| Identification of specific molecules involved in cancer and the development of inhibitors to block these molecules-targeted therapies-is an exciting and interesting area of current cancer research. polyunsaturated fatty acids (PUFAs) (such as, arachidonic acid, AA) and their metabolites (such as, prostaglandin E2, PGE2) were found in many disease, including cancer. However, how they participate in the cancer cells metabolism and What kind of role play in the metabolism is still not clear. In this study, cancer cell, breast cancer sample and transfer delta-6 dehydrogenase genes in mice model as research object, at the molecular level, cell and animal level, through observing the change of AA, AA metabolites PGE2 and AA synthesis of related desatuarase (D6D) and a series of gene related to the occurrence and the development of cancer, to explore whether D6D correlated with cancerogenesis. This will lay the foundation for further clinical studies in vivo and clinic study. Main research methods and the results are as follows:1. Effect of anti-cancer drug on PUFAs, their metabolites and related gene in cancer cellsMethods:The proliferation of cells was detected by XTT, and the profile of fatty acids was examined by Gas chromatography (GC). In addition, stearoyl-CoA desaturase-1 (SCD1), delta-5-desaturase (D5D), D6D and cyclooxygenase II (COX-2) mRNA expression were measured by the real-time reverse transcription. D5D and D6D protein expression were measured by Western Blotting.The PGE2 production was determined by enzyme-linked immunosorbent assay.Results:a. After being treated with CPT (0.0625-2μM), the Inhibiton ratio of cancer cells gradually increased (P<0.05 or P<0.01); After being treated with Ber (0.0625-2μM), the Inhibiton ratio of cancer cells gradually increased (P<0.05 or P<0.01).b. After being treated with Ber (8μg/ml or16μg/ml) and CPT (0.25μMor0.5μM), respectively, the percent of PUFAs in these five cancer cell lines change, but the common characteristics is:the chang of AA is significant in all cancer cell lines (P<0.05).c. Compared to control group, the expression of D5D and D6D mRNA were significantly increased in Ber and CPT treated group (P<0.05 or P<0.01); CPT treated in HT-29 cell group, the expression of SCD1 mRNA were significantly increased (P<0.01); CPT treated in B16 cell group, the expression of SCD1 mRNA were significantly decreased (P<0.05); Ber or CPT treated in other cell groups, the expression of SCD1 mRNA were not changed; Ber and CPT treated in MDA-MB-231, MCF-7 and ZR-75-1 cell group, the expression of COX-2 mRNA were significantly increased (P<0.01); Ber and CPT treated in HT-29 and B16 cell group, the expression of SCD1 mRNA were significantly decreased (P<0.05 or P<0.01).d. Ber or CPT treated in all cell groups, the expression of D5D and D6D protein were obversely increased.e. Ber or CPT treated in other four cell groups, the product of PGE2 were raised except for B16 cell.Conclusions:the improvement of expression of D5D and D6D induced the increase of AA, and the product of PGE2 were obversely raised; the reduce of expression of D5D and D6D induced the decrease of AA, and the product of PGE2 were obversely reduced. This suggests D5D and D6D can be as the biological targets of the found and treatment of cancer biology.2. Change of D6D in human Breast cancertissueMethods:the profile of fatty acids was examined by GC. the activity of D6D was measured by the ratio of LA/AA. D6D mRNA expression were measured by the real-time reverse transcription. D5D and D6D protein expression were measured by Western Blotting. The PGE2 production was determined by enzyme-linked immunosorbent assay.Results:a. The conversion of LA and ALA, as indicated by the ratio of LA/AA and ALA/EPA, were significantly enhanced in tumor tissues comparing to normal and adjacent tissues, suggest the acivity of D6D was significantly improved.b. Expression of D6D mRNA was up-regulated by 64 folds and 50 folds in human breast cancer comparing to normal and adjacent tissues; Expression of D6D protein was up-regulated in human breast cancer comparing to normal and adjacent tissues; Expression of D6D mRNA and protein were changed in normal tissues comparing to adjacent tissues.c. The product of PGE2 was significantly raised in human breast cancer comparing to normal and adjacent tissues; The product of PGE2 was changed in normal tissues comparing to adjacent tissues.Conclusions:State the up-regulation of activity and expression of D6D induced the increase of AA, at the same time, the product of PGE2 was also obversely improved, suggest D6D can be as the biology target of treating cancer.3. Role of D6D in vitro and in melanoma growth in vivoMethods:B16 cell treated with growth factors, including epidermal growth factor (EGF), fibroblast growth factor (FGF), et al, Oxidation reaction type (H2O2), radiation (UV light and X-array), Acidic conditions (pH 6.5) and hypoxia conditions. After B16 cell was treated by these factors, the profile of fatty acids was examined by GC, the activity of D6D was measured by the ratio of LA/AA, D6D and PPARa mRNA expression were measured by the real-time reverse transcription, D6D protein expression were measured by Western Blotting. Inoculate Cancer cells into mouse, tumor growth will be monitored everyday by measuring the length (L) and width (w) of the tumor with a caliper and calculating tumor volume on the basis of the following formula:volume= (1/2)Lw2, to assess he incidence of tumor formation and tumor growth rate. cancer-related genes were analysed by PCR array. The PGE2 production was determined by ELISA,Results:a. The activity of D6D is significantly higher in growth and death-challenging factors than in control, and the activity of D6D is increased.b. The expression of D6D mRNA was up-regulated by growth and death-challenging factors, The expression of D6D protein was up-regulated by growth and death-challenging factors except for Acidic conditions (pH 6.5).c. PPAR-a was significantly up-regulated in B16 cells by both growth and death-challenging factors, However, the regulation of PPAR-a mRNA expression by individual factors was not proportional to the corresponding levels of D6D expression. d. Over-expression of D6D promoted B16 cell growth while knock-down of D6D inhibited B16 cell growth in vitro.e. Over-expression of D6D promoted B16 cell migration while knock-down of D6D inhibited B16 cell migration in vitro.f. Over-expression of D6D promoted B16 melanoma growth in vivo.g. AA and PGE2 was higher in D6D-over B16 melanoma than in Vector-control.h. Expression of several genes that are involved in tumorigenesis, such as anti-apoptotic genes (bax, bcl-2, bcl-211 and survivin), oncogenes (brcal,fos,jun, met and myc), transcriptional factors (nfkbland nfkbia), and angiogenesis related genes (pdgfa, pdgfb, vegfa, vegfb and vegfc) were up-regulated, while the cancer-suppressor gene pten was down-regulated by 64 folds.Conclusions:Testify AA, D6D and PGE2 Closely related with cancer, and D6D can be used as a new anti-cancer targets.In summary, the study suggest PUFAs involve in the cancerogenesis and the up-regulation of the activity and expression of D6D can promote cancerogenesis and migration, then, D6D act as a new anti-cancer target. |
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