Effect of dequalinium on K1735-M2 melanoma cell growth, directional migration and invasion in vitro

Helige, Christine; Smolle, Josef; Zellnig, Günther; Fink‐Puches, Regina; Kerl, Helmut; Tritthart, H. A.

doi:10.1016/0959-8049(93)90589-8

Cited by 27 publications

(19 citation statements)

References 12 publications

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“…This result may explain the ability of DECA to delay the morphological response by fibroblasts treated with phorbol ester (10), to act as an anti-tumor agent (8,25), and to inhibit cell motility and invasion (26). That DECA did not entirely abolish TPA-induced translocation but merely suppressed it may be explained by the artificially strong stimulus provided by TPA (27) that also produces direct interactions of PKC with membrane lipids (28).…”

Section: Discussionmentioning

confidence: 88%

Photoinduced Inactivation of Protein Kinase C by Dequalinium Identifies the RACK-1-binding Domain as a Recognition Site

Rotenberg

Sun

1998

Journal of Biological Chemistry

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Protein kinase C (PKC), 1 a monomeric Ca 2ϩ -and phospholipiddependent serine/threonine protein kinase, plays a critical role in growth factor-activated signaling and malignant transformation (1, 2). A current focus of investigation is to identify a specific inhibitor of PKC that is targeted to a structural element of the protein that influences PKC behavior in cells. Because the regulatory domain of PKC distinguishes it from other protein kinases, it is this segment of PKC, contained in the N-terminal portion of the protein, that attracts the greatest interest. This domain houses the binding regions for PKCactivating ligands such as phosphatidylserine (PS), diacylglycerol (DAG), and phorbol esters (C1 region) as well as for Ca 2ϩ (C2 region).1,1Ј-Decamethylenebis-4-aminoquinaldinium diiodide (DECA; dequalinium) ( Fig. 1) is a dicationic lipophilic PKC inhibitor whose mechanism of action remains unknown. Identified as a potent anti-tumor agent in several animal models (8) and as a drug that is selectively accumulated by cancer cells (9), DECA inhibits PKC␤1 activity in vitro and in murine embryo fibroblasts at low micromolar concentrations (10). Observations that associate DECA action with both the catalytic and regulatory domains of PKC (10) relegate this compound to a general category of PKC inhibitor defined as "mixed-type" (11). However, previous findings have shown that, in its interaction with the regulatory domain, DECA is not competitive with PS (10) and phorbol ester or Ca 2ϩ . 2A candidate DECA recognition site in the regulatory domain with functional significance is the RACK-1-binding site. Identified as the receptor for activated C kinase, RACK-1 is a PKC-binding protein found in the Triton-X-insoluble material of the particulate fraction (3-7). It is believed to recognize specific sites in the C2 region of PKC␤ that lie immediately C-terminal to the phorbol ester-binding domain (6). When bound to the C2 region of PKC␣, this adaptor protein has been proposed to stabilize the activated form of the enzyme, but acts as neither substrate nor inhibitor. Synthetic peptides modeled after the RACK-1-binding domain in the C2 domain of PKC␤ were observed to block the association of PKC with RACK-1 in vitro and to inhibit PKC translocation in cardiac myocytes and Xenopus oocytes (6).The photochemically induced behavior of DECA makes possible a novel approach to elucidate the inhibitory mechanism of this interesting compound. Irradiation by long-wave UV light (365 nm) in the presence of a target protein can cause covalent modification of that protein, as originally described with the mitochondrial F 1 -ATPase (12). The present study harnesses the photolyzable behavior of DECA to show that the RACK-1-binding domain of PKC is a recognition site for DECA. EXPERIMENTAL PROCEDURESMaterials-Recombinant human PKC␣ and PKC␤1 were purchased from PanVera Corp. (Madison, WI); dioleoylphosphatidylserine and DAG were obtained from Avanti Polar Lipids (Alabaster, AL); and TPA was purchased from LC Services (Woburn, MA). [␥-32...

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Section: Discussionmentioning

confidence: 88%

Photoinduced Inactivation of Protein Kinase C by Dequalinium Identifies the RACK-1-binding Domain as a Recognition Site

Rotenberg

Sun

1998

Journal of Biological Chemistry

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“…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 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.…”

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confidence: 99%

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

et al. 2000

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Dequalinium (DECA) is a potent antitumor agent and inhibitor of protein kinase C (PKC). Previously it was shown that PKC␣ 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 2ϩ -dependent PKC catalytic activity, signifying inactivation of a conventional PKC isoform. Because PKC␣ is the only conventional PKC isoform detected in B16 F10 cells, a stably transfected clone expressing a kinase-defective mutant of PKC␣ was developed that exhibited a substantial loss of adhesion and motility and was refractory to further inhibition by DECA. These findings identify PKC␣ 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 (␣, ␤, and ␥) are activated by Ca 2ϩ , diacylglycerol, and phosphatidylserine (PS) or by PS plus 12-O-tetradecanoylphorbol-13-acetate (TPA; a potent and specific phorbol ester); the novel isoforms (␦, ⑀, , and ) are insensitive to Ca 2ϩ but can be activated by diacylglycerol, PS, and TPA; and the atypical isoforms (/ and ) 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 / and ) translocate to the membrane where they interact with specific substrates to initiate effects on cell behavior.Recent studies have implicated a critical role for PKC␣ 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 charact...

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“…For the present work, mouse melanoma K1735-M2 cells, a highly invasive melanoma cell line (Helige et al, 1993), were used to investigate berberine effects on cell proliferation and in situ mitochondrial physiology. In addition, isolated mitochondrial fractions were used to investigate direct berberine effects.…”

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confidence: 99%

Mitochondrially Targeted Effects of Berberine [Natural Yellow 18, 5,6-dihydro-9,10-dimethoxybenzo(g)-1,3-benzodioxolo(5,6-a) quinolizinium] on K1735-M2 Mouse Melanoma Cells: Comparison with Direct Effects on Isolated Mitochondrial Fractions

Pereira¹,

Branco²,

Matos³

et al. 2007

J Pharmacol Exp Ther

137

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Berberine [Natural Yellow 18, 5,6-dihydro-9,10-dimethoxybenzo(g)-1,3-benzodioxolo(5,6-a)quinolizinium] is an alkaloid present in plant extracts and has a history of use in traditional Chinese and Native American medicine. Because of its ability to arrest the cell cycle and cause apoptosis of several malignant cell lines, it has received attention as a potential anticancer therapeutic agent. Previous studies suggest that mitochondria may be an important target of berberine, but relatively little is known about the extent or molecular mechanisms of berberine-mitochondrial interactions. The objective of the present work was to investigate the interaction of berberine with mitochondria, both in situ and in isolated mitochondrial fractions. The data show that berberine is selectively accumulated by mitochondria, which is accompanied by arrest of cell proliferation, mitochondrial fragmentation and depolarization, oxidative stress, and a decrease in ATP levels. Electron microscopy of berberine-treated cells shows a reduction in mitochondria-like structures, accompanied by a decrease in mitochondrial DNA copy number. Isolated mitochondrial fractions treated with berberine had slower mitochondrial respiration, especially when complex I substrates were used, and increased complex I-dependent oxidative stress. It is also demonstrated for the first time that berberine stimulates the mitochondrial permeability transition. Direct effects on ATPase activity were not detected. The present work demonstrates a number of previously unknown alterations of mitochondrial physiology induced by berberine, a potential chemotherapeutic agent, although it also suggests that high doses of berberine should not be used without a proper toxicology assessment.Berberine (Fig.

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Effect of dequalinium on K1735-M2 melanoma cell growth, directional migration and invasion in vitro

Cited by 27 publications

References 12 publications

Photoinduced Inactivation of Protein Kinase C by Dequalinium Identifies the RACK-1-binding Domain as a Recognition Site

Photoinduced Inactivation of Protein Kinase C by Dequalinium Identifies the RACK-1-binding Domain as a Recognition Site

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

Mitochondrially Targeted Effects of Berberine [Natural Yellow 18, 5,6-dihydro-9,10-dimethoxybenzo(g)-1,3-benzodioxolo(5,6-a) quinolizinium] on K1735-M2 Mouse Melanoma Cells: Comparison with Direct Effects on Isolated Mitochondrial Fractions

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