Expression Of TNFRs In Hypopharyngeal Carcinoma And Application Of Gene-switch In Regulating Their Expression For Sensitization To TNF And DDP

Hypopharyngeal carcinoma is a kind of tumor with highly malignantproperties including easy diffusion to adjacent structures and early lymphnode metastasis. Squamous cell carcinoma (SCC）is the most commonpathological type. Because of its special biological characters, the prognosis ofhypopharyngeal SCC (HPSCC) is poor, with an overall5-year survival rate ofapproximately20%to40%. Surgical operation is still restricted to a subset ofabout60%of all cases because early-stage cases of HPSCC are difficult toidentify. Despite improvements in diagnostic tools, surgical techniques, andconcurrent chemo-radiation therapy, the5-year survival rate of HPSCCremains nearly unimproved in the past two decades. Therefore, it is urgent toseek more effective treatment strategies to improve the therapeutic outcomesof HPSCC.It has been confirmed that the molecular biological aspects playimportant parts in the process of the tumorigenesis and progression of cancers.Nowadays, more and more researchers focus on gene therapy, trying toestablish methodologies to prevent cancers from occuring. The ideal genetherapy should reside on regulating target genes in specific organs accordingto the severity of the diseases. However, it is a diffcult problem on how toregulate the expression of a certain gene in accordance with the amount inneed at punctual time. The traditional gene inducible systems relying on thethermal shock protein systems, hormone systems and the heavy metalssystems have many drawbakcs. For example, they possess commonshortcomings like high background, polytrophic effect and environmentalpollution, etc. These disadvantages limit their applications and development.The gene inducible system created by Gossen, also known as Tetracyclin inducible gene expression system (Tet-on/off system), overcomes thedisadvantages of the traditional inducible systems. This system canspecifically regulate exogenous gene expression through E.coli Tnolregulatory elements. Tet system is considered to be the most ideal induciblegene expression system. It has many advantages such as high efficiency,specificity and restriction. It has been applied to many areas of research, suchas the study of gene function and the treatment of disease. However, therehave been are no relevant reports on study of HPSCC at present.Tumor Necrosis Factor-α (TNF-α) was discovered30years ago. It isregarded as the strongest antitumor cytokine identified till present. TNF-αcankill the tumor cells directly or exert its antitumor effects by activating theimmune system, without influencing on normal cell functions simultaneously.However, the half-life of TNF-αis short. The low-dose TNF-αhas nosignificant antitumor effect but rather promote the cell proliferation in sometumors. Continuous injection of large doses of TNF-α for therapeuticpurposes would eventually cause fever, chills, fatigue, headache, shock andother side effects. Therefore, the clinical application of TNF-αin cancerpatients and its practical effects remains fairly questionable and very limited.TNF-α exerts its biological effects via two known transmembranereceptors, TNFR1(p55) and TNFR2(p75), both of which are composed ofsignal peptide, cytoplasmic domains, transmembrane region and extracellulardomains. The cytoplasmic domains of TNFR1and TNFR2are obviouslydifferent, indicating that TNFR1and TNFR2mediate different intacellularsignaling pathways. Functionally, TNFR1can mediate both apoptosis andnecrosis, and promote the proliferation of cells as well by activating NF-κ B.TNFR2also mediate two pathways. It can either promote the apoptosisthrough or independent of TNFR1, or promote the proliferation of cells. Theproliferation or apoptosis pathways mediated by TNFR are still not fullyunderstood. Previous studies have shown that TNFR levels of cell surface canalter the sensitivity of cells to TNF-α. The higher the TNFR expression, themore cytotoxic effect of TNF-α is noticed. Therefore, TNFRs play important roles in the regulation of TNF-αeffects.It has been reported that TNF-α has synergistic effects withchemotherapy drugs in induing cell death in many tumors, such as soft tissuesarcoma, melanoma, gastric cancer, lung cancer and some other malignancies.However, there have been no similar reports on head and neck cancers.Besides, the mechanism by which TNF-α and chemotherapy drug interactwith each other is not completely clear. In management of hypopharyngealcancer, how to reduce application dosage of TNF-α and chemotherapy drugsto avoid sides effects with simultaneous improvement of their treatmenteffects remains a core problem to solve. The expression levels of TNFRs oncertain tumor cells may determine the outcomes of above treatments.In this study, we used immunohistochemistry and Western blot to detectthe expression of TNFR1and TNFR2in HPSCC tissues and analyzed therelationship between TNFR expression and associated clinicopathologicfactors. Stable Tet-on-TNFR2Fadu cell lines were established by lentiviraltransfection. The proliferation-inhibiting and apoptosis-inducing effects ofTNF-α and cisplatin (DDP) in cells with different TNFR-expression levelswere investigated, for a deep insight into relationship between TNF-αandDDP resistance of hypopharyngeal cancer and the expression of TNFRs. Thepresent investigation is divided into three parts as follows.Part one: Expression of TNFR1and TNFR2and its clinocopathologicsignificance in HPSCCObjective: To investigate TNFR1and TNFR2expression and thus therelationship between TNFR expression and clinicopathologic features inHPSCC, for preliminary definition of their roles in tumorigenesis anddevelopment.Methods:1The expression levels of TNFR1and TNFR2were detected byimmunohistochemical staining in45HPSCC specimens and correspondingtumor-adjacent hypopharyngeal tissues, and their relationships withclinicopathologic characteristics were evaluated. 2The expression of TNFR1and TNFR2was also detected by WesternBlot in3HPSCC specimens and tumor-adjacent hypopharyngeal tissues.Results:1Immunohistochemistry analysis.1.1The expression of TNFR in HPSCC and paracancerous hypopharyngealmucosal tissues.Positive staining of both TNFR1and TNFR2was seen in all45HPSCCspecimens, and there was no significant difference of the expression levelsbetween TNFR1and TNFR2among the45HPSCC samples. However,expression of TNFR1and TNFR2was observed in50%and10%of the pairedtumor-adjacent hypopharyngeal tissues, respectively. Further statisticalanalyses showed that the average optical density (AOD) values of bothTNFR1and TNFR2in HPSCC were significantly higher than those in normalhypopharyngeal tissues (P<0.01). These data indicate that the expression ofboth TNFR1and TNFR2is upregulated in HPSCC.1.2Relationship between TNFR expression and clinicopathologic factors inHPSCC.The data revealed that both TNFR1and TNFR2expression had nothingto do with age (TNFR1, t=0.125，P=0.901. TNFR2, t=0.149, P=0.883), sex(TNFR1, t=0.060, P=0.952. TNFR2, t=0.959，P=0.343) and tumor location(TNFR1, F=0.169, P=0.845. TNFR2, F=1.888, P=0.164).However, TNFR1expression was significantly correlated withhistological grade (t=3.822，P＜0.01), tumor clinical stage (t=4.751，P＜0.01),T stage (t=5.087，P＜0.01), and lymph node metastasis (t=4.189，P＜0.01).We next analyzed the correlation of the expression status of TNFR2withclinicopathologic factors. Unlike TNFR1, TNFR2expression status was notsignificantly associated with any of the above mentioned clinicopathologicfactors in HPSCC(P＞0.05).2Correlation of TNFR1expression with TNFR2expression in HPSCC.The distinct correlation of the expression levels of TNFR1and TNFR2with the clinicopathologic factors suggests that there exist a correlation between the expression levels of TNFR1and TNFR2in HPSCC. Indeed, ourstatistical analyses demonstrated a reverse correlation between the expressionof TNFR1and TNFR2in HPSCC (rs=-0.305, P＜0.05). The ratio of TNFR1and TNFR2AOD was significantly correlated with histological grade, tumorclinical stage, T stage, and lymph node metastasis (all P＜0.01), and hadnothing to do with age, sex and tumor location(P＞0.05).3Western blotting showed that compared with tumor-adjacenthypopharyngeal tissues, the protein expression of TNFR1(t=5.079，P＜0.05)and TNFR2(t=17.333，P＜0.01) were both significantly increased in HPSCC.Conclusions: The expression of both TNFR1and TNFR2wassignificantly higher in HPSCC than in tumor-adjacent hypopharyngeal tissues.TNFR1expression and the ratio of TNFR1and TNFR2was significantlycorrelated with histological grade, tumor clinical stage, T stage, and lymphnode metastasis. But TNFR2expression status was not significantly associatedwith any of the clinicopathologic factors in HPSCC. There is a reversecorrelation between the expression of TNFR1and TNFR2in HPSCC. TNFRplay an important role in the occurrence and development of HPSCC.Part two: Establishment of a stable Tet-on-TNFR2system cell linemediated by lentivirus in FaDu cellObjective: To establish a stable Tet-on-TNFR2cell line transfected withlentivirus in FaDu cell, and to provide a cell line for further study on therelationship between TNFR expression and the sensitivity of TNF and DDP.Methods:1Construction of a Tet-on-TNFR2recombinant lentiviral vectorThe open reading frame (ORF) sequences of TNFR2gene was purchasedfrom Shanghai Hanbio Biological Company. Amplification of TNFR2ORFsequence was carried out through PCR. Then, the products and Tet-onlentiviral vector carriers were subjected to XhoI and EcoRI double enzymedigestion. The plasmid acquired from enzyme-linked products was trasformedin competent cells overnight, after which time the formed bacteria clones werepicked and identified by PCR. The PCR products were sent for sequencing tests for final confirmation.2Preparation before packaging plasmids2.1The lentivirus system contains lentiviral plasmid pHBTet-on-Puro-TNFR2(empty vector control pHBTet-on-Puro), and auxiliary packaging plasmidspspax2and pMD2G. Among them, pHBTet-on-Puro-TNFR2with Puromycinresistance and drug-induced UBC promoter can induce the TNFR2geneexpression by Doxorubicin(Dox).2.2The lentiviral plasmid (pHBTet-on-Puro-TNFR2) preparation plasmidpspax2and pMD2G were extracted without endotoxin to obtain the highpurity lentiviral vector and auxiliary packaging plasmids.3The lentivirus vectors pHBTet-on-Puro-TNFR2and lentivirus auxiliarypackaging plasmids were co-transfected into293T cells for72h. Then thesupernatant was collected, concentrated and resuspended with500ul DMEMfor acquisition of infection viral paticles.4FaDu cells were infected with lentivirus vectors. Puromycin was used toselect the stable Tet-on-Puro-TNFR2viral system infected cells for a week.5The stable Tet-on-Puro-TNFR2cells were detected by quantitativereal-time PCR (QPCR) and Western blotting.Results:1Though identification the recombinant plasmid sequence wasconsistent with the experimental design.2Stable Tet-on-Puro-TNFR2FaDu cell clones were selected by2.0μg/ml of puromycin.3QPCR showed that the mRNA level of TNFR2in Tet-on-Puro FaDucell without induction by Dox was0.126±0.046. The mRNA level of TNFR2in Tet-on-Puro FaDu cells induced by Dox for24h was0.215±0.124. Therewas no significant difference between the two groups (t=1.606, P＞0.05). ThemRNA level of TNFR2in stable Tet-on-Puro-TNFR2FaDu cells withoutinduction by Dox was0.149±0.056. Wheras, the mRNA level of TNFR2was3.518±0.733in the same cells induced by Dox for24h. The mRNA level ofTNFR2was significantly increased in stable Tet-on-Puro-TNFR2FaDu cell (t=8.125, P＜0.05) after Dox induction.4Western blot showed that TNFR2protein was significantly increasedin stable Tet-on-Puro-TNFR2FaDu cells stimulated with Dox for24h than innomal Fadu cells with or without Dox induction and Tet-on-Puro FaDu cellswith or without Dox stimulation.Conclusions:The stable Tet-on-Puro-TNFR2Fadu cell line was successfullyestablished by lentivirus transfection, which provides a new cell line forfurther investigation on the function of TNFRs.Part Three: Regulation of TNFR expression by Tet-on system to increasethe sensitization of FaDu cells to TNF and DDPObjective: To observe the proliferation and apoptosis rates induced bycisplatin (DDP) and TNF-ɑ in Fadu cells and changes in the proliferation andapoptosis rates after neutralizing the effect of TNFR1or up-regulating TNFR2expression by Tet-on system for definition of the relationship between theTNFR expression and sensitivity to TNF-ɑ and DDP.Methods:This part of investigation mainly foucused on evaluation of proliferationand apoptosis changes in Fadu cells when TNF-ɑ and DDP were applicatedunder different TNFR expression conditions. Inhibition of proliferation wasdetected by MTT method. The apoptosis rate was detected by flow cytometrythrough Annexin V/PI double staining.1The study of proliferation-inhibiting and apoptosis-inducing effects ofTNF-ɑ, DDP and their combinations in nomal TNFR-expressing Fadu cells.2The study of difference in proliferation inhibition and apoptosisinduced by TNF-ɑ, DDP or both combined in Fadu cells after neutralizingTNFR1.3The study of changes in proliferation and apoptosis rates induced byTNF-ɑ, DDP and both combined upon Fadu cells after upregulating theexpression of TNFR2by Tet-on systerm.4The study of changes in proliferation and apoptosis induced by TNF-ɑ, DDP and both combined after upregulating the expression of TNFR2by Tet-on systerm and neutralizing TNFR1with the blocking antibody.Results:1The results of MTT1.1TNF-ɑ can promote cell proliferation at low concentrations (0.1-10ng/ml)and can inhibit the tumor growth at high concentrations (50-100ng/ml) instable Tet-on-Puro Fadu cell (Group A). TNF-ɑ combined with DDP cansignificantly enhance the antitumor effect of DDP.1.2In stable Tet-on-Puro Fadu cells, neutralizing TNFR1with its blockingantibody (Group B) can reverse the proliferation-promoting effects oflow-dose (0.1-10ng/ml) TNF-ɑ prominently (t=4.390，t=5.886，t=5.258, all P<0.05); high-dose TNF-ɑ (50-100ng/ml) induced no significant difference inproliferation inhibition compared to Group A (t=2.657, t=1.083, t=1.113, allP>0.05). TNF-ɑ in low concentration combined with DDP can significantlyenhance the antitumor effect of DDP, but high concentration was nosignificant difference compared with Group A (P>0.05).1.3In stable Tet-on-Puro-TNFR2Fadu cells induced by Dox (Group C), lowdoses of TNF-ɑ can inhibit the proliferation of cells significantly compared toGroup A (P <0.01). The effects of high concentration TNF-ɑ (50-100ng/ml)were of no significant difference with Group A (P>0.05). In this group, theeffect of each concentration TNF-ɑ combined with DDP increased. The effectwas statistically difference (P <0.05).1.4In stable Tet-on-Puro-TNFR2expressing Fadu cells induced by Dox,with simultaneous neutralization of TNFR1(Group D), TNF-ɑ cansignificantly inhibit the proliferation of cells compared with Group A, GroupB (except TNF-ɑ0.1ng/ml) and C (except TNF-ɑ0.1ng/ml). The antitumoreffects of TNF-ɑ combined with DDP were significantly greater compared tothe three groups (all P＜0.05), except for that of TNF-ɑ at50-100ng/ml inGroup C.2The apoptosis results by Annexin V/PI double staining in flow cytometry2.1The apoptosis rate induced by10ng/ml TNF-ɑ revealed no significant difference (t=0.918，P＞0.05) between Group B and Group A. The apoptosisrate was obviously increased in Group C compared to Group A (t=13.504，P＜0.01). The apoptosis rate was significantly increased in Group D comparedto Group A and Group B (t=12.424，t=10.058，P＜0.05). But there was nosignificant change in apoptosis rates compared to Group C (t=0.624，P＞0.05).2.2The apoptosis rate induced by10ng/ml TNF-ɑ and6μg/ml DDP bearedno significant difference between Group B and Group A(all P＞0.05).Apoptosis rate in Group C increased significantly compared to Group A(t=8.498，P＜0.05). The apoptosis rate of Group D was significantly higherthan Group A and Group B (all P<0.05). There was no significant differencein apoptosis rate in comparison with Group C (t=0.795，P＞0.05).Conclusions:1In Fadu cells without changes in TNFR expression, TNF-ɑ inducescell proliferation at low concentrations (0.1-10ng/ml), while highconcentration TNF-ɑ (50-100ng/ml) exhibits effects of proliferation inhibitionon tumor cells. TNF-ɑ and DDP have a synergistic effect in inducingproliferation inhibition.2Neutralizatiion of TNFR1with its blocking antibody reversesproliferation-promoting effects of TNF-ɑ and can increase the antitumoreffects of TNF-ɑ combined with DDP at low concentrations, but does notincrease the apoptosis rate induced by TNF-ɑ and/or DDP in Fadu cells.3Upregulation of TNFR2in Fadu cells obviously promotes theproliferation inhibition induced by TNF-ɑ at low concentrations and TNF-ɑ ateach concentrations plus DDP, and can obviously promotes apoptosis inducedby TNF-ɑ and TNF-ɑ plus DDP.4Upregulation of TNFR2with simultaneous neutralization of TNFR1in Fadu cells can significantly increase the therapeutic effect of TNF-ɑ andDDP.5In hypopharyngeal carcinoma Fadu cells, it is highly likely thatTNFR1mediates proliferation pathway whereas TNFR2mediates apoptosispathway. Regulating the expression levels of TNFR1and TNFR2may shed light on improving the therapeutic effects of TNF-ɑ and DDP throughinterfering with the crosstalk between the two receptors.