Yonsei Med J.  2010 Sep;51(5):708-716. 10.3349/ymj.2010.51.5.708.

Pseudomonas aeruginosa Exotoxin A Reduces Chemoresistance of Oral Squamous Carcinoma Cell via Inhibition of Heat Shock Proteins 70 (HSP70)

  • 1Department of Oral Anatomy, School of Dentistry, Research Institute for Oral Biotechnology, Pusan National University, Yangsan, Korea. ki91000m@pusan.ac.kr
  • 2Department of Anatomy, College of Medicine, Kosin University, Busan, Korea.
  • 3Department of Oral and Maxillofacial Surgery, School of Dentistry, Research Institute for Oral Biotechnology, Pusan National University, Yangsan, Korea.
  • 4Basic Research Laboratory, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA.


Oral squamous carcinoma (OSCC) cells exhibit resistance to chemotherapeutic agent-mediated apoptosis in the late stage of malignancy. Increased levels of heat shock proteins 70 (HSP70) in cancer cells are known to confer resistance to apoptosis. Since recent advances in the understanding of bacterial toxins have produced new strategies for the treatment of cancers, we investigated the effect of Pseudomonas aeruginosa exotoxin A (PEA) on HSP70 expression and induction of apoptosis in chemoresistant OSCC cell line (YD-9).
The apoptotic effect of PEA on chemoresistant YD-9 cells was confirmed by MTT, Hoechst and TUNEL stains, DNA electrophoresis, and Western blot analysis.
While YD-9 cells showed high resistance to chemotherapeutic agents such as etoposide and 5-fluorouraci (5-FU), HSP70 antisense oligonucelotides sensitized chemoresistant YD-9 cells to etoposide and 5-FU. On the other hand, PEA significantly decreased the viability of YD-9 cells by deteriorating the HSP70-relating protecting system through inhibition of HSP70 expression and inducing apoptosis in YD-9 cells. Apoptotic manifestations were evidenced by changes in nuclear morphology, generation of DNA fragmentation, and activation of caspases. While p53, p21, and E2F-1 were upregulated, cdk2 and cyclin B were downregulated by PEA treatment, suggesting that PEA caused cell cycle arrest at the G2/M checkpoint.
Therefore, these results indicate that PEA reduced the chemoresistance through inhibition of HSP70 expression and also induced apoptosis in chemoresistant YD-9 cells.


Pseudomonas aeruginosa exotoxin A; chemoresistance; oral squamous cell carcinoma cells; heat shock protein 70; apoptosis

MeSH Terms

ADP Ribose Transferases/*pharmacology
Antineoplastic Agents/*pharmacology
Apoptosis/drug effects
Bacterial Toxins/*pharmacology
Blotting, Western
Carcinoma, Squamous Cell/drug therapy/*metabolism
Cell Cycle/drug effects
Cell Line, Tumor
Chromatography, Liquid
Cyclin B/metabolism
Cyclin-Dependent Kinase 2/metabolism
Drug Resistance, Neoplasm/*drug effects
E2F1 Transcription Factor/metabolism
HSP70 Heat-Shock Proteins/genetics/*metabolism
In Situ Nick-End Labeling
Mouth Neoplasms/drug therapy/*metabolism
Tandem Mass Spectrometry
Tumor Suppressor Protein p53/metabolism
Virulence Factors/*pharmacology


  • Fig. 1 Effects of PEA treatment on the viability of chemoresistant YD-9 cells. (A) After 24 h incubation with etoposide (2 - 10 µg/mL) and (B) 5-FU (1 - 2,000 µM), cell viabilities were determined by MTT assay. YD-9 cell line showed resistance to etoposide and 5-FU, (C) whereas PEA significantly decreased the proliferation of YD-9 cells. Four independent assays were performed and data shown are the mean ± SD of the means obtained from triplicates of each assay.

  • Fig. 2 Detection of HSP70 and the effect of PEA on HSP70 expression in YD-9 cells. (A) After incubation with15 nM PEA for indicated periods, identification of differentially expressed proteins using LC-MS/MS indicated HSP70. (B) Western blot for HSP70 from YD-9 and HGF-1 cells was performed. The expression level of HSP70 in YD-9 cells was higher than HGF-1 cells. (C) After incubation with indicated doses of PEA for 24 hours, Western blot analysis showed a decrease of HSP70 expression. (D) After incubation with 15 nM of PEA for indicated time periods, Western blot analysis showed a time-dependent continual decrease in HSP70 expression.

  • Fig. 3 Restoration of sensitivity to etoposide and 5-FU by inhibition of HSP70. (A) After incubation with HSP70 antisense oligonucleotide for 24 hours as described in the material and methods section, YD-9 cells were treated with various concentrations of etoposide and 5-FU. (B) After incubation with 15 nM PEA for 24 hours, YD-9 cells were treated with various concentrations of etoposide and 5-FU. Cell viabilities were determined by an MTT assay. Four independent assays were performed and data shown are the mean ± SD of the means obtained from triplicates of each assay.

  • Fig. 4 Demonstration of apoptosis in YD-9 cells treated with 15 nM PEA. (A) Nuclear condensation and fragmentation were clearly shown at 24 hours after treatment with 15 nM PEA. (B) A TUNEL assay showed apoptotic cells in YD-9 cells treated with PEA. (C) DNA electrophoresis showed a DNA ladder in YD-9 cells treated with PEA.

  • Fig. 5 Demonstration of PEA induced the cleavage of caspase-3, capase-6, DFF45, and lamin A. (A) Pro-form of caspase-3 (32 kDa) was degraded, and 17 kDa and 12 kDa cleaved forms were produced after PEA treatment. Caspase-6 pro-enzyme (34 kDa) was degraded, and 16 kDa cleaved forms were produced after PEA treatment. (B) Three cleaved products of DFF45 (30, 20, and 11 kDa) were demonstrated in YD-9 cells treated with PEA, which are evident in later time points (48 and 72 hours). Two cleaved products of lamin A (45 and 28 kDa) are demonstrated in YD-9 cells treated with PEA, which are shown at 24 hours after PEA treatment.

  • Fig. 6 Involvement of E2F-1, Apaf-1, and caspase-9 in PEA-induced apoptosis. YD-9 cells were cultured in the presence of 15 nM PEA for the indicated time, and whole cell lysates were subjected to Western blot analysis of E2F-1 (56 kDa), Apaf-1 (130 kDa), and caspase-9 (full-length, 49 kDa; cleaved, 37 kDa).

  • Fig. 7 Western blot analysis of cell cycle regulation proteins in YD-9 cells treated with 15 nM PEA. YD-9 cells were treated with 15 nM PEA for the indicated time. Total cellular lysate proteins were resolved by SDS-PASE, transferred to nitrocellulose membranes, and probed for p53, p21, cdk1, and cyclin B.


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