Mechanistic studies of anticancer aptamer AS1411 reveal a novel role for nucleolin in regulating Rac1 activation.

AS1411 is a G-rich quadruplex-forming oligodeoxynucleotide that binds specifically to nucleolin, a protein found on the surface and in the cytoplasm of most malignant cells but absent from the surface/cytoplasm of most normal cells. AS1411 has shown promising clinical activity and is being widely used as a tumor-targeting agent, but its mechanism of action is not fully understood. Previously, we showed that AS1411 is taken up in cancer cells by macropinocytosis (fluid phase endocytosis) and subsequently stimulates further macropinocytosis by a nucleolin-dependent mechanism. In the current study, we have investigated the significance and molecular mechanisms of AS1411-induced macropinocytosis. Our results indicate that the antiproliferative activity of AS1411 in various cell lines correlated with its capacity to stimulate macropinocytosis. In DU145 prostate cancer cells, AS1411 induced activation of EGFR, Akt, p38, and Rac1. Activation of Akt and p38 were not critical for AS1411 activity because Akt activation was not observed in all AS1411-responsive cell lines and knockdown of p38 had no effect on AS1411's ability to inhibit proliferation. On the other hand, activation of EGFR and Rac1 appeared to play a role in AS1411 activity in all cancer cell lines examined (DU145, MDA-MB-468, A549, LNCaP) and their inhibition significantly reduced AS1411-mediated macropinocytosis and AS1411 antiproliferative activity. Interestingly, downregulation of nucleolin expression by siRNA also produced a substantial increase in activated Rac1, revealing a previously unknown role for nucleolin as a negative regulator of Rac1 activation. Our results are consistent with a model whereby AS1411 binding to nucleolin leads to sustained activation of Rac1 and causes methuosis, a novel type of nonapoptotic cell death characterized by hyperstimulation of macropinocytosis. We speculate that methuosis is a tumor/metastasis suppressor mechanism that opposes the malignant functions of Rac1 and that cancer cells may overexpress nucleolin to surmount this barrier.

A novel small molecule that induces oxidative stress and selectively kills malignant cells.

We have synthesized a novel molecule named XB05 (1-bromo-1,1-difluoro-non-2-yn-4-ol) and evaluated its effects in a variety of human cell lines. XB05 displayed potent antiproliferative activity against cell lines derived from leukemia or solid tumors, but had less effect on nonmalignant cells. To identify factors that contribute to the cancer selectivity of XB05, we chose three cell lines that had high sensitivity to XB05 (U937 leukemia), moderate sensitivity (A549 lung cancer), or low sensitivity (Hs27 nonmalignant skin fibroblasts), and proceeded to assess cell death and oxidative stress in these cells. XB05 was found to induce cell death via both apoptotic and nonapoptotic mechanisms in U937 and A549 cells, whereas it had no cytotoxicity against Hs27 cells at comparable concentrations. Treatment with XB05 caused an increase in reactive oxygen species in all cell lines tested, but levels were higher in malignant compared to nonmalignant cells. XB05 treatment also induced DNA damage exclusively in the malignant cells. Differences in antioxidant responses were observed between cell lines. For example, XB05 caused a decrease in levels of glutathione and nuclear Nrf2 in the most sensitive cells (U937), whereas the least sensitive cells (Hs27) displayed increased glutathione levels and no change in nuclear Nrf2. XB05 could react in vitro with cysteine and glutathione, but had much lower reactivity compared to typical thiol-reactive electrophiles, diethyl maleate and maleimide. In summary, XB05 is a novel compound that selectively kills malignant cells, most likely by disrupting cellular redox homeostasis, making it a promising candidate for development as a chemotherapeutic agent.

Physangulidine A, a withanolide from Physalis angulata, perturbs the cell cycle and induces cell death by apoptosis in prostate cancer cells.

Recently, our group reported the discovery of three new withanolides, physangulidines A-C, from Physalis angulata. In this study, the biological effects of physangulidine A (1), which was the most active and abundant of the three new constituents, are described. It was found that 1 significantly reduces survival in clonogenic assays for two hormone-independent prostate cancer cell lines. Flow cytometry and confocal microscopy studies in DU145 human prostate cancer cells indicated that 1 induces cell cycle arrest in the G(2)/M phase and causes defective mitosis. It was determined also that 1 produces programed cell death by apoptosis, as evidenced by biochemical markers and distinct changes in cell morphology. These results imply that the antimitotic and proapoptotic effects of 1 may contribute significantly to the biological activities and potential medicinal properties of its plant of origin.

A new paradigm for aptamer therapeutic AS1411 action: uptake by macropinocytosis and its stimulation by a nucleolin-dependent mechanism.

AS1411 is a first-in-class anticancer agent, currently in phase II clinical trials. It is a quadruplex-forming oligodeoxynucleotide that binds to nucleolin as an aptamer, but its mechanism of action is not completely understood. Mechanistic insights could lead to clinically useful markers for AS1411 response and to novel targeted therapies. Previously, we proposed a model where cell surface nucleolin serves as the receptor for AS1411, leading to selective uptake in cancer cells. Here, we compare uptake of fluorophore-labeled AS1411 (FL-AS1411) in DU145 prostate cancer cells (sensitive to AS1411) and Hs27 nonmalignant skin fibroblasts (resistant to AS1411). Uptake of FL-AS1411 occurred by endocytosis in both cell types and was much more efficient than an inactive, nonquadruplex oligonucleotide. Unexpectedly, uptake of FL-AS1411 was lower in cancer cells compared with Hs27 cells. However, the mechanism of uptake was different, occurring by macropinocytosis in cancer cells, but by a nonmacropinocytic pathway in Hs27 cells. Additionally, treatment of various cancer cells with AS1411 caused hyperstimulation of macropinocytosis, provoking an increase in its own uptake, whereas no stimulation was observed for nonmalignant cells. Nucleolin was not required for initial FL-AS1411 uptake in DU145 cells but was necessary for induced macropinocytosis and FL-AS1411 uptake at later times. Our results are inconsistent with the previous mechanistic model but confirm that nucleolin plays a role in mediating AS1411 effects. The data suggest a new model for AS1411 action as well as a new role for nucleolin in stimulating macropinocytosis, a process with potential applications in drug delivery.

Cell-surface nucleolin is a signal transducing P-selectin binding protein for human colon carcinoma cells.

We have previously shown that P-selectin binding to Colo-320 human colon carcinoma cells induces specific activation of the alpha(5)beta(1) integrin with a concomitant increase of cell adhesion and spreading on fibronectin substrates in a phosphatidylinositol 3-kinase (PI3-K) and p38 MAPK-dependent manner. Here, we identified by affinity chromatography and characterized nucleolin as a P-selectin receptor on Colo-320 cells. Nucleolin mAb D3 significantly decreases the Colo-320 cell adhesion to immobilized P-selectin-IgG-Fc. Moreover, nucleolin becomes clustered at the external side of the plasma membrane of living, intact cells when bound to cross-linked P-selectin-IgG-Fc chimeric protein. We have also found P-selectin binding to Colo-320 cells induces tyrosine phosphorylation specifically of cell-surface nucleolin and formation of a signaling complex containing cell-surface nucleolin, PI3-K and p38 MAPK. Using siRNA approaches, we have found that both P-selectin binding to Colo-320 cells and formation of the P-selectin-mediated p38 MAPK/PI3-K signaling complex require nucleolin expression. These results show that nucleolin (or a nucleolin-like protein) is a signaling receptor for P-selectin on Colo-320 cells and suggest a mechanism for linkage of nucleolin to P-selectin-induced signal transduction pathways that regulate the adhesion and the spreading of Colo-320 on fibronectin substrates.