| 【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 tissueswe 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 cellsThe 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 cancerCUTL1could 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. |
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