Interferon-gamma-induced nitric oxide inhibits the proliferation of murine renal cell carcinoma cells

Abstract

Renal cell carcinoma (RCC) remains one of the most resistant tumors to systemic chemotherapy, radiotherapy, and immunotherapy. Despite great progress in understanding the basic biology of RCC, the rate of responses in animal models and clinical trials using interferons (IFNs) has not improved significantly. It is likely that the lack of responses can be due to the tumor's ability to develop tumor escape strategies. Currently, the use of targeted therapies has improved the clinical outcomes of patients with RCC and is associated with an increase of Th1-cytokine responses (IFNγ), indicating the importance of IFNγ in inhibiting tumor proliferation. Thus, the present study was designed to investigate a new mechanism by which IFNγ mediates direct anti-proliferative effects against murine renal cell carcinoma cell lines. When cultured RCC cell lines were exposed to murine recombinant IFNγ, a dose dependent growth inhibition in CL-2 and CL-19 cells was observed; this effect was not observed in Renca cells. Growth inhibition in CL-2 and CL-19 cell lines was associated with the intracellular induction of nitric oxide synthase (iNOS) protein, resulting in a sustained elevation of nitric oxide (NO) and citrulline, and a decrease in arginase activity. The inhibition of cell proliferation appears to be due to an arrest in the cell cycle. The results indicate that in certain RCC cell lines, IFNγ modulates L-arginine metabolism by shifting from arginase to iNOS activity, thereby developing a potent inhibitory mechanism to encumber tumor cell proliferation and survival. Elucidating the cellular events triggered by IFNγ in murine RCC cell lines will permit anti-tumor effects to be exploited in the development of new combination therapies that interfere with L-arginine metabolism to effectively combat RCC in patients.

Keywords: Interferon-gamma; L-arginine; arginase 2; cell proliferation.; nitric oxide; nitric oxide synthase; polyamines; renal cell carcinoma.

Figure 1

Characterization of iNOS expression and iNOS activity in murine RCC cell lines cultured in normal conditions. (A) Representative Western blot analysis for iNOS (130 kDa) and GAPDH (35 kDa) in CL-2, CL-19 and Renca cells cultured in normal conditions for 24, 48, and 72 h. Twenty five μg of total protein was loaded per line. RAW 264.7 cells stimulated with LPS plus murine IFNγ were used as positive control (+Ctrl) for iNOS protein. (B) Nitrite production in the 3 cell lines was determined by Greiss reaction in culture media supernatants collected after 24, 48, and 72 h in culture. Results are the mean ± SE of triplicates of five different separate experiments. (C) Reverse transcription-polymerase chain analysis of iNOs mRNA expression. The result of a representative experiment is shown; similar results were obtained in at least three experiments. The expected sizes of amplified fragments were 459 bp for iNOS and 436 bp for β-actin.

Figure 2

IFNγ-induced iNOS expression and iNOS activity in murine RCC cell lines. For reference, Figure 1A can be used as untreated control for these experiments. (A) Time course of iNOS protein expression by Western blot after stimulation of cells for 24, 48, and 72 h with 10 U/ml of murine IFNγ. (B) Nitrite production in culture supernatants following stimulation with IFNγ. Values for nitrite production are expressed by mean ± SE for three different experiments performed five different times. (C) Pattern of 24, 48, and 72 h iNOS mRNA induction (RT-PCR) in response to IFNγ stimulation. Each Western blot and RT-PCR presented is from single experiment that is representative of three to five separate experiments.

Figure 3

Effect of IFNγ and LNMMA on CL-2, CL-19 and Renca cell proliferation. (A) Three hundred thousand cells from CL-2, CL-19 and Renca were seeded in six well plates and cultured in presence or absence of IFNγ (10U/ml) or iNOS inhibitor LNMMA (1mM). Cell proliferation was measured at 24, 48, and 72 h using the MTT assay. The results are expressed as O.D (570) means ± SE of triplicate determinations from five independent experiments. Significant differences in proliferation were observed between control and IFNγ treated CL-2 (* p= 0.0084) and CL-19 (**p=0.0001) cells respectively. (B) Morphological changes and inhibition of cell proliferation in the CL-2, CL-19 and Renca cells before and after treatment with IFNγ. Microscopic images of mRCC cells after treatment with IFNγ were taken at 48h. Photographs were taken under phase contrast at 100x magnification.

Figure 4

Nitrite (NO^-2) production measured in mRCC cells after treatment with IFNγ and IFNγ plus LNMMA. Average nitrite concentration between untreated and treated cells with IFNγ over time in (A) CL-2 (* p= 0.003) and (B) CL-19 (** p= 0.037) at 48 and 72 h respectively. Significant reduction of nitrite production (*** p<0.0001) in CL-2 and CL-19 cells after the addition of iNOS inhibitor LNMMA. (C) Nitrite values in Renca cells after IFNγ treatment. Results are the mean ± SE of triplicates of five different separate experiments.

Figure 5

Effect of IFNγ treated cells on apoptosis and cell cycle. (A) Cells were plated for 1 day and then cultured in media containing IFNγ. DNA was extracted and subjected to DNA fragmentation ladder analysis. Data are representative of three independent experiments with similar results. (B) Analysis of cell cycle progression in untreated and treated mRCC cell lines with IFNγ. After 24, 48, and 72 h cells were analyzed for DNA content by flowcytometry. A representative experiment performed at 48 h indicate the distribution of the cells in G1, S, and G2/M phases of the cell cycle.

Figure 6

The effects of IFNγ on the induction of arginase activity in the mRCC cell lines. Cells were treated with 10 U/ml of IFNγ for 24, 48, and 72 h and tested for arginase activity (conversion of L-argine to L-ornitine by arginase). As compared to untreated cells (Ctrl) significant reduction of ARG activity is observed: CL-2 (* p= 0.02) CL-19 (** p= 0.0008) and Renca (*** p= 0.03) at 72 h. The data represent the mean ± SE of triplicate determinations from five separate experiments.

Figure 7

L-arginine, L-ornithine, and citrulline levels. Culture supernatants of CL-2, CL-19 and Renca cells untreated and treated with IFNγ were collected at 24, 48, and 72 h and analyzed by HPLC after deproteinization with methanol and derivatization with OPA. Standards for L-arginine, L-ornithine, and citrulline in methanol were run with each experiment. Asterisks show significant differences between untreated and treated cells. (A) CL-19 L-arginine (* p= 0.0002), (B) CL-19. L-ornithine (** p=0.0183), and (C) CL-2 and CL-19 citrulline (*** p=0.0001). The results are expressed as mean ± SE of duplicate determinations from four independent experiments.

Figure 8

Cytokine profile in supernatants of mRCC cell lines. The cell lines were seeded in 6 well plates and treated with IFNγ (10U/ml) for 24, 48, and 72 h. Supernatants were collected and assayed using a Th1/Th2 panel Bio-Plex cytokine assay. IL-1β, IL-6, VEGF, and TGFβ levels are shown. Treatment with IFNγ has a significant inhibitory effect in CL-2 cells production of IL-1β and IL-6 in CL-2 (* p<0.001; ** p=0.002) and IL-1β in CL-19 (* p=0.009). Data are representative of experiments at 48h of at least 3 independent experiments.