Anti-Tumor Activity By Nordihydroguaiaretic Acid In Osteosarcoma Cells In Vitro

Osteosarcoma is the most common primary malignantneoplasm of bone that progresses rapidly and has a poor prognosis. Since the introduction of chemotherapy, survival in localised high-grade osteosarcoma has improved considerably. However, there is still no worldwide consensus on a standard chemotherapy approach. Standard treatment includes the use of’up-front’multiagent chemotherapy, definitive surgery of the primary tumor, and postoperative chemotherapy. Currently,chemotherapy treatment for osteosarcoma includes epirubicin, cisplatin, etoposide, methotrexate, and cyclophosphamide. These drugs are known to cause serious systemic toxicity, when used either as single agent or in combination with other drugs.Moreover, it has been shown that osteosarcoma cells are not highly susceptible to most chemotherapeutic agents.Therefore, it is an urgent need to develop more available chemotherapy strategies or find safety and effective chemotherapy agents for the treatment of osteosarcoma.Interestingly, a recent study demonstrated that Nordihydroguaiaretic acid (NDGA) and its derivatives had potent anti-tumor activity on osteosarcoma cells through unidentified mechanisms.The use of plants for medicinal purposes predates written human history. Creosote bush, Larrea tridentata, is known as chaparral or greasewood in the United States and as gobernadora or hediondilla in Mexico. Creosote bush is an important plant with a long history of medicinal use. Nordihydroguaiaretic acid (NDGA), the main metabolite of the creosote bush, has shown to have promising applications in the treatment of multiple diseases. Several medicinal properties have been supported in cell culture and animal studies as well as historical reports. However, the safety and possible toxicity from its application must still be determined in clinical studies. Over the years, this compound has been studied and has gained popularity and interest due to its antineoplastic, antiviral and anti-inflammatory characteristics. The molecular mechanisms and medical applications of NDGA have attracted much attention and hundreds of papers have been published in the last few years. NDGA has been identified to have a significant role in cancer therapy including that of reast, prostate, lung, esophageal and skin cancers. Models of carcinogenesis have emonstrated the capacity of NDGA to inhibit the growth of several human cancer types both in cell cultures and in animal models. These encouraging results suggest a possible herapeutic chemotherapy role for NDGA.In lung cancer, there is evidence to suggest that NDGA exerts a chemo protective activity;NDGA suppresses lung cancer cell growth in cell lines exposed to NDGA.Furthermore, one study demonstrated that adding0.1%NDGA to the drinking water of thymic mice bearing non-small cell lung cancer tumors significantly inhibits tumor growth ompared with control mice. In addition, NDGA has not only been shown to suppress reast cancer cell growth, it has a synergistic effect with retinoic acid on the inhibition of ammary tumor cell transformation and proliferation. Furthermore, NDGA has beenshown to be able to induce apoptosis in several human pancreatic and cervical cancer cell ines, which is thought to be due to its activity as a lipooxygenase inhibitor. Similarly, NDGA has been reported to promote cell death of trastuzumab-naive and trastuzumabrefractory uman epidermal growth factor receptor2(HER2)-overexpressing breast cancer ells by inducing DNA fragmentation. It has also been demonstrated that combination reatment with NDGA and trastuzumab suppressed proliferation and survival of rastuzumab-refractory cells to a greater degree than either agent alone, suggesting that DGA increases the sensitivity of refractory cells to trastuzumab, a monoclonal antibody to HER2. Due to the tendency of some breast cancers to develop resistance to trastuzumab, the possibility of developing adjunctive treatments has been explored, and NDGA may prove useful in this approach. Furthermore, NDGA has been shown to significantly inhibit damaging Ultraviolet B irradiation induced signaling pathways in human keratinocytes by two suggested modes of action. First, NDGA acts as an antioxidant to prevent the harmful effect of reactive oxygen species. Secondly, NDGA affects gene expression and differentiation, which is likely through its effects on leukotriene synthesis. Moreover, when topical NDGA is used in the treatment of actinic keratoses, it has been shown to be superior to5-fluorouracil.The complete mechanisms by which NDGA is anti-tumorigenic and anti-proliferative are still being elucidated. Preliminary in vivo studies have revealed that NDGA suppresses tumor growth by inhibiting metabolic enzymes as well as RTK phosphorylation, which is overexpressed in certain cancer cells. Meanwhile, NDGA has been shown to modulate tumor cell sensitivity to vinblastine by a mechanism independent of interference with the multidrug resistance (MDR) gene product.The mammalian target of rapamycin (mTOR) serves as a central regulator of cell growth, proliferation, survival, and metabolism by integrating both intracellular and extracellular signal.14The functions of mTOR are elicited by the context of two multiprotein complexes termed mTOR complex1(mTORC1) and mTORC2. The major downstream effectors of mTORC1are the ribosomal subunit S6kinase1(S6K1) and the eukaryotic initiation factor4E binding protein1(4E-BP1), two regulators of protein translation initiation and cell growth.mTORC2phosphorylates and activates protein kinase B(PKB)/Akt, an important pro-survival factor in cells. Signaling through the mTOR pathway contributes to growth, progression, and chemoresistance of many cancers.Accordingly, inhibitors have been developed as potentially valuable therapeutics. Recent evidences from basic and clinical research revealed that mTOR signaling plays important role in osteosarcoma carcinogenesis and progression. Up-regulation of mTOR/S6signaling pathway in human osteosarcoma and the correlation of mTOR and S6expression patterns with the oncological progression of osteosarcoma patients have been reported. mTOR and its downstream product are present and active in canine osteosarcoma cells.To determine whether mTOR signaling is a critical target of NDGA in osteosarcoma, the effects of NDGA on mTOR signaling and its targets were examined in this study.Our results revealed that NDGA decreased phosphorylation of both mTORC1downstream targets S6(S235/236),4E-BP1(T37/46), and mTORC2targets Akt (S473) in osteosarcoma cells. It is well established that inactivation of mTORC1induces arrest of the cell cycle in the G1phase by down-regulation of cyclin D1levels in some cell types. In consistent with NDGA-induced inhibition of mTORC1signaling, NDGA dose dependenly decreased the levels of cyclin D1and induced G1cell cycle arrest in osteosarcoma cells, in which the percentage of cells in G0/G1phase increased by one-third. mTORC2could be activated by growth factors and serves as kinase of prosurvival factor Akt (S473). We found that NDGA inhibited phosphorylation of Akt (S473) and induced apoptosis in osteosarcoma cells as demonstrated by cleavage, activation of caspases-3, and increase in Annexin V-FITC/PI-binding in NDGA-treated cells. Activation of caspase-3in osteosarcoma cell lines suggested that extrinsic and intrinsic pathways of apoptosis possibly participate in the reduction of osteosarcoma cells by NDGA. The inhibition of pro-survival factor and activation of the apoptosis-related process by NDGA indicated that NDGA is strong pro-apoptotic reagent in osteosarcoma cells. Altogether, our findings demonstrated that both mTORC1(growth-promoting) and mTORC2(anti-apoptotic) signaling pathways were inhibited by NDGA and suggested that mTOR signaling may be critical target of NDGA in osteosarcoma cells.In this study, we investigated the anti-tumor effects of the NDGA on osteosarcoma cells and the involvement of mTORsignaling in this process.We demonstrate that inhibition of mTOR signaling by NDGA may contribute to its anti-tumor activities in osteosarcoma cells.1.NDGA Prevented Cell Proliferation in Osteosarcoma CellsTo examine the effect of NDGA on proliferation of human osteosarcoma cells, MG63, U2Os and Saos-2cells were treated with NDGA at concentrations ranging from10to100uM for additional72h or at the concentration of lOOuM for1-4days, proliferation of MG63, U2Os and Saos-2cells was inhibited by NDGA dose and time dependently.2.NDGA reduced colony formation in Osteosarcoma CellsWe next examined the ability of MG63, U2Os and Saos-2cells to form colonies in the presence of NDGA. We found that NDGA reduced colony formation at concentration as low as10uM. The number of colonies was decreased by NDGA treatment in a dose (10-50mM) dependent manner. These results suggest that NDGA prevents colony formation of human osteosarcomacells dose and time dependently.3. NDGA reduced Adhesion, migration and invasion in Osteosarcoma Cells Adhesion, migration and invasion of Nordihydroguaiaretic acid treated Osteosarcoma cells were measured by Cell Counting Kit-8assay, wound assay and transwell chamber method respectively. Western blotting analysis was used to evaluate the expression of MMPs protein. Results showed Nordihydroguaiaretic acid inhibited the adhesion, migration and invasion of Osteosarcoma cells in a dose-depended manner (P<0.05). The protein level of MMP-2and MMP-9was down-regulated.4. NDGA Inhibited mTORC1Signaling and Induced Cell CycleArrest in Osteosarcoma CellsMTORC1signaling plays key role in regulation of protein translation and cell growth. We next examined the effect of NDGA on mTORC1signaling in osteosarcoma cells.20-150uM of NDGA treatment for24h inhibited phosphorylation of S6(S235/236) dose dependently, while the protein levels of S6was not altered by NDGA. Cyclin Dl plays a key role in G1to S phase transition in cell cycle progression. Initiation of cyclin Dl translation is dependent on activation of mTORCl. We further examined if inhibition of mTORC1signaling by NDGA would down-regulate cyclin Dl expression in osteosarcoma cells.20-150uM of NDGA treatment for24h decreased protein levels of cyclin D1dose dependently in Osteosarcoma cells.To determine whether inhibition of mTORC1and decrease of cyclin D1by NDGA induce G1cell cycle arrest, standard cell cycle analysis was performed on the osteosarcoma cell lines treated with NDGA.Flow cytometric analysis revealed an increase in the percentage of hypo-diploid cells in G1phase in Osteosarcoma cell lines treated with NDGA. These results suggest that NDGA could inhibit mTORC1signaling, down-regulate cyclin D1and induce G1cell cycle arrest in osteosarcoma cells.5. NDGA Inhibited mTORC2Signaling and Induced Apoptosis in Osteosarcoma CellsMTORC2could be activated by growth factors and serves as kinase of pro-survival factor Akt (S473). We next examined the effect of NDGA on mTORC2signaling in osteosarcoma cells. Our results demonstrated that NDGA reduced phosphorylation of Akt (473) dose dependently in Osteosarcoma cells. Further study revealed that NDG treatment induced cleavage and activation of caspase-3.Inhibition of Akt and activation caspase-3by NDGA implicated its pro-apoptotic role in osteosarcoma cells.the percentage of apoptotic cells increased markedly and dose dependently after treated with NDGA at concentrations of100-200mM, as stained by Hoechst33342. Percentage of cells undergone both apoptosis and death increased by NDGA treatment as detected by the binding of annexin V-FITC/PI, the results were examined by flow cytometry.Taken together, our results suggested that NDGA inhibited mTORC2, activated pro-apoptotic factor caspase-3and induced both apoptosis and cell death in osteosarcoma cells.In a summary, our results suggested that NDGA is a promising agent for treatment of osteosarcoma and mTOR (mTORC1and mTORC2) signaling may contribute to anti-tumor effects of NDGA in osteosarcoma cells. More in vivo study and clinical evidence are needed to verify the use of NDGA in the treatment of osteosarcoma.