| Penaeidin-2is an important antimicrobial peptide derived from the Pacific white shrimp, Penaeus vannamei, and possesses both antibacterial and antifungal activities. Recent studies suggest that recombinant penaeidins shows similar activities to the native Penaeidin-2protein. Current research on AMPs have shown that some of cationic antimicrobial peptides have antitumor activity, Yet to date, there have been no studies on the potential antitumor effects of rPen-2.Renal cell carcinoma (RCC) is the most common malignant tumor of the adult kidney, accounting for approximately3%of all adult cancer cases. Because of the resistance of RCC to radiotherapy and chemotherapy, the identification and development of novel therapeutic strategies and drugs are required.Here, we evaluated the potential of recombinant penaeidin-2(rPen-2) in the selective killing of kidney cancer cell lines ACHN and A498, and its mechanism of action.3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays revealed that rPen-2has a higher cytotoxic andanti-proliferative effect on cancer cells when compared with a normal kidney cell line, HK-2. A dose-dependent inhibition was observed with maximum growth inhibition by rPen-2on the cancer cells at100μg/mL. The inhibitory efficiency of treatment increased over time. The maximal growth inhibition of ACHN and A498were62.4%and70.4%, respectively, at48h, this was in sharp contrast to HK-2cells which showed only13.2%growth inhibition.DNA-specific fluorescent dye staining and flow cytometric analysis showed a high percentage of apoptosis in tumor cells. Hoechst33258staining showed more apoptotic activity in tumor cells than those of control HK-2after treatment with100μg/mL rPen-2for24h. AO/EB double-staining was used to distinguish early apoptotic and late apoptotic cells. The cancer cells presented both early apoptosis and late apoptosis, and the plasma membrane of these cells seems to be damaged. To further confirm apoptotic cell death, we performed FACS analysis after Annexin V/PI staining and observed similar results. FACS analyses revealed6.2-15.8%,28-62.9%, and41.7-71.5%death in HK-2, ACHN, and A498cells, respectively. Among dead cells after24to48h of rPen-2treatment,6.7-12.0%,22.7-33.7%, and25.3-38%were in early apoptosis (Annexin V single stained), respectively, while0.4-3.2%,1.7-32.5%, and3.1-36.2%were in late stage apoptosis (Annexin V/PI double stained), respectively. Using spectrophotometry detected lactate dehydrogenase(LDH)releasing, LDH releasing rate of HK-2, ACHN,A498, which treated with100μg/mL rPen-2for24h, are3.4%-5.3%,25.6%-32.3%,33.8%-35.3%. All these results revealed selective killing effect of rPen-2to kidney cancer cells, this killing effect include leading cancer cells apoptosis and lysis.The dynamic binding process of rPen-2with the plasma membrane was displayed by laser confocal scanning microscopy. Using confocal microscopy, we observed that rPen-2combined with the cell membrane of HK-2cells after1h, but the conjunction was weak during the entire process. However, an abundance of rPen-2was observed to combine with the plasma membrane of tumor cells and entered into the nucleus rapidly.Laser confocal scanning microscopy demonstrated that the plasma membrane were the key site where rPen-2interacted with and destroyed tumor cells, rPen-2enter the cells and locate in cytoplasm or nucleus indicate that there maybe some other mechanisms in rPen-2killing tumor cells.Scanning electron microscopy (SEM) showed the morphologic changes of the cell membranes of kidney cancer cells treated with rPen-2.In comparison to the smooth membrane surface of control cells, plasma membrane of cancer cells appeared morphologic transformations such as fold, perforation and so on.In this study, we explores the effect of rPen-2on kidney cancer cells for the first time, find rPen-2can selectively kill kidney cancer cells and harmless to human renal tubular epithelial cells. Because of antimicrobial peptides mainly attack the plasma membrance, rPen-2could avoid the chemotherapy resistance effectively. This research provided a new way for development of new drug to treat RCC or other cancers. Antimicrobial peptides(AMPs) are important components of the innate defense systems, widely exist in various life forms.AMPs have high activity against both Gram positive bacteria and Gram-negative bacteria, they also possesse antibacterial,antifungal, antitumor,antiviral and antiparasite activities.Researches show that, AMPs kill the bacteria and microbes mainly by attacking the membranes, this mode of action is effective both to the resistent and sensitive bacteria. Antimicrobial peptides also have other mechanism of action,such as attacking chromosome and ribosomes after entering cells.However, the bioactivities of AMPs are not only killing function, AMPs also play roles of chemokines, taking part in immune responses in higher animals. Compared with the traditional antibiotics, antimicrobial peptides could avoid the development of resistance in bacteria, and act synergistic effect with the antibiotics. Therefore, antimicrobial peptides become a hot research topic in the development of new drugs.Neisseria gonorrhoeae is the pathogens of the gonorrhoeae. Gonorrhoeae is one of the main sexually transmitted diseases(STDs) in our country of which the morbidity is in the second place in STDs.In recent years, with the antibiotics being widely used, many drug-resistent strains of Neisseria gonorrhoeae have been found in clinic, the strains are generally resistant to the conventional antibiotics such as penicillins, macrolides, quinolones and so on. There are even super strains resistent to all antibiotics, drug-resistent strains of Neisseria gonorrhoeae is a serious threat to human health. Therefore, the development of new anti-Neisseria gonorrhoeae drugs is imperative. The plutella xylostella antimicrobial peptides pxCECAl are applied in this research. This cationic antimicrobial peptide first isolated from the plutella xylostella by our laboratory, it have characteristics of high expression and high bioactivities, which is easy to be purificated. The previous researches our laboratory show that the plutella xylostella antimicrobial peptides pxCECAl has antibiotics activities to Escherichia coli, Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus (MRSA), Bacillus subtilis and Bacillus pumilus. Using the plutella xylostella antimicrobial peptides pxCECAl treat the multidrug-resistent strains of Neisseria gonorrhoeae, the result show that the minimal inhibitory concentrations(MIC) is4μg/mL, the pxCECA1has a good effect at relatively low drug concentrations. The growth inhibition test and sterilization experiment show that the antimicrobial activity of the pxCECA1to the multidrug-resistent strains of Neisseria gonorrhoeae in a dose-dependent manne, and the pxCECA1can directly kill bacteria rather than only inhibit them, which will help decrease the resistance. The MIC that the multidrug-resistent strains of Neisseria gonorrhoeae treat with the pxCECA1in combination with ceftriaxone sodium shows that the two drugs have synergistic effect on antimicrobial activity,the doses of the two drug can be highly reduced. The pxCECA1in combination with ceftriaxone sodium not only make dosage reduction, but also prevent the development of antibiotic resistance.This research provide a new idea to develop therapeutic drugs for the multidug-resistent strains of Neisseria gonorrhoeae in clinic. |
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