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Pseudomonas aeruginosa exotoxin A reduces chemoresistance of oral squamous carcinoma cell via inhibition of heat shock proteins 70 (HSP70).

Park SR, Lee KD, Kim UK, Gil YG, Oh KS, Park BS, Kim GC - Yonsei Med. J. (2010)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Oral Anatomy, School of Dentistry, Research Institute for Oral Biotechnology, Pusan National University, Yangsan, Korea.

ABSTRACT

Purpose: 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).

Materials and methods: The apoptotic effect of PEA on chemoresistant YD-9 cells was confirmed by MTT, Hoechst and TUNEL stains, DNA electrophoresis, and Western blot analysis.

Results: 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.

Conclusion: Therefore, these results indicate that PEA reduced the chemoresistance through inhibition of HSP70 expression and also induced apoptosis in chemoresistant YD-9 cells.

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Related in: MedlinePlus

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.
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Figure 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.

Mentions: In an attempt to identify changes of protein expression by PEA treatment, identification of proteins using LC-MS/MS was performed. Among many differentially expressed proteins, HSP70 was identified and exhibited a significant decrease in response to PEA treatment (Fig. 2A). The level of HSP70 expression was compared by Western blotting on YD-9 and human normal gingival fibroblast (HGF-1). The result illustrated in Fig. 2B showed an increase in HSP70 expression in YD-9 cells. Densiometric analysis indicated that HSP70 expression was five times higher in YD-9 compared to what is obtained from HGF-1. Overexpression of HSP70 decreased in various concentrations of PEA after 24 hours incubation (Fig. 2C) and at various time points after incubation with 15 nM PEA (Fig. 2D).


Pseudomonas aeruginosa exotoxin A reduces chemoresistance of oral squamous carcinoma cell via inhibition of heat shock proteins 70 (HSP70).

Park SR, Lee KD, Kim UK, Gil YG, Oh KS, Park BS, Kim GC - Yonsei Med. J. (2010)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2908850&req=5

Figure 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.
Mentions: In an attempt to identify changes of protein expression by PEA treatment, identification of proteins using LC-MS/MS was performed. Among many differentially expressed proteins, HSP70 was identified and exhibited a significant decrease in response to PEA treatment (Fig. 2A). The level of HSP70 expression was compared by Western blotting on YD-9 and human normal gingival fibroblast (HGF-1). The result illustrated in Fig. 2B showed an increase in HSP70 expression in YD-9 cells. Densiometric analysis indicated that HSP70 expression was five times higher in YD-9 compared to what is obtained from HGF-1. Overexpression of HSP70 decreased in various concentrations of PEA after 24 hours incubation (Fig. 2C) and at various time points after incubation with 15 nM PEA (Fig. 2D).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Oral Anatomy, School of Dentistry, Research Institute for Oral Biotechnology, Pusan National University, Yangsan, Korea.

ABSTRACT

Purpose: 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).

Materials and methods: The apoptotic effect of PEA on chemoresistant YD-9 cells was confirmed by MTT, Hoechst and TUNEL stains, DNA electrophoresis, and Western blot analysis.

Results: 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.

Conclusion: Therefore, these results indicate that PEA reduced the chemoresistance through inhibition of HSP70 expression and also induced apoptosis in chemoresistant YD-9 cells.

Show MeSH
Related in: MedlinePlus