The Role And Mechanism Of FEN1 In Cisplatin Resistance And Sensitization Effect Of Curcumin In Breast Cancer

Background:Breast cancer is one of the most common cancers which threat the health of women.According to the statistics on cancer incidence and mortality in the United States in 2004,the most common incident form of cancer was breast(29% of all incident cases),followed by lung and colorectal cancer,and breast cancer has the second great mortality(15%).Cisplatin is the first generation of platinum drugs and also a kind of cell cycle spe cific drugs.The main target of cisplatin is nucleophilic DNA,protein and RNA in the cell.As a broad-spectrum anticancer drug for clinical treatment of tumor,cisplatin is powerful to various solid tumours including breast,ovarian,testicular cancer and lung cancer,etc.However,cisplatin resistance frequently occurs in patients and the mechanism is not very clear.One of the most important mechanisms is due to the enhanced DNA repair function which offsets the effect of cisplatin to inhibit DNA repair.Efficient DNA damage repair partly depends on the function of structure-specific nuclease family members,which can remove the damaged bases or nucleotides or various DNA intermediate structures.FEN1(Flap endonuclease 1)is an important member of these family which plays a key role in DNA replication and repair.In gastric cancer SGC-7901 cells,the down-regulation of FEN1 expression was reported to increase the sensitivity of cisplatin treatment.At the same time,FEN1 is highly expressed in breast cancer cells,overexpression of FEN1 may be associated with the mutidrug resistance in breast cancer.But the detail role and mechanism of FEN1 in cisplatin resistance remains unclear.Curcumin is a kind of polyphenols extracted from turmeric root which has b een found to process obvious biological functions including free radical scavenging,antioxidant,anticancer and lipid peroxidation inhibitory in various tumor researches.Curcumin also can be used as a tumor chemotherapy sensitization agent to induce apoptosis of breast cancer cells and inhibit the growth of tumor in vitro and in vivo.Our previous study demonstrated that curcumin inhibited cell proliferation through down-regulation of FEN1 expression in breast cancer cells.Based on these data,it is speculated that curcumin may enhance the sensitivity of breast cancer cells to cisplatin by down-regulation of FEN1 expression.In the study,three kinds of breast cell lines MCF-7,MCF-7DDP(cisplatin-resistant MCF-7),MDA-MB-231 and nude mouse xenograft model were used to research the role and mechanism of FEN1 in cisplatin resistance and sensitization effect of curcumin in breast cancer.Contents and methods:Part I The role of FEN1 in the sensitivity of breast cancer cells to cisplatin1.Effect of cisplatin on proliferation of breast cancer cells.2.Effect of cisplatin on FEN1 expression in breast cancer cells.3.Identification of phenotype and FEN1 expression of MCF-7 and MCF-7DDP cells.4.Changes of the sensitivity of MCF-7 cells to cisplatin after FEN1 is overexpressed.5.Changes of the sensitivity of MCF-7DDP cells to cisplatin after FEN1 expression is silenced.Part II Curcumin enhances the sensitivity of breast cancer cells to cisplatin by down-regulation of FEN1 expression1.Effect of curcumin on proliferation and FEN1 expression in breast cancer cells.2.Effect of cisplatin combined with curcumin on proliferation,apoptosis and FEN1 expression in breast cancer cells.3.Effect of cisplatin combined with curcumin on tumor growth and FEN1 expression in nude mouse xenograft model.Part III The molecular mechanism of curcumin to down-regulate expression of FEN11.Changes of ERK phosphorylation and FEN1 expression in MCF-7 and MCF-7DDP cells treated with cisplatin,ERK inhibitor U0126 alone or their combination.2.Changes of ERK phosphorylation and FEN1 expression in cells treated with cisplatin,curcumin alone or their combination.3.Hierarchy of FEN1 regulation in MCF-7 and MDA-MB-231 cells treated with cisplatin,curcumin alone or their combination.4.Changes of FEN1 expression induced by the ubiquitin-proteasome pathway in MDA-MB-231 cells treated with cisplatin combined with curcumin or MG132,a proteasome inhibitor.Results:Part I The role of FEN1 in the sensitivity of breast cancer cells to cisplatin1.Low concentrations of cisplatin had no effect on cell proliferation,how ever,high concentrations of cisplatin effectively inhibited the cell proliferation in a dose-dependent manner.2.FEN1 protein expression was up-regulated in a dose-dependent manner in cells treated with invalid inhibitory concentration or low concentration of cisplatin.3.Expression of FEN1 in MCF-7DDP cells was obviously higher than MCF-7 cells.4.Overexpression of FEN1 decreased the sensitivity of MCF-7 cells to cisplatin.5.Knock-down of Fen1 expression restored the sensitivity of MCF-7DDP cells to cisplatin.Part II Curcumin enhances the sensitivity of breast cancer cells to cisplatin by down-regulation of FEN1 expression1.Curcumin inhibited cell proliferation through down-regulated the expression of FEN1.2.Combination of cisplatin and curcumin inhibited cell proliferation,promoted apoptosis and enhanced the sensitivity of cisplatin in breast cancer cells through down-regulation of FEN1 expression.3.Combination of cisplatin and curcumin significantly inhibited tumor growt h and decreased FEN1 expression in a nude mouse xenograft model.Part III The molecular mechanism of curcumin to down-regulate expression of FEN11.The inhibition of phosphorylation of ERK was involved in curcumin enhanc ing the sensitivity of MCF-7 and MCF-7DDP cells to cisplatin through down-regulation of FEN1 expression.2.The increase of FEN1 ubiquitination was involved in curcumin enhancing the sensitivity of MDA-MB-231 cells to cisplatin through down-regulation of FEN1 expression.Conclusion:Overexpression of FEN1 is involved in cisplatin resistance in breast cancer cells.Curcumin enhances the sensitivity of breast cancer cells to cisplatin through down-regulation of FEN1 expression,which is associated with the inhibiti on of phosphorylation of ERK or the increase of ubiquitination of FEN1.Significance:Our current study suggests for the first time that overexpression of FEN1 play a key role in cisplatin therapy resistance and FEN1 gene may be a new target for curcumin to enhance the sensitivity of cisplatin.Our results provide a new mechanism for the research on cisplatin resistance and curcumin as a cisplatin sensitizing agent in breast cancer cells.At the same time,because a way to reduce FEN1 expression can enhanc e the sensitivity of breast cancer cells to cisplatin,our study may be useful to search for new strategies to enhance the sensitivity of breast cancer cells to clinical treatment with cisplatin.