Effects Of Local Hyperthermia On Promoting Apoptosis And Proinflammatory Cytokines Of HPV Infected Keratinocytes

IntroductionHuman Papillomaviruses(HPV)are small closed circular double-stranded DNA virus,which infect cutaneous and muco-cutaneous epithelia.HPV has significant host specificity.A vast majority of these infections are either clinically unapparent or benign proliferative conditions as warts or condylomas.More than 120 HPV genotypes have been identified.At the present stage,traumatic therapies(freezing,electrocautery,laser, chemistry corrosion,etc)were used to treat warts.The purpose of these means was to eliminate the tissues infected by HPV.They were hard to be accepted by patients because they were painful,recovered slowly and usually accompanied with scar.Local hyperthermia has been used to treat many superficial and deep benign and malignant tumors both in human and animals.Reports indicated that hyperthermia plays an important role in controlling tumors and infectious diseases.Some scholars utilized local hyperthermia to treat warts and they gained good efficacy.However there were much remained about the mechanism of action.Condyloma acuminatum(CA) is a very recalcitrant epithelial benign growth caused by Human papilloma virus(HPV) infection.Its incidence rate increased year by year.CA is hazardous to society and family because it's infectiousness is serious.CA is recalcitrant and its recurrence rate is very high.It has been thought highly of because its dissemination is difficult to be controlled.Some scholars considered that the infective tissues were more sensitive to hyperthermia and difficult to recover after thermal damage than normal skin.They thought the effects of hyperthermia on immune function were one of the mechanisms of warts extinction.Alexander supposed that hyperthermia could destroy HPV directly or indirectly.Gatto found that after 43℃hyperthermia the form of keratinocytes altered partly without alteration of their bioactivity.At or above 44℃hyperthermia,the death rate of keratinocytes increased.We presumed that the possible mechanisms of hyperthermia action on warts were as follow:Promoting apoptosis of HPV infective keratinocytes;promoting local immunity to clean virus;acting on HPV derectively.HPV can't be cultured in vitro.We use CA as viral vector to observe the effects of local hyperthermia on HPV infected skin.The present study investigated the effect of controlled local hyperthermia on inducing apoptosis of keratinocyts in CA,the possible signaling pathways involved in, as well as the expression of some cytokines secreted by keratinocyts.Materials and methods1.Patients and specimens12 CA patients were enrolled(4 men,8 women).The mean age of patients was 32.6 years(range:22～48 years).The mean duration of illness was 2.1 months(range: 1～6 months).Upon obtaining informed consents,incisional biopsy was taken.4 pieces of normal skin were obtained from individuals undergoing plastic surgery in our hospital.2.Hyperthermia device and local hyperthermia treatmentWe designed and manufactured a hyperthermia device(patent pending).The hyperthermia device has an infrared emitting source.The heat generated by the device could act on skin surface without direct contact.The heated surface temperature was controlled and stabilized at desired degrees(pre-setup temprature℃±0.1℃)by an infrared temperature monitor and a feedback circuit.Each biopsy specimen was approximately equally divided into 4 portions:one portion was frozen directly and stored at -70℃;each of the remaining three dividends was put into a 20mm flat-bottom culture dish,immersed(with top of the tissue slightly above the surface of the medium) in RPMI 1640 culture solution with the dermis downward.The samples were heated to surface temperature of 37℃,42℃,45℃,for 30 minutes,respectively.The samples were then completely immersed in medium and incubated at 37℃with 5%CO₂ incubator for 12 hours and then refrigerated at -70℃.3.HPV typingTotal DNA was extracted from specimens according to the instructions from Universal Genomic DNA Extraction Kit Ver.3.0.PCR amplification was performed using HPV6,11 type-specific primers.Amplification products were subjected to electrophoresis on 1.5%agarose and stained with ethidium bromide.4.In Situ Detection of ApoptosisApoptosis was detected by enzymatic labeling of DNA strand breaks using TUNEL.TUNEL labeling was performed using an in situ Cell Death Detection Kit. Quantitation of apoptotic cells was accomplished by counting the number of apoptotic bodies.Apoptosis indices were the mean value of five microscopic fields randomly selected.5.Fluorescent Real-time(Quantitative)PCRThe expression of DR4,DR5,Fas,Bax,Bcl-2,CCL-20,TNF-αand IL-1αwere detected by fluorescent real-time quantitative PCR.Total RNA was extracted from specimens using Trnzol reagent.Complementary DNA was synthesized from 2μg of the total RNA using a First Strand cDNA Synthesis Kit according to the manufacturer's protocol.The cDNA obtained was used as a template for subsequent PCR amplification using SYBR-Green PCR kit and relative quantitative analysis was done with double standard curve method.Amplification conditions were as follow:initial denaturation at 95℃for 2 min,followed by 40 cycles of denaturation at 95℃for 15s,annealing at 56 ℃for 30s,extension at 68℃for 40s.All assays were performed in triplicate.6.ImmunohistochemistryThe expression of Fas,Bax,Bcl-2 protein were detected by immunochemistry using SP-9000 kit.7.Statistical analysisANOVA and independent-sample t test were used to analyze the differences of numbers of apoptotic cells,expression of apoptosis related genes and cytokines. Correlations between apoptosis and apoptosis related genes expression were analyzed by two-variable correlation analysis.The results of immunohistochemistry were analyzed by Mann Whitney Test.A p value＜0.05 was considered to be statistically significant.Results1.Apoptosis in different groupsThere were more apoptotic keratinocytes in CA lesions than in normal skin,at any given temperature points.Nevertheless,the apoptosis index of CA treated with hyperthermia at 42℃and 45℃were significantly higher than those of untreated and 37℃groups(p＜0.01).Specimens subjected to high hyperthermia temperature showed apoptotic cells in full thickness of the epidermis.Higher hyperthermia was more efficient to induce apoptosis in both normal and CA lesions.There was no correlation between HPV types and apoptosis indices of CA at any given hyperthermia points.In normal skin,apoptotic cells increased with the elevation of temperature.2.The expression of apoptosis related genesIn the lesions of CA,transcripts from DR4,DR5,Fas and Bax all increased very significantly,especially in higher hyperthermia temperature.On the other hand,the levels of anti-apoptotic Bcl-2 transcripts were decreased with elevation of hyperthermia temperatures.In normal skin,though there were increased number of apoptotic cells with increment of hyperthermia temperature,transcripts of the DR4,DR5,and Bcl-2 remained relatively stable.Fas and Bax transcripts were significantly elevated in accordance with increased hyperthermia in normal skin.While Two-variable correlation analysis showed that apoptosis index was positively correlated with DR4, DR5,Fas and Bax mRNA expression,in CA.Apoptosis index was negatively correlated with Bcl-2 expression in CA.In normal skin,Apoptosis index was positively correlated with Fas and Bax mRNA expression.3.The expression of apoptosis proteinIn CA lesions,the expression of Fas,Bax,Bcl-2 protein increased than normal skin.Fas,Bax protein increased very significantly in higher hyperthermia.On the other hand,there was no obviously Bcl-2 protein expression after hyperthermia.The expression of Fas and Bax protein were significantly elevated in accordance with increased hyperthermia in normal skin.4.The expression of CytokinesIn CA lesions,the expression of CCL-20,TNF-α,IL-1αmRNA were higher than that in normal skin.The levels of CCL-20 and IL-1αmRNA were decreased with elevation of hyperthermia temperatures.While TNF-αmRNA kept stable.The levels of CCL-20 and IL-1αmRNA increased at 45℃hyperthermia in normal skin.Conclusions1.Local hyperthermia can increase the apoptosis of keratinocyte in CA and normal skin,especially above 42℃.2.Fas,Bcl-2,Bax mRNA and protein increase in CA compared with normal skin. In CA,hyperthermia can increase the expression of Fas,Bax DR4 and DR5,while the expression Bcl-2 decreased.In normal skin,the apoptosis were correlated to the elevation of Fas and Bax expression.3.CCL-20,TNF-αand IL-1αmRNA in CA were significantly higher than that in normal skin.CCL-20 and IL-1αmRNA decreased with the elevation of hyperthermia temperatures in CA.But they increased in normal skin at 45℃.Hyperthermia has no obviously effect on TNF-αmRNA expression.