Khat induces G1-phase arrest and increased expression of stress-sensitive p53 and p16 proteins in normal human oral keratinocytes and fibroblasts

Khat is a psychostimulant plant used by over 10 million people daily, mainly in eastern Africa and the Middle East. Previous studies have suggested an association between khat use and oral lesions such as hyperkeratosis and oral cancer. This study investigated the effects of an extract of khat on primary normal human oral keratinocytes (NOK) and normal human oral fibroblasts (NOF). Low (sublethal) concentrations of khat inhibited the proliferation of both cell types in a dose-dependent and time-dependent manner. Both NOK and NOF treated with khat accumulated in the G1-phase of the cell cycle and showed increased expression of the stress-sensitive p53 protein after 24 h. Normal human oral keratinocytes showed a profound increase in p16^INK4A (p16) after 24 h and showed morphological changes suggesting cell differentiation. Normal human oral fibroblasts showed growth inhibition and increased expression of p21^WAF1/CIP1 (p21) within 24 h. The concentrations of khat tested in this study were within the range of those found in the oral cavity of khat chewers. The results show that stress induced by khat modulates the cell cycle in oral keratinocytes and fibroblasts. It is further speculated whether khat could have similar effects in vivo , especially in keratinocytes.     

CXCR-4 knockdown by small interfering RNA inhibits cell proliferation and invasion of oral squamous cell carcinoma cells

Background:  Oral squamous cell carcinomas (OSCCs) are characterized by a high degree of local invasion and a high rate of metastases to cervical lymph nodes. Downregulation of CXCR-4 by siRNA inhibits invasion and growth of breast and colon cancer cells. However, there have been no reports on the downregulation of CXCR-4 by small interfering RNA (siRNA) in oral cancer cells.
Methods:  We generated two stable CXCR-4-knockdown clones (KBsi and KOSCC-25Bsi) from the KB and KOSCC-25B OSCC cell lines by lentiviral delivery. In vitro invasion and cell proliferation assays were used to investigate the effect of CXCR-4 downregulation on cell proliferation and invasiveness in KBsi and KOSCC-25Bsi. Immunohistochemistry was performed to evaluate the correlation between CXCR-4 expression and proliferation in 26 OSCC tissue samples.
Results:  CXCR4-knockdown OSCC cells showed reduced invasiveness. The invasiveness of KBsi decreased to 29.5% of the vector-infected controls, and KOSCC-25Bsi decreased to 38.1% of the control vector-infected cells ( P  <   0.05). The CXCR4-knockdown OSCC cells grew significantly slower than the vector-infected control cells. KBsi and KOSCC-25Bsi cells proliferated at 69.5% and 71.7%, respectively, of the rate of control vector-infected cells ( P  <   0.05). CXCR-4-positive group had significantly higher PCNA labeling index than CXCR-4-negative group in OSCC tissue samples.
Conclusion:  These results suggest that the downregulation of CXCR-4 induces anti-proliferative and anti-invasive effects in OSCC and that CXCR-4 might be a useful target molecule for the treatment of OSCC.     

miR-127-3p对口腔鳞状细胞癌细胞增殖、迁移及Zwint-1基因表达的影响

目的 探讨miR-127-3p调控Zeste white 10(ZW10)相互作用着丝粒蛋白1(Zwint-1)对口腔鳞状细胞癌(OSCC)细胞增殖、迁移及侵袭的影响.方法 体外培养OSCC细胞系PE/CA-PJ15、CAL27,对其转染并分为空白对照组(NG组)、阴性转染组(NC组)、过表达miR-127-3p组(m...     

Delivery of cytolethal distending toxin B induces cell cycle arrest and apoptosis in gingival squamous cell carcinoma in vitro

The cytolethal distending toxin (Cdt) from Actinobacillus actinomycetemcomitans consists of three proteins, CdtA, CdtB, and CdtC, which are responsible for cell cycle arrest and apoptosis. In the present study, local delivery systems of recombinant CdtB and CdtB-expressing plasmid were established using Ca9-22, human gingival squamous cell carcinoma cell line. When CdtB was delivered to Ca9-22 cells using a BioPORTER, a 32-kDa protein was detected by Western blotting, and G2 cell cycle arrest and apoptosis occurred. In addition, the CdtB delivered upregulated the expression of phosphorylated p53 and the cyclin-dependent kinase inhibitor p21(CIP1/WAF1) in Ca9-22 cells, suggesting that these intracellular molecules might contribute to the induction of G2 cell cycle arrest and apoptosis. When the CdtB-expressing plasmid was transfected into Ca9-22 cells by lipofection or electroporation, CdtB (32 kDa) was clearly detected. Further, TdT-mediated dUTP nick end labeling positive cells were observed after transfection of the CdtB-expressing plasmid. These findings indicated that delivery of the CdtB protein and transfection of the cdtB gene induced cell cycle arrest and apoptosis in Ca9-22 cells in vitro, and we conclude that it may be possible to induce apoptosis in human gingival squamous cell carcinoma by electroporation of the cdtB gene.     

流体剪切力对大鼠成骨细胞增殖及细胞周期的影响

目的：观察流体剪切力对大鼠成骨细胞增殖及细胞周期的影响。方法：大鼠成骨细胞受强度为13dyne／cm^2的流体剪切力刺激60min,应用四甲基偶氮唑蓝（MTT）法和流式细胞仪检测细胞的增殖能力和细胞周期。结果：流体剪切力作用后,大鼠成骨细胞增殖能力提高,细胞活性增强。加力组S期细胞百分比（14．67±1．18）％,较对照组S期细胞百分比（8．05±1．08）％约增高82．2％（P〈0．01）。结论：流体剪切力具有提高大鼠成骨细胞增殖能力以及增强其细胞活性的作用。     

碱性成纤维细胞生长因子对人牙周膜细胞增殖和细胞周期的影响

目的：探讨碱性成纤维细胞生长因子（bFGF)对体外培养的人牙周膜细胞（PDLC）增殖和细胞周期的影响，且呈剂量依赖性；FCM对细胞周期时相分析结果显示，经bFGF作用后，人PDLC的DNA合成量（S%）显著升高，G1%降低，反映细胞增殖活力的增殖指数PrI值（S G2M）%增高。结论：bFGF能促进人PDLC的增殖和DNA合成，这一作用可能是通过促使处于G1期的PDLC进入S期来实现的。     

Sinuleptolide inhibits proliferation of oral cancer Ca9-22 cells involving apoptosis,oxidative stress,and DNA damage

ObjectiveSinuleptolide, a soft corals-derived bioactive norditerpenoid, is a marine natural product with a potent anti-inflammatory effect. We evaluate the potential anti-oral cancer effects of sinuleptolide and investigate the possible mechanisms involved.DesignsCell viability, cell cycle, apoptosis, reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and DNA damage analyses were performed.ResultsIn a cell viability assay, we found that sinuleptolide is dose-responsively antiproliferative against oral gingival cancer Ca9-22 cells but less harmful to normal human gingival fibroblast (HGF-1) cells (P < 0.001). In cell cycle analysis, sinuleptolide induced subG1 accumulation at a higher dose and led to G2/M arrest of Ca9-22 cells (P < 0.005). Apoptosis was significantly increased in sinuleptolide-treated Ca9-22 cells based on annexin V and poly(ADP-ribose) polymerase (PARP) expressions (P < 0.05–0.0001). Based on flow cytometer analysis, sinuleptolide also induced the generation of ROS and decreased MMP in a dose-responsive manner (P < 0.05–0.0001). DNA damage increased dose-responsively after sinuleptolide treatments (P < 0.001) based on comet and γH2AX assays.ConclusionSinuleptolide can induce an antiproliferation of oral cancer Ca9-22 cells involving apoptosis, oxidative stress and DNA damage, suggesting that sinuleptolide represents a potential chemotherapeutic drug for oral cancer treatment.     

Effects of nicotine on proliferation, cell cycle, and differentiation in immortalized and malignant oral keratinocytes

BACKGROUND: Numerous epidemiological studies have reported that tobacco smoking is a major risk factor for oral cancer, but relatively little is known about the effect of nicotine, a major product of cigarette smoking, on immortalized oral keratinocytes and cancer cells. METHODS: We investigated the effects of nicotine on the growth and differentiation of immortalized human oral keratinocytes (IHOK), primary oral cancer cells (HN4), metastatic oral cancer cells (HN12), and human skin keratinocytes (HaCaT), in the monolayer and in the three-dimensional (3D) raft cultures using the MTT assay, Western blotting, and cell cycle analysis. RESULTS: Nicotine inhibited the proliferation of immortalized and malignant keratinocytes in dose- and time-dependent manners as determined by MTT assay. The 3D organotypic culture showed that nicotine at high concentration (300 microM) inhibits epithelial maturation, surface keratinization, and decreased epithelial thickness. Flow cytometry showed that nicotine inhibited cell cycle progression by inducing G(0)/G(1) arrest of HaCaT, IHOK, HN4, and HN12 cells without causing apoptosis. Nicotine treatment increased p21 expression in immortalized cells (HaCaT, IHOK) and oral cancer cells (HN4, HN12), but decreased pRb and p53 expression in oral cancer cells. Moreover, after high-dose nicotine treatment, the involucrin expression increased markedly in immortalized cells, but not in oral cancer cells. CONCLUSIONS: We demonstrated that nicotine inhibits growth through cell cycle arrest at G(0)/G(1) phase probably by increasing the expression of p21(WAF1/CIP1). Nicotine also affects epithelial differentiation in immortalized and malignant oral keratinocytes. Malignant oral keratinocytes appear to be more resistant to the effects of nicotine on epithelial growth and differentiation as compared to the immortalized cells.     

Cell proliferation activity and the expression of cell cycle regulatory proteins in oral lichen planus

BACKGROUND: In oral lichen planus (OLP), destruction of the basal cell layer, which is one of the characteristic histological features, is seen and many changes in cell proliferation, cell repair and cell death occur in the injured mucosal epithelium. METHODS: We studied mucosal tissues from 19 patients of OLP and 10 controls, with immunohistochemistry for Ki-67, p53, cyclin dependent kinase inhibitors (CDKI) and cyclins. Mitotic count was calculated. TUNEL assay was also performed for evaluation of apoptotic cell death. RESULTS: Mitotic count, Ki-67 and cyclin D1 labeling indices in the basal and parabasal layers of OLP mucosa were elevated in comparison with those of controls. p53, p21Cip1 and TUNEL indices of OLP mucosa were also increased. CONCLUSIONS: These complex changes, which concomitantly occur in the injured mucosal epithelium, could contribute to the development and maintenance of characteristic mucosal epithelial architectures seen in OLP.     

Porphyromonas gingivalis-induced autophagy suppresses cell proliferation through G1 arrest in oral cancer cells

Objectives

We investigated the response of oral cancer cells to intracellular invasion of Porphyromonas gingivalis to define changes in the biological characteristics of oral cancer cells evoked by the presence of oral pathogenic bacteria within a tumour microenvironment.

Designs

The proliferative activity, cell cycle, and autophagic response were evaluated in oral cancer cells infected with P. gingivalis 381. ROS generation was detected in these cells by DCFDA assay, and its role in the responses of oral cancer cells to P. gingivalis infection was further investigated.

Resutls

P. gingivalis inhibited proliferation of oral cancer cells by inducing G1 cell cycle arrest, but had no effect on apoptosis. Following infection with P. gingivalis, the expression of cyclin D1 and cdk4 was decreased in oral cancer cells, whereas p21, a Cdk inhibitor, was upregulated, in comparison with non-infected controls. Autophagy was prominently enhanced in these infected cells, presumably contributing to the suppressed proliferation. Further experiments revealed that such autophagic response was activated by the formation of reactive oxygen species, as evidenced by the lack of autophagic response and cell proliferation upon removal of reactive oxygen species.

Conclusions

These findings provide a novel insight into the mechanism by which cancer cells are influenced by tumour microenvironment including oral bacteria.     

1,25(OH)2Vitamin D3 induces elevated expression of the cell cycle inhibitor p18 in a squamous cell carcinoma cell line of the head and neck

BACKGROUND: 1Alpha,25-dihydroxyvitamin D(3) [1,25(OH)(2) Vitamin D(3)] induces growth inhibition in squamous cell carcinoma (SCC) cell lines of the head and neck by arresting the cells in the G0/G1 phase of the cell cycle, probably due to an enhanced expression of p21, which could be demonstrated in other cell lines (JPPA, SCC9) before. In SCC25, a SCC cell line isolated from tongue, growth inhibition but no overexpression of p21 was detected. The retinoblastoma gene, as a direct target of G1 cyclin-CDK complexes, showed an obvious shift from the hyperphosphorylated to the hypophosphorylated form under 1,25(OH)(2)Vitamin D(3), which indicates that the growth inhibition takes place in the G0/G1 phase. To explore the possible pathway of growth inhibition in SCC25 we investigated other cell cycle inhibitors (p18, p19, p27). METHODS: Synchronized cells were treated with 1,25(OH)(2)Vitamin D(3) over 96 h. The cell cycle status and expression of cell cycle-regulating proteins was determined by fluorescence-activated cell sorting (FACS) and Western blotting. An overexpression of p18 in 1,25(OH)(2)Vitamin D(3) vs. ethanol-treated cells was determined until 30 h in SCC25. No influence was detectable on the expression of p27 and p19. CONCLUSION: One mechanism by which 1,25(OH)(2)Vitamin D(3) controls cell growth might be the upregulation of p21. As p21 was unsusceptible to 1,25(OH)(2)Vitamin D(3) in SCC25, other inhibiting proteins were necessary to be tested. The proven upregulation of p18 seems to be the responsible step for growth inhibition of 1,25(OH)(2)Vitamin D(3) in SCC25.     

生物钟基因Per1对人口腔鳞状细胞癌细胞增殖、凋亡、细胞周期和体内成瘤的影响及机理

目的 探讨生物钟基因Per1对细胞周期相关基因的调控作用,以及对人口腔鳞状细胞癌细胞SCC15增殖、凋亡、细胞周期和体内成瘤的影响。方法 构建3组针对Per1 RNA的短发夹RNA（shRNA）慢病毒重组质粒,转染SCC15细胞,用蛋白质印迹法（Western blot）及实时荧光定量PCR（qRT-PCR）检测筛选RNA干扰作用最强组为实验组,对照组（Control-shRNA）为对任何基因均无干扰效应的shRNA序列的质粒,空白组为未做任何处理的SCC15细胞。用qRT-PCR检测各组细胞中细胞周期相关基因Per1、p53、Cyclin D1、Cyclin E、Cyclin A2、Cyclin B1、CDK1、CDK2、CDK4、CDK6、p16、p21、Wee1、cdc25、E2F、Rb1 mRNA的表达;用流式细胞仪检测各组细胞增殖、凋亡和细胞周期分布;将实验组和空白组细胞分别接种于裸鼠背部皮下,观察成瘤情况。结果 成功构建了3组Per1-shRNA慢病毒质粒,通过qRT-PCR和Western blot证明Per1-shRNA-Ⅰ组沉默效果最佳,作为实验组。Per1-shRNA-Ⅰ组中Cyclin D1、Cyclin E、Cyclin B1、CDK1和Wee1 mRNA的表达水平高于Control-shRNA组和SCC15组（P<0.05）,p53、Cyclin A2、p16、p21和cdc25 mRNA的表达水平降低（P<0.05）;Control-shRNA组和SCC15组中各基因mRNA的表达水平无差异（P>0.05）。CDK2、CDK4、CDK6、E2F和Rb1 mRNA的表达水平在3组中均无统计学差异（P>0.05）。Per1-shRNA-Ⅰ组中细胞增殖指数高于Control-shRNA组和SCC15组（P<0.05）,凋亡指数降低（P<0.05）,S期的细胞数降低（P<0.05）,G2/M期的细胞数增加（P<0.05）。Control-shRNA组和SCC15组细胞的增殖指数和凋亡指数无统计学差异（P>0.05）。Per1-shRNA-Ⅰ组细胞体内成瘤能力显著增强（P<0.05）。结论 生物钟基因Perl是重要的抑癌基因,Perl能调控下游众多的细胞周期基因,其表达变化影响细胞周期进程、增殖和凋亡的平衡失调及体内成瘤能力,对Per1深入研究有可能进一步明确癌症的发生发展机制,为癌症的治疗提供新的有效分子靶点。     

Expression of p21 (Waf1/Cip1) in head and neck cancer in relation to proliferation, differentiation, p53 status and cyclin D1 expression

p21 (Waf1/Cip1) is a critical downstream effector in the p53-dependent pathway of growth control and causes growth arrest through inhibition of cyclin-dependent kinases. In this study 67% of 43 head and neck squamous cell carcinoma (HNSCC) and 60% of 15 tumour-adjacent oral dysplasias overexpressed p21 by immunohistochemical staining. Overexpression of p21 in HNSCC was independent of the presence of functional p53, as assessed by analysis of mutations and loss of heterozygosity and by immunohistochemistry. Rather, the expression pattern of p21 was associated with differentiation. Furthermore, in most tumours, the p21 positive cells did not incorporate bromodeoxyuridine (BrdU), which indicates inhibition of proliferation by p21 in these cells. In some tumours, p21 was also expressed in proliferating cells. In these latter tumour cells, cyclin D1 was frequently expressed as well. Therefore, we suggest that expression of cyclin D1 might overcome the inhibitory effect of p21 in these cells.     

circ_0005379通过调控miR-17-5p/酰基辅酶A氧化酶1轴抑制口腔鳞状细胞癌的发展进程

探讨circ_0005379对口腔鳞状细胞癌（OSCC）细胞增殖、凋亡、迁移和侵袭的影响及作用机制。     

沉默Icmt基因对舌鳞癌CAL-27和SCC-4细胞增殖、凋亡及细胞周期的影响

目的:探讨异戊二烯基半胱氨酸羧基甲基转移酶(isoprenylcysteine carboxyl methyltransferase,Icmt)对舌鳞癌CAL-27和SCC-4细胞增殖、凋亡和细胞周期的影响及其相关机制.方法:针对人Icmt基因序列设计并构建3条小干扰RNA(small interfering RNA,...     

Lnc RNA KCNQ1OT1靶向miR-506-3p调控舌鳞状细胞癌细胞增殖、侵袭和迁移的机制研究

目的 探讨长链非编码RNA(Lnc RNA)KCNQ1OT1通过靶向调控miR-506-3p对舌鳞状细胞癌(tongue squamous cell carcinomas,TSCC)细胞增殖、侵袭和迁移作用机制.方法 实时荧光定量PCR(RT-qPCR)检测正常牙龈上皮细胞(human gin-gival epithe...