Figure 1.
DSPP upregulation in oral-cancer cells, OSC2 and SCC25, and the dysplastic oral keratinocyte cells, DOK.
(A) Western blot (WB) and densitometric analyses show significant upregulation of DSPP in OSC2, SCC25, and DOK cells compared with HOK negative controls. There is a basal (<10%) level of DSPP expression in primary HOK cells. MCF7 cell line known to express DSPP was used as positive control. Normalization was with β-actin. (B) Semiquantitative RT-PCR analysis shows DSPP-mRNA expression in OSC2, SCC25, and DOK cells consistent with WB results in (A) with undetectable DSPP-mRNA levels in HOK cell. Normalization was with GAPDH. Cells used in study: OSC2, a human OSCC cell line derived from regional cervical lymph node metastasis of a primary human tongue squamous cell carcinoma; SCC25, a primary tongue squamous cell carcinoma; DOK, a human oral epithelial dysplastic cell line derived from dorsal tongue; and MCF7 (acronym of Michigan Cancer Foundation – 7), a human breast cancer cell line isolated from a 69-year-old Caucasian female.
Figure 2.
Transient lentiviral-mediated DSP-shRNA knockdown results in altered morphology of OSC2 cells.
(A) OSC2 cells prior to lentiviral-transduced transfection with DSP-ShRNA exhibiting characteristic morphology of viable epithelial tumor cells in culture. (B, E) Scrambled (control) sequence-transfected OSC2 cells at 24- and 48 h exhibiting viable epithelial cells and morphology comparable to (A). (C, F; illustrative areas) DSP-shRNA transfection OSC2 cells exhibiting significantly increased number of cells with loss of cell-cell contact, more rounded and irregular outline, prominent nuclear blebbing, and cellular disintegration consistent with those of effete and dying cells at 24- and 48-h, respectively. (D) copGFP transfected OSC2 cells confirm very high transfection efficiency (green fluorescence).
Figure 3.
Generation of stable DSPP-silenced OSC2 cell lines.
Stable lines selection of lentiviral-transduced shRNA was accomplished via puromycine selection. (A) WB and corresponding densitometric analyses of level of DSPP-silencing following selection of six stable lines show level of silencing ranging from ∼5% (line [L] 4) to ∼95% (L2) compared to scrambled sequence (shC) control. (B) qRT-PCR analysis show significantly reduced DSPP-mRNA in DSPP-silenced L1 (>90%) and L2 (>95%) cells (exhibiting the most knockdown on WB) compared to shC control. (C) Immunfluorescence confocal microscopy verifies DSPP-silencing as significantly reduced DSPP signal (green fluorescence) in L2.
Figure 4.
DSPP-silencing results in dowregulation of Ki-67, PCNA, p53, and inhibition of cell proliferation and colony-formation.
(A) WB of ki-67, PCNA, and p53 following DSPP-silencing in OSC2 cells show significant downregulation for each of these proteins. (B) The proliferation status of DSPP-silenced OSC2 cells compared to parental OSC2 and shC control carried out via MTT assay shows decreased cell proliferation of 53% for L2 (demonstrating the most degree of silencing) compared with <5% for L4 (least silencing), and ∼30% for L6 (moderate silencing) and shC control (p<0.05; n = 3). (C) L2 cells formed smaller and significantly fewer (<25%) colonies in soft agar compared with parental OSC2 and shC control cells (*p<0.05).
Figure 5.
DSPP silencing decreased OSC2 migration and invasion.
(A, B) Modified Boyden-Chamber experiment shows that DSPP-silencing in (L2) decreased invasion (A) and migration (B) of OSC2 cells by ∼25%, respectively, compared to shC control and parental OSC2 cells (for each comparison using Dunn methods of multiple comparisons).
Figure 6.
DSPP-silencing reduces G0/G1 arrest in OSC2 cells.
(A) Flow cytometric analysis shows proportion of DSPP-silenced L2 cells in G0/G1 phase significantly increased (79.51%) compared with parental OSC2 (44.7%) and shC controls (45.98%). (B) Conversely, DNA laddering experiments shows no significant difference in rate of apoptosis between L2 cells and controls. This result indicates that DSPP-silencing alone does not increase the rate of apoptosis in OSC2 cells.
Figure 7.
DSPP-silencing sensitizes OSC2 cells to cisplatin-induced apoptosis.
(A) Treatment of DSPP-silenced L2, parental OSC2, and shC control cells with 50 uM for 24 h followed by Annexin V/FITC flow cytometry analysis show a 56.19% increase in apoptotic rate (measured by sub-G1 cellular DNA content) for L2 cells compared with 41.28% in parental with cisplatin and OSC2 and 43.69% in shC control with cisplatin. (B) Trypan blue staining bar chart quantitation indicates comparable apoptotic rates to that in (A). (C) WB showing significant down-regulation of EGFR in DSPP-silenced OSC2 cells, including L2. Symbols: Cis = cisplatin; L2 = DSPP-silenced OSC2 cells line 2; OSC2 = parental (without knockdown) osc2 cells, M1 = live cells; M2 = deal cells.
Figure 8.
DSPP-silencing downregulates SIBLING-partnering MMPs in OSC2 cells.
(A) WB and (B) corresponding densitometric analyses show significant down-regulation of pro- and activated MMP-2, MMP-3, and MMP-9 in all but one (L4) of the six DSPP-silenced stable lines. (C) Least square regression analyses show significantly reduced levels of pro-and activated MMP-2 {(y = 0.850x, p<0.001) (y = 1.156x, p<0.001)}; MMP-3 {(y = 0.0.994x, p<0.001) (y = 1.324x, p = 0.004)}, and MMP-9 {(y = 1.248x, p = 0.005, y = 0.809, p = 0.013)}, and a direct correlation between the degree of DSPP-silencing and MMP suppression; however, the level of MMP suppression did not differ significantly between pro- and cleaved forms. (D) Densitometric analysis of WB of VEGF levels showed down-regulation ranging from <5% (L4) to 86% (L2) with the level of down-regulation directly proportional to the degree of DSPP-silencing.