Synergistic antitumor activity of epidermal growth factor receptor tyrosine kinase inhibitor gefitinib and IFN-alpha in head and neck cancer cells in vitro and in vivo.

PURPOSE: Epidermal growth factor receptor (EGFR) overexpression has been implicated in the development of head and neck squamous cell carcinomas (HNSCC) and represents a potential therapeutic target for this disease. We have reported previously that growth inhibitory concentrations of IFN-alpha enhance the expression and activity of EGFR and that this effect could represent an escape mechanism to the growth inhibition and apoptotic cell death induced by IFN-alpha. In this study, we investigate whether the combination of IFN-alpha and gefitinib (Iressa, AstraZeneca Pharmaceuticals, Macclesfield, United Kingdom), a selective EGFR tyrosine kinase inhibitor, might have a cooperative antitumor effect on HNSCC-derived cell lines. EXPERIMENTAL DESIGN: The interaction of IFN-alpha and gefitinib was evaluated in vitro on HNSCC-derived cell lines by median drug effect analysis calculating a combination index with CalcuSyn software and in vivo by using HNSCC xenografts in nude mice. The mechanism of gefitinib and IFN-alpha interactions was also studied by analysis of cell cycle kinetics, apoptosis assays, and Western blotting of EGFR signal transduction components. RESULTS: Simultaneous exposure to gefitinib and IFN-alpha produced synergistic antiproliferative and proapoptotic effects compared with single drug treatment. Furthermore, daily treatment of gefitinib (50 mg/kg p.o.) in combination with an IFN-alpha regimen (50,000 units s.c. three times weekly) induced tumor growth delay and increased survival rate on established HNSCC xenografts in nude mice. Moreover, the concomitant treatment with gefitinib suppressed the stimulation of extracellular signal-regulated kinase phosphorylation/activity induced by IFN-alpha both in vitro and in vivo. CONCLUSION: The observed cooperative antitumor effects could be, at least in part, explained by the inhibition exerted by gefitinib of an IFN-alpha-induced EGF-dependent survival pathway, which involves extracellular signal-regulated kinase activation. These results provide a rationale for the clinical evaluation of gefitinib in combination with IFN-alpha in HNSCC.     

Growth stimulatory effect of interleukin (IL)-1 alpha produced by oral squamous carcinoma cell lines

The expression of IL-1 alpha and its effect on the cell growth were examined in six human oral squamous carcinoma cell lines. All the cell lines expressed IL-1 alpha mRNA and protein at various levels. Particularly, HSC-2 and HSC-3 cells showed high level of the mRNA expression and secreted large amounts of IL-1 alpha into the culture fluid. Scatchard plot analysis of IL-1 alpha binding revealed that HSC-2 cells had high-and low-affinity receptors, whereas IL-1 alpha receptors on HSC-3 cells were of undetectable level. The cell growth of HSC-2 and HSC-3 cells was stimulated by IL-1 alpha and inhibited by anti-IL-1 alpha antibody or IL-1 receptor antagonist. The expression of IL-1 alpha mRNA by these cell lines was induced by either IL-1 alpha, epidermal growth factor (EGF) or transforming growth factor alpha (TGF-alpha). On the other hand, IL-1 alpha promoted the mRNA expression of TGF-alpha and EGF receptor. These findings indicate that IL-1 alpha acts as an autocrine growth stimulator for oral squamous carcinoma cells in vitro and its interaction with EGF/TGF-alpha/receptor system may play a role in this enhanced growth by IL-1 alpha.     

Combined inhibition of c-Src and epidermal growth factor receptor abrogates growth and invasion of head and neck squamous cell carcinoma

PURPOSE: Increased expression and/or activation of epidermal growth factor receptor (EGFR) is associated with tumor progression and poor prognosis in many cancers, including head and neck squamous cell carcinoma (HNSCC). Src family kinases, including c-Src, mediate a variety of intracellular or extracellular signals that contribute to tumor formation and progression. This study was undertaken to elucidate the role of c-Src in the growth and invasion of HNSCC and to determine the effects of combined targeting of EGFR and Src kinases in HNSCC cell lines. EXPERIMENTAL DESIGN: HNSCC cells were engineered to stably express a dominant-active form of c-Src and investigated in cell growth and invasion assays. The biochemical effects of combined treatment with the Src inhibitor AZD0530, a potent, orally active Src inhibitor with Bcr/Abl activity, and the EGFR kinase inhibitor gefitinib were examined, as well as the consequences of dual Src/EGFR targeting on the growth and invasion of a panel of HNSCC cell lines. RESULTS: HNSCC cells expressing dominant-active c-Src showed increased growth and invasion compared with vector-transfected controls. Combined treatment with AZD0530 and gefitinib resulted in greater inhibition of HNSCC cell growth and invasion compared with either agent alone. CONCLUSIONS: These results suggest that increased expression and activation of c-Src promotes HNSCC progression where combined targeting of EGFR and c-Src may be an efficacious treatment approach.     

Honokiol inhibits the growth of head and neck squamous cell carcinoma by targeting epidermal growth factor receptor

Here, we report the chemotherapeutic effect of honokiol, a phytochemical from Magnolia plant, on human head and neck squamous cell carcinoma (HNSCC). Treatment of HNSCC cell lines from different sub-sites, SCC-1 (oral cavity), SCC-5 (larynx), OSC-19 (tongue) and FaDu (pharynx) with honokiol inhibited their cell viability, which was associated with the: (i) induction of apoptosis, (ii) correction of dysregulatory cell cycle proteins of G0/G1 phase. Honokiol decreased the expression levels of epidermal growth factor receptor (EGFR), mTOR and their downstream signaling molecules. Treatment of FaDu and SCC-1 cell lines with rapamycin, an inhibitor of mTOR pathway, also reduced cell viability of HNSCC cells. Administration of honokiol by oral gavage (100 mg/kg body weight) significantly (P < 0.01-0.001) inhibited the growth of SCC-1 and FaDu xenografts in athymic nude mice, which was associated with: (i) inhibition of tumor cell proliferation, (ii) induction of apoptosis, (iii) reduced expressions of cyclins and Cdks, and (iv) inhibition of EGFR signaling pathway. Molecular docking analysis of honokiol in EGFR binding site indicated that the chemotherapeutic effect of honokiol against HNSCC is mediated through its firm binding with EGFR, which is better than that of gefitinib, a commonly used drug for HNSCC treatment.     

Antitumor effects of IDN5109 on head and neck squamous cell carcinoma

Taxanes, a new class of antitumor drugs, are effective against a large number of human tumors, although there are problems with drug resistance. The novel taxane, IDN5109, is characterized by its high tolerability, antitumor efficacy, ability to overcome multidrug resistance, and oral bioavailabilty. We investigated the cellular response of IDN5109 to head and neck squamous cell carcinoma (HNSCC), and compared the antitumor activity of IDN5109 with that of paclitaxel. This is the first demonstration of antitumor effects of IDN5109 on HNSCC. In in vitro experiments, IDN5109 showed antiproliferative effects against HNSCC cell lines. After treatment with IDN5109, Bcl-2 and Bcl-XL were down-regulated, Bax was up-regulated, and caspase-3 was activated. After treatment with IDN5109, concentrations of both VEGF and IL-8 in the culture supernatant of HNSCC cells decreased. In in vivo experiments, the oral administration of IDN5109 showed antitumor effects against HNSCC tumor xenografts. Immunohistochemistry showed that IDN5109 inhibited tumor angiogenesis and induced apoptosis in HNSCC cells, producing a decreased blood vessel density and increased apoptosis index. On the basis of these results, IDN5109 is useful as a chemotherapeutic agent against HNSCC.     

单克隆抗体CH12抑制头颈部鳞状细胞癌裸鼠种植瘤的生长

目的:观察嵌合型单克隆抗体CH12对头颈部鳞状细胞癌(head and neck squamous cell carcinomas,HNSCC)裸鼠种植瘤生长的抑制作用,为进一步研究CH12单抗在肿瘤治疗中的作用提供参考数据。方法:Western blotting检测5种HNSCC细胞系A253、CAL27、Detroit 562、FaDu和RPMI 2650中表皮生长因子受体(epidermal growth factor receptor,EGFR)的表达,流式细胞术检测CH12单抗同这5种细胞系的结合能力。皮下接种CAL27和A253细胞,建立HNSCC裸鼠种植瘤模型。模型鼠腹腔注射CH12单抗,以PBS作为阴性对照,观察肿瘤生长情况,绘制肿瘤生长曲线。结果:EGFR在CAL27、A253、FaDu及Detroit 562细胞中均有不同程度的表达,其中CAL27细胞中EGFR的表达水平最高,A253细胞次之。CH12单抗与5种HNSCC细胞系的结合能力由高到低依次为CAL27、FaDu、A253、Detroit 562和RPMI 265细胞。CH12单抗对CAL27和A253细胞裸鼠种植瘤的生长均有显著抑制作用,抑瘤率分别为56.8%(P=0.022)和59.7%(P=0.015)。结论:单克隆抗体CH12对EGFR高表达的HNSCC细胞种植瘤的生长具有明显的抑制作用。     

EphB4 provides survival advantage to squamous cell carcinoma of the head and neck

The receptor tyrosine kinase EphB4 and its ligand EphrinB2 play critical roles in blood vessel maturation, and are frequently overexpressed in a wide variety of cancers. We studied the aberrant expression and biological role of EphB4 in head and neck squamous cell carcinoma (HNSCC). We tested the effect of EphB4-specific siRNA and antisense oligonucleotides (AS-ODN) on cell growth, migration and invasion, and the effect of EphB4 AS-ODN on tumor growth in vivo. All HNSCC tumor samples express EphB4 and levels of expression correlate directly with higher stage and lymph node metastasis. Six of 7 (86%) HNSCC cell lines express EphB4, which is induced either by EGFR activation or by EPHB4 gene amplification. EphrinB2 was expressed in 65% tumors and 5 of 7 (71%) cell lines. EphB4 provides survival advantage to tumor cells in that EphB4 siRNA and AS-ODN significantly inhibit tumor cell viability, induce apoptosis, activate caspase-8, and sensitize cells to TRAIL-induced cell death. Furthermore, EphB4-specific AS-ODN significantly inhibits the growth of HNSCC tumor xenografts in vivo. Expression of EphB4 in HNSCC tumor cells confers survival and invasive properties, and thereby provides a strong rationale for targeting EphB4 as novel therapy for HNSCC.     

Type I collagen degradation by invasive oral squamous cell carcinoma

Expression of extracellular matrix-degrading proteases is required for tumor cell invasion. In the present study we examined the production of type I collagen-degrading matrix metalloproteinases (MMPs) in the invasive oral squamous cell carcinoma-derived cell line HSC-3. In the absence of serum or exogenous growth factors, HSC-3 cells displayed no collagen degradation activity. Addition of serum slightly increased collagen proteolysis. However, addition of epidermal growth factor (EGF) resulted in nearly complete degradation of the collagen matrix. Zymography showed that MMP-2 and -9 are secreted by HSC-3 cells. EGF stimulated secretion of an additional gelatinase with a molecular weight similar to that of MMP-1. Immunoblotting of conditioned medium confirmed that EGF and, to a lesser degree type I collagen, increased production of MMP-1. Finally, in situ hybridization revealed intense expression of MMP-1 in oral squamous cell carcinoma specimens. Together, these results indicate that MMP-1 is expressed, induced by EGF, and required for type I collagen degradation in oral squamous cell carcinoma.     

Combined inhibition of PLC{gamma}-1 and c-Src abrogates epidermal growth factor receptor-mediated head and neck squamous cell carcinoma invasion

PURPOSE: Mortality from head and neck squamous cell carcinoma (HNSCC) is usually associated with locoregional invasion of the tumor into vital organs, including the airway. Understanding the signaling mechanisms that abrogate HNSCC invasion may reveal novel therapeutic targets for intervention. The purpose of this study was to investigate the efficacy of combined inhibition of c-Src and PLCgamma-1 in the abrogation of HNSCC invasion. EXPERIMENTAL DESIGN: PLCgamma-1 and c-Src inhibition was achieved by a combination of small molecule inhibitors and dominant negative approaches. The effect of inhibition of PLCgamma-1 and c-Src on invasion of HNSCC cells was assessed in an in vitro Matrigel-coated transwell invasion assay. In addition, the immunoprecipitation reactions and in silico database mining was used to examine the interactions between PLCgamma-1 and c-Src. RESULTS: Here, we show that inhibition of PLCgamma-1 or c-Src with the PLC inhibitor U73122 or the Src family inhibitor AZD0530 or using dominant-negative constructs attenuated epidermal growth factor (EGF)-stimulated HNSCC invasion. Furthermore, EGF stimulation increased the association between PLCgamma-1 and c-Src in HNSCC cells. Combined inhibition of PLCgamma-1 and c-Src resulted in further attenuation of HNSCC cell invasion in vitro. CONCLUSIONS: These cumulative results suggest that PLCgamma-1 and c-Src activation contribute to HNSCC invasion downstream of EGF receptor and that targeting these pathways may be a novel strategy to prevent tumor invasion in HNSCC.     

Role of GRB2‐associated binder 1 in epidermal growth factor receptor‐induced signaling in head and neck squamous cell carcinoma

The epidermal growth factor receptor (EGFR) plays an important role in the pathogenesis of head and neck squamous cell carcinoma (HNSCC). Despite the high expression of EGFR in HNSCC, EGFR inhibitors have only limited success as monotherapy. The Grb2‐associated binder (GAB) family of adaptor proteins acts as docking/scaffolding molecules downstream of tyrosine kinase receptors. We hypothesized that GAB1 may amplify EGFR‐induced signaling in HNSCCs and therefore could play a role in the reduced sensitivity of HNSCC to EGFR inhibitors. We used representative human HNSCC cell lines overexpressing wild type EGFR, and expressing GAB1 but not GAB2. We demonstrated that baseline Akt and MAPK signaling were reduced in HNSCC cells in which GAB1 expression was reduced. Furthermore, the maximal EGF‐induced activation of the Akt and MAPK pathway was reduced and delayed, and the duration of the EGF‐induced activation of these pathways was reduced in cells with GAB1 knock‐down. In agreement with this, HNSCC cells in which GAB1 levels were reduced showed an increased sensitivity to the EGFR inhibitor gefitinib. Our work demonstrates that GAB1 plays an important role as part of the mechanism of by which EGFR induces induced activation of the MAPK and AKT pathway. Our results identify GAB1 as an amplifier of the EGFR‐initiated signaling, which may also interfere with EGFR degradation. These findings support the emerging notion that reducing GAB1 function may sensitize HNSCC to EGFR inhibitors, hence representing a new therapeutic target HNSCC treatment in combination with EGFR targeting agents.     

Epidermal growth factor-induced cyclooxygenase-2 enhances head and neck squamous cell carcinoma metastasis through fibronectin up-regulation

Epidermal growth factor receptor (EGFR) activation is a major cause of metastasis in many cancers, such as head and neck squamous cell carcinoma (HNSCC). However, whether the induction of cyclooxygenase-2 (COX-2) mediates EGF-enhanced HNSCC metastasis remains unclear. Interestingly, we found that EGF induced COX-2 expression mainly in HNSCC. The tumor cell transformation induced by EGF was repressed by COX-2 knockdown, and this repression was reversed by simultaneously treating the cells with EGF and prostaglandin E₂ (PGE₂). The down-regulation of COX-2 expression or inhibition of COX-2 activity significantly blocked EGF enhancement of cell migration and invasion, but the addition of PGE₂ compensated for this inhibitory effect in COX-2-knockdown cells. COX-2 depletion inhibited EGF-induced matrix metalloproteinase (MMP)-1, MMP-2, MMP-3, MMP-9, and fibronectin expression and Rac1/cdc42 activation. The inhibitory effect of COX-2 depletion on MMPs and the fibronectin/Rac1/cdc42 axis were reversed by co-treatment with PGE₂. Furthermore, depletion of fibronectin impeded the COX-2-enhanced binding of HNSCC cells to endothelial cells and tumor cells metastatic seeding of the lungs. These results demonstrate that EGF-induced COX-2 expression enhances HNSCC metastasis via activation of the fibronectin signaling pathway. The inhibition of COX-2 expression and activation may be a potential strategy for the treatment of EGFR-mediated HNSCC metastasis.     

Epidermal growth factor‐induced COX‐2 regulates metastasis of head and neck squamous cell carcinoma through upregulation of angiopoietin‐like 4

Epidermal growth factor receptor (EGFR) expression and activation are the major causes of metastasis in cancers such as head and neck squamous cell carcinoma (HNSCC). However, the reciprocal effect of EGF‐induced COX‐2 and angiopoietin‐like 4 (ANGPTL4) on HNSCC metastasis remains unclear. In this study, we revealed that the expression of ANGPTL4 is essential for COX‐2‐derived prostaglandin E₂ (PGE₂)‐induced tumor cell metastasis. We showed that EGF‐induced ANGPTL4 expression was dramatically inhibited with the depletion and inactivation of COX‐2 by knockdown of COX‐2 and celecoxib treatment, respectively. Prostaglandin E₂ induced ANGPTL4 expression in a time‐ and dose‐dependent manners in various HNSCC cell lines through the ERK pathway. In addition, the depletion of ANGPTL4 and MMP1 significantly impeded the PGE₂‐induced transendothelial invasion ability of HNSCC cells and the binding of tumor cells to endothelial cells. The induction of molecules involved in the regulation of epithelial‐mesenchymal transition was also dependent on ANGPTL4 expression in PGE₂‐treated cells. The depletion of ANGPTL4 further blocked PGE₂‐primed tumor cell metastatic seeding of lungs. These results indicate that the EGF‐activated PGE₂/ANGPTL4 axis enhanced HNSCC metastasis. The concurrent expression of COX‐2 and ANGPTL4 in HNSCC tumor specimens provides insight into potential therapeutic targets for the treatment of EGFR‐associated HNSCC metastasis.     

shRNA干扰RACK1表达增强口腔鳞癌细胞放射敏感性的研究

目的 探讨下调RACK1基因表达对口腔鳞癌细胞生长及放射敏感性的影响。方法 构建RACK1基因的shRNA载体,通过脂质体转染技术将其转入口腔鳞癌HSC-3细胞,G418筛选得到稳定转染细胞。荧光定量RT-PCR和Western blot分别检测细胞中RACK1mRNA和RACK1蛋白表达水平;CCK8实验检测细胞生长能力;流式细胞仪检测细胞凋亡;细胞体外侵袭实验检测细胞侵袭能力;克隆形成实验检测下调RACK1表达联合X射线照射对细胞增殖能力的影响。构建裸鼠移植瘤模型,观察下调RACK1基因表达联合X射线照射对口腔鳞癌的生长抑制作用。结果 RT-PCR和Western blot结果显示转染后HSC-3细胞RACK1mRNA相对表达量明显降低(P<0.05),RACK1蛋白表达水平明显降低(P<0.05)。CCK8实验结果显示下调RACK1表达可抑制HSC-3细胞生长(P<0.05),联合X射线照射使细胞凋亡率明显升高(P<0.05),外侵袭穿透细胞数显著减少(P<0.05)。克隆形成实验结果显示shRACK1组的存活分数低于对照组,放射增敏比为1.37(D0值比)。裸鼠移植瘤实验显示shRACK1组照射后瘤体生长缓慢,瘤体体积减小幅度低于对照组(P<0.05),瘤体质量低于对照组(P<0.05)。结论 下调RACK1表达可以增强口腔鳞癌细胞的放射敏感性,为提高口腔鳞癌放射敏感性研究提供新思路。     

Abrogation of VEGF expression in human head and neck squamous cell carcinoma decreases angiogenic activity in vitro and in vivo

Angiogenesis is increased in various human cancers, including head and neck squamous cell carcinoma (HNSCC), and correlates with tumor progression and metastasis. Vascular endothelial growth factor (VEGF) has been shown to be a key regulator of angiogenesis. We determined whether VEGF antisense oligonucleotide treatment can decrease angiogenic activity of HNSCC cell lines in vitro and of HNSCC xenografts in vivo. Established human HNSCC cell lines were screened for VEGF expression at both mRNA and protein levels. By using a 21-mer antisense phosphorothioate oligonucleotide targeting the translation start site of human VEGF mRNA, we examined modulation of VEGF expression in cell line supernatants by capture ELISA, and in cell lysates by Western blotting. Human umbilica vein endothelial cells (HUVEC) were grown in conditioned medium produced from the treated tumor cells. Endothelial cell (EC) proliferation was determined by cell count and EC migration was measured using a modified Boyden chamber. Mice with HNSCC xenografts were treated with PBS, VEGF antisense or sense oligonucleotides (10 mg/kg; i.p. injection), respectively and tumor volumes were measured for 5 weeks. VEGF antisense oligonucleotide treatment resulted in a significant reduction of VEGF protein expression compared to sense control. Although the growth rate of the tumor cell lines was not affected, addition of conditioned medium from VEGF antisense-treated tumor cells resulted in decrease of endothelial cell proliferation and migration. VEGF antisense oligonucleotide treatment of HNSCC xenografts resulted in a significant tumor growth suppression. These results suggest that downmodulation of VEGF using antisense oligonucleotides may be a potential therapy for the inhibition of angiogenesis in HNSCC.     

Enhanced antiangiogenic therapy of squamous cell carcinoma by combined endostatin and epidermal growth factor receptor-antisense therapy.

PURPOSE: We tested the combined effects of antiangiogenic endostatin and epidermal growth factor receptor (EGFR) antisense gene therapy on squamous cell carcinoma (SCC). EXPERIMENTAL DESIGN and Results: The 1483 cell line of human head and neck SCC (HNSCC) and SCC-VII/SF murine SCC cells was used to establish tumors in nude mice and immunocompetent C3H mice, respectively. Tumor-bearing mice were treated with endostatin (20 mg/kg/day, s.c.), liposomal EGFR-antisense expression plasmid (25 microg/mouse, three times/week, intratumoral), a combination of both agents, or liposomal EGFR-sense plasmid as a control. Endostatin or EGFR-antisense alone significantly, yet partially, inhibited the growth of 1483 and SCC-VII/SF tumors, and a combination of both treatments completely blocked tumor growth. Immunohistochemistry analysis demonstrated that a complete suppression of tumor angiogenesis was achieved by the combination treatment. Down-regulation of vascular endothelial growth factor was shown in EGFR-antisense-treated tumors. These results suggest that the EGFR-antisense treatment, in addition to its inhibitory activity on tumor cell proliferation, might have a synergistic effect with endostatin on SCC-induced angiogenesis. In vitro studies demonstrated that EGFR inhibition by antisense oligonucleotides or EGFR-specific tyrosine kinase inhibitor down-regulated the production of VEGF in HNSCC cells. Additional experiments demonstrated that these EGFR inhibition approaches also directly suppressed the growth of endothelial cells. CONCLUSION: A combination of endostatin and EGFR targeting strategies profoundly inhibited the angiogenesis and growth of SCC in vivo. EGFR-antisense therapy might have multiple inhibitory effects against both tumor cells and endothelial cells, leading to enhanced antitumor efficacy. Such a combination strategy might represent a novel and promising approach for HNSCC therapy.     

In vivo and in situ imaging of head and neck squamous cell carcinoma using near-infrared fluorescent quantum dot probes conjugated with epidermal growth factor receptor monoclonal antibodies in mice

In this study, we applied near-infrared fluorescent quantum dots (NIRF-QDs) for non-invasive in vivo and in situ imaging of head and neck squamous cell carcinoma (HNSCC). The U14 squamous cancer cell line with high expression of epidermal growth factor receptor (EGFR) was implanted subcutaneously into the head and neck regions of nude mice to establish HNSCC models. NIRF-QDs with an emission wavelength of 800 nm (NIRF-QD800) were conjugated with EGFR monoclonal antibodies to develop the QD800-EGFR Ab probe. In vivo and in vitro studies demonstrated that the QD800-EGFR Ab probe can specifically bind EGFR expressed on U14 cells. U14 squamous cell carcinoma in the head and neck can be clearly visualized by in vivo imaging after intravenous injection of QD800-EGFR Ab probes. The results suggested that in situ imaging using NIRF-QD-EGFR Ab probes has unique advantages and prospects for the investigation of tumor development, early diagnosis and personalized therapy of HNSCC.