Targeting SERCA2 By XL, A Heterocyclic Cyclohexanone Curcumin Analog, In The Treatment Of Human Colorectal Cancer

Curcumin has numerous pharmacological, chemopreventative and chemotherapeutic actions in many cancers. However, curcumin has shown limited clinical efficacy, due to its low bioavailability and low stability in physiological media. Therefore, there is a need for the development of new and efficacious cyclohexanone analogs of curcumin.for colorectal cancer (CRC). In this study, curcumin analog XL showed potent cytotoxicity towards human colon cancer cells and inhibited xenotransplanted tumor growth. Most notably, XL featured binding to sarco/endoplasmic reticulum calcium-ATPase (SERCA)2 in a novel binding site and suppressed its Ca2+ -ATPase activity both in vitro and in vivo. This was accompanied by the induction of endoplastmic reticulum stress leading to apoptosis and G2/M cycle arrest in human colon carcinoma SW480 cells.In the first chapter of this study, we investigated the cytotoxicity of XL as well as its effect on cell proliferation and apoptosis. After screening from a series of second generation heterocyclic cyclohexanone curcumin analogs, we identified XL with significant dose-dependent suppression of proliferation in a panel of human colon cancer cell lines compared with curcumin. Further more, XL induces apoptosis related to ER stress in SW480 cells. Such apoptosis was verified by immunoblotting. XL increased the cleavage of PARP and caspase 3 in a dose-dependent manner. Interestingly, the increase in CHOP and DR5 protein levels were observed. The up-regulation of DR5 was confirmed on the surface of SW480 cells after XL treatment, whereas there was no change in the cell surface expression of Fas. Moreover, XL treatment caused the accumulation of ubiquitinated proteins and increased GRP78, ATF4 and CHOP expression in SW480 cells.These all revealed that XL induced ER-stress assoaciated apoptosis. It has been demonstrated that treating cells by drugs that target ER calcium influx (thapsigargin) results in ER stress, which induces G2 cell-cycle arrest via mRNA translation of the p53 isoform p53/47. Measurement of DNA content showed that XL indeed increased the percentage of cells in G2/M phase and decreased that in G0/G1 phase of the cell cycle in a dose-dependent manner. Like thapsigargin, XL induced p53/p47 mRNA translation.In the second chapter, we further investigated XL activity in vivo. Administration of XL inhibited tumor growth compared with controls, and XL did not have any effect on the weight of liver and spleen. In addition, Ca2+-ATPase activity was significantly inhibited in the XL-treated tumors compared with olive oil-treated tumors, confirming SERCA2 inhibition in vivo. The cleavage of PARP and caspase 3 as well as CHOP expression in the tumors were greatly elevated in the XL treatment group. Moreover, the immunohistochemical and tunel staining assay showed apparent apoptosis induction in tumor tissues from XL-treated group compared with olive oil-treated group. Immunostaining also revealed that CD31+ blood vessels in XL-treated mice were greatly decreased compared with controls.In the third chapter, we further investigated the target of drug action for XL. We and other investigators have demonstrated that curcumin, is a potent inhibitor of SERCA2. Since XL showed the similar activity as curcumin, we measured the activity of Ca2+ -ATPase in S W480 cells after treatment with XL and observed that XL significantly inhibited Ca2+-ATPase activity as well as inducing a steady rise in cytosolic Ca2+ levels. We detected SERCA2b expression in SW480 cells, however, there was hardly detectable SERCA1 or SERCA3 expression, moreover, XL showed no effect on SERCA2 expression at either mRNA or protein levels in SW480 cells. These results suggested that XL inhibited SERCA2 activity in SW480 cells without affecting its expression. To test the possibility of the SERCA2 protein as the target of XL in SW480 cells, because XL is a compound with fluorescence, immunofluorescence staining revealed the colocalization of XL and SERCA2 in SW480 cells.Futhermore, stably knocking-down or overexpression of SERCA2 all impaired the sensitivity of SW480 cells to XL. To further explore the potential mode of binding of XL to the SERCA2 protein, we employed molecular docking using AutoDock 4.2. XL appears to bind the cleft between the loop connecting M3 and M4 and the L78 loop on the lumenal side, possibly sealing the access pathway at the very surface through hydrogen bonds, hydrophilic interactions and VanderWaal’s forces with a group of residues. This novel binding site was also supported by evidence that XL showed synergistic cytotoxicity with thapsigargin, which binds the E2 form of SERCA in the transmembrane domain in SW480 cells. To verify the importance of these residues in the binding of XL, we generated SERCA2 mutants carrying H278â†' A and K876â†'A mutations and overpressed the mutant SERCA2 in HEK cells. These mutations didin’t reduce ER-stress assoaciated apoptosis as well as the steady rise in cytosolic Ca2+ levels induced by XL.In summary, our study revealed XL excerted much stronger activity than curcumin in vitro and in vivo. According to searching for upstream molecular of ER-stress, we identifies curcumin analog XL as a lead compound targeting SERCA2 with a novel binding site and demonstrates the efficacy of SERCA2 as a therapeutic target and XL as a therapeutic agent for the treatment of CRC.