Effect of osteopontin on the drug transporter expression in prostate cancer cells

IS Hsieh1, WM Fu1, RS Yang2, WJ Chuang3, HC Liou1. 1College of Medicine, National Taiwan University, Pharmacology, 100, Taiwan, 2National Taiwan University Hospital, Orthopedic Surgery, 100, Taiwan, 3College of Medicine, National Cheng Kung University, Biochemistry and Institute of Basic Medical Science, 701, Taiwan

Resistance of chemotherapy is always a major problem in progressive cancer disease and tumor cells begin to amplify its proliferation, metastasis and invasion to distant organs, leading to low outcome and survival rate. Under the threatening of chemotherapy, the cancer cells will turn on self-protection mechanism and overexpress the ABC drug transporters that actively pump out a variety of amphipathic compounds from cells and thus decrease the therapeutic effects of chemotherapeutic agents, i.e., induction of multidrug resistance (MDR). Recent studies indicate that drug resistance can be rapidly induced by some tumor-associated microenvironment factors, such as SDF-1, IL-6, VEGF, integrin, etc. Osteopontin (OPN), an extracellular matrix protein, has a functional RGD domain for binding to integrin and plays an important role in tumor microenvironment. It is involved in tumor survival, metastasis and invasion via PI3K-Akt and NF-κB pathways.

Here we found that OPN expression was up-regulated by hypoxic condition in PC-3 prostate tumor cells. OPN increased the mRNA and protein expression of P-gp, a subfamily of ABC transporter, in a concentration- and time-dependent manner. This regulation of drug transporters by OPN increased the function of cell protective mechanism and this protective function of OPN was antagonized by pretreatment with αvβ3 integrin monoclonal antibody. The fluorescent property of daunomycin provides the advantage for the measurement of drug pumping-out activity. Using confocal microscope and flow cytometric analysis to examine the intracellular fluorescent intensity of daunomycin and evaluate the accumulation of the drug, OPN was found to increase the drug pumping out activity. OPN inhibited daunomycin-induced cell death was further antagonized by the concomitant treatment of αvβ3 integrin monoclonal antibody. Since OPN upregulates drug transporter to pump out daunomycin, we then examined whether the long-term treatment of daunomycin affected the expression of OPN. Daunomycin treatment further enhanced the expression of OPN. To examine the effects of endogenous OPN expression on daunomycin-induced cell death, knockdown of OPN was performed by using shRNA transfection. Knockdown of endogenous OPN in PC-3 cells enhanced the apoptosis induced by daunomycin. Furthermore, knockdown of OPN also potentiated the cell death caused by other P-gp substrate drugs, including paclitaxel, doxorubicin, actinomycin-D and rapamycin. To confirm the effect of OPN on the sensitivity to chemotherapeutic drug, the in vivo Xenograft model in NOD-SCID mice was performed. The animal study show that the OPN-knockdown group was more sensitive to the cytotoxic effect of daunomycin (tumor volume was 2428 ± 360 mm3 and 909 ± 289 mm3 for empty vector and OPN knockdown group, respectively). Values are expressed as mean ± SEM. Results were analyzed with one-way analysis of variance (ANOVA), followed by Neuman-Keuls. Significance was defined as p < 0.05.

In conclusion, OPN plays an important role in regulating drug resistance via the increase of transporter- P-gp expression and knockdown of OPN enhances the cytotoxic action of daunomycin in vitro and in vivo. OPN can be a potential therapeutic target for cancer therapy to inhibit drug resistance.

Abbreviations:

ABC transporter : ATP-binding cassette transporter; P-gp : p-glycoprotein; OPN : osteopontin; MDR : multidrug resistance; SDF-1 : stromal cell-derived factor-1; IL-6 : Interleukin-6; VEGF : Vascular endothelial growth factor; PI3K : phosphatidylinositol-3-kinase; NF-κB : nuclear factor kappa B