| Head and neck squamous cell carcimoma (HNSCC) is a common malignant tumor in human body. It exhibits rapid growth and strong invasiveness. Combined radiotherapy and chemotherapy with operation has become a chief method and tendency for this disease. Management of HNSCC is a puzzled question in tumor therapy right along for its low sensitivity and strong resistance to radiotherapy and chemotherapy. Recently gene therapy brings new chance for tumor treatment. Gene therapy, with the breakthrough of insight into oncogenes and anti- oncogenes, has become one of the most fundamental and bright methods in tumor therapy although there is still a lot to do in recognizing its particular mechanism. Due to the advantages of gene therapy, which include uneasy to produce drug resistance, unlimited by tumor cell life cycle, strong specificity, excellent therapeutic effect for primary tumor and metastatic tumor and so on, investigation on this aspect and its correlated domains is becoming more and more attractive. The key point mentioned above is the target of study, therefore the probation of tumor signal transduction pathway is the basis for this purpose.Abnormality of cell signal transduction and regulation is intimately related to the pathogenesis and promotion of tumor. In some tumors, signal transduction pathways center on several determinate nuclear factors, and form the final switch of cell malignant transformation. Signal transducer and Activator of transcription 3(Stat3) signal pathway discovered recently is constantly activated and abnomally modulate cells in great quantities of human tumors. It plays an important role in the occurrence, development and evolution of human malignant tumor. Today, study on Stat3 is a hot spot in signal transduction pathway investigation.Up to now, it is unclear whether Stat3 tumor signal transduction pathway exists in laryngeal squamous carcinoma, and whether it participates in transformation and development of this disease. For approaching the molecule mechanism of Stat3 signal pathway in the occurrence, development and infiltration transfusion of laryngeal squamous carcinoma, and selecting ideal gene therapy target, Immunohistochemistry technique, Western Blotting, MTT and flow cytometry were used to investigated the activated state and clinical significance of Stat3 signal pathway in laryngeal squamous carcinoma tissue and Hep-2 cell line. This study was carried out to recognize the relationgship and mechanism of Stat3 in laryngocarcinoma biological behaviour and radiotherapy and chemotherapy resistance of laryngocarcinoma from different aspects, to search clinical significance of Stat3 protein expression level in laryngocarcinoma tissue, and to summarize whether Stat3 can be considered as tumor marker for early diagnosis, preoperative auxiliary diagnosis and pathogenetic condition monitoring.After Stat3 antisense oligonucleotides transfection into laryngocar- cinoma cell line, we studied the relationship between laryngocarcinoma cell multiplication level overtime and plasmid concentration, in attempt to know whether the expression and activation of Stat3 protein was down regulated when the laryngocarcinoma cell cycle was blocked. CyclinD1, CyclinE, CDK2, CDK4, CDK6 protein expression and G1/S cell ratio were determined, which can help to undermine the potential mechanism of Stat3 signal transduction pathway in controling the cancer cell transformation from G1 to S phase.We used interact technique to block activated Stat3 signal pathway and its impact on the hyperplasia and apoptosis of laryngeal quamous carcinoma cell. We also used this technique to research the possibility of reducing laryngocarcinoma chemotherapy resistance and enhancing its sensitivity to DDP. Therefore, the study of Stat3 signal transduction pathway and cell cycle regulation had important significance to oncogene Stat3 target therapy. It could offer theoretic and experimental evidence in discovering high-performance and low-toxicity anticancer drugs and designing original conjunctival therapeutic plan. Based on the completion of this study, we could further understand the mechanism of pathogenesis, infiltration and metastasis of laryngocarcinoma, reveal the therapy effect and mechanism of therapy targeting Stat3 signal transduction pathway. This might be helpful in enhancing combined therapeutic efficacy in HNSCC and in improving its prognosis. This experiment was separated into 3 parts:Part One The relationship between Stat3 constitutively activate and biological behaviour of laryngeal squamous carcinoma Objective: Study the expression and correlation of Stat3, p-Stat3, Cyclin Dl and Bcl-xl in laryngeal squamous carcinoma tissues and laryngeal squamous carcinoma cell lines, and observe the correlation of Stat3 constitutive activation and clinical pathology of laryngeal squamous carcinoma.Methods1 Immunohistochemistry SP technique was used for detecting the expression of Stat3 protein and phosphorylation Stat3 (P-Stats) protein in 50 laryngeal squamous carcinoma specimens, their corresponding para-cancer laryngeal mucosa tissues and Hep-2 cells.2 Western-blot technique was used for detecting the protein expression of Stat3, p-Stat3, and their downstream target gene product Cyclin Dl,Bcl-xl in 50 laryngeal squamous carcinoma tissues, their corresponding normal laryngeal mucosa tissues adjacent to cancers and Hep-2 cells.Results1 Constitutively activated Stat3 signal existed in laryngeal squamous carcinoma tissue1.1 Expressed signal of Stat3 protein was expressed in both cytoplasm and cell nucleus. Stat3 protein positive cells account for 72% tumor cells in laryngocarcinoma tissues and 44% in normal tissues, the former of which is obviously higher in laryngocarcinoma group than that in control group(P<0.05).1.2 P-Stat3 protein was phosphorylated Stat3 protein (activated Stat3 protein), its expression intensity represented extent of the activation of Stat3. Its positive staining mainly located in nucleus. P-Stat3 protein positive rate was 58% (29 samples) in laryngocarcinoma tissues and 0% in normal tissues. There was a significant difference between these two group (P=0.000).1.3 Based on Western-blot by quantification of Stat3 was 86.9738±1.58576 and 36.1033±0.52674 in cancer tissues and in normal tissues respectively. There exist significant difference between these two groups (P=0.000). The expression of p-Stat3 in cancer tissues was 56.9734±3.93939 and 16.5233±0.52913 in normal tissues, there is significant difference between these two groups (P=0.000). The expression of Cyclin Dl in cancer tissues was 24.4376±5.6231 and 5.9435±1.7529 in normal tissues, there is significant difference (P=0.0001) between them. The expression of Bcl-xl in cancer tissues was 33.7834±3.5673 and 14.6884±2.1485 in normal tissues, the difference between them was also very significant (P=0.0001). In laryngocarcinoma sample tissue, the protein expression level of Stat3, p-Stat3, Cyclin Dl and Bcl-xl were significantly higher than that in para-cancer mucosa. The expression of the above protein in tumor tissue was 2.4, 3.4, 5.1 and 2.3 times higher than that in para-tumor mucosa.2 The relationship between Stat3 expression and clinical pathologic characters of laryngeal squamous carcinoma2.1 The expression of Stat3 in laryngocarcinoma tissue did not correlate with the patients'age (P=0.716), sex (P=0.853), tumor position (P=0.725) and T-stage (P=0.751), but related to clinical stage (P=0.014), pathologic grade (P=0.032), lymph node metastasis (P=0.025). The lower the pathologic grade is the higher the Stat3 protein positive rate will be (P=0.0169). The advanced clinical stage is obviously consistent with high Stat3 protein positive rate (P=0.0056). The expression of Stat3 protein augmented in patients who suffered from lymph node metastasis (P=0.0083).2.2 The expression of p-Stat3 in laryngocarcinoma tissue had no relationship with the patients'age (P=0.875), sex (P=0.816), tumor position (P=0.797) and T-stage (P=0.670), but related to clinical stage (P=0.026), pathologic grade (P=0.019), lymph node metastasis (P=0.034). The low pathologic grade is associated with high p-Stat3 protein positive rate (P=0.0004). The late clinical stage is accompanied by high p-Stat3 protein positive rate (P=0.0208). The expression of p-Stat3 protein increased in the patients who suffered from lymph node metastasis (P=0.0144).2.3 The expression of Cyclin Dl in laryngocarcinoma tissue had no relationship with patients'age (P=0.875), sex (P=0.871), tumor position (P=0.889) and T-stage (P=0.612), but related to clinical stage (P=0.015), pathologic grade (P=0.027) and lymph node metastasis (P=0.034).2.4 The expression of Bcl-xl in laryngocarcinoma tissue had no relationship with the patients'age (P=0.873), sex (P=0.882), tumor position (P=0.923) and T-stage (P=0.686), but related to clinical stage (P=0.023), pathologic grade (P=0.038), lymph node metastasis (P=0.033).3 Pearson' s correlation analysis was used for analyzing the correlation and variability of expression of p-Stat3, Cyclin Dl and Bcl-xl in laryngocarcinoma tissue. The expression of p-Stat3 and Cyclin D1 exhibit linear correlation (r=0.437,P<0.05), but p-Stat3 and Bcl-xl did not.4 Constitutively activated Stat3 signal existed in Hep-2 cells Results from immunocytochemistry and Western Blotting showed that Stat3 and p-Stat3 protein were all expressed in Hep-2 cells. Stat3 protein mainly located in cytoplasm, scarcely in nucleus. P-Stat3 protein mainly located in nucleus, but compared with the expression of Stat3, p-Stat3 protein was less, and Stat3 signal pathway downstream target gene Cyclin Dl and Bcl-xl protein were all expressed in Hep-2 cells.This indicates that Stat3 signaling was extensively activated in Hep-2 cells.5 The correlation and variability of expression of Stat3, p-Stat3, Cyclin Dl and Bcl-xl in Hep-2 cells.Western Blotting showed the change of Cyclin Dl expression tendency was similar to that of Stat3, but Bcl-xl expression did not have obvious change. Part Two The regulation of Stat3 signal transduction pathway by G1 to S phase in laryngocarcinoma cellObjective: After Stat3 antisense oligonucleotides transfection into laryn- gocarcinoma cell line, we studied the relationship between the laryngocarcino- ma cell cycle blocked and the protein expression of Stat3, CDK and CDI. To undermine the potential mechanism of Stat3 signal transduction pathway in controling the cancer cell transformation from G1 to S phase.Methods1 Stat3 sense oligonucleotides (Stat3 S-ON) and Stat3 antisense oligonucleotides (Stat3 AS-ON) were packaged with lipids then transfected into Hep-2 cells. Cells transfected with lipids acted as non- transfection group cells (control group).2 MTT was used for detecting the hyperplasia condition in Stat3 AS-ON and Stat3 S-ON transfected group at different concentration (100,150,200,300,400 nmol/L) treated after 24h, 48h and 72h.3 Flow cytometry was used for detecting the cell cycle condition at final concentration (200nmol/L) of Stat3 AS-ON and Stat3 S-ON after 24h, 48h and 72h.4 Western Blotting was used for detecting the protein expression level of cell cyclin CyclinD1 and CyclinE, cyclin dependent kinase CDK2, CDK4 and CDK6 72h after Stat3 AS-ON transfection.Results1 The state of Hep-2 cell transfected with oligonucleotide: we observed different cells transfected with dose of plasmid under green fluorescence filter, lipidosome-oligonucleotide compound got into cytoplasm in 30min after transfection, 80%-90% cells were observed to show green fluorescence in cell nucleus after 1h, meanwhile strong green fluorescence was also seen in cytolymph. More than 90% cells were showed green fluorescence in cell nucleus after 2h.2. Stat3 signal transduction pathway contributes to the change of laryngocarcinoma cell from G1 phase to S phase. 2.1 The cell multiplication level decreased in Hep-2 cells transfected with Stat3 AS-ON, but in corresponding control group and Stat3 S-ON transfected group this change was not obvious.2.2 72h after Stat3 AS-ON transfection Hep-2 cells, the ratio of G1 phase cells rised from 55.7% to 62.9%, for S phase cells the ratio decreased from 33.6% to 15.9%. It indicated Stat3 AS-ON (200nmol/L) might inhibite translation from G1 phase to S phase.3 The regulatory effect of Stat3 signal transduction pathway to Cyclin and CDK in G1~S period laryngocarcinoma cells. 72h after Stat3 AS-ON transfected Hep-2 cell, CyclinD1 and CyclinE and cell cyclin dependent kinase CDK2, CDK4 and CDK6 all show regulation to different degree.4 The regulatory effect of Stat3 signal transduction pathway to cell cyclin dependent kinase inhibitor (CKI). 72h after Stat3 AS-ON transfection into Hep-2 cell, the expression of CKI p21 and p27 protein increased. Part Three Study of the molecular mechanism about Stat3 AS-ON combined chemotherapy in regulation of the hyperplasia and apoptosis of laryngeal carcinoma cellsObjective: After blocking activated Stat3 signal pathway and its impact on the hyperplasia and apoptosis of laryngeal quamous carcinoma cell with interact technique, we research the possibility of reducing laryngocarcinoma chemotherapy resistance and enhancing its sensitivity to DDP. Methods1 Stat3 sense oligonucleotides (Stat3 S-ON) and Stat3 antisense oligonucleotides (Stat3 AS-ON) were packaged with lipid body and transfected into laryngocarcinoma cell line Hep-2 cells. Experiments were divided into Stat3 S-ON group, Stat3 AS-ON group, DDP group and DDP+Stat3 AS-ON group. Group transfected with lipid body was taken as non-transfection control. Hep-2 cells added serum-free medium (SFM) acted as blank.2 The Hep-2 cells of both transfected group and control group were treated with diffenert concentration (100,150,200,300,400 nmol/L) of Stat3 AS-ON, Stat3 S-ON and DDP (the final mass concentration was 4μg/ml). Then we detected the hyperplasia condition at 0h, 24h, 48h and 72h by MTT.3 The Hep-2 cells in both transfected group and control group were treated with final concentration 200 nmol/L Stat3 AS-ON, Stat3 S-ON and final mass concentration 4μg/ml DDP. Then we detected the cell cycle change at 0h, 24h, 48h and 72h by flow cytometry.4 The Hep-2 cells in both transfected group and control group were treated by Stat3 AS-ON, Stat3 S-ON final concentration 200 nmol/L and DDP at final mass concentration 4μg/ml. Then we detected the cell apoptosis at 0h, 24h, 48h and 72h by flow cytometry.5 The Hep-2 cells in both transfected group and control group were treated by Stat3 AS-ON, Stat3 S-ON at final concentration 200 nmol/L and DDP at final mass concentration 4μg/ml for 72h. We detected the protein expression level of Stat3, P-Stat3 and its target gene product Bcl-xL, CyclinD1 by Western Blotting.Results1 Stat3 constantly expressed and phosphorylated in laryngocarcinoma cell line Hep-2 cells. Western Blotting showed that Stat3 protein constantly expressed and had some degree's phosphorylation.2 Stat3 AS-ON inhibited the hyperplasy activity of Hep-2 cells.2.1 MTT showed that control group and S-ON group could not inhibit the hyperplasy of tumor cells.2.2 Fourty-eight hours after transfection, the hyperplasy inhibition effect of became more stronger with the increasing concentration of AS-ON ranging from 0 to 200 nmol/L. The optimal concentration of 96 well plate in this experiment was 200nmol/L, but it could not completely inhibit the hyperplasy of tumor cells.2.3 Detected at different time after antisense transfection, antisensenucleic acids began to show some hyperplasy inhibition effect at 12th hour. The effect became visible at 36th hour. Cell survival rate constantly decreased (F=5.26, P=0.004).2.4 DDP group showed some proliferating inhibit effect.2.5 When Hep-2 cells were treated with Stat3 AS-ON and DDP, the hyperplasy level was decreased obviously.3 The block effect of Stat3 AS-ON on cell cycle of Hep-2 cell when Hep-2 cells were treated by Stat3 AS-ON and DDP for 72h, G1 phase cell ratio rised from 55.7% to 74.9%. Meanwhile S phase cell ratio decreased from 33.6% to 6.9%. S phase cell ratio in Stat3 S-ON group, lipid body group and control group were 28%~34%. 74.9% cells were blocked in G0/G1 phase in Stat3AS-ON+DDP group, it was higher than that in Stat3 S-ON group, lipid body group and control group.4 Stat3 AS-ON and DDP promoted the apoptosis of Hep-2 laryngeal carcinoma cells. By combined Stat3 AS-ON and DDP treatment on Hep-2 cells for 72h, the apoptosis rates in Stat3AS-ON+DDP group, DDP group, Stat3 AS-ON group, Stat3 S-ON group, lipid body group and control group were 32.9%, 13.5%, 28.1%, 3.18%, 2.35% and 1.78%, respectively. Statistical analysis results showed that apoptosis of Hep-2 cells increased after treated with antisense oligonucleotides. There was significant difference between Stat3 AS-ON+DDP group and Stat3 S-ON group (F=4.717, P=0.008), between Stat3 AS-ON+DDP group and lipid body group (F=5.554, P=0.007), and between Stat3 AS-ON+DDP group and control group (F=4.796, P=0.006).The differences among Stat3 S-ON group, lipid body group and control group had no statistical significance.5 Stat3 AS-ON and DDP could down regulate the activity and expression of Stat3 signal transduction pathway members. The expression and activity of Stat3, p-Stat3 protein and its target gene product Bcl-xL and CyclinD1 decreased after treat with Stat3 AS-ON and DDP in Hep-2 laryngeal carcinoma cells for 72h.Part Four The study of JAK inhibitor AG490 combined chemotherapy on inhibition of Stat3 signal transduction pathway in laryngeal carcinoma cells Objective: Treated with AG490(the inhibitor of JAK-STAT signaling transduction) and DDP in Hep-2 cell line, to study the effect of the inhibitor on signaling transduction and chemotherapy on tumour cells.Methods1 JAK inhibitor AG490 and DDP were respectively or combinedly used for treating laryngocarcinoma cell line Hep-2. The experiment was divided into AG490 group, DDP+AG490 group and DDP group. Hep-2 cells added to serum-free medium (SFM) acted as blank group.2 MTT was used for detecting the cell hyperplasy on the 0, 1, 2, 3 days after cells were treated with AG490 and/or DDP.3 Flow cytometry was used for detecting the cell cycly on the 0, 1, 2, 3 days after treating cells with AG490 and/or DDP.4 Flow cytometry was used for detecting the cell apoptosis on the 0, 1, 2, 3 days after treatment of cells with AG490 and/or DDP partly.5 Western Blotting was used for detecting the protein expression level of Stat3, P-Stat3 and its target gene product Bcl-xL, CyclinD1 after Hep-2 cells were treated with AG490 and/or DDP for 72h.Results1 Stat3 is constantly expressed and phosphorylated in laryngeal carcinoma cell line Hep-2 cells.2 AG490 and DDP inhibited the proliferation of Hep-2 cells. MTT showed that control group did not have any tumor inhibition effect. Cell proliferation was detected at different times after treatment with AG490. At 12th hour, inhibitory effects of proliferation began to appear. At 36th hour visible hyperplasy occurred with a constant decrease of cell viability (F=5.26, P=0.004). DDP group displayed some hyperplasy depressive effect. The cell hyperplasy level of Hep-2 cells treated with AG490 and DDP decreased obviously. The difference of cell proliferation level between AG490 + DDP group and DDP group was statistically significant (F=4.856, P=0.008).3 The inhibited effect of AG490 to the cell cycle of Hep-2 cells. 72h after treatment with AG490 and DDP, G1 phase cell rate rised from 55.5% to 70.7%, S phase cell rate decreased from 33.1% to 25.7%. Cell proliferation was inhibited. In AG490+DDP group, 70.7% cells were blocked at G0-G1 phase, which was higher than that of AG490, DDP and control group. Their difference had statistical significance.4 AG490 and DDP promoted the apoptosis of Hep-2 cells. AG490 and DDP were jointly used to treat on Hep-2 cells for 72h. The percentage of apoptotic cells rised from 2.34% to 31.32%. The apoptosis rate in AG490+DDP group, DDP group, AG490 group and control group was 31.32%,23.5%,14.1%,5.14%, respectively. Statistical analysis showed that apoptosis of Hep-2 cells increased after treated with AG490 and DDP. Comparing AG490+DDP group with control group, the difference between them had statistical significance (F=4.824, P=0.005).5 AG490 and DDP could breakdown the expression and activity of Stat3 signal transduction pathway members. The expression and activity of Stat3 protein and its target gene product Bcl-xL and CyclinD1 decreased after treated with AG490 and DDP for 72h in laryngocarcinoma cells.Conclusion1. For the first time we detected the activating status of Stat3 signal pathway in laryngeal squamous carcinoma tissue and cell line. We confirmed the existence of constitutively activated Stat3 signal pathway in laryngeal squamous carcinoma from different aspects, including protein level, location, gene level expression, and so on.2. The expression of Stat3 protein and p-Stat3 protein were different between laryngeal squamous carcinoma tissue and normal laryngeal mucosa tissue. The expression of p-Stat3 possesses heterology in nature. The abnormal activation of p-Stat3 in cancer cells is asynchronized. The expression of Stat3 and p-Stat3 protein was not related to the patients'age, sex, T-stage and tumor position, but related to cell differentiation level, clinical stage, and lymph node metastasis. The expression of Bcl-xl protein was associated with tumor differentiation level. In the process of the differentiation depression, Bcl-xl had a tendency to be down- regulated. It is suggested that activated Stat3 signal pathway plays important role in the occurrence, development, infiltration and metastasis in laryngeal squamous carcinoma.3. Antisense interaction technique was used for blocking the constitutive activation of Stat3 signal pathway with specificity and high efficientcy, inducing the apoptosis of tumor cells, blocking the cell cycle, and inhibiting the hyperplasy of laryngeal squamous carcinoma cells. All of these showed, Stat3 signal transduction pathway had important regulation effect on G1~S phase laryngocarcinoma cells. It controlled the abnormal hyperplasy of laryngeal carcinoma cells through dominating the balance between CDK/Cyclin compound and CKI members, and thus mediated cell malignant transformation. Activated Stat3 signal pathway was an ideal molecular target in gene therapy of laryngocarcinoma.4. When the Stat3 signaling pathway was blocked by JAK inhibitor, the expression of Stat3 downstream anti-apoptosis gene Bcl-xL and CyclinD1 was blocked with subsequent apoptosis of tumor cells. The expression of Bcl-xL protein gradually decreased after treated with AG490, resulting in obvious increase of apoptosis. These indicate that AG490 can promote apoptosis of Hep-2 cells which is related to JAK-STAT3-Bcl-xL pathway.5. Blocking constitutive activation of Stat3 signaling pathway, the survival and growth of canceration transformant cells displayed a irreversible dependence on Stat3 signal pathway. It revealed that drug resistance of laryngocarcinoma cells might be related to the abnormal activation of Stat3. AG490 could enhance the tumor cell sensitivity to chemotherapeutics. Both of these two approaches produced a cooperative anti-tumor effect.6. Antisense Stat3 could not completely inhibited the hyperplasy of tumor cells, and the apoptotic-inducing effect of Stat3 AS-ON was limited. We could not promote its killing effect only by raising the concentration of antisensenucleic acids unconditionally. Stat3 AS-ON could coordinate with therapeutical effect of DDP, which targeting indicates Stat3 interaction technique might become a new strategy of gene therapy in laryngocarcinoma.
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