Identification of potential biomarkers and survival analysis for oral squamous cell carcinoma: A transcriptomic study.

OBJECTIVE: Oral squamous cell carcinoma (OSCC) is one of the most common neoplasms worldwide. The current study aimed to identify potential biomarkers associated with OSCC survival. MATERIALS AND METHODS: Differentially expressed genes (DEGs) in atypical OSCC cases were identified using two public datasets: The Cancer Genome Atlas and the Gene Expression Omnibus database. Receiver operating characteristic (ROC) analysis was performed to identify the cutoff, and the candidate DEGs related to survival. Kaplan-Meier and Cox regression analysis using the categorized genes were employed to identify genes that impact the overall survival in OSCC. RESULTS: A total of 263 OSCC samples and 105 healthy tissues were used to identify 295 upregulated and 131 downregulated genes expressed only in non-smokers. ROC analyses identified 25 candidate genes associated with death. Survival analyses demonstrated that the following DEGs, namely CSTA, FGFR2, MMP19, OLR1, PCSK1, RAMP2, and CGB5, are potential OSCC prognostic factors. CONCLUSION: We found that CSTA, FGFR2, MMP19, OLR1, PCSK1, RAMP2, and CGB5 are associated with a low survival rate in OSCC. However, further studies are needed to validate our findings and facilitate the development of these factors as potential biomarkers for OSCC survival.

Photodynamic therapy mediated by nanoparticles Aluminum Chloro Phthalocyanine in oral squamous carcinoma cells.

The aim of this study is to investigate the antineoplastic potential of photodynamic therapy (PDT) mediated by an aluminum-phthalocyanine chloride nanoemulsion (AlPc-NE), against an oral squamous cell carcinoma (OSCC) cell line in vitro. Both OSCC (SCC9) and A431 cell lines were studied in vitro. Four study groups were used: Group 1 (phosphate-buffered saline [PBS]), Group 2 (PBS + 28.3 J/cm2 irradiation), Group 3 (AlPc-NE alone), and Group 4 (AlPc-NE + 28.3 J/cm2 irradiation). To test the effect of PDT with AlPc-NE, cell viability, migration, and cell death assays were performed. Moreover, the expressions of Ki-67 and TP53 were evaluated using immunoassays. The results showed that PDT mediated by all AlPc-NE concentrations evaluated (i.e., 0.7, 0.35, and 0.17 nM AlPc) significantly reduced the viability of SCC9 cells. Migration and cell death assays also revealed that PDT with AlPc-NE significantly reduced the rate of migration and increased cell death compared to the control groups. In addition, it was found that PDT with AlPc-NE reduced Ki-67 and mutated TP53 immunoexpression. PDT with AlPc-NE is effective in reducing the viability and migration of SCC9. Moreover, PDT with AlPc-NE nanoemulsions reduces the cell proliferation and expression of mutant TP53.

Effect of low-level light therapy before radiotherapy in oral squamous cell carcinoma: An in vitro study.

Radiation therapy for head and neck squamous cell carcinoma (HNSCC) is associated with several complications. Although photobiomodulation (PBM) has radioprotective effects in normal tissue, it could also enhance the growth of neoplastic cells. Thus, the present study aimed to investigate the cellular response of oral squamous cell carcinoma with pre-exposure to low-level phototherapy before radiotherapy. SCC9, Cal-27, A431, and HaCaT cell lines were subjected to low-level light therapy and radiotherapy. The cells were treated with a single energy density (300 J/cm2) of a light-emitting diode (660 nm) prior to ionizing radiation at different doses (0, 2, 4, and 6 Gy). After 24 h, wound scratch, proliferation, clonogenic cell survival, cell death, and reactive oxygen species (ROS) analyses were performed to evaluate cell response. The cell lines pre-exposed to PBM at the analyzed dosage were radiosensitive. The treatment significantly reduced cell proliferation and clonogenic cell survival. Migration and cell death assays also revealed positive results, with the treatment group showing lower rate of migration and higher cell death than did the control group. Moreover, PBM effectively increased the intracellular levels of ROS. PBM at 300 J/cm2 is a promising radiosensitizing modality to reduce the radiation dose and avoid the intolerable side effects of radiotherapy for HNSCC, thus increasing the probability of successful treatment. However, further studies are needed to support and confirm the results.

Prion protein is associated with a worse prognosis of head and neck squamous cell carcinoma.

BACKGROUND: Head and neck squamous cell carcinoma (HNSC) etiopathogenesis remains unclear, and the biological changes with the activation of heat shock proteins (HSPs) and prion protein (PRNP) promoted by hypoxia in HNSC are undetermined. This study investigates hypoxia's effect in lymph node metastasis by PRNP expression changes and its main partners. METHODS: The study combined a theoretical/cell culture study with a case-control study. First, bioinformatics and cell culture were performed. A case-control study was performed in a second step by comparing HNSC patients with and without lymph node metastasis. ANALYSES: The Cancer Genome Atlas (TCGA) data source validates the theory in the global population study. RESULTS: Bioinformatics analysis suggests that hypoxia-inducible factor-1α (HIF1A) is associated with HSPA4, HSP90AA1 and PRNP expression. TCGA data validate the hypothesis that higher HSP90AA1, HSPA4 and PRNP are related to metastases and low survival. Herein, the cell study demonstrated that muted PRNP did not respond to hypoxia. DISCUSSION: Our results collectively provide the first evidence that PRNP promotes HNSC lymph node metastasis progression through the upregulation of HSPA4, HSP90AA1 and HIF1A. Our findings may provide a molecular basis for the promoting Role of PRNP in HNSC progression.

High levels of ANXA2 are characteristic of malignant salivary gland tumors.

OBJECTIVE: Malignant salivary gland tumors (MSGTs) present different phenotypic characteristics and various clinical outcomes, which proved to be a diagnostic challenge. Considering the heterogeneity of MSGT, this study aims to identify molecule related to the nature of MSGT. METHODS: For screening, proteomic analysis comparing MSGT with pleomorphic adenoma (PA) and salivary gland was performed. The MSGT-associated protein which presented in the higher number in the Gene Expression Omnibus (GEO) database was selected. To validate the data, immunohistochemistry (IHC) was performed in 14 patients with PA, 22 patients with MSGT, and 14 controls. RESULTS: 16 proteins were associated with MSGT. ANXA2 was the primary protein, according to GEO database analyses. ANXA2 was most expressed in the cell membrane. However, some ANXA2 staining was also observed in the cytoplasm and nucleus. ANXA2 was highly expressed in MSGT in comparison with control. Also, ANXA2 has a higher expression in adenocarcinoma not otherwise specified (ANOS) and myoepithelial carcinoma (MC) in comparison with PA. CONCLUSION: In conclusion, this study demonstrated that MSGT presented higher levels of ANXA2 in comparison with normal salivary glands. Also, ANXA2 might be interesting as a molecular marker of ANOS and MS.

p16 (CDKN2A) SNP rs11515 was not associated with head and neck carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is considered a serious public health problem in many countries. Recently, genetic variations have been considered as important factors to cancer susceptibility and prognosis. More specifically, genetic polymorphisms have been associated with the development and prognosis of HNSCC. The purpose of the current study was to investigate an association among p16 (CDKN2A) gene polymorphism at rs11515, age, and HNSCC aggressiveness. PCR-RFLP analysis was used to investigate the p16 (CDKN2A) gene in 96 patients with HNSCC and in 100 individuals without HNSCC. A case group was categorized by age in younger (<60 years) and older (≥ 60 years) patients. Differences between the case and control groups were determined using Fisher and chi-squared tests. Time of survival was calculated from the date of diagnosis to the date of last follow-up visit or to the date of death using the Kaplan-Meier estimator and comparing this to the log-rank test. Statistical significance was set at p<0.05. In the present study, no association was established between HNSCC and rs11515 polymorphism, as indicated in a previous study. We found that HNSCC individuals with large-sized tumors and with metastatic disease presented worse overall survival, consistent with fundamental concepts that establish the effects of tumor size and lymph node metastasis to HNSCC outcomes. This study identified that there is no difference in the distribution of rs11515 between the control and HNSCC groups. In addition, no differences between rs11515 genotypes and clinicopathological parameters were observed.

The antineoplastic potential of crotoxin isolated from Crotalus durissus terrificus snake venom on oral squamous cell carcinoma.

This study investigated the antineoplastic effects of crotoxin isolated from snake venom of the South American Crotalus durissus terrificus in oral cancer cell lines and in an animal model of chemically induced oral cancer. We analyzed cell viability and death, clonogenic formation, DNA fragmentation, migration assay, and gene expression of MMP2, MMP9, COL1A1, and CASP3. In the animal model, after induction of oral cancer by 4-nitroquinoline-1-oxide carcinogen, mice were treated with crotoxin to investigate its effects on tumor development in tongue and oral mucosa. Crotoxin inhibited cell proliferation, viability, colony formation, and migration, favoring cell death. Furthermore, crotoxin increased caspase-3 expression, decreased Ki-67 protein and mRNA expression of MMP2, MMP9, and COL1A1. Mice treated with crotoxin at 10 µg/kg did not alter biochemical parameters total cholesterol, very-low-density lipoprotein, high-density lipoprotein, liver transaminases, glycemia, creatinine, and urea. Crotoxin treatment significantly reduced the frequency of oral squamous cell carcinoma lesions by 50%. Thus, this study highlights crotoxin as a promising chemotherapeutic substance, considering its effects on controlling the neoplastic cell population, reducing cell migration, and inhibiting tumor development. Clinical studies are necessary to understand better the impact of crotoxin as a potential adjuvant therapeutic agent for oral cancer patients.

Gallic acid has an inhibitory effect on skin squamous cell carcinoma and acts on the heat shock protein HSP90AB1.

Differences in the features of aggressiveness of non-melanoma skin cancer (NMSC) subtypes, between basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) are relevant characteristics. Comparing the characteristics between NMSC subtypes might help identify molecules associated with cancer metastasis and invasion. Considering these facts, the current study aimed to identify a molecular target for inhibiting skin cancer metastasis and invasion. Proteomic analysis suggested that heat shock protein 90 kDa, alpha, class B member 1 (HSP90AB1), pentaxin (PTX3), caspase-14 (CASP14), S100, actin-1, and profilin were the primary targets related to metastasis and invasion. However, after a differential expression comparison between BCC and SCC, HSP90AB1 was identified as the best target to repress metastasis and invasion. Based on molecular docking results, gallic acid (GA) was selected to inhibit HSP90AB1. A specific Hsp90ab1 siRNA targeting was designed and compared to GA. Interestingly, GA was more efficient in silencing HSP90AB1 than siRNAhsp90ab1. Hence, our data suggest that HSP90AB1 is a crucial biomarker for identifying invasion and metastasis and that its inhibition may be a viable strategy for treating skin cancer.

Pathways Related to the Anti-Cancer Effects of Metabolites Derived from Cerrado Biome Native Plants: An Update and Bioinformatics Analysis on Oral Squamous Cell Carcinoma.

BACKGROUND: Oral cancer is a significant health problem worldwide. Oral squamous cell carcinoma (OSCC) is a malignant neoplasm of epithelial cells that mostly affects different anatomical sites in the head and neck and derives from the squamous epithelium or displays similar morphological characteristics. Generally, OSCC is often the end stage of several changes in the stratified squamous epithelium, which begin as epithelial dysplasia and progress by breaking the basement membrane and invading adjacent tissues. Several plant-based drugs with potent anti-cancer effects are considered inexpensive treatments with limited side effects for cancer and other diseases. OBJECTIVE: The aim of this review is to explore whether some Brazilian plant extracts or constituents exhibit anti-tumorigenic activity or have a cytotoxic effect on human oral carcinoma cells. METHODS: Briefly, OSCC and several metabolites derived from Brazilian plants (i.e., flavonoids, vinblastine, irinotecan, etoposide and paclitaxel) were used as keywords to search the literature on PubMed, GenBank and GeneCards. RESULTS: The results showed that these five chemical compounds found in Cerrado Biome plants exhibit anti-neoplastic effects. Evaluating the compounds revealed that they play a main role in the regulation of cell proliferation. CONCLUSION: Preserving and utilising the biodiversity of our planet, especially in unique ecosystems, such as the Cerrado Biome, may prove essential to preserving and promoting human health in modern contexts.

Might anxiety disorders promote head and neck cancer development?

Cancer patients present a higher risk of experiencing anxiety disorders (AD). However, it is not clear if AD might be associated with cancer development. Thus, our study aimed to evaluate if AD might be related to head and neck squamous cell carcinoma (HNSCC) development. The combination of an applied animal basic study and a retrospective diagnostic case and control study in patients was performed. As a result, we obtained that stress reduced the locomotor activity of the animals in the group stress and stress + 4NqO (p < 0.0001). The stress showed no influence on the progression of neoplasia in mice. In the same way, the case group did not present differences in anxiety scores in comparison to control. Moreover, no association between HNSCC staging and anxiety scores was observed. In conclusion, our in vivo findings in humans and animals have shown that there is no relationship between AD and oral squamous cell carcinoma.

Radiation Therapy Reduced Blood Levels of LDH, HIF-1α, and miR-210 in OSCC.

Radiation Therapy (RT) is a treatment option for a large number of neoplasias. However, the effect of RT on the level of hypoxia markers is poorly understood. The present study aimed to investigate the effect of RT on the levels of hypoxic markers in Oral squamous cell carcinoma (OSCC). Evaluation of HIF-1α and miR-210 levels in OSCC was performed. Then a proteomic analysis was performed to identify candidate hypoxic targets of RT. To validate proteomic studies, the effect of RT on HIF-1α, miR-210, PDH-A and LDH-A levels under hypoxia was assessed by qRT-PCR. The impact of RT in hypoxia markers was evaluated in patients to confirm in vitro results. An increase in the HIF-1α levels was observed in OSCC. RT reduced OSCC cell proliferation and migration. Interestingly, hypoxia could revert the effect of radiation on OSCC phenotype. However, proteomics analyses suggested that LDH is one of the critical targets of RT even in hypoxia. Moreover, RT decreased HIF-1α, miR-210, and LDH even in hypoxia. The current study demonstrated that hypoxia could revert the effects of RT in the OSCC context. However, RT reduces the levels HIF-1α, miR-210 and LDH in vivo and in vitro. The consequences of RT in blood should be carefully investigated.

Is HIF1-a deregulated in malignant salivary neoplasms?

BACKGROUND: There is significant controversy in the literature regarding the relationship between hypoxia and salivary gland neoplasms (SGNs). OBJECTIVE: The current study aims to investigate levels of hypoxia markers in both benign and malignant salivary neoplasms. PATIENTS AND METHODS: The current study sample is comprised of a total of 62 samples. HIF-1α expression was evaluated by immunohistochemistry. Additionally, HIF-1α mRNA and miR-210 levels were assessed using qRT-PCR. RESULTS: No differences in HIF-1α expression were observed among the control group, benign and malignant SGNs. Similarly, HIF-1α mRNA levels were similar between benign and malignant SGNs. Also, there was no difference in miR-210 expression between case and control groups. CONCLUSION: The angiogenic markers, miR-210 and HIF-1α, do not appear to distinguish malignancy in salivary glands.

The antineoplastic potential of crotoxin isolated from Crotalus durissus terrificus snake venom on oral squamous cell carcinoma

This study investigated the antineoplastic effects of crotoxin isolated from snake venom of the South American Crotalus durissus terrificus in oral cancer cell lines and in an animal model of chemically induced oral cancer. We analyzed cell viability and death, clonogenic formation, DNA fragmentation, migration assay, and gene expression of MMP2, MMP9, COL1A1, and CASP3. In the animal model, after induction of oral cancer by 4-nitroquinoline-1-oxide carcinogen, mice were treated with crotoxin to investigate its effects on tumor development in tongue and oral mucosa. Crotoxin inhibited cell proliferation, viability, colony formation, and migration, favoring cell death. Furthermore, crotoxin increased caspase-3 expression, decreased Ki-67 protein and mRNA expression of MMP2, MMP9, and COL1A1. Mice treated with crotoxin at 10 μg/kg did not alter biochemical parameters total cholesterol, very-low-density lipoprotein, high-density lipoprotein, liver transaminases, glycemia, creatinine, and urea. Crotoxin treatment significantly reduced the frequency of oral squamous cell carcinoma lesions by 50%. Thus, this study highlights crotoxin as a promising chemotherapeutic substance, considering its effects on controlling the neoplastic cell population, reducing cell migration, and inhibiting tumor development. Clinical studies are necessary to understand better the impact of crotoxin as a potential adjuvant therapeutic agent for oral cancer patients.

Hypoxia reduces the E-cadherin expression and increases OSCC cell migration regardless of the E-cadherin methylation profile.

OBJECTIVE: The purpose of the current study is to investigate the association between E-cadherin methylation status, hypoxia and OSCC. METHODS: HaCat and SCC9 cell lines were submitted to hypoxic treatment, followed by methylation profile analysis (MS-PCR) and analysis of the expression of mRNA gene E-cadherin (RT-PCR). Study group samples comprise individuals affected by potentially malignant lesions Potential Malignant Oral Lesion (PMOL, n=18) and oral squamous cell carcinoma (OSCC, n=28). The control group oral mucosa (OM, n=15) of patients with an oral mucocele. Cell migration ability was evaluated a scratch wound assay in SCC9 and HaCat cell lines RESULTS: E-cadherin mRNA expression in the cell lines SCC9 and HaCat was significantly reduced under hypoxia, regardless of the methylation profile, when compared to the control group. No differences in methylation profile of the E-cadherin were observed among the groups OM, PMOL and OSCC. HaCat and SCC9 presented increases in cell migration rates under hypoxia. CONCLUSION: The current study demonstrates that hypoxia reduces E-cadherin expression and increase cell migration, regardless of the methylation profile. Additionally, no differences in E-cadherin methylation patterns were observed among OM, PMOL and OSCC.

Metformin increases PDH and suppresses HIF-1α under hypoxic conditions and induces cell death in oral squamous cell carcinoma.

BACKGROUND: Metformin is a biguanide, belonging to the oral hypoglycemic agents and is a widely used in the treatment of type 2 diabetes. Evidence indicate that Metformin inhibits cell proliferation in several human cancers and inhibits the Warburg phenomenon in tumor cells. RESULTS: Low PDH levels were observed in OSCC, and Metformin promotes an increase in PDH levels in hypoxic conditions. Metformin also reduced HIF-1α mRNA and protein levels. Metformin demonstrated antiproliferative effects, inhibited migration, increased the number of apoptotic cells and increased the transcription of caspase 3. OBJECTIVE: The present study aims to explore the effects of Metformin in hypoxic conditions. Specifically, we focused on pyruvate dehydrogenase (PDH), (hypoxia-inducible factor 1α) HIF-1α levels and the oral squamous cell carcinoma (OSCC) cell phenotype. Additionally, we also investigated a theoretical consequence of Metformin treatment. METHODS: PDH levels in patients with OSCC and oral dysplasia were evaluated. Metformin was administered in vitro to test the effect of Metformin under hypoxic conditions. The results were complemented by Bioinformatics analyses. CONCLUSIONS: In conclusion, our current findings show that Metformin reduces HIF-1α gene expression and increases PDH expression. Metformin inhibits cell proliferation and migration in the OSCC cell line model. Additionally, Metformin enhances the number of apoptotic cells and caspase 3 levels. Interestingly enough, Metformin did not increase the mutant p53 levels under hypoxic conditions.

Leptin receptor polymorphism Gln223Arg (rs1137101) in oral squamous cell carcinoma and potentially malignant oral lesions.

The purpose of this study was to assess the LEPR gene Gln223Arg polymorphism (rs1137101) in oral squamous cell carcinoma (OSCC) and in potentially malignant oral lesions (PMOL) in comparison to normal oral mucosa in a Brazilian population. Smokers (n = 89) were selected from a representative sample of 471 individuals from the general population of Montes Claros, Brazil. Participants were age and gender matched to patients with OSCC (n = 25) and oral epithelial dysplasia (n = 25). We investigated the LEPR Gln223Arg polymorphism (A>G; rs1137101) in these groups. Genotype variants were assessed by RFLP-PCR, using MspI (HPAII) restriction endonuclease. The institutional review board of the Universidade Estadual de Montes Claros approved the study (process number 2667/2011). Written informed consent for this study was obtained from all participants. The GG genotype (Arg223Arg) appears to be the more relevant polymorphic variant in OSCC. It occurred, approximately, twice as frequently in OSCC patients than in the general population. In contrast, the A allele in its homozygosis form (Gln223Gln) is significantly associated with the development of PMOL; 80% of the samples from the PMOL group exhibit AA genotype. Our findings suggest new insights regarding LEPR gene variations in the development of OSCC and PMOL.