Open Access

Glycerol as a chemical chaperone enhances radiation-induced apoptosis in anaplastic thyroid carcinoma cells

  • Ken Ohnishi1,
  • Ichiro Ota2,
  • Katsunari Yane2,
  • Akihisa Takahashi1,
  • Kazue Yuki2,
  • Mie Emoto2,
  • Hiroshi Hosoi2 and
  • Takeo Ohnishi1Email author
Molecular Cancer20021:4

https://doi.org/10.1186/1476-4598-1-4

Received: 21 July 2002

Accepted: 4 October 2002

Published: 4 October 2002

Abstract

Introduction

Anaplastic thyroid carcinoma, which is one of the most aggressive, malignant tumors in humans, results in an extremely poor prognosis despite chemotherapy and radiotherapy. The present study was designed to evaluate therapeutic effects of radiation by glycerol on p53-mutant anaplastic thyroid carcinoma cells (8305c cells). To examine the effectiveness of glycerol in radiation induced lethality for anaplastic thyroid carcinoma 8305c cells, we performed colony formation assay and apoptosis analysis.

Results

Apoptosis was analyzed with Hoechst 33342 staining and DNA ladder formation assay. 8305c cells became radiosensitive when glycerol was added to culture medium before X-ray irradiation. Apoptosis was induced by X-rays in the presence of glycerol. However, there was little apoptosis induced by X-ray irradiation or glycerol alone. The binding activity of whole cell extracts to bax promoter region was induced by X-rays in the presence of glycerol but not by X-rays alone.

Conclusion

These findings suggest that glycerol is effective against radiotherapy of p53-mutant thyroid carcinomas.

Keywords

anaplastic thyroid carcinomaglycerolapoptosisp53

Background

Anaplastic thyroid carcinoma, which is a relatively uncommon malignancy comprising 5–14 % of thyroid cancers [1], is recognized as one of the most aggressive malignant tumor in humans and fails to respond to the available chemotherapeutic agents or radiation [2, 3]. Most patients die within a year after diagnosis [4, 5]. Thus, the improvement of therapeutic effects is needed to restore chemo- and radiosensitivity to anaplastic thyroid carcinomas which have a high frequency of mutant p53 (mp53). Recently, p53 gene therapy has been developed [68] and applied to patients carrying mp53[911]. However, overexpression of wild-type p53 (wtp53) risks inducing suppression of proliferation in normal cells as well as targeted cancer cells. The suppression of proliferation of blood cells possibly gives negative effects on the physical condition of patients. Thus, there are still difficulties in the clinical application of p53 gene therapy. We have reported a new cancer therapy strategy against mp53 cancer cells, i.e. chemical chaperon therapy. This therapy is based on glycerol-mediated conformational change of mp53 molecules. The restoration of the normal function in mp53 leads to enhanced radiosensitivity and induction of apoptosis [12]. Glycerol appears to confer wtp53 function on mp53 [13]. The function of glycerol as a chemical chaperone has been reported elsewhere [14, 15]. This novel tool for enhancing the radiosensitivity of cancer cells bearing mp53 is worth investing as p53-targeted cancer therapy, since p53 mutations are frequently found in human cancer cells [16, 17]. In the present study, we report the apoptotic effect of glycerol combined with radio-treatment in anaplastic thyroid carcinoma cells carrying mp53.

Results

To evaluate the effectiveness of glycerol in radiation induced lethality for anaplastic thyroid carcinoma 8305c cells, we examined colony formation assay. As shown in Fig. 1, when glycerol was added to culture medium before X-ray irradiation at a final concentration of 0.6 M, 8305c cells became about 3 times more X-ray-sensitive at the D10 dose. In contrast, there was no enhanced radiation sensitivity observed in 8305c cells after the addition of 0.3 M glycerol alone (data not shown). These results suggest that glycerol has an ability to enhance the radiosensitivity of 8305c cells. Figure. 2A shows the time-course of the frequency of X-ray induced apoptotic cells. Though the frequency did not increase after X-ray irradiation without the glycerol treatment, the frequency was augmented in a time-dependent manner in the presence of glycerol. Typical apoptotic bodies detected by Hoechst 33342 staining analysis are shown in Fig. 2C and 2D. The destruction of nuclei was observed 48 h after X-ray irradiation, especially with glycerol (Fig. 2D). Apoptotic bodies appeared at a higher frequency in the presence of glycerol (Fig. 2D) than in the absence of glycerol (Fig. 2C). Increased incidence of apoptotic body was not observed after glycerol treatment alone. We next examined the induction of DNA fragmentation after X-ray irradiation using agarose gel electrophoresis of DNA extracted from 8305c cells (Fig. 3). Clear DNA ladders in the nucleosomal size were observed 24–48 h after X-ray irradiation combined with glycerol (Fig. 3A). However, a faint DNA ladder was observed 48 h after X-ray irradiation alone (Fig. 3B). No DNA ladder was observed 48 h after glycerol treatment alone (Fig. 3A) and thereafter. These results indicate that glycerol sensitized radiation-induced apoptosis in anaplastic thyroid carcinoma in 8305c cells. DNA (p53 consensus sequence, p53CON) binding activity of whole cell extracts from X-ray (6 Gy) irradiated cells was analyzed with gel mobility-shift assay (Fig. 4). Whole cell extracts from non-irradiated cells showed no DNA binding activity. Similarly, the extracts from X-ray-irradiated cells showed no DNA binding activity 6 h after the irradiation. However, the extracts from glycerol-treated cells showed DNA binding activity and the binding activity was also observed in the extracts from the cells irradiated with X-rays 6 h after the irradiation.
Figure 1

Clonogenic surviving fractions after X-ray. Opened circles, without glycerol; Closed circles, with 0.6 M glycerol.

Figure 2

The time course of apoptotic body detected with Hoechst33342 staining after X-ray irradiation (6 Gy). A, 8305c cells were treated with (closed circles) or without (opened circles) 0.6 M glycerol. B-D, photographs of apoptotic body in 8305c cells. B, control; C, 48 h after X-ray irradiation alone; D, 48 h after X-ray irradiation combined with 0.6 M glycerol; Bars, 25 μm.

Figure 3

DNA fragmentation detected with agarose gel electrophoresis of DNA extracted from X-ray (6 Gy) irradiated cells. A and B represent with and without glycerol (0.6 M), respectively. C, no irradiation; Lanes 12, 24. 36 and 48, incubation periods (h) after X-ray irradiation. Lane M, øX174 DNA Hae III fragments as size markers.

Figure 4

DNA (p53CON) binding activity of whole cell extracts from X-ray (6 Gy) irradiated cells analyzed with gel mobility-shift assay. Non-irradiated or glycerol-treated cells were collected with X-ray-irradiated cells 6 h after the irradiation.

Discussion

Recently, the mutation of p53 has been reported to play an important role in anaplastic change in differentiated thyroid carcinomas [19, 20]. Supporting this, 8305c cells show a point mutation at codon 273 of the p53 gene. The p53 gene plays a pivotal role in the pathway that controls cell growth, proliferation and apoptosis [2124]. The lack of the p53-regulated pathway is a common feature in a large number of tumors, suggesting that it may be central to the pathogenesis of human cancers. We have recently reported that the effectiveness by radiotherapy or hyperthermic therapy depends on p53-status in human squamous cell carcinoma (SAS cells) [12, 25]. That is, SAS cells transfected with mp53 are more radiation or heat resistant than neo control cells. The similar p53-dependent radiosensitivity has also been reported elsewhere [26]. The present results may provide useful information for radiotherapy of mp53 cancer cells.

The present observed X-ray sensitivity enhanced by glycerol may result from the enhancement of p53-independent X-ray-induced apoptosis. However, this presumption is not possible from the following reason. We have reported that glycerol can restore wtp53 function to mp53 cancer cells [12, 13, 27]. The present data in 8305c cells strongly supports the chemical chaperone effect of glycerol. Glycerol possibly enhanced X-ray-sensitivity in 8305c cells through restoration of mp53 to wtp53 functions. The present data from gel mobility-shift assay suggest that the restoration is brought through conformational change of mp53 to wtp53. The mp53 molecules from cells treated with glycerol and X-rays might affect the transactivation of p53-down stream genes, especially apoptosis-related genes such as bax. In relation to this, we have reported that glycerol has an ability to enhance bax expression in mp53 human glioblastoma cells through phosphorylation of p53 at serine 15 by PI3-K family [28]. The conformational change of mp53 might be induced by glycerol alone because the DNA binding activity of mp53 was restored after glycerol treatment alone (Fig. 4). However, in this case, the transcriptional activity of mp53 is not likely to be restored. Possibly, the mp53 molecules from cells treated with glycerol alone is not modified by X-ray-induced phosphorylation or acetylation. Therefore, it seems that the cells treated with glycerol alone did not show apoptosis, as shown in Fig. 2 and 3. We have previously reported that the DNA binding activity of mp53 was almost not restored after glycerol treatment alone in p53 mutated at codon 248 [12]. Therefore, the effect of glycerol on mp53 may depend on mutation site of p53 and amino acid sequence affecting its three-dimensional conformation. This result suggests that the investigation of mutation site of p53 is an important process for the cancer therapies based on p53 status.

New strategy based on the functional restoration of mp53 is recently being developed. Recent studies have reported new compounds which rescue mp53 conformation and function [29, 30]. The compounds can induce apoptosis in mp53 human tumor cells through restoration of the transcriptional transactivation function to mp53. The compounds may serve as anticancer drugs targeting mp53. Compared with these compounds, glycerol seems to be the most hopeful molecule for cancer therapy, because it has been widely used as a clinical reagent already.

Conclusions

The present study suggests that chemical chaperones, such as a glycerol, are useful for radiotherapy of p53-mutant thyroid carcinomas.

Methods

Cells

The cells used in this study were a human anaplastic thyroid carcinoma-derived cell line, 8305c bearing a point mutation at codon 273 in the p53 gene [18]. 8305c cells were obtained from JCRB (HSRRB, Osaka, Japan). Cells were maintained in continuous culture at 37°C in a 5 % CO2 humidified atmosphere. Cells were grown in RPMI 1640 medium supplemented with 10% heat-inactivated FBS. Penicillin (100 U/ml) and streptomycin (100 μg/ml) were added to the media.

Glycerol treatment

Glycerol was added into the culture medium at a final concentration of 0.6 M at 48 h before irradiation by X-ray source (MBR-1520R, Hitachi, Tokyo, Japan). Thereafter the cells were incubated at 37°C in the presence of glycerol until sampling of apoptosis assay. For survival assay, the medium with glycerol was changed to glycerol-free medium after 10 h incubation and thereafter cells were incubated for seven to ten days at 37°C in glycerol free-medium.

Cell survival assay

To measure the radiosensitivity of the cells, cell survival after X-ray irradiation was measured by plating cells into 25 cm2 flasks containing medium. Thereafter, colonies were rinsed with PBS, fixed with methanol, stained with 2% Giemsa solution (Merck, Woodbridge, NJ, USA). Colonies containing at least 50 cells were counted. Surviving fractions were measured in 3 independent duplicate experiments. Bars (standard errors) are shown when they exceed the symbols.

Analysis of apoptosis

We performed the Hoechst 33342 staining and the agarose gel electorophoresis for analyses of apoptosis, using the 8305c cells. Two or more independent experiments were repeated. Induction of apoptosis was analyzed by detection of apoptotic bodies and DNA fragmentation. To detect apoptotic bodies, cells were fixed with 1% glutaraldehyde (Nacalai Tesque, Kyoto, Japan) in PBS at 4°C, washed with PBS, stained with 0.2 mM Hoechst 33342 (Sigma Chemical Co.), and then observed under a fluorescence microscope. Apoptotic bodies were counted at 3 different fields of the microscopic observation. One hundred cells were judged under one field. For detection of DNA fragmentation in the nucleosomal size range, cells were suspended in lysis buffer containing 10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 10 mM EDTA and 1% sodium N-lauroylsarcosinate, incubated with 100 μg/ml protease K at 37°C overnight, and then centrifuged at 18,500 × g for 30 min. The resulting supernatants were incubated with 100 μg/ml RNase A at 37°C for 1 h. The DNA in the solution was precipitated with ethanol at -20°C, dissolved in 10 mM Tris-HCl, pH 8.0, and 1 mM EDTA, and electrophoresed for 30 min at 100 V through 3% NuSieve 3:1 gels containing 40 mM Tris-acetate, pH 7.8, 2 mM EDTA and 0.5 μg/ml ethidium bromide. After electrophoresis, the gels were photographed under ultraviolet light.

Gel mobility-shift assay

Whole cell extracts were prepared from X-ray-irradiated or non-irradiated cells (about 2 × 107 cells) according to the method described elsewhere [12]. The binding activity of p53 to p53 consensus sequence (p53CON) was measured by a gel mobility-shift assay [12] using a synthetic double-stranded DNA fragment encoding the p53CON (5'-GGACATGCCCGGGCATGTCC-3', Japan Bioservice, Niiza, Saitama, Japan) based on a specific sequence located upstream to the bax gene as a probe.

Declarations

Acknowledgment

This work was supported by Grants-in-Aid from the Ministry of Education, Science, Sports, Technology and Culture of Japan.

Authors’ Affiliations

(1)
Department of Biology, Nara Medical University
(2)
Department of Otorhinolaryngology, Nara Medical University

References

  1. Venkatesh YS, Ordonez NG, Schultz PN, Hickey RC, Goepfert H, Samaan NA: Anaplastic carcinoma of the thyroid. A clinicopathologic study of 121 cases. Cancer. 1990, 66: 321-330.View ArticlePubMedGoogle Scholar
  2. Tan RK, Finley RK, Driscoll D, Bakamjian V, Hicks WL, Shedd DP: Anaplastic carcinoma of the thyroid: a 24-year experience. Head Neck. 1995, 17: 41-47.View ArticlePubMedGoogle Scholar
  3. Sweeney PJ, Haraf DJ, Recant W, Kaplan EL, Vokes EE: Anaplastic carcinoma of the thyroid. Ann Oncol. 1996, 7: 739-744.View ArticlePubMedGoogle Scholar
  4. Jereb B, Stjernsward J, Lowhagen T: Anaplastic giant-cell carcinoma of the thyroid. A study of treatment and prognosis. Cancer. 1975, 35: 1293-1295.View ArticlePubMedGoogle Scholar
  5. Aldinger KA, Samaan NA, Ibanez M, Hill CS: Anaplastic carcinoma of the thyroid: a review of 84 cases of spindle and giant cell carcinoma of the thyroid. Cancer. 1978, 41: 2267-2275.View ArticlePubMedGoogle Scholar
  6. Fujiwara T, Grimm EA, Mukhopadhyay T, Zhang WW, Owen-Schaub LB, Roth JA: Induction of chemosensitivity in human lung cancer cells in vivo by adenovirus-mediated transfer of the wild-type p53 gene. Cancer Res. 1994, 54: 2287-2291.PubMedGoogle Scholar
  7. Lowe SW, Bodis S, McClatchey A, Remington L, Ruley HE, Fisher DE, Housman DE, Jacks T: p53 status and the efficacy of cancer therapy in vivo. Science. 1994, 266: 807-810.View ArticlePubMedGoogle Scholar
  8. Spitz FR, Nguyen D, Skibber JM, Cusack J, Roth JA, Cristiano RJ: In vivo adenovirus-mediated p53 tumor suppressor gene therapy for colorectal cancer. Anticancer Res. 1996, 16: 3415-3422.PubMedGoogle Scholar
  9. Fujiwara T, Cai DW, Georges RN, Mukhopadhyay T, Grimm EA, Roth JA: Therapeutic effect of a retroviral wild-type p53 expression vector in an orthotopic lung cancer model. J Natl Cancer Inst. 1994, 86: 1458-1462.View ArticlePubMedGoogle Scholar
  10. Roth JA: Modification of tumor suppressor gene expression in non-small cell lung cancer (NSCLC) with a retroviral vector expressing wildtype (normal) p53. Hum Gene Ther. 1996, 7: 861-874.View ArticlePubMedGoogle Scholar
  11. Roth JA, Nguyen D, Lawrence DD, Kemp BL, Carrasco CH, Ferson DZ, Hong WK, Komaki R, Lee JJ, Nesbitt JC, Cai D: Retrovirus-mediated wild-type p53 gene transfer to tumors of patients with lung cancer. Nat Med. 1996, 2: 985-991.View ArticlePubMedGoogle Scholar
  12. Ohnishi K, Ota I, Takahashi A, Ohnishi T: Glycerol restores p53-dependent radiosensitivity of human head and neck cancer cells bearing mutant p53. Brit J Cancer. 2000, 83: 1735-1739. 10.1054/bjoc.2000.1511PubMed CentralView ArticlePubMedGoogle Scholar
  13. Ohnishi T, Matsumoto H, Wang X, Takahashi A, Tamamoto T, Ohnishi K: Restoration by glycerol of p53-dependent apoptosis in cells bearing the mutant p53 gene. Int J Radiat Biol. 1999, 75: 1095-1098. 10.1080/095530099139557View ArticlePubMedGoogle Scholar
  14. Welch WJ, Brown CR: Influence of molecular and chemical chaperones on protein folding. Cell Stress Chap. 1996, 1: 109-115.View ArticleGoogle Scholar
  15. Thomas PJ, Qu BH, Pedersen PL: Defective protein folding as a a basis of human disease. TIBS. 1995, 20: 456-459. 10.1016/S0968-0004(00)89100-8PubMedGoogle Scholar
  16. Hollstein M, Sidransky D, Vogelstein B, Hallis CC: p53 mutations in human cancers. Science. 1991, 253: 49-53.View ArticlePubMedGoogle Scholar
  17. Jia LQ, Osada M, Ishioka C, Gamo M, Ikawa S, Suzuki T, Shimodaira H, Niitani T, Kudo T, Kuroki T: Screening the p53 status of human cell lines using a yeast functional assay. Mol Carcinog. 1997, 19: 243-253. 10.1002/(SICI)1098-2744(199708)19:4<243::AID-MC5>3.0.CO;2-DView ArticlePubMedGoogle Scholar
  18. Ito T, Seyama T, Mizuno T, Tsuyama N, Hayashi T, Hayashi Y, Dohi K, Nakamura N, Akiyama M: Unique association of p53 mutations with undifferentiated but not with differentiated carcinomas of the thyroid gland. Cancer Res. 1992, 52: 1369-1371.PubMedGoogle Scholar
  19. Fagin JA, Matsuo K, Karmakar A, Chen DL, Tang SH, Koeffler HP: High prevalence of mutations of the p53 gene in poorly differentiated human thyroid carcinomas. J Clin Invest. 1993, 91: 179-184.PubMed CentralView ArticlePubMedGoogle Scholar
  20. Ito T, Seyama T, Mizuno T, Tsuyama N, Hayashi T, Hayashi Y, Dohi K, Nakamura N, Akiyama M: Unique association of p53 mutations with undifferentiated but not with differentiated carcinomas of the thyroid gland. Cancer Res. 1992, 52: 1369-1371.PubMedGoogle Scholar
  21. El-Deiry WS, Tokino T, Velculescu VE, Levy DB, Parsons R, Trent JM, Lin D, Mercer WE, Kinzler KW, Vogelstein B: WAF1, a potential mediator of p53 tumor suppression. Cell. 1993, 75: 817-825.View ArticlePubMedGoogle Scholar
  22. Harper JW, Adami GR, Wei N, Keyomarsi K, Elledge SJ: The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell. 1993, 75: 805-816.View ArticlePubMedGoogle Scholar
  23. Lowe SW, Ruley HE, Jacks T, Housman DE: p53-dependent apoptosis modulates the cytotoxicity of anticancer agents. Cell. 1993, 74: 957-967.View ArticlePubMedGoogle Scholar
  24. Polyak K, Xia Y, Zweier JL, Kinzler KW, Vogelstein B: A model for p53-induced apoptosis. Nature. 1997, 389: 300-305. 10.1038/38525View ArticlePubMedGoogle Scholar
  25. Ota I, Ohnishi K, Takahashi A, Yane K, Kanata H, Miyahara H, Ohnishi T, Hosoi H: Transfection with mutant p53 gene inhibits heat-induced apoptosis in a head and neck cell line of human squamous cell carcinoma. J Radiat Oncol Biol Phys. 2000, 47: 495-501. 10.1016/S0360-3016(00)00437-5. 10.1016/S0360-3016(00)00437-5View ArticleGoogle Scholar
  26. Lowe SW: Cancer therapy and p53. Curr Opin Oncol. 1995, 7: 547-553.View ArticlePubMedGoogle Scholar
  27. Ohnishi T, Ohnishi K, Wang X, Takahashi A, Okaichi K: Restoration of mutant TP53 to normal TP53 function by glycerol as a chemical chaperone. Radiat Res. 1999, 151: 498-500.View ArticlePubMedGoogle Scholar
  28. Ohnishi T, Ohnishi K, Takahashi A: Glycerol restores heat-induced p53-dependent apoptosis of human glioblastoma cells bearing mutant p53. BMC Biotechnol. 2002, 2: 6- 10.1186/1472-6750-2-6PubMed CentralView ArticlePubMedGoogle Scholar
  29. Foster BA, Coffey HA, Morin MJ, Rastinejad F: Pharmacological rescue of mutant p53 conformation and function. Science. 1999, 286: 2507-2510. 10.1126/science.286.5449.2507View ArticlePubMedGoogle Scholar
  30. Bykov VJ, Issaeva N, Shilov A, Hultcrantz M, Pugacheva E, Chumakov P, Bergman J, Wiman KG, Selivanova G: Restoration of the tumor suppressor function to mutant p53 by a low-molecular-weight compound. Nat Med. 2002, 8: 282-288. 10.1038/nm0302-282View ArticlePubMedGoogle Scholar

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