Fruticulosin: A novel type 2 ribosome-inactivating protein from Abrus fruticulosus seeds that exhibits toxic and antileishmanial activity.

Plant ribosome-inactivating proteins (RIPs) are a family of toxins that inhibit protein synthesis. In this study, we have isolated a novel type 2 ribosome-inactivating protein (RIP) present in seeds of the Abrus fruticulosus, named of fruticulosin. Fruticulosin, shows characteristics common to other type 2 RIPs, as specificity by galactosides (d-galactose, N-acetyl-d-galactosamine, and d-lactose), mass of approximately 60 kDa and presence of the of disulfide bonds. The N-terminal amino acid sequence (26 residues) of A-chain fruticulosin, determined by Edman degradation, revealed high similarity of the A-chain with those of other type 2 RIPs. The secondary structure of fruticulosin was analysed by circular dichroism, which showed that fruticulosin contains α-helices (22.3%), ß-sheets (43.5%), and random coils and corners (34.2%). Furthermore, fruticulosin showed high toxicity in Artemia sp. (3.12 µg/mL), inhibited in vitro protein synthesis by a cell-free system and showed RNA N-glycosidase activity. Fruticulosin presented biological activities such as agglutination and antileishmanial activity on promastigote forms of Leishmania major.

Formulation of nanoparticles ribosome inactivating proteins from Mirabilis jalapa L. (RIP MJ) conjugated AntiEpCAM antibody using low chain chitosan-pectin and cytotoxic activity against breast cancer cell line.

Ribosome Inactivating Proteins (RIPs) isolated from Mirabilis jalapa L. (MJ protein) leaves showed high cytotoxic effect on malignant. Chitosan nanoparticles have frequently been used in protein delivery applications. The aim of this study was to develop targeted drug delivery system of RIP MJ for breast cancer therapy with chitosan nanoparticles conjugated antiEpCAM antibody. RIP MJ nanoparticles were prepared using low viscous chitosan and pectin using polyelectrolit complex method, followed by conjugation process with antiEpCAM antibody. Characterization of this formula was then carried out for its entrapment efficiency, particles size, zeta potential, morphology using transmission electron microscope (TEM) and cytotoxic assay against T47D and Vero cell line. The optimal concentration of MJ protein; low viscous chitosan; pectin for preparing AntiEpCAM conjugated of RIP MJ nanoparticles was 0.1%; 0.01%;1% (m/v) respectively and showed satisfactory formula with the average particle size of 376.8±105.2nm, polydispersity index (PI) 0.401, zeta potential 43,71 mV, high entrapment efficiency 98,97±0,12%. Transmission electron microscope (TEM) imaging showed a spherical and homogenous structure for nanoparticles. The in vitro cytotoxicity analysis showed that RIP MJ nanoparticle had more cytotoxic effect compared to unformulated RIP against T47D cell-lines. AntiEpCAM conjugated RIP MJ nanoparticles however, increased cytotoxic effect of RIPs on Vero cell-lines not for T47D cell-lines. Chitosan-Pectin nanoparticles suitable for delivering protein to target cancer cells.

Elderberries: a source of ribosome-inactivating proteins with lectin activity.

Sambucus (Adoxaceae) species have been used for both food and medicine purposes. Among these, Sambucus nigra L. (black elder), Sambucus ebulus L. (dwarf elder), and Sambucus sieboldiana L. are the most relevant species studied. Their use has been somewhat restricted due to the presence of bioactive proteins or/and low molecular weight compounds whose ingestion could trigger deleterious effects. Over the last few years, the chemical and pharmacological characteristics of Sambucus species have been investigated. Among the proteins present in Sambucus species both type 1, and type 2 ribosome-inactivating proteins (RIPs), and hololectins have been reported. The biological role played by these proteins remains unknown, although they are conjectured to be involved in defending plants against insect predators and viruses. These proteins might have an important impact on the nutritional characteristics and food safety of elderberries. Type 2 RIPs are able to interact with gut cells of insects and mammals triggering a number of specific and mostly unknown cell signals in the gut mucosa that could significantly affect animal physiology. In this paper, we describe all known RIPs that have been isolated to date from Sambucus species, and comment on their antiviral and entomotoxic effects, as well as their potential uses.

Balsamin, a novel ribosome-inactivating protein from the seeds of Balsam apple Momordica balsamina.

Plant seeds, a rich source of proteins, are considered important for their application as functional ingredients in a food system. A novel ribosome-inactivating protein (RIP), balsamin was purified from the seeds of Balsam apple, Momordica balsamina. Balsamin was purified by ion exchange chromatography on CM Sepharose and gel filtration on superdex-75. It has a molecular weight of 28 kDa as shown by SDS-PAGE analysis. Balsamin inhibits protein synthesis in a rabbit reticulocyte lysate-based cell free translation assay with an IC(50) of 90.6 ng ml(-1). It has RNA N-glycosidase activity and releases a 400-base long fragment termed the Endo fragment from 28S rRNA in the same manner as does saporin-6 from Saponaria officinalis. The N-terminal sequence analysis of the first 12 amino acids of balsamin revealed that it shares 83% similarity with type I RIP α-MMC from Momordica charantia and 50% similarity with ß-MMC (from Momordica charantia), bryodin I (from Bryonia dioica) and luffin a (from Luffa cylindrica). Balsamin was further characterized by mass spectrometry. CD spectroscopic studies indicate that secondary structure of balsamin contains helix (23.5%), ß-strand (24.6%), turn (20%) and random coil (31.9%). Thus RIPs activity expressed in vegetables like Momordica sp. advocates its usage in diet.

Synthesis and purification of a toxin-linked conjugate targeting epidermal growth factor receptor in Escherichia coli.

Aberrant epidermal growth factor receptor (EGFR) signaling is a common feature of multiple tumor types, including glioblastoma (GBM). As such, EGFR has emerged as an attractive target for antitumor therapy. In the present study, we sought to develop an immunotoxin capable of specifically targeting EGFR-expressing cells and mediating inhibition of cell growth and cell killing. The Luffin P1 (LP1) ribosome inactivating protein was chosen to generate a fusion protein, antiEGFR/LP1, based upon its potent protein synthesis inhibition and small size (5 kDa). LP1 was fused to the C-terminus of an anti-EGFR single-chain antibody (scFv). The recombinant antiEGFR/LP1 protein was expressed in Escherichia coli, and refolded and purified on an immobilized Ni(2+)-affinity chromatography column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting analysis revealed that antiEGFR/LP1 was sufficiently expressed. Confocal microscopy and flow cytometry demonstrated that antiEGFR/LP1 bound specifically to EGFR-positive cells (U251), as almost no binding to EGFR-negative (Jurkat cells) was observed under identical time and dosage conditions. Finally, the MTT cell viability assay showed that antiEGFR/LP1 elicited obvious cytotoxicity toward EGFR-positive tumor cells. Collectively, these results suggest that antiEGFR/LP1 is biologically active and specific toward EGFR-positive tumor cells and may represent an effective EGFR-targeted cancer therapy.

Molecular cloning and functional analysis of a recombinant ribosome-inactivating protein (alpha-momorcharin) from Momordica charantia.

Alpha-momorcharin (α-MC), a member of the ribosome-inactivating protein (RIP) family, has been used not only as antiviral, antimicrobial, and antitumor agents, but also as toxicant to protozoa, insects, and fungi. In this study, we expressed the protein in Escherichia coli Rosetta (DE3) pLysS strain and purified it by nickel-nitrilotriacetic acid affinity chromatography. A total of 85 mg of homogeneous protein was obtained from 1 l culture supernatant of Rosetta (DE3) pLysS, showing a high recovery rate of 73.9%. Protein activity assay indicated that α-MC had both N-glycosidase activity and DNA-nuclease activity, the former releasing RIP diagnostic RNA fragment (Endo's fragment) from rice rRNAs and the latter converting supercoiled circular DNA of plasmid pET-32a(+) into linear conformations in a concentration-dependent manner. Specially, we found that α-MC could inhibit the mycelial growth of Fusarium solani and Fusarium oxysporum with IC(50) values of 6.23 and 4.15 µM, respectively. Results of optical microscopy and transmission electron microscopy demonstrated that α-MC caused extensive septum formation, loss of integrity of the cell wall, separation of the cytoplasm from the cell wall, deformation of cells with irregular budding sites, and apoptosis in F. solani. Moreover, α-MC was active against Pseudomonas aeruginosa with an IC(50) value of 0.59 µM. The α-MC protein carries a high potential for the design of new antifungal drugs or the development of transgenic crops resistant to pathogens.

The antimutagenic effect of mistletoe lectin (Viscum album L. var. coloratum agglutinin).

A galactose- and N-acetyl-D-galactosamine-specific lectin (Viscum album L. var. coloratum agglutinin, VCA), which is known for its anticancer activity, was isolated from mistletoe. In this study, we investigated the antimutagenic potentials of VCA by using the pre-incubation method of the Ames test (Salmonella typhimurium TA98 and TA100) in the presence or absence of S9 mixture. Viscum album L. var. coloratum agglutinin was assessed for its antimutagenic properties against the mutagens 2-aminoanthracene (2AA) and furylfuramide (AF-2) for strain TA98, and sodium azide (NaN(3) ) and 2-aminoanthracene (2AA) for strain TA100. The concentrations used for this test compound were 100, 200 and 400 µg per plate. Viscum album L. var. coloratum agglutinin showed moderate, but not negligible, protective effects regarding the antimutagenic properties against the direct-acting mutagens NaN(3) and AF-2. Furthermore, VCA was more effective in preventing the mutagenicity of the indirect-acting mutagen 2-AA (in the presence of S9) when tested with both TA98 and TA100. In conclusion, this report has shown broad ranging antimutagenic effects of VCA to numerous mutagens in TA98 and TA100 Salmonella typhimurium strains. Although the data presented here cannot be applied in vivo, they can support other antimutagenic and anticarcinogenic findings for VCA.

Immunoaffinity purification of α-momorcharin from bitter melon seeds (Momordica charantia).

α-Momorcharin (α-MMC), a type I ribosome-inactivating protein (RIP), has shown therapeutic potential such as anti-tumor and anti-viral agent. Traditional process of α-MMC purification from bitter melon seeds was time consuming and low efficient. To take this challenge, we made an affinity matrix by coupling the monoclonal antibody (McAb) with Sepharose 4B. Using this attractive strategy, 196 mg of α-MMC was obtained from 100 g of bitter melon seeds as the starting material. The yield of the protein was 2.7%. The homogeneity and properties of the protein were assessed by SDS-PAGE, acidic PAGE, RP-HPLC and N-terminal sequence as well as Western blot. Purified α-MMC showed remarkable inhibition to the melanoma cell line JAR and EMT-62058. In addition, it also displayed obvious inhibition on hepatitis B virus (HBV). This work provided a simple, rapid and efficient approach for α-MMC purification from Momordica charantia.

Antiviral Activity of Ribosome-Inactivating Proteins.

Ribosome-inactivating proteins (RIPs) are rRNA N-glycosylases from plants (EC 3.2.2.22) that inactivate ribosomes thus inhibiting protein synthesis. The antiviral properties of RIPs have been investigated for more than four decades. However, interest in these proteins is rising due to the emergence of infectious diseases caused by new viruses and the difficulty in treating viral infections. On the other hand, there is a growing need to control crop diseases without resorting to the use of phytosanitary products which are very harmful to the environment and in this respect, RIPs have been shown as a promising tool that can be used to obtain transgenic plants resistant to viruses. The way in which RIPs exert their antiviral effect continues to be the subject of intense research and several mechanisms of action have been proposed. The purpose of this review is to examine the research studies that deal with this matter, placing special emphasis on the most recent findings.

Atomic-resolution structures of type I ribosome inactivating protein alpha-momorcharin with different substrate analogs.

Alpha-momorcharin (Alpha-MMC) from the seed of bitter melon is a type I ribosome inactivating protein (RIP) that removes a specific adenine from 28S rRNA and inhibits protein biosynthesis. Here, we report seven crystal complex structures of alpha-MMC with different substrate analogs (adenine, AMP, cAMP, dAMP, ADP, GMP, and xanthosine) at 1.08 Å to 1.52 Å resolution. These structures reveal that not only adenine, but also guanine and their analogs can effectively bind to alpha-MMC. The side chain of Tyr93 adopts two conformations, serving as a switch to open and close the substrate binding pocket of alpha-MMC. Although adenine, AMP, GMP, and guanine are located in a similar active site in different RIPs, residues involved in the interaction between RIPs and substrate analogs are slightly different. Complex structures of alpha-MMC with different substrate analogs solved in this study provide useful information on its enzymatic mechanisms and may enable the development of new inhibitors to treat the poisoning of alpha-MMC.

Transition state analogues rescue ribosomes from saporin-L1 ribosome inactivating protein.

Ribosome inactivating proteins (RIPs) catalyze the hydrolytic depurination of one or more adenosine residues from eukaryotic ribosomes. Depurination of the ribosomal sarcin-ricin tetraloop (GAGA) causes inhibition of protein synthesis and cellular death. We characterized the catalytic properties of saporin-L1 from Saponaria officinalis (soapwort) leaves, and it demonstrated robust activity against defined nucleic acid substrates and mammalian ribosomes. Transition state analogue mimics of small oligonucleotide substrates of saporin-L1 are powerful, slow-onset inhibitors when adenosine is replaced with the transition state mimic 9-deazaadenine-9-methylene-N-hydroxypyrrolidine (DADMeA). Linear, cyclic, and stem-loop oligonucleotide inhibitors containing DADMeA and based on the GAGA sarcin-ricin tetraloop gave slow-onset tight-binding inhibition constants (K(i)*) of 2.3-8.7 nM under physiological conditions and bind up to 40000-fold tighter than RNA substrates. Saporin-L1 inhibition of rabbit reticulocyte translation was protected by these inhibitors. Transition state analogues of saporin-L1 have potential in cancer therapy that employs saporin-L1-linked immunotoxins.

Structural basis for sugar recognition, including the Tn carcinoma antigen, by the lectin SNA-II from Sambucus nigra.

Bark of elderberry (Sambucus nigra) contains a galactose (Gal)/N-acetylgalactosamine (GalNAc)-specific lectin (SNA-II) corresponding to slightly truncated B-chains of a genuine Type-II ribosome-inactivating protein (Type-II RIPs, SNA-V), found in the same species. The three-dimensional X-ray structure of SNA-II has been determined in two distinct crystal forms, hexagonal and tetragonal, at 1.90 A and 1.35 A, respectively. In both crystal forms, the SNA-II molecule folds into two linked beta-trefoil domains, with an overall conformation similar to that of the B-chains of ricin and other Type-II RIPs. Glycosylation is observed at four sites along the polypeptide chain, accounting for 14 saccharide units. The high-resolution structures of SNA-II in complex with Gal and five Gal-related saccharides (GalNAc, lactose, alpha1-methylgalactose, fucose, and the carcinoma-specific Tn antigen) were determined at 1.55 A resolution or better. Binding is observed in two saccharide-binding sites for most of the sugars: a conserved aspartate residue interacts simultaneously with the O3 and O4 atoms of saccharides. In one of the binding sites, additional interactions with the protein involve the O6 atom. Analytical gel filtration, small angle X-ray scattering studies and crystal packing analysis indicate that, although some oligomeric species are present, the monomeric species predominate in solution.

Ribosome-inactivating proteins isolated from dietary bitter melon induce apoptosis and inhibit histone deacetylase-1 selectively in premalignant and malignant prostate cancer cells.

Epidemiologic evidence suggests that a diet rich in fruits and vegetables is associated with a reduced risk of prostate cancer (PCa) development. Although several dietary compounds have been tested in preclinical PCa prevention models, no agents have been identified that either prevent the progression of premalignant lesions or treat advanced disease. Momordica charantia, known as bitter melon in English, is a plant that grows in tropical areas worldwide and is both eaten as a vegetable and used for medicinal purposes. We have isolated a protein, designated as MCP30, from bitter melon seeds. The purified fraction was verified by SDS-PAGE and mass spectrometry to contain only 2 highly related single chain Type I ribosome-inactivating proteins (RIPs), alpha-momorcharin and beta-momorcharin. MCP30 induces apoptosis in PIN and PCa cell lines in vitro and suppresses PC-3 growth in vivo with no effect on normal prostate cells. Mechanistically, MCP30 inhibits histone deacetylase-1 (HDAC-1) activity and promotes histone-3 and -4 protein acetylation. Treatment with MCP30 induces PTEN expression in a prostatic intraepithelial neoplasia (PIN) and PCa cell lines resulting in inhibition of Akt phosphorylation. In addition, MCP30 inhibits Wnt signaling activity through reduction of nuclear accumulation of beta-catenin and decreased levels of c-Myc and Cyclin-D1. Our data indicate that MCP30 selectively induces PIN and PCa apoptosis and inhibits HDAC-1 activity. These results suggest that Type I RIPs derived from plants are HDAC inhibitors that can be utilized in the prevention and treatment of prostate cancer.

[Study on the immunotoxicity of ribosome inhibiting protein of Momordica charantia].

OBJECTIVE: To separate and purify ribosome inhibiting protein (RIP) from Momordica charantia (bitter melon) seeds and to evaluate its acute toxicity and immunotoxicity in animal. METHODS: Ion exchange chromatography and gel filtration chromatography were applied in the separating and purifying of RIP from Momordica charantia seeds. Then the acute toxicity testing of RIP in mice was conducted to obtain its half lethal dose (LD50). Active systemic anaphylaxis(ASA)test in guinea pig and passive cutaneous anaphylaxis test (PCA) in rat were performed to evaluate its immunotoxicity. RESULTS: The LD50 (iv) in mice of RIP was 25.2 mg/kg in ASA, guinea pigs of the higher and lower RIP group all appeared stong allergic responses and most of them died quickly. In PCA, obvious blue dye in skin were observed in SD rats of the RIP group. CONCLUSION: RIP getting from Momordica charantia seeds had a relatively strong immunotoxicity in animals.

Cinnamomin: separation, crystallization and preliminary X-ray diffraction study.

Cinnamomin from Cinnamonum camphora seeds, a type II ribosome-inactivating protein that interferes with protein biosynthesis in mammalian cells, can induce the apoptosis of carcinoma cells and be used as an insecticide. A rapid and improved method has been developed for the extraction and purification of cinnamomin from camphora seed. Purification of cinnamomin is achieved with two successive steps of hydrophobic interaction chromatography carried out on a fast protein liquid chromatography (FPLC) system. Crystals suitable for X-ray diffraction analysis were obtained by vapor diffusion method. A complete data set at 2.8 A resolution has been collected. Data indexation and refinement indicate that the crystal is orthorhombic with space group P2(1)2(1)2(1) and unit cell dimensions a = 52.39 A, b = 126.33 A, c = 161.45 A. There are two molecules per asymmetric unit. Initial phasing by molecular replacement method yielded a solution, which will contribute to the structure determination. A molecular model will further the understanding of the mechanism of cinnamomin function. The latter will be combined with bio-informatics to facilitate the medical and other applications of cinnamomin.

Cloning and expression of antiviral/ribosome-inactivating protein from Bougainvillea xbuttiana.

A full-length cDNA encoding ribosome-inactivating/antiviral protein (RIP/AVP)from the leaves of Bougainvillea x buttiana was isolated. The cDNA consisted of 1364 nucleotides with an open reading frame (ORF)of 960 nucleotides encoding a 35.49 kDa protein of 319 amino acids. The deduced amino acid sequence has a putative active domain conserved in RIPs/AVPs and shows a varying phylogenetic relationship to the RIPs from other plant species. The deduced protein has been designated BBAP1 (Bougainvillea x buttiana antiviral protein1). The ORF was cloned into an expression vector and expressed in E.coli as a fusion protein of approximately 78 kDa. The cleaved and purified recombinant BBAP1 exhibited ribosome-inhibiting rRNA N-glycosidase activity,and imparted a high level of resistance against the tobacco mosaic virus (TMV).

Marmorin, a new ribosome inactivating protein with antiproliferative and HIV-1 reverse transcriptase inhibitory activities from the mushroom Hypsizigus marmoreus.

Ribosome inactivating proteins (RIPs) are enzymes that inactivate ribosomes by eliminating one or more adenosine residues from rRNA, a 9,567-Da RIP with a novel N-terminal sequence was isolated from fresh fruiting bodies of the mushroom Hypsizigus marmoreus. The protein was unadsorbed on DEAE-cellulose, adsorbed on Affi-gel blue gel, and appeared as a single peak upon gel filtration on Superdex 75. The protein, designated as marmorin, inhibited proliferation of hepatoma Hep G2 cells and breast cancer MCF-7 cells, HIV-1 reverse transcriptase activity, and translation in the rabbit reticulocyte lysate system with an IC50 of 0.15 microM, 5 microM, 30 microM, and 0.7 nM, respectively. Compared to RIPs from hairy gourd, bitter gourd, ridge gourd, garden pea, and the mushroom Flammulina velutipes, marmorin was more potent in its antiproliferative activity toward hepatoma (HepG2) and breast cancer (MCF-7) cells, similar in inhibitory potency toward HIV-1 reverse transcriptase (with the exception that it was more potent than ridge gourd RIP and bitter gourd RIP), and less potent in translation-inhibitory potency. Marmorin was devoid of antifungal, protease, RNase, mitogenic, anti-mitogenic, nitric oxide-inducing, hemagglutinating, and trypsin inhibitory activities.

Isolation and characterization of two Korean mistletoe lectins.

Two isolectins (KML-IIU and the KML-IIL) were individually isolated from the previously reported Korean mistletoe lectin, KML-C, by using an immunoaffinity column. Molecular weights of the KML-IIU and the KML-IIL were 64 kDa and 60 kDa respectively. Both of the lectins were composed of heterogeneous A and B subunits linked with a disulfide bond, and showed the same carbohydrate-binding specificities for Gal and GalNAc. However, they are different not only in biophysical properties (glycosylation and amino acid compositions) but also bioactivities (cell killing and cytokine induction). The KML-IIL showed 17-145 times stronger in cytotoxicities to various human and mouse cancer cell lines than the KML-IIU. The KML-IIL also induced TNF-alpha secretion from mouse peritoneal macrophages 4.5 times better than the KML-IIU. The results demonstrated isolectins in Korean mistletoe were varied in bioactivities and the KML-IIL may be developed as an anti-cancer agent.

Genome-wide screening of Oryza sativa ssp. japonica and indica reveals a complex family of proteins with ribosome-inactivating protein domains.

Ribosome-inactivating proteins (RIPs) are cytotoxic enzymes capable of halting protein synthesis by irreversible modification of ribosomes. Although RIPs are widespread they are not ubiquitous in the plant kingdom. The physiological importance of RIPs is not fully elucidated, but evidence suggests a role in the protection of the plant against biotic and abiotic stresses. Searches in the rice genome revealed a large and highly complex family of proteins with a RIP domain. A comparative analysis retrieved 38 RIP sequences from the genome sequence of Oryza sativa subspecies japonica and 34 sequences from the subspecies indica. The RIP sequences are scattered over different chromosomes but are mostly found on the third chromosome. The phylogenetic tree revealed the pairwise clustering of RIPs from japonica and indica. Molecular modeling and sequence analysis yielded information on the catalytic site of the enzyme, and suggested that a large part of RIP domains probably possess N-glycosidase activity. Several RIPs are differentially expressed in plant tissues and in response to specific abiotic stresses. This study provides an overview of RIP motifs in rice and will help to understand their biological role(s) and evolutionary relationships.

Α-MMC and MAP30, two ribosome-inactivating proteins extracted from Momordica charantia, induce cell cycle arrest and apoptosis in A549 human lung carcinoma cells.

α­Momorcharin (α­MMC) and momordica anti­human immunodeficiency virus protein (MAP30), produced by Momordica charantia, are ribosome­inactivating proteins, which have been reported to exert inhibitory effects on cultured tumor cells. In order to further elucidate the functions of these agents, the present study aimed to investigate the effects of α­MMC and MAP30 on cell viability, the induction of apoptosis, cell cycle arrest, DNA integrity and superoxide dismutase (SOD) activity. α­MMC and MAP30 were purified from bitter melon seeds using ammonium sulfate precipitation in combination with sulfopropyl (SP)­sepharose fast flow, sephacryl S­100 and macro­Cap­SP chromatography. MTT, flow cytometric and DNA fragmentation analyses were then used to determine the effects of α­MMC and MAP30 on human lung adenocarcinoma epithelial A549 cells. The results revealed that A549 cells were sensitive to α­MMC and MAP30 cytotoxicity assays in vitro. Cell proliferation was significantly suppressed following α­MMC and MAP30 treatment in a dose­ and time­dependent manner; in addition, the results indicated that MAP30 had a more potent anti­tumor activity compared with that of α­MMC. Cell cycle arrest in S phase and a significantly increased apoptotic rate were observed following treatment with α­MMC and MAP30. Furthermore, DNA integrity analysis revealed that the DNA of A549 cells was degraded following treatment with α­MMC and MAP30 for 48 h. The pyrogallol autoxidation method and nitrotetrazolium blue chloride staining were used to determine SOD activity, the results of which indicated that α­MMC and MAP30 did not possess SOD activity. In conclusion, the results of the present study indicated that α­MMC and MAP30 may have potential as novel therapeutic agents for the prophylaxis and treatment of cancer.