**6. Molecular chaperone 90kD heat shock protein**

As reviewed above, several ways to potentiate the cytotoxicity of TMZ have been reported, and thus many pathways can be targeted in an effort to sensitize tumor cells to chemotherapeutic agents. A molecular chaperone, 90kD heat shock protein (hsp90) has recently attracted attention as a sensitizing agent because it is expressed at 2–10-fold higher levels in tumor tissue than in normal tissue, and is associated with many proteins (termed client proteins) involved in cell cycle regulation, cell survival and oncogenesis. Taken together, these studies indicated that many hsp90 client proteins are involved in cytoprotective mechanisms against cellular stressors such as DNA damage, suggesting that hsp90 might be important in the survival of tumor cells after exposure to DNA-damaging chemotherapeutic agents. Therefore we hypothesized that hsp90 inhibitors might act as antitumor agents against gliomas and might potentiate the cytotoxicity of DNA-damaging agents.

To investigate this hypothesis, we used 17-AAG, a geldanamycin derivative, as a pharmacological inhibitor of hsp90 and examined whether an hsp90-targeted strategy could be useful for chemosensitizing glioma cells to the DNA-damaging agents BCNU, cisplatin, and TMZ (Ohba et al., 2010).

The clonogenicity of cells treated with cisplatin, BCNU, or TMZ was depressed with 17- AAG. This 17-AAG–induced potentiation of the effects of these chemotherapeutic agents was recognized at a lower concentration than that needed to induce cytotoxicity with 17- AAG alone, and was more remarkable in the cisplatin- and BCNU-treated cells than in the

Biological Responses of Glioma Cell to Chemotherapeutic Agents 75

tumor cells has a 100-fold higher binding affinity for the hsp90 inhibitor 17-AAG than does hsp90 derived from normal cells. These data suggest that the effect of hsp90 inhibitors on normal cells may be much smaller than that on tumor cells and that an hsp90 inhibitor

The mechanism of the 17-AAG–induced enhancement of the cytotoxicity of DNAcrosslinking agents has not yet been completely elucidated, and suppression of other survival-promoting factor(s) could be involved in enhancement of DNA-damaging agents,

We reviewed our studies focusing chemosensitization of gliomas. We propose that combination of conventional chemotherapy using DNA-damaging agents and molecular targeted therapy could be a potentially useful new antiglioma therapeutic strategy. However, enhancement of the effect of chemotherapeutic agents clearly depends on the mechanism by these compounds exhibit cytotoxicity. Therefore the development of a safe

Hirose, Y., Berger, M. S. & Pieper, R. O. (2001a). p53 Effects Both the Duration of G2/M

Hirose, Y., Berger, M. S. & Pieper, R. O. (2001b). Abrogation of the Chk1-mediated G2

Hirose, Y., Katayama, M., Stokoe, D., Haas-Kogan, D. A., Berger, M. S. & Pieper, R. O.

Hirose, Y., Katayama, M., Mirzoeva, O. K., Berger, M. S. & Pieper, R. O. (2005). Akt

Ohba. S., Hirose, Y., Kawase, T. & Sano, H. (2009). Inhibition of c-Jun N-terminal kinase

*Neuro-oncology,* Vol.95, No.3 (December 2009), pp307-316, ISSN 0167-594X Ohba, S., Hirose, Y., Yoshida, K., Yazaki, T. & Kawase, T. (2010). Inhibition of 90-kD heat

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Arrest and the Fate of Temozolomide-treated Human Glioblastoma Cells. *Cancer* 

Checkpoint Pathway Potentiates Temozolomide-induced Toxicity in a p53 independent manner in Human Glioblastoma Cells. *Cancer Res*earch Vol. 61, No.15

(2003). The p38 Mitogen-activated Protein Kinase Pathway Links the DNA Mismatch Repair System to the G2 Checkpoint and to Resistance to Chemotherapeutic DNA-methylating Agents. *Molecular and Cellular Biology,* Vol.23,

Activation Suppresses Chk2-mediated, Methylating Agent-induced G2 Arrest and Protects from Temozolomide-induced Mitotic Catastrophe and Cellular Senescence. *Cancer Research* Vol.65, No.11 (June 2005), pp4861-4869, ISSN 0008-

enhances temozolomide-induced cytotoxicity in human glioma cells. *Journal of* 

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might therefore be useful for selectively killing tumor cells.

and effective therapeutic regimen will require further investigation

(August 2001), pp5843-6849, ISSN 0008-5472

No.22 (November 2003), pp8306-8315, ISSN 0270-7306

however.

**7. Conclusion** 

**8. References** 

5472

0022-3085

TMZ-treated cells. Isobologram analyses revealed that the interactions between 17-AAG and cisplatin or BCNU were synergistic, whereas the interaction between 17-AAG and TMZ was no more than additive (Figure 9).

The FACS analyses revealed that the population of cells with a sub-G1 DNA content was increased by combined treatment with 17-AAG and either cisplatin or BCNU; furthermore, the combined treatment remarkably increased the number of annexin V–positive and PInegative cells. These results demonstrated that the 17-AAG– induced enhancement of the DNA crosslinking agents–induced cytotoxicity was either associated in part or entirely with an increase in apoptotic cell death.

Fig. 9. Effect of Hsp90 inhibitor 17-AAG on cytotoxicity induced by various DNA damaging agents.

A reasonable concern is that hsp90 inhibitors may act not only on tumor cells, but also on normal cells; however, the authors of previous studies have shown that hsp90 is expressed at 2–10-fold higher levels in tumor than in normal tissue, and that hsp90 derived from tumor cells has a 100-fold higher binding affinity for the hsp90 inhibitor 17-AAG than does hsp90 derived from normal cells. These data suggest that the effect of hsp90 inhibitors on normal cells may be much smaller than that on tumor cells and that an hsp90 inhibitor might therefore be useful for selectively killing tumor cells.

The mechanism of the 17-AAG–induced enhancement of the cytotoxicity of DNAcrosslinking agents has not yet been completely elucidated, and suppression of other survival-promoting factor(s) could be involved in enhancement of DNA-damaging agents, however.
