Brusatol improves the efficacy of an anti-mouse-PD-1 antibody via inhibiting programmed cell death 1 ligand 1 expression in a murine head and neck squamous cell carcinoma model.

OBJECTIVE: Combing PD-1/PD-L1 immune checkpoint inhibitors with natural products has exhibited better efficacy than monotherapy. Hence, the purpose of this research was to examine the anti-cancer effects of brusatol, a natural quassinoid-terpenoid derived from Brucea javanica, when used in conjunction with an anti-mouse-PD-1 antibody in a murine head and neck squamous cell carcinoma (HNSCC) model and elucidate underlying mechanisms. DESIGN: A murine HNSCC model and an SCC-15 cell xenograft nude mouse model were established to investigate the anti-cancer effects of brusatol and anti-PD-1 antibody. Mechanistic studies were performed using immunohistochemistry. Cell proliferation, migration, colony formation, and invasion were evaluated by MTT, migration, colony formation, and transwell invasion assays. PD-L1 levels in oral squamous cell carcinoma (OSCC) cells were assessed through qRT-PCR, flow cytometry, and western blotting assays. The impact of brusatol on Jurkat T cell function was assessed by an OSCC/Jurkat co-culture assay. RESULTS: Brusatol improved tumor suppression by anti-PD-1 antibody in HNSCC mouse models. Mechanistic studies revealed brusatol inhibited tumor cell growth and angiogenesis, induced apoptosis, increased T lymphocyte infiltration, and reduced PD-L1 expression in tumors. Furthermore, in vitro assays confirmed brusatol inhibited PD-L1 expression in OSCC cells and suppressed cell migration, colony formation, and invasion. Co-culture assays indicated that brusatol's PD-L1 inhibition enhanced Jurkat T cell-mediated OSCC cell death and reversed the inhibitory effect induced by OSCC cells. CONCLUSIONS: Brusatol improves anti-PD-1 antibody efficacy by targeting PD-L1, suggesting its potential as an adjuvant in anti-PD-1 immunotherapy.

HMGB1 Signaling-Mediated Tumor Immunity in Cancer Progress.

Tumor immunity is a cycle that begins with the release of antigens from tumor cells and ends with the destruction of tumor cells. High mobility group box 1 (HMGB1) is a nonhistone protein widely present in the nucleus of mammalian cells and can be released by immune cells or tumor cells. As a proinflammatory mediator or alarm protein, the activity and function of HMGB1 are determined by the environment, binding receptors, redox status and posttranslational modifications (PTMs), and HMGB1 plays a key role in inflammation and tumor immune processes. In this review, we summarize in detail the current studies on the dual role of HMGB1 in tumor immunity, focusing mainly on immunosuppressive effects, such as regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs), as well as antitumor immunoenhancement effects, such as immunogenic cell death (ICD). Finally, we discuss the potential and challenges of HMGB1 in antitumor immunotherapy.

Knockdown of HMGB1 inhibits the crosstalk between oral squamous cell carcinoma cells and tumor-associated macrophages.

Tumor-associated macrophages (TAMs), the major component of the tumor microenvironment (TME), play distinctly different roles in different tumors. High mobility group box 1 (HMGB1), a nonhistone protein in the nucleus, can perform functions during inflammation and cancers. However, the role of HMGB1 in the crosstalk between oral squamous cell carcinoma (OSCC) cells and TAMs remains unclear. Here, we established a coculture system of TAMs and OSCC cells to explore the bidirectional effect and potential mechanism of HMGB1 in OSCC cell-TAM interactions. Our results showed that HMGB1 was significantly upregulated in OSCC tissues and positively associated with tumor progression, immune cell infiltration and macrophage polarization. Then, knocking down HMGB1 in OSCC cells inhibited the recruitment and polarization of cocultured TAMs. Moreover, the knockdown of HMGB1 in macrophages not only suppressed polarization, but also inhibited cocultured OSCC cell proliferation, migration and invasion in vitro and in vivo. Mechanistically, macrophages secreted higher levels of HMGB1 than OSCC cells, and dampening endogenous HMGB1 reduced HMGB1 secretion. Both OSCC cell-generated and macrophage-endogenous HMGB1 may regulate TAM polarization by promoting receptor TLR4 expression and NF-κB/p65 activation and enhancing IL-10/TGF-ß expression. HMGB1 in OSCC cells may regulate macrophage recruitment via IL-6/STAT3. In addition, TAM-derived HMGB1 may affect aggressive phenotypes of cocultured OSCC cells by regulating the immunosuppressive microenvironment through the IL-6/STAT3/PD-L1 and IL-6/NF-κB/MMP-9 pathways. In conclusion, HMGB1 may regulate the crosstalk between OSCC cells and TAMs, including modulating macrophage polarization and attraction, enhancing cytokine secretion, and remodeling and creating an immunosuppressive TME to further affect OSCC progression.

Chemoprevention of 4NQO-Induced Mouse Tongue Carcinogenesis by AKT Inhibitor through the MMP-9/RhoC Signaling Pathway and Autophagy.

Oral cancer (OC), the most common cancer in the head and neck, which has a poor prognosis, histopathologically follows a stepwise pattern of hyperplasia, dysplasia, and cancer. Blocking the progression of OC in the precancer stage could greatly improve the survival and cure rates. AKT protein plays a critical role in the signal transduction of cancer cells, and we found that AKT was overexpressed in human OC samples through analysis of TCGA database. Therefore, this study is aimed at investigating the chemopreventive effect of an AKT inhibitor (MK2206 2HCl) on OC. In vivo, we established a 4-nitroquinoline-1-oxide- (4NQO-) induced mouse tongue carcinogenesis model to investigate the potential chemopreventive effect of MK2206 2HCl on mouse OC resulting from 4NQO. The results showed that MK2206 2HCl could significantly reduce the incidence rate and growth of OC, inhibit the transformation of dysplasia to cancer in the 4NQO-induced mouse tongue carcinogenesis model, and simultaneously markedly suppress cell proliferation, angiogenesis, and mast cell (MC) infiltration in 4NQO-induced mouse tongue cancers. In vitro, our results revealed that MK2206 2HCl could also inhibit oral squamous cell carcinoma (OSCC) cell malignant biological behaviors, including cell proliferation, colony formation, cell invasion, and migration, while promoting apoptosis. Mechanistic studies revealed that MK2206 2HCl suppressed matrix metalloproteinase 9 (MMP-9) and RhoC expression and promoted autophagy gene LC3 II expression. In summary, our findings demonstrated the chemopreventive effect of MK2206 2HCl on the 4NQO-induced mouse tongue carcinogenesis model, which likely has an underlying mechanism mediated by the MMP-9/RhoC signaling pathway and autophagy.

MMP-9 Knockdown Inhibits Oral Squamous Cell Carcinoma Lymph Node Metastasis in the Nude Mouse Tongue-Xenografted Model through the RhoC/Src Pathway.

Oral squamous cell carcinoma (OSCC) is one of the most common types of cancers in developing countries. A major contributor to the high mortality rate of OSCC is the tendency of oral cancer cells to metastasize to lymph nodes around the head and neck during the early stages of cancer development. Matrix metalloproteinase 9 (MMP-9), an endopeptidase, can degrade the extracellular matrix and basement membrane and plays a key role in tumor invasion and metastasis. In vitro, cell migration ability was conducted by scratching assays. We also investigated the interaction abilities between OSCC cells and vascular endothelial cells (ECs) by an adhesion assay and transendothelial migration assay. And we established a BALB/c nude mouse tongue-xenografted metastasis model to investigate the role of MMP-9 and explore its potential underlying mechanism in OSCC growth, lymph node metastasis, and angiogenesis in vivo. The results showed that knockdown of MMP-9 could significantly suppress OSCC cell migration, proliferation, interactions between endothelial cells, xenografted tumor growth, and angiogenesis and simultaneously markedly inhibited OSCC cell metastasis to mouse lymphonodi cervicales superficiales, axillary lymph nodes, and even distant inguinal lymph nodes. Mechanistic studies revealed that knockdown of MMP-9 also led to a decreased expression of RhoC, Src, and F-actin by RT-PCR, western blotting, and immunohistochemistry. And the bioinformatic analysis showed that MMP-9, RhoC, and Src mRNA expression was positively and linearly correlated in OSCC on TCGA database. Together, our findings indicated that MMP-9 plays a very important role in OSCC growth, migration, angiogenesis, and lymph node metastasis, and its potential mechanism may be mediated by RhoC and Src gene expression.

Knockdown of RhoC Inhibits Oral Squamous Cell Carcinoma Cell Invasion and Metastasis via Regulation of HMGA2.

Ras homolog family member C (RhoC) is an important component of intracellular signal transduction and its overexpression has been reported to be involved in regulating tumor proliferation, invasion, and metastasis in various malignant tumors. However, its role and underlying mechanism in oral squamous cell carcinoma (OSCC) still remain obscure. In our study, RhoC expression, its relation with clinical stages, and survival rate in OSCC were analyzed using datasets from The Cancer Genome Atlas (TCGA). Next, a RhoC knockdown cell model was established in vitro, and the effects of RhoC knockdown in OSCC cells were detected by the MTT assay, colony formation assay, transwell invasion assay, scratch assay, and F-actin phalloidin staining. An in vivo tongue-xenografted nude mouse model was established to measure the effects of knockdown of RhoC on tumor cell growth and lymph node metastasis. A mechanism study was conducted by real-time PCR and immunocytochemistry. The results of TCGA analysis showed that RhoC was overexpressed in OSCC tumor tissues. In vitro assays indicated that knockdown of RhoC did not have much effect on OSCC cell growth but significantly suppressed cell colony formation, invasion, and migration abilities, and F-actin polymerization was also reduced. The tongue-xenografted in vivo model demonstrated that knockdown of RhoC suppressed OSCC cell growth and inhibited metastasis to the superficial cervical lymph nodes. Further mechanism studies showed that knockdown of RhoC downregulated HMGA2 expression, and HMGA2 expression was highly correlated with RhoC expression in OSCC tumor tissues via the analysis of TCGA datasets. Overall, our study showed that knockdown of RhoC inhibited OSCC cells invasion and migration in vitro and OSCC cell growth and lymph node metastasis in vivo. Moreover, the potential mechanisms involved in these activities may be related to the regulation of HMGA2 expression. The RhoC gene could serve as a promising therapeutic target for OSCCs in the future.

Knockdown of Matrix Metallopeptidase 9 Inhibits Metastasis of Oral Squamous Cell Carcinoma Cells in a Zebrafish Xenograft Model.

Destruction of extracellular matrix (ECM) is one of the basic steps of tumor invasion and metastasis. Matrix metalloproteinase (MMP) 9, a kind of zinc-ion-dependent endopeptidase, can degrade almost all protein components in the ECM, destroy the histological barrier of tumor cell invasion, and play a key role in tumor invasion and metastasis. The role of MMP-9 in tumor invasion and metastasis has attracted increasing attention and is considered the main proteolytic enzyme in this process. Although the overexpression of MMP-9 was detected in Oral squamous cell carcinoma (OSCC) tissues, further basic studies in vivo and in vitro are needed to investigate the role of MMP-9 in OSCCs and provide scientific validation. In this research, we developed a novel OSCC zebrafish xenograft model to study the role of the MMP-9 gene in oral carcinogenesis. Firstly, the MMP-9/shRNA lentiviral clone and control virus were constructed and transfected into OSCC cells. Then, the decreasing expression of MMP-9 was verified by RT-PCR and immunocytochemistry. Cell proliferation was detected by MTT assay. Colony formation was evaluated by colony formation assay. Cell invasion was evaluated using transwell invasion assay in vitro. In addition, OSCC cells with MMP-9/shRNA knockdown and control vector were injected into zebrafish and an OSCC tumor model in zebrafish was established to evaluate invasion and metastasis in vivo. Knockdown of MMP-9 gene by shRNA could inhibit OSCC cell growth and clone formation and markedly suppress cell invasion in vitro. And the knockdown of the MMP-9 gene could also significantly decrease the metastatic distance and number of metastatic tumor cells or lesions in vivo and suppress the metastasis rate in xenografted zebrafish. Taken together, these evidences indicated that the knockdown of MMP-9 might suppress OSCC cell invasion and metastasis in vivo and in vitro. The MMP-9 gene may be a promising therapeutic target for OSCCs in the future.

High­dose folic acid improves endothelial function by increasing tetrahydrobiopterin and decreasing homocysteine levels.

The aim of this study was to investigate the effect of folic acid (FA) on tetrahydrobiopterin (BH4), neopterin, nitric oxide (NO) and homocysteine (Hcy) levels in endothelial cells. Human umbilical vein endothelial cells (HUVECs) were cultured in vitro in the presence or absence of Hcy. The effect of various doses of FA on Hcy, BH4, neopterin and NO concentrations in HUVECs was then assessed. In the 5 and 10 nmol/l FA treatment groups, FA was found to significantly increase the levels of BH4 (10.56±3.86 and 11.23±2.1919 pmol/g vs 6.32+2.87 nmol/g; P<0.05 vs. control) and NO production (37.86±12.34 nmol/l, 38.45±11.23 nmol/l vs 26.21±9.24 nmol/l; P<0.001 vs. paired Hcy group), but reduce the levels of Hcy (132.87±29.67 and 140.87±26.76 nmol/l vs. 165.23±30.56 nmol/l; P<0.05 vs. Hcy group). No significant differences were observed in neopterin levels among the different groups of HUVECs. In conclusion, high doses of FA may be capable of protecting endothelial cells through reducing levels of Hcy and increasing BH4 and NO production.