) Human breast cancer is the predominant malignancy and the leading cause of cancer death in women from Western society. Recently, the association between human prolactin (hPRL) activity and breast cancer has been re-emphasized. In our recent studies, we have successfully developed a hPRL receptor specific antagonist by a single amino acid substitution mutation within hPRL molecule at the position 129 from Gly to Arg (hPRL-G129R). Using cell-based assays, we have demonstrated that hPRL-G129R was able to bind to hPRL receptor and block hPRL signal transduction. More importantly, we have shown that hPRL-GI29R was able to inhibit breast cancer cell proliferation through induction of apoptosis. In this study, we attempt to produce and purify hPRL-GI29R, along with hPRL (as control), and use them for in vivo studies. There are two specific aims of this proposal.
Specific aim (1) is to produce and purify greater than 70mg of hPRL and hPRL-GI29R at greater than 95 percent purity by FPLC. After hPRL and hPRL-GI29R are produced, they will be tested in vitro for their biological activities by (a) receptor binding assay; (b) STAT 5 (an intracellular signaling molecule for PRL) phosphorylation assay; and (c) apoptosis assay to ensure their quality.
Specific aim (2) is to carry out in vivo studies of hPRL-G129R using SCID mice bearing human breast cancer as model system. We choose to use two human breast cancer cells (T-47D and MCF- 7; from ATCC) and two modes of tumor inoculation for each cell lines will be used for this study (s.c to monitor the tumor development and i.p. monitor the survival rate) to ensure the in vivo experimental results. Two days after tumor cell injection, purified hPRL or hPRL-GI29R will be injected i.p. at two dose groups (1ug/g body weight and 4ug/g body weight) daily for two weeks. Following tumor development, the tumors after s.c. injection will be dissected and weighed while mice with i.p. injection of tumor cells will be followed for survival. We hope that hPRL antagonist could be used to improve the outcome of human breast cancer therapy in the near future.
