The Expression Of Metallothionein 1H And Its Role In Drug Resistance In Non-small Cell Lung Cancer

Lung cancer is one of the most common malignant tumors in today's world andthe leading cause of cancer-related death. Non-small-cell lung cancer accounts for70-80ï¼…of lung cancer. Chemotherapy is one of the main treatments. Recent years,platinum-based adjuvant chemotherapy has improved effectively patients' outcomesbut the treatment often fails because of the development of drug resistance and thislimits the wide use of cisplatin. Accordingly, how to improve the sensitivity ofchemotherapeutic drugs, reduce side effects and improve the curative effect is one ofthe most key problems. The development of lung cancer is a complicated course withmulti-gene change. Genetic alterations precede morphological changes. Acoordingly,the identification of molecular biomarkers to differentiate tumor from normal cellsand predict tumor behavior and therapy targeting abnormal genes are of greatimportande in clinical practice. With the development of research on gene therapy,gene therapy in combination with chemotherapy is becoming one of comprehensivetreatments.Metallothioneins(MTs) are a group of ubiquitous low molecular mass,cysteine-rich intracellular metal-binding proteins. MTs are involved in trace metalhomeostasis and metabolism, the detoxification of toxic metals, scavenging of freeradicals. Recent years, the relationship between MT and tumors has become a focus of research. MTs are known to play putative roles in cancer cell proliferation, apoptosis,differentiation, drug resistance and prognosis. In human, MT genes are located onchromosome 16q13 and may involve at least 10 identified functional genes: MT2A,MT1A,MT1B,MT1E,MT1F,MT1G,MT1H,MT1X,MT3 and MT4. Researchsrevealed different MT genes in humans possibly play different functional roles duringdevelopment or under various physiological conditions. MT isoforms may haveunique functions.The specific functional roles of each of the MT isoforms are not precisely known.So far, there are some data about MT2A isoform but is a lack of data on the functionof MT1H. In the present study, expressions of MT protein and MT1H mRNA in 68cases of non-small cell lung carcinoma and 25 cases of lung benign disease aredetected by immunohistochemistry and semi-quantitative RT-PCR, respectively andthen the influences of exogenous MT1H on the cell biological behavior andchemosensitivity of A549 cells are investigated. SiRNA targeting MT1H is used todown-regulate MT1H expression and evaluate the role of MT1H in drug resistance ofA549/DDP cells. The expressions of MT1H in peripheral bloods of 44 cases withNSCLC before and after chemotherapy are investigated to explore the relationshipwith cisplatin resistance.Partâ… : Metallothionein 1H expressions in NSCLC andlung adenocarcinoma cell linesMethods1. The expressions of MT1H mRNA were detected in 68 cases of non-small cell lungcarcinoma, 25 cases of lung benign disease, A549 cells and A549/DDP cells bysemi-quantitative RT-PCR.2. The expressions of MT protein were detected in 68 cases of non-small cell lungcarcinoma and 25 cases of benign lung tissues by immunohistochemistry.3. Statistical analysis: All dates were analyzed for statistical significance usingChi-square by SPSS10.0 statistical package. The relation of two variables was analyzed by the pearson's correlation analysis,α=0.05 was considered statisticallysignificant.Results1. MT1H mRNA was expressed in 28 of 68 patients with NSCLC but not in benignlung tissues.2. The positive rate of MT protein in NSCLC (69.1%) was significantly higher thanthat in benign lung tissues (28.0%)(P＜0.05).3. The rate of MT1H mRNA in the histological grade 3 tumors (51.2%) wassignificantly higher than that in the histological grade 1 or 2 tumors (25.9%)(P＜0.05).But no significant correlation was found with patient's gender, age, histologic type,TNM staging and lymph node metastasis, respectively (P＞0.05).4. The rate of MT protein in the histological grade 3 tumors (85.4%) was significantlyhigher than that in the histological grade 1 or 2 tumors (44.4%)(P＜0.05). The rate ofMT protein in the groups with＞60(83.3%) was significantly higher than that in thegroups≤60 (57.9%)(P＜0.05). But no significant correlation was found with patient'sgender, histologic type, TNM staging and lymph node metastasis,respectively(P＞0.05).5. There was significant correlation between MT1H mRNA and MT protein (P＜0.05).6. RT-PCR analysis revealed that MT1H mRNA was highly expressed in A549/DDPbut not in A549 cells.Pattâ…¡: Expression of MT1H gene and effect on theproliferation, cell cycle and cisplatin-resistance in A549 cellsChapter 1: Construct of MT1H eukaryotic expression plasmid and itsexpression in A549 cellsMethods1. Target gene fragment was obtained by PCR amplification from plasmidpACT2-MT1H containing human MT1H cDNA and inserted into pcDNA3.1(-) eukaryotic expression vector to construct recombinant plasmid pcDNA3.1(-)-MT1H.2. The recombinant plasmid pcDNA3.1(-)-MT1H was stably transfected into A549cells having no basal expression of MT1H by lipofectamine 2000. The cells weretransfected with blank vector as control group. The positive monoclone was screenedby G418.3. RT-PCR and Western blot assays were used to identify the expression of MT1HmRNA and protein in A549, respctively.Results:1. About 192bp fragment was released while the MT1H eukaryotic expression vectorwas digested with XbaI and BamHI and was the same as what was expected.Recombinant plasmid was sequenced, and the result was the same as MT1H cDNAsequence published in Genbank. This confirmed that eukaryotic expression vectorpcDNA3.1(-)-MT1G was constructed successfully.2. RT-PCR and Western blot results showed that MT1H mRNA and MT1H fusedprotein were expressed in MT1H transfected group but weren't expressed inuntransfected and blank vector transfected groups. MT1H was stably expressed inA549 cells.Chapter 2: Effect of MT1H gene on the proliferation and cell cycle inA549 cellsMethods1. MTT assay was performed to detect the effect of MT1H on cell proliferationcapacity.2. Flow cytometry (FCM) was preformed to detect the effect of MT1H on cell cycle.3. RT-PCR was performed to detect the effect of MT1H on the expression ofCyclinD1.4. Statistical analysis: All dates were analyzed by SPSS10.0 statistical package. Themean of two groups uses student's t-test and the mean among more groups usesANVOA.α=0.05 was considered statistically significant. Results1. Proliferation capacity significantly enhanced in cells transfected with pcDNA3.1(-)-MT1H compared with the control groups. Significant difference was found fromthe 3th day (P＜0.05).2. FCM showed that cell number of G0/G1 phase obviously decreased while cellnumber of S phase increased in cells transfected with pcDNA3.1(-)-MT1H comparedwith the control groups. Significant difference was found (P＜0.05).3. The expressions of CyclinD1 mRNA were 0.27±0.047 and 0.52±0.060 in groupstransfected with blank vector and with pcDNA3.1(-)-MT1H respectively. Comparedwith control group, CyclinD1 expression was elevated significantly in the grouptransfected with pcDNA3.1(-)-MT1H(P＜0.05).Chapter 3: Effect of MT1H gene on drug resistance in A549 cellsMethods1. MTT assay was performed to detect the sensitivity to cisplatin of untransfectedgroup, group transfected with blank vector and group transfected with MT1H. IC₅₀values of cells to cisplatin were calculated at the same time.2. Three groups above were treated with 5μg/ml or 10μg/ml cisplatin for 24 h,respectively. The cells were stained with FITC-annexin V and PI and then cellapoptosis rates were analyzed by FCM.3. Statistical analysis: All dates were analyzed by SPSS10.0 statistical package. Themean of two groups uses student's t-test and the mean among more groups usesANVOA.α=0.05 was considered statistically significant.Results1. The viability of the cells transfected with MT1H was higher than that of controlgroups at 24 h after exposure to cisplatin. IC₅₀ values of untransfected group andgroups transfected with blank vector and with MT1H were 4.3±0.5,4.0±0.5,14.7±0.6ug/ml, respectively. IC₅₀ value of group transfected with MT1H was higher than thatof control groups (P＜0.05). 2. At 24 h after exposure to 10μg/ml cisplatin, the apoptosis rate of untransfectedgroup and group transfected with MT1H were 27.45±2.64%,12.71±0.61%,respectively. At 24 h after exposure to 5μg/ml cisplatin, the apoptosis rate ofuntransfected group and group transfected with MT1H were 12.95±1.64%,7.11±0.41%, respectively. Compared with control group, the apoptosis induced by cisplatin waslower in group transfected with MT1H (P＜0.05).Partâ…¢: Reversal of drug resistance in A549/DDP cells bydown-regulation of MT1H interfered with MT1H siRNAMethods1. A549/DDP cells were transfected with siRNA targeting MT1H as transfectiongroup and with control siRNA as control group.2. At 48 h after transfection, the mRNA and protein levels of MT1H were detected byRT-PCR and Dot blot, immunohistochemistry, respectively.3. A549/DDP cells were transfected with MT1H siRNA or with control siRNA for 24h and then treated with cisplatin of different concentration for 24 h, respectively. Cellviability was detected by MTT and IC₅₀ values of cells to cisplatin were calculated.4. Untransfected group, groups transfected with MT1H siRNA or with control siRNAwere treated with cisplatin for 24 h. The cells were stained with FITC-annexin V andPI and cell apoptosis rates were analyzed by FCM.5. Three groups above were treated with cisplatin for 24 h and then cell apoptosisrates were detected by FCM.6. The expressions of Bcl-2 and Bax proteins in the cells transfected with or withoutMT1H siRNA were detected by immunohistochemistry, respectively.7. Statistical analysis: All dates were analyzed by SPSS 10.0 statistical package. Themean of two groups uses student's t-test and the mean among more groups usesANVOA.α=0.05 was considered statistically significant.Results1. The mRNA and protein levels of MT1H were significantly down-regnlated at 48 h after transfection in MT1H siRNA group compared with the control group.2. The viability of the cells transfected with MT1H siRNA was lower than that ofcontrol groups at 24 h after exposure to cisplatin. IC₅₀ values of untransfected groupand groups transfected with control siRNA or with MT1H were 37.9±1.6,36.4±1.8,15.2±1.1μg/ml, respectively. IC₅₀ value of group transfected with MT1H siRNA waslower than that of control groups (P＜0.05). The relative reversal efficiency is 67.6%.3. Compared with untransfected group (5.95±0.51%) and group transfected withcontrol siRNA (7.80±0.46%), the apoptosis induced by cisplatin was elevated ingroup transfected with MT1H siRNA at 24 h after exposure to cisplatin.4. At 24 h after exposure to cisplatin, AI values of untransfected group, grouptransfected with control siRNA or with MT1H siRNA were 12.6±2.3%,16.3±2.6%,38.4±5.7%, respectively. The apoptosis rate of group transfected with MT1H siRNAwas higher than that of control groups (p＜0.05).5. The expressions of Bcl-2 in untransfected group and group transfected with MT1HsiRNA were 2.28±0.21,0.99±0.14 and the expressions of Bax were 1.12±0.17,1.35±0.13, respectively. Bcl-2 expression was greatly down-regulated but Bax hadlittle change after transfection of MT1H siRNA.Partâ…£: Relationship between the expression of MT1H mRNA inperipheral blood and cisplatin-resistance in NSCLCMethods1.46 advanced NSCLC patients pathologically confirmed were studied. All patientsreceived cisplatin-contained regimen chemotherapy as the first line treatment.Response was assessed after two cycles of chemotherapy. Peripheral blood sampleswere obtained from each patient before chemotherapy and after two cycles (before thethird cycle). The expressions of MT1H mRNA in bloods were detected by RT-PCR,and then the relationship between the expression of MT1H mRNA and response tochemotherapy was analyzed.2. Statistical analysis: All dates were analyzed for statistical significance using student's t-tset by SPSS10.0 statistical package,α=0.05 was considered statisticallysignificant.Results1. MT1H mRNA was expressed in all the samples. 26 of 46 patients were respondersto chemotherapy and 20 were non-responders. Median MT1H mRNA levels(relativetoβ-actin) were 0.48±0.27 and 0.43±0.17 in the responders and non-respondersbefore chemotherapy. There was no significant difference (P＞0.05). This suggestedMT1H was not associated with intrinsic cisplatin resistance.2. Median MT1H mRNA levels were 0.46±0.23, 0.56±0.22 in before and afterchemotherapy, respectively. There was significant difference between two groups(p＜0.05). MT1H mRNA levels were increased following chemotherapy. Furthermore,there was significant difference between in the responders but no significantdifference in the non-responders before and after chemotherapy. This suggestedMT1H was associated with acquired cisplatin resistance.Conclusions1. The expressions of MT protein and MT1H mRNA in NSCLC tissues and benignlung tissues have significant difference. MT protein expression in NSCLC isassociated with histological grade and patients age. MT1H mRNA expression isassociated with histological grade. The reveals MT and MT1H are involved in thecarcinogenesis and development of NSCLC.2. MT1H could promote proliferation of lung adenocareinoma cell A549 byup-regulating expression of CyclinD1 to facilitate cells into S phase.3. MT1H may promote drug resistance in A549 cells. The mechanism may be relatedto down-regulation of DDP-induced apoptosis.4. Down-regulation of MT1H interfered with MT1H siRNA could effectively reversethe drug resistance in A549/DDP cells by down-regulating the expression of Bcl-2and increasing DDP induced apoptosis in A549/DDP cells. The maybe a verypromising strategy for lung carcinoma therapy. 5. MT1H mRNA expression in peripheral blood is not associated with responsebefore chemotherapy. MT1H mRNA levels are elevated significantly afterchemotherapy and associated with response. This suggests MT1H is associated withacquired cisplatin-resistance but not intrinsic resistance in NSCLC.