ABSTRACT
@#Apoptosis is an important means to regulate cell proliferation and maintain homeostasis. Recent researches have shown that the B-cell lymphoma-2 (BCL-2) family not only plays a dominant role in the regulation of normal cell apoptosis, but also plays a crucial role in the formation of tumor genesis, progression and subsequent drug resistance mediated by the escape mode of apoptosis. The phenomenon that BCL-2 family antagonized the apoptosis induced by antitumor drugs and then acquired drug resistance has been reported in the clinical treatment of hematologic lymphatic system tumors, breast cancer, lung cancer, gastric cancer and other diseases. Thus, specific inhibitors targeting anti-apoptotic members of the BCL-2 family have emerged with the development of research. In this paper, we systematically reviewed the regulation of apoptosis mediated by BCL-2 family and the drug resistance mediated by BCL-2 family. Meanwhile, we summarized the research advances of BCL-2 family specific inhibitors to provide new strategy for solving the problems on tumor therapeutic resistance and for finding new therapeutic targets in the future.
ABSTRACT
Aim To study the apoptosis of human hep-atoma cell line ( HepG2 ) induced by different polar parts of Arnebia euchroma ( Royle ) Johnst ( AE ) and to verify its anti-hepatoma effect by a mouse orthotopic liver cancer model so as to explore the anti-cancer effect of AE extract. Methods Firstly, MTT method and Annexin V-FITC/PI double staining method were used to detect the anti-proliferative and pro-apoptotic effects of each polar part of AE on HepG2 cells, and Western blot was used to detect the expression of Bcl-2 apoptosis family proteins incells. Based on the above experimental results, the effective parts with significant pro-apoptotic effect were screened out for anti-in situ liver cancer experiments in mice, and the organ indexes, liver function indexes and tissue sections of mice with orthotopic liver cancer before and after administration were evaluated. Results With the decrease of the polarity of AE extract,the anti-proliferation and pro-apoptotic effects on HepG2 cells were enhanced, and the anti-proliferation and apoptosis-inducing effects of AE petroleum ether fraction ( AEP) were the most significant. When AEP dose was 1.56 (μg • L
ABSTRACT
Aim: To investigate the promoting effect of Bcl-2/Bcl-xL inhibitor ABT-737 on apoptosis of gastric cancer cells induced by small molecule Mcl-1 inhibitor UMI-77, and to explore its possible mechanism. Methods: The response of gastric cancer MGC-803 and HGC-27 cells to different concentrations of UMI-77 was detected by MTS assay. In the UMI-77-resistant cell lines, the effect of treatment with UMI-77/ABT-737 alone or in combination on cell viability was detected by MTS assay. The apoptotic rate and the changes of the mitochondrial membrane potential were analyzed by flow cytometry. The cleavage of caspase-9, caspase-3 and PARP-1, as well as the expression level of Bcl-2 family members and IAP proteins, were determined by Western blot. Results: Compared with MGC-803 cells, HGC-27 cells were resistant to UMI-77. Treatment with ABT-737 alone in HGC-27 cells also induced minimal level of cell death. While treatment with both agents induced much greater decreased cell viability. All the dead cells were positive for Annexin V and mitochondrial membrane potential collapsed. Caspase-9, caspase-3 and its substrate PARP-1 were cleaved. All of these proved that the sensitization effect was achieved by activating the mitochondrial apoptotic pathway. Protein levels of XIAP, cIAP1 and cIAP2 decreased after treatment with UMI-77 plus ABT-737. It also resulted in the increase of NOXA and Bcl-2 along with the decline of PUMA and Mcl-1. Conclusions: The combination of UMI-77 and ABT-737 could significantly increase the sensitivity of gastric cancer cells to the Mcl-1 small molecule inhibitor UMI-77.
ABSTRACT
Objective: To explore the apoptotic effect of baicalein, a coumarin flavonone, on human ovarian carcinoma HO-8910 cells, as well as the mechanisms. Methods: HO-8910 cells were treated with esculetin at a series of concentrations for different times. Expression of apoptosis related Bax/Bcl-2, and Caspases proteins in esculetin treated HO-8910 cells were detected by Western blotting. Cell growth and apoptosis were measured by MTT test and flow cytometry in vitro. Results: Cell viability assay showed that esculetin had obvious anti-proliferation effects on HO-8910 cells in a dose- and time-dependent manner. Compared with control group, the group treated with esculetin showed a significant increase in apoptosis rate (P < 0.05, P < 0.01). The results demonstrated that esculetin up-regulated the Bax/Bcl-2 ratio and cleaved Caspase-3, cleaved Caspase-9 expression in a dose-dependent manner. Conclusion: In summary, baicalein exerts anti-growth and induced-apoptosis activity against ovarian cancer HO-8910 cells through activating Caspase and Bcl-2 family proteins, therefore presenting as a promising therapeutic agent for the treatment of ovarian cancer.
ABSTRACT
Objective: To investigate the effects of artesunate combined with bortezomib on the proliferation, apoptosis and autophagy of human acute myeloid leukemia cell lines MV4-11, and its mechanisms. Methods: MTT method was used to determine the anti-proliferation effect of different concentrations of artesunate, bortezomib and their combination on MV4-11 cells. The cell apoptosis were analyzed by flow cytometry. The expression of cleaved-Caspase-3, Bcl-2 family protein (Bcl-2, Mcl-1, Bim, Bax) and autophagy-related protein LC3B were assayed by Western blot. Results: Artesunate displayed a proliferation inhibition effect on MV4-11 with dose- and time-dependent manner, the IC(50) of artesunate on MV4-11 after 48 hours was 1.44 μg/ml. Bortezomib displayed a proliferation inhibition effect on MV4-11 with dose-dependent manner, the IC(50) of bortezomib on MV4-11 after 48 hours was 8.97 nmol/L. The combination of artesunate (0.75, 1.0 μg/ml) and Bortezomib (6, 8 nmol/L) showed higher inhibition on MV4-11 than artesunate or bortezomib alone in the same concentration gradient after 48 hours (P<0.05) . The cooperation index of the two drugs were all less than 1. The 48 h apoptotic rate of artesunate (1.5 μg/ml) on MV4-11 was (15.27±2.18) %, (19.85±3.23) % of bortezomib (8 nmol/L) , (81.67±5.96) % of combination of the two drugs, significantly higher than the single group (P<0.05) . When combination of the two drugs on MV4-11 after 24 hours, the levels of pro-apoptotic protein Bim and the cleaved activation of Caspase-3 and autophagy-related protein LC3B were up-regulated and the anti-apoptotic protein Bcl-2 expressions was down-regulated. Conclusion: Combination of artesunate with bortezomib shows a significant synergistic effects on proliferation, apoptosis and autophagy of MV4-11 cell lines, which may be associated with Bcl-2 family proteins expression.
Subject(s)
Humans , Apoptosis , Artesunate , Autophagy , Bortezomib , Cell Line, Tumor , Cell Proliferation , Leukemia, Myeloid, AcuteABSTRACT
Objective@#To investigate the effects of artesunate combined with bortezomib on the proliferation, apoptosis and autophagy of human acute myeloid leukemia cell lines MV4-11, and its mechanisms.@*Methods@#MTT method was used to determine the anti-proliferation effect of different concentrations of artesunate, bortezomib and their combination on MV4-11 cells. The cell apoptosis were analyzed by flow cytometry. The expression of cleaved-Caspase-3, Bcl-2 family protein (Bcl-2, Mcl-1, Bim, Bax) and autophagy-related protein LC3B were assayed by Western blot.@*Results@#Artesunate displayed a proliferation inhibition effect on MV4-11 with dose- and time-dependent manner, the IC50 of artesunate on MV4-11 after 48 hours was 1.44 μg/ml. Bortezomib displayed a proliferation inhibition effect on MV4-11 with dose-dependent manner, the IC50 of bortezomib on MV4-11 after 48 hours was 8.97 nmol/L. The combination of artesunate (0.75, 1.0 μg/ml) and Bortezomib (6, 8 nmol/L) showed higher inhibition on MV4-11 than artesunate or bortezomib alone in the same concentration gradient after 48 hours (P<0.05) . The cooperation index of the two drugs were all less than 1. The 48 h apoptotic rate of artesunate (1.5 μg/ml) on MV4-11 was (15.27±2.18) %, (19.85±3.23) % of bortezomib (8 nmol/L) , (81.67±5.96) % of combination of the two drugs, significantly higher than the single group (P<0.05) . When combination of the two drugs on MV4-11 after 24 hours, the levels of pro-apoptotic protein Bim and the cleaved activation of Caspase-3 and autophagy-related protein LC3B were up-regulated and the anti-apoptotic protein Bcl-2 expressions was down-regulated.@*Conclusion@#Combination of artesunate with bortezomib shows a significant synergistic effects on proliferation, apoptosis and autophagy of MV4-11 cell lines, which may be associated with Bcl-2 family proteins expression.
ABSTRACT
Objective To investigate the effects of gemcitabine and ABT-199 on proliferation inhibition and apoptosis induction of Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL) cell line SUP-B15, and to explore its synergistic mechanism. Methods SUP-B15 cells in logarithmic growth phase were treated with gemcitabine (0.025 and 0.050 μmol/L), ABT-199 (0, 0.5, 1.0, 2.0, 4.0, 8.0 μmol/L) or two drugs for 24 h. Cell proliferation was detected by CCK-8 method, apoptosis was detected by flow cytometry (FCM), mitochondrial membrane potential was detected by JC-1 method, and expression of mitochondrial apoptosis pathway-related protein was analyzed by Western blot. Results The 50 ％ inhibitory concentration (IC50) of SBT-B15 cells treated with ABT-199 for 24 h was (4.13±0.89) μmol/L. However, gemcitabine (0.025, 0.050 μmol/L) significantly enhanced the inhibitory effect of ABT-199 on proliferation of SUP-B15 cells, the IC50 values were (2.23 ±0.73) and (1.15 ±0.45) μmol/L, respectively. The results of FCM assay showed that compared with the monotherapy group [(7.33±1.54)％], 0.025 umol/L gemcitabine combined with ABT-199 (1.0 and 2.0 μmol/L) acted on SUP-B15 cells for 24 h, the proportions of apoptotic cells were (32.42±1.45) ％and (44.33±1.86) ％, the difference was statistically significant (F=70.78, P<0.001);compared with the monotherapy group [(9.60 ±2.76) ％], 0.05 μmol/L gemcitabine combined with ABT-199 (1.0 and 2.0 μmol/L) acted on SUP-B15 cells for 24 h, the proportion of apoptotic cells increased to (47.63 ± 3.81) ％ and (58.73 ±4.33) ％, respectively, and the difference was statistically significant (F= 79.21, P<0.001). The JC-1 experiment showed that treated with ABT-199 and gemcitabine for 12 h, the percentage of depolarizing cell was significantly higher than that in single agent group, and the difference was statistical significant (P<0.001). Western blot showed that the anti-apoptotic proteins bcl-2, bcl-xL and Mcl-1 decreased after treated by gemcitabine combined with ABT-199 for 12 h. Conclusion Gemcitabine could enhance the proliferation inhibition and induce apoptosis of Ph+ALL cells by ABT-199, and its mechanism may be related to down-regulation of anti-apoptosis-related proteins.
ABSTRACT
Objective To explore the apoptotic effect of polyporusterone A on estrogen receptor (ER)-positive and ER-negative human breast cancer cells and the possible mechanism. Methods Three human breast cancer cell lines (MDA-MB-453, MCF-7, and BT474) were chosen in this study. MTT assay was performed to measure the relative cell viabilities. Flow cytometry was used to analyze the apoptosis and cell cycle. Western blot analysis was used to determine the expression levels of Bcl-2 family proteins. Results Cell proliferation of ER-negative human breast cancer cells was significantly inhibited by polyporusterone A in a dose-and time-dependent manner, while no significant effect was observed on the proliferation of strogen receptor (ER)-positive human breast cancer MCF-7 and BT474 cells. Moreover, polyporusterone A could reversibly arrest the MDA-MB-253 cells in G1 or G2/M phase. Flow cytometry results showed that 50 μmol/L polyporusterone A induced MDA-MB-253 cells apoptosis after treatment for 24 h, while no apoptosis occurred in MCF-7 and BT474 cell lines. Western blot results showed that 50 μmol/L polyporusterone A up-regulated the protein expression of Bad and Bax, but down-regulated the expression of Bcl-2 and Bcl-w protein. Conclusion Polyporusterone A can inhibit the proliferation of ER-negative breast cancer cells and promote apoptosis, which may be associated with the regulation of the expression of Bcl-2 family proteins.
ABSTRACT
Objective: To explore the inductive effect of galangin on HPV-positive human cervical cancer cells and the possible mechanism. Methods: Two HPV-positive human cervical cancer cell lines (SiHa cell and HeLa cell) and one HPV-negative human cervical cancer cell line (C-33-A cell) were given different concentration of galangin (20, 40, and 80 μmol/L) for 24, 48, and 72 h. Three human cervical cancer cell lines and relative cell viabilities were determined by the MTT method. Apoptosis and cell cycle were analyzed by flow cytometry. Western blotting analysis was used to determine the protein expression levels of Bcl-2 family proteins. Results: Cell proliferation of two HPV-positive human cervical cancer cells was significantly inhibited by galangin in a dose- and time-dependent manner, and galangin had no effect on cell proliferation of HPV-negative human cervical cancer cells. Cell cycle detection results showed that galangin could reversibly arrest the two HPV-positive cell lines, either in G1 or in G2/M phases. Flow cytometry results showed that beyond certain galangin concentration or/and over 24 h exposure, the cells underwent apoptosis. The data of Western blotting showed that 40 μmol/L galangin up-regulated the expression levels of Bad, Bid, and Bax, but down-regulated Bcl-2 and Bcl-w. Conclusion: Galangin can inhibit the proliferation of HPV-positive cervical cancer cells and promote apoptosis, which may be associated with the regulation of Bcl-2 family proteins expression.
ABSTRACT
[Objective] To investigate the effects of platycodin D(PD) on the proliferation and apoptosis of human stomach cancer SGC7901 and the related mechanism.[Methods] SGC7901 was cultured in virto and was treated with 5～20μm·L-1 concentrations of PD.Cell proliferation was examined by MTT assay.Cell apoptosis was detected by Annexin V FITC/PI double staining.The change of mitochondrial trans-membrane potential was measured by JC-1 staining.The potein expression of cleaved caspase-3,cleaved caspase-9,cleaved PARP,bcl-2,bax,p-ERK,ERK,p-JNK,JNK,p-p38 and p38 detected by Western blot.[Results] MTT results showed that PD inhibited the growth of SGC7901 cells in a dose-dependent manner at 24h and 48h.SGC7901 cells treated with PD for 24h showed significantly enhanced apoptosis and weakened mitochondrial membrane potential compared with the control cells.Western blot results showed that PD could up-regulate expression of cleaved PARP,cleaved caspase-3,cleaved caspase-9,bax,p-JNK,p-p38 protein,decreased bcl-2,p-ERK protein,the expression of ERK,JNK,p38 protein did not change significantly.[Conclusion] PD may inhibit the proliferation and induce the apoptosis of SGC7901 cells.These findings indicated that PD inhibited cell proliferation by inhibiting the ERK signaling.PD effect on bax and bcl-2 by activation of JNK and p38 signaling pathway resulted in the decrease of mitochondrial membrane potential and activation of caspase,which induced the apoptosis of cancer cells.
ABSTRACT
The present study evaluated the effects of Androctonus amoreuxi scorpion venom, Cerastes cerastes snake venom and their mixture on prostate cancer cells (PC3). An MTT assay was used to determine the anti-proliferative effect of the venoms, while quantitative real time PCR was used to evaluate the expression of apoptosis-related genes (Bax and Bcl-2). Furthermore, colorimetric assays were used to measure the levels of malondialdehyde (MDA) and antioxidant enzymes. Our results show that the venoms significantly reduced PC3 cell viability in a dose-dependent manner. On the other hand, these venoms significantly decreased Bcl-2 gene expression. Additionally, C. cerastes venom significantly reduced Bax gene expression, while A. amoreuxi venom and a mixture of A. amoreuxi & C. cerastes venoms did not alter Bax expression. Consequently, these venoms significantly increased the Bax/Bcl-2 ratio and the oxidative stress biomarker MDA. Furthermore, these venoms also increased the activity levels of the antioxidant enzymes, catalase, superoxide dismutase, glutathione peroxidase, glutathione reductase, and glutathione-S-transferase. Overall, the venoms have cytotoxic and anti-proliferative effects on PC3 cells.
Subject(s)
Humans , Apoptosis , Catalase , Cell Survival , Gene Expression , Genes, bcl-2 , Glutathione Peroxidase , Glutathione Reductase , Hand , Malondialdehyde , Oxidative Stress , Prostate , Prostatic Neoplasms , Real-Time Polymerase Chain Reaction , Scorpion Venoms , Scorpions , Snake Venoms , Snakes , Superoxide Dismutase , Venoms , Viper Venoms , ViperidaeABSTRACT
<p><b>OBJECTIVE</b>To investigate the effect of Xuezhikang (, XZK) on renal cell apoptosis in diabetic rats and the possible mechanism.</p><p><b>METHODS</b>Sixty-six rats were randomly divided into 3 groups: the normal, model and XZK groups. In each group, the rats were further randomly divided into 3-month and 6-month subgroups, respectively. Diabetes of rats was induced by a single intraperitoneal injection of 1% streptozocin at 60 mg/kg body weight. Rats in the XZK group received gastric perfusion of XZK (1200 mg/kg body weight) everyday for 3 or 6 months, while rats in the normal and model groups received equal volume of saline. Twenty-four hours' urine was collected for urinary albumin excretion (UAE) measurement. Periodic acid-Schiff (PAS) and Masson's trichrome staining were used for saccharides and collagen detection. Cell apoptosis of renal cortex was investigated by TdT-mediated dUTP nick end labeling (TUNEL) staining. Bax and Bcl-2 expressions were detected by immunohistochemistry and Western blot, respectively. Cytochrome C (Cyt C) and caspase-9 concentration were detected by Western blot.</p><p><b>RESULTS</b>Compared with the model group, XZK treatment could significantly decrease the kidney hypertrophy index, 24 h UAE, renal cell apoptosis, cytoplasmic Cyt C level and active caspase-9 level, as well as suppress the increment of Bax and up-regulate the expression of Bcl-2, leading to the suppression of Bax/Bcl-2 ratio at 3 and 6 months (P<0.05 or P<0.01). Moreover, XZK treatment could alleviate the deposition of PAS-stained saccharides and Masson's trichromestained collagen to different extent.</p><p><b>CONCLUSIONS</b>Renal cell apoptosis was observed in diabetic kidney, in which mitochondrial apoptotic pathway might be involved. XZK treatment could attenuate pathological changes in diabetic kidney and reduce renal cell apoptosis, probably via the suppression of Bax/Bcl-2 ratio, which lead to inhibition of Cyt C release and following caspase-9 activation.</p>
Subject(s)
Animals , Male , Albuminuria , Blood , Apoptosis , Blood Glucose , Metabolism , Caspase 9 , Metabolism , Cytochromes c , Metabolism , Diabetes Mellitus, Experimental , Blood , Drug Therapy , Metabolism , Pathology , Drugs, Chinese Herbal , Pharmacology , Therapeutic Uses , Hypertrophy , In Situ Nick-End Labeling , Kidney , Pathology , Kidney Glomerulus , Pathology , Lipids , Blood , Mesangial Cells , Pathology , Proto-Oncogene Proteins c-bcl-2 , Metabolism , Rats, Sprague-Dawley , Streptozocin , bcl-2-Associated X Protein , MetabolismABSTRACT
Mycobacterium tuberculosis (Mtb) causing tuberculosis as an intracellular pathogen initially infects alveolar macrophages following aerosol inhalation. Thus, macrophages play a critical role in the establishment of Mtb infection and macrophage cell death, a common outcome during Mtb infection, may initiate host- or pathogen-favored immune responses, resulting in facilitating protection or pathogenesis, respectively. In addition, virulent Mtb strains are known to inhibit apoptosis and consequently down-regulates immune response using a variety of strategies. In many recent studies have shown that virulent Mtb can either augment or reduce apoptosis by regulating expression of pro-apoptotic and anti-apoptotic proteins belonging to Bcl-2 family proteins. In this review, we will discuss and dissect the apoptotic pathways of Bcl-2 family proteins in Mtb-infected macrophages.
Subject(s)
Humans , Apoptosis , Apoptosis Regulatory Proteins , Cell Death , Inhalation , Macrophages , Macrophages, Alveolar , Mycobacterium tuberculosis , Mycobacterium , TuberculosisABSTRACT
Mitochondrial quality control is the important mecha-nism that regulates the morphology,quantity and quality of mito-chondrial in cell to maintain cellular homeostasis and thus,cell survival and health.It has been revealed that members of Bcl-2 family are linked to mitochondrial function and integrity.Bcl-2 family proteins are the key regulators of mitochondrial quality control,participating in the signaling pathways regulating the crosstalk between mitophagy and apoptosis,as well as mitochon-drial fission and fusion.This paper mainly reviews their impact on mitochondrial quality and the major signaling pathways regula-ted by Bcl-2 family proteins.
ABSTRACT
Objective:Guanxinshutong on cerebral ischemia reperfusion ( I/R) in rats learning and memory ability and brain Bcl-2,Bax protein expression to explore the protection mechanisms of Guanxinshutong on cerebral I /R injury in brain tissue.Methods:Healthy Wistar rats for the study were randomly divided into sham operation group ,I/R model group,high dose Guanxinshutong (1 g/kg) and low dose Guanxinshutong (0.5 g/kg) group,n=15.cerebral I/R model was estabhished by using the left commom cartid artery ligation method,modeling each group were fed daily before saline ,saline,Guanxinshutong (1 g/kg) and Guanxinshutong (0.5 g/kg), continuous 7 days;3 d after water maze training while continuing administration ,continuous 7 d,the eight day ,Morris water maze test;after the test,the rats were sacrificed blood and brain tissue ,serum by ELISA of Bcl-2 and Bax protein levels change;using HE staining of rat brain pathology;using immunohistochemistry assay in rat brain organization of Bcl-2 and Bax protein levels changes.Results:Compared with the sham group ,Guanxinshutong high-dose group test latency slightly longer timers after platform and platform quadrant dwell decreased slightly ,serum and brain tissue slightly elevated levels of Bax protein ,Bcl-2 protein levels decreased slightly but not significantly different ( P>0.05 ) ,low-dose group Guanxinshutong through latency test in rats ,after the number of platforms and platform quadrant dwell time decreased , serum and brain tissue elevated levels of Bax protein , Bcl-2 protein content decreased significantly different(P<0.01),cerebral I/R model group was significantly prolonged latency test ,after the number of platforms and platform quadrant dwell time significantly reduced serum and brain tissue was significantly increased Bax protein ,Bcl-2 protein content decreased with significant differences ( P<0.01 ).Conclusion: Guanxinshutong can significantly improve spatial memory , and by reducing Bax protein content and increased Bcl-2 protein,inhibition of cerebral I/R injury of apoptosis,and thus the brain tissue I/R has a protective effect.
ABSTRACT
Nitric oxide (NO) is recognized as a mediator and regulator of inflammatory responses. NO is produced by nitric oxide synthase (NOS), and NOS is abundantly expressed in the human dental pulp cells (HDPCs). NO produced by NOS can be cytotoxic at higher concentrations to HDPCs. However, the mechanism by which this cytotoxic pathway is activated in cells exposed to NO is not known. The purpose of this study was to elucidate the NO-induced cytotoxic mechanism in HDPCs. Sodium nitroprusside (SNP), a NO donor, reduced the viability of HDPCs in a dose- and time-dependent manner. We investigated the in vitro effects of nitric oxide on apoptosis of cultured HDPCs. Cells showed typical apoptotic morphology after exposure to SNP. Besides, the number of Annexin V positive cells was increased among the SNP-treated HDPCs. SNP enhanced the production of reactive oxygen species (ROS), and N-acetylcysteine (NAC) ameliorated the decrement of cell viability induced by SNP. However, a soluble guanylate cyclase inhibitor (ODQ) did not inhibited the decrement of cell viability induced by SNP. SNP increased cytochrome c release from the mitochondria to the cytosol and the ratio of Bax/Bcl-2 expression levels. Moreover, SNP-treated HDPCs elevated activities of caspase-3 and caspase-9. While pretreatment with inhibitors of caspase (z-VAD-fmk, z-DEVD-fmk) reversed the NO-induced apoptosis of HDPCs. From these results, it can be suggested that NO induces apoptosis of HDPCs through the mitochondria-dependent pathway mediated by ROS and Bcl-2 family, but not by the cyclic GMP pathway.
Subject(s)
Humans , Acetylcysteine , Annexin A5 , Apoptosis , Caspase 3 , Caspase 9 , Cell Survival , Cyclic GMP , Cytochromes c , Cytosol , Dental Pulp , Guanylate Cyclase , Mitochondria , Nitric Oxide , Nitric Oxide Synthase , Nitroprusside , Reactive Oxygen Species , Tissue DonorsABSTRACT
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 microM 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.
Subject(s)
Animals , Humans , Rats , Apoptosis , Blotting, Western , Bromodeoxyuridine , Caspase 9 , Caspases , Cell Cycle Checkpoints , Cell Survival , Cyclin D1 , DNA , DNA Fragmentation , Down-Regulation , Hand , Inhibitory Concentration 50 , Neuroblastoma , Proteins , StilbenesABSTRACT
Objective: To investigate the role of apoptosis in triptolide-induced acute nephrotoxicity and the possible mechanisms in vivo. Methods Thirty male SD rats were randomly divided into control and two testing groups. The rats of two testing groups were ip injected with triptolide solution at doses of 1 and 2 mg/kg of body weight, respectively, and the rats of the control group were ip injected with 0.9% physiological saline instead. Testing rats were killed 48 h after the injection; Blood samples were collected and both kidneys were removed. The BUN and Scr concentrations in plasma were measured, and renal histology was examined by HE staining. TUNEL staining was performed to evaluate apoptosis of renal tissue. Renal expression of apoptosis related proteins Bcl-2, Bax, Bid, Bad, Fas, and FasL proteins, as well as corresponding regulating genes were assessed by immuno-histochemical staining and real-time PCR. Results: After injection, a large number of apoptotic tubular cells appeared corresponding to the increases of BUN and Scr concentrations. Furthermore, increased expression of Bax, Bid, Bad, Fas, and FasL was detected at both protein and mRNA levels, but the expression of Bcl-2 did not differ among the three groups. Conclusion: These results suggest that apoptosis of tubular cells plays a key role in the pathogenesis of triptolide-induced acute nephrotoxicity, and Bcl-2 family, as well as Fas and FasL, is involved in this process.
ABSTRACT
Apoptosis deregulation might have a role in the pathophysiology of polycythemia vera (PV). This study evaluated Bcl-2 molecule expression in CD34+ cells and leukocytes in 12 PV patients. Gene expression was investigated by real time PCR using SybrGreen Quantitect kit and protein expression was evaluated by western-blotting. JAK2 V617F mutation was detected according to Baxter et al (2005). CD34+ cells from PV patients presented higher levels of A1 and Mcl-1 expression (median: 22.6 and 5.2, respectively) in comparison with controls (0.9 and 0.5, p=0.004 and p=0.020); while Bcl-2 and Bcl-xL expression decreased in PV patients (0.18 and 1.19) compared with controls (1.39 and 2.01, p=0.006 and p=0.020). CD34+ cells in PV patients showed an elevated Bid expression (14.4) in comparison with healthy subjects (1.0; p=0.002). Patients' leukocytes showed an A1 augmentation (7.41, p=0.001) and a reduced expression of Bax (0.19; p=0.040) and Bad (0.2; p=0.030). There was no correlation between JAK2 V617F allele burden and molecular expression. PV patients showed alterations in Bcl-2 members' expression, which may interfere with control of apoptotic machinery and contribute to disease pathogenesis.
A desregulação da apoptose parece participar da fisiopatologia da policitemia vera (PV). Este estudo avaliou a expressão das moléculas da família Bcl-2 em células hematopoéticas CD34 + e leucócitos de 12 pacientes com PV. Foram realizados: a quantificação da expressão gênica por PCR em tempo real utilizando kit Sybrgreen Quantitect, avaliação da expressão de proteínas por western-blot e detecção da mutação JAK2 V617F segundo Baxter et al. (2005). Células CD34 + dos pacientes com PV apresentaram maior expressão de A1 e Mcl-1 (mediana: 22,6 e 5,2, respectivamente) em comparação com controles (0,9 e 0,5, p = 0,004 e p = 0,020) e expressão de Bcl-2 e Bcl-xL diminuída nestes pacientes (0,18 e 1,19) em relação aos controles (1,39 e 2,01, p = 0,006 e p = 0,020). Células CD34 + dos pacientes com PV mostraram expressão elevada de bid (14,4) em comparação aos controles (1,0; p = 0,002). Leucócitos dos pacientes mostraram aumento de A1 (7,41, p = 0,001) e expressão reduzida do Bax (0,19; p = 0,04) e Bad (0,2; p = 0,030). Não houve correlação entre percentagem de alelos JAK2 V617F mutados e expressão molecular. Pacientes com PV apresentaram alterações na expressão de moléculas Bcl-2 que podem interferir no controle da apoptose e contribuir para a patogênese da doença.
Subject(s)
Humans , Polycythemia Vera/classification , Apoptosis/physiology , Genes, bcl-2 , MutationABSTRACT
OBJECTIVE: Promoter methylation of Bcl-2 family genes in cancer cells were studied to verify possible correlation between DNA methylation pattern of Bcl-2 family members and cancer. METHODS: The genomic DNAs were extracted from different cancer cell lines, HeLa, CaSki and K562, and ovarian cancer tissue from patients. The cytosine residues were converted to uracil by sodium bisulfite treatment. MSP (methylation specific PCR) was performed to determine the methylation status of Bcl-2, Mcl-1, Noxa, and Harakiri promoters. Using primers that distinguish methylated DNA from unmethylated DNA after bisulfite modification of DNA, MSP was conducted to observe the methylation pattern of Bcl-2 family genes in different cancer cells. RESULTS: The promoter regions of Bcl-2 family genes including Mcl-1, Bcl-2, and Noxa were not methylated in cancer cells, whereas the proapoptotic Bcl-2 family gene Harakiri was detected as methylated in the cancer cell lines and hypomethylated in the ovarian cancer tissue. CONCLUSION: The present study demonstrated the differential methylation profiles of Bcl-2 family genes in cancerous cells, which suggests a possible connection between the methylation pattern of some of Bcl-2 family genes and ovarian cancer.