Antitumor Activity, Structure-activity Relationship, And Action Mechanism Of Two Kinds Of Antitumor Peptides

Chemotherapy plays an important role in the treatment of various cancers. However, the use of conventional chemotherapeutic agents is often associated with deleterious side effects caused by inadversent drug induced damage to healthy cells and tissues. Moreover, with the frequently use of conventional chemotherapeutic agents, tumor cells also become resistant to chemotherapy. Thus, the development of new antitumor drugs that lack side effects and are unaffected by common mechanisms of chemoresistance would be a major advance in tumor treatment. Usually, there are two strategies to achieve this purpose: first, chemo-modifying the structure of conventional drugs; second, looking for new drugs. In the first part of this study, the analogs of a clinical used antitumor peptide drug actinomycin D were synthesized and their antitumor activity and the mechanism of action were studied. In the second part of this study, the antitumor effects and the novel mode of action of a antimicrobial peptide polybia-MPI against multidrug resistant tumor cells were studied.1. The synthesis and the mechanism of antitumor effects of actinomycin D analogsActinomycin D (AMD), a clinical used antitumor peptide, consists of a planar phenoxazone ring and two cyclic pentapeptides. Although AMD possesses valuable biological activity, its clinical usefulness is limited by its narrow spectrum of antitumor effects and its extreme cytotoxicity.In this study, we synthesized 12 analogs of AMD. The antimicrobial activity and cytotoxic activities of these compounds in vitro were investigated. The results showed that most D-valine-substituted analogs had much lower antimicrobial activity and cytotoxic activities in vitro than AMD itself, but three N-methyl-L-valine-substituted analogs had comparable or even more remarkable cytotoxic activities in vitro than AMD. Acute toxicities and antitumor effects of the N-methyl-L-valine-substituted analogs in mice were also examined. The result showed that the acute toxicity of [D-Val², L-MeLeu⁵]₂AMD is comparable to AMD itself and that of [D-Val², L-MeIle⁵]₂AMD is slightly more toxic, about 1.25-fold than AMD. However, the acute toxicity of [D-Val², D-MeLeu⁵]₂AMD is about 2-fold lower than AMD. This suggest that the N-methyl-D-amino acid replacement in the cyclic ring might play a vital role in determining their decreased acute toxicities, and perhaps the N-methyl-D-leucine substituent is more favorable, though there may be a slightly loss of antitumor activity. This finding may be helpful for the design and develop more potent antitumor agents together with low acute toxicity and the N-methyl-D-leucine substituent have the potential to be used as antitumor drug leads.[D-Val², L-MeLeu⁵]₂ AMD (Leu⁵AMD) is the most powerful analog in this study, and the mechanism of it on the proliferation of human gastric carcinoma cell line SGC-7901 was further studied. The results showed that Leu5AMD inhibited the proliferation and induces apoptosis in SGC-7901 cells in a dose-dependent manner. Apoptosis induced by Leu⁵AMD was further confirmed by annexin V-FITC/PI dual staining assay. After treatment with Leu⁵AMD, the loss of mitochondrial potential and the decrease of bcl-2 gene expression were observed in apoptotic cells, suggesting that Leu⁵AMD may be involved in mitochondria and bcl-2 related apoptotic pathway.2. The mechanism antitumor effects and the novel mode of action of a novel antimicrobial peptide polybia-MPI.A novel antimicrobial peptide, polybia-MPI, was purified from the venom of the social wasp Polybia paulista. It has potent antimicrobial activity against both Gram-positive and Gramnegative bacteria, but causing no hemolysis to rat erythrocytes. To date, there is no report about its antitumor effects on any tumor cell lines. In this study we synthesized polybia-MPI and studied its antitumor efficacy and cell selectivity. Our results revealed that polybia-MPI exerts cytotoxic and antiproliferative efficacy by pore formation. It can selectively inhibit the proliferation of prostate and bladder cancer cells, but has lower cytotoxicity to normal murine fibroblasts. In addition, to investigate the structure-activity relationship of polybia-MPI, three analogs in which Leu⁷, Ala⁸ or Asp⁹ replaced by L-pro were designed and synthesized. L-pro substitution of Leu⁷ or Asp⁹ significantly reduces the content of a-helix conformation, and L-pro substitution of Ala8 can disrupt the a-helix conformation thoroughly. The L-pro substitution induces a significant reduction of antitumor activity, indicating that theα-helix conformation of polybia-MPI is important for its antitumor activity.As the frequent emergency of resistant tumor cells during treatment, the development of new agents with new modes of action attracts a great deal of interest. Polybia-MPI was a short cationic a-helical amphiphilic peptide that has selective toxicity toward cancer cells but no hemolytic activity. Its target selectivity is based on the binding preference to membranes containing anionic phospholipids by electrostatic driving. Its ability to make PI and trypan blue permeate into tumor cells at the same rate (within minutes), suggests a killing mechanism that involves plasma membrane perturbation. SEM and confocal microscopy experiments verified that the cell died as a result of acute injury and bursting, suggesting necrosis. As compared to the conventional chemotherapy, polybia-MPI targets at the cell membrane rather than enters into the cell to exert its action. So it is difficult for tumor cells to develop resistance to polybia-MPI during treatment and its action is not affected by the common multi-drug resistant mechanism. Although this is an initial study that looked at its in vitro activity rather than the in vivo activity, with the increasing resistance of conventional chemotherapy, polybia-MPI may offer a novel therapeutic strategy in the treatment of multi-drug resistant cancer.