Identification and Biological Evaluation of a Novel Small-Molecule Inhibitor of Ricin Toxin.

The plant-derived toxin ricin is classified as a type 2 ribosome-inactivating protein (RIP) and currently lacks effective clinical antidotes. The toxicity of ricin is mainly due to its ricin toxin A chain (RTA), which has become an important target for drug development. Previous studies have identified two essential binding pockets in the active site of RTA, but most existing inhibitors only target one of these pockets. In this study, we used computer-aided virtual screening to identify a compound called RSMI-29, which potentially interacts with both active pockets of RTA. We found that RSMI-29 can directly bind to RTA and effectively attenuate protein synthesis inhibition and rRNA depurination induced by RTA or ricin, thereby inhibiting their cytotoxic effects on cells in vitro. Moreover, RSMI-29 significantly reduced ricin-mediated damage to the liver, spleen, intestine, and lungs in mice, demonstrating its detoxification effect against ricin in vivo. RSMI-29 also exhibited excellent drug-like properties, featuring a typical structural moiety of known sulfonamides and barbiturates. These findings suggest that RSMI-29 is a novel small-molecule inhibitor that specifically targets ricin toxin A chain, providing a potential therapeutic option for ricin intoxication.

Cytotoxic effects of recombinant proteins enhanced by momordin Ic are dependent on cholesterol and ganglioside GM1.

Plant-derived triterpenoid saponins have been shown to play a powerful role in enhancing the cytotoxic activity of protein therapeutics. However, the mechanism of how saponins are acting is not clearly understood. In this study, momordin Ic (MIC), a triterpenoid saponin derived from Kochia scoparia (L.) Schrad., specifically enhance the antiproliferative effect of recombinant MAP30 (a type I ribosome inactivating protein, RIP) in breast cancer cells. Subsequently, the possible mechanism of how MIC enhanced the cytotoxicity of MAP30 was analyzed in detail. We observed the level of intracellular labeled MAP30 using fluorescence microscopy and flow cytometry. And a reporter protein, GAL9, was used to monitor the role of MIC in promoting endosomal escape. We found endosomal escape does not play a role for the enhancer effect of MIC while the effect of MIC on MAP30 is cholesterol dependent and that ganglioside GM1, a lipid raft marker, can competitively inhibit cytotoxicity of MAP30 enhanced by MIC. Finally, we provided some insights into the correlation between the sugar side chain of MIC and its role in enhancing of RIP cytotoxicity and altering of drug cell tropism.

Recombinant anti-HIV MAP30, a ribosome inactivating protein: against plant virus and bacteriophage.

The ribosome inactivating proteins (RIPs) efficiently decrease the microbial infections in plants. Momordica charantia MAP30 is a type I RIP that has not been investigated against plant viruses or bacteriophages. To evaluate of these activities, the recombinant MAP30 (rMAP30) was produced in the hairy roots of Nicotiana tabacum. Inoculation of 3 µg of transgenic total protein or 0.6 µg of rMAP30 against 0.1 µg of TMV reduced the leaf necrotic spots to 78.23% and 82.72%, respectively. The treatment of 0.1 µg of CMV with rMAP30 (0.6 µg) showed the reduction in the leaf necrotic spots to 85.8%. While the infection was increased after rMAP30 dilution. In the time interval assays, the leaves were first inoculated with 1 µg of rMAP30 or 0.1 µg of purified TMV or CMV agent for 6 h, then virus or protein was applied in order. This led the spot reduction to 35.22% and 67% for TMV, and 38.61% and 55.31% for CMV, respectively. In both the pre- and co-treatments of 1:10 or 1:20 diluted bacteriophage with 15 µg of transgenic total protein, the number and diameter of the plaques were reduced. The results showed that the highest inhibitory effect was observed in the pre-treatment assay of bacteriophage with transgenic total protein for 24 h. The decrease in the growth of bacteriophage caused more growth pattern of Escherichia coli. The results confirm that rMAP30 shows antibacterial activity against Streptococcus aureus and E. coli, antifungal activity against Candida albicans, and antiviral activity against CMV and TMV. Moreover, rMAP30 exhibits anti-phage activity for the first time. According to our findings, rMAP30 might be a valuable preservative agent in foods and beverages in the food industry as well as an antiviral and antimicrobial mixture in agriculture.

Heterophyllin: A New Adenia Toxic Lectin with Peculiar Biological Properties.

Ribosome-inactivating proteins (RIPs) are plant toxins that were identified for their ability to irreversibly damage ribosomes, thereby causing arrest of protein synthesis and induction of cell death. The RIPs purified from Adenia plants are the most potent ones. Here, we describe a novel toxic lectin from Adenia heterophylla caudex, which has been named heterophyllin. Heterophyllin shows the enzymatic and lectin properties of type 2 RIPs. Interestingly, in immunoreactivity experiments, heterophyllin poorly cross-reacts with sera against all other tested RIPs. The cytotoxic effects and death pathways triggered by heterophyllin were investigated in three human-derived cell lines: NB100, T24, and MCF7, and compared to ricin, the most known and studied type 2 RIP. Heterophyllin was able to completely abolish cell viability at nM concentration. A strong induction of apoptosis, but not necrosis, and the involvement of oxidative stress and necroptosis were observed in all the tested cell lines. Therefore, the enzymatic, immunological, and biological activities of heterophyllin make it an interesting molecule, worthy of further in-depth analysis to verify its possible pharmacological application.

Specificity of viscumin revised. As probed with a printed glycan array.

Viscumin, a lectin used in anti-cancer therapy, was originally considered as ßGal recognizing protein; later, an ability to bind 6'-sialyl N-acetyllactosamine (6'SLN) terminated gangliosides was found. Here we probed viscumin with a printed glycan array (PGA) containing a large number of mammalian sulfated glycans, and found a strong binding to glycans with 6-O-SuGal moiety as lactose, N-acetyllactosamine (LN), di-N-acetyllactosamine (LacdiNAc), and even 6-O-SuGalNAcα (but not SiaTn). Also, the ability to bind some of αGal terminated glycans, including Galα1-3Galß1-4GlcNAc, was observed. Unexpectedly, only weak interaction was detected with parent neutral ß-galactosides including LN-LN-LN and branched (LN)2LN oligolactosamines; in the light of these data, one should not confidently classify viscumin as a ß-galactoside-binding lectin. Carrying out PGA in the presence of neutral or sulfated/sialylated glycan, together with sequential elution from lactose-sepharose and consideration of the protein structure, lead to the conclusion that two glycan-binding sites of viscumin have different specificities, one of which prefers charged sulfated and sialylated moieties.

Raddeanin A synergistically enhances the anti-tumor effect of MAP30 in multiple ways, more than promoting endosomal escape.

Biomacromolecules such as proteins and nucleic acids are very attractive due to their high efficiency and specificity as cancer therapeutics. In fact, the endocytosed macromolecules are often trapped in the endosomes and cannot exhibit pharmacological effects well. Many strategies have been used to address this bottleneck, and one promising approach is to exploit the endosomal escape-promoting effect of triterpenoid saponins to aid in the release of biomacromolecules. Here, Raddeanin A (RA, an oleanane-type triterpenoid saponin) was proved to significantly promote endosomal escape as it recruited Galectin-9, an endosomal escape event reporter. As expected, RA effectively enhanced the anti-tumor effect of MAP30 (a type I ribosome-inactivating protein derived from Momordica charantia). However, based on the results of fluorescent colocalization, RA did not significantly promote MAP30 release from endosomes, suggesting that RA enhances MAP30 activity not only by promoting endosomal escape. Furthermore, it was found that the inhibitors of micropinocytosis and caveolae could almost completely inhibit the cytotoxicity of MAP30 combined with RA without affecting the cytotoxicity of MAP30 alone, indicating that RA may regulate the endocytic pathway of MAP30. Meanwhile, the effect of RA is related to the intra vesicular pH and cholesterol content on cell membrane, and is also cell-type dependent. Therefore, RA enhanced the anti-tumor effect of MAP30 in multiple ways, not just by promoting endosomal escape. Our findings will help to further decipher the possible mechanisms by which triterpenoid saponins enhance drug activity, and provide a new perspective for improving the activity of endocytosed drugs.

Viscotoxin and lectin content in foliage and fruit of Viscum album L. on the main host trees of Hyrcanian forests.

Mistletoe (Viscum album L.) is a hemiparasitic plant that absorbs water and nutrients from the host tree. Mistletoe contains two groups of cytotoxic, immunomodulatory and antitumor proteins, viscotoxins and lectins. This study evaluated the quantity and quality of viscotoxins and total lectins in the stems with leaves (foliage) and fruit of mistletoe on Parrotia persica and Carpinus betulus in September with immature green berries and in December with mature white berries. Viscum album L. plants were harvested from host species located in the Hyrcanian forests of Iran in 2019. The highest level of viscotoxins was detected in the December foliage of V. album settled on C. betulus (9.25 mg/g dry weight [DW]), and the highest content of lectins was found in the December foliage of V. album settled on P. persica (0.79 mg/g DW) and C. betulus (0.73 mg/g DW) respectively. The immature green berries of V. album from both host species contained much higher concentrations of viscotoxins and lectins than the mature white berries. Four isoforms of viscotoxins, viscotoxin A1, A2, A3 and B could be identified in all samples of both host species. Viscotoxin A3 was the predominant viscotoxin isoform followed by viscotoxin A1.

Differences in Medium-Induced Conformational Plasticity Presumably Underlie Different Cytotoxic Activity of Ricin and Viscumin.

Structurally similar catalytic subunits A of ricin (RTA) and viscumin (MLA) exhibit cytotoxic activity through ribosome inactivation. Ricin is more cytotoxic than viscumin, although the molecular mechanisms behind this difference are still poorly understood. To shed more light on this problem, we used a combined biochemical/molecular modeling approach to assess possible relationships between the activity of toxins and their structural/dynamic properties. Based on bioassay measurements, it was suggested that the differences in activity are associated with the ability of RTA and MLA to undergo structural/hydrophobic rearrangements during trafficking through the endoplasmic reticulum (ER) membrane. Molecular dynamics simulations and surface hydrophobicity mapping of both proteins in different media showed that RTA rearranges its structure in a membrane-like environment much more efficiently than MLA. Their refolded states also drastically differ in terms of hydrophobic organization. We assume that the higher conformational plasticity of RTA is favorable for the ER-mediated translocation pathway, which leads to a higher rate of toxin penetration into the cytoplasm.

Sequence, Structure, and Binding Site Analysis of Kirkiin in Comparison with Ricin and Other Type 2 RIPs.

Kirkiin is a new type 2 ribosome-inactivating protein (RIP) purified from the caudex of Adenia kirkii with a cytotoxicity compared to that of stenodactylin. The high toxicity of RIPs from Adenia genus plants makes them interesting tools for biotechnology and therapeutic applications, particularly in cancer therapy. The complete amino acid sequence and 3D structure prediction of kirkiin are here reported. Gene sequence analysis revealed that kirkiin is encoded by a 1572 bp open reading frame, corresponding to 524 amino acid residues, without introns. The amino acid sequence analysis showed a high degree of identity with other Adenia RIPs. The 3D structure of kirkiin preserves the overall folding of type 2 RIPs. The key amino acids of the active site, described for ricin and other RIPs, are also conserved in the kirkiin A chain. Sugar affinity studies and docking experiments revealed that both the 1α and 2γ sites of the kirkiin B chain exhibit binding activity toward lactose and D-galactose, being lower than ricin. The replacement of His246 in the kirkiin 2γ site instead of Tyr248 in ricin causes a different structure arrangement that could explain the lower sugar affinity of kirkiin with respect to ricin.

Characterization of Sialic Acid Affinity of the Binding Domain of Mistletoe Lectin Isoform One.

Sialic acid (Sia) is considered as one of the most important biomolecules of life since its derivatives and terminal orientations on cell membranes and macromolecules play a major role in many biological and pathological processes. To date, there is only a limited number of active molecules that can selectively bind to Sia and this limitation has made the study of this glycan challenging. The lectin superfamily is a well-known family of glycan binding proteins, which encompasses many strong glycan binding peptides with diverse glycan affinities. Mistletoe lectin (ML) is considered one of the most active members of lectin family which was initially classified in early studies as a galactose binding lectin; more recent studies have suggested that the peptide can also actively bind to Sia. However, the details with respect to Sia binding of ML and the domain responsible for this binding are left unanswered because no comprehensive studies have been instigated. In this study, we sought to identify the binding domain responsible for the sialic acid affinity of mistletoe lectin isoform I (MLI) in comparison to the binding activity of elderberry lectin isoform I (SNA), which has long been identified as a potent Sia binding lectin. In order to execute this, we performed computational carbohydrate-protein docking for MLB and SNA with Neu5Ac and ß-Galactose. We further analyzed the coding sequence of both lectins and identified their glycan binding domains, which were later cloned upstream and downstream to green fluorescent protein (GFP) and expressed in Escherichia coli (E. coli). Finally, the glycan affinity of the expressed fusion proteins was assessed by using different biochemical and cell-based assays and the Sia binding domains were identified.

Expression and purification of a recombinant ELRL-MAP30 with dual-targeting anti-tumor bioactivity.

MAP30 (Momordica antiviral protein 30kD) is a single-chain Ⅰ-type ribosome inactivating protein with a variety of biological activities, including anti-tumor ability. It was reported that MAP30 would serve as a novel and relatively safe agent for prophylaxis and treatment of liver cancer. To determine whether adding two tumor targeting peptides could improve the antitumor activities of MAP30, we genetically modified MAP30 with an RGD motif and a EGFRi motif, which is a ligand with high affinity for αvß3 integrins and with high affinity for EGFR. The recombinant protein ELRL-MAP30 (rELRL-MAP30) containing a GST-tag was expressed in E. coli. The rELRL-MAP30 was highly expressed in the soluble fraction after induction with 0.15 mM IPTG for 20 h at 16 °C. The purified rELRL-MAP30 appeared as a band on SDS-PAGE. It was identified by western blotting. Cytotoxicity of recombinant protein to HepG2, MDA-MB-231, HUVEC and MCF-7 cells was detected by MTT analysis. Half maximal inhibitory concentration (IC50) values were 54.64 µg/mL, 70.13 µg/mL, 146 µg/mL, 466.4 µg/mL, respectively. Proliferation inhibition assays indicated that rELRL-MAP30 could inhibit the growth of Human liver cancer cell HepG2 effectively. We found that rELRL-MAP30 significantly induced apoptosis in liver cancer cells, as evidenced by nuclear staining of DAPI. In addition, rELRL-MAP30 induced apoptosis in human liver cancer HepG2 cells by up-regulation of Bax as well as down-regulation of Bcl-2. Migration of cell line were markedly inhibited by rELRL-MAP30 in a dose-dependent manner compared to the recombinant MAP30 (rMAP30). In summary, the fusion protein displaying extremely potent cytotoxicity might be highly effective for tumor therapy.

Ebulin l Is Internalized in Cells by Both Clathrin-Dependent and -Independent Mechanisms and Does Not Require Clathrin or Dynamin for Intoxication.

Ebulin l is an A-B toxin, and despite the presence of a B chain, this toxin displays much less toxicity to cells than the potent A-B toxin ricin. Here, we studied the binding, mechanisms of endocytosis, and intracellular pathway followed by ebulin l and compared it with ricin. COS-1 cells and HeLa cells with inducible synthesis of a mutant dynamin (K44A) were used in this study. The transport of these toxins was measured using radioactively or fluorescently labeled toxins. The data show that ebulin l binds to cells to a lesser extent than ricin. Moreover, the expression of mutant dynamin does not affect the endocytosis, degradation, or toxicity of ebulin l. However, the inhibition of clathrin-coated pit formation by acidification of the cytosol reduced ebulin l endocytosis but not toxicity. Remarkably, unlike ricin, ebulin l is not transported through the Golgi apparatus to intoxicate the cells and ebulin l induces apoptosis as the predominant cell death mechanism. Therefore, after binding to cells, ebulin l is taken up by clathrin-dependent and -independent endocytosis into the endosomal/lysosomal system, but there is no apparent role for clathrin and dynamin in productive intracellular routing leading to intoxication.

Kirkiin: A New Toxic Type 2 Ribosome-Inactivating Protein from the Caudex of Adenia kirkii.

Ribosome-inactivating proteins (RIPs) are plant toxins that irreversibly damage ribosomes and other substrates, thus causing cell death. RIPs are classified in type 1 RIPs, single-chain enzymatic proteins, and type 2 RIPs, consisting of active A chains, similar to type 1 RIPs, linked to lectin B chains, which enable the rapid internalization of the toxin into the cell. For this reason, many type 2 RIPs are very cytotoxic, ricin, volkensin and stenodactylin being the most toxic ones. From the caudex of Adenia kirkii (Mast.) Engl., a new type 2 RIP, named kirkiin, was purified by affinity chromatography on acid-treated Sepharose CL-6B and gel filtration. The lectin, with molecular weight of about 58 kDa, agglutinated erythrocytes and inhibited protein synthesis in a cell-free system at very low concentrations. Moreover, kirkiin was able to depurinate mammalian and yeast ribosomes, but it showed little or no activity on other nucleotide substrates. In neuroblastoma cells, kirkiin inhibited protein synthesis and induced apoptosis at doses in the pM range. The biological characteristics of kirkiin make this protein a potential candidate for several experimental pharmacological applications both alone for local treatments and as component of immunoconjugates for systemic targeting in neurodegenerative studies and cancer therapy.

Recombinant pebulin protein, a type 2 ribosome-inactivating protein isolated from dwarf elder (Sambucus ebulus L.) shows anticancer and antifungal activities in vitro.

In this study, encoding sequence of a new type 2 RIP (pebulin) was isolated and cloned from dwarf elder (Sambucus ebulus L.) native to the northern regions of Iran. The nucleotide sequence of pebulin was ligated to the pET-28a(+) expression plasmid and cloned into the E. coli strain BL21 (DE3) in order to express heterologously of recombinant protein. The recombinant pebulin protein was mainly produced in the form of insoluble inclusion bodies probably because to absence of N-glycosylation process in E. coli. Therefore, in order to increase the expression of recombinant protein in soluble form, co-expression of the target protein with the pG-Tf2 chaperone plasmid and incubation of bacterial culture under low temperature were used to enhance solubility and accumulation of recombinant protein. After purification of the recombinant protein using affinity chromatography method, the bioactivity of pebulin was analyzed by hemagglutination, anticancer, and antifungal assays. The results of the hemagglutination assay showed that purified pebulin agglutinated erythrocytes in all human blood groups. In addition, pebulin considerably inhibited the proliferation of cancer cell lines MCF-7 and HT-29 in a time- and dose-dependent manner and indicated remarkably growth-inhibiting effect against the plant pathogenic fungi such as Alternaria solani and Fusarium oxysporum.

MAP30 Inhibits Bladder Cancer Cell Migration and Invasion In Vitro Through Suppressing Akt Pathway and the Epithelial/Mesenchymal Transition Process.

The antitumor activity of Momordica anti-human immunodeficiency virus protein of 30 kDa (MAP30) has been proved. However, the role of MAP30 on tumor metastasis has not yet been identified. For this purpose, we investigated this effect and underlying mechanism of MAP30 in bladder cancer (BC). Here, we reported that MAP30 significantly inhibited the cell proliferation and clone formation of 5637 and T24 cells in vitro by promoting apoptosis and cell cycle arrest. We also found MAP30 inhibited cell migration and invasion by suppressing the epithelial/mesenchymal transition (EMT) process. Moreover, the Affymetrix GeneChip assay revealed that MAP30 significantly changed gene expression profile in T24 cells, especially the genes in cell cycle regulation pathways. After the Ingenuity Pathway Analysis, we predicted that NUPR1 was the most important upstream regulator. Subsequently, we verified that the AKT and EMT signaling pathways were inhibited by MAP30 treatment in T24 cells. In conclusion, MAP30 treatment inhibited the progression of human BC, especially cell migration and invasion through suppressing AKT pathways.

Intracellular Transport of Ribosome-Inactivating Proteins Depends on Annexin 13.

In the present study, we assessed the role of annexin 13 membrane-binding protein (ANXA13) in the intracellular transport of vesicles containing type II ribosome-inactivating proteins (RIP-IIs). A modified human intestinal epithelial cell line HT29 was used, in which the expression of ANXA13 was significantly reduced. The cytotoxic effect of ricin and viscumin was evaluated by modification of 28S ribosome RNA. The observed differences in the activity of toxins on the parental and modified HT29 lines indicate that ANXA13 plays a different role in the intracellular transport of vesicles containing the RIP-IIs.

Relationship between the Expression Level of PSMD11 and Other Proteasome Proteins with the Activity of Ricin and Viscumin.

The role of proteasome proteins and proteins of the ERAD system in the cytotoxicity of type II ribosome-inactivating proteins ricin and viscumin was investigated. For this, the cell line of colorectal adenocarcinoma HT29, as well as the HT29-sh002 line obtained on its basis, were used. On the basis on the proteome analysis of these lines and the estimation of the proportion of inactivated ribosomes, it was shown that the contribution of the proteasome to the degradation of the catalytic subunits of toxins is different. The role of the Cdc37 co-chaperone in maintaining the stability of A subunit of viscumin in the cytoplasm is shown.

MAP30 protein from Momordica charantia is therapeutic and has synergic activity with cisplatin against ovarian cancer in vivo by altering metabolism and inducing ferroptosis.

Increasing evidence shows that Traditional Chinese Medicine (TCM) has an obvious appeal for cancer treatment, but there is still a lack of scientific investigation of its underlying molecular mechanisms. Bitter melon or bitter gourd (Momordica charantia) is an edible fruit that is commonly consumed, and it is used to cure different diseases in various ancient folk medical practices. We report that a bioactive protein, MAP30, isolated from bitter melon seeds exhibited potent anticancer and anti-chemoresistant effects on ovarian cancer cells. Functional studies revealed that MAP30 inhibited cancer cell migration, cell invasion, and cell proliferation in various ovarian cancer cells but not normal immortalized ovarian epithelial cells. When administered with cisplatin, MAP30 produced a synergistic effect on cisplatin-induced cell cytotoxicity in ovarian cancer cells. When low doses of cisplatin and MAP30 were co-injected intraperitoneally, a remarkable reduction of tumor dissemination and tumor growth was observed in an ovarian cancer ascites mouse model. Notably, blood tests confirmed that MAP30 did not cause any adverse effects on liver and kidney functions in the treated mice. MAP30 activated AMP-activated protein kinase (AMPK) signaling via CaMKKß and induced cell cycle arrest in the S-phase. MAP30 modulated cell metabolism of ovarian cancer cells via suppression of GLUT-1/-3-mediated glucose uptake, adipogenesis, and lipid droplet formation in tumor development and progression. MAP30 also induced an increase in intracellular Ca2+ ion concentration, which triggered ROS-mediated cancer cell death via apoptosis and ferroptosis. Collectively, these findings suggest that natural MAP30 is a non-toxic supplement that may enhance chemotherapeutic outcomes and benefit ovarian cancer patients with peritoneal metastases.

Transiently Expressed Mistletoe Lectin II in Nicotiana benthamiana Demonstrates Anticancer Activity In Vitro.

Mistletoe (Viscum album) extracts have been used as alternative and complementary therapeutic preparations in multiple cancers for decades. Mistletoe lectins (ML-I, ML-II, and ML-III) are considered to be the main anticancer components of such preparations. In the present study, ML-II was transiently expressed in Nicotiana benthamiana using the pEAQ-HT expression system. Expression levels of up to 60 mg/kg of the infiltrated plant tissue were obtained, and a three-fold increase was achieved by adding the endoplasmic reticulum (ER) retention signal KDEL to the native ML-II sequence. The native protein containing His-tag and KDEL was purified by immobilized metal affinity chromatography (IMAC) and gel filtration. We found that the recombinant ML-II lectin was glycosylated and retained its carbohydrate-binding activity. In addition, we demonstrated that plant produced ML-II displayed anticancer activity in vitro, inhibiting non-small cell lung cancer H460 and A549 cells with EC50 values of 4 and 3.5 µg/mL, respectively. Annexin V-448A and PI double staining revealed that cell cytotoxicity occurred via apoptosis induction. These results indicate that ML-II transiently expressed in N. benthamiana plants is a promising candidate as an anticancer agent, although further optimization of production and purification methods is required to enable further in vitro testing, as well as in vivo assays.

Antitumor mechanism of MAP30 in bladder cancer T24 cells, and its potential toxic effects in mice.

To investigate the antitumor mechanism of MAP30 in human bladder cell line (T24) and its potential toxic effects in mice.  In this study, the biological behavior of MAP30's influence on bladder cell was investigated to reveal the antitumor mechanism and role of MAP30 in bladder cancer. MAP30 gene sequence optimized by gene synthesis codon was inserted into the prokaryotic expression vector pET-28a to produce a large amount of target protein in Escherichia coli. The protein product was obtained after purification. Membrane hydration method was used to prepare MAP30 liposome in order to enhance its membrane permeability. The effects of MAP30 on the viability, apoptosis and migration of T24 cell were assessed using 3­(4,5­dimethyl­thiazol­2­yl)­2,5­diphenyl­2H­tetrazolium bromide (MTT), flow cytometric and TUNEL assays, respectively. Mice were transfected with bladder cancer cells for 48 h. The expressions of apoptotic and non-apoptotic proteins were determined using Western blotting. Changes in tumor volume and occurrence of metastasis were assessed using luciferase assay. After 7 days, liver and kidney were excised for histological examination. The levels of reactive oxygen species (ROS), malondialdehyde (MDA), and reduced glutathione (GSH), and activities of catalase and glutathione peroxidase (GPx) were determined in serum or homogenate using enzyme-linked immunosorbent assay (ELISA). The yield of MAP30 after purification was significantly increased. The results of MTT assay showed that MAP30 significantly and concentration-dependently inhibited the proliferation and migration of T24 cells (p < 0.05). The prepared liposomes had uniform hydrated particle size of 132.6 nm, with encapsulation efficiency of 78 %. The inhibitory effect of MAP30 liposome on T24 cells was significantly higher than that of MAP30, and MAP30 significantly increased the number of apoptotic cells (p < 0.05). Western blotting showed that MAP30 significantly promoted the expression of caspase 3 (p < 0.05), but did not significantly affect the expressions of bcl-2 and bax (p > 0.05). It also significantly down-regulated the expressions of NF-kB, JNK and MMP2 (p < 0.05). Tumor formation was significantly inhibited, and tumor volume reduced in bladder cancer-bearing mice after treatment with MAP30 (p < 0.05). Histological examination showed that MAP30 induced mild histological changes in the liver and kidney of mice, and significantly increased the level of MDA at day 1 (p < 0.05). It also significantly and time-dependently increased ROS, but reduced GSH levels and activities of catalase and GPx (p < 0.05). However, MAP30 had no significant effect on DNA (p > 0.05). The apoptotic effect of MAP30 in T24 cells is mediated via activation of caspase-3 signaling pathway. The protein produces mild histological changes in the liver and kidney of mice, but has no significant effect on DNA.