| Breast cancer is one of the leading causes of mortality among women throughout the world and it ranks second following lung cancer in cancer deaths.The breast cancer incidence has increased dramatically in china with 3%to 4%annual increments during the past two decades.While treatments commonly used in clinical such as surgery, chemotherapy,radiotherapy and other means provided a high survival rate for patients, but long-term chemotherapy which caused strong side effect,degrading the life quality of patients enormously.So taking a series of adjuvant treatment to reduce the drug use and increasing therapeutic effect is very important..Cyclosporin A(CsA) is a noncytotoxic immunosuppressant that was first discovered in 1970 and it initially was used for immunosuppression following organ and marrow transplantation.Subsequently,it has been applied in virtually all branches of medicine where autoimmune or inflammatory processes play a role in the pathology. However,recent studies have explained that CsA is also associated with cancer.CsA is a substrate and inhibitor of P-glycoprotein(P-gp).It has been used as one of the first-generation multidrug resistance(MDR) modulators to reverse MDR and improve chemotherapy.Combining with CsA increases the plasma concentration of Chemotherapy drugs and decrease the clearance of P-gp substrates,such as digoxin and etoposide.On the other hand,CsA is an inhibitor but not a substrate for breast cancer resistance protein(BCRP).Tumorigenesis is characterized by enhanced activities of glycolytic enzyme as well as distinct changes in the glycolytic isoenzyme.Tumor cells must occupy a high rate of nutrients and maintain a balance between the use of nutrients for energy production and for anabolic processes.In contrast,less nutrient uptake is required in most adult tissues and a greater fraction of available nutrients are used for energy production rather than macromolecule synthesis.This difference between metabolism of tumor cells and their normal counterparts was first pointed out by Warburg.The pyruvate kinase isoenzyme M2(PKM2) is one of the most important regulators of the balance between glycolytic energy regeneration(for example,ATP synthesis) and the synthesis of cell building blocks(for example,protein,lipid and nucleic acid synthesis) in tumor cells.There are four pyruvate kinase isoenzymes(M1,M2,L,and R) are known.PKM2 is the embryonic form which is progressively replaced by PKM1 in brain and muscle,PKL in kidney and liver,and PKR in erythrocytes during differentiation.When adult tissue cells in quiescence re-enter the cell cycle,the tissue-specific isoenzymes are replaced by PKM2.[7]In most,if not all,PKM2 in tumor cells is overexpressed.Knockdown of PKM2 and replacement of PKM2 by PKM1 were shown to reduce the Carcinogenicity of human tumor cell lines to form tumors in nude mouse xenografts.These findings are consistent with the idea that glucose is used for anabolic processes preferentially rather than oxidative phosphorylation in tumor cells,and that this metabolic switch may be required to support cell growth.Microarray studies have shown that pyruvate kinase which regulates the rate-limiting final step of glycolysis is one of the most upregulated gene sets in cancer.In this study,we first found that CsA can down-regulate the expression and activity of PKM2,and significantly inhibited the proliferation of the breast tumor cell and induced necrosis.We have built PKM2 expression plasmid and transferred the plasmid to tumor cells.Compared with the control group,over-expression of PKM2 resulted in a decline in sensitivity of CsA,indicating part of the mechanism relating to inhibition of CsA on tumor cell proliferation,suggesting that CsA has a potential application value in the tumor-assisted therapy.The main work can be divided into two parts:Partâ… :CsA inhibited breast tumor cell proliferation and induced cell necrosis CsA with different concentrations treated MCF-7,MDA-MB-435,MDA-MB-231 and normal mammary primary cells,M3TT assay the growth of these cells showed that CsA has a high inhibition on the growth of tumor cells than their normal breast cells. After treatment of MCF-7 cells with CsA,cycle G1 phase of MCF-7 increased significantly,while showed a reduction in access to S phase,and cell cycle arrested in G1 phase.The study showed cell division was inhibited due to the reduction of synthesis of biological macromolecules.We used Annexin-â…¤and PI respectively, marking breast cancer cells treated with CsA and analyzed cell death by flow cytometry, and found that CsA don't induce apoptosis,but directly induce tumor cell necrosis, and have a direct killing effect.Partâ…¡:CsA inhibit tumor cell growth by down-regulating the expression and activity of PKM2 of breast tumor cellWe used quantitative PCR technique to detect the difference of pyruvate kinase at the mRNA level between normal breast cells and breast tumor cells,and used western blot to detect differences of PKM2 at the protein level.The results showed that normal breast tissue cells have a smaller expression of PKM2 but a larger PKM1 expression, while tumor cells have a bigger expression of PKM2 but a smaller PKM1 expression.We used CsA with different concentrations to treat MCF-7,MDA-MB-435, MDA-MB-231 and normal mammary primary cells,then detected of PKM2 in mRNA level by quantitative PCR and detected PKM2 in protein level by western blot,the results confirmed that CsA can reduce the expression of PKM2.At the same time,using NP40 to lysis MCF-7 cells treated with CsA,then detecte the activity ofpyruvate kinase in cell lysate,which have come to the same result.Pyruvate kinase is a key enzyme in glycolysis process,which directly related to ATP generation,so we have adopted a luciferase fluorescence to assay the amount of ATP in cells treated with CsA,and found that ATP have a reduced yields in MCF-7 cells treated with CsA.In order to exclude that the reason of decrease ATP production is for impaired mitochondrial function,we used JC-1 to detecte mitochondrial membrane potential of MCF-7,the results showed that after treatment with CsA,mitochondrial function of MCF-7 cell is not impaired,indicating the reduction of ATP production in MCF-7 cells is caused by decreased activity of pyruvate kinase.In order to further confirm PKM2 play an important role in cell proliferation,we constructed a PKM2 eukaryotic expression vector PKM2-pEGFP.After the vector was transfected into the cells,these cells can express exogenous PKM2,we found that after transfection of PKM2 expression vector,inhibition of CsA on MCF-7 have reduced, which confirmed that CsA can reduce expression of PKM2 and inhibit proliferation of tumor cell..Conclusions1.PKM2 in breast tumor cells have a high expression,while in normal breast tissues have a low levels.2.Cyclosporin A can inhibit the proliferation of breast tumor cells and cause necrosis.3.Cyclosporin A can inhibit the expression of PKM2 and decrease activity of PKM2 in cells,leading to a lack of cellular ATP,which causes cell death.4.In vitro into PKM2 can reduce the damage of CsA to the cells,indicating that CsA may inhibit tumor proliferation by reducing the expression of PKM2.The above results show that CsA have a new mechanism for cancer treatment, suggesting that may have potential applications in the adjuvant treatment of cancer, which opening up new methods and new ways to provide a new thought for the treatment of malignant tumors. |
