Cells and culture conditions
Androgen-dependent LNCaP prostate cancer cells, androgen-independent PC-3 and DU-145 prostate cancer cells, and renal carcinoma 786-O cells were obtained from ATCC (Rockville, MD). The 769-P, HRC-51, HRC-45, HRC-78, and SK-45 human renal cell carcinoma cell lines were a kind gift of Dr. Joseph Testa (Fox Chase Cancer Center, Philadelphia, PA). The SK-26B cell line was obtained through the generosity of Dr. Finke (The Cleveland Clinic Foundation, Cleveland OH). Initial stocks were cryopreserved, and at every six-month interval a fresh aliquot of frozen cells was utilized for the experiments. No authentication was done by the authors. Cells were cultured in RPMI 1640 (Bio-Whittaker, Walkersville, MD) supplemented with 10% FCS (Hyclone, Logan, UT), gentamicin (50 mg/l), sodium pyruvate (1 mM) and non-essential amino acids (0.1 mM) under conditions indicated in the figure legends.
Antibodies and Reagents
Antibodies to PTEN, Akt, phospho-Akt(S473), phospho-Akt(T308), phospho-Tuberin/TSC2(T1462), Tuberin/TSC2, phospho-4EBP1(T37/46) and GAPDH were obtained from Cell Signaling Technology (Beverly, MA). Sunitinib and temsirolimus were obtained from LC Laboratories (Woburn, MA). GDC-0980 was obtained from ChemieTek (Indianapolis, IN). GSK690693 was obtained from Selleck (Houston, TX).
Stable Transfection with PTEN
To generate the PTEN expression vector PTEN ORF was amplified with specific forward 5‵-atcttgaagcttgccaccatgacagccatcatcaaag-3‵ and reverse 5‵-atcttggaattctcagacttttgtaatttgtgtatgctgatc-3‵ primers carrying HindIII and EcoR1 restriction sites (underlined) using as a template pOTB7 vector (clone ID#3937787) containing full-length PTEN cDNA (Thermo Scientific, Huntsville, AL) and then cloned into HindIII/EcoR1 sites of pcDNA3.1-puro vector. The sequence of the pcDNA3.1-PTEN-puro construct was validated in the DNA Sequencing Facility of the Fox Chase Cancer Center. Cells were transfected with either the PTEN expression vector or the pcDNA3.1-puro control vector using the TransIT-Prostate transfection kit (Mirus Bio, Madison, WI). Selection was performed using puromycin (1 μg/ml).
Western Blot Analysis
Whole cell lysates preparation and Western Blot Analysis were performed as described previously (7
Measurement of apoptosis
DNA fragmentation was detected using the APO-BRDU kit (The Phoenix Flow Systems, Inc., San Diego, CA) according to manufacturer's instructions.
Measurement of IL-6 and IL8
IL-6 and IL-8 levels in cell culture supernatants were measured using ELISA kits (R&D Systems, Minneapolis, MN).
Assessment of in vivo tumor growth
For in vivo studies, 1 × 106 PC-3-PTEN or PC-3-puromycin cells were inoculated s.c. in the flank region of 6 week-old male C.B17/Icr-scid mice using a 27-gauge needle. All animal procedures were done in accordance with institutional guidelines on animal care and with appropriate institutional certification. Animals were fed an autoclaved AIN-93M diet (Harlan Teklad, Madison, WI) and water ad libitum. Two weeks after the injection of tumor cells, animals were randomly assigned to the control or experimental groups (n=5 mice/group). The mice were treated with either vegetable oil (control) or sunitinib (40 mg/kg in vegetable oil, 5 days/week, p.o.).
To examine the effect of co-administration of sunitinib and temsirolimus on tumor growth in vivo in PTEN-deficient cells, subcutaneous tumors were established using PTEN-negative PC-3 cells. Two weeks after tumor cell injection animals were randomly assigned to the control or experimental groups (n=7 mice/group). The mice were treated with (i) vegetable oil (control); (ii) sunitinib (40 mg/kg in vegetable oil, 5 days/week, p.o.); (iii) temsirolimus (10 mg/kg, once a week, i.v.); or with sunitinib and temsirolimus combined. Tumors were measured twice weekly and their volumes were calculated by the formula: [volume = 0.52 × (width)2 × length]. To examine sunitinib levels in plasma and tumor tissue, sunitinib (40 mg/kg in vegetable oil) was administered p.o. daily to C.B17/Icr-scid mice with PC-3 xenografts for 7 consecutive days. Animals were euthanized via CO2 asphyxiation 3 hours following last sunitinib administration. Levels of sunitinib in plasma and tumor samples were examined at Wolfe Laboratories (Watertown, MA).
Statistical analysis was performed using a two-sided Student's t-test. A p-value of <0.05 was considered statistically significant.