Genotoxic and cytotoxic activity of host defense peptides against human soft tissue sarcoma in an in vitro model.

Soft tissue sarcomas (STSs) are a heterogeneous group of rare, mesenchymal tumors. Treatment with common chemotherapeutic drugs is consistently associated with low response rates and high rates of adverse toxic effects. Host defense peptides (HDPs) are used as part of innate immunity, and many of them act by directly lysing the target cell membrane. Studies have demonstrated high selectivity of HDP analogs against malignant cells because of a relative abundance of negative charges in malignant cell membranes, compared to normal cells. Our aim was to assess the toxic efficacy of [D]-K(6)L(9), [D]-K(3)H(3)L(9), and Protegrin-1 against the fibrosarcoma cell line, HT1080, and primary human fibroblasts to analyze the potential of these peptides as therapeutic options against STSs. Cell proliferation of the fibrosarcoma cell line, HT1080, and human fibroblasts was determined in vitro after treatment with [D]-K(6)L(9), [D]-K(3)H(3)L(9), and Protegrin-1. Genotoxicity was examined on the basis of the mild alkali version of single-cell gel electrophoresis (comet assay). Doxorubicin, a commonly used STS chemotherapeutic agent, served as the control. The native HDP, Protegrin-1, could show a cytotoxic tendency against malignant cells, but no selectivity in genotoxic trials. The synthetic peptide, [D]-K(6)L(9), could not show any selective oncolytic activity against sarcoma cells. [D]-K(3)H(3)L(9) has shown a tendency for toxic selectivity against malignant cells. There is a potential of developing suitable oncolytic candidates with selectivity against malignant cells. [D]-K(3)H(3)L(9) showed the first promising results, but there has to be further investigation to improve the therapeutic properties of HDPs.

Suppression of soft tissue sarcoma growth by a host defense-like lytic peptide.

BACKGROUND: Soft tissue sarcoma (STS) is an anatomically and histologically heterogeneous neoplasia that shares a putative mesenchymal cell origin. The treatment with common chemotherapeutics is still unsatisfying because of association with poor response rates. Although evidence is accumulating for potent oncolytic activity of host defense peptides (HDPs), their potential therapeutic use is often limited by poor bioavailability and inactivation in serum. Therefore, we tested the designer host defense-like lytic D,L-amino acid peptide [D]-K3H3L9 on two STS cell lines in vitro and also in an athymic and syngeneic mouse model. In recent studies the peptide could show selectivity against prostate carcinoma cells and also an active state in serum. METHODS: In vitro the human synovial sarcoma cell line SW982, the murine fibrosarcoma cell line BFS-1 and primary human fibroblasts as a control were exposed to [D]-K3H3L9, a 15mer D,L-amino acid designer HDP. Cell vitality in physiological and acidic conditions (MTT-assay), cell growth (BrdU) and DNA-fragmentation (TUNEL) were investigated. Membrane damage at different time points could be analyzed with LDH assay. An antibody against the tested peptide and recordings using scanning electron microscopy could give an inside in the mode of action. In vivo [D]-K3H3L9 was administered intratumorally in an athymic and syngeneic (immunocompetent) mouse model with SW982 and BFS-1 cells, respectively. After three weeks tumor sections were histologically analyzed. RESULTS: The peptide exerts rapid and high significant cytotoxicity and antiproliferating activity against the malignant cell lines, apparently via a membrane disrupting mode of action. The local intratumoral administration of [D]-K3H3L9 in the athymic and syngeneic mice models significantly inhibited tumor progression. The histological analyses of the tumor sections revealed a significant antiproliferative, antiangiogenic activity of the treatment group. CONCLUSION: These findings demonstrate the in vitro and in vivo oncolytic activity of [D]-K3H3L9 in athymic and syngeneic mouse models.

Frequent concomitant inactivation of miR-34a and miR-34b/c by CpG methylation in colorectal, pancreatic, mammary, ovarian, urothelial, and renal cell carcinomas and soft tissue sarcomas.

The microRNA encoding genes miR-34a and miR-34b/c represent direct p53 target genes and possess tumor suppressive properties as they mediate apoptosis, cell cycle arrest, and senescence. We previously reported that the miR-34a gene is subject to epigenetic inactivation by CpG methylation of its promoter region in primary prostate cancer and melanomas, and in 110 different cancer cell lines of diverse origin. Here we analyzed the methylation status of miR-34a and miR-34b/c in additional primary tumors of divergent sites. We found methylation of miR-34a or miR-34b/c in formalin-fixed, paraffin-embedded (FFPE) tumor samples from 178 patients with the following frequencies: colorectal cancer (74% miR-34a, 99% miR-34b/c; n = 114), pancreatic cancer (64%, 100%; n = 11), mammary cancer (60%, 90%; n = 10), ovarian cancer (62%, 69%; n = 13), urothelial cancer (71%, 57%; n = 7), and renal cell cancer (58%, 100%; n = 12). Furthermore, soft tissue sarcomas showed methylation of miR-34 gene promoters in FFPE samples (64%, 45%; n = 11), in explanted, cultured cells (53%, 40%; n = 40), and in frozen tissue samples (75%, 75%, n = 8). In the colorectal cancer samples a statistically significant correlation of miR-34a methylation and the absence of p53 mutation was detected. With the exception of sarcoma cell lines, the inactivation of miR-34a and miR-34b/c was concomitant in most cases. These results show that miR-34 inactivation is a common event in tumor formation, and suggest that CpG methylation of miR-34a and miR-34-b/c may have diagnostic value. The mutual exclusiveness of miR-34a methylation and p53 mutation indicates that miR-34a inactivation may substitute for loss of p53 function in cancer.

Oncolytic designer host defense peptide suppresses growth of human liposarcoma.

Sarcomas display a rare and heterogeneous group of tumors. Treatment options are limited. Host defense peptides (HDPs), effector molecules of the innate immune system, might provide a more effective treatment option. The aim of our study was to analyze the oncolytic activity and mode of action of a designer HDP. In vitro, the human liposarcoma cell line SW-872 and primary human fibroblasts as a control were exposed to [D]-K(3)H(3)L(9), a 15-mer D,L-amino acid designer peptide. Cell growth (MTT assay), proliferation (BrdU assay) and genotoxicity (TUNEL assay) were analyzed. The mode of action was examined via fluorescence-activated cell sorter (FACS) analysis and confocal laser scanning microscopy. In vivo, [D]-K(3)H(3)L(9) (n = 7) was administered intratumorally in a SW-872 xenograft mouse model (Foxn1nu/nu). Phosphate buffered saline served as a control (n = 5). After 4 weeks, tumor sections were histologically analyzed with respect to proliferation, cytotoxicity, vessel density and signs of apoptosis and necrosis, respectively. In vitro, [D]-K(3)H(3)L(9) highly significantly (p < 0.01) inhibited cell metabolism and proliferation. TUNEL assay revealed corresponding genotoxicity. FACS analysis suggested induction of necrosis as a cause of cell death. The mean tumor volume of the control group exponentially increased sevenfold, whereas the mean tumor growth was negligible in the treatment group. Macroscopically, [D]-K(3)H(3)L(9) induced full tumor remission in 43% of treated animals and partial remission in 43%. Vessel density was significantly reduced by 52%. Morphological analyses supported the hypothesis of cancer cell killing by necrosis. In summary, [D]-K(3)H(3)L(9) exerts very promising oncolytic activity on liposarcoma cells. Our study demonstrates the potential of HDPs as a novel therapeutic option in future soft tissue sarcoma therapy.

Nitric oxide therapy: fact or fiction?

Nitric oxide (NO) has been the topic of many studies regarding its therapeutic benefit in patients with cardiac disease. Recent studies are now revealing potential advantages for healthy individuals and endurance athletes. This article discusses current research focused on NO augmentation in relation to muscular strength and endurance. Arginine, an NO precursor, has been more extensively studied as a supplement for performance enhancement. Its role in cardiovascular endurance and strength training is assessed in individuals with various athletic backgrounds. The therapeutic role of NO in the treatment of tendinopathies is also reviewed.