| Background: Persistent human papillomavirus(HPV)infection can cause verrucous lesion of the skin and mucous membrane and a variety of benign or malignant diseases such as genital warts,cervical,vulvar,anal,and head and neck cancers.Cervical cancer is the 2nd most common cancer in woman worldwide,mostly caused by HPV subtypes 16 and 18,which are responsible for around 70% of cervical cancers.A prophylactic vaccine,based on the papillomavirus like particles(VLPs),has been available to the public since 2006,but the vaccine is not effective for those already infected.Condyloma Acuminata(CA)is also the result of infection with Human Papillomavirus.CA is in the second place of the sexually transmitted diseases(STDs)in China,account for about 16% of all sexually transmitted diseases.More than 40 kinds of HPV genotypes are associated with the genital warts,HPV6 and 11 account for approximately 90%.However,there are no drugs that eradicate genital warts.An ideal therapeutic vaccine should elicit high quality,and sufficient numbers of cytotoxic T lymphocytes(CTLs)that are able to migrate to the tumor site,overcome the immune suppressive environment of the tumor and kill tumor or viral infected cells.HPV16 E7 long peptide vaccine with antiIL-10 receptor antibodies increases the vaccine induced CTLs and infiltrate into tumor compared with immunization without IL-10 signaling blockade,but inhibits tumor growth weakly,which reminds that the tumor microenvironment probably stunt the efficacy of the vaccine.Synthesized natural caerin peptides from amphibian skin secretions increase acute inflammation by Tec kinase signaling pathway and LXR/RXR pathway and promote tumor cells apoptosis.In this study,I assume that caerin peptides are able to inhibit the proliferation of tumor cell in vivo and boost the efficacy of a HPV16 E7 peptide-based vaccine containing IL-10 signaling inhibitor.I investigated whether caerin peptides inhibit proliferation of HPV+ cell in vitro and in vivo.The next,I prepared a temperature-sensitive caerin peptides gel to explore whether topical application of F1/F3 gel inhibits TC-1 tumor growth,then tested whether caerin peptides were able to boost the efficacy of a HPV16 E7 peptide-based vaccine containing IL-10 signaling inhibitor.Results: 1.The antiproliferative activities of caerin 1.1(simplified as F1)and caerin 1.9(simplified as F3)against TC-1 cells were evaluated by MTT assays.Negative controls included(1)P3 peptide and(2)cells without any peptides treatment had not antiproliferative effect.The additive effect of caerin 1.1 and caerin 1.9(mass ratio 50:50,simplified as F1/F3)was evaluated.The inhibition effect of F1 or F3 was concentration dependent as F1/F3 at 5 g/m L did exhibit inhibition of the proliferation of TC-1 cells(P<0.05)and at 10 g/m L(P<0.01)compared to untreated cells.In addition,F1/F3 do not inhibit the growth of another noncancer cell line HUVC at the same concentration(P>0.05,ns).2.The apoptosis of TC-1 cells exposed to different concentrations of caerin 1.1,caerin 1.9,and P3 treatments for 24 h were measured by flow cytometric analysis after double-staining with Annexin V-FITC/PI.Caerin 1.1 and caerin 1.9 caused significantly more TC-1 cell apoptosis at 5 g/ml and 10 g/ml,compared to P3 and the untreated control.3.Caerin 1.1 and 1.9 inhibit TC-1 growth in vivo.I investigate whether caerin 1.1 and 1.9 were able to inhibit TC-1 cell growth in vivo.C57BL/6 mice were subcutaneously transplanted with TC-1 cells.Four days later,30 ?g of caerin 1.1 and 1.9,P3 or PBS was injected directly into the tumor daily for 7 consecutive days.Results showed that caerin peptides,but not P3 peptide or PBS were able to inhibit the TC-1 cell growth.4.Temperature-sensitive gel of caerin peptides were prepared,and the physical characteristics of F1/F3 gel were sterile,weak acid and low/no hydrophilia.5.I investigate whether F1/F3 in PBS was statistically different from F1/F3 in gel.According to MTT analysis,F1/F3 gel has the ability to inhibit TC-1 tumor cell growth.At 5 ?g/ml and 10 ?g/ml,there is no statistical difference between F1/F3 in PBS and F1/F3 in the gel(F1/F3 gel vs F1/F3 P> 0.05,ns).In addition,when the concentration is at 15 ?g/ml,there was a statistical difference between F1/F3 gel and F1/F3(F1/F3 gel vs F1/F3,P<0.05),F1/F3 gel has a higher effective cytotoxicity against tumor cells.Gel matrix and P3 gel cannot inhibit TC-1 tumor cell growth.6.Furthermore,the bioactivity of the F1/F3 gel stored at room temperature and 4 ? was tested by MTT assay.After storage at room temperature for 30 days and 4? for 5 months,the F1/F3 gel was able to inhibit TC-1 cell growth at concentration as low as 5 ?g/ml.7.Next,I explored whether the F1/F3 gel inhibited TC-1 growth in vivo to confirm that F1/F3 peptides maintained their bioactivity after gel preparation.Five days after TC-1 cells were transplanted subcutaneously into C57BL/6 mice,F1/F3 gel at concentration equal to free F1/F3 were injected into the TC-1 tumors daily for 7 days.Then,3 days later,mice were sacrificed,and tumors were isolated and weighed.Tumor sizes and weights were significantly lower in mice that received an intra-tumor injection with either F1/F3 gel or free F1/F3 peptides,compared to those from mice that received localized injection of gel dissolved in PBS(P<0.01).No differences were noted between the effects of F1/F3 peptides and F1/F3 gel.8.To investigate whether topical application of F1/F3 gel was able to inhibit TC-1 growth in vivo,TC-1 cells were subcutaneously transplanted into C57BL/6 mice.After 3 days,TC-1 tumor-bearing mice were divided into seven groups to receive no treatment,gel only,P3 gel,F1/F3 gel,5% imiquimod cream,5% imiquimod plus gel only,or 5% imiquimod alternating with F1/F3 gel.These treatments were applied topically to the skin above the TC-1 tumor.The gels were applied daily for 7 days consecutively,except for 5% imiquimod,which was applied every other day as recommended by the manufacturer.Mice were sacrificed 2 days after the final treatment,and the isolated tumors were weighed.Topical application of both F1/F3 gel and imiquimod inhibited TC-1 growth compared with the untreated control group.The tumor weights from mice in the F1/F3 gel group were significantly less than those from mice that received gel only or P3 control gel(P<0.05).Interestingly,TC-1 tumor-bearing mice treated with F1/F3 gel alternating with 5% imiquimod had lower tumor weights than those treated with F1/F3 gel or 5% imiquimod cream alone,although these differences were not statistically significant.9.To investigate the mechanism of caerin gel inhibiting tumor cell growth.I examined the numbers of tumor-infiltrating T cells and NK cells in TC-1 tumors in mice treated with P3 gel,F1/F3 gel,and untreated control groups by using flow cytometry.The results showed that the numbers of both T cells and NK cells infiltrating to TC-1 tumors were significantly increased in mice treated with F1/F3 gel compared to those mice that remained untreated or treated with P3 control gel(P<0.01),P3 gel had no difference compared with untreated group.10.Caerin peptides increase the efficacy of a HPV16 E7 peptide-based vaccine containing IL-10 signaling inhibitor.TC-1 tumor bearing mice were divided randomly into 4 groups: 1)Ex/MPLA/anti-IL-10 R antibody immunization(Ex represents 4 overlapping peptides of entire HPV16E7 protein)and caerin 1.1/1.9 tumor local injection;2)Ex/MPLA/anti-IL-10 R antibody immunization plus PBS tumor local injection;3)caerin 1.1/1.9 tumor local injection and 4)PBS tumor local injection.TC-1 tumor bearing mice were immunized twice 7?days apart,followed by intra-tumor injection of caerin 1.1/1.9 or PBS.The result clearly showed that immunization with Ex/MPLA/anti-IL-10 antibody plus local injection of caerin peptides significantly increased the survival time of TC-1 tumor bearing mice.Immunization with Ex/MPLA/anti-IL-10 R antibody alone,or tumor local injection of caerin peptides alone did not increase the survival time of TC-1 tumor bearing mice compared with PBS treated control group.Conclusion and Research significance:1.Caerin peptides inhibit proliferation of TC-1 tumor cells and have no cytotoxicity to normal cells.2.The temperature-sensitive gel of caerin peptides had anti-tumor activity in vivo and in vitro.3.The temperature-sensitive gel of caerin peptides are stable.4.The temperature-sensitive gel of caerin peptides applied topically to the skin above the TC-1 tumor is able to inhibit TC-1 tumor growth.5.One of the mechanisms of caerin peptides temperature-sensitive gel inhibit TC-1 tumor growth is by recruiting more T cells and NK cells to the tumor site.6.Caerin peptides combined the therapeutic vaccine Ex/MPLA/?-IL10 R has stronger anti-tumor effect.Significance and Prospects: The development of temperaturesensitive caerin peptides gel may provide a better choice for the treatment of condyloma acuminatum.Caerin peptides were able to improve the efficacy of the HPV therapeutic vaccine.The study lay a solid foundation for further improving the efficacy of therapeutic vaccines. |
PDF Full Text Request