivo, LLC cells were introduced into C57BL/6 mice via tail vein injection of LLC. Seven days after injection, mice were then randomly selected for i.p. injection at intervals of 2 days of PBS or Reishi F3 fraction, or oral administration of Reishi F3 fraction. We found that Reishi F3 fraction (i.p.) significantly inhibited lung tumor multiplicity (26.8±4.9 v.s. 7.0±1.4, p<0.001) and tumor volume of lung lesion (146.5±65.9 v.s. 78.2±8.0, p<0.05) compared with only PBS treatment (Fig. 4A). Similar differences were seen in Reishi F3 fraction (oral), and this group significant inhibited lung tumor multiplicity (9.7±4.0 v.s. 26.8±4.9, p<0.05) and tumor volume of lung lesion (15.9+11.2 v.s. 146.5±65.9, p<0.05) compared with only PBS treatment (Fig. 4A). Furthermore, this model provides the basis for combining Reishi F3 fraction with standard chemotherapeutic agents. In this study we choose the optimal schedule of cis-diamminedichloroplatinum(II) (CDDP; cisplatin^®) administration combined with Reishi F3 fraction at day7. However, the combination of CDDP and Reishi F3 fraction (i.p.) further reduced lung tumor multiplicity (5±2.8 vs. 12.8+6.3, p<0.05) and tumor volume of lung lesion (7.0+1.8 vs. 141.9+65.2, p<0.05) compared with CDDP treatment alone (Fig. 4A).

[0055] To further analyze the LLC metastasis in the testing mice, experiments of histopathologic staining was done. Sections of lung stained with H&E showed the morphology of tumor metastasis to the alveolar lung tissue at day28, which disappeared after Reishi F3 fraction treatment (Fig. 4B). We found that Reishi F3 fraction (i.p.) or Reishi F3 fraction (oral) combined with CDDP inhibited tumor metastasis to the alveolar lung tissue compared with only CDDP treatment (Fig. 4B). Next, we also found that Reishi F3 fraction (i.p.) or Reishi F3 fraction (oral) combined with CDDP inhibited tumor metastasis to the liver tissue compared with only CDDP treatment (Fig. 4C). Taken together, these data suggest that the combination of Reishi F3 fraction and effective CDDP may also reduce distant metastases.

[0056] In order to increase the effectiveness of the methods of the present invention, it may be desirable to combine the anticancer compositions with other agents also effective in the treatment of hyperproliferative disease. Such combination treatments may occur within administration of the therapeutic methods of the present invention, for instance combining gene therapy and an anticancer drug within the same in situ injection protocol. Alternatively, combination treatments may be utilized within the scope of the present invention by administering one or more therapeutic agents in situ by the methods of the present invention in addition to, for example, administering a therapeutic agent systemically. [0057] Tumor cell resistance to chemotherapy and radiotherapy agents represents a major problem in clinical oncology. One goal of current cancer research is to find ways to improve the efficacy of chemo- and radiotherapy by combining it with gene therapy. For example, the herpes simplex- thymidine kinase (HS-tK) gene, when delivered to brain tumors by a retroviral vector system, successfully induced susceptibility to the antiviral agent ganciclovir (Culver, et al, 1992). In the context of the present invention, it is contemplated that Reishi F3 therapy could be used similarly in conjunction with chemotherapeutic, radiotherapeutic, or immunotherapeutic intervention, in addition to other pro-apoptotic or cell cycle regulating agents.

[0058] Alternatively, the Reishi F3 composition therapy may precede or follow the other agent treatment by intervals ranging from minutes to weeks. In embodiments where the other agent and expression construct are applied separately to the cell, one would generally ensure that a significant period of time did not expire between the times of each delivery, such that the agent and expression construct would still be able to exert an advantageously combined effect on the cell. In such instances, it is contemplated that one may contact the cell with both modalities within about 12-24 h of each other and, more preferably, within about 6-12 h of each other. In some situations, it may be desirable to extend the time period for treatment significantly, however, where several d (2, 3, 4, 5, 6 or 7) to several wk (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respective administrations.

[0059] The Reishi F3 compositions can be administered in conjunction with other forms of cancer treatment such as surgery. Approximately 60% of persons with cancer will undergo surgery of some type, which includes preventative, diagnostic or staging, curative and palliative surgery. Curative surgery is a cancer treatment that may be used in conjunction with other therapies, such as the treatment of the present invention, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy and/or alternative therapies.

[0060] Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and/or destroyed. Tumor resection refers to physical removal of at least part of a tumor. In addition to tumor resection, treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and microscopically controlled surgery (Mohs' surgery). It is further contemplated that the present invention may be used in conjunction with removal of superficial cancers, precancers, or incidental amounts of normal tissue.

[0061] Upon excision of part of all of cancerous cells, tissue, or tumor, a cavity may be formed in the body. Treatment may be accomplished by perfusion, direct injection or local application of the area with an additional anti-cancer therapy. Such treatment may be repeated, for example, every 1 , 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, or 12 months. These treatments may be of varying dosages as well.

[0062] It is contemplated that other agents may be used in combination with the Reishi F3 fraction compositions of the present invention to improve the therapeutic efficacy of treatment. These additional agents include immunomodulatory agents, agents that affect the up-regulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, inhibitors of cell adhesion, or agents that increase the sensitivity of the hyperproliferative cells to apoptotic inducers. Immunomodulatory agents include tumor necrosis factor; interferon alpha, beta, and gamma; IL-2 and other cytokines; F42K and other cytokine analogs; or MIP-1, MIP-1B, MCP-1, RANTES, and other chemokines. It is further contemplated that the up regulation of cell surface receptors or their ligands such as Fas/Fas ligand, DR4 or DR5/TRAIL would potentiate the apoptotic inducing abilities of the present invention by establishment of an autocrine or paracrine effect on

hyperproliferative cells. Increases intercellular signaling by elevating the number of GAP junctions would increase the anti-hyperproliferative effects on the neighboring hyperproliferative cell population. In other embodiments, cytostatic or differentiation agents can be used in combination with the present invention to improve the anti-hyerproliferative efficacy of the treatments.

Inhibitors of cell adhesion are contemplated to improve the efficacy of the present invention.

Examples of cell adhesion inhibitors are focal adhesion kinase (FAKs) inhibitors and Lovastatin. It is further contemplated that other agents that increase the sensitivity of a hyperproliferative cell to apoptosis, such as the antibody c225, could be used in combination with the present invention to improve the treatment efficacy.

[0063] The Reishi F3 Fraction can be co-formulated or co-administered with several

chemotherapeutic agents that are in use in the treatment of cancer, including alkylating agents, antimetabolites antagonists, anticancer antibiotics, and plant-derived anticancer agents. Examples of "alkylating agents" include nitrogen mustard, nitrogen mustard-N-oxide hydrochloride, chlorambutyl, cyclophosphamide, ifosfamide, thiotepa, carboquone, improsulfan tosylate, busulfan, nimustine hydrochloride, mitobronitol, melphalan, dacarbazine, ranimustine, estramustine phosphate sodium, triethylenemelamine, carmustine, lomustine, streptozocin, pipobroman, etoglucid, carboplatin, cisplatin, miboplatin, nedaplatin, oxaliplatin, altretamine, ambamustine, dibrospidium hydrochloride, fotemustine, prednimustine, pumitepa, ribomustin, temozolomide, treosulphan, trophosphamide, zinostatin stimalamer, carboquone, adozelesin, cystemustine, and bizelesin. Examples of "antimetabolites" include mercaptopurine, 6-mercaptopurine riboside, thioinosine, methotrexate, enocitabine, cytarabine, cytarabine ocfosfate, ancitabine hydrochloride, 5-FU drugs (e.g., fluorouracil, tegafur, UFT, doxifluridine, carmofur, gallocitabine, emmitefur), aminopterine, leucovorin calcium, tabloid, butocine, folinate calcium, levofolinate calcium, cladribine, emitefur, fludarabine, gemcitabine, hydroxycarbamide, pentostatin, piritrexim, idoxuridine, mitoguazone, thiazophrine, and ambamustine, etc. Examples of "anticancer antibiotics" include actinomycin-D, actinomycin-C, mitomycin-C, chromomycin-A3, bleomycin hydrochloride, bleomycin sulfate, peplomycin sulfate, daunorubicin hydrochloride, doxorubicin hydrochloride, aclarubicin hydrochloride, pirarubicin hydrochloride, epirubicin hydrochloride, neocarzinostatin, mithramycin, sarcomycin, carzinophilin, mitotane, zorubicin hydrochloride, mitoxantrone hydrochloride, and idarubicin hydrochloride, etc. Examples of "plant-derived anticancer agents" include etoposide phosphate, vinblastine sulfate, vincristine sulfate, vindesine sulfate, teniposide, paclitaxel, docetaxel, and vinorelbine, etc.

[0064] An exemplary drug for co-administration with the Reishi F3 compositions is cisplatin. Cisplatin has been widely used to treat cancers such as metastatic testicular or ovarian carcinoma, advanced bladder cancer, head or neck cancer, cervical cancer, lung cancer or other tumors.

Cisplatin (CDDP) can be administered alone or in combination with Reishi F3 agents, with efficacious doses used in clinical applications of 5-20 mg/m² for 5 days every three weeks for a total of three courses. Exemplary doses may be 0.50 mg/m², 1.0 mg/m², 1.50 mg/m², 1.75 mg/m²,

2 2 2 2 2 2 2

2.0 mg/m , 3.0 mg/m , 4.0 mg/m , 5.0 mg/m , 10 mg/m , 20 mg/m , 50 mg/m . Of course, all of these dosages are exemplary, and any dosage in-between these points is also expected to be of use in the invention. Cisplatin is not absorbed orally and must therefore be delivered via injection intravenously, subcutaneously, intratumorally or intraperitoneally.

[0065] Hormonal therapy may also be used in conjunction with the present invention or in combination with any other cancer therapy previously described. The use of hormones may be employed in the treatment of certain cancers such as breast, prostate, ovarian, or cervical cancer to lower the level or block the effects of certain hormones such as testosterone or estrogen. This treatment is often used in combination with at least one other cancer therapy as a treatment option or to reduce the risk of metastases.

[0066] By the term "administering," it is meant that the Reishi F3 compositions are delivered to the host in such a manner that it can achieve the desired purpose. As mentioned the compositions can be administered by an effective route, such as orally, topically, rectally, etc. The compositions can be administered to any host in need of treatment, e.g., vertebrates, such as mammals, including humans, male humans, female humans, primates, pets, such as cats and dogs, livestock, such as cows, horses, birds, chickens, etc. [0067] An effective amount of the Reishi F3 compositions are administered to such a host.

Effective amounts are such amounts which are useful to achieve the desired effect, preferably a beneficial or therapeutic effect as described above. Such amount can be determined routinely, e.g., by performing a dose-response experiment in which varying doses are administered to cells, tissues, animal models to determine an effective amount in achieving an effect. Amounts are selected based on various factors, including the milieu to which the Reishi extract is administered (e.g., a patient with cancer, animal model, tissue culture cells, etc.), the site of the cells to be treated, the age, health, gender, and weight of a patient or animal to be treated, etc. Useful amounts include, 10 milligrams- 100 grams, preferably, e.g., 100 milligrams-10 grams, 250 milligrams-2.5 grams, 1 gm, 2 gm, 3 gm, 500 milligrams- 1.25 grams, etc., per dosage of different forms of the compositions such as the Reishi F3 powder, Reishi F3 paste, or Reishi F3 infusions and beverages prepared to contain the effective ingredients of Reishi F3, and injections, depending upon the need of the recipients and the method of preparation.

[0068] The present invention can be utilized with a variety of excipients. The Reishi F3 compositions of the present invention can be in any form which is effective, including, but not limited to dry powders, grounds, emulsions, extracts, and other conventional compositions.

[0069] The Reishi F3 compositions of the present invention can be administered in any form by any effective route, including, e.g., oral, parenteral, enteral, intraperitoneal, topical, transdermal (e.g., using any standard patch), ophthalmic, nasally, local, non-oral, such as aerosol, inhalation, subcutaneous, intramuscular, buccal, sublingual, rectal, vaginal, intra-arterial, and intrathecal, etc. It can be administered alone, or in combination with any ingredient(s), active or inactive, including in a medicinal form, or as a food or beverage additive.

[0070] In preferred embodiments of the invention, the compositions are administered orally in any suitable form, including, e.g., pill, capsule, granule, tablet or a suspension.

[0071] The compositions can be combined with any pharmaceutically acceptable carrier. By the phrase, "pharmaceutically acceptable carriers," it is meant any pharmaceutical carrier, such as the standard carriers described, e.g., Remington: The Science and Practice of Pharmacy. Lippincott Williams & Wilkins; Twenty first Edition (2005). Examples of suitable carriers are well known in the art and can include, but are not limited to, any of the standard pharmaceutical carriers such as a phosphate buffered saline solutions, phosphate buffered saline containing Polysorb 80, water, emulsions such as oil/water emulsion and various types of wetting agents. Other carriers may also include sterile solutions, tablets, coated tablets pharmaceutical and capsules. Typically such carriers contain excipients such as such as starch, milk, sugar, certain types of clay, gelatin, stearic acid or salts thereof, magnesium or calcium stearate, talc, vegetable fats or oils, gums, glycols. Such carriers can also include flavor and color additives or other ingredients. Compositions comprising such carriers are formulated by well known conventional methods. Generally excipients formulated with the compositions are suitable for oral administration and do not deleteriously react with it, or other active components.

[0072] Suitable pharmaceutically acceptable carriers include but are not limited to water, salt solutions, alcohols, gum arabic, vegetable oils, benzyl alcohols, gelatin, carbohydrates such as lactose, amylose or starch, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid monoglycerides and diglycerides, pentaerythritol fatty acid esters, hydroxy methylcellulose and the like. Other additives include, e.g., antioxidants and preservatives, coloring, flavoring and diluting agents, emulsifying and suspending agents, such as acacia, agar, alginic acid, sodium alginate, bentonite, carbomer, carrageenan, carboxymethylcellulose, cellulose, cholesterol, gelatin, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose, octoxynol 9, oleyl alcohol, povidone, propylene glycol monostearate, sodium lauryl sulfate, sorbitan esters, stearyl alcohol, tragacanth, xanthan gum, and derivatives thereof, solvents, and miscellaneous ingredients such as microcrystalline cellulose, citric acid, dextrin, dextrose, liquid glucose, lactic acid, lactose, magnesium chloride, potassium metaphosphate, starch, and the like.

[0073] The compositions can also be formulated with other active ingredients, such as antioxidants, vitamins (A, C, ascorbic acid, B's, such as B l, thiamine, B6, pyridoxine, B complex, biotin, choline, nicotinic acid, pantothenic acid, B12, cyanocobalamin, and/or B2, D, D2, D3, calciferol, E, such as tocopherol, riboflavin, K, Kl, K2).

EXAMPLES

[0074] Without intent to limit the scope of the invention, exemplary instruments, apparatus, methods and their related results according to the embodiments of the present invention are given below. Note that titles or subtitles may be used in the examples for convenience of a reader, which in no way should limit the scope of the invention. Moreover, certain theories are proposed and disclosed herein; however, in no way they, whether they are right or wrong, should limit the scope of the invention so long as the invention is practiced according to the invention without regard for any particular theory or scheme of action.

EXAMPLE 1: Cell lines

[0075] A549, a human lung adenocarcinoma cell line, was obtained from Bioresource Collection and Research Center (BCRC, Hsinchu, Taiwan). Cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and 2mM L-glutamine (Life Technologies, Inc., MD). Adherent cells were detached by incubation with trypsin-EDTA. Murine Lewis lung carcinoma (LLC, H-2b, ATCC: CRL-1642) was obtained from BCRC and cultured in RPMI 1640 and Dulbecco's modified Eagle's medium (GIBCO-Life Technologies) supplemented with 1.5 g/L of NaHC03 and 10% fetal bovine serum (FBS). All cells were maintained at 37°C in a humidified atmosphere of 5% C0₂-95% air.

EXAMPLE 2: Reagents and antibodies

[0076] Recombinant human TGF-βΙ (R&D), anti-E-cadherin (BD bioscience), anti-N-cadherin (BD bioscience), anti-vimentin (Sigma), anti-y-catenin (Santa Cruz), anti-phospho-smad2/3 (Santa Cruz), anti-snail (Abeam), anti-slug (Cell Signaling), anti-TGF- RII (Santa Cruz), anti- -actin (Santa Cruz).

EXAMPLE 3: Mice

[0077] Male C57BL/6 (H-2b; 6-8 weeks of age) mice were purchased from National Laboratory Animal Center (Taipei, Taiwan). Mice were raised under a license from the Institutional Animal Care and Use Committee at the National Yang-Ming University.

EXAMPLE 4: Preparation of Reishi F3 Fraction Powder

[0078] Preparation of Reishi Crude Extract: Reishi (Ganoderma lucidum) fruit body was pulverized into approximately 5mm pellets, these fruit body pellets were added into 0.04N NaOH solution (1 kg pellet/12 liter solution) which was pre-heated to 80°C, and the mixture was stirred gently for 3 hours at 80°C. The solution was cooled to room temperature and the pH was adjusted to 7.2±0.2 by 32% HCl. Celite (1 kg/100 liter solution) was added to the solution, stirred, then filtered through filter-press system to obtain the clear Reishi Crude Extract. The HPLC (high pressure liquid chromatography) pattern, which shows many different molecular weight size peaks, of this Reishi Crude Extract is shown in Fig. 5.

[0079] Preparation of Reishi F3 Fraction: The Reishi Crude Extract was further purified by using 100KD tangential flow filtration system (Millipore Corporation, USA) to remove the small molecular weight fractions, components and NaCl. The HPLC pattern of the purified Reishi F3 Fraction is shown in Fig 6.

[0080] Drying of Reishi F3 Fraction Powder: The water was removed from the purified solution of Reishi F3 Fraction by a standard lyophilization process. [0081] The Reishi F3 Fraction disclosed herein has been shown to be identical to the fraction-3, 155 to 205 mL in Fig 1 of US Patent No. 7,135, 183, incorporated herein in its entirety by reference. The methods used to compare these fractions were HPLC finger-print pattern and cytokine expression pattern.

[0082] Reishi F3 Fraction composition: The Reishi F3 Fraction Powder was dissolved in double- distilled H₂0 or PBS. The solution was stirred at 25°C for 30 min, and centrifuged (1800 x g) at 4°C for 10 min. The supernatant (dissolution rate is 50%) was filtered using 0.22μιη sterile filters (Millipore, Inc., Temecula, CA) as described (Hsu H-Y, et al. (2004) Extract of Reishi

Polysaccharides Induces Cytokine Expression via TLR4-Modulated Protein Kinase Signaling Pathways. J Immunol 173(10):5989-5999).

EXAMPLE 5: Preparation of whole cell extracts

[0083] For analysis of protein expression, cells (4 x 10⁵ on a 6 cm tissue culture plate) were cultured and then treated with Reishi F3 fraction (60, 120 and 180 μg/ml) or vehicle. Cells were rinsed one time with cold PBS containing 1% a₃V04, and lysed in situ with 40 μΐ lysis buffer, the cell lysed was kept on ice for 10 min, and then centrifuged for 12000 X rpm for 10 min at 4°C. The protein concentration of supernatant solution was determined using the Bradford protein assay (Bio-Rad, Hercules, CA) with bovine serum albumin as the standard.

EXAMPLE 6: Western Blot Analysis

[0084] Cells were harvested after the incubation period and lysis buffer was added. Equal amounts of proteins were s loaded onto 10% SDS-PAGE and transferred into Immobilon® PVDF membrane via wet transfer blotting system (Blot Electrophoretic Transfer Cell, Bio-Red; transfer buffer: 25 mM Tris-base, 192 mM glycine, 0.1% SDS and 20% methanol, at 250 niA for 100 min) for Western blot analysis.

EXAMPLE 7: In vitro cell migration, invasion and soft-agar colony formation assay

[0085] Cell migration assay were done using 6.5-mm Costar Transwell^® chambers (8 μιη pore size; Corning, NY). After LLC cells were treated with Reishi F3 fraction

were seeded onto the Transwell^® chambers. Cell invasion assay using the Transwell^® filters were coated with appropriate Matrigel^® (Becton Dickinson, Franklin Lakes, NJ). After 24h incubation, the filter was gently removed from the chamber and the noninvasive cells on the upper surface were removed by wiping with a cotton swab. The cells were fixed with methanol for 10 minutes. Cells that migrated to the lower side of the membranes were stained with Liu stain (Handsel Technologies, Inc., Taipei, Taiwan). Cells were examined under microscopy at 100X magnification. For studies of soft-agar colony formation assay, LLC cells were plated in 6-well plates (1500 cells per well) with 0.33% agar on a 0.5% agar (Noble agar, Becton Dickinson) under layer containing DMEM containing 10% FBS. Cells were cultured for 16 days and colonies were counted.

EXAMPLE 8: In vivo metastasis assay

[0086] LLC cells (2 x 10⁵ cells) were injected into the lateral tail vein of C57BL/6 mice. Mice were randomized to six treatment groups of PBS, Reishi F3 fraction (ip), Reishi F3 fraction (oral), cis- diamminedichloroplatinum (II) (CDDP or cisplatin)/Reishi F3 fraction (ip), or CDDP/Reishi F3 fraction (ip). Mice were treated with CDDP (0.5 mg/g) i.p. injection on day 7. At the same time, mice were treated with Reishi F3 fraction (0.075 mg/g) by i.p. injection at intervals of 2 days, or daily oral administration with Reishi F3 fraction (0.15 mg/g). Mice were sacrificed after 4 weeks, and all organs were examined for metastasis formation. The lungs were removed and fixed in 10% paraformaldehyde. The number and tumor volume of lung tumor colonies were counted. Tumor volume was calculated as length x width x height x 0.5236 (in mm³) with reference to a previous report (Santini D, Vincenzi B, Tonini G, Scarpa S, & Baldi A (2003) Correspondence Re: E. Corey et al, Zoledronic Acid Exhibits Inhibitory Effects on Osteoblastic and Osteolytic Metastases of Prostate Cancer. Clin. Cancer Res., 9: 295-306, 2003. Clinical Cancer Research 9(8):3215-3215). Representative lung tumors were removed, fixed, and embedded in paraffin. Embedded tissues were stained with H&E for histological analysis.

EXAMPLE 9: Statistical Analysis

[0087] Data were expressed as mean ± SD and statistical significances were examined by Student's t-test.

[0088] All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

[0089] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claim.

Claims

CLAIMS What is claimed is:

1. A pharmaceutical composition comprising:

one or more sub-fractions of an extract of Ganoderma lucidum (Reishi), wherein the one or more sub-fractions are present in an amount sufficient to reduce migration and invasion of non- small cell lung cancer (NSCLC) cells; and

optionally, a pharmaceutically acceptable excipient.

2. A pharmaceutical composition comprising:

one or more sub-fractions of an extract of Ganoderma lucidum (Reishi), wherein the one or more sub-fractions are present in an amount sufficient to modulate epithelial-mesenchymal transition (EMT); and

optionally, a pharmaceutically acceptable excipient.

3. The composition of claim 1, wherein the reduction in migration and invasion of cancer cells relates to immune-modulation by the Reishi extract.

4. The composition of claims 1, 2 or 3, wherein the one or more sub-fractions are obtainable by a method comprising the step of extracting homogenized tissue of Ganoderma lucidum with aqueous alkaline solution.

5. The composition of claim 4, wherein the alkaline extract is neutralized to a pH of about 7.2 ± 0.2 and filtered to obtain a clear Reishi Crude Extract.

6. The composition of claim 4 or 5, wherein the clear Reishi Crude Extract is subjected to tangential flow filtration such as to remove low molecular weight components and obtaining a purified Reishi extract.

7. The composition of claim 4, wherein the Reishi extract displays an HPLC profile according to Figure 6.

8. The composition of claims 4 or 7, wherein the Reishi extract comprises a polysaccharide or glycopeptide comprising terminal fucose residues.

9. The composition of claims 1, 2 or 3, wherein the epithelial-mesenchymal transition (EMT), or migration and invasion of non-small cell lung cancer (NSCLC) cells are associated with a solid tumor selected from non-small cell lung cancer (NSCLC), pancreatic cancer, colorectal cancer, and hepatocellular cancer.

10. A method for preventing, treating or reducing a cancer-associated event in a cell, the method comprising:

contacting the cell with a composition comprising a Reishi extract, wherein the composition comprises an amount of Reishi F3 fraction sufficient for (a) modulating epithelial-mesenchymal transition (EMT), or (b) reducing migration and invasion of lung cancer cells, or both.

1 1. The method of claim 10, wherein the Reishi extract is an alkaline extract.

12. The method of claim 10, wherein the Reishi extract is purified to having a HPLC profile according the Figure 6.

13. The method of claim 10, wherein the cell is a mammalian cell.

14. The method of claim 13, wherein the mammalian cell is in a human.

15. The method of claim 13, wherein the mammalian cell is in a non-human animal.

16. The method of claim 10, wherein the cancer cell comprises neuroblastoma, melanoma, non- Hodgkin's lymphoma, Epstein-Barr related lymphoma, Hodgkin's lymphoma, retinoblastoma, small cell lung cancer, brain tumors, leukemia, epidermoid carcinoma, prostate cancer, renal cell carcinoma, transitional cell carcinoma, breast cancer, ovarian cancer, lung cancer colon cancer, liver cancer, stomach cancer, and other gastrointestinal cancers.

17. The method of claim 10, wherein the cancer cell comprises a solid tumor selected from non- small cell lung cancer (NSCLC), pancreatic cancer, colorectal cancer, breast cancer and hepatocellular cancer.

18. The method of claim 10, wherein modulating epithelial-mesenchymal transition (EMT) comprises reduction of one or more symptoms selected from:

loss of epithelial-cell markers;

loss of cell polarity and cell-junction proteins by epithelial tumor cells; and

acquisition of protein mesenchymal-cell markers.

19. The method of claim 10, wherein modulating epithelial-mesenchymal transition (EMT) comprises depletion of EMT with one or more effects selected from: conversion of fibroblastic to epithelial morphology;

up-regulation of major epithelial cell markers expression; and

down-regulation of mesenchymal cell markers expression.

20. The method of claim 18 or 19, wherein the EMT is induced by EMT -related signaling pathways driven via receptors such as platelet derived growth factor receptor (PDGFR); fibroblast growth factor receptor (FGFR); cMET; TGFBR; IGF-1R; and kinases selected from PI3K, AKT and mTOR.

21. The method of claim 18 or 19, wherein the EMT is induced by TGF-β 1.

22. The method of claim 21, wherein the reduction of EMT comprises suppression of TGF-βΙ- mediated signal transduction comprising one or more effects selected from (i) reduction of TGF-βΙ production, (ii) down-regulation of TGF-β receptor II expression, (iii) decrease of Smad2/3 phosphorylation, and (iv) decrease of Snail protein expression.

23. The method of claim 18 or 19, wherein the epithelial-cell markers are selected from E-cadherin and γ-catenin.

24. The method of claim 18 or 19, wherein the protein mesenchymal-cell markers are selected from vimentin, fibronectin, and N-cadherin.

25. The method of claim 10, wherein reducing migration and invasion of lung cancer cells comprises an anti-metastatic effect.

26. The method of claim 25, wherein the lung cancer cells are non-small cell lung cancer ( SCLC) cells.

27. The method of claim 25, wherein the anti-metastatic effect comprises reducing a Mesenchymal Epithelial Transition (MET) and thereby reducing proliferation and growth of epithelial tumor cells at sites distal from the primary tumor.

28. The method claim 27, wherein the Reishi F3 fraction is administered in conjunction with a therapeutically effective amount of a chemotherapeutic agent selected from cisplatin, doxorubicin, Taxol, daunorubicin, mitomycin, actinomycin D, bleomycin, VP 16, tumor necrosis factor, vincristine, vinblastine, carmustine, melphalan, cyclophosphamide, chlorambucil, bisulfan, lomustine, penicillin, erythromycin, amoxicillin, cefazolin, imipenem, aztreonam, sulbactam, linezolid, gentamicin, sulfamethoxazole, vancomycin, ciprofloxacin, fusidic acid, trimethoprim, metronidazole, clindamycin, mupirocin, amphotericin B, rifampin, fluconazoleor, or a combination thereof.

29. The method claim 27, wherein the Reishi F3 fraction is administered in conjunction with a therapeutically effective amount of czs-diamminedichloroplatinum (II) (CDDP or cisplatin).

30. The method of claim 29, wherein the cisplatin is administered intraperitoneally.

31. The method of claim 29, wherein administration of Reishi F3 fraction with cisplatin reduces distant metastases by one or more of (a) reducing lung tumor multiplicity, and (b) reducing tumor volume,

wherein the tumor multiplicity or volume is reduced by an amount greater than that achieved by administration of cisplatin alone.

32. The method claim 27, wherein the Reishi F3 fraction is administered in conjunction with a an anti-cancer therapy selected from a drug, a hormone, a gene therapy composition, a radionuclide, a nutraceutical, a surgical procedure, a radiation procedure or a combination thereof.

33. The method of claim 32, wherein the Reishi F3 fraction is administered prior to or following the anti-cancer therapy.

34. The method of any of claims 10 through 33, wherein the Reishi F3 fraction is administered orally, intravenously, subcutaneously, intramuscularly, intraperitoneally, intranasally or transdermally, concurrently or sequentially.