Abstract
Diacylglycerol (DAG)-mediated signaling pathways, such as those mediated by protein kinase C (PKC), are central in regulating cell proliferation and apoptosis. DAG-responsive C1 domains are therefore considered attractive drug targets. Our group has designed a novel class of compounds targeted to the DAG binding site within the C1 domain of PKC. We have previously shown that these 5-(hydroxymethyl)isophthalates modulate PKC activation in living cells. In this study we investigated their effects on HeLa human cervical cancer cell viability and proliferation by using standard cytotoxicity tests and an automated imaging platform with machine vision technology. Cellular effects and their mechanisms were further characterized with the most potent compound, HMI-1a3. Isophthalate derivatives with high affinity to the PKC C1 domain exhibited antiproliferative and non-necrotic cytotoxic effects on HeLa cells. The anti-proliferative effect was irreversible and accompanied by cell elongation. HMI-1a3 induced down-regulation of retinoblastoma protein and cyclins A, B1, D1, and E. Effects of isophthalates on cell morphology, cell proliferation and expression of cell cycle-related proteins were different from those induced by phorbol 12-myristate-13-acetate (PMA) or bryostatin 1, but correlated closely to binding affinities. Therefore, the results strongly indicate that the effect is C1 domain-mediated.
Highlights
The protein kinase C (PKC) family of serine/threonine kinases consists of ten known isozymes that can be divided into three classes based on their regulatory domain structure and activation properties [1]
The well-characterized C1 domain ligands phorbol 12-myristate-13-acetate (PMA) and bryostatin 1 were used as reference compounds
In efforts to develop drugs to treat PKC-related diseases, targeting the C1 domain has some advantages over the traditional approach of targeting the catalytic site
Summary
The protein kinase C (PKC) family of serine/threonine kinases consists of ten known isozymes that can be divided into three classes based on their regulatory domain structure and activation properties [1]. PKC C1 domains are cysteine-rich sequences that are approximately 50 amino acids long and are located within the regulatory region of the enzyme. They are folded into zinc fingerlike structures that respond to increased DAG levels at the plasma membrane, leading to relocation and activation of PKC isoforms. DAG is generated by phospholipase C-mediated hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2) after activation of G protein-coupled receptors or receptor tyrosine kinases [2]. It can be produced indirectly from phosphatidylcholine via phospholipase D and phosphatic acid phosphatase [2]. Phorbol esters are diterpene-structured natural compounds that mimic DAG actions but with significantly higher potency [3]
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