Combination treatment with TRA-8 anti death receptor 5 antibody and CPT-11 induces tumor regression in an orthotopic model of pancreatic cancer

Abstract

Purpose: Evaluate the response of human pancreatic cancer cell lines and orthotopic tumors to TRA-8, an agonistic antibody to death receptor 5, in combination with irinotecan (CPT-11).

Experimental design: MIA PaCa-2 and S2VP10 cells were treated with TRA-8 and/or CPT 11. Cell viability was determined by ATP assay. JC-1 mitochondrial depolarization and Annexin V assays confirmed cell death by apoptosis. Immunoblotting was used to evaluate protein changes. MIA PaCa-2 cells were injected into the pancreas of severe combined immunodeficient mice. Mice underwent abdominal ultrasound to quantitate tumor size before and after treatment with twice weekly injections of 200 microg TRA-8 and/or 25 mg/kg CPT-11 for one or two treatment cycles, each lasting 2 weeks.

Results: MIA PaCa-2 cells were more sensitive to TRA-8 and showed additive cytotoxicity, whereas S2VP10 cells showed synergistic cytotoxicity when treated with TRA-8 and CPT-11. Cell death occurred via apoptosis with increased cleavage of caspase-3, caspase-8, and caspase-9 and proapoptotic proteins Bid and poly(ADP)ribose polymerase after combination treatment compared with either agent alone. XIAP and Bcl-XL inhibitors of apoptosis were down-regulated. After a single cycle of in vivo combination therapy, tumor sizes had diminished significantly (P<0.001) at 8 days posttreatment compared with no treatment, CPT-11, and TRA-8; and there was a 50-day increase in survival with combination treatment over untreated controls (P=0.0002), 30 days over TRA-8, and a 36-day increase over CPT-11 monotherapy (P=0.0003). With two cycles of TRA-8/CPT-11 treatment, mean survival time increased significantly (P<0.001) to 169 days versus untreated controls, TRA-8 or CPT-11 (76, 121, or 108 days, respectively).

Conclusions: Combination TRA-8 and CPT-11 therapy produced enhanced cytotoxicity and survival in the MIA PaCa-2 orthotopic model of pancreatic cancer.

Fig. 1

Viability of pancreatic cancer cells in vitro after treatment with CPT-11 and TRA-8. An ATP-based cell viability assay was used to assess cytotoxicity. MIA PaCa-2 cells treated with CPT-11 and TRA-8 showed additive reduction in cell viability compared with either agent alone. S2VP10 cells treated with the combination of CPT-11 and TRA-8 showed a synergistic reduction in cell viability compared with treatment with either agent alone.

Fig. 2

Analysis of apoptosis using Annexin V – FITC and flow cytometry in MIA PaCa-2 and S2VP10 cells after treatment with CPT-11 and TRA-8. Cells were treated with CPT-11 for 24 h, then TRA-8 was added. Cells were stained with Annexin V – FITC and PI after 2 h treatment with TRA-8. Combination treatment with CPT-11 and TRA-8 produced enhanced apoptosis as indicated by a shift to cells that were FITC⁺/PI⁻, which was greater than with either agent alone.

Fig. 3

Analysis of mitochondrial membrane depolarization using JC-1 assay on TRA-8– and CPT-11 – treated pancreatic cell lines. MIA PaCa-2 (A) and S2VP10 (B) cells were treated with 30 µmol/L CPT-11 for 24 h, then 300 ng/mL TRA-8 was added. Cells were stained with JC-1 dye 24 h after adding TRA-8 and analyzed using flow cytometry. Fluorescence intensity in the red and green channels was plotted, and the ratio of red to green fluorescence was calculated for each condition. Combination treatment with CPT-11 and TRA-8 produced a greater reduction in mitochondrial membrane potential, as indicated by a shift from red fluorescence to green fluorescence, than treatment with CPT-11 or TRA-8 alone.

Fig. 4

Western blot analysis of apoptosis regulatory proteins in MIA PaCa-2 and S2VP10 cells treated with CPT-11, gemcitabine, TRA-8, or the combination of TRA-8 and the chemotherapy drugs. Cells were treated with CPT-11 (10 or 300 µmol/L for S2VP10 and MIA PaCa-2, respectively) or 30 nmol/L gemcitabine for 24 h. Cells were then treated with 24 ng/mL TRA-8 for 3 h. Untreated cells were used as controls. Equivalent amounts of protein were analyzed by Western blot and actin levels were compared with control for protein loading (not shown). Combination treatment produced enhanced caspase-3, caspase-8, and caspase-9 activation as shown by cleavage of their proforms. Bid, a caspase-8 substrate, and poly(ADP)ribose polymerase, a caspase-3 substrate, also showed enhanced cleavage. Bcl-XL was down-regulated in MIA PaCa-2 cells receiving CPT-11 and combination treatment. XIAP was down-regulated in S2VP10 cells receiving combination treatment with TRA-8 and CPT-11.

Fig. 5

Intrapancreatic MIA PaCa-2 tumor growth measured by ultrasound imaging. The mean tumor size in each treatment group were equivalent before initiation of treatment (day 21). Combination therapy with one cycle of CPT-11 and TRA-8 produced a 90.2% mean decrease in tumor size at day 41, whereas there was no change in mean tumor size in the group treated with TRA-8 alone. At day 83 after tumor cell injection, the group treated with CPT-11 and TRA-8 showed the least amount of tumor growth.

Fig. 6

Survival study comparing SCID mice with intrapancreatic MIA PaCa-2 tumors treated with one cycle of therapy with CPT-11 (25 mg/kg i.p. on days 15, 19, 22, and 26 postimplant), TRA-8 (200 µg i.p. on days 14, 18, 21, and 25 postimplant), or the combination of CPT-11 and TRA-8. Mean survival was significantly increased in the combination therapy group. Arrows, start and end of treatment.

Fig. 7

Ultrasound imaging of SCID mice with orthotopic MIA PaCa-2 tumors and two cycles of therapy. Mice underwent ultrasound imaging with measurements of tumor size on days 24, 73, and 83. They were treated with one cycle of 200 µg TRA-8 i.p. on days 25, 28, 32, and 35 and a second cycle on days 53, 56, 60, and 63; one cycle of 25 mg/kg CPT-11 i.p. on days 26, 29, 33, 36 and a second cycle on days 54, 57, 61, and 64; or a combination of TRA-8 and CPT-11 with the same dose and schedule as either agent alone.

Fig. 8

Survival of SCID mice with orthotopic MIA PaCa-2 tumors treated with two cycles of TRA-8, CPT-11, or the combination of the two agents. Arrows, start and end of each treatment cycle.