Figure 3. Effect of Targeting the Glutaminase II Pathway in Pancreatic Cancer.

(A-B) Assessment of GTK Expression in Pancreatic Cancer Cells. GTK expression was assessed by western blotting in (A) A6L, A32, E3, JD13D, P8, P10, P198, and P215 pancreatic cancer cell lines, (B) P198 shGTK-KD, and P198 shControl. Tubulin served as a loading control. (C) Effect of Glutaminase 1 Inhibitor, BPTES, on Cell Numbers of P198 shGTK-KD and P198 shControl in vitro. Pancreatic cancer cells P198 shControl and P198 shGTK-KD were grown in an incubator at 37°C in 5% CO₂ and 95% air (vol/vol) in DMEM containing 10% FBS, 1% penicillin-streptomycin, and 1μg/mL puromycin. Cells were treated with DMSO vehicle control or 10 μM BPTES. Cell numbers were assessed at 48, 72, 96, and 120 hours after treatment (n = 3 samples per group and per time point). This experiment was replicated three times with similar results. (D) P198 shGTK-KD and P198 shControl Xenograft Tumors. 5 × 10⁶ P198 shGTK-KD and P198 shControl cells were subcutaneously injected into the backs of mice (n = 20 tumors per group and per time point). Tumor formation and size progression were measured over 52 days and tumor sizes in the mice at day 52 are pictured. (E) Effect of Glutamine Antagonist, JHU083 (1 mg/kg five days a week for three weeks) on Patient-Derived Pancreatic Cancer (JHU094) Orthotopic Tumors. Patient-derived pancreatic cancer (JHU094) tumors were implanted into the pancreas of mice. Tumor weights of Day 0 (the starting treatment day) were assessed from twelve mice that were euthanized at Day 0 and their tumors were extracted from the pancreas and weighed. Mice received 1 mg/kg (0.022 mg of JHU083 in 100 μL of vehicle control per mouse) of JHU083, by intraperitoneal injection (IP) or 100 μL of vehicle control five days per week for three weeks. The vehicle control consisted of 95% (vol/vol) HEPES buffered saline in ethanol. Tumors were then excised and weighed. All values are shown as mean ± SEM (n = 20 tumors per group for every time point for xenograft tumors, 10 per group for JHU083 treated tumors). NS, not significant, *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t-test) where indicated.