| Background:Osteosarcoma is a highly malignant bone cancer associated with locally aggressive growth and early metastatic potential that typically occurs in children and adolescents. It is characterized by frequent vascular invasion, locally aggressive growth to the adjacent soft tissue, high rates of local recurrence and early metastasis. Neoadjuvant chemotherapy followed by aggressive surgical techniques and post-operative chemotherapy have improved survival; however, patients with metastatic disease at diagnosis or those who have recurrent disease and drug resistance have an extremely poor prognosis, with only20%surviving at5years. Although the drugs for osteosarcoma metastasis and resistance were developed, the clinical efficacy is still not satisfied. This disappointing outcome strongly suggests that the evaluation of novel, effective therapeutic agents is urgently needed to prevent osteosarcoma progression and improve patient survival rates.Curcumin is a naturally occurring phenolic compound shown to modulate expression of genes involved in cell proliferation, apoptosis, invasion, metastasis, angiogenesis, and resistance to chemotherapy. Curcumin have a wide variety of antitumor activities in many different cancers, such as colorectal carcinoma, head and neck squamous cell carcinoma, pancreatic cancer. It is reported that curcumin inhibited cell growth and induced apoptosis in pancreatic cancer cells through the down-regulation of cyclin D1and Bcl-XL.It is also reported that curcumin exhibited anti-invasive and anti-metastatic effects in part by the suppression of matrix metalloproteinase-9(MMP-9) and vascular endothelial growth factor (VEGF).Diallyl trisulfide (DATS) is a naturally occurring organosulfur compound derived from Allium vegetable shown to reduce the risk of cardiovascular disease and diabetes, stimulate immune system, protect against infections, and have anti-aging as well as anti-cancer effects. DATS is suggested to inhibit cell proliferation, cause G2/M phase cell cycle arrest and induce apoptosis of human prostate cancer cells via cyclin B1downregulation and Bcl-2and caspase-3inhibition. Moreover, DATS could also inhibit migration and invasion in human colon cancer cells through the inhibition of matrix metalloproteinase-2,-7, and-9expressions. It is also reported that DATS inhibits human prostate cancer cell metastasis and angiogenesis by VEGF down-regulation. The data suggested that DATS could inhibit cell proliferation, apoptosis, invasion, metastasis and angiogenesis, as well as have a wide variety of antitumor activities in many different cancers. Our previous studies have also shown that DATS can induce G0/G1cell cycle arrest and apoptosis, thereby inhibiting the survival of osteosarcoma SaOS-2cell, indicating that DATS may also be involved in anti-osteosarcoma effects.Taken together, natural plant products curcumin and DATS shown to modulate cell proliferation, apoptosis, invasion, metastasis, and angiogenesis, play inhibitory roles in a variety of tumors. However, their effects on osteosarcoma are unclear. Thereafter, we intend to investigate the effects of natural plant products curcumin and DATS on the biological behavior of osteosarcoma cell. Our data will provide the evidence that natural plant products curcumin and DATS may be an effective approach for the treatment of osteosarcoma.Objective:The purpose of the current study was to investigate the effects of curcumin and DATS on the proliferation, cell cycle, invasion, metastasis, and angiogenesis of osteosarcoma cells.Materials and methods:1. Cell proliferation assay:Osteosarcoma cells were treated with different concentrations of curcumin or DATS for24h,48h and72h. MTT assay was performed to detect the osteosarcoma cell viability after the natural plant products treatment.2. Flow cytometry assay:Osteosarcoma cells were cultured on12-well plates and treated with different concentrations of curcumin or DATS. After48h incubation, cells cycle distribution was analyzed by flow cytometry using propidium iodide (PI) staining. Flow cytometry assay was conducted to determine the osteosarcoma cell cycle phase after the natural plant products treatment.3. Wound-healing assay:The wound was generated when the cells reached around90%confluency by scratching the surface of the plates with a pipette tip. The cells were incubated in the absence and presence of DATS for24h and36h. Wound-healing assay was used to examine the migration ability of osteosarcoma cells after the natural plant products treatment.4. Invasion assay:The cells were incubated with different concentrations of curcumin or DATS for24h. Next, viable cells confirmed by trypan blue exclusion were seeded in serum-free medium onto the upper chamber, while complete medium was added in the lower compartment as a chemoattractant. After48h of incubation, abilities of invasion were quantified by counting the number of invaded cells from Transwell chamber.5. Matrigel in vitro HUVECs tube formation assay:The DATS-treated cells were cultured in serum-free medium for24h. The conditioned media were centrifuged and collected. HUVECs were trypsinized and seeded in Matrigel-coated well with conditioned medium from DATS-treated or control cells. The chamber was incubated for12h. The angiogenesis capacity induced by osteosarcoma cells was analyzed by tube formation.Results:1. Curcumin and DATS suppressed osteosarcoma cell proliferation:U2OS, SaOS-2and MG-63cells were treated with7.5,15,22.5,30,37.5μM of curcumin or25,50,100μM of DATS for24,48,72h, and cell viability was determined by MTT assay. We found that curcumin and DATS could significantly inhibit the proliferation of osteosarcoma cells in a dose-dependent and time-dependent manner.2. Curcumin and DATS induced osteosarcoma cell cycle arrested at G2/M and G0/G1phase, respectively:Cell cycle distribution of U2OS, SaOS-2and MG-63cells, following a48h treatment with15μM of curcumin or25,50μM of DATS, was determined by propidium iodide (PI) staining. The results showed that curcumin or DATS-treated osteosarcoma cells were arrested at at G2/M and G0/G1phase, respectively, in a concentration-dependent manner.3. DATS decreased osteosarcoma cell migration:U2OS, SaOS-2and MG-63cells were treated with25uM of DATS for24,36h, and cell migration abilities were determined by wound-healing assay. Data demonstrated that DATS treatment significantly decreased the capacity of wound healing in U2OS, SaOS-2and MG-63cells compared with those cells without DATS treatment, in a time-dependent manner.4. Curcumin and DATS inhibited osteosarcoma cell invasion:U2OS, SaOS-2and MG-63cells were treated with7.5.15,22.5μM of curcumin or25,50,100μM of DATS for24h, an in vitro invasion assay was conducted to test the effects of curcumin and DATS on cell invasion. The treatment of U2OS and MG-63cells for48h with curcumin resulted in a dose-dependent inhibition of cell invasion. Furthermore, DATS markedly inhibited the capacity of U2OS and SaOS-2cell invasion in a concentration-dependent manner.5. DATS reduced tube formation of HUVECs induced by conditioned media from osteosarcoma cells:U2OS, SaOS-2and MG-63cells were treated with25μM of DATS for24h, and the tube formation assay was performed in growth factor reduced-Matrigel in vitro. We found that conditioned media from U2OS, SaOS-2and MG-63cells were able to significantly induce the tube formation of HUVECs. Moreover, conditioned media from U2OS, SaOS-2and MG-63cells treated with25μM of DATS inhibited tube formation compared to the medium from cells treated with DM SO.Conclusions:1. Curcumin could induce G2/M cell cycle arrest, inhibit the proliferation and invasion of osteosarcoma cells in vitro.2. DATS could induce G0/G1cell cycle arrest, suppress the proliferation, migration, invasion, as well as angiogenesis of osteosarcoma cells in vitro.3. Curcumin and DATS shown to inhibit the tumorigenesis and aggressiveness of osteosarcoma, may be an effective approach for the treatment of osteosarcoma. Background:Notch signaling pathway plays a pivotal role in fundamental processes of cell-fate determination, including stem cell maintenance, differentiation, proliferation and apoptosis, and thereby may contribute to the carcinogenesis. Deregulated Notch-1receptor expression has been reported in a growing number of solid human tumors, such as pancreas, cervix, and colon, as well as osteosarcoma. It has also been reported that manipulation of Notch-1showed a crucial role for Hes-1in osteosarcoma invasion and metastasis. Moreover, inhibition of Notch-1pathway using γ-secretase inhibitor suppressed osteosarcoma growth in vitro and in vivo. Therefore, the down-regulation of Notch-1signaling may be an effective approach for osteosarcoma therapy.Our previous studies have shown that natural plant products curcumin and DATS could induce cell cycle arrest, suppress the proliferation, migration, invasion, as well as angiogenesis of osteosarcoma U2OS, SaOS-2, MG-63cells in vitro. However, the precise molecular mechanisms by which curcumin and DATS exert their antitumor activity remain unclear. The fact that the downregulation of Notch-1contributes to curcumin and D ATS-induced inhibition of proliferation, invasion and angiogenesis in osteosarcoma cells remains to be further explored.It is reported that curcumin inhibited cell growth and induced apoptosis in pancreatic cancer cells through suppression of Notch-1signaling pathway, which correlated with the down-regulation of cyclin D1and Bcl-XL. Our previous study found that curcumin and DATS could decrease Notch-1expression in osteosarcoma SaOS-2cells, suggesting that curcumin and DATS may inhibit Notch-1signaling pathway in osteosarcoma cells.It is also reported that MMP and VEGF plays an important role in the invasion, metastasis and angiogenesis of tumor cells. Specifically, it has been reported that down-regulation of Notch-1reduced MMPs and VEGF expression, resulted in the inhibition of pancreatic cancer invasion and metastasis. Thereafter, Notch-1may have cross-talk with MMPs and VEGF. which is critically involved in the processes of tumor cell invasion and metastasis.All of those reports led us to conduct the further study to determine whether curcumin and DATS could inhibit the Notch-1signaling and its target genes in osteosarcoma cells and how it is related to other Notch-responsive genes. Based on our previous work, we further explored the underlying mechanisms by which curcumin and DATS exert their antitumor activity, which would provide a novel target for the treatment of osteosarcoma.Objective: The aim of this study was to determine the effect of curcumin and DATS on Notch-1signaling pathway, MMPs and VEGF in osteosarcoma cells, and clarify the role of the Notch-1signal pathway in the anti-osteosarcoma effects of curcumin and DATS.Materials and methods:1. Real-time reverse transcription-PCR (RT-PCR) analysis:Osteosarcoma cells treated with different concentrations of curcumin or DATS were collected, total RNA was isolated by TRIzol, reverse transcription was performed with M-MuLV reverse transcriptase. Real time RT-PCR was used to detect the mRNA expression of Notch-1, Hes-1, Hey-1, Hey-2, VEGF, MMP-2and MMP-9before or after the natural plant products treatment.2. Western blot:Osteosarcoma cells treated with different concentrations of curcumin or DATS were collected, total proteins were extracted by RIPA. Western blot was peformed to investigate the protein expression of Notch-1, Hes-1, cyclin D1, VEGF, MMP-2and MMP-9before or after the natural plant products treatment.3. Gelatin zymography analysis:Osteosarcoma cells were treated with different concentrations of curcumin or DATS. Protein concentrations of cell-free culture supernatants were determined with the bicinchoninic acid assay protein reagent kit. An equal amount of protein was seperated on10%SDS-polyacrylamide gel containing1mg/ml gelatin A. Gelatin zymography was conducted to determine the activities of MMP-2and MMP-9of the supernatants before or after the natural plant products treatment.4. Enzyme-linked immunosorbent assay (ELISA):The supernatants of osteosarcoma cells treated with different concentrations of curcumin or DATS were collected. ELISA was applied to examine the VEGF activity of the supernatants before or after the natural plant products treatment.5. Cell transfection:Osteosarcoma cells were stably transfected with Notch-1intracellular domain (ICN) cDNA or Notch-1small interfering RNA (siRNA) by using the Lipofectamine2000reagent. After48h transfection, cells were selected in G418containing medium for at least2weeks. The protein expression of Notch-1, Hes-1, cyclin D1, VEGF, MMP-2and MMP-9before or after transfection was examined by western blot. The activities of MMP-2and MMP-9of the supernatants before or after transfection were detected by gelatin zymography. The VEGF activity of the supernatants before or after transfection was determined by ELISA.6. Cell viability assay:Transfected or untransfected osteosarcoma cells were treated with different concentrations of curcumin or DATS for48h. MTT assay was performed to detect the osteosarcoma cell viability before or after transfection.7. Invasion assay:Transfected or untransfected osteosarcoma cells were incubated with different concentrations of curcumin or DATS for24h. Next, viable cells confirmed by trypan blue exclusion were seeded in serum-free medium onto the upper chamber, while complete medium was added in the lower compartment as a chemoattractant. After48h of incubation, abilities of invasion were quantified by counting the number of invaded cells from Transwell chamber. Invasion assay was conducted to determine the osteosarcoma cell invasion before or after transfection.8. Matrigel in vitro HUVECs tube formation assay:Transfected or untransfected osteosarcoma cells treated with DATS were cultured in serum-free medium for24h. The conditioned media were centrifuged and collected. HUVECs were trypsinized and seeded in Matrigel-coated well with conditioned medium from DATS-treated or control cells. The chamber was incubated for8h. Tube formation assay was applied to examine the osteosarcoma cell angiogenesis before or after transfection.Results:1. Curcumin and DATS inhibit Notch-1signaling pathway, MMPs and VEGF in osteosarcoma cells1.17.5,15,22.5μM of curcumin reduced Notch-1and its target gene expression in U2OS and MG-63cells. Compared with control, there was a reduction of Notch-1, Hes-1, Hey-1, Hey-2, MMP-2and MMP-9mRNA levels after curcumin treatment by real-time RT-PCR. Western blot results demonstrated that the protein level of Notch-1, Hes-1, Cyclin D1, MMP-2and MMP-9expression was also inhibited by treatment with curcumin. Gelatin zymography analysis demonstrated that both MMP-2and MMP-9activities were reduced in curcumin-treated cells compared with control cells.1.225,50μM of DATS reduced Notch-1and its target gene expression in U2OS, SaOS-2and MG-63cells. Compared with control, there was a reduction of Notch-1, Hes-1, Hey-1, Hey-2, VEGF, MMP-2and MMP-9mRNA levels after DATS treatment by real-time RT-PCR. Western blot results demonstrated that the protein level of Notch-1, Hes-1, Cyclin D1, VEGF, MMP-2and MMP-9expression was also inhibited by treatment with DATS. Gelatin zymography analysis demonstrated that both MMP-2and MMP-9activities were reduced in DATS-treated cells compared with control cells. DATS could lead to a decrease in the levels of VEGF secreted in the culture medium as assessed by ELISA assay.2. Upregulation of Notch-1decreased anti-osteosarcoma effects of curcumin and DATS2.1Western blot showed Notch-1protein levels were significantly increased after transfection with a pMSCV-ICN plasmid, suggesting that the pMSCV-ICN plasmid effectively upregulates the expression level of Notch-1in U2OS cells. Moreover, Notch-1overexpression significantly increased Hes-1protein levels.2.2Notch-1overexpression significantly increased U2OS cell growth, and this overexpression rescued curcumin-and DATS-induced proliferation inhibition.2.3Notch-1overexpression significantly increased U2OS cell invasion, and this overexpression rescued curcumin-and DATS-induced cell invasion inhibition.2.4Notch-1overexpression significantly increased MMP-2and MMP-9activities of U2OS cell, and this overexpression abrogated curcumin-induced inhibition of MMP-2and MMP-9activities.3. Downregulation of Notch-1enhanced anti-osteosarcoma effects of curcumin and DATS 3.1Western blot showed Notch-1protein levels were significantly decreased after transfection with Notch-1siRNA suggesting that Notch-1siRNA effectively downregulated the expression level of Notch-1in U2OS cells. Moreover, Notch-1downregulation significantly inhibited Hes-1, Cyclin D1, VEGF, MMP-2and MMP-9protein levels.3.2Notch-1downregulation significantly inhibited U2OS cell growth, and this downregulation enhanced curcumin-and DATS-induced proliferation inhibition.3.3Notch-1downregulation significantly inhibited U2OS cell invasion, and this down-regulation enhanced curcumin-and DATS-induced cell invasion3.4Notch-1downregulation significantly inhibited U2OS cell supernatant-induced tube formation, and this down-regulation enhanced DATS-induced tube formation inhibition.3.5Notch-1downregulation significantly inhibited MMP-2and MMP-9activities of U2OS cell supernatant, and this down-regulation enhanced curcumin-and DATS-induced inhibition of MMP-2and MMP-9activities.3.6Notch-1downregulation significantly inhibited VEGF secretion of U2OS cell, and this down-regulation enhanced DATS-induced inhibition of VEGF secretion.Conclusions:1. Curcumin inhibits proliferation and invasion of osteosarcoma cells through inactivation of the Notch-1signaling pathway and MMPs.2. DATS suppresses proliferation, migration, invasion and angiogenesis of osteosarcoma cells via inhibition of the Notch-1signaling pathway, MMPs and VEGF. Background:Recent studies have demonstrated that a class of small molecules--microRNA (miRNA) plays an important role in fundamental processes of cell-fate determination, including cell proliferation, apoptosis, invasion and angiogenesis, and thereby may contribute to the tumorigenesis and aggressiveness of osteosarcoma. Our previous studies have shown that DATS could suppress proliferation, migration, invasion and angiogenesis of osteosarcoma cells, and miRNA may be involved in the anti-osteosarcoma effects of DATS. MiRNA are recently found as a class of small, non-protein-coding RNA molecules that are well conserved in eukaryotic cells. MiRNAs can bind to the3’untranslated region (UTR) of cognate mRNAs through fully complementary or imperfect base-pairing repressing the translation or decreasing the stability of the bound mRNAs.Dysregulation of miRNAs have been reported in a variety of solid tumors, including osteosarcoma. Moreover, miRNAs have been involved in tumor cell growth, proliferation, invasion and metastasis. Indeed, miR-21is significantly overexpressed in osteosarcoma tissues and knockdown of miR-21greatly decreases osteosarcoma cell invasion and migration via RECK upregulation. Upregulation of miR-34a inhibits the proliferation and metastasis of osteosarcoma cells in vitro and in vivo through inactivation of c-Met. Additionally, down-regulation of miR-143/145correlates with the lung metastasis of human osteosarcoma cells and over-expression of miR-143/145suppresses cell invasion and angiogenesis via down-regulation of their target gene MMP-13and VEGF. Therfore, some miRNAs are reported as oncomirs which could function as either oncogenes or tumor suppressors, which may play key roles in the pathogenesis and development of osteosarcoma.Emerging evidence suggest that there is a cross-talk between miRNAs and the Notch-1signaling pathway in tumor development and progression. Recent studies have shown that miR-34a participates in the regulation of p53and Notch pathways consistent with tumor suppressor activity, suggesting that further mechanistic understanding of Notch regulation by miRNAs and finding novel agents that could inactivate Notch would become a promising strategy for the treatment of aggressive osteosarcoma.In our previous studies, we found that DATS treatment concomitantly attenuated Notch-1expression and up-regulated miR-34a in osteosarcoma cells, which led us to conduct the current study to determine how microRNAs regulates Notch-1signaling pathway in anti-osteosarcoma effects of DATS that are typically lost in osteosarcoma.Objective:The aim of the current study was to investigate the miRNAs regulated by the Notch-1signaling pathway in osteosarcoma. Furthermore, the role of miR-34a and miR-200b in regulation of Notch-1expression and their mechanisms in anti-osteosarcoma effects of DATS were also determined.Materials and methods:1. miRNA Real time RT-PCR:DATS-treated osteosarcoma cells were collected, total RNA was isolated by TRIzol, reverse transcription was performed with All-in-One miRNA kits. MiRNA Real time RT-PCR was used to detect the mRNA expression of miR-21, miR-34a, miR-143, miR-145and miR-200b,c before or after the natural plant products treatment.2. Notch-1siRNA cell transfection:Osteosarcoma cells were transfected with Notch-1siRNA using the Lipofectamine2000reagent. MiRNA Real time RT-PCR was used to detect the mRNA expression of miR-21, miR-34a, miR-143, miR-145and miR-200b,c before or after transfection.3. Mimics cell transfection:Osteosarcoma cells were transfected with miR-34a or miR-200b mimics using the Lipofectamine2000reagent. MiRNA Real time RT-PCR was used to detect the mRNA expression of miR-34a and miR-200b before or after transfection.4. Western blot:Osteosarcoma cells transfected with miR-34a or miR-200b mimics were collected, total proteins were extracted by RIPA. Western blot was peformed to investigate the protein expression of Notch-1, VEGF, MMP-2and MMP-9before or after transfection.5. Gelatin zymography analysis:Osteosarcoma cells were transfected with miR-34a or miR-200b mimics. Protein concentrations of cell-free culture supernatants were determined with the bicinchoninic acid assay protein reagent kit. An equal amount of protein was seperated on10%SDS-polyacrylamide gel containing1mg/ml gelatin A. Gelatin zymography was conducted to determine the activities of MMP-2and MMP-9of the supernatants before or after transfection.6. ELISA:The supernatants of osteosarcoma cells transfected with miR-34a or miR-200b mimics were collected. ELISA was applied to examine the VEGF activity of the supernatants before or after transfection.7. Cell viability assay:Osteosarcoma cells were transfected with miR-34a or miR-200b mimics. MTT assay was performed to detect the osteosarcoma cell viability before or after transfection.8. Invasion assay:Osteosarcoma cells were transfected with miR-34a or miR-200b mimics. Next, viable cells confirmed by trypan blue exclusion were seeded in serum-free medium onto the upper chamber, while complete medium was added in the lower compartment as a chemoattractant. After48h of incubation, abilities of invasion were quantified by counting the number of invaded cells from Transwell chamber. Invasion assay was conducted to determine the osteosarcoma cell invasion before or after transfection.9. Matrigel in vitro HUVECs tube formation assay:Osteosarcoma cells transfected with miR-34a or miR-200b mimics were cultured in serum-free medium for24h. The conditioned media were centrifuged and collected. HUVECs were trypsinized and seeded in Matrigel-coated well with conditioned medium from DATS-treated or control cells. The chamber was incubated for8h. Tube formation assay was applied to examine the osteosarcoma cell angiogenesis before or after transfection.Results:1. Effects of DATS on the expression of miRNAs in osteosarcoma cells:The results revealed that50μM of DATS treatment increased the relative expressions of miR-143and miR-145while decreased the relative expression of miR-21in U2OS, SaOS-2and MG-63cells. DATS treatment increased the relative expression of miR-34a in U2OS cells but not in SaOS-2and MG-63cells. Interestingly, miR-200b and miR-200c expression were very low in U2OS and MG-63cells and DATS was able to up-regulate their expression.2. Effects of Notch-1siRNA on the expression of miRNAs in osteosarcoma cells: We found that Notch-1down-regulation led to reexpression of miR-34a, miR-143, miR-145, miR-200b and miR-200c which are typically lost in osteosarcoma cells as well as resulted in a decrease in miR-21expression in U2OS cells.3. Effects of miR-34a and miR-200b on the expression of Notch-1, VEGF, MMP-2and MMP-9in osteosarcoma cells:We conducted transfection experiment using U2OS cells by miR-34a or miR-200b mimics. As expected, the transfection of miR-34a and miR-200b mimics increased the relative levels of miR-34a and miR-200b, respectively, which consequently decreased the relative levels of Notch-1protein in U2OS cells. Reexpression of miR-34a and miR-200b also inhibited the expression and activities of VEGF, MMP-2and MMP-9in U2OS cells.4. Effects of miR-34a and miR-200b on osteosarcoma cell survival, invasion and angiogenesis:Reexpression of miR-34a and miR-200b significantly decreased the cell survival and invasion of U2OS cells as well as reduced the HUVECs tube formation.Conclusions:1. DATS could inhibit osteosarcoma cell growth, invasion and HUVECs tube formation via a novel mechanism targeting Notch-miRNA regulatory circuit.2. Reexpression miR-34a and miR-200b could inhibit osteosarcoma cell growth, invasion and reduced the HUVECs tube formation through inhibition of the Notch-1signaling pathway, MMPs and VEGF. |
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