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。     

Functional characterization of cultured cells derived from an intraepidermal carcinoma of the skin (IEC-1)

We have successfully isolated a cell line (IEC-1) from an intraepidermal carcinoma of the skin of a patient and compared its behavior, in vitro, to normal human epidermal keratinocytes (HEK) and squamous cell carcinoma cell lines (SCCs). HEK differentiation comprises an initial growth arrest followed by an induction of squamous differentiation-specific genes such as transglutaminase type 1 (TG-1). Using thymidine uptake and TG-1 induction as markers of proliferation and differentiation, respectively, we were able to show that HEKs and the IEC-1 cells undergo growth arrest and induce TG-1 mRNA expression in response to various differentiation-inducing stimuli, while neoplastic SCC cell lines did not. However, differentiation in HEKs was an irreversible process whereas differentiation of the IEC-1 cells was reversible. Furthermore, growth of IEC-1 cells in organotypic raft cultures revealed differences in their ability to complete a squamous differentiation program compared with that of normal HEKs. The IEC-1 cells also exhibited a transitional phenotype with respect to replicative lifespan; HEKs had a lifespan of 4-6 passages, IEC-1 cells of 15-17 passages, and SCC cells were immortal. These alterations in IEC-1 cell behavior were not associated with functional inactivation or mutations of the p53 gene. These data indicate that the IEC-1 cells, derived from a preneoplastic skin tumor, exhibit differences in their ability to undergo terminal differentiation and have an extended replicative lifespan.     

p21活化激酶1在口腔癌细胞侵袭和迁移中的作用

p21活化激酶1(PAK1)与多种癌症类型的进展有关,然而,其在口腔癌中的作用仍不明确。本研究以人舌鳞状细胞癌(oral squamous cell carcinoma,OSCC)HSC4细胞系为研究材料,考察了PAK1在细胞系中的定位及血清生长因子对PAK1定位的影响。通过转染PAK1 siRNA来敲低OSCC细胞中的PAK1,并分析PAK1敲低对细胞侵袭和迁移性的影响。研究发现PAK1在人舌鳞状细胞癌细胞系HSC4中主要定位于细胞核,而血清生长因子可调节PAK1在不同亚细胞区域中的易位或积累,并且还可能影响OSCC细胞的细胞骨架结构。此外,敲低PAK1可显著抑制OSCC细胞的迁移和侵袭能力,表明PAK1在OSCC发生发展过程中起着至关重要的作用。     

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.     

Potential involvement of MYC- and p53-related pathways in tumorigenesis in human oral squamous cell carcinoma revealed by proteomic analysis

Oral squamous cellular carcinoma is a malignant tumor with poor prognosis. Discovery of early markers to discriminate between malignant and normal cells is of high importance in clinical diagnosis. Subcellular fractions from 10 oral squamous cell carcinoma and corresponding control samples, enriched in mitochondrial and cytosolic proteins, as well as blood from the tumor were analyzed by proteomics, two-dimensional gel electrophoresis, followed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Three-hundred and fifty different gene products were identified. Twenty proteins showed deranged levels in oral squamous cell carcinoma in comparison with the control samples and are potentially involved in tumor growth and metastasis. Of these, 16 proteins were upregulated. By applying pathway analysis, we found 8 of the upregulated gene products to be linked to three main locus genes, p53, MYC, and MYCN, and could be candidate biomarkers for OSCC. The findings of this pilot study show that OSCC gene ontology combined with proteomic analysis is a powerful tool in systems biology for the elucidation of the complexity of expression profiles in cellular processes. Application of such pathway analysis has the potential to generate new insights into complex molecular mechanisms underlying disease related processes and could therefore significantly contribute to the efficient performance of the entire discovery process.     

Role of EZH2 in oral squamous cell carcinoma carcinogenesis

Oral squamous cell carcinoma (OSCC) is a common human malignancy with high incidence rate and poor prognosis. Although the polycomb group protein enhancer of zeste homolog 2 (EZH2) plays a crucial role in cell proliferation and differentiation during the occurrence and development progress of several kinds of malignant tumors, the impact of EZH2 on the development and progression of OSCC is unclear. In this study, we demonstrate that EZH2 is overexpressed in OSCC cells and clinical tissue. With in vitro RNAi analysis, we generated stable EZH2 knocking down cell lines from two OSCC cell lines, with two sh-RNAs targeting to EZH2, respectively. We found that knocking down of EZH2 could decrease the proliferation ability and induce apoptosis of OSCC cells. Moreover, we demonstrated that of EZH2 inhibition decreased the migration and metastasis of OSCC cells. In conclusion, the results of the current study demonstrated an association between EZH2 expression and OSCC cell development. We recommend that EZH2 acts as an oncogene and plays an important role in OSCC carcinogenesis.     

MTFP1 overexpression promotes the growth of oral squamous cell carcinoma by inducing ROS production

Mitochondrial fission process 1 (MTFP1) is a novel nuclear‐encoded protein that promotes mitochondrial fission. Increasing lines of evidence indicate that increased mitochondrial fission is involved in carcinogenesis and tumor progression. However, the expression and biological effects of MTFP1 in cancer development is still unclear, especially in oral squamous cell carcinoma (OSCC). In this study, we first evaluated the expression of MTFP1 in 12‐paired OSCC tumor and peritumor tissues. We then explored the effects of MTFP1 knockdown or overexpression on cell growth by cell proliferation, colony formation, cell cycle, and cell apoptosis assays. Furthermore, the mechanisms by which MTFP1 promoted OSCC cell growth were explored. Our results showed that MTFP1 is frequently overexpressed in OSCC tissues. Functional experiments revealed that MTFP1 promoted the growth of OSCC cells by inducing the progression of cell cycle and suppressing cell apoptosis. Mechanistically, MTFP1 overexpression‐mediated mitochondrial fragmentation and subsequent ROS production was found to be involved in the promotion of OSCC cell growth. Collectively, our study demonstrates that MTFP1 plays a critical oncogenic role in OSCC carcinogenesis, which may serve as a potential therapeutic target in the treatment of this malignance.     

hsa_circRNA_100533 regulates GNAS by sponging hsa_miR_933 to prevent oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) is the most common malignant tumor of the head and neck region. Circular RNA (circRNA), as one kind of noncoding RNA, involves in biological processes in diverse cancers. circRNA functions mainly as the microRNA (miRNA) sponge, competitively binding to miRNAs to regulate target gene expressions. However, the expression profiles and roles of circRNAs in OSCC are still unexplored. circRNA microarrays and quantitative real-time polymerase chain reaction was used to identify the hsa_circRNA_100533 downregulated in OSCC tissues and cell lines. Bioinformatics methods were used to predict the interactions among circRNAs, miRNA, and target genes. Based on the luciferase reporter assay and AGO2 RIP assay, we found that hsa_circRNA_100533 binds to miRNAs as a miRNA sponge. hsa_circRNA_100533 inhibited cell proliferation, migration, and promoted cell apoptosis in OSCC cell lines, which could be blocked by hsa-miR-933 overexpression. hsa_circRNA_100533 binds to hsa-miR-933 as a miRNA sponge to regulate GNAS expression, and to modulate cell proliferation, migration, and apoptosis. In summary, the hsa_circRNA_100533-miR-933-GNAS axis affect the proliferation and apoptosis of OSCC cells through the mechanism of competing endogenous RNAs. hsa_circRNA_100533 may function as promising diagnostic biomarkers and effective therapeutic targets for OSCC.     

Curcumin analog HO‐3867 triggers apoptotic pathways through activating JNK1/2 signalling in human oral squamous cell carcinoma cells

Human oral squamous cell carcinoma (OSCC) is the common head and neck malignancy in the world. While surgery, radiotherapy and chemotherapy are emerging as the standard treatment for OSCC patients, the outcome is limited to the recurrence and side effects. Therefore, patients with OSCC require alternative strategies for treatment. In this study, we aimed to explore the therapeutic effect and the mode of action of the novel curcumin analog, HO‐3867, against human OSCC cells. We analysed the cytotoxicity of HO‐3867 using MTT assay. In vitro mechanic studies were performed to determine whether MAPK pathway is involved in HO‐3867 induced cell apoptosis. As the results, we found HO‐3867 suppressed OSCC cells growth effectively. The flow cytometry data indicate that HO‐3867 induce the sub‐G1 phase. Moreover, we found that HO‐3867 induced cell apoptosis by triggering formation of activated caspase 3, caspase 8, caspase 9 and PARP. After dissecting MAPK pathway, we found HO‐3867 induced cell apoptosis via the c‐Jun N‐terminal kinase (JNK)1/2 pathway. Our results suggest that HO‐3867 is an effective anticancer agent as its induction of cell apoptosis through JNK1/2 pathway in human oral cancer cells.     

RANKL triggers resistance to TRAIL-induced cell death in oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) occurs as a malignancy of the oral cavity. RANK ligand (RANKL) is essential for osteoclast formation/bone resorption. Recently, we showed autoregulation of receptor activator of nuclear factor-κB ligand (RANKL) stimulates OSCC cell proliferation. OSCC cells show resistance to tumor necrosis factor related apoptosis inducing ligand (TRAIL) treatment. Therefore, we hypothesize that RANKL promotes resistance for TRAIL induction of OSCC apoptotic cell death. In this study, SCC14A and SCC74A cells cultured with TRAIL revealed high-level expression of RANKL which increased resistance to TRAIL inhibition of tumor cell proliferation. RANKL stimulation inhibited terminal deoxynucleotidyl transferase dUTP nick end labeling positive staining in TRAIL-treated cells. CRISPR/Cas-9 knockout of RANKL (RANKL-KO) increased caspase-9, caspase-3 activity and cytochrome c release in OSCC cells. RANKL inhibited proapoptotic proteins BAD and BAX expression. TRAIL treatment suppressed the SQSTM1/p62 and RANKL restored the expression. Interestingly, RANKL alone significantly increased proteasome activity. RANKL-KO in OSCC cells inhibited autophagic activity as evidenced by decreased light chain 3B-II and beclin-1 expression. Thus, RANKL stimulation of OSCC tumor cells triggered resistance for TRAIL-induced OSCC cell death. Taken together, blockade of RANKL may inhibit OSCC tumor progression and enhance the potential of TRAIL induced OSCC tumor cell apoptosis.     

Generation of Reactive Oxygen Species by Polyenylpyrroles Derivatives Causes DNA Damage Leading to G2/M Arrest and Apoptosis in Human Oral Squamous Cell Carcinoma Cells

Oral squamous cell carcinoma (OSCC) accounts for 5.8% of all malignancies in Taiwan and the incidence of OSCC is on the rise. OSCC is also a common malignancy worldwide and the five-year survival rate remains poor. Therefore, new and effective treatments are needed to control OSCC. In the present study we have investigated the efficacy and associated mechanisms of polyenylpyrroles and their analogs in both in vitro cell culture and in vivo nude mice xenografts. Auxarconjugatin B (compound 1a) resulted in cell cycle arrest in the G2/M phase and caspase-dependent apoptosis in OEC-M1 and HSC-3 cells by activating DNA damage and mitochondria dysfunction through the loss of mitochondrial membrane potential, release of cytochrome c, increase in B-cell lymphoma-2-associated X protein level, and decrease in B-cell lymphoma-2 level. Compound 1a-induced generation of intracellular reactive oxygen species through cytochrome P450 1A1 was identified as a major mechanism of its effect for DNA damage, mitochondria dysfunction and apoptosis, which was reversed by antioxidant N-acetylcysteine as well as cytochrome P450 1A1 inhibitor and specific siRNA. Furthermore, compound 1a-treated nude mice showed a reduction in the OEC-M1 xenograft tumor growth and an increase in the caspase-3 activation in xenograft tissue. These results provide promising insights as to how compound 1a mediates cytotoxicity and may prove to be a molecular rationale for its translation into a potential therapeutic against OSCC.     

TCRP1 promotes radioresistance of oral squamous cell carcinoma cells via Akt signal pathway

Tongue cancer resistance-associated protein 1 (TCRP1) is a novel gene located on human chromosome 11q13.4 which has been reported as a candidate related to chemotherapeutic resistance to cisplatin. Results suggest that TCRP also contribute to radioresistance in oral squamous cell carcinoma (OSCC) cells. We previously established exogenous overexpression of TCRP1 cell line Tca8113/TCRP1 and TCRP1 knockdown cell line Tca8113/PYM-siRNA and paired control cell lines, which provides a cell model system to investigate the roles and mechanisms of TCRP1-mediated radioresponse in OSCC. In this study, we first compared the radiosensitivity of up/down-regulating expression of TCRP1 cell lines and paired control cell lines by a clonogenic survival assay, Hoechst 33258 staining, cell growth assay, and comet assay. The results indicated that TCRP1 played a significant role in mediating OSCC radioresistance through decreased cells apoptosis and increased cellular proliferation and long-term survival. The further study found that TCRP1 function by up-regulating Akt activity and levels and then elevating the level of NF-κB. In summary, these results provided strong evidence for the linkage between TCRP1 and radiation sensitivity and may provide theoretical base of TCRP1 as a potential molecular mark of estimating the response for irradiation in OSCC.     

Platycodin D,a triterpenoid saponin from Platycodon grandiflorum,suppresses the growth and invasion of human oral squamous cell carcinoma cells via the NF‐κB pathway

This work was undertaken to explore the effects of platycodin D, a triterpenoid saponin from Platycodon grandiflorum, on the growth and invasiveness of human oral squamous cell carcinoma (OSCC). Platycodin D caused a significant, concentration‐dependent inhibition of cell viability and induced significant apoptosis in OSCC cells. Moreover, platycodin D significantly inhibited OSCC cell invasion. At the molecular level, platycodin D increased the amounts of IκBα protein and reduced the expression of phosphorylated NF‐κB p65, MMP‐2, and MMP‐9. Ectopic expression of constitutively active NF‐κB p65 prevented platycodin D‐mediated induction of apoptosis and suppression of invasion in OSCC cells. In vivo studies confirmed that platycodin D retarded the growth of subcutaneous SCC‐4 xenograft tumors and reduced phosphorylation of NF‐κB p65. Altogether, platycodin D shows inhibitory activity on OSCC growth and invasion through inactivation of the NF‐κB pathway and might provide therapeutic benefits in the treatment of OSCC.     

p120-catenin suppresses proliferation and tumor growth of oral squamous cell carcinoma via inhibiting nuclear phospholipase C-γ1 signaling

p120-catenin (p120) serves as a stabilizer of the calcium-dependent cadherin-catenin complex and loss of p120 expression has been observed in several types of human cancers. The p120-dependent E-cadherin-β-catenin complex has been shown to mediate calcium-induced keratinocyte differentiation via inducing activation of plasma membrane phospholipase C-γ1 (PLC-γ1). On the other hand, PLC-γ1 has been shown to interact with phosphatidylinositol 3-kinase enhancer in the nucleus and plays a critical role in epidermal growth factor-induced proliferation of oral squamous cell carcinoma (OSCC) cells. To determine whether p120 suppresses OSCC proliferation and tumor growth via inhibiting PLC-γ1, we examined effects of p120 knockdown or p120 and PLC-γ1 double knockdown on proliferation of cultured OSCC cells and tumor growth in xenograft OSCC in mice. The results showed that knockdown of p120 reduced levels of PLC-γ1 in the plasma membrane and increased levels of PLC-γ1 and its signaling in the nucleus in OSCC cells and OSCC cell proliferation as well as xenograft OSCC tumor growth. However, double knockdown of p120 and PLC-γ1 or knockdown of PLC-γ1 alone did not have any effect. Immunohistochemical analysis of OSCC tissue from patients showed a lower expression level of p120 and a higher expression level of PLC-γ1 compared with that of adjacent noncancerous tissue. These data indicate that p120 suppresses OSCC cell proliferation and tumor growth by inhibiting signaling mediated by nuclear PLC-γ1.     

Expression of human apolipoprotein(a) kringles in colon cancer cells suppresses angiogenesis-dependent tumor growth and peritoneal dissemination

BACKGROUND: Anti-angiogenesis therapy has been regarded as a promising treatment of cancer based on the fact that most tumors and their metastasis are angiogenesis-dependent. Gene therapy can potentially expand the horizons of tumor angiogenesis therapy by virtue of its ability to produce high concentrations of therapeutic agents in a local area for a sustained period. The present study was performed to evaluate the therapeutic potential of gene therapy for the treatment of cancer and metastasis. METHODS: The murine colon carcinoma cell line CT26 was manipulated ex vivo to express an anti-angiogenic molecule, LK68, consisting of human apolipoprotein(a) kringle domains, KIV(9)-KIV(10)-KV, using retrovirus-mediated gene transfer. Its effects on colon tumor growth and metastasis were evaluated in experimental animal models established by injecting LK68-expressing and control CT26 cells subcutaneously or into the peritoneal cavity of BALB/c mice, respectively. RESULTS: Expression of LK68 significantly suppressed colon tumor growth in mice, but did not influence the growth of tumor cells in vitro. Immunohistochemical analysis of tumor tissues revealed a significant reduction in microvessel density in LK68-expressing tumors. Thus, the suppression of tumor growth appears to result mainly from inhibition of tumor angiogenesis. This decrease in vessel density is correlated with a notable increase in tumor cell apoptosis in vivo, but has no influence on proliferation. Moreover, expression of LK68 prevents peritoneal dissemination, and consequently improves overall host survival. CONCLUSIONS: These results collectively indicate that a gene therapy strategy using LK68 cDNA is useful for the treatment for both colon tumor growth and peritoneal dissemination.     

Identification of novel keratinocyte differentiation modulating compounds by high-throughput screening

The authors have designed high-throughput screens to identify compounds that promote or inhibit terminal differentiation of primary human epidermal keratinocytes. Eleven known inhibitors of signaling pathways and approximately 4000 compounds of diverse structure were screened using an In-Cell Western system based on immunofluorescent staining of the terminal differentiation marker, involucrin. Staurosporine, a nonspecific protein kinase C inhibitor, and H89, a protein kinase A inhibitor, promoted expression of involucrin. Conversely, U0126, a MEK inhibitor, and SAHA or SBHA, 2 histone deacetylase inhibitors, reduced the expression of involucrin during calcium-induced stratification. In addition, the authors found 1 novel compound that induced keratinocyte differentiation and 2 novel compounds that were inhibitory to calcium-induced differentiation. The differentiation-inducing compound also inhibited growth of a human squamous cell carcinoma line by stimulating both differentiation and apoptosis. Because the compound affected the tumor cells at a lower concentration than primary keratinocytes, it may have potential as an antitumor therapy.     

Quantitative phosphoproteomic analysis reveals γ‐bisabolene inducing p53‐mediated apoptosis of human oral squamous cell carcinoma via HDAC2 inhibition and ERK1/2 activation

γ‐Bisabolene, one of main components in cardamom, showed potent in vitro and in vivo anti‐proliferative activities against human oral squamous cell carcinoma (OSCC). γ‐Bisabolene activated caspases‐3/9 and decreased mitochondrial memebrane potential, leading to apoptosis of OSCC cell lines (Ca9‐22 and SAS), but not normal oral fibroblast cells. Phosphoproteome profiling of OSCC cells treated with γ‐bisabolene was identified using TiO2‐PDMS plate and LC‐MS/MS, then confirmed using Western blotting and real‐time RT‐PCR assays. Phosphoproteome profiling revealed that γ‐bisabolene increased the phosphorylation of ERK1/2, protein phosphatases 1 (PP1), and p53, as well as decreased the phosphorylation of histone deacetylase 2 (HDAC2) in the process of apoptosis induction. Protein–protein interaction network analysis proposed the involvement of PP1‐HDAC2‐p53 and ERK1/2‐p53 pathways in γ‐bisabolene‐induced apoptosis. Subsequent assays indicated γ‐bisabolene eliciting p53 acetylation that enhanced the expression of p53‐regulated apoptotic genes. PP1 inhibitor‐2 restored the status of HDAC2 phosphorylation, reducing p53 acetylation and PUMA mRNA expression in γ‐bisabolene‐treated Ca9‐22 and SAS cells. Meanwhile, MEK and ERK inhibitors significantly decreased γ‐bisabolene‐induced PUMA expression in both cancer cell lines. Notably, the results ascertained the involvement of PP1‐HDAC2‐p53 and ERK1/2‐p53 pathways in mitochondria‐mediated apoptosis of γ‐bisabolene‐treated cells. This study demonstrated γ‐bisabolene displaying potent anti‐proliferative and apoptosis‐inducing activities against OSCC in vitro and in vivo, elucidating molecular mechanisms of γ‐bisabolene‐induced apoptosis. The novel insight could be useful for developing anti‐cancer drugs.