Mucoricin is a ricin-like toxin that is critical for the pathogenesis of mucormycosis.

Fungi of the order Mucorales cause mucormycosis, a lethal infection with an incompletely understood pathogenesis. We demonstrate that Mucorales fungi produce a toxin, which plays a central role in virulence. Polyclonal antibodies against this toxin inhibit its ability to damage human cells in vitro and prevent hypovolemic shock, organ necrosis and death in mice with mucormycosis. Inhibition of the toxin in Rhizopus delemar through RNA interference compromises the ability of the fungus to damage host cells and attenuates virulence in mice. This 17 kDa toxin has structural and functional features of the plant toxin ricin, including the ability to inhibit protein synthesis through its N-glycosylase activity, the existence of a motif that mediates vascular leak and a lectin sequence. Antibodies against the toxin inhibit R. delemar- or toxin-mediated vascular permeability in vitro and cross react with ricin. A monoclonal anti-ricin B chain antibody binds to the toxin and also inhibits its ability to cause vascular permeability. Therefore, we propose the name 'mucoricin' for this toxin. Not only is mucoricin important in the pathogenesis of mucormycosis but our data suggest that a ricin-like toxin is produced by organisms beyond the plant and bacterial kingdoms. Importantly, mucoricin should be a promising therapeutic target.

Rhizopus oligosporus and Lactobacillus plantarum Co-Fermentation as a Tool for Increasing the Antioxidant Potential of Grass Pea and Flaxseed Oil-Cake Tempe.

Tempe-type fermentation originating from Indonesia can enhance the antioxidant activity of plant material. However, this biological potential depends on substrates and applied microorganisms. This study aimed to determine whether co-fermentation with Rhizopus oligosporus and Lactobacillus plantarum improved antioxidant activity of tempe obtained from grass pea seeds with flaxseed oil-cake addition (up to 30%). For this purpose, substances reacting with Folin-Ciocalteu reagent and free radicals scavenging potential were measured in water-soluble fractions and dialysates from simulated in vitro digestion. Additionally, the water-soluble phenolic profile was estimated. The higher level of water-extractable compounds with antioxidant activity was determined in co-fermentation products than in fungal fermentation products. Moreover, the fermentation process with the use of L. plantarum contributed to a greater accumulation of some phenolic acids (gallic acid, protocatechuic acid) in tempe without having a negative effect on the levels of other phenolic compounds determined in fungal fermented tempe. During in vitro digestion simulating the human digestive tract, more antioxidant compounds were released from products obtained after co-fermentation than fungal fermentation. An addition of 20% flaxseed oil-cake and the application of bacterial-fungal co-fermentation, can be considered as an alternative tool to enhance the antioxidant parameters of grass pea tempe.

Signaling pathways associated with macrophage-activating polysaccharide isolated from the fermentation liquor of Rhizopus nigricans.

Our previous reports showed that the structural features and immunologic enhancement of polysaccharide (EPS1-1) from Rhizopus nigricans. However, the molecular mechanism in cellular immunomodulatory of EPS1-1 remains unclear. Here the experiments for the molecular mechanisms of EPS1-1 on the peritoneal macrophages were performed. The results demonstrated that the expression of TLR4 was significantly improved by EPS1-1. Subsequently, the phosphorylation of p38MAPK, ERK1/2, JNK and IKKα/ß were promoted. Moreover, EPS1-1 enhanced the expressions of IL-2, TNF-α and iNOS in EPS1-1-induced macrophages which were pretreated with MAPK signaling pathway inhibitors, and reduced the blocking effects of the inhibitors to the expressions of p-p38MAPK, p-ERK1/2 and p-IKKα/ß. Therefore, these results illustrated that EPS1-1 could improve the immune functions of peritoneal macrophages by promoting the gene expressions of IL-2, TNF-α and iNOS via the MAPK and NF-κB signaling pathways.

Exopolysaccharides isolated from Rhizopus nigricans induced colon cancer cell apoptosis in vitro and in vivo via activating the AMPK pathway.

Colorectal cancer (CRC) is a leading cause of cancer-related human deaths. The exopolysaccharide (EPS1-1), isolated from Rhizopus nigricans, has been described as exhibiting anti-tumor and pro-apoptotic activity against CRC, although the underlying mechanism is poorly understood. Herein, we investigate how EPS1-1 induces apoptosis of CRC cells in vitro and in vivo. Our results show that, in vitro, EPS1-1 suppressed cell growth and facilitated apoptosis in a dose- and time-dependent manner by activating the AMP-activated protein kinase (AMPK) pathway in mouse colon cancer CT26 cells. However, treatment with small interfering RNAs (siRNAs) targeting AMPKα or with compound C, an AMPK inhibitor, interfered with the pro-apoptosis effects of EPS1-1. We also show that EPS1-1 initiated the release of reactive oxygen species (ROS) and liver kinase B1 (LKB1), both of which are necessary signals for AMPK activation. Furthermore, EPS1-1-mediated apoptosis is regulated by inactivation of mammalian target of rapamycin complex 1 (mTORC1) and activation of the jun-NH2 kinase (JNK)-p53 signaling axis dependent on AMPK activation. In vivo, azoxymethane/dextran sulfate sodium (AOM/DSS)-treated CRC mice, when administered EPS1-1, exhibited activation of the AMPK pathway, inhibition of mTORC1, and accumulation of p53 in tumor tissues. Collectively, these findings suggest that EPS1-1-induced apoptosis relies on the activation of the AMPK pathway. The present study provides evidence suggesting that EPS1-1 may be an effective target for development of novel CRC therapeutic agents.

Genome Mining and Heterologous Expression Reveal Two Distinct Families of Lasso Peptides Highly Conserved in Endofungal Bacteria.

Genome mining identified the fungal-bacterial endosymbiosis Rhizopus microsporus-Mycetohabitans (previously Burkholderia) rhizoxinica as a rich source of novel natural products. However, most of the predicted compounds have remained cryptic. In this study, we employed heterologous expression to isolate and characterize three ribosomally synthesized and post-translationally modified peptides with lariat topology (lasso peptides) from the endosymbiont M. rhizoxinica: burhizin-23, mycetohabin-16, and mycetohabin-15. Through coexpression experiments, it was shown that an orphan gene product results in mature mycetohabin-15, albeit encoded remotely from the core biosynthetic gene cluster. Comparative genomics revealed that mycetohabins are highly conserved among M. rhizoxinica and related endosymbiotic bacteria. Gene knockout and reinfection experiments indicated that the lasso peptides are not crucial for establishing symbiosis; instead, the peptides are exported into the environment during endosymbiosis. This is the first report on lasso peptides from endosymbiotic bacteria.

[Preparation and antitumor activity of sulfated exopolysaccharide from Rhizopus nigricans].

OBJECTIVE: To explore the effect of the composition ratio on substitution of sulfate group in sulfated exopolysaccharide (EPS) from Rhizopus nigricans and how sulfate modification affects the anti-tumor activity of EPS. METHODS: We used a chlorosulfonic acid-pyridine method to modify EPS and analyzed the effect of esterification ratio on the degree of sulfate substitution using barium chloride turbidimetry. The sulfate groups binding with EPS were analyzed with infrared spectrum analysis. CCK-8 assay was used to evaluate the inhibitory effect of EPS sulfate (SEPS) on the proliferation of human colon cancer HCT 116 cells, and annexin V-FITC/PI double staining was used to assess the pro-apoptotic effect of SEPS in the cells. RESULTS: The esterifying agent and EPS at the composition ratios of 1:1 and 2:1 resulted in sulfate substitution of 0.98% (SEPS-1) and 1.18% (SEPS-2), respectively, and the substitution was improved by increasing the ratio of the esterifying agent (P < 0.05). Infrared spectrum analysis showed that the S=O stretching vibration absorption peak of -OSO3- appeared near 1249 cm-1, indicating that the sulfate group combined with EPS to form sulfate. CCK-8 assay showed that SEPS-1 produced stronger inhibitory effects on the proliferation of HCT 116 cells than EPS within the concentration range of 0.02-0.10 mg/L (P < 0.05). At the concentrations of 0.04-0.08 mg/L, SEPS-2 showed a lower anti-tumor activity than SEPS-1 (P < 0.05). SEPS-1 also showed stronger pro-apoptotic effect than EPS, and as its concentration increased, SEPS-1 dose-dependently increased the ratio of early apoptotic cells and necrotic cells; the cells treated with 0.06, 0.08 and 0.10 mg/mL SEPS-1 showed early apoptotic rates of 6.38%, 11.8% and 12.5%, and late apoptotic and necrotic rates of 5.26%, 8.04% and 6.80%, respectively. CONCLUSIONS: The composition ratio of the esterifying agent has a direct impact on the degree of substitution of EPS, which can be improved by increasing the ratio of the esterifying agent. Sulfate modification of EPS can enhance its antitumor activity, which, however, is not directly related with the degree of substitution.

In Situ Ring Contraction and Transformation of the Rhizoxin Macrocycle through an Abiotic Pathway.

A Rhizopus sp. culture containing an endosymbiont partner ( Burkholderia sp.) was obtained through a citizen-science-based soil-collection program. An extract prepared from the pair of organisms exhibited strong inhibition of Ewing sarcoma cells and was selected for bioassay-guided fractionation. This led to the purification of rhizoxin (1), a potent antimitotic agent that inhibited microtubule polymerization, along with several new (2-5) and known (6) analogues of 1. The structures of 2-6 were established using a combination of NMR data analysis, while the configurations of the new stereocenters were determined using ROESY spectroscopy and comparison of GIAO-derived and experimental data for NMR chemical shift and 3 JHH coupling values. Whereas compound 1 showed modest selectivity for Ewing sarcoma cell lines carrying the EWSR1/ FLI1 fusion gene, the other compounds were determined to be inactive. Chemically, compound 2 stands out from other rhizoxin analogues because it is the first member of this class that is reported to contain a one-carbon-smaller 15-membered macrolactone system. Through a combination of experimental and computational tests, we determined that 2 is likely formed via an acid-catalyzed Meinwald rearrangement from 1 because of the mild acidic culture environment created by the Rhizopus sp. isolate and its symbiont.

Beneficial effects of extracellular polysaccharide from Rhizopus nigricans on the intestinal immunity of colorectal cancer mice.

An extracellular polysaccharide (EPS1-1) extracted from the fermentation broth of Rhizopus nigricans has been proved to enhance the immunity of immunosuppressed mice. The purpose of this study was to investigate the beneficial effects of EPS1-1 on the intestinal immunity of mice with colorectal cancer induced by azoxymethane (AOM) and dextran sodium sulfate (DSS). The results showed that EPS1-1 could resist hydrolysis in an artificial stomach. Oral EPS1-1 modulated gut microbiota and increased the concentration of total short-chain fatty acids (SCFAs) in the feces of colorectal cancer mice compared with the AOM/DSS only-treated mice. Furthermore, EPS1-1 increased the villus length, ratio of villus length and crypt depth in colonic tissues, and improved the number of acid mucus-secreting goblet cells in mice with colorectal cancer. These findings suggest that EPS1-1 might play an important role in the improvement of intestinal function in mice with colorectal cancer, which indicate its strong potential as efficient bio-secure immunotherapy for clinical applications or adjuvant drug.

The impact of Rhizopus oryzae cultivation on rice bran: Gamma-oryzanol recovery and its antioxidant properties.

This study evaluated the effect of the solid state cultivation (SSC) time of rice bran by Rhizopus oryzae on γ-oryzanol recovery and its antioxidant properties. Gamma-oryzanol was extracted with organic solvents and its extracts were characterized by GC-FID and HPLC-UV. The antioxidant capacity was assessed by DPPH and ABTS+ assays, ß-carotene/linoleic acid system, and reduction of oxidation in lipid system. The biomass showed the γ-oryzanol recovery increased by 51.5% (20.52mg/g), and 5.7% in polyunsaturated fatty acids. The γ-oryzanol major components changing in their profile. The γ-oryzanol extract from biomass (72h) showed the greatest DPPH inhibition (59.0%), while 90.5% inhibition of oxidation of ß-carotene/linoleic acid system, and 30% reduction of the indicators of oxidation in olive oil was observed in the one cultivated at 96h, these behaviors were confirmed by PCA analyses. SSC provides an increase in the γ-oryzanol recovery followed by improving of the functional properties of rice bran.

Epitope Mapping of Rhi o 1 and Generation of a Hypoallergenic Variant: A CANDIDATE MOLECULE FOR FUNGAL ALLERGY VACCINES.

Efficacy of allergen-specific immunotherapy is often severely impaired by detrimental IgE-mediated side effects of native allergen during vaccination. Here, we present the molecular determinants for IgE recognition of Rhi o 1 and eventually converting the allergen into a hypoallergenic immunogen to restrain health hazards during desensitization. Rhi o 1 is a respiratory fungal allergen. Despite having cross-reactivity with cockroach allergen, we observed that non-cross-reactive epitope predominantly determined IgE binding to Rhi o 1. Denaturation and refolding behavior of the allergen confirmed that its IgE reactivity was not essentially conformation-dependent. A combinatorial approach consisting of computational prediction and a peptide-based immunoassay identified two peptides ((44)TGEYLTQKYFNSQRNN and (311)GAEKNWAGQYVVDCNK) of Rhi o 1 that frequently reacted with IgE antibodies of sensitized patients. Interestingly, these peptides did not represent purely linear IgE epitopes but were presented in a conformational manner by forming a spatially clustered surface-exposed epitope conferring optimal IgE-binding capacity to the folded allergen. Site-directed alanine substitution identified four residues of the IgE epitope that were crucial for antibody binding. A multiple mutant (T49A/Y52A/K314A/W316A) showing 100-fold lower IgE binding and reduced allergenic activity was generated. The TYKW mutant retained T-cell epitopes, as evident from its lymphoproliferative capacity but down-regulated pro-allergic IL-5 secretion. The TYKW mutant induced enhanced focusing of blocking IgG antibodies specifically toward the IgE epitope of the allergen. Anti-TYKW mutant polyclonal IgG antibodies competitively inhibited binding of IgE antibodies to Rhi o 1 up to 70% and suppressed allergen-mediated histamine release by 10-fold. In conclusion, this is a simple yet rational strategy based on epitope mapping data to develop a genetically modified hypoallergenic variant showing protective antibody response for immunotherapeutic applications.

Immune-enhancing activity of extracellular polysaccharides isolated from Rhizopus nigricans.

Extracellular polysaccharides (EPS1-1) was extracted from fermentation liquor of Rhizopus nigricans and evaluated its immune-enhancing activities in vitro and in vivo. Results suggested that the proliferation of lymphocyte was stimulated after treated with EPS1-1. Moreover, the activities of macrophages were enhanced by increasing the activities of phagocytosis and acid phosphatase, the production of NO and the mRNA levels of IL-2, TNF-α and iNOS. Furthermore, EPS1-1 could significantly boost the immunity of normal and immunosuppressed mice, which included the increase of loaded swimming time, footpad swelling, organ index and the secretion of IL-2 and TNF-α in serum, thus suggesting that EPS1-1 could improve the body immunity through cellular immunity and humoral immunity. These findings provided further insights into the potential use of EPS1-1 as immunopotentiator or new function food.

Anti-tumor activity of exopolysaccharide from Rhizopus nigricans Ehrenb on S180 tumor-bearing mice.

In this study, the effect of antitumor and immune activities of extracellular polysaccharides (EPS) from Rhizopus nigricans Ehrenb were investigated using S180 bearing mice. The results revealed that EPS in the concentration range 50-1000 µg/mL can inhibited S180 cell proliferation in a dose dependent manner. EPS at the highest dose of 1000 µg/mL showed significantly antitumor activity against S180 with inhibition rate of 47.53%. However, EPS significantly simulated spleen lymphocytes in the concentration of 500 µg/mL, and the increase proliferation ability showed a dose-dependent effect with EPS at the dose of 50-500 µg/mL. In comparison with the control groups, the weights of tumor were declined and the inhibition rates of tumor were remarkably decreased in the treated groups. Pretreatment with EPS at the dose of 75 mg/kg/day, the inhibition rate was decreased by 44.38% (P<0.05). EPS increased the concentrations of IL-2 and TNF-a. The pathological changes of model control group were very obvious. Meanwhile, the prophylactic administration of EPS could more efficiently inhibit the growth of S180 tumor than direct administration of EPS. EPS could prolong the survival period of S180 tumor bearing mice, and the doses 75 mg/kg/day of EPS and combined with cyclophosphamide (20 mg/kg/day) were 43.36% and 36.28% respectively compared to control groups (P<0.05). The results suggested EPS confirmed in vivo anti-tumor effects observed in vitro, and the mechanism of anti-tumor effect of EPS may be at least in part mediated by increased immune activity in host.

Purification, partial characterization and antitumor effect of an exopolysaccharide from Rhizopus nigricans.

In this study, a homogeneous exopolysaccharide (EPS1-1) was purified from the fermentation broth of Rhizopus nigricans. EPS1-1 was composed of glucose, mannose, galactose and fructose in the molar ratio of 5.89:3.64:3.20:1.00 with weight average molecular weight of 9.7×10(3)g/mol. EPS1-1 could significantly inhibit proliferation of human colorectal carcinoma HCT-116 cells in vitro. EPS1-1 also induced S phase cell cycle arrest and increased sub-G0/G1 population, a hallmark of apoptosis. The results of morphological characterization and flow cytometry showed that EPS1-1 induced apoptotic cell death in HCT-116 cells. EPS1-1 caused dissipation of mitochondrial membrane potential, accumulation of reactive oxygen species, up-regulation of Bax and p53 mRNA expression and down-regulation of Bcl-2 mRNA expression, which suggested that mitochondrial pathway was involved in the EPS1-1-induced apoptosis. These findings bring new insights into the potential use of EPS1-1 as antitumor drug against human colorectal carcinoma.

Enhanced acid tolerance of Rhizopus oryzae during fumaric acid production.

Ensuring a suitable pH in the culture broth is a major problem in microorganism-assisted industrial fermentation of organic acids. To address this issue, we investigated the physiological changes in Rhizopus oryzae at different extracellular pH levels and attempted to solve the issue of cell shortage under low pH conditions. We compared various parameters, such as membrane fatty acids' composition, intracellular pH, and adenosine triphosphate (ATP) concentration. It was found that the shortage of intracellular ATP might be the main reason for the low rate of fumaric acid production by R. oryzae under low pH conditions. When 1 g/l citrate was added to the culture medium at pH 3.0, the intracellular ATP concentration increased from 0.4 to 0.7 µmol/mg, and the fumaric acid titer was enhanced by 63% compared with the control (pH 3.0 without citrate addition). The final fumaric acid concentration at pH 3.0 reached 21.9 g/l after 96 h of fermentation. This strategy is simple and feasible for industrial fumaric acid production under low pH conditions.

Rhizopus oryzae lipase immobilized on hierarchical mesoporous silica supports for transesterification of rice bran oil.

The tunable textural properties of self-oriented mesoporous silica were investigated for their suitability as enzyme immobilization matrices to support transesterification of rice bran oil. Different morphologies of mesoporous silica (rod-like, rice-like, and spherical) were synthesized and characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), and nitrogen adsorption-desorption isotherms. The surface area, pore size, and ordered arrangement of the pores were found to influence the immobilization and activity of the enzyme in the mesopores. The immobilization in rod-like silica was highest with an immobilization efficiency of 63 % and exhibited minimal activity loss after immobilization. Functionalization of the mesoporous surface with ethyl groups further enhanced the enzyme immobilization. The free enzyme lost most of its activity at 50 °C while the immobilized enzyme showed activity even up to 60 °C. Transesterified product yield of nearly 82 % was obtained for 24 h of reaction with enzyme immobilized on ethyl-functionalized SBA-15 at an oil:methanol ratio of 1:3. Fourier transform infrared spectroscopy (FT-IR) and Gas chromatography-mass spectrometry (GC-MS) were used to characterize the transesterified product obtained. The reusability of the immobilized enzyme was studied for 3 cycles.

Polysaccharides from Rhizopus nigricans mycelia induced apoptosis and G2/M arrest in BGC-823 cells.

In this study, we reported that polysaccharides (RPS) from the mycelia of liquid-cultured Rhizopus nigricans inhibited the proliferation and clonogenic potential of human gastric cancer BGC-823 cells. Results of acridine orange/ethidium bromide and Hoechst 33258 staining showed that treated cells displayed typical morphological characteristics of apoptosis such as condensation of chromatin, nuclear pyknosis and formation of apoptotic bodies. Flow cytometry analyses and colorimetric assay indicated that RPS induced mitochondria-mediated apoptosis which was associated with collapse of mitochondrial membrane potential, activation of caspase-9 and caspase-3, generation of intracellular reactive oxygen species and elevation of intracellular calcium in BGC-823 cells. Moreover, cell-cycle analysis revealed that RPS arrested cells in the G2/M phase of the cell cycle. These results provided further insights into the potential use of RPS as an anti-cancer agent against human gastric cancer.

Aqueous extracts of Rhizopus oryzae induced nitric oxide production in rat hepatocyte cell line RLN-10.

Aqueous extracts of Rhizopus oryzae (Aq-ROU) have a broad range of physiological activity. Here we identified a new physiological effect of Aq-ROU in rat hepatocyte cell line RLN-10. Aq-ROU induced the accumulation of nitrite, a stable metabolite nitric oxide (NO), in cell culture medium and induced potent diaminofluorescein-FM diacetate staining in the cells. Real-time reverse transcriptase (RT)-PCR analysis showed marked inducible NO synthase gene expression. Additionally, markedly enhanced expression of p22(phox) and temporally increased expression of NADPH oxidase1 indicated that superoxide was produced. Nuclear translocation of nuclear factor-kappa (NF-κ) B p65 increased remarkably following Aq-ROU and following lipopolysaccharide treatment, a potent activator of NF-κB. Ammonium pyrrolidine-1-carbodithioate, an inhibitor of NF-κB, inhibited NO production following Aq-ROU treatment. Our data indicate that Aq-ROU induces NO production and potentially the production of superoxide, which may contribute to the broad range of physiological effects observed for Aq-ROU ingested by animals.

Structural determination of aspericins A-C, new furan and pyran derivates from the marine-derived fungus Rhizopus sp. 2-PDA-61, by 1D and 2D NMR spectroscopy.

Three new furan and pyran derivatives named aspericins A-C (1-3), as well as a known asperic acid (4), have been isolated from the marine-derived fungus Rhizopus sp. 2-PDA-61. The complete (1)H and (13)C NMR assignments for the new compounds were carried out using (1)H, (13)C, DEPT, COSY, HMQC, HMBC, and NOESY NMR experiments. Compounds 1-3 were evaluated for their cytotoxic activities on P388, A549, HL-60, and BEL-7420 cell lines by the MTT and SRB methods.

Sterols isolated from Nuruk (Rhizopus oryzae KSD-815) inhibit the migration of cancer cells.

An activity-guided fractionation method was used to isolate anticancer components from Nuruk (Rhizopus oryzae KSD-815:KSD-815). Dried powder of KSD-815 was extracted with 80% methanol and partitioned successively using nhexane, ethyl acetate, n-butanol, and water. The n-hexane and n-butanol fractions showed a strong antimigratory effect on human cancer cells. Both of these fractions were subjected to separation and purification procedures using silica gel, octadecyl silica gel, and Sephadex LH-20 column chromatographies to afford four purified compounds. These were identified as ergosterol peroxide (1), stigmast- 5-en-3beta,7beta-diol (2), ergosta-7,22-dien-3beta,5alpha,6beta,9alpha-tetraol (3), and daucosterol (4), respectively, by spectroscopic methods such as nuclear magnetic resonance spectrometry, mass spectrometry, and infrared spectroscopy, and comparison with those in the literature. Compounds 1-4 were isolated from KSD-815 for the first time. Compounds 1 and 4 inhibited the migration of MDA-MB-231 cells at concentrations lower than 20 micronM.

Six new ergosterols from the marine-derived fungus Rhizopus sp.

Six new ergosterols, including 3beta-hydroxyl-(22E, 24R)-ergosta-5,8,22-trien-7,15-dione (1), 3beta-hydroxyl-(22E, 24R)-ergosta-5,8,14,22-tetraen-7-one (2), 3beta,15beta-dihydroxyl-(22E, 24R)-ergosta-5,8(14),22-trien-7-one (3), 3beta,15alpha-dihydroxyl-(22E, 24R)-ergosta-5,8(14),22-trien-7-one (4), 3beta-hydroxyl-(22E, 24R)-ergosta-5,8(14),22-trien-7,15-dione (5), and 5alpha,8alpha-epidioxy-23,24(R)-dimethylcholesta-6,9(11),22-trien-3beta-ol (6), have been isolated from the marine-derived fungus Rhizopus sp., along with four known ones (7-10). The structures of the new compounds were determined on the basis of extensive spectroscopic data. All compounds were evaluated for their cytotoxic activities on P388, A549, HL-60, and BEL-7420 cell lines by the MTT and SRB methods.