Photo-Induced Inactivation of Protein Kinase Ca by Dequalinium Inhibits Motility of Murine Melanoma Cells

Dequalinium (DECA) is a potent antitumor agent and inhibitor of protein kinase C (PKC). Previously it was shown that PKCa activity in vitro could be irreversibly inhibited when treated with DECA at low micromolar concentrations and irradiated with 366 nm of light. This approach was used to probe the role of intracellular PKC activity in the motility of metastatic murine melanoma B16 F10 cells and as a target for DECA analogs with increasing PKC inhibitory potencies. Pretreatment of a monolayer of B16 F10 cells with 250 nM of a DECA analog in the presence of UV irradiation for 5 min resulted in 1) complete inhibition of cell motility for up to 4 h in a time-lapse motility assay and 40 to 60% inhibition of cell migration in a Boyden chamber, and 2) inhibition by 40 to 60% of intracellular phosphatidylserine/Ca-dependent PKC catalytic activity, signifying inactivation of a conventional PKC isoform. Because PKCa is the only conventional PKC isoform detected in B16 F10 cells, a stably transfected clone expressing a kinase-defective mutant of PKCa was developed that exhibited a substantial loss of adhesion and motility and was refractory to further inhibition by DECA. These findings identify PKCa catalytic activity both as a mechanistic component of cell motility and adhesion and as a critical intracellular target of DECA. These studies further suggest that the combined use of UV with nanomolar concentrations of DECA offers an effective chemotherapeutic approach to inhibit metastatic behavior of melanoma cells. Protein kinase C (PKC) (EC 2.7.1.37) is a serine/threonine protein kinase that participates in numerous signaling pathways. PKC consists of a family of related isoforms variously expressed by cells. Each isoform can be assigned to one of three subcategories based on the means by which it is activated: the conventional PKC isoforms (a, b, and g) are activated by Ca, diacylglycerol, and phosphatidylserine (PS) or by PS plus 12-O-tetradecanoylphorbol-13-acetate (TPA; a potent and specific phorbol ester); the novel isoforms (d, e, h, and u) are insensitive to Ca but can be activated by diacylglycerol, PS, and TPA; and the atypical isoforms (l/i and z) are insensitive to conventional activators and TPA but can stimulated by phospholipids such as phosphatidylinositol (Kazanietz et al., 1993). When cells are treated with TPA, certain isoforms (except l/i and z) translocate to the membrane where they interact with specific substrates to initiate effects on cell behavior. Recent studies have implicated a critical role for PKCa in cell adhesion and motility, two highly integrated activities that are fundamental to metastatic potential (Palecek et al., 1997). Previous work with mouse melanoma cells (Gopalakrishna and Barsky, 1988) showed that PKC has a mechanistic role in the metastatic activity of B16 cells in a syngeneic C57BL/6 mouse model (Fidler, 1975). In that work, highly metastatic B16 F10 cells exhibited proportionately higher PKC activity in the membrane fraction than did B16 F1 cells which had characteristically low metastatic activity. Furthermore, B16 F1 cells having low metastatic activity were rendered highly metastatic by treatment with TPA. The potent and specific nature of TPA action implicated the TPAresponsive PKC isoforms in the metastatic activity of this melanoma cell model. Other laboratories have since recognized a role for PKC in promoting the adhesion and motility of B16 cells (Dumont et al., 1992; Dumont and Bitonti, 1994; Lewis et al., 1996; La Porta and Comolli, 1997). Intervention in the metastatic activity of B16 melanoma cells was demonstrated with PKC-targeted inhibitors (Dumont et al., 1992; Liu et al., 1992), an example of which was the light-activated inhibitor, calphostin C (Liu et al., 1992). In the present study, dequalinium (DECA) was examined This work was supported by grants from the National Institutes of Health (CA60618), the Elsa U. Pardee Foundation, the Professional Staff Congress of the City University of New York Research Foundation, and the Austrian Fond SFB, F208, Biological communication systems. Aspects of this work appeared in the Proceedings of the American Association for Cancer Research, Vol. 88 (abstract 1551) and Vol. 90 (abstract 3702). ABBREVIATIONS: PKC, protein kinase C; DECA, 1,19-decamethylene-bis-4-aminoquinaldinium di-iodide; PS, phosphatidylserine; TPA, 12-Otetradecanoylphorbol-13-acetate; DMSO, dimethyl sulfoxide. 0026-895X/00/040729-09$3.00/0 MOLECULAR PHARMACOLOGY Vol. 58, No. 4 Copyright © 2000 The American Society for Pharmacology and Experimental Therapeutics 13050/852447 Mol Pharmacol 58:729–737, 2000 Printed in U.S.A. 729 at A PE T Jornals on July 7, 2017 m oharm .aspeurnals.org D ow nladed from as a UV light-activated tool by which to explore a specific role for PKCa in the motility of melanoma cells and as a prototype for the future design of antimetastatic agents. DECA was previously shown to be an antitumor agent (Weiss et al., 1987; Christman et al., 1990) that is selectively accumulated by cancer cells because of the higher electrochemical potentials maintained across mitochondrial and plasma membranes (Chen, 1989). As shown for several cancer cell lines, a dicationic molecule such as DECA can be accumulated up to 25,000-fold relative to its exogenous concentration (Chen, 1989). The drug is taken up by the mitochondria, which serve as storage depots that slowly release the drug into the cytoplasm. Previous studies showed that low micromolar levels of DECA are required to inhibit the motility and invasiveness of human melanoma cells in vitro (Fink-Puches et al., 1993; Helige et al.,1993; Hofmann-Wellenhof et al., 1995). An attractive feature of the B16 F10 cell system, however, is that, unlike most other cancer cells, they do not accumulate DECA in their mitochondria (Bernal et al., 1983), thus concentrating much of the drug in the cytosol where many PKC isoforms are localized. DECA is a noncompetitive PKC inhibitor exhibiting IC50 5 10 mM (Rotenberg et al., 1990). A fortuitous property of DECA is that it can be rendered chemically reactive by irradiation with UV light (366 nm), producing covalent modification at its enzyme target site (Rotenberg and Sun, 1998). The property of photo-induced covalent modification by DECA was first demonstrated with the mitochondrial F1 ATPase, which underwent covalent modification with coincident inactivation of activity (Zhuo and Allison, 1988). Similarly, when PKC is treated with low micromolar concentrations of DECA and UV light in vitro, there is a dosedependent, loss of catalytic activity that cannot be reversed by dilution (Rotenberg and Sun, 1998). In the present work photo-induced inactivation is demonstrated with intracellular PKC in cells that have been treated with the drug and irradiated directly with long-wave UV light. A novel aspect is that UV-induced inhibition of both PKC activity and cell motility can be observed with nanomolar concentrations of DECA or DECA analogs recently developed by this laboratory (Qin et al., 2000). These studies implicate PKCa as a critical component in the motility of metastatic melanoma cells and establish it as an important target for antimetastatic agents. Experimental Procedures Materials. Cell culture media, phosphate-buffered saline, and growth serum were purchased from Gibco-BRL (Gaithersburg, MD). Matrigel was acquired from BD Biosciences (Bedford, MA). PS was obtained from Avanti Polar Lipids, Inc. (Alabaster, AL), TPA was purchased from LC Services (Woburn, MA), and Go6976 was acquired from CalBiochem (San Diego, CA). Nitrocellulose membranes were obtained from Amersham Pharmacia Biotech (Piscataway, NJ), [g-P]ATP (3000 Ci/mmol) was from NEN-DuPont (Wilmington, DE), and protein dye reagent was from Bio-Rad (Hercules, CA). The SER peptide (RFARKGSLRQKNV) was synthesized by N. Pileggi (Protein Core Facility, Columbia University, New York). All curvefitting and graphical representations were prepared with CA-Cricket Graph III software purchased from Computer Associates International, Inc. (Islandia, NY). Cell Culture. Murine melanoma B16 F10 cells were cultured in 10-cm dishes to 75 to 80% confluence in RPMI medium containing 10% fetal bovine serum, 1% penicillin/streptomycin, and 0.125 mg/ml