Apoptosis induction by combination of drugs or a conjugated molecule associating non-steroidal anti-inflammatory and nitric oxide donor effects in medullary thyroid cancer models: implication of the tumor suppressor p73

Abstract

Background: Medullary thyroid cancer (MTC) is a C-cell neoplasm. Surgery remains its main treatment. Promising therapies based on tyrosine kinase inhibitors demand careful patient selection. We previously observed that two non-steroidal anti-inflammatory drugs (NSAID), indomethacin, celecoxib, and nitric oxide (NO) prevented tumor growth in a model of human MTC cell line (TT) in nude mice.

Methods: In the present study, we tested the NO donor: glyceryl trinitrate (GTN), at pharmacological dose, alone and in combination with each of the two NSAIDs on TT cells. We also assessed the anti-proliferative potential of NO-indomethacin, an indomethacin molecule chemically conjugated with a NO moiety (NCX 530, Nicox SA) on TT cells and indomethacin/GTN association in rMTC 6-23 cells. The anti-tumoral action of the combined sc. injections of GTN with oral delivery of indomethacin was also studied on subcutaneous TT tumors in nude mice. Apoptosis mechanisms were assessed by expression of caspase-3, TAp73α, TAp73α inhibition by siRNA or Annexin V externalisation.

Results: The two NSAIDs and GTN reduced mitotic activity in TT cells versus control (cell number and PCNA protein expression). The combined treatments amplified the anti-tumor effect of single agents in the two tested cell lines and promoted cell death. Moreover, indomethacin/GTN association stopped the growth of established TT tumors in nude mice. We observed a significant cleavage of full length PARP, a caspase-3 substrate. The cell death appearance was correlated with a two-fold increase in TAp73α expression, with inhibition of apoptosis after TAp73α siRNA addition, demonstrating its crucial role in apoptosis.

Conclusion: Association of NO with NSAID exhibited amplified anti-tumoral effects on in vitro and in vivo MTC models by inducing p73-dependent apoptotic cell death.

Keywords: Apoptosis; Medullary thyroid carcinoma; NO-donors; Non-steroidal anti-inflammatory drugs; TT cells; rMTC 6–23 cells.

Fig. 1

a Evolution of the number of viable, control or treated TT cells: 25 μM celecoxib, (cele), 100 μM GTN or a combination of celecoxib plus GTN, during 4 days. b Evolution of viable TT cell number during 4-day treatments with 100 μM indomethacin (indo), 100 μM GTN or a bi-therapy of indomethacin plus GTN. c Effects of the conjugated molecule (NO-donor conjugated to indomethacin, NCX 530), 100 μM (NO-indo100) or 200 μM NO-indomethacin (NO-indo200), during the first 4 days of treatment. Representative graphs of three (a, b) or two (c) independent experiments performed in triplicates. Adherent cells were dissociated with trypsin-EDTA and count with an hemocytometer with blue trypan exclusion. * = P < 0.05, ** = P < 0.01, *** = P < 0.001 versus control (Fisher test)

Fig. 2

a Evolution of viable TT cell number in control wells and in wells treated by NSAID/GTN combination: 25 μM celecoxib plus 100 μM GTN (cele + GTN), or 100 μM indomethacin plus 100 μM GTN (indo + GTN), or 150 μM NCX 530 (NO-indo), during 8 days. b Evolution of viable TT cell populations after cessation of treatments with NSAID/GTN bi-therapies or NCX 530 (25 μM celecoxib, 100 μM indomethacin, 100 μM GTN or 150 μM NCX 530). Representative graphs of two independent experiments performed in triplicates. Adherent cells were dissociated with trypsin-EDTA and count with a hemocytometer. *** = P < 0.001 versus control for each treatment (Fisher tests)

Figure 3

Expression of various proteins in TT cells after 3 days of exposition to 25 μM celecoxib (cele), 100 μM indomethacin (indo), 100 μM GTN or NSAID/GTN combinations. a PCNA protein. For each category, n = 6 to 3. b PARP heavy chain. For each category, n = 4 to 3. c Tumor suppressor p53. For each category, n = 4 to 2. d TAp73α. For each category, n = 7 to 4. Graphs obtained from associated results of two experiments on TT cells. Proteins from TT cell lysates were separated by SDS-PAGE using 10 % acrylamide gel and transferred onto nitro cellulose membranes. Immunoblots were probed with specific antibodies and a α-tubulin (internal control) antibody. * = P < 0.05, *** = P < 0.001 versus control (Fisher tests)

Fig. 4

a Effect of p73 siRNA pre-treatment on TAp73 and PARP expressions in TT cells receiving 25 μM celecoxib plus 100 μM GTN (combi) during 2 days and 8 h. b Effect of p73 siRNA pre-treatment on TAp73 expression in the same TT cells. c Effect of p73 siRNA pre-treatment on full length PARP expression in these cells. Graphs obtained from associated results of two experiments on TT cells. For each category, n = 6 to 4. Proteins from TT cell lysates were separated by SDS-PAGE using 10 % acrylamide gel and transferred onto nitro cellulose membranes. Immunoblots were probed with specific antibody and a α-tubulin (internal control) antibody. * = P < 0.05, ** = P < 0.01 (Fisher tests)

Fig. 5

Evolution of the number of viable, control or treated rMTC 6–23 cells: 200 μM indomethacin (indo), 100 μM GTN, or the combination of indomethacin plus GTN, during 6 days. Experiment performed in triplicates. Adherent cells were dissociated with trypsin-EDTA and their number was evaluated with a cell counter (blue trypan exclusion). Drug combination amplified the anti-proliferative effect of each drug (P < 0.01 versus indomethacin alone, P < 0.0001 versus GTN alone, at D6) and reduced the cell number at D6 versus D0 (P < 0.05, Fisher tests)

Fig. 6

In vivo effect of GTN plus indomethacin association on growth of TT xenografts in nude mice. Indomethacin was administrated in drinking water (2 mg/kg of body weight × day). Mice treated by GTN received Nitronal sc injections each two days (100 μl during the first week and 150 μl/20 g body weight the second week of treatment). a Effect on tumor volumes. The GTN-indomethacin/GTN association stopped the growth of TT tumors from D7 to D12. Significant differences were revealed between tumor volumes by ANOVAs (P < 0.0001 combination group versus control, P < 0.05 combination group versus GTN treated group; control group, n = 5, GTN treated group, n = 5 and indomethacin/GTN association, n = 4). * = P < 0.05, ** = P < 0.01 (Fisher tests versus control at D9 or D12). b A microphotography of immunostaining of caspase 3 protein revealed numerous apoptotic cells (dark dots) in indomethacin/GTN treated tumors. No staining was observed in the two other groups of tumor