BPR1K653, a Novel Aurora Kinase Inhibitor, Exhibits Potent Anti-Proliferative Activity in MDR1 (P-gp170)-Mediated Multidrug-Resistant Cancer Cells

Over-expression of Aurora kinases promotes the tumorigenesis of cells. The aim of this study was to determine the preclinical profile of a novel pan-Aurora kinase inhibitor, BPR1K653, as a candidate for anti-cancer therapy. Since expression of the drug efflux pump, MDR1, reduces the effectiveness of various chemotherapeutic compounds in human cancers, this study also aimed to determine whether the potency of BPR1K653 could be affected by the expression of MDR1 in cancer cells.BPR1K653 specifically inhibited the activity of Aurora-A and Aurora-B kinase at low nano-molar concentrations in vitro. Anti-proliferative activity of BPR1K653 was evaluated in various human cancer cell lines. Results of the clonogenic assay showed that BPR1K653 was potent in targeting a variety of cancer cell lines regardless of the tissue origin, p53 status, or expression of MDR1. At the cellular level, BPR1K653 induced endo-replication and subsequent apoptosis in both MDR1-negative and MDR1-positive cancer cells. Importantly, it showed potent activity against the growth of xenograft tumors of the human cervical carcinoma KB and KB-derived MDR1-positive KB-VIN10 cells in nude mice. Finally, BPR1K653 also exhibited favorable pharmacokinetic properties in rats.BPR1K653 is a novel potent anti-cancer compound, and its potency is not affected by the expression of the multiple drug resistant protein, MDR1, in cancer cells. Therefore, BPR1K653 is a promising anti-cancer compound that has potential for the management of various malignancies, particularly for patients with MDR1-related drug resistance after prolonged chemotherapeutic treatments

Natural Endogenous Human Matriptase and Prostasin Undergo Zymogen Activation via Independent Mechanisms in an Uncoupled Manner.

The membrane-associated serine proteases matriptase and prostasin are believed to function in close partnership. Their zymogen activation has been reported to be tightly coupled, either as a matriptase-initiated proteolytic cascade or through a mutually dependent mechanism involving the formation of a reciprocal zymogen activation complex. Here we show that this putative relationship may not apply in the context of human matriptase and prostasin. First, the tightly coupled proteolytic cascade between matriptase and prostasin might not occur when modest matriptase activation is induced by sphingosine 1-phospahte in human mammary epithelial cells. Second, prostasin is not required and/or involved in matriptase autoactivation because matriptase can undergo zymogen activation in cells that do not endogenously express prostasin. Third, matriptase is not required for and/or involved in prostasin activation, since activated prostasin can be detected in cells expressing no endogenous matriptase. Finally, matriptase and prostasin both undergo zymogen activation through an apparently un-coupled mechanism in cells endogenously expressing both proteases, such as in Caco-2 cells. In these human enterocytes, matriptase is detected primarily in the zymogen form and prostasin predominantly as the activated form, either in complexes with protease inhibitors or as the free active form. The negligible levels of prostasin zymogen with high levels of matriptase zymogen suggests that the reciprocal zymogen activation complex is likely not the mechanism for matriptase zymogen activation. Furthermore, high level prostasin activation still occurs in Caco-2 variants with reduced or absent matriptase expression, indicating that matriptase is not required and/or involved in prostasin zymogen activation. Collectively, these data suggest that any functional relationship between natural endogenous human matriptase and prostasin does not occur at the level of zymogen activation

Prostasin does not undergo zymogen activation in response to exposure to a mildly acidic buffer in cells expressing no matriptase.

<p>ACHN human renal adenocarcinoma cells were treated with PBS (lanes 1) or pH 6.0 buffer for 20 min (lanes 2). Cell lysates were analyzed by Western blot analysis for prostasin species (<i>Pros</i>.) or for HAI-1 (<i>HAI-1</i>). The prostasin-HAI-1 complex (Prost.-HAI-1 complex), HAI-1 and prostasin zymogen are indicated. This experiment was conducted at least twice with the same profile observed. Representative data are shown.</p

Matriptase can undergo autoactivation in cells that do not express prostasin.

<p>Human hematological cancer cell lines Raji, Namalwa, and Ramos, human prostate cancer cell lines DU145 and PC3, and human hepatoma Hep3B cells were treated with PBS as the non-activation control (lanes 1) or pH 6.0 buffer for 20 min to induce matriptase activation. Cell lysates were prepared and analyzed by Western blot analysis for matriptase species using the matriptase specific mAb M24. The matriptase species observed include matriptase zymogen (band <i>a</i>), matriptase-HAI-2 complex (band <i>b</i>), and matriptase-HAI-1 complex (band <i>c</i>). This study was conducted multiple times (n>5) with the same profile observed. Representative data are shown.</p

Sphingosine 1-phosphate induces matriptase activation without inducing prostasin zymogen activation.

<p>Human mammary epithelial cells 184 A1N4 were treated with fresh medium, which contains sphingosine 1-phosphate derived from FBS, for the indicated times. The cells were harvested and analyzed by Western blot for matriptase species (<i>A</i>) and prostasin (<i>B</i>.). The size of matriptase-HAI-1 complex (MTP-HAI-1) and prostasin-HAI-1 complex (Pros.-HAI-1) were indicated. The experiment was conducted twice with the same profile observed. Representative data are shown.</p

Human mammary and prostate epithelial cells simultaneously activate matriptase and prostasin in response to transient exposure to a pH 6.0 buffer.

<p>Human prostate epithelial cells RWPE-1 (<i>A</i>.), mammary epithelial cells 184 A1N4 (<i>B</i>.), MTSV 1.1B (<i>C</i>.) and MTSV 1.7 (<i>D</i>.) were exposed to PBS as a non-activation control (N, lanes 1 and 3) or phosphate buffer pH 6.0 for 20 min (A, lanes 2 and 4). Cell lysates were prepared and analyzed by Western blot for species containing matriptase (MTP, lanes 1 and 2, bands <i>a</i> and <i>c</i>) or prostasin (Pros., lanes 3 and 4, bands <i>b</i>, <i>d</i>, and <i>e</i>). This experiment was conducted at least twice with the same profile observed. Representative data are shown.</p

Human Caco-2 cells express prostasin predominantly in the activated form and matriptase primarily in the zymogen form.

<p>Human Caco-2 enterocytes were cultured and harvested at indicated dates post confluency. Cell lysates were analyzed by Western blot for prostasin species (<i>A</i>.) and matriptase species (<i>B</i>.). The composition of the various forms of prostasin and matriptase are indicated. The experiment was conducted twice with the same profile observed. Representative data are shown.</p