Yonsei Med J.  2011 Sep;52(5):773-778. 10.3349/ymj.2011.52.5.773.

The Bacterial Protein Azurin Enhances Sensitivity of Oral Squamous Carcinoma Cells to Anticancer Drugs

• Choi JH
• Lee MH
• Cho YJ
• Park BS
• Kim S
• Kim GC

• KMID: 1108070
• DOI: http://doi.org/10.3349/ymj.2011.52.5.773

Abstract

PURPOSE
Surgical therapy is the primary treatment for oral cancer, but it can cause facial distortion. Therefore, if anticancer drugs are effective against oral cancer, they may be used preferentially. However, oral squamous carcinoma cells (OSCCs) are resistant to these drugs, so finding a way to enhance the sensitivity of these cells to anticancer drugs is important. The bacterial protein azurin is known to selectively enter cancer cells and induce apoptosis. In this study, we show the anticancer effect of azurin in OSCC.
MATERIALS AND METHODS
OSCC cell line (YD-9) was subjected to azurin treatment. Cell viability, morphology and protein expression levels were monitored after treatment of azurin. Cells were also subjected to combination treatment of azurin with either 5-fluorouracil or etopside.
RESULTS
Azurin-treated cells showed decreased cell viability accompanied by apoptotic phenotypes including morphological change, DNA breakage, and increases in p53 and cyclin B1 protein levels. Combination treatment of azurin with other anti-tumor agents caused an increase in sensitivity to anticancer drugs in azurin-treated YD-9 cells.
CONCLUSION
Azurin has a strong synergistic anticancer effect on oral cancer cells when it is used along with anticancer drugs.

Keyword

Azurin; oral squamous carcinoma; osteosarcoma; 5-fluorouracil; etoposide

MeSH Terms

Antineoplastic Agents/*administration & dosage
Apoptosis/drug effects
Azurin/*administration & dosage/genetics
Carcinoma, Squamous Cell/*drug therapy/metabolism/pathology
Cell Line, Tumor
Cyclin B1/metabolism
Drug Synergism
Etoposide/administration & dosage
Fluorouracil/administration & dosage
Humans
Mouth Neoplasms/*drug therapy/metabolism/pathology
Tumor Suppressor Protein p53/metabolism

Figure

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  Fig. 1 Extraction and purification of azurin from bacteria: (A) A schematic diagram of GST-tagged full length azurin (B) Expression of purified azurin protein: GST-azurin protein was amplified in bacteria and purified, as indicated in Materials and Methods. M: protein marker.

• 

  Fig. 2 Azurin inhibits the viability of YD-9 cells. (A) YD-9 cells were treated with various concentrations of azurin as indicated. After 48 h of incubation, cell growth was tested. Data shown are the average of 4 independent experiments, with a mean value of p<0.03. (B) YD-9 and MG-63 cells were treated with 200 µg/mL azurin and incubated for 24, 48, and 72 h as indicated, and cell viability was tested. Mean value, p<0.04.

• 

  Fig. 3 Azurin triggers apoptosis of YD-9 cells. Morphological changes in YD-9 cells after 24 h incubation in the presence or absence of 200 µg/mL azurin, as observed with simple microscopy (A) and after cell staining with hemacolor (B) or Hoechst 33352 (C). (D) DNA fragmentation of 200 µg/mL azurin-treated cells occurred in a time-dependent manner. (E) YD-9 cells were exposed to 200 µg/mL azurin for indicated times. Total protein (30 µg) was subjected to SDS-PAGE and western blotting. Data shown is the representative of 3 independent experiments. M, DNA marker; C, Control (lysate of untreated cells).

• 

  Fig. 4 Azurin enhances sensitivity of YD-9 and MG-63 cells to 5-fluorouracil and etoposide. YD-9 and MG-63 cells were treated with 10-1,000 µM of 5-fluorouracil (5-FU) (A) or 2-10 µg/mL etoposide (B) along or not along with 200 µg/mL azurin as indicated for 24 h and subjected to a cell growth assay. Data shown are representative of 4 independent experiments, with p<0.02.

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