The Over Expression Of Fatty Acid Synthase On Multiple Myeloma And It's Molecular Mechanism As A Therapeutic Target

Multiple myeloma is a B-cell malignancy of neoplastic plasma cells that generally produce a monoclonal immunoglobulin protein. The estimated incidence of multiple myeloma accounts for 10% of hematological malignancies and 1% of all malignancies. The median age of patients with multiple myeloma is 65 years. Intricate interactions with the marrow microenvironment,myeloma plasma cells receive critical survival signals that lead to this tumor resistant to chemotherapy. Standard therapy with melphan-prednisome or VAD has been pallliative. High-dose melphan followed by autologous stem cell transplant prolongs survival to a median of 4 to 5 years compared to 3 years with conventional chemotherapy. Thus there is a need for novel therapeutic approaches to treat this cancer.One therapeutic target that has not previously been considered for multiple myeloma treatment is the pathway of the endogenous synthesis of fatty acid. Fatty acid synthase (FAS) , the principle enzyme in the endogenous synthsis of fatty acid, is implicated in tumorigenesis through its role in cell proliferation and membrane lipid incorporation of neoplastic cells. Carcinoma cells are dependent on endogenous fatty acid synthesis for growth in vitro. FAS is highly expressed in a wide variety of human malignancies and has been associated with aggressiveness, however, normal tissues have low levels of FAS. The differential expression of FAS between normal tissues and cancer has led to the notion that FAS may be a target for anticancer therapy.To investigate the potential targeting of FAS for the treatment of multiple myeloma, we first determined whether FAS was overexpressed in human multiple myeloma . By immunohistochemistry ,we found 19 of 27 human multiple myeloma bone marrow samples to express significantly increased levels of FAS compared with iron deficiency anemia (IDA) bone marrow samples. We note the intensive brown staining of FAS located in the cytoplasm of MM tumor cells, whereas IDA patients' bone marrow cells have undetectablelevel of FAS. By RT-PCR , We initially assessed the presence of the mRNA encoding FAS in human myeloma cell lines (U266,RPMI8226 )and 22 of 27 multiple myeloma patients' bone marrow plasma cells compared with healthy donor PBMNCs. By Immunoblot analysis we determine the level of FAS protein expression, total proteins (100μg) were extracted from 27 multiple myeloma patients' bone marrow sample,U266,RPMI8226 and healthy donor PBMNCs. FAS protein was detected in 16 of 27 patients and myeloma cell lines but not detected in healthy donor PBMNCs.Encouraged by the findings that human multiple myeloma bone marrow samples and cell lines all overexpress FAS, we treated human multiple myeloma cell lines (U266, RPMI8226) in vitro with cerulenin , a native inhibitor of FAS. Cells proliferation was measured by methyl thiazolyl tetrazolium(MTT) analysis and apoptosis was evaluated by flow cytometry. After treating with cerulenin(the concentration from 5ug/ml to 640ug/ml) ,the proliferation inhibition rate of U266 cells was assayed by MTT analysis. Cell apoptosis and cycle distribution were evaluated by flow cytometer(FCM). When U266 cells were treated with 20ug/ml cerulenin for 12 h and 24h, the early apoptosis rate revealed by Annexin V/PI are 56.9% and 69.3% respectively, but the blank controls are 4.3% and 1.8% (p<0.01).The cells cycle DNA analysis show the cycle is blocked in S phase. Cerulenin-induced apoptosis was subsequently investigated by Annexin V-binding in cell line as well as in normal fibroblasts after treatment with 20ug/ml of cerulenin. U266 cells were highly sensitive to cerulenin treatment and exhibited more than 75% specific apoptosis after 12h of treatment. No apoptotic effects were observed in normal human fibroblasts. The extent of apoptosis was determined by cytofluorometry after staining with Annexin V FITC and propidium iodide. These results suggest that FAS may be an effective target for pharmacological therapy in human multiple myeloma.To investgate the expression of apoptosis related genes inducing by cerulenin on multiple myeloma cells and further explore it's molecular mechanism,We Analysis the expression changes of 96 apoptosis related genes by superArray cDNA on U266 cells which treated with cerulenin (20ug/ml) for 12 h.. Semi-quantitive RT-PCR was employed to confirm the representative expression changes genes, Rip2,Caspase9 and TRAF2.After treated with cerulenin for 12h,44 apoptosis related genes expression changed, 41 genesexpression increase over 2 fold times and 3 decrease over 2 fold times. Multi-apoptosis related gene families and multi-related genes have changed during the course. The expression of Caspase 9 increase markedly, it indicate that mitochondria pathway played the key position role in Cerulenin inducing apoptosis on U266 cells, otherwise, TRAF2 expression change suggest nuclear factor(NF) participate in Cerulenin inducing apoptosis on U266 cells. The death acceptor signaling pathway and the death acceptor non-dependence signaling pathway co-regulate in Cerulenin inducing apoptosis.To study the effect and mechanism of epigallocate-chin-3 gallate (EGCG,reported may be a natural inhibitor of fatty-acid synthase) on myeloma cell line U266. Proliferation inhibition rate of U266 cells was assayed by MTT after treating with EGCG (the concentration from 5ug/ml to 400 ug/ml).Apoptosis ,cell cycle and DNA content analysis by flow cytometer(FCM). Treat with 5ug/ml-400ug/ml of EGCG for 24h,the proliferation of U266 cells was obviously inhibited with dosage related effect. The inhibition rate of human skin fibroblast cells are all lower than 30%.When U266 cells are treated for 12h with 50ug/ml of EGCG,the lately stage apoptosis rate revealed by Annxin V/PI using FCM are 46,9%.Cell cycle DNA analysis show the cycle is blocked in S phase and the apoptosis peak occur in front of G1 phase. EGCG could induce U266 cells apoptosis and inhibit U266 cells proliferation. EGCG might be a new potential option for multiple myeloma therapy. To compare the expression of apoptosis related genes inducing by cerulenin with EGCG on multiple myeloma cells, we analysis the expression differences of 96 apoptosis related genes by superArray cDNA on U266 cells. The death acceptor signaling pathway and the death acceptor non- dependence signaling pathway co-regulate in Cerulenin and EGCG inducing apoptosis. Some differences exist in their signaling pathway. Conclution1 .By immunohistochemistry, RT-PCR and immunoblot analysis we find both FAS mRNA and protein was significantly overexpressed in majority of multiple myeloma patients and two myeloma cell lines.2. FAS inhibitor(cerulenin) could induce U266 cells apoptosis and inhibit U266 cells proliferation. FAS might be a new potential target for multiple myeloma therapy.3. The expression of Caspase 9 increase markedly, it indicate that mitochondria pathwayplayed the key role in Cerulenin inducing apoptosis on U266 cells, otherwise TRAF2 expression change suggest that nuclear factor(NF) participate in Cerulenin inducing apoptosis on U266 cells.4. EGCG could induce U266 cells apoptosis and inhibit U266 cells proliferation. EGCG might be a new potential option for multiple myeloma therapy.5. In conclution we believe that multiple myeloma should be included among tumors potentially sensitive to anti-FAS agents and as a potential therapeutic target.