1. Targeting of reactive oxygen species can be a potential therapeutic strategy for cancer treatment Ying-Ray Lee 1, San-Yuan Chen 2, and Hau-Ren Chen 3 1 Department of Medical Research, Ditmanson Medical Foundation Chiayi Christian Hospital, Chiayi, Taiwan 2 Department of Chinese Medicine, Ditmanson Medical Foundation Chiayi Christian Hospital, Chiayi, Taiwan 3 Department of Life Science, National Chung Cheng University, Chia-Yi, Taiwan Introduction: Chronic inflammation is induced by chemical, biological, and physical factors and is in turn associated with an increased risk of several human cancers formation. Chronic inflammation has been linked to various steps involved in carcinogenesis, including cellular transformation, promotion, survival, proliferation, invasion, angiogenesis, and metastasis. The link of inflammation and cancer has been demonstrated by anti-inflammatory therapies that show efficacy in cancer prevention and treatment. During inflammation, mast cells and leukocytes are recruited to the site of inflammation, which leads to increase uptake of oxygen, and thus, an increased release and accumulation of active oxygen species (ROS) at the site of damage. Therefore, inflammatory cells may increase DNA damage by activating pro-carcinogens to DNA-damaging species by ROS-dependent mechanisms. ROS is defined as an imbalance between production of free radicals and reactive metabolites. Oxidative stress interacts with three stages of this carcinogenesis. During the initiation stage, ROS may produce DNA damage by introducing gene mutations and structural alterations of the DNA. In the promotion stage, ROS can contribute to abnormal gene expression, blockage of cell- to cell communication, and modification of second messenger systems, thus resulting in an increase of cell proliferation or a decrease in apoptosis of the initiated cell population. Finally, oxidative stress may also participate in the progression stage of the cancer process by adding further DNA alterations to the initiated cell population. It is well known that elevated ROS in the cells can cause DNA damage, and thus contribute to tumor development through the regulation of cellular proliferation, angiogenesis, and metastasis. Moreover, ROS is observed in various types of cancer cells, and this tends to make these cells and tumors more resistant to chemotherapy. However, enhancing of ROS in the cancer cells can be used as a novel therapeutic strategy in multiple tumors recently. Hence, this study aimed to develop a novel therapeutic agent by regulating ROS in betel nut chewing-mediated oral squamous cell carcinoma (OSCC) cells. We explored the chemotherapeutic effects of piplartine (PL) in human OSCCs and present evidence that PL inhibits the growth of human OSCC cells through cell cycle arrest, senescence, and ROS-mediated caspase-dependent apoptosis. Figure 1. Growth inhibition of human oral squamous cell carcinoma cells. Human OSCC cell lines, OC2 and OCSL, were treated without or with piplartine. The cell viabilities were examined by CCK-8 analysis and each data point represents the results of 5 repeated experiments. Results: Conclusions: II. Piplartine induced cell cycle arrest in human oral squamous cell carcinoma cells Figure 2. Piplartine induced cell cycle arrest in human oral squamous cell carcinoma cells. (A) OC2 and (B) OCSL cells were treated without or with piplartine, and the cell cycle was analyzed by flow cytometry. IV. Piplartine induced autophagy in human oral squamous cell carcinoma cells V. Piplartine mediated apoptosis is through ROS induction Figure 5. Inhibition of ROS suppressed piplartine mediated cellular apoptosis in human oral squamous cell carcinoma cells. (A) OC2 and (B) OCSL cells were incubated with piplartine and/or with N-acetyl-L- cysteine (NAC; an inhibitor of ROS), and the cell viability was determined by CCK-8 analysis. I. Piplartine inhibited cellular growth in human oral squamous cell carcinoma cells II. Piplartine induced cellular apoptosis in human oral squamous cell carcinoma cells Figure 3. Piplartine induced cell apoptosis in human oral squamous cell carcinoma cells. (A) OC2 and OCSL cells were treated without or with piplartine (20uM), and the apoptotic cells was examined by flow cytometry. (B) Piplartine mediated apoptosis was suppressed by co- treatment with pan-caspase inhibitor (Z-VAD-FMK). DMSO was used as a negative control and etoptoside (50uM) was used as a positive control. Figure 4. Autophagy induction in human oral squamous cell carcinoma cells under piplartine treatment. Human OSCC cell lines, (A) OC2 and (B) OCSL, were incubated without or with piplartine. The expression of LC3-II (a marker of autophagy) was determined Western blot. Beta- actin was used as a loading control. 1.Piplartine suppresses tumour growth of human oral squamous cell carcinoma by regulating the cell cycle and inducing apoptosis and autophagy. 2.Piplartine induces a caspase-dependnet apoptosis. 3.Inhibition of ROS induction can reduce piplartine mediated human oral squamous cell carcinoma cellular death. 4.Piplartine is a potential chemotherapy agent for human oral squamous cell carcinoma.