Badania Zeolitu i jego wlasciwosci (anty nowo tworowe)

J Mol Med (2001) 78:708–720 DOI 10.1007/s001090000176

ORIGINAL ARTICLE

Krešimir Pavelic ́ · Mirko Hadžija · Ljiljana Bedrica Jasminka Pavelic ́ · Ivan –Dikic ́ · Maša Katic ́ Marijeta Kralj · Maja Herak Bosnar
Sanja Kapitanovic ́ · Marija Poljak-Blaži

Sˇimun Križanac · Ranko Stojkovic ́ · Mislav Jurin Boris Subotic ́ · Miroslav Cˇ olic ́

Natural zeolite clinoptilolite: new adjuvant in anticancer 

Received: 17 April 2000 / Accepted: 15 October 2000 / Published online: 24 January 2001 © Springer-Verlag 2001

Abstract Natural silicate materials, including zeolite clinoptilolite, have been shown to exhibit diverse biologi- cal activities and have been used successfully as a vac- cine adjuvant and for the treatment of diarrhea. We report a novel use of finely ground clinoptilolite as a potential adjuvant in anticancer therapy. Clinoptilolite treatment of mice and dogs suffering from a variety of tumor types led to improvement in the overall health status, prolongation of life-span, and decrease in tumors size. Local applica- tion of clinoptilolite to skin cancers of dogs effectively reduced tumor formation and growth. In addition, toxi- cology studies on mice and rats demonstrated that the treatment does not have negative effects. In vitro tissue culture studies showed that finely ground clinoptilolite inhibits protein kinase B (c-Akt), induces expression of p21WAF1/CIP1 and p27KIP1 tumor suppressor proteins, and blocks cell growth in several cancer cell lines. These data indicate that clinoptilolite treatment might affect cancer growth by attenuating survival signals and inducing tu- mor suppressor genes in treated cells.

K. Pavelic ́ (✉) · M. Hadžija · J. Pavelic ́ · M. Katic ́ · M. Kralj M.H. Bosnar · S. Kapitanovic ́ · M. Poljak-Blaži · R. Stojkovic ́ M. Jurin · B. Subotic ́
Rud-er Boškovic ́ Institute, Division of Molecular Medicine and Division of Material Chemistry, Bijenicˇka 54, Zagreb, Croatia e-mail: pavelic@rudjer.irb.hr

Tel.: +3851-46-80094, Fax: +3851-46-80094

L. Bedrica
Veterinary Faculty, University of Zagreb, Heinzelova 55, 10001 Zagreb, Croatia

I. –Dikic ́
Ludwig Institute for Cancer Research, Biomedical Center, Husargatan 3, Uppsala, Sweden

Sˇ. Križanac
Institute of Pathology, Faculty of Medicine, University of Zagreb, Sˇalata 11, Zagreb, Croatia

M . Cˇ o l i c ́
Molecular Technologies Inc., 6512 Segovia, Goleta, CA 93117, USA

Keywords Clinoptilolite · Adjuvant · Anticancer · Treatment

KRESˇIMIR PAVELIC ́
received his M.D./Ph.D. de- gree in medicine and experi- mental oncology from the Uni- versity of Zagreb, Croatia. He received research fellowship at the RPMI in Buffalo, N.Y., University of Cincinnati, Ohio, and Fullbright fellow- ship at the Mayo Clinic, Ro- chester, Minn., USA. He is di- rector of the Division of Mo- lecular Medicine at Rud-er Boškovic ́ Institute and Direc- tor of the National Cancer Re- search Program of the Repub- lic of Croatia. Dr. Pavelic ́ is also Professor of Molecular Biology at the Department of Pharmacy and Biochemistry at the University of Zagreb. His research interests include mo- lecular medicine, particularly cancer genetic.

MIROSLAV CˇOLIC ́
received his Ph.D. in applied surface chemistry with minors in molecular biology and bio- physics from the University of California at Berkeley, USA. He is presently Vice President of the Research and Develop- ment Division, Molecutec Corporation, Goleta, Califor- nia. His research interests in- clude free radicals chemistry and biology, environmental chemistry, biomedical effects of dietetic products, and small molecules drug discovery.

Abbreviations EGF: Epidermal growth factor ·
FBS: Fetal bovine serum · MAPK: Mitogen-activated protein kinases · PDGF: Platelet-derived growth factor · SDS: Sodium dodecyl sulfate

Introduction

Zeolites are hydrated natural and synthetic microporous crystals with well-defined structures containing AlO4 and SiO4 tetrahedra linked through the common oxygen atoms [1]. Zeolites have been extensively used in various industrial applications based on their properties to act as catalysts, ion exchangers, adsorbents, and detergent builders [2, 3, 4, 5, 6]. It is also known that silicates and aluminosilicates possess biological activity, either posi- tive or negative. Talc and silica have been used in skin care for many decades, while well defined structures and catalytic activity make aluminosilicates an attractive model system for protein and enzyme mimetics [7]. Re- cent results have also demonstrated that natural, biologi- cally nontoxic clinoptilolite from Cuba deposits is very effective as glucose adsorbent, and this has been sug- gested as a potential medication for individuals suffering from diabetes mellitus [8].

The best known positive biological activity of natural clinoptilolite is its action as antidiarrheal drug (see [9] and references therein). Clinoptilolite lowers the inci- dence of death and sickness (diarrheal syndrome) pro- duced by intestinal diseases in swine, rats, and calves (see [9] and references therein). Based on these results a comprehensive study was carried out on antidiarrheal drugs based on natural clinoptilolite as an active materi- al, in the therapy of acute diarrheal diseases in humans [9]. The research lead to approval of the antidiarrheal drug Enterex for use in humans. In addition, accumulat- ing evidence has indicated that zeolites play an impor- tant role in regulating the immune system. Ueki et al. [10] and Aikoh et al. [11] have reported that silica, sili- cates, and aluminosilicates act as nonspecific immuno- stimulators similarly to superantigens. Superantigens are a class of immunostimulatory and disease-causing pro- teins of bacterial and viral origin with the ability to acti- vate relatively large fractions (5–20%) of the T cell pop- ulation. Activation requires simultaneous interaction of the superantigens with Vβ domain of T cell receptor and with major histocompatibility complex class II mole- cules on the surface of antigen presenting cells [10]. Pro- inflammatory macrophages, which belong to class II MHC antigen-presenting cells, are activated by fibro- genic silicate particulates [12, 13, 14, 15]. Indeed, exper- iments carried out by Ueki and coworkers [10] have shown that removal of MHC class II DP/DR positive cells results in a lack of macrophage stimulation by as- bestos.

Direct interaction of silicate particles with cells other than lymphocytes has also been identified and described. It seems that mineral particles can trigger alterations in gene expression by initiating signaling events upstream

of gene transactivation [16]. Exposure of cells to silicate particles has been shown to lead to activation of mito- gen-activated protein kinases (MAPK), protein kinase C, and stress-activated protein kinases [17]. Important tran- scription factors such as activator protein 1 and nuclear factor κB are also activated, and expression of proin- flammatory cytokines such as interleukin 1α, interleukin 6, and tumor necrosis factor α is enhanced [18]. Modifi- cations in receptor activation kinetics or activity of inte- grins may be responsible for the observed behavior. Al- ternatively, particles engulfed by phagocytosis have been reported to stimulate production of reactive oxygen spe- cies [19]. It was recently shown that redox regulation of gene expression is a general phenomenon in most cells.

The above knowledge of zeolites and other silicates led us to test the biological activity of natural clinoptil- olite. Mechanical treatment of natural clinoptilolite was used to produce small-sized particles (MZ) that were tested for possible toxicity and anticancer activity in vi- vo. Here we provide evidence that orally administered natural clinoptilolite is nontoxic and useful in cancer treatment in animal models. Additional in vitro tissue culture experiments with various cancer cell lines indi- cated that MZ treatment modifies intracellular signaling pathways leading to inhibition of survival signals and in- duction of tumor suppressor genes.

Materials and methods

Natural clinoptilolite

The fine powder of natural clinoptilolite was obtained by tribome- chanical micronization. Chemical composition of the MZ was de- termined by the atomic absorption spectroscopy. Qualitative and quantitative phase analyses of the MZ were performed by powder X-ray diffractometry using a Siemens 5000D diffractometer with CuKα radiation in the region 2θ=4–80°. Thermogravimetric and differential thermogravimetric analysis of the MZ was performed using a TA 4000 System (Mettler-Toledo) apparatus. The heating rate was 10 K/min in nitrogen atmosphere. Particle size distribu- tion curves of the MZ were taken by a Mastersize XLB (Malvern) laser light-scattering particle-size analyzer.

Cell lines and proliferation assay

Effect of MZ on in vitro cell proliferation was studied on several human cell lines: diploid fibroblasts (Hef522), cervical carcinoma (HeLa), colon carcinomas (CaCo-2, HT-29, and SW 620), mam- mary carcinomas (MCF-7 and SkBr-3), and one mouse fibrosarco- ma cell line. The cells were maintained by culturing in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin in a humidified atmosphere with 5% CO2 at 37°C. For the purpose of proliferation assay experiments the cells were plated at a concentration of 1×104 cells/ml onto 96- microwell plates (200 μl/well). After overnight incubation the standard medium was replaced with the medium which was pre- treated with either 0.5, 5, or 50 mg/ml MZ. For this purpose the medium and MZ were mixed, and after 18 h of shaking MZ was pelleted by centrifugation (5000 g for 10 min).

The cells were then incubated for additional 72 h, when cell viability (cell growth) was measured using MTT assay which de- tects dehydrogenase activity in viable cells. For this purpose the medium was discarded, and MTT was added to each well at con-

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centration of 20 μg/40 μl. After 4 h of incubation at 37°C the pre- cipitates were dissolved in 160 μl DMSO. The absorbance was measured on an enzyme-linked immunosorbent assay reader at 570 nm. The cell proliferation is expressed as a percentage of ab- sorbance, recorded in cell line treated with particular concentra- tion of MZ, in relation to the absorbance of control, nontreated, cells which was expressed as 100%.

Analysis of p21WAF1/CIP1 and p27KIP1

Experiments with p21WAF1/CIP1 and p27KIP1 were carried out on hu- man adenocarcinoma (CaCo-2) and human cervical carcinoma (HeLa) cell lines. The cells, originally grown in tissue culture flasks, were collected and seeded onto glass slides. After 24 h the medium was replaced either with the fresh standard medium (con- trol cells) or with the medium pretreated with 50 mg/ml MZ. After 72 h of incubation the cells were washed with PBS and fixed in methanol with 3% hydrogen peroxide (Kemika, Zagreb, Croatia).

Proteins, p21WAF1/CIP1 and p27KIP1, expression was analyzed immunocytochemically. Nonspecific binding was blocked by ap- plying normal rabbit serum (1:10) for 30 min. Primary antibodies p21 (5 μg/ml, PharMingen) and p27 (2 μg/ml, Transduction Labo- ratories) were allowed to bind overnight at 4°C. Slides were washed three times in PBS. Secondary antibody (rabbit anti- mouse; Dako, Denmark) was applied for 1 h at room temperature. Finally, peroxidase-antiperoxidase (Dako) conjugate diluted 1:100 in PBS was applied for 1 h at room temperature. After washing with PBS the slides were stained with 0.025% diaminobenzidine tetrahydrochloride (Sigma) containing 4% H2O2 for 7 min and counterstained with hematoxylin for 30 s. The slides were ana- lyzed with a light microscope (Olympus). The level of nonspecific background staining was established for each measurement using control cells processed in the same way but without exposure to the primary antibody.

The concentration of antigen was assessed by estimating the relative visual intensity of a chromogenic label, and the results are expressed on a three-point scale as follows: –, negative staining; +, weak staining and ++, moderate staining.

Biochemical studies of signaling pathways

The following were used: epidermal growth factor (EGF; Inter- gen), platelet-derived growth factor (PDGF) BB (Amgen), protein ladder markers (10–200 kDa; Life Technologies), leupeptin and a miniprotease inhibitor kit (Boehringer-Mannheim), Pefabloc (Fluka), aprotinin (Trasylol, Bayer), and nitrocellulose membranes (Millipore). Affinity-purified rabbit polyclonal anti-Akt, anti- pAkt, anti-JNK, anti-pJNK and anti-pERK2 (MAPK) antibodies were purchased from New England Biolabs. The rabbit polyclonal anti-ERK2 (C-14) antibodies were from Santa Cruz Biotechnolo- gy. Secondary antibodies, peroxide-conjugated swine anti-rabbit were from New England Biolabs, peroxide-conjugated sheep anti- mouse immunoglobulin from Amersham/Pharmacia, and perox- ide-conjugated protein A from Kirkegaard and Perry Laboratories.

Murine fibrosarcoma cells were grown in Petri dishes (6 cm in diameter) in RPMI medium with 10% FBS up to the 80% conflu- ence. Before starting the experiments the cells were starved for 24 h. Subsequently the cells were treated with MZ pretreated me- dium with or without 10% FBS for 0, 5, 30, and 60 min or with EGF (100 μg/ml) and PDGF (40 μg/ml). After the indicated time of treatment the cells were washed with PBS and scraped into ice- cold lysis buffer containing 50 mM hydroxyethylpiperazine ethane sulfonic acid, pH 7.2, 150 mM NaCl, 1 mM EDTA, 20 mM NaF, 2 mM sodium orthovanadate, 1% (w/v) Triton X-100, 10% (w/v) glycerol, and protease inhibitors (1 mM Pefabloc, 10 μg/ml leu- peptin, and 1% Trasylol). Following 45 min at 4°C with gentle rocking a soluble fraction was prepared by centrifugation at 4°C for 15 min at 13,000 g. Equal amounts of cell lysates (measured by the Bradford assay) were mixed with 3× sodium dodecyl sul- fate (SDS) sample buffer and heated for 2 min at 98°C. Proteins

were separated by SDS polyacrylamide gel electrophoresis and transferred onto nitrocellulose membrane. Immunoblots were blocked with 5% bovine serum albumin in TBS (10 mM Tris-HCl, pH 7.4; 150 mM NaCl) for 1 h, incubated for 1 h with primary an- tibodies (anti-pAkt, anti-pJNK, anti-pERK2) in TBS, washed six times for 10 min each in TBS 0.05% Triton X-100, and then incu- bated for 1 h with appropriate secondary antibody. Following fur- ther washes, immunoblots were visualized by using enhanced che- miluminescence reagents. To reprobe blots they were incubated in stripping buffer (62.5 mM Tris-HCl, pH 6.7; 2% SDS; 100 mM 2-mercaptoethanol) at 58°C for 25 min, washed extensively with TBS, reblocked as described above, and reblotted with the appro- priate antibodies.

Isolation of apoptotic DNA fragments

HeLa cells (1×105) were grown in a 10-ml flask for 24 h, after which the medium was discarded and replaced with the MZ pre- treated medium (see above). After 24 h the cells were tripsinized, pelleted by centrifugation (1200 g), and washed twice in PBS. Afterwards the cells were resuspended 10 s in 100 μl lysis buffer (1% NP-40 in 20 mM EDTA, 50 mM Tris-HCl, pH 7.5) and cen- trifuged 5 min at 3000 g. The supernatant was transferred to a new Eppendorf tube while the pellet was incubated once more with 100 μl lysis buffer and centrifuged as before. The supernatants were pooled together and incubated 2 h in 1% SDS and RNase (5 μg/μl) at 56°C, after which the proteinase K was added in final concentration 2.5 μg/μl overnight. DNA fragments were pelleted by addition of 1/2 volume of 10 M ammonium acetate and 2.5 vol- ume of prechilled absolute ethanol. After centrifugation (30 min, 12,000 g), the pellet was washed with 70% ethanol, centrifuged 10 min at 12000 g, dried, and dissolved in 20 μl TE buffer (10 mM Tris-HCl pH 7.4; 1 mM EDTA pH 8). The DNA was visualized on 1.5% agarose gel.

Animals

Mice

CBA/HZgr and C57BL/6 mice of both sexes were used. Toxicity study experiments were performed on the CBA/HZgr strain, while experiments with tumors were performed on both strains. For non- clinical tolerance testing male mice of the BALB/c strain were used. At the beginning of the experiments the animals were about 4 months old, weighing 25–28 g. Until beginning the experiments the mice were maintained in standard conditions with unrestricted access to food and water.

Rats

Wistar rats of both sexes from the animal breeding colony at the Institute for Medical Research, Zagreb, Croatia were used for tox- icity and nonclinical tolerance testing studies. At the beginning of the experiments they were 2–3 months old, weighing in average 300 g (males) and 200 g (females).

Dogs

Twenty-two dogs were used in the experiments. They were of var- ious breeds, weighing from 3 to 42 kg. The animals were of both sexes, 5–14 years old. The data on the 14 dogs in which disease improvement was observed, are presented in Table 2.

Application of mechanically treated natural clinoptilolite (MZ)

Because of the insolubility of the tested substance, it was adminis- tered to the animals either orally by gavage or in their diet (mice,

rats), supplemented as powder to the conventional food, or in cap- sules (dogs) which were again admixed to food. When testing the growth of mammary aplastic carcinoma or mammary aplastic car- cinoma metastases formation MZ and standard food for laboratory mice (Pliva, Zagreb, Croatia) were mixed in the ratio 20%:80%. Each mouse on average ate about 4 g food daily, thus consuming about 800 mg MZ. When testing the growth of melanoma, MZ was given to mice orally (gavage) at doses of 20, 30, and 40 mg/mice five times per day (tested doses were 100, 150, and 200 mg/mice, respectively). In toxicity studies MZ was applied in diet mixed with standard food.

Tumors

Mammary carcinoma occurred spontaneously in CBA/HZgr mice, maintained in the animal breeding section of the Division of Mo- lecular Medicine, Rud-er Boškovic ́ Institute, Zagreb, Croatia. The tumor is a highly anaplastic carcinoma with very high incidence of mitoses; it does not form any glandular structures and leads to spontaneous metastases in the lungs. After transplantation of 1×106 viable tumor cells into the animals a growing tumor is ob- tained which causes the mouse’s death after about 4 weeks. For the purpose of the experiments tumor cell suspension was always prepared from in vivo growing tumor.

Melanoma B16, originally obtained from Holt Radium Insti- tute, Manchester, United Kingdom, has been maintained at the Rud-er Boškovic ́ Institute since 1975 by subcutaneous inoculations of suspension containing 2×106 tumor cells into flanks of C57BL/6 mice.

Spontaneous tumors in dogs were of various origins, sizes, and locations. The data on 14 tumors are presented in Table 2. In an- other 8 tumors, not presented in Table 2, there were two lympho- mas, two autoimmune hemolytic anemias, and one each of pros- tate tumor, osteosarcoma, mammary fibrochondroadenocarcino- ma, and epulis.

To obtain tumor cells in suspension large pieces of tumor re- moved from the mice were cut up in very small pieces (Hank’s so- lution). The particles were allowed to settle, and the supernatant (cell suspension) was removed and spun down at 150 g for 10 min. The pellet was resuspended and cell viability was tested by Trypan blue exclusion test: more than 90% of tumor cells were scored as viable. To obtain locally growing tumor, an inoculum of 0.1 ml, containing 1×106 viable tumor cells, was injected subcuta- neously into the right thigh of recipient mice. Tumor growth was checked each day after tumor cell inoculation into the mice. When the tumor was established, its size was measured by a caliper. Three diameters were measured, and tumor volume was calculat- ed. To obtain experimental lung metastases 0.25 ml, containing 1×105 mammary aplastic carcinoma cells, was injected into mouse tail vein. The mice were killed 18 days later. The lungs were re- moved, washed in water, separated into lobules, and immersed in a fixative. Macroscopically visible nodules on lung’s surface were counted.

Toxicology studies

Preclinical toxicology was performed according to standards and regulations of the Organization for Economic Cooperation and Development principles of food laboratory practice (Paris 1998). The testing was approached by setting the “limit” test – applying the high doses of MZ, 2×200 and 2×500 mg/mouse per day orally (gavage) for 6, 14, and 30 days. Since the MZ did not cause the death of mice in a “limit” test, an “up and down” test was performed on mice, with daily doses ranging from 60 to 400 mg/mouse (MZ given orally, gavage, for 30 days). Again, no toxicity was observed. Therefore a classical acute, subchronic and chronic toxicity study of mice and rats of both sexes (separately) was performed.

Mice

The mice were of the CBA/HZgr strain. MZ was given in a diet (powdered MZ mixed with standard food at the ratio of 25:75%). The duration of study was as follows: acute toxicity, 1 month; sub- chronic toxicity, up to 3 months; chronic toxicity, up to 6 months. Animals were monitored for: phenotypic changes, changes in be- havior, and survival (every day), changes in body weight (week- ly), amount of food and water consumed (checked on days 14 and 28 when mice were kept for 24 h in metabolic cages, five mice per cage), changes in hematological and serum clinical chemistry pa- rameters (erythrocytes, leukocytes, plateletes, hematocrit, hemo- globin, glucose, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, bilirubin, inorganic phosphorous, and calcium; after 1, 3, and 6 months); and urine clinical chemistry pa- rameters (glucose, proteins, urobilinogen, bilirubin, nitrites, ery- throcytes, leukocytes, pH, and specific gravity; urine was collect- ed while the animals were kept, once a month for 24 h, in meta- bolic cages). Pathohistological analysis of liver, spleen, kidney, brain, lung, testes, ovary, duodenum, eye, stomach, large and small intestine, muscles, myocard, pancreas, thymus and axillary lymph node was carried out on killed experimental and control mice.

Rats

Wistar rats were used. MZ was given in a diet (mixed with stan- dard food at ratios of 25:75 and 50:50). The duration of study was as follows: acute toxicity, 1 month; subchronic toxicity, 3 months; chronic toxicity, 12 months. Animals were monitored for: pheno- typic changes, changes in behavior and survival (every day), changes in body weight (every 4 days), amount of food (every day) and water consumed (every 4 days), and changes in hemato- logical and serum clinical chemistry parameters (the same as for mice; once a month). Pathohistological analysis of liver, spleen, lung, kindey, testes, ovary and brain, was performed on killed ex- perimental and control rats after 1, 6, and 12 months.

The reproductive/developmental toxicity was tested on mice (CBA/HZgr) due to their short gestation period and larger litter size. MZ was given in a diet (powdered MZ mixed with standard food at the ratio of 25:75%). For reproductive toxicity study ten male and ten female mice were fed with the food supplemented with the MZ for 50 and at least 14 days, respectively, before mat- ing. The treatment continued during the prepregnancy and preg- nancy period (one cycle) and to the point of weaning offspring. The same pair of animals was fed with the MZ and monitored dur- ing four consecutive cycles (approximately 4–5 months). The same schedule was applied for control, nontreated, animals. The parental generation was monitored for duration of cycle period (prepregnancy and pregnancy period), fertility (presence or ab- sence of litter in particular cycle), delivery incidence, mortality, and pathohistological appearance of ovaries, after 4th cycle. Num- ber of total and viable pups born as well gain in pups body weight and pups mortality until weaning was also scored.

For teratology study healthy, untreated pregnant mice were fed with MZ mixed to the conventional food from day 6 through day 16 of gestation and the mice were killed 1 day before parturition. The fetuses were analyzed for microscopic pathology.

Local tolerance was evaluated to ascertain whether the test substance is tolerated at the sites in the body which may come into contact with the product as a result of its administration.

Repeated-dose dermal tolerance testing was performed on male Wistar rats and male BALB/c mice. MZ was applied on the shaved skin of the whole dorsal region of animals in three ways: (a) as original powder, (b) mixed with neutral creme at the ratio of 1:1, (c) mixed with paraffin oil at the ratio of 1:1. The animals were treated twice a day during 28 days. Macroscopic changes in the treated skin were examined daily. The left dorsal region of the animal was used as control. For microscopic analysis of the possi- ble changes the skin samples were collected 1 day after the last treatment.

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Results

Properties of mechanically treated natural clinoptilolite

Mechanically treated natural clinoptilolite (MZ) con- tained approximately 85 wt.% clinoptilolite. The remain- ing 15% consisted of silica, montmorillonite and mainly mordenite zeolite. The chemical composition of the natu- ral clinoptilolite is presented in Table 1. Differential thermal analysis (differential thermogravimetric) of the MZ shows that the maximum rate of water desorption occurred at 50°C, indicating that the change in sample weight during heating to 50°C corresponds to the remov- al of loosely held moisture within the solid microstruc-

Fig. 1 A Differential thermogravimetric curve of MZ; dm/dt dif- ferential change (dm) in the mass of sample in differential time in- terval (dt); T temperature of heating. B Weight loss during con- trolled heating of the MZ from ambient temperature (T=25°C) up to T=800°C. C Distribution of differential particle sizes by num- ber of MZ. ND Number percentage of particles of the correspond- ing diameter D. D Cumulative particle size distribution by number

ture. Analysis of the water desorption curve shows that the MZ contains approx. 16 wt.% of water (loosely held moisture + zeolitic water) of which approx. 2 wt.% is loosely held moisture (Fig. 1A, B). No phase transforma- tion was observed during the heating of MZ to 800°C. Particle size analysis of the MZ showed that maximum frequency of particles (approx. 13%) appeared at 1.5 μm with average size of 2.9 μm. In 25% of particles the size was up to 1.5 μm, in 50% up to 2 μm, and in 75% up to 3 μm (Fig. 1C, D).

Table 1 Chemical composition of the mechanically treated natural clinoptilolite (MZ)

Oxide

SiO2
Al2O3
Fe2O3
Na2O
K2O
MgO
CaO
MnO
TiO
Water (800°C)

wt.%

50–55 9.3–11.4 2.2–2.8 0.8–1.1 2.9–4.3 0.8–1–2

13.7–17.2 0.07–0.90 0.14–0.22

14–16

of the MZ. ΣN tween D=0 and D

2

D

Percentage of the particles having diameters be-

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Fig. 2 Effect of the medium pretreated with 0.5, 5.0, and 50.0 mg/ml MZ on growth of various cell lines. Vertical bars Standard deviations; all white bars (50 mg/ml) are statistically dif- ferent in comparison to control (P<0.001, Student’s t test)

The effect of MZ on proliferation of cell lines grown in vitro

Figure 2 presents the cell proliferation state of Hef522, HeLa, CaCo-2, SW620, HT-29, MCF-7, SKBR-3, and mouse fibrosarcoma cells after 3 days of treatment. The growth of all cell lines except Hef522 and SW620 was significantly inhibited with the dose of 50 mg/ml. The strongest inhibition (for 50%) was seen on mouse fibro- sarcoma cells, the growth of SW620 cells was un- changed, and that of Hef522 cells was slightly stimulat- ed. Similar results were observed measuring [3H]thymi- dine incorporation assay in the presence of 10% FBS in mouse fibrosarcoma cells (data not shown).

Analysis of intracellular signaling pathways in MZ-treated cells

Since previous studies have indicated that exposure of cells to silicate particles leads to activation of MAPK, protein kinase C, and stress-activated protein kin- ases/JNK [17], we further analyzed whether MZ treat- ment also affects mitogenic and survival signaling path- ways in these cell models.

The most significant results were detected measuring the activity of Akt protein. Akt, or protein kinase B, has been recently shown to mediate survival signals down- stream of phosphoinositide-3 kinase by phosphorylating Bad proteins. We have observed an increase in Akt phos- phorylation in response to serum, EGF, or insulin treat- ment. The addition of the MZ pretreated medium con- taining 10% FBS to the cells decreased Akt phosphoryla- tion in comparison to the cells treated with only serum containing medium, while the addition of growth factors EGF and PDGF restored its activity (Fig. 3A) and over- came the effects of MZ on cell growth. Determination of the activity of Akt at various times after the addition of MZ pretreated medium with 10% FBS showed slight de- crease in pAkt level after 5 min. This decrease was more

Fig. 3 A Activity of Akt protein 5 min after addition of the MZ pretreated medium to murine fibrosarcoma cells. B Decreased Akt protein activity at various times after treatment of murine fibrosar- coma cells with MZ pretreated medium. C Effect of serum free MZ-pretreated medium on the activity of MAPK in murine fibro- sarcoma cells. WB Western blot; FBS fetal bovine serum; MZ me- chanically activated clinoptilolite zeolite; pAkt phosphorylated Akt; EGF epidermal growth factor; PDGF platelet-derived growth factor; MAPK mitogen-activated protein kinase; pMAPK phos- phorylated mitogen-activated protein kinase

pronounced after 30 and 60 min of treatment (Fig. 3B). However, the addition of MZ pretreated medium without serum to the cells increased activity of Akt compared on- ly to the serum-starved cells. Overnight treatment of the cells with EGF also increased Akt activity. However, combined overnight treatment of the cells with EGF and MZ pretreated medium decreased Akt activity, indicating that inhibition of Akt might be linked to MZ inhibition of the EGF-triggered pathways.

MAP kinase activity was increased in serum-starved cells in response to EGF, PDGF, or serum. Addition of only MZ pretreated medium to the serum-starved cells increased MAPK activity only temporarily (after 5 min);

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Fig. 4 Immunohistochemical analysis of p27KIP1 on control HeLa cells (A) and HeLa cells after incubation with the MZ pretreated medium (B). Brown staining Cells expressing p27KIP1

in the next 30 min MAPK activity returned to the normal level (Fig. 3C). In contrast, addition of MZ pretreated medium plus 10% serum slightly decreased MAPK ac- tivity compared only to serum-treated cells or cells incu- bated only with MZ pretreated medium. These results are in agreement with those of the previously performed thymidine test.

Medium pretreated with MZ added to the cells either alone or in combination with serum caused no change in JNK activity (data not shown).

The effect of MZ on expression of inhibitors of cy- cline-dependent kinases, p21WAF1CIP1 and p27KIP1, was tested using immunocytochemical method, in HeLa and CaCo-2 cells. Treatment with MZ induced the expres- sion of p21WAF1/CIP1 in CaCo-2 cells and p27KIP1 in HeLa cells, while nontreated cells were negative for expression of p21WAF1/CIP1/p27KIP1 (Fig. 4).

Induction of programmed cell death-apoptosis

To evaluate whether the inhibition of cell growth by MZ is due to programmed cell death, i.e., apoptosis, an at- tempt was made to isolate small DNA fragments. Large amount of small (degraded) DNA fragments in DNA iso- late would indicate that MZ induces programmed cell death in treated cells. The result of small DNA fragment isolation from HeLa cell is shown in Fig. 5. DNA isolat- ed from MZ treated cells exhibited significant degrada- tion (lane 3a bulk of low-molecular, degraded DNA indi- cated with an arrow) in comparison to DNA from un- treated cells (lane 2). The DNA degradation in MZ treat- ed cells is most probably due to induced programmed cell death (apoptosis).

Toxicology

Oral (in diet) administration of MZ to mice and rats for 6 and 12 months, respectively, caused no changes that could be considered a toxic effect of treatment. The MZ

Fig. 5 Apoptotic DNA fragments in 1.5% agarose gel. Lane 1 DNA molecular weight marker IX (Øx 174/HindIII); lane 2 DNA isolated from untreated HeLa cells; lane 3 DNA isolated from the MZ-treated HeLa cells; degraded, low-molecular DNA fragments

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Fig. 6 Tumor growth following injection of 1×106 mammary aplastic carcinoma cells into the right thigh of CBA/HZgr mice. The animals were exposed to 20% of MZ in the food either from the day of tumor transplantation (n=14) or 15 days prior to tumor transplantation (n=14). Control mice received standard food. Verti- cal bars Standard deviation. The differences between control and both experimental groups were statistically significant (P<0.001, Student’s t test) for the days 25, 30, and 35

equalized (regulated) and shortened the prepregnancy period. The number of pups per litter was increased in MZ-treated mice. Probably for this reason the gain in pups’ body weight until weaning was decreased. As a fi- nal consequence higher mortality of pups between days 8 and 21 of the neonatal period was observed. However, there are no differences between control and treated ani- mals that would suggest reproductive toxicity attribut- able to the MZ administration. The MZ did not elicit tox- icity during the period of organogenesis. The test sub- stance, MZ, was not toxic or allergenic for the skin.

Effect of MZ on tumor growth in animal models

Previous studies in cultured cells have suggested that MZ inhibits growth of cancer cells in vitro. To study the effect of MZ in vivo studies on mice, rats, and dogs were undertaken. Subsequent studies were performed on mu- rine transplantable tumors, melanoma B16, and mamma- ry carcinoma. Mammary aplastic carcinoma cells were injected into the right thigh of two groups of mice. One group (n=14) was fed with food supplemented with MZ starting from 15 days prior tumor transplantation until the animal’s death; the other group (n=14) was fed with MZ from the day of tumor transplantation until the ani- mal’s death. A group of five tumor-bearing mice receiv- ing standard food was used as control. Tumor growth was significantly inhibited in both groups of animals fed with MZ supplemented food (Fig. 6). The tumor growth curves for individual animals were uniform, particularly when MZ was given prior to the tumor transplantation. However, there was no difference in mice survival among the groups.

Melanoma B16 cells were inoculated subcutaneously in C57BL mice on day 0. For the next 30 days the mice were given MZ orally five times per day. Tumor volume was recorded; it was markedly lower in 5 of 80 mice (daily dose 150 mg/mouse) than in the control group

Fig. 7 Growth rate (A) of melanoma B16 treated with 150 mg MZ/mouse per day and survival (B) of melanoma-bearing mice treated with three different doses of MZ

(Fig. 7A). Despite the fact that the tumors started to grow more rapidly after the therapy with MZ was abro- gated (between days 30 and 60 after tumor transplanta- tion), the mice lived a statistically significantly longer period when treated with 200 and 150 mg MZ than con- trol animals (Fig. 7B). The mice used for experimental mammary aplastic carcinoma lung metastases formation were fed with MZ diet from 15 days prior to tumor cell injection until to the end of the experiment, i.e., 18 days after tumor transplantation. The controls consumed stan- dard food. Each of these two groups comprised 20 ani- mals. About 20–40 nodules per animal were scored, but there was no difference between the groups (data not shown).

There was no effect of MZ treatment on in vivo growth of two mammary carcinomas which differed from that showed in Fig. 6 (data not shown).

Of 22 dogs suffering from various kinds of spontane- ous tumors that were treated with MZ, 14 responded to therapy, i.e., the tumor disappeared completely, or the tumor size was significantly reduced (presented in Table 2). Among three dogs which had prostate tumor there was one that was stated sonography showed to have (in addition to prostate tumor) a prostate cyst (case 3). The dog was conspicuously quiet, without ap- petite, and hardly moved. When the usual therapy did not work, MZ therapy was started. After only 2 days of treatment the dog became active; on the third day it be- gan eating normally, and on the fourth day the dog uri- nated normally, blood-free urine. On day 10 the cyst and the tumor were reduced in size, and after 1 month they had disappeared completely. Although the prostate be-

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Table 2 The effect of MZ treatment on growth of spontaneous tu- mors of dogs (→ values before and after treatment with MZ, a.t. after beginning of treatment, HMT hematocrit, ALT alanine amino-

transferase, AST aspartate aminotransferase, ALP alkaline phos- phatase, GGT γ-glutamyl transferase, L number of leukocytes)

No. Breed

1. 1  Schnauzer

2. 2  Poodle

3. 3  German shepherd

4. 4  Mixed German

   shepherd

5. 5  English cocker spaniel

6. 6  Poodle

Age Weight Sex (years)a (kg)

8 15 M

12 16 M

8 42 M

14 20 F

8 15 F

11 F

Diagnosis

Prostate adenocarcinomab

Prostate adenocarcinoma (4×3 cm) and testis tumor
(20 cm)

Prostate adenocarcinoma (5×5 cm) and cyst

Mammary adenocarcinoma, multiple – 5 nodes (0.5–3 cm)

Mammary adenocarcinoma, multiple – 4 nodes (0.5–3 cm)

Mammary adenocarcinoma, multiple – 4 nodes (0.5–3 cm)

Mammary adenocarcinoma, multiple – 4 nodes (0.5–3 cm)

Mammary adenocarcinoma, multiple – 4 nodes (0.5–3 cm)

Mammary adenocarcinoma, multiple – 4 nodes (0.5–3 cm)

Skin adenocarcinoma (tail)

Previous treatment

Castration

–

Antibiotics

–

–

–

–

–

Antibiotics

Surgically removed, resection wound did not heal

MZ treatment

3×200 mg/day, 28 days

3×200 and 2×200 mg/day, 6 months

3×1200 mg/day

3×400 mg/day, 1 month

3×400 mg/day, 58 days

3×400 mg/day, 2.5 months

5×400 mg/day, 3 months

3×400 mg/day, 4 months

5×400 mg/day, 10 months

6×400 mg/day, 62 days, and local appl. of powdered substance

Biochemical and hematological changes

HMT 61→45; ALT 103→62

AST 55→10; GGT 4→1

Bilirubin 25.8→6.2; AST 497→16; ALT 433→43; ALP 79→33

No changes

No changes

No changes

No changes

No changes

No changes

Glucose 6.9→3.8; AST 50→38

Therapeutic effects

7 days a.t. general improvement; withdraw of catheter;

14 days a.t. no signs of disease

90 days a.t. reduction in tumor mass (testis) to 1/3

29 days a.t. tumor disappeared

10 days a.t. all nodes disappeared; 12 months later no signs of disease

58 days a.t. all tumor nodes reduced in size 50%

2–3 months (smaller nodules); 4–6 months (larger nodules)

2–3 months (smaller nodules); 4–6 months (larger nodules)

2–3 months (smaller nodules); 4–6 months (larger nodules)

2–3 months (smaller nodules); 4–6 months (larger nodules)

3 days a.t. normal healing started and completed 7 days later

7. 7  Dobermann 8 F pinscher

8. 8  English 9 F cocker

   spaniel

9. 9  Airedale 9 F terrier

10. 10  German 8 38 M shepherd

Table 2 (continued)
No. Breed Age

(years)a

11. 11  Mixed 10 German

    shepherd

12. 12  Malamute 12

13. 13  German 5 pinch

14. 14  Berner 8 sennenhund

a At the beginning of therapy b Hormone dependent

Weight Sex (kg)

35 M

40 M

3 M

40 M

Diagnosis

Carcinoma planocellulare of the skin (tail), 3 tumor

Carcinoma planocellulare of the tongue

Hypertrophy and hyperplasia of salivary gland

Lung cancer

Previous treatment

Two nodes surgically removed

Surgically removed, resection wound did not heal

Antibiotics

–

MZ treatment

4×100 mg/day, 93 days

3×100 mg/day, 32 days

3×100 mg/day, 147 days

4×400 mg/day, 35 days

Biochemical and hematological changes

Urea 17.5→6.3

–

Urea 9.5→7.5; AST 40→27; ALT 54→36; L 3.1→12

AST 35→16; bilirubin 8.5→2.8

Therapeutic effects

remaining node disappeared 67 days a.t.

3 days after treatment wound started to heal, and

2 days later no further signs of wound visible; dog started to eat

7 days a.t. node became softer and smaller (75%);
14 days later no signs of hypertrophy

7 days a.t. general improvement; 7 days later no signs of tumor (obtained by X-ray)

717

came only insignificantly smaller, the dog showed no signs of illness. At this point it is interesting to note that the very high pretherapy serum values for aspartate ami- notransferase (497 μmol/l) and alanine aminotransferase (433 μmol/l) decreased after 1 month of MZ therapy to normal levels (16 and 43 μmol/l) and remained in the normal range for entire observation period (5 months).

Another dog (case 2) had, in addition to prostate tu- mor, a testis tumor. The testis was approximately 20 cm in diameter when the therapy with MZ was started. After 1 month therapy the testis size was reduced by one-third. After 2 months of therapy the testis was reduced in size to one-half and after 3 months to one-third of its pre- treatment size (Fig. 8A). However, the prostate remained equally large.

The third dog (case 1) diagnosed to have prostate ade- nocarcinoma came to the clinic in a very bad general condition. It urinated only with great difficulty. After 1 month of classical therapy no improvement was ob- served. A catheter was placed in the dog’s urethra. The therapy was continued for a further 2 weeks but did not work. The dog was ante finem and the owners asked for euthanasia. Classical therapy was then replaced by MZ

therapy (3×200 mg/day). After 1 week a general im- provement was observed, and the catheter was removed. After 14 days of therapy no signs of disease were still visible. The therapy continued for an additional 14 days, with daily health improvement. Then the owners decided on castration (in most cases castration eliminates prob- lems related to the prostate), and the therapy with MZ was stopped. Eight months later the dog is still alive without any major health problems.

Three dogs suffered from skin tumors. One of these (case 11) had three lesions nodules on the skin above the tail. Two were removed, and the third, the smallest, was left. Histologically the tumor was diagnosed as carcino- ma planocellulare. After 1 month of therapy with MZ the cherry-sized tumor was reduced in size by one-third. Over following 5 weeks the lesion disappeared com- pletely. The dog is still (7 months latter) under therapy. The presently 11-year-old dog is very vivacious and in unusually good condition.

Another dog (case 10) suffered from adenocarcinoma on the skin of the tail, which was surgically removed. However, even 2 weeks after surgery the wound did not heal, and amputation was considered. The dog was then

718

Fig. 8
perplasia (B) in dogs. A Poodle, 12 years old, case 2. B Pinch, 5 years old, case 13. Arrow Day of therapy cessation. All other de- tails are indicated in Table 1

given MZ in capsules, and powdered MZ was also scat- tered on the wound. The wound healed within 1 week.

The third dog (case 12) had a growth on its tongue of approx. 2 cm diameter. Histologically it was carcinoma planocellulare. After surgical removal of the tumor the wound did not heal. The dog was given MZ orally in capsules, and powdered MZ was also applied locally. Five days later the biopsy wound was no longer visible.

A 5-year-old dog (case 13), diagnosed to a have en- larged (walnut-size node) left salivary gland, was treated with conventional therapy for 4 months, without success. During that time the gland become larger and larger, and the dog developed serious problems with swallowing and salivation. After only 1 week of MZ therapy the node become softer and smaller by one-third. After a fur- ther 1 week the node disappeared completely, and only the capsule was palpable (Fig. 8B).

Mammary adenocarcinomas, in the form of multiple nodules (in sizes between that of green beans and large walnuts), were diagnosed in six female dogs. After the therapy with MZ was started, the nodules disappeared completely: in one dog after 10 days, with no signs of disease even after 12 months; in four dogs after 2–3 months (smaller nodules) and 4–6 months (larger nodules), with no signs of disease thereafter, at the pres- ent, 2 months; and in one dog the nodules were reduced in size to 50% after 58 days of treatment.

In one case of a dog (case 14) with lung cancer, again, after only 14 days of treatment with MZ (4×400 mg/day) signs of tumor disappeared completely.

In addition to the effects of MZ expressed on the pri- mary disease, all dogs, even those in which primary dis- ease was not cured, responded to MZ therapy in only about 7 days with general constitutional and behavioral improvement lasting even after the therapy was inter- rupted. The same was observed for some hematological and serum clinical parameters measured before and after the therapy. Hematocrit decreased to the normal range in case 1. Very high total serum bilirubin values fell to the normal range in cases 3 and 14, while serum urea con- centration change was noted in cases 11 and 13. The most pronounced improvement was noted for aspartate aminotransferase, alanine aminotransferase, and alkaline leukocyte phosphatase, with pretherapy values normal- ized after the therapy was started in almost all cases (nos. 1, 2, 3, 10, 13, and 14; Table 2).

Discussion

Numerous natural compounds are commonly used for the treatment of various diseases, including green tea and soybean extracts (for review see [20]). Recent findings indicate that dietetic products and antioxidant com- pounds also have a beneficial effect particularly in can- cer patients. In many cases the exact mechanism of their action is not fully understood. In this report we studied the effect of natural clinoptilolite zeolite particles on de- velopment of several cancer models in vivo and in vitro. We found that mechanically activated clinoptilolite zeo- lites act as anticancer therapeutic agents in in vivo ani- mal studies and in tissue culture cell models. Clinoptilol- ite applied orally in mice and dogs suffering from a vari- ety of tumor types led to a significant shrinkage of some tumors and improvement in overall health status in some animals.

The range of effects was diverse, ranging from nega- tive antitumor response, to normalization of biochemical parameters, prolongation of life span, and decrease in tu- mor size. The best results in animal models were ob- served in the treatment of skin cancer in dogs, suggest- ing that adsorption of some active components is respon- sible for MZ activity (direct contact action). Comple- mentary studies performed in tissue culture indicated that MZ treatment affects proliferation and survival of several cancer cell lines. Addition of MZ inhibited cell proliferation in a concentration-dependent manner, in part due to induction of inhibitors of cycline dependent kinases, inhibition of B/Akt expression and induction of programmed cell death.

The work described here was performed with the non- toxic natural, high silica content zeolite, clinoptilolite. The zeolite particles were negatively charged in the en- tire pH range studied (pH 1–11). Electron microscopy showed the absence of fibers, and most particles were round with very rough surface (data not shown). The ab-

Growth rate of testicular tumor (A) and salivary gland hy-

sence of fibrous, positively charged particles was en- couraging since such particles are present in asbestos and erionite zeolites, which are highly carcinogenic and mutagenic. In addition, activated zeolite particles did not catalyze the production of hydroxyl radicals, unlike as- bestos or erionite (data not shown). It seems that absence of fibrous particles capable of producing hydroxyl radi- cals makes this zeolite sample nontoxic and noncarcino- genic, at least when applied orally.

Silicate and aluminosilicate particulates can interact directly with specific cells and modify their intracellular pathways, leading to the regulation of gene expression. MZ was particularly successful in inhibiting protein ki- nase B/Akt in in vitro experiments with cancer cells. Such inactivation resulted in growth inhibition and in- crease in apoptosis of cancer cells. Inhibition of Akt by MZ treatment was shown only in the presence of serum. This indicated that adsorption of serum components can be one of the mechanisms of MZ action in these experi- ments. Indeed, the addition of EGF to serum-free medi- um led to activation of Akt, which was also blocked by MZ pretreatment. Adsorption of molecules involved in signal transduction cascades, such as inositol phospha- tides and calcium, might also contribute to its therapeutic efficiency. Preliminary lipid adsorption studies show that MZ are strong lipid sorbents. Similar results are ob- served with adsorption of proteins. Modifications of membrane ordering and interactions of other proteins with membrane proteins might also be involved [21], since membrane translocation is needed for activation of protein kinase B/Akt. It has also recently been shown that the activation of phosphoinositide-3 kinase and Akt is responsible for the ability of transformed epithelial cells to survive without cell attachment. Recent results indicate that constitutive activation of phosphoinositide- 3 kinase in five small-cell lung cancers cell lines studied was responsible for fast growth and anchorage indepen- dence of small-cell lung cancer cells [22]. In accordance with this, MZ treatment leads to inhibition of protein ki- nase B/Akt pathways and subsequent apoptosis in our cell model. Akt has recently been demonstated to inacti- vate an important cyclin inhibitor and tumor suppressor molecule, p27KIP1 [22].

Here we provide evidence that MZ treatment increases levels of p21WAF1CIP1 and p27KIP1 in tumor cell models. It is not yet clear whether inhibition of Akt is involved in regulation of expression of p21WAF1CIP1 and p27KIP1 cell cycle inhibitors. Preliminary results also show that MZ adsorbs and deactivates nitric oxide and other oxidants. In addition, it has recently been reported that antioxi- dants stimulate the activation of cyclin inhibitor p21WAF1/CIP1 [23]. This molecule is responsible for the arrest of cell growth, and its expression in adenocarcino- mas of lung is positively correlated with optimistic sur- vival prognosis. The present study observed that activat- ed clinoptilolite induces tumor suppressor molecules (both p21 and p27).

The mechanisms of action of MZ in vivo remain largely unknown at this time. The results presented here

indicate that inhibition of proliferation and survival of cancer cells may be part of mechanisms involved in anti- cancer effect of MZ compounds. More studies on several other aspects of their action including possible immuno- modulatory action of MZ will be performed in the future. Taken together, this report characterizes cellular effects of the MZ compounds in tissue culture cell models and provides data supporting a role of natural zeolite as an anticancer therapeutic agent in in vivo tumor models.

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