缺氧诱导因子1α对口腔鳞状上皮癌细胞株接受放射和化学药物治疗感受性的影响
결양유도인자1α대구강린상상피암세포주접수방사화화학약물치료감수성적영향
Influence of hypoxia-inducible factor-1 alpha on the susceptibility of oral squamous cell carcinomas cell lines against chemotherapeutic drugs and radiation
  • 万方数据
    中国临床康复 중국림상강복
    CHINESE JOURNAL OF CLINICAL REHABILITATION
    2006年 47期 221-225 ,共5页

    周璿%陈世璋

    주선%진세장

    口腔%癌,鳞状细胞%放射治疗%化学疗法,辅助 口腔%癌,鱗狀細胞%放射治療%化學療法,輔助
    구강%암,린상세포%방사치료%화학요법,보조
    背景:缺氧诱导因子1α是一种转录因子,缺氧环境下对哺乳动物细胞起重要的调控作用.然而缺氧诱导因子1α对口腔鳞状上皮癌细胞株的放疗和化疗感受性的作用有待研究.目的:观察缺氧诱导因子1α对口腔鳞状上皮癌细胞株接受放射和化学药物治疗感受性的影响.设计:观察对比实验.单位:首都医科大学附属北京朝阳医院.材料:口腔鳞状上皮癌细胞株-2,口腔鳞状上皮癌细胞株-4,口腔鳞状上皮癌细胞株-5,和口腔鳞状上皮癌细胞株-6来自人口腔鳞状上皮癌细胞(由日本高知大学医学部口腔外科建株).方法:本实验于2004-09/2006-08于首都医科大学附属北京朝阳医院完成.将口腔鳞状上皮癌细胞株-2,口腔鳞状上皮癌细胞株-4,口腔鳞状上皮癌细胞株-5,和口腔鳞状上皮癌细胞株-6采用含小牛血清,左旋谷酰胺,青霉素,链霉素的改良DMEM培养液中培养.①从未处理和用30 Gyγ射线,100 μmol/L顺铂或100 μmol/L 5-氟脲嘧啶处理后的口腔鳞状上皮癌细胞株中提取细胞核蛋白.采用免疫印迹法检测缺氧诱导因子1α蛋白表达水平,同时采用定量PCR检测缺氧诱导因子1αmRNA的表达.②不同干预条件下细胞增殖抑制情况:将口腔鳞状上皮癌细胞株单细胞悬液以1×104/孔接种于96孔板中,每孔分别加入终浓度为100μmol/L的顺铂或5-氟尿嘧啶,或用30 Gyγ射线处理细胞,培养48 h后,采用用噻唑蓝比色法检测细胞增殖抑制情况.③不同干预条件下细胞凋亡分析:口腔鳞状上皮癌细胞株细胞用30 Gyγ射线,100 μmol/L顺铂或100 μmol/L 5-氟脲嘧啶处理24h后,用Propidium iodide和Annexin V-FITC染色,用流式细胞仪测定凋亡细胞数(仅分析γ射线,顺铂处理组).④质粒构建和转染:将人缺氧诱导因子1αcDNA克隆至pcDNA3.1/V5-His TOPO表达载体.用脂质转染法将缺氧诱导因子1α cDNA暂时转染口腔鳞状上皮癌细胞株-2细胞.将缺氧诱导因子1α siRNA(有义序列为5'CUGAUGACCAGCAACUUGAtt 3')暂时转染口腔鳞状上皮癌细胞株-5细胞,设立空白对照,同时给予细胞增殖抑制情况观察,以及细胞凋亡分析(仅分析顺铂处理组),方法同前.主要观察指标:①不同口腔鳞状上皮癌细胞株缺氧诱导因子1α的mRNA和蛋白表达情况.②不同干预条件下口腔鳞状上皮癌细胞株增殖抑制及细胞凋亡情况.③缺氧诱导因子1α过表达或缺氧诱导因子1α基因敲除的转染细胞的蛋白水平检测.④转染细胞和对照口腔鳞状上皮癌细胞株细胞增殖抑制情况和细胞凋亡分析结果.结果:①不同口腔鳞状上皮癌细胞株间缺氧诱导因子1α mRNA和蛋白表达结果:口腔鳞状上皮癌细胞株-2,口腔鳞状上皮癌细胞株-4,口腔鳞状上皮癌细胞株-5和口腔鳞状上皮癌细胞株-6细胞缺氧诱导因子1α mRNA的相对表达水平分别为1.0,2.2,4.3和4.0.缺氧诱导因子1α的细胞总蛋白和细胞核蛋白的表达在口腔鳞状上皮癌细胞株-2和口腔鳞状上皮癌细胞株-4细胞中较弱,而在口腔鳞状上皮癌细胞株-5和口腔鳞状上皮癌细胞株-6细胞中较强.②不同干预条件下口腔鳞状上皮癌细胞株细胞增殖抑制及被诱导凋亡情况:经γ射线,顺铂及5-氟脲嘧啶处理后,口腔鳞状上皮癌细胞株-2细胞数分别下降至(39.5±3.2)%,(39.2±1.2)%和(47.9±3.6)%,口腔鳞状上皮癌细胞株-4细胞数分别下降至(53.9±6.6)%,(54.3±1.4)%,(54.8+3.8)%.口腔鳞状上皮癌细胞株-5细胞数分别下降至(74.1±3.8)%,(76.5±9.1)%,(69.6±7.7)%,口腔鳞状上皮癌细胞株-6细胞数分别下降至(71.4±7.4)%,(84.4±8.8)%,(82.0±4.5)%.顺铂处理后,口腔鳞状上皮癌细胞株-2,口腔鳞状上皮癌细胞株-4,口腔鳞状上皮癌细胞株-5和口腔鳞状上皮癌细胞株-6细胞的凋亡细胞数分别为(50.9±1.3)%,(67.3±2.2)%,(12.2±0.8)%和(38.6±0.9)%.γ射线处理后,口腔鳞状上皮癌细胞株-2,口腔鳞状上皮癌细胞株-4,口腔鳞状上皮癌细胞株-5和口腔鳞状上皮癌细胞株-6细胞的凋亡细胞数分别为(21.2±1.1)%,(14.6±0.9)%,(9.7±1.0)%和(10.4±0.8)%.③不同干预条件下转染细胞增殖抑制及凋亡情况:经γ射线,顺铂及5-氟脲嘧啶处理后,转染了缺氧诱导因子1α表达载体的口腔鳞状上皮癌细胞株-2细胞数高于对照组(t=-4.693,-8.617,-6.721,P＜0.01);转染了缺氧诱导因子1α siRNA的口腔鳞状上皮癌细胞株-5细胞数低于对照组细胞(t=5.800,5.595,4.253,P＜0.05～0.01).用顺铂处理24 h后,转染缺氧诱导因子-1α的口腔鳞状上皮癌细胞株-2细胞凋亡数为(34.0±1.9)%,低于空白对照[(49.6±3.4)%,t=6.937,P＜0.01].转染缺氧诱导因子1α siRNA的口腔鳞状上皮癌细胞株-5细胞凋亡数为(27.7±2.3)%.高于空白对照[(11.4±2.1)%,t=-8.941,P＜0.01].结论:缺氧诱导因子1α表达与口腔鳞癌细胞对放、化疗感受性之间有负相关性.抑制癌细胞的缺氧诱导因子1α表达能增强癌细胞对放疗、化疗的感受性.
    배경:결양유도인자1α시일충전록인자,결양배경하대포유동물세포기중요적조공작용.연이결양유도인자1α대구강린상상피암세포주적방료화화료감수성적작용유대연구.목적:관찰결양유도인자1α대구강린상상피암세포주접수방사화화학약물치료감수성적영향.설계:관찰대비실험.단위:수도의과대학부속북경조양의원.재료:구강린상상피암세포주-2,구강린상상피암세포주-4,구강린상상피암세포주-5,화구강린상상피암세포주-6래자인구강린상상피암세포(유일본고지대학의학부구강외과건주).방법:본실험우2004-09/2006-08우수도의과대학부속북경조양의원완성.장구강린상상피암세포주-2,구강린상상피암세포주-4,구강린상상피암세포주-5,화구강린상상피암세포주-6채용함소우혈청,좌선곡선알,청매소,련매소적개량DMEM배양액중배양.①종미처리화용30 Gyγ사선,100 μmol/L순박혹100 μmol/L 5-불뇨밀정처리후적구강린상상피암세포주중제취세포핵단백.채용면역인적법검측결양유도인자1α단백표체수평,동시채용정량PCR검측결양유도인자1αmRNA적표체.②불동간예조건하세포증식억제정황:장구강린상상피암세포주단세포현액이1×104/공접충우96공판중,매공분별가입종농도위100μmol/L적순박혹5-불뇨밀정,혹용30 Gyγ사선처리세포,배양48 h후,채용용새서람비색법검측세포증식억제정황.③불동간예조건하세포조망분석:구강린상상피암세포주세포용30 Gyγ사선,100 μmol/L순박혹100 μmol/L 5-불뇨밀정처리24h후,용Propidium iodide화Annexin V-FITC염색,용류식세포의측정조망세포수(부분석γ사선,순박처리조).④질립구건화전염:장인결양유도인자1αcDNA극륭지pcDNA3.1/V5-His TOPO표체재체.용지질전염법장결양유도인자1α cDNA잠시전염구강린상상피암세포주-2세포.장결양유도인자1α siRNA(유의서렬위5'CUGAUGACCAGCAACUUGAtt 3')잠시전염구강린상상피암세포주-5세포,설립공백대조,동시급여세포증식억제정황관찰,이급세포조망분석(부분석순박처리조),방법동전.주요관찰지표:①불동구강린상상피암세포주결양유도인자1α적mRNA화단백표체정황.②불동간예조건하구강린상상피암세포주증식억제급세포조망정황.③결양유도인자1α과표체혹결양유도인자1α기인고제적전염세포적단백수평검측.④전염세포화대조구강린상상피암세포주세포증식억제정황화세포조망분석결과.결과:①불동구강린상상피암세포주간결양유도인자1α mRNA화단백표체결과:구강린상상피암세포주-2,구강린상상피암세포주-4,구강린상상피암세포주-5화구강린상상피암세포주-6세포결양유도인자1α mRNA적상대표체수평분별위1.0,2.2,4.3화4.0.결양유도인자1α적세포총단백화세포핵단백적표체재구강린상상피암세포주-2화구강린상상피암세포주-4세포중교약,이재구강린상상피암세포주-5화구강린상상피암세포주-6세포중교강.②불동간예조건하구강린상상피암세포주세포증식억제급피유도조망정황:경γ사선,순박급5-불뇨밀정처리후,구강린상상피암세포주-2세포수분별하강지(39.5±3.2)%,(39.2±1.2)%화(47.9±3.6)%,구강린상상피암세포주-4세포수분별하강지(53.9±6.6)%,(54.3±1.4)%,(54.8+3.8)%.구강린상상피암세포주-5세포수분별하강지(74.1±3.8)%,(76.5±9.1)%,(69.6±7.7)%,구강린상상피암세포주-6세포수분별하강지(71.4±7.4)%,(84.4±8.8)%,(82.0±4.5)%.순박처리후,구강린상상피암세포주-2,구강린상상피암세포주-4,구강린상상피암세포주-5화구강린상상피암세포주-6세포적조망세포수분별위(50.9±1.3)%,(67.3±2.2)%,(12.2±0.8)%화(38.6±0.9)%.γ사선처리후,구강린상상피암세포주-2,구강린상상피암세포주-4,구강린상상피암세포주-5화구강린상상피암세포주-6세포적조망세포수분별위(21.2±1.1)%,(14.6±0.9)%,(9.7±1.0)%화(10.4±0.8)%.③불동간예조건하전염세포증식억제급조망정황:경γ사선,순박급5-불뇨밀정처리후,전염료결양유도인자1α표체재체적구강린상상피암세포주-2세포수고우대조조(t=-4.693,-8.617,-6.721,P＜0.01);전염료결양유도인자1α siRNA적구강린상상피암세포주-5세포수저우대조조세포(t=5.800,5.595,4.253,P＜0.05～0.01).용순박처리24 h후,전염결양유도인자-1α적구강린상상피암세포주-2세포조망수위(34.0±1.9)%,저우공백대조[(49.6±3.4)%,t=6.937,P＜0.01].전염결양유도인자1α siRNA적구강린상상피암세포주-5세포조망수위(27.7±2.3)%.고우공백대조[(11.4±2.1)%,t=-8.941,P＜0.01].결론:결양유도인자1α표체여구강린암세포대방、화료감수성지간유부상관성.억제암세포적결양유도인자1α표체능증강암세포대방료、화료적감수성.
    BACKGROUND: The transcription factor, hypoxia-inducible factor-1al pha (HIF-1α), is the key regulator that controls the hypoxic response of mammalian cells. However, the role of HIF-1α in the therapeutic efficacy of chemo-radiotherapy in oral squamous cell carcinomas (OSC) is poorly understood. OBJECTIVE: To investigate the effect of HIF-α on the susceptibility of OSC cell lines against chemotherapeutic drugs and radiation. DESIGN: AN observational comparative experiment. SETTING: Beijing Chaoyang Hospital Affiliated to Capital Medical University. MATERIALS: OSC-2, OSC-4, OSC-5, and OSC-6 cell lines were estab lished from oral squamous cell carcinoma (The cell lines were from the De partment of Oral Oncology, Kochi Medical School, Japan). METHODS: The experiments were completed in Beijing Chaoyang Hospi tal affiliated to Capital Medical University from September 2004 to August 2006. The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing fetal bovine serum (FBS), L-glutamine, penicillin and streptomycin. ① Total cell lysates were extracted from untreated OSC cells and those treated with 30 Gy γ-rays, 100 μmol/L cis-dichlorodiamine plat inum (CDDP), or 100 μmol/L 5-fluorouracil (5-FU). The expressions of HIF-1α protein and mRNA were determined with western blotting analysis and real-time polymerase chain reaction (PCR). ② Inhibition of cell prolif eration after different interventions: OSC cells were seeded in 96-well mi croplates (1.0×104) and cultured for 48 hours after the irradiation with 30 Gy γ-rays or in the presence of 100 μmol/L CDDP or 100 mmol/L 5-FU, and then the inhibition of cell proliferation was detected with 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. ③ Apoptosis after different interventions: OSC cell lines were cultured for 24 hours after the irradiation with 30 Gy γ-rays or in the presence of 100 μmol/L CDDP or 100 μmol/L 5-FU. The cells were then stained with annexin V-FITC and propidium iodide, and the numbers of apoptotic cells were analyzed by a FACScan cytometer (only for the OSC cells treated with γ-rays and CDDP). ④ Plasmid construction and transfection: Human HIF 1α cDNA was cloned into the pcDNA3.1/V5-His TOPO expression vector. OSC-2 cells were temporarily transfected with HIF-1α cDNA. OSC-5 cells were temporarily transfected with HIF-1α siRNA (Sense sequence is 5' CUGAUGACCAGCAACUUGAtt 3'). The blank control group was also set, the inhibition of cell proliferation was observed, and the apoptosis was ana lyzed (only in the CDDP-treated group) by using the methods mentioned above. MAIN OUTCOME MEASURES: ① Expressions of HIF-1α protein and mRNA in different OSC cell lines; ② Inhibition of proliferation and the apoptosis of OSC cell lines after different interventions; ③ Overexpression and knockdown of HIF-1α by expression vector and siRNA; ④ Influence of overexpression and knockdown of HIF-1α on the susceptibility of OSC cell lines against γ-rays and chemotherapeutic drugs. RESULTS: ① Expressions of HIF-1α protein and mRNA in different OSC cell lines: The relative expression levels of HIF-1α mRNA were 1.0, 2.2, 4.3 and 4.0 in OSC-2, OSC-4, OSC-5 and OSC-6 cells, respectively. Simi larly, the expression of HIF-1α total cell lysates proteins and nuclear proteins were much lower in OSC-2 and OSC-4 cells than in OSC-5 and OSC6 cells. ② Inhibition of proliferation and the apoptosis in OSC cell lines after different interventions: After the treatment with g-rays, CDDP, and 5-FU, the cell numbers decreased obviously to (39.5±3.2)%, (39.2±1.2)%,and(47.9±3.6)% in OSC-2 cells, (53.9±6.6)%, (54.3±1.4)%, (54.8±3.8)%in OSC-4 cells. Those decreased to (74.1±3.8)%, (76.5±9.1)%, (69.6±7.7)%in OSC-5 cells and (71.4±7.4)%, (84.4±8.8)%, (82.0±4.5)% in OSC-6 cells.After the OSC cells were treated of with CDDP, the numbers of apoptotic cells were (50.9±1.3)%, (67.3±2.2)%, (12.2±0.8)% and (38.6±0.9)% in OSC-2, OSC-4, OSC-5 and OSC-6 cells. G-rays induced the apoptosis were (21.2±1.1)%, (14.6±0.9)%, (9.7±1.0)% and (10.4±0.8)% in OSC-2, OSC-4,OSC-5 and OSC-6 cells. ③ Inhibition of proliferation and the apoptosis in the transfected cell after different interventions: The cell numbers of HIF-1 α-transfected OSC-2 cells treated with γ-rays, CDDP and 5-FU were much more than those in the control group (t=-4.693 ,-8.617,-6.721, P ＜ 0.01),whereas the cell numbers of OSC-5 cells transfected with siRNA were obviously fewer than those in the control group (t=5.800, 5.595, 4.253, P ＜ 0.05-0.01). After the incubation of HIF-1α-transfected OSC-2 cells with CDDP for 24 hours, apoptosis was detected in (34.0±1.9)% of the cells, which was lower than that in the control group [(49.6±3.4)%, t=6.937, P ＜ 0.01]. After the incubation of HIF-1α siRNA transfected OSC-5 cells with CDDP for 24hours, apoptosis was detected in (27.7±2.3)% of the HIF-1α siRNA transfected OSC-5 cells, which was obviously higher than that in the blank control group [(11.4±2.1)%, t=-8.941, P ＜ 0.01].CONCLUSION: The expression level of HIF-1α correlates negatively with the susceptibility of OSC cells against chemotherapeutic drugs and radiation. The down-regulation of HIF-1α expression enhances the susceptibility of OSC cells against chemotherapeutic drugs and radiation. Therefore, the down-regulation of HIF-1α may be a potentially effective strategy for cancer treatment.
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