Dataset Information

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 ?-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 ?- 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.

SUBMITTER: Seo KS

PROVIDER: S-EPMC3901760 | biostudies-literature | 2014 Jan

REPOSITORIES: biostudies-literature

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Publications

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

Seo Kang-Sik KS Kim Jong-Seok JS Park Ji-Hoon JH Song Kyoung-Sub KS Yun Eun-Jin EJ Park Jong-Il JI Kweon Gi Ryang GR Yoon Wan-Hee WH Lim Kyu K Hwang Byung-Doo BD

BMC cancer 20140122

<h4>Background</h4>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.<h4>Methods</h4>Cell viability was examined by thiazolyl blue tetrazolium (MT ...[more]

PMID: 24447339

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Project description:BackgroundMammalian target of rapamycin (mTOR), involved in PI3K/AKT/mTOR pathway, is known to play a central role in regulating the growth of cancer cells. The PI3K/AKT/mTOR pathway enhances tumor survival and proliferation through suppressing autophagy, which sustains energy homeostasis by collecting and recycling cellular components under stress conditions. Conversely, inhibitors of the mTOR pathway such as RAD001 induce autophagy, leading to promotion of tumor survival and limited antitumor efficacy. We thus hypothesized that the use of autophagy inhibitor in combination with mTOR inhibition improves the cytotoxicity of mTOR inhibitors in bladder cancer.Materials and methodsThe cytotoxicity of RT4, 5637, HT1376, and T24 human bladder cancer cells treated with RAD001 alone or combined with autophagy inhibitors (3-methyladenine (3-MA), bafilomycin A1 (Baf A1), chloroquine, or hydroxychloroquine) was assessed using the WST-8 cell viability kit. The autophagy status in cells was analyzed by the detection of microtubule-associated light chain 3 form II (LC3-II), using immunofluorescent staining and Western blot. Acidic vesicular organelle (AVO) formation in treated cells was determined by acridine orange vital staining. Inhibition of mTOR pathway by RAD001 was monitored by using a homemade quantitative polymerase chain reaction gene array, while phospho-mTOR was detected using Western blot. Induced apoptosis was determined by measurement of caspase 3/7 activity and DNA fragmentation in cells after treatment.ResultsAdvanced bladder cancer cells (5637, HT1376, and T24) were more resistant to RAD001 than RT4. Autophagy flux detected by the expression of LC3-II showed RAD001-induced autophagy. AVO formation was detected in cells treated with RAD001 and was inhibited by the addition of 3-MA or Baf A1. Cotreatment of RAD001 with autophagy inhibitors further reduced cell viability and induced apoptosis in bladder cancer cells.ConclusionOur results indicate that simultaneous inhibition of the mTOR and autophagy pathway significantly enhances apoptosis, and it is suggested to be a new therapeutic paradigm for the treatment of bladder cancer.

Dataset Information

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

Background

Methods

Results

Conclusions

Publications

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

Similar Datasets

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