Pei-Ling Hsieh

Andrographolide impedes cancer stemness and enhances radio-sensitivity in oral carcinomas via miR-218 activation

Current evidence suggests that oral cancer stem cells (OCSCs) possess high tumorigenic and metastatic properties as well as chemo- and radioresistance. In this study, we demonstrated that andrographolide, the main bioactive component in the medicinal plant Andrographis, significantly reduced oncogenicity and restored radio-sensitivity of ALDH1+CD44+ OCSCs. Mechanistic studies showed that andrographolide treatment increased the expression of microRNA-218 (miR-218), leading to the downregulation of Bmi1. We showed that knockdown of miR-218 in ALDH1−CD44− non-OCSCs enhanced cancer stemness, while silencing of Bmi1 significantly counteracted it. Furthermore, we found tumor growth was reduced in mice bearing xenograft tumors after andrographolide treatment via activation of miR-218/Bmi1 axis. Together, these data demonstrated that the inhibition of tumor aggressiveness in OCSCs by andrographolide was mediated through the upregulation of miR-218, thereby reducing Bmi1 expression. These findings suggest that andrographolide may be a valuable natural compound for anti-CSCs treatment of OSCC.

Hinokitiol suppresses cancer stemness and oncogenicity in glioma stem cells by Nrf2 regulation

PurposeGlioma is one of the lethal malignancies with poor prognosis. In addition, glioma stem cells (GSCs) have been considered as the crucial player that attributed to the tumorigenesis and drug resistance. In the current study, we investigated the therapeutic effect of hinokitiol, a natural bioactive compound of aromatic tropolone, on the characteristics of GSCs and the possible mechanism.MethodsU87MG and T98G glioma cells were used to isolate GSCs. CD133 positivity and ALDH1 activity of GSCs following hinokitiol treatment were assessed by flow cytometry analysis. Secondary sphere formation, migration, invasion, and colony-forming assays were performed to examine the self-renewal capacity and oncogenicity in GCS after hinokitiol administration. The expression of Nrf2 was evaluated by RT-PCR and western blot analyses.ResultsWe demonstrated that hinokitiol effectively inhibited the CD133 positivity and ALDH1 activity along with the reduced self-renewal, migration, invasion, and colony formation properties of GSCs. In addition, hinokitiol repressed the gene and protein expression of Nrf2, which has been shown to be critical for those GSCs features. Furthermore, we showed that administration of exogenous Nrf2 counteracted the inhibitory effect of hinokitiol on self-renewal and invasiveness of GSCs.ConclusionThese evidences suggest that treatment of hinokitiol significantly attenuates the hallmarks of GSCs due to downregulation of Nrf2 expression. Hence, hinokitiol may serve as a promising agent for the therapy of glioma.

Hinokitiol ablates myofibroblast activation in precancerous oral submucous fibrosis by targeting Snail

Oral submucous fibrosis (OSF) is a precancerous condition with symptoms of limited mouth opening and areca nut chewing habit has been implicated in its pathogenesis. Hinokitiol, a natural tropolone derived from Chamacyparis taiwanensis, has been reported to improve oral lichen planus and inhibit various cancer cells. Here, we showed that hinokitiol reduced the myofibroblast activities in fBMFs and prevented the arecoline‐induced transdifferentiation. Treatment of hinokitiol dose‐dependently downregulated the myofibroblast markers as well as various EMT transcriptional factors. In particular, we identified that Snail was able to bind to the E‐box in the α‐SMA promoter. Our data suggested that exposure of fBMFs to hinokitiol mitigated the hallmarks of myofibroblasts, while overexpression of Snail eliminated the effect of hinokitiol. These findings revealed that the inhibitory effect of hinokitiol on myofibroblasts was mediated by repression of α‐SMA via regulation of Snail and showed the anti‐fibrotic potential of hinokitiol in the treatment of OSF.

Inhibitory effect of GMI, an immunomodulatory protein from Ganoderma microsporum, on myofibroblast activity and proinflammatory cytokines in human fibrotic buccal mucosal fibroblasts

Oral submucous fibrosis (OSF) has been indicated as one of the oral potentially malignant disorders. Epidemiological studies have attributed this pathological fibrosis to the habit of areca nuts chewing, which causes chronic inflammation and persistent activation of myofibroblasts in the oral cavity. Hence, it is crucial to find an effective intervention to ameliorate inflammation in order to prevent the malignant progression of OSF. In this study, we assessed the anti‐inflammatory effect of the immunomodulatory protein, GMI, extracted from Ganoderma microsporum on the expression proinflammatory cytokines and the myofibroblast characteristics in human fibrotic buccal mucosal fibroblasts (fBMFs). Our results demonstrated that the expression level of interleukin (IL)‐6 and IL‐8 were decreased after exposure of GMI and the myofibroblast activities, including collagen gel contraction, migration, invasion, and wound healing abilities were inhibited as well. Furthermore, we confirmed these findings in the arecoline‐stimulated BMFs. Consistent with the above findings, the expression of the myofibroblast marker α‐smooth muscle actin and other fibrogenic markers, such as type I collagen, fibronectin, and vimentin in fBMFs were all reduced in a dose‐dependent manner. Collectively, our data suggested that GMI suppressed the proinflammatory cytokines and myofibroblast features in fBMFs, and could serve as a promising and natural antifibrosis agent.

GMI ablates cancer stemness and cisplatin resistance in oral carcinomas stem cells through IL-6/Stat3 signaling inhibition

Cancer stem cells (CSCs) have been identified to exert tumor-initiating ability, resulting in the recurrence, metastasis and chemoresistance of oral squamous cell carcinomas. In the present study, we showed that GMI, an immunomodulatory protein from Ganoderma microsporum, induc ed a cytotoxic effect in oral carcinomas stem cells (OCSCs). Treatment of GMI dose-dependently inhibited the expression of CSC markers, including ALDH1 activity and CD44 positivity. Moreover, GMI suppressed the self-renewal property, colony formation, migration, and invasion abilities as well as potentiated chemo-sensitivity in OCSCs. Our results suggested that the tumor suppressive effect of GMI was mediated through inhibition of IL-6/Stat3 signaling pathway. Furthermore, tumor growth was reduced in mice bearing xenograft tumors after oral administration of GMI. Taken together, we demonstrated the anti-CSC effect of GMI in oral cancer and GMI may serve as a natural cisplatin adjuvant to prevent cancer recurrence.

LncRNA GAS5-AS1 inhibits myofibroblasts activities in oral submucous fibrosis

BACKGROUND/PURPOSE Emerging research findings suggest that long non-coding RNAs (lncRNAs) are key regulators to fibrosis formation. Nevertheless, the role of lncRNA GAS5-AS1 in the progression of precancerous oral submucous fibrosis (OSF) remains to be elucidated. METHODS Quantitative real-time PCR were used to examine the expression of GAS5-AS1 in OSF tissues. The activities of myofibroblasts, including collagen contractility and cell migration, as well as the marker α-smooth muscle actin (SMA) were assessed following overexpression of GAS5-AS1. Also, we analyzed the expression of Smad activity in order to gain insight into the downstream regulator. RESULTS The level of GAS5-AS1 was found significantly downregulated in the OSF tissues and fibrotic buccal mucosal fibroblasts (fBMFs). Ectopic expression of GAS5-AS1 significantly reduced the abilities of collagen gel contraction and migration in fBMFs or arecoline-treated BMFs. Moreover, we have shown that overexpression of GAS5-AS1 inhibited the expression of p-Smad and the marker of myofibroblasts. CONCLUSION We showed the reduced expression of GAS5-AS1 in OSF tissues and demonstrated its effect on the myofibroblast activities and the level of p-Smad and α-SMA, indicating its potential contribution in OSF pathogenesis.

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.

Berberine-targeted miR-21 chemosensitizes oral carcinomas stem cells

Cancer recurrence and chemoresistance are two major obstacles to the treatment of oral squamous cell carcinomas (OSCC). And cancer stem cells (CSCs) have been found to possess tumor initiating, self-renewal and metastasis abilities, resulting in the relapse and chemoresistance of OSCC. In the present study, we investigated the anti-CSCs effect of berberine, a phenanthrene alkaloid isolated from the Berberis genus. Our results demonstrated that berberine dose dependently downregulated the oncogenicity in vitro, including ALDH1 activity, self-renewal property, and colony formation and invasion abilities as well as potentiated chemosensitivity of OSCC-CSCs. In addition, tumor growth in mice was attenuated after oral gavage treatment of berberine. We showed that the expression of miR-21 was suppressed following administration of berberine in OSCC-CSCs. And inhibition of endogenous miR-21 reduced the characteristics of CSCs, including self-renewal, migration, invasion capabilities and ALDH1 activity. Taken together, we demonstrated the anti-CSC effect of berberine in oral cancer and its potential to serve as adjuvant to traditional chemotherapy to improve treatment effect.

miR-200c inhibits the arecoline-associated myofibroblastic transdifferentiation in buccal mucosal fibroblasts

BACKGROUND/PURPOSE MicroRNA-200c (miR-200c) recently emerged as an important regulator of tumorigenesis and cancer metastasis, however, its role in regulating oral submucous fibrosis (OSF) remains unknown. In this study, we investigated the functional role of miR-200c in myofibroblastic differentiation activity and identified its potential target. METHODS qRT-PCR was applied to assess the expression of miR-200c in OSF tissues and fibrotic buccal mucosal fibroblasts (fBMFs). Arecoline, a major areca nut alkaloid, was utilized to explore whether the expression of miR-200c would alter following stimulation. Collagen gel contraction, migration and invasion capabilities were examined in arecoline-stimulated BMFs as wells as in fBMFs. Luciferase reporter assay was conducted to show the relationship between miR-200c and ZEB1. RESULTS Our results showed that the expression of miR-200c was downregulated in OSF specimen and fBMFs. Arecoline treatment dose-dependently reduced the relative expression of miR-200c in normal BMFs. Overexpression of miR-200c impeded the arecoline-induced collagen gel contraction, migration, invasion and wound healing capacities. Moreover, ectopic expression of miR-200c in fBMFs successfully reduced the increased collagen gel contractility and invasion abilities. Our results demonstrated that ZEB1 was a direct target of miR-200c, and overexpression of miR-200c inhibited the expression of ZEB1 and α-SMA. CONCLUSION These findings suggest that downregulation of miR-200c in OSF may be involved in the pathogenesis of areca nut-associated OSF through regulation of ZEB1.

miR-145 mediates the anti-cancer stemness effect of photodynamic therapy with 5-aminolevulinic acid (ALA) in oral cancer cells

5-Aminolevulinic acid-mediated photodynamic therapy (ALA-PDT) has been used in the treatment of various precancerous and malignant lesions. Our previous work has demonstrated that ALA-PDT possesses the potential to serve as an adjuvant therapy against head and neck cancer via eliminating the cancer stem cells (CSCs) property. This study aimed to further investigate the possible molecular mechanism underlying the effect of ALA-PDT. Our results revealed that ALA-PDT upregulated the expression of microRNA-145 (miR-145) in two oral cancer cell lines. Overexpression of miR-145 in oral CSCs further enhanced the treatment effect of ALA-PDT with lower self-renewal, invasion capacities and reduced CD44 expression, while inhibition of miR-145 exhibited the opposite phenomena. These findings suggest that the anti-CSCs effect of ALA-PDT is due to an elevation of miR-145.