Ubiquitin-specific protease 14 regulates cell proliferation and apoptosis in oral squamous cell carcinoma

Ubiquitin-specific protease 14, a deubiquitinating enzyme, has been implicated in the tumorigenesis and progression of several cancers, but its role in oral squamous cell carcinoma remains to be elucidated. The aim of this study was to explore the expression pattern and roles of Ubiquitin-specific protease 14 in the occurrence and development of oral squamous cell carcinoma. Interestingly, Ubiquitin-specific protease 14 was overexpressed in oral cancer tissues and cell lines at both mRNA and protein levels. b-AP15, a specific inhibitor of Ubiquitin-specific protease 14, significantly inhibited the growth of cancer cells and increased cell apoptosis in a dose-dependent manner. Moreover, knockdown of Ubiquitin-specific protease 14 by shRNA significantly inhibited the proliferation and migration of cancer cells in vitro. Finally, using a xenograft mouse model of oral squamous cell carcinoma, knockdown of Ubiquitin-specific protease 14 markedly inhibited tumor growth and triggered the cancer cell apoptosis in vivo, supporting previous results. In conclusion, for the first time we have demonstrated the expression pattern of Ubiquitin-specific protease 14 in oral squamous cell carcinoma and verified a relationship with tumor growth and metastasis. These results may highlight new therapeutic strategies for tumor treatment, application of Ubiquitin-specific protease 14 selective inhibitor, such as b-AP15, or knockdown by shRNA. Collectively, Ubiquitin-specific protease 14 could be a potential therapeutic target for oral squamous cell carcinoma patients.     

Activation of Toll-like receptor 3 induces apoptosis of oral squamous carcinoma cells in vitro and in vivo

Toll-like receptors are well known as molecular sensors of pathogen-associated molecular patterns. They control activation of the innate immune response and subsequently shape the adaptive immune response. Recent publications have demonstrated that Toll-like receptors also play important roles in multiple human cancers, yet their function in oral squamous cell carcinoma remains unclear. In this study, we showed that both oral squamous cell carcinoma cell lines and tissues from oral squamous carcinoma patients express relatively high levels of Toll-like receptor 3. We also found that synthetic dsRNA-polyinosinic-polycytidilic acid, a Toll-like receptor 3 ligand, induced apoptosis of oral squamous carcinoma cells mainly via Toll-like receptor 3, through interferon-β production and activation of caspases 3 and 9. Moreover, in an oral squamous cell carcinoma xenograft mouse model, we demonstrated for the first time that activation of Toll-like receptor 3 inhibited oral squamous cell carcinoma tumor growth in vivo. Therefore, the direct proapoptotic activity of Toll-like receptor 3 in human oral squamous carcinoma cells may make this protein a viable therapeutic target in the treatment of oral squamous cell carcinoma.     

The stem cell code in oral epithelial tumorigenesis: ‘The cancer stem cell shift hypothesis’

Tumors of the oral cavity provide an ideal model to study various stages of epithelial tumor progression. A group of cancer cells termed cancer stem cells (CSCs) eludes therapy, persists and initiates recurrence augmenting malignant spread of the disease. Hitherto, accurate identification and separation of such minimal residual cells have proven futile due to lack of identifiable traits to single out these cells from the heterogeneous tumor bulk. In this review we have compiled comprehensive evidence from comparative phenotypic and genotypic studies on normal oral mucosa as well as tumors of different grades to elucidate that differential expression patterns of putative stem cells markers may identify ‘minimal residual disease’ in oral squamous cell carcinoma. We propose the “cancer stem cell shift hypothesis” to explain the exact identity and switch-over, tumor-promoting mechanisms adapted by putative CSCs with correlation to tumor staging.     

芳姜黄酮对人皮肤鳞状细胞癌A431细胞迁移、侵袭及凋亡的影响和机制研究

为研究芳姜黄酮(Ar-Turmerone)对人鳞状细胞癌A431细胞增殖、迁移、侵袭和凋亡的影响及机制。实验采用CCK-8法检测抑制率,吉姆萨染色观察细胞形态,划痕实验和Transwell小室实验研究细胞迁移和侵袭能力的变化,流式细胞仪检测细胞凋亡率。此外,通过实时荧光定量聚合酶链反应(Real-time PCR)与蛋白质印迹法(western blot)法检测mRNA和蛋白表达。siRNA阻断Notch1,Hes1和PTEN,检测相应的下游mRNA和蛋白的表达变化,流式细胞仪检测细胞凋亡率。结果发现,芳姜黄酮可以抑制A431细胞增殖,使细胞形态发生改变,抑制细胞体外迁移和侵袭能力,促进细胞凋亡。经过芳姜黄酮处理后,Notch1,Hes1,PTEN的mRNA和蛋白表达升高。沉默Notch1,Hes1 mRNA和蛋白表达低于单纯给药组,而沉默Hes1,PTEN mRNA和蛋白表达也低于单纯给药组;沉默PTEN后,与单纯给药组相比,细胞死亡率降低。总之,芳姜黄酮可以抑制人鳞状细胞癌A431细胞的增殖并促进其凋亡,且具有抑制体外迁移和侵袭的作用,其促进细胞凋亡的机制是通过Notch1/Hes1/PTEN途径实现的。     

MicroRNA-181a enhances the chemoresistance of human cervical squamous cell carcinoma to cisplatin by targeting PRKCD

MicroRNAs(miRNAs) are involved in regulating the response of cancer cells to various therapeutic interventions, but their involvement in the chemoresistance of human cervical squamous cell carcinoma is not fully understood. We found miR-181a was significantly up-regulated in specimens from patients with chemoresistant cervical squamous cell carcinoma. In this study, we aimed to clarify the role of miR-181a in regulating the chemoresistance of cervical cancer. Two human cervical squamous cancer cell lines, SiHa and Me180, were used. Enforced expression of miR-181a enhanced chemoresistance to cisplatin in cervical cancer cells through apoptosis reversion. In a nude mouse xenograft model, the overexpression of miR-181a markedly inhibited the therapeutic response to cisplatin. PRKCD, a target gene of miR-181a and a promoter of apoptosis, was negatively regulated by miR-181a. We found that the effect of miR-181a on chemoresistance was mediated by PRKCD. Additionally, silencing of PRKCD yielded an effect similar to that of miR-181a up-regulation and inhibited apoptosis in cervical cancer cells. Our findings suggest that miR-181a may function as an oncogene and induce chemoresistance in cervical squamous cell carcinoma cells at least in part by down-regulating PRKCD, thus may provide a biomarker for predicting chemosensitivity to cisplatin in patients with cervical squamous cancer.     

紫铆因对食管鳞癌细胞增殖和存活影响的初步研究

研究紫铆因对人食管鳞癌细胞增殖和存活的影响。通过MTS和软琼脂集落实验检测紫铆因对食管鳞癌增殖的抑制,生化分析仪检测紫铆因对食管鳞癌糖酵解的影响,并利用免疫印迹检测紫铆因对食管鳞癌细胞增殖和凋亡激活相关蛋白分子的表达。结果发现紫铆因剂量依赖性抑制KYSE150和Eca109细胞增殖,下调EGFR信号通路活化,抑制HK2的表达及糖酵解。一定浓度的紫铆因能诱导食管鳞癌细胞发生凋亡,caspase3和PARP被剪切,Bcl-2和Mcl-1表达下调,但Bcl-XL未见明显改变。结果证明紫铆因抑制食管鳞癌的增殖,可能与EGFR信号通路和糖酵解被抑制,及促存活蛋白Bcl-2和Mcl-1的表达下调有关。     

Proteasome inhibitor PS-341 induces apoptosis through induction of endoplasmic reticulum stress-reactive oxygen species in head and neck squamous cell carcinoma cells

PS-341, also known as Velcade or Bortezomib, represents a new class of anticancer drugs which has been shown to potently inhibit the growth and/or progression of human cancers, including head and neck squamous cell carcinoma (HNSCC). Although it has been logically hypothesized that NF-kappaB is a major target of PS-341, the underlying mechanism by which PS-341 inhibits tumor cell growth is unclear. Here we found that PS-341 potently activated the caspase cascade and induced apoptosis in human HNSCC cell lines. Although PS-341 could inhibit NF-kappaB activation, the inhibition of NF-kappaB was not sufficient to initiate apoptosis in HNSCC cells. Using biochemical and microarray approaches, we found that proteasome inhibition by PS-341 induced endoplasmic reticulum (ER) stress and reactive oxygen species (ROS) in HNSCC cells. The inhibition of ROS significantly suppressed caspase activation and apoptosis induced by PS-341. Consistently, PS-341 could not induce the ER stress-ROS in PS-341-resistant HNSCC cells. Taken together, our results suggest that in addition to the abolishment of the prosurvival NF-kappaB, PS-341 might directly induce apoptosis by activating proapoptotic ER stress-ROS signaling cascades in HNSCC cells, providing novel insights into the PS-341-mediated antitumor activity.     

CMTM5 exhibits tumor suppressor activity through promoter methylation in oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) is one of the most common types of malignancies in the head and neck region. CKLF-like MARVEL transmembrane domain-containing member 5 (CMTM5) has been recently implicated as a tumor suppressor gene in several cancer types. Herein, we examined the expression and function of CMTM5 in oral squamous cell carcinoma. CMTM5 was down-regulated in oral squamous cell lines and tumor samples from patients with promoter methylation. Treatment with the demethylating agent 5-aza-2′-deoxycytidine restored CMTM5 expression. In the OSCC cell lines CAL27 and GNM, the ectopic expression of CMTM5-v1 strongly inhibited cell proliferation and migration and induced apoptosis. In addition, CMTM5-v1 inhibited tumor formation in vivo. Therefore, CMTM5 might act as a putative tumor suppressor gene through promoter methylation in oral squamous cell carcinoma.     

H-7 and fetal calf serum (FCS) act synergistically to increase apoptosis in the KB line of human oral carcinoma cells

There is a high incidence of oral squamous cell carcinoma (SCC) worldwide. The survival rate is among the lowest of the major cancers and has not improved significantly over the past two decades. The KB line of human oral carcinoma cells is a useful experimental system for studies of the biology of oral SCC. In a previous study, we reported inhibition of KB cell proliferation and stimulation of desmosome formation in confluent cultures treated with 20 microM H-7 (1-(5-isoquinolinylsulfonyl)-2-methylpiperazine). In the present study, the effects of this protein kinase C (PKC) inhibitor on the survival of KB cells were investigated. Apoptotic cells were detected using a combination of Hoechst 33258 nuclear stain, TUNEL technique and ultrastructural analysis. Our results indicated that H-7 significantly increased apoptosis in KB cells in a dose-dependent manner. Maximal stimulation occurred at 100 microM, the highest dose of H-7 tested. Apoptotic cells exhibited nuclear fragmentation, chromatin condensation and apoptotic bodies. Interestingly, H-7 and fetal calf serum (FCS) acted synergistically to increase apoptosis in KB cells, suggesting that there is a serum activated subpopulation of H-7 target cells in the cultures. The underlying mechanism of activation remains to be elucidated. Our study suggests that the PKC inhibitor H-7 is a potentially useful cytostatic agent for oral carcinoma cells.     

MiR-21 is involved in cervical squamous cell tumorigenesis and regulates CCL20

MicroRNA 21 (miR-21) has been implicated in various aspects of carcinogenesis. However, its function and molecular mechanism in cervical squamous carcinoma have not been studied. Using TaqMan quantitative real-time PCR and Northern blot, we confirmed that miR-21 is significantly overexpressed in human cervical squamous cancer tissues and cell lines. Remarkably, we showed that the level of miR-21 correlates with the tumor differentiation and nodal status by ISH. Furthermore, we demonstrated that miR-21 regulates proliferation, apoptosis, and migration of HPV16-positive cervical squamous cells. In order to identify candidate target genes for miR-21, we used gene expression profiling. By luciferase reporter assays, we confirmed that CCL20 is one of its target genes, which is related to the HPV16 E6 and E7 oncogenes. Our results suggest that miR-21 may be involved in cervical squamous cell tumorigenesis.     

The treatment with differentiation- and apoptosis-inducing agent, vesnarinone, of a patient with oral squamous cell carcinoma

A patient with histopathological recurrent oral cancer with well-differentiated squamous cell carcinoma, was treated with differentiation- and apoptosis-inducing agent, vesnarinone, per os at a dose of 180 mg/day for 56 days and then at a dose of 60 mg/day for 93 days. The vesnarinone administration caused complete remission of the tumour. It has been found by immunohistochemical staining and PCR-SSCP analysis that the recurrent tumour has wild type p53 gene and relative high level of LeY expression as well as DNA fragmentation in the cancer cells, as assessed by nick-end labelling. These findings suggest that the cure of oral squamous cell carcinoma observed in this case might be associated with induction of differentiation and apoptosis of cancer cells by vesnarinone.     

Vimentin-mediated regulation of cell motility through modulation of beta4 integrin protein levels in oral tumor derived cells

Vimentin expression correlates well with migratory and invasive potential of the carcinoma cells. The molecular mechanism by which vimentin regulates cell motility is not yet clear. Here, we addressed this issue by depleting vimentin in oral squamous cell carcinoma derived cell line. Vimentin knockdown cells showed enhanced adhesion and spreading to laminin-5. However, we found that they were less invasive as compared to the vector control cells. In addition, signaling associated with adhesion behavior of the cell was increased in vimentin knockdown clones. These findings suggest that the normal function of β4 integrin as mechanical adhesive device is enhanced upon vimentin downregulation. As a proof of principle, the compromised invasive potential of vimentin depleted cells could be rescued upon blocking with β4 integrin adhesion-blocking (ASC-8) antibody or downregulation of β4 integrin in vimentin knockdown background. Interestingly, plectin which associates with α6β4 integrin in the hemidesmosomes, was also found to be upregulated in vimentin knockdown clones. Furthermore, experiments on lysosome and proteasome inhibition revealed that perhaps vimentin regulates the turnover of β4 integrin and plectin. Moreover, an inverse association was observed between vimentin expression and β4 integrin in oral squamous cell carcinoma (OSCC). Collectively, our results show a novel role of vimentin in modulating cell motility by destabilizing β4 integrin-mediated adhesive interactions. Further, vimentin-β4 integrin together may prove to be useful markers for prognostication of human oral cancer.     

Low-dose radiation hypersensitivity in human tumor cell lines: effects of cell-cell contact and nutritional deprivation

The hyper-radiosensitivity at low doses recently observed in vitro in a number of cell lines is thought to have important implications for improving tumor radiotherapy. However, cell-cell contact and the cellular environment influence cellular radiosensitivity at higher doses, and they may alter hyper-radiosensitivity in vivo. To confirm this supposition, we investigated the effects of cell density, multiplicity and nutritional deprivation on low-dose hypersensitivity in vitro. Cell survival in the low-dose range (3 cGy to 2 Gy) was studied in cells of two human glioma (BMG-1 and U-87) and two human oral squamous carcinoma (PECA-4451 and PECA-4197) lines using a conventional macrocolony assay. The effects of cell density, multiplicity and nutritional deprivation on hyper-radiosensitivity/induced radioresistance were studied in cells of the BMG-1 cell line, which showed prominent hypersensitivity and induced radioresistance. The induction of growth inhibition, cell cycle delay, micronuclei and apoptosis was also studied at the hyper-radiosensitivity-inducing low doses. Hyper-radiosensitivity/induced radioresistance was evident in the cells of all four cell lines to varying extents, with maximum sensitivity at 10-30 cGy, followed by an increase in survival up to 50 cGy-1 Gy. Both the glioma cell lines had more prominent hyper-radiosensitivity than the two squamous carcinoma cell lines. Low doses inducing maximum hyper-radiosensitivity did not cause significant growth inhibition, micronucleation or apoptosis in BMG-1 cells, but a transient G(1)/S-phase block was evident. Irradiating and incubating BMG-1 cells at high density for 0 or 4 h before plating, as well as irradiating cells as microcolonies, reduced hyper-radiosensitivity significantly, indicating the role of cell-cell contact-mediated processes. Liquid holding of BMG-1 cells in HBSS + 1% serum during and after irradiation for 4 h significantly reduced hyper-radiosensitivity, suggesting that hyper-radiosensitivity may be due partly to active damage fixation processes at low doses. Therefore, our findings suggest that the damage-induced signaling mechanisms influenced by (or mediated through) cell-cell contact or the cellular environment, as well as the lesion fixation processes, play an important role in hyper-radiosensitivity. Further studies are required to determine the exact nature of the damage that triggers these responses as well as for evaluating the potential of low-dose therapy.     

口腔鳞癌细胞系NTCR中侧群细胞的相关研究

目的:口腔鳞癌是口腔颌面部常见的恶性肿瘤之一,本研究以侧群细胞为肿瘤干细胞突破口,通过检测、分选口腔鳞癌细胞系NTCR中侧群细胞(side population,SP)细胞亚群,深入研究不同细胞亚群的体内、外相关生物学特性,寻找口腔鳞癌中肿瘤干细胞存在的证据。方法:选取口腔鳞癌细胞系NTCR作为研究对象,Hoechst 33342染色后行流式细胞仪检测,分选口腔鳞状细胞癌中的SP细胞和非SP细胞,进行体外培养、长期分化和体内成瘤实验,对2种亚群细胞的体内和体外生物学特性进行检测和比较。结果:口腔鳞状细胞癌细胞系NTCR中含有9.3%SP细胞,其SP细胞在细胞的增殖能力、自我更新能力及裸鼠体内成瘤能力等方面与干细胞特性相似。结论:SP细胞可以认为是肿瘤干细胞的富集。进一步深入研究,有可能作为口腔鳞癌诊断、治疗和预后的靶标。     

Comparative proteomics approach to screening of potential diagnostic and therapeutic targets for oral squamous cell carcinoma

This work demonstrates that a comprehensive strategy of proteomics identification combined with further validation and detailed functional analysis should be adopted in the field of cancer biomarker discovery. A comparative proteomics approach was utilized to identify differentially expressed proteins in 10 oral squamous carcinoma samples paired with their corresponding normal tissues. A total of 52 significantly and consistently altered proteins were identified with eight of these being reported for the first time in oral squamous carcinoma. Of the eight newly implicated proteins, RACK1 was chosen for detailed analysis. RACK1 was demonstrated to be up-regulated in cancer at both the mRNA and protein levels. Immunohistochemical examination showed that the enhanced expression of RACK1 was correlated with the severity of the epithelial dysplasia as well as clinical stage, lymph node involvement, and recurrence, which are known indicators of a relatively poor prognosis in oral squamous carcinoma patients. RNA interference specifically targeted to silence RACK1 could initiate apoptosis of oral squamous carcinoma cells. Taken together, the results indicate that RACK1 is up-regulated in oral squamous carcinoma, not only being closely related to cell proliferation and apoptosis but also linked to clinical invasiveness and metastasis in carcinogenesis. The observations suggest that RACK1 may be a potential biomarker for early diagnosis, prognosis, and monitoring in the therapy of oral squamous carcinoma. Further this comprehensive strategy could be used for identifying other differentially expressed proteins that have potential to be candidate biomarkers of oral squamous carcinoma.     

PAX9 reactivation by inhibiting DNA methyltransferase triggers antitumor effect in oral squamous cell carcinoma

Aberrant DNA hypermethylation is associated with oral carcinogenesis. Procaine, a local anesthetic, is a DNA methyltransferase (DNMT) inhibitor that activates anticancer mechanisms. However, its effect on silenced tumor suppressor gene (TSG) activation and its biological role in oral squamous cell carcinoma (OSCC) remain unknown. Here, we report procaine inhibited DNA methylation by suppressing DNMT activity and increased the expression of PAX9, a differentiation gene in OSCC cells. Interestingly, the reactivation of PAX9 by procaine found to inhibit cell growth and trigger apoptosis in OSCC in vitro and in vivo. Likely, the enhanced PAX9 expression after exposure to procaine controls stemness and differentiation through the autophagy-dependent pathway in OSCC cells. PAX9 inhibition abrogated procaine-induced apoptosis, autophagy, and inhibition of stemness. In OSCC cells, procaine improved anticancer drug sensitivity through PAX9, and its deficiency significantly blunted the anticancer drug sensitivity mediated by procaine. Additionally, NRF2 activation by procaine facilitated the antitumor response of PAX9, and pharmacological inhibition of NRF2 by ML385 reduced death and prevented the decrease in the orosphere-forming potential of OSCC cells. Furthermore, procaine promoted antitumor activity in FaDu xenografts in athymic nude mice, and immunohistochemistry data showed that PAX9 expression was significantly enhanced in the procaine group compared to the vehicle control. In conclusion, PAX9 reactivation in response to DNMT inhibition could trigger a potent antitumor mechanism to provide a new therapeutic strategy for OSCC.