| Objective:To investigate the efficacy and mechanisms of Notch signaling inhibition as an adjuvant to docetaxel in castration-resistant prostate cancer(CRPC) using a γ-secretase inhibitor(GSI), PF-03084014. Methods:(1) In vitro: Human prostate cancer cell lines DU145 and PC3,docetaxel resistant type DU145 R, PC3 R cells were treated with PF-03084014,Docetaxel, Docetaxel and PF-03084014 or vehicle(DMSO). Cell viability assays were measured after the treatment. The expression of NICD was also examined by western-blot.(2) In vivo: SCID mice were injected subcutaneously with the four cell lines. When cohorts of tumors reached 100-200 mm3, mice were randomly assigned to control and three treatment groups:(1) vehicle;(2) PF-03084014(150mg/kg, dissolved in 0.05% Methyl cellulose, daily, p.o., 7-days-on/7-days-off schedule for 4 week);(3) docetaxel(5mg/kg for DU145 and PC3; 10mg/kg for DU145 R and PC3 R, i.p, weekly for 4 weeks);(4) PF-03084014+ docetaxel(same dosage as above for 4 weeks). Tumor size and body weight were measured twice a week. Intratibial injection of DU145 R cells formed bone metastasis model, After 8 weeks, mice were randomly assigned to 4 groups(5 mice per group). Control and treatments were the same as for the subcutaneous groups. Radiographs was performed pre- and post-treatment.After the treatment procedure, tumor tissues were harvested and stored as needed. Immunohistochemistry was used to quantify ki67, cleaved-caspase 3 and CD31 expression. Real-time polymerase chain reaction(Real-time PCR) was used to quantify m RNA expressions of HEY1 and HEY2. Western Blot were used to quantify protein expressions of NICD, Cyclin E, BCL-2, BCL-xl, BAK, BAX, MEK1/2, phosphor-MEK1/2( ser217/221), ERK1/2, phosphor-ERK1/2( T202/Y204), AKT, phosphor-AKT(ser473), PI3 K, phosphor-PI3K(tyr455/199), EGFR, P52,E-cadherin, Snail, Slug, MDR1, NANOG. Single cell suspensions were created using collagenase V from harvested tumor tissues. After staining, alkaline dehydrogenase(ALDH) expression was measured by flow cytometry analysis. The second-generation prostasphere formation was also performed in serum free medium. Results:(1) In vitro: PF-03084014 inhibited prostate cancer growth in DU145, PC3 and DU145 R, PC3 R cell lines via dosage and time dependence. To docetaxel resistant DU145 R, PC3 R cells, PF-03084014(10μM for 48hr) sensitized these cells to docetaxel again.(2) In soft tissue mice model, PF-03084014 alone decreased the prostate cancer tumor volume in DU145 and PC3 by 39.7% and 28.9%, respectively; 5mg/kg docetaxel alone inhibited tumor growth in DU145 and PC3 by 36.1%, and 38.5%, respectively; the combination of the two agents increased the anti-tumor efficacy compared to either agent alone with DU145 and PC-3, decreased by 64.1% and 56.6%, respectively. To the docetaxel resistant DU145 R, PC3 R cell lines, PF-03084014 alone decreased DU145 R and PC3 R by 45% and 35%, respectively. Docetaxel alone decreased DU145 R and PC3 R by 36% and 18%, respectively. The combination of both agents increased the anti-tumor efficacy compared to either agent alone for the PC3 R and DU145 R cells(74.4%, 56.7%, respectively). Mouse body weights were not impacted by treatment.In DU145 tumor xenografts, PF-03084014 alone decreased Ki67 by 42.5%, increased cleaved-caspase 3 by 91% and decreased microvessel density by 84.4%;The combination reduced Ki67 expression by 68.3% and microvessel density by 88.4%; whereas, it increased cleaved-caspase 3 expression by 290.6%. PCR results showed that PF-03084014 decreased HEY1 and HEY2 m RNA expression in both DU145 and DU145 R tumor tissues compared to vehicle. Also, PF-03084014 alone suppressed NICD-1 expression in DU145 and DU145 R tumor tissues.In DU145 and DU145 R tumor xenografts, PF-03084014 treatment decreased the cell cycle promoting protein Cyclin E with/without the presence of docetaxel. PF-03084014 had a global effect on expression of the apoptotic pathway BCL-2 protein family. Generally, pro-apoptotic proteins, BAK and BAX were elevated, and anti-apoptotic proteins, BCL-XL and BCL-2 were decreased. PF-03084014 impaired MEK and ERK phosphorylation. Phosphorylation of AKT was inhibited in DU145 cells and PI3 K phosphorylation was decreased in DU145 R cells. As to proteins associated with epithelial-mesenchymal transition(EMT) and drug efflux proteins, PF-03084014 treatment increased E-cadherin expression, decreased vimentin expression and EMT transcription factors: snail, slug; PF-03084014 treatment decreased MDR1 expression. EGFR was decreased in both cell lines after PF-03084014 treatment. The NFk B-family protein P52 was also decreased by PF-03084014 treatment.(3) PF-03084014 treatment decreased the proportion of ALDHhigh cells compared to vehicle-treated cells in all cell lines. The stem cell transcription factor nanog was decreased after PF-03084014 treatment, but increased by docetaxel alone and decreased by the combination of both. Decreased prostasphere formation was induced from pre-treated docetaxel plus PF-03084014 cells when compared to docetaxel treatment only(TSFE: 9.6% vs. 3.8%, p=0.0317).But The mean sphere diameter between the groups was not different(59.59 nm vs. 55.39 nm, p=0.5231). The tumors in the mice that had been treated with the combination of PF-03084014 and docetaxel grew slower with a longer tumor doubling time than those treated with docetaxel alone(37.07 days vs. 15.54 days, p<0.01).(4) Under bone circumstances,PF-03084014 alone inhibited the DU145 R tumor growth, docetaxel alone did not. However, the combination of PF-03084014 and docetaxel induced a markedly increased tumor response than either drug alone. PF-03084014 induced a 19.8% reduction in Ki67 positivity compared to vehicle-treated mice; while docetaxel alone had no effect; however, the combination of both drugs reduced Ki67 by 48%. PF-03084014 induced an increase in Apoptag staining of 1.9-fold and the combination of both PF-03084014 and docetaxel increased Apoptag staining by 6.7-fold. Conclusion:These results reveal that PF-03084014 enhances docetaxel-mediated tumor response and provides a rationale to explore GSIs as adjunct therapy in conjunction with docetaxel for men with CRPC. |
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