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Objective To investigate the effect of sulforaphane (SFN) on the proliferation and self-renewal of lung cancer stem cells and its regulatory mechanism. Methods MTT method was used to detect the effect of SFN on the proliferation of lung adenocarcinoma cell lines H460 and A549; tumor sphere formation experiment was used to detect the ability of tumor sphere formation; Western blot was applied to explore the expression of stemness-related proteins (such as β-catenin, Klf4, c-myc) in lung adenocarcinoma cells before and after SFN treatment; NGS sequencing was used to analyze the effect of SFN on the expression profile of tumor cell miRNAs. qRT-PCR verified the changes in the transcription level of key miRNAs by SFN. Western blot was used to detect the effect of SFN on the expression of DNMTs in tumor cells. We constructed miR-200c promoter-GFP plasmid, and applied IF, methylation PCR and DNA sequencing methods to detect the effect of SFN on the methylation level of tumor spheres and miRNA promoter. Results The miRNAs expression profile of lung adenocarcinoma tumor spheres changed significantly after SFN (5.0μmol/L) treatment, and miRNA-200c increased the most. Compared with the control group, the expression of β-catenin, Klf4, c-myc and Vimentin genes in H460 and A549 cells of SFN-S group decreased, and the protein expression levels of DNMT1 and DNMT3a were also significantly decreased. Compared with the control group, H460 and A549 cells stably expressing pEGFP-R200c plasmid in SFN-S group significantly reduced tumor sphere diameter, while tumor sphere fluorescence intensity increased, and GFP protein expression was up-regulated. There were 9 CpG-rich regions in the miR-200c promoter region in the above-mentioned pEGFP-R200c plasmid cell line, and the methylation levels were 88.9%, 44.4% and 38.8% in the control group, SFN-S group and 5-Aza-dC group, respectively. Conclusion SFN may downregulate the expression of stem-related genes in lung cancer stem cells by epigenetically decreasing the methylation level of miR-200c promoter and promoting the transcription of miR-200c.
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Objective@#To evaluate the clinical efficacy and side effects of cyberknife therapy in the treatment of retroperitoneal lymph node metastatic tumor.@*Methods@#Among the 81 patients presenting with postoperative retroperitoneal lymph node metastases, 33 cases suffered from abdominal pain or low back pain, 7 had unilateral hydronephrosis and ureteral dilatation, and 8 developed unilateral or bilateral lower limb swelling. Using stereotactic radiotherapy with a cyberknife, DT was delivered at 33-45 Gy/3-6 F. The improvement of symptoms, objective tumor response rate, and irradiation-induced side effects were observed.@*Results@#At 4 weeks after treatment, pain and swelling of the lower extremities were completely mitigated, and hydronephrosis was fully healed in all patients. Enhanced CT or MRI was performed every 2 to 3 months. After 6-month follow-up, the complete response (CR) rate was calculated as 77%(62/81), 21%(17/81) for the partial response (PR) rate, 3%(2/81) for the stable disease (SD) rate and the effective rate (CR+ PR) was 98%.No case progressed. The main side effect was intestinal injury, including grade 1 in 16%(13/81), grade 2 in 9%(7/81). Multivariate analysis demonstrated the CR rate was not correlated with the type of the primary tumor or the size of the tumor (P>0.05), but was significantly associated with the distance of>0.5 cm between the tumor and intestine and the BED (α/β=10)>70 Gy of the tumor irradiation (P<0.01). The incidence of irradiation-related side effects was correlated with the maximum diameter of tumor>5 cm and the distance of<0.5 cm between the tumor and intestine (P<0.01).@*Conclusion@#Cyberknife is an efficacious and safe approach in the treatment of retroperitoneal lymph node metastatic tumors.
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Objective To investigate the effect of sulfuraphane (SFN) on proliferation of glioma stem cell line SWO-38, and its mechanism threreof. Methods Cell proliferation of SWO-38 treated with SFN was measured by cell prolifera?tion assay. Clone formation experiment, tumor sphere formation experiment and Western blotting method were applied to de?tect the ability of cell clone formation and tumor sphere formation, and the expression of stemness relative genes, such asβ-catenin, Oct4, Sox-2 and c-Myc. The effects of SFN and/or miR-124 inhibitor (miR-124i) on the expression of stemness rel?ative genes were compared. Changes of miRNAs (miRNA-9, 21, 221, 124, 128 and 181) induced by SFN were measured by real time quantity PCR. Results SFN suppressed the proliferation of SWO-38 cells in a dose-dependent manner, in which IC50 was (26.41±2.13)μmol/L. SFN also decreased the ability of forming cell clone and tumor sphere, as well as the expres?sion of stemness relative genes (β-catenin, Oct4, Sox-2 and c-Myc) in a dose-dependent manner. At the same time, SFN led to the change in many miRNAs, during which SFN increased the transcription of miR-124 (-5.9-fold) and miR-128 (-2.6-fold), and decreased the transcription of miR-9, miR-21 and miR-221. Compared to the blank control, the expression levels ofβ-catenin, Oct4, and Sox-2 were significantly increased in miR-124i group. On the other hand, the expressions of above genes were also higher in combined group, which was treated with miR-124i and SFN than those in SFN group, but lower than those of miR-124i group. Conclusion SFN can efficiently inhibit the proliferation of giloma stem cells in SWO-38 cell line through miR-124/(β-catenin/Sox-2/Oct4) signaling pathway.
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Objective To explore the effect of methotrexate packaged by tumor derived microparticles (T-MP MTX) combined with radiotherapy on lung cancer stem cell (CSC) in vitro. Methods T-MP MTX was prepared from non-small cell lung cancer A549 cells. Proliferative changes of A549 cells, bronchial epithelial cells H460 and 16HBE cells treated by T-MP MTX were assayed by MTT method. Cell cycles of A549 cells in blank group and T-MP MYX group were examined by fluorescence activated cell sorting (FACS). The effect of T-MP MTX combined with radiotherapy on CSCs was assessed by tumor sphere formation experiment and animal experiment. The expressions of stemness relative genes (such as β-catenin, Nanog, SOX-2 and KLF4) were measured by Western blot. Results T-MP MTX dose-dependently inhibited the cell growth in A549 cells, but didn't in H460 cells and 16HBE cells. The S cycle ratio of A549 cells in blank group and T-MP MYX group measured by FACS were (15.83±3.14)%and (47.47±6.69)%, respectively. S cycle ratio of T-MP MYX group was notably higher compared with that of blank group (t=7.411, P=0.002). Further study revealed that the number of tumor sphere in blank group, control group, 2 Gy group, 4 Gy group and 6 Gy group was (268.9±22.4), (172.4±18.7), (48.3±5.1), (16.3±3.5) and (5.1±3.1), respectively. The number of tumor sphere in other groups was decreased compared with that in blank group (F=228.291, P=0.000). The numbers of tumor sphere in 2 Gy group, 4 Gy group and 6 Gy groups was also reduced compared with that in control group. Importantly, the number of tumor sphere in these groups were decreased dramatically as the dose of radiotherapy increased (F=95.142, P=0.000). The results of tumor sphere volume were similar with the number of tumor sphere. Western blot experiment showed that T-MP MTX treatment in A549 cells decreased the expression of stemness relative genes (β-catenin, Nanog, SOX-2 and KLF4), and its role was reinforced when radiotherapy was combined. Animal experiment implied that activity of luciferase in T-MP MTX group was decreased compared with that in blank group (P=0.000), and the activity of luciferase in T-MP MTX plus 2 Gy group was reduced significantly (t=6.887, P=0.002). Conclusions T-MP MTX has a potential to sensibilize radiotherapy, and it will synergistically inhibit the proliferation of CSCs when combined with radiotherapy. Moreover, its mechanism may be related with T-MP MTX activating CSCs from hypometabolism state and blocking process of cell cycle.
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Objective To investigate the effects of MTA1 knock down on migration and invasion of NPC cell 5-8F in vitro. Methods RNAi (Si-MTA1-01 and Si-MTA1-02) that can transiently silenced MTA1 was designed, synthesized and transfected into 5-8F cells by lipofectamine 2000. Control group (transfection with nonsense sequence) was also estab-lished. The efficiency of MTA1 depletion was determined by q-PCR and Western blot. Wound-healing assay ,Matrigel inva-sion assay and thesolid-phase adhesion assay were performed to investigate the effect of MTA1 knockdown on 5-8F cell me-tastasis. Results Transiently knock down of MTA1 decreased MTA1 transcription and expression in 5-8F cells compared to shRNA-con cells, showing by Real-time PCR and western blot. The invasion and migration of the cells transfected with siRNA-MTA1 were much weaker than the control group (P＜0.05). Conclusion silencing MTA 1 gene can effectively in-hibit the migration and invasion of nasopharyngeal carcinoma cell, and might be a promising target for NPC treatment.