Elevated vimentin expression in buccal mucosal fibroblasts by arecoline in vitro as a possible pathogenesis for oral submucous fibrosis

Areca quid chewing is strongly correlated with oral submucous fibrosis (OSF) in Taiwan. The cytotoxicity of arecoline, a major areca nut alkaloid, on human oral fibroblasts has been extensively studied. To date, however, there has been little research exploring the possible effects of arecoline on cytoskeleton components. In this study, in addition to conducting a cytotoxicity assay, we examine the effect of arecoline on vimentin, an intermediate filament, and its expression in human buccal mucosal fibroblasts on exposure to various levels of arecoline (0-200 microg/ml) for 48 h. At a concentration above 50 microg/ml, arecoline demonstrated dose-dependent cytotoxicity (P<0.05) for cultured fibroblasts. Using sodium dodecyl sulphate-polyacrylamide gel electrophoresis, we demonstrated dose-dependent elevation of 57 kDa cytoskeletal-protein levels for arecoline. Evidence from immunoblotting assay indicated this 57 kDa cytoskeletal protein was vimentin. The increase in vimentin with arecoline exposure corresponded to that noted for fibroblasts cultured from OSF patients. Immunohistochemical assay also revealed that vimentin expression was much higher for OSF specimens than for normal buccal mucosa. We suggest these results may advance understanding of the possible pathogenesis for submucous fibrosis through the transformation of normal buccal mucosa as a result of areca quid chewing.     

Retracted: Arecoline induces epithelial-mesenchymal transformation and promotes metastasis of oral cancer by SAA1 expression

Arecoline, the main alkaloid of areca nut, is well known for its role in inducing submucosal fibrosis and oral squamous cell carcinoma (OSCC), however the mechanism remains unclear. The aim of this study was to establish an arecoline-induced epithelial-mesenchymal transformation (EMT) model of OSCC cells and to investigate the underlying mechanisms. CAL33 and UM2 cells were induced with arecoline to establish an EMT cell model and perform RNA-sequence screening. Luminex multiplex cytokine assays, western blot, and RT-qPCR were used to investigate the EMT mechanism. Arecoline at a concentration of 160 μg/ml was used to induce EMT in OSCC cells, which was confirmed using morphological analysis, transwell assays, and EMT marker detection. RNA-sequence screening and Luminex multiplex cytokine assays showed that many inflammatory cytokines (such as serum amyloid A1 [SAA1], interleukin [IL]-6, IL-36G, chemokine [CCL]2, and CCL20) were significantly altered during arecoline-induced EMT. Of these cytokines, SAA1 was the most highly upregulated. SAA1 overexpression induced EMT and promoted the migration and invasion of CAL33 cells, while SAA1 knockdown attenuated arecoline-induced EMT. Moreover, arecoline enhanced cervical lymph node metastasis in an orthotopic xenograft model of the tongue established using BALB/c nude mice. Our findings revealed that arecoline induced EMT and enhanced the metastatic capability of OSCC by the regulation of inflammatory cytokine secretion, especially that of SAA1. Our study provides a basis for understanding the mechanism of OSCC metastasis and suggests possible therapeutic targets to prevent the occurrence and development of OSCC associated with areca nut chewing.     

Areca nut extract and arecoline induced the cell cycle arrest but not apoptosis of cultured oral KB epithelial cells: association of glutathione, reactive oxygen species and mitochondrial membrane potential

There are 600 million betel quid (BQ) chewers in the world. BQ chewing is a major etiologic factor of oral cancer. Areca nut (AN) and arecoline may inhibit the growth of oral mucosal fibroblasts (OMF) and keratinocytes. In this study, AN extract (100-800 microg/ml) and arecoline (20-120 microM) inhibited the growth of oral KB cells by 36-90 and 15-75%, respectively. Exposure to arecoline (> 0.2 mM) for 24 h induced G(2)/M cell cycle arrest of OMF and KB cells. Areca nut extract (> 400 microg/ml) also induced G(2)/M arrest of KB cells, being preceded by S-phase arrest at 7-h of exposure. No evident sub-G(0)/G(1) peak was noted. Marked retraction and intracellular vacuoles formation of OMF and KB cells were observed. Glutathione (GSH) level, mitochondrial membrane potential (Deltabetam) and H(2)O(2) production of KB cells were measured by flow cytometry. GSH level [indicated by 5-chloromethyl-fluorescein (CMF) fluorescence] was depleted by 24-h exposure of KB cells to arecoline (0.4-1.2 mM) and AN extract (800-1200 microg/ml), with increasing the percentage of cells in low CMF fluorescence. By contrast, arecoline (0.1-1.2 mM) and AN extract (800-1200 microg/ml) induced decreasing and increasing H(2)O(2) production (by 2',7'-dichloro- fluorescein fluorescence), respectively. Hyperpolarization of Deltabetam (increasing of rhodamine uptake) was noted by 24-h exposure of KB cells to arecoline (0.4-1.2 mM) and AN extract (800-1200 microg/ml). AN extract (100- 1200 microg/ml) and arecoline (0.1-1.2 mM) induced little DNA fragmentation on KB cells within 24 h. These results indicate that AN ingredients are crucial in the pathogenesis of oral submucous fibrosis (OSF) and oral cancer by differentially inducing the dysregulation of cell cycle control, Deltabetam, GSH level and intracellular H(2)O(2) production, these events being not coupled with cellular apoptosis.     

Increased tissue inhibitor of metalloproteinase-1 expression and inhibition of gelatinase A activity in buccal mucosal fibroblasts by arecoline as possible mechanisms for oral submucous fibrosis

Oral submucous fibrosis (OSF) is a pre-malignant fibrotic lesion of the mouth in areca quid chewers. It is probably a consequence of disturbances in the hemeostatic equilibrium between synthesis and degradation of extracellular matrix molecules (ECM). To date, there has been little research about the role of tissue inhibitors of metalloproteinase (TIMPs) and matrix metalloproteinases (MMPs) in the pathogenesis of OSF. In the present study, we examined the activity of TIMPs from cells cultured from OSF and normal buccal mucosa. OSF specimens were found to have higher TIMP-1 expression than normal buccal mucosal fibroblasts (BMFs) by Western blots. To verify whether arecoline, a major areca nut alkaloid, could affect TIMP or MMP production by human BMFs, Western blots and gelatine zymography were used. Arecoline was found to elevate TIMP-1 expression at the concentration level under 20 microg/ml in a dose-dependent manner. The amount of TIMP-1 was about 2.7 fold at a concentration level of 10 microg/ml compared with control. From gelatin zymograms, the main gelatinolytic proteinase secreted by the human BMFs was MMP-2, and only minimal amounts of MMP-9 could be detectable from zymogram. In addition, arecoline was found to inhibit MMP-2 secretion and production at the concentration level of 40 microg/ml. The gelatinolytic activity of MMP-2 was about 54% at a concentration level of 80 microg/ml compared with control. Taken together, it was found that arecoline acted not only as an inhibitor on gelatinolytic activity of MMP-2, but also a stimulator for TIMP-1 activity. These synergistic effects may contribute to the ECM components accumulation in the areca quid associated OSF.     

Ripe areca nut extract induces G1 phase arrests and senescence-associated phenotypes in normal human oral keratinocyte

Around 200-600 million Asians chew areca (also called betel), which contains a mixture of areca nut and other ingredients. Epidemiological evidences indicated that areca use is tightly linked to oral carcinogenesis. This study investigated the effects of ripe areca nut extract (ANE) on cultured normal human oral keratinocyte (NHOK). Acute subtoxic ANE treatment inhibited DNA synthesis and induced cell cycle arrest at G1 phase in early passage (< 4th passage) cells. This was accompanied by a slight increase in the sub-G1 cellular fraction. O6-Methylguanine-DNA methyltransferase (MGMT), Hsp27 and p38MAPK was upregulated. p16 and p21 were remarkably upregulated early and declined afterwards. In contrast, the increase of dephosphorylated Rb seemed to be secondary to the episodes of p16 and p21 upregulation. To simulate the chronic areca exposure in vivo, constant ANE treatment in serial NHOK culture was performed. It resulted in a significant decrease in the population doubling, increase in senescence-associated beta-galactosidase (SA-beta-Gal) and decrease in cell proliferation in NHOK of late passages (> or = 4th passage). Induction of senescence-associated phenotypes, G2/M accumulation and genomic instability following long-term ANE treatment were also observed in a low-grade oral carcinoma cell. ANE-treated NHOK also had a higher nuclear factor-kappaB (NF-kappaB) fraction and a lower cytosolic IkappaBalpha level relative to the control in late passages. Moreover, electrophoretic mobility shift assay (EMSA) indicated that ANE treatment shifted the NF-kappaB complex from high mobility position to lower mobility position in late-passaged NHOK. ANE treatment also upregulated IL-6 and cyclooxygenase-2 (COX-2) mRNA expressions in late-passaged NHOK. In summary, our findings suggest that ANE induces the cell cycle arrest at G1/S phase and the occurrence of senescence-associated phenotypes of NHOK. The upregulation of p38MAPK, p16, p21, NF-kappaB, IL-6 and COX-2 are likely to participate in the control of these impacts.     

Areca nut extract treatment down-regulates involucrin in normal human oral keratinocyte through P13K/AKT activation

Areca (betel) is an important etiological factor linked to the high prevalence of oral carcinoma and other oral diseases in South Asians. Involucrin is a key component of the cornified envelop and a differentiation marker of keratinocyte. In this study, we found that 5 microg/ml non-toxic areca nut extract (ANE) treatment resulted in the 0.5-fold down-regulation of involucrin and disruption in involucrin distribution in normal human oral keratinocyte (NHOK). Progressive down-regulation of involucrin during oral carcinogenesis was noted. Activation of AKT by 1.7-fold and up-regulation of COX-2 by 2-fold were elicited following ANE treatment in NHOK. Treatment with PI3K/AKT blockers reverted the down-regulation of involucrin. ANE also down-regulated involucrin by 0.6-fold and disturbed both cornified envelope and cell aggregation in calcium-induced differentiated NHOK. However, such phenomena seemed to be independent from the ANE-associated COX-2 activation. The ANE-associated down-regulation of involucrin through AKT pathway could underlie the areca-associated epithelial pathogenesis.     

Arecoline-mediated inhibition of AMP-activated protein kinase through reactive oxygen species is required for apoptosis induction

Arecoline is the major alkaloid of areca nut (AN) and known to induce reactive oxygen species (ROS) production and apoptosis. The metabolic sensor AMP-activated protein kinase (AMPK), activated by ROS, also regulates apoptosis. This study used several types of cells as the experimental model to analyze the roles of ROS and AMPK in arecoline-induced apoptosis. We found that arecoline dose-dependently increased intracellular ROS level, and two antioxidants, N-acetyl-L-cysteine (NAC) and glutathione, attenuated arecoline-induced apoptotic cell death. Interestingly, arecoline dose- and time-dependently inhibited rather than stimulated AMPK-Thr(172) phosphorylation, and both NAC and glutathione relieved this inhibition. The AMPK activator, 5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR), also restored the phosphorylation level of AMPK-Thr(172) and attenuated apoptotic cell death under arecoline insult. In contrast, the AMPK inhibitor, compound C, and RNA interference of AMPK expression increased the cytotoxicity of arecoline. Collectively, these results suggest that arecoline may inhibit AMPK through intracellular ROS, responsible for the execution of apoptosis.     

The upregulation of heat shock protein 70 expression in areca quid chewing-associated oral squamous cell carcinomas

Heat shock protein 70 (HSP70) is an important stress-induced protein. Areca quid chewing is a major risk factor of oral squamous cell carcinoma (OSCC). The aim of this study was to compare HSP70 expression in normal human oral epithelium and OSCC and further to explore the potential mechanisms that may lead to induce HSP70 expression. 41 OSCC and 10 normal epithelium specimens were examined by immunohistochemistry and analyzed by the clinico-pathological profiles. The oral epithelial cell line GNM cells were challenged with arecoline, a major areca nut alkaloid, by using Western blot analysis. Furthermore, glutathione precursor N-acetyl-l-cysteine (NAC), AP-1 inhibitor curcumin, extracellular signal-regulated protein kinase inhibitor PD98059, and protein kinase C inhibitor staurosporine were added to find the possible regulatory mechanisms. The results from immunohistochemistry demonstrated that HSP70 expression was significantly higher in OSCC specimens (p<0.05). No significant difference in HSP70 expression was observed with respect to age, sex, T category, and stage (p>0.05). The low HSP70 expression was associated with lymph node metastasis (p=0.005). The high HSP70 expression was found in poor differentiated tumor groups (p=0.036). Arecoline was found to elevate HSP70 expression in a dose- and time-dependent manner (p<0.05). The addition of NAC, curcumin, PD98059, and staurosporine markedly inhibited the arecoline-induced HSP70 expression (p<0.05). Taken together, HSP70 expression is significantly upregulated in areca quid chewing-associated OSCC. HSP70 could be used clinically as a marker for tumors possessing the potential for differentiation as well as lymph node metastasis. In addition, arecoline-induced HSP70 expression was downregulated by NAC, curcumin, PD98059, and staurosporine.     

The association between c-fos and c-jun expression and estrogen and progesterone receptors is lost in human endometrial cancer.

In normal human endometrium, expressions of the proto-oncogenes c-fos and c-jun parallel. We have previously shown that the expression of c-jun is related to proliferation and estrogen receptor (ER) status in endometrial epithelial cells. In this study, we analyzed endometrial cancer tissues for c-fos and c-jun messenger RNA (mRNA) expression by Northern blotting. Proto-oncogene expression was compared with ER and progesterone receptor (PR) status and with the proliferation marker Ki-67, as well as with histological grade and the use of hormone-replacement therapy (HRT). Messenger RNA for c-fos was detected in 35 of 37 cancer tissues and mRNA for c-jun in 37 of 40 tissue samples studied. No correlation was observed between the relative mRNA levels of c-fos and c-jun, suggesting distinct control mechanisms, if any, in endometrial cancer. In contrast to normal endometrium, there was no correlation between the proto-oncogene expression and Ki-67, ER or PR immunoreactivity. Neither were there any correlations between c-fos or c-jun expression and the histological grade of the tumor or preceding HRT. Our results reveal the loss of association between proto-oncogene expression and ovarian steroid receptors or cell proliferation in malignant endometrium. This gives further support to the hypothesis that alterations in estrogen and progesterone signalling pathways are involved in the pathogenesis of endometrial cancer.     

人膀胱癌耐药细胞株原癌基因c-jun、c-fos表达与γ-谷氨酰半胱氨酸合成酶及谷胱甘肽的关系

 目的　研究原癌基因c jun和c fos的表达对谷胱甘肽介导的膀胱癌多药耐药的影响。 方法　应用逆转录聚合酶链式反应 (RT PCR)和蛋白免疫印迹法 (WesternBlotting) ,检测c jun、c fos基因在人膀胱癌细胞株BIU87及其阿霉素诱导的多药耐药亚株BIU87/A的表达 ,并分析其与两株细胞的γ 谷氨酰半胱氨酸合成酶 (γ GCS)表达和细胞内谷胱甘肽 (GSH)水平的关系。结果　BIU87/A细胞γ GCSmRNA的相对表达水平明显高于BIU87(P <0 .0 5 ) ,与此相应 ,BIU87/A细胞内GSH水平也高于BIU87(P <0 .0 1) ;c jun基因mRNA及其蛋白在BIU87/A的表达均高于BIU87,而c fos基因在BIU87和BIU87/A的表达未见明显差异 (P >0 .0 5 )。结论　原癌基因c jun上调γ GCS表达和GSH的合成可能是BIU87/A细胞多药耐药形成的重要机制之一 ,而c fos对BIU87/A的耐药形成可能不起主要作用。     

The immediate early gene response to a differentiative stimulus is disrupted by the v-abl and v-ras oncogenes.

The immediate early gene activation in response to a differentiative stimulus was investigated in the hematopoietic progenitor cell line 32DC13(G). A transient and coordinated increase in mRNA levels for c-fos, c-jun, jun-B, TIS-7/PC-4, TIS-8/egr-1, and TIS-11 occurred during the first 2 h of treatment with granulocyte colony stimulating factor (G-CSF), which ultimately induces the 32DC13(G) cells to terminally differentiate into neutrophilic granulocytes. This pattern of mRNA induction was disrupted by v-abl and v-ras, two oncogenes known to interfere with G-CSF-induced differentiation of 32DC13(G) cells. Induction of the mRNAs for c-jun and TIS-7/PC-4 was blocked by the presence of v-abl, whereas v-ras caused constitutive expression of c-fos mRNA and blocked the c-jun, jun-B and TIS-7/PC-4 mRNA response. Release of the differentiation block in the ras-transformed 32DC13(G) cells by co-treatment with retinoic acid and G-CSF partially restored the normal c-fos and c-jun mRNA induction pattern, suggesting that the proper activation of these genes may be important for myeloid differentiation.