Sarcoma-180 Cells Research Articles

Dose-Dependent and Time-Dependent Antitumor Activity of <i>Pleurotus ostreatus</i> Polysaccharides in Vitro

Elucidation of the<i> in vitro</i> antitumor effects of <i>Pleurotus ostreatus</i> polysaccharides is very important for their future clinical application. In this paper, we use Sarcoma-180 (S180) cells to test the antitumor activity of the mushroom polysaccharides. The S180 cells in freshly prepared mouse ascites were directly treated by water-soluble polysaccharides from mycelium of <i>Pleurotus ostreatus</i> and immediately detected by MTT [3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di- phenytetrazoliumromide] method. The results can be directly visualized with naked eyes through the color changes. The<i> in vitro</i> antitumor activity of <i>P. ostreatus</i> polysaccharides on S180 cells are dose-dependently and time-dependently. <i>P. ostreatus</i> strains p11, p23, P44, p105 and p176 showed significant inhibition to S180 cell activity under high polysaccharides concentration (1000μg/ml ~ 1250μg/ml), and there are non-significant effects under the middle concentration (750μg/ml). On the contrary, the S180 cell activity were significantly enhanced under low concentration (250μg/mL-500μg/ml). The effects of polysaccharides generally increased during 4-8 treat hours, but remained stable more than 7 hours. However, the strains P23, P44 and P105 enhanced the S180 cell activity for short time processing (4-5h), but inhibited the cell activity for long time processing (6-8h). Among the tested mushroom strains, P44 exhibited the highest inhibition rate (68.4%) under high concentration of 1250µg/ml and treat time of 7-8 hours, P23 had the highest enhancement (119.5%) under low concentration of 250µg/ml and treat time of 8 hours. The antitumor activity of <i>P. ostreatus</i> polysaccharides was dependent on its concentration and treat time, indicating the complicated antitumor mechanism of mushroom polysaccharides and the strict study should be conducted before their clinical application.

Investigating In Vitro and In Vivo Anti-Tumor Activity of Curvularia-Based Platinum Nanoparticles.

The use of platinum (Pt)-based anticancer drugs, although widespread in clinical practice, is severely limited due to toxic side-effects. One of the strategies for making Pt-based chemotherapy more effective is the synthesis and use of Pt nanoparticles (PtNPs). However, increasing evidence suggestD that nanoplatinum also pose potential risk to human health. This study examined the toxicity and anticancer activity of mycosynthesized PtNPs against sarcoma-180 (S-180) cells in vitro and in vivo. Curvularia affinis Boedijn, a phyto-pathogenic fungi isolated from rice, was used to synthesize PtNPs (named as CaPtNP). Well dispersed, mostly spherical CaPtNPs, with sizes ranging from 3-9 nm were characterized by Transmission electron microscopy (TEM), field emission scanning electron microscopy, X-ray diffraction, atomic force microscopy, and Fourier transform infrared spectrometry. Two concentrations of the CaPtNPs (2.31 and 4.63 ng/mL) were selected based on in vitro cytotoxicity assay on erythrocytes and peripheral blood mononuclear cells. The selected doses were found to induce significant in vitro and in vivo anti-proliferative and pro-apoptotic activity in S-180 cells. Elevated levels of pro-apoptotic markers (p53, Bax/Bcl2 ratio, Cyt c, caspase-3, cleaved PARP) and reduced BrdU incorporation validated the anticancer activity of CaPtNPs. The antitumor activity was further confirmed in S-180 transplanted tumor bearing mice. Moreover, examination of the impact of sub-chronic exposure (three months) of CaPtNPs on the ultra-structural features of renal and hepatic tissue by TEM revealed no significant toxicological manifestation in these organs. The CaPtNPs were also found to reduce oxidative stress and improve liver function in tumor bearing mice compared with untreated controls. Thus, this green CaPtNPs was well tolerated in mice and displayed significant antitumor property.

Inhibition of Radioactive Depletion of Hemocytes and Antitumor Effects of Flavonoid

Natural products are able to inhibit radiation effects and exert an antitumor effect with fewer adverse reactions; however, their antitumor effects are less than those of widely-used synthetic drugs. Flavonoid is a natural material that has attracting attention, and we extracted this material with alcohol and investigated the effect of continuous flavonoid administration on radioactivity-induced reduction of hemocytes, in addition to the antioxidant and antitum or effects of flavonoid. Following a 1-week adjustment period, flavonoid was administered intraperitoneally to male ICR mice at a dose of 100 mg/kg every other day for 2 weeks. Following administration, 2 Gy whole-body irradiation was performed and the counts of leukocytes, lymphocytes, and granulocytes and monocytes in the peripheral blood were determined 1, 3, 7, 15 and 30 days after irradiation. These cells were considered since they are closely associated with immunity to radioactivity. In a second experiment, flavonoid was similarly administered to the mice for 2 weeks after a 1-week adjustment period, and 2 Gy whole-body irradiation was performed. The antioxidant effects in hemocytes were then investigated using 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH), a radical generator. In a third experiment, 1×106 Sarcoma-180 cells were inoculated into the right thigh of mice, which were divided into four groups: control, flavonoid-treated, 6 Gy irradiated and Flavonoid-treated + 6 Gy irradiated groups, and changes in tumor size were measured for 20 days. Statistical analysis was conducted using ANOVA for multiple groups. In the three experiments, administration of Flavonoid inhibited the reduction of hemocytes caused by whole-body irradiation, showed antioxidant effects against radioactivity, and inhibited tumor growth, respectively. In conclusion, our data suggest that the antioxidant effect of Flavonoid inhibits hemocyte reduction caused by whole-body irradiation and enhances immunological inhibition of tumor growth.

PHYTOCHEMICAL SCREENING, ANTIOXIDANT, ANTI-CYTOTOXIC AND ANTICANCER EFFECTS OF GALINSOGA PARVIFLORA AND VERNONIA POLYANTHES (ASTERACEAE) EXTRACTS

The study was designed to investigate the chemical composition and the biological effects of G. parviflora and V. polyanthes ethanolic extracts in vitro. Total content of phenols, flavonoids and tannins was quantified by spectrophotometry; chemical characterization was permed by mass spectrometry (ESI (-) FT-ICR MS and APCI (+) FT-ICR MS analysis). Antioxidant activities were determined by FRAP and Fe2+ chelating methods. Extracts cytotoxicity was evaluated in human lymphocytes, sarcoma-180 (S-180) and human gastric adenocarcinoma (AGS) cells, by MTT assay. V. polyanthes presented higher total content of tannins and G. parviflora presented higher amount of phenols and flavonoids. Chemical characterization showed the presence of flavonoids, phenolic acids and sesquiterpene lactones in V. polyanthes extract, and steroids, phenolic acids and fatty acids (Poly Unsaturated Fatty Acids - PUFA) in G. parviflora extract. V. polyanthes extract stood out in the Fe2+ chelation test. G. parviflora extract did not present outstanding antioxidant results in the tested protocols. Both species showed a tendency to promote cytotoxicity in human lymphocyte cells. Regarding the antiproliferative effect, both species were able to reduce S-180 cell viability and G. parviflora extract showed high antiproliferative potential in the assay with AGS cells. These findings reinforce the medicinal use of these plants, as well as suggest their potential use for the development of new drugs and for the treatment of cancers.

Ruthenium(II)/Benzonitrile Complex Induces Cytotoxic Effect in Sarcoma-180 Cells by Caspase-Mediated and Tp53/p21-Mediated Apoptosis, with Moderate Brine Shrimp Toxicity.

Ruthenium(II)/benzonitrile complexes have demonstrated promising anticancer properties. Considering that there are no specific therapies for treating sarcoma, we decided to evaluate the cytotoxic, genotoxic, and lethal effects of cis-[RuCl(BzCN)(phen)(dppb)]PF6 (BzCN = benzonitrile; phen = 1,10-phenanthroline; dppb = 1,4-bis-(diphenylphosphino)butane), as well as the mechanism of cell death induction that occurs against murine sarcoma-180 tumor. Thus, MTT assay was applied to assess the ruthenium cytotoxicity, showing that the compound is a more potent inhibitor for the sarcoma-180 tumor cell viability than normal cells (lymphocytes). The comet assay indicated low genotoxic for normal cells. cis-[RuCl(BzCN)(phen)(dppb)]PF6 also showed moderate lethality in Artemia salina. The complex induced cell cycle arrest in the G0/G1 phase in sarcoma-180 cells. In addition, the complex caused S180 cells to die by apoptosis by an increase in Annexin-V-positive cells and morphological changes typical of apoptotic cells. Additionally, cis-[RuCl(BzCN)(phen)(dppb)]PF6 increased the gene expression of Bax, Casp3, and Tp53 in S180 cells. By using a western blot, we observed an increased protein level of TNF-R2, Bax, and p21. In conclusion, cis-[RuCl(BzCN)(phen)(dppb)]PF6 is active and selective for sarcoma-180 cells, leading to cell cycle arrest at the G0/G1 and cell death through a caspases-mediated and Tp53/p21-mediated pathway.

IN VIVO ANTITUMOR ACTIVITY OF PHYTOCHEMICAL PITC-2 OBTAINED FROM TISSUE CULTURED PLANT PLUCHEA INDICA ON SARCOMA-180 SOLID TUMOR MICE MODEL

Objective: PITC-2 was isolated from the methanolic root extract of tissue cultured medicinal plant Pluchea indica (L.) Less. PITC-2 is a thiophene derivative which is 2-(Prop-1-ynyl)-5(5,6-dihydroxyhexa-1,3-diynyl)-thiophene. The main objective of the study is to evaluate the in vivo antitumor activity of PITC 2 against sarcoma-180 cancer cell in Swiss albino mice.Methods: The antitumor activity was evaluated by treatment with PITC-2 at a dose of 2.5 and 5 mg/kg b.w for 21 days on sarcoma-180 mice model. Cell viability was studied using 3-(4, 5- dimethylthiazol -2-yl)-2, 5-diphenyl tetrazolium bromide assay and cell apoptosis, G1 cell cycle arrest and reduction in tumor cell proliferation were evaluated by histopathological analysis and Bcl-2, cyclic-D1, and Ki-67 protein expression through immunohistochemistry study.Results: Precisely, PITC-2 had a cytotoxic effect on various in vitro cancer cells. Significant decreases in solid tumor volume and weight along with increase lifespan also observed. The histopathological and immunohistopathological examination indicates that PITC-2 induces apoptosis, typical morphological changes and suppresses tumor cell proliferation along with G1 cell cycle arrest through the downregulation of the intratumoral expression of Bcl-2, cyclic D1, and Ki-67 and thus highlighting antiproliferative and apoptotic properties against sarcoma-180 in vivo solid tumor model.Conclusion: The present results clearly demonstrate that PITC-2 significantly inhibits sarcoma-180 cell growth in a dose-dependent manner in in vivo mice model. Besides this, the study reveals a comprehensive perception of the possible mechanism behind the antitumor activity of PITC-2 by significant changes in the morphological, hematological, biochemical parameters in sarcoma-180 cells.

Effect of lutein and doxorubicin combinatorial therapy on S180 cell proliferation and tumor growth.

Multidrug resistance and toxicity significantly compromise the therapeutic efficacy for sarcomas. We aimed at evaluating the effect of lutein-doxorubicin (DOX) combinatorial therapy on inhibiting S180 (Sarcoma 180) cell proliferation and tumor growth. S180 cells in logarithmic growth phase were treated with lutein, DOX, or lutein-DOX combinatorial therapy for 48 h. The cell survival rate was determined by MTT assay. Apoptosis was detected by flow cytometry. The expression of PCNA, P53, and NF-κB was assessed by Western blot. Further, mice bearing S180 tumors received lutein, DOX, or lutein-DOX combinatorial therapy by oral gavage. Lutein-DOX combinatorial therapy significantly decreased the proliferation of S180 cells (p<0.01) in vitro. Also, the expression of proliferating cell nuclear antigen (PCNA) (p<0.05) and the apoptosis-relevant gene p53 were decreased, which resulted in increased cell apoptosis (p<0.05). The level of nuclear factor kappa B (NF-κB) was also decreased by the combinatorial therapy. Lutein-DOX combinatorial therapy reduced the cytotoxicity of DOX and reduced the inflammatory response. The inhibitory effect of lutein-DOX combinatorial therapy on cell proliferation was confirmed in vivo. The growth rate and size of the tumor at 30 d after treatment were significantly lower than those of the control group and DOX single therapy. Lutein and DOX synergistically inhibit sarcoma cell proliferation and tumor growth. This novel therapeutic regimen could potentially improve clinical outcome of sarcoma patients.

Pharmaceutical Production of Anti-tumor and Immune-potentiating Enterococcus faecalis-2001 β-glucans: Enhanced Activity of Macrophage and Lymphocytes in Tumor-implanted Mice.

Enterococcus faecalis 2001 is a probiotic lactic acid bacterium and has been used as a biological response modifier (BRM). From physiological limitation of bacterial preservation in storage and safety, the live E. faecalis 2001 has been heat-treated and the BRM components containing high level of β-glucan, named EF-2001, were prepared. The heat-treated EF-2001 has been examined for the antioxidative potential for radical scavenging and anti-tumor activities as well as immune-enhancing response in mice. Lymphocyte versus polymorphonuclear leukocyte ratio was increased in mice upon treatment with EF-2001. The number of lymphocytes was increased in the EF-2001-treated group. In the mice bearing two different Ehrlich solid and Sarcoma-180 carcinomas, the treatment with EF-2001 resulted in anti-tumor action. Tumor-suppressive capacity upon treatment with EF-2001 was significantly increased compared to normal controls. During the time interval administration of 5 weeks between the priming and secondary administration of EF-2001, the expression and production levels of TNF-α were also observed in the EF- 2001-administered mice. Additionally, anti-tumor activity examined with the intravenous administration of EF 2001 with a 34 times interval was also observed, as the growth of Sarcoma180 cells was clearly inhibited by the EF-2001. From the results, it was suggested that the immune response is enhanced due to antioxidative activity caused by the EF-2001 and anti-tumor activity by NK cells and TNF-α.

Purification, characterization, and antitumor activity of a novel glucan from the fruiting bodies of Coriolus Versicolor.

Cancer is one of the most common causes of deaths worldwide. Herein, we report an efficient natural anticancer glucan (CVG) extracted from Coriolus Versicolar (CV). CVG was extracted by the hot water extraction method followed by ethanol precipitation and purified using gas exclusion chromatography. Structural analysis revealed that CVG has a linear α-glucan chain composed of only (1→ 6)-α-D-Glcp. The antitumor activity of CVG on Sarcoma-180 cells was investigated in vitro and in vivo. Mice were treated with three doses of CVG (40, 100, 200 mg/kg body weight) for 9 days. Tumor weight, relative spleen, thymus weight, and lymphocyte proliferation were studied. A significant increase (P< 0.01) in relative spleen and thymus weight and a decrease (P< 0.01) in tumor weight at the doses of 100 and 200 mg/kg were observed. The results obtained demonstrate CVG has antitumor activity towards Sarcoma-180 cells by its immunomodulation activity.

Poly-L-Lysine Inhibits Tumor Angiogenesis and Induces Apoptosis in Ehrlich Ascites Carcinoma and in Sarcoma S-180 Tumor

Background:This study focuses on the role of Poly-L-lysine (PLL), an essential amino acid, on molecular changes of tumor angiogenesis suppression, pro-apoptotic and anti-apoptotic gene expression after treatment on Ehrlich ascites carcinoma (EAC) and solid sarcoma-180 tumor cells bearing mice.Materials and Methods:The cell viability was carried out using MTT assay. The antitumor activity was evaluated by treatment with PLL at 20 and 40mg/kg/b.w doses for 14 days in EAC ascites tumor and 21 days for Sarcoma-180 solid tumor model. Several tumor evaluation studies, haematological and biochemical parameters were estimated. Importantly, the tumor cell apoptosis was assessed using microscopic observations, DNA fragmentation assay, Flow cytometric analysis, cell-cycle and electron-microscopic study, following which, the expression of several signal proteins related to pro-apoptosis, anti-apoptosis and tumor angiogenesis were quantified using western blotting and immunohistochemistry study.Results:Precisely, PLL had cytotoxic effect on K562; A549; U937 and B16F10 cancer cells. Significant decreases in liquid and solid tumors and increased life span of treated mice were observed (P<0.05). Typical morphological changes, apoptosis bleb phenomenon and sub-G1 cell cycle arrests revealed that PLL promoted apoptotic cell death. Western blot and immunohistochemistry confirms, PLL activated apoptotic signalling cascades through down regulation of Bcl-2 and CD31 protein and up-regulation of Bax and p53 proteins. The anti-angiogenic effects were also accompanied with decreased VEGF expression and reduced peritoneal-angiogenesis and microvessel density.Conclusions:The antitumor and antitumor-angiogenic activity of PLL was confirmed from all the results via up and down regulation of relevant signal proteins reported in this publication.

Replica exchange molecular dynamics simulations reveal the structural and molecular properties of levan-type fructo-oligosaccharides of various chain lengths

BackgroundLevan and levan-type fructo-oligosaccharides (LFOs) have various potential applications in pharmaceutical and food industries due to their beneficial properties such as their low intrinsic viscosity and high water solubility. Previous studies showed that they exhibited prebiotic effects, anti-inflammatory and anti-tumor activities against Sarcoma-180 tumor cells of human. Despite their various potential applications, the structural and molecular properties of LFOs of various chain lengths are not well understood.ResultsWe employed the replica-exchange molecular dynamics simulations method (REMD) in AMBER14 to elucidate structural and molecular properties of LFOs with chain lengths of 5 (LFO5), 10 (LFO10) and 15 (LFO15) residues in two models of generalized Born implicit solvent (GBHCT and GBOBC1). For LFO10 and LFO15, four distinct conformations (helix-like, partial helix, zig-zag and random structures) were characterized by their upper-middle and lower-middle torsions. For LFO5, two distinct conformations (partial helix and random structures) were characterized by their middle torsion and molecular angle of residues 1, 3 and 5. To determine hydrogen bonds important for the formation of helix-like structures of LFO10 and LFO15, occurrence frequencies of hydrogen bonds were analyzed, and the O6(i)--H3O(i+1) hydrogen bond was found with the highest frequency, suggesting its importance in helix formation. Among three dihedral angles between two fructosyl units [ϕ (O5’-C2’-O6-C6), ψ (C2’-O6-C6-C5) and ω (O6-C6-C5-C4)], dihedral angle distributions showed that ω was the most flexible dihedral angle and probably responsible for conformational differences of LFOs.ConclusionsOur study provides important insights into the structural and molecular properties of LFOs, which tend to form helical structures as the chain length increases from 5 to 15 residues. This information could be beneficial for the selection of LFOs with appropriate lengths and properties for pharmaceutical and biological applications.Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-016-1182-7) contains supplementary material, which is available to authorized users.

Synthesis and cytotoxic activity of novel 11-methyl-6H-pyrido[4,3-b]carbazole derivatives linked to amine, N-methylurea, and N-methyl-N-nitrosourea moieties with various types of carbamoyl tethers at the C-5 atom

Thirty-five types of novel pyridocarbazoles (5-(N-alkyl)carbamoyl-11-methyl-6H-pyrido[4,3-b]carbazoles and 5-(N-alkyl)carbamoyl-2,11-dimethyl-6H-pyrido[4,3-b]carbazol-2-ium chloride derivatives), that were conjugated with amine, N-methylurea, and N-methyl-N-nitrosourea moieties through alkyl-, oxyalkyl-, and iminoalkylcarbamoyl linkers, were synthesized by a series of reactions of methyl 11-methyl-6H-pyrido[4,3-b]carbazole-5-carboxylates with polymethylenediamine (n=2–5), p-nitrophenyl N-methylcarbamate, and N-methyl-N-nitrosocarbamate in high yields, and their cytotoxic activities were evaluated against Sarcoma-180, NIH3T3, HeLa S-3, and L1210 cell lines. These compounds exhibited potent cytotoxic activity (IC50=1.6–50 μM) and odd-even alternation effect. 9-Methoxy-2,11-dimethyl-5-((2-(3-methyl-3-nitrosoureido)ethyl)carbamoyl)-6H-pyrido[4,3-b]carbazol-2-ium chloride exhibited the most potent cytotoxic activity (IC50=0.15 μM) and cell selectivity against HeLa S-3. Some of the un-charged 5-(N-alkyl)carbamoyl-6H-pyrido[4,3-b]carbazole derivatives, which were conjugated with the amine and N-methyl-N-nitrosourea moieties through a dimethylene spacer, also exhibited potent cytotoxic activity (IC50=0.43–2.4 μM) and remarkable cell selectivity as well as ellipticine, 9-hydroxyellipticine, and the starting methyl ester of the pyridocarbazole-5-carboxylate (IC50=0.30–2.2 μM).

Antitumor effect of sonodynamically activated pyrrolidine tris-acid fullerene

In this study, the sonodynamically induced antitumor effect of pyrrolidine tris-acid fullerene (PTF) was investigated. Sonodynamically induced antitumor effects of PTF by focused ultrasound were investigated using isolated sarcoma-180 cells and mice bearing ectopically-implanted colon 26 carcinoma. Cell damage induced by ultrasonic exposure was enhanced by 5-fold in the presence of 80 µM PTF. The combined treatment of ultrasound and PTF suppressed the growth of the implanted colon 26 carcinoma. Ultrasonically induced 2,2,6,6-tetramethyl-4-piperidone-1-oxyl (4oxoTEMPO) production in the presence and absence of PTF was assessed, and it was shown that 80 µM PTF enhanced 4oxoTEMPO production as measured by ESR spectroscopy. Histidine, a reactive oxygen scavenger, significantly reduced cell damage and 4oxoTEMPO generation caused by ultrasonic exposure in the presence of PTF. These results suggest that singlet oxygen is likely to be involved in the ultrasonically induced cell damage enhanced by PTF.

Parkia javanica Extract Induces Apoptosis in S-180 Cells via the Intrinsic Pathway of Apoptosis

ABSTRACTParkia javanica is a leguminous tree, various parts of which are used as food and folklore medicine by the ethnic groups of northeastern India. The present study investigates the in vitro and in vivo anticancer effect of aqueous methanol extract of P. javanica fruit (PJE). HPLC analysis was done to establish the fingerprint chromatogram of PJE and its in vitro radical scavenging activity was measured. PJE caused significant cytotoxicity in sarcoma-180 (S-180), A549, AGS, and MDA-MB435S cancer cells in vitro. Exploration of the mechanistic details in S-180 cells suggested that the reduced cell viability was mediated by induction of apoptosis. Increased expression of proapoptotic proteins such as p53, p21, Bax/Bcl2, cytochrome c (Cyt c), caspase 9, and cleaved poly(ADP-ribose) polymerase, and decrease in proliferative and antiapoptotic markers (Ki-67, Proliferating Cell Nuclear Antigen [PCNA], Bcl-2) validated the anticancer effect of PJE. A decline in the relative fluorescence emission upon staining S-180 cells with Rhodamine 123 (Rh 123), enhanced expression of cytosolic Cyt c and mitochondrial Bax, and inhibition of apoptosis in the presence of caspase-9 inhibitor in PJE-treated cells indicated intrinsic pathway of apoptosis. Liver function test and hepatic antioxidant enzymes demonstrated non-toxicity of PJE. Finally, the detection of PJE in sera by HPLC confirmed its bioavailability.

Creatine supplementation with methylglyoxal: a potent therapy for cancer in experimental models.

The anti-cancer effect of methylglyoxal (MG) is now well established in the literature. The main aim of this study was to investigate the effect of creatine as a supplement in combination with MG both in vitro and in vivo. In case of the in vitro studies, two different cell lines, namely MCF-7 (human breast cancer cell line) and C2C12 (mouse myoblast cell line) were chosen. MG in combination with creatine showed enhanced apoptosis as well as higher cytotoxicity in the breast cancer MCF-7 cell line, compared to MG alone. Pre-treatment of well-differentiated C2C12 myotubes with cancerogenic 3-methylcholanthrene (3MC) induced a dedifferentiation of these myotubes towards cancerous cells (that mimic the effect of 3MC observed in solid fibro-sarcoma animal models) and subsequent exposure of these induced cancer cells with MG proved to be cytotoxic. Thus, creatine plus ascorbic acid enhanced the anti-cancer effects of MG. In contrast, when normal C2C12 muscle cells or myotubes (mouse normal myoblast cell line) were treated with MG or MG plus creatine and ascorbic acid, no detrimental effects were seen. This indicated that cytotoxic effects of MG are specifically limited towards cancer cells and are further enhanced when MG is used in combination with creatine and ascorbic acid. For the in vivo studies, tumors were induced by injecting Sarcoma-180 cells (2×10(6) cells/mouse) in the left hind leg. After 7days of tumor inoculation, treatments were started with MG (20mg/kg body wt/day, via the intravenous route), with or without creatine (150mg/kg body wt/day, fed orally) and ascorbic acid (50mg/kg body wt/day, fed orally) and continued for 10 consecutive days. Significant regression of tumor size was observed when Sarcoma-180 tumor-bearing mice were treated with MG and even more so with the aforesaid combination. The creatine-supplemented group demonstrated better overall survival in comparison with tumor-bearing mice without creatine. In conclusion, it may be stated that the anti-cancer effect of MG is enhanced by concomitant creatine supplementation, both in chemically transformed (by 3MC) muscle cells in vitro as well as in sarcoma animal model in vivo. These data strongly suggest that creatine supplementation may gain importance as a safe and effective supplement in therapeutic intervention with the anti-cancer agent MG.

Soy-Based Multiple Amino Acid Oral Supplementation Increases the Anti-Sarcoma Effect of Cyclophosphamide.

The use of a mixture of amino acids caused a selective apoptosis induction against a variety of tumor cell lines, reduced the adverse effects of anti-cancer drugs and increased the sensitivity of tumor cells to chemotherapeutic agents. We evaluated the effects and underlying mechanisms of soy-derived multiple amino acids’ oral supplementation on the therapeutic efficacy of low-dose cyclophosphamide (CTX) and on tumor growth, apoptosis, and autophagy in severe combined immunodeficiency (SCID) mice that were injected with sarcoma-180 (S-180) cells. 3-methyladenine or siRNA knockdown of Atg5 was used to evaluate its effect on sarcoma growth. A comparison of mice with implanted sarcoma cells, CTX, and oral saline and mice with implanted sarcoma cells, CTX, and an oral soy-derived multiple amino acid supplement indicated that the soy-derived multiple amino acid supplement significantly decreased overall sarcoma growth, increased the Bax/Bcl-2 ratio, caspase 3 expression, and apoptosis, and depressed LC3 II-mediated autophagy. Treatment with 3-methyladenine or Atg5 siRNA elicited similar responses as CTX plus soy-derived multiple amino acid in downregulating autophagy and upregulating apoptosis. A low dose of CTX combined with an oral soy-derived multiple amino acid supplement had a potent anti-tumor effect mediated through downregulation of autophagy and upregulation of apoptosis.

IN VITRO AND IN VIVO EVALUATION OF 2-CHLOROE THYLN ITROSOUREA DERIVATIVES AS ANTITUMOR AGENTS

To evaluate potential of Naphthal-NU, Napro-NU and 5-Nitro-naphthal-NU, 2-chloroethylnitrosourea compounds with substituted naphthalimide in the pre-clinical studies. In vitro cytotoxicity of three nitrosoureas was determined in human and mouse tumor cell lines by MTT assays. In vivo anti-tumor potential was evaluated in Sarcoma-180 (S-180) and Ehrlich's carcinoma (EC) solid tumors. Apoptosis in S-180 cells was analyzed by using Annexin V-Propidium Iodide (PI). Histological analysis of liver and kidney was performed at optimum dose (50 mg/kg). Expression status of CD4(+), CD8(+) and CD25(+) cells in treated mouse were also examined. Significant tumor growth retardation by the compounds was noted in early and advanced disease groups, as the life span of drug treated mice increased considerably. Drug induced killing was observed by induction of apoptosis. Naphthal-NU and 5-Nitro-naphthal-NU were effective to normalize the tumor induced structural abnormalities of liver and kidney. The compounds have no immunotoxic effect on CD4(+) and CD8(+) T cells and down regulate CD4(+)CD25(+) regulatory T cells. Overall data holds promise for the antitumor activity with lower toxicity of the compounds that can be utilized for the treatment of human malignant tumors.

Cytoxicity and apoptotic mechanism of ruthenium(II) amino acid complexes in sarcoma-180 tumor cells.

Over the past several decades, much attention has been focused on ruthenium complexes in antitumor therapy. Ruthenium is a transition metal that possesses several advantages for rational antitumor drug design and biological applications. In the present study, five ruthenium complexes containing amino acids were studied in vitro to determine their biological activity against sarcoma-180 tumor cells. The cytotoxicity of the complexes was evaluated by an MTT assay, and their mechanism of action was investigated. The results demonstrated that the five complexes inhibited the growth of the S180 tumor cell line, with IC50 values ranging from 22.53 µM to 50.18 µM, and showed low cytotoxicity against normal L929 fibroblast cells. Flow cytometric analysis revealed that the [Ru(gly)(bipy)(dppb)]PF6 complex (2) inhibited the growth of the tumor cells by inducing apoptosis, as evidenced by an increased number of Annexin V-positive cells and G0/G1 phase cell cycle arrest. Further investigation showed that complex 2 caused a loss of mitochondrial membrane potential; activated caspases 3, caspase-8, and caspase-9 and caused a change in the mRNA expression levels of caspase 3, caspase-9 as well as the bax genes. The levels of the pro-apoptotic Bcl-2 family protein Bak were increased. Thus, we demonstrated that ruthenium amino acid complexes are promising drugs against S180 tumor cells, and we recommend further investigations of their role as chemotherapeutic agents for sarcomas.

영지(Ganoderma lucidum)의 β-Glucan에 의한 Sarcoma-180 육종암 생장 억제

버섯 다당류 <TEX>${\beta}$</TEX>-glucan의 항암 활성을 확인하기 위하여 영지균사체로부터 단백다당류(GLP)를 분리하고 Sarcoma-180 육종암을 이식시킨 마우스에 복강 투여하여 항암활성을 확인하였다. 육종암이 서혜부에 이식된 마우스에 GLP를 20 mg/kg의 농도로 10일간 복강투여 한 후 30일차에 확인한 결과, Sarcoma-180 육종암은 대조군대비 71.4% 억제되었으며, 마우스의 혈청, 종양조직 및 간조직 내의 TNF-<TEX>${\alpha}$</TEX>의 농도는 대조군보다 높게 나타났다. 따라서 GLP는 생체 내 TNF-<TEX>${\alpha}$</TEX>의 양적증가를 유도하며, 종양괴사 또는 에폽토시스와 연관된 육종암의 생장억제가 확인되었다. 이때 생장이 억제된 육종암 세포의 미세구조를 관찰한 결과, 상대적으로 큰 핵과 세포의 에폽토시스에서 전형적으로 보여지는 염색질 응축이 관찰되었으며, 핵막은 특징적으로 뭉쳐져 불규칙한 모양을 나타내었다. 따라서 영지에서 분리된 GLP는 종양 세포의 에폽토시스를 유도하여 종양의 성장을 억제하는 것으로 여겨진다. Mushroom-derived <TEX>${\beta}$</TEX>-glucan, a polysaccharide (GLP) isolated from the mycelium of Ganoderma lucidum, was previously shown to have inhibitory effects against tumor-bearing mice in vivo. We investigated the apoptotic effect of mushroom-derived <TEX>${\beta}$</TEX>-glucan in a sarcoma-180 tumor cell- bearing mice model using an ELISA to determine the levels of tumor necrosis factor-<TEX>${\alpha}$</TEX> (TNF-<TEX>${\alpha}$</TEX>) in the mice. The morphology of the tumor cells was assessed with transmission electron microscopy (TEM). GLP was injected into the tumor-bearing mice at a dose (i.p.) of 20 mg/kg for 10 days. After 30 days, the tumor mass from the inguinal region was collected, weighed, and assayed using TEM and a TNF-<TEX>${\alpha}$</TEX> ELISA kit. The tumors that developed in the mice treated with GLP were 71.4% smaller than those in the control group, showing the ability of GLP to inhibit tumor growth. The levels of TNF-<TEX>${\alpha}$</TEX> in the serum of the sarcoma-180 bearing mice were 12 times greater than in the serum of the nonbearing tumor mice. An ultrastructural study demonstrated that the GLP-treated sarcoma-180 tumor cells were condensed, with rearranged chromatin. In addition, the marginated chromatin in nucleus induced the nuclear compartment, and there were many vacuolization in the cell. GLP could be an effective apoptosis-inducing compound in sarcoma-type cancers.

Anti-tumor effects and apoptosis induction by Realgar bioleaching solution in Sarcoma-180 cells in vitro and transplanted tumors in mice in vivo.

Realgar which contains arsenic components has been used in traditional Chinese medicine (TCM) as an anticancer drug. However, neither Realgar nor its formula are soluble in water. As a result, high dose of Realgar has to be administered to achieve an effective blood medicine concentration, and this is associated with adverse side effects. The objective of the present study was to increase the solubility of a formula using hydrometallurgy technology as well as investigating its effects on in vitro and in vivo cell proliferation and apoptosis in Sarcoma-180 cell line. Antiproliferative activity of Realgar Bioleaching Solution (RBS) was evaluated by MTT assay. Further, effects of RBS on cell proliferation and apoptosis were studied using flow cytometry and transmission electron microscopy. Kunming mice were administered RBS in vivo, where arsenic specifically targeted solid tumors. The results indicated that RBS extract potently inhibited the tumor growth of Sarcoma-180 cell line in a dose-dependent manner. Flow cytometry and transmission electron microscopy further indicated that RBS significantly induced cell apoptosis through the inhibition of cell cycle pathway in a dose-dependent manner. Further, on RBS administration to mice, arsenic was specifically targeted to solid tumors RBS could substitute for traditional Realgar or its formula to work as a potent tool in cancer treatment.