Bone marrow mesenchymal stem cells promote head and neck cancer progression through Periostin‐mediated phosphoinositide 3‐kinase/Akt/mammalian target of rapamycin

Figures

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  Figure 1. Mesenchymal stem cells conditioned media (MSC‐CM) promoted proliferation and inhibited apoptosis of head and neck cancer (HNC) cells. (A) Cell proliferation was assessed using CCK‐8 assay. (B) Cell apoptosis assay was assessed by flow cytometric analysis of Annexin V and propidium iodide. (C) Flow cytometric analysis of cell cycle distribution. (D) Real‐time PCR analysis of P21 and P16 mRNA expression. (**P < .01)

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  Figure 2. Mesenchymal stem cells conditioned media (MSC‐CM) promoted migration of head and neck cancer (HNC) cells. (A,B) Transwell assay shows that MSC‐CM promoted the migration of CAL 27 and HN4 cells. (C,D) Wound‐healing assay shows that MSC‐CM promoted the migration of CAL 27 and HN4 cells. (**P < .01)

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  Figure 3. Mesenchymal stem cells conditioned media (MSC‐CM) promoted epithelial‐mesenchymal transformation (EMT) of head and neck cancer (HNC) cells. CAL 27 or HN4 cells were treated with MSC‐CM or control media for 72 h, then gene and protein expression levels of EMT‐related molecules were detected. (A) Real‐time PCR assay of Snail, Twist, E‐cadherin, N‐cadherin and Vimentin. (B) Western blot and quantification analysis of E‐cadherin, N‐cadherin and Vimentin. (*P < .05 and **P < .01)

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  Figure 4. Mesenchymal stem cells conditioned media (MSC‐CM) activated the phosphoinositide 3‐kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway and promoted Periostin (POSTN) in head and neck cancer (HNC) cells. CAL 27 and HN4 cells were cultured in MSC‐CM or control media for 72 h. (A) Western blot assay of p‐PI3K, p‐Akt and p‐mTOR. (B) Real‐time PCR analysis of POSTN mRNA expression. (C) Western blot analysis of POSTN. (*P < .05 and **P < .01)

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  Figure 5. Periostin (POSTN)‐activated phosphoinositide 3‐kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway promoted head and neck cancer (HNC) cells. (A) POSTN was silenced effectively by siRNA. (B) PI3K/Akt/mTOR pathway was inhibited when POSTN was silent. (C) Cell proliferation ability tested by CCK‐8 assay. (D) mRNA expression of P16 and P21. (E) Cell migration ability tested by Transwell assay. (F) Western blot analysis of E‐cadherin, N‐cadherin and Vimentin. (G) Western blot assay of p‐PI3K and p‐mTOR. (H) Cell proliferation ability. (I) mRNA expression of P16 and P21. (J) Cell migration ability. (**P < .01)

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  Figure 6. Bone marrow mesenchymal stem cells (BMMSC) promoted tumorigenesis in murine models of head and neck cancer (HNC). CAL 27 in control medium, mesenchymal stem cells conditioned media (MSC‐CM), and mixed cells of CAL 27 and MSC were injected into the middle of the tongue of BALB/C mice. (A,B) Tumor invasion was assessed using H&E staining and immunohistochemistry (IHC) staining of Ki67. (C) Numbers of neck lymph node metastases were assessed. (D) Western blot analysis of Periostin (POSTN) in tumor tissues. (E) Western blot analysis of E‐cadherin and N‐cadherin in tumor tissues. (*P < .05 and **P < .01)

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  Figure 7. Periostin (POSTN) and N‐cadherin were highly expressed in head and neck cancer (HNC) cancer samples. Positive staining of POSTN and N‐cadherin was mainly located in the cell cytoplasm which showed brown granules. (A) Immunohistochemistry (IHC) staining of POSTN in well, moderate and poorly differentiated HNC tissues. (B) IHC staining of N‐cadherin in well, moderate and poorly differentiated HNC tissues