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Four weeks after the first administration of siRNAs, the mice were sacrificed humanely, the GFP-SAS xenografts, lungs, livers and kidneys were dissected, and Akt1 protein expression levels were determined by western blotting. Total RNA was extracted from tissues by homogenization in 0. Hydroxymethylbilane synthase HMBS was used as an internal control.

All in vitro experiments were performed in triplicate and repeated 3 times. Using Human Genome Survey Arrays, we determined the gene expression profiles in 10 primary OSCC tissues, 10 human OSCC cell lines, 3 normal oral mucosal tissues and a human immortalized non-neoplastic keratinocyte cell line. The expression of 7, genes was undetectable in all normal oral mucosal tissues and in a non-neoplastic epithelial cell line. A Experimental plan for microarray analysis. In all normal oral mucosal tissues and a non-neoplastic epithelial cell line, 7, genes were not expressed.

To confirm the microarray data, we examined the expression of Akt1 protein in human OSCC cells and tissues by western blot analysis. Subsequently, we compared the expression levels of Akt1 protein in primary OSCCs with adjacent normal oral mucosal tissues from the same patient and found that Akt1 protein was more highly expressed in the tumor tissues than in the normal tissues Fig.

Left hand panels show a HE-stained section and right hand panels show a section stained for Akt1 immunohistochemically. Bottom panels show that diffusely positive staining for Akt is focused in cancer cells. Top panels show normal epithelial cells are negative for cytoplasmic Akt1 expression. We also examined their effects on the invasive growth of OSCC cells using the collagen gel culture system.

The effects on Akt1 protein expression were evaluated by western blotting and showed that all siAkt1 constructs suppressed expression. B The effects of siAkt and 58 on cell growth were assessed in cells seeded in complete medium and both siRNA constructs significantly inhibited cell growth.

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C The effects of siAkt and 58 on the invasive growth were examined in the collagen gel culture system and they significantly suppressed the invasive growth. We assessed the growth inhibitory effect of siAkt1 in vivo using a mouse model. During the administration of siAkt1, no reduction in food intake or body weight was observed in the mice.

The mice were then injected i. B Western blots showing the expression levels of Akt1 protein in excised tissues. Treatment with siAkt1 reduced the expression of Akt1 protein in the tumor tissues but not in lung, liver and kidney.

Only siAkt can target both human and mouse Akt1. We also checked whether the systemic delivery of atelocollagen-mediated siRNA induced interferon responses in mice.

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We compared this to a positive control group injected with poly I:C , which induced a strong interferon response, but the siRNAs did not Fig. These results indicated that atelocollagen specifically delivered siRNAs into tumor tissues without inducing an interferon response. Interferon responses to atelocollagen-mediated siRNA systemic delivery. After i. Poly I:C was used as a positive control.


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Two of these primary cultured cells were derived from tumors of the tongue and two from lower gingival tumors. A Primary cell cultures derived from OSCC tumors from 4 patients were transfected with siAkt1, cultured for 48 h, and lysed. The inhibitory effects of siAkt and on Akt1 expression were confirmed by western blotting. Cell growth was evaluated after 3 days using WST-8 assays.

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To understand the molecular mechanisms involved in the growth inhibitory effect of siAkt1, we carried out an oligonucleotide-based microarray analysis after transfecting siAkt1 into GFP-SAS cells. Among these genes, cancer-related genes were identified by functional analysis using IPA software Table I. In microarray analysis, we identified Akt1 as the only candidate gene commonly overexpressed in OSCC tissues and cell lines. Overexpression of Akt1 protein in OSCC was also confirmed by western blot analysis and immunohistochemistry. In many human malignancies, activated Akt promotes cell proliferation, survival and metastasis of cancer cells by regulating the activity of a number of downstream molecules, such as mammalian target of rapamycin mTOR , glycogen synthase kinase 3 GSK3 and Bclassociated death promoter BAD 15 , Akt activation has also been reported to be a significant prognostic indicator in OSCC, with its inhibition being a possible molecular approach to treatment MK is a highly potent, allosteric pan-Akt inhibitor.

The phenotypes of knockout mice have suggested a critical role for Akt1 in cell survival, a role for Akt2 in glucose metabolism and a role for Akt3 in brain development In cancers, overexpression of specific Akt isoforms, with or without gene amplification, has been reported. For example, upregulation has been seen in the expression of Akt1 in gastric and breast cancers, of Akt2 in ovarian, pancreatic, liver, and colorectal cancers, and of Akt3 in melanoma and breast cancers.

Furthermore, a somatic activating mutation in Akt1 has been identified in ovarian, breast, and colorectal cancers. Although the correlation between the development of specific cancers and the hyperactivation of specific Akt isoforms remains unclear, a deficiency of Akt1 has been shown to be sufficient to inhibit endometrial and prostatic neoplasia in phosphatase and tensin homolog PTEN mice Most recently, Naruse and colleagues reported that phosphorylated mTOR was overexpressed in approximately half of OSCC cases and that its expression level was correlated with the TN classification and survival rate.

Our study has also shown that atelocollagen-mediated, systemic administration of siAkt1 suppressed the expression of Akt1 in tumor tissues but not in normal tissues and the growth of human OSCC cells in nude mice, without severe side effects such as lung, liver, or renal damage. This selective targeting may be due to the vascular abnormalities of tumor tissues, called the enhanced permeability and retention EPR effect Furthermore, atelocollagen is a highly biocompatible biomaterial having been used in the field of healthcare as a component of local hemostatic agents and for tissue regeneration and is unlikely to induce immune reactions in vivo.

These results suggest that nucleic acid-based drugs, such as atelocollagen-complexed siRNA, may provide novel therapeutic opportunities for human malignancies, with minimal risks of adverse events. Int J Cancer. View Article : Google Scholar. Cancer Res. Oral Oncol. N Engl J Med. Oncol Rep. Int J Oral Maxillofac Surg. Nat Med. Nucleic Acids Res. Biochem Biophys Res Commun. Cancer Cell.

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1: Oral Structures and Tissues

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Tissues, Part 2 - Epithelial Tissue: Crash Course A&P #3

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However, regeneration of the supporting structures of the tooth remains an elusive goal and a challenge. The formation of new bone and cementum with supportive periodontal ligament is the ultimate objective, but current regeneration therapies are incapable of achieving this in a predictable way.

Oral epithelial stem cells in tissue maintenance and disease: the first steps in a long journey

The regeneration of periodontal tissue requires a combination of fundamental events, such as appropriate level and sequencing of regulatory signals, the presence of progenitor cells, an extracellular matrix or carrier and an adequate blood supply. The identification of mesenchymal stem cells from bone marrow started a new era in regenerative medicine. The aim of this review was to describe the main sources of mesenchymal stem cells from tissues in the oral cavity and the potential of these cells in regenerative therapy.

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