Enhanced Anticancer Effect Of Arginine Conjugation With Conjugated Linoleic Acid On Human Bladder Cancer Cells

It has been reported that conjugated linoleic acid (CLA) can inhibit cancer cells growth and induce apoptosis in vitro and vivo. A recent landmark study demonstrated that CLA treatment promoted apoptosis in breast, bladder, lung, colon cancer cell lines. Carcinoma of urinary bladder is a common mankind malignant tumor, the life and health of the human being are seriously threatened by it. However, CLA was rapidly decomposed to form furan fatty acids when it was oxidized in air. The susceptive oxidation and low solubility limited the clinical application of CLA. Arginine (Arg), a water-soluble amino acid, is used to connect with conjugated linoleic acid by amido linkage. This studuy was to investigate whether Arginine-Conjugated Linoleic Acid (Arg-CLA) displays anti-cancer properties in human bladder cancer T24cells, and to assess apoptotic mechanism in bladder cancer T24cells.The chemical synthesis of arg attachment to the carboxyl end was confirmed using an infrared spectroscope, and the amido linkage was identified both1552.13and1640.79cm-1. HPLC was used to evaluate the characterization of PCLA.The apoptosis effect of ACLA and CLA on human bladder cancer T24cells was determined. T24cells were treated by various concentration of ACLA and CLA treatment for24,48, and72hours and analyzed by MTT assay. As a result, ACLA treatment resulted in a dose-dependent (50-200μM) and time-dependent (24-72hours) inhibition of cellular proliferation and cell viability.The apoptotic effect of ACLA was also confirmed by analyzing cell morphology and by PI staining and the annexin V method. The extent of apoptosis was quantified by flow-cytometric analysis of ACLA-treated cells labeled with PI and annexin V. Alterations in signaling events were determined in Western blot analysis probing for phosphorylated Akt protein, indicative of activation. The role of caspase-3, PPARy, Bcl-2family in apoptosis was analyzed by Western blotting. As a result, the cellular motility was evidently inhibited in dose-dependent and time-dependent manner by ACLA. As shown by PI staining and the annexin V method, we found that ACLA caused a dosage dependent increase in T24cell apoptosis. Western blot analysis indicated that treatment of T24cells with ACL A resulted in a dose-dependent activation of PPARy and caspase-3proteins48hours after ACLA treatment. ACL A treatment resulted in an appreciable down-regulation of protein expression of phospho-Akt, a decrease in antiapoptotic Bcl-2and a concomitant increase in proapoptotic Bax proteins in T24cells.These results indicated that ACLA can effectively induce the apoptosis of T24cells. The effect of ACLA may be associated with the regulation of p-Akt, Bcl-2, Bax, Caspase-3protein expression.