Jin Hee Shin

Histone Deacetylase Inhibitor–Mediated Radiosensitization of Human Cancer Cells: Class Differences and the Potential Influence of p53

Histone deacetylase inhibitors (HDI) are emerging as potentially useful components of the anticancer armamentarium and as useful tools to dissect mechanistic pathways. HDIs that globally inhibit histone deacetylases (HDAC) have radiosensitizing effects, but the relative contribution of specific HDAC classes remains unclear. Newly characterized HDIs are now available that preferentially inhibit specific HDAC classes, including SK7041 (inhibits class I HDACs) and splitomicin (inhibits class III HDACs). We investigated in human cancer cells the relative radiosensitizations that result from blocking specific HDAC classes. We found that trichostatin A (TSA; inhibitor of both class I and II HDACs) was the most effective radiosensitizer, followed by the class I inhibitor SK7041, whereas splitomicin (inhibitor of class III) had least effect. Interestingly, radiosensitization by TSA in cell lines expressing p53 was more pronounced than in isogenic lines lacking p53. Radiosensitization of cells expressing p53 by TSA was reduced by pifithrin-α, a small-molecule inhibitor of p53. In contrast, the radiosensitization by TSA of cells expressing low levels of p53 was enhanced by transfection of wild-type p53–expressing vector or pretreatment with leptomycin B, an inhibitor of nuclear export that increased intracellular levels of p53. These effects on radiosensitization were respectively muted or not seen in cells treated with SK7041 or splitomicin. To our knowledge, this may be among the first systematic investigations of the comparative anticancer effects of inhibiting specific classes of HDACs, with results suggesting differences in the degrees of radiosensitization, which in some cell lines may be influenced by p53 expression.

Epigenetic modulation of radiation response in human cancer cells with activated EGFR or HER-2 signaling: Potential role of histone deacetylase 6

BACKGROUND AND PURPOSE Histone deacetylase inhibitors (HDIs) are prototypes of agents targeting epigenetic modifications and have received considerable attention for their promise as targeted anticancer drugs. We examined the effects and potential mechanism(s) of combining LBH589 and irradiation in human cancer cells having activated EGFR or HER-2 signaling, focusing on the role of HDAC6. METHODS AND MATERIALS We evaluated whether the HDI, LBH589, would radiosensitize a panel of human tumor cell lines having activated EGFR or HER-2 signaling. A mechanistic role for the HDAC6 isotype was investigated using RNA interference and ectopic overexpression HDAC6. RESULTS The HDI, LBH589, enhanced the radiosensitivity of the human carcinoma cell lines we tested. Radiosensitization was accompanied by abrogation of radiation-induced G2/M arrest and was associated with aberrant mitotic features and prolonged gammaH2AX foci. Radiation-induced apoptosis was also increased. LBH589 radiosensitized cells with activated EGFR or HER-2 signaling to a greater degree than the HDIs SK7041 or TSA. However radiosensitization by the three HDI was equivalent in cells without activation of this signaling. LBH589 led acetylation of histone H3 and HSP90. This was associated with down-regulation of the client oncoproteins EGFR, HER-2, and decreased phosphorylation of Akt and ERK. Specific inhibition of HDAC6 by RNAi increased radiosensitivity as well as increasing acetylation of HSP90 and reducing the association of HSP90 with its client proteins. Conversely, ectopic overexpression of HDAC6 isotype increased the levels of p-EGFR and p-AKT expression, and reduced LBH589-mediated radiosensitization. CONCLUSIONS These findings define a unique mechanism for counteracting pro-survival signaling from EGFR or HER-2 that is present in many tumor cells.

Enhancement of Radiation Effects by Flavopiridol in Uterine Cervix Cancer Cells.

PURPOSE To determine the effects of combinations of radiation and flavopiridol, an inhibitor of cyclin-dependent kinases and global transcription, in a human uterine cervix cancer cell line. MATERIALS AND METHODS Human uterine cervix cancer cells (HeLa), cultured to the mid-log phase, were exposed to X-rays, flavopiridol, and combinations of X-rays and flavopiridol in various sequences. The end point in this study was the clonogenic survival, which was measured via clonogenic assays. In order to determine the intrinsic cytotoxicity of flavopiridol, 0, 5, 12.5, 25, 37.5, 50 and 100 nM of flavopiridol were added to cell culture media. In the combination treatment, four different schedules of flavopiridol and irradiation combinations were tested: treatment of flavopiridol for 24 hours followed by irradiation, simultaneous administration of flavopiridol and irradiation, and irradiation followed by flavopiridol (for 24 hours) at intervals of 6 and 24 hours. The fraction of cells surviving after the combination treatment with 2 Gy of radiation (SF2) was compared with that of the fraction of cells surviving after treatment with irradiation alone. RESULTS The cytotoxicity of flavopiridol was found to be dose-dependent, with an IC50 of 80 nM. No cytotoxic enhancements were observed when flavopiridol and radiation were administered simultaneously. Flavopiridol, administered either 24 hours before or 6 hours after irradiation, exerted no sensitizing effects on the cells. Only one protocol resulted in a radiosensitizing effect: the administration of flavopiridol 24 hours after irradiation. CONCLUSION Flavopiridol enhanced the effects of radiation on a uterine cervix cancer cell line in vitro, and this enhancement was both sequence- and time-dependent.

A Histone Deacetylase Inhibitor, Trichostatin A, Enhances Radiosensitivity by Abrogating G2/M Arrest in Human Carcinoma Cells

PURPOSE Histone deacetylase inhibitors (HDIs) are emerging as potentially useful components in anticancer therapy. In this study, we tried to confirm the radiosensitizing effect of trichostatin A (TSA) on a panel of human carcinoma cell lines and elucidate its mechanism of interaction. MATERIALS AND METHODS A549, HeLa and Caski cells were exposed to TSA for 18 hr prior to irradiation, and the cell survival then measured using a clonogenic assay. Western blot and flow cytometric analyses, for histone acetylation, and cell cycle and apoptosis, respectively, were also performed. RESULTS TSA increased the acetylation of histone H3. The pretreatment of TSA consistently radiosensitized all three cell lines. The SF2 (surviving fraction at 2 Gy) of TSA-treated cells was significantly lower than that of mock treated cells. The SER (sensitizer enhancement ratio) increased in all 3 cell lines, in concentration dependent manners. The TSA treated cells showed abrogation of radiation-induced G2/M arrest, in a concentration dependent manner. CONCLUSION The pretreatment of TSA enhanced the radiosensitivity of a panel of human carcinoma cells, which was attributed, in part, to the abrogation of radiation-induced G2/M arrest.

In Vitro and In Vivo Radiosensitizing Effect of Valproic Acid on Fractionated Irradiation.

Purpose This study was conducted in order to validate the radiosensitization effect of valproic acid, a biologically available histone deacetylase inhibitor, for fractionated radiation. Materials and Methods Radiosensitization effect of valproic acid was tested for the A549 cell line and U87MG cell line in vitro. Fractionated irradiation of 12 Gy in four fractions was administered on D2-5 with valproic acid, 150 mg/Kg, ip, bid for six consecutive days (D1-6) to A549 and U87MG tumors implanted in BALB/c-nude mice. A growth delay curve was formulated. Results Radiosensitization effect of valproic acid was found for both cell lines; A549 at 1.5 mM and 3.0 mM concentration and U87MG at 3.0 mM concentration. In growth delay analysis, a statistically significant radiosensitization effect was observed for both tumors (p < 0.001 for both tumors). Difference for change in slope for control and valproic acid versus radiotherapy and radiotherapy plus valproic acid showed borderline significance for the U87MG cell line (p=0.065), indicating beyond additive effect, whereas this difference was statistically insignificant for A549 tumor (p=0.951), indicating additive effect. Conclusion Results of this study indicate that a radiosensitizing effect for fractionated radiotherapy of valproic acid for A549 and U87MG tumors in vivo is evident and that it may be more than additive for U87MG tumors. Further exploitation of histone deacetylase inhibitors in clinical trials is warranted.

Sequence-Dependent Radiosensitization of Histone Deacetylase Inhibitors Trichostatin A and SK-7041.

Purpose This preclinical study is to determine whether the capacity of histone deacetylase (HDAC) inhibitors to enhance radiation response depends on temporal sequences of HDAC inhibition and irradiation. Materials and Methods The effects of HDAC inhibitors trichostatin A (TSA) and SK-7041 on radiosensitivity in human lung cancer cells were examined using a clonogenic assay, exposing cells to HDAC inhibitors in various sequences of HDAC inhibition and radiation. We performed Western blot of acetylated histone H3 and flow cytometry to analyze cell cycle phase distribution. Results TSA and SK-7041 augmented radiation cell lethality in an exposure time-dependent manner when delivered before irradiation. The impact of TSA and SK-7041 on radiosensitivity rapidly diminished when HDAC inhibition was delayed after irradiation. Radiation induced the acetylation of histone H3 in cells exposed to TSA, while irradiation alone had no effect on the expression of acetylated histone H3 in TSA-naïve cells. Preirradiation exposure to TSA abrogated radiation-induced G2/M-phase arrest. When delivered after irradiation, TSA had no effect on the peak of radiation-induced G2/M-phase arrest. Conclusion TSA and SK-7041 enhances radiosensitivity only when delivered before irradiation. Unless proven otherwise, it seems prudent to apply scheduling including preirradiation HDAC inhibition so that maximal radiosensitization is obtained.