Auranofin Inhibition of Thioredoxin Reductase Sensitizes Lung Neuroendocrine Tumor Cells (NETs) and Small Cell Lung Cancer (SCLC) Cells to Sorafenib as well as Inhibiting SCLC Xenograft Growth

Abstract

Thioredoxin Reductase (TrxR) is a key enzyme in hydroperoxide detoxification through peroxiredoxin enzymes and in thiol-mediated redox regulation of cell signaling. Because cancer cells produce increased steady-state levels of reactive oxygen species (ROS; i.e., superoxide and hydrogen peroxide), TrxR is currently being targeted in clinical trials using the anti-rheumatic drug, auranofin (AF). AF treatment decreased TrxR activity and clonogenic survival in small cell lung cancer (SCLC) cell lines (DMS273 and DMS53) as well as the lung atypical (neuroendocrine tumor) NET cell line H727. AF treatment also significantly sensitized DMS273 and H727 cell lines in vitro to sorafenib, a multi-kinase inhibitor that was shown to decrease intracellular glutathione. The pharmacokinetic and pharmacodynamic properties of AF treatment in a mouse SCLC xenograft model was examined to maximize inhibition of TrxR activity without causing toxicity. AF was administered intraperitoneally at 2 mg/kg or 4 mg/kg (IP) once (QD) or twice daily (BID) for 1 to 5 days in mice with DMS273 xenografts. Plasma levels of AF were 10-20 μM (determined by mass spectrometry of gold) and the optimal inhibition of TrxR (50 %) was obtained at 4 mg/kg once daily, with no effect on glutathione peroxidase 1 activity. When this daily AF treatment was extended for 14 days a significant prolongation of median survival from 19 to 23 days (p=0.04, N=30 controls, 28 AF) was observed without causing changes in animal bodyweight, CBCs, bone marrow toxicity, blood urea nitrogen, or creatinine. These results show that AF is an effective inhibitor of TrxR both in vitro and in vivo in SCLC, capable of sensitizing NETs and SCLC to sorafenib, and supports the hypothesis that AF could be used as an adjuvant therapy with agents known to induce disruptions in thiol metabolism to enhance therapeutic efficacy.

Figure 1:. AF decreases TrxR activity and clonogenic survival of lung NET and NECs. AF enhances clonogenic cell death with sorafenib treatment.

(A) Small cell lung cancer DMS273 and DMS53 and bronchial carcinoid H727 cell line were treated with 1 μM AF for 24 h (gray bars) then collected for TxrR enzyme activity. All activity was normalized to H727 control cells. (B) Same cell lines and treatment as in (A) with the clonogenic assay. Normalized to each respective control. * p<0.05 compared to untreated cells 2-way ANOVA with Fishers LSD n=3. (C) Exponentially growing DMS273 and H727 cells were treated for 48 h with sorafenib at 2.5 or 5 μM followed by harvest and glutathione assay. (D,E) DMS273 and H727 cells were treated with 48 h sorafenib and the last 24 h with 1 μM AF followed by clonogenic assay. Clonogenic survival colony counts were normalized to killing with treatment of AF alone. n ≥ 3 independent experiments 2-way ANOVA Fishers LSD * significantly different than control (p<0.05), # significantly different than either drug alone (p<0.05).

Figure 2 -. Dose escalation of AF selectively decreases TrxR activity in SCLC xenografts, kidneys and liver, but does not affect Gpx1 activity.

(A, B, C) Female athymic nude mice (6 per group) were xenografted with 5 ×10⁶ DMS273 cells (2 tumors/mouse one on each flank) and given AF IP injections for 24 h or 5 days either QD or BID. Kidney, liver, and tumor samples were collected for TrxR activity. (D) Plasma samples were collected 9 hours after final BID dose (5 days of therapy) and gold content determined. (E) Gpx1 activity was determined in xenograft tumors after 5 days of AF therapy. * = p-value < 0.05, ** = p-value < 0.01, *** = p-value < 0.001. Analyzed by Mix Effects ANOVA with Fishers LSD comparisons.

Figure 3. AF decreases TrxR activity in DMS273 tumors in vivo independent of tumor size.

2 mice each with 2 flank DMS273 xenografts in the first cohort and 4 mice with 1 flank DMS273 xenografts in the third cohort, were treated with AF 4 mg/kg IP QD for 14 days then euthanized and evaluated for TrxR enzyme activity assays and results are plotted in aggregate (A) or as a function of tumor size at the end of treatment (B). * p<0.05 compared to untreated cells two-ways ANOVA with Fishers LSD comparisons.

Figure 4 -. 4 mg/kg QD AF IP for 14 days is non-toxic to bone marrow, liver and kidneys of mice.

Female nude mice bearing DMS273 xenografts were treated with either vehicle or 4 mg/kg AF I.P every day for 14 days. Mice were euthanized when tumors were ≥ 1000 mm³. This experiment was done on 3 separete cohorts (9–10 animals/cohort) of animals for a total of 28 AF treated mice and 30 vehicle control treated mice. (A) Mice were monitored daily and the % of the initial weight was plotted as a function of time from the beginning of AF treatment. (B, C) Bone marrow from 9 AF treated mice (5 from the first cohort, 4 from the second cohort) and 13 control mice (7 from the first cohort, 6 from the second cohort) was harvested from the femurs at euthanasia and subjected to flow cytometry for HSCs (Lineage (Lin)− Sca-1+ c-Kit+, LSK) shown in (B) and HSPCs (Lin− cKit+ Sca1+ CD135−) populations shown in (C). (D-I) Blood was drawn from 7 control mice and 5 AF treated mice from the third cohort) via cardiac puncture at euthanasia and evaluated for clinical chemistry endpoints by Antech Diagnostics.

Figure 5 -. 4 mg/kg QD AF IP for 14 days prolongs survival in nude mice with DMS273 xenografts.

Tumors from all mice shown in Figure 4A, were measured via calipers and tumor volumes calculated. When tumor volumes measured 1000 mm³ mice were considered to have reached euthanasia criteria and Kaplan-Meier curves (three cohorts combined) were used to estimate overall survival. Log-rank (Mantel Cox) test was used to determine significance. (p<0.04)