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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

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.
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Figure 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.

Mentions: We examined whether the reduced viability of YD-9 cells was caused by apoptosis. Hoechst staining showed nuclear condensation and fragmentation in YD-9 cells after 24 hours treatment of 15 nM PEA (Fig. 4A). In the TUNEL assay, a large number of cells treated with 15 nM PEA for 24 hours exhibited a positive response (Fig. 4B). Cells treated with 15 nM PEA showed DNA degradation characteristics of an apoptotic ladder (Fig. 4C). To examine if the apoptosis was mediated by caspases, we carried out Western blotting experiments which indicated that PEA activated two effectors, caspases caspase-3 and -6. In addition to the degradation of caspase-3 and -6, cleaved products of each caspase were obtained (Fig. 5A). In the experiment to study the effects of PEA on caspase target proteins such as DFF45 and lamin A, the cleaved products of DFF45 (30 and 11 kDa) and lamin A (45 and 28 kDa) were observed (Fig. 5B).


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)

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

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

Figure 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.
Mentions: We examined whether the reduced viability of YD-9 cells was caused by apoptosis. Hoechst staining showed nuclear condensation and fragmentation in YD-9 cells after 24 hours treatment of 15 nM PEA (Fig. 4A). In the TUNEL assay, a large number of cells treated with 15 nM PEA for 24 hours exhibited a positive response (Fig. 4B). Cells treated with 15 nM PEA showed DNA degradation characteristics of an apoptotic ladder (Fig. 4C). To examine if the apoptosis was mediated by caspases, we carried out Western blotting experiments which indicated that PEA activated two effectors, caspases caspase-3 and -6. In addition to the degradation of caspase-3 and -6, cleaved products of each caspase were obtained (Fig. 5A). In the experiment to study the effects of PEA on caspase target proteins such as DFF45 and lamin A, the cleaved products of DFF45 (30 and 11 kDa) and lamin A (45 and 28 kDa) were observed (Fig. 5B).

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