CerS6 regulates cisplatin resistance in oral squamous cell carcinoma by altering mitochondrial fission and autophagy.

Chemoresistance remains a challenge in the effective treatment of solid tumors, including oral squamous cell carcinoma (OSCC). Mitochondrial dynamics and autophagy have recently been implicated in the chemoresistance of cancer cells. The neutralization of ceramide is also associated with multidrug resistance, and ceramide synthase 6 (CerS6) is known to induce apoptosis. However, whether CerS6 regulates chemoresistance in OSCC is not clearly understood. Therefore, we investigated the role of CerS6 in the susceptibility of OSCC cells to cisplatin. In this study, we observed that cisplatin-resistant OSCC cells process lower levels of fission-state mitochondria and cell apoptosis than cisplatin-sensitive cells, and autophagy was activated in cisplatin-resistant OSCC cells. We found lower CerS6 expression in cisplatin-resistant OSCC cells. Overexpression of CerS6 with lentivirus-encoded CerS6 complementary DNA in cisplatin-resistant OSCC cells increased cisplatin sensitivity. Overexpression of CerS6 enhanced mitochondrial fission and apoptosis and attenuated cisplatin-induced autophagy in cisplatin-resistant OSCC cells. Further investigation indicated that CerS6 might function through altering calpain expression to enhance cisplatin sensitivity. Cisplatin-resistant OSCC cells xenografted onto a nude mouse model confirmed that CerS6 enhanced cisplatin chemotherapy sensitivity to reduce tumor volume. These data indicate that CerS6 could mediate an effective response to cisplatin in chemoresistant OSCC.

Influence of selenium on radiogenic collagen destruction and the degree of collagen tissue maturation in stage III oral squamous cell carcinoma patients undergoing therapeutic irradiation.

INTRODUCTION: We set out to assess whether selenium, an antioxidant mineral could influence radiogenic collagen maturation. MATERIALS AND METHODS: The study comprise of normal (Group I), untreated oral carcinoma cases (Group II) (n = 20), cases who underwent radiotherapy (Group IIa) n = 10 and cases supplemented with selenium along with radiotherapy (Group IIb) n = 10. RESULTS: Spectrophotometric estimation and luminescence spectral assignment of collagen showed improved collagen maturation status. Measurement of the mature collagen cross-links hydroxylysylpyridinoline and lysylpyridinoline by high-performance liquid chromatography on irradiated tissues showed a considerable decrease in the selenium Group IIb (P < 0.05) indicating a decrease in collagen fragments. Electron microscopic studies showed significant morphological alteration in the selenium group. The micro nucleus frequency, decreased in radiation group (P < 0.05) compared with untreated (P < 0.05). While much more decrease observed in the selenium group (P < 0.05). DISCUSSION: The results represent the effect of selenium treatment with a bearing on carcinogenic process to curtail it, thus enhancing the maturity of collagen.

Isolation of a hemidesmosome-rich fraction from a human squamous cell carcinoma cell line.

Hemidesmosomes are cell-to-matrix adhesion complexes anchoring keratinocytes to basement membranes. For the first time, we present a method to prepare a fraction from human cultured cells that are highly enriched in hemidesmosomal proteins. Using DJM-1 cells derived from human squamous cell carcinoma, accumulation of hemidesmosomes was observed when these cells were cultured for more than 10 days in a commercial serum-free medium without supplemental calcium. Electron microscopy demonstrated that numerous electron-dense adhesion structures were present along the basal cell membranes of DJM-1 cells cultured under the aforementioned conditions. After removing cellular materials using an ammonia solution, hemidesmosomal proteins and deposited extracellular matrix were collected and separated by electrophoresis. There were eight major polypeptides, which were determined to be plectin, BP230, BP180, integrin α6 and ß4 subunits, and laminin-332 by immunoblotting and mass spectrometry. Therefore, we designated this preparation as a hemidesmosome-rich fraction. This fraction contained laminin-332 exclusively in its unprocessed form, which may account for the promotion of laminin deposition, and minimal amounts of Lutheran blood group protein, a nonhemidesmosomal transmembrane protein. This hemidesmosome-rich fraction would be useful not only for biological research on hemidesmosomes but also for developing a serum test for patients with blistering skin diseases.

Autophagy inhibition contributes to radiation sensitization of esophageal squamous carcinoma cells.

Radiotherapy is a useful component of treatment strategies for esophageal cancer. The role of autophagy in response to ionizing radiation was investigated in human esophageal squamous carcinoma cells. Cell viability and clonogenic survival assay were used to evaluate the radiosensitivity of autophagy inhibitor (3-MA) on esophageal squamous carcinoma cells. The percentage of apoptotic cells and cell cycle analysis were assessed by flow cytometry; DAPI staining was used to detect apoptotic cells. The expression of beclin-1 and LC3 was measured using a Western blot. The ultrastructural analysis was under the electron microscope. 6 Gy irradiation induced a massive accumulation of autophagosomes accompanied by strong upregulation of beclin-1 and LC3-II expression in TE-1 cells. Compared with radiation alone, 3-MA combined with radiation significantly decreased cell viability, as well as autophagic ratio, beclin-1, and LC3-II protein level. Inhibition of autophagy increased radiation-induced apoptosis and the percentage of G2/M-phase cells. Blockade of autophagy with 3-MA enhanced cytotoxicity of radiotherapy in human esophageal squamous carcinoma cells. It suggests that inhibition of autophagy could be used as adjuvant therapy to treat esophageal squamous cell carcinoma.

Bioenergetics of lung tumors: alteration of mitochondrial biogenesis and respiratory capacity.

Little is known on the metabolic profile of lung tumors and the reminiscence of embryonic features. Herein, we determined the bioenergetic profiles of human fibroblasts taken from lung epidermoid carcinoma (HLF-a) and fetal lung (MRC5). We also analysed human lung tumors and their surrounding healthy tissue from four patients with adenocarcinoma. On these different models, we measured functional parameters (cell growth rates in oxidative and glycolytic media, respiration, ATP synthesis and PDH activity) as well as compositional features (expression level of various energy proteins and upstream transcription factors). The results demonstrate that both the lung fetal and cancer cell lines produced their ATP predominantly by glycolysis, while oxidative phosphorylation was only capable of poor ATP delivery. This was explained by a decreased mitochondrial biogenesis caused by a lowered expression of PGC1alpha (as shown by RT-PCR and Western blot) and mtTFA. Consequently, the relative expression of glycolytic versus OXPHOS markers was high in these cells. Moreover, the re-activation of mitochondrial biogenesis with resveratrol induced cell death specifically in cancer cells. A consistent reduction of mitochondrial biogenesis and the subsequent alteration of respiratory capacity was also observed in lung tumors, associated with a lower expression level of bcl2. Our data give a better characterization of lung cancer cells' metabolic alterations which are essential for growth and survival. They designate mitochondrial biogenesis as a possible target for anti-cancer therapy.

Extracellular environment as one mediator of blue light-induced mitochondrial suppression.

OBJECTIVES: The current study tested the hypothesis that the extracellular environment mediates mitochondrial suppression of oral epithelial cells and fibroblasts by blue light. METHODS: We exposed Balb fibroblasts (Balb), normal human epidermal keratinocytes (NHEK), and oral squamous carcinoma cells (OSC2) to blue light (30-120J/cm2) in different cell-culture media and in phosphate buffered saline (PBS). Mitochondrial activity (MTT method) was used to assess cellular response 72 h post-light exposure. Cell-culture media were replaced or supplemented before or after light exposure to assess the variables of exposure time and medium degradation as mediators of blue light-induced effects. RESULTS: Mitochondrial activity of NHEK was not suppressed by exposure to blue light regardless of extracellular conditions. The mitochondrial activity of OSC2 and Balb cells was suppressed most when cells were exposed to light in cell-culture medium (versus PBS). Blue light suppressed mitochondrial activity more when irradiated medium remained in contact with the cells at least 1h, indicating a time-dependence of the medium effects. Neither a replacement nor a supplementation of medium components reduced blue light-induced mitochondrial suppression. SIGNIFICANCE: Our results suggest that tissue environments influence cellular responses to blue light and that these environments should be considered when assessing any biological effects of blue light during the photopolymerization of restorative resins.

Monoclonal antibody to fibulin-1 generated by genetic immunization.

Fibulin-1 (Fbln-1) is an extracellular matrix (ECM) and plasma glycoprotein. Considering the growing evidence indicating that Fbln-1 plays a role in cancer we sought to develop monospecific antibodies to better facilitate further studies of the function of Fbln-1 in breast cancer. Using a plasmid expression vector encoding full-length human Fbln-1D as an immunogen and CpG oligodeoxyribonucleotides as adjuvant a monoclonal antibody (MAb) against Fbln-1 was produced. This MAb, designated MEM-2 was of IgM isotype and reacted with bacterially expressed Fbln-1. Furthermore, MEM-2 reacted with Fbln-1 expressed in the ECM released by cultured human breast carcinoma SKBR-3 cells in ELISA, and also with Fbln-1 present in SKBR-3 cell extract in immunoprecipitation and Western blotting. MEM-2 also reacted with Fbln-1 in human breast carcinoma specimens. These findings illustrate the utility of genetic immunization as a means of generating monoclonal antibodies to tumor-related ECM proteins. MEM-2 represents a useful new tool for the study of Fbln-1 in breast cancer.

Estudio clinico, histopatológico y ultraestructural del músculo de la lengua en pacientes con carcinoma epidermoide / A clinical, histopathological and ultrastructural study of tongue muscle in patients with squamous cell carcinoma

En el presente trabajo se realizó un estudio mediante microscopÝa de luz y electrónica de transmisión con el fin de estudiar las alteraciones musculares producidas en el músculo de la lengua en pacientes con carcinoma epidermoide. Las anomalÝas estructuras observadas incluyeron, alteraciones tanto en el sistema contrßctil como sarcotubular, por otro lado se observó atrofia y necrosis segmentaria, alteración de mitocondrias y proliferación de organelos, asÝ como vaculolas, lisosomas y grßnulos de lipofucsina. El infiltrado mononuclear estuvo representado principalmente por neutrófilos, macrófagos y mastocitos. Este estudio representa el primer reporte sobre las alteraciones ultraestructurales a nivel del músculo de la lengua producida por esta patologÝa maligna

Changes in the ultrastructure of human cells related to certain biological responses under hyperthermic culture conditions.

It has been reported that human cancer cells are more sensitive to high temperatures than normal human cells, and that cell proliferation and viability are affected by the temperature environment. In this study, we proceeded further, and turning our attention to the close relationship between cell morphology and temperature, used two human cancer cell lines and two normal cell strains to investigate how intracellular fine structure changes in a high temperature environment. The results showed that 1) both of the human cancer cell lines were more sensitive to high temperature than the normal human cell strains, and a difference between the temperature sensitivity of the human cancer cell lines was also confirmed. 2) There is no clear difference between the manner in which normal human cells and malignant human cells are affected by hyperthermia. 3) Among other cell structures, effects on the membrane system were observed as early changes in cell structure. The mitochondria were particularly affected, followed by the rER. 4) Changes in the nucleoplasm, as well as the nuclear membrane (inner membrane), and then the intranuclear chromatin, etc., were observed as late changes. 5) Changes in mitochondria were observed in the early stage, but temporarily tended to recover, and were then fatally affected again in the late stage. We discuss the relationship between cell proliferation, cell viability, and cell ultrastructure based on the above results.

Identification of a point mutation in the folate receptor gene that confers a dominant negative phenotype.

UM-SCC-38 cells, a squamous cell carcinoma cell line of the head and neck, express limited amounts of folate receptor alpha antigen which is not capable of binding either folic acid or 5-methyltetrahydrofolic acid. Three distinct mutations in the open reading frame of the folate receptor were identified. We now show that the three mutants are nonfunctional with respect to folic acid binding because the protein products do not bind folate. Additionally, a study of MA104 cells (a receptor-positive cell line) transfected with each mutant was done. Expression of one mutant, FR-67, results in a dominant negative phenotype because folate binding is significantly reduced although membrane antigen is significantly increased. Coexpression of FR-67 and the normal protein in MA104 cells also results in large, bright clusters of receptor protein inside the cell around the nucleus when visualized using indirect immunofluorescence. These clusters are not found in cells that express either normal or FR-67 protein alone. In conclusion, this study provides the first evidence of a mutant folate receptor protein capable of affecting normal receptor function in a dominant negative manner.

Carcinogênese química cutânea e subcutânea em gerbil (Meriones unguiculatus) / Chemical cutaneous and subcutaneous carcinogenesis in gerbil (Meriones unguiculatus)

O comportamento do gerbil (Meriones unguiculatus) como modelo em oncologia experimental é estudado. Aplicaçöes tópicas de 3-metilcolantreno (MC), sobre a pele do dorso, induziram pailomas, carcinomas epidermoides, carcinomas basocelulares e lesöes hiperpigmentadas semelhantes ao nevus azul. Ao contr rio, somente lesöes pigmentadas foram observadas nos animais tratados com MC (iniciaçäo) associado ao óleo de croton (promoçäo). Além disoo, fibrossarcomas e um linfoma foram induzidos com injeçöes subcutâneas de MC.

Anti-tumor and differentiation-inducing activity of N,N-dimethylformamide (DMF) in head-and-neck cancer xenografts.

The anti-tumor activity of the putative differentiation-inducing agent dimethylformamide (DMF) was assessed in 7 head-and-neck xenograft (HNX) lines transplanted into nude mice. The drug was administered intra-peritoneally at the maximum tolerated dose. A significant growth-inhibitory effect was observed in 3 out of 7 tumor lines tested. When compared with 5 conventional drugs active in patients with squamous-cell carcinoma of the head and neck (HNSCC), DMF was as effective as the most active drugs (cisplatin and bleomycin). The most sensitive xenograft line, the poorly differentiated tumor HNX-14C, was used to test the hypothesis that differentiation induction might play a role in the anti-tumor activity of DMF. Light microscopic examination did not show clear-cut alteration of differentiation characteristics such as keratin and keratin pearl formation. Furthermore, we used a monoclonal antibody to study the expression of cytokeratin 10 which is useful as a differentiation marker of human HNSCC tumors. Keratin 10, not present in HNX-14C tumors grown under control conditions, became expressed in some cells upon DMF treatment. Further evidence for a differentiation-inducing activity of DMF was found in electron-microscopic studies. In treated HNX-14C tumors, in addition to cells with normal ultrastructural features, better-differentiated cells were observed, as manifested by an increase in the number of tonofilaments and desmosomes. The results show that DMF has a potential value for the treatment of patients with head-and-neck cancer, and that differentiation induction might play a role in the anti-tumor action of the drug.

Mutually antagonistic effects of hydrocortisone and retinyl acetate on envelope competence in cultured malignant human keratinocytes.

Serially propagated SCC-13 keratinocytes, derived from a human squamous cell carcinoma, are greatly influenced by culture conditions in their ability to form ionophore-inducible cross-linked envelopes. Supplementation of the growth medium with fetal bovine serum at concentrations ranging from 0.5 to 20 percent had little effect on competence to form envelopes in confluent cultures. At each serum concentration, however, addition of hydrocortisone to the medium led to an increase in competence of almost fourfold, from approximately 20 to nearly 80 percent. With the serum supplementation held at 5 percent, addition of retinyl acetate to the medium suppressed competence in a concentration-dependent manner over the range of 1 to 100 ng/ml. At the highest concentration employed, competence was reduced over fourfold in the presence of hydrocortisone and virtually eliminated in its absence. When the cells were grown using serum depleted of endogenous vitamin A, a majority were competent in the absence of hydrocortisone. Under this condition, retinyl acetate suppressed competence over fivefold in the absence of hydrocortisone, but not at all in its presence. We conclude that hydrocortisone stimulates envelope competence primarily by antagonizing the suppressive effect of vitamin A. The SCC-13 cell line may prove valuable in studying mechanisms of retinoid and corticosteroid therapeutic action on diseased human keratinocytes.

Inhibition of growth of 3-methylcholanthrene-induced mouse skin tumor by protease inhibitor [N,N-dimethylcarbamoylmethyl 4-(4-guanidinobenzoyloxy)-phenylacetate] methanesulfate.

3-Methylcholanthrene-induced tumor was challenged with a low molecular synthetic protease inhibitor, [N,N-dimethylcarbamoylmethyl 4-(4-guanidinobenzoyloxy)-phenylacetate] methanesulfate. This drug at a dosage of 10 mg/kg or 20 mg/kg was administered by ip injection to 20 mice each harboring a solitary tumor twice daily for 10 weeks. This protease inhibitor significantly inhibited tumor growth and prolonged the survival time of the tumor-bearing mice (P less than 0.001). The results suggest the involvement of kinin-forming proteases, such as trypsin, plasmin and kallikrein, in the tumor growth.