PMA synergistically enhances apicularen A-induced cytotoxicity by disrupting microtubule networks in HeLa cells

Abstract

BackgroundCombination therapy is key to improving cancer treatment efficacy. Phorbol 12-myristate 13-acetate (PMA), a well-known PKC activator, increases the cytotoxicity of several anticancer drugs. Apicularen A induces cytotoxicity in tumor cells through disrupting microtubule networks by tubulin down-regulation. In this study, we examined whether PMA increases apicularen A-induced cytotoxicity in HeLa cells.MethodsCell viability was examined by thiazolyl blue tetrazolium (MTT) assays. To investigate apoptotic potential of apicularen A, DNA fragmentation assays were performed followed by extracting genomic DNA, and caspase-3 activity assays were performed by fluorescence assays using fluorogenic substrate. The cell cycle distribution induced by combination with PMA and apicularen A was examined by flow cytometry after staining with propidium iodide (PI). The expression levels of target proteins were measured by Western blotting analysis using specific antibodies, and α-tubulin mRNA levels were assessed by reverse transcription polymerase chain reaction (RT-PCR). To examine the effect of combination of PMA and apicularen A on the microtubule architecture, α-tubulin protein and nuclei were visualized by immunofluorescence staining using an anti-α-tubulin antibody and PI, respectively.ResultsWe found that apicularen A induced caspase-dependent apoptosis in HeLa cells. PMA synergistically increased cytotoxicity and apoptotic sub-G1 population induced by apicularen A. These effects were completely blocked by the PKC inhibitors Ro31-8220 and Go6983, while caspase inhibition by Z-VAD-fmk did not prevent cytotoxicity. RNA interference using siRNA against PKCα, but not PKCβ and PKCγ, inhibited cytotoxicity induced by combination PMA and apicularen A. PMA increased the apicularen A-induced disruption of microtubule networks by further decreasing α- and β-tubulin protein levels in a PKC-dependent manner.ConclusionsThese results suggest that the synergy between PMA and apicularen A is involved by PKCα activation and microtubule disruption, and that may inform the development of novel approaches to treat cancer.

Sphingosine kinase-2 Inhibitor ABC294640 Enhances Doxorubicin-Induced Apoptosis of NSCLC Cells via Altering Survivin Expression

“…In the current study, PMA increased cell population in S phase and decreased Sub G1 phase of the cell cycle by combining with DOX which may be the reason for the observed decrease in DOXinduced cell death in A549 cells (as the another NSCLC cell line). However, this is in contrast to few others where they have shown synergistic effects between PMA and anticancer agents in the induction of cytotoxicity accompanied by an increase in apoptotic sub-G1population [35].…”

“…A large body of evidence has indicated that PMA can exhibit either proliferative or anti-proliferative effects via the differential ability of protein kinase C (PKC) isoforms in regulation of the cell cycle [34]. Stimulation of PKC by PMA, can alter anticancer drug effect depending on the type of chemotherapeutic agent, phase of the cells in cell cycle at the time of activation, the specific PKC isoforms activated, and the type of cell lines [35,36]. Exposure of a panel of human NSCLC cells (e. g. H358, H441 and H322 cells) to PMA has been shown to cause distinctive responses of the cells for arrest or progression into the different phase of the cell cycle depending on which phase in the cell cycle and/or which PKC isoforms becomes activated [34].…”

Extracellular DNA traps in a ctenophore demonstrate immune cell behaviors in a non-bilaterian

“…Importantly, exposure to PMA can also trigger non-ETotic cell death pathways 60 , raising the possibility that the extracellular DNA we observed was a result of cell rupture and release of DNA secondary to cell death. To distinguish between these possible scenarios, we used a combination of two DNA dyes: Hoechst, which is a membrane-permeable dye that labels DNA in both live cells and dead cells, and SytoxGreen, a non-permeable cell stain that selectively labels DNA in cells with compromised cell membranes, as observed in cells undergoing cell death (Fig.…”

Extracellular DNA traps in a ctenophore demonstrate conserved immune cell behaviors in a non-bilaterian

“…Importantly, exposure to PMA can also trigger non-ETotic cell death pathways 58 , raising the possibility that the extracellular DNA we observed was a result of cell rupture and release of DNA secondary to cell death. To distinguish between these possible scenarios, we used a combination of two DNA dyes: Hoechst, which is a membrane-permeable dye that labels DNA in both live cells and dead cells, and SytoxGreen, a non-permeable cell stain that selectively labels DNA in cells with compromised cell membranes, as observed in cells undergoing cell death ( Fig.…”