Induction of the Endoplasmic Reticulum Stress and Autophagy in Human Lung Carcinoma A549 Cells by Anacardic Acid

Anacardic acid (AA, 2-hydroxy-6-pentadecylbenzoic acid), a constituent of the cashew-nut shell, has a variety of beneficial effects on the treatment of cancer and tumors. However, the fact that AA induces ER stress and autophagy in cancer cell is not known. We investigated the effect of AA on ER-stress and autophagy-induced cell death in cancer cells. Because of our interest in lung cancer, we used the non-small cell lung adenocarcinoma A549 cells treated with 3.0 μg/ml of AA for this research. In this research we found that AA induces intracellular Ca²⁺ mobilization and ER stress. AA induced the ER stress-inducing factors, especially IRE1α, and the hallmarks of UPR, Grp78/Bip and GADD153/CHOP. AA inhibited the expression of p-PERK and its downstream substrate, p-elF2α. We also demonstrated that AA induces autophagy. Up-regulation of autophagy-related genes and the appearance of autophagosome in transfected cells with green fluorescent protein (GFP)-LC3 and GFP-Beclin1 plasmids showed the induction of autophagy in AA-treated A549 cells. The morphological analysis of intracellular organelles by TEM also showed the evidence that AA induces ER stress and autophagy. For the first time, our research showed that AA induces ER stress and autophagy in cancer cells.     

Mitoferrin-2-dependent Mitochondrial Iron Uptake Sensitizes Human Head and Neck Squamous Carcinoma Cells to Photodynamic Therapy

Photodynamic therapy (PDT) is a promising approach to treat head and neck cancer cells. Here, we investigated whether mitochondrial iron uptake through mitoferrin-2 (Mfrn2) enhanced PDT-induced cell killing. Three human head and neck squamous carcinoma cell lines (UMSCC1, UMSCC14A, and UMSCC22A) were exposed to light and Pc 4, a mitochondria-targeted photosensitizer. The three cell lines responded differently: UMSCC1 and UMSCC14A cells were more resistant, whereas UMSCC22A cells were more sensitive to Pc 4-PDT-induced cell death. In non-erythroid cells, Mfrn2 is an iron transporter in the mitochondrial inner membrane. PDT-sensitive cells expressed higher Mfrn2 mRNA and protein levels compared with PDT-resistant cells. High Mfrn2-expressing cells showed higher rates of mitochondrial Fe²⁺ uptake compared with low Mfrn2-expressing cells. Bafilomycin, an inhibitor of the vacuolar proton pump of lysosomes and endosomes that causes lysosomal iron release to the cytosol, enhanced PDT-induced cell killing of both resistant and sensitive cells. Iron chelators and the inhibitor of the mitochondrial Ca²⁺ (and Fe²⁺) uniporter, Ru360, protected against PDT plus bafilomycin toxicity. Knockdown of Mfrn2 in UMSCC22A cells decreased the rate of mitochondrial Fe²⁺ uptake and delayed PDT plus bafilomycin-induced mitochondrial depolarization and cell killing. Taken together, the data suggest that lysosomal iron release and Mfrn2-dependent mitochondrial iron uptake act synergistically to induce PDT-mediated and iron-dependent mitochondrial dysfunction and subsequent cell killing. Furthermore, Mfrn2 represents a possible biomarker of sensitivity of head and neck cancers to cell killing after PDT.     

内质网膜复合亚基3调控内质网应激影响人畸胎瘤细胞的存活

内质网膜蛋白复合物(endoplasmic reticulum membrane complex,EMC)在跨膜蛋白质的生物发生和膜整合中发挥重要作用.内质网膜复合亚基3 (endoplasmic reticulum membrane complex 3,EMC3)是EMC的重要组成部分,但其在生殖细胞中发挥的作用未见...     

Deguelin Induces Both Apoptosis and Autophagy in Cultured Head and Neck Squamous Cell Carcinoma Cells

Head and neck squamous cell carcinoma (HNSCC) represents more than 5% of all cancers diagnosed annually in United States and around the world. Despite advances in the management of patients with this disease, the survival has not been significantly improved, and the search for potential alternative therapies is encouraging. Here we demonstrate that deguelin administration causes a significant HNSCC cell death. Deguelin induces both cell apoptosis and autophagy by modulating multiple signaling pathways in cultured HNSCC cells. Deguelin inhibits Akt signaling, and down-regulates survivin and cyclin-dependent kinase 4 (Cdk4) expressions, by disrupting their association with heat shock protein-90 (Hsp-90). Deguelin induces ceramide production through de novo synthase pathway to promote HNSCC cell death. Importantly, increased ceramide level activates AMP-activated protein kinase (AMPK), which then directly phosphorylates Ulk1 and eventually leads to cell autophagy. We found that a low dose of deguelin sensitized HNSCC cells to 5-FU. Finally, using a nude mice Hep-2 xenograft model, we also showed a significant anti-tumor ability of deguelin in vivo. Together, we suggest that deguelin may represent a novel and effective chemo-agent against HNSCC.     

The Natural Pesticide Dihydrorotenone Induces Human Plasma Cell Apoptosis by Triggering Endoplasmic Reticulum Stress and Activating p38 Signaling Pathway

Dihydrorotenone (DHR) is a natural pesticide widely used in farming industry, such as organic produces. DHR is a potent mitochondrial inhibitor and probably induces Parkinsonian syndrome, however, it is not known whether DHR is toxic to other systems. In the present study, we evaluated the cytotoxicity of DHR on human plasma cells. As predicted, DHR impaired mitochondrial function by decreasing mitochondrial membrane potential in plasma cells. Because mito-dysfunction leads to unfolded protein response (UPR) and endoplasmic reticulum (ER) stress, we examined the signature proteins in ER stress, including GRP78, ATF4, and CHOP. After DHR treatment, these proteins were significantly upregulated. It is reported that activation of the mitogen-activated protein kinases p38 and JNK are involved in endoplasmic reticulum stress. However, in the subsequent study, DHR was found to activate p38 but not the JNK signaling. When pre-treated with p38 inhibitor SB203580, activation of p38 and cell apoptosis induced by DHR was partially blocked. Thus, we found that DHR induced human plasma cell death by activating the p38 but not the JNK signaling pathway. Because plasma cells are very important in the immune system, this study provided a new insight in the safety evaluation of DHR application.     

Decreased Plakophilin-1 Expression Promotes Increased Motility in Head and Neck Squamous Cell Carcinoma Cells

Desmosomes are prominent cell-cell adhesive junctions found in a variety of epithelial tissues, including the oral epithelium. The transmembrane core of the desmosome is composed of the desmosomal cadherins that interact extracellularly to mediate cell-cell adhesion. The cytoplasmic domain of desmosomal cadherins interact with plaque proteins that in turn interact with the keratin intermediate filament cytoskeleton. Plakophilin 1 is a major desmosomal plaque component that functions to recruit intermediate filaments to sites of cell-cell contact via interactions with desmoplakin. Decreased assembly of desmosomes has been reported in several epithelial cancers. We examined plakophilin-1 expression in an esophageal squamous cell carcinoma tissue microarray and found that plakophilin-1 expression inversely correlates with tumor grade. In addition, we sought to investigate the effect of plakophilin-1 expression on desmosome assembly and cell motility in oral squamous cell carcinoma cell lines. Cell lines expressing altered levels of plakophilin-1 were generated and the ability of these cells to recruit desmoplakin to sites of cell-cell contact was examined. Our results show that decreased expression of plakophilin-1 results in decreased desmosome assembly and increased cell motility and invasion. These data lead us to propose that loss of plakophilin-1 expression during head and neck squamous cell carcinoma (HNSCC) progression may contribute to an invasive phenotype.     

SILAC-Based Mass Spectrometry Analysis Reveals That Epibrassinolide Induces Apoptosis via Activating Endoplasmic Reticulum Stress in Prostate Cancer Cells

Epibrassinolide (EBR) is a polyhydroxylated sterol derivative and biologically active compound of the brassinosteroids. In addition to well-described roles in plant growth, EBR induces apoptosis in the LNCaP prostate cancer cells expressing functional androgen receptor (AR). Therefore, it is suggested that EBR might have an inhibitory potential on androgen receptor signaling pathway. However, the mechanism by which EBR exerts its effects on LNCaP is poorly understood. To address this gap in knowledge, we used an unbiased global proteomics approach, i.e., stable-isotope labeling by amino acids in cell culture (SILAC). In total, 964 unique proteins were identified, 160 of which were differentially expressed after 12 h of EBR treatment. The quantification of the differentially expressed proteins revealed that the expression of the unfolded protein response (UPR) chaperone protein, calreticulin (CALR), was dramatically downregulated. The decrease in CALR expression was also validated by immunoblotting. Because our data revealed the involvement of the UPR in response to EBR exposure, we evaluated the expression of the other UPR proteins. We demonstrated that EBR treatment downregulated calnexin and upregulated BiP and IRE1α expression levels and induced CHOP translocation from the cytoplasm to nucleus. The translocation of CHOP was associated with caspase-9 and caspase-3 activation after a 12 h EBR treatment. Co-treatment of EBR with rapamycin, an upstream mTOR pathway inhibitor, prevented EBR-induced cell viability loss and PARP cleavage in LNCaP prostate cancer cells, suggesting that EBR could induce ER stress in these cells. In addition, we observed similar results in DU145 cells with nonfunctional androgen receptor. When proteasomal degradation of proteins was blocked by MG132 co-treatment, EBR treatment further induced PARP cleavage relative to drug treatment alone. EBR also induced Ca²⁺ sequestration, which confirmed the alteration of the ER pathway due to drug treatment. Therefore, we suggest that EBR promotes ER stress and induces apoptosis.     

The Small Molecule Inhibitor QLT0267 Radiosensitizes Squamous Cell Carcinoma Cells of the Head and Neck

Background

The constant increase of cancer cell resistance to radio- and chemotherapy hampers improvement of patient survival and requires novel targeting approaches. Integrin-Linked Kinase (ILK) has been postulated as potent druggable cancer target. On the basis of our previous findings clearly showing that ILK transduces antisurvival signals in cells exposed to ionizing radiation, this study evaluated the impact of the small molecule inhibitor QLT0267, reported as putative ILK inhibitor, on the cellular radiation survival response of human head and neck squamous cell carcinoma cells (hHNSCC).

Methodology/Principal Findings

Parental FaDu cells and FaDu cells stably transfected with a constitutively active ILK mutant (FaDu-IH) or empty vectors, UTSCC45 cells, ILK ^{floxed/floxed(fl/fl)} and ILK ^−/− mouse fibroblasts were used. Cells grew either two-dimensionally (2D) on or three-dimensionally (3D) in laminin-rich extracellular matrix. Cells were treated with QLT0267 alone or in combination with irradiation (X-rays, 0–6 Gy single dose). ILK knockdown was achieved by small interfering RNA transfection. ILK kinase activity, clonogenic survival, number of residual DNA double strand breaks (rDSB; γH2AX/53BP1 foci assay), cell cycle distribution, protein expression and phosphorylation (e.g. Akt, p44/42 mitogen-activated protein kinase (MAPK)) were measured. Data on ILK kinase activity and phosphorylation of Akt and p44/42 MAPK revealed a broad inhibitory spectrum of QLT0267 without specificity for ILK. QLT0267 significantly reduced basal cell survival and enhanced the radiosensitivity of FaDu and UTSCC45 cells in a time- and concentration-dependent manner. QLT0267 exerted differential, cell culture model-dependent effects with regard to radiogenic rDSB and accumulation of cells in the G2 cell cycle phase. Relative to corresponding controls, FaDu-IH and ILK ^fl/fl fibroblasts showed enhanced radiosensitivity, which failed to be antagonized by QLT0267. A knockdown of ILK revealed no change in clonogenic survival of the tested cell lines as compared to controls.

Conclusions/Significance

Our data clearly show that the small molecule inhibitor QLT0267 has potent cytotoxic and radiosensitizing capability in hHNSCC cells. However, QLT0267 is not specific for ILK. Further in vitro and in vivo studies are necessary to clarify the potential of QLT0267 as a targeted therapeutic in the clinic.     

DNA-Bound Platinum Is the Major Determinant of Cisplatin Sensitivity in Head and Neck Squamous Carcinoma Cells

Purpose

The combination of systemic cisplatin with local and regional radiotherapy as primary treatment of head and neck squamous cell carcinoma (HNSCC) leads to cure in approximately half of the patients. The addition of cisplatin has significant effects on outcome, but despite extensive research the mechanism underlying cisplatin response is still not well understood.

Methods

We examined 19 HNSCC cell lines with variable cisplatin sensitivity. We determined the TP53 mutational status of each cell line and investigated the expression levels of 11 potentially relevant genes by quantitative real-time PCR. In addition, we measured cisplatin accumulation and retention, as well as the level of platinum-DNA adducts.

Results

We found that the IC₅₀ value was significantly correlated with the platinum-DNA adduct levels that accumulated during four hours of cisplatin incubation (p = 0.002). We could not find a significant correlation between cisplatin sensitivity and any of the other parameters tested, including the expression levels of established cisplatin influx and efflux transporters. Furthermore, adduct accumulation did not correlate with mRNA expression of the investigated influx pumps (CTR1 and OCT3) nor with that of the examined DNA repair genes (ATR, ATM, BRCA1, BRCA2 and ERCC1).

Conclusion

Our findings suggest that the cisplatin-DNA adduct level is the most important determinant of cisplatin sensitivity in HNSCC cells. Imaging with radio-labeled cisplatin might have major associations with outcome.     

Head and Neck Squamous Cell Carcinoma: Prognosis using molecular approach

Head and neck squamous cell carcinoma (HNSCC) is the fifth most prevalent cancer worldwide. Apart from various known clinicopathogical factors, it is still a major concern as many genetic and epigenetic alterations bring about the possibility of this deadly disease. The aim of this review is to explore the possible role of DNA repair pathways and the polymorphic status of DNA repair genes (XPA, XPC, XPD, XRCC1 and XRCC3) in the onset of HNSCC, along with sequence variations in genes such as Glutathione S-transferases (GSTT1, M1 and P1) that are significantly associated with HNSCC risk. We also focus on the p53 gene mutation induced by various etiological agents and threat factors with its implications towards HNSCC, and emphasise the current therapeutic interventions in treating HNSCC.     

Challenges in EGFRvIII Detection in Head and Neck Squamous Cell Carcinoma

ObjectiveHead and neck squamous cell carcinoma (HNSCC) accounts for more than 5% of all cancers worldwide. The mortality rate of HNSCC has remained unchanged (approximately 50%) over the last few decades. Ubiquitous overexpression of wild type EGFR in many solid tumors has led to the development of EGFR targeted therapies. EGFR can be constitutively activated via several mechanisms including the truncated, EGFR variant III isoform (EGFRvIII). EGFRvIII lacks exons 2–7 and has been reported to be present in up to 20–40% of HNSCC. EGFRvIII has been shown to contribute to cetuximab resistance. The mechanisms leading to EGFRvIII expression in HNSCC are unknown. The present investigation was undertaken to determine the etiology of EGFRvIII in HNSCC.ResultsUnlike glioma, EGFRvIII expression in HNSCC did not correlate with EGFR amplification. We found evidence of genomic deletion of the exon 2–7 in 6 of 7 HNSCC cases examined, however, the presence of genomic deletion did not always result in mRNA expression of EGFRvIII. RNA sequencing with automated alignment did not identify EGFRvIII due to microhomology between intron 1 and exon 8. RNA sequencing analyzed by manual alignment methods did not correlate well with RT-PCR and PCR findings.ConclusionThese findings suggest that genomic deletion as well as additional regulatory mechanisms may contribute to EGFRvIII expression in HNSCC. Further, large scale automated alignment of sequencing are unlikely to identify EGFRvIII and an assay specifically designed to detect EGFRvIII may be necessary to detect this altered form of EGFR in HNSCC tumors.     

Decitabine Rescues Cisplatin Resistance in Head and Neck Squamous Cell Carcinoma

Cisplatin resistance in head and neck squamous cell carcinoma (HNSCC) reduces survival. In this study we hypothesized that methylation of key genes mediates cisplatin resistance. We determined whether a demethylating drug, decitabine, could augment the anti-proliferative and apoptotic effects of cisplatin on SCC-25/CP, a cisplatin-resistant tongue SCC cell line. We showed that decitabine treatment restored cisplatin sensitivity in SCC-25/CP and significantly reduced the cisplatin dose required to induce apoptosis. We then created a xenograft model with SCC-25/CP and determined that decitabine and cisplatin combination treatment resulted in significantly reduced tumor growth and mechanical allodynia compared to control. To establish a gene classifier we quantified methylation in cancer tissue of cisplatin-sensitive and cisplatin-resistant HNSCC patients. Cisplatin-sensitive and cisplatin-resistant patient tumors had distinct methylation profiles. When we quantified methylation and expression of genes in the classifier in HNSCC cells in vitro, we showed that decitabine treatment of cisplatin-resistant HNSCC cells reversed methylation and gene expression toward a cisplatin-sensitive profile. The study provides direct evidence that decitabine restores cisplatin sensitivity in in vitro and in vivo models of HNSCC. Combination treatment of cisplatin and decitabine significantly reduces HNSCC growth and HNSCC pain. Furthermore, gene methylation could be used as a biomarker of cisplatin-resistance.     

Inverse Immunological Responses Induced by Allergic Rhinitis and Head and Neck Squamous Cell Carcinoma

Several epidemiological studies have investigated the relation between allergy and cancer with contradicting conclusions, and reports on immunological differences are scarce. By focusing on inflammation, the present study was designed to compare the immune response induced by allergic rhinitis (AR) and head and neck squamous cell carcinoma (HNSCC). Blood and serum was obtained from patients with symptomatic seasonal AR, and newly detected HNSCC, as well as healthy controls. Peripheral blood mononuclear cells (PBMC) and polymorphonuclear leukocytes (PMN) were isolated and cultured with or without the toll-like receptor ligands, Pam₃CSK₄, LPS, R837, and CpG. Cellular activation and cytokine release were assessed with ELISA, Luminex Multiplex Immunoassay, flow cytometry, and real-time RT-PCR. Sera from HNSCC patients showed elevated levels of innate immune cytokines, and exhibited a response profile consistent with an increased innate immune reaction. In contrast, sera and stimulated PBMC from AR patients displayed increased concentrations of T cell related cytokines, consistent with an adaptive immune response. The presented data demonstrate that AR and HNSCC induce two distinct immunological processes, indicating an inverse association between the immunological responses seen in patients with allergy and cancer of the upper airway.     

Bitter Melon Reduces Head and Neck Squamous Cell Carcinoma Growth by Targeting c-Met Signaling

Head and neck squamous cell carcinoma (HNSCC) remains difficult to treat, and despite of advances in treatment, the overall survival rate has only modestly improved over the past several years. Thus, there is an urgent need for additional therapeutic modalities. We hypothesized that treatment of HNSCC cells with a dietary product such as bitter melon extract (BME) modulates multiple signaling pathways and regresses HNSCC tumor growth in a preclinical model. We observed a reduced cell proliferation in HNSCC cell lines. The mechanistic studies reveal that treatment of BME in HNSCC cells inhibited c-Met signaling pathway. We also observed that BME treatment in HNSCC reduced phosphoStat3, c-myc and Mcl-1 expression, downstream signaling molecules of c-Met. Furthermore, BME treatment in HNSCC cells modulated the expression of key cell cycle progression molecules leading to halted cell growth. Finally, BME feeding in mice bearing HNSCC xenograft tumor resulted in an inhibition of tumor growth and c-Met expression. Together, our results suggested that BME treatment in HNSCC cells modulates multiple signaling pathways and may have therapeutic potential for treating HNSCC.     

Upregulation of SQLE Contributes to Poor Survival in Head and Neck Squamous Cell Carcinoma

Recently, increasing attention has been paid to the role of Squalene epoxidase (SQLE) in several types of cancers. However, its functional role in tumor progression of head and neck squamous cell carcinoma (HNSCC) is still unclear. We performed bioinformatic analyses and relative experiments to assess the potential mechanism of SQLE-mediated HNSCC malignancy. And the results showed that SQLE was significantly upregulated in tumor samples compared with peritumor samples. Mechanistically, miR-584-5p downregulation may lead to the upregulation of SQLE in HNSCC. Moreover, high SQLE expression in HNSCC was associated with TNM stage, distant metastasis, and poor survival, indicating that SQLE be involved in the progression of HNSCC. Furtherly, SQLE boosted proliferation, migration, invasion of HNSCC cells in vitro and in vivo. Bioinformatic studies showed that PI3K/Akt signaling participated in HNSCC progression mediated by SQLE overexpression, which is confirmed by in vitro and in vivo analysis. Particularly, treatment with terbinafine, an inhibitor of SQLE widely used in the treatment of fungal infections, showed a therapeutic influence on HNSCC. Our findings demonstrate that SQLE plays a vital role in HNSCC progression, providing research evidence for SQLE as a prospective HNSCC therapeutic target and for terbinafine as a candidate drug of HNSCC treatment in the future