Potent antitumor effects of a novel actinomycin D analog Leu5AMD.

Leu5AMD ([D-Val2, L-MeLeu5]2 AMD) is a novel actinomycin D (AMD) analog, in which both N-methylvalines were replaced by N-methylleucines. In the present study, an attempt has been made to investigate the effects of Leu5AMD on the proliferation of human gastric carcinoma cell line SGC-7901. 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. In addition, the in vivo antitumor effects of Leu5AMD on S-180 bearing mice and the acute toxicity on healthy mice were investigated. Treatment with Leu5AMD markedly suppressed the growth of Sarcoma xenograft. These results suggest that Leu5AMD may be used as a promising chemotherapeutical agent for patients affected by gastric carcinoma and other solid cancer.

Antitumor effects, cell selectivity and structure-activity relationship 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 Gram-negative 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 alpha-helix conformation, and L-Pro substitution of Ala8 can disrupt the alpha-helix conformation thoroughly. The L-Pro substitution induces a significant reduction of antitumor activity, indicating that the alpha-helix conformation of polybia-MPI is important for its antitumor activity. In summary, polybia-MPI may offer a novel therapeutic strategy in the treatment of prostate cancer and bladder cancer, considering its relatively lower cytotoxicity to normal cells.

Pharmacological effects of the dansylated neuropeptide FF analogues on body temperature and morphine analgesia.

In our previous work, the two putative agonists (dansyl-GSRFamide and dansyl-PQRFamide) and the two putative antagonists (dansyl-GSRamide and dansyl-PQRamide) on neuropeptide FF (NPFF) receptors were synthesized to evaluate the importance of Phe(8) of NPFF. In the present study, these putative NPFF agonists/antagonists containing different N-terminal sequences were further examined for their pharmacological profiles in thermoregulatory and nociceptive tests. The results indicated that the two dansylated agonists potently possessed similar thermoregulation (rank order of potencies: dansyl-GSRFamide>>NPFF>dansyl-PQRFamide) and different modulation of opioid-induced analgesia; in contrast, both of the two putative antagonists exhibited marked hypothermia (rank order of potencies: dansyl-PQRamide>dansyl-GSRamide) and facilitation of morphine analgesia (rank order of potencies: dansyl-PQRamide > dansyl-GSRamide). These data reveal that the difference of the N-terminal residues of the two putative agonists causes their dissociation of pharmacological pro- and anti-opioid effects. In addition, their N-terminal part is important to determine the potency of the dansylated agonists/antagonists. Our work might be helpful to develop a highly potent and fluorescent NPFF ligand.

Membrane active antitumor activity of NK-18, a mammalian NK-lysin-derived cationic antimicrobial peptide.

As the increasing emergence of multi-drug resistant tumor cells, there is an urgent need for developing new chemotherapeutic agents. NK-lysin was a novel effector of cytotoxic T cells and natural killer (NK) cells and had broad antimicrobial activity. In this study, we developed a core region of NK-lysin termed NK-18, and studied its antitumor activity and possible action mode. Our results showed that NK-18 (with 18 amino acids) possesses potent antitumor activity against bladder and prostate cancer cells by disrupting the integrity of cell membrane, but has negligible hemolysis activity against mouse erythrocytes. In addition, CD spectra was employed to study its conformation in membrane mimicking environment. NK-18 takes a standard α-helical conformation in membrane mimicking environment, which could be accounted for its more potent antitumor activity compared with its low α-helical content homologous derivatives. These findings together with its shorter amino acid sequence and lower synthesis cost suggest that NK-18 could present an alternative therapeutic strategy to cancer chemotherapy and play a promising role in fighting the multi-drug resistant tumors.

In vitro and in vivo antitumor effects of novel actinomycin D analogs with amino acid substituted in the cyclic depsipeptides.

The actinomycin D (AMD) analogs in which the D-valine residues (the second amino acid residue in the cyclic depsipeptide of AMD) and the N-methyl-L-valine residues (the fifth amino acid residue in the cyclic depsipeptide of AMD) were replaced with D-Phe or l- and D-forms N-methylvalines, N-methylisoleucine, N-methylleucine, N-methylphenylalanine, N-methylalanine, and sarcosine were synthesized. 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 compound 4 L-methylleucine(5)-AMD analog is comparable to AMD itself and that of compound 3(L-Methylisoleucine(5)-AMD analog) is slightly more toxic, about 1.25-fold than AMD. However, the acute toxicity of compound 5 D-methylleucine5-AMD analog is about 2-fold lower than AMD. This suggested that the N-methyl-D-amino acid replacement in the cyclic ring might play a vital role in their decreased acute toxicities, and perhaps the N-methyl-D-leucine substituent is more favorable, though there may be a slight loss of antitumor activity. This finding may be helpful for the design and development of more potent antitumor agents together with low acute toxicity, and suggests that the N-methyl-D-leucine substituent has the potential to be used as antitumor drug lead.

A novel analog of antimicrobial peptide Polybia-MPI, with thioamide bond substitution, exhibits increased therapeutic efficacy against cancer and diminished toxicity in mice.

Polybia-MPI (MPI), a short cationic α-helical antimicrobial peptide, exhibited excellent anticancer activity and selectivity in vitro in our previous studies. To improve its in vivo application, we synthesized an analog (MPI-1) of MPI by replacing the C terminal amide -[CO-NH(2)] with thioamide -ψ[CS-NH(2)]. Although there is just one atom difference, the MPI-1 exhibited some surprising properties. In vitro studies revealed that MPI-1 exhibited relatively high lytic activity over MPI, whereas its stability to enzymatic degradation in serum was improved remarkably. Despite the enhanced toxicity in vitro, MPI-1 exhibited significantly lower mortality to mice than MPI at 75 mg/kg. Importantly, in vivo anticancer activity study indicated that MPI-1 could remarkably suppress the growth of sarcoma xenograft tumors more efficiently than MPI. Therefore, the significantly improved anticancer activity and predominantly lower in vivo toxicity might allow MPI-1 to be a good candidate for future anticancer treatment.

Novel mode of action of polybia-MPI, a novel antimicrobial peptide, in multi-drug resistant leukemic cells.

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 alpha-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.

Correlation of COX-2 and K-ras expression to clinical outcome in gastric cancer.

Recent studies have suggested that K-ras play an important role in the induction of COX-2 expression in tumor cells. In the present study, tumor samples of 89 gastric cancer patients were prepared in tissue microarrays and they were stained by immunohistochemistry with antibodies against COX-2 and K-ras. We investigated the relationship between the protein expressions of COX-2 and K-ras in gastric cancer and their significance as prognostic markers in gastric cancer patients. The over expression rate of COX-2 and K-ras in gastric cancer was 61.8% and 61.8% (55/89) of all the patients, respectively. There was a significant positive correlation between COX-2 and K-ras expression in gastric cancer. COX-2 and K-ras positivity were correlated with depth of invasion and lymph node metastasis, respectively. K-ras positivity was correlated with growth pattern. Patients with COX-2 and K-ras positive tumors had a poorer prognosis than those with COX-2 and K-ras negative tumors. Over expression of COX-2 and K-ras were closely correlated to prognostic of patients with gastric cancer and they educed synergistic effect with carcinogenesis and development in gastric cancer.

Expression and clinical significance of vascular endothelial growth factor, cyclooxygenase-2, and Bcl-2 in borderline ovarian tumors.

OBJECTIVE: The objectives were to study the expression of vascular endothelial growth factor (VEGF), cyclooxygenase-2 (Cox-2), and Bcl-2 in borderline ovarian tumors (BOTs) and the relationship within them, and to investigate the correlation between expression of VEGF, Cox-2, and Bcl-2, and the clinicopathologic features of BOTs. METHODS: An immunohistochemical technique was used to investigate the expression of VEGF ,Cox-2, and Bcl-2 in 69 borderline, 18 benign, and 27 malignant human ovarian tumor tissues. RESULTS: Expression rate of VEGF protein (59.4%) in BOTs was higher than in benign tumors (27.8%) and was lower than in ovarian carcinomas (92.6%), and there was a significant difference between BOTs and benign ovarian tumors (p < 0.05), and carcinoma (p < 0.01). Significant correlation was observed between the positive expression rate for VEGF and clinical stage of BOTs (p < 0.05). The statistical analysis did not show a close correlation between the expression of VEGF and tissue type, and peritoneal implants in BOTs (p > 0.05). The expression rate of Cox-2 was significantly higher in ovarian carcinomas (81.5%) than in BOTs (57.9%) and in benign ovarian tumors (38.9%) (p < 0.05). Significant correlation was observed between the positive expression rate for Cox-2 and the clinical stage of BOTs (p < 0.05). The statistical analysis showed no close correlation between the expression of Cox-2 and tissue type, and peritoneal implants in BOTs (p > 0.05). There was a significant difference between the expression of Bcl-2 in ovarian carcinomas and BOTs than that in benign ovarian tumors (p < 0.05). The positive expression rate of Bcl-2 was not related to clinical stages and peritoneal implants (p > 0.05). Statistical analysis showed a positive correlation between the expression of Cox-2 and VEGF, and Bcl-2 in BOTs. CONCLUSIONS: Overexpression of VEGF, Cox-2, and Bcl-2 in BOTs may play an important role in the oncogenesis and progression of BOTs. It is feasible to detect VEGF, Cox-2, and Bcl-2 in the diagnosis and to predict the prognosis of BOTs.