Bortezomib suppresses acute myelogenous leukaemia stem-like KG-1a cells via NF-κB inhibition and the induction of oxidative stress.

Acute myelogenous leukaemia (AML) originates and is maintained by leukaemic stem cells (LSCs) that are inherently resistant to antiproliferative therapies, indicating that a critical strategy for overcoming chemoresistance in AML therapy is to eradicate LSCs. In this work, we investigated the anti-AML activity of bortezomib (BTZ), emphasizing its anti-LSC potential, using KG-1a cells, an AML cell line with stem-like properties. BTZ presented potent cytotoxicity to both solid and haematological malignancy cells and reduced the stem-like features of KG-1a cells, as observed by the reduction in CD34- and CD123-positive cells. A reduction in NF-κB p65 nuclear staining was observed in BTZ-treated KG-1a cells, in addition to upregulation of the NF-κB inhibitor gene NFΚBIB. BTZ-induced DNA fragmentation, nuclear condensation, cell shrinkage and loss of transmembrane mitochondrial potential along with an increase in active caspase-3 and cleaved PARP-(Asp 214) level in KG-1a cells. Furthermore, BTZ-induced cell death was partially prevented by pretreatment with the pancaspase inhibitor Z-VAD-(OMe)-FMK, indicating that BTZ induces caspase-mediated apoptosis. BTZ also increased mitochondrial superoxide levels in KG-1a cells, and BTZ-induced apoptosis was partially prevented by pretreatment with the antioxidant N-acetylcysteine, indicating that BTZ induces oxidative stress-mediated apoptosis in KG-1a cells. At a dosage of 0.1 mg/kg every other day for 2 weeks, BTZ significantly reduced the percentage of hCD45-positive cells in the bone marrow and peripheral blood of NSG mice engrafted with KG-1a cells with tolerable toxicity. Taken together, these data indicate that the anti-LSC potential of BTZ appears to be an important strategy for AML treatment.

Exploring the combination of tumor-stroma ratio, tumor-infiltrating lymphocytes, and tumor budding with WHO histopathological grading on early-stage oral squamous cell carcinoma prognosis.

BACKGROUND: While the relevance of the World Health Organization histopathological grading system as a prognostic tool for oral squamous cell carcinoma has received many critics, other histopathological features such as tumor-stroma ratio, tumor-infiltrating lymphocytes, and tumor budding are displaying promising results. Here, we evaluated the prognostic impact of the incorporation of tumor-stroma ratio, tumor-infiltrating lymphocytes, and tumor budding into World Health Organization histopathological grading for patients with oral squamous cell carcinoma. METHODS: A total of 95 patients with early-stage oral squamous cell carcinoma were enrolled in the study, and World Health Organization tumor grading, tumor-stroma ratio, tumor-infiltrating lymphocytes, and tumor budding were evaluated in surgical slides stained with hematoxylin and eosin. Survival analyses for cancer-specific survival and disease-free survival were performed using Cox regression models, and receiver operating characteristic curves were applied for assessment of the performance of the combinations. RESULTS: Tumor-stroma ratio (stroma-rich) was significantly and independently associated with both shortened cancer-specific survival and poor disease-free survival, individually and in combination with World Health Organization histopathological grading. The combination of tumor-stroma ratio with World Health Organization grading did not improve the discriminatory ability compared to tumor-stroma ratio alone. Although low tumor-infiltrating lymphocytes were associated with shortened cancer-specific survival, the association did not withstand multivariate analysis. However, in combination with World Health Organization grading, low tumor-infiltrating lymphocytes were independently associated with poor cancer-specific survival. The combination of tumor-infiltrating lymphocytes and World Health Organization histopathological grading displayed a better discrimination of poor cancer-specific survival than tumor-infiltrating lymphocytes alone, but not at a significant level. CONCLUSION: Our findings support tumor-stroma ratio as a potential prognostic marker for patients with oral squamous cell carcinoma, and the incorporation of tumor-infiltrating lymphocytes into the World Health Organization grading system improves the prognostic ability of the tumor grading alone.

Overexpression of heat-shock protein 47 impacts survival of patients with oral squamous cell carcinoma.

BACKGROUND: The expression of heat-shock protein 47 (HSP47) has been linked to collagen synthesis control and implicated in fibrotic disorders, but more recent studies have demonstrated its role in solid tumors. In this study, we explored the prognostic impact of HSP47 in oral squamous cell carcinomas (OSCC) and determined the in vitro effects of its loss-of-function on viability, proliferation, migration, invasion, and resistance to cisplatin of OSCC cells. METHODS: The HSP47 expression in tumor samples was assessed by immunohistochemistry in two independent cohorts totaling 339 patients with OSCC, and protein levels were associated with clinicopathological features and survival outcomes. The OSCC cell lines HSC3 and SCC9 were transduced with lentivirus expressing short hairpin RNA to stably silence HSP47 and used in assays to measure cellular viability, proliferation, migration, and invasion. RESULTS: HSP47 was overexpressed in OSCC samples, and its overexpression was significantly and independently associated with poor disease-specific survival and shortened disease-free survival in both OSCC cohorts. The knockdown of HSP47 showed no effects on cell viability or cisplatin sensitivity, but impaired significantly proliferation, migration, and invasion of OSCC cells, with stronger effects on SCC9 cells. CONCLUSION: Our results show a significant prognostic impact of HSP47 overexpression in OSCC and reveal that HSP47 inhibition impairs the proliferation, migration, and invasion of OSCC cells. HSP47 may represent a potential therapeutic target for OSCC.

Macrophages and endothelial cells orchestrate tumor-associated angiogenesis in oral cancer via hedgehog pathway activation.

The present study aimed to evaluate the role of Hedgehog (Hh) molecule expression in association with the clinical aspects of oral squamous cell carcinoma (OSCC), as well as angiogenesis and CD163+ macrophages. Twenty-eight cases of OSCC, nine cases of tumor-free resection margins (TM), and four cases of non-neoplastic oral mucosa (NNM) were submitted to immunohistochemistry to detect proteins Sonic Hedgehog (SHH), Indian Hedgehog (IHH), GLI1, CD163, and CD105. Protein colocalization with respect to SHH/CD163, IHH/CD163, GLI1/CD163, and GLI1/CD105 was assessed by immunohistochemical double staining. In tumor parenchyma, SHH and IHH were present in the cytoplasm of neoplastic cells, while GLI1 was observed in cytoplasm and nucleus. Endothelial cells were found to express SHH, IHH, and GLI1 within CD105+ vessels, and a positive correlation between infiltrating macrophage density (IMD) and microvascular density (MVD) was observed in cases of OSCC and TM. When compared to TM and NNM, the OSCC cases demonstrated higher immunoreactivity for SHH (p = 0.01), IHH (p = 0.39), GLI1 (p = 0.03), IMD (p = 0.0002), and MVD (p = 0.0002). Our results suggest the participation of the Hh pathway in OSCC by way of autocrine and paracrine signaling, in addition to the participation of both SHH and IHH ligands. Endothelial cells were also found to exhibit positivity with respect to Hh pathway components and we surmise that these molecules may play a role in tumor angiogenesis. CD163+ macrophages were also observed to express IHH, a ligand of this pathway, in addition to being associated with tumor neovascularization.

Elevated VEGFA mRNA levels in oral squamous cell carcinomas and tumor margins: a preliminary study.

BACKGROUND: New blood vessel formation events are essential for tumor clonal expansion and progression. Despite the importance of vascular endothelial growth factor A (VEGFA) for tumor angiogenesis, few studies have evaluated the expression profile of this gene in oral squamous cell carcinoma (OSCC) and tumor margins (TM). This study aimed to evaluate the expression of the VEGFA gene and its protein in OSCC and TM. METHODS: Gene expression was evaluated in cryopreserved samples of OSCCs (n = 44), TM (n = 7), and normal mucosa from healthy patients (n = 3; NM). Quantitative PCRs were performed using the TaqMan system for the VEGFA gene, and gene expression was determined using the 2(-∆∆CQ) method. For immunohistochemical evaluation, 27 OSCC samples were embedded in a tissue microarray (TMA) block and reactions were performed using the Advance system. RESULTS: VEGFA transcript levels were 1.7-fold higher in OSCC than in NM samples. VEGFA mRNA was overexpressed in TM samples compared to NM (3.4-fold) and OSCC (2.0-fold) samples. VEGFA transcript level was overexpressed in T3-T4 tumors, metastatic lymph nodes, and stage III-IV OSCCs. Immunoreactivity of the VEGFA protein was detected in the cytoplasm of parenchymal and stromal cells, mainly in endothelial cells and fibroblasts. CONCLUSION: Our results show that VEGFA was overexpressed in aggressive OSCCs and that VEGFA expression may be an important prognostic factor in this type of cancer. Finally, tumor margins may be involved in the production of angiogenic molecules.

In silico approaches for drug repurposing in oncology: a scoping review.

Introduction: Cancer refers to a group of diseases characterized by the uncontrolled growth and spread of abnormal cells in the body. Due to its complexity, it has been hard to find an ideal medicine to treat all cancer types, although there is an urgent need for it. However, the cost of developing a new drug is high and time-consuming. In this sense, drug repurposing (DR) can hasten drug discovery by giving existing drugs new disease indications. Many computational methods have been applied to achieve DR, but just a few have succeeded. Therefore, this review aims to show in silico DR approaches and the gap between these strategies and their ultimate application in oncology. Methods: The scoping review was conducted according to the Arksey and O'Malley framework and the Joanna Briggs Institute recommendations. Relevant studies were identified through electronic searching of PubMed/MEDLINE, Embase, Scopus, and Web of Science databases, as well as the grey literature. We included peer-reviewed research articles involving in silico strategies applied to drug repurposing in oncology, published between 1 January 2003, and 31 December 2021. Results: We identified 238 studies for inclusion in the review. Most studies revealed that the United States, India, China, South Korea, and Italy are top publishers. Regarding cancer types, breast cancer, lymphomas and leukemias, lung, colorectal, and prostate cancer are the top investigated. Additionally, most studies solely used computational methods, and just a few assessed more complex scientific models. Lastly, molecular modeling, which includes molecular docking and molecular dynamics simulations, was the most frequently used method, followed by signature-, Machine Learning-, and network-based strategies. Discussion: DR is a trending opportunity but still demands extensive testing to ensure its safety and efficacy for the new indications. Finally, implementing DR can be challenging due to various factors, including lack of quality data, patient populations, cost, intellectual property issues, market considerations, and regulatory requirements. Despite all the hurdles, DR remains an exciting strategy for identifying new treatments for numerous diseases, including cancer types, and giving patients faster access to new medications.

Rapid Detection of SARS-CoV-2 Based on the LAMP Assay Associated with the CRISPRCas12a System.

BACKGROUND: The global public health system has been severely tested by the COVID-19 pandemic. Mass testing was essential in controlling the transmission of the SARS-CoV-2; however, its implementation has encountered challenges, particularly in low-income countries. The urgent need for rapid and accurate tests for SARS-CoV-2 has proven to be extremely important. Point-of-care tests using the CRISPR system for COVID-19 have shown promise, with a reported high sensitivity and rapid detection. The performance of a CRISPR-based SARS-CoV-2 testing system was reported in this study. METHODS: A total of 29 nasopharyngeal samples were evaluated, including 23 samples from individuals suspected of COVID-19, and six samples positive for H3N2 or respiratory syncytial virus. Two reference samples with known concentrations of SARS-CoV-2 RNA (3000 RNA copies/mL) or viral titer determined by plaque assay (105 PFU/mL) were also evaluated. The LAMP technique was employed to amplify the ORF1ab gene and the results were analyzed using a Gemini XPS fluorescence reader. RESULTS: The RT-LAMP-CRISPR/Cas12 assay showed 100% concordance compared to RT-PCR. The RT-PCR presented a detection limit of 0.01 PFU/mL and the CRISPR/Cas12 system showed a limit of 15.6 PFU/mL. The RT-PCR sensitivity was approximately 8 RNA copies/µL and CRISPR/Cas12 at 84 RNA copies/µL. CONCLUSION: The RT-LAMP-CRISPR/Cas12a assay offered a promising alternative for the detection of SARS-CoV-2 and reinforces that CRISPR-based diagnostic techniques can be an alternative for fast and accurate assays.

SARS-CoV-2 Detection via RT-PCR in Matched Saliva and Nasopharyngeal Samples Reveals High Concordance in Different Commercial Assays.

BACKGROUND: Self-collected saliva samples can increase the diagnostic efficiency and benefit healthcare workers, patient care, and infection control. This study evaluated the performance of self-collected saliva samples compared to nasopharyngeal swabs using three commercial kits for the qualitative detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). METHODS: Matched nasopharyngeal and saliva samples were collected from 103 patients with either asymptomatic or symptomatic COVID-19. Both samples were evaluated using three commercial kits (TaqCheck, Allplex, and TaqPath). To evaluate sample stability, viral RNA extraction was performed in the presence or absence of an RNA-stabilizing solution. Storage conditions, including the duration, temperature, and stability after freezing and thawing of the samples, were also evaluated. RESULTS: All the saliva samples showed 100% concordance with the nasopharyngeal swab results using TaqCheck and Allplex kits, and 93% using TaqPath kit. No difference was observed in the samples that used the RNA-stabilizing solution compared to the group without the solution. The Ct values of the freeze-thawed samples after 30 days were higher than those on day 0; however, the results were consistent the fresh samples. CONCLUSION: The high concordance of SARS-CoV-2 detection via reverse transcription-polymerase chain reaction (RT-PCR) in matched saliva and nasopharyngeal samples using different commercial assays reinforces the concept that self-collected saliva samples are non-invasive, rapid, and reliable for diagnosing SARS-CoV-2 infection.

In silico approaches for drug repurposing in oncology: Protocol for a scoping review of existing evidence.

Drug repurposing has been applied in the biomedical field to optimize the use of existing drugs, leading to a more efficient allocation of research resources. In oncology, this approach is particularly interesting, considering the high cost related to the discovery of new drugs with therapeutic potential. Computational methods have been applied to predict associations between drugs and their targets. However, drug repurposing has not always been promising and its efficiency has yet to be proven. Therefore, the present scoping review protocol was developed to screen the literature on how in silico strategies can be implemented in drug repurposing in oncology. The scoping review will be conducted according to the Arksey and O'Malley framework (2005) and the Joanna Briggs Institute recommendations. We will search the PubMed/MEDLINE, Embase, Scopus, and Web of Science databases, as well as the grey literature. We will include peer-reviewed research articles involving in silico strategies applied to drug repurposing in oncology, published between January 1, 2003, and December 31, 2021. Data will be charted and findings described according to review questions. We will report the scoping review using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Extension for Scoping Review guidelines (PRISMA-ScR).

Stress induced phosphoprotein 1 overexpression controls proliferation, migration and invasion and is associated with poor survival in oral squamous cell carcinoma.

Objective: Although there have been remarkable achievements in the molecular landscape of oral squamous cell carcinoma (OSCC) in recent years, bringing advances in the understanding of its pathogenesis, development and progression, little has been applied in the prognosis and choosing the optimal treatment. In this study, we explored the influence of the stress induced phosphoprotein 1 (STIP1), which is frequently reported to be highly expressed in many cancers, in OSCCs. Methods: STIP1 expression was assessed in the TCGA database and in two independent cohorts by immunohistochemistry. Knockdown strategy was applied in OSCC cell lines to determine the impact of STIP1 on viability, proliferation, migration and invasion. The zebrafish model was applied for studying tumor formation and metastasis in vivo. The association of STIP1 and miR-218-5p was explored by bioinformatics and mimics transfection. Results: STIP1 was highly expressed in OSCCs and significantly associated with shortened survival and higher risk of recurrence. STIP1 down-regulation decreased proliferation, migration and invasion of tumor cells, and reduced the number of metastases in the Zebrafish model. STIP1 and miR-218-5p were inversely expressed, and the transfection of miR-218-5p mimics into OSCC cells decreased STIP1 levels as well as proliferation, migration and invasion. Conclusion: Our findings show that STIP1 overexpression, which is inversely associated with miR-218-5p levels, contributes to OSCC aggressiveness by controlling proliferation, migration and invasion and is a determinant of poor prognosis.

The Impact of Histopathological Features on the Prognosis of Oral Squamous Cell Carcinoma: A Comprehensive Review and Meta-Analysis.

OBJECTIVE: Over many decades, studies on histopathological features have not only presented high-level evidence of contribution for treatment directions and prognosis of oral squamous cell carcinoma (OSCC) but also provided inconsistencies, making clinical application difficult. The 8th TNM staging system of OSCC has acknowledged the importance of some histopathological features, by incorporating depth of invasion (DOI) to T category and extranodal extension (ENE) to N category. The aim of this systematic review with meta-analysis is to determine the most clinically relevant histopathological features for risk assessment and treatment planning of OSCC and to elucidate gaps in the literature. METHODS: A systematic review was conducted using PRISMA guidelines, and the eligibility criteria were based on population, exposure, comparison, outcome, and study type (PECOS). PubMed, Cochrane, Scopus, and Web of Science were searched for articles exploring the impact of histopathological features on OSCC outcomes with Cox multivariate analysis. Pooled data were subjected to an inverse variance method with random effects or fixed effect model, and the risk of bias was evaluated using quality in prognosis studies (QUIPS). Quality of evidence was assessed with the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) criteria. RESULTS: The study included 172 articles published from 1999 to 2021. Meta-analyses confirmed the prognostic potential of DOI, ENE, perineural invasion, lymphovascular invasion, and involvement of the surgical margins and brought promising results for the association of bone invasion, tumor thickness, and pattern of invasion with increased risk for poor survival. Although with a small number of studies, the results also revealed a clinical significance of tumor budding and tumor-stroma ratio on predicted survival of patients with OSCC. Most of the studies were considered with low or moderate risk of bias, and the certainty in evidence varied from very low to high. CONCLUSION: Our results confirm the potential prognostic usefulness of many histopathological features and highlight the promising results of others; however, further studies are advised to apply consistent designs, filling in the literature gaps to the pertinence of histopathological markers for OSCC prognosis. SYSTEMATIC REVIEW REGISTRATION: International Prospective Register of Systematic Reviews (PROSPERO), identifier CRD42020219630.

Inhibition of CAL27 Oral Squamous Carcinoma Cell by Targeting Hedgehog Pathway With Vismodegib or Itraconazole.

Oral Squamous Cell Carcinoma (OSCC) presents an important challenge for the health systems worldwide. Thus, unraveling the biological mechanisms involved in OSCC pathogenesis is essential to the discovery of new drugs with anticancer potential. The Hedgehog (HH) pathway has shown promising results as a therapeutic target both in vitro and in vivo. This study aimed to investigate the effects of vismodegib and itraconazole on the expression of Hedgehog (HH) genes (PTCH1, SMO, and GLI1), cell cycle and cell death in OSCC cells. Alamar Blue assay was used to assess the cytotoxicity of vismodegib and itraconazole in a panel of oral cancer cell lines, including CAL27. The expression of HH signaling components after treatment with vismodegib and itraconazole, at concentrations of 25 or 50 µg/ml was evaluated by qPCR. Cell cycle and apoptosis were evaluated by flow cytometry after 72 h treatment with 50 µg/ml of vismodegib or itraconazole. HH signaling was activated in OSCC cell lines CAL27, SCC4, SCC9, and HSC3. Vismodegib and itraconazole significantly reduced CAL27 cell viability after 48 h of treatment. Gene expression of PTCH1, SMO, and GLI1 decreased in response to 24 h of treatment with vismodegib or itraconazole. Furthermore, CAL27 cells exhibited alterations in morphology, cell size, and cellular granularity. An increase in the DNA fragmentation was observed after treatment and both inhibitors induced apoptosis after 72 h. In conclusion, SMO inhibitors vismodegib and itraconazole demonstrably reduced the expression of HH genes in CAL27 OSCC cell line. In addition, treatment with vismodegib and itraconazole reduced cellular viability and altered the morphology of CAL27 cells, and also induced apoptosis.

Hedgehog pathway activation in oral squamous cell carcinoma: cancer-associated fibroblasts exhibit nuclear GLI-1 localization.

The purpose of this study was to evaluate the expression of Hedgehog (HH) signaling molecules (SHH and GLI-1) by cancer-associated fibroblasts (CAF) in oral squamous cell carcinoma (OSCC). Immunohistochemistry was used to detect molecular HH signaling and CAF-related protein expression, including α-SMA and S100A4, in 70 samples of human OSCC. The colocalization of α-SMA and S100A4 with SHH was also evaluated by double-staining. In vitro study was performed using primary normal oral fibroblast (NOF) and CAF through immunofluorescence and Western Blot for CAF-proteins, SHH, and GLI-1. Forty-five cases (64.28%) were positive for α-SMA exclusively in tumor stroma, and S100A4 was identified in the cytoplasm of CAFs in 94.28% (n = 66) of the cases. With respect to stromal cells, 64 (91.43%) OSCC cases were positive for SHH, and 31 were positive for GLI-1 (44.29%); positive correlations were found between SHH and α-SMA (p < 0.0001, φ = 0.51), as well as between SHH and S100A4 (p = 0.087, φ = 0.94). Protein expression of SHH and GLI-1 was observed in primary CAFs and NOFs. Although SHH was found to be localized in the cellular cytoplasm of both cell types, GLI-1 was present only in the nuclei of CAF. Our results indicate that CAFs are not only potential sources of HH ligands in tumor stroma, but may also respond to HH signaling through nuclear GLI-1 activation. We further observed that elevated SHH expression by OSCC cells was associated with higher CAF density, reinforcing the chemoattractant role played by these molecules.

Enhanced Expression of Hedgehog Pathway Proteins in Oral Epithelial Dysplasia.

The aim of this study was to characterize the profile of the proteins involved in the Hedgehog signaling pathway to aid in the understanding of the pathogenesis of oral epithelial dysplasia (OED). The proteins SHH, PTCH1, HHIP, SUFU, GLI1, and cyclin D1 were evaluated by immunohistochemistry in 25 cases of OED, 4 of non-neoplasic oral mucosa, 8 of inflammatory fibrous hyperplasia and 5 of hyperkeratosis. SHH proteins were predominant in OED cases. Although PTCH1 protein was observed in all cases, this molecule was more highly expressed in OED. The inhibitor protein SUFU was present in OED and HHIP protein was overexpressed in OED. GLI1 proteins were predominantly found in the nuclei of epithelial cells in OED. Basal and suprabasal cells in the epithelial lining were positive for cyclin D1 only in OED. In conclusion, comparative analysis of the proteins involved in the Hedgehog pathway suggests that enhanced expression of these proteins can play an important role in the biological behavior of OED.