Cell viability was assessed by measuring the absorbance at 570?nm in an ELISA plate reader. DNA fragmentation assay The cells were lysed using buffer IgM Isotype Control antibody (PE-Cy5) containing 300?mM TrisCHCl (pH?7.5), 100?mM NaCl, 10?mM EDTA, 200?mM sucrose and 0.5% SDS. after staining with propidium iodide (PI). The expression levels of target proteins were measured by Western blotting analysis using specific antibodies, and -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, -tubulin protein and nuclei were visualized by immunofluorescence staining using an anti–tubulin antibody and PI, respectively. Results We 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 PKC, but not PKC and PKC, inhibited cytotoxicity induced by combination PMA and apicularen A. PMA increased the apicularen A-induced disruption of microtubule networks by further decreasing – Tiagabine and -tubulin protein levels in a PKC-dependent manner. Conclusions These results suggest that the synergy between PMA and apicularen A is involved by PKC activation and microtubule disruption, and that may inform the development of novel approaches to treat cancer. alkaloids inhibit tumor cell proliferation by inducing the depolymerizaiton of microtubules , and taxanes induce apoptosis by promoting microtubule assembly . Apicularen A disrupts microtubule networks by inhibiting tubulin synthesis . Efforts to develop more effective cancer therapy combinations with microtubule-interfering agents are underway. The finding that PMA increases the antitumor activity of paclitaxel, a chemotherapeutic agent that inhibits tubulin polymerization, and in a xenograft model of prostate cancer  prompted us to test whether PMA increases Tiagabine apicularen A-induced cell death. The results of the present study demonstrate that PMA-mediated PKC activation strongly increases apicularen A-induced apoptotic cell death and disruption of microtubule networks in HeLa cells. Methods Cell culture Human HeLa cervical cancer cells (ATCC, Rockville, MD) were cultured in Dulbeccos modified Eagles medium supplemented with 10% fetal bovine serum and antibiotics. Cells were maintained at 37C, 5% CO2 and 95% air. Antibodies and chemicals Apicularen A was provided by Dr. Ahn (Division of Ocean Science, Korea Maritime University, Busan, Korea) and dissolved in dimethyl sulfoxide. Phorbol 12-myristate 13-acetate (PMA), thiazolyl blue tetrazolium bromide (MTT), anti–tubulin and anti–tubulin antibodies were purchased from Sigma (St Louis, MO, USA). Anti-PARP and anti-actin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-caspase-3 antibody was purchased from R&D Systems (Wiesbaden, Germany). Z-VAD-fmk, Ro31-8220 and Go6983 were purchased from Calbiochem (San Diego, CA, USA). All other reagents were molecular biology grade. Cell viability assay Cell viability was assessed by thiazolyl blue tetrazolium (MTT) assay. Exponentially growing cells were exposed to apicularen A in the presence or absence of PMA for 24 and 48 hours. MTT solution was added to each well (0.5?mg/ml) and incubated for 2 hours. Cell viability was assessed by measuring the absorbance at 570?nm in an ELISA plate reader. DNA fragmentation assay The cells were lysed using buffer containing 300?mM TrisCHCl (pH?7.5), 100?mM NaCl, 10?mM EDTA, 200?mM sucrose and 0.5% SDS. Intracellular DNA was extracted with phenol/chloroform (1:1) and chloroform/isoamylalcohol (24:1). DNA was precipitated and digested in 10?mM TrisCHCl (pH?8.0), 1?mM EDTA and 40?g/ml RNase A for 1 hour at 37C. Then, DNA (10?g) was resolved by electrophoresis in a 1.2% agarose gel supplemented with ethidium bromide (0.2?g/ml), and Tiagabine DNA fragmentation was examined by.