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

Abstract

BACKGROUND:

Combination 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.

METHODS:

Cell 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.

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 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.

CONCLUSIONS:

These 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.

• Authors: Kang-Sik Seo, Jong-Seok Kim, Ji-Hoon Park, Kyoung-Sub Song, Eun-Jin Yun, Jong-Il Park, Gi Ryang Kweon, Wan-Hee Yoon, Kyu Lim, Byung-Doo Hwang
• Journal: BMC cancer (BMC Cancer) Vol. 14 Pg. 36 (Jan 22 2014) ISSN: 1471-2407 [Electronic] England
• PMID: 24447339 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Bridged Bicyclo Compounds, Heterocyclic
• Protein Kinase Inhibitors
• Tubulin
• Tubulin Modulators
• apicularen A
• PRKCA protein, human
• Protein Kinase C-alpha
• CASP3 protein, human
• Caspase 3
• Tetradecanoylphorbol Acetate

Topics

• Antineoplastic Combined Chemotherapy Protocols (pharmacology)
• Apoptosis (drug effects)
• Bridged Bicyclo Compounds, Heterocyclic (pharmacology)
• Caspase 3 (metabolism)
• Cell Cycle Checkpoints
• Cell Survival (drug effects)
• Drug Synergism
• Female
• HeLa Cells
• Humans
• Microtubules (drug effects, genetics, metabolism, pathology)
• Protein Kinase C-alpha (antagonists & inhibitors, genetics, metabolism)
• Protein Kinase Inhibitors (pharmacology)
• RNA Interference
• Tetradecanoylphorbol Acetate (pharmacology)
• Time Factors
• Transfection
• Tubulin (genetics, metabolism)
• Tubulin Modulators (pharmacology)
• Uterine Cervical Neoplasms (genetics, metabolism, pathology)