Podophyllotoxin Nanostructured Lipid Carriers Induced VK2/E6E7 Cells Apoptosis Through Caspase-dependent Pathway

BackgroundCondyloma acuminatum(CA)is one of the most common sexually transmitted diseases(STDs)caused by human papillomavirus(HPV)and it often occurs in the anus and external genitalia.According to reports,the incidence ranks first of STDs in the United States and Europe,and in our country it ranks only second to gonorrhea.HPVs are small DNA virus with high degree of species specificity:human skin keratinocytes and mucosal squamous cells are their natural host.HPVs also take on special nature of only proliferation to a certain degree of differentiation of epithelial cells.Meantime HPVs do not lead to viremia,without blood diffusion,and are not easily identified by the immune system.HPVs are divided into more than 100 kinds of genotypes by molecular biology techniques,and different types of HPVs infection can cause different clinical manifestations.CA is mainly caused by HPV-6,11,16,18,31,32,33,35,39,42,43,44,51,52,53,and 54.According to the relationship between HPV and cancer,and HPVs can be further divided into low-risk and high-risk type.Low-risk types mainly include 6,11,40,42,43,44,54,61,70,72,51 and ep6108,with relevant to some benign lesions of CA and low-grade cervical intraepithelial neoplasia lesions(CIN Ⅰ).While there are 15 kinds of high-risk types,HPV16,18,31,33,35,39,45,51,52,56,55,59,68,73 and 82 respectively,closely related to cervical cancer and high-grade cervical intraepithelial neoplasia lesions(CIN Ⅱ/Ⅲ),especially HPV16,18.But there are a number of genotypes not yet reached a consensus in their classification due to the very limited epidemiological data.Epidemiological survey showed that the HP Vs have high infection rate in the group of sexually active population,and 75%to 80%will be infected with HPVs in a period of their lifetime.In the crowd of HPV-infected patients,part of the population show the recessive infection and subclinical infection of HP Vs,because of the difference of the immune function and the existence of HP Vs immune escape mechanism.At present,the presence of subclinical infection of HPVs is important reason of recurrence and spread.HP Vs carriers of subclinical infection and recessive infection still have infectivity with sexual contact,so that HPVs widely spread.Coupled with the lack of antiviral,the infection rate of HPVs and morbidity of CA have increased year by year.The subclinical and recessive infection of HPVs without clear clinical manifestation can easily be ignored,and because of this the diagnosis and treatment of HPVs infection are more and more difficult.HPVs DNA contains E6,E7 gene,especially high-risk types—HPV16,18.The E6,E7 gene can transfect cells of host,and the consequences of transfection lead to the host’s chromosome instability,DNA replication,transcription disorder,and so much as the incidence of tumors.At the same time E6,E7 gene expression product can also combine with that of tumor suppressor genes p53 and pRb,promote their degradation,and reduce the function of tumor suppressor,finally make cells transformation and cancerization.Therefore,infection with high-risk type HPVs for a long time,continuously or repeatedly may lead to precancerous lesions and invasive carcinoma.Currently,the exact treatment of CA and how to reduce and prevent cancer recurrence are the major issues of the medical profession that need to be solved urgently.The major clinical methods for CA include local medication,physical methods,surgery,systemic therapy,photodynamic therapy and vaccine therapy emerging in recent years.Traditional therapies,including topical application of 0.5%Podophyllotoxin(POD)tincture,interferon,cryotherapy,microwave therapy,etc,have high relapse rate and adverse reactions rate.While photodynamic therapy(PDT)is expensive,and it has local irritation.Moreover,the HPV vaccinecan only prevent HPV6,11,16,18,and it is ineffective to current infection.The difficult thretment of CA causes a heavy financial and psychological burden to patients and the patients’families,and resulting in negative social impact.0.5%POD tincture is one of the most effective first-line drugs recommended by the WHO for the treatment of CA.There are large skin and mucosa irritations,such as pain,erosion and edema,when 0.5%POD tincture is used on glans penis,anus,vagina,cervix and other mucosa.System absorption too much POD may cause serious side effects,including dizziness,nausea,vomiting,diarrhea,abdominal pain,thrombocytopenia,leucopenia,abnormal liver function,ataxia and peripheral nerve palsy that limits its further application in clinical.On other hand,0.5%POD tincture cannot eradicate virus,consequently CA may easily relapse.In order to overcome the limitations of POD application on mucosa,to reduce adverse effects and prevent relapse,our team led by Professor Zeng successfully developed nanostructured carrier preparations of POD,including 0.5%liposomes of POD,solid lipid nanoparticles(SLN).Subsequently we found that POD on nanoparticles parcel had the following merits:nuniform distribution of nanoparticles,small particle size,good stability,and high coating rate,good skin targeting and little system absorbs.Through experimentation on animals,we confirmed POD nanoparticles preparations could inhibit proliferation of human keratinocyte.In clinic trial we also found cure rate to CA of POD nanoparticles preparations was similar to traditional preparations,but the former had significantly lower recurrence rate and adverse reactions.Nanostructured lipid carriers(NLC)are new nanostructured drug carrier systems.Compared to SLN,it has a higher physical stability,not easy to be degraded,better biological tissue compatibility,targeting,and controlled release.We had prepared drug concentration of 0.5%of podophyllotoxin nano lipid carrier(POD-NLC)successfully,using the method of modified low temperature curing-emulsion evaporation,and then we found POD-NLC could induce apoptosis and inhibit proliferation of immortalized human cervical epithelial cells(H8 cells)and immortalized human vaginal epithelial cells(VK2/E6E7 cells),but the mechanism of apoptosis and proliferation inhibition of POD-NLC on these cells has not yet had an in-depth study.On the basis of the long-term studies of our group,we plan to preopare 0.5%POD-NLC,apply it on VK2/E6E7 cells,and explore the possible apoptosis and proliferation inhibition mechanisms by POD-NLC on VK2/E6E7 cells in vitro,in order to understand the application potential in vaginal HPV infection prevention and treatmenr of this new nano-preparation,provide theoretical basis to new drug development and research for the treatment of CA and a reference for clinical research of new nano drug carriers.Objectives1.To prepare 0.5%podophyllotoxin nanolipid carrier(POD-NLC)using the method of modified low temperature curing-emulsion evaporation,to culture immortalized human vaginal epithelial cells(VK2/E6E7 cells)in vitro,to detect and analyze the expression of apoptosis-related gene,and to observe the effect of POD-NLC on microtubule and micro filament of VK2/E6E7 cells.2.To preliminarily explore the possible mechanisms of apoptosis by POD-NLC on VK2/E6E7 cells in vitro.Methods1.The effect of POD-NLC on the expression of apoptosis-related gene of VK2/E6E7 cells in vitro.(1)Prepared 0.5%POD-NLC and blank-NLC in our lab;(2)Treated VK2/E6E7 cells with different concentrations(0.25,1μg/mL)of POD-NLC,lμg/mL POD tincture and blank-NLC in vitro.The relative expression level of bcl-2,bax,caspace-3,8,9,10 and p53 mRNA were assessed by Real-time Quantitative PCR(qPCR).Experiment repeated three times;(3)Determine the relative expression level of bcl-2 and bax protein by Western Blot.Experiment repeated three times.2.The effect of POD-NLC on microtubule(MT)and micro filament(MF)of VK2/E6E7 cells.Tag β-actin,α-tubulin and cell nucleus,using confocal laser scanning microscope(CLSM)observe the effect of different concentrations(0.25,1μg/mL)of POD-NLC,lμg/mL POD tincture and blank-NLC on microtubule and micro filament of VK2/E6E7 cells.3.StatisticsStatistical software SPSS13.0 was used to conduct statistical analysis for all the experimental data.The data were interpreted by X±SD and analyzed by One-Way ANOVA.Multiple comparisons were addressed by Bonferroni test or by Welch and Tamhane’s T2 test,when heterogeneity of variance.When P≤0.05,there was significant difference.Results1.VK2/E6E7 cells were treated for 24 hours with different concentrations(0.25,lμg/mL)of POD-NLC,lμg/mL POD tincture and blank-NLC.The results of qPCR showed that compared with control group,POD-NLC could increase the levels of bax,caspase-8,10 and p53 mRNA in a dose-dependent manner(P<0.01)and 1μg/mL POD-NLC had greater effect than 1μg/mL POD tincture(P<0.01),thereinto caspase-8 mRNA had obviously increased.High and low concentrations of POD-NLC and POD tincture could increase the levels of caspase-9 mRNA(P<0.01),but there was no statistically significant difference between the groups(P>0.05).High concentrations of POD-NLC could raise the levels of caspase-3 mRNA too(P<0.05),but there was no statistically significant difference among low concentrations of POD-NLC,POD tincture and control group(P>0.05).Meanwhile the expression of bcl-2 mRNA decreased(P<0.01),but there was no statistically significant difference between the groups(P>0.05).There was no significant difference between blank-NLC and control group(P<0.05).2.VK2/E6E7 cells were treated for 24 hours with different concentrations(0.25,1μg/mL)of POD-NLC,and 1μg/mL POD tincture.Compared with control group,the expression of bax protein significantly increased with the increasing dosage of POD-NLC examined by Western Blot(P<0.01),and the effect of 1μg/mL POD-NLC on bax protein was greater than 1μg/mL POD tincture(P<0.05).Meantime bcl-2 protein significantly decreased by 1μg/mL POD-NLC and 1μg/mL POD tincture(P<0.05),while there was no significant difference between the two groups(P>0.05).3.After VK2/E6E7 cells treated with different concentrations(0.25,1μg/mL)of POD-NLC,POD tincture and blank-NLC for 24h,microtubules and microfilament were visualized by CLSM.Microtubule showed green fluorescence,microfilament red fluorescence,and the nucleus blue.In control and blank-NLC group,microtubules and microfilaments distributed around the cytoplasm evenly with regular shape and full structure,and generally presented complete cellular morphology,meantime,cell nucleus structure was complete.In contrast,after treated with 0.25μg/mLPOD-NLC,there were certain degrees of damage on microtubules in VK2/E6E7 cells,and decrease in the number,at the same time,microfilaments were destroyed,and cell morphology changed.While with 1μg/mLPOD-NLC,the vast majority of microtubules were damaged,and the bundle structure disappeared,just remaining a little dotted irregular fluorescence.Besides,microfilaments were obviously damaged,polygonal or fusiform cell shape disappeared,making the cells round sphere or ellipsoid,microvilli and connections between cells disappeared,and the nuclear shape also damaged,which were similar to the effects on VK2/E6E7 by POD tincture.The destruction of microtubule and micro filament by high concentrations of POD-NLC was more apparent than the equal concentration of POD tincture.Conclusions1.POD-NLC could change multiple apoptosis-related genes(bax,bcl-2,caspase-3,8,9,10,p53)to induced apoptosis in VK2/E6E7 cells.POD-NLC has a greater effect on bax,caspase-8,10,and p53 than POD tincture.Based on the literatures and our experimental data,we hypothesize POD-NLC may induce apoptosis of VK2/E6E7 cells through death receptor pathway(POD-NLC→VK2/E6E7 cells→aspase-8,10 expression→activation of caspase-3→apoptosis),and by the classical mitochondrial pathway(POD-NLC-+VK2/E6E7 cells→p53 expression→bcl-2 decrease,bax increase→activation of caspase-9→activation of caspase-3→apoptosis).Death receptor pathway may be the main way of apoptosis.And effect of POD-NLC on inducing apoptosis is greater than POD tincture.In addition,increased expression of p53 of VK2/E6E7 cells by POD-NLC hints that POD-NLC may reverse carcinogenesis of HPV on cells.2.POD-NLC destroys the structure of microtubules and microfilaments,destabilizes mitotic spindles and interferes with mitoses of cells,so as to inhibit cell proliferation.And the effect of POD-NLC on destruction of microtubules and microfilaments is greater than POD tincture.