转录因子CUTL1在胃癌多药耐药中的作用及机制研究
摘要
【背景】
胃癌是是最常见的恶性肿瘤之一，其死亡率在世界范围内位于恶性肿瘤的第二位。随着医学研究的不断进步和发展，胃癌的治疗方法和策略也得到不断完善，但化疗仍然是胃癌治疗的主要手段之一。而多药耐药（multidrug resistance, MDR）的出现，往往使得化疗失败，导致胃癌患者预后较差，5年生存率不足40%。因此，阐明胃癌多药耐药的机制势在必行。国内外学者，已经发现了大量的胃癌多药耐药相关基因和分子。我们实验室也建立了胃癌耐药细胞亚系，并应用抑制消减杂交、差异显示PCR、部分随机siRNA文库和miRNA芯片等策略，成功筛选并鉴定出一系列耐药相关的基因和miRNA。但由于胃癌的耐药机制非常复杂，这些结果尚不能完全阐明胃癌多药耐药的本质。因此，采用新策略继续深入研究胃癌多药耐药机制具有非常重要的意义。
转录因子是一类重要的基因调控分子，参与多种细胞生物学过程，如：细胞分化、增殖、凋亡、细胞周期进展和肿瘤发生发展等。近年来，转录因子在多药耐药中的作用逐渐得到公认。新近出现的转录因子活性谱芯片（oligonucleotide array-based transcription factor assay, OATFA），为体外研究转录因子DNA结合活性提供了新的思路和策略。我们课题组前期利用此芯片，在胃癌阿霉素耐药细胞SGC7901/ADR和亲本细胞SGC7901中筛选出差异表达的转录因子，其中CUTL1差异最明显，推测CUTL1有可能参与胃癌多药耐药。
CUTL1，属于转录因子同源结构域家族，其在进化上高度保守。CUTL1的表达水平和活性受到广泛的调节，包括转录起始的改变，蛋白酶解加工，磷酸化和乙酰化等，并且这些调节均通过对细胞周期的调控发挥作用。同时，CUTL1可以通过转录激活或转录抑制的方式，调节基因的表达，从而参与多种细胞生物学功能，如细胞分化、细胞增殖、细胞周期进展、细胞迁移和侵袭等。文献报道，CUTL1可以通过抑制凋亡而促进胰腺癌多药耐药的发生。因此，我们认为深入研究转录因子CUTL1在胃癌多药耐药中的作用和机制，具有非常重要的意义，有可能为临床逆转多药耐药提供新的思路和靶标。
【目的】
从多水平多角度研究转录因子CUTL1在胃癌多药耐药中的作用，并初步探讨其可能的分子机制。
【方法】
1、利用MTT方法检测脱药培养2周的胃癌阿霉素耐药细胞、给药培养的耐药细胞和亲本细胞对化疗药物的敏感性；2、PCR、Western blot和EMSA实验检测SGC7901/ADR细胞和SGC7901细胞中CUTL1的表达量和DNA结合活性；3、收集新鲜的胃癌活检组织标本，利用EMSA检测胃癌组织标本中CUTL1的结合活性，利用组织培养药物敏感性检测技术判断新鲜胃癌组织标本对化疗药物的敏感性，结合两者，从组织水平分析CUTL1活性与药物敏感性的相关性；4、扩增和鉴定CUTL1正义和反义载体，并分别感染和转染SGC7901/ADR和SGC7901细胞，通过RT-PCR和Westernblot检测感染和转染后细胞中CUTL1的表达量；5、MTT方法检测感染和转染后细胞对常见化疗药物的敏感性；6、通过流式细胞术分析CUTL1对胃癌细胞凋亡的影响；7、建立裸鼠成瘤模型，进一步在体内证实CUTL1对药物敏感性的影响；8、通过报告基因实验、qPCR、Western blot和MTT等方法对CUTL1的靶基因BMP6进行功能验证，以初步探讨CUTL1参与胃癌多药耐药的可能分子机制。
【结果】
1、转录因子CUTL1在胃癌耐药细胞中的表达量和活性降低
我们分别给药培养和脱药培养2周阿霉素耐药胃癌细胞SGC7901/ADR，然后利用MTT方法检测其对化疗药物的敏感性，结果显示，脱药培养2周后，SGC7901/ADR与给药培养的细胞对化疗药物的敏感性无明显差别，而二者均明显低于SGC7901的药物敏感性。
我们利用RT-PCR、Western blot和EMSA方法检测SGC7901/ADR和SGC7901细胞中CUTL1的表达量和表达活性，结果显示，SGC7901/ADR细胞中CUTL1的表达量和表达活性均明显低于SGC7901细胞，具有统计学意义（p<0.05）。
2、 CUTL1活性与胃癌组织的多药耐药性呈负相关
我们利用EMSA实验检测33例新鲜胃癌组织标本中CUTL1的活性，以滞后条带灰度值的平均值为准，其中16例被纳入CUTL1高活性组，17例纳入CUTL1低活性组。HDRA对33例胃癌组织标本对化疗药物的敏感性进行检测，以抑制率大于50%为标准，结果显示，17例标本对化疗药物呈内源性的耐药，而且其中13例的CUTL1活性较低，提示CUTL1高活性组中的抑制率明显高于CUTL1低活性组，p<0.05，具有统计学意义，可见CUTL1活性与胃癌组织的多药耐药性呈负相关。
3、CUTL1在胃癌细胞多药耐药中的作用
我们成功扩增了CUTL1正义和反义载体，并分别感染和转染SGC7901/ADR细胞和SGC7901/MKN45细胞，分别命名为：SGC7901/ADR-CUTL1，SGC7901/ADR-VC，SGC7901-shRNA和SGC7901-VC，MKN45-shRNA和MKN45-VC。RT-PCR、Westernblot结果均提示CUTL1在SGC7901/MKN45-shRNA中的表达明显低于SGC7901-VC，MTT结果提示SGC7901-shRNA对多种化疗药物的敏感性明显低于SGC7901-VC.CUTL1在SGC7901/ADR-CUTL1中的表达明显高于SGC7901/ADR-VC，其对药物的敏感性也高于SGC7901/ADR-VC。
AnnexinV/PI染色法检测细胞受药物诱导调亡的变化，发现上调CUTL1表达能够明显增加SGC7901/ADR细胞对ADR的药物敏感性,降低CUTL1表达能够明显降低SGC7901/MKN45细胞对ADR的药物敏感性。SGC7901/ADR-CUTL1成瘤后，给予化疗药物，肿瘤的抑制率明显高于SGC7901/ADR-VC组，而SGC7901-shRNA组的肿瘤抑制率低于SGC7901-VC。4.CUTL1可能通过BMP6参与胃癌多药耐药
CUTL1可以抑制BMP6的表达，并且CUTL1可以与BMP6启动子结合并抑制其转录。siRNA阻断BMP6的表达后，可以增强CUTL1呈低表达的胃癌耐药细胞对化疗药物的敏感性，部分逆转胃癌耐药细胞的多药耐药表型。因此，CUTL1可能通过调节BMP6而参与胃癌多药耐药。
【结论】
我们率先发现转录因子CUTL1在胃癌耐药细胞中表达量和DNA结合活性明显降低。并且，CUTL1的表达量和结合活性与胃癌组织和细胞对药物的敏感性呈正相关，上调CUTL1的表达可增加胃癌细胞对药物的敏感性，并逆转多药耐药表型，而下调CUTL1可以增加多药耐药性。且CUTL1可能通过调节BMP6而参与胃癌多药耐药。这一研究将为胃癌治疗提供新的思路和靶点。
【Backgroud】
Gastric cancer is one of the most common malignant cancers, and it is thesecond most common cause of cancer death worldwide. Although the strategiesand methods for the treatment of gastric cancer have been improved graduallyalong with the development of medicine, chemotherapy is still one of the mostimportant methods. Multidrug resistance hampered the clinical efficacy ofchemotherapy. So the outcome of patients with advanced gastric cancer is verypoor, and the survival rate for5years is less than40%. Therefore, it is crucial toclarify the mechamisms of multidrug resistance. A number of genes andmolecules related with MDR have been identified. In our previous studies, wesuccessfully screened and identified through suppression subtractivehybridization, differential display-PCR, partially random siRNA library andmicro RNA array. However, the precise mechaniams of MDR are stillunclarified because of its complex nature. So it is important to go on the studies of MDR through novel strategies.
Transcription factors (TFs) play important roles in multiple physiological andpathological processes, including cell differentiation, cell proliferation, cellapoptosis, carcinogenesis, and so on. In recent years, the role of transcriptionfactors in MDR has been accepted by researchers. Oligonucleotide Array-basedTranscription Factor Assay (OATFA) is a newly established and quite sensitivetechnology for the detection of TFs DNA binding activity in vitro, which couldallow simultaneously analyze the activity of multiple active TFs and thusfacilitate a high-throughput profiling. In our previous study, we screened andobtained differential expression TFs through OATFA, CUTL1showed dramaticchanges. So we supposed that CUTL1might be an important regulator in MDRin gastric cancer.
CUTL1, whose proteins are evolutionarily conserved, belongs to thehomeodomain transcrption factor family. The expression and activity of CUTL1are generally regulated, including the alteration of transcription initation,proteolytic processing, phosphorylation and acetylation, and so on. In addtion,the regulation of CUTL1was related with cell cycle progression. However,CUTL1could regulate the exprssion of downstream genes through acting astranscription suppressor or transcription activator. So CUTL1participats inmultiple biological process, such as: cell differentiation, cell proliferation, cellapoptosis, carcinogenesis, and so on. Recently, researched reported that CUTL1might mediated MDR by inhibiting cell apoptosis in pancreatic cancer.Therefore, it is significant that we investigate the role of CUTL1in MDR ingastric cancer, which might provide novel target for reversing the MDR ofgastric cancer.
【Objectives】
We evaluated the role of CUTL1in regulating drug resistance in gastriccancer in multi-levels, and we primarily investigated the possible mechamismsof CUTL1in MDR.
【Methods】
1. MTT assay was performed to detect the sensitivity of SGC7901/ADR-r cells(cultured withour drug for2weeks), SGC7901/ADR-a cells (cultured with ADR0.6μg/ml) and SGC7901cells.2. RT-PCR, Western blot and EMSA werecarried out to evaluate the expression and DNA binding activity of CUTL1inSGC7901/ADR and SGC7901cells.3. Fresh gastric cancer tissues werecollected. EMSA was performed to detect the activity of CUTL1in gastriccancer tissues, and histoculture drug response assay (HDRA) was carried out toevaluate the sensitivity of gastric cancer tissues to chemotherapeutic agents.Then analyze the correlation between the CUTL1activity and the drugsensitivity.4. CUTL1sense and shRNA vectors were amplified and identified.SGC7901/ADR cell were infected by CUTL1retrovirus vector, and SGC7901and MKN45cells were transfected by CUTL1shRNA vector, respectively.5.MTT was preformed to detect the sensitivity of infected and transfected cells tochemotherapeutic agents.6. Flow cytometry was carried out to evaluate theeffcts of CUTL1on cell apoptosis.7. Nude tumorigenesis model wereconstructed to evaluate in vivo CUTL1effects in relation to chemotherapy.8.Candidate target gene of CUTL1, BMP6was identified by repoter gene assay,Western blot, PCR, and MTT assay.
【Results】
1. The experssion and acitivity of CUTL1in SGC7901/ADR cells weredecreased.
we first characterized the drug resistance phenotype of SGC7901/ADR cells cultured under drug free conditions for two weeks. After two-week drug freeculture, SGC7901/ADR cells still retained resistance to ADR, at the similarlevel observed for those cells that were continually cultured with ADR (p>0.05),but in contrast to its parental SGC7901cells (p<0.05). We then tested the cellsfor drug resistance to these drugs after culturing cells under drug free condition,as we did for ADR. Similar results were found for these drugs.
RT-PCR and Western blot were carried out to study the expression of CUTL1in relation to drug-resistance in gastric cancer cells. Compared withdrug-sensitive SGC7901cells, the expression of CUTL1in SGC7901/ADR cellsshowed significant reduction (P<0.05). In agreement with PCR and Western blot,results from EMSA assay also indicated that CUTL1activity in SGC7901/ADRwas60%less than that in SGC7901cells (P<0.05). All together, these resultsdemonstrated that reduced CUTL1expression and activity is related to drugresistance.
2. CUTL1activity was inversely related to multiple drug-resistances incancer tissues
we interrogated the expression patterns of CUTL1in human gastric cancerlesions for evidence of human relevance, specifically drug sensitivity as criteriafor clinicopathological validation. To this end, we collected fresh tissuesspecimens from33patients with gastric cancer and performed EMSA andHDRA to measure CUTL1activity in relation to drug response. Based on themean value from EMSA,16samples came out with high CUTL1activity and17with low CUTL1activity. Clinicopathological parameters were well balanced inboth. No significant differences between two groups were observed in age,gender, tumor depth, TNM stage, or tumor differentiation. Histoculture drugresponse assay (HDRA) was applied to determine chemosensitivity of these clinical tissues. Chemosensitivity was regarded as positive when the inhibitionratio is50%or higher tested by HDRA. Among these33patients,17samplesshowed intrinsic drug resistance. Moreover,13samples from these groupdemonstrated lower CUTL1activity. Statistical analysis for the relation ofHDRA with EMSA demonstrated that reduced CUTL1activity is associatedwith drug resistance in cancer tissues.
3. Role of CUTL1related to drug resistance in gastric cancer cells
The expression of CUTL1in SGC7901/ADR-CUTL1cells was detected byRT-PCR and Western blot respectively. Significant increase of CUTL1expression at mRNA level and protein level was observed inSGC7901/ADR-CUTL1cells compared to SGC7901/ADR-VC cells. Next, weinvestigated the functional activity of CUTL1in relation to drug resistance. Thiswas done using MTT assay by measuring sensitivity of SGC7901/ADR-CUTL1cells or SGC7901/ADR-VC cells to multiple anticancer drugs including ADR,5-Fu, MMC and CDDP. The IC50of SGC7901/ADR-CUTL1cells was muchless than that of SGC7901/ADR-VC cells for chemotherapeutic agents. As aresult of increased sensitivity to chemotherapy drugs, the ratio of apoptoticSGC7901/ADR-CUTL1cells was significantly increased (>3folds) comparedwith that observed in SGC7901/ADR-VC cells. Moreover, the inhibition ratio oftumor size of SGC7901/ADR-CUTL1was higher than the group injected withSGC7901/ADR-VC cells. Overall, the results indicated that overexpressingactive CUTL1reversed the drug resistance, and thus increased the apoptosisinduced by these drugs. More importantly, the reversed drug resistance was notonly limited to ADR, but also to other structurally unrelated drugs, whichsuggested that CUTL1plays an important role in the occurrence of multipledrug resistance.
To further evaluate the functional importance of CUTL1in the regulation ofdrug resistance, a separate series of experiments was performed wherein shRNAmethodology was used to knock down the expression of CUTL1, and effectsupon drug-resistance were measured. Two CUTL1-specific shRNA vectors,namely CUTL1-shRNA1and CUTL1-shRNA2, were designed and constructedrespectively. Stable cell lines were established from parental SGC7901andMKN45cells and designated as SGC7901(or MKN45)-shRNA1, SGC7901(orMKN45)-shRNA2, SGC7901(or MKN45)-VC (vector control) respectively.RT-PCR and Western blot analysis were performed after CUTL1siRNAtargeting. As CUTL1-shRNA1more effectively downregulated the expressionof CUTL1in SGC7901and MKN45, SGC7901-shRNA1and MKN45-shRNA1cells were used in further cellular assays.
As anticipated, SGC7901-shRNA1cells showed decreased sensitivity to allabove anticancer drugs as indicated. Accordingly, the proportion of apoptoticcells in SGC7901-shRNA1cells was significantly decreased (>2folds)compared with that in SGC7901-VC cells, as demonstrated by flow cytometry.Similar results were obtained in MKN45-shRNA1cells. Moreover, theinhibition ratio of tumor size in the group injected with SGC7901-shRNA1cellswas significantly higher than that of the group injected with SGC7901-VCcells.Therefore, knock down of CUTL1resulted in a significant multiple drugresistance phenotypes. These findings strengthened our hypothesis that CUTL1is related to multiple drug resistance.
4. CUTL1might mediate MDR through regulating the expression ofBMP6in gastric cancer
CUTL1could suppress the experssion of BMP6at mRNA and protein level.And reporter gene assay showed CUTL1could bind with the promoter of BMP6, by which CUTL1would inhibit BMP6transcription. MTT assay revealed thatthe sensitivity of cancer cells with low CUTL1expression was significantlyincreased by konckdown of BMP6by siRNA. So these data demonstrated thatCUTL1might particiapte the MDR through regulating BMP6in gastric caner.However, there are still a lot of studies for us to perform.
【Conclusion】
We found the expresion and DNA binding activity of transcription factorCUTL1was significantly in gastric drug resistant cells. And the exprssion andactivity of CUTL1was inversely associated with the resistance both in gastriccancer cells and tissues. Overexprssion of CUTL1might increase the sensitivityof cancer cells to chemotherapeutic agents, and reverse the drug resistantphenotype. However, knockdown of CUTL1could increase the drug resistanceof cell. Moreover, CUTL1might mediate MDR through modulating theexpression of BMP6in gastric cancer.
胃癌是是最常见的恶性肿瘤之一，其死亡率在世界范围内位于恶性肿瘤的第二位。随着医学研究的不断进步和发展，胃癌的治疗方法和策略也得到不断完善，但化疗仍然是胃癌治疗的主要手段之一。而多药耐药（multidrug resistance, MDR）的出现，往往使得化疗失败，导致胃癌患者预后较差，5年生存率不足40%。因此，阐明胃癌多药耐药的机制势在必行。国内外学者，已经发现了大量的胃癌多药耐药相关基因和分子。我们实验室也建立了胃癌耐药细胞亚系，并应用抑制消减杂交、差异显示PCR、部分随机siRNA文库和miRNA芯片等策略，成功筛选并鉴定出一系列耐药相关的基因和miRNA。但由于胃癌的耐药机制非常复杂，这些结果尚不能完全阐明胃癌多药耐药的本质。因此，采用新策略继续深入研究胃癌多药耐药机制具有非常重要的意义。
转录因子是一类重要的基因调控分子，参与多种细胞生物学过程，如：细胞分化、增殖、凋亡、细胞周期进展和肿瘤发生发展等。近年来，转录因子在多药耐药中的作用逐渐得到公认。新近出现的转录因子活性谱芯片（oligonucleotide array-based transcription factor assay, OATFA），为体外研究转录因子DNA结合活性提供了新的思路和策略。我们课题组前期利用此芯片，在胃癌阿霉素耐药细胞SGC7901/ADR和亲本细胞SGC7901中筛选出差异表达的转录因子，其中CUTL1差异最明显，推测CUTL1有可能参与胃癌多药耐药。
CUTL1，属于转录因子同源结构域家族，其在进化上高度保守。CUTL1的表达水平和活性受到广泛的调节，包括转录起始的改变，蛋白酶解加工，磷酸化和乙酰化等，并且这些调节均通过对细胞周期的调控发挥作用。同时，CUTL1可以通过转录激活或转录抑制的方式，调节基因的表达，从而参与多种细胞生物学功能，如细胞分化、细胞增殖、细胞周期进展、细胞迁移和侵袭等。文献报道，CUTL1可以通过抑制凋亡而促进胰腺癌多药耐药的发生。因此，我们认为深入研究转录因子CUTL1在胃癌多药耐药中的作用和机制，具有非常重要的意义，有可能为临床逆转多药耐药提供新的思路和靶标。
【目的】
从多水平多角度研究转录因子CUTL1在胃癌多药耐药中的作用，并初步探讨其可能的分子机制。
【方法】
1、利用MTT方法检测脱药培养2周的胃癌阿霉素耐药细胞、给药培养的耐药细胞和亲本细胞对化疗药物的敏感性；2、PCR、Western blot和EMSA实验检测SGC7901/ADR细胞和SGC7901细胞中CUTL1的表达量和DNA结合活性；3、收集新鲜的胃癌活检组织标本，利用EMSA检测胃癌组织标本中CUTL1的结合活性，利用组织培养药物敏感性检测技术判断新鲜胃癌组织标本对化疗药物的敏感性，结合两者，从组织水平分析CUTL1活性与药物敏感性的相关性；4、扩增和鉴定CUTL1正义和反义载体，并分别感染和转染SGC7901/ADR和SGC7901细胞，通过RT-PCR和Westernblot检测感染和转染后细胞中CUTL1的表达量；5、MTT方法检测感染和转染后细胞对常见化疗药物的敏感性；6、通过流式细胞术分析CUTL1对胃癌细胞凋亡的影响；7、建立裸鼠成瘤模型，进一步在体内证实CUTL1对药物敏感性的影响；8、通过报告基因实验、qPCR、Western blot和MTT等方法对CUTL1的靶基因BMP6进行功能验证，以初步探讨CUTL1参与胃癌多药耐药的可能分子机制。
【结果】
1、转录因子CUTL1在胃癌耐药细胞中的表达量和活性降低
我们分别给药培养和脱药培养2周阿霉素耐药胃癌细胞SGC7901/ADR，然后利用MTT方法检测其对化疗药物的敏感性，结果显示，脱药培养2周后，SGC7901/ADR与给药培养的细胞对化疗药物的敏感性无明显差别，而二者均明显低于SGC7901的药物敏感性。
我们利用RT-PCR、Western blot和EMSA方法检测SGC7901/ADR和SGC7901细胞中CUTL1的表达量和表达活性，结果显示，SGC7901/ADR细胞中CUTL1的表达量和表达活性均明显低于SGC7901细胞，具有统计学意义（p<0.05）。
2、 CUTL1活性与胃癌组织的多药耐药性呈负相关
我们利用EMSA实验检测33例新鲜胃癌组织标本中CUTL1的活性，以滞后条带灰度值的平均值为准，其中16例被纳入CUTL1高活性组，17例纳入CUTL1低活性组。HDRA对33例胃癌组织标本对化疗药物的敏感性进行检测，以抑制率大于50%为标准，结果显示，17例标本对化疗药物呈内源性的耐药，而且其中13例的CUTL1活性较低，提示CUTL1高活性组中的抑制率明显高于CUTL1低活性组，p<0.05，具有统计学意义，可见CUTL1活性与胃癌组织的多药耐药性呈负相关。
3、CUTL1在胃癌细胞多药耐药中的作用
我们成功扩增了CUTL1正义和反义载体，并分别感染和转染SGC7901/ADR细胞和SGC7901/MKN45细胞，分别命名为：SGC7901/ADR-CUTL1，SGC7901/ADR-VC，SGC7901-shRNA和SGC7901-VC，MKN45-shRNA和MKN45-VC。RT-PCR、Westernblot结果均提示CUTL1在SGC7901/MKN45-shRNA中的表达明显低于SGC7901-VC，MTT结果提示SGC7901-shRNA对多种化疗药物的敏感性明显低于SGC7901-VC.CUTL1在SGC7901/ADR-CUTL1中的表达明显高于SGC7901/ADR-VC，其对药物的敏感性也高于SGC7901/ADR-VC。
AnnexinV/PI染色法检测细胞受药物诱导调亡的变化，发现上调CUTL1表达能够明显增加SGC7901/ADR细胞对ADR的药物敏感性,降低CUTL1表达能够明显降低SGC7901/MKN45细胞对ADR的药物敏感性。SGC7901/ADR-CUTL1成瘤后，给予化疗药物，肿瘤的抑制率明显高于SGC7901/ADR-VC组，而SGC7901-shRNA组的肿瘤抑制率低于SGC7901-VC。4.CUTL1可能通过BMP6参与胃癌多药耐药
CUTL1可以抑制BMP6的表达，并且CUTL1可以与BMP6启动子结合并抑制其转录。siRNA阻断BMP6的表达后，可以增强CUTL1呈低表达的胃癌耐药细胞对化疗药物的敏感性，部分逆转胃癌耐药细胞的多药耐药表型。因此，CUTL1可能通过调节BMP6而参与胃癌多药耐药。
【结论】
我们率先发现转录因子CUTL1在胃癌耐药细胞中表达量和DNA结合活性明显降低。并且，CUTL1的表达量和结合活性与胃癌组织和细胞对药物的敏感性呈正相关，上调CUTL1的表达可增加胃癌细胞对药物的敏感性，并逆转多药耐药表型，而下调CUTL1可以增加多药耐药性。且CUTL1可能通过调节BMP6而参与胃癌多药耐药。这一研究将为胃癌治疗提供新的思路和靶点。
【Backgroud】
Gastric cancer is one of the most common malignant cancers, and it is thesecond most common cause of cancer death worldwide. Although the strategiesand methods for the treatment of gastric cancer have been improved graduallyalong with the development of medicine, chemotherapy is still one of the mostimportant methods. Multidrug resistance hampered the clinical efficacy ofchemotherapy. So the outcome of patients with advanced gastric cancer is verypoor, and the survival rate for5years is less than40%. Therefore, it is crucial toclarify the mechamisms of multidrug resistance. A number of genes andmolecules related with MDR have been identified. In our previous studies, wesuccessfully screened and identified through suppression subtractivehybridization, differential display-PCR, partially random siRNA library andmicro RNA array. However, the precise mechaniams of MDR are stillunclarified because of its complex nature. So it is important to go on the studies of MDR through novel strategies.
Transcription factors (TFs) play important roles in multiple physiological andpathological processes, including cell differentiation, cell proliferation, cellapoptosis, carcinogenesis, and so on. In recent years, the role of transcriptionfactors in MDR has been accepted by researchers. Oligonucleotide Array-basedTranscription Factor Assay (OATFA) is a newly established and quite sensitivetechnology for the detection of TFs DNA binding activity in vitro, which couldallow simultaneously analyze the activity of multiple active TFs and thusfacilitate a high-throughput profiling. In our previous study, we screened andobtained differential expression TFs through OATFA, CUTL1showed dramaticchanges. So we supposed that CUTL1might be an important regulator in MDRin gastric cancer.
CUTL1, whose proteins are evolutionarily conserved, belongs to thehomeodomain transcrption factor family. The expression and activity of CUTL1are generally regulated, including the alteration of transcription initation,proteolytic processing, phosphorylation and acetylation, and so on. In addtion,the regulation of CUTL1was related with cell cycle progression. However,CUTL1could regulate the exprssion of downstream genes through acting astranscription suppressor or transcription activator. So CUTL1participats inmultiple biological process, such as: cell differentiation, cell proliferation, cellapoptosis, carcinogenesis, and so on. Recently, researched reported that CUTL1might mediated MDR by inhibiting cell apoptosis in pancreatic cancer.Therefore, it is significant that we investigate the role of CUTL1in MDR ingastric cancer, which might provide novel target for reversing the MDR ofgastric cancer.
【Objectives】
We evaluated the role of CUTL1in regulating drug resistance in gastriccancer in multi-levels, and we primarily investigated the possible mechamismsof CUTL1in MDR.
【Methods】
1. MTT assay was performed to detect the sensitivity of SGC7901/ADR-r cells(cultured withour drug for2weeks), SGC7901/ADR-a cells (cultured with ADR0.6μg/ml) and SGC7901cells.2. RT-PCR, Western blot and EMSA werecarried out to evaluate the expression and DNA binding activity of CUTL1inSGC7901/ADR and SGC7901cells.3. Fresh gastric cancer tissues werecollected. EMSA was performed to detect the activity of CUTL1in gastriccancer tissues, and histoculture drug response assay (HDRA) was carried out toevaluate the sensitivity of gastric cancer tissues to chemotherapeutic agents.Then analyze the correlation between the CUTL1activity and the drugsensitivity.4. CUTL1sense and shRNA vectors were amplified and identified.SGC7901/ADR cell were infected by CUTL1retrovirus vector, and SGC7901and MKN45cells were transfected by CUTL1shRNA vector, respectively.5.MTT was preformed to detect the sensitivity of infected and transfected cells tochemotherapeutic agents.6. Flow cytometry was carried out to evaluate theeffcts of CUTL1on cell apoptosis.7. Nude tumorigenesis model wereconstructed to evaluate in vivo CUTL1effects in relation to chemotherapy.8.Candidate target gene of CUTL1, BMP6was identified by repoter gene assay,Western blot, PCR, and MTT assay.
【Results】
1. The experssion and acitivity of CUTL1in SGC7901/ADR cells weredecreased.
we first characterized the drug resistance phenotype of SGC7901/ADR cells cultured under drug free conditions for two weeks. After two-week drug freeculture, SGC7901/ADR cells still retained resistance to ADR, at the similarlevel observed for those cells that were continually cultured with ADR (p>0.05),but in contrast to its parental SGC7901cells (p<0.05). We then tested the cellsfor drug resistance to these drugs after culturing cells under drug free condition,as we did for ADR. Similar results were found for these drugs.
RT-PCR and Western blot were carried out to study the expression of CUTL1in relation to drug-resistance in gastric cancer cells. Compared withdrug-sensitive SGC7901cells, the expression of CUTL1in SGC7901/ADR cellsshowed significant reduction (P<0.05). In agreement with PCR and Western blot,results from EMSA assay also indicated that CUTL1activity in SGC7901/ADRwas60%less than that in SGC7901cells (P<0.05). All together, these resultsdemonstrated that reduced CUTL1expression and activity is related to drugresistance.
2. CUTL1activity was inversely related to multiple drug-resistances incancer tissues
we interrogated the expression patterns of CUTL1in human gastric cancerlesions for evidence of human relevance, specifically drug sensitivity as criteriafor clinicopathological validation. To this end, we collected fresh tissuesspecimens from33patients with gastric cancer and performed EMSA andHDRA to measure CUTL1activity in relation to drug response. Based on themean value from EMSA,16samples came out with high CUTL1activity and17with low CUTL1activity. Clinicopathological parameters were well balanced inboth. No significant differences between two groups were observed in age,gender, tumor depth, TNM stage, or tumor differentiation. Histoculture drugresponse assay (HDRA) was applied to determine chemosensitivity of these clinical tissues. Chemosensitivity was regarded as positive when the inhibitionratio is50%or higher tested by HDRA. Among these33patients,17samplesshowed intrinsic drug resistance. Moreover,13samples from these groupdemonstrated lower CUTL1activity. Statistical analysis for the relation ofHDRA with EMSA demonstrated that reduced CUTL1activity is associatedwith drug resistance in cancer tissues.
3. Role of CUTL1related to drug resistance in gastric cancer cells
The expression of CUTL1in SGC7901/ADR-CUTL1cells was detected byRT-PCR and Western blot respectively. Significant increase of CUTL1expression at mRNA level and protein level was observed inSGC7901/ADR-CUTL1cells compared to SGC7901/ADR-VC cells. Next, weinvestigated the functional activity of CUTL1in relation to drug resistance. Thiswas done using MTT assay by measuring sensitivity of SGC7901/ADR-CUTL1cells or SGC7901/ADR-VC cells to multiple anticancer drugs including ADR,5-Fu, MMC and CDDP. The IC50of SGC7901/ADR-CUTL1cells was muchless than that of SGC7901/ADR-VC cells for chemotherapeutic agents. As aresult of increased sensitivity to chemotherapy drugs, the ratio of apoptoticSGC7901/ADR-CUTL1cells was significantly increased (>3folds) comparedwith that observed in SGC7901/ADR-VC cells. Moreover, the inhibition ratio oftumor size of SGC7901/ADR-CUTL1was higher than the group injected withSGC7901/ADR-VC cells. Overall, the results indicated that overexpressingactive CUTL1reversed the drug resistance, and thus increased the apoptosisinduced by these drugs. More importantly, the reversed drug resistance was notonly limited to ADR, but also to other structurally unrelated drugs, whichsuggested that CUTL1plays an important role in the occurrence of multipledrug resistance.
To further evaluate the functional importance of CUTL1in the regulation ofdrug resistance, a separate series of experiments was performed wherein shRNAmethodology was used to knock down the expression of CUTL1, and effectsupon drug-resistance were measured. Two CUTL1-specific shRNA vectors,namely CUTL1-shRNA1and CUTL1-shRNA2, were designed and constructedrespectively. Stable cell lines were established from parental SGC7901andMKN45cells and designated as SGC7901(or MKN45)-shRNA1, SGC7901(orMKN45)-shRNA2, SGC7901(or MKN45)-VC (vector control) respectively.RT-PCR and Western blot analysis were performed after CUTL1siRNAtargeting. As CUTL1-shRNA1more effectively downregulated the expressionof CUTL1in SGC7901and MKN45, SGC7901-shRNA1and MKN45-shRNA1cells were used in further cellular assays.
As anticipated, SGC7901-shRNA1cells showed decreased sensitivity to allabove anticancer drugs as indicated. Accordingly, the proportion of apoptoticcells in SGC7901-shRNA1cells was significantly decreased (>2folds)compared with that in SGC7901-VC cells, as demonstrated by flow cytometry.Similar results were obtained in MKN45-shRNA1cells. Moreover, theinhibition ratio of tumor size in the group injected with SGC7901-shRNA1cellswas significantly higher than that of the group injected with SGC7901-VCcells.Therefore, knock down of CUTL1resulted in a significant multiple drugresistance phenotypes. These findings strengthened our hypothesis that CUTL1is related to multiple drug resistance.
4. CUTL1might mediate MDR through regulating the expression ofBMP6in gastric cancer
CUTL1could suppress the experssion of BMP6at mRNA and protein level.And reporter gene assay showed CUTL1could bind with the promoter of BMP6, by which CUTL1would inhibit BMP6transcription. MTT assay revealed thatthe sensitivity of cancer cells with low CUTL1expression was significantlyincreased by konckdown of BMP6by siRNA. So these data demonstrated thatCUTL1might particiapte the MDR through regulating BMP6in gastric caner.However, there are still a lot of studies for us to perform.
【Conclusion】
We found the expresion and DNA binding activity of transcription factorCUTL1was significantly in gastric drug resistant cells. And the exprssion andactivity of CUTL1was inversely associated with the resistance both in gastriccancer cells and tissues. Overexprssion of CUTL1might increase the sensitivityof cancer cells to chemotherapeutic agents, and reverse the drug resistantphenotype. However, knockdown of CUTL1could increase the drug resistanceof cell. Moreover, CUTL1might mediate MDR through modulating theexpression of BMP6in gastric cancer.
引文
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