Expression And Significance Of Key Enzymes Of Aerobic Glycolysis In Malignant Pleural Effusion And Plasma Of Lung Cancer

Background: Pleural effusion is a frequent clinical pleuroiasis,and manydiseases can cause pleural effusion,malignant diseases such as bronchogeniccarcinoma、breast cancer、malignant pleural mesothelioma and metastatic tumorof pleura.benign diseases such as tuberculous pleuritis、pneumonia、purulentpleurisy、heart failure、hypoproteinemia、rheumatism、rheumatoid and systemiclupus eythematosus, the main causes of pleural effusion are tuberculosis andprimary bronchial lung cancer.The median survival period of cancer patientswith malignant pleural effusion is3-12months.however benign pleural effusioncan be cured by antituberculosis、anti-infection、hormone、immunosuppressorand symptomatic treatment.Therefore,it is very important to differentiate benignand malignant pleural effusion for further treatment and prognosis. The mediansurvival of cancer with malignant pleural effusion is very short,so usingnoninvasive or minimally invasive test is very important.The sensitivity ofcytologic examination of pleural effusion is variable with limited sensitivity andnot predictive of prognosis.Recent studies have focused on the use of tumormarkers as a diagnostic tool for malignant pleural effusion. However,thesensitivity and specificity of currently available tumor markers are limited. Inthe1920s, German biochemist Otto Warburg first characterized aerobicglycolysis of cancer metabolism.That is, tumor cells prefer to metabolizeglucose to lactic acid and to obtain energy at a high rate even in the presence ofoxygen but not tricarboxylic acid cycle. The theory was named as Warburgeffect or aerobic glycolysis which is the most common metabolic hallmark ofmalignant tumors. Aerobic glycolysis is believed an important driver for cancercell growth、proliferation、malignancy、metastasis、survival and therapeutic resistance.Based on the Wauburg effect of tumor cells, we designed FDG-PET(fluorine deoxidization glucose positron emission tomography) which has beenapplied in clinical extensively. So far it is one of the best tests to diagnosetumor and guide treatment. Cancer cell acquire energy by aerobic glycolysis andthe three rate-limiting enzymes of aerobic glycolysis HK-2、PFK-1and PKM2are highly expressed in many cancers such as lung cancer、breast cancer、cervical cancer、liver cancer、malignant melanoma,etc.So the detections of HK-2、PFK-1and PKM2in pleural effusion and plasma may have adjuvant role indifferenciating benign and malignant pleural effusion and plasma. Many canersadopt aerobic glycolysis to obtain energy,it is necessary to establish a new cellline for tumor metabolic research.Objective: to evaluate the differential diagnostic value of measuringHK-2、PFK-1and PKM2in pleural effusions；to evaluate the early diagnosticvalue of measuring HK-2、PFK-1and PKM2in plasma；to establish a new humanlung adenocarcinoma cell line and offer an ideal experimental model in vitro forthe prevention and treatment of lung cancinoma in china.Methods and Results:Part1. Expression and significance of key enzymes of aerobic glycolysisin malignant pleural effusion of lung cancerall the pleural effusions in this study were collected and sorted into twogroups respectively by histopathological diagnosis and/or cytologic pathologydiagnosis of the hospitalization.One group is benign diagnosed tuberculosis、pneumonia、 heart failure and liver cirrhosis(n=34),the other is malignantdiagnosed lung cancer (n=46). HK-2、PFK-1and PKM2in pleural effusions weremeasured by ELISA respectively.1、The level of HK-2in malignant pleural effusions is2298.97±1918.62Mu/L,the benign group is1631.73±1340.58Mu/L.The difference has statisticalsignificances(U=436.0，W=1031.0，P＝0.001). HK-2is significantly higher inthe malignant pleural effusions group compared with the benign group.2、The level of PFK-1in malignant pleural effusions is4165.43±2714.07U/L,the benign group is3184.97±2233.96U/L.The difference has statisticalsignificances(U=463.0,W=1058.0，P＝0.002). PFK-1is significantly higher in the malignant pleural effusions group compared with the benign group.3、 The level of PKM2in malignant pleural effusions is36.93±28.98IU/mL,the benign group is23.11±18.12IU/mL.The difference has statisticalsignificance(U=461.0，W=1056.0，P＝0.002). PKM2is significantly higher inthe malignant pleural effusions group compared with the benign group.4、 The level of HK-2in lung adenocarcinoma pleural effusions is2257.89±1809.81Mu/L, in lung squamous carcinoma is3038.57±3158.19Mu/L,in small cell lung cancer is1586.65±347.73Mu/L.No significantdifferences among the three types of lung cancer were found in pleuraleffusions(P＞0.05).5、 The level of PFK-1in lung adenocarcinoma pleural effusions is4251.96±2846.75U/L, in lung squamous carcinoma is4134.15±2581.33U/L,insmall cell lung cancer is3121.55±842.03U/L.No significant differences amongthe three types of lung cancer were found in pleural effusions(P＞0.05).6、 The level of PKM2in lung adenocarcinoma pleural effusions is35.19±28.35IU/mL, in lung squamous carcinoma is58.21±35.69IU/mL,in smallcell lung cancer is23.41±4.27IU/mL.No significant differences among the threetypes of lung cancer were found in pleural effusions(P＞0.05).Part2. Expression and significance of key enzymes of aerobic glycolysisin plasma of lung cancerall the plasmas in this study were collected and sorted into two groupsrespectively by histopathological diagnosis and/or cytologic pathologydiagnosis of the hospitalization.Benign group diagnosed tuberculosis、pneumonia (n=29), and malignant group diagnosed lung cancer (n=37),Inmalignant group,23patients had pleural effusions and matched plasmas. HK-2、PFK-1and PKM2in plasmas were measured by ELISA respectively.1、The level of HK-2in plasmas of malignant group is2065.88±1331.54Mu/L,the benign group is1378.06±956.16Mu/L.The difference has statisticalsignificances(U=265.0，W=700.0，P＝0.000).HK-2is significantly higher in themalignant group compared with the benign group.2、 The level of PFK-1in plasmas of malignant group is3639.46±2063.83U/L,the benign group is2702.01±1897.96U/L.The difference has statistical significances(U=320.0， W=755.0， P＝0.005). PFK-1issignificantly higher in the malignant group compared with the benign group.3、 The level of PKM2in plasmas of malignant group is32.09±30.28IU/mL,the benign group is19.31±14.38IU/mL.The difference hasstatistical significances(U=299.0，W=734.0，P＝0.002). PKM2is significantlyhigher in the malignant group compared with the benign group.4、The level of HK-2in plasmas of lung adenocarcinoma is2026.89±1169.92Mu/L, in lung squamous carcinoma is2403.51±2090.48Mu/L,in small cell lungcancer is1628.09±550.83Mu/L.No significant differences among the three typesof lung cancer were found in plasmas(P＞0.05).5、 The level of PFK-1in plasmas of lung adenocarcinoma is4047.96±2245.85U/L, in lung squamous carcinoma is2422.69±926.03U/L,insmall cell lung cancer is2802.10±199.91U/L.No significant differences amongthe three types of lung cancer were found in plasmas(P＞0.05).6、The level of PKM2in plasmas of lung adenocarcinoma is34.16±30.90IU/mL, in lung squamous carcinoma is30.80±34.88IU/mL,in small cell lungcancer is16.53±3.54IU/mL.No significant differences among the three types oflung cancer were found in plasmas(P＞0.05).7、In malignant group,HK-2、PFK-1and PKM2of pleural effusions andmathched plasmas had not differences(P＞0.05）.Part3. cancer cell cultivation of malignant pleural effusions of lungcancerPleural effusions of lung cancer patients which were diagnosed byhistology and/or cytology were collected,then we isolated and cultivated cancercells in malignant pleural effusions.The tumor cells were identified byobservation of light microscopy、 cell HE staining、 cell growth curve、xenotransplantation experiment、tissue HE staining and karyotype analysis. Inthe observation of168hours,cancer cell still remained proliferated continuouslyand showed up the incubation phase and exponential growth phase,flat phase、degradation phase and decline phase were not appeared in7days.The cells wereseparated and cultivated growed stably in vitro.Its cell morphology and invasivegrowth indicated this cell line conformed malignant cell characteristics,nude mice that inoculated the neoplastic cells growed tumor nodules.the neoplasticcell morphology was similar to the patients’ pathological biopsy.Conclusions:1. Detection the contents of HK-2、PFK-1and PKM2in pleural effusionscould be useful as a complementary marker in the differential diagnosis ofmalignant pleural effusions from benign group.2. Detection the contents of HK-2、PFK-1and PKM2in plasmas could beuseful in the early diagnosis of tumor.3. The contents of HK-2、PFK-1and PKM2were not correlated withpathological type of lung cancer.4. In malignant group,HK-2、PFK-1and PKM2of pleural effusion andmatched plasma had not differences.5. H114cell line meets the standards of establishing cell lines,it’s a newhuman lung adenocarcinoma cell line.