RNAi-Mediated Knockdown of Cottontail Rabbit Papillomavirus Oncogenes Using Low-Toxicity Lipopolyplexes as a Paradigm to Treat Papillomavirus-Associated Cancers.

The cottontail rabbit papillomavirus (CRPV)-associated VX2 carcinoma of the New Zealand White rabbit serves as a model system for human papillomavirus (HPV)-associated head and neck squamous cell carcinomas (HNSCCs). The aim of this study was to evaluate the tumor-inhibiting effect of RNAi-mediated knockdown of the CRPV oncogenes, E6 and E7, using siRNA-loaded lipopolyplexes (LPPs). VX2-carcinoma-derived cells were cultured for up to 150 passages. In addition, CRPV E6 and E7 oncogenes were transiently expressed in COS-7 cells. Efficiency and safety of LPPs were evaluated in both VX2 cells and the COS-7 cell line. Both of these in vitro CRPV systems were validated and characterized by fluorescence microscopy, Western blot, and RT-qPCR. Efficient knockdown of CRPV E6 and E7 was achieved in VX2 cells and COS-7 cells pretransfected with CRPV E6 and E7 expression vectors. Knockdown of CRPV oncogenes in VX2 cells resulted in reduced viability, migration, and proliferation and led to a G0/G1 block in the cell cycle. CRPV E6 and E7 siRNA-loaded LPPs could represent promising therapeutic agents serving as a paradigm for the treatment of papillomavirus-positive cancers and could be of value for the treatment of CRPV-associated diseases in the rabbit such as papillomas and cancers of the skin.

Lipid-Coated Polymeric Nanoparticles for the Photodynamic Therapy of Head and Neck Squamous Cell Carcinomas.

Next to alcohol and tobacco abuse, infection with human papillomaviruses (HPVs) is a major risk factor for developing head and neck squamous cell carcinomas (HNSCCs), leading to 350,000 casualties worldwide each year. Limited therapy options and drug resistance raise the urge for alternative methods such as photodynamic therapy (PDT), a minimally invasive procedure used to treat HNSCC and other cancers. We prepared lipid-coated polymeric nanoparticles encapsulating curcumin as the photosensitizer (CUR-LCNPs). The prepared CUR-LCNPs were in the nanometer range (153.37 ± 1.58 nm) and showed an encapsulation efficiency of 92.69 ± 0.03%. Proper lipid coating was visualized using atomic force microscopy (AFM). The CUR-LCNPs were tested in three HPVpos and three HPVneg HNSCC lines regarding their uptake capabilities and in vitro cell killing capacity, revealing a variable but highly significant tumor cell inhibiting effect in all tested HNSCC cell lines. No significant differences were detected between the HPVpos and HPVneg HNSCC groups (mean IC50: (9.34 ± 4.73 µmol/L vs. 6.88 ± 1.03 µmol/L), suggesting CUR-LCNPs/PDT to be a promising therapeutic option for HNSCC patients independent of their HPV status.

Generation of a Syngeneic Heterozygous ACVRL1(wt/mut) Knockout iPS Cell Line for the In Vitro Study of HHT2-Associated Angiogenesis.

Hereditary hemorrhagic telangiectasia (HHT) type 2 is an autosomal dominant disease in which one allele of the ACVRL1 gene is mutated. Patients exhibit disturbances in TGF-beta/BMP-dependent angiogenesis and, clinically, often present with severe nosebleeds as well as a reduced quality of life. The aim of our study was to use CRISPR/Cas9 to knockout ACVRL1 in normal induced pluripotent stem cells (iPSCs) and evaluate the effects on TGF-beta- and BMP-related gene expression as well as angiogenesis. The CRISPR/Cas9 knockout of the ACVRL1 gene was carried out in previously characterized wild-type (ACVRL1wt/wt) iPSCs. An HHT type 2 iPS cell line was generated via a single-allele knockout (ACVRL1wt/mut) in wild-type (ACVRL1wt/wt) iPSCs, resulting in a heterozygous 17 bp frameshift deletion in the ACVRL1 gene [NG_009549.1:g.13707_13723del; NM_000020.3:c.1137_1153del]. After the generation of embryoid bodies (EBs), endothelial differentiation was induced via adding 4 ng/mL BMP4, 2% B27, and 10 ng/mL VEGF. Endothelial differentiation was monitored via immunocytochemistry. An analysis of 151 TGF-beta/BMP-related genes was performed via RT-qPCR through the use of mRNA derived from single iPS cell cultures as well as endothelial cells derived from EBs after endothelial differentiation. Differential TGF-beta/BMP gene expression was observed between ACVRL1wt/wt and ACVRL1wt/mut iPSCs as well as endothelial cells. EBs derived from CRISPR/Cas9-designed ACVRL1 mutant HHT type 2 iPSCs, together with their isogenic wild-type iPSC counterparts, can serve as valuable resources for HHT type 2 in vitro studies.

The importin beta superfamily member RanBP17 exhibits a role in cell proliferation and is associated with improved survival of patients with HPV+ HNSCC.

BACKGROUND: More than twenty years after its discovery, the role of the importin beta superfamily member Ran GTP-binding protein (RanBP) 17 is still ill defined. Previously, we observed notable RanBP17 RNA expression levels in head and neck squamous cell carcinoma (HNSCC) cell lines with disruptive TP53 mutations. METHODS: We deployed HNSCC cell lines as well as cell lines from other tumor entities such as HCT116, MDA-MB-231 and H460, which were derived from colon, breast and lung cancers respectively. RNAi was used to evaluate the effect of RanBP17 on cell proliferation. FACS analysis was used for cell sorting according to their respective cell cycle phase and for BrdU assays. Immunocytochemistry was deployed for colocalization studies of RanBP17 with Nucleolin and SC35 (nuclear speckles) domains. TCGA analysis was performed for prognostic assessment and correlation analysis of RanBP17 in HNSCC patients. RESULTS: RNAi knockdown of RanBP17, significantly reduced cell proliferation in HNSCC cell lines. This effect was also seen in the HNSCC unrelated cell lines HCT116 and MDA-MB-231. Similarly, inhibiting cell proliferation with cisplatin reduced RanBP17 in keratinocytes but lead to induction in tumor cell lines. A similar observation was made in tumor cell lines after treatment with the EGFR kinase inhibitor AG1478. In addition to previous reports, showing colocalization of RanBP17 with SC35 domains, we observed colocalization of RanBP17 to nuclear bodies that are distinct from nucleoli and SC35 domains. Interestingly, for HPV positive but not HPV negative HNSCC, TCGA data base analysis revealed a strong positive correlation of RanBP17 RNA with patient survival and CDKN2A. CONCLUSIONS: Our data point to a role of RanBP17 in proliferation of HNSCC and other epithelial cells. Furthermore, RanBP17 could potentially serve as a novel prognostic marker for HNSCC patients. However, we noted a major discrepancy between RanBP17 RNA and protein expression levels with the used antibodies. These observations could be explained by the presence of additional RanBP17 splice isoforms and more so of non-coding circular RanBP17 RNA species. These aspects need to be addressed in more detail by future studies.

Cucumber-Derived Exosome-like Vesicles and PlantCrystals for Improved Dermal Drug Delivery.

(1) Background: Extracellular vesicles (EVs) are considered to be efficient nanocarriers for improved drug delivery and can be derived from mammalian or plant cells. Cucumber-derived EVs are not yet described in the literature. Therefore, the aim of this study was to produce and characterize cucumber-derived EVs and to investigate their suitability to improve the dermal penetration efficacy of a lipophilic active ingredient (AI) surrogate. (2) Methods: The EVs were obtained by classical EVs isolation methods and by high pressure homogenization (HPH). They were characterized regarding their physico-chemical and biopharmaceutical properties. (3) Results: Utilization of classical isolation and purification methods for EVs resulted in cucumber-derived EVs. Their dermal penetration efficacy for the AI surrogate was 2-fold higher when compared to a classical formulation and enabled a pronounced transdermal penetration into the viable dermis. HPH resulted in submicron sized particles composed of a mixture of disrupted plant cells. A successful isolation of pure EVs from this mixture was not possible with classical EVs isolation methods. The presence of EVs was, therefore, proven indirectly. For this, the lipophilic drug surrogate was admixed to the cucumber juice either prior to or after HPH. Admixing of the drug surrogate to the cucumber prior to the HPH resulted in a 1.5-fold increase in the dermal penetration efficacy, whereas the addition of the AI surrogate to the cucumber after HPH was not able to improve the penetration efficacy. (4) Conclusions: Results, therefore, indicate that HPH causes the formation of EVs in which AI can be incorporated. The formation of plant EVs by HPH was also indicated by zeta potential analysis.

Comparison of Whiskbroom and Pushbroom darkfield elastic light scattering spectroscopic imaging for head and neck cancer identification in a mouse model.

The early detection of head and neck cancer is a prolonged challenging task. It requires a precise and accurate identification of tissue alterations as well as a distinct discrimination of cancerous from healthy tissue areas. A novel approach for this purpose uses microspectroscopic techniques with special focus on hyperspectral imaging (HSI) methods. Our proof-of-principle study presents the implementation and application of darkfield elastic light scattering spectroscopy (DF ELSS) as a non-destructive, high-resolution, and fast imaging modality to distinguish lingual healthy from altered tissue regions in a mouse model. The main aspect of our study deals with the comparison of two varying HSI detection principles, which are a point-by-point and line scanning imaging, and whether one might be more appropriate in differentiating several tissue types. Statistical models are formed by deploying a principal component analysis (PCA) with the Bayesian discriminant analysis (DA) on the elastic light scattering (ELS) spectra. Overall accuracy, sensitivity, and precision values of 98% are achieved for both models whereas the overall specificity results in 99%. An additional classification of model-unknown ELS spectra is performed. The predictions are verified with histopathological evaluations of identical HE-stained tissue areas to prove the model's capability of tissue distinction. In the context of our proof-of-principle study, we assess the Pushbroom PCA-DA model to be more suitable for tissue type differentiations and thus tissue classification. In addition to the HE-examination in head and neck cancer diagnosis, the usage of HSI-based statistical models might be conceivable in a daily clinical routine.

Sensitivity of Papilloma Virus-Associated Cell Lines to Photodynamic Therapy with Curcumin-Loaded Liposomes.

Photodynamic therapy (PDT) is a minimally invasive therapeutic approach used in the treatment of various medical conditions and cancerous diseases, involving light, a photosensitizing substance, and oxygen. Curcumin, a naturally occurring compound, carries antitumor activities and potentially could be exploited as a photosensitizer in PDT. Only little is known about liposomal-encapsulated curcumin that could help in increasing the efficacy, stability, and bioavailability of this compound. This study investigates the in vitro effects of curcumin-loaded liposomes in combination with PDT. Three papilloma virus-associated cell lines were treated with curcumin-loaded liposomes corresponding to a curcumin concentration of 0-100 µmol/L for 4 h followed by illumination at 457 nm (blue) for 45, 136, and 227 s at a fluence of 220.2 W/m2 (100 mA) corresponding to 1, 3 and 5 J·cm-2. After 24 h, the biological outcome of the treatment was assessed with the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), SYTO9/PI (propidium iodide), Annexin V-FITC (fluorescein isothiocyanate)/PI, clonogenic survival, and scratch (wound closure) assays. Photoactivation of curcumin-loaded liposomes led to a significant reduction in colony formation and migratory abilities, as well as to an increase in tumor cell death. The results point to the combination of curcumin-loaded liposomes with PDT as a potentially useful tool for the treatment of papillomavirus-associated malignancies.

Alternative splicing of the TNFSF13B (BAFF) pre-mRNA and expression of the BAFFX1 isoform in human immune cells.

Human B cell activating factor (TNFSF13B, BAFF) is a tumor necrosis factor superfamily member. Binding its unique receptor (TNFRSF13C, BAFF-R) mediates gene expression and cell survival in B cells via activation of NFκB pathway. Furthermore, there is data indicating a role in T cell function. A functionally inhibitory isoform (ΔBAFF) resulting from the deletion of exon 3 in the TNFSF13B pre-RNA has already been reported. However, data on the complexity of post-transcriptional regulation is scarce. Here, we report molecular cloning of nine TNFSF13B transcript variants resulting from alternative splicing of the TNFSF13B pre-mRNA including BAFFX1. This variant is characterized by a partial retention of intron 3 of the TNFSF13B gene causing the appearance of a premature stop codon. We demonstrate the expression of the corresponding BAFFX1 protein in Jurkat T cells, in ex vivo human immune cells and in human tonsillar tissue. Thereby we contribute to the understanding of TNFSF13B gene regulation and reveal that BAFF is regulated through a post-transcriptional mechanism to a greater extent than reported to date.

Evaluation of digital image analysis as a supportive tool for the stratification of head and neck vascular anomalies.

BACKGROUND: The histological differentiation of individual types of vascular anomalies (VA), such as lymphatic malformations (LM), hemangioma (Hem), paraganglioma (PG), venous malformations (VeM), arteriovenous malformations (AVM), pyogenic granulomas (GP), and (not otherwise classified) vascular malformations (VM n.o.c.) is frequently difficult due to the heterogeneity of these anomalies. The aim of the study was to evaluate digital image analysis as a method for VA stratification METHODS: A total of 40 VA tissues were examined immunohistologically using a selection of five vascular endothelial-associated markers (CD31, CD34, CLDN5, PDPN, VIM). The staining results were documented microscopically followed by digital image analyses based quantification of the candidate-marker-proteins using the open source program ImageJ/Fiji. RESULTS: Differences in the expression patterns of the candidate proteins could be detected particularly when deploying the quotient of the quantified immunohistochemical signal values. Deploying signal marker quotients, LM could be fully distinguished from all other tested tissue types. GP achieved stratification from LM, Hem, VM, PG and AVM tissues, whereas Hem, PG, VM and AVM exhibited significantly different signal marker quotients compared with LM and GP tissues. CONCLUSION: Although stratification of different VA from each other was only achieved in part with the markers used, the results of this study strongly support the usefulness of digital image analysis for the stratification of VA. Against the background of upcoming new diagnostic techniques involving artificial intelligence and deep (machine) learning, our data serve as a paradigm of how digital evaluation methods can be deployed to support diagnostic decision making in the field of VAs.

Aberrant Expression of Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) in Warthin Tumors.

The Warthin tumor represents the second most frequent benign tumor of the parotid gland and is characterized by the presence of oncocytes rich in structurally and functionally altered mitochondria. Next to its role in metabolism, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is also implicated in cellular mitophagy. Immunohistochemistry was carried out on Warthin tumor and normal control (parotid gland with striated ducts) tissues, using anti-GAPDH specific antibodies followed by digital image analysis. Laser capture microdissection was used to isolate the oncocytic tumor cell and normal control striated duct compartments for RNA extraction and qPCR. Warthin tumor oncocytes exhibited a markedly spotted GAPDH staining pattern exhibiting cells with cytoplasmic and nuclear, only nuclear or none GAPDH staining. A significantly lower (p < 0.0001) total GAPDH signal was detected in Warthin tumor oncocytes. Similarly, significantly lower (p < 0.005) GAPDH mRNA levels were seen in oncocytes compared with normal ductal cells. To exclude the possibility of this GAPDH staining pattern being a general feature of oncocytic neoplasms of different organs, we tested a cohort of renal oncocytoma and oncocytic chromophobe carcinoma; none showed this type of staining. The observed progressive GAPDH loss in Warthin tumor oncocytes could be implicated in the pathogenesis of Warthin tumors.

Influence of buffy coat-derived putative endothelial progenitor cells on tumor growth and neovascularization in oral squamous cell carcinoma xenografts.

OBJECTIVES: The aim of this murine in vivo study was to investigate whether buffy coat-derived putative endothelial progenitor cells (BCEPC) alter tumor growth and neovascularization in oral squamous cell carcinomas (OSCC). MATERIALS AND METHODS: A murine xenograft model using the PCI-13 oral cancer cell line was deployed of which n = 24 animals received 2 × 106 BCEPC by transfusion whereas the control group (n = 24) received NaCl (0.9%) instead. Tumor size, volume, and capillary density were determined by sonography and measurement with a caliper. Immunohistochemical analysis was carried out with antibodies specific for Cytokeratins, Flt-4, Podoplanin, and Vimentin. RESULTS: In the experimental group, systemic application of BCEPC significantly increased tumor volume to 362.49% (p = 0.0012) and weight to 352.38% (p = 0.0018) as well as vascular densities to 162.15% (p = 0.0021) compared with control tumors. In addition, BCEPC-treated xenografts exhibited higher Cytokeratin expression levels by a factor of 1.47 (p = 0.0417), Podoplanin by a factor of 3.3 (p = 0.0020) and Vimentin by a factor of 2.5 (p = 0.0001), respectively. CONCLUSIONS: Immunohistochemical investigations support the notion that BCEPC transfusion influences neovascularization and lymphatic vessel density, thereby possibly promoting tumor progression. Future studies, which will include gene expression analysis, should help to define the possible role of BCEPC during OSCC progression in more detail. CLINICAL RELEVANCE: Endothelial progenitor cells (EPCs) could serve as a target structure for the treatment of OSCC and possibly other solid tumors.

Virome Analysis Reveals No Association of Head and Neck Vascular Anomalies with an Active Viral Infection.

BACKGROUND/AIM: Vascular anomalies encompass different vascular malformations [arteriovenous (AVM), lymphatic (LM), venous lymphatic (VLM), venous (VM)] and vascular tumors such as hemangiomas (HA). The pathogenesis of vascular anomalies is still poorly understood. Viral infection was speculated as a possible underlying cause. MATERIALS AND METHODS: A total of 13 human vascular anomalies and three human skin control tissues were used for viral analysis. RNA derived from AVM (n=4) and normal skin control (n=3) tissues was evaluated by RNA sequencing. The Virome Capture Sequencing Platform for Vertebrate Viruses (VirCapSeq-VERT) was deployed on 10 tissues with vascular anomalies (2×AVM, 1×HA, 1×LM, 2×VLM, 4×VM). RESULTS: RNA sequencing did not show any correlation of AVM with viral infection. By deploying VirCapSeq-VERT, no consistent viral association was seen in the tested tissues. CONCLUSION: The analysis does not point to the presence of an active viral infection in vascular anomalies. However, transient earlier viral infections, e.g. during pregnancy, cannot be excluded with this approach.

The Auricular VX2 Carcinoma Is a Suitable Animal Model for Identifying Biomarkers for HNSCC Therapy Response.

BACKGROUND/AIM: The rabbit auricular VX2 carcinoma is an established animal model for human head and neck squamous cell carcinoma (HNSCC). Previously, we observed that intraperitoneal oxidative (O3/O2) stress induced tumor remission. Our aim was to evaluate candidate genes associated with tumor regression. MATERIALS AND METHODS: For identification of tumor remission-related genes, microarray analysis was performed with subsequent validation by polymerase chain reaction (PCR), in situ hybridization, immunohistochemistry and western blot analysis. RESULTS: Microarray analysis indicated a prominent reduction of epidermal growth factor receptor (Egfr, Erbb1) expression levels in regressing tumors. Quantitative PCR confirmed a significant (p<0.005) down-regulation of Erbb1-3 mRNA in regressing VX2 tumors. Histological localization of Erbb1-3 mRNA transcript and protein indicated reduced Erbb gene expression occurring at the level of individual VX2 tumor cells rather than solely being an effect of tumor shrinkage. This study highlights changes in the Erbb gene signature of regressing VX2 carcinomas as a predictor for therapy response. The VX2 carcinoma animal model, therefore, appears suitable for the identification and evaluation of new diagnostic, prognostic and therapeutic biomarkers prior to their application in patients with HNSCC.

Modulation of Tumor Cell Metabolism by Laser Photochemotherapy with Cisplatin or Zoledronic Acid In Vitro.

BACKGROUND/AIM: Laser photochemotherapy is a new approach in cancer treatment using low-level laser therapy (LLLT) to enhance the effect of chemotherapy. MATERIALS AND METHODS: In order to evaluate the effect of LLLT on tumor cells, HeLa cells were treated with cisplatin or zoledronic acid (ZA) followed by LLLT. Cell viability was evaluated with 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide assay. Oxidative phosphorylation and glycolysis were measured using extracellular flux analysis. Immunocytochemistry of heat-shock protein 70 (HSP70) and western blot analysis were performed. RESULTS: LLLT alone increased viability and was associated with lower oxidative phosphorylation but higher glycolysis rates. Cisplatin and ZA alone lowered cell viability, glycolysis and oxidative phosphorylation. This effect was significantly enhanced in conjunction with LLLT and was accompanied by reduced oxidative phosphorylation and collapse of glycolysis. CONCLUSION: Our observations indicate that LLLT may raise the cytotoxicity of cisplatin and ZA by modulating cellular metabolism, pointing to a possible application in cancer treatment.

Ectonucleotidase CD39 expression in regional metastases in head and neck cancer.

INTRODUCTION: CD39 is the rate-limiting enzyme in the generation of immunosuppressive adenosine and its expression and activity are significant in tumor progression. Squamous cell carcinoma of the head and neck (HNSCC) shows an overall poor prognosis due to high local recurrence rates and early metastatic spread. MATERIAL AND METHODS: Primary tumor specimens and lymph node specimens harvested during neck dissection of 65 patients with a diagnosis of HNSCC were subjected to immunohistochemical and H-score analysis of CD39 expression. Demographics, histopathology and subsequent outcome were analyzed. RESULTS: The primary cancer was squamous cell carcinoma in all patients (male/female 55:10). H-score for CD39 expression in the primary lesion and metastatic lymph nodes was significantly higher in advanced compared to early stages with no significant differences among different tumor locations. High intratumoral and intrametastatic CD39 expression was associated with an inferior patients' overall survival at a mean follow-up of 83.4 months (6-204 months). CONCLUSION: CD39 expression in HNSCC correlated positively with tumor stage and appears to predict poor prognosis. Therefore, CD39 expression in primary lesions and metastatic lymph nodes seems to identify patients at high risk in HNSCC of all tumor sites. Immunotherapeutic approaches targeting CD39 might be promising for this patient population.

Foxp3 expression in lymph node metastases in patients with head and neck cancer.

INTRODUCTION: The prevalence and activity of regulatory T cells in patients with cancer correlates with poor prognosis. These cells are characterized by their expression of Forkhead box protein-3 (Foxp3). Squamous cell carcinoma is the most prevalent type of cancer in the head and neck region with overall poor survival rates, also due to early spread of metastatic cells. MATERIAL AND METHODS: Primary tumor specimens as well as lymph node specimens harvested during neck dissection of 65 patients with a diagnosis of HNSCC were subjected to immunohistochemical and H-score analysis of Foxp3 expression. Demographics, diagnoses, histopathology and subsequent outcome were analyzed. RESULTS: The primary cancer was squamous cell carcinoma in all patients (male/female 55:10) with the following tumor locations: oral cavity n = 16, oropharynx n = 28, hypopharynx n = 11 and larynx n = 10 (Stage III n = 18; Stage IVA n = 45; Stage IVB n = 2). The H-score for Foxp3 expression in the primary lesion as well as metastatic lymph nodes was significantly higher in advanced stages compared to early stages with differences among tumor locations, which were not significant. High Foxp3 expression was associated with inferior overall survival rates at a mean follow-up of 83.4 months (6-204 months) Conclusions: Foxp3 expression in HNSCC varied from the anatomical site and correlated positively with tumor stage and was associated with poor prognosis. Therefore, Foxp3 expressions in primary lesions as well as lymphogenic metastases appear to predict high-risk HSNCC patients. Novel therapeutic approaches targeting Foxp3+ cells might seem promising for this patient population.

Indication for differential sorting of the rat v-SNARE splice isoforms VAMP-1a and -1b.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins are essential constituents of the intracellular trafficking machinery. The variable C-terminus in the 2 rat VAMP-1 splice isoforms VAMP-1a and -1b potentially acts as a sorting signal, because similar changes at the C-terminal end of a human VAMP-1 splice isoform resulted in its sorting to mitochondria. To evaluate the differences in the subcellular localization of these two v-SNARE proteins, VAMP-1a and -1b proteins tagged with green fluorescent protein (GFP) and red fluorescent protein (RFP) were expressed in HeLa, COS-7, and MDCK cells and evaluated by conventional confocal as well as total internal reflection fluorescence microscopy. Regions consistent with the endoplasmic reticulum and Golgi apparatus demonstrated a major overlap of both signals. In the periphery, vesicular structures were observed that mainly expressed one of the 2 isoforms. Within our experimental settings, we could not observe sorting of any of the 2 isoforms to mitochondria or peroxisomes, whereas both isoforms were found expressed in a minor subset of singular vesicles, which sporadically appeared to co-localize with the exocyst marker EXOC3/Sec6. Because vesicular structures were seen that expressed only one of the two splice variants, it is possible that VAMP-1a and VAMP-1b are sorted to distinct cellular compartments that require further characterization.

The enhancing effect of a laser photochemotherapy with cisplatin or zolendronic acid in primary human osteoblasts and osteosarcoma cells in vitro.

BACKGROUND: Photodynamic therapies (PDT) have become increasingly popular in the adjuvant treatment of different tumour entities. Chemotherapeutic agents, such as cisplatin may be used in combination with low-level laser therapy (LLLT) as laser photochemotherapy. The aim of this study was to investigate the effect of LLLT on cell bioviability of normal and malignant bone cells under chemotherapeutic conditions with either cisplatin or zolendronic acid in vitro. METHODS: Primary human osteoblasts (HOB) and an osteosarcoma cell line (Saos-2) were treated with different concentrations of zolendronic acid or cisplatin and irradiated twice with a diode laser (wavelength 670 nm, 120 s, energy outputs of 100mW/cm2 , continuous wave mode). Cell viability was tested by XTT-assay and via histomorphological analysis. RESULTS: LLLT alone increased bioviability for both cell lines. LLLT lowered HOB viability at the three highest concentrations of cisplatin and zolendronic acid. For Saos-2, LLLT reduced cell viability at every concentration of cisplatin. In cases of incubation with zolendronic acid, similar to osteoblasts, LLLT lowered cell viability at the highest concentration only. CONCLUSIONS: Based on the conditions of this study, laser photochemotherapy may be able to raise the cytotoxicity of cisplatin and zolendronic acid in benign and malignant bone cells. This could be of interest in the development of new therapeutic treatment modalities against neoplastic bone diseases like osteosarcoma.

ΔNp63 activates the Fanconi anemia DNA repair pathway and limits the efficacy of cisplatin treatment in squamous cell carcinoma.

TP63, a member of the p53 gene family gene, encodes the ΔNp63 protein and is one of the most frequently amplified genes in squamous cell carcinomas (SCC) of the head and neck (HNSCC) and lungs (LUSC). Using an epiallelic series of siRNAs with intrinsically different knockdown abilities, we show that the complete loss of ΔNp63 strongly impaired cell proliferation, whereas partial ΔNp63 depletion rendered cells hypersensitive to cisplatin accompanied by an accumulation of DNA damage. Expression profiling revealed wide-spread transcriptional regulation of DNA repair genes and in particular Fanconi anemia (FA) pathway components such as FANCD2 and RAD18 - known to be crucial for the repair of cisplatin-induced interstrand crosslinks. In SCC patients ΔNp63 levels significantly correlate with FANCD2 and RAD18 expression confirming ΔNp63 as a key activator of the FA pathway in vivo Mechanistically, ΔNp63 bound an upstream enhancer of FANCD2 inactive in primary keratinocytes but aberrantly activated by ΔNp63 in SCC. Consistently, depletion of FANCD2 sensitized to cisplatin similar to depletion of ΔNp63. Together, our results demonstrate that ΔNp63 directly activates the FA pathway in SCC and limits the efficacy of cisplatin treatment. Targeting ΔNp63 therefore would not only inhibit SCC proliferation but also sensitize tumors to chemotherapy.

A cisplatin-resistant head and neck cancer cell line with cytoplasmic p53(mut) exhibits ATP-binding cassette transporter upregulation and high glutathione levels.

PURPOSE: Head and neck squamous cell carcinoma (HNSCC) cell lines with cytoplasmically sequestered mutant p53 (p53(mut_c)) are frequently more resistant to cisplatin (CDDP) than cells with mutant but nuclear p53 (p53(mut_n)). The aim of the study was to identify underlying mechanisms implicated in CDDP resistance of HNSCC cells carrying cytoplasmic p53(mut). METHODS: Microarray analysis, quantitative reverse transcription polymerase chain reaction, Western blot analysis and immunocytochemistry were used to identify and evaluate candidate genes involved in CDDP resistance of p53(mut_c) cells. RNAi knockdown or treatment with inhibitors together with flow cytometry-based methods was used for functional assessment of the identified candidate genes. Cellular metabolic activity was assessed with the XTT assay, and the redox capacity of cells was evaluated by measuring cellular glutathione (GSH) levels. RESULTS: Upregulation of ABCC2 and ABCG2 transporters was observed in CDDP-resistant p53(mut_c) HNSCC cells. Furthermore, p53(mut_c) cells exhibited a pronounced side population that could be suppressed by RNAi knockdown of ABCG2 as well as treatment with the ATP-binding-cassette transporter inhibitors imatinib, MK571 and tariquidar. Metabolic activity and cellular GSH levels were higher in CDDP-resistant p53(mut_c) cells, consistent with a higher capacity to fend off cytotoxic oxidative effects such as those caused by CDDP treatment. Finally, ABCC2/G2 inhibition of HNSCC cells with MK571 markedly enhanced CDDP sensitivity of HNSCC cells. CONCLUSIONS: The observations in this study point to a major role of p53(mut_c) in conferring a stem cell like phenotype to HNSCC cells that is associated with ABCC2/G2 overexpression, high GSH and metabolic activity levels as well as CDDP resistance.