Angelica polymorpha Maxim Induces Apoptosis of Human SH-SY5Y Neuroblastoma Cells by Regulating an Intrinsic Caspase Pathway

Rahman, Md. Ataur,Bishayee, Kausik,Huh, Sung-Oh Korean Society for Molecular and Cellular Biology 2016 Molecules and cells Vol.39 No.2

Angelica polymorpha Maxim root extract (APRE) is a popular herbal medicine used for treating stomachache, abdominal pain, stomach ulcers, and rheumatism; however the effect of APRE on cancer cells has not yet been explored. Here, we examined APRE cytotoxicity seen on target neuroblastoma cells (NB) using cell viability assays, DAPI visualization of fragmented DNA, and Western blotting analysis of candidate signaling pathways involved in proliferation and apoptosis. We demonstrated that APRE reduced cell viability in NB to a greater extent than in fibroblast cells. In addition, we found that APRE could inhibit the three classes of MAPK proteins and could also down-regulate the PI3K/AKT/GSK-$3{\beta}$ activity all being relevant for proliferation and survival. APRE could also up-regulate Bax expression and down-regulate Bcl-2 and Mcl-1. With APRE treatment, depolarization of mitochondria membrane potential and activation of caspase-3 was demonstrated in the SH-SY5Y cells. We could not found increased activity of death receptor and caspase-8 as markers of the extrinsic apoptosis pathway for the APRE treated cells. In presence of a caspase-3 siRNA and a pan-caspase inhibitor, APRE could not reduce the viability of NB cells to a significant degree. So we predicted that with APRE, the intrinsic pathway was solely responsible for inducing apoptosis as we also showed that the non-caspase autophagy pathway or ER stress-ROS mediated pathways were not involved. These findings demonstrate that an intrinsic mitochondria-mediated apoptosis pathway mediates the apoptotic effects of APRE on SH-SY5Y cells, and that APRE shows promise as a novel agent for neuroblastoma therapy.

Antiproliferative and Cytotoxic Effects of Resveratrol in Mitochondria-Mediated Apoptosis in Rat B103 Neuroblastoma Cells

Rahman, Md. Ataur,Kim, Nam-Ho,Kim, Seung-Hyuk,Oh, Sung-Min,Huh, Sung-Oh The Korean Society of Pharmacology 2012 The Korean Journal of Physiology & Pharmacology Vol.16 No.5

Resveratrol, a natural compound, has been shown to possess anti-cancer, anti-aging, anti-inflammatory, anti-microbial, and neuroprotective activities. In this study, we examined the antiproliferative and cytotoxicity properties of resveratrol in Rat B103 neuroblastoma cells; although it's molecular mechanisms for the biological effects are not fully defined. Here, we examined the cellular cytotoxicity of resveratrol by cell viability assay, antiproliferation by BrdU assay, DNA fragmentation by DNA ladder assay, activation of caspases and Bcl-2 family proteins were detected by western blot analyses. The results of our investigation suggest that resveratrol increased cellular cytotoxicity of Rat B103 neuroblastoma cells in a dose-and time-dependent manner with $IC_{50}$ of 17.86 ${\mu}M$ at 48 h. On the other hand, incubation of neuroblastoma cells with resveratrol resulted in S-phase cell cycle arrests which dose-dependently and significantly reduced BrdU positive cells through the downregulation of cyclin D1 protein. In addition, resveratrol dose-dependently and significantly downregulated the expression of anti-apoptotic protein includes Bcl-2, Bcl-xL and Mcl-1 and also activates cleavage caspase-9 and-3 via the downregulation of procaspase-9 and -3 in a dose-dependent manner which indicates that involvement of intrinsic mitochondria-mediated apoptotic pathway. In conclusion, resveratrol increases cellular cytotoxicity and inhibits the proliferation of B103 neuroblastoma cells by inducing mitochondria-mediated intrinsic caspase dependent pathway which suggests this natural compound could be used as therapeutic purposes for neuroblastoma malignancies.

Antiproliferative and Cytotoxic Effects of Resveratrol in Mitochondria- Mediated Apoptosis in Rat B103 Neuroblastoma Cells

Md. Ataur Rahman,김남호,김승혁,Sung-Min Oh,허성오 대한약리학회 2012 The Korean Journal of Physiology & Pharmacology Vol.16 No.5

Resveratrol, a natural compound, has been shown to possess anti-cancer, anti-aging, anti-inflammatory, anti-microbial, and neuroprotective activities. In this study, we examined the antiproliferative and cytotoxicity properties of resveratrol in Rat B103 neuroblastoma cells; although it’s molecular mechanisms for the biological effects are not fully defined. Here, we examined the cellular cytotoxicity of resveratrol by cell viability assay, antiproliferation by BrdU assay, DNA fragmentation by DNA ladder assay, activation of caspases and Bcl-2 family proteins were detected by western blot analyses. The results of our investigation suggest that resveratrol increased cellular cytotoxicity of Rat B103 neuroblastoma cells in a dose-and time-dependent manner with IC50 of 17.86 μM at 48 h. On the other hand, incubation of neuroblastoma cells with resveratrol resulted in S-phase cell cycle arrests which dose-dependently and significantly reduced BrdU positive cells through the downregulation of cyclin D1 protein. In addition, resveratrol dose-dependently and significantly downregulated the expression of anti-apoptotic protein includes Bcl-2, Bcl-xL and Mcl-1 and also activates cleavage caspase-9 and-3 via the downregulation of procaspase-9 and -3 in a dose-dependent manner which indicates that involvement of intrinsic mitochondria-mediated apoptotic pathway. In conclusion, resveratrol increases cellular cytotoxicity and inhibits the proliferation of B103 neuroblastoma cells by inducing mitochondria-mediated intrinsic caspase dependent pathway which suggests this natural compound could be used as therapeutic purposes for neuroblastoma malignancies.

Dog sperm cryopreservation in trisaccharides supplemented glycerol-free Tris: effect of different freezing methods on sperm parameters and gene expression related to motility and apoptosis

Md. Ataur Rahman,Sang-Hyoun Park,Yubyeol Jeon,A.H. Nabeel,EunJi Kim,Il-Jeoung Yu 한국수정란이식학회 2018 한국수정란이식학회 학술대회 Vol.2018 No.11

The present study was undertaken to evaluate the effect of trisaccharides supplementation in glycerol-free tris (GFT) for the cryopreservation of dog spermatozoa. In the first experiment (E1), dog spermatozoa were resuspended with 50, 75, 100 or 125 mM of raffinose, melezitose or maltotriose and cooled at 4 ℃ for 10 min. To determine the effect of different cooling time, the spermatozoa resuspended with 100 mM of raffinose, melezitose or maltotriose were cooled during 10, 20, 30 or 40 min at 4 ℃ (second experiment; E2). The straws were then aligned horizontally for 10 min on the rack and then plunged into LN2. In the third experiment (E3), to determine the effect of different vapor freezing time, the spermatozoa resuspended with 100 mM raffinose were cooled at 4 ℃ for 20 min and frozen in LN2 for 5, 10, 15 or 20 min and then plunged into LN2. In the fourth experiment (E4), to compare different freezing methods [cooling plus vapor freezing (CV), cooling plus step-down freezing (CS) and direct step-down freezing (SD)], the spermatozoa resuspended with 100 mM raffinose were cooled for 20 min and frozen in LN2 vapor for 5 min in case of CV method. In case of CS method, spermatozoa were cooled for 20 min at 4℃ and then frozen by the step-down freezing method. The straws were then aligned horizontally at 18, 15, 5, and 2 cm respectively from the surface of LN2 for 1, 1, 1.4, and 5 min, respectively in an L shaped straw holder and then plunged into LN2. For SD method, the straws were directly aligned horizontally at the same levels as CS from the surface of LN2 for 1, 1, 1.9, and 5 min, respectively and then plunged into LN2. After thawing at 37℃ for 25 sec, the spermatozoa were then incubated for 30 min in the freezing extender (E1) or in the 50 mM sucrose supplemented GFT (E2, E3, and E4) at 24℃. Following post-thaw incubation, sperm progressive motility and viability were assessed in E1, E2, E3, and E4. In addition, acrosome integrity, and gene expression related to apoptosis (BAX, BCL2, and Caspase10) and sperm motility (SMCP) were evaluated in E4. The results demonstrated that, in E1, using 75 mM trisaccharides resulted in significantly (p<0.05) higher sperm motility in all sugar groups. Using 100 mM melezitose significantly (p<0.05) improved the post-thaw viability than the 100 mM raffinose. The viability in 100 mM maltotriose was similar with 100 mM raffinose and melezitose group. In E2, the different cooling time has no significant effect on post-thaw sperm progressive motility in all the sugar types. In addition, the viability was variable among the different groups. In E3, liquid nitrogen vapor freezing for 5 min resulted in improved motility and viability. The sperm progressive motility was significantly (p<0.05) higher in CV and SD group compared to CS group and the sperm viability was significantly (p<0.05) higher in CV group compared to the other groups in E4. However, the acrosomal integrity of spermatozoa in the group CV was significantly (p<0.05) higher than the group CS and SD. In addition, the expression of SMCP gene was significantly (p<0.05) higher in the CV group than the CS group. In contrast, the expression of Caspase10 significantly (p<0.05) lower in the group CV and SD than the group CS. Furthermore, the ratio of gene expression of BAX and BCL2 was significantly (p<0.05) lower in the group CV than the group CS. Therefore, cryopreservation of dog spermatozoa in 100 mM of raffinose supplemented GFT cooled for 20 min and vapor freezing for 5 min provides better progressive sperm motility, viability, and acrosome integrity with higher expression of SMCP gene and lower expression of caspase10 and BAX/BCL2 ratio following post-thaw incubation in 50 mM sucrose supplemented GFT for 30 min at 24℃.

5-Hydroxytryptamine 6 Receptor (5-HT₆R)-Mediated Morphological Changes via RhoA-Dependent Pathways

Rahman, Md. Ataur,Kim, Hanna,Lee, Kang Ho,Yun, Hyung-Mun,Hong, Jung-Hwa,Kim, Youngjae,Choo, Hyunah,Park, Mikyoung,Rhim, Hyewhon Korean Society for Molecular and Cellular Biology 2017 Molecules and cells Vol.40 No.7

The $5-HT_6R$ has been considered as an attractive therapeutic target in the brain due to its exclusive expression in the brain. However, the mechanistic linkage between $5-HT_6Rs$ and brain functions remains poorly understood. Here, we examined the effects of $5-HT_6R$-mediated cell morphological changes using immunocytochemistry, Western blot, and live-cell imaging assays. Our results showed that the activation of $5-HT_6Rs$ caused morphological changes and increased cell surface area in HEK293 cells expressing $5-HT_6Rs$. Treatment with 5-HT specifically increased RhoA-GTP activity without affecting other Rho family proteins, such as Rac1 and Cdc42. Furthermore, live-cell imaging in hippocampal neurons revealed that activation of $5-HT_6Rs$ using a selective agonist, ST1936, increased the density and size of dendritic protrusions along with the activation of RhoA-GTP activity and that both effects were blocked by pretreatment with a selective $5-HT_6R$ antagonist, SB258585. Taken together, our results show that $5-HT_6R$ plays an important role in the regulation of cell morphology via a RhoA-dependent pathway in mammalian cell lines and primary neurons.

Gintonin stimulates autophagic flux in primary cortical astrocytes

Rahman, Md. Ataur,Hwang, Hongik,Nah, Seung-Yeol,Rhim, Hyewhon The Korean Society of Ginseng 2020 Journal of Ginseng Research Vol.44 No.1

Background: Gintonin (GT), a novel ginseng-derived exogenous ligand of lysophosphatidic acid (LPA) receptors, has been shown to induce cell proliferation and migration in the hippocampus, regulate calcium-dependent ion channels in the astrocytes, and reduce β-amyloid plaque in the brain. However, whether GT influences autophagy in cortical astrocytes is not yet investigated. Methods: We examined the effect of GT on autophagy in primary cortical astrocytes using immunoblot and immunocytochemistry assays. Suppression of specific proteins was performed via siRNA. LC3 puncta was determined using confocal microscopy. Results: GT strongly upregulated autophagy marker LC3 by a concentration- as well as time-dependent manner via G protein-coupled LPA receptors. GT-induced autophagy was further confirmed by the formation of LC3 puncta. Interestingly, on pretreatment with an mammalian target of rapamycin (mTOR) inhibitor, rapamycin, GT further enhanced LC3-II and LC3 puncta expression. However, GT-induced autophagy was significantly attenuated by inhibition of autophagy by 3-methyladenine and knockdown Beclin-1, Atg5, and Atg7 gene expression. Importantly, when pretreated with a lysosomotropic agent, E-64d/peps A or bafilomycin A1, GT significantly increased the levels of LC3-II along with the formation of LC3 puncta. In addition, GT treatment enhanced autophagic flux, which led to an increase in lysosome-associated membrane protein 1 and degradation of ubiquitinated p62/SQSTM1. Conclusion: GT induces autophagy via mTOR-mediated pathway and elevates autophagic flux. This study demonstrates that GT can be used as an autophagy-inducing agent in cortical astrocytes.

Therapeutic implication of autophagy in neurodegenerative diseases

( Md. Ataur Rahman ),( Hyewhon Rhim ) 생화학분자생물학회(구 한국생화학분자생물학회) 2017 BMB Reports Vol.50 No.7

Autophagy, a catabolic process necessary for the maintenance of intracellular homeostasis, has recently been the focus of numerous human diseases and conditions, such as aging, cancer, development, immunity, longevity, and neurode-generation. However, the continued presence of autophagy is essential for cell survival and dysfunctional autophagy is thought to speed up the progression of neurodegeneration. The actual molecular mechanism behind the progression of dysfunctional autophagy is not yet fully understood. Emerging evidence suggests that basal autophagy is necessary for the removal of misfolded, aggregated proteins and damaged cellular organelles through lysosomal mediated degradation. Physiologically, neurodegenerative disorders are related to the accumulation of amyloid β peptide and α -synuclein protein aggregation, as seen in patients with Alzheimer`s disease and Parkinson`s disease, respectively. Even though autophagy could impact several facets of human biology and disease, it generally functions as a clearance for toxic proteins in the brain, which contributes novel insight into the pathophy-siological understanding of neurodegenerative disorders. In particular, several studies demonstrate that natural compounds or small molecule autophagy enhancer stimuli are essential in the clearance of amyloid β and α-synuclein deposits. Therefore, this review briefly deliberates on the recent implications of autophagy in neurodegenerative disorder control, and emphasizes the opportunities and potential therapeutic application of applied autophagy. [BMB Reports 2017; 50(7): 345-354]

Gintonin stimulates autophagic flux in primary cortical astrocytes

Md. Ataur Rahman,황홍익,나승열,Hyewhon Rhim 고려인삼학회 2020 Journal of Ginseng Research Vol.44 No.1

Background: Gintonin (GT), a novel ginseng-derived exogenous ligand of lysophosphatidic acid (LPA)receptors, has been shown to induce cell proliferation and migration in the hippocampus, regulatecalcium-dependent ion channels in the astrocytes, and reduce b-amyloid plaque in the brain. However,whether GT influences autophagy in cortical astrocytes is not yet investigated. Methods: We examined the effect of GT on autophagy in primary cortical astrocytes using immunoblotand immunocytochemistry assays. Suppression of specific proteins was performed via siRNA. LC3 punctawas determined using confocal microscopy. Results: GT strongly upregulated autophagy marker LC3 by a concentration- as well as time-dependentmanner via G proteinecoupled LPA receptors. GT-induced autophagy was further confirmed by theformation of LC3 puncta. Interestingly, on pretreatment with an mammalian target of rapamycin (mTOR)inhibitor, rapamycin, GT further enhanced LC3-II and LC3 puncta expression. However, GT-inducedautophagy was significantly attenuated by inhibition of autophagy by 3-methyladenine and knockdownBeclin-1, Atg5, and Atg7 gene expression. Importantly, when pretreated with a lysosomotropicagent, E-64d/peps A or bafilomycin A1, GT significantly increased the levels of LC3-II along with theformation of LC3 puncta. In addition, GT treatment enhanced autophagic flux, which led to an increase inlysosome-associated membrane protein 1 and degradation of ubiquitinated p62/SQSTM1. Conclusion: GT induces autophagy via mTOR-mediated pathway and elevates autophagic flux. This studydemonstrates that GT can be used as an autophagy-inducing agent in cortical astrocytes.

Induction of apoptosis by Dioscorea nipponica Makino extracts in human SH-SY5Y neuroblastoma cells via mitochondria-mediated pathway

Rahman, Md. Ataur,Yang, Haijie,Kim, Nam-Ho,Huh, Sung-Oh 한국통합생물학회 2014 Animal cells and systems Vol.18 No.1

Dioscorea nipponica, a perennial herb growing in mountainous areas, has been used as a folk medicine for asthma, rheumatoid arthritis, bronchitis, and other disease in Korea, whereas the effect of this plant on cancer cells has not been clearly clarified. Cellular cytotoxicity was examined by cell viability assay and DNA fragmentation by DNA ladder assay. Activation of different protein expression was detected by western blot analyses. We first demonstrated that D. nipponica extract (DNE) reducing the SH-SY5Y cell viability with $IC_{50}$ of $27.57{\mu}g/mL$ at 24 h. However, DNE downregulated the expression of antiapoptotic protein includes B-cell lymphoma-2 (Bcl-2), B-cell lymphoma-extra large (Bcl-xL), and myeloid cell leukemia 1 (Mcl-1) which also activated cleaved caspase-9 and caspase-3 in a dose- and time-dependent manner. Consequently, DNE-induced cytotoxicity was not mediated by the Fas/FasL, phosphatidylinositide-3 kinase/AKT/glycogen synthase kinase-$3{\beta}$ (PI3K/AKT/GSK-$3{\beta}$), and mitogen activated protein (MAP) kinases signaling pathway. In addition, DNE-induced cells shown damage morphology which had become cell rounding, neurite retraction, membrane blebbing, is right spell blebbing, and shrunken in a dose- and time-dependent manner that clearly indicate this morphological change might be due to the process of apoptosis which shown fragmented DNA. These results indicate that apoptotic effects of DNE on SH-SY5Y cells are mediated by intrinsic mitochondrial caspases signaling pathway, suggesting that DNE might be effective as an anticancer agent for neuroblastoma malignancies.

Cytotoxic effect of gambogic acid on SH-SY5Y neuroblastoma cells is mediated by intrinsic caspase-dependent signaling pathway.

Rahman, Md Ataur,Kim, Nam-Ho,Huh, Sung-Oh Dr. W. Junk B. V. Publishers ; Kluwer Academic Pub 2013 MOLECULAR AND CELLULAR BIOCHEMISTRY - Vol.377 No.1

<P>Gambogic acid (GA) is the dry resin of Garcinia hanburyi (Guttiferae) with potent anti-tumor activity, various bioactivities, including detoxification, homeostasis, anti-inflammatory, and parasiticide, whereas the effect of this natural compound on cancer cells has not been clearly clarified. Here, we examined cellular cytotoxicity by cell viability assay and DNA fragmentation by DNA-ladder assay. Activation of different protein expressions were detected by western blot analyses. We first demonstrated that GA reduces the human SH-SY5Y neuroblastoma cell viability with IC50 of 1.28 μM at 6 h which has less toxicity in fibroblast cells. However, lower concentration GA significantly downregulated the expression of anti-apoptotic protein including Bcl-2, Bcl-xL, and Mcl-1, which also dramatically activated cleaved caspase-9 and -3 in a dose- and time-dependent manner. Consequently, GA-induced cytotoxicity was not mediated by the Fas/FasL and PI3 K/AKT/GSK-3β signaling pathway. In addition, GA-induced cells showed damage morphology which had become cell rounding, neurite retraction, membrane blebbing and shrunken in a dose- and time-dependent manner that clearly indicates this morphological change might be due to the process of apoptosis which shows fragmented DNA. Therefore, the findings presented in this study demonstrate that apoptotic effects of GA on SH-SY5Y cells are mediated by intrinsic mitochondrion-dependent caspase pathway which suggests this natural compound might be effective as an anti-cancer agent for neuroblastoma malignancies.</P>