Targeted inhibition of inducible nitric oxide synthase inhibits growth of human melanoma in vivo and synergizes with chemotherapy

Abstract

Purpose: Aberrant expression of inflammatory molecules, such as inducible nitric oxide (NO) synthase (iNOS), has been linked to cancer, suggesting that their inhibition is a rational therapeutic approach. Whereas iNOS expression in melanoma and other cancers is associated with poor clinical prognosis, in vitro and in vivo studies suggest that iNOS and NO can have both protumor and antitumor effects. We tested the hypothesis that targeted iNOS inhibition would interfere with human melanoma growth and survival in vivo in a preclinical model.

Experimental design: We used an immunodeficient non-obese diabetic/severe combined immunodeficient xenograft model to test the susceptibility of two different human melanoma lines to the orally-given iNOS-selective small molecule antagonist N(6)-(1-iminoethyl)-l-lysine-dihydrochloride (L-nil) with and without cytotoxic cisplatin chemotherapy.

Results: L-nil significantly inhibited melanoma growth and extended the survival of tumor-bearing mice. L-nil treatment decreased the density of CD31+ microvessels and increased the number of apoptotic cells in tumor xenografts. Proteomic analysis of melanoma xenografts with reverse-phase protein array identified alterations in the expression of multiple cell signaling and survival genes after L-nil treatment. The canonical antiapoptotic protein Bcl-2 was downregulated in vivo and in vitro after L-nil treatment, which was associated with increased susceptibility to cisplatin-mediated tumor death. Consistent with this observation, combination therapy with L-nil plus cisplatin in vivo was more effective than either drug alone, without increased toxicity.

Conclusions: These data support the hypothesis that iNOS and iNOS-derived NO support tumor growth in vivo and provide convincing preclinical validation of targeted iNOS inhibition as therapy for solid tumors.

Figure 1. Human melanoma lines constitutively express iNOS and make NO in vitro and in vivo

A. Human melanoma lines mel624, A375, and mel526, and the iNOS-negative colon cancer line WiDR were assessed by Western blot for iNOS protein expression. B. 5 × 10⁶ A375 melanoma cells were injected subcutaneously into recipient NOD/SCID mice and allowed to form 0.5 cm tumors. Xenografts were then harvested and sections stained by immunohistochemistry for iNOS and the stable NO reaction product nitrotyrosine. Representative MRI (tumor in false color) and immunohistochemistry results are shown.

Figure 2. The iNOS-selective competitive antagonist L-nil blocks iNOS-derived NO production in vitro and in vivo without affecting cell viability

A. Human melanoma lines mel624, A375, mel526; or colon cancer line WIDR or WIDR transfected with iNOS plasmid were incubated for 72 hours with the indicated concentrations of L-nil, and NO released into the supernatant measured using the nitrite reconversion method. B. The effect of L-nil on NO production by WIDR+iNOS plasmid was used to determine the IC₅₀ of L-nil in the cell culture system. C. Serum was collected 7 hours after intraperitoneal LPS injection of C57BL/6 mice (4 per group) pre-treated for 48 hours with 0.1% L-nil in drinking water (L-nil) or plain drinking water (control) Nitrite levels were determined using the Griess assay. D. Mel624 or A375 melanoma cells were cultured for the indicated length of time in medium containing varying concentrations of L-nil and proliferation was measured by XTT assay.

Figure 3. Orally-administered selective iNOS antagonists suppress the growth of human melanoma and extend survival without overt toxicity in an immunodeficient mouse xenograft model

A. 5×10⁶ mel624 cells were injected subcutaneously on day 0. Starting on day 3, mice were treated with 0.15% L-nil in drinking water, or plain water control, for 28 days. Tumor sizes are expressed as mm²; each black line represents an individual mouse, median size is with a bold line. Right panels show representative MRI pictures of day 22 tumors from L-nil-(top) and control-(bottom) treated mice. Data is representative of three experiments. B. Kaplan-Meier survival estimates for control-and 0.15% L-nil-treated mel624-bearing mice. C. Using the experimental design described in 3A, mel624-bearing mice were treated with 0.15% L-nil or 0.2% 1,3-PBIT in drinking water or plain drinking water control for 28 days starting on day 3. Tumor sizes are expressed as mean +/− SEM. Data is representative of two experiments. D. Mice were weighed after 21 days treatment with 0.15% L-nil or 0.2% PBIT in drinking water, or plain water control.

Figure 4. Oral L-nil treatment inhibits intratumoral nitrotyrosine formation, and decreases tumor microvessel density

Mel624-bearing mice were treated for 14 days with 0.15% L-nil in drinking water or plain water control prior to sacrifice, harvest of xenografts, and hematoxalin and eosin staining (top), or immunohistochemical staining for iNOS, nitrotyrosine (NT), or the vascular marker CD31. Representative images are shown at the indicated original magnifications, except the insert pictures of representative CD31-stained vessels at 400X. Computer-assisted quantitation of images is shown to the right.

Figure 5. L-nil downregulates Bcl-2 expression and induces intratumoral apoptosis in vivo

A. Mel624 xenografts were harvested from mice after treatment with 0.15% L-nil or plain water control for 14 days. Paraffin-embedded slides were subjected to TUNEL analysis of apoptotic cells (top), or double-staining for TUNEL and the vascular marker CD31(bottom). Representative images are shown on the left and quantitation of TUNEL staining on the right. B. Western blot analysis mel624 xenograft lysates was performed to confirm downregulation of Bcl-2 expression in tumors from L-nil treated mice (2 replicates of 2 tumors per group). The bar graph shows quantitation of Bcl-2 levels (normalized to Erk2 protein expression) in tumors from control and L-nil treated mice. C. 5×10⁴ A375 cells were cultured in 48 well plates for 3 days with the indicated concentration of L-nil, at which time the medium and inhibitor were replenished and cells cultured for an additional 2 days prior to immunostaining for intracellular Bcl-2 and analysis by flow cytometry.

Figure 6. L-nil enhances sensitivity of human melanoma cells to anti-tumor effect of cisplatin in vitro and in vivo

A. A375 and mel624 cells were treated with the indicated concentrations of cisplatin for 48 hours and proliferation was measured by XTT assay. Data are presented as mean +/− SEM and are representative of three independent experiments. B. 2×10⁴ A375 melanoma cells were cultured in the indicated concentration of L-nil for 3 days. On day 3, the medium was discarded and cells were incubated for another 48 hours in fresh media with the indicated concentration of L-nil and/or cisplatin. Cells were then trypsinized and cell death was assessed by annexin V staining and flow cytometry. Data are presented as mean +/− SEM and are representative of 3 independent experiments. *p=0.012, **p=0.003) C. SCID-NOD mice were injected subcutaneously with 5×10⁶ A375 or mel624 cells, and treated with 0.2% L-nil for the indicated time, starting on day 3. Cisplatin was given as three intraperitoneal injections of 2.5 mg/kg (A375) or 6 mg/kg (mel624) spaced three days apart, beginning on day 13 (mel624) or day 25 (A375). The numbers to the right of each curve give the fraction of surviving mice. For A375 p<0.001 for CDDP+L-nil vs. CDDP alone; p=0.009 for CDDP+L-nil vs. L-nil alone. For mel624 p<0.001 for CDDP+L-nil vs. CDDP alone; p=0.010 for CDDP+L-nil vs. L-nil alone on day 30.