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.

Is Mistletoe Treatment Beneficial in Invasive Breast Cancer? A New Approach to an Unresolved Problem.

BACKGROUND/AIM: In this retrospective study, we compared breast cancer patients treated with and without mistletoe lectin I (ML-I) in addition to standard breast cancer treatment in order to determine a possible effect of this complementary treatment. PATIENTS AND METHODS: This study included 18,528 patients with invasive breast cancer. Data on additional ML-I treatments were reported for 164 patients. We developed a "similar case" method with a distance measure retrieved from the beta variable in Cox regression to compare these patients, after stage adjustment, with their non-ML-1 treated counterparts in order to answer three hypotheses concerning overall survival, recurrence free survival and life quality. RESULTS: Raw data analysis of an additional ML-I treatment yielded a worse outcome (p=0.02) for patients with ML treatment, possibly due to a bias inherent in the ML-I-treated patients. Using the "similar case" method (a case-based reasoning approach) we could not confirm this harm for patients using ML-I. Analysis of life quality data did not demonstrate reliable differences between patients treated with ML-I treatment and those without proven ML-I treatment. CONCLUSION: Based on a "similar case" model we did not observe any differences in the overall survival (OS), recurrence-free survival (RFS), and quality of life data between breast cancer patients with standard treatment and those who in addition to standard treatment received ML-I treatment.

Homeopathic mistletoe adverse reaction mimics nodal involvement in 18F-FDG PET/CT performed for evaluation of response to chemotherapy in lymphoma.

Some patients use complementary medicine. We present a patient with Hodgkin's lymphoma, scanned with 18F-FDG PET/CT for evaluation of response after chemotherapy, who was self-administering mistletoe as a homeopathic medicine product. The careful review of the images of the entire scan and patient collaboration in anamnesis were crucial to avoid a false positive result. A review of the published scientific data on the effects of mistletoe is also presented.

Expression of Momordica charantia MAP30 and its antitumor effect on bladder cancer cells.

BACKGROUND: Momordica charantia (MC) is an edible medicinal plant that is known for its diversified biological functions. Momordica Antiviral Protein 30kD (MAP30) is a type I single chain ribosome-inactivating protein (RIP) isolated from the mature fruit and seeds of MC. Since MAP30 content in MC is limited, the study aim was to generate the recombinant MAP30 protein using prokaryotic expression system and determine its apoptotic/growth inhibitory effects on bladder cancer 5637 cells. METHODS: MAP30 gene was amplified by PCR from MC genomic DNA and identified by sequencing. The target gene was inserted into pET-28a (+) vector and transformed into E. coli BL21 (DE3) cells. Positive clones were selected by PCR. Recombinant protein was efficiently expressed under induction with 1.0 mM Isopropylthio-ß-D-galactoside (IPTG) at 30° C for 4 hours. Cytotoxicity studies were performed using MTT assay by treating 5637 bladder cancer cells with 100 µg/mL, 200 µg/mL, and 400 µg/mL concentrations of MAP30 for 24 hours and 48 hours, respectively. Flow cytometry was used to measure the apoptosis of MAP30-treatedcells in time course experiments. RESULTS: Full-length MAP30 gene was successfully expressed in Escherichia coli (E. coli) BL21 strain and MAP30 recombinant protein inhibited the growth of bladder cancer 5637 cells at 200 µg/mL and 400 µg/mL concentrations by inducing apoptosis of target cells in a dose- and time-dependent manner. CONCLUSION: It was, therefore, concluded that the MAP30 recombinant protein displayed potent antitumor activity in vitro.

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.

Tumor-associated CD75s- and iso-CD75s-gangliosides are potential targets for adjuvant therapy in pancreatic cancer.

Pancreatic adenocarcinoma confers one of the highest mortality rates in malignant human tumors with very poor prognosis. Because as yet no treatments are available that produce a substantial survival benefit for this fatal neoplasia, new therapeutic concepts are urgently required to support cancer standard treatment. In search of tumor-associated gangliosides with therapeutic background, we probed a random collection of cancerous and adjacent normal postoperative tissue samples from 38 patients for the expression of CD75s- and iso-CD75s-gangliosides. We exhaustively analyzed the expression of CD75s-1-ganglioside (IV(6)Neu5Ac-nLc4Cer) and structurally closely related iso-CD75s-1-ganglioside (IV(3)Neu5Ac-nLc4Cer) by means of immunohistology of cryosections and semiquantitative TLC of tissue lipid extracts combined with mass spectrometry. CD75s-1- and iso-CD75s-1-ganglioside showed an elevated expression in 42% and 66% of the tumors, respectively, indicating a significant association with neoplastic transformation (P = 0.001). Thus, increased expression of CD75s-1- and iso-CD75s-1-gangliosides renders these cell surface molecules promising candidates for oncologic applications. Further statistical analysis revealed a significant enhancement of CD75s-1-ganglioside in the group of less differentiated tumors (grade >2) suggesting this ganglioside as a potential marker for poor differentiation. The CD75s-specific antitumor drug rViscumin, which represents the recombinant counterpart of the ribosome-inactivating lectin viscumin, has successfully passed clinical phase I trials and provides an opportunity for treating pancreatic cancer. Consequently, if an enhanced expression is existent in malignant tissues, we propose the targeting of CD75s-gangliosides with rViscumin as a novel potential strategy in adjuvant treatment of pancreatic malignancies.