Introduction

Key points: Question: What are the most effective treatments in late stage of gynecological cancers? Finding: Since our patients have failed chemotherapy and radiation therapy, we are looking for new method to treat those clinical situations. We used Comosain in cancer treatment through our experience and literature evidence, which showed Comosain with remarkable effectiveness against most tumors. Meaning: In our clinical trial, we entered 14 patients into low dose cohort and 21 patients into high dose group. We transferred low dose group into high dose cohort due to ineffectiveness after 6 weeks. Total number of 35 high dose group patients were discussed and analyzed.

Purpose

Administered of oral Glyco-polypeptides (Comosain) in cancer treatment in nonclinical trials has been reported as early in 1968 by Wolf M, & Ransberger K [1]. In vitro and animal studies have suggested of anti-metastatic effect for Glycopolypeptides (Comosain). Batkin & Taussig in 1988 reported that orally administered Glyco-polypeptides (Comosain) reduced the incidence of pulmonary metastasis in Lewis lung cancer cells in mice [2,3]. In recent years, 1988 Batkin & Taussig suggested the antitumor mechanisms are due to fibrinolytic effect in Glyco- polypeptides (Comosain) [4]. Taussig & Batkin in 1988 discovered that Glyco-polypeptides (Comosain) has anti-Platelet aggregation effects [5]. Taussig and Batkin in 1985 also discovered Inhibition the growth of tumor cells such as Lewis lung carcinoma, V-8 lymphoma, MC1-1 acites, KATO-gastric carcinoma cells [5]. Maurer & Hozumi, in 1994 Discovered Glyco- polypeptides (Comosain) Induced Differentiation in leukemic cells [6]. Hale, & Haynes in 1992 and Cantrell et al. in 1996 had suggested that due to Major Histocompatibility Protein Kinases, such as MMAPK (Major Mitogen Activating Protein Kinase) and TPK (Tyrosine Phosphorylation Kinase) inhibitors were activated by Glyco-polypeptides (Comosain) [7]. T-cell activation and cascade production of Interleukin 2, 6, 8, and TNF-a (Tumor Necrotizing Factors) via CD-2, CD-3 surface antigen of WBC [8-14].

Garbin, Harrach, Eckert, & Maurer in 1994 [15] and Hale, & Haynes in 1992 [7] also suggested that Glyco-polypeptides(Comosain) will reduce surface antigens of CD-44, CD-44v, CD44s, CD45, & CD 47 in tumor cells of breast carcinoma. From the experimental studies above, we conclude that activation of Glycopolypeptides (Comosain) in lymphocytes and T-cells have antimetastatic effects both in vitro and in vivo.

In the present study, we compared the modulation of low dose cohort and high dose cohort of Comosain administration to the patients with stage 3, and stage 4, refractory solid tumors, which including various types of carcinoma of lung, prostate and bladder. All patients failed previously on at least two regimens of chemotherapy and /or failed on radiation therapy, The treatment were carried out for at least 24 to 28 weeks, the complete blood count, liver, renal function, hematopoietic elements, tumor markers were evaluated at an interval of every 2 to 4 weeks, the computerized tomography scan were performed at an interval of every 3 to 4 months. The size of tumors were measured, the tumor markers were recorded for the evaluation of Complete Response (CR), Partial Response, (PR), Stable Disease (SD), and Progressive Disease (PD) according to the Standard Response Criteria of National Cancer Institute (NCI). The Common toxicity were recorded by using NCI’s Standard Toxicity Criteria. The results of CR and PR were promising and astonishing when Comosain were administered in patients of high dose group [16-20].

Materials and Methods

Glyco-polypeptides (Comosain) were purchased from Natural Organics Laboratories, Amityville, N.Y., Capsules to contain Glyco-polypeptides (Comosain) were purchased from Capusugel Co. Greenwood, North Carolina. Comosain were analyzed by using SDS-Polyacryl- Amide Gel Electrophoresis (SDS-PAGE), Cation Exchange Chromatography (CEC), Florescence High Performance Liquid Chromatography (FPLC) to determinate the purity and separation of Glyco-polypeptides (Comosain) fraction of F1, F2, F3, F4, F5, F6, F9 in stem.

Glyco-polypeptides (Comosain) were detected by Amperometric detection [9]. Monosaccharides fraction are L-fructose, D-galactosamine, D-glucosamine, D-xylose, D-mannose, D-glucose, D-galactose, D-fructose, and Deoxyribose [21].

Clinical Application and Study protocol

Patients Eligibility and Selection (Total number of patients: 35)

1. Patients with stage III and IV solid cancer of lung, prostate and bladder with tissue proof of well-documented malignancies, whether by tissue biopsies and have not been helped by conventional radiation therapy and/or chemotherapies for at least two separated regimens are eligible for this study [22,23].
2. Or patients must have no available therapy known to provide clinical benefit. For example, the lung, prostate and bladder cancer patients must have failed at least 2 chemotherapy regiments in the metastatic setting [24,25].

Additionally

 Patient’s age is between 18 and 95+ years, not taking anticoagulants, have no history of abdominal fistula, gastro-enteral perforation, peptic ulcer diseases, or intra-abdominal abscess within 4 months prior to study enrollment, and patient has not had major surgery within 4 weeks prior to study enrollment, and other requirements are same as NCI’s criteria. Also, Patient does not have uncontrolled hypertension, diabetes, or cardiac arrhythmia, and not allergic to Glyco-polypeptides (Comosain) -containing products, not pregnant or breastfeeding. Patient’s WBC count <3K/uL, hemoglobin <9.0 g/dL, platelet counts must be <100,000/ uL, and INR <1.5 have no significant abnormal hepatic and/or renal function [26-29].

Patient’s tumors are measurable between 0.2-10+ cm in size and number between 1-15+. All measurable tumors that have spread to the bones, liver, lung, kidney, and abdomen will be included in the data analysis. Patients who are eligible for this study will be randomly assigned to either the low dose cohort or the high dose cohort by a coin toss. Each study subject will be assigned a patient number for the purpose of this study [30-35].

Methods of Study

The Dose of Comosain at 50 mg/kg/day is extrapolated from in vivo animal studies and determined to be safe by a Safety study on healthy human subjects. The High Dose cohort will be given Comosain at 50 mg /kg/day (at a body weight of 50-60 kg) to a maximum of 2400 mg /day and divided into 2 doses/day of 1200 mg/dose, and taken with meals. Low dose Cohort patients will be given Comosain at 10mg/kg/day that is 500 mg/day, divided into two doses of 250 mg /dose and taken with meals [36-40].

High Dose Cohort: The number of patients will be at least 21. Low dose Cohort: The number of patients will be at least 14.

1. Blood/laboratory tests will be scheduling every 2-4 weeks, which include CBC, Chemistry-7, Chemistry-24, liver and renal function, CEA, CA125, CA153, CA199, PSA, TSH, alfa-Feto- Protein and other tumor markers [41,42].
2. Radiological tests will be assessed every 3-4 months. Each patient will be also assessed every 4 weeks for any side effects that they may have experienced.
3. Using NCI standard toxicity criteria for hematology, renal, and hepatic system evaluation.
4. Adverse events, serious adverse events reporting information also using NCI criteria [43-45].

Duration and Route of Administration

The patients will be evaluated by blood tests and/or CT scans at the end of each 6 weeks cycle and at six months for signs of disease progression. If the disease did not progress, then treatment will continue, and the patient will be evaluated every six months thereafter until the investigator determines otherwise. If the disease did progress, then the patient will be taken off the study. On the Humanitarian base, the low dose group patients will be transferred to the high dose group due to lack of efficacy in the treatment [4650].

Results

Results 1

At the end of six months, each patient will be determined whether or not to continue with this therapy and assess the efficacy of the therapy by using NCI Standard response Criteria:

I. Evaluation of Target Lesions

1. Complete Response (CR): Disappearance of all Target lesions, and lymph nodes must be reduced <10mm.
2. Partial Response (PR): At least a 30% decrease in the sum of the diameters of target lesions compared with the baseline sum diameters [51,52].
3. Progressive Disease (PD): At least a 20% increase in the sum of the diameters of target lesions compared with the smallest sum on study. In addition, the sum must demonstrate an absolute increase of at least 5 mm. The appearance of new lesions is also considered progressions.
4. Stable Disease (SD): Neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for PD, taking as reference to the smallest sum diameters while on study [53].

II. Evaluation of Non-Target Lesions

1. Complete Response (CR): Disappearance of all nontarget lesions and normalization of tumor marker level. All lymph nodes must be <10mm short axis [54].
2. Non-CR/ Non-PD: Persistence of one or more non-target lesion(s) and /or maintenance of tumor marker level above the normal limits.
3. Progressive Disease (PD): Appearance of one or more new lesions and/ or unequivocal progression of existing non-target lesions. Unequivocal progression should not normally trump target lesion status [55,56].

Results 2

Age distribution, all the patients are mainly in their 6 decade and above. The disease classification and distribution are as following: lung carcinoma account for 45.7% (16/35), in prostate carcinoma the incidence is 42.8% (15/35), in bladder carcinoma the incidence are 11.4% (4/35) (Table 1) [57-61].

The tumor markers such as CEA,CA-125, CA-199, PSA, and alpha-feto-protein are been monitored, their values are corresponding to the tumor masses, they return to normal value when tumor have CR, and when the tumor progress the tumor marker value are elevated [62-65].

The serious adverse effect in toxicity in both groups are not observed, there were no serious hematopoietic or hepato-renal toxicity, no anaphylactic reaction or life threaten events. There were rarely minor side effects such as nausea, vomiting, diarrhea, palpitation, headache, insomnia, pruritus, urticaria, and skin rash. We conclude that Glyco-polypeptides (Comosain, Ananases) administered in an amount of 2500 to 3000 mg/day to the patients with average body weight are effective and non-toxic [66-69].

Conclusion

In summary, Glyco-polypeptides (Comosain) administration in double-blind study showed effectiveness only in patients with high dose cohort of 50 mg/kg/day regimen [70,71]. The low dose cohort showed no efficacy at all. Both groups did not show serious adverse effects such as leukopenia, anemia, hepato-renal toxicity, anaphylactic reaction, and life-threaten events. Minor adverse effects such as nausea, vomiting, diarrhea, urticaria, insomnia, palpitation, pruritus, and headache occurred rarely. The remarkable cancercidal effects probably due to massive production of Interleukin-II, VI, VIII, and tumor necrotizing factors from CD2, CD-3 in monocytes and lymphocytes. The fibrinolytic effects on tumor surface antigens of CD-44, CD-44V, CD-44S, CD-45, and CD-47 which induce dehydration, necrosis, and possible calcification in the tumor cells. This action mechanism of Glycopolypeptides (Comosain) is mainly attributed to inhibition of 2 kinases: Major Mitogen Activating Protein Kinases and Tyrosine Phosphorylation Kinases [72,73]. In the WBC culture test with concentration of Glyco-polypeptides (Comosain) in an amount of 1 mg/ml will increase the production of Interleukin II by 400 times/106 WBC, Interleukin-6 by 650 time/10⁶ WBC, and the TNF by 42 times/10⁶ WBC.

The results in the high dose group patients showed remarkable CR rates of 74%, PR rate of 20%, SD rate of 0%, PD rate of 5.7%. Dr. HR Maurer in his complimentary tumor therapy also showed Glyco-polypeptides (Comosain) in an amount of 1000-to-3000 mg/day for the period of 1 to 3 years has no severe side effects nor any life threaten events.

Acknowledgement

The authors sincerely thank Dr. Harvowitz at Cal. Tech Institute for her expertise assistance in molecular protein sequence analysis and assay in SDS-PAGE mass spectroscopy. Dr. M. Fishbein of Department of Pathology at UCLA for his expertise assistance in Pathology. Editorial assistance of Miss Beth Shirokawa, Miss Janet Shih and Miss Qiting Ma are gratefully acknowledged. From Department of Molecular Biology, Biochemistry, Surgical Oncology, Garfield Medical

Group, Oeyama-Moto Cancer Research Foundation, and King / Drew Medical University, Los Angeles, California. This research was supported in part by a grant from The Oeyama-Moto Cancer Research Foundation Grant # (OMCR2002).

Case 1: Lung cancer pre and post treatments.

Case 2: Lung cancer pre and post treatments.

Case 3: Lung cancer pre and post treatments.

Case 4: Prostate cancer pre and post treatments.

Case 1: Breast cancer pre and post treatment (Kidney mets).

Case 2: Breast cancer pre and post treatment (Bone Mets).

Case 3: Breast cancer pre and post treatment (Liver Mets, 9.2 cm shrinkage to 2.6 cm).

Case 4: Ovarian cancer pre and post treatment. Ovarian Cancer 4th.

References

1. Harrach T, Eckert K, Maurer HR, Machleidt I, Machleidt W, et al. (1998) Isolation and characterization of two forms of an acidic bromelain stem J Protein Chem 17: 351-361.
2. Barnes S et al. (1995) Effect of genistein on in vitro and in vivo models of cancer. J Nur 125: 777S-783S.
3. So FV, Guthrie N, Chambers AF, Moussa M, Carroll KK (1996) Inhibition of human breast cancer cell proliferation and delay of mammary tumorigenesis by flavonoids and citrus juices. Nutr Cancer 26: 167-181.
4. Batkin S, Taussig SJ, Szekerezes J (1988) Antimetastatic effect of bromelain with or without its proteolytic and anticoagulant activity. J Cancer Res Clin Oncol 114: 507-508.
5. Birch M, Miychell S, Hart IR (1991) Isolation and characterization of human melanoma cell variants expressing high and low levels of CD Cancer Res 51: 6660-6667.
6. Cantrell D (1996) T cell antigen receptor signal transduction pathways. Annu Rev Immunol 14: 259-274.
7. Castillo MH, Perkins E, Campbell JH, Hassett JM, Kandaswami C, et al. (1989) The effect of Bio-flavonoid-Quercetin on squamous cell carcinoma of head and neck origin. Am J Surg 158: 351-355.
8. Cooreman W (1978) “Bromelain” In: Pharmacological EnzymesProperties and Assay Method. Ruyssen R, Lauwers A (Editors). Pp: 107-121. Story-Scientia Scientific Publishing Co., Gent, Belgium.
9. Denning SM, Le PT, Singer KH, Haynes BF (1990) Antibodies against the CD44 p80, Lymphocyte homing receptor molecule augment human peripheral blood T-cell activation. J Immunol 144: 7-15.
10. Desser L, Rehberger A, Paukovits, W (1994) Proteolytic enzymes and amylase induce cytokine production in human peripheral blood mononuclear cells in vitro. Cancer Biother 9: 253-263.
11. Desser L, Rehberger A, Kokron E, Paukovits W (1993) Cytokine synthesis in human peripheral blood mononuclear cells after oral administration of polyenzyme preparation. Oncology. 50: 403-407.
12. Eckert K, Grunberg E, Garbin F, Maurer HR (1997) Preclinical studies with prothymosin a-1 on mononuclear cells from tumor patients. Int J Immunopharmacol 19: 493-500.
13. Eckert K, Grabowska E, Strange R, Schneider U, Maurer HR, et (1999) Effects of oral bromelain administration on the impaired immunocytoxicity of mononuclear cells from mammary tumor patients. Oncol Rep 6: 1191-1199.
14. So FV, Guthrie N, Chambers AF, Moussa M, Carroll KK (1996) Inhibition of breast cancer cell proliferation and delay of mammary tumor-genesis by flavonoids and citrus juices. Nutr Cancer 26: 167
15. Iyu F, Lysogorskaya EN, Oksenoit ES, Rudenskaya GN, Stepanov VM (1984) In L-pyroglutamyl-L-Phenyl-Alanayl-L-Leucin-P-Nitroaniline, A chromogenic substrate for thio-proteinase assay. Anal Biochem 143: 293-297.
16. Gallatin WM, Wayner EA, Hoffman PA, John TSt, Carter WG (1989) Structure homology between lymphocyte receptor for high endothelium and class III extracellular matrix receptor. Proc Natl Acad Sci USA 86: 4654-4658.
17. Garbin F, Harrach T, Eckert K, Maurer H (1994) Bromelain proteinase F9 augments human lymphocyte- mediated growth inhibition of various tumor cells in vitro. Int J Oncol 5: 197-203.
18. Garbin F, Harrach T, Eckert K, Buttner P, Maurer HR (1994) Bromelain Proteinase F9 augments human lymphocyte-mediated growth inhibition of various tumor cells in vitro. Inte J Oncol 5: 197-203.
19. Garbin F, Eckert K, Buttner P, Garbe C, Maurer HR (1994) Prothymosin a-1 augments deficient antitumor activity of monocyte from melanoma patients in vitro. Anticancer Res 14: 2405-2411.
20. Grabowska E, Eckert K, Fichiner I, Schultze-Forster K, Maurer H (1997) Bromelain proteases suppress growth, invasion and lung metastasis of B16F10 mouse melanoma cell. Int J Oncol 11: 243-248.
21. Gunthert U, Hofman M, Rudy W, Reber S, Zoller M, et al. (1991) A new variant of glycoprotein CD44 Confers metastatic potential to rat carcinoma cells. Cell. 65:13-24.
22. Harrach T, Gebauer F, Eckert K, Kunze R, Maurer H (1994) Bromelain proteinases modulate the CD44 expression on human Molt 4/8 leukemia and SK-Mel28 melanoma cells in vitro. Int J Oncol 5: 485
23. Harrach T, Eckert K, Schulze-Forster K, Nuck R, Grunow D, et al. (1995) Isolation and partial characterization of basic proteinases from stem bromelain. J protein chem 14: 41-52.
24. Harrach T, Eckert K, Maurer HR, Machleidt I, Machleidt W, et al. (1998) Isolation and characterization of two forms of an acidic bromelain stem J Protein Chem 17: 351-361.
25. Haynes BF, Telen MJ, Hale LP, Denning SM (1989) CD 44: a molecule involved in leucocyte adherence and T-cell activation. Immunol Today 10: 423-428.
26. Heinckie RM, van der Wal L, Yokoyama M (1971) Effect of bromelain (ananase) on human platelet aggregation, Experientia 28: 844-845.
27. Hofman M, Rudy W, Gunthert U, Zimmer SG, Zawadzki V, et al. (1993) A link between ras and metastatic behavior of tumor cells: ras induces CD44 promoter activity and leads to low level expression of metastatic specific variants of CD44 in CREF cells. Cancer Res 53: 1516-1521.
28. Huet S, Groux B, Caillou H, Valenton, Prieur AM, et al. (1989) CD44 contributes to T cell activation. J Immunol 143: 789-801.
29. Ishihara H, Takahashi N, Oguri S, Tejima S (1979) Complete structure of the carbohydrate moiety of stem bromelain. J Biol Chem 254: 10715-10719.
30. Kandaswami C, Perkins E, Soloniuk DS, Drzewiecki G, Middleton Jr E (1991) Anti-proliferative effects of citrus flavonoids on human squamous cell carcinoma in vitro. Cancer Lett 56: 147-152.
31. Kleef R, Delohery TM, Bovbjerg DH (1996) Selective modulation of cell adhesion molecules on lymphocytes by bromelain protease-5. 64: 339-346.
32. Lee KL, Albee KL, Bernasconi RJ, Edmunds T (1997) Complete amino acid sequence of ananain and comparison with bromelain and other plant cysteine proteinases. Biochem J 327: 199-202.
33. Liao B (1984) Reported plant extracts derived from Anans comosus as an anti-cancer agents in animal experiments in breast and colonic carcinomas. Internal Communication, Non- Publication Materials.
34. Liao BS, Liao A, Liao A, Liao J, Liao BA, et al. (2003) Bioflavonoids derived from Fructus crateagus served as anti-cancer and antilipidemic agents. United States Patent Application Publication.
35. De Gaetano L (1978) Bromelain and its platelet aggregation effect. Drug Exp Clin Res 1: 49-53.
36. Hale LP, Haynes BF (1992) Bromelain treatment of human T cells removes CD44, CD45RA, E2/MIC2, CD6, CD7, CD8, and Leu 8/ LAM1 surface molecules and markedly enhances CD2-mediated T cell activation. J Immunol 149: 3809-3816.
37. Manach C, Regerat F, Texier O, Agullo G, Demigne C, et al. (1996) Bioavailability, metabolism, and physiological impact of 4-oxo Nutrition Reseach. 16: 517-544.
38. Mantovani A, Bottazzi B, Colotta F, Sozzani S, Ruco L (1992) The origin and function of tumor associated macrophages. Immunol Today 13: 265-270.
39. Matsumoto G, Nghiem MP, Nozaki N, Schmits R, Penninger JM (1998) Cooperation between CD44 and LFA-1/CD11a adhesion receptors in lymphokine-Activated killer cell cytotoxicity. Journal of Immunology 160: 5781-5789.
40. Maurer H, Eckert K, Szekerezes J (1989) Bromelain in the complementary tumor therapy. J Oncol 31: 66-73.
41. Maurer HR, Hozumi M, Honma Y, Okabe-Kado J (1988) Bromelain induces the differentiation of leukemic cells in vitro: An explanation for its cytostatic effect? Planta Med 54: 377-381.
42. Metzig C, Grabowska E, Eckert K, Rehse K, Maurer HR (1999) Bromelain proteases reduce human platelet aggregation in vitro, adhesion to bovine endothelial cells and thrombus formation in rat vessels in vivo. In Vivo 13: 7-12.
43. Morita A, Uchida D (1979) Inhibition of platelet aggregation in vitro with bromelain Treatment. Arch Int Pharmacodyn 239: 340-350.
44. Mynott A, Engwerda C (2010) T-cell receptor (TCR.)/ CD3, CD2 activated by bromelain through the Mitogen Activated Protein (MAP) Kinase Pathway. US Patent 7: 833-963.
45. Napper AD, Bennet SP, Holdridge MB, Leonard MJ, Rogers EE, et al. (1994) Further purification and characterization of multiple forms of Bromelainases derived cysteine proteinases Ananain and Comosain. Biochemic J 301: 727-735.
46. Netti C, Bandi GL, Pecile A (1972) Anti-inflammatory action of proteolytic enzymes of animal vegetable or bacterial origin administered orally compared with that of known anti-phlogistic compounds. Farmaco Prat 27: 453-466.
47. Ota S, Muta E, Katahira Y, Okamoto Y (1985) Reinvestigation of fractionation and some properties of the proteolytically active components of stem and fruit bromelain. J Biochem 98: 219-228.
48. Peterson G, Barnes S (1991) Genistein Inhibition of the growth of human breast cancer cells: Independence from estrogen receptors and multi-drug resistance gene. Biochem Biophy Res Commun 179: 661-667.
49. Picker LJ, De los Toyos J, Telen MJ, Haynes BF, Butcher EC (1989) Monoclonal antibodies against the CD44 [In(Lu)-related p80], and Pgp-1 antigens in man recognize the Hermes class of lymphocyte homing receptors. J Immunol 142: 2046-2051.
50. Pirotta (1978) Prolong the prothrombin and partial thromboplastin time in relative high doses of bromelain administration. Drug Exp Clin Res 4: 1-20.
51. Ranelletti FO, Ricci R, Larocca LM, Maggiano N, Capelli A, et al. (1992) Growth Inhibitory effect of quercetin and presence of Type- II estrogen-binding sites in human colon cancer cell lines and primary colorectal tumors. Int J Cancer 50: 486-492.
52. Renzini et al. (1972) Bromelain increases permeability of antibiotic drugs. Drug Res 2: 410- 412.
53. Revilla E, Ryan JM (2000) Analysis of several phenolic compounds with potential antioxidant properties in grape extracts and wines by high-performance liquid chromatography- photodiode array detection without sample preparation. J Chromotogr 881: 461-469.
54. Rollwagen FM, Baqar S (1996) Oral cytokine administration. Immunology Today 17: 548-550.
55. Rowan AD, Buttle DJ, Barrett AJ (1990) The cysteine proteinases of the pineapple plant. Biochem J 266: 869-875.
56. Rowan AD, Buttle DJ, Barrett AJ, (1988) Ananain: a novel cysteine proteinase found in pineapple stem. Arch Biochem Biophys 267: 262
57. Rowan AD, Buttle DJ (1994) Pineapple cysteine endopeptidases. Methodology Enzymol 244: 555- 568.
58. Saija A, Scalese M, Lanza M, Marzullo D, et al. (1995) Flavonoids as antioxidant agents: importance of their interaction with biomembranes. Free Radic Biol Med 19: 481-486.
59. Scambia G, Ranelletti FO, Panici PB, Piantelli M, Bonanno G, et al. (1991) Quercetin inhibits the growth of a multidrug-resistant estrogenreceptor-negative MCF-7 human breast-cancer cell line expressing type II estrogen-binding sites. Cancer Chemother Pharmacol 28: 255
60. Scambia G, Ranelletti FO, Panici PB, Piantelli M, Rumi C, et al. (1990) Type II estrogen binding sites in a lymphoblastoid cell line and growthinhibitory effect of estrogen, anti-estrogen and bioflavonoids. Int J Cancer 46: 1112-1116.
61. Seltzer A (1964) Bromelain reduces blood level of prostaglandine E-2, and thromboxane-A-2 in exudates during acute inflammation. EENT Monthly 43: 54-57.
62. Singhal RL, Yeh YA, Prajda N, Olah E, Sledge GW, et al. (1995) Qucercetin down-regulates signal transduction in human breast cancer cells. Biochem Biophys Res Commun 208: 425-431.
63. Smyth R, Brennan R (1962) Plasmin activation and its relation with bromelain. Arch Int Pharmacodyn 136: 230-236.
64. Taussig SJ, Szekerczes J, Batkin S (1985) Inhibition of Tumour Growth in vitro by Bromelain, an Extract of the Pineapple Plant (Ananas comosus). Planta Med 6: 538-539.
65. Taussig SJ, Batkin S (1988) Bromelain, the enzyme complex of pineapple (Ananas comosus) and its clinical application. An update. J Ethnopharmacol 22: 191-203.
66. Batkin S, Taussig SJ, Szekerczes J (1988) Antimetastatic effect of bromelain with or without its proteolytic and anticoagulant activity. J Cancer Res Clin Oncol 114: 507-508.
67. Tinozzi V (1978) Bromelain in the tissue permeability of antibiotic drug. Drug Exp Clin Res 1: 39-44.
68. Uhlig, Seifert (1981) Bromelain and blood level of fibrinogen, and its fibrinolytic effects. Fortschritte Der Medizin 15: 554-556.
69. van de Winkel JG, van Ommen R, Huizinga TW, de Raad MA, Tuijnman WB, et al. (1989) Proteolysis induces increased binding affinity of the monocyte type II FcR for human IgG. J Immunol 143: 571-578.
70. Verma AK, Johnson JA, Gould MN, Tanner MA (1988) Inhibition of 7,12-dimethylbenz(a)anthracene- and N-nitrosomethylurea-induced rat mammary cancer by dietary flavonol quercetin. Cancer Res 48: 5754-5758.
71. Yasuda Y, Takahashi N, Murachi T (1970) The composition and structure of carbohydrate moiety of stem Bromelain Biochemistry 9: 25-32.
72. Yoshioka S, Izutsu K, Aso Y, Takeda Y (1991) Inactivation kinetic enzyme pharmaceuticals in aqueous solution. Pharma Res 4: 480
73. Zhao J, Wang J, Chen Y, Agarwal R (1999) Anti-tumor-promoting activity of a polyphenolic fraction isolated from grape seeds in the mouse skin two-stage initiation-promotion protocol and identification of procyanidin B5-3’-gallate as the most effective antioxidant constituent. Carcinogenesis 20: 1737- 1745.