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Cancer Letters 155 (2000) 67±70

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Potential utility of antineoplaston A-10 levels in breast cancer q Farid Badria a, Mohamed Mabed b,*, Wael Khafagy c, Liala Abou-Zeid d b

a Department of Pharmacognosy, Faculty of Pharmacy, Mansoura University, Mansoura, Egypt Hematology and Medical Oncology Unit, Faculty of Medicine, Mansoura University, Mansoura, Egypt c General Surgery Department, Faculty of Medicine, Mansoura University, Mansoura, Egypt d Medicinal Chemistry Department, Faculty of Pharmacy, Mansoura University, Mansoura, Egypt

Received 15 January 2000; received in revised form 27 February 2000; accepted 27 February 2000

Abstract Antineoplastons, ®rst described by Burzynski, are naturally occurring peptides and amino acid derivatives, which control neoplastic growth. Antineoplaston A-10 (3-phenylacetyl amino-2,6-pepridinedione) is the ®rst chemically identi®ed antineoplaston. Here we describe the potential utility of antineoplaston A-10 as a predictive test for breast cancer. Antineoplaston A-10 level was measured in the urine of 31 breast cancer patients and 17 normal women using high performance thin layer chromatography (HPTLC). Signi®cantly lower antineoplaston A-10 levels were detected among patients with breast cancer with a P value , 0.001.These data suggest a strong inverse association of urinary antineoplaston A-10 level with breast cancer. Such ®nding was the stimulus for further investigations of antineoplaston A-10 levels in some benign as well as other malignant diseases to determine the utility of this approach as a predictive test for women who are at risk of developing breast cancer. q 2000 Published by Elsevier Science Ireland Ltd. All rights reserved.6 Keywords: Breast cancer; Antineoplaston A-10; Screening

1. Introduction Investigation for the presence of physiologically or pathologically active peptides in urine has been going on for the past 100 years. Biologically active polypeptides have been isolated from urine, which have demonstrated hormone like activity or regulation of biologic function. Examples of biologically active peptides isolated from urine include growth factors, pituitary hormones and kinins [1,2]. Bruzynski's earliest studies described methods for the isolation q

These data were presented at the second UICC Cancer Management Meeting, Antwerp-Belgium, April 14±18, 1999. * Corresponding author. Tel.: 12-050-351784; fax: 12-050367016. E-mail address: mohmabed@mum.mans.eun.eg (M. Mabed).

and quantitative measurement of peptides from the blood of human with renal diseases, heart diseases and obesity. Effects of peptides from human urine on cancer cells were later tested for their capacity to inhibit tumor cell growth. Early reports of an effect of these fractions on cancer cells in vitro appeared in 1973 and 1976 [3,4]. The active principles were therefore named antineoplastons. Two types of antineoplastic compounds were found, the ®rst consisted of strongly acidic peptides acted speci®cally on different kinds of tumor cells while the other type had both neutral and slightly acidic properties and displayed a broad spectrum activity. The later preparations were designed antineoplaston A and further separated into fractions A-1, A-2, A-3, A-4 and A-5 that were reported to have low cytotoxicity. A-10 was the active component present in the urinary antineoplastons and

0304-3835/00/$ - see front matter q 2000 Published by Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(00)00408-0


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F. Badria et al. / Cancer Letters 155 (2000) 67±70

it was identi®ed as 3-phenylacetyl amino-2,6-pepridinedione [5,6]. Therefore, urine is a convenient and economic source for isolation of biologically active peptides. The overall objective of our program is the identi®cation of antineoplaston A-10 present in human urine under normal and pathological conditions and determination of its potential utility as a predictive test for women who are at high risk of developing breast cancer. 2. Patients and methods 2.1. Subjects The participants were 31 women between 30 and 67 years of age who had histologically con®rmed diagnosis of breast cancer. Only those cases without previous treatment for breast cancer were included in the study. The tumor stage was classi®ed following the International Union Against Cancer/TNM staging system [7]. All of the 17 age-matched controls had no history of cancer or any other breast disease. 2.2. Puri®cation, detection and determination of A-10 Urine (25 ml) was puri®ed by Amberlite XAD-2 resin by washing with water (100 ml £ 2), 100 ml from methanol: water (1:1), and ®nally 100 ml pure methanol. The last two fractions were evaporated under reduced pressure at 358C. The produced resi-

dues were spotted on thin layer chromatography (TLC) and high performance thin layer chromatography (HPTLC) and developed in a solvent system (1-butanol: glacial acetic acid: water; 4:1:1 v/v). The developed plates were visualized under short UV lamp. The Rf was determined as 0.63 in comparison with an authentic sample from A-10 [8]. Each urine sample was treated under the same conditions. The ®nal residues from the all samples were spotted on HPTLC and the concentration of each spot was determined by CAMAG TLC scanner (Wilmington, NC). 2.3. Statistical analysis Student t-test is used to examine the difference between urinary antineoplaston A-10 level in breast cancer patients relative to their healthy controls. 3. Results The study included 31 patients with histologically con®rmed diagnosis of breast cancer. Mean age was 41.5 years range (30±67 years). Five were stage I, ten stage IIa, nine stage IIIa and seven stage IV. As shown in Fig. 1, signi®cantly lower antineoplaston A-10 level has been detected among breast cancer patients relative to their healthy controls with a P value ,0.001 (Student t-test). No other signi®cant relations as regards age, histologic grade or stage of the disease were observed in this study.

Fig. 1. Mean urinary antineoplaston A-10 level in 31 breast cancer patients and 17 healthy controls (P , 0:001).


F. Badria et al. / Cancer Letters 155 (2000) 67±70

4. Discussion The evaluation of tumor markers has not been shown to be of bene®t in the pre-operative evaluation of breast cancer patients [9]. Although carcinoembryonic antigen (CEA) may be useful in monitoring response to therapy, it is infrequently elevated in primary breast cancer [10]. The marker that has caused the most interest is CA 15-3. CA 15-3 is elevated in only 20% of women with primary breast cancer but elevations between 61 and 84% have been recorded for women with metastatic breast lesions, particularly those with bone metastases [11,12]. Elevation of CA 15-3 was observed in approximately 20% of women with benign breast lesions and in patients with benign gastrointestinal diseases, diminishing the usefulness of this marker as a screening test or for initial diagnosis. However, it is exceptionally good marker to monitor patients with recurrent disease. It falls during successful therapy and elevations occur before clinical evidence of recurrence [13,14]. In addition to CA15-3, there are many other breast cancer membrane antigens under investigation as serum markers, including CA 549, CA M26, CA 27.29, MCA and CASA [15±18]. The use of two markers or more resulted in a better combined sensitivity at the expense of specitivity. The best sensitivity for any two markers was 88% for CA 15-3 and CEA [19±21]. There may be an advantage in using panels in the follow-up of breast cancer patients at high risk for recurrence or during therapy [22], but it is dif®cult to assess the effectiveness because the optimal marker panel may differ among patients. Overall, the value of tumor markers in primary breast cancer has not been clearly established. The available markers have no real utility in the preoperative evaluation of the patients. The identi®cation of a highly speci®c and sensitive marker is currently an active area of research. Antineoplastons are naturally occurring cytodifferentiating agents. Chemically, they are medium and small sized peptides, amino acid derivatives and organic acids, which exist in blood, tissues and urine. Antineoplaston A-10 is the ®rst chemically identi®ed antineoplaston. The reported inhibitory effect of A-10 on various tumor cells suggests a potential bene®t for the treatment of human cancers with such agent [23,24]. In the study of Tusda et al. 1995

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[25], the antineoplastons were less toxic than conventional chemotherapeutic agents and they were useful in maintenance therapy for cancer patients. Also, when combined with a small dose of cis-diamminedichloroplatinum (CDDP) in the tumor growth study of human hepatocellular carcinoma, transplanted tumor growth was ef®ciently inhibited more than each agent alone [26]. A-10 signi®cantly inhibited the growth curve of human breast cancer serially transplanted to athymic mice [27,28]. Such activities of A-10 coupled with the low toxicity and novelty of basic A-10 structure provide an exciting possibility of developing a new class of clinically useful antineoplastic drugs with minimal side effects. Our study describes its potential utility as a predictive test for breast cancer. Signi®cantly lower A-10 level was found in breast cancer patients relative to their healthy control. Hendry et al. 1987 [29] have shown that A-10 may interact reversibly with DNA and thereby compete with carcinogens that form covalent linkage with DNA. It may follows that the decrease of urinary A10 level re¯ects its decrease at such critical points and allows such interactions to occur and permits the growth of tumors induced by various carcinogens. Overall, these data show a strong inverse association of urinary antineoplaston A-10 level with breast cancer. Further evaluation of antineoplaston A-10 levels in benign as well as other malignant diseases is under investigation to determine the utility of this approach as a predictive test for women who are at risk of developing breast cancer.

References [1] I. Miwa, E.G. Erdos, T. Seki, Presence of three peptides in urinary kinin (substance Z) preparations, Life Sci. 7 (1968) 1339±1343. [2] E. Wollheim, H.P. Nast, A humoral factor in the genesis of essential hypertension, Klin. Wochenschr. 49 (1971) 426±433. [3] S.R. Bruzynski, J. Georgiades, Effects of urinary peptides on DNA RNA and protein synthesis in normal and neoplastic cells, Fed. Proc. 32 (1973) 677. [4] S.R. Bruzynski, T.L. Loo, D.H. Ho, P.N. Rao, J. Georgiades, H. Kratzenstein, Biologically active peptides in human urine, Physiol. Chem. Phys. 8 (1976) 13±22. [5] S.R. Bruzynski, Z. Stolzmann, B. Szopa, E. Stolzmann, O.P. Kaltenberg, Antineoplaston A in cancer therapy, Physiol. Chem. Phys. 9 (1977) 485±500. [6] S.R. Bruzynski, Puri®ed Antineoplaston Fractions and Meth-


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[7] [8] [9] [10] [11] [12] [13]

[14] [15]

[16] [17]

[18]

[19]

F. Badria et al. / Cancer Letters 155 (2000) 67±70 ods of Treating Neoplastic Disease, 1985 US patent 4558057; December 10. L.H. Sobin, C. Wittekind, UICC/TNM Classi®cation of Malignant Tumors, 5th Edition, Wiley-Liss, New York, 1997. L. Abou-Zeid, Antiestrogen and/or antiprogestin structurally related new drugs of chemotherapeutic value, Ph.D Thesis, Mansoura University, Egypt, (1995). G. Crombach, Considerations in rational use of tumor markers in breast carcinoma, Schweiz. Rundsch. Med. Prax. 87 (1998) 589±594. Y. Lee, Carcinoembryonic antigen as a monitor of recurrent breast cancer, J. Surg. Oncol. 20 (1982) 109±114. D.F. Hayes, V.R. Zurawski Jr, W.D. Kufe, J. Clin. Oncol. 4 (1986) 1542±1550. R. Colomer, A. Ruibal, L. Salvador, Circulating tumor marker levels in advanced breast carcinoma correlate with the extent of metastatic disease, Cancer 64 (1989) 1676±1681. A.M. Dnistrian, M.K. Schwartz, E.J. Greeberg, C.A. Smith, D.C. Schwartz, CA 15-3 and carcinoembryonic antigen in the clinical evaluation of breast cancer, Clin. Chim. Acta. 200 (1991) 81±94. F. Sa®, I. Kohler, E. Rottinger, H. Beger, The value of tumor marker CA 15-3 in diagnosing and monitoring breast cancer, Cancer 68 (1991) 574±582. A.M. Dnistrian, M.K. Schwartz, E.J. Greenberg, C.A. Smith, D.C. Schwartz, Evaluation of CA M26 CA M29, CA 15-3 and CEA as circulating tumor markers in breast cancer patients, Tumor Biol. 12 (1991) 82±90. A.M. Dnistrian, M.K. Schwartz, E.J. Greenberg, C.A. Smith, R. Dorsa, D.C. Schwartz, CA 549 as a marker in breast cancer, Int. J. Biol. Markers 6 (1991) 139±143. T. Heinze, P. Schurenkmper, C. Minguillon, W. Lichtenegger, Mammary serum antigen (MSA) CA 549, CA 15-3 and CEA in breast cancer, preoperative sensitivity and correlation to prognostic factors, Anticancer Res. 17 (1997) 2953±2954. M. Meisel, J. Weise, G. Schwesinger, W. Straube, Cancer associated serum antigen (CASA) levels in patients with breast carcinoma and 3 control groups without breast cancer, Arch. Gynecol. Obstet. (1998) 159±162. A. Martoni, C. Zamagni, B. Bellanova, L. Zanichellii, F. Vecchi, N. Cacciari, E. Strocchi, F. Pannuti, CEA, MCA, CA 15-3 and CA 549 and their combinations in expressing and monitoring metastatic breast cancer: a prospective comparative study, Eur. J. Cancer 31 (1995) 1615±1621.

[20] M. Torres, C. Pacheco, A. Valverde, A.C. Rebollo, A. Moral, J.A. Vallejo, A. Mateo, CA 549 and SP2 in postoperative breast cancer patients Comparison with CA 15-3, CEA and TPA, Int. J. Biol. Markers 10 (1995) 94±99. [21] R. Molina, J. Jo, X. Filella, G. Zanon, J. Pahiso, M. Munoz, B. Farrus, M.L. Latre, C. Escriche, J. Estape, A.M. Ballesta, Cerb B-2 oncoprotein CEA and CA 15-3 in patients with breast cancer: Prognostic value, Breast Cancer Res. Treat. 51 (1998) 109±119. [22] A. Nicolini, P. Ferrari, A. Sagripanti, A. Carpi, The role of tumor markers in predicting skeletal metastases in breast cancer patients with equivocal bone scintigraphy, Br. J. Cancer 79 (1999) 1443±1447. [23] H. Tsuda, A. Iemura, M. Sata, M. Uchida, K. Yamana, H. Hara, Inhibitory effect of antineoplaston A-10 and AS2-1 on human hepatocellular carcinoma, Kurume Med. J. 43 (2) (1996) 137±147. [24] Y. Sugita, H. Tsuda, H. Maruiwa, M. Hirohata, M. Shigemori, H. Hara, The effect of antineoplaston, a new antitumor agent on malignant brain tumors, Kurume Med. J. 42 (3) (1995) 133±140. [25] H. Tsuda, H. Hara, N. Eriguchi, H. Nishida, H. Yashida, T. Kumabe, Y. Sugita, Toxicology study on antineoplaston A-10 and AS2-1 in cancer patients, Kurume Med. J. 42 (4) (1995) 241±249. [26] H. Tsuda, S. Sugihara, H. Nishida, H. Hara, N. Eriguchi, K. Ishii, S. Sasaki, S. Yoshimura, N. Tanaka, The inhibitory effect of the combination of antineoplaston A-10 injection with a small dose of cis-diamminedichloroplatinum on cell and tumor growth of hepatocellular carcinoma, Jpn. J. Cancer Res 83 (5) (1992) 527±531. [27] K. Hashimoto, T. Koga, Y. Shintomi, M. Tanaka, T. Kakegawa, H. Tsuda, H. Hara, The anticancer effect of antineoplaston A-10 on human breast cancer serially transplanted to athymic mice, Nippon Gan Chiryo Gakkai Shi 25 (1) (1990) 1±5. [28] H. Tsuda, Inhibitory effect of antineoplaston A-10 on breast cancer transplanted to athymic mice and human hepatocellular carcinoma cell lines, Kurum Med. J. 37 (2) (1990) 97± 104. [29] L.B. Hendry, T.G. Muldoon, S.R. Burzynski, J.A. Copland, A.F. Lehner, Stereochemical modeling studies of the interaction of antineoplaston A-10 with DNA, Drugs Exp. Clin. Res. 1 (1987) 77±81.


antineoplastons in breast cancer