Exploring the therapeutic mechanisms of resveratrol for treating arecoline-induced malignant transformation in oral epithelial cells: insights into hub targets.

BACKGROUND: Betel nut chewing is a significant risk factor for oral cancer due to arecoline, its primary active component. Resveratrol, a non-flavonoid polyphenol, possesses anti-cancer properties. It has been shown to inhibit arecoline-induced oral malignant cells in preliminary experiments but the underlying mechanism remains unclear. This research therefore aimed to explore the potential therapeutic targets of resveratrol in treating arecoline-induced oral cancer. METHODS: Data mining identified common targets and hub targets of resveratrol in arecoline-induced oral cancer. Gene set variation analysis (GSVA) was used to score and validate the expression and clinical significance of these hub targets in head and neck cancer (HNC) tissues. Molecular docking analysis was conducted on the hub targets. The effect of resveratrol intervention on hub targets was verified by experiments. RESULTS: Sixty-one common targets and 15 hub targets were identified. Hub targets were highly expressed in HNC and were associated with unfavorable prognoses. They played a role in HNC metastasis, epithelial-mesenchymal transition, and invasion. Their expression also affected immune cell infiltration and correlated negatively with sensitivity to chemotherapeutic agents such as bleomycin and docetaxel. Experiments demonstrated that resveratrol down-regulated the expression of the hub targets, inhibited their proliferation and invasion, and induced apoptosis. CONCLUSION: Resveratrol inhibits the arecoline-induced malignant phenotype of oral epithelial cells by regulating the expression of some target genes, suggesting that resveratrol may be used not only as an adjuvant treatment for oral cancer, but also as an adjuvant for oral cancer prevention due to its low toxicity and high efficacy. © 2024 Society of Chemical Industry.

Surgical treatment compared with "wait and see" in patients affected by oral leukoplakia to prevent oral cancer: Preliminary data from a multicenter randomized controlled trial.

OBJECTIVE: Oral leukoplakia (OL) is one of the most common and investigated oral potentially malignant disorders (OPMD). Preventing OSCC occurrence should be the primary outcome in the clinical management of OL. Surgical removal of OL is performed by most clinicians, although its effectiveness in reducing OSCC onset has still not been established by randomized controlled trials (RCT). Wait and see approach is characterized by frequent clinical examinations and periodical biopsies of OL, avoiding unnecessary surgical procedures. This is the first multicenter RCT in literature aiming at comparing the effectiveness of surgical removal and the "wait and see" approach in preventing OSCC onset in patients affected by dysplastic and non-dysplastic OL. METHODS: Two Italian referral care centres for oral diseases were involved in this multicenter two-arm RCT comparing the surgical removal of OL (group A) and the "wait and see" approach (group B), with the aim of reducing oral cancer onset. RESULTS: This report shows preliminary data on the first 161 patients, with a mean follow-up of 19.14 ± 11.25 months. Eight cases of OSCC occurred (6 out 8 involving the tongue): one case in group A and seven cases in group B. Moreover, OL recurred in 13 (20%) cases after surgical excision. CONCLUSIONS: Within the limitations of this preliminary report, these initial data underline the increased risk of OSCC onset in the case of OL of the tongue in the presence of epithelial dysplasia in group B ("wait and see") compared to group A (surgery). This RCT is currently ongoing at the same clinical departments, with the aim of enrolling 310 patients and collecting data at 5-year follow-up, in order to achieve conclusive results, in an evidence-based medicine approach.

Enhancing oral squamous cell carcinoma detection: a novel approach using improved EfficientNet architecture.

PROBLEM: Oral squamous cell carcinoma (OSCC) is the eighth most prevalent cancer globally, leading to the loss of structural integrity within the oral cavity layers and membranes. Despite its high prevalence, early diagnosis is crucial for effective treatment. AIM: This study aimed to utilize recent advancements in deep learning for medical image classification to automate the early diagnosis of oral histopathology images, thereby facilitating prompt and accurate detection of oral cancer. METHODS: A deep learning convolutional neural network (CNN) model categorizes benign and malignant oral biopsy histopathological images. By leveraging 17 pretrained DL-CNN models, a two-step statistical analysis identified the pretrained EfficientNetB0 model as the most superior. Further enhancement of EfficientNetB0 was achieved by incorporating a dual attention network (DAN) into the model architecture. RESULTS: The improved EfficientNetB0 model demonstrated impressive performance metrics, including an accuracy of 91.1%, sensitivity of 92.2%, specificity of 91.0%, precision of 91.3%, false-positive rate (FPR) of 1.12%, F1 score of 92.3%, Matthews correlation coefficient (MCC) of 90.1%, kappa of 88.8%, and computational time of 66.41%. Notably, this model surpasses the performance of state-of-the-art approaches in the field. CONCLUSION: Integrating deep learning techniques, specifically the enhanced EfficientNetB0 model with DAN, shows promising results for the automated early diagnosis of oral cancer through oral histopathology image analysis. This advancement has significant potential for improving the efficacy of oral cancer treatment strategies.

Fractionated irradiation induces radioresistant oral carcinoma cells with enhanced malignant phenotypes.

OBJECTIVE: The fact that certain oral carcinoma patients experience radiotherapy failure implies that a more radioresistant and aggressive phenotype of surviving cancer cells potentially occurs during treatment. Our study aimed to establish radioresistant oral cancer cells through a fractionated irradiation protocol that mimics clinically relevant radiotherapy dosing strategies and to investigate all-round alterations in the malignant phenotype. METHODS: Radioresistant oral carcinoma cells were generated by exposing Cal27 and Detroit 562 cells to 60 Gy radiation in 10 dose-escalating fractions and verified by cell immunofluorescence. Specific markers related to the epithelial-mesenchymal transition (EMT) process and the cancer stem cell (CSC) phenotype were assessed by Western blotting. Cell invasion and migration were evaluated using Matrigel-coated transwell and wound healing assays, respectively. Nontargeted metabolomics was used to mechanistically delineate the potential metabolic patterns linked to EMT and CSCs; the CSC phenotype was also examined by sphere formation assays and cell immunofluorescence. RESULTS: Radioresistant oral carcinoma cell lines were successfully established and validated. These cells exhibited enhanced EMT and increase in both cell invasion and migration. These radioresistant cells further demonstrated a high metabolic profile, notably marked by lipid metabolism reprogramming and functional enrichment of ATP-binding cassette (ABC) transporters. Consistently, enhanced CSC phenotype in radioresistant cells was confirmed by elevated expression of stemness markers and increased sphere-forming capacity. CONCLUSION: Radioresistant oral carcinoma cells subjected to fractionated radiation exhibit an augmented malignant phenotype. The metabolic characteristics linked to enhanced EMT and CSC phenotypes provide potential targets for improving radiotherapy in oral carcinoma.

Oral mucosa - an examination map for confocal laser endomicroscopy within the oral cavity: an experimental clinical study.

OBJECTIVES: Confocal laser endomicroscopy (CLE) is an optical method that enables microscopic visualization of oral mucosa. Previous studies have shown that it is possible to differentiate between physiological and malignant oral mucosa. However, differences in mucosal architecture were not taken into account. The objective was to map the different oral mucosal morphologies and to establish a "CLE map" of physiological mucosa as baseline for further application of this powerful technology. MATERIALS AND METHODS: The CLE database consisted of 27 patients. The following spots were examined: (1) upper lip (intraoral) (2) alveolar ridge (3) lateral tongue (4) floor of the mouth (5) hard palate (6) intercalary line. All sequences were examined by two CLE experts for morphological differences and video quality. RESULTS: Analysis revealed clear differences in image quality and possibility of depicting tissue morphologies between the various localizations of oral mucosa: imaging of the alveolar ridge and hard palate showed visually most discriminative tissue morphology. Labial mucosa was also visualized well using CLE. Here, typical morphological features such as uniform cells with regular intercellular gaps and vessels could be clearly depicted. Image generation and evaluation was particularly difficult in the area of the buccal mucosa, the lateral tongue and the floor of the mouth. CONCLUSION: A physiological "CLE map" for the entire oral cavity could be created for the first time. CLINICAL RELEVANCE: This will make it possible to take into account the existing physiological morphological features when differentiating between normal mucosa and oral squamous cell carcinoma in future work.

Spindle Cell Carcinoma of Buccal Mucosa: An Unusual Presentation of Squamous Cell Carcinoma.

Oral squamous cell carcinoma (OSCC) is the most common carcinoma in the H&N (head and neck) region, in which squamous cells show variability in differentiation like basaloid, glandular, and spindle cells. Spindle cell carcinoma (SpCC) is an unusual variant of SCC that is aggressive in nature and has the ability to recur and metastasize. The presence of malignant mesenchymal and squamous epithelial cells gives it a biphasic nature. So, we present a case of SpCC of buccal mucosa in a 45-year-old male who had an ulcerated growth on his left buccal mucosa that had been present for two years.

The Use of Immune Regulation in Treating Head and Neck Squamous Cell Carcinoma (HNSCC).

Immunotherapy has emerged as a promising new treatment modality for head and neck cancer, offering the potential for targeted and effective cancer management. Squamous cell carcinomas pose significant challenges due to their aggressive nature and limited treatment options. Conventional therapies such as surgery, radiation, and chemotherapy often have limited success rates and can have significant side effects. Immunotherapy harnesses the power of the immune system to recognize and eliminate cancer cells, and thus represents a novel approach with the potential to improve patient outcomes. In the management of head and neck squamous cell carcinoma (HNSCC), important contributions are made by immunotherapies, including adaptive cell therapy (ACT) and immune checkpoint inhibitor therapy. In this review, we are focusing on the latter. Immune checkpoint inhibitors target proteins such as programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) to enhance the immune response against cancer cells. The CTLA-4 inhibitors, such as ipilimumab and tremelimumab, have been approved for early-stage clinical trials and have shown promising outcomes in terms of tumor regression and durable responses in patients with advanced HNSCC. Thus, immune checkpoint inhibitor therapy holds promise in overcoming the limitations of conventional therapies. However, further research is needed to optimize treatment regimens, identify predictive biomarkers, and overcome potential resistance mechanisms. With ongoing advancements in immunotherapy, the future holds great potential for transforming the landscape of oral tumor treatment and providing new hope for patients.

EMT Dynamics in Lymph Node Metastasis of Oral Squamous Cell Carcinoma.

BACKGROUND: Epithelial-mesenchymal transition (EMT) enables tumor cell invasion and metastasis. Many studies have demonstrated the critical role of EMT in lymph node metastasis in oral squamous cell carcinoma (OSCC). During EMT, epithelial cancer cells lose intercellular adhesion and apical-basal polarity and acquire mesenchymal properties such as motility and invasiveness. A significant feature of EMT is cadherin switching, involving the downregulation of E-cadherin and upregulation of N-cadherin. The TGF-ß/SMAD pathway can also induce EMT. We aimed to evaluate EMT markers as predictors of lymph node metastasis in OSCC. METHODS: We performed genetic profiling of 159 primary OSCCs from TCGA and analyzed the expression of EMT markers, including cadherin switch genes (CDH1, CDH2), and TGF-ß/SMAD pathway genes. Samples were divided into advanced (stage III-IV) and early (stage I-II) stage groups. Differential expression analysis was performed, as well as an independent validation study containing fresh OSCC samples. RESULTS: TGF-ß/SMAD pathway genes such as SMAD6 were upregulated in advanced stage tumors. N-cadherin and SNAIL2 were overexpressed in node-positive tumors. Keratins were downregulated in these groups. CONCLUSION: Our findings demonstrate that EMT marker expression correlates with lymph node metastasis in OSCC. Developing therapies targeting regulators such as N-cadherin may prevent metastasis and improve outcomes.

Hesperidin Inhibits Oral Cancer Cell Growth via Apoptosis and Inflammatory Signaling-Mediated Mechanisms: Evidence From In Vitro and In Silico Analyses.

Background Oral carcinoma presents a significant health challenge, prompting the need for innovative therapeutic approaches. Elevation of inflammatory mediators, including tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6), has promoted cellular proliferation, inhibited apoptosis, and fostered oral cancer progression through complex signaling pathways. Hesperidin, a flavanone glycoside found in citrus fruits, is of keen interest in this study as it has been proven to have multiple health benefits through in vivo and in vitro studies. However, the mechanism behind the anticancer activity of hesperidin in oral carcinoma remains obscure. Aim The study aimed to explore the anticancer potential of hesperidin on human oral cancer cells (KB cells) by modulating pro-inflammatory and apoptotic signaling mechanisms. Methods Cancer cell growth inhibitory activity was assessed using the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay. Gene expression analysis was performed using real-time RT-PCR analysis. In addition, in silico docking analysis was conducted to confirm the binding affinity of hesperidin with pro-inflammatory and apoptosis signaling molecules. The data were analyzed using one-way ANOVA and the "t" test. Results Utilizing the MTT assay, a dose-dependent cytotoxic effect of hesperidin was unveiled, with a remarkable IC50 value indicative of its potent inhibition of cell proliferation. Complementing these findings (p<0.05), qRT-PCR analysis demonstrated hesperidin's regulatory influence on key molecular targets within the KB cell line. Hesperidin treatment resulted in a noteworthy reduction in TNF-α, interleukin-1 beta (IL-1-ß), IL-6, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and B-cell lymphoma 2 (Bcl-2) mRNA expression levels (p<0.05), highlighting its inhibitory role in cell proliferation, migration, and inflammation processes. Simultaneously, hesperidin promoted the expression of BAX mRNA (p<0.05), indicating an enhancement in cell death. Molecular docking simulations further revealed robust binding affinities between hesperidin and target proteins, suggesting its potential to disrupt cellular functions and inflammatory signaling pathways in oral cancer cells. Conclusion The cytotoxic effects on the KB cell line and its anti-inflammatory properties position hesperidin as a compelling candidate for further exploration in the quest for effective oral carcinoma treatments. These findings shed light on the intricate molecular mechanisms underlying hesperidin's promise as a therapeutic agent against oral carcinoma.

Therapeutic efficacy of platelet-rich fibrin on surgical site wound healing in patients undergoing oral carcinoma resection: A meta-analysis.

Oral carcinoma, a prevalent malignancy of the oral cavity, often results in surgical site wounds post-resection. The therapeutic efficacy of platelet-rich fibrin (PRF) in wound healing and scar formation has garnered significant attention. This meta-analysis aimed to evaluate the role of PRF in promoting surgical site wound healing and reducing scar formation following oral carcinoma resection. A systematic search, adhering to PRISMA guidelines, was conducted across multiple databases. The primary outcomes assessed were the Landry, Turnbull and Howley (LTH) wound healing index and the Manchester scar scale (MSS). Statistical evaluations were performed using RevMan 5.4 software. Six studies were incorporated, involving 93 patients treated with PRF and 97 in the control group. For the LTH index, significant improvements in wound healing were observed in the PRF group with I2 = 74%, (Random: SMD: 3.70, 95% CIs: 2.66 to 4.75, p < 0.01). The Manchester scar scale assessment, which included 60 PRF-treated patients and 60 controls, indicated a significant reduction in scar formation in the PRF group I2 = 79%, (Random: SMD: 9.13, 95% CIs: 6.06 to 12.20, p < 0.01). PRF demonstrates promising therapeutic potential in enhancing surgical site wound healing and reducing scar formation post oral carcinoma resection. The application of PRF has been associated with improved wound healing metrics and diminished scar severity. However, further high-quality studies are warranted to confirm these findings.

Tumor budding to investigate local invasion, metastasis, and prognosis of head and neck carcinoma: A systematic review.

The aim of this systematic review is to shed light on the role of tumor budding (TB) in the biology, behavior, and prognosis of head and neck squamous cell carcinoma (HNSCC). A search was run in PubMed, Scopus, and Embase databases following PRISMA guidelines. After full-text screening and application of inclusion/exclusion criteria, 36 articles were included. Several investigations support the prognostic role of TB, which might play a role in selecting rational treatment strategies. To achieve this goal, further research is needed for greater standardization in TB quantification. Although TB is not included as a negative prognostic factor in the current management guidelines, it might be reasonable to consider a closer follow-up for HNSCC cases with high histopathological evidence of TB.

Photodynamic Therapy with an Association of Methylene Blue and Toluidine Blue Promoted a Synergic Effect against Oral Squamous Cell Carcinoma.

Among the most malignant cancers, oral squamous cell carcinoma (OSCC) stands out as the most common malignant head and neck tumor. Despite advances in the field of treatment, the prognosis of patients with OSCC remains poor. Aiming to overcome the limitations of the currently existing therapies against OSCC, the present work aims to investigate the potential of photodynamic therapy (PDT) with phenothiazine derivatives used alone or in combination. The incorporation of methylene blue (MB) and toluidine blue (TB) was evaluated in OSCC cell lines (HSC-3 and SCC-9) and a nontumor cell line (Hfib). Both compounds exhibited concentration and time-dependent incorporation, with higher rates observed in tumor cells. Regarding dark-phase cytotoxic activity, SCC-9 cells were the most sensitive cell line with an IC50 value of 362.6 µM and 41.4 µM for MB and TB, respectively. Using PDT, all lineages showed greater sensitivity, presenting lower IC50 values when compared to the dark phase values. The combination index values of 0.69 (dark phase) and 0.73 (clear phase) associated with concave isobolograms, in both phases, revealed that MB and TB have synergistic effects when combined against SCC-9 cells. These findings suggest that MB or TB assisted with PDT holds promise for OSCC treatment.

Cold Atmospheric Plasma Jet Irradiation Decreases the Survival and the Expression of Oncogenic miRNAs of Oral Carcinoma Cells.

Despite recent advancements, therapies against advanced oral squamous cell carcinoma (OSCC) remain ineffective, resulting in unsatisfactory therapeutic outcomes. Cold atmospheric plasma (CAP) offers a promising approach in the treatment of malignant neoplasms. Although the effects of CAP in abrogating OSCC have been explored, the exact mechanisms driving CAP-induced cancer cell death and the changes in microRNA (miRNA) expression are not fully understood. We fabricated and calibrated an argon-CAP device to explore the effects of CAP irradiation on the growth and expression of oncogenic miRNAs in OSCC. The analysis revealed that, in OSCC cell lines following CAP irradiation, there was a significant reduction in viability; a downregulation of miR-21, miR-31, miR-134, miR-146a, and miR-211 expression; and an inactivation of the v-akt murine thymoma viral oncogene homolog (AKT) and extracellular signal-regulated kinase (ERK) signals. Pretreatment with blockers of apoptosis, autophagy, and ferroptosis synergistically reduced CAP-induced cell death, indicating a combined induction of variable death pathways via CAP. Combined treatments using death inhibitors and miRNA mimics, alongside the activation of AKT and ERK following the exogenous expression, counteracted the cell mortality associated with CAP. The CAP-induced downregulation of miR-21, miR-31, miR-187, and miR-211 expression was rescued through survival signaling. Additionally, CAP irradiation notably inhibited the growth of SAS OSCC cell xenografts on nude mice. The reduced expression of oncogenic miRNAs in vivo aligned with in vitro findings. In conclusion, our study provides new lines of evidence demonstrating that CAP irradiation diminishes OSCC cell viability by abrogating survival signals and oncogenic miRNA expression.

Familial oral lichen planus: A new risk group for oral cancer?

OBJECTIVE: The familial type of oral lichen planus (OLP) is rare, with a paucity of data regarding its clinical significance. Our objective was to characterize patients with familial OLP. METHODS: Families with at least two members diagnosed with OLP were included. Clinical and demographic data and medical history were recorded. RESULTS: Twenty families, 19 Jewish and 1 Arab, were identified. Of the Jewish families, 57.8% were non-Ashkenazi, originating mainly from central Asia. Of those with OLP there were 14 males and 23 females with an average age of 49.1. Dyslipidemia, cardiovascular, and thyroid disorders (27.7%, 22.2%, and 16.6%, respectively) were the most common comorbidities. Five patients from five distinct families had oral cancer, two with second primary. CONCLUSIONS: To the best of our knowledge, this is the largest study describing familial OLP. The predominant and common ethnicity of the families with multiple members diagnosed with OLP may imply an ethnic tendency. The higher tendency of hypothyroidism and the high percentage of OSCC among familial OLP patients might be connected to familial OLP and the latter suggests that this population is predisposed to malignant transformation. Thus, this group should be considered as a high-risk group.

Circular RNA hsa-circ-0006203 - hsa-circ-0004872 as novel detecting biomarkers in oral cancer.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is the most common cancer of the head and neck region. The circular RNA (circRNA) is known to serve an important role in the carcinogenesis of different types of cancer. However, the circRNA role of OSCC remains unclear. MATERIAL AND METHODS: OSCC tissues and adjacent normal tissues were obtained to detect circRNAs expression by the next generation sequencing (NGS), and OSCC tissues were selected to verify the differentially significant circRNAs by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). To further investigate the role of hsa-circ-0006203 - hsa-circ-0004872, the primer design and RT-PCR were performed. The expression levels were detected by RT-qPCR. RESULTS: The NGS results demonstrated that circRNAs were abundantly expressed in OSCC, and two circRNAs were significantly differentially expressed. hsa-circ-0006203 - hsa-circ-0004872 were significantly downregulated in OSCC tissue samples and was statistically correlated with pathological differentiation. CONCLUSION: In summary, the results of the present study revealed that OSCC tissues have abundant circRNAs and, to the best of our knowledge, it was our team who firstly explore the regulatory role of the hsa-circ-0006203 - hsa-circ-0004872 network in OSCC. The results indicated that hsa-circ-0006203 - hsa-circ-0004872 may be a potential biomarker for OSCC.

Molecular docking analysis of cetuximab with NOTCH signalling pathway targets for oral cancer.

Notch signaling is an evolutionarily ancient mechanism which intricated in cell-cell communication and it plays a crucial role in various developments in malignancies. Inactivating mutations of NOTCH targets are present in about 10 % of cases of squamous cell carcinoma of the skin, oral cavity, and esophagus that rendering it one of the most frequently mutated genes in oral squamous cell carcinoma. Therefore, it is of interest to document the molecular docking analysis of cetuximab with the NOTCH signaling targets such as NOTCH1, NICD, and HES1. These results suggest that targeting the NOTCH signaling with cetuximab might leads to the better outcome for suppression of invasion and metastasis in oral carcinoma.

Molecular docking analysis of proflavin with the Wnt pathway targets for OSCC.

Wnt signaling pathway plays a critical role in tumor progression and metastasis of various cancer types. Therefore, it is of interest to document the molecular docking analysis of proflavin with the Wnt pathway targets (GSK3ß, ß-catenin, and VIM) for oral squamous cell carcinoma (OSCC). These results suggest that targeting the wnt signaling downstream targets with proflavine might leads to the better outcome for therapeutic outcome for the inhibition of invasion and metastasis in oral squamous cell carcinoma.

Personalized Medicine in Oral Oncology: Imaging Methods and Biological Markers to Support Diagnosis of Oral Squamous Cell Carcinoma (OSCC): A Narrative Literature Review.

For decades, oral squamous cell carcinoma (OSCC) has been one of the most prevalent and mortal cancers worldwide. The gold standard for OSCC diagnosis is still histopathology but this narrative multidisciplinary review has the aim to explore the literature about conventional OSCC prognostic indicators related to the pTNM stage at the diagnosis such as the depth of invasion and the lymphovascular invasion associated with distant metastasis as indicators of poor life expectancy. Despite its multifactorial nature and recognizable precursors, its diagnosis at the early stages is still challenging. We wanted to highlight the importance of the screening as a primary weapon that a stomatologist should consider, intercepting all at-risk conditions and lesions associated with OSCC and its early stages. This narrative review also overviews the most promising imaging techniques, such as CT, MRI, and US-echography, and their application related to clinical and surgical practice, but also the most-investigated prognostic and diagnostic tissue and salivary biomarkers helpful in OSCC diagnosis and prognostic assessment. Our work highlighted remarkable potential biomarkers that could have a leading role in the future. However, we are still far from defining an appropriate and concrete protocol to apply in clinical practice. The hope is that the present and future research will overcome these limitations to benefit patients, clinicians, and welfare.

Suppression of Krüppel-like factor 5 basal expression by CREB1 binding to far distal element.

BACKGROUND: Krüppel-like factor 5 (KLF5) is a transcription factor regulating the proliferation and differentiation of epithelial cells, and its uncontrolled expression is closely associated with carcinoma progression. Sp3 binding to the minimal essential region (MER) of KLF5 gene is critical for KLF5 basal expression, but the expression control mechanism is unknown. OBJECTIVE: This study aimed to identify a regulatory region for KLF5 basal expression and the binding protein in carcinoma cells by analyzing the promoter upstream region. METHODS: Reporter assays determined the silencer region. The protein binding to the region was identified by database analysis and ChIP assay. The protein mediating the interaction between the region and the MER was confirmed through chromosome conformation capture (3 C) on ChIP assay. The effects of the protein on KLF5 expression were analyzed using qRT-PCR and western blot. RESULTS: Reporter assay localized the 425-region from upstream KLF5 gene as the silencer. Database analysis and ChIP assay found CREB1 binding to the 425-region. CREB1 siRNA or mutation of CREB1-binding site in the 425-region increased luciferase activities and decreased the binding to 425-region. 3 C on ChIP assay showed that CREB1 mediated interaction of the 425-region and the MER. CREB1 overexpression decreased endogenous KLF5 expression and luciferase activity. CONCLUSIONS: The 425-region is the silencer of KLF5 basal expression, and CREB1 binding suppresses the expression.