Dataset Information

DSPP-MMP20 gene silencing downregulates cancer stem cell markers in human oral cancer cells.

ABSTRACT:

Background

Recent findings indicate that dentin sialophosphoprotein (DSPP) and matrix metalloproteinase (MMP) 20 interact in oral squamous cell carcinoma (OSCC). The objective of this study was to determine the effects of DSPP/MMP20 gene silencing on oral cancer stem cell (OCSC) markers.

Methods

The expression of well-established OCSC markers: ABCG2; ALDH1; CD133; CD44; BMI1; LGR4, and Podoplanin in DSPP/MMP20-silenced OSCC cell line, OSC2, and controls were assayed by western blot (WB), and flow cytometry techniques. The sensitivity of OSC2 cells to cisplatin following DSPP/MMP20 silencing was also determined.

Results

DSPP/MMP20 silencing resulted in downregulation of OCSC markers, more profoundly ABCG2 (84%) and CD44 (81%), following double silencing. Furthermore, while treatment of parent (pre-silenced) OSC2 cells with cisplatin resulted in upregulation of OCSC markers, DSPP/MMP20-silenced OSC2 cells similarly treated resulted in profound downregulation of OCSC markers (72 to 94% at 50 ?M of cisplatin), and a marked reduction in the proportion of ABCG2 and ALDH1 positive cells (~?1%).

Conclusions

We conclude that the downregulation of OCSC markers may signal a reduction in OCSC population following MMP20/DSPP silencing in OSCC cells, while also increasing their sensitivity to cisplatin. Thus, our findings suggest a potential role for DSPP and MMP20 in sustaining OCSC population in OSCCs, possibly, through mechanism(s) that alter OCSC sensitivity to treatment with chemotherapeutic agents such as cisplatin.

SUBMITTER: Nikitakis NG

PROVIDER: S-EPMC6040065 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Publications

DSPP-MMP20 gene silencing downregulates cancer stem cell markers in human oral cancer cells.

Nikitakis Nikolaos G NG Gkouveris Ioannis I Aseervatham Jaya J Barahona Kelvin K Ogbureke Kalu U E KUE

Cellular & molecular biology letters 20180711

<h4>Background</h4>Recent findings indicate that dentin sialophosphoprotein (DSPP) and matrix metalloproteinase (MMP) 20 interact in oral squamous cell carcinoma (OSCC). The objective of this study was to determine the effects of DSPP/MMP20 gene silencing on oral cancer stem cell (OCSC) markers.<h4>Methods</h4>The expression of well-established OCSC markers: ABCG2; ALDH1; CD133; CD44; BMI1; LGR4, and Podoplanin in DSPP/MMP20-silenced OSCC cell line, OSC2, and controls were assayed by western blo ...[more]

PMID: 30002682

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Project description:BackgroundWe determined recently that dentin sialophosphoprotein (DSPP), a member of the SIBLING (Small integrin-binding ligand N-linked glycoproteins) family of phosphoglycoproteins, is highly upregulated in human oral squamous cell carcinomas (OSCCs) where upregulation is associated with tumor aggressiveness. To investigate the effects of DSPP-silencing on the tumorigenic profiles of the oral cancer cell line, OSC2, short-hairpin RNA (shRNA) interference was employed to silence DSPP in OSC2 cells.Methodology/principal findingsMultiple regions of DSPP transcript were targeted for shRNA interference using hDSP-shRNA lentiviral particles designed to silence DSPP gene expression. Control shRNA plasmid encoding a scrambled sequence incapable of degrading any known cellular mRNA was used for negative control. Following puromycin selection of stable lines of DSSP-silenced OSC2 cells, phenotypic hallmarks of oral carcinogenesis were assayed by western blot and RT-PCR analyses, MTT (cell-viability), colony-formation, modified Boyden-Chamber (migration and invasion), and flow cytometry (cell-cycle and apoptosis) analyses. DSPP-silenced OSC2 cells showed altered cell morphology, reduced viability, decreased colony-formation ability, decreased migration and invasion, G0/G1 cell-cycle arrest, and increased tumor cell sensitivity to cisplatin-induced apoptosis. Furthermore, MMP-2, MMP-3, MMP-9, VEGF, Ki-67, p53, and EGFR were down-regulated. There was a direct correlation between the degree of DSPP-silencing and MMP suppression, as indicated by least squares regression: MMP-2 {(y?=?0.850x, p<0.001) (y?=?1.156x, p<0.001)}, MMP-3 {(y?=?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)}.Conclusions/significanceDSPP-silencing in OSC2 cell decreased salient hallmarks of oral tumorigenesis and provides the first functional evidence of a potential key role for DSPP in oral cancer biology. The down-regulation of MMP-2, MMP-3, MMP-9, p53 and VEGF in DSPP-silenced OSC2 cells provides a significant functional/molecular framework for deciphering the mechanisms of DSPP activities in oral cancer biology.

Project description:The aim of the present study was to analyze the acquisition of the differentiated phenotype in the human gastric signet ring cell adenoma cancer KATO‑III cell line in vitro. The morphology of KATO‑III cells was explored by microcinematography. Different cytokines secreted by both adherent and non‑adherent KATO‑III cells into medium were observed. The cancer stem cell phenotypes were identified by reverse transcription‑quantitative polymerase chain reaction using primers (E‑Cad, Slug, Snail, vimentin, NANOG, NESTIN, OCT3/4 and C‑X‑C motif chemokine receptor 4) or antibodies [cluster of differentiation (CD)90 and CD117] by flow cytometry (FACS). The influence of the induction media for the differentiation of mesenchymal cells was studied through viability and proliferation assays, by evaluating gene expression and the expression of markers via FACS. Cell viability and cell cycle distribution were evaluated following the treatment of KATO‑III with acetyl salicylic acid and using the induction media as an inhibitor of epithelial‑mesenchymal transition (EMT) and heparanase. A total of 3 phenotypes of KATO‑III were observed (adherent, non‑adherent and cell cluster), which have internal potential for cell transition into one of the other phenotypes. KATO‑III was differentiated into adipocyte‑, chondrocyte‑, osteocyte‑ and neurocyte‑like cells by the induction media. Identification of the induced cells was conducted using cell dyes. Reduced mRNA expression of EMT‑associated molecules, stem cell markers and heparanase was observed with acetyl salicylic acid and induction media. An inhibitory effect of acetyl salicylic acid and the induction media was also noted in regard to cell proliferation. In addition, acetyl salicylic acid induced G0/G1 phase cell cycle arrest in KATO‑III cells. In conclusion, the induction of the differentiation of cancer stem cells into non‑proliferating cells offers the possibility for novel drug design to overcome the issues associated with metastasis, drug resistance and systemic toxicity with improved therapeutic efficacy.

Dataset Information

DSPP-MMP20 gene silencing downregulates cancer stem cell markers in human oral cancer cells.

Background

Methods

Results

Conclusions

Publications

DSPP-MMP20 gene silencing downregulates cancer stem cell markers in human oral cancer cells.

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