ABSTRACT
Photodynamic therapy (PDT) is a promising treatment in cancer therapy, with a photosensitizer activated by visible light. Aloe-emodin (AE) is a promising photosensitive agent. In this study, the photosensitizing effects and possible mechanisms of AE-PDT in MG63 cells were evaluated. The efficiency of AE-PDT was analyzed by MTT assay. The mode of cell death was investigated by Hoechst 33,342 staining and flow cytometer. The intracellular distribution of AE was detected with confocal microscopy. The formation of reactive oxygen species (ROS) was detected by DCFH-DA. The mitochondrial membrane potential (MMP) was measured by Rhodamine 123. The expression of proteins including cytochrome c, caspase-3, -9, and -12, CHOP and GRP78 was detected by western blot. Apoptosis is the primary mode of cell death in our study, which occurs in a manner of depending on AE concentration and irradiation dose. Confocal microscopy showed that AE was primarily localized on the mitochondria and endoplasmic reticulum (ER) of MG63 cells. AE-PDT resulted in rapid increases of intracellular ROS production, which reached a peak at 2 h, followed by declining of mitochondrial membrane potential, releasing of cytochrome c from mitochondria into the cytoplasm, and up-regulation of caspase-3, -9, and -12, CHOP and GRP78. These results suggest that death of MG63 cells induced by AE-PDT is triggered by ROS. Meanwhile, Mitochondria and ER serve as the subcellular targets, which are responsible for AE-PDT-induced death of MG63 cells.
Subject(s)
Anthraquinones/pharmacology , Endoplasmic Reticulum Stress/drug effects , Mitochondria/drug effects , Mitochondria/metabolism , Photochemotherapy , Photosensitizing Agents/pharmacology , Signal Transduction/drug effects , Apoptosis/drug effects , Biomarkers , Cell Line, Tumor , Cell Survival/drug effects , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum Chaperone BiP , Endoplasmic Reticulum Stress/genetics , Gene Expression , Humans , Membrane Potential, Mitochondrial/drug effects , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , Osteosarcoma/genetics , Osteosarcoma/metabolism , Reactive Oxygen Species/metabolismABSTRACT
Gastric carcinoma (GC) has high incidence and mortality rates in China. Surgery and chemotherapy are the main treatments. Photodynamic therapy (PDT) has become a new treatment modality, appearing in recent experimental studies and clinical trials in various tumors. This study explores the combined effect of gene transfection with PDT on GC cells using aloe emodin (AE)-encapsulated nanoliposomes, which acted as gene carrier as well as one photosensitizer (PS). AE-encapsulated nanoliposomes (nano-AE) were prepared by reverse evaporation method. Electron microscopy and nano-ZS90 analyzer were used to detect its morphology, size, and wavelength. Western blot was used to detect the expression of the caspase-3 after transfection. MTT assay and flow cytometry were employed to determine the cytotoxic and apoptotic rates, respectively. Hoechst 33342 staining was adopted to detect the morphological changes in death gastric cancer cells. Cellular reactive oxygen species (ROS) contents were measured by DCFH-DA staining. Outcomes demonstrated that the nano-AE has good properties as gene delivery carriers as well as a PS. The group in which the recombinant plasmid of r-caspase-3 was transfected had higher protein expression of the caspase-3 than controls, meanwhile the proliferation rates of the transfected cells were inhibited by the nano-AE-mediated PDT in an energy-dependent manner. In addition, in the transfected cells, the death rate increased to 77.3% as assessed 12 h after PDT (6.4 J/cm(2) ). Hochest 33342 staining also revealed that the death rate increased significantly in the transfected group compared with other groups. Compared to control groups, the production of ROS in nano-AE PDT group had quadrupled in SGC-7901 cells as early as 1 h after PDT, while it is similar to the group of nano-AE transfection and PDT. Nano-AE-mediated r-caspase-3 gene transfection coupled with PDT could inhibit the proliferation rate and increase the apoptotic rate remarkably in human gastric cancer cells.
Subject(s)
Anthraquinones/administration & dosage , Caspase 3/genetics , Drug Compounding , Liposomes , Nanocomposites , Photochemotherapy , Stomach Neoplasms/genetics , Apoptosis/drug effects , Cell Line, Tumor , Humans , Light , Liposomes/chemistry , Nanocomposites/chemistry , Nanocomposites/ultrastructure , Particle Size , Reactive Oxygen Species/metabolism , Stomach Neoplasms/drug therapy , Stomach Neoplasms/metabolism , TransfectionABSTRACT
The objective of this study is to use high (49/s) and low (9/s) stimulation rates of the BAEP to investigate the possible mechanism responsible for BPPV. A total of 81 patients (55 women and 26 men, mean age ± SD = 54.6 ± 15.0) with idiopathic BPPV, as well as 106 control subjects (70 women and 36 men, mean age ± SD = 51.2 ± 16.3) participated in the study. The results of high (49/s) and low (9/s) stimulation rates of the BAEP test were compared and analyzed. The difference in BAEP wave I peak latencies between low and high stimulation rate (DPL I) and BAEP wave I peak latency in high stimulation (HPL I) of affected ears (0.24 ± 0.14 and 1.91 ± 0.21 ms) in BPPV patients were significantly prolonged when compared with the controls (0.10 ± 0.08 and 1.76 ± 0.18 ms) and unaffected ears (0.12 ± 0.10 and 1.82 ± 0.21 ms) (p < 0.001). The abnormal rate of DPL I in the affected ear (52/83, 62.65 %) was significantly higher than that in the unaffected ear (7/79, 8.86 %) and the normal left ear (4/106, 3.77 %). We suggest that ischemia of the inner ear might be one of the causes of BPPV and that DPL I may be used to assess the ischemic degree in subjects over 20 years old.
Subject(s)
Benign Paroxysmal Positional Vertigo/physiopathology , Evoked Potentials, Auditory, Brain Stem/physiology , Adult , Case-Control Studies , Ear, Inner , Female , Humans , Male , Middle AgedABSTRACT
Although BMP9 is highly capable of promoting osteogenic differentiation of mesenchymal stem cell (MSCs), the molecular mechanism involved remains to be fully elucidated. Here, we explore the possible involvement and detail role of JNKs (c-Jun N-terminal kinases) in BMP9-induced osteogenic differentiation of MSCs. It was found that BMP9 stimulated the activation of JNKs in MSCs. BMP9-induced osteogenic differentiation of MSCs was dramatically inhibited by JNKs inhibitor SP600125. Moreover, BMP9-activated Smads signaling was decreased by SP600125 treatment in MSCs. The effects of inhibitor are reproduced with adenoviruses expressing siRNA targeted JNKs. Taken together, our results revealed that JNKs was activated in BMP9-induced osteogenic differentiation of MSCs. What is most noteworthy, however, is that inhibition of JNKs activity resulted in reduction of BMP9-induced osteogenic differentiation of MSCs, implying that activation of JNKs is essential for BMP9 osteoinductive activity.
Subject(s)
Cell Differentiation/drug effects , Growth Differentiation Factors/pharmacology , JNK Mitogen-Activated Protein Kinases/metabolism , Mesenchymal Stem Cells/cytology , Osteogenesis/drug effects , Animals , Anthracenes/pharmacology , Cells, Cultured , HCT116 Cells , Humans , JNK Mitogen-Activated Protein Kinases/antagonists & inhibitors , JNK Mitogen-Activated Protein Kinases/genetics , Mesenchymal Stem Cells/metabolism , Mice , Phosphorylation/drug effects , RNA Interference , RNA, Small Interfering , Signal Transduction/drug effects , Smad Proteins/metabolismABSTRACT
Although BMP6 is highly capable of inducing osteogenic differentiation of mesenchymal progenitor cells (MPCs), the molecular mechanism involved remains to be fully elucidated. Using dominant negative (dn) mutant form of type I and type II TGFß receptors, we demonstrated that three dn-type I receptors (dnALK2, dnALK3, dnALK6), and three dn-type II receptors (dnBMPRII, dnActRII, dnActRIIB), effectively diminished BMP6- induced osteogenic differentiation of MPCs. These findings suggested that ALK2, ALK3, ALK6, BMPRII, ActRII and ActRIIB are essential for BMP6-induced osteogenic differentiation of MPCs. However, MPCs in this study do not express ActRIIB. Moreover, RNA interference of ALK2, ALK3, ALK6, BMPRII and ActRII inhibited BMP6-induced osteogenic differentiation in MPCs. Our results strongly suggested that BMP6-induced osteogenic differentiation of MPCs is mediated by its functional TGFß receptors including ALK2, ALK3, ALK6, BMPRII, and ActRII.
Subject(s)
Bone Morphogenetic Protein 6/metabolism , Cell Differentiation , Mesenchymal Stem Cells/metabolism , Osteogenesis , Receptors, Transforming Growth Factor beta/metabolism , Activin Receptors, Type I/antagonists & inhibitors , Activin Receptors, Type I/genetics , Activin Receptors, Type I/metabolism , Activin Receptors, Type II/antagonists & inhibitors , Activin Receptors, Type II/genetics , Activin Receptors, Type II/metabolism , Animals , Bone Morphogenetic Protein 6/genetics , Bone Morphogenetic Protein Receptors, Type I/antagonists & inhibitors , Bone Morphogenetic Protein Receptors, Type I/genetics , Bone Morphogenetic Protein Receptors, Type I/metabolism , Bone Morphogenetic Protein Receptors, Type II/antagonists & inhibitors , Bone Morphogenetic Protein Receptors, Type II/genetics , Bone Morphogenetic Protein Receptors, Type II/metabolism , Cell Line , HEK293 Cells , Humans , Mesenchymal Stem Cells/cytology , Mice , RNA Interference , RNA, Small Interfering/metabolism , Signal Transduction , Smad Proteins/metabolism , TransfectionABSTRACT
Although previous studies have demonstrated that BMP9 is highly capable of inducing osteogenic differentiation of mesenchymal stem cells, the molecular mechanism involved remains to be fully elucidated. In this study, we showed that BMP9 simultaneously promotes the activation of Smad1/5/8, p38 and ERK1/2 in C3H10T1/2 cells. Knockdown of Smad4 with RNA interference reduced nuclear translocation of Smad1/5/8, and disrupted BMP9-induced osteogenic differentiation. BMP9-induced osteogenic differentiation was blocked by p38 inhibitor SB203580, whereas enhanced by ERK1/2 inhibitor PD98059. SB203580 decreased BMP9-activated Smads singling, and yet PD98059 stimulated Smads singling in C3H10T1/2 cells. The effects of inhibitor were reproduced with adenovirus expressing siRNA targeted p38 and ERK1/2, respectively. Taken together, our findings revealed that Smads, p38 and ERK1/2 are involved in BMP9-induced osteogenic differentiation. Also, it is noteworthy that p38 and ERK1/2 may play opposing regulatory roles in mediating BMP9-induced osteogenic differentiation of C3H10T1/2 cells.