Severe Hyperthermia Induces Apoptosis Mediated by Caspases Activation and Suppression of Hsp90-alpha Expression in Osteosarcoma Cells

Mohammed Ali Nashiry, Gabriele Ruth Anisah Froemming, Yeap Swee Keong, Aletza Binti Mohd Ismail, Aisha Mohd Din, Alyaa Mahmood Al-Khateeb


BACKGROUND: Hyperthermia is used as an adjuvant treatment to sensitize cancer cells to subsequent radiotheraphy or chemotherapy. The aim of this study was to study the effect of severe hyperthermia on osteosarcoma cells and its underlying causes.

METHODS: Short-term (1 h) severe hyperthermia (45°C) was applied to osteoblast-like osteosarcoma cells (MG-63) and the heat shock response and cell death mechanisms were investigated after recovery at 37°C for 72 h.

RESULTS: Cell viability was significantly reduced (p<0.05) and apoptosis was significantly induced by severe hyperthermia in MG-63 cells (p<0.001). Caspase 3/7, 4 and 12 activities were significantly increased after 72 h of recovery at 37°C, indicating that severe hyperthermia induced endoplasmic reticulum (ER) stress and apoptosis (p<0.05). Heat shock protein 90 alpha (Hsp90α) was significantly down regulated at the protein level after recovery, in association with apoptosis induction (p<0.01). Additionally, caspase 8 and 9 were activated, possibly as a result of ER stress or other stimuli while, B-cell leukemia 2 family protein (BCL-2) mRNA was down regulated (p<0.01).

CONCLUSION: Severe hyperthermia could cause prolonged cell cytotoxicity by inducing apoptosis in association with inhibition of Hsp90α. This indicates the therapeutic potential of severe hyperthermia for the treatment of osteosarcoma.

KEYWORDS: hyperthermia, apoptosis, endoplasmic reticulum, stress, heat shock proteins, osteosarcoma

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Mohanty S, Inchara Y, Crasta JA, Ananthamurthy A. An unusual case of primary osteosarcoma of the rib in an adult. Indian J Med Paediatr Oncol. 2010; 31: 18-20, CrossRef.

López-Martínez J, García-Sandoval P, Fernández-Hernández J, Valcárcel-Díaz A. Functionality and osteointegration of bone allografts in long bone osteosarcomas. Acta Ortop Mex. 2012; 26: 30-4, PMID.

Hogendoorn P, Athanasou N, Bielack S, De Alava E, Dei Tos AP, Ferrari S, et al. Bone sarcomas: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol. 2010; 21 (Suppl 5): v204-13, CrossRef.

Wang YH, Xiong J, Wang SF, Yu Y, Wang B, Chen YX, et al. Lentivirus-mediated shRNA targeting insulin-like growth factor-1 receptor (IGF-1R) enhances chemosensitivity of osteosarcoma cells in vitro and in vivo. Mol Cell Biochemy. 2010; 341: 225-33, CrossRef.

Kobayashi E, Hornicek FJ, Duan Z. MicroRNA involvement in osteosarcoma. Sarcoma. 2012; 2012: 359739, CrossRef.

Trieb K, Blahovec H, Kubista B. Effects of hyperthermia on heat shock protein expression, alkaline phosphatase activity and proliferation in human osteosarcoma cells. Cell Biochem Funct. 2007; 25: 669-72, CrossRef.

Hou CH, Lin FL, Hou SM, Liu JF. Hyperthermia induces apoptosis through endoplasmic reticulum and reactive oxygen species in human osteosarcoma cells. Int J Mol Sci. 2014; 15: 17380-95, CrossRef.

Rong Y, Mack P. Apoptosis induced by hyperthermia in Dunn osteosarcoma cell line in vitro. Int J Hyperthermia. 2000; 16: 19-27, CrossRef.

Nakano H, Tateishi A, Miki H, Imamura T, Cho S, Abe S, et al. Hyperthermic isolated regional perfusion for the treatment of osteosarcoma in the lower extremity. Am J Surg. 1999; 178: 27-32, CrossRef.

Kalamida D, Karagounis IV, Mitrakas A, Kalamida S, Giatromanolaki A, Koukourakis MI. Fever-range hyperthermia vs. hypothermia effect on cancer cell viability, proliferation and HSP90 expression. PloS One. 2015; 10: e0116021, CrossRef.

Shellman YG, Howe WR, Miller LA, Goldstein NB, Pacheco TR, Mahajan RL, et al. Hyperthermia induces endoplasmic reticulummediated apoptosis in melanoma and non-melanoma skin cancer cells. J Invest Dermatol. 2008; 128: 949-56, CrossRef.

Marcu MG, Doyle M, Bertolotti A, Ron D, Hendershot L, Neckers L. Heat shock protein 90 modulates the unfolded protein response by stabilizing IRE1α. Mol Cell Biol. 2002; 22: 8506-13, CrossRef.

Gallerne C, Prola A, Lemaire C. Hsp90 inhibition by PU-H71 induces apoptosis through endoplasmic reticulum stress and mitochondrial pathway in cancer cells and overcomes the resistance conferred by Bcl-2. Biochim Biophys Acta. 2013; 1833: 1356-66, CrossRef.

Sreedhar AS, Kalmár É, Csermely P, Shen YF. Hsp90 isoforms: functions, expression and clinical importance. FEBS Let. 2004; 562: 11-5, CrossRef.

Csermely P, Schnaider T, So C, Prohászka Z, Nardai G. The 90-kDa molecular chaperone family: structure, function, and clinical applications. A comprehensive review. Pharmacol Ther. 1998; 79: 129-68, CrossRef.

Nakajima K, Yanagawa T, Watanabe H, Takagishi K. Hyperthermia reduces migration of osteosarcoma by suppression of autocrine motility factor. Oncol Rep. 2012; 28: 1953-8, CrossRef.

Dresios J, Aschrafi A, Owens GC, Vanderklish PW, Edelman GM, Mauro VP. Cold stress-induced protein Rbm3 binds 60S ribosomal subunits, alters microRNA levels, and enhances global protein synthesis. Proc Natl Acad Sci. 2005; 102: 1865-70, CrossRef.

Zeng Y, Kulkarni P, Inoue T, Getzenberg RH. Down-regulating cold shock protein genes impairs cancer cell survival and enhances chemosensitivity. J Cell Biochem. 2009; 107: 179-88, CrossRef.

Sakurai T, Itoh K, Higashitsuji H, Nonoguchi K, Liu Y, Watanabe H, et al. Cirp protects against tumor necrosis factor-α-induced apoptosis via activation of extracellular signal-regulated kinase. Biochim Biophys Acta. 2006; 1763: 290-5, CrossRef.

Goping S, Gross A, Lavoie JN, Nguyen M, Jemmerson R, Roth K, et al. Regulated targeting of BAX to mitochondria. J Cell Biol. 1998; 143: 207-15, CrossRef.

Kuwana T, Newmeyer DD. Bcl-2-family proteins and the role of mitochondria in apoptosis. Curr Opin Cell Biol. 2003; 15: 691-9, CrossRef.

Wei MC, Zong WX, Cheng EHY, Lindsten T, Panoutsakopoulou V, Ross AJ, et al. Proapoptotic BAX and BAK: a requisite gateway to mitochondrial dysfunction and death. Science. 2001; 292: 727-30, CrossRef.

Nuñez G, Clarke MF. The Bcl-2 family of proteins: regulators of cell death and survival. Trends Cell Biol. 1994; 4: 399-403, CrossRef.

Reed JC. Bcl-2 and the regulation of programmed cell death. J Cell Biol. 1994; 124: 1-6, CrossRef.

Hitomi J, Katayama T, Eguchi Y, Kudo T, Taniguchi M, Koyama Y, et al. Involvement of caspase-4 in endoplasmic reticulum stressinduced apoptosis and Aβ-induced cell death. J Cell Biol. 2004; 165: 347-56, CrossRef.

Shiraishi H, Okamoto H, Yoshimura A, Yoshida H. ER stress-induced apoptosis and caspase-12 activation occurs downstream of mitochondrial apoptosis involving Apaf-1. J Cell Sci. 2006; 119: 3958-66, CrossRef.

Rosati E, Sabatini R, Rampino G, De Falco F, Di Ianni M, Falzetti F, et al. Novel targets for endoplasmic reticulum stress-induced apoptosis in B-CLL. Blood. 2010; 116: 2713-23, CrossRef.

Sano R, Reed JC. ER stress-induced cell death mechanisms. Biochim Biophys Acta. 2013; 1833: 3460-70, CrossRef.

Schmitt E, Gehrmann M, Brunet M, Multhoff G, Garrido C. Intracellular and extracellular functions of heat shock proteins: repercussions in cancer therapy. J Leukoc Biol. 2007; 81: 15-27, CrossRef.

Quanz M, Herbette A, Sayarath M, de Koning L, Dubois T, Sun JS, et al. Heat shock protein 90α (Hsp90α) is phosphorylated in response to DNA damage and accumulates in repair foci. J Biol Chem. 2012; 287: 8803-15, CrossRef.

Taiyab A, Sreedhar AS, Rao CM. Hsp90 inhibitors, GA and 17AAG, lead to ER stress-induced apoptosis in rat histiocytoma. Biochem Pharmacol. 2009; 78: 142-52, CrossRef.

Guo Y, Ziesch A, Hocke S, Kampmann E, Ochs S, De Toni EN, et al. Overexpression of heat shock protein 27 (HSP 27) increases gemcitabine sensitivity in pancreatic cancer cells through S-phase arrest and apoptosis. J Cell Mol Med. 2015; 19: 340-50, CrossRef.

Trieb K, Lechleitner T, Lang S, Windhager R, Kotz R, Dirnhofer S. Heat shock protein 72 expression in osteosarcomas correlates with good response to neoadjuvant chemotherapy. Hum Pathol. 1998; 29: 1050-5, CrossRef.

Ehlén Å, Brennan DJ, Nodin B, O'connor DP, Eberhard J, AlvaradoKristensson M, et al. Expression of the RNA-binding protein RBM3 is associated with a favourable prognosis and cisplatin sensitivity in epithelial ovarian cancer. J Transl Med. 2010; 8: 78, CrossRef.

Jögi A, Brennan DJ, Rydén L, Magnusson K, Fernö M, Stål O, et al. Nuclear expression of the RNA-binding protein RBM3 is associated with an improved clinical outcome in breast cancer. Mod Pathol. 2009; 22: 1564-74, CrossRef.

Jonsson L, Bergman J, Nodin B, Manjer J, Pontén F, Uhlén M, et al. Low RBM3 protein expression correlates with tumour progression and poor prognosis in malignant melanoma: an analysis of 215 cases from the Malmö Diet and Cancer Study. J Transl Med. 2011; 9: 114, CrossRef.

Jonsson L, Gaber A, Ulmert D, Uhlén M, Bjartell A, Jirström K. High RBM3 expression in prostate cancer independently predicts a reduced risk of biochemical recurrence and disease progression. Diagnostic Pathology. 2011; 6: 91, CrossRef.

Zeng Y, Wodzenski D, Gao D, Shiraishi T, Terada N, Li Y, et al. Stress response protein RBM3 attenuates the stem-like properties of prostate cancer cells by interfering with CD44 variant splicing. Cancer Res. 2013; 73: 4123-33, CrossRef.

Aisha M, Nor-Ashikin M, Sharaniza A, Nawawi H, Kapitonova M, Froemming G. Short-term moderate hypothermia stimulates alkaline phosphatase activity and osteocalcin expression in osteoblasts by upregulating Runx2 and osterix in vitro. Exp Cell Res. 2014; 326: 46-56, CrossRef.


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