Jaewang Lee

Accelerated oral wound healing using a pre-vascularized mucosal cell sheet

Cell sheets with pre-vascularization have recently been developed but remain relatively untested in oral wound healing. Therefore, we examined the potential utility of our newly developed pre-vascularized mucosal cell sheets in oral wound healing. Mucosal keratinocytes, fibroblasts, and endothelial progenitor cells were primarily cultured for in vitro cell expansion from mucosa and blood of Sprague-Dawley rats. Mucosal cell sheets were generated using cultured keratinocytes and plasma fibrin (K sheet) or keratinocytes and a mixture of fibrin, fibroblasts, and endothelial cells (PV sheet). Autologous sheets were transplanted on deep wounds in the buccal region of rats. The gross and histological characteristics of wound healing were compared among control wound, K sheet, and PV sheet groups. We successfully cultured and expanded keratinocytes, fibroblasts, and endothelial progenitor cells in vitro for generating mucosal cell sheets with or without pre-vascularization. In the in vivo oral wound model, compared with the control wound, the PV sheet group exhibited rapid wound closure more prominently than the K sheet group. The histological healing in the PV sheet group was similar to that in rat normal buccal mucosa without fibrosis. The pre-vascularized mucosal cell sheet exhibited in vivo efficacy in oral wound healing by promoting accelerated healing.

RITA plus 3-MA overcomes chemoresistance of head and neck cancer cells via dual inhibition of autophagy and antioxidant systems

Reactivation of p53 and induction of tumor cell apoptosis (RITA) is a small molecule that blocks p53–MDM2 interaction, thereby reactivating p53 in tumors. RITA can induce exclusive apoptosis in cancer cells independently of the p53 pathway; however, the resistance of cancer cells remains a major drawback. Here, we found a novel resistance mechanism of RITA treatment and an effective combined treatment to overcome RITA resistance in head and neck cancer (HNC) cells. The effects of RITA and 3-methyladenine (3-MA) were tested in different HNC cell lines, including cisplatin-resistant and acquired RITA-resistant HNC cells. The effects of each drug alone and in combination were assessed by measuring cell viability, apoptosis, cell cycle, glutathione, reactive oxygen species, protein expression, genetic inhibition of p62 and Nrf2, and a mouse xenograft model of cisplatin-resistant HNC. RITA induced apoptosis of HNC cells at different levels without significantly inhibiting normal cell viability. Following RITA treatment, RITA-resistant HNC cells exhibited a sustained expression of other autophagy-related proteins, overexpressed p62, and displayed activation of the Keap1-Nrf2 antioxidant pathway. The autophagy inhibitor 3-MA sensitized resistant HNC cells to RITA treatment via the dual inhibition of molecules related to the autophagy and antioxidant systems. Silencing of the p62 gene augmented the combined effects. The effective antitumor activity of RITA plus 3-MA was also confirmed in vivo in mouse xenograft models transplanted with resistant HNC cells, showing increased oxidative stress and DNA damage. The results indicate that RITA plus 3-MA can help overcome RITA resistance in HNC cells. Condensed abstract This study revealed a novel RITA resistant mechanism associated with the sustained induction of autophagy, p62 overexpression, and Keap1-Nrf2 antioxidant system activation. The combined treatment of RITA with the autophagy inhibitor 3-methyladenine overcomes RITA resistance via dual inhibition of autophagy and antioxidant systems in vitro and in vivo.

Promotion of oral surgical wound healing using autologous mucosal cell sheets

OBJECTIVES Severe oral mucosal and tissue defects can lead to pain, infection, and later undesirable healing of scarring and adhesion, resulting in a poor quality of life. In vitro-engineered oral mucosal equivalents for covering such defects are an alternative to avoiding the donor site morbidity of conventional skin or tissue grafts. We examined the efficacy of our newly developed three-dimensional mucosal cell sheets in an in vivo tongue wound model mimicking the surgical extirpation of tongue cancer. MATERIALS AND METHODS Small oral mucosal and autologous fibrin samples were obtained from surgical patients and Sprague-Dawley rats. The fibrin was mixed with fibroblasts and seeded with keratinocytes that had been primarily cultured for in vitro cell expansion. The three-dimensional autologous cell sheets, cultured in air-lift interface inserts, were transplanted into deep wounds of the rat ventral tongue. Gross and microscopic findings of the postsurgical wounds were compared between wound control and cell sheet groups. RESULTS The cell sheets were flexible, expandable, and easy to transfer, and had histological characteristics similar to that of the normal oral mucosa, with high p63 positivity. They promoted oral wound healing with earlier re-epithelialization and less fibrosis than that in the wound control. The cell sheet-healed tongue had similar histology to that of a normal tongue. CONCLUSIONS Our engineered cell sheets have potential applicability for the rapid healing of oral mucosal and soft tissue defects, without scarring, adhesion, and functional deficits. CONDENSED ABSTRACT The efficacy of in vitro-engineered mucosal equivalents, using completely autologous mucosa and plasma, was examined. Transplantation of the autologous cell sheets into deep wounds of the rat ventral tongue promoted oral wound healing with earlier re-epithelialization and less fibrosis than that in controls. Healed and normal tongues showed similar histology.

Development of an in vitro cell-sheet cancer model for chemotherapeutic screening

Epithelial cancer grows in vivo in a microenvironment that comprises tumour, stroma, and immune cells. A three-dimensional (3D) culture model might be able to mimic the tumour microenvironment in vivo; therefore, we developed a new 3D epithelial cancer model using in vitro cell-sheet engineering and compared the results of treatment with several chemotherapeutic drugs among the 3D cell-sheet model, spheroid culture, and 2D cell culture. Methods: The cell sheet comprised keratinocytes and a plasma fibrin matrix containing fibroblasts. Cancer spheroids with or without cancer-associated fibroblasts (CAFs) were interposed between the keratinocytes and fibrin layer. Cell growth, viability, and hypoxia were measured using the cell counting kit-8, LIVE/DEAD assay, and propidium iodide and LOX-1 staining. The morphology, invasion, and mRNA and protein expression were compared among the different cell culture models. Results: Enhanced resistance to sorafenib and cisplatin by cancer spheroids and CAFs was more easily observed in the 3D than in the 2D model. Invasion by cancer-CAF spheroids into the fibrin matrix was more clearly observed in the 3D cell sheet. The expansion of viable cancer cells increased in the 3D cell sheet, particularly in those with CAFs, which were significantly inhibited by treatment with 10 μM sorafenib or 20 μM cisplatin (P < 0.05). TGF-β1, N-cadherin, and vimentin mRNA and protein levels were higher in the 3D cell-sheet model. Conclusions: The 3D cell sheet-based cancer model could be applied to in vitro observation of epithelial cancer growth and invasion and to anticancer drug testing.

Hederagenin Induces Apoptosis in Cisplatin-Resistant Head and Neck Cancer Cells by Inhibiting the Nrf2-ARE Antioxidant Pathway

Acquired resistance to cisplatin is the most common reason for the failure of cisplatin chemotherapy. Hederagenin, triterpenoids extracted from ivy leaves, exhibits antitumor activity in various types of cancer. However, the therapeutic potential of hederagenin in head and neck cancer (HNC) has remained unclear. Therefore, we examined the effects of hederagenin in cisplatin-resistant HNC cells and characterized its molecular mechanisms of action in this context. We evaluated the effects of hederagenin treatment on cell viability, apoptosis, reactive oxygen species (ROS) production, glutathione levels, mitochondrial membrane potential (ΔΨm), and protein and mRNA expression in HNC cells. The antitumor effect of hederagenin in mouse tumor xenograft models was also analyzed. Hederagenin selectively induced cell death in both cisplatin-sensitive and cisplatin-resistant HNC cells by promoting changes in ΔΨm and inducing apoptosis. Hederagenin inhibited the Nrf2-antioxidant response element (ARE) pathway and activated p53 in HNC cells, thereby enhancing ROS production and promoting glutathione depletion. These effects were reversed by the antioxidant trolox. Hederagenin activated intrinsic apoptotic pathways via cleaved PARP, cleaved caspase-3, and Bax. The selective inhibitory effects of hederagenin were confirmed in cisplatin-resistant HNC xenograft models. These data suggest that hederagenin induces cell death in resistant HNC cells via the Nrf2-ARE antioxidant pathway.

Use of oral mucosal cell sheets for accelerated oral surgical wound healing

We developed a highly efficient in vitro‐engineered mucosa equivalent using completely autologous mucosa and blood and investigated its feasibility and efficacy for oral surgical wound healing.

Nrf2 inhibition reverses resistance to GPX4 inhibitor-induced ferroptosis in head and neck cancer

&NA; Glutathione peroxidase 4 (GPX4) is a regulator of ferroptosis (iron‐dependent, non‐apoptotic cell death); its inhibition can render therapy‐resistant cancer cells susceptible to ferroptosis. However, some cancer cells develop mechanisms protective against ferroptosis; understanding these mechanisms could help overcome chemoresistance. In this study, we investigated the molecular mechanisms underlying resistance to ferroptosis induced by GPX4 inhibition in head and neck cancer (HNC). The effects of two GPX4 inhibitors, (1S, 3R)‐RSL3 and ML‐162, and of trigonelline were tested in HNC cell lines, including cisplatin‐resistant (HN3R) and acquired RSL3‐resistant (HN3‐rslR) cells. The effects of the inhibitors and trigonelline, as well as of inhibition of the p62, Keap1, or Nrf2 genes, were assessed by cell viability, cell death, lipid ROS production, and protein expression, and in mouse tumor xenograft models. Treatment with RSL3 or ML‐162 induced the ferroptosis of HNC cells to varying degrees. RSL3 or ML‐162 treatment increased the expression of p62 and Nrf2 in chemoresistant HN3R and HN3‐rslR cells, inactivated Keap1, and increased expression of the phospho‐PERK–ATF4–SESN2 pathway. Transcriptional activation of Nrf2 was associated with resistance to ferroptosis. Overexpression of Nrf2 by inhibiting Keap1 or Nrf2 gene transfection rendered chemosensitive HN3 cells resistant to RSL3. However, Nrf2 inhibition or p62 silencing sensitized HN3R cells to RSL3. Trigonelline sensitized chemoresistant HNC cells to RSL3 treatment in a mouse model transplanted with HN3R. Thus, activation of the Nrf2–ARE pathway contributed to the resistance of HNC cells to GPX4 inhibition, and inhibition of this pathway reversed the resistance to ferroptosis in HNC. Graphical abstract A proposed model of Nrf2‐induced resistance to GPX4 inhibition‐induced ferroptosis in cancer cells. The GPX4 inhibitors RSL3 and ML‐162 induce endoplasmic reticulum stress and subsequently p62 expression via the PERK–ATF4–SESN2 pathways. Nrf2 is activated by p62–Keap1 interaction and antioxidant response elements (ARE) related to iron and antioxidant systems is increased, resulting in a decreased labile iron pool, thus contributing to the resistance to ferroptosis. Figure. No caption available. HighlightsRSL3 and ML‐162 inhibited GPX4, inducing the ferroptotic death of head and neck cancer (HNC) cells to varying degrees.Resistance to GPX4 inhibition is related to the increased expression of p62.The p62–Keap1–Nrf2 antioxidant system is also involved in the RSL3 resistance mechanism.Nrf2 inhibition sensitized chemoresistant HNC cells to RSL3 treatment.Trigonelline reversed RSL3‐induced resistance to ferroptosis in HNC via inhibition of the Nrf2 system, both in vitro and in vivo.

CISD2 inhibition overcomes resistance to sulfasalazine-induced ferroptotic cell death in head and neck cancer

Sulfasalazine has been repurposed to induce ferroptotic cancer cell death via inhibition of xc--cystine/glutamate antiporter (xCT). However, cancer cells are capable of developing mechanisms to evade cell death. Therefore, we sought to determine the molecular mechanisms underlying resistance to sulfasalazine-induced ferroptosis in head and neck cancer (HNC). The effects of sulfasalazine and pioglitazone were tested in various HNC cell lines. The effects of these drugs and inhibition and overexpression of CISD2 gene were determined by evaluating viability, cell death, lipid ROS production, mitochondrial iron, and mouse tumor xenograft models. SAS induced ferroptotic cell death in HNC at different levels. CISD2 expression showed an association between its expression and ferroptosis resistance. CISD2 overexpression conferred resistance to ferroptosis by sulfasalazine. Silencing CISD2 gene rendered resistant HNC cells susceptible to sulfasalazine-induced ferroptosis, with increased levels of lipid ROS and mitochondrial ferrous iron. Pioglitazone induced over-accumulation of mitochondrial iron and ROS and sensitized resistant HNC cells to sulfasalazine treatment in vitro and in a mouse tumor-xenograft model. CISD2 inhibition overcomes HNC resistance to ferroptotic cell death induced by sulfasalazine via increased accumulation of mitochondrial ferrous iron and lipid ROS.

Plasticity of oral mucosal cell sheets for accelerated and scarless skin wound healing

OBJECTIVES Wound healing is generally faster and associated with less scarring in the oral mucosa than in the skin. Although rarely studied, oral mucosa equivalents may contribute to rapid, scarless cutaneous wound healing. Therefore, we examined the potential utility of our newly developed oral mucosal cell sheet in skin wound healing. MATERIALS AND METHODS Oral mucosa and skin samples were obtained from surgical patients and Sprague-Dawley rats. Keratinocytes and fibroblasts were primarily cultured for in vitro cell expansion. Mucosa and skin equivalents were produced with a mixture of cultured fibroblasts and autologous fibrin from plasma and seeding keratinocytes. Mucosal and skin cell sheets were transplanted in full-thickness excisional wounds of rat skin with control wounds. Gross, histological, and molecular characteristics of wound healing according to different postsurgical days were compared in control and cell sheet-covered wounds. RESULTS Keratinocytes and fibroblasts derived from the oral mucosa were cultured faster than those derived from the skin. The in vitro-engineered oral mucosa and skin equivalents were successfully produced using complete autologous mucosa or skin and plasma fibrin, showing similarity to the histological characteristics of the skin or mucosa. In the in vivo rat model, the oral mucosal and skin cell sheet promoted wound healing with early wound closure and less scarring. The cell sheet-treated wounds showed lower TGF-β1, α-smooth muscle actin, and fibronectin mRNA expression than the control wounds. CONCLUSIONS The oral mucosal cell sheet demonstrated in vivo tissue plasticity through good adaptation to skin wounds, contributing to accelerated and scarless healing.

Abstract 1210: Hederagenin overcomes the cisplatin resistance of head and neck cancer by targeting the antioxidant defense mechanisms

Background: Cisplatin is a first-line chemotherapeutic agent for head and neck cancer (HNC). Acquired resistance to cisplatin is the most common reason for the failure of cisplatin chemotherapy. Hederagenin is extracted from the leaves of Cyclocarya paliurus, also known as sweet tea, which is one of the most popular teas utilized in traditional Chinese medicine. There is accumulating evidence it exhibited significant cytotoxicity against several types of cancers, however, which has been rarely tested in HNC cells. Herein, we examined the potential utility of hederagenin for overcoming cisplatin resistance in HNC cells and further clarified its molecular mechanisms of action. Methods: Parental and cisplatin-resistant HNC cells, and Nrf2 or SLC7A11 gene overexpressed HNC cell lines and other human HNC cells were used. The cells were used to examine the effects of hederagenin treatment in HNC cell lines by measuring cell viability, cell cycle, cell death, reactive oxygen species (ROS) production, glutathione level, mitochondrial membrane potential (ΔΨm), and protein expression. The anti-tumor effect of hederagenin in mouse tumor xenograft models was also tested. Results: Hederagenin induced cell death in both cisplatin-sensitive and -resistant HNC cells. Hederagenin treatment resulted in effective death of cisplatin-resistant HNC cells in a form of apoptotic cell death with the ΔΨm change. Hederagenin inhibited Nrf2 and SLC7A11 resulting in an increased ROS accumulation in HNC cells, of which effects were reversed by the pretreatment of antioxidant trolox. In addition, hederagenin caused the apoptosis effectively induced by targeting the antioxidant defense mechanisms in HNC cells. Subsequently, hederagenin activated signaling pathways of cell death involving caspase-3, PUMA, and PARP in the cells. The growth inhibitory effects of hedragenin were also confirmed in the tumor xenograft model implanted with cisplatin-resistant HNC cells. Conclusions: Our data suggests that hederagenin can overcome the resistance of cisplatin in resistant HNC cells via targeting antioxidant systems. Citation Format: Daiha Shin, Eun Hye Kim, Hyejin Jang, Jaewang Lee, Ji Won Kim, Jae Ryung Lee, Minsu Kwon, Seungho Baek, Jong-Lyel Roh. Hederagenin overcomes the cisplatin resistance of head and neck cancer by targeting the antioxidant defense mechanisms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1210. doi:10.1158/1538-7445.AM2017-1210