Abstract: The purpose of this paper, the cytotoxic effects of some biological and chemical agents on G1, S ,G2, M and G0 phases of the adenohypohyse carcinoma (AHC), kidney adenocarcinoma (KAC) and skin carcinoma (SC) cells obtained with chemical carcinogens DMBA (Dimethylbenzanthracene), cadmium chloride is to study. Experimental anticancerogenic study. The present study was conducted in Biology Laboratory of Faculty Sciences and Arts, University of Dumlupinar, Kutahya between january 2001 and july 2002. Adenohypophyse carcinoma, kidney adenocarcinoma and skin carcinoma tumours were obtained by injecting 1 ml DMBA/Kg/day (Dimethylbenzanthracene) to the female rabbits (Lepus capensis) at 36-42 days. Some agents were tested on cancer cells. Adenohypophyse carcinoma, kidney adenocarcinoma and skin carcinoma tumours and some biological and chemical anticancerogen agents. Tortoise (Testudo greaca) shell, sponge (Geolica cydonum), medusa (Aurelia aurita), meat fly (Calliphora erythrocephala larva, frog (Rana ridibunda) larva, juniper (Juniperus communis) berries and mistletoe (Viscum album) extracts and cis platin chloride have decreased by killing AHC, KAC and SC cells (p<0.05, p 0.01, p<0.001 ) in G2, M and G0 phases. In G0 phase, 3.0 µmol ml-1 dose of biological extracts and EtAzhexAzhepSi have inhibited in significantly the AHC cells, respectively at 94, 93, 91, 86, 69 and 65% levels (p<0.001). CsCl and CsCl+MgCl2 were more effected on AHP cells, at 94 and 96% levels (p<0.001). Genistein, genistin, glycitein, glycitin, daitzein and daitzin; were decreased in significantly the AHC cells (p<0.05, p<0.01, p<0.001). Cesium chloride; cesium chloride and magnesium chloride mixture were most affected as the shrinkage on KAC cells, at 95 and 97% rations (p<0.001). CsCl and MgCl2 were most effected on SC cells than other agens (95 and 96%, p<0.001). Inhibition rates of tortoise shell, sponge, frog larva, eat fly larva,medusa and juniper berries extracts on SK cells were respectively 83, 88, 65, 78, 89 and 88% in G0 phase (p<0.001). These data suggest that some biological extracts and chemicals tested may be useful chemotherapeutic agents to inhibit the growth of cancer cells and this study would be a light for new anticancerogenic experiments and preventing the variously cancer on human.
INTRODUCTION
It couldn’t be met the literature about shell-cancer relations. Tortoise species have very longevity and and their shell are very much resistant against the environmental and the meterological conditions. These animals are hibernate and they have antifreeze substances as the glycoprotein. Due to the antifreeze substances, tortoise shell and their bloods and their extracts may kill the cancer cells or some of them may decrease.
The sponges have not the blood and lymph vessels, but their chemical defenses in the water are very powerful. In general, the sponges have various anticancerogen substances as Agosterol A, Jasplakinolide, Naamidine A, Spongistatins Agelastins, Crellatatin, Bolinequineno, Haliclona, Cyclamines, Scalarence, Sestertatins, Spongouridine, Spongosine, Spongothymidine[1,2]. These sponge chemicals are cytotoxic for cancer cells. Therefore, sponge chemicals may be used in cancer therapy and preventing of microbial infections. The Japanese sponge Halicondria okadai has halicondrins and these chemicals in vitro towards p388 and B 16 melanoma. Also, these chemicals are potent and strong against p388 leukemia and lung tumor[3-5]. However, another sponge chemicals are spongistatins and spongistatins have been isolated from Spirastrella sipinispirulifera and this cemical is very cytotoxic. The spongistatins are inhibitors of tubulin polymerisation. Also, Jasplakinolide obtained fom Jaspis sp. is cytotoxic substance and antifungal[7-16]. Mycapeoxide B obtained fom Mycale sp. exhibits antiviral and anticancer activities[7,14,17].
During this study, the literatures with related to the larval therapy of various cancer cells couldn’t met. In prestudies, it was observed that the frog and meat fly larvae have proteolytic enzymes and therefore these larvae extracts may be cytotoxic effect on cancer cells and fresh extracts of larvae may digest the tumors. The medusa has a powerful the proteolytic enzymes and their digestive systems are very fastly and have a large digestive capacity for small invertebrates and fishes. But, the literature with related to the cancer of the medusa couldn’t met.
The juniper berries are used to break the kidney stones and the antihyperglycemia in alternative medicine. Juniper berries extracts are cytotoxic for kidneys when it is taken in much quantities but their anticancerogen effects are unknown.
The mistletoes are live as exoparasite on various tree and this plant has been using use as antihyperglycemic in Turkey. It couldn’t be found any a research about these plants as related with the cancer.
Soybean isoflavonoids; genistin, genistein, glycitein, glycitin, daitzein, daitzin; are isolated as chemical and these chemicals are known as phytoestrogens and a few of these have anticancerogen. At the same time, isoflavonoids have oestrogenic effect in humans, but this activity is much lower than that of human oestrogens[18,19]. Soybean isoflavonoids are prevent the development of cancer in many different organ systems and are decreased the cancer cells[20-26].
Dimethylsilane polyamines have cytotoxic effects and these chemicals are polyamine analogues. Most dimethylsilane polyamines accumulate to higher levels in spermidine-depleted cell than in cells with a normal polyamine pattern. AzhexSi, AzhexSi-DFMO, Azhex-AzhepSi, Et Azhex-AzhepSi, AzhepSi, DL 54 and Hexamine analoguse of ployamines have cytotoxic efects and these are decreased the various cancer cells and are inhibited the growing of cells[27-38].
However, little information is available on the in vitro effects of chronic treatment with isoflavonoids, dimethylsilane polyamines and Tortoise (Testudo greaca) shell, sponge (Geolica cydonum), medusa (Aurelia aurita), meat fly (Calliphora erythrocephala larva, frog (Rana ridibunda) larva, juniper (Juniperus communis) berries and mistletoe (Viscum album) extracts.Therefore, the present study was conducted.
MATERIALS AND METHODS
Subjects: In this study, tortise (Testudo greaca), frog (Rana ridibunda) larvae, meat fly (Calliphora erythrocephal A.) larvae, juniper (Juniperus communis) berries, mistletoe (Viscum album) were obtained from foresty and wet areas. Sponge (Geodica cydonium) and medusa (Aurelia aurita) were collected from Bodrum and Marmaris coasts of Turkey in the Mediterranean. Genistein was from, Genay, France, Daitzein, Daitzin, Genistin, Glycitein and Glycitin were purchased from Fujicco Co. Ltd., Kobe, Japan. Cesium chloride and magnesium chlorure, Cis Platin were Merck, Dimethylsilane polyamines were purchased from Merck (Darmstadt, Germany) and Sigma Chemical Co. (St Louis, Mo). Also, other chemicals and DMBA were purchased from Merck.
The present study was conducted in biology laboratory of our faculty between January 2001 and July 2002 years as below :
Preparing of agent extracts: Concentraions of the extracts and chemicals were prepared as 1 and 3 μml/ml to the pre-experimental studies.
Two adult male tortoise were anesthesized with the phenobarbitalnatrium and later they were decapitaded with 5 ml of anesthetic matter. Anesthetic matter was injected as intra arteria jugularis.Plastron and carapax of the tortoise were sawn and its internal organs were taken out.The shells were cleaned from the meats and the fats and sterilized with 70% of ethyl alcohol and were dried. These shells were broken with the cracking machine and were ground with the grinder. The grinding particules were 43 micron diameter.The meal of the shell was boiled in 10% of KOH and was extracted by hexane and methanol and was sterilized in the autoclave.
Porifera specimens were washed with distilled water and cut into slices and these were washed over and over again and they were dried in etuve and were ground.The diameters of the slices were 43 micron and were extracted by hexane and methanol and the slices were sterilized.The larva specimens were collected from watery habitats and they were washed with the distilled water and were killed with the ether inhalation.The larvae were homogenised with the cold bidistilled water as v/v and and were extracted by hexane and methanol.
The meat flies were cultured in bowine meat medium and their eggs were cracked at 18°C in 24 h and the white pups were come tolight approximately at 5 days. These pups were killed with the ether inhalation and were homogenised with the cold bidistilled water as v:v. and were extracted by hexane and methanol. The medusa specimens collected were put in the glass flasks and immediately they were allowed by theirselves melting and all medusa specimens were homogenised at the coast and were extracted by hexane and methanol.The fresh berries were washed with the bidistilled water and were dried between the filter papers and later berries were ground with the grainer. The meal of the berries was diluted with the bidistilled water and also was sterilized in autoclave.
The present mistletoes were collected from pines (Pinus nigra) and were washed with the bidistilled water and were dried between the filtered pappers. Later the fresh mistletoes were homogenised at the equal volumes, that is v:v va ration and were extracted by hexane and methanol and were sterilized in autoclave. All of the materials were sterilized and their micro organism controls were made.
Preparing of tumor cells and inhibition of cancer cells: Adenohypophyse carcinoma, kidney adenocarcinoma and skin carcinoma tumours were obtained by injecting 1.0 ml 3.0 DMBA/Kg/day (DMBA:Dimethylbenzanthracene) Adenohypohyse, Kidney and kidney glands and Internal and external skin areas of the female rabbits (Lepus capensis) at 36-42 days and as one dosage in day. The animal number, injecting area of DMBA and type of cancer were given at below. After approximately 36-42 days, rabbits were anesthesized with phenobarbitalnatrium injecting and were taken their organs and the animals were decapitated. In these operations, lung carcinoma, breast adeno carcinoma, leukemia cells were obtained in U.V. sterilized chamber. All of the tumors were taken in the sterilised glass flasks. Immediately, tumors were homogenised with 0.09% of NaCl at equal volumes, that is, 1 v:1 v in sterilized conditions. Later, the homogenised cancer cells were cultured in RPMI-1640 medium (Sigma-Aldrich.St. Louis, Mo.) supplemented with 10% v/v fetal bovine serum (Gibco, Paisley, UK), 1.0 v/v L-glutamine and 1.0% v/v penicillin, streptomycine and neomycine (PSN) antibiotic mix. Biorack does not provide a 5.0% C02 atmosphere; therefore, for experiments in the GCAK cassettes, the RPMI- 1640 mix supplemented with 25 mμ hepes, 12 mM Na2CO3 and 1 mM sodium pyruvate, all from Sigma. These supplements replace the requirement for atmospheric CO2 by buffering the medium and providing a source of soluble bicarbonate.Additional serum was added to the cells at transfer to 37°C as indicated in the experimental protocol to increase the serum concentration to 10%. Experiments were performed in both the GCAK-1 and 2 units.The culture chamber volume and material composition of both cassetta types are identical. Each cassette chamber was loaded with approximately 0.5 ml of cell suspension using an eppendorf multinjector. Viable and total cell counts were determined from trypan blue exclusion counts under the microscope using a heamocytometer. Two counts of at least 200 cells were performed for each sample in the cassettes. The initial viability of the cells was determined before loading of the cells into the cassettes. The initial viability of the cancer cells was determined before of the cells at 37°C. Moreover, caspase method was applicated. As components, the caspase substrate reagent kit (cat. A304R1G-3) was used. This kit contains three vials (each vials contain 500 μl) plus one additional substrate vial containing at least 750 μl of 10 μl M substrate in RPMI 1640 medium. Additionally, 1 bottle (60 ml) of flow cytometry dilution buffer is included. Three caspase substrate reagent kit (cat. A304R1G-6) contains six vials (500 μl) plus two additional substrate vials (750 μl) of 10 μM substrate RPMI 1640 medium and 2 bottles of the flow cytometry dilution buffer. All of the medium were removed in order to minimize substrate dilution. 50 to 75 μl of 10 μM substrate solution were added to each of centrifuged cell pellets.The cell number in these solutions was between 0.5 and 1.0 million per sample.The vortex was not used. The incubation was made in 5% CO2 incubator at 37°C for 60 min before cytometric analysis. The present cancer cells were once by adding 1 ml of ice cold flow cytometry dilution buffer, centrifuging and removing all of buffer. The cell suspensions were kept on ice until analysis by flow cytometry. All samples were analysed within 60 to 90 min after the end of the 37°C incubation. After collecting PI(+) cells if a drop of a 5-10 mg ml-1 propodium iodide (PI) solution was added and samples are rerun on the flow cytometer. Reanalysis was make within 5 minutes of PI addition. The fluoresence of dead cells, i.e., those that has shown PI (+), has shown lower fluorescence intensity in the FL 1 channel than was apoptotic PI (-) or non-apoptotic cells. The cytotoxic effects were tested on the cell analysis, that is, G1, S, G2, M and G0 phases. These phases, G1, S, G2 ,M and G0 were established with the classic histopathological assays. Each treatment was tested twenty times[1-41].
Statistical analysis: ANOVA and means±SEM of percent values were calculated; p and±SEM values were given in Tables[4-15].
RESULTS
In this study, each agent has inhibited in the various levels the cancer cells. Results were given Table 1, 2 and 3. Here results were given to the effects of 3.0 μmol ml-1 of agents, because this dose was more important in phases. According to Table 1, the inhibition of adenohypophyse carcinoma cells.
Phase G1: Medusa, spongia, tortoise shell, meat fly lava and juniper beries extracts have decreased at 47, 44, 38, 26 and 26% levels, respectively (p<0.01, p<0.001).Genistein, genistin and daitzein, have made the cytotoxic effect at 25, 15 and 15% levels, respecively (p<0.01). CsCl and CsCl + MgCl2. Mixture have decreased AHC cells at 58 and 60% levels (p<0.01).MgCl2 has increased the effect of CsCl. Dimethylsilane polyamines have not inhibited in the significantly than theirselves controls.
Phase S: The effects in G2 phase have found similarly to that of G1. Medusa, sponge, tortoise shell, juniper berries and meat fly larva extracts have made the shrikage on AHC cells at 52, 42, 35, 35 and 25% levels, respectively (p<0.01, p<0.001). Genistein has the most inhibited than other isoflavonoids, at 21% ration (p< 0.01).Also, CsCl and CsCl+MgCl2 have decreased the AHC cells at 59 and 67% levels (p<0.01). But, dimethylsilane polyamines have not made an important effect in S phase.
Phase G2: Medusa, sponge, tortoise shell, juniper berries and meat fly larva extracts were more effected in phase G2, at 79, 72, 68, 63 and 49% rations, respectively (p<0.01, p<0.001). Genistein and daitzein have inhibited at 29 and 26% levels (p<0.01, p<0.001).CsCl and CsCl + MgCl2 mixture were more effected than other agents(p<0.001). AzhexAzhepSi was effected at 42% level (p<0.01).
Phase M: Mistletoe extract exterior, other biological extracts have made the cytotoxic effects, that is, inhibition percentages from maximum to minumum in Table 2 : medusa: 86%, sponge : 85%, tortoise shell : 80%, juniper berries : 77%, meat fly larva : 45% and frog larva : 41% (p<0.01, p<0.001). Isoflavonoids, enistein, daitzin, daitzein, genistin, were effected at 35, 23, 22 and 21% levels, respectively, than controls (p<0.01). CsCl and CsCl+ MgCl2 were effected at 82 and 81% rations (p<0.001). EtAzhexAzhepSi, EtAzhepSi, Hexamine, DL54 and AzhexAzhepSi from dimethylsilane polyamines were effected on AHC cells at 68, 48, 47, 46 and 44% levels (p<0.01, p<0.001).
Phase G0: Inhibition percentages were given in parenthesis. The glycitin exterior, other all agents were the most effected, especially CsCl (94%) and CsCl+MgCl2 (96%), medusa (94%), sponge (93%), tortoise shell (91%), juniper berries (86%), meat fly larva extracts (69%) and EtAzhexAzhepSi (65%) (p<0.001). AHC cells have been decreased in significantly in M and G0 phases (p<0.01, p<0.001).
According to Table 2; inhibition of kidney adenocarcinoma (KAC) cells :
Phase G1: Sponge and medusa extracts were more effected than other biological extracts and their inhibition percentages were respectively 44 and 46% (p<0.01). Genistein from isoflavonoids was also effected at 27 level (p<0.01). CsCl and CsCl+MgCl2 were the most effectted than other agents (p<0.01). Dimethylsilane polyamines were not in significantly.
Phase S: Frog larva and mistletoe extracts exterior, other all biological extracts have decreased in significantly the KAC cells (p<0.01, p<0.001), especially medusa and sponge extracts were more effected than othes (p<0.001). Genistin and daitzein were effected at 17 and 16% levels (p<0.01). CsCl and CsCl+ MgCl2 mixture were the most effected, that is, their inhibition percentages were 61 and 65% (p<0.01).
Phase G2: The inhbition rates in phase G2 have shown the increaes.Mistletoe extract exterior, other all biological extracts have increase the shrinkage in KAC cells.The inhibition percentages of medusa, tortoise shell, sponge, juniper berries and meat fly larvaextract were more important than other biological extracts and their inhibition percentages were, respectively 81, 70, 69, 68 and 54% ( p<0.01).
| Table 1: | In vitro inhibition percentages of adenohypophyse cells. C: Control (normal cells without cancer) Dosages : 1 and 3 μ mol ml-1 |
| *: p<0.05, **: p<0.01, ***: p<0.001 ( than that of the controls ). ±SEM values of all results have been found between 1.23 and 2.17 | |
| Table 2: | In vitro inhibition percentages of kidney adenocarcinoma cells. C.: Control (normal cells without cancer) Dosages: 1 and 3 μ mol ml-1 |
| *: p<0 05, **: p<0.01, ***: p<0.001 ( than that of the controls). ±SEM values of all results have been found between is between 2.23 and 3.10 | |
| Table 3: | In vitro inhibition percentages of skin carcinoma cells. C.: Control (normal cells without cancer) Dosages : 1 and 3 μ mol ml-1 |
| *: p<0 05, **: p<0.01, ***: p<0.001 ( than that of the controls). ±SEM values of all results have been found between is between 3.45 and 4.27 | |
Genistein, daitzein, daitzin were inhibited KAC cells as, respectively 32, 26 and 22% (p<0.01). CsCl and CsCl+MgCl2 mixture were effected at 78 and 78% levels (p<0.01). AzhexAzhepSi was effected than other polyamins (p<0.01).
Phase M: Inhibition percentageswere given in the paranthesis. The most favourities of this phase were medusa (88%), sponge (81%), tortoise shell (79%) and juniper berries (68%) extyracts (p<0.001), genistein (36%), daitzin (29%), daitzein (24%), genistin (23%) and CsCl (86%), CsCl + MgCl2 mixture (87%), EtAzhexAzhepSi (71%), EtAzhepSi (51%), DL54 (47%), AzhexAzhepSi (71%) and Hexamine (47%) (p<0.01, p<0.001).
Phase G0: The present agents have inhibited in significantlt the KAC cells in this phase. The glycitin exterior, other all agents have killed in the significantly the KAC cells (p<0.01, p<0.001). Medusa (91%), sponge (89%), tortoise shell (88%), juniper beries (87%), meat fly larva (73%) and and frog larva (59%) extracts were the most effected agents ontheKAC cells (p<0.01, p<0.001). Genistein (60%), genistin (49%), glycitein (25%), daitzin and daitzein (38%) isoflavonoids have decreased in the significantly the KAC cells than that of other isoflavonoids (p<0.01, p<0.001).The inhibition percentages of CsCl and CsCl + MgCl2 agents were 95 and 97% levels (p<0.001). All dimethylsilane polyamines were effected in phae G0 (p<0.001).
According to Table 3; inhibition of skin carcinoma (SK) cells :
Phase G1: The most effected agents from extracts were sponge (48%), medusa (42%), tortoise shell (37%), meat fly larva (27%) and juniper berries (26%) (p<0.01, p<0.001). Genistein, genistin and daitzein have inhibited SC cells at 29, 23 and 27% levels, respectively (p<0.05, p<0.01, p<0.001). CsCl and CsCl + MgCl2 have effected as 55 and 66% (p<0.01). Dimethylsilane polyamines couldn’t have inhibited in significantly than their controls.
Phase S: Medusa (56%), sponge (46%), tortoise shell and juniper berries (36%) extracts have shown in significantly apoptotic effects (p<0.01, p<0.001). Inhibition percentages of genistein, daitzein and genistin were found as 33, 29 and 25% (p<0.05, p<0.01).CsCl and CsCl +MgCl2 have made 61 and 65% of the shrinkages (p<0.01, p<0.001). But, dimethylsilane polyamines were not effected in significanytly in phase S.
Phase G2: The mistletoe etract and AzhexSi exterior, other all extracts and chemical agents have increased the apoptotic action of the SC cells. Inhibþition percentages were given in the paranthesis. Mdeusa (80%), sponge (68%), tortoise shell (67%), juniper berries (71%), meat fly larva (57%) and frog larva (35%) extracts were more effected than that of G1 and S phases (p<0.01, p<0.001).
Genistein, genistin, glycitein, daitzein and daitzin have the apoptotic functions at 38, 28, 21, 26 and 27% levels (p<0.01, p<0.001). Inhibition rates of CsCl, CsCl + MgCl2 and AzhexAzhepSi have found as respectively 78, 77 and 58% (p<0.01, p<0.001).
Phase M: The mainly effected agents SK cells in phase M were the mistletoe exterior, all extracts; isoflavonoids; CsCl, CsCl + MgCl2 mixture; Azhepsi exterior other polyamines (p<0.01, p<0.001).
Medusa (88%) and CsCl+MgCl2 (87%) were the most effected (p<0.001). The present extracts, the mistletoe extract exterior, were more effceted than that of isoflavonoids.
Phase G0: All of agents have decreased in significantly the SC cells (p<0.01, p< 0.001).CsCl +MgCl2 was the most effected than other agents, as 96% level (p<0.001). Tortoise shell (83%), sponge (88%), frog larva (65%), meat fly larva (78%), medusa (89%) and juniper berries (88%) extracts have made in the significantly the shrinkages onSC cells (p<0.001). Genistein (60%), genistin (51%), glycitin (38%), daitzein (43%) and daitzin (40%) were more effected than that of other phases (p<0.01, p<0.001). Dimrthylsilane polyamines were more effected than other phase (p<0.01, p<0.001).
DISCUSSION
Several phytochemicals and micronutrients that are present in animal and vegetable extracts are known to exert cancer chemopreventive effects in several organs.Among them, the soybean isoflavonoid genistein received much attention due to its potential anticarcinogenic, antiproliferative effects and its potential role in several signal transduction pathways. The present study was designed to investigate the effect of isoflavonoids on dimethylbenzanthracene (DMBA) induced adenohypophyse carcinoma, kidney adenocarcinoma and skin carcinoma carcinogenesis. The results of this investigation emphasize that the biological effects of isoflavonoids, particularly genistein may be organ specific, inhibiting cancer development in some sites yet showing no effect or an enhancing effect on the tumorigenesis at other sites, such as the colon.
The inhibition of tumor cells levels by genistein indicates its possible antioxidant potential, which is independent of the observed present tumor enhancement, yet this agent may also possess several biological effects that overshadow its antioxidant potential. The exact mechanism(s) of malign or benign tumor enhancement by genistein remain to be elucidated; it is likely that its present tumor-enhancing effects may, at least in part, be related to inhibition of prostaglandin catabolic enzyme activities. The incidence of variously cancer is lowest in countries in which soy product consumption is high. Dietary factors are thought to be important in prostate cancer growth and progression because of the increased incidence of clinical disease and the dietary changes that accompany immigration from a country with low incidence to one with high incidence. Soybeans contain a number of polycyclic isoflavonoids, such as genistein, that are capable of inhibiting key enzymes controlling cell signaling and replication. The in vitro growth of several rat and human some cancer cell lines has been reported to be inhibited by genistein and soy has been demonstrated to inhibit chemically induced prostate cancer formation in rats[20,21,25,42].
Cuticula structure of tortoise shell contain plypeptide, phenol, boron ,Na+, K+ and Ca++ and sulfur substances collected from Kütahya (Turkey).Therefore, polypeptide, phenol and boron can cause the apoptosome formations. Because, shell extract made the spherical apoptosome during experiments. These cases observed in laboratory. On the other hand, medusa extracts have the various enzymes and because of this reason it cause the apoptosis. Also, frog and meat larvae extracts have digestive enzymes. These larvae species are metamorphosis, but their extracts didn’t make this effect on cancer cells. The incidence of variously cancer is lowest in countries in which soy product consumption is high.
Dietary factors are thought to be important in prostate cancer growth and progression because of the increased incidence of clinical disease and the dietary changes that accompany immigration from a country with low incidence to one with high incidence. Soybeans contain a number of polycyclic isoflavonoids, such as genistein, that are capable of inhibiting key enzymes controlling cell signaling and replication.
The in vitro growth of several rat and human some cancer cell lines has been reported to be inhibited by genistein and soy has been demonstrated to inhibit chemically induced prostate cancer formation in rats[20,21,25,42].
Sponge species contain antimicribial and toxic substances.Each sponge species has different and sometime similar substances. Their pharmacceutical active, physiologically compatible derivatives is useful for treating the various neoplastic disease such as acute myelocytic leukemia, acute lymphocytic leukemia, malignant melanoma, adenocarcinoma of lung, neuroblastoma, breast carcinoma, ovarian carcinoma, gastric carcinoma, bladder carcinoma, hematologic malignancies. But this will be dependent upon the dosage. Endication and contrendication are very importantas pharmacologically. Juniper berries are primarily used in the treatment urinary tract infections such as cystitis and urethritis. Their berries are also often used in the treatment of rheumatism arthritis and gout and arthritic conditions associated with the accumulation of acid waste. In Turkey, as a vapour bath, juniperus is helpful in the treatment of bronchitis and lung infections, antiviral activities exhibited by the volatile oil. Therfore, juniper berries may have made the apoptosis and the apoptosome on cancer cells.
According to the results of this study, the compositions of the present agents can preferably presented for administration to humans and vertebrata in unit dosage forms, such as tablets, capsules, pills, powders, granule, suppositories, strerile parenteral solutions or suspensions and oral solutions or suspensions containing suitable quantities of an active ingredient[4-15, 17, 40-44].
Several dosage forms are shown in the following examples:
| Composition I | |
| Hard-Gelatin Capsules (for oral use) | |
| Total sponge extract | 30 mg |
| Corn starch | 20 gm |
| Talch | 20 gm |
| Magnesium stearate | 3 gm |
| Magnesium chloride | 100 mg |
Capsules may be useful for a neoplastic disease, oral, one or two capsules one to four times a day. All agents may named as ingredients and these may prepared in different dosage.
Using the procedure above, capsules are similarly prepared containing an active ingredient in various dosage, for example 5,10,15 and 20 μg amounts by substituting 5, 10, 15 and 20 mg of an active ingredient for the 30 mg used above.
| Composition II | |
| Tablets | |
| Active ingredinet ,micronized (for example, one of sponge, tortoise, isoflavonoids, CsCl) | 30 mg |
| Lactose | 250 gm |
| Corn starch | 40 gm |
| Magnesium stearate | 3 gm |
| Light liquid petrolatu | 4 gm |
| Magnesium chloride | 100 mg |
| Composition III | |
| Oral suspension (for one thousand ml) | |
| Active ingredient, micronized(for example, juniper berries) | 6 mg |
| Citric acid | 2 gm |
| Benzoic acid | 1 gm |
| Sucrose | 800 gm |
| Tragacanth | 3 gm |
| Magnesim chloride | 100 mg |
| Lemon oil | 3 gm |
| Deionized water | 1000 ml |
| Composition IV | |
| Parenteral product (for neoplastic disease) | |
| Active ingredinet, micronized (for example. CsCl+MgCl2+genistein +daitzein+tortoise shell+sponge) | 25 mg |
| Polysorbate -80 | 3 gm |
| Methylparaban | 3 gm |
| Propylparaban | 0.2 gm |
| Water for injection | 1000 ml |
All the ingredients except the active ingredient, are dissolved in the water and the solution sterilised by filtration. To he sterile solution is added the sterilised active ingredient, finaly divided by means of an air micronizer and the final suspension is filled into sterile vials and the vials sealed.
In this study, Cesium chloride and mixture of cesium chloride and magnesium chloride have decreased the amont of cancer cells tested (p<0.01 and p<0.001). Because, cesium compouns have toxic effects and the shrinkage affects[39,42,44]. Therefore, cesium chloride may use together or instead of magnesium chloride above formulations.On the other hand, both cesium chloride and spongistatin may decreaed amount of cancer cells[42,44].