Metastatic Oral Cancer Cell Research Articles

Isolation and phenotypic characterization of cancer stem cells from metastatic oral cancer cells.

To isolate cancer stem cells (CSC) from a metastatic oral squamous cell carcinoma (OSCC) cell line and investigate their invitro and invivo phenotypic characteristics. Subpopulations with individual staining intensities for CD44 and CD326 were isolated from the OSCC cell line LN-1A by FACS: CD44Low/CD326- (CSC-M1), CD44Low/CD326High (CSC-E), and CD44High/CD326- (CSC-M2). Proliferation, clonogenic potential, adhesion, migration, epithelial-mesenchymal transition markers, and sensitivity to cisplatin and TVB-3166 were analyzed invitro. Tumor formation and metastasis were assessed by subcutaneous and orthotopic inoculations into BALB/c mice. E-cadherin levels were higher in CSC-E cells while vimentin and Slug more produced by CSC-M2 cells. CSC-M1 and CSC-M2 subpopulations showed higher proliferation, produced more colonies, and have stronger adhesion to the extracellular matrix. All cell lines established tumors; however, CSC-E and CSC-M2 formed larger masses and produced more metastases. The CSC subpopulations here described show increased cancer capabilities invitro, tumorigenic and metastatic potential invivo, and may be exploited in the search for novel therapeutic targets for OSCC.

Triple knockdown of CDC37, HSP90-alpha and HSP90-beta diminishes extracellular vesicles-driven malignancy events and macrophage M2 polarization in oral cancer

ABSTRACT Evidence has been accumulating to indicate that extracellular vesicles (EVs), including exosomes, released by cancer cells can foster tumour progression. The molecular chaperones – CDC37, HSP90α and HSP90β play key roles in cancer progression including epithelial-mesenchymal transition (EMT), although their contribution to EVs-mediated cell–cell communication in tumour microenvironment has not been thoroughly examined. Here we show that triple depletion of the chaperone trio attenuates numerous cancer malignancy events exerted through EV release. Metastatic oral cancer-derived EVs (MEV) were enriched with HSP90α HSP90β and cancer-initiating cell marker CD326/EpCAM. Depletion of these chaperones individually induced compensatory increases in the other chaperones, whereas triple siRNA targeting of these molecules markedly diminished the levels of the chaperone trio and attenuated EMT. MEV were potent agents in initiating EMT in normal epithelial cells, a process that was attenuated by the triple chaperone depletion. The migration, invasion, and in vitro tumour initiation of oral cancer cells were significantly promoted by MEV, while triple depletion of CDC37/HSP90α/β reversed these MEV-driven malignancy events. In metastatic oral cancer patient-derived tumours, HSP90β was significantly accumulated in infiltrating tumour-associated macrophages (TAM) as compared to lower grade oral cancer cases. HSP90-enriched MEV-induced TAM polarization to an M2 phenotype, a transition known to support cancer progression, whereas the triple chaperone depletion attenuated this effect. Mechanistically, the triple chaperone depletion in metastatic oral cancer cells effectively reduced MEV transmission into macrophages. Hence, siRNA-mediated knockdown of the chaperone trio (CDC37/HSP90α/HSP90β) could potentially be a novel therapeutic strategy to attenuate several EV-driven malignancy events in the tumour microenvironment. Abbreviations CDC37: cell division control 37; EMT: epithelial-mesenchymal transmission; EV: extracellular vesicles; HNSCC: head and neck squamous cell carcinoma; HSP90: heat shock protein 90; TAM: tumour-associated macrophage

HSP‐enriched properties of extracellular vesicles involve survival of metastatic oral cancer cells

Cancer cells often secrete extracellular vesicles (EVs) that carry heat shock proteins (HSPs) with roles in tumor progression. Oral squamous cell carcinoma (OSCC) belongs to head and neck cancers (HNC) whose lymph-node-metastases often lead to poor prognosis. We have examined the EV proteome of OSCC cells and found abundant secretion of HSP90-enriched EVs in lymph-node-metastatic OSCC cells. Double knockdown of HSP90α and HSP90β, using small interfering RNA significantly reduced the survival of the metastatic OSCC cells, although single knockdown of each HSP90 was ineffective. Elevated expression of these HSP90 family members was found to correlate with poor prognosis of HNC cases. Thus, elevated HSP90 levels in secreted vesicles are potential prognostic biomarkers and therapeutic targets in metastatic OSCC.

Abstract 2091: Metastatic oral cancer cell adhesion to vascular and lymphatic endothelial cells is sialyl Lewis A/X-dependent

Abstract Background: The vast majority of mortalities from cancer, including oral cancer, are due to metastatic disease. Previous studies have shown that sialylated cell surface glycoconjugates mediate the extravasation of circulating tumor cells from several different tumor types. The aim of this study was to investigate whether metastatic oral squamous cell carcinoma (OSCC) cells overexpress and employ sialyl Lewis A/X (sLeA/X) to adhere to E-selectin on endothelial cells. Materials and methods: Twenty-one matched-pairs of primary and nodal metastatic OSCC tissue sections were evaluated immunohistochemically using anti-sLeA (clone KM231) and anti-sLeX (clone KM93) monoclonal antibodies (mAbs). q-PCR was performed to measure relative transcript expression of a panel of glycogens potentially involved in sLeA/X synthesis in OSCC cells. sLeA/X cell surface expression was assessed by flow cytometry and fluorescence immunocytochemistry. sLeA/X involvement in OSCC cell adhesion behavior was evaluated using static and flow adhesion assays employing recombinant E-selectin (rE-selectin) and TNF-α-stimulated human dermal microvascular endothelial cells (HuDMEC) or human lymphatic endothelial cells (HLEC), respectively. Several approaches to prevent OSCC cell binding to rE-selectin or HuDMEC were tested including the use of anti-sLeA/X mAbs, neuraminidase, fucosidase, IELLQAR®, chondroitin sulfate, swainsonine and FUT3 siRNA. Results: Immunohistochemistry analysis showed sLeA/X to be mainly expressed by well- to moderately-differentiated OSCC. sLeA/X expression on primary OSCC correlates with lymph node metastasis but not tumor differentiation status. The metastatic OSCC cells, TR146, displayed higher sLeA/X expression than the non-metastatic OSCC cells, SCC4 and Cal27, and this was at least partially related to up-regulated gene expression of fucosyltransferase III. Knocking down FUT3 expression in TR146 cells reduced sLeX but not sLeA levels. TR146 cells adhered to rE-selectin and TNF-α-stimulated HuDMEC/HLEC in significantly greater numbers than SCC4 cells. Attachment of TR146 cells to rE-selectin was significantly reduced following incubation with anti-sLeA/X mAbs, neuraminidase and FUT3 siRNA. Treatment of TR146 cells with neuraminidase also caused a significant decrease in cell adhesion to HuDMEC under hydrodynamic flow conditions. Conclusions: Elevated levels of sLeX in metastatic OSCC cells results from increased FUT3 expression. Metastatic OSCC cell binding to HuDMEC/HLEC is sLeA/X-mediated and may be a target for anti-metastasis therapy. Citation Format: Ahmed Alkishi, Syed Ali Khurram, Martin Thornhill, Craig Murdoch. Metastatic oral cancer cell adhesion to vascular and lymphatic endothelial cells is sialyl Lewis A/X-dependent. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2091. doi:10.1158/1538-7445.AM2014-2091

Role of Activation-Induced Cytidine Deaminase in the Development of Oral Squamous Cell Carcinoma

PurposeIn humans, activation-induced cytidine deaminase (AID) expression results due to inflammation and this deaminase activity is also involved in carcinogenesis. The aim of this study is to investigate the correlation between AID expression and the clinical classification of oral cancer tissues.Experimental DesignThe current study investigated the correlation between AID expression and the clinical classification of oral cancer tissues from 27 patients who underwent surgical resection using immunohistochemistry. Specific AID expression and its induction by cytokine stimulation were investigated in cultured HSC oral cancer cell lines by reverse transcriptase PCR.ResultsAID expression was detected in 10 of 27 specimens (37.0%). AID expression was more frequently detected in early-stage cancer, especially in early stage T, than in late-stage cancer (T1/T2 vs. T3/4; P = 0.0493, N0 vs. N1/2/3; P = 0.0793). HSC-2, a nonmetastatic oral cancer cell line, abundantly expressed endogenous AID, whereas no such expression was observed in HSC-3, a metastatic oral cancer cell line. Moreover, AID expression was substantially induced in HSC-2 cells by stimulation of an inflammation-related cytokine, TNF-α.ConclusionsAberrant AID expression in the oral epithelium would contribute to the initiation of oral squamous cell carcinoma. Avoiding persistent AID inducible condition such as frequent cleaning of oral cavity would play an important role for the prevention of developing oral cancer.

The "One-Two Punch" of Isoprenoids to Inflammation

Copyright: © 2013 Mo H. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. A large body of evidence from recent studies has shown that tocotrienols, the vitamin E molecules with an unsaturated farnesyl side chain, have potential in the prevention and/or treatment of cancer, metabolic syndrome, obesity, diabetes, osteoporosis, and neurodegeneration [1]. These activities of tocotrienols may converge on the emerging finding that tocotrienols suppress the activation of nuclear factor kappa B (NFκB), a major mediator in chronic inflammation that is gaining recognition as one of the main mechanisms underlying these chronic diseases. Tocotrienol-mediated inhibition of NFκB DNA binding activity or suppression of tumor necrosis factor αand lipopolysaccharide-induced NFκB expression has been demonstrated in human breast cancer cells [2,3], colon carcinoma cells [4], malignant melanoma cells [5], pancreatic cancer cells [6], gastric cancer cells [7], metastatic oral cancer cells [8], adipocytes [9], and macrophages [10]. Moreover, the in vitro NFκB-suppressive activity of tocotrienols was borne out in gastric cancer [7] and pancreatic cancer [11] in nude mice. Readers are referred to recent reviews [12-14] for more details on the anti-inflammatory activity of tocotrienols.

Mechanism of sappanchalcone-induced growth inhibition and apoptosis in human oral cancer cells

Sappanchalcone, a flavonoid extracted from Caesalpinia sappan, exhibits cytoprotective activity, but the molecular basis for the anticancer effect of sappanchalcone has not been reported. In this study, we examined whether sappanchalcone could inhibit the growth of human primary and metastatic oral cancer cells, and we analyzed the signaling pathway underlying the apoptotic effects of the compound in this process using 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide (MTT) assays, fluorescence microscopy, flow cytometry, and Western blotting. Sappanchalcone-treated oral cancer cells showed an increased cytosolic level of cytochrome c, downregulated Bcl-2 expression, upregulated Bax and p53 expression, caspase-3 and -9 activation, and poly (ADP-ribose) polymerase cleavage. Furthermore, sappanchalcone induced activation of p38, extracellular signal-regulated kinase (ERK), c-Jun amino-terminal kinase (JNK), and Nuclear factor k B (NF-κB), as demonstrated by the phosphorylation of each mitogen-activated protein kinases (MAPKs), the degradation of inhibitor of NF-κα (IκB-α), increased expression of nuclear p65, and NF-κB-DNA binding. Inhibition of the expression of p38, ERK, JNK, and NF-κB by pharmacological inhibitors reversed sappanchalcone-induced growth inhibition and apoptosis. These results provide the first evidence that sappanchalcone suppresses oral cancer cell growth and induces apoptosis through the activation of p53-dependent mitochondrial, p38, ERK, JNK, and NF-κB signaling. Thus, it has potential as a chemotherapeutic agent for oral cancer.

Isoliquiritigenin 2′-methyl ether induces growth inhibition and apoptosis in oral cancer cells via heme oxygenase-1

We previously reported that a chloroform extract of Caesalpinia sappan L. induces apoptosis in oral cancer cells but not in normal epithelial cell lines. In the present study, we explored the effects of a single compound isolated from C. sappan heartwood, isoliquiritigenin 2′-methyl ether (ILME), on cultured primary and metastatic oral cancer cell lines using MTT assays, fluorescence microscopy, flow cytometry, and Western blotting. ILME inhibited the growth of the oral cancer cells in a time- and dose-dependent manner. The major mechanism of growth inhibition was apoptosis induction, as shown by flow cytometric analysis of sub-G 1-phase arrest and by annexin V-FITC and propidium iodide staining. ILME time-dependently activated NF-κB transcription factors, phospholated the MAP kinases JNK (c-Jun N-terminal kinase) and ERK (extracellular signal-regulated kinase). Furthermore, ILME treatment upregulated HO-1 expression though activation of Nrf2 (NF-E2-related factor 2) pathway, and induced the expression of heme oxygenase-1 (HO-1). Tin protoporphyrin, an HO-1 inhibitor, dose-dependently attenuated the growth-inhibitory effect of ILME and blocked ILME-induced expression of the p21 and p53 cell cycle-regulatory proteins. These results provide the first evidence that the anti-oral cancer effects of ILME may involve a mechanism in which HO-1 is upregulated via a pathway involving MAP kinases, NF-κB, and Nrf2. Thus, ILME could be considered to be a potential chemotherapeutic target for anti-oral cancer treatment strategies.

UCN-01 (7-hydroxystaurosporine) induces apoptosis and G1 arrest of both primary and metastatic oral cancer cell lines in vitro

Our aim was to clarify the in vitro antiproliferative effects of UCN-01 on human oral squamous cell carcinoma (OSCC) cell lines. Cell growth was measured by MTT assay, and cell cycling was assessed by flow cytometry. Changes in the levels of protein and protein phosphorylation were analyzed by Western blotting. In addition, tumor cell apoptosis was assessed by propidium iodide (PI) and annexin double-staining. UCN-01 significantly inhibited the proliferation of all the OSCC cell lines, with a 50% inhibition concentration of about 300 nmol/L, and induced G1 arrest in these cell lines in a dose-dependent manner. Primary and metastatic oral cancer cell lines had different sensitivities to UCN-01. Our results showed that HSC-3 cells (primary-type OSCC) are less sensitive than LMF4 cells (metastatic-type OSCC) to UCN-01. In addition, the induction of p21 in OSCCs was found to be important for the suppression of tumor growth. The results of this study suggest that UCN-01 induces apoptosis and G1 arrest in OSCCs, albeit with different sensitivity of the primary and metastatic cell lines to UCN-01.

Iron chelator‐induced growth arrest and cytochrome c‐dependent apoptosis in immortalized and malignant oral keratinocytes

Many studies have shown the anti-proliferative effects of iron deprivation on cancer cells, but the effects of iron-chelators on oral cancer have not been clearly elucidated. To investigate the effects of an iron chelator, desferrioxamine (DFO), on the growth of immortalized human oral keratinocytes (IHOK), primary oral cancer cells (HN4), metastatic oral cancer cells (HN12) and human skin keratinocytes (HaCaT) in the MTT assay, three-dimensional (3D) raft cultures, Western blotting, cell cycle analysis, nuclear staining and cytochrome c expression for apoptosis signaling pathway were used. Desferrioxamine inhibited the growth of immortalized IHOK and HaCaT and malignant HN4 and HN12 keratinocytes in a time- and dose-dependent manner according to the MTT assay. The 3D organotypic culture also revealed that DFO-treated cells showed less epithelial maturation, less surface keratinization and decreased epithelial thickness. The major mechanism of growth inhibition with the micromolar DFO treatment was by the induction of apoptosis, which was supported by nuclear DAPI staining, DNA fragmentation analysis and flow cytometric analysis for sub-G(1) phase arrest and Annexin V-FITC (fluorescein isothiocyanate) staining. Furthermore, Bax expression increased together with p53 and p21(WAF1/CIP1), while the Bcl-2 expression decreased in the immortalized and malignant keratinocytes treated with DFO. Time-dependent cytochrome c from mitochondria was observed in DFO-treated IHOK and oral cancer cells and was accompanied by the activation of caspase-3 in IHOK cells. These results demonstrate that DFO has growth inhibitory effects on immortalized and malignant oral keratinocytes through the induction of apoptosis and suggest that further evaluation of DFO as a potential therapeutic agent for human oral precancerous lesions is warranted.

Extract ofCoptidis rhizoma induces cytochrome-c dependent apoptosis in immortalized and malignant human oral keratinocytes

Coptidis rhizoma (C. rhizoma) had been demonstrated as an antioxidant and anticancer agent, however, its antioral cancer mechanism still remains unclear. Using water extracts of C. rhizoma, growth and apoptosis-related experiments for the treatment of multi-stage of oral cancer were carried out on immortalized human oral keratinocytes (IHOK), primary oral cancer cells (HN4), metastatic oral cancer cells (HN12) and human skin keratinocytes (HaCaT) by MTT assay, three-dimensional (3-D) raft cultures, western blotting, cell cycle analysis, nuclear staining and cytochrome c expression related to the apoptosis signaling pathway. C. rhizoma inhibited the proliferation of immortalized and malignant oral keratinocytes in a dose- and time-dependent manner. In 3-D organotypic culture, C. rhizoma-treated cells showed less maturation than the control cells, displaying low surface keratinization and decreased epithelial thickness. The major mechanism of growth inhibition by C. rhizoma appears to be the induction of apoptosis, which is supported by the results of the cell cycle analysis, FITC-annexin V staining, DNA fragmentation assay and DAPI staining. The induction of apoptosis by C. rhizoma was more prominent in immortalized keratinocytes than in malignant oral keratinocytes. Cytochrome-c release from mitochondria, accompanied by the activation of caspase-3, was observed in C. rhizoma-treated IHOK and oral cancer cells. These results suggest that C. rhizoma has apoptotic effects in immortalized and malignant oral keratinocytes via the mitochondrial signaling pathway.

Silencing of cystatin M in metastatic oral cancer cell line MDA-686Ln by siRNA increases cysteine proteinases and legumain activities, cell proliferation and in vitro invasion

Cystatins are inhibitors of lysosomal cysteine proteinases. Cystatin M demonstrates more diverse tissue distribution, target specificity and biological function than other cystatins from the same family. We utilized small interference RNAs (siRNA) to silence cystatin M gene expression in a metastatic oral cancer cell line (MDA-686Ln) that expresses a high level of cystatin M. We tested four different siRNAs targeted to different sites of the cystatin M mRNA, and found three out of the four siRNAs were effective in suppressing cystatin M expression by > 50% at both mRNA and protein levels, as measured by quantitative real-time RT-PCR and Western blotting. We used siRNA-#1, which demonstrated highest efficiency of silencing cystatin M, to evaluate the phenotypic outcome of silencing cystatin M in MDA-686Ln cells. Cystatin M inhibition significantly increased the enzymatic activities of cathepsins B and L and legumain while reducing cysteine protease inhibitor activity both in the media and intracellularly. MDA-686Ln cells treated with siRNA#1 demonstrated markedly increased proliferation rate, in vitro motility and Matrigel invasiveness. Collectively, our data show that silencing of cystatin M in tumor cells not only increases their invasion and motility via cysteine–proteinase-dependent pathways, but also renders them hyperproliferative through a currently unknown mechanism.

Effects of nicotine on proliferation, cell cycle, and differentiation in immortalized and malignant oral keratinocytes

Numerous epidemiological studies have reported that tobacco smoking is a major risk factor for oral cancer, but relatively little is known about the effect of nicotine, a major product of cigarette smoking, on immortalized oral keratinocytes and cancer cells. We investigated the effects of nicotine on the growth and differentiation of immortalized human oral keratinocytes (IHOK), primary oral cancer cells (HN4), metastatic oral cancer cells (HN12), and human skin keratinocytes (HaCaT), in the monolayer and in the three-dimensional (3D) raft cultures using the MTT assay, Western blotting, and cell cycle analysis. Nicotine inhibited the proliferation of immortalized and malignant keratinocytes in dose- and time-dependent manners as determined by MTT assay. The 3D organotypic culture showed that nicotine at high concentration (300 microM) inhibits epithelial maturation, surface keratinization, and decreased epithelial thickness. Flow cytometry showed that nicotine inhibited cell cycle progression by inducing G(0)/G(1) arrest of HaCaT, IHOK, HN4, and HN12 cells without causing apoptosis. Nicotine treatment increased p21 expression in immortalized cells (HaCaT, IHOK) and oral cancer cells (HN4, HN12), but decreased pRb and p53 expression in oral cancer cells. Moreover, after high-dose nicotine treatment, the involucrin expression increased markedly in immortalized cells, but not in oral cancer cells. We demonstrated that nicotine inhibits growth through cell cycle arrest at G(0)/G(1) phase probably by increasing the expression of p21(WAF1/CIP1). Nicotine also affects epithelial differentiation in immortalized and malignant oral keratinocytes. Malignant oral keratinocytes appear to be more resistant to the effects of nicotine on epithelial growth and differentiation as compared to the immortalized cells.

Differential susceptibility of metastatic and primary oral cancer cells to TRAIL-induced apoptosis

TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) preferentially induces apoptosis of cancer cells without toxicity in normal cells. TRAIL plays an important role in host immune surveillance against tumor metastasis. Cathepsin B (CB) is a mediator of apoptosis whose activity is regulated by its inhibitors, known as cystatins. We examined the TRAIL-mediated cytotoxicity rates of clonally-related primary and metastatic oral cancer (OC) cells and correlated them with the expression levels of TRAIL receptors, cathepsin B and cystatins A, B, C and M. Two pairs of primary (686Tu and 101A) and metastatic (686Ln and 101B) OC cell lines were treated with various concentrations (5 to 1000 ng/ml) of recombinant human TRAIL protein for 14 h, and cell viability and apoptotic rate were determined. In both pairs of cell lines, primary OC cells revealed greater susceptibility to TRAIL than their metastatic counterparts. The protein synthesis inhibitor cycloheximide markedly increased the TRAIL sensitivity of these cell lines, whereas the CB-specific chemical inhibitor CA-074 markedly reduced the sensitivity of primary OC cells to TRAIL. DNA laddering and M30 CytoDEATH immunodetection assays confirmed that TRAIL-induced OC cell death is an apoptotic process. Expression levels of TRAIL death (DR4 and DR5) and decoy (DcR1 and DcR2) receptors were not different between primary and metastatic OC cells. However, expression levels of cystatins were higher in metastatic OC cells than in their respective primary cells, whereas CB levels remain unchanged. Cathepsin B is a mediator of TRAIL-induced apoptosis in OC cells. Elevated levels of cystatins in metastatic OC cells may cause their greater resistance to TRAIL-induced apoptosis. Our data suggest that high expression of cystatins in OC cells may confer a metastatic phenotype by enhancing their resistance to TRAIL.

Elevated Cystatin expression in metastatic oral cancer cells confers greater resistance to TRAIL-induced apoptosis

Background TRAIL (tumor necrosis factor–related apoptosis-inducing ligand) preferentially induces apoptosis of cancer cells without toxicity in normal cells. TRAIL plays an important role in host immunosurveillance against tumor metastasis. Cathepsin B (CB) is a mediator of apoptosis whose activity is regulated by inhibitors known as cystatins (CY). Objective To relate the TRAIL sensitivity of clonally related primary and metastatic oral cancer (OC) cells with their CB and CY levels. Study design Two pairs of primary (686Tu and 101Tu) and metastatic (686Ln and 101Ln) OC cell lines were treated with various concentrations (10-1000 ng/mL) recombinant human TRAIL protein for 14 hours. Cell viability was quantified by MTT assay. Apoptosis rate among TRAIL-treated cells was measured using the TUNEL and M30 CytoDEATH immunodetection assays. CB and CY (A, B, C, and M) levels in these cells lines were analyzed by RT-PCR and Western blots. Results Primary cells revealed greater susceptibility to TRAIL-induced killing (ED 50 of 50 and 250 ng/mL for 686Tu and 101Tu, respectively) than their metastatic clones (ED 50 of 250 and 1000 ng/mL for 686Ln and 101Ln, respectively). Primary cells in the presence of CB-specific chemical inhibitor CA074 revealed markedly increased resistance to TRAIL (ED 50 > 1000 ng/mL). Expression levels of CY were markedly higher in metastatic OC cells than in their respective primary cells whereas CB levels remain unchanged. Conclusion CB is a mediator of TRAIL-induced apoptosis in OC cells. Elevated levels of cystatins in metastatic OC cells correlate with their greater resistance to TRAIL-induced apoptosis. Our data suggest that overexpression of CY in OC cells may confer a metastatic phenotype by enhancing their resistance to TRAIL.