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.

Antiproliferative potential from aqueous Viscum album L. preparations and their main constituents in comparison with ricin and purothionin on human cancer cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Mistletoe has been used since ancient times in Europe mostly for medicinal purposes. Since 1917, mistletoe preparations have been applied in cancer therapy and today are the most frequently used complementary medicine in tumor treatment. The main cytotoxic constituents of Viscum album are lectins and viscotoxins. AIM OF THE STUDY: The aim of this in vitro study was to investigate the antiproliferative potential of Viscum album preparations from different host trees and to assess the impact of mistletoe lectin 1 (ML-1) and viscotoxin A (VT-A) in comparison to a structurally similar lectin and thionin. MATERIALS AND METHODS: By means of widely accepted 2D Alamar Blue Assay, based on population counting of living cells using a fluorescent cell viability dye, the potential impact to inhibit tumor cell of the mistletoe preparations (Iscucin®) and their single compounds (ML-1 and VT-A) on the cell growth of six human cancer cell lines were evaluated. Also the mixture of ML-1 and VT-A corresponding to the contents in the specific mistletoe preparations were monitored. Ricin and purothionin were used as reference lectin and reference thionin, respectively. RESULTS: The lung carcinoma cell line HCC827 was very sensitive to the Iscucin® preparations. Very strong antiproliferative effects were found with Iscucin®Salicis and Tiliae and a strong with Iscucin®Crataegi, Mali and Populi. The IC50 concentrations of the Iscucin® preparations correlated with their respective ML-1 contents, but the ML-1 levels were much lower than the IC50 concentration of isolated ML-1 (1 ng/ml - 56 ng/ml). ML-1 was much more effective than ricin. Iscucin® preparations, ML-1 and ricin showed antiproliferative activity on human tumor cells. VT-A and purothionin had no effect on cell viability in the concentration ranges tested. CONCLUSION: The complete mistletoe extract is more potent to inhibit tumor cell proliferation than isolated ML-1 at an equivalent concentration level. Phenolic compounds found in all Iscucin® preparations might contribute to uphold the cytotoxic activity of ML-1 by antioxidative action. However, further studies are necessary to evaluate the role of VT-A and possible synergistic actions to the antiproliferative effect of aqueous mistletoe extracts.

Viscumins functionally modulate cell motility-associated gene expression.

In Europe extracts from Viscum album L., the European white-berry mistletoe, are widely used as a complementary cancer therapy. Viscumins (mistletoe lectins, ML) have been scrutinized as important active components of mistletoe and exhibit a variety of anticancer effects such as stimulation of the immune system, induction of cytotoxicity, reduction of tumor cell motility as well as changes in the expression of genes associated with cancer development and progression. By microarray expression analysis, quantitative RT-PCR and RT-PCR based validation of microarray data we demonstrate for the Viscum album extract Iscador Qu and for the lectins Aviscumine and ML-1 that in glioma cells these drugs differentially modulate the expression of genes involved in the regulation of cell migration and invasion, including processes modulating cell architecture and cell adhesion. A variety of differentially expressed genes in ML treated cells are associated with the transforming growth factor (TGF)-ß signaling pathway or are targets of TGF-ß. ML treatment downregulated the expression of TGF-ß itself, of the TGF-ß receptor II (TGFBR2), of the TGF-ß intracellular signal transducer protein SMAD2, and of matrix-metalloproteinases (MMP) MMP-2 and MMP-14. Even if the changes in gene expression differ between Aviscumine, Iscador Qu and ML-1, the overall regulation of motility associated gene expression by all drugs showed functional effects since tumor cell motility was reduced in a ML-dependent manner. Therefore, ML containing compounds might provide clinical benefit as adjuvant therapeutics in the treatment of patients with invasively growing tumors such as glioblastomas.

Articulatin-D induces apoptosis via activation of caspase-8 in acute T-cell leukemia cell line.

Leukemia is among the most aggressive and prevalent human malignant carcinoma. Chemotherapy is the preferred therapeutic strategy; however, recurrence of cancer and non-selective cytotoxicity are the major concerns. Unlike synthetic chemotherapeutic agents, mistletoe ribosome-inactivating protein (RIP) displays anti-tumor function in various types of cancers. However, its effect on leukemia cells is little explored. In this study, we assessed the impact of Viscum articulatum RIP (Articulatin-D) on the survival of acute T-cell leukemia cells and the involved molecular and cellular mechanisms. Cell proliferation assay showed that Articulatin-D suppressed the viability of leukemia cells selectively. We further confirmed that the elevation of mitochondrial membrane potential and exposure of phosphatidylserine are the early events of apoptosis induction in Articulatin-D-treated Jurkat cells. Subsequently, we found that Articulatin-D treatment induces apoptosis in Jurkat cells in a time- and concentration-dependent manner. In conclusion, we provided evidence that Articulatin-D efficiently activates caspase-8 involved in extrinsic pathway of apoptosis induction, which ultimately results in caspase-3-dependent DNA fragmentation of Jurkat cells. Further evaluation of Articulatin-D in cell culture and animal models may provide novel information on selective cytotoxicity to acute T-cell leukemia and its involvement in targeting tumor cell survival pathways.

MAP30 inhibits autophagy through enhancing acetyltransferase p300 and induces apoptosis in acute myeloid leukemia cells.

Momordica anti-human immunodeficiency virus protein of 30 kDa (MAP30) has been shown to exhibit potent antitumor activities against several solid tumors. In the present investigation we demonstrated that MAP30 significantly inhibited the proliferation of acute myeloid leukemia (AML) HL-60 and THP-1 cell lines and patient AML cells through autophagy inhibition and apoptosis induction. Intriguingly, MAP30-induced cell death and apoptosis were partially rescued in combination with an autophagy activator rapamycin, and aggravated in combination with an autophagy inhibitor bafilomycin A1 in HL-60 cells, suggesting that autophagy is a pro-survival signal and its inhibition contributes to the induction of apoptosis in MAP30­induced cell death. Further mechanism analysis demonstrated that MAP30 enhanced p300, and C646, a selective inhibitor of p300, markedly promoted autophagy and partially rescued the MAP30-induced cell death in HL-60 cells and patient AML cells. Collectively, our findings suggest that apoptosis and autophagy act cooperatively to elicit MAP30-induced cell death and MAP30 may be a potential antitumor drug candidate against AML.

Use of ribosome-inactivating proteins from Sambucus for the construction of immunotoxins and conjugates for cancer therapy.

The type 2 ribosome-inactivating proteins (RIPs) isolated from some species belonging to the Sambucus genus, have the characteristic that although being even more active than ricin inhibiting protein synthesis in cell-free extracts, they lack the high toxicity of ricin and related type 2 RIPs to intact cells and animals. This is due to the fact that after internalization, they follow a different intracellular pathway that does not allow them to reach the cytosolic ribosomes. The lack of toxicity of type 2 RIPs from Sambucus make them good candidates as toxic moieties in the construction of immunotoxins and conjugates directed against specific targets. Up to now they have been conjugated with either transferrin or anti-CD105 to target either transferrin receptor- or endoglin-overexpressing cells, respectively.

A cytotoxic type-2 ribosome inactivating protein (from leafless mistletoe) lacking sugar binding activity.

Articulatin-D, a 66 kDa ribosome inactivating protein (RIP) comprised of 29 kDa A-chain linked to 35 kDa B-chain, is purified from leafless mistletoe (Viscum articulatum) parasitic on Dalbergia sp. from Western Ghats (India). N-terminal sequence and LC-MS/MS analyses of A- and B-chain confirmed that articulatin-D is a type-2 RIP having high homology with other mistletoe lectins. Translation inhibition and diagnostic N-glycosidase activity of articulatin-D illustrate the presence of catalytically active A-chain. Its inability to: (i) bind to acid treated Sepharose CL-6B column, (ii) agglutinate trypsin-treated and untreated RBCs of human (A, B, O, AB), mice, rat, rabbit, buffalo, porcine, pigeon, cock, fish, sheep and goat even with 10mg/ml of purified articulatin-D, (iii) show change in circular dichroism spectra after addition of sugar to the native protein, (iv) bind to different sugars (galactose, lactose, gal-NAc, rhamnose, arabinose, fucose and mannose) immobilized on Sepharose 4B matrix, and (v) show change in enthalpy during titration with galactose confirm that the B-chain of articulatin-D lacks sugar binding activity. Despite this, articulatin-D is highly toxic as characterized with low IC(50) against different cancer cell lines (Jurkat: 0.31 ± 0.02 nM, MOLT-4: 0.51 ± 0.03 nM, U-937: 0.64 ± 0.07 nM, HL-60: 0.79 ± 0.11 nM, Raji: 1.45 ± 0.09 nM). Toxicity of RIPs has been ascribed to the absence/presence of B-chain with sugar binding activity. Identification of articulatin-D, the first cytotoxic RIP with B-chain lacking sugar binding activity opens new vistas in understanding cytotoxic action of RIPs.

The preclinical and clinical activity of aviscumine: a potential anticancer drug.

Extracts from the European mistletoe plant Viscumalbum have been studied for decades for their direct and indirect anticancer activity. Therefore, scientists were interested in identifying the active compound (mistletoe lectin) in these extracts and making it available as a highly purified molecule for drug development. Recombinant mistletoe lectin (INN: aviscumine) was produced in Escherichiacoli. It has been shown to have immunomodulatory and cytotoxic activity in invitro and in animal models and can target tumour cells. Clinical phase I studies also demonstrated immunomodulatory activity, which appears to have a positive effect on disease stabilisation. This review explores the current knowledge base for aviscumine's mechanism of action, efficacy and side-effects in both preclinical studies and clinical trials, and it considers aviscumine's potential as a cancer therapy.

Down-regulation of some miRNAs by degrading their precursors contributes to anti-cancer effect of mistletoe lectin-I.

BACKGROUND AND PURPOSE: Mistletoe lectin-I (ML-I), the main anti-cancer component of mistletoe extracts, was originally thought to act exclusively on 28S rRNA. Here, we investigate the down-regulating effect and mechanism of CM-1, an ML-I isolated from Chinese mistletoe, on some miRNAs. EXPERIMENTAL APPROACH: The anti-cancer effects of CM-1 were assessed in vitro and in vivo in colorectal cancer cells. The miRNAs down-regulated by CM-1 were identified by miRNA microarray assay and validated by qRT-PCR analysis. The suppression of host gene transcription or by degradation of precursors was determined by qRT-PCR and enzyme activity assays respectively. The qRT-PCR, Western blot and immunohistochemistry were used to examine the expression of their target gene and related downstream effector. Cell proliferation was assayed in stably transfected HEK-293 cells with different levels of these miRNAs. KEY RESULTS: CM-1 showed prominent anti-neoplastic activity towards CLY and HT-29 cells both in vitro and in vivo. The miR-135a&b were the miRNAs most down-regulated by CM-1. Their host gene transcription was largely up-regulated, while their precursors were degraded directly by CM-1. The expression of their target gene adenomatous polyposis coli and the phosphorylation of related effector ß-catenin were both significantly up-regulated. The IC(50) values of CM-1 on derivative HEK-293 cells with high miR-135a&b levels were 2-4 times lower than that of control cells. CONCLUSIONS AND IMPLICATIONS: CM-1 down-regulated some miRNAs by degrading their precursors, which contributes to its prominent anti-cancer activity.

Mistletoe lectin-I augments antiproliferative effects of the PPARgamma agonist rosiglitazone on human malignant melanoma cells.

As malignant melanoma cells are highly resistant to conventional chemotherapy, survival rates after tumor spread remain poor and hence there is an urgent need for new therapeutic options. For both mistletoe lectin-I (ML-I) and the thiazolidinediones as synthetic ligands of the peroxisome proliferator-activated receptor gamma (PPARgamma) an antiproliferative effect on malignant melanoma cells has previously been shown. Hence, the aim of this study was to investigate whether the combination of ML-I and the PPARgamma ligand rosiglitazone is more efficacious in the treatment of malignant melanoma cells than either agent alone. Proliferation of three human melanoma cell lines treated with ML-I, rosiglitazone and the combination of both was measured in a broad concentration range (0.0001-100 microg/mL) using the XTT cell proliferation assay. Combined application tremendously increased the antiproliferative effect on all three melanoma cell lines compared with single agent treatment. In comparison with the single use of rosiglitazone, the combination with ML-I significantly increased the inhibition of cell growth by 51-79% and in comparison with the single use of ML-I by 9-32%, respectively. In conclusion, this study shows that the combination of ML-I with rosiglitazone significantly augments their antiproliferative effect on malignant melanoma cells in comparison with their single agent application, which might be a promising tool for further therapeutic studies.

In silico modulation of apoptotic Bcl-2 proteins by mistletoe lectin-1: functional consequences of protein modifications.

The mistletoe lectin-1 (ML-1) modulates tumor cell apoptosis by triggering signaling cascades through the complex interplay of phosphorylation and O-linked N-acetylglucosamine (O-GlcNAc) modification in pro- and anti-apoptotic proteins. In particular, ML-1 is predicted to induce dephosphorylation of Bcl-2-family proteins and their alternative O-GlcNAc modification at specific, conserved Ser/Thr residues. The sites for phosphorylation and glycosylation were predicted and analyzed using Netphos 2.0 and YinOYang 1.2. The involvement of modified Ser/Thr, and among them the potential Yin Yang sites that may undergo both types of posttranslational modification, is proposed to mediate apoptosis modulation by ML-1.