| Conjugated linoleic acid (CLA) refers to a group of polyunsaturated fatty acids that exist as positional and geometric isomers of linoleic acid (LA; 18:2). Many researchers showed that CLA possessed many physiological functions such as: antiatherosclerosis, antidiabetogenesis, stimulating immunity, increasing bone density, reducing body fat etc. In particular, the anticarcinogenesis ability of CLA had increasingly received considerable attention.CLA is the most powerful anticancer fatty acid and can alter initiation, promotion, progression, and metastasis of malignant tumors. CLA could reduce the size of many solid tumors in experimental animals by inhibiting proliferation and derivation of tumor cell, such as:breast carcinoma, skin cancer, proventriculus cancer, thymic carcinoma and colon carcinoma. In addition, CLA inhibited the growth of mammary (SKBr3, SKBR3, MCF-7, MCF-10A, MDA-MB-231, MDA-MB468), bronchogenic (A-427, SK-LU-1, A-549), hepatoma (HepG2), gastric (SGC-7901), colon (SW480), colorectal (HT-29), prostate (PC-3, DU145), glioblastoma (M21-HPB), neurospongioma (A 172) cell lines in vitro.CLA is a collective term for isomers. The most abundant isomer found in nature, especially in meat and dairy products, is the cis-9, trans-11 (c9, t11) CLA isomer (>80%). Synthetical CLA is a mixture of equal proportions (>40%) of the two major forms, cis9, trans11-CLA (c9, t11-CLA) and trans10, cisl2-CLA, (t10, c12-CLA), and a number of minor isomers (i.e. t7, t9; c9, e11; t9, t11; c10,c12; t10, t12; t11, t13 and c11, c13-CLA). Distinct effects on apoptosis have been reported for the CLA mixture and for each of the two major isomers. c9, t11-CLA is known to be mainly effective isomer. Therefore, people showed concern on how to rasie the content of c9, tll-CLA.Chemotherapy is a very important method to deal with cancer. Multidrug resistance (MDR) is a significant reason for chemotherapy failure and leads to the decreased survival in cancer. The tumor MDR is induced by one drug and results in the tumor resistance to other drugs and the failure of chemical therapy. The mechanism of MDR is complicate, and one of the most important reasons is the overexpression of P-gp. The P-gp was found to be overexpressed on many cancer cells such as hepatoma, gastric, mammary, ovarian, endometrial, non-small cell carcinoma, acute myeloblastic leukemia, colon cell lines. The P-gp can pumps multiple types of drugs out of the cell using the energy generated from ATP, and confers multidrug resistance on cancer cells.Combination anti-cancer drug with MDR reverser is a good method to inhibite multidrug resistance in clinic and increase sensitivity on cancer cells. In many reports, borneol has less toxity and can inhibit the function of P-gp to transport the lipophilia drugs out of the tumor cells, so borneol could be used to reduce MDR.Study objects1 To incorporating the inhibitory effect of borneol to P-gp and the anticancer effect of CLA, conjugated linoleic acid borneol ester (BCLA) was synthesized in order to enhance drug concentration in the tumor cells.2 For high price and less content in nature, CLA could most be obtained from chemistry synthesis. Synthesized CLA was more complicated and had less physiological activity than those in nature, and c9, t11-CLA isomer was thought to had more activity than other isomers. Using different selectivity on the CLA isomers, lipase was used to separate c9, t11-CLA isomer for strengthening the purity and physiological activity of the CLA.3 To investigate the inhibitory effect of BCLA on some kinds of cancer cells.Methods and resultsIn the present study, a new derivative of BCLA was obtained by introducing borneol into the carboxy site of CLA in a solvent-free system. The reaction product was isolated by acid-base titration to eliminate the residual fatty acid, then distilled under vacuum to remove the remaining borneol. Its structure has been confirmed by GC-MS,1H-NMR,13C-NMR.During the reaction, in combination with one-factor-at-a-time optimization on lipase and reaction time, the mainly factors such as temperature, lipase amount, water content, time were optimized by response surface methodology (RSM). The highest conversion yield of 79.2±0.5% was obtained in three independent replicates under optimum reaction conditions, as follows:Lipase AYS, enzyme amount 40%(w/w by weight of borneol), reaction time 48h, temperature 40℃, water content 30%(w/w by weight of borneol), and molar ratio 2.5 (CLA/borneol).Meanwhile, CLA isomers were separated by enzyme-catalyzed method. Four product isomers were achieved by GC-MS analysis as follow:isoborneol-c9, t11-CLA 35.8%, isoborneol-t10, c12-CLA 6.3%, borneol-c9, t11-CLA 43.4%, borneol-t10, c12-CLA 14.5%. The amount of c9, t11-CLA (79.2%) was approximately four times than t10, c12-CLA (20.8%).Furthermore, the anti-cancer effects of BCLA were evaluated. HepG2 cells, Huh7 cells and Lovo cells were incubated in vitro with different concentrations (6.5、12.5、25、50、100、200μM) of BCLA、CLA and borneol for 24h. Cells morphological changes were detected under light microscope; Inhibition of cell proliferation was measured by the MTT assays. A remarkable difference in chemosensitivity to BCLA, CLA and borneol, were found in three cell lines. We found that BCLA and CLA could inhibit the proliferation of cancer cells, and the inhibitory effects corresponded with cell type and drug concentration. The results showed that the order of inhibitory effects on HepG2 cells was BCLA> CLA, antitumor effects on Lovo cells was CLA > BCLA, cytotoxicity to Huh7 cells depended on their concentration. At low dose, inhibitory effect of BCLA on Huh7 cells was greater than CLA, but CLA seemed to be more effective at high dose. Borneol had no evident inhibitory activity, but it was dose dependent. The IC50 value of BCLA, CLA and borneol for HepG2 cells was 114.84±11.11μM,156.2±5.85μM,468.87±16.09μM. The IC50 value of BCLA, CLA and borneol for Huh7 cells was 153.89±3.19μM,99.33±1.82μM,271.88±22.99μM. The IC50 value of BCLA, CLA and borneol for Lovo cells was 148.23±6.58μM, 54.97±1.37μM,537.41±6.81μM, respectively. HepG2 cells, Huh7 cells and Lovo cells growth were inhibited by BCLA.ConclusionIn the experiment, BCLA was obtained successfully. The content of c9, tll-CLA raised up to 79.2%. The ratio of c9, t11-CLA and t10, c12-CLA in BCLA is similar with CLA in nature. BCLA could mock the pharmacological property and feature of CLA in nature. The anticancer results showed that CLA and borneol played a part in cancer cells, respectively. The anticancer effect of BCLA is different depending on cell type. CLA and borneol had antitumor synergism in this paper. |
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