Isophthalate-induced cell elongation and inhibition of cell proliferation are not mediated by PKC

Virpi Talman, Elina Ekokoski, Raimo K Tuominen. University of Helsinki, Faculty of Pharmacy, Division of Pharmacology and toxicology, FI-00014, Finland

Derivatives of isophthalic acid, such as HMI-1a3, are potential drug candidates that target the C1 domain of protein kinase C (PKC). These compounds bind to PKC at submicromolar concentrations and modify PKC-dependent ERK1/2 activation in HeLa human cervical cancer cells (Boije af Gennäs et al. 2009). They also inhibit proliferation and induce elongation of HeLa cells in a concentration-dependent manner (Talman et al. 2011). The aim of this study was to clarify whether the effects of HMI-1a3 on cell morphology and proliferation were mediated by PKC- or ERK-dependent pathways. HeLa cells (CCL-2 from ATCC) were co-incubated with HMI-1a3 (0.1-20 µM) and either the PKC activator PMA (10-100nM), the pan-PKC inhibitor Gö6983 (10 nM-10 µM), or the MEK inhibitor U0126 (10 µM). Furthermore, the effects of HMI-1a3 were also studied under conditions where two PKC isoforms with high expression levels in HeLa cells, PKCα and PKCδ, were separately knocked down using siRNA. PKC isozyme-specific siRNA molecules (Dharmacon RNA Technologies) were transfected using X-tremeGENE siRNA transfection reagent (Roche) according to manufacturer’s instructions. Cell viability was assessed using a standard MTT assay. HeLa cell proliferation rate and morphology were analyzed by real-time imaging using a continuous cell culturing platform with integrated optics and an automated phase-contrast camera (Cell-IQ®, Chipmantech, Tampere, Finland). Images were quantified with the Cell-IQ Analyzer® software using a protocol specifically created for HeLa cells (Talman et al. 2011). Effects of drug treatments were normalized to vehicle treatment, which was set to 100%. HMI-1a3 inhibited HeLa cell proliferation in a concentration-dependent manner: proliferation was totally blocked at concentrations ≥10 µM. Surprisingly, neither inhibition/activation of PKC, nor inhibition of MEK1/2, had any effect on the cytotoxic response to HMI-1a3 (n=3). Furthermore, exposure of HeLa cells to Gö6983 alone had no effect on their proliferation or morphology, and exposure to Gö6983 did not alter the HMI-1a3-evoked response (n=3). Both siRNA transfections reduced the expression of the particular PKC isoform by approximately 90% and slowed down HeLa cell proliferation by 25-50%. Despite the slower proliferation rate of the siRNA-transfected cells, down-regulation of PKCα or PKCδ expression had no influence on the HMI-1a3-evoked concentration-dependent inhibitory response in proliferation or on morphological changes evoked by the isophthalate (n=3). In conclusion, these data suggest that the HMI-1a3-induced cytotoxicity, cell elongation and cell cycle arrest in HeLa cells are not mediated by PKC or ERK cascades.

References:

Boije af Gennäs G, Talman V, Aitio O, Ekokoski E, Finel M, Tuominen RK, Yli-Kauhaluoma J (2009) Design, synthesis and biological activity of isophthalic acid derivatives targeted to the C1 domain of PKC. J Med Chem. 52(13):3969-81.

Talman V, Tuominen RK, Boije af Gennäs G, Yli-Kauhaluoma J, Ekokoski E (2011) C1 domain-targeted Isophthalate Derivatives Induce Cell Elongation and Cell Cycle Arrest in HeLa Cells. PLoS ONE 6(5): e20053. doi:10.1371/journal.pone.0020053