Robert Mandic

The role of matrix metalloproteinases in squamous cell carcinomas of the head and neck.

The ability of tumors to infiltrate the surrounding tissue is one of the major characteristics of a malignancy. This process is based on the tumors ability to destroy the extracellular matrix (ECM) including the basement membrane (BM). Several previous studies identified matrix metalloproteinases (MMPs), a family of zinc-dependent endopeptidases to be keyplayers in this process. Since then multiple investigations focused on the expression and activation levels of their extracellular regulators and to a lesser extent of their transcriptional modulators. However, the exact diagnostic and prognostic values of these regulators still remain unclear. Squamous cell carcinomas of the head and neck (HNSCC) are known for their infiltrative growth and there is strong evidence that at least some members of the MMP-family play a crucial role in this process. It turned out that MMP-2, -9, -13 and to a lesser extent MMP-7 are related to the metastatic potential of HNSCC but further studies will be required to establish the exact role of MMPs in HNSCC. This Review will discuss the current literature concerning the role of MMPs in HNSCC.

Subunit H of the V-ATPase Binds to the Medium Chain of Adaptor Protein Complex 2 and Connects Nef to the Endocytic Machinery

Nef is an accessory protein of human and simian immunodeficiency viruses (HIV and SIV) that is required for efficient viral infectivity and pathogenicity. It decreases the expression of CD4 on the surface of infected cells. V1H is the regulatory subunit H of the vacuolar membrane ATPase (V-ATPase). Previously, the interaction between Nef and V1H has been found to facilitate the internalization of CD4, suggesting that V1H could connect Nef to the endocytic machinery. In this study, we demonstrate that V1H binds to the C-terminal flexible loop in Nef from HIV-1 and to the medium chain (μ2) of the adaptor protein complex 2 (AP-2) in vitro and in vivo. The interaction sites of V1H and μ2 were mapped to a central region in V1H from positions 133 to 363, which contains 4 armadillo repeats, and to the N-terminal adaptin-binding domain in μ2 from positions 1 to 145. Fusing Nef to V1H reproduced the appropriate trafficking of Nef. This chimera internalized CD4 even in the absence of the C-terminal flexible loop in Nef. Finally, blocking the expression of V1H decreased the enhancement of virion infectivity by Nef. Thus, V1H can function as an adaptor for interactions between Nef and AP-2.

Pathogenesis of lymphangiomas

Based on various hypotheses concerning lymphangiogenesis published in the literature, different putative mechanisms of lymphangioma development are discussed including failure of the lymphatic system to connect with or separate from the venous system, abnormal budding of the lymphatic system from the cardinal vein, or acquired processes such as traumata, infections, chronic inflammations, and obstructions. Increasingly, the possible influence of lymphangiogenic growth factors on the development of lymphangiomas is discussed. The proved expression of different growth factors in the endothelium of lymphangiomas leads to new hypotheses regarding the pathogenesis of lymphangiomas. Thus, further studies on the lymphangiogenesis and the development of lymphangiomas will have to clarify as to whether lymphangiomas are true malformations or neoplastic in nature.

Microcystic Lymphatic Malformations of the Tongue Diagnosis, Classification, and Treatment

OBJECTIVE To describe a classification of microcystic lymphatic malformations of the tongue and to investigate different treatment methods. DESIGN Retrospective review of patients treated for microcystic lymphatic malformations of the tongue. Lymphatic malformations were classified into the following 4 groups according to their extent: isolated superficial microcystic lymphatic malformations of the tongue (stage I); isolated lymphatic malformations of the tongue with muscle involvement (stage II; stage IIA, involving a part of the tongue; stage IIB, involving the entire tongue); microcystic lymphatic malformations of the tongue and the floor of mouth (stage III); and extensive microcystic lymphatic malformations involving the tongue, floor of mouth, and further cervical structures (stage IV). PATIENTS Twenty patients with microcystic lymphatic malformation of the tongue. MAIN OUTCOME MEASURES Medical records were reviewed for demographic data and extent and treatment of the lymphatic malformations. RESULTS Three patients had stage I disease; 5 patients, stage II; 3 patients, stage III; and 9 patients, stage IV. In 6 patients, the lymphatic malformations could be completely removed by carbon dioxide laser surgery; the remaining 13 patients had persistent disease. CONCLUSIONS The initial stage seems to predict outcome. Carbon dioxide laser therapy provides good results primarily in stages I and IIA lymphatic malformations. In advanced lymphatic malformations (stages IIB, III, and IV), an interdisciplinary approach is necessary, because complete surgical excision is often impossible owing to the diffuse growth behavior, and therefore recurrence and persistence are common.

Intraglandular application of botulinum toxin leads to structural and functional changes in rat acinar cells.

Intraglandular injection of botulinum toxin (BoNT) leads to a transient denervation of the submandibular gland and this is associated with reduced salivary secretion. The purpose of the present study was to verify whether temporary acinar atrophy occurs simultaneously with chemical denervation of the glands.

A basal-cell-like compartment in head and neck squamous cell carcinomas represents the invasive front of the tumor and is expressing MMP-9

Head and neck squamous cell carcinomas (HNSCCs) are the most frequent malignancies of the upper aerodigestive tract. The cancer stem cell (CSC) hypothesis concludes that CSCs constitute the dangerous tumor cell population due to their ability of self-renewal and being associated with relapse of tumor disease, invasiveness and resistance to chemo(radio)therapy. The aim of this study was to look for CSC candidates and expression of MMP-9 that previously was implicated in HNSCC invasiveness. Immunohistochemical, immunofluorescence and Western blot analysis were performed on HNSCC tumor specimens using antibodies specific for MMP-9, CD44, ALDH1 and CK14. Gelatinolytic activity was assessed by zymography. Pearson correlation analysis was used for statistical comparison. Immunohistochemical analysis found CD44 and MMP-9 to co-localize in tumor cells at the invasive front. Western blot analysis demonstrated a significant correlation (p=0.0047) between CD44 and MMP-9 in the tested tissues. In addition gelatinolytic activity of HNSCC tissues was found to significantly correlate (p=0.0010) with MMP-9 expression. The CD44(+) invasive front of the tumor was also positive for ALDH1 and CK14, all of them being typically expressed by cells in the basal cell layer of normal stratified squamous epithelia that also harbors the epithelial stem cells. The observations point to a role of a MMP-9 positive basal-cell-like cell layer in the process of HNSCC invasiveness. This compartment likely contains CSCs since it is expressing the putative CSC markers CD44, ALDH1 and CK14. This cell layer therefore should be considered a major therapeutic target in the treatment of head and neck cancer.

Reduced Cisplatin Sensitivity of Head and Neck Squamous Cell Carcinoma Cell Lines Correlates with Mutations Affecting the COOH-Terminal Nuclear Localization Signal of p53

Purpose: Head and neck squamous cell carcinomas (HNSCC) are the most frequent malignancies of the upper aerodigestive tract. Cisplatin resistance is a major problem in the treatment of a large number of HNSCC cancer patients. In this study, nine randomly selected HNSCC cell lines were investigated regarding expression, presence of mutations, nucleocytoplasmic distribution of p53, and sensitivity to cisplatin. Experimental Design: Protein expression was evaluated by Western blot analysis. The whole open reading frame of p53 was determined by reverse transcription-PCR sequencing. Nucleocytoplasmic distribution was evaluated by confocal laser scanning microscopy. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide viability assay was used to test for cisplatin sensitivity. Results: p53 mutations were found in all nine investigated HNSCC cell lines. Nuclear p53 signal was detected in six cell lines, whereas three cell lines exhibited total loss of nuclear p53 signal. Nuclear signal depended on the presence or absence of the COOH-terminal nuclear localization signal in p53. Cisplatin sensitivity was highly reduced in the group with loss of nuclear p53 signal compared with those with detectable nuclear signal. Transfection of wild-type and mutant p53 into a rat embryonic cell system showed highly reduced activity of the nuclear localization signal mutant p53 protein. Conclusion: Taken together, these data suggest that “loss of nuclear p53 signal” correlates with cisplatin resistance in HNSCC. If these results can be validated on a larger number of tumor samples, including fresh tumor tissues, it potentially could help in sparing a subgroup of HNSCC patients the side effects associated with unnecessary chemotherapy by identifying cisplatin nonresponders before chemotherapy induction.

Treatment with ozone/oxygen-pneumoperitoneum results in complete remission of rabbit squamous cell carcinomas

Head and neck squamous cell carcinomas (HNSCC) represent a group of metastasizing tumors with a high mortality rate in man and animals. Since the biomolecule ozone was found to inhibit growth of various carcinoma cells in vitro we here applied the highly aggressive and lethal VX2 carcinoma HNSCC tumor model of the New Zealand White rabbit to test whether ozone exerts antitumorous effects in vivo. Therapeutic insufflation of medical ozone/oxygen (O3/O2) gas mixture into the peritoneum (O3/O2‐pneumoperitoneum) at an advanced stage of tumor disease led to a survival rate of 7/14 rabbits. Six of the seven surviving rabbits presented full tumor regression and the absence of local or distant lung metastases. Insufflation of pure oxygen (O2) resulted in a survival rate of 3/13 animals accompanied by full tumor remission in 2 of the 3 surviving animals. Of the 14 sham‐treated animals only 1 had spontaneous tumor remission and survived. No adverse effects or changes in standard blood parameters were observed after repeated intraperitoneal insufflations of the O3/O2 or O2 gas. Animals with O3/O2‐induced tumor eradication developed tolerance against reimplantation of the VX2 tumor. This could be reversed by immune suppression with a combination of dexamethasone and cyclosporin A suggesting an antitumorous effect of O3/O2‐mediated activation of the bodys own immunosurveillance. Although the exact mechanisms of action are still unclear the present data point to O3/O2‐pneumoperitoneum as a promising new strategy in anticancer therapy.

Treatment of HNSCC cell lines with the EGFR‐specific inhibitor cetuximab (Erbitux®) results in paradox phosphorylation of tyrosine 1173 in the receptor

Overexpression of the epidermal growth factor receptor (EGFR, ErbB1, HER1) is frequent in head and neck squamous cell carcinomas (HNSCCs) and correlates with disease progression. Inhibition of EGFR with the kinase inhibitor AG1478 abolished receptor phosphorylation and reduced cell proliferation. However, treatment of HNSCC cells with cetuximab (Erbitux®), a monoclonal antibody designed to block the EGFR ligand binding site, led to paradox EGFR activation due to hyperphosphorylation of tyrosine 1173, however, with a concomitant reduction in Erk1/2 phosphorylation levels. No pronounced influence on cell proliferation levels could be observed after treatment with this antibody. Since cetuximab appears able to activate EGFR in HNSCC cell lines, it is necessary to rethink the exact mechanisms by which cetuximab that recently was approved for the treatment of advanced head and neck cancer, inhibits tumor growth.

Botulinum toxin prevents radiotherapy-induced salivary gland damage

Radiotherapy of head and neck malignancies results in severe damage to salivary glands. Irradiation-induced sialadenitis with xerostomia leads to a significant deterioration of the quality of life which lasts life-long. Here we show in a preliminary study that intraglandular application of botulinum toxin performed prior to radiation reduces significantly the radiation induced toxicity of the glandular tissue in rats.