Oral Squamous Cell Carcinoma Xenograft Research Articles

Inhibition of LSD1 epigenetically attenuates oral cancer growth and metastasis.

Lysine-specific demethylase 1 (LSD1) is a nuclear histone demethylase and a member of the amine oxidase (AO) family. LSD1 is a flavin-containing AO that specifically catalyzes the demethylation of mono- and di-methylated histone H3 lysine 4 through an FAD-dependent oxidative reaction. LSD1 is inappropriately upregulated in lung, liver, brain and esophageal cancers, where it promotes cancer initiation, progression, and metastasis. However, unlike other lysine-specific demethylases, the role and specific targets of LSD1 in oral squamous cell carcinoma (OSCC) pathogenesis remain unknown. We show that LSD1 protein expression was increased in malignant OSCC tissues in a clinical tissue microarray, and its expression correlated with progressive tumor stages. In an orthotopic oral cancer mouse model, LSD1 overexpression in aggressive HSC-3 cells promoted metastasis whereas knockdown of LSD1 inhibited tumor spread, suggesting that LSD1 is a key regulator of OSCC metastasis. Pharmacological inhibition of LSD1 using a specific small molecule inhibitor, GSK-LSD1, down-regulated EGF signaling pathway. Further, GSK-LSD1 attenuates CTGF/CCN2, MMP13, LOXL4 and vimentin expression but increased E-cadherin expression in pre-existing, patient-derived tonsillar OSCC xenografts. Similarly, GSK-LSD1 inhibited proliferation and CTGF expression in mesenchymal cells, including myoepithelial cells and osteosarcoma cells. In addition, gene set enrichment analysis revealed that GSK-LSD1 increased p53 expression and apoptosis while inhibiting c-myc, β-catenin and YAP-induced oncogenic transcriptional networks. These data reveal that aberrant LSD1 activation regulates key OSCC microenvironment and EMT promoting factors, including CTGF, LOXL4 and MMP13.

The role (if any) of chemotherapy in hepatocellular carcinoma

The study by Masatoshi Kudo and colleagues 1 Kudo M Moriguchi M Numata K et al. S-1 versus placebo in patients with sorafenib-refractory advanced hepatocellular carcinoma (S-CUBE): a randomised, double-blind, multicentre, phase 3 trial. Lancet Gastroenterol Hepatol. 2017; (published online April 6.)http://dx.doi.org/10.1016/S2468-1253(17)30072-9 Google Scholar published in The Lancet Gastroenterology & Hepatology is an appreciated effort to overcome the unmet needs in treatment of advanced hepatocellular carcinoma. The study is a well-designed, randomised, double-blind, multicentre phase 3 trial comparing S-1 with placebo after progression on or intolerance to sorafenib in Japanese patients with advanced hepatocellular carcinoma. The results unfortunately did not show an improvement in survival with S-1 (11·1 months for S-1 and 11·2 months for placebo) despite encouraging phase 1/2 data (progression-free survival of 3·7 months and overall survival of 16·6 months). 2 Furuse J Okusaka T Kaneko S et al. Phase I/II study of the pharmacokinetics, safety and efficacy of S-1 in patients with advanced hepatocellular carcinoma. Cancer Sci. 2010; 101: 2606-2611 Crossref PubMed Scopus (35) Google Scholar A post-hoc exploratory predictive analysis detected an enriched population with significant benefit (overall survival of 14·0 months for S-1 versus 12·3 months for placebo, p=0·016). These patients had Child-Pugh A cirrhosis, alpha-fetoprotein less than 400 ng/mL, and stage III or IV disease. However, there was no mention of correlation between outcome and prior sorafenib exposure, or if S-1 activity is potentiated by sorafenib-induced tumour necrosis, 3 Itashiki Y Harada K Ferdous T Yoshida H Effects of tumor necrosis factor-related apoptosis-inducing ligand alone and in combination with fluoropyrimidine anticancer agent, S-1, on tumor growth of human oral squamous cell carcinoma xenografts in nude mice. Anticancer Res. 2007; 27: 2365-2375 PubMed Google Scholar although adding S-1 to sorafenib in the first-line setting did not show added benefit compared with historical results with sorafenib alone (overall survival 10·5 months). 4 Ooka Y Chiba T Ogasawara S et al. A phase I/II study of S-1 with sorafenib in patients with advanced hepatocellular carcinoma. Invest New Drugs. 2014; 32: 723-728 Crossref PubMed Scopus (10) Google Scholar S-1 versus placebo in patients with sorafenib-refractory advanced hepatocellular carcinoma (S-CUBE): a randomised, double-blind, multicentre, phase 3 trialS-1 did not prolong overall survival in patients with sorafenib-refractory advanced hepatocellular carcinoma. Further research is needed to identify subgroups of patients who might benefit from S-1. Full-Text PDF

Synergistic effect of honokiol and 5‐fluorouracil on apoptosis of oral squamous cell carcinoma cells

5-Fluorouracil (5-FU) is an essential chemotherapeutic agent for oral squamous cell carcinoma (OSCC). However, toxic side effects have limited its role in OSCC therapy. The aim of this study was to explore whether combination therapy with 5-FU and honokiol (HNK), a small natural organic molecule shown to induce apoptosis in OSCC cells, could enhance the anticancer activity of 5-FU without notably increasing its toxicity. 5-FU and/or HNK were used to treat OSCC cells both in vitro and in vivo. The therapeutic effect and underlying mechanisms were evaluated by cell viability assay, flow cytometry, OSCC xenograft mouse model, and Western blot. Tumor tissue apoptosis was detected by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. Toxicity was assessed following hematoxylin and eosin staining. Exposure to HNK + 5-FU produced a synergistic cytotoxic effect on OSCC cells. Both HNK and 5-FU could induce apoptosis through the mitochondria-mediated intrinsic pathway, and their specific signaling pathways were different. In the mouse OSCC xenograft model, treatment with 5-FU + HNK substantively retarded tumor growth, as compared to treatment with either drug individually. TUNEL analysis further confirmed that the superior in vivo antitumor efficacy of 5-FU + HNK was associated with enhanced stimulation of cell apoptosis. Notably, HNK did not increase the toxicity of 5-FU. These findings suggest that HNK and 5-FU exert a synergistic therapeutic effect on OSCC by inducing apoptosis. HNK might thus enhance the clinical therapeutic efficacy of 5-FU without increasing its toxicity.

Granzyme B‐truncated VEGF fusion protein represses angiogenesis and tumor growth of OSCC

To evaluate the antitumor effects of fusion protein hGrB-TV of human granzyme B (hGrB) and truncated vascular endothelial growth factor (tVEGF) on human oral squamous cell carcinoma (OSCC) invitro and invivo. The fusion protein hGrB-TV was expressed and purified from E.coli bacteria by affinity chromatography. The cytotoxcity of hGrB-TV on VEGFR-2 (Flk-1)(+) OSCCcells was analyzed invitro. The antitumor therapeutic study was conducted on OSCC xenografts invivo. The purified hGrB-TV fusion protein was selectively internalized into VEGFR-2 (Flk-1)(+) OSCC cellsand endothelial cells. It can cleave inactive caspase 3 into its active p20 form. The hGrB-TV showed dose-dependent cytotoxicity on VEGFR-2(+) SCC-9 cells. The morphological changes and cytolysis were appeared within dozen minutes. However, no cytotoxicity was observed on VEGFR-2(-) cells. The hGrB alone or tVEGF alone did not have any toxicity on SCC-9 cells. In addition, hGrB-TV treatment completely destroyed the vasculature of the chick chorioallantoic membrane (CAM) invivo and consequently led to chick embryo development arrest. Most importantly, the fusion protein hGrB-TV inhibited tumor angiogenesis and growth of humanOSCC xenografts in nude mice without any apparent toxicity. The fusion protein hGrB-TV specifically inhibits angiogenesis and tumor growth of OSCC; hGrB-TV is a powerful and safe therapeutic molecule for tumor therapy.

Overexpression of proteasomal activator PA28α serves as a prognostic factor in oral squamous cell carcinoma.

BackgroundDespite recent advances in oral squamous cell carcinoma (OSCC) diagnosis and therapy, disease recurrence remains common and is strongly associated with mortality. It is therefore critical to identify new targets for both treatment and diagnostic purposes. We aimed at investigating the role of PA28α, a proteasomal activator in OSCC.MethodsThe expression of PA28α was examined in a panel of OSCC cell lines and tissues, associated with oncomine analysis. In a large OSCC patient cohort, the prognostic value of PA28α expression was evaluated. Primary clinical end points were recurrence-free and overall survival rate. Functional involvement of PA28α in OSCC was examined in both in vitro and in vivo models upon specific siRNA knockdown.ResultsPA28α was found to be overexpressed in OSCC cell lines and tumor tissues. High expression of PA28α was significantly associated with recurrence and poorer overall survival. Specific knockdown of PA28α inhibited OSCC cell proliferation, migration, invasion in vitro and reduced the growth of OSCC xenografts in vivo. Multivariate Cox regression analyses revealed PA28α as independent prognostic predictors.ConclusionsThese results suggest that PA28α is involved in OSCC oncogenesis and may serve as a potential prognostic factor.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-016-0309-z) contains supplementary material, which is available to authorized users.

Blocking TNF-α inhibits angiogenesis and growth of IFIT2-depleted metastatic oral squamous cell carcinoma cells

Our previous study demonstrated that the depletion of interferon-induced protein with tetratricopeptide repeats 2 (IFIT2) promoted metastasis and was associated with a poor prognosis in patients with oral squamous cell carcinoma (OSCC). Our current study explores the major downstream signaling involved in IFIT2 depletion-induced OSCC metastasis. To this end, we used two cell lines (designated sh-control-xeno and sh-IFIT2-xeno) derived from human OSCC xenografts expressing sh-control and sh-IFIT2, respectively, and one metastatic OSCC subline (sh-IFIT2-meta) from an IFIT2-depleted metastatic tumor. We found that the sh-IFIT2-meta cells proliferated more slowly than the sh-control-xeno cells but exhibited higher migration and chemoresistance. Using microarray technology and Ingenuity Pathway Analysis, we found that TNF-α was one of the major downstream targets in IFIT2-depleted OSCC cells. Quantitative real-time PCR, western blotting, and ELISA results confirmed that TNF-α was upregulated in the sh-IFIT2-meta cells. Blocking TNF-α abolished the angiogenic activity induced by the sh-IFIT2-meta cells. Furthermore, the human-specific TNF-α antibody golimumab significantly inhibited in vivo angiogenesis, tumor growth and metastasis of sh-IFIT2-meta cells. These results demonstrate that IFIT2 depletion results in TNF-α upregulation, leading to angiogenesis and metastasis of OSCC cells.

Role of transcription factor Sp1 in the 4-O-methylhonokiol-mediated apoptotic effect on oral squamous cancer cells and xenograft

Recently, biphenolic components derived from the Magnolia family have been studied for anti-cancer, anti-stress, and anti-inflammatory pharmacological effects. However, the pharmacological mechanism of action of 4-O-methylhonokiol (MH) is not clear in oral cancer. The aim of this study was to investigate the role of MH in apoptosis and its molecular mechanism in oral squamous cell carcinoma (OSCC) cell lines, HN22 and HSC4, as well as tumor xenografts. Here, we demonstrated that MH decreased cell growth and induced apoptosis in HN22 and HSC4 cells through the regulation of specificity protein 1 (Sp1). We employed several experimental techniques such as MTS assay, DAPI staining, PI staining, Annexin-V/7-ADD staining, RT-PCR, western blot analysis, immunocytochemistry, immunohistochemistry, TUNEL assay and in vivo xenograft model analysis. MH inhibited Sp1 protein expression and reduced Sp1 protein levels via both proteasome-dependent protein degradation and inhibition of protein synthesis in HN22 and HSC4 cells; MH did not alter Sp1 mRNA levels. We found that MH directly binds Sp1 by Sepharose 4B pull-down assay and molecular modeling. In addition, treatment with MH or knocking down Sp1 expression suppressed oral cancer cell colony formation. Moreover, MH treatment effectively inhibited tumor growth and Sp1 levels in BALB/c nude mice bearing HN22 cell xenografts. These results indicated that MH inhibited cell growth, colony formation and also induced apoptosis via Sp1 suppression in OSCC cells and xenograft tumors. Thus, MH is a potent anti-cancer drug candidate for oral cancer.

CMTM3 inhibits cell growth and migration and predicts favorable survival in oral squamous cell carcinoma.

Downregulation of CKLF-like MARVEL transmembrane domain-containing member 3 (CMTM3) has been reported in a number of human tumors. However, the role of CMTM3 in oral squamous cell carcinoma (OSCC) remains largely unknown. In this study, we showed that the expression of CMTM3 was significantly reduced in OSCC cell lines and primary tumor specimens (P < 0.001). Methylation-specific PCR showed hypermethylation in CMTM3 promoter in a significant proportion of tumor tissues (61 %). The expression of CMTM3 was associated with T stage, lymph node metastasis, tumor node metastasis (TNM) stage, and recurrence of OSCC patients (P < 0.05, n = 201). More importantly, CMTM3 expression was associated with the prognosis of OSCC patients (P < 0.001) and was an independent prognostic factor (hazard ratio = 0.593, 95 % confidence interval, 0.272-1.292; P = 0.039). Overexpression of CMTM3 inhibited the growth and migration of OSCC cells. In vivo experiments also showed that the growth of OSCC xenografts in nude mice was significantly inhibited by CMTM3 overexpression. These findings indicate that downregulation of CMTM3 due to promoter hypermethylation contributed to the proliferation and migration of OSCC cells and suggest that CMTM3 is an independent prognostic factor for the evaluation of the survival of OSCC patients.

Nimotuzumab increases the anti-tumor effect of photodynamic therapy in an oral tumor model.

Oral squamous cell carcinoma (OSCC) represents 90% of all oral cancers and is characterized with poor prognosis and low survival rate. Epidermal growth factor receptor (EGFR) is highly expressed in oral cancer and is a target for cancer therapy and prevention. In this present work, we evaluate the efficacy of photodynamic therapy (PDT) in combination with an EGFR inhibitor, nimotuzumab in oral cancer cell lines and OSCC xenograft tumor model. PDT is a promising and minimally invasive treatment modality that involves the interaction of a photosensitizer, molecular oxygen and light to destroy tumors. We demonstrated that EGFR inhibitors nimotuzumab and cetuximab exhibits anti-angiogenic properties by inhibiting the migration and invasion of oral cancer cell lines and human endothelial cells. The EGFR inhibitors also significantly reduced tube formation of endothelial cells. Chlorin e6-PDT in combination with nimotuzumab and cetuximab reduced cell proliferation in different oral cancer and endothelial cells. Furthermore, our in vivo studies showed that the combination therapy of PDT and nimotuzumab synergistically delayed tumor growth when compared with control and PDT treated tumors. Downregulation of EGFR, Ki-67 and CD31 was observed in the tumors treated with combination therapy. Analysis of the liver and kidney function markers showed no treatment related toxicity. In conclusion, PDT outcome of oral cancer can be improved when combined with EGFR inhibitor nimotuzumab.

WP1066 sensitizes oral squamous cell carcinoma cells to cisplatin by targeting STAT3/miR-21 axis.

Accumulating evidence reveals that activation of STAT3 and miR-21 contributes to chemoresistance in multiple tumors. We examined the expression of STAT3 and miR-21 in 43 oral squamous cell carcinoma (OSCC) tumors and classified them into cisplatin sensitive or resistant group. Tca8113 and Tca8113/DDP cells were treated with cisplatin (DDP), WP1066 (STAT3 inhibitor) or in combination. MTT, colony formation, wound healing, 3-D culture, and transwell chamber assays were used to evaluate the malignant phenotype of OSCC cells. We evaluated the effect of WP1066 on the expression of STAT3 and miR-21. A Tca8113/DDP OSCC xenograft tumor model was established to evaluate the therapeutic effect of WP1066 in combination with DDP. The expression of STAT3/miR-21 was significantly increased in DDP-resistant OSCC samples and Tca8113/DDP cells compared to its parental cell. Treatment of DDP combined with WP1066 efficiently inhibited Tca8113 and Tca8113/DDP cell proliferation, migration and invasion. STAT3 mediated OSCC cell survival and DDP resistance through upregulating the expression of miR-21 and downregulating miR-21 downstream targets, including PTEN, TIMP3 and PDCD4. WP1066 plus DDP treatment could inhibit Tca8113 and Tca8113/DDP cell growth by inhibiting STAT3 phosphorylation and miR-21 expression. These results indicated that STAT3/miR-21 axis could be a candidate therapeutic target for OSCC chemoresistance.

Induction of Peroxiredoxin 1 by Hypoxia Regulates Heme Oxygenase-1 via NF-κB in Oral Cancer

Overexpression of peroxiredoxin 1 (Prx1) has been observed in numerous cancers including oral squamous cell carcinoma (OSCC). The precise molecular mechanism of up-regulation of Prx1 in carcinogenesis, however, is still poorly understood. The objective of this study is to investigate the relationship between Prx1 and hypoxia, and potential mechanism(s) of Prx1 in OSCC cell line SCC15 and xenograft model. We treated wild-type and Prx1 knockdown SCC15 cells with transient hypoxia followed by reoxygenation. We detected the condition of hypoxia, production of reactive oxygen species (ROS), and expression and/or activity of Prx1, heme oxygenase 1 (HO-1) and nuclear factor-kappa B (NF-κB). We found that hypoxia induces ROS accumulation, up-regulates Prx1, increases NF-κB translocation and DNA binding activity, and down-regulates HO-1 in vitro. In Prx1 knockdown cells, the expression level of HO-1 was increased, while NFκB translocation and DNA binding activity were decreased after hypoxia or hypoxia/reoxygenation treatment. Moreover, we mimicked the dynamic oxygenation tumor microenvironment in xenograft model and assessed the above indices in tumors with the maximal diameter of 2 mm, 5 mm, 10 mm or 15 mm, respectively. Our data showed that tumor hypoxic condition and expression of Prx1 are significantly associated with tumor growth. The expression of HO-1 and NF-κB, and NF-κB DNA binding activity were significantly elevated in 15 mm tumors, and the level of 8-hydroxydeoxyguanosine was increased in 10 mm and 15 mm tumors, compared to those in size of 2 mm. The results from this study provide experimental evidence that overexpression of Prx1 is associated with hypoxia, and Prx1/NF-κB/HO-1 signaling pathway may be involved in oral carcinogenesis.

Combined cetuximab and celecoxib treatment exhibits a synergistic anticancer effect on human oral squamous cell carcinoma invitro and invivo.

The aim of the present study was to evaluate the potency of epidermal growth factor receptor(EGFR) pathway inhibition achieved by combining cetuximab(CET), an anti-EGFR monoclonal antibody, and celecoxib(CXB), a cyclooxygenase-2 (COX-2) inhibitor, in oral squamous cell carcinoma (OSCC) invitro and invivo. The OSCC cell line, HSC3, was treated with CET(0-400µg/ml), CXB(0-40µM), or a combination of both at a range of concentrations. Cell proliferation, apoptosis, migration and invasion were determined to assess the anticancer effects invitro. The invivo effects of CET and CXB on tumor cell growth were examined using an OSCC xenograft nude mouse model. In addition, downstream protein expression levels of EGFR, p-EGFR, PI3K, p-PI3K, AKT and p-Akt were evaluated by western blot analysis. It was found that the combination of low concentrations of CET and CXB significantly suppressed the proliferation, migration and invasion of the HSC3 tumor cells and decreased PEG2 production and VEGF expression invitro, and inhibited tumor growth invivo compared to the action of either agent alone. The results also showed that this combination significantly induced apoptosis and increased caspase-3 and caspase-8 activity compared to the action of either agent alone (P<0.01). Furthermore, the combination treatment significantly reduced the expression of p-EGFR, p-PI3K and p-Akt in the HSC3 cell line, which may contribute to the inhibition of tumor growth. Taken together, our findings revealed that the additive combination of CET and CXB is a potential drug candidate for the treatment of OSCC.

Boron Carbide Particle as a Boron Compound for Boron Neutron Capture Therapy

Background: In boron neutron capture therapy (BNCT), the intravenous administration of boron-containing compounds such as boronophenylalanine (BPA) and borocaptate sodium (BSH) is followed by irradiation with thermal neutrons. In the present study, we determined whether a boron-rich boron carbide (B4C) nanoparticle could be used for BNCT. Methods: B4C nanoparticles were obtained by the laser fragmentation of boron particles in ethyl acetate. Neutron irradiation was delivered via a neutron beam at the Kyoto University Research Reactor (KUR). Results: Cell viability was lower in oral squamous cell carcinoma (SCC) cells exposed to neutrons in the presence of B4C particles than in those exposed to neutrons only. The combination of B4C particles and neutron irradiation also decreased the formation of colonies.Injecting B4C particles into the oral SCC xenografts of nude mice increased the concentration of 10B in the tumors, but not in the kidney, liver, or spleen. The body weights of animals were not affected by neutron irradiation to B4C-injected tumors.Tumor volumes were lowerTumor volumes were lower in the B4C- mediated BNCT group than in the neutron group. Conclusion: These results indicate that B4C particles can be used locally as a boron compound in BNCT for oral SCC.

Overexpression of LIM and SH3 Protein 1 Leading to Accelerated G2/M Phase Transition Contributes to Enhanced Tumourigenesis in Oral Cancer

BackgroundLIM and SH3 protein 1 (LASP-1) is a specific focal adhesion protein involved in several malignant tumors. However, its role in oral squamous cell carcinoma (OSCC) is unknown. The aim of this study was to characterize the role and molecular status/mechanism of LASP-1 in OSCC.MethodsWe evaluated LASP-1 mRNA and protein expressions in OSCC-derived cell lines and primary OSCCs. Using an shRNA system, we analyzed the effect of LASP-1 on the biology and function of the OSCC cell lines, HSC-3 and Ca9-22. The cells also were subcutaneously injected to evaluate tumor growth in vivo. Data were analyzed by the Fisher’s exact test or the Mann-Whitney U test. Bonferroni correction was used for multiple testing.ResultsSignificant up-regulation of LASP-1 was detected in OSCC-derived cell lines (n = 7, P<0.007) and primary OSCCs (n = 50, P<0.001) compared to normal controls. LASP-1 knockdown cells significantly inhibited cellular proliferation compared with shMock-transfected cells (P<0.025) by arresting cell-cycle progression at the G2 phase. We observed dramatic reduction in the growth of shLASP-1 OSCC xenografts compared with shMock xenografts in vivo.ConclusionOur results suggested that overexpression of LASP-1 is linked closely to oral tumourigenicity and further provide novel evidence that LASP-1 plays an essential role in tumor cellular growth by mediating G2/M transition.

Combined zoledronic acid and meloxicam reduced bone loss and tumour growth in an orthotopic mouse model of bone‐invasive oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) is common in cats and humans and invades oral bone. We hypothesized that the cyclooxygenase (COX)-2 inhibitor, meloxicam, with the bisphosphonate, zoledronic acid (ZOL), would inhibit tumour growth, osteolysis and invasion in feline OSCC xenografts in mice. Human and feline OSCC cell lines expressed COX-1 and COX-2 and the SCCF2 cells had increased COX-2 mRNA expression with bone conditioned medium. Luciferase-expressing feline SCCF2Luc cells were injected beneath the perimaxillary gingiva and mice were treated with 0.1 mg kg(-1) ZOL twice weekly, 0.3 mg kg(-1) meloxicam daily, combined ZOL and meloxicam, or vehicle. ZOL inhibited osteoclastic bone resorption at the tumour-bone interface. Meloxicam was more effective than ZOL at reducing xenograft growth but did not affect osteoclastic bone resorption. Although a synergistic effect of combined ZOL and meloxicam was not observed, combination therapy was well-tolerated and may be useful in the clinical management of bone-invasive feline OSCC.

PP062: Detection of peroxiredoxin 1 and hypoxia in a xenograft model of human oral cancer

Hypoxia is closely related to malignant progress, angiogenesis, invasion, metastasis and tolerance of treatment in malignant tumor. But the precise molecular mechanism of hypoxia is not clear in oral squamous cell carcinoma (OSCC). In this study, for the first time we investigated interrelationship between hypoxia and Prx1 during tumor growth in human OSCC xenograft model. Eighty tumors were divided into four groups according to a mean tumor diameter of 2, 5, 10, and 15 mm. 2 mm group was set as control. 5, 10, and 15 mm were experimental groups. Pimonidazole staining, immunohistochemistry, QRT-PCR, Western blot, ELISA were used to detect hypoxia, MVD, 8-OHdG, Prx1, HO-1 and NF-B genes. The results showed the extent of tumor hypoxia increased compared with control with tumor size increasing (p

Novel HSP90 Inhibitor NVP-AUY922 Enhances the Anti-tumor Effect of Temsirolimus Against Oral Squamous Cell Carcinoma

Heat shock protein 90 (HSP90) and mammalian target of rapamycin (mTOR) are involved in the molecular pathogenesis of advanced oral squamous cell carcinoma. HSP90 inhibitors are capable of effectively interfering with multiple signaling pathways, including the mTOR signaling pathway. However, the combined effects of HSP90 and mTOR inhibitors on oral squamous cell carcinoma are still unknown. In this study, we investigated the dual treatment of the novel HSP90 inhibitor NVP-AUY922 and temsirolimus against oral squamous cell carcinoma. The effect of the combination of NVP-AUY922 and temsirolimus on oral squamous cell carcinoma in vitro and in vivo was determined by MTS assay and mouse xenograft models. The effect of the combination on angiogenesis was determined by tube formation assay and angioreactor. The combination treatment of NVP-AUY922 and temsirolimus significantly inhibited the proliferation of SAS oral squamous cell carcinoma cells in vitro and suppressed the growth of oral squamous cell carcinoma xenografts in vivo. We have clearly shown that the combination treatment of NVP-AUY922 and temsirolimus inhibited vascular formation both in vitro and in vivo. Moreover, the combination treatment of NVP-AUY922 and temsirolimus prolonged the survival rate in mice xenografted with oral squamous cell carcinoma. Here, we showed the activity of a combination of mTOR and HSP90 inhibitors for the treatment of advanced oral squamous carcinoma.

In vitro and in vivoanti-tumor effect of metformin as a novel therapeutic agent in human oral squamous cell carcinoma

BackgroundMetformin, which is widely used as an antidiabetic agent, has recently been reported to reduce cancer risk and improve prognosis in certain malignancies. However, the specific mechanisms underlying the effect of metformin on the development and progression of several cancers including oral squamous cell carcinoma (OSCC) remain unclear. In the present study, we investigated the effects of metformin on OSCC cells in vitro and in vivo.MethodsOSCC cells treated with or without metformin were counted using a hemocytometer. The clonogenic ability of OSCC cells after metformin treatment was determined by colony formation assay. Cell cycle progression and apoptosis were assessed by flow cytometry, and the activation of related signaling pathways was examined by immunoblotting. The in vivo anti-tumor effect of metformin was examined using a xenograft mouse model. Immunohistochemistry and TUNEL staining were used to determine the expression of cyclin D1 and the presence of apoptotic cells in tumors from mice treated with or without metformin.ResultsMetformin inhibited proliferation in the OSCC cell lines CAL27, WSU-HN6 and SCC25 in a time- and dose-dependent manner, and significantly reduced the colony formation of OSCC cells in vitro. Metformin induced an apparent cell cycle arrest at the G0/G1 phase, which was accompanied by an obvious activation of the AMP kinase pathway and a strongly decreased activation of mammalian target of rapamycin and S6 kinase. Metformin treatment led to a remarkable decrease of cyclin D1, cyclin-dependent kinase (CDK) 4 and CDK6 protein levels and phosphorylation of retinoblastoma protein, but did not affect p21 or p27 protein expression in OSCC cells. In addition, metformin induced apoptosis in OSCC cells, significantly down-regulating the anti-apoptotic proteins Bcl-2 and Bcl-xL and up-regulating the pro-apoptotic protein Bax. Metformin also markedly reduced the expression of cyclin D1 and increased the numbers of apoptotic cells in vivo, thus inhibiting the growth of OSCC xenografts.ConclusionsOur data suggested that metformin could be a potential candidate for the development of new treatment strategies for human OSCC.

Enhanced antitumor effect of cisplatin in human oral squamous cell carcinoma cells by tumor suppressor GRIM‑19.

Gene associated with retinoid‑interferon‑induced mortality19 (GRIM‑19) is a novel candidate tumor suppressor gene located on the human chromosome 19p13.1 region. Our previous study demonstrated that the upregulation of GRIM‑19 in human oral squamous cell carcinoma (OSCC) cells significantly inhibited tumor cell growth invitro and invivo. In the present study, the combined effects of cationic liposome (LP)‑mediated GRIM‑19 gene (LP‑pGRIM‑19) and the low‑dose chemotherapeutic drug, cisplatin (CDDP), on tumor cell growth invitro and invivo were examined, and the molecular mechanism of their mutual action was investigated by cell proliferation, colony formation, apoptosis, migration, invasion and western blotting assays invitro, and a node nude tumor model. It was demonstrated that cationic LP‑pGRIM‑19 gene therapy sensitized the response of breast cancer cells to CDDP, and that LP‑pGRIM‑19 in combination with CDDP significantly induced apoptosis and inhibited proliferation, colony formation, migration and invasion of the cells, compared with CDDP treatment alone. In addition, systemic treatment with a combination of intravenous injection of LP‑pGRIM‑19 and intraperitoneal injection of low‑dose CDDP into subcutaneous HSC3 human OSCC xenograft mice resulted in a significant inhibition of tumor growth (P<0.05). Further investigations indicated that the enhancement of CPPP‑mediated antitumor effects by GRIM‑19 may be associated with the upregulation of phosphorylated p53 and the downregulation of Bcell lymphoma‑2, cyclinD1, vascular endothelial growth factor, matrix metalloproteinase (MMP)‑2 and MMP‑9, the proteins of which are involved in the activation of signal transducer and activator of transcription3. The results of the present study suggested that the combination of GRIM‑19 gene therapy with low‑dose CPPP‑based chemotherapy may be a potent therapeutic strategy for the treatment of OSCC.

Anti-tumor effect of a novel FAK inhibitor TAE226 against human oral squamous cell carcinoma

Focal adhesion kinase (FAK) overexpression is frequently found in invasive and metastatic cancers, but its role in oral squamous cell carcinoma is not yet well understood. In order to seek therapies targeting oral squamous cell carcinoma, we developed the novel FAK Tyr(397) inhibitor TAE226 and investigated its anti-tumor effects and mechanisms. Expression of phosphorylated FAK Tyr(397) was examined by immunohistochemical and immunoblot analysis. The effect of TAE226 on in vitro and in vivo studies were confirmed by proliferation, cell cycle, apoptosis and angiogenesis analysis. We found that phosphorylated FAK was highly expressed in human tongue oral squamous cell carcinoma in patients. Importantly, TAE226 greatly suppressed the proliferation, migration and invasion of human oral squamous cell carcinoma SAS cells with an apparent structural change of actin fiber and a loss of cell adhesion. In addition, TAE226 inhibited the expression of phospho-FAK Tyr(397) and phospho AKT Ser(473), resulting in caspase-mediated apoptosis. Furthermore, oral administration of TAE226 in mice suppressed the growth and angiogenesis of oral squamous cell carcinoma xenografts in vivo. Our results provide compelling evidence that FAK is critically involved in oral squamous cell carcinoma and that the FAK inhibitor TAE226 can potentially be effectively used for the treatment of oral squamous cell carcinoma.