Qingqiong Luo

In vitro and in vivo anti-tumor effect of metformin as a novel therapeutic agent in human oral squamous cell carcinoma.

BackgroundMetformin, which is widely used as an antidiabetic agent, has recently been reported to reduce cancer risk and improve prognosis in certain malignancies. However, the specific mechanisms underlying the effect of metformin on the development and progression of several cancers including oral squamous cell carcinoma (OSCC) remain unclear. In the present study, we investigated the effects of metformin on OSCC cells in vitro and in vivo.MethodsOSCC cells treated with or without metformin were counted using a hemocytometer. The clonogenic ability of OSCC cells after metformin treatment was determined by colony formation assay. Cell cycle progression and apoptosis were assessed by flow cytometry, and the activation of related signaling pathways was examined by immunoblotting. The in vivo anti-tumor effect of metformin was examined using a xenograft mouse model. Immunohistochemistry and TUNEL staining were used to determine the expression of cyclin D1 and the presence of apoptotic cells in tumors from mice treated with or without metformin.ResultsMetformin inhibited proliferation in the OSCC cell lines CAL27, WSU-HN6 and SCC25 in a time- and dose-dependent manner, and significantly reduced the colony formation of OSCC cells in vitro. Metformin induced an apparent cell cycle arrest at the G0/G1 phase, which was accompanied by an obvious activation of the AMP kinase pathway and a strongly decreased activation of mammalian target of rapamycin and S6 kinase. Metformin treatment led to a remarkable decrease of cyclin D1, cyclin-dependent kinase (CDK) 4 and CDK6 protein levels and phosphorylation of retinoblastoma protein, but did not affect p21 or p27 protein expression in OSCC cells. In addition, metformin induced apoptosis in OSCC cells, significantly down-regulating the anti-apoptotic proteins Bcl-2 and Bcl-xL and up-regulating the pro-apoptotic protein Bax. Metformin also markedly reduced the expression of cyclin D1 and increased the numbers of apoptotic cells in vivo, thus inhibiting the growth of OSCC xenografts.ConclusionsOur data suggested that metformin could be a potential candidate for the development of new treatment strategies for human OSCC.

Role of toll-like receptor 4 on the immune escape of human oral squamous cell carcinoma and resistance of cisplatin-induced apoptosis.

BackgroundToll-like receptor 4 (TLR4) is expressed on immune cells as a sensor that recognizes lipopolysaccharide (LPS), a microbial conserved component. It has recently been determined that the expression of TLR4 is also found in various types of tumor cells. Cisplatin is a widely used chemotherapeutic agent for oral squamous cell carcinoma (OSCC) treatment. However, the mechanisms responsible for cisplatin resistance are not well understood.ResultsThe present study was designed to elucidate the role of TLR4 expression in human OSCC regarding immune escape and apoptotic resistance to cisplatin. TLR4 and the myeloid differentiation primary response gene 88 (MyD88) were highly expressed in OSCC cell lines. Upon LPS stimulation both NF-κB and p38 MAPK pathways were activated in OSCC cell lines, followed by the production of large quantities of IL-6, IL-8 and VEGF compared with human immortalized oral epithelia cells (HIOECs). OSCC cell lines were found to be resistant to cisplatin-mediated apoptosis after pretreatment with LPS.ConclusionsOur results suggested that TLR4 was functionally expressed in human OSCC cells and development of resistance to cisplatin in human OSCC might occur through the mechanism involving TLR4 and its signaling pathway. Suppression of TLR4 and its signaling pathway might thus elevate sensitivity to cisplatin and potentially help improve the prognosis of patients with OSCC.

Overexpression of SENP3 in oral squamous cell carcinoma and its association with differentiation

Small ubiquitin-like modifier (SUMO) modification is an important post-translational protein modification that can be reversed by SUMO-specific proteases (SENPs); however, the physiological function of SENPs remains largely unexplored, and little is known about the regulation of SENPs themselves. As one of the crucial members of the SUMO system, SENP3 is essential for rRNA processing and cell proliferation. In the present study, we analysed the expression of SENP3 in human oral squamous cell carcinoma (OSCC) and investigated the correlation between its expression and clinicopathological parameters in OSCC patients. The expression of SENP3 was higher in OSCC tissues than that in the normal mucosa adjacent to the tumor, and a modest increase in reactive oxygen species (ROS) regulated SENP3 stability and localization. ROS induced SENP3 redistribution from the nucleoli to the nucleoplasm. Taken together, these results indicated that the expression level of SENP3 may be associated with the differentiation of OSCC and that SENP3 may play an important role in the development of OSCC under oxidative stress.

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 pharmacological NF-κB inhibitor BAY11-7082 induces cell apoptosis and inhibits the migration of human uveal melanoma cells.

Uveal melanomas are highly metastatic and have high rate of recurrence due to the lack of effective systemic therapy. The identification of important survival pathways in uveal melanomas provides novel therapeutic targets for effective treatment. In the present study, we found that the NF-κB signaling pathway was constitutively and highly activated in uveal melanoma cells. Treatment with the pharmacological NF-κB specific inhibitor BAY11-7082 markedly decreased the nuclear translocation of NF-κB. In a dose-dependent setting, BAY11-7082 inhibited the proliferation and growth of uveal melanoma cells by inducing apoptosis without effect on cell cycle. The migration capacity of uveal melanoma cells was also significantly suppressed by BAY11-7082 treatment. Mechanistically, BAY11-7082 increased the activity of caspase 3 and reduced the expression of anti-apoptotic protein Bcl-2, but did not influence the expression of pro-apoptotic protein Bax. Furthermore, BAY11-7082 induced uveal melanoma cell apoptosis and inhibited xenograft tumor growth in vivo. Collectively, the present study identified NF-κB as an important survival signal for uveal melanoma cells and suggested that administration of specific NF-κB inhibitor BAY11-7082 could serve as an effective treatment for patients with uveal melanoma.

Protective effect of galangin in Concanavalin A-induced hepatitis in mice

Galangin is an active pharmacological ingredient from propolis and Alpinia officinarum Hance, and has been reported to have anti-inflammatory and antioxidative properties. The present study aims to reveal the effect of galangin on Concanavalin A (ConA)-induced hepatitis (CIH), a well-established animal model of immune-mediated liver injury, and to clarify the related mechanism. C57BL/6 mice were pretreated with galangin followed by ConA challenge. Results indicated that galangin inhibited ConA-induced liver damage. Mice pretreated with galangin showed more reduction of liver damage when compared with control mice pretreated with vehicle solution. In galangin-pretreated mice with induced CIH, increases in serum levels of several inflammatory cytokines, including tumor necrosis factor-α, interferon-γ, and interleukin-12 were dramatically attenuated, and chemokines and adhesion molecules like interferon inducible protein-10, macrophage inflammatory protein-1α, and inter-cellular adhesion molecule-1 messenger RNA expressions in liver were decreased. Moreover, CIH mice pretreated with galangin showed less leukocyte infiltration and T-cell activation in the liver. Further, the mechanism of the anti-inflammatory effects of galangin may be attributed to its modulation of crucial inflammatory signaling pathways, including nuclear factor kappa B and interferon-gamma/signal transducer and activator of transcription 1. Collectively, these findings suggest the preventive and therapeutic potential of galangin in immune-mediated liver injury in vivo.

Oridonin induces G2/M cell cycle arrest and apoptosis in human oral squamous cell carcinoma

Abstract Oridonin, an active diterpeniod isolated from Rabdosia rubescens, has been reported for its anti‐tumor activity on several cancers, however, its effect on oral squamous cell carcinoma (OSCC) remains unclear. In this study, we demonstrated for the first time that oridonin inhibited the growth of OSCC cells both in vitro and in vivo. Oridonin decreased the proliferation and clonal formation of cultured OSCC cells in a dose‐dependent manner. Further study indicated that oridonin induced G2/M phase arrest in OSCC cells, which was associated with the downregulation of proteins related to G2/M transition including cdc25C, cdc2 and cyclin B1, as well as the upregulation of p53 and phosphorylated‐cdc2. In addition, we discovered that oridonin induced OSCC cell apoptosis by activating the intrinsic apoptotic pathway, which was indicated by the increased expression of cleaved‐caspase 3, cleaved‐caspase 9 and proapoptotic protein Bax and reduced expression of caspase 9 and antiapoptotic protein Bcl‐xl. Finally, oridonin suppressed the growth of OSCC in an xenograft mouse model. Immunohistochemical analysis showed a reduction of cyclin B1‐positive cancer cells and an increase of TUNEL‐positive cancer cells in oridonin‐treated mice. Therefore, oridonin may be a potentially effective agent for the treatment of OSCC in future.

MicroRNA-22 suppresses cell proliferation, migration and invasion in oral squamous cell carcinoma by targeting NLRP3

MicroRNAs (miRNAs) have been implicated as important regulators of carcinogenesis and tumor development. Recently, microRNA‐22 (miR‐22) has been reported to be a cancer‐related miRNA in several types of tumors. In this study, we aimed to investigate the role of miR‐22 in oral squamous cell carcinoma (OSCC). We found that miR‐22 expression was significantly decreased in OSCC tissues compared with that in the adjacent noncancerous tissues. Furthermore, lentivirus‐mediated miR‐22 overexpression markedly reduced OSCC cell viability, migration and invasion, whereas miR‐22 inhibitor promoted these parameters. Mechanistically, NLR family pyrin domain containing three (NLRP3) was identified as a direct target of miR‐22. miR‐22 expression was inversely correlated with NLRP3 expression both in OSCC tissues and cell lines. Moreover, overexpression of miR‐22 in OSCC cells could reverse the tumor‐promoting effect of the activated NLRP3 inflammasome and vice versus. Therefore, our results indicate that miR‐22 may play a suppressive role in OSCC by targeting NLRP3, which offer new insights into the molecular mechanisms of the growth and metastasis of OSCC.

The role of NLRP3 inflammasome in 5-fluorouracil resistance of oral squamous cell carcinoma

Background5-Fluorouracil (5-FU) is a widely used drug for the therapy of cancer. However, the chemoresistance of tumor cells to 5-FU usually limits its clinical effectiveness. In this study, we explored the role of NLRP3 inflammasome in 5-FU resistance of oral squamous cell carcinoma (OSCC).MethodsThe mRNA and protein expression levels of NLRP3, Caspase1 and IL-1β in resected OSCC specimens or cell lines were measured respectively by quantitative real time-PCR (qRT-PCR) and western blot. NLRP3 and Ki-67 expression in paraffin-embedded OSCC tissues was determined by immunohistochemistry. The correlation between 5-FU treatment and the expression and activation of NLRP3 inflammasome was further examined by evaluating NLRP3 and IL-1β expression in OSCC cell lines without or with NLRP3 knocked down. Cell viabilities of OSCC cells were determined by the MTT assay. Apoptosis and intracellular reactive oxygen species (ROS) of OSCC cells induced by 5-FU were measured by the flow cytometer. The carcinogen-induced tongue squamous carcinoma mice model was established by continuous oral administration of 4-nitroquinoline 1-oxide in wild-type BALB/c, Nlrp3−/− and Caspase1−/− mice. Tumor incidence were observed and tumor area were evaluated.ResultsIn the clinical analysis, expression and activation of NLRP3 inflammasome was clearly increased in OSCC tissues of patients who received 5-FU-based chemotherapy. Multivariate Cox regression analysis revealed that this high expression was significantly correlated with tumor stage and differentiation, and was associated with poor prognosis. Moreover, 5-FU treatment increased expression and activation of NLRP3 inflammasome in OSCC cells in a cell culture system and xenograft mouse model. Silencing of NLRP3 expression significantly inhibited OSCC cell proliferation and enhanced 5-FU-induced apoptosis of OSCC cells. Further investigation showed that intracellular ROS induced by 5-FU promoted the expression and activation of NLRP3 inflammasome and increased the production of interleukin (IL)-1β, which then mediated the chemoresistance. With the carcinogen-induced OSCC model, we found less and later tumor incidence in Nlrp3−/− and Caspase1−/− mice than wild-type mice. And greater decrease of tumor area was observed in the gene deficient mice treated with 5-FU.ConclusionsOur findings suggest that NLRP3 inflammasome promoted 5-FU resistance of OSCC both in vitro and in vivo, and targeting the ROS/NLRP3 inflammasome/IL-1β signaling pathway may help 5-FU-based adjuvant chemotherapy of OSCC.