A distinct role for interleukin-6 as a major mediator of cellular adjustment to an altered culture condition.

Tissue microenvironment adjusts biological properties of different cells by modulating signaling pathways and cell to cell interactions. This study showed that epithelial-mesenchymal transition (EMT)/ mesenchymal-epithelial transition (MET) can be modulated by altering culture conditions. HPV E6/E7-transfected immortalized oral keratinocytes (IHOK) cultured in different media displayed reversible EMT/MET accompanied by changes in cell phenotype, proliferation, gene expression at transcriptional, and translational level, and migratory and invasive activities. Cholera toxin, a major supplement to culture medium, was responsible for inducing the morphological and biological changes of IHOK. Cholera toxin per se induced EMT by triggering the secretion of interleukin 6 (IL-6) from IHOK. We found IL-6 to be a central molecule that modulates the reversibility of EMT based not only on the mRNA level but also on the level of secretion. Taken together, our results demonstrate that IL-6, a cytokine whose transcription is activated by alterations in culture conditions, is a key molecule for regulating reversible EMT/MET. This study will contribute to understand one way of cellular adjustment for surviving in unfamiliar conditions.

Areca nut exposure increases secretion of tumor-promoting cytokines in gingival fibroblasts that trigger DNA damage in oral keratinocytes.

Molecular crosstalk between cancer cells and fibroblasts has been an emerging hot issue in understanding carcinogenesis. As oral submucous fibrosis (OSF) is an inflammatory fibrotic disease that can potentially transform into squamous cell carcinoma, OSF has been considered to be an appropriate model for studying the role of fibroblasts during early stage carcinogenesis. In this sense, this study aims at investigating whether areca nut (AN)-exposed fibroblasts cause DNA damage of epithelial cells. For this study, immortalized hNOF (hTERT-hNOF) was used. We found that the levels of GRO-α, IL-6 and IL-8 increased in AN-exposed fibroblasts. Cytokine secretion was reduced by antioxidants in AN-exposed fibroblasts. Increase in DNA double strand breaks (DSB) and 8-oxoG FITC-conjugate was observed in immortalized human oral keratinocytes (IHOK) after the treatment of cytokines or a conditioned medium derived from AN-exposed fibroblasts. Cytokine expression and DNA damage were also detected in OSF tissues. The DNA damage was reduced by neutralizing cytokines or antioxidant treatment. Generation of reactive oxygen species (ROS) and DNA damage response, triggered by cytokines, were abolished when NADPH oxidase (NOX) 1 and 4 were silenced in IHOK, indicating that cytokine-triggered DNA damage was caused by ROS generation through NOX1 and NOX4. Taken together, this study provided strong evidence that blocking ROS generation might be a rewarding approach for cancer prevention and intervention in OSF.

Cytoskeletal alteration modulates cancer cell invasion through RhoA-YAP signaling in stromal fibroblasts.

Cancer-associated fibroblasts(CAFs) participate in carcinogenesis through interaction with cancer cells. This study aimed to investigate the mechanism of cytoskeletal alteration of CAFs and its role in invasion of oral squamous cell carcinoma(OSCC).Immortalized normal fibroblasts(hTERT-hNOFs) co-cultured with OSCC cells showed myofibroblastic and senescent phenotypes like CAFs. Thus, this study substituted hTERT-hNOFs for CAFs. Next, the cytoskeletal alteration and its molecular mechanism were investigated in hTERT-hNOFs co-cultured with OSCC. As results, we found that RhoA regulated cytoskeletal organization in fibroblasts surrounding OSCC cells. Furthermore, as a downstream transcriptional factor of RhoA, YAP was mainly localized in the nucleus of hTERT-hNOFs co-cultured with OSCC. Consequently, we examined whether nuclear YAP localization of fibroblasts could influence cancer progression. YAPS127A fibroblasts manifesting nuclear localization of YAP induced cytoskeletal alteration and increased gel contractility and matrix stiffness, and thereby enhances the invasiveness of OSCC cells. In conclusion, the modification of tumor microenvironment, such as cytoskeletal change and matrix remodeling via RhoA-YAP in CAFs, modulates OSCC invasion. These understandings will provide the development of novel approaches for CAFs-based cancer therapy.

CXCL1 induces senescence of cancer-associated fibroblasts via autocrine loops in oral squamous cell carcinoma.

Cancer-associated fibroblasts (CAFs) have emerged as one of the main factors related to cancer progression, however, the conversion mechanism of normal fibroblasts (NOFs) to CAFs has not been well elucidated. The aim of this study was to investigate the underlying mechanism of CAF transformation from NOFs in oral squamous cell carcinoma (OSCC). This study found that NOFs exposed to OSCC cells transformed to senescent cells. The cytokine antibody array showed the highest secretion levels of IL-6 and CXCL1 in NOFs co-cultured with OSCC cells. Despite that both IL-6 and CXCL1 induced the senescent phenotype of CAFs, CXCL1 secretion showed a cancer-specific response to transform NOFs into CAFs in OSCC, whereas IL-6 secretion was eventuated by common co-culture condition. Further, CXCL1 was released from NOFs co-cultured with OSCC cells, however, CXCL1 was undetectable in mono-cultured NOFs or co-cultured OSCC cells with NOFs. Taken together, this study demonstrates that CXCL1 can transform NOFs into senescent CAFs via an autocrine mechanism. These data might contribute to further understanding of CAFs and to development of a potential therapeutic approach targeting cancer cells-CAFs interactions.

Apoptosis-related microRNA-145-5p enhances the effects of pheophorbide a-based photodynamic therapy in oral cancer.

MicroRNAs (miRNAs) regulate key biological processes, and their aberrant expression has been related to cancer development. Photodynamic therapy (PDT) has emerged as one of the most promising modalities for cancer treatment. However, the application of PDT has been limited to superficially localized human cancerous and precancerous lesions. To increase the usefulness of both PDT and miRNAs in cancer therapy, this study investigated whether apoptosis-related miRNA expression is influenced by PDT in oral cancer and whether miRNAs can enhance PDT efficacy. To achieve this goal, we performed a miRNA array-based comparison of apoptosis-related miRNA expression patterns following PDT using pheophorbide a (Pa) as a photosensitizer. After Pa-PDT, 13.1% of the miRNAs were down-regulated, and 16.7% of the miRNAs were up-regulated. Representative miRNAs were selected according to expression difference: miR-9-5p, miR-32-5p, miR-143-3p, miR-145-5p, miR-192-5p, miR-193a-5p, miR-204-5p, miR-212-3p, miR-338-3p, and miR-451a. Among them, only miR-145-5p showed the consistent reduction repeatedly in all cell lines after Pa-PDT. Further, the combined treatment of a miR-145-5p mimic and Pa-PDT increased phototoxicity, reactive oxygen species generation, and apoptotic cell death, suggesting that miRNAs expression could be a useful marker for enhancing the therapeutic effect of Pa-PDT. This study will provide a promising strategy for introducing miRNA as cancer therapy.