The Effect And Mechanism Of Chronic Intermittent Hypoxia On Resistance To Endocrine Therapy

It has been reported that intermittent hypoxia, which is likely more prevalent than acute hypoxia in breast cancers, can cause persistent depression of estrogen receptor alpha (ER-a) expression in breast cancer cell lines. However, it remains suspension whether intermittent hypoxia may be associated with resistance to endocrine therapy.Effects of chronic intermittent hypoxia on the proliferation, cell cycle distribution and in vitro invasion of human breast cancer cells were investigated by CCK-8, FCM and transwell. Differences in sensitivity to estradiol and fulvestrant (ICI 182,780, Tocris) between cells under normoxia and hypoxia were detected by CCK-8. RT-PCR and Western blot were used to examine the genetic and proteinic changes in ER-mediated signaling pathway and hypoxia regulatory pathway to explore the potential mechanism of resistance to endocrine therapy caused by chronic intermittent hypoxia.1. Effects of cinobufacini on the proliferation, cell cycle distribution and in vitro invasion in different human breast cancer cells cultured respectively under normoxia and hypoxia were investigated by CCK-8, FCM and transwell. RT-PCR and Western blot were used to examine the genetic and proteinic levels of various cyclins to explore the potential mechanisms of cinobufacini.2. ER positive human breast cancer cells cultured under normoxia and hypoxia were treated respectively with cinobufacini and Bortezomib (LC Laborataries) to determine their effets on the improvement in resistance to fulvestrant (ICI 182,780, Tocris). RT-PCR and Western blot were used to examine the genetic and proteinic changes in ER-mediated signaling pathway and hypoxia regulatory pathway.to explore the potential mechanisms of their improvements in resistance to fulvestrant (ICI 182,780, Tocris).3. In ER positive human breast cancer cells cultured under normoxia and hypoxia, the expression of HIF-1αwas blocked by a reformed type of small interfering RNA (siRNA), stealth RNAi (Invitrogen) to determine its effet on the improvement in resistance to fulvestrant (ICI 182,780, Tocris). RT-PCR, real-time Q-PCR and Western blot were used to examine the genetic and proteinic changes in ER-mediated signaling pathway and hypoxia regulatory pathway.to explore the potential mechanisms of its improvement in resistance to fulvestrant(ICI 182,780, Tocris).1. ER positive human breast cancer cells cultured under normoxia and hypoxia were implanted orthotopically into BALB/c female nude mice to determine the effect of chronic intermittent hypoxia on the tumor growth.2. Fulvestrant (Astrazeneca) was administered to BALB/c female nude mice implanted by ER positive human breast cancer cells cultured under normoxia and hypoxia respectively. Tumor growth was tested to demonstrate the effect of chronic intermittent hypoxia on resistance to endocrine therapy in vivo.3. Changes in ER-mediated signaling pathway and hypoxia regulatory pathway were detected by immunohistochemistry, and the relationship among them was analysized.1. Chronic intermittent hypoxia significantly promoted cancer cell proliferation in both MCF-7 and MDA-MB-231 cell lines (P< 0.05) rather than in ZR-75-1 cell line.2. Chronic intermittent hypoxia significantly increased in vitro invasion capability of MCF-7 and MDA-MB-231 cell lines (P< 0.05).3. The sensitivity to estrogens declined dramatically in hypoxia MCF-7 cells compared to normoxia ones.4. The IC50 of fulvestrant was 60nM for the MCF-7 cell line cultured under normoxia, while the corresponding IC50 was 800nM for that under hypoxia. Therefore, MCF-7 breast cancer cells under normoxic condition were capable of an intact response to fulvestrant, whereas the inhibitory effect of fulvestrant was significantly reduced in those under intermittent hypoxia.5. At both mRNA and protein levels, chronic intermittent hypoxia up-regulated the expression levels of HIF-1α, CA-Ⅸand GAPDH, while down-regulated those of FIH-1 and ER-αin MCF-7 cell line.1. Cinobufacini inhibited the cell growth of both normoxia MCF-7 and MDA-MB-231 (IC50= 0.57 mg/ml and IC50= 0.31 mg/ml, respectively), and this inhibitory effect was time-and dose-dependent (P< 0.05).2. Cinobufacini significantly decreased in vitro invasion capability of MCF-7 and MDA-MB-231 cells (P< 0.05).3. Cinobufacini induced S-phase arrest of MDA-MB-231 cells in a concentration-dependent way (P< 0.0001).4. At both mRNA and protein levels, cinobufacini down-regulated the expression of cyclin A1, cyclin D1 and cyclin E1, while up-regulated that of p21 in MDA-MB-231 cell line. However, there was no discernible change in the expression of cyclin B1 across the time.5. Cinobufacini inhibited the hypoxia MCF-7 cell growth (IC50= 0.54 mg/ml).6. Cinobufacini significantly restored the response to fulvestrant in hypoxia MCF-7 cells, which induced the up-regulation of ER-αand FIH-1 as well as the down-regulation of HIF-1αand CA-Ⅸ.7. Bortezomib, a proteasome inhibitor, significantly restored the response to fulvestrant in hypoxia MCF-7 cells, which induced the up-regulation of ER-αand FIH-1 as well as down-regulation of HIF-1αand CA-Ⅸ.8. To further clarify whether intermittent hypoxia was associated with resistance to endocrine therapy, the expression of HIF-1αwas blocked by a reformed type of siRNA, StealthTM RNAi (Invitrogen), which can reduce the cytotoxic interferon response unlike conventional siRNA. Interestingly, knock-down of HIF-1αdid result in the restoration of not only the ER-αexpression but also the response to fulvestrant.1. There was no difference of tumor formation rate between mice implanted orthotopically with hypoxia and normoxia cells for either MCF-7 or MDA-MB-231 cell lines. However, chronic intermittent hypoxia significantly promoted tumor growth (P< 0.05).2. When compared to controls, fulvestrant was effective in suppressing tumor growth of mice implanted orthotopically with normoxia MCF-7 cells rather than hypoxia ones (P< 0.05).3. Chronic intermittent hypoxia was associated with increasing HIF-1αand CA-Ⅸprotein levels as well as decreasing FIH-1 and ERαexpression inn xenograft tumors (P< 0.05).These data provide functional evidence that intermittent hypoxia may confer resistance to endocrine therapy in ER-positive breast cancers through the crosstalk between HIF-1αand ER signaling, which holds a promise to overcome endocrine resistance.