Function And Mechanism Of Drosophila Melanogaster Deoxyribonucleoside Kinase Reversing Gemcitabine Resistance In Breast Cancer Cells

Objectives: The number of new cancers and cancer-related death in China ranks first in the world,breast cancer has replaced lung cancer and became the world's most common cancer.As a water-soluble cytosine nucleoside derivative,gemcitabine(2',2'-difluorodeoxycytidine,d Fd C)is a cell cycle-specific anti-metabolic drug and one of the most primary chemotherapy drugs for breast cancer patients.It mainly inhibits the synthesis of tumor cell DNA.When its triphosphate binds to DNA,it prevents deoxynucleotides from synthesizing DNA,and thereby inhibits the DNA chain.Its function depends on deoxycytidine kinase(d CK)which is the phosphorylation rate-limiting enzyme.Both in vivo and in vitro experiments have confirmed that d CK activity is positively correlated with dFdC sensitivity.Multisubstrate deoxyribonucleoside kinase of Drosophila melanogaster(Dm-dNK)is a potential suicide gene,which acts on nucleic acid analogues together with the kinase in the cell to convert it into phosphorylation activation.Then phosphorylated nucleic acid analogues mix into DNA or RNA strands to prevent cell division and cause cell death.Compared to common deoxyribonucleotide kinases,Dm-dNK has wider substrate specificity and higher catalytic efficiency,and can effectively phosphorylate most natural deoxyribonucleotides and clinically commonly used nucleoside analogs.The preliminary studies used retrovirus,adenovirus or lentivirus as the carrier,carrying Dm-dNK in combination with a variety of precancerous drugs,and achieved obvious tumor killing effects in a variety of tumor cells.The purpose of this study was to study the potential reversal of gemcitabine resistance by Dm-dNK and explore its mechanism.Methods: In the first part of the study,we used the concentration gradient method to construct the gemcitabine-resistant breast cancer cells which were defined as MDA-MB-231 R,MCF-7R,and SKBR-3R.The MTT assay was used to observe the cell inhibition rate of each cell line.Each drug-resistant cell line was infected with the lentivirus carrying Dm-dNK.The MTT experiment was used to compare the cell viability after adding gemcitabine.In the second part of the study,we mutated the 247-position arginine of Dm-dNK to serine and defined it as Dm-dNKmut.After infection with lentivirus carrying wild-type or mutant Dm-dNK,RT-PCR and western blot were used to detect and evaluate Dm-dNK transcription and protein expression levels.DAPI staining was used to detect the intracellular localization of wild-type and mutant Dm-dNK,and the enzyme activity test was used to detect the enzyme activity of each group of cells.MTT experiment was used to compare the viability of MDA-MB-231 R infecting lentivirus with Dm-dNK or Dm-dNKmut.The drug resistance-related protein P-gp,CDA and d CK were compared in parental and drug-resistant cells.The flow cytometry technique compared the apoptosis rate of each group of MDA-MB-231 R infected with lentivirus,Dm-dNK or Dm-dNKmut.Western blot was used to compare the expression levels of P-gp protein before and after MDA-MB-231 R infection with Dm-dNK or Dm-dNKmut.In the third part,we proportionally mixed the Dm-dNK-positive MDA-MB-231 R cells and non-transfected cells.The MTT test was used to evaluate the bystander effect.The same method was used to evaluate the bystander effect of mutant Dm-dNK.The breast cancer drug-resistant cell MDA-MB-231 R was subcutaneously injected into the right axilla of each BALB/C nude mouse,and divided into 5 groups,Group 1: d Fd C;Group 2: Lv-Dm-dNK+PBS;Group 3: Lv-Dm-dNKmut+PBS;Group 4: Lv-Dm-dNK+d Fd C;Group 5: Lv-Dm-dNKmut+d Fd C.One-way analysis of variance was used to compare the tumor growth curves in each group.Results: In the first part of the study,we successfully constructed d Fd C-resistant breast cancer cell lines MDA-MB-231 R,MCF7R,and SKBR-3R through the low-concentration gradient increasing method.The sensitivity of the drug-resistant cell lines to d Fd C was significantly lower than that of the parental cells.After transfecting Dm-dNK-carrying lentivirus,the sensitivity to gemcitabine was significantly enhanced than that of the parental cells.In the second part of the study,the level of RNA transcription and protein translation together with enzyme activity were similar in MDA-MB-231 R cells transfected with Dm-dNK and Dm-dNKmut,but the cell location is different.The sensitivity to gemcitabine was greatly increased after infecting Dm-dNK or Dm-dNKmut,and there is no significant difference between Dm-dNK and Dm-dNKmut groups.Compared with the parental cells,the expression of P-gp and CDA in MDA-MB-231 R was significantly up-regulated,while the expression of d CK was lower than that in the parental cells.After infecting Dm-dNK wild or mutant type,the expression level of P-gp decreased,and the rate of apoptosis increased significantly.In the third part,we found that Dm-dNK and its mutants can also induce a strong bystander effect in MDA-MB-231 R cells.At the same time,it was verified in vivo that Dm-dNK wild and mutant type can also successfully reverse gemcitabine resistance.Conclusion: The method of increasing concentration gradient can be used to construct various types of breast cancer cell lines resistant to gemcitabine.Dm-dNK and Dm-dNKmut are expressed in the nucleus and cytoplasm respectively,and the enzyme activity and phosphorylation ability are equally present in drug-resistant cell lines.Dm-dNK and its mutant type can effectively reverse the resistance of breast cancer cells to gemcitabine by increasing the phosphorylation efficiency,increasing the rate of apoptosis,and reducing P-gp level.Dm-dNK and Dm-dNKmut can also exhibit significant bystander effect in MDA-MB-231 R,improving the reversing drug-resistance efficiency.In vivo experiments have shown that the group of Dm-dNK and Dm-dNKmut can significantly reduce the tumor volume combined with d Fd C compared with other groups.