Possible link between hypoxia inducible factor-1α and breast cancer resistant protein in human breast cancer cell lines

Hueng-Chuen Fan1,2, Jing Jin1, Chia-Ling Yang1, Shin-Nan Cheng2, Hee-Kyoung Kang1, Tai-Ping Fan1. 1Department of Pharmacology, University of Cambridge, UK and 2Department of Pediatrics, Tri-Service General Hospital, Taipei, Taiwan, ROC.

Hypoxia is known to confer resistance to some drugs leading to failure of chemotherapy, probably due to the up-regulation of d rug resistant proteins, such as breast cancer resistance protein (BCRP), via hypoxia inducible factor-1α (HIF-1α) ( Krishnamurthy et al., 2004). HIF-1α has been regarded as a master regulator that activates pathways regulating physiological responses to various levels of hypoxia. In cancer cells, HIF-1α supports their adaptation to hypoxia, e.g. production of vascular endothelial growth factor (VEGF), thereby enhancing tumo u r growth and progression ( Semenza, 2002). Here we examine how hypoxia affects the responsiveness of cancer cells to drug treatment.

BCRP is overexpressed in mitoxantrone (MX)-resistant MCF-7/MX cells. By RT-PCR, we found lower mRNA levels of HIF-1α and VEGF in these cells , but higher mRNA levels of negative regulators of HIF-1α including phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and von Hippel-Lindau (VHL) when compared with MX-sensitive MCF-7/S cells. K inetic studies on the effect of hypoxia (1% O2) on HIF-1α mRNA level i n MCF-7/S cells revealed an increase in response to hypoxia and reached a peak at 3h, but subsequently declined at 18h. The BCRP mRNA level steadily increased within the first 3h, and then declined after 9h. In MCF-7/MX cells, the initial low HIF-1α mRNA level peaked at 3h and then declined to the basal level at 18h. The BCRP mRNA remained constant during hypoxia, but dropped to an undetectable level by 18h . MTT assay showed that 18h hypoxia rendered MCF-7/S cells resistant to the cytotoxic effects of MX, but did not affect the chemosensitivity of MCF-7/MX cells to the drug. The results of Western blot suggest a BCRP-like protein to be responsible for the increased chemoresistance of MCF-7/S cells to MX under 18h hypoxia. Since the r egulation of HIF-1α has been linked to the generation of reactive oxygen species (ROS) ( Chandel et al.,1998) , the effects of hypoxia on the production of ROS in both cell lines were investigated. By FACScan, a biphasic effect of hypoxia on ROS production was evident in MCF-7/S cells. In contrast, there was no obvious change of ROS production in MCF-7/MX cells, except at 18 h where its level doubled.

The data presented here do not confirm the relationship between HIF-1α and BCRP as proposed by Krishnamurthy (2004). Significantly, these data provide a platform for comparative studies on chemosensitive and chemoresistant cancer cells, and their modification by oxygen tension and chemotherapeutics. It is hoped that further studies of this kind would lead to the development of novel drugs for circumventing clinical drug resistance.

Chandel, NS et al. (1998) Proc Natl Acad Sci USA 95 (20):11715-20.
Krishnamurthy, P et al. (2004) J Biol Chem 279(23):24218-25.
Semenza, GL (2002) Trends Mol Med 8(4 Suppl):S62-7.

HCF and TPF acknowledge the financial support from Ministry of National Defense, Taiwan, ROC and NuLiv Science Taiwan, respectively.