Figure 1.
Combined effects of DFMO+diclofenac inhibit the growth of A431 xenograft tumors.
(A) Tumor growth curve and end-point tumor volume at day 15 in A431 xenografts (n = 10) treated with DFMO and diclofenac as individual agent and in combination. @ - significant when DFMO alone compared to vehicle-treated control, # - significant when diclofenac alone compared to vehicle-treated control, $ - significant when DFMO+diclofenac compared to vehicle, & and ? - significant when DFMO+diclofenac compared to single treatment of DFMO and diclofenac respectively. All values are mean±SEM; *, p<0.05, **, p<0.005, ***, p<0.0001. (B) A431 cells (2.5×106) were injected subcutaneously into the right and left flank of nude mice. Representative animal from different groups after 15 days of drug treatment compared to the vehicle-treated control. The tumors are marked in dotted red lines.
Figure 2.
DFMO+diclofenac decreases proliferation and induce cell death by apoptosis in human epidermoid xenografts.
(A) Tissue sections from A431 xenograft tumors were stained for PCNA or TUNEL. DFMO+diclofenac treatment on expression levels of PCNA and TUNEL-positive apoptotic cells as compared to vehicle and their individual treatments. (B) Western blot analysis was done by randomly selecting two individual samples from each group. The effect of ODC and COX-2 inhibitors treated as single agents and in combination on the expression of cyclin D1 and apoptotic marker proteins. The bar diagram represents relative expression level of these proteins & the error bars demonstrate the standard error between two individual samples selected from each group. (cyclin D1: @ = NS, # = 0.04, $ = 0.001, & = 0.01, ? = 0.05; Bcl-2: @ = NS, # = 0.04, $ = 0.01, & = 0.05, ? = 0.05; cleaved caspase-3: @ = 0.005, # = 0.007, $ = 0.002, & = 0.004 ? = NS). @ - significant when DFMO alone compared to vehicle-treated control, #- significant when diclofenac alone compared to vehicle-treated control, $ - significant when DFMO+diclofenac compared to vehicle, & and ?-significant when DFMO+diclofenac compared to single treatment of DFMO and diclofenac respectively. Identical β-actin loading controls are denoted by symbol ‘†’ ‘¥’ ‘‡’ and ‘€’ in various figures.
Figure 3.
DFMO+diclofenac decrease the expression levels of ODC and COX-2 through Akt and ERK signaling axis.
(A) Western blot analysis showing expression of ODC and COX-2. The bar diagram represents relative expression levels of these proteins (ODC: @ = 0.04, # = NS, $ = 0.01, & = NS, ? = 0.02; COX-2: @ = NS, # = 0.04, $ = 0.008, & = 0.003 ? = NS). No significant changes were noted in the levels of COX-1 (data not shown) upon DFMO or diclofenac treatments or both in these tumors. (B) DFMO+diclofenac treatment exert some target-unrelated effects. Individual and combinatorial treatments of DFMO and diclofenac on the levels of p-ERK (@ = 0.05, # = 0.03, $ = 0.001, & = 0.002 and ? = 0.05) and p-MAPKAP-2 (@ = NS, # = 0.03, $ = 0.008, & = 0.04 and ? = 0.05), an important mediators of tumor growth. (C) Effect of DFMO and diclofenac treatments on the levels of p-Akt ser 473 (@ = NS, # = 0.05, $ = 0.02, & = 0.005 and ? = 0.005) and thr 308 (@ = NS, # = 0.05, $ = 0.02, & = 0.03 and ? = 0.04) when treated alone and in combination. @ - significant when DFMO alone compared to vehicle-treated control, # - significant when diclofenac alone compared to vehicle-treated control, $ - significant when DFMO+diclofenac compared to vehicle, & and ? - significant when DFMO+diclofenac compared to single treatment of DFMO and diclofenac respectively.
Figure 4.
Combinatorial treatment of DFMO and diclofenac decreases EMT processes.
(A) The effect of DFMO+diclofenac treatment on levels of epithelial marker, E-cadherin and mesenchymal markers, vimentin and fibronectin in A431 xenograft tumors. (B) Western blot analysis of EMT transcription factors snail (@ = NS, # = 0.03, $ = 0.004, & = 0.009, ? = 0.01), slug (@ = NS, # = NS, $ = 0.008, & = 0.005, ? = 0.03) and twist (@ = NS, # = 0.05, $ = 0.01, & = 0.009, ? = 0.05) upon DFMO+diclofenac treatment. Following this, the levels of matrix metalloproteinase’s MMP-2 & -9 was also investigated (@ = NS, # = 0.05, $ = 0.008, & = 0.02, ? = 0.05) and -9 (@ = NS, # = 0.009, $ = 0.007, & = 0.004). @ - significant when DFMO alone compared to vehicle-treated control, # - significant when diclofenac alone compared to vehicle-treated control, $ - significant when DFMO+diclofenac compared to vehicle, & and ? - significant when DFMO+diclofenac compared to single treatment of DFMO and diclofenac respectively.
Figure 5.
DFMO+diclofenac treatment reduces proliferation and migration in A431 cells.
(A) Clonogenic cell survival assay in DFMO+diclofenac treated A431 cells after 10 days as compared to their single agent treatments and vehicle-treated control. (B) Western blot analysis of p-Akt (ser-473), p-ERK, cyclin D1, Bcl-2 and COX-2 in the combined treatment group of DFMO and diclofenac as compared to control and their individual treatments. These cells retained their epithelial phenotype, as marked by increased E-cadherin expression. (C) Cell migration assay in DFMO+diclofenac combination treatment as compared to control or individual treatments after 12 h. (D) Representative pictures of A431 cells treated with the ODC and COX-2 inhibitors after 24 h of treatment. (E) Cell cycle analysis show significant accumulation of cells in G2/M phase with a concomitant reduction in G1 phase in DFMO+diclofenac treated A431 cells.
Figure 6.
Akt inhibition reduces growth of human epidermal xenografts.
(A) Tumor growth curve and end-point tumor volume at day 15 in A431 xenografts (n = 10) treated with target-specific Akt inhibitor. (B) Effect of Akt inhibition on the levels of p-AKT ser-473 and thr-308 (p = 0.03). All values are mean ± SEM; *, p<0.05, **, p<0.005, ***, p<0.0001. (C) Akt inhibition restores epithelial phenotype in xenograft tumors. Western blot analysis of EMT transcription factors (slug, twist) and mesenchymal markers (N-cadherin, Fibronectin) in API-59CJ-Ome treated tumors.
Figure 7.
Akt over-expression resists the effect of DFMO+diclofenac.
Treatment of myr-flag-Akt overexpressing cells with DFMO and diclofenac resisted the changes in the levels of p-Akt ser-473 and cyclin D1 as compared to empty vector control and wild type A431 cells.
Figure 8.
Flow diagram showing effects of ODC and COX-2 combined inhibition on the molecular targets involved in human SCC pathogenesis.
ODC and COX-2 over-expression leads to increase in polyamine biosynthesis and PGE2 levels respectively, activating Akt and ERK-dependent signaling pathways which modulate tumor growth and invasion through enhanced EMT resulting in aggressive growth of SCCs.