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

Figures & data

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) and 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 cell line’s respective control. * p < .05 compared to untreated cells two-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 the treatment of AF alone. n ≥ 3 independent experiments two-way ANOVA Fishers LSD * significantly different than control (p < .05), # significantly different than either drug alone (p < .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 d either QD or BID. Kidney, liver, and tumor samples were collected for TrxR activity. (d) Plasma samples were collected 9 h after the final BID dose (5 d of therapy), and gold content was determined. (e) Gpx1 activity was determined in xenograft tumors after 5 d of AF therapy. * = p-value <.05, ** = p-value <.01, *** = p-value <.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. Two mice each with two flank DMS273 xenografts in the first cohort and four mice with one flank DMS273 xenografts in the third cohort were treated with AF 4 mg/kg IP QD for 14 d and 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 < .05 compared to untreated cells two-way ANOVA with Fishers LSD comparisons.

Figure 4. 4 mg/kg QD AF IP for 14 d is nontoxic to bone marrow, liver, and kidneys of mice. Female nude mice-bearing DMS273 xenografts were treated with either vehicle or 4 mg/kg AF IP every day for 14 d. Mice were euthanized when tumors were ≥1000 mm³. This experiment was done on three separate 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 seven control mice and five AF-treated mice from the third cohort) via cardiac puncture at euthanasia and evaluated for clinical chemistry endpoints by Antech Diagnostics.

Table 1. CBC analysis of peripheral blood during AF treatment. Number of mice in each group in parentheses.

	WBC (X10^3/µL)	Neutrophils (X10^3/µL)	Lymphocytes (X10^3/µL)	RBC (X10^6/µL)	Hemoglobin (g/dl)	Hematocrit (%)	MCV (fl)	Platelets (X10^3/µL)
Control (n = 16)	8.34 (4.39)	2.00 (1.56)	1.34 (1.43)	9.15 (1.58)	13.9 (2.54)	52.6 (8.07)	57.6 (2.06)	1300 (115)
AF 4 mg/kg (n = 17)	7.14 (3.50)	1.77 (1.17)	1.24 (1.43)	8.82 (1.14)	13.0 (1.84)	50.5 (5.83)	57.3 (1.60)	1410 (419)

Figure 5. 4 mg/kg QD AF IP for 14 d prolongs survival in nude mice with DMS273 xenografts. Tumors from all mice shown in Figure 4a were measured via calipers, and tumor volumes were 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 the overall survival. Log-rank (Mantel Cox) test was used to determine significance (p < .04).

Data availability statement

The authors confirm that the data supporting the findings of this study are available within the article or its supplementary materials. Any data that support the findings of this study that are not found within the article are available from the corresponding author, [MAF], upon reasonable request.