cyclin-d1 and Neoplasms--Squamous-Cell

This study investigated the effect of RhoA silencing through RNA interference on proliferation and growth of tongue cancer cells, as well as explored the possible mechanisms of this effect.. SSC-4 tongue cancer cells were cultured in vitro and then transfected with small interfering RNA to knock down RhoA expression. The tested cells were divided into three groups: experimental group (experimental group 1: transfected with RhoA-siRNA-1; experi-mental group 2: transfected with RhoA-siRNA-2), negative control group (transfected by random sequence NC-siRNA), and blank control group (transfected with Lipofectamine). The expression levels of RhoA mRNA were respectively measured by quantitative real-time polymerase chain reaction and western blot assay. Moreover, the expression levels of cyclin D1, p21, and p27 and RhoA protein were evaluated by Western blot assay. Proliferation and growth potentiality were analyzed through evaluation of doubling times and methyl thiazolyl tetrazolium assessment.. The expression levels of RhoA gene and protein of experimental groups significantly decreased following siRNA transfection compared with those in the negative and blank control groups. The expression of cyclin D1 decreased significantly and that of p21 and p27 increased significantly. The doubling time was extended and the growth potentiality decreased.. The results indicated that RhoA silencing can inhibit proliferation of tongue cancer cells, whereas RhoA affects cell proliferation by regulating the cell cycle pathway. Thus, RhoA is a potential target in gene therapy for tongue cancer.. 目的 通过RNA干扰技术沉默RhoA基因从而探讨RhoA对舌癌细胞增殖和生长的影响及其作用机制。 方法 体外培养舌鳞状细胞癌SCC-4细胞，以小分子干扰RNA转染沉默RhoA基因的表达。实验分为3组：实验组（又分为实验1组和实验2组，脂质体分别转染对应序列1的RhoA-siRNA和序列2的RhoA-siRNA）、阴性对照组（脂质体转染NC-siRNA）和空白对照组（不转染siRNA）。采用实时定量聚合酶链反应技术检测SCC-4细胞转染后RhoA mRNA的表达，Western blot检测RhoA、Cyclin D1、p21和p27蛋白的表达，四唑盐比色法检测舌癌细胞生长水平和倍增时间。 结果 与阴性对照组和空白对照组相比，实验组舌癌细胞的RhoA基因及蛋白表达降低，p21、p27蛋白表达升高，Cyclin D1蛋白表达降低，细胞倍增时间延长，增殖能力降低（P<0.05）。 结论 沉默RhoA基因可以抑制舌癌细胞的增殖和生长，RhoA基因通过调控细胞周期信号转导途径影响舌癌细胞的增殖，RhoA基因可以成为舌癌基因治疗的靶点。.

Topics: Cell Line, Tumor; Cell Proliferation; Cyclin D1; Gene Silencing; Humans; Neoplasms, Squamous Cell; Real-Time Polymerase Chain Reaction; rhoA GTP-Binding Protein; RNA, Messenger; RNA, Small Interfering; Tongue Neoplasms; Transfection

Photodynamic therapy (PDT) is widely used to treat non-melanoma skin cancer. However, some patients affected with squamous cell carcinoma (SCC) do not respond adequately to PDT with methyl-δ-aminolevulinic acid (MAL-PDT) and the tumors acquire an infiltrative phenotype and became histologically more aggressive, less differentiated, and more fibroblastic. To search for potential factors implicated in SCC resistance to PDT, we have used the SCC-13 cell line (parental) and resistant SCC-13 cells obtained by repeated MAL-PDT treatments (5th and 10th PDT-resistant generations). Xenografts assays in immunodeficient mice showed that the tumors generated by resistant cells were bigger than those induced by parental cells. Comparative genomic hybridization array (aCGH) showed that the three cell types presented amplicons in 3p12.1 CADM2, 7p11.2 EFGR, and 11q13.3 CCND1 genes. The 5th and 10th PDT-resistant cells showed an amplicon in 5q11.2 MAP3K1, which was not present in parental cells. The changes detected by aCGH on CCND1, EFGR, and MAP3K1 were confirmed in extracts of SCC-13 cells by reverse-transcriptase PCR and by western blot, and by immunohistochemistry in human biopsies from persistent tumors after MAL-PDT. Our data suggest that genomic imbalances related to CCND1, EFGR, and particularly MAP3K1 seem to be involved in the development of the resistance of SCC to PDT.

Topics: Aged, 80 and over; Aminolevulinic Acid; Animals; Biopsy; Cell Line, Tumor; Comparative Genomic Hybridization; Cyclin D1; Drug Resistance, Neoplasm; ErbB Receptors; Female; Humans; Male; MAP Kinase Kinase Kinase 1; Mice, Nude; Neoplasm Invasiveness; Neoplasms, Squamous Cell; Photochemotherapy; Photosensitizing Agents; Skin Neoplasms; Xenograft Model Antitumor Assays

Cancer is a public health problem in the world accounting for most of the deaths. Currently, common treatment of cancer such as chemotherapy works by killing fast-growing cancer cells. Unfortunately, chemotherapy cannot tell the difference between cancer cells and fast-growing healthy cells, including red and white blood cells. As a result, one of the most serious potential side effects of some types of chemotherapy is a low white blood cell count that makes it unreliable (Parkin et al. [34]; Pauk et al. [3]). Even though intense research has been going on in recent years, successful therapeutic targets against this disease have been elusive. In this study, we evaluate the anti-proliferative activity of Euphorbia mauritanica and Kedrostis hirtella in lung cancer. In our assessment it was observed that E. mauritanica and K. hirtella were able to induce cell death at 5 μg/ml in A549 cells over 22 h and at 10 μg/ml over 24 h in the Lqr1 cell line. Molecular analysis of DNA fragmentation and Annexin V were used to examine the type of cell death induced by E. mauritanica and K. hirtella extracts. These results showed an increase in necrotic and apoptotic characteristics with both nuclear DNA fragmentation and smear. Therefore, these results suggest that E. mauritanica and K. hirtella may play a role in inducing cell death in lung cancer cells. However, further studies need to be conducted to ascertain these results.

Topics: Apoptosis; Carrier Proteins; Cell Line, Tumor; Cell Proliferation; Cell Survival; Chromatography, Thin Layer; Chrysobalanaceae; Cyclin D1; DNA Fragmentation; DNA-Binding Proteins; Drug Screening Assays, Antitumor; Euphorbia; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Necrosis; Neoplasms, Squamous Cell; Plant Extracts; Staurosporine; Tumor Suppressor Protein p53; Ubiquitin-Protein Ligases

Head and neck cancer is the sixth most common cancer worldwide and laryngeal cancer represents the largest subgroup. However, the molecular mechanism underlying its malignant behavior and progression is not clarified. Accumulating evidence has shown that Notch1 signaling pathway plays a central role in carcinogenesis, but its potential role in regulating the development of laryngeal carcinoma, has not been characterized. Here, we identified that Notch1 signaling pathway was activated in laryngeal carcinoma accompanied with up-regulation of Notch1 and Hes1 expression. Overexpression of Notch1 in laryngeal carcinoma cell line Hep-2 led to suppression of tumor cellular proliferation and arrested cell cycle in the G0/G1 phase and induced cell apoptosis, which were coupled with the down-regulation of cyclin D1, cyclin E, cdk2 and bcl-2 and up-regulation of caspase-3, caspase-9 and p53. Most importantly, up-regulation of Notch1 expression also reduced the migration of Hep-2 cells, which was closely associated with down-regulation of MMP-2 and MMP-9. The finding may lay a foundation for further investigations into the Notch1 signaling pathway as a potential target for laryngeal carcinoma.

Topics: Apoptosis; Basic Helix-Loop-Helix Transcription Factors; Blotting, Western; Caspase 3; Caspase 9; Cell Cycle; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase 2; Disease Progression; Flow Cytometry; Gene Expression Regulation, Neoplastic; Homeodomain Proteins; Humans; Laryngeal Neoplasms; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Neoplasms, Squamous Cell; Proto-Oncogene Proteins c-bcl-2; Receptor, Notch1; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Transcription Factor HES-1; Transfection; Tumor Suppressor Protein p53

To clarify the relationship between cyclin D1 and cisplatin resistance of Tca8113/cis diamminedichloroplatinum (CDDP) in vitro and in vivo.. We applied the transfection method with plasmids pcDNA3.1-antisense-cyclin D1 by Lipofectamine 2000. Tca8113/CDDP cells were used as control. MTT assay was used to identify the proliferation and sensibility of those cells to cisplatin. Subsequently, 18 nude mice were subcutaneously injected by those cells and divided into 3 groups with 6 mice in each group. Every mouse was treated by cisplatin with 5 mg . kg(-1) . d(-1) for 5 days. The sizes of tumor were measured every other day and were described with the growth curves. After 20 days, tumors were anatomized and weighed. The tumor inhibition ratios were calculated and the HE slides were observed to determine the cell sensibility to cisplatin.. The transfected cells with pcDNA3.1-antisense-cyclin D1grew more slowly than other cells and showed higher sensibility to cisplatin in vitro. The tumors developed by cells with pcDNA3.1-antisense-cyclin D1 were smaller than the The tumor inhibition ratio was 74% (P < 0.05). The necrosis area was larger in the tumors developed by the transfected cells with pcDNA3.1-antisense-cyclin D1 than other groups.. Antisense oligonucleotides of cyclin D1 can improve the sensibility of Tca8113/CDDP cells to cisplatin and inhibit the growth of tumors.

Topics: Animals; Cell Line, Tumor; Cisplatin; Cyclin D1; Drug Resistance, Neoplasm; Female; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Mouth Neoplasms; Neoplasms, Squamous Cell; Oligonucleotides, Antisense

Integrin-linked kinase (ILK) functions in cooperative integrin-growth factor receptor-mediated signaling to control cell survival and proliferation. The effect of tyrosine kinase (tk) inhibition of the epidermal growth factor receptor (EGFR) on radiation survival and growth was evaluated in human FaDu squamous cell carcinoma cells expressing different forms of ILK.. ILK-wild-type (wk) and -hyperactive kinase (hk) transfected cells were grown on fibronectin (Fn) under serum presence or depletion, irradiated (0-6Gy) and/or treated with the EGFR-tk inhibitor BIBX1382BS.. ILK-wk and -hk transfectants showed significant radiosensitization compared to vector control cells. Antisurvival and antiproliferative effects of EGFR-tk inhibition plus/minus irradiation were counteracted by adhesion to Fn relative to the control substratum, poly-L-lysine. Similar to vector controls, ILK transfectants exhibited a strong decrease in cell proliferation but no enhanced radiation sensitivity after EGFR-tk inhibition. This decrease was accompanied by changes in cyclin D1 and phosphorylated MAPK persisting to day 10 following transient drug exposure.. Our data demonstrate a prosurvival role of adhesion and an antisurvival role of ILK upon irradiation. Inhibition of EGFR-tk using BIBX1382BS does not affect the intrinsic cellular radiosensitivity of cells grown on fibronectin. Thus, simultaneous targeting of adhesion and growth factor receptor-mediated signaling might potently improve anticancer strategies.

Topics: Cell Adhesion; Cell Growth Processes; Cell Survival; Cyclin D1; DNA Damage; DNA, Neoplasm; ErbB Receptors; Fibronectins; G1 Phase; Humans; Mitogen-Activated Protein Kinases; Neoplasms, Squamous Cell; Organic Chemicals; Protein Serine-Threonine Kinases; Transfection

Head and neck squamous cell carcinoma (HNSCC) has a relatively high mortality rate and poor prognosis. Recently, we showed that overexpression of phosphorylated (p) nuclear factor-kappaB (NF-kappaB) in squamous cell carcinoma of the tonsil (SCCT) and high grade dysplasia is associated with a poor prognosis. Because the mammalian target of the rapamycin (mTOR) pathway contributes to the activation of NF-kappaB through immunophilin/mTOR signaling, we investigated: (a) the immunohistochemical expression and state of activation and potential clinical significance of components of the mTOR signal transduction pathway in SCCT patients (morphoproteomics); and (b) the inhibitory effects of rapamycin on the growth and state of activation of mTOR in 2 HNSCC cell lines (pharmacoproteomics). Archival biopsy materials from 39 patients with SCCT were studied by immunohistochemistry for the expression of p-mTOR (Ser 2448), and p-p70S6K (Thr 389), and/or cyclin D1. Results for SCCT were compared with adjacent non-neoplastic epithelium, when present, and with normal tonsillar epithelium from approximately age-matched controls; clinical outcomes were also assessed. SCCT showed mTOR (Ser 2448) expression in 93% (30/32 cases) with 2+ or 3+ plasmalemmal and/or cytoplasmic intensity in 84% vs 42% in surface epithelium from normal tonsils (p <0.001). The mean combined expression score (signal intensity x percentage of positive cells) for p-p70S6K was significantly greater in the SCCT group vs adjacent non-neoplastic squamous epithelium and normal tonsillar epithelium of the control group (p <0.05). A relationship existed between higher p-p70S6K expression levels in the non-neoplastic squamous epithelium adjacent to the SCCT and increased risk of death from disease (hazard ratio = 7.9; 95% confidence interval (CI) = 2.1 to 29.9; p = 0.002). There was also a relationship between nuclear expression of cyclin D1 in SCCT and shortened recurrence-free survival (p = 0.015). Two human HNSCC cell lines, SCC-15 and FaDu, were incubated with and without rapamycin to assess its impact on growth and on the expression of p-mTOR. Rapamycin in a dose-dependent fashion inhibited growth more in SCC-15, which correlated with a greater reduction in constitutively activated p-mTOR (Ser 2448) as shown by Western blotting. In conclusion, these morphoproteomic and pharmacoproteomic data collectively provide a rationale for selecting mTOR effectors as therapeutic targets in HNSCC.

Topics: Antineoplastic Agents; Cell Line, Tumor; Cyclin D1; Humans; Immunohistochemistry; Neoplasms, Squamous Cell; Protein Kinases; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; Sirolimus; Tonsillar Neoplasms; TOR Serine-Threonine Kinases

Squamous cell carcinoma (ESCC) is the most frequent histological subtype in esophageal cancer, although the incidence of esophageal adenocarcinoma (EAC) is increasing faster than any other malignancy in the western world. New developments in the understanding of molecular mechanisms in esophageal cancer comprise analysis of the genetic tumor profiles by CGH (comparative genomic hybridization), the detection of tumor suppressor gene inactivation, and the analysis of proto-oncogenes. Especially the inactivation of the p53 gene proved to be of particular importance for the development of esophageal cancer. Also p15 and p16 have been identified to be involved in the pathogenesis of esophageal cancer by influencing the cyclin kinase inhibitor cascade and DNA mismatch repair processes. Amplification of cyclin D1 results in growth advantage for tumor cells and enhances tumorigenesis; gene amplification and overexpression of cyclin D1 were frequently demonstrated especially in ESCC. Regarding the dysplasia-metaplasia-carcinoma sequence of Barrett's esophagus, inhibition of apoptosis by overexpression of bcl-2 proteins occurs as an early event.

Topics: Adenocarcinoma; Barrett Esophagus; Biomarkers, Tumor; Cyclin D1; Esophageal Neoplasms; Gene Expression Regulation, Neoplastic; Growth Substances; Humans; Molecular Biology; Neoplasms, Squamous Cell; Risk Factors; Smoking; Tumor Suppressor Proteins

Squamous cancers of the oral cavity and esophagus are common worldwide, but no good genetically based animal model exists. A number of environmental factors as well as genetic alterations have been identified in these cancers, yet the specific combination of genetic events required for cancer progression remains unknown. The Epstein-Barr virus ED-L2 promoter (L2) can be used to target genes in a specific fashion to the oral-esophageal squamous epithelium. To that end, we generated L2-cyclin D1 (L2D1(+)) mice and crossbred these with p53-deficient mice. Whereas L2D1(+) mice exhibit a histologic phenotype of oral-esophageal dysplasia, the combination of cyclin D1 expression and p53 deficiency results in invasive oral-esophageal cancer. The development of the precancerous lesions was significantly reversed by the application of sulindac in the drinking water of the L2D1(+)/p53(+/-) mice. Furthermore, cell lines derived from oral epithelia of L2D1(+)/p53(+/-) and L2D1(+)/p53(-/-) mice, but not control mice, formed tumors in athymic nude mice. These data demonstrate that L2D1(+)/p53(+/-) mice provide a well-defined, novel, and faithful model of oral-esophageal cancer, which allows for the testing of novel chemopreventive, diagnostic, and therapeutic approaches.

Topics: Animals; Antineoplastic Agents; Cyclin D1; Disease Models, Animal; ErbB Receptors; Esophageal Neoplasms; Genotype; Herpesvirus 4, Human; Humans; Male; Mice; Mice, Inbred C57BL; Mice, Nude; Mice, Transgenic; Mouth Neoplasms; Neoplasms, Squamous Cell; Promoter Regions, Genetic; Sulindac; Tumor Cells, Cultured; Tumor Suppressor Protein p53

We previously described the oral-esophageal tissue-specific expression of cyclin D1 with the Epstein-Barr virus ED-L2 promoter in transgenic mice, and resulting dysplasia. Given the evidence for an interplay between environmental and genetic factors in esophageal squamous carcinogenesis, the aim of this study was to determine the potential cooperation of the nitrosamine compound N-nitrosomethylbenzylamine (NMBA), an esophageal specific carcinogen, in the cyclin D1 transgenic mice. NMBA was first demonstrated to induce dysplasia in two strains of inbred mice, C57BL/6 and FVB/N. Subcutaneous NMBA was then administrated to wild type and transgenic mice beginning at 4 weeks of age. Mice were monitored for the duration of the study for general appearance, activity and weight, and were euthanized at 12 and 15 months. Histopathologic analysis revealed increased severity of dysplasia in cyclin D1 mice treated with NMBA compared with treated age-matched wild-type mice and untreated mice. There was also increased proliferating cell nuclear antigen (PCNA) expression in the esophagi of NMBA treated cyclin D1 mice. Taken together, these findings suggest that a genetic alteration, specifically cyclin D1 overexpression and a chemical carinogen, NMBA, may cooperate to increase the severity of esophageal squamous dysplasia, a prominent precursor to carcinoma.

Topics: Animals; Carcinogens; Cell Division; Cyclin D1; Dimethylnitrosamine; Epithelium; Esophageal Neoplasms; Gene Expression; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neoplasms, Squamous Cell