Targeting oral cancer stemness and chemoresistance by isoliquiritigenin-mediated GRP78 regulation.

Cancer stem cells (CSCs) are cells that drive tumorigenesis, contributing to metastasis and cancer recurrence as well as resistance to chemotherapy of oral squamous cell carcinomas (OSCC). Therefore, approaches to target CSCs become the subject of intense research for cancer therapy. In this study, we demonstrated that isoliquiritigenin, a chalcone-type flavonoid isolated from licorice root, exhibited more toxicity in oral cancer stem cells (OSCC-CSCs) compared to normal cells. Treatment of isoliquiritigenin not only inhibited the self-renewal ability but also reduced the expression of CSC markers, including the ALDH1 and CD44. In addition, the capacities of OSCC-CSCs to invade, metastasize and grow into a colony were suppressed by isoliquiritigenin. Most importantly, we showed that isoliquiritigenin potentiated chemotherapy along with downregulated expression of an ABC transporter that is associated with drug resistance, ABCG2. Moreover, a combination of isoliquiritigenin and Cisplatin significantly repressed the invasion and colony formation abilities of OSCC-CSCs. Our results suggested that administration of isoliquiritigenin reduced the protein expression of mRNA and membrane GRP78, a critical mediator of tumor biology. Overexpression of GRP78 reversed the inhibitory effect of isoliquiritigenin on OSCC-CSCs. Furthermore, isoliquiritigenin retarded the tumor growth in nude mice bearing OSCC xenografts. Taken together, these findings showed that isoliquiritigenin is an effective natural compound that can serve as an adjunct to chemotherapy for OSCC.

DHFR and MDR1 upregulation is associated with chemoresistance in osteosarcoma stem-like cells.

Tumor-initiating cells (TICs) are defined as a specialized subset of cells with tumor-initiating capacity that can initiate tumor growth, tumor relapse and metastasis. In the present study, osteosarcoma TICs (OS-TICs) were isolated and enriched from the osteosarcoma U2OS and MG-63 cell lines using sphere formation assays and serum-depleted media. These enriched OS-TICs showed the expression of several typical cancer stemness markers, including octamer-binding transcription factor 4, Nanog homeobox, cluster of differentiation (CD)117, Nestin and CD133, and the expression of ATP binding cassette subfamily G member 2, multidrug resistance protein 1 (MDR1) and dihydrofolate reductase (DHFR). Notably, in vitro and in vivo tumorigenic properties were enhanced in these OS-TICs. Additionally, methotrexate and doxorubicin are the most widely used anticancer agents against osteosarcoma, and the observed enhanced chemoresistance of OS-TICs to these two agents could be associated with the upregulation of DHFR and MDR1. These findings suggest that the upregulation of DHFR and MDR1 is associated with the development of chemoresistance of OS-TICs.

Targeting CD133 in the enhancement of chemosensitivity in oral squamous cell carcinoma-derived side population cancer stem cells.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is one of the most common cancers in the world. Previously, we enriched a subpopulation of OSCC-derived cancer stem cells (OSCC-CSCs), and identified CD133 as an OSCC-CSC marker. METHOD: We determined the function of CD133 on chemosensitivity of oral cancer CSCs by silencing CD133. RESULTS: Initially, we observed that the expression profile of CD133 in OSCC-side population (OSCC-SPs) cells, which exerted properties of CSCs, was significantly upregulated than that of major population (MPs) cells of OSCCs. The cell viability experiments showed that SPs were more chemoresistant compared with major populations. Importantly, targeting CD133 ameliorated the drug resistance of OSCC-SPs to cisplatin treatment. Targeting CD133 and cisplatin co-treatment led to the maximal inhibition on tumor initiating properties in OSCC-SPs. CONCLUSION: Side population cells with CSCs properties existed in OSCCs, and silencing CD133 exhibited a prominent therapeutic effect in enhancing the sensitivity of chemotherapy in OSCC through elimination of CSCs. © 2015 Wiley Periodicals, Inc. Head Neck 38: E231-E238, 2016.

Elevation of Twist expression by arecoline contributes to the pathogenesis of oral submucous fibrosis.

BACKGROUND/PURPOSE: Oral submucous fibrosis (OSF), a chronic progressive scarring disease, has been considered as a precancerous condition of oral mucosa. In this study, we investigated the functional role of Twist, an epithelial-mesenchymal transition (EMT) transcriptional factor, in myofibroblastic differentiation activity of OSF. METHODS: Arecoline, a major areca nut alkaloid, was used to explore whether expression of Twist could be changed dose-dependently in human primary buccal mucosal fibroblasts (BMFs). Collagen gel contraction and migration capability in arecoline-stimulated BMFs and primary oral submucous fibrosis-derived fibroblasts (OSFs) with Twist knockdown was presented. RESULTS: We observed that the treatment of arecoline dose-dependently increased Twist expression transcript and protein levels in BMFs. The myofibroblast activity including collagen gel contraction and migration capability also induced by arecoline, while knockdown of Twist reversed these phenomena. Importantly, inhibition of Twist led to the suppression collagen contraction and wound healing capability of primary cultivated OSFs. Clinically, Twist transcript and protein expression was higher in areca quid chewing-associated OSF tissues than in normal oral mucosa tissues. CONCLUSION: This evidence suggests that upregulation of Twist might be involved in the pathogenesis of areca quid-associated OSF through dysregulation of myofibroblast activity.

Activation of microRNA-494-targeting Bmi1 and ADAM10 by silibinin ablates cancer stemness and predicts favourable prognostic value in head and neck squamous cell carcinomas.

Tumor initiating cells (TICs) possessing cancer stemness were shown to be enriched after therapy, resulting in the relapse and metastasis of head and neck squamous cell carcinomas (HNC). An effective therapeutic approach suppressing the HNC-TICs would be a potential method to improve the treatments for HNC. We observed that the treatment of silibinin (SB) dose dependently down-regulated the ALDH1 activity, CD133 positivity, stemness signatures expression, self-renewal property, and chemoresistance in ALDH1+CD44+ HNC-TICs. Using miRNA-microarray and mechanistic studies, SB increased the expression of microRNA-494 (miR-494) and both Bmi1 and ADAM10 were identified as the novel targets of miR-494. Moreover, overexpression of miR-494 results in a reduction in cancer stemness. However, knockdown of miR-494 in CD44-ALDH1- non-HNC-TICs enhanced cancer stemness and oncogenicity, while co-knockdown of Bmi1 and ADAM10 effectively reversed these phenomena. Mice model showed that SB treatment by oral gavage to xenograft tumors reduced tumor growth and prolonged the survival time of tumor-bearing mice by activation of miR-494-inhibiting Bmi1/ADAM10 expression. Survival analysis indicated that a miR494highBmi1lowADAM10low phenotype predicted a favourable clinical outcome. We conclude that the inhibition of tumor aggressiveness in HNC-TICs by SB was mediated by up-regulation miR-494, suggesting that SB would be a valuable anti-cancer drug for treatment of HNC.

Knockdown of S100A4 impairs arecoline-induced invasiveness of oral squamous cell carcinomas.

OBJECTIVES: Metastasis is the most common cause of oral squamous cell carcinoma (OSCC)-related death. The physiological function of S100A4 in the pathogenesis of areca quid chewing-associated OSCC has not been uncovered. METHOD: OSCC tissues from areca quid chewers were analyzed by immunohistochemistry for S100A4 expression. The functions of S100A4 in invasiveness of arecoline-treated oral epithelial (OE) cells were determined by loss function approaches. RESULTS: Expression of S100A4 was positively correlated with clinical grading and lymph node metastasis of OSCC. Upregulated S100A4 is correlated with poor survival outcome of OSCC patients. Arecoline led to dose-dependent elevation of S100A4 expression in oral epithelial (OE) cells. Down-regulation of S100A4 significantly reversed arecoline-induced oncogenecity in OE cells. The additions of pharmacological agents LY294002, SP600125, and CAY10585 were found to inhibit arecoline-induced S100A4 expression in OE cells. CONCLUSION: Arecoline-induced S100A4 expression was down-regulated by LY294002, SP600125, or CAY10585 treatment. Targeting S100A4 might offer a new strategy for the treatment of OSCC patients with metastasis.

Sox2 expression involvement in the oncogenicity and radiochemoresistance of oral cancer stem cells.

OBJECTIVES: Sox2, a high-mobility-group DNA binding protein, is part of the key set of transcription factors that are involved in the maintenance of pluripotency and self-renewal in undifferentiated stem cells. A recent study has further suggested cancer stem cells (CSCs) are key contributors to radiochemoresistance and are responsible for oral squamous cell carcinoma (OSCC) progression. The aim of this study was to determine the emerging role of Sox2 in radiochemosensitivity of oral CSCs. METHODS: We determined the function of Sox2 on oncogenicity and radiochemosensitivity of OSCC by overexpression or silencing Sox2 in vitro and in vivo. RESULTS: Initially, Sox2 expression was increased in OSCC cell lines and OSCC specimens. Upregulated Sox2 is correlated with poor survival outcome of OSCC patients. Overexpression of Sox2 was demonstrated to enhance invasiveness, anchorage-independent growth, xenotransplantation tumourigenicity in OSCC cells. Targeting Sox2 to spheroid cells (SC) and ALDH1+CD44+ cells from OSCC significantly inhibited their CSCs and tumorigenic abilities. Down regulation of SOX2 in OSCC-SC was found to repress invasiveness and diminish epithelail-mesenchymal transition (EMT) traits. Furthermore, silencing Sox2 effectively suppressed the expression of drug-resistance and anti-apoptotic genes and increased the sensitivity of the cells to radiation combined cisplatin treatment. Finally, the in vivo therapeutic efficacy of targeting Sox2 synergistically suppressed tumorigenesis and improved the survival rate when used in combination with radiotherapy and cisplatin in OSCC-SC-transplanted immunocompromised mice. CONCLUSION: Sox2-mediated CSCs property is associated with the regulation of EMT and Sox2 s as therapeutic target in OSCC.

ZEB1 as an indicator of tumor recurrence for areca quid chewing-associated oral squamous cell carcinomas.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is the sixth most prevalent malignancy worldwide and the third most common cancer in developing nation. Most OSCC patients relapse within months after receiving treatment. Therefore, searching the biomarkers of recurrence is urgently required to improve OSCC patient survival. METHODS: We set out to explore whether expression of ZEB1 could be triggered in oral epithelial cells (SG and FaDu) by arecoline in vitro. Control and ZEB1-knockdown arecoline-stimulated SG and FaDu were subjected to migration/invasiveness/anchorage-independent growth assay. Primary and recurrent OSCC tissues from areca quid chewers were analyzed using real-time RT-PCR analysis for ZEB1 expression. RESULTS: Arecoline led to dose-dependent elevation of ZEB1 expression in SG and FaDu cells. Downregulation of ZEB1 by lentiviral infection significantly reversed arecoline-induced oncogenicity including migration ability, cell invasiveness, and anchorage-independent growth in SG and FaDu cells. Clinically, the level of ZEB1 expression was higher in recurrent OSCC tumor samples but lower in primary lesions. CONCLUSIONS: Targeting ZEB1 might offer a new strategy for the treatment of OSCC patients. ZEB1 can serve as a progression and relapse marker in OSCC patients.

Concurrent expression of Oct4 and Nanog maintains mesenchymal stem-like property of human dental pulp cells.

Human dental pulp stem cells (DPSCs), unique mesenchymal stem cells (MSCs) type, exhibit the characteristics of self-renewal and multi-lineage differentiation capacity. Oct4 and Nanog are pluripotent genes. The aim of this study was to determine the physiological functions of Oct4 and Nanog expression in DPSCs. Herein, we determined the critical role of an Oct4/Nanog axis modulating MSCs properties of DPSCs by lentiviral-mediated co-overexpression or co-knockdown of Oct4/Nanog in DPSCs. MSCs properties including osteogenic/chondrogenic/adipogenic induction differentiation was assayed for expression of osteogenic/chondrogenic/adipogenic markers by quantitative real-time RT-PCR analysis. Initially, we observed that the expression profile of Oct4 and Nanog in dental pulp cells, which exerted properties of MSCs, was significantly up-regulated compared to that of STRO-1-CD146- dental pulp cells. Down-regulation of Oct4 and Nanog co-expression significantly reduced the cell proliferation, osteogenic differentiation capability, STRO-1, CD146, and Alkaline phosphatase (ALP) activity of DPSCs. In contrast, co-overexpression of Oct4 and Nanog enhanced the expression level of STRO-1 and CD146, proliferation rate and osteogenic/chondrogenic/adipogenic induction differentiation capability, and expression of osteogenic/chondrogenic/adipogenic induction differentiation markers. Our results suggest that Oct4-Nanog signaling is a regulatory switch to maintain properties in DPSCs.

Enhanced chemosensitivity by targeting Nanog in head and neck squamous cell carcinomas.

Chemo-resistance is the major cause of high mortality in head and neck squamous cell carcinomas (HNSCC) in which HNSCC-derived cancer stem cells (CSCs) may be involved. Previously, we enriched a subpopulation of HNSCC-derived spheroid cells (SC) (HNSCC-SC) and identified Nanog as a CSCs marker. The aim of this study was to determine the role of Nanog in the chemosensitivity of HNSCC. The functional and clinicopathological studies of Nanog were investigated in HNSCC cells and specimens. Nanog expression was increased in HNSCC cell lines as compared to a normal oral epithelial cell line. Nanog upregulation in clinical tissues from HNSCC patients with recurrent and metastatic specimens relative to the mRNA levels in the samples from normal or primary tissues were examined. Targeting Nanog in HNSCC-SC significantly inhibited their tumorigenic and CSCs-like abilities and effectively increased the sensitivity of HNSCC-SC to chemotherapeutic drug cisplatin treatment. Targeting Nanog in HNSCC-SC showed a synergistic therapeutic effect with cisplatin. Our results suggest that targeting Nanog may have promising therapeutic potential for HNSCC.

Oct4 mediates tumor initiating properties in oral squamous cell carcinomas through the regulation of epithelial-mesenchymal transition.

BACKGROUND: Overexpression of Oct4, an important transcription factor of embryonic stem cells (ESC), has been reported in several cancers. The aim of this study was to determine the emerging role of Oct4 in oral squamous cell carcinoma (OSCC) both in vitro and in vivo. METHODOLOGY/PRINCIPAL FINDING: Tumourigenic activity and molecular mechanisms of Oct4 overexpression or knockdown by lentiviral infection in OSCC was investigated in vitro and in vivo. Initially, we demonstrated that Oct4 expression was increased in OSCC cell lines as compared to a normal oral epithelial cell line SG. Overexpression of Oct4 was demonstrated to enhance cell proliferation, invasiveness, anchorage-independent growth and xenotransplantation tumourigenicity. These findings were coupled with epithelial-mesenchymal transition (EMT) transformation in OSCCs. In contrast, the silence of Oct4 significantly blocked the xenograft tumorigenesis of OSCC-derived cancer stem cells (OSCC-CSCs) and significantly improved the recipient survival. Clinically, the level of Oct4 expression was higher in recurrent and metastatic OSCC specimens but lower in primary OSCC specimens. CONCLUSION/SIGNIFICANCE: Our results suggest that Oct4-mediated tumorigenecity is associated with the regulation of EMT. Oct4 might be a therapeutic target for OSCC.

Decreased SPLUNC1 expression is associated with Pseudomonas infection in surgically treated chronic rhinosinusitis patients who may require repeated sinus surgery.

OBJECTIVES/HYPOTHESIS: Chronic rhinosinusitis colonized with Pseudomonas aruginosa is difficult to treat and is related to biofilm formation. Repeated sinus surgery is often required for these patients. Short palate, lung, and nasal epithelial clone 1 (SPLUNC1) is an epithelium-secreted protein that is involved in innate immunity and has anti-Pseudomonas and antibiofilm functions. This study examined if SPLUNC1 expression was related to sinusitis with bacterial culture positive for Pseudomonas and the possibility of using SPLUNC1 to predict treatment outcomes for sinusitis. STUDY DESIGN: Nonrandomized retrospective study. METHODS: This was a retrospective study of patients at a tertiary referral center. Pseudomonas aruginosa infection was compared to clinical variables such as SPLUNC1 mRNA expression levels, immunohistochemical (IHC) stain intensity, Lund-Mackay sinus computed tomography scores, rapid recurrent sinusitis, requirement for repeat sinus surgery, Phadiatop test results, age, gender, nasal polyp(s), and patients' presence/absence of diabetes mellitus. Comparisons between groups were performed using the χ(2) test or Fisher exact test when one confronter was <5. The statistical analyses were carried out with SPSS version 13. RESULTS: P. aeruginosa sinus infections were associated with lower sinus mucosa SPLUNC1 expression (P = .0018), weaker SPLUNC1 IHC staining intensity (P = .011), and poor postoperative outcome (i.e., need repeated sinus surgery) (P < .001). Other factors were not associated with Pseudomonas sinus infection. CONCLUSIONS: Sinusitis with positive P. aeruginosa bacterial culture is associated with decreased SPLUNC1 sinus mucosa expression. Repeated sinus surgeries are more frequently needed for these patients.

Quercetin in elimination of tumor initiating stem-like and mesenchymal transformation property in head and neck cancer.

BACKGROUND: Previously, we enriched a subpopulation of head and neck cancer-derived tumor initiating cells (HNC-TICs) presented high tumorigenic, chemo-radioresistant, and coupled with epithelial-mesenchymal transition (EMT) properties. The purpose of this study was to investigate the therapeutic effect and molecular mechanisms of quercetin on HNC-TICs. METHOD: ALDH1 activity of head and neck cancer cells with quercetin treatment was assessed by the Aldefluor assay flow cytometry analysis. Self-renewal, invasiveness, and EMT capability of HNC-TICs with different doses of quercetin was presented. RESULTS: We first observed that the treatment of quercetin significantly downregulated the ALDH1 activity of head and neck cancer cells in a dose-dependent manner (p < .05). Moreover, quercetin reduced self-renewal property and stemness signatures expression in head and neck cancer-derived sphere cells. The migration ability of head and neck cancer-derived sphere cells was lessened under quercetin treatment partially due to the decreased productions of Twist, N-cadherin, and vimentin. CONCLUSION: Quercetin suppressing HNC-TICs characteristics may therefore be valuable therapeutics clinically in combination with standard treatment modalities.

Epithelial-mesenchymal transition transcription factor ZEB1/ZEB2 co-expression predicts poor prognosis and maintains tumor-initiating properties in head and neck cancer.

OBJECTIVES: Both epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC) properties may be involved in metastasis, which contributes to the high mortality rate of patients with head and neck cancers (HNCs). However, the mechanisms through which the EMT transcription factors ZEB1 and ZEB2 regulate HNC are still unclear. METHODS: Tumor initiating capability of HNC-CH133(+) cells with ZEB1/2 knockdown or co-overexpression was presented in vitro and in vivo. RESULTS: In the present study, we demonstrated that ZEB1/ZEB2 expression was significantly increased in HNC-CD133(+) CSC-like cells compared with HNC-CD133(-) cells. The small interfering RNA (siRNA)-mediated co-knockdown of ZEB1 and ZEB2 (siZEB1/2) in HNC-CH133(+) cells suppressed their CSC-like properties, including self-renewal ability, the expression of stemness markers, and drug resistance. In contrast, the co-overexpression of ZEB1/ZEB2 in HNC-CD133(-) cells enhanced their sphere-forming ability and increased the percentage of CD44-positive cells and side population cells. In vivo studies showed that the delivery of siZEB1/2 to xenograft tumors in nude mice reduced tumor growth and the rate of distant metastasis. In clinical samples, the levels of ZEB1/ZEB2 expression were low in local lesions but high in metastatic lymph nodes in HNC tissues. Patients with tumors that co-expressed ZEB1(high) and ZEB2(high) had especially poor survival rates. CONCLUSION: Therapies targeting ZEB1/ZEB2 in HNC-CD133(+) cells may provide a new approach for HNC therapy in the future.

Impairment of tumor-initiating stem-like property and reversal of epithelial-mesenchymal transdifferentiation in head and neck cancer by resveratrol treatment.

SCOPE: Recent reports have demonstrated that head and neck cancer-derived tumor-initiating cells (HNC-TICs) presented high tumorigenic, chemoradioresistant, metastatic properties, and were coupled with gain of epithelial-mesenchymal transition (EMT) characteristics. The aim of this study was to investigate the chemotherapeutic effect and regulatory mechanisms of resveratrol on HNC-TICs. METHODS AND RESULTS: We first observed that the treatment of resveratrol significantly downregulated the ALDH1 activity and CD44 positivity of head and neck cancer (HNC) cells in a dose-dependent manner (p < 0.05). Moreover, resveratrol treatment reduced self-renewal property and stemness genes signatures (Oct4, Nanog, and Nestin) expression in sphere-forming HNC-TICs. Additionally, the repressive effect of resveratrol on in vitro malignant properties including invasiveness/anchorage-independent growth was mediated by regulating productions of EMT markers Slug, ZEB1, N-cadherin, E-cadherin, and Vimentin. Importantly, an in vivo nude mice model showed that resveratrol treatment to xenograft tumors by oral gavage reduced tumor growth, stemness, and EMT markers in vivo. Lastly, synergistic effect of resveratrol and conventional chemotreatment attenuated tumor-initiating cells property in HNC-TICs. CONCLUSIONS: Our results demonstrated that resveratrol would be a valuable therapeutics clinically in combination with conventional chemotherapy treatment modalities for malignant HNCs by elimination of tumor-initiating stem-like and EMT properties.

Nuclear localization signal-enhanced RNA interference of EZH2 and Oct4 in the eradication of head and neck squamous cell carcinoma-derived cancer stem cells.

Metastasis is the major cause of high mortality in head and neck squamous cell carcinoma (HNSCC), in which HNSCC-derived cancer stem cells (CSCs) may be involved. Several reports have coupled non-viral gene delivery with RNA interference (RNAi) to target specific genes in cancer cells. However, the delivery efficiency of RNAi is limited and remained to be improved. Moreover, the therapeutic effect of non-viral gene delivery approaches on HNSCC-derived CSCs is still uncertain. In this study, we found that EZH2 and Oct4 are upregulated in HNSCC-derived ALDH1+/CD44+ CSC-like cells. Polyurethane-short branch PEI (PU-PEI)-based administration of double-stranded DNA (dsDNA) encoding small interfering RNA (siRNA) against EZH2 and Oct4 (siEZH2/siOct4) led to partial anti-cancer capacity and mild suppression of CSC-like properties. By pre-conjugation of nuclear localization signal (NLS) to siRNA-expressing dsDNA, the anti-cancer efficacy was enhanced due to elevated nuclear delivery. Notably, the NLS-preconjugated siEZH2/siOct4 constructs remarkably repressed epithelial-mesenchymal transdifferentiation (EMT) and radioresistance in ALDH1+/CD44+ CSC-like cells, in which Wnt5A and CyclinD1 may be involved respectively. We furthermore demonstrated that this improved method was capable of reducing tumor growth and metastasis in vivo. Our findings may provide a feasible non-viral gene delivery method to eradicate HNSCC-derived CSCs and improve HNSCC therapy.

Let-7d functions as novel regulator of epithelial-mesenchymal transition and chemoresistant property in oral cancer.

Oral squamous cell carcinoma (OSCC) is a prevalent cancer worldwide. Let-7 family has been shown to function as a tumor suppressor through regulating multiple oncogenic signaling. Recent study reported that combined underexpression of miR-205 and let-7d showed negative correlation with the survival prognosis of head and neck cancer patients. However, the let-7d-involved mechanism in regulating OSCC is still unclear. In this study, we first demonstrated that let-7d expression was significantly decreased while Twist and Snail expression was increased in OSCC cancer cell lines and primary cultures as compared to normal human oral keratinocyte cells. To further investigate the role of let-7d in OSCC, we applied the SPONGE method to knock down let-7d in OECM-1 and two primary OSCC cell types. The results showed that knockdown of let-7d promote epithelial-mesenchymal transition (EMT) traits and migratory/invasive capabilities in OSCC cells. Furthermore, down-expression of let-7d significantly activated Twist and Snail expressions and chemo-resistant abilities of OSCC cells. Notably, overexpression of let-7d effectively reversed the EMT phenotype, blocked migratory/invasive abilities, and further increased the chemosensitivity in oral cancer tumor initiating ALDH1+ cells. In sum, these results show that let-7d negatively modulates EMT expression and also plays a role in regulating chemo-resistant ability in oral cancer.

The influence of occlusal stimuli on basic fibroblast growth factor expression in the periodontal healing of replanted teeth.

Occlusal stimuli and the periodontal healing of replanted teeth have been reported to be related. However, the mechanism for preventing dentoalveolar ankylosis remains unclear. Basic fibroblast growth factor (bFGF/FGF-2) is considered as a key factor in wound healing. The purpose of this study was to evaluate the relationship between occlusal stimuli, bFGF, and the periodontal healing after tooth replantation. Five-week-old male rats were divided into non-occluded, occluded, and recovery groups. The right maxillary first molars were replanted in all groups, and the left maxillary first molars in the 2-week occluded group without replantation were served as nontreated. An anterior bite plate was attached to the maxillary and mandibular incisors to produce occlusal hypofunction in the non-occluded group and was then removed after 1 week in the recovery group. Histological observations were performed after 1 and 2 weeks of the experimental period. After 2 weeks, the non-occluded group had detectable ankylosis and obvious periodontal tissue stricture. Meanwhile, the occluded and recovery groups showed enlarged and thickened periodontia without ankylosis. The number of bFGF-positive cells in the occluded and recovery groups significantly increased as compared to in the non-occluded group. These results suggest that occlusal stimuli enhance the production of bFGF in the periodontal healing of replanted teeth and prevent dentoalveolar ankylosis.