Human Tongue Squamous Cell Carcinoma Research Articles

Silencing Dicer expression enhances cellular proliferative and invasive capacities in human tongue squamous cell carcinoma

microRNAs (miRNAs) are aberrantly expressed in cancer. An enzyme essential for miRNA processing is Dicer, whose expression is deregulated in diverse types of cancer and correlates with tumor progression. However, whether the regulation of Dicer expression affects tongue squamous cell carcinoma is unknown. In the present study, we investigated how silencing the expression of Dicer alters cell proliferation, cell cycle patterns, and cell migration and invasion in the Tca-8113 tongue squamous cell carcinoma cell line. Dicer expression levels were determined using quantitative PCR and western blot analysis in normal oral gingival epithelial cells and in two tongue squamous cell carcinoma lines, Tca-8113 and UM-1. Tca-8113 cells were transfected with Dicer siRNA or a negative control siRNA. Cell proliferation was determined using the MTT assay and the cell cycle was examined using flow cytometry. Cell migration and invasion changes were evaluated using wound-healing, adherence and Transwell assays. Dicer was expressed at lower levels in the tongue squamous cell carcinoma cell lines Tca-8113 and UM-1 compared to normal gingival epithelial cells, and less Dicer was expressed in UM-1 cells compared to Tca-8113 cells. Notably, Tca-8113 cells transfected with Dicer siRNA had significantly higher proliferative and invasive abilities than cells transfected with the negative control siRNA or non-transfected cells. Silencing Dicer may promote the progression of tongue squamous cell carcinoma. Dicer could serve a promising biomarker and a potential therapeutic target for tongue squamous cell carcinoma.

Neuropilin-1 and Oral Squamous Cell Carcinoma

Angiogenesis is one of the most important prognostic factors in oral squamous cell carcinoma (SCC). Neuropilins (NRPs) are transmembrane receptors that bind vascular endothelial growth factor (VEGF) and class 3 semaphorins (SEMA3). Upregulation of neuropilin-1 (NRP1) and its effect on angiogenesis, lymphangiogenesis, and the metastatic process have been shown in some human carcinomas. Western blotting, enzyme-linked immunosorbent assay (ELISA), and immunohistochemistry (IHC) were performed on selected human tongue SCC (HTSCC) cell lines and specimens of HTSCC and dysplasia to evaluate NRP1 or VEGF expression.

Truncation of inhibitor of growth family protein 5 effectively induces senescence, but not apoptosis in human tongue squamous cell carcinoma cell line

In these studies, inhibitor of growth protein 5 (ING5) and various fragments of it were overexpressed in the human tongue squamous cell carcinoma cell line, HSC-3. The roles of ING5 in HSC-3 cells were then identified in vitro. Our results indicate that intact ING5 can inhibit proliferation and induce apoptosis in HSC-3 cells. Moreover, two truncated fragments of ING5 (aa 1-184 and aa 107-226) can induce cellular senescence. To analyze the signaling pathway involved, western blotting was performed. In these assays, two truncated fragments of ING5 were found to inhibit the cyclin E and CDK2 expression. These results are consistent with the S phase arrest observed with the overexpression of truncated ING5. However, the mechanisms of ING5-induced cellular senescence remain unclear, and extensive investigations are required in future studies.

Relation between the expression of mitotic centromere–associated kinesin and the progression of squamous cell carcinoma of the tongue

Mitotic centromere-associated kinesin (MCAK) is a microtubule depolymerase indispensable for microtubule binding during spindle formation. The purpose of this study was to investigate the association of MCAK expression with squamous cell carcinoma of the oral tongue (SCCOT). Immunohistochemistry was used in 47 cases of SCCOT. Determination of proliferation and migratory capabilities was performed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and Transwell chamber assay, respectively, on cells from the human tongue squamous cell carcinoma cell line Tca8113 that were transfected with MCAK small interfering RNA (siRNA). MCAK expression level in oral tongue cancer tissue is significantly higher (P < .01) than that of corresponding normal tissue. In addition, high expression of MCAK is significantly associated with lymph node metastasis (P < .05) and tumor staging (P < .01). Moreover, gene silencing of MCAK suppresses proliferation and migration of Tca8113 cells (P < .05; P < .01). The expression of MCAK may be associated with the progression of SCCOT.

Kaempferol Reduces Matrix Metalloproteinase-2 Expression by Down-Regulating ERK1/2 and the Activator Protein-1 Signaling Pathways in Oral Cancer Cells

BackgroundKaempferol has been proposed as a potential drug for cancer chemoprevention and treatment because it is a natural polyphenol contained in plant-based foods. Recent studies have demonstrated that kaempferol protects against cardiovascular disease and cancer. Based on this finding, we investigated the mechanisms by which kaempferol produces the anti-metastatic effect in human tongue squamous cell carcinoma SCC4 cells.Methodology/Principal FindingsIn this study, we provided molecular evidence associated with the anti-metastatic effect of kaempferol by demonstrating a substantial suppression of SCC4 cell migration and invasion. This effect was associated with reduced expressions of MMP-2 and TIMP-2 mRNA and protein levels. Analysis of the transcriptional regulation indicated that kaempferol inhibited MMP-2 transcription by suppressing c-Jun activity. Kaempferol also produced an inhibitory effect on the phosphorylation of ERK1/2.ConclusionsThese findings provide new insights into the molecular mechanisms involved in the anti-metastatic effect of kaempferol, and are valuable in the prevention of oral cancer metastasis.

Plakoglobin Represses SATB1 Expression and Decreases In Vitro Proliferation, Migration and Invasion

Plakoglobin (γ-catenin) is a homolog of β-catenin with dual adhesive and signaling functions. Plakoglobin participates in cell-cell adhesion as a component of the adherens junction and desmosomes whereas its signaling function is mediated by its interactions with various intracellular protein partners. To determine the role of plakoglobin during tumorigenesis and metastasis, we expressed plakoglobin in the human tongue squamous cell carcinoma (SCC9) cells and compared the mRNA profiles of parental SCC9 cells and their plakoglobin-expressing transfectants (SCC9-PG). We observed that the mRNA levels of SATB1, the oncogenic chromatin remodeling factor, were decreased approximately 3-fold in SCC9-PG cells compared to parental SCC9 cells. Here, we showed that plakoglobin decreased levels of SATB1 mRNA and protein in SCC9-PG cells and that plakoglobin and p53 associated with the SATB1 promoter. Plakoglobin expression also resulted in decreased SATB1 promoter activity. These results were confirmed following plakoglobin expression in the very low plakoglobin expressing and invasive mammary carcinoma cell line MDA-MB-231 cells (MDA-231-PG). In addition, knockdown of endogenous plakoglobin in the non-invasive mammary carcinoma MCF-7 cells (MCF-7-shPG) resulted in increased SATB1 mRNA and protein. Plakoglobin expression also resulted in increased mRNA and protein levels of the metastasis suppressor Nm23-H1, a SATB1 target gene. Furthermore, the levels of various SATB1 target genes involved in tumorigenesis and metastasis were altered in MCF-7-shPG cells relative to parental MCF-7 cells. Finally, plakoglobin expression resulted in decreased in vitro proliferation, migration and invasion in different carcinoma cell lines. Together with the results of our previous studies, the data suggests that plakoglobin suppresses tumorigenesis and metastasis through the regulation of genes involved in these processes.

Novel role for the transient receptor potential channel TRPM2 in proliferation and apoptosis of human tongue carcinoma cell

Objective: To investigate the expression of Transient Receptor Potential Melastatin type 2 (TRPM2) in the human tongue squamous cell carcinoma tissues and SCC cell lines. The effect of activated and down-regulation of TRPM2 on SCC cell lines were observed.

HS-1200과 cisplatin의 병용처리가 사람구강암세포에 미치는 세포자멸사 효과에 대한 연구

담즙산은 지방의 흡수와 콜레스테롤의 항상성을 조절하는 유전자의 전사에 관여하는 필수 콜레스테롤의 생성물이다. 담즙산 합성유도체인 HS-1200이 여러 가지 암세포에서 세포자멸사(apoptosis)를 유도한다는 것이 알려져 있다. 본 연구는 사람혀 편평세포암종세포(SCC25 cells)에서 담즙산 합성유도체인 HS-1200과 대표적인 항암제인 cisplatin의 병용처리 후 세포자멸사 증가효과가 있는지 알아보기 위해 수행하였다. HS-1200과 cisplatin의 병용처리가 단독처리에 비해서 효과적인 세포생존율 감소가 있는지 확인하기 위해서 MTT법을 시행하였고, 세포자멸사의 유도와 증가를 알기 위해서는 DNA 전기영동법, Hoechst 염색법, DNA hypoploidy법 을 사용하였다. 그리고 세포자멸사에 관계하는 단백질의 발현 변화와 세포내에서의 이동을 밝혀내기 위해서 Western blot 분석과 면역형광 염색법을 수행하였다. 더 나아가서 proteasome 활성도와 사립체막 전위 변화를 측정하였다. 본 연구에서는 HS-1200과 cisplatin을 병용처리한 SCC25 세포에서 핵의 농축, DNA분절, MMP와 proteasome 활성도의 감소, Bax의 증가와 Bcl-2의 감소, DNA양의 감소, cytochrome c의 세포질로의 유리, AIF와 DFF40(CAD)의 핵으로의 이동, caspase-9, caspase-7, caspase-3, PARP 그리고 DFF45(ICAD)의 활성화와 같은 다양한 세포자멸사 증거를 보였다. 반면에 상기 물질들에 단독처리 된 SCC25 세포에서는 세포자멸사 현상이 거의 없었다. 24시간 동안 <TEX>$25{\mu}M$</TEX>의 HS-1200, <TEX>$4{\mu}g/ml$</TEX>의 cisplatin 을 각기 단독처리 한 결과에서는 세포자멸사를 거의 유도하지 못했으나, 병용처리한 결과에는 아주 탁월하고 명확한 세포자멸사의 유도를 보였다. 그러므로 본 실험결과는 HS-1200과 cisplatin 의 병용요법이 사람구강편평세포암종 환자를 위해 새로운 치료전략으로서의 가능성을 보여준다고 생각한다. Bile acids are polar derivatives of cholesterol essential for the absorption of dietary lipids and regulate the transcription of genes that control cholesterol homeostasis. Recently it have been identified the synthetic chenodeoxycholic acid (CDCA) derivatives HS-1200 and cisplatin showed apoptisis-inducing activity on various cancer cells in vivo and in vitro. This study was undertaken to investigate the synergistic apoptotic effect of co-treatment with HS-1200 and cisplatin on human tongue squamous cell carcinoma cells (SCC25 cells). To investigate whether the co-treatment with HS-1200 and cisplatin compared to each single treatment efficiently reduces the viability of SCC25 cells, MTT assay was conducted. The induction and augmentation of apoptosis were confirmed by DNA electrophoresis, Hoechst staining and an analysis DNA hypoploidy. Westen blot analysis and immunofluorescent staining were also performed to evaluate the expression levels and the translocation of apoptosis-related proteins following this co-treatment. Furthermore, proteasome activity and mitochondrial membrane potential (MMP) change were also assayed. In this study, co-treatment with HS-1200 and cisplatin on SCC25 cells showed several lines of apoptotic manifestation such as nuclear condensations, DNA fragmentation, reduction of MMP and proteasome activity, the increase of Bax and the decrease of Bcl-2, decrease of DNA content, the release of cytochrome c into cytosol, translocation of AIF and DFF40 (CAD) onto nuclei, and activation of caspase-9, caspase-7, caspase-3, PARP and DFF45 (ICAD) whereas each single treated SCC25 cells did not show these patterns. Although the single treatment of <TEX>$25{\mu}M$</TEX> HS-1200 and <TEX>$4{\mu}g/ml$</TEX> cisplatin for 24 h did not induce apoptosis, the co-treatment of these reagents prominently induced apoptosis. Therefore our data provide the possibility that the combination therapy with HS-1200 and cisplatin could be considered as a novel therapeutic strategy for human squamous cell carcinoma.

Signal transducers and activators of transcription-3 modulates human squamous cell carcinoma invasion via targeting mircoRNA-21 in vitro

To investigate the effect and mechanism of signal transducers and activators of transcription 3 (STAT-3) modulates human tongue squamous cell carcinoma invasion ability via targeting mircoRNA-21. Tscca and Tca8113P160 human tongue squamous cell carcinoma cell lines were used.WP1066 (STAT-3 inhibitor) , the small molecule inhibitor of STAT-3 was used to suppress the STAT-3 expression. The half maximal inhibitory concentration (IC50 value) of WP1066 in the two cell lines was determined by methyl thiazolyl tetrazolium (MTT) assay. The expression level of STAT-3 and phosphorylation of STAT-3 (pSTAT-3) was examined by Western blotting. Real-time PCR was used to detect the mircoRNA-21 expression after treated with WP1066. Matrigel matrix and transwell assay were used to determine cancer cell colony formation and invasion ability after treated with WP1066. Tumor invasion related proteins in Tscca and Tca8113P160 cell lines were measured by Western blotting. Luciferase reporter gene assay was conducted to detect the relationship between STAT-3 and mircoRNA-21. The IC50 to WP1066 in Tscca cell was 3.1 and 3.5 µmol/L for Tca8113P160 cell respectively. STAT-3/pSTAT-3 protein level was suppressed significantly (Tscca: STAT-3: F = 887.154, P = 0.000; pSTAT-3: F = 332.212, P = 0.000; Tca8113P160: STAT-3: F = 322.895, P = 0.000; pSTAT-3:F = 788.357, P = 0.000). mircoRNA-21 expression was down-regulated (Tscca:F = 32.157, P = 0.000; Tca8113P160: F = 11.349, P = 0.007). The diameters of culture clone in cell treated with WP1066 were less than control groups (Tscca:F = 15.751, P = 0.004; Tca8113P160: F = 12.964, P = 0.007). The number of tongue cancer cell migrating through the transwell membrane in WP1066 treated group was less than in control groups (Tscca: F = 1688.926, P = 0.000; Tca8113P160: F = 327.528, P = 0.000). In addition, MMP-2/9 protein expression was decreased in both of the cell lines treated with WP1066, while TIMP-3 was up regulated dramatically. STAT-3 could modulate mircoRNA-21 directly. Reduction of STAT-3 can inhibit tongue cancer cell invasion ability via targeting mircoRNA-21.

The radiosensitization effect of mTOR inhibitior in human tongue squamous cell carcinoma.

e13585 Background: mTOR plays a critical role in malignant phenotype in many tumors. Everolimus [RAD001 (40-O-(2-hydroxyethyl)-rapamycin)] is a rapamycin rapalog that is being developed as an antitumor agent and is approved for various cancers. Recently, RAD001 has also been reported to increase the radiosensitization following for amplifying the autophagic pathway; however, it could promote an alternative mechanism that leads to apoptosis. In HNSCC, radiation therapy is a common form of cancer treatment, but locoregional recurrence and/or distant metastasis is the most cause of failure after RT and it results in HNSCC significant morbidity. Evidence from previous studies showed that RAD001 enhanced the cytotoxic effects of radiation in HNSCC cell lines. The molecular mechanism of radiosensitization by RAD001 in HNSCC has not cleared.Our aim is to evaluate the role of molecular mechanism of RAD001-mediated radiosensitization in HNSCC. Methods: Radiosensitivity effects of RAD001 were assays on human oral squamous cell carcinoma cell lines (SCC4 and DuFa). RAD001 (30 and 300 nM)and radiation (0~8 Gy) alone and in combination were evaluated for antitumor activity in the clonogenic assay. Next, we also evaluated the RAD001 on radiation-induced signal transduction by western blot analysis. Results: Here, we show that 300nM RAD001 plus 6 Gy cohort with significant HNSCC cell line growth suppression compared to 6 Gy alone (p=0.004) or 30n MRAD001 plus 6 Gy (p=0.008). RAD001 treatment effectively reduces mTOR downstream effector p-S6 protein compared with the vehicle or irradiated alone. RAD001 + 6 Gy significantly increases p53 and PARP cleavage, which facilitates apoptosis. Unexpectedly, autophagy pathway, such as beclin 1 and LC3II were not affected by RAD001. Addiationlly, we discover that Akt phosphorylation (Ser473) is up-regulated after RAD001/and irradiation of HNSCC cells. Conclusions: These findings suggest radiosensitization by the RAD001 involves the induction of apoptosis in HNSCC. Notwithstanding, Akt signaling is increased under RAD001 and it could attenuate anticancer efficacy instead, contribute to the development of resistance.

Proteomic analysis of protein expression profiles during hyperthermia-induced apoptosis in Tca8113 cells

The aim of the present study was to explore protein expression profiles during cancer cell apoptosis induced by hyperthermia. A hyperthermia-induced apoptosis model was established using a Tca8113 cell line derived from a human tongue squamous cell carcinoma, which underwent fluorescent differential display two-dimensional (2D) gel electrophoresis at 2, 6, 8, 12 and 24 h following the induction of hyperthermia. Proteins were identified by mass spectrometry analysis. Expression changes in the proteins were detected by western blot analysis. A total of 107 proteins were detected that exhibited different expression levels in the hyperthermia-treated cells compared with the controls, and 57 of these proteins were identified. Expression changes in the representative proteins were further verified by western blot analysis. These 57 proteins were identified according to the following functional groups: energy metabolism-related enzymes, cytoskeleton-related proteins, chaperones, transcription factors, protein synthesis-related proteins and cell division- and proliferation-related proteins. These groups included 44 upregulated and 13 downregulated proteins. Among the 44 upregulated proteins, 27 were upregulated continuously, eight were upregulated at an early time-point and nine were upregulated at a middle to late time-point. Among the 13 downregulated proteins, five were downregulated continuously, six were downregulated at an early time-point and two were downregulated at a middle to late time-point. These results indicate that hyperthermia-induced Tca8113 cell apoptosis is controlled by multiple factors, which include time and regulatory proteins.

Dinuclear and heptanuclear complexes of copper(II) with 7-azaindole ligand: Synthesis, characterization, magnetic properties, and biological activity

Three new complexes of Cu(II) with 7-azaindole have been synthesized and characterized, a dicopper compound, [Cu(C7H5N2)2(H2O)]2·2CH3CN, 1, and two heptacopper compounds [Cu7(C7H5N2)6(μ3-OH)6(μ2-H2O)2(μ2-CH3OH)4](CH3COO)2·2C7H8·6CH3OH, 2, and [Cu7(C7H5N2)5(CH3COO)(μ3-OH)6(μ2-H2O)4(μ2-CH3OH)2](CH3COO)2, 4. The structure of 2 is monoclinic and it crystallizes in the P21/c space group: a=13.475(4)Å; b=12.945(4)Å; c=23.392(7)Å; β=91.232(6)°. It contains a unique Cu7O12 core in which a central Cu(II) is situated at an inversion center and is bonded to 6 other Cu(II) ions via bridging oxygen atoms from OH−, H2O, and CH3OH groups. Anionic 7-azaindole ligands bridge between adjacent outer Cu(II) ions and all Cu(II) ions have distorted octahedral coordination geometries. Variable temperature magnetic susceptibility measurements revealed the presence of antiferromagnetic exchange interactions between Cu(II) ions which leads to an S=5/2 ground state at 1.8K.Cytotoxicity and cell proliferation activities of the Cu compounds using human tongue squamous cell carcinoma and normal cells revealed that the compounds stimulated proliferation in both types of cells.

Abstract 2958: Knockdown of Src expression dissolves resistance to TRAIL-induced apoptosis in oral cancer cells.

Abstract A radical operation for advanced oral cancer results in malfunction of chewing, swallowing, and pronouncing etc. Nevertheless, the most standard therapy for oral cancer is a surgical resection because of its good outcome compared to radiation and chemotherapy. It is expected that treatment is less-invasive and effective against not only primary lesion but also metastatic sites or cells with metastatic potential. Because the acquisition of resistance to anoikis and apoptosis is considered as an essential event to successfully metastasize, we focused on the apoptotic signaling pathways mediated by natural death ligands. In this study, we have used oral squamous cell carcinoma (OSCC) cell lines with distinct metastatic potential and attempted to disclose their apoptosis sensitivity. More than half of the cells died under the low attachment culture condition in all OSCC cell lines with no relation between their metastatic potential, while other kind of cancer cell lines showed anoikis resistance. SAS cells established from human tongue squamous cell carcinoma showed modest sensitivity to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), whereas SAS-T5 and SAS-L1 cells, a clone with high metastatic potential showed robust resistance. Fas ligand (Anti-Fas/CD95 monoclonal antibody) showed the similar trend of apoptosis induction as well as TRAIL, but TNF-α had no effect in these cells. We confirmed that the expression levels and the localization of TRAIL receptors were not correlated with TRAIL sensitivity. One of the mechanisms underlying the differences in resistance between these cell lines was an activity of Src kinase. Phosphorylated Src (Tyr 416), the active form, was upregulated in SAS-T5 and SAS-L1 cells compared to parental SAS cells. Src inhibitor PP2 suppressed cell proliferation in response to dependence on Src signaling. Downregulation of Src with siRNA made metastatic cells dissolve the resistance to TRAIL-induced apoptosis. These results suggest that combination therapy with TRAIL plus medicine inhibiting Src kinase may be a treatment strategy for oral squamous cell carcinoma. Citation Format: Tomohiro Hamakawa, Satoshi Hino, Koh-ichi Nakashiro, Hiroyuki Hamakawa. Knockdown of Src expression dissolves resistance to TRAIL-induced apoptosis in oral cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2958. doi:10.1158/1538-7445.AM2013-2958

Scutellarin inhibits the growth and invasion of human tongue squamous carcinoma through the inhibition of matrix metalloproteinase-2 and -9 and αvβ6 integrin

Scutellarin can inhibit the growth and migration of tongue cancer cells in vitro and can regulate cell adhesion; such agents will be the next generation anticancer therapeutics. In this study, we evaluated the antitumor effects of the scutellarin on human tongue squamous carcinoma (SAS) cell line and investigated its molecular mechanism. To explore the possible mechanisms underlying the antitumor effect of scutellarin, we studied its impact on the growth and invasion of SAS cells xenografted into nude mice, on the expression of MMP-2, MMP-9, integrin αvβ6, and c-JUN, and also on changes in collagen fibers. We observed that the growth of xenograft SAS tumors in nude mice was significantly inhibited by the administration of scutellarin without major adverse effects. Results showed that scutellarin mediated inhibition of tumor cell proliferation, induced apoptosis, and regulated expression of matrix metallo-proteinase (MMP)-2 and -9, and integrin αvβ6 at the mRNA and protein levels in vivo. Moreover, scutellarin regulated the expression of collagen fibers in the tumor microenvironment (surrounding the tumor), thereby inhibiting cell invasion and metastasis. Our in vitro and in vivo studies have shown that scutellarin reduces the expression of MMP-2 and -9, and integrin αvβ6 in SAS cells, possibly through the regulation of expression of transcription factor AP-1. These results suggest that scutellarin can have an anti-tumor therapeutic effect by inhibition of the ability of SAS cells to invade and metastasize.

Potentiation of Scutellarin on Human Tongue Carcinoma Xenograft by Low-Intensity Ultrasound

Scutellarin 7-O-β-d-glucuronide (scutellarin) has shown great potential as a chemotherapeutic agent for cancer treatment, but only at high dosage. Here we investigate the possibility of using low intensity ultrasound to reduce the scutellarin dosage. Ultrasound intensities of 1.0 W/cm2 and 0.05 W/cm2 were used for in vivo and in vitro experiments, respectively, and a very low dosage of scutellarin (15 nM) was used. Tumor-bearing Balb/c mice and SAS human-tongue squamous carcinoma cell suspensions were used for the in vivo and in vitro experiments, respectively. Each kind of subjects was divided into control, ultrasound-alone, scutellarin-alone, and combined ultrasound-scutellarin treatment groups. Only the combined treatment showed strong anticancer effects. In the in vivo case, the combined treatment significantly delayed tumor growth, initiated cellular chromatin changes (including a decrease in the number of cytoplasmic organelles and fragmentation of condensed nuclear chromatin), inhibited tumor angiogenesis and lymphangiogenesis, stopped cancer-cell proliferation, decreased MMP-2 and MMP-9 expression levels and caused cancer-cell apoptosis. In the in vitro case, the combined treatment produced cancer cell-shape irregularity in a manner seriously fractured microvilli, inhibited cancer-cell migratory and invasion activities, and induced cancer-cell apoptosis. Because the combined treatment did not increase intracellular ROS production, scutellarin is not a sonosensitizer so that the anticancer effect is not through sonodynamic therapy. Low-intensity ultrasound is merely increasing the permeability of scutellarin into cancer cells. Based on our results, one may perform localized chemotherapy using much reduced dosage of the drug with the help of low intensity ultrasound, which will greatly minimize side effects.

Establishment of a normalized revascularization mouse model using tumor transplantation

Tumor blood vessels play an important role in tumor progression and metastasis and have recently been shown to differ in structure from normal blood vessels. Thus, normalization of tumor blood vessels using molecular targeted therapy has been attracting attention as a potential new treatment for cancer. However, some studies have suggested that this therapy is unable to cause significant tumor shrinkage and instead promotes drug resistance. Therefore, we hypothesized that regeneration of normal blood vessels in tumors may lead to improvement of hypoxic conditions in tumors. Furthermore, regeneration of normal blood vessels may contribute to potential improvements in the delivery of anticancer drugs to hypoxic tumors. In this report, we sought to characterize whether transplanting normal endothelial cells into tumor-bearing mice would trigger vascular remodeling. Tumor cells (SAS; human tongue squamous cell carcinoma) were injected into the dorsal subcutis of SCID mice. After 2 weeks, the tumor-bearing mice were injected cisplatin intraperitoneally to regress tumors and tumor vessels as a first step. An additional 2 weeks later, to induce normal angiogenesis in the tumor, human endothelial cells (HMVECs) were transplanted into necrotic regions of the tumor as a second step. Microscopic observations revealed that the transplanted human endothelial cells formed anastomoses with the host mouse vasculature, and perfused vessels were detectable after 7 days. Thus, this regenerated blood vessel mouse model is a useful model for the future development of new cancer therapies, construction of reliable drug delivery systems, and improvement of hypoxic conditions.

Prognostic Implications of MicoRNA miR-195 Expression in Human Tongue Squamous Cell Carcinoma

BackgroundmiR-195 is aberrantly expressed in multiple types of disease. But little is known about the dysregulation of miR-195 in tongue squamous cell carcinoma (TSCC). In this study, we investigated the roles of miR-195 in the development and progression of TSCC.MethodsUsing quantitative reverse transcription-polymerase chain reaction (qRT-PCR), we evaluated miR-195 expression in TSCC samples from 81 patients. Overall survival of these patients was examined using Kaplan–Meier curves with log-rank tests and the Cox proportional hazards model. The expression of two known miR-195 target genes, Cyclin D1 and Bcl-2, was also examined in the TSCC samples by immunohistochemistry. The effects of miR-195 overexpression on cell cycle progression and apoptosis and its effects on the expression of Cyclin D1 and Bcl-2 were examined in transfected TSCC cell lines (SCC-15 and Cal27) using fluorescence-activated cell sorting assays, luciferase reporter assays, and Western blots.ResultsReduced miR-195 expression was associated with tumor size and the clinical stage of TSCC tumors. Kaplan–Meier survival analysis indicated that the TSCC patients with reduced expression of miR-195 had poor overall survival and in multivariable analyses low levels of miR-195 emerged as an independent prognostic factor for this clinical outcome. Levels of miR-195 expression were inversely correlated with the expression of Cyclin D1 and Bcl-2. Overexpression of miR-195 inhibited cell cycle progression, promoted apoptosis, and reduced Cyclin D1 and Bcl-2 expression in two TSCC cell lines.ConclusionsmiR-195 may have potential applications as a prognostic factor for TSCC patients.

Mechanisms Underlying Cancer Progression Caused by Ezrin Overexpression in Tongue Squamous Cell Carcinoma

BackgroundEzrin is a member of the ezrin, radixin, and moesin family that provides a functional link between the plasma membrane and the cortical actin cytoskeleton. A correlation between ezrin overexpression and aggressive cancer behavior has been recently reported in various tumor types. However, its roles in the mechanisms underlying progression of tongue squamous cell carcinoma (SCC) are unclear.MethodWe used human tongue SCC and noncancerous tissue microarrays to immunohistochemically analyze the ezrin expression level and its relationship with proliferative activity. The human tongue SCC cell line HSC-3 was used to determine the effects of ezrin RNA interference (RNAi) on cancer cells during MTT; wound healing and invasion assays; immunofluorescence of the actin cytoskeleton; and western blotting of E-cadherin, N-cadherin, β-catenin, and the active and total RhoA/Rac1/cdc42.ResultsEzrin was overexpressed in 46.4% of the tumors examined in human tongue SCC tissue microarrays. Ezrin expression was correlated with the Ki-67 index. Ezrin depletion by RNAi in the HSC-3 cells significantly reduced cell proliferation, migration, and invasiveness and disturbed actin reorganization during podia formation. Its effects on RhoA/Rac1/cdc42 expression were not significant, whereas it enhanced E-cadherin and β-catenin expression and decreased N-cadherin expression.ConclusionsEzrin is often overexpressed in primary tongue SCCs and may have an important role in their growth, migration, and invasiveness possibly via its relationship with the E-cadherin/β-catenin complex and the cadherin switch. Thus, ezrin could be a therapeutic target in tongue SCC.

Plakoglobin interacts with the transcription factor p53 and regulates the expression of 14-3-3σ

Plakoglobin (γ-catenin), a constituent of the adherens junction and desmosomes, has signaling capabilities typically associated with tumor/metastasis suppression through mechanisms that remain undefined. To determine the role of plakoglobin during tumorigenesis and metastasis, we expressed plakoglobin in human tongue squamous cell carcinoma (SCC9) cells and compared the mRNA profiles of parental SCC9 cells and their plakoglobin-expressing transfectants (SCC9-PG). We detected several p53-target genes whose levels were altered upon plakoglobin expression. In this study, we identified the p53 regulated tumor suppressor 14-3-3σ as a direct plakoglobin-p53 target gene. Coimmunoprecipitation experiments revealed that plakoglobin and p53 interact, and chromatin immunoprecipitation and electrophoretic mobility shift assays revealed that plakoglobin and p53 associate with the 14-3-3σ promoter. Furthermore, luciferase reporter assays showed that p53 transcriptional activity is increased in the presence of plakoglobin. Finally, knockdown of plakoglobin in MCF-7 cells followed by luciferase assays confirmed that p53 transcriptional activity is enhanced in the presence of plakoglobin. Our data suggest that plakoglobin regulates gene expression in conjunction with p53 and that plakoglobin may regulate p53 transcriptional activity, which may account, in part, for the tumor/metastasis suppressor activity of plakoglobin.

Oral cancer-associated fibroblasts inhibit heat-induced apoptosis in Tca8113 cells through upregulated expression of Bcl-2 through the Mig/CXCR3 axis

The aim of this study was to detect oral cancer-associated fibroblast-secreted cytokines and their regulation of heat-induced apoptosis in the human tongue squamous cell carcinoma cell line Tca8113. We isolated cancer-associated fibroblasts (CAFs) from human tongue squamous cell carcinoma and normal fibroblasts (NFs) from normal oral mucosa. The expression profiles of cytokines secreted by CAFs and those secreted by NFs were detected using the RayBio® human cytokine antibody microarray. The conditioned medium was prepared by mixing the CAF or NF supernatant cell culture medium with fresh complete medium. The expression levels of Bax, Bcl-2 and CXCR3 in Tca8113 cells were detected by western blot analysis. The heat-induced apoptosis ratio of the Tca8113 cells was detected by propidium iodide staining combined with flow cytometry. The quantity of the Mig factor, one of the chemokines secreted by CAFs, was clearly increased 19-fold when compared with the level in NFs. The conditioned medium of NFs had no obvious effect on the expression levels of Bax/Bcl-2 and the heat-induced apoptosis ratio in the Tca8113 cells. However, the expression levels of Bcl-2 were significantly upregulated and heat-induced Tca8113 cell apoptosis was inhibited in the CAF-conditioned medium. After adding neutralizing antibodies against Mig or its receptor CXCR3, the enhanced expression of the Bcl-2 protein and the inhibited heat-induced apoptosis of Tca8113 cells in the CAF-conditioned medium were significantly attenuated. CAFs may increase the expression levels of Bcl-2 through the paracrine secretion of Mig and reduce the thermosensitivity of Tca8113 cells.