1.The influence of microtubule intervention drugs on the energy metabolism of myocardial cells after hypoxia.
Miao TENG ; Yue-Sheng HUANG ; Ji ZHENG ; Yong-Ming DANG ; Qiong ZHANG
Chinese Journal of Burns 2007;23(3):164-167
OBJECTIVETo investigate the influence of microtubule intervention drugs on the energy metabolism of myocardial cells after hypoxia.
METHODSThe primary passage of cultured myocardial cells from neonatal rats were divided into A (with hypoxia), B (with hypoxia and administration of 10 micromol/ml colchicine), C (with hypoxia and administration of 5 micromol/ml taxol), D (with hypoxia and administration of 10 micromol/ml taxol) and E (with hypoxia and administration of 15 micromol/ml taxol) groups. The creatine kinase (CK) activity and contents of ATP and ADP were assayed with colorimetry and HPLC, respectively, and the vitality of myocardial cells were determined by trypan blue method at 0.5, 1.0, 3.0, 6.0, 12.0, 24.0 post-hypoxia hours (PHH).
RESULTSThe mortality was obviously higher in B and E groups than those in A group( P < 0.05) at each time-points, but that in C and D groups were markedly lower than those in A group during 6.0 to 24.0 PHH (P < 0.01). The CK activity was significantly higher in B group than that in A group during 1.0 to 24.0 PHH, while that in E group was evidently higher, but it was lower in C and D groups than that in A group at each time-points (P < 0.05 or 0.01). The ATP contents in C group during 0.5 to 6.0 PHH were [(49.9 +/- 2.8), (40.7 +/- 2.0), (25.8 +/- 1.9), (19.1 +/- 1.2) microg/10(6) cells, respectively], which were obviously higher than those in A group [(42.9 +/- 5.8), (29.5 +/- 1.8), (18.2 +/- 0.9), (14.1 +/- 0.7) microg/10(6) cells, respectively, P < 0.05 or P < 0.01, and those in E group at each time-point were significantly lower than those in A and D groups (P < 0.01). The changes in the contents of ADP were on the contrary to the above.
CONCLUSIONMicrotubule-destabilizing drugs and high concentration microtubule-stabilizing drugs can sharply decrease ATP content in myocardiocytes under hypoxic conditions, while suitable amount of microtubule-stabilizing drugs can protect myocardiocytes by promoting its energy production.
Animals ; Cell Hypoxia ; Cells, Cultured ; Colchicine ; pharmacology ; Energy Metabolism ; drug effects ; Microtubules ; drug effects ; metabolism ; Myocytes, Cardiac ; drug effects ; metabolism ; Paclitaxel ; pharmacology ; Rats ; Rats, Sprague-Dawley
2.Dynamic long-term microstructural and ultrastructural alterations in sensory nerves of rats of paclitaxel-induced neuropathic pain.
Yuan WU ; Jun LI ; Junfei ZHOU ; Yi FENG
Chinese Medical Journal 2014;127(16):2945-2952
BACKGROUNDPaclitaxel, as a first line anti-neoplastic compound, frequently produces long-term pain after tumors have been treated. Clinical manifestations are varied and non-specific. Pathology of the nervous system during the development of the neuropathic pain is unclear. Thus, early diagnosis and treatment is often unsatisfying for patients. This study aimed to promote considerate understanding of the structural alteration of sensory nerves.
METHODSAll rats were simply randomized into 3 groups: paclitaxel group, vehicle group and saline group. An established rat model of paclitaxel-induced peripheral neuropathy (2 mg/kg) was chosen for our research, behavior tests were operated during the procedure of 56 days. All rats were sampled on days 0, 3, 7, 28 and 56. The hind paw plantar skin, sciatic nerves, dorsal root ganglion and attached fibers, and lumbar spinal cord were processed for light and electron microscopy. The differences among 3 groups were analyzed with one-way analysis of variance (ANOVA).
RESULTSWe affirmed that paclitaxel-induced mechano-allodynia and mechano-hyperalgesia occured after a 3-7-day delay, and this pain peaked at day 28 and persisted to day 56. Paclitaxel and vehicle treatment both evoked thermal-hyperalgesia. Paclitaxel-induced axonal and myelin sheath degeneration was evident. At days 3 and 7, significant increases in atypical mitochondria in both myelinated axons and C-fibers of paclitaxel-treated nerves indicated that injured mitochondria correlated to specific paclitaxel-induced neuropathic pain, and the abnormity sustained till day 56. Microtubule was unaffected in myelinated axons or C-fibers in paclitaxel- or vehicle-treated rats. Significant increase of G ratio was evident with paclitaxel injection at days 7 and 28.
CONCLUSIONOur research suggests a causal role for axonal degeneration, abnormalities in axonal mitochondria, and structural modification of axonal microtubules in paclitaxel-induced neuropathic pain, and the abnormal mitochondria could be connected to the chronic neuropathic pain.
Animals ; Antineoplastic Agents, Phytogenic ; adverse effects ; Axons ; drug effects ; metabolism ; Male ; Microtubules ; drug effects ; metabolism ; Mitochondria ; drug effects ; metabolism ; Neuralgia ; chemically induced ; Paclitaxel ; adverse effects ; Random Allocation ; Rats ; Rats, Sprague-Dawley
3.The influence of microtubule intervention drugs on glycolytic key enzymes in myocardial cells after hypoxia.
Miao TENG ; Yue-Sheng HUANG ; Yong-Ming DANG ; Ya-Dong FANG ; Qiong ZHANG
Chinese Journal of Burns 2008;24(2):102-106
OBJECTIVETo investigate the influence of microtubule intervention drugs on glycolytic key enzymes in myocardial cells after hypoxia.
METHODSThe primary passage of cultured myocardial cells from neonatal rats were divided into A group (with hypoxia), B group (with hypoxia and administration of l0 micromol/L colchicine), C group (with hypoxia and administration of 5 micromol/L taxol), D group (with hypoxia and administration of 10 micromol/L taxol), E group (with hypoxia and administration of 15 micromol/L taxol). The morphology of microtubule was observed with laser scanning microscope (LSM). The cell vitality was assayed by cell counting kit (CCK). The activities of hexokinase (HK), pyruvate kinase (PK), phosphofructokinase (PFK) and lactate dehydrogenase (LDH) were assayed with colorimetry.
RESULTSIn group B and E, the microtubule structure was damaged heavily, and the cell vitality was decreased significantly [The cell vitality was (89.99 +/- 3.47)% in B group and (84.56 +/- 6.61)% in E group, respectively, at 1.0 post hypoxia hour (PHH), and hoth values were obviously lower than that in A group (97.44 +/- 1.76)%, P < 0.01]. The HK, PK and PFK activities decreased obviously. The activities of HK, PK and PFK in group C were similar to those of the A group. Compared with that in other groups, the degree of damage of microtubule structure in D group was milden. The activities of HK, PK and PFK in D group during 0.5 - 6.0 PHH were significantly higher than those in A group. The activity of LDH in each group was increased after hypoxia.
CONCLUSIONProper concentration of microtubule-stabilizing drugs can alleviate the damages to microtubule structure, and enhance the activity of glycolytic key enzymes of myocardial cells at early stage of hypoxia.
Animals ; Cell Hypoxia ; Cells, Cultured ; Glycolysis ; drug effects ; Hexokinase ; metabolism ; L-Lactate Dehydrogenase ; metabolism ; Microtubules ; drug effects ; metabolism ; Myocytes, Cardiac ; enzymology ; metabolism ; Phosphofructokinase-1 ; metabolism ; Pyruvate Kinase ; metabolism ; Rats ; Rats, Sprague-Dawley
4.Biological characteristics of microtubule and related drug research.
Jian-nong LI ; Jian-dong JIANG
Acta Pharmaceutica Sinica 2003;38(4):311-315
Amino Acids
;
isolation & purification
;
Animals
;
Antineoplastic Agents, Phytogenic
;
pharmacology
;
Binding Sites
;
Colchicine
;
pharmacology
;
Humans
;
Microtubules
;
drug effects
;
physiology
;
Paclitaxel
;
pharmacology
;
Tubulin
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chemistry
;
isolation & purification
;
metabolism
;
Vinblastine
;
pharmacology
5.Progress in the study of tubulin inhibitors.
Hai SHANG ; Li PAN ; Shu YANG ; Hong CHEN ; Mao-sheng CHENG
Acta Pharmaceutica Sinica 2010;45(9):1078-1088
Microtubule is one of the key components of the cytoskeleton and plays an important role in the maintenance of cell shape and the process of signal transduction and mitosis. Due to the extreme importance of microtubule in the process of mitosis, tubulin becomes one of the most important targets for development of new anticancer drugs and tubulin inhibitors are used for the treatment of cancer nowadays. These inhibitors have antitumor activity by inhibiting or promoting the assembly of tubulin to microtubules and interfering the process of cell mitosis. This review summarized the research progress of the tubulin inhibitors, especially the introduction of the tubulin inhibitors of pharmacological activities and the progress of clinical research. Also, the development trend of these inhibitors is discussed.
Antineoplastic Agents
;
chemical synthesis
;
chemistry
;
pharmacology
;
Humans
;
Microtubules
;
drug effects
;
metabolism
;
Mitosis
;
drug effects
;
Molecular Structure
;
Neoplasms
;
drug therapy
;
Stilbenes
;
chemical synthesis
;
chemistry
;
pharmacology
;
Structure-Activity Relationship
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Tubulin
;
metabolism
;
Tubulin Modulators
;
chemical synthesis
;
chemistry
;
pharmacology
6.The colchicine derivative CT20126 shows a novel microtubule-modulating activity with apoptosis.
Sung Kuk KIM ; Sang Min CHO ; Ho KIM ; Heon SEOK ; Soon Ok KIM ; Taeg Kyu KWON ; Jong Soo CHANG
Experimental & Molecular Medicine 2013;45(4):e19-
New colchicine analogs have been synthesized with the aim of developing stronger potential anticancer activities. Among the analogs, CT20126 has been previously reported to show immunosuppressive activities. Here, we report that CT20126 also shows potential anticancer effects via an unusual mechanism: the modulation of microtubule integrity and cell cycle arrest at the G2/M phase before apoptosis. When we treated COS-7 cells with CT20126 (5 muM), the normal thread-like microtubules were disrupted into tubulin dimers within 10 min and thereafter repolymerized into short, thick filaments. In contrast, cells treated with the same concentration of colchicine exhibited microtubule depolymerization after 20 min and never underwent repolymerization. Furthermore, optical density (OD) analysis (350 nm) with purified tubulin showed that CT20126 had a higher repolymerizing activity than that of Taxol, a potent microtubule-polymerizing agent. These results suggest that the effects of CT20126 on microtubule integrity differ from those of colchicine: the analog first destabilizes microtubules and then stabilizes the disrupted tubulins into short, thick polymers. Furthermore, CT20126 induced a greater level of apoptotic activity in Jurkat T cells than colchicine (assessed by G2/M arrest, caspase-3 activation and cell sorting). At 20 nM, CT20126 induced 47% apoptosis among Jurkat T cells, whereas colchicine induced only 33% apoptosis. Our results suggest that the colchicine analog CT20126 can potently induce apoptosis by disrupting microtubule integrity in a manner that differs from that of colchicine or Taxol.
Acetylation/drug effects
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Animals
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Apoptosis/*drug effects
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COS Cells
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Caspase 3/metabolism
;
Cattle
;
Cell Division/drug effects
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Cercopithecus aethiops
;
Colchicine/*analogs & derivatives/chemistry/pharmacology
;
Enzyme Activation/drug effects
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G2 Phase/drug effects
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Humans
;
Jurkat Cells
;
Microtubules/*metabolism
;
Poly(ADP-ribose) Polymerases/metabolism
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Tubulin/metabolism
;
Tubulin Modulators/chemistry/*pharmacology
7.Inhibition of cell proliferation by a resveratrol analog in human pancreatic and breast cancer cells.
Young Bin HONG ; Hyo Jin KANG ; Hee Jeong KIM ; Eliot M ROSEN ; Sivanesan DAKSHANAMURTHY ; Riccardo RONDANIN ; Riccardo BARUCHELLO ; Giuseppina GRISOLIA ; Simoni DANIELE ; Insoo BAE
Experimental & Molecular Medicine 2009;41(3):151-160
Resveratrol has been reported to possess cancer preventive properties. In this study, we analyzed anti-tumor activity of a newly synthesized resveratrol analog, cis-3,4',5-trimethoxy-3'-hydroxystilbene (hereafter called 11b) towards breast and pancreatic cancer cell lines. 11b treatments reduced the proliferation of human pancreatic and breast cancer cells, arrested cells in the G2/M phase, and increased the percentage of cells in the subG1/G0 fraction. The 11b treatments also increased the total levels of mitotic checkpoint proteins such as BubR1, Aurora B, Cyclin B, and phosphorylated histone H3. Mechanistically, 11b blocks microtubule polymerization in vitro and it disturbed microtubule networks in both pancreatic and breast cancer cell lines. Computational modeling of the 11b-tubulin interaction indicates that the dimethoxyphenyl group of 11b can bind to the colchicine binding site of tubulin. Our studies show that the 11b treatment effects occur at lower concentrations than similar effects associated with resveratrol treatments and that microtubules may be the primary target for the observed effects of 11b. These studies suggest that 11b should be further examined as a potentially potent clinical chemotherapeutic agent for treating pancreatic and breast cancer patients.
Antineoplastic Agents/*pharmacology
;
Binding Sites
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Breast Neoplasms
;
Cell Cycle/drug effects
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Cell Line, Tumor
;
Cell Proliferation/*drug effects
;
Colchicine/chemistry/pharmacology
;
Cyclin B/metabolism
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G2 Phase/drug effects
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Humans
;
Microtubules/drug effects/metabolism
;
Models, Molecular
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Pancreatic Neoplasms
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Protein-Serine-Threonine Kinases/metabolism
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Stilbenes/*pharmacology
;
Tubulin/metabolism
8.Overexpression of synuclein-gamma confers resistance to antimicrotubule drugs against human hepatoma cells.
Shi-Xiang CHENG ; Sai ZHANG ; Hao ZHANG ; Dan-Qing SONG ; Yu-Ping WANG ; Yu-Huan LI ; Xue-Fu YOU ; Yue-Ming WANG ; Jian-Dong JIANG
Acta Pharmaceutica Sinica 2010;45(6):724-729
Liver cancer is one of the most common neoplastic diseases with high mortality in China. Currently, antimicrotubule drugs such as paclitaxel (PTX) and vincristine (VCR), are used as the common agents in the clinical chemotherapy for liver cancer. However, the responses of patients to these drugs vary markedly. Successful identification of intracellular factors influencing liver cancer's sensitivity to antimicrotubule drugs would be of great clinical importance. In this study, by engineering human hepatoma cell HepG2 to overexpress synuclein-gamma (SNCG), we investigated if SNCG is a molecular factor associated with the sensitivity to antimicrotubule drug treatment. Real-time RT-PCR and Western blotting assays showed SNCG was successfully overexpressed in HepG2/ SNCG cells compared with HepG2/Neo cells. The overexpressed SNCG altered the proliferation activity in HepG2 cells, which was 66% higher than that of HepG2/Neo cells through MTT method. The overexpressed SNCG also reduced sensitivity of HepG2 cells to antimicrotubule drugs: after PTX or VCR treatment, the proportion of HepG2/SNCG cells in G2/M arrest was significantly lower than that in HepG2/Neo cells. Correspondingly, HepG2/SNCG cells showed significantly lower mitotic index than HepG2/Neo cells. Meanwhile, HepG2/SNCG cells showed higher resistance to PTX and VCR than HepG2/Neo cells, with resistance index 21 and 15 respectively. Our studies suggested that the overexpression of SNCG could confer resistance to antimicrotubule drugs in hepatoma cells; and it indicated that SNCG may be as a potential response marker for antimicrotubule drugs in liver cancer chemotherapy.
Antineoplastic Agents, Phytogenic
;
pharmacology
;
Cell Cycle
;
Cell Proliferation
;
Drug Resistance, Neoplasm
;
Gene Expression Regulation, Neoplastic
;
Genetic Vectors
;
Hep G2 Cells
;
drug effects
;
metabolism
;
Humans
;
Microtubules
;
drug effects
;
Mitosis
;
drug effects
;
Mitotic Index
;
Paclitaxel
;
pharmacology
;
Plasmids
;
RNA, Messenger
;
metabolism
;
Transfection
;
Vincristine
;
pharmacology
;
gamma-Synuclein
;
biosynthesis
;
genetics
;
physiology
9.End-binding protein 1 stimulates paclitaxel sensitivity in breast cancer by promoting its actions toward microtubule assembly and stability.
Youguang LUO ; Dengwen LI ; Jie RAN ; Bing YAN ; Jie CHEN ; Xin DONG ; Zhu LIU ; Ruming LIU ; Jun ZHOU ; Min LIU
Protein & Cell 2014;5(6):469-479
Paclitaxel is a microtubule-targeting agent widely used for the treatment of many solid tumors. However, patients show variable sensitivity to this drug, and effective diagnostic tests predicting drug sensitivity remain to be investigated. Herein, we show that the expression of end-binding protein 1 (EB1), a regulator of microtubule dynamics involved in multiple cellular activities, in breast tumor tissues correlates with the pathological response of tumors to paclitaxel-based chemotherapy. In vitro cell proliferation assays reveal that EB1 stimulates paclitaxel sensitivity in breast cancer cell lines. Our data further demonstrate that EB1 increases the activity of paclitaxel to cause mitotic arrest and apoptosis in cancer cells. In addition, microtubule binding affinity analysis and polymerization/depolymerization assays show that EB1 enhances paclitaxel binding to microtubules and stimulates the ability of paclitaxel to promote microtubule assembly and stabilization. These findings thus reveal EB1 as a critical regulator of paclitaxel sensitivity and have important implications in breast cancer chemotherapy.
Antineoplastic Agents, Phytogenic
;
pharmacology
;
therapeutic use
;
Apoptosis
;
drug effects
;
Breast Neoplasms
;
drug therapy
;
metabolism
;
pathology
;
Cell Cycle Checkpoints
;
drug effects
;
Cell Line, Tumor
;
Female
;
Humans
;
MCF-7 Cells
;
Microtubule-Associated Proteins
;
antagonists & inhibitors
;
genetics
;
metabolism
;
Microtubules
;
chemistry
;
metabolism
;
Paclitaxel
;
pharmacology
;
therapeutic use
;
RNA Interference
;
RNA, Small Interfering
;
metabolism
10.Pleckstrin homology domain of phospholipase C-gamma1 directly binds to 68-kDa neurofilament light chain.
Sung Kuk KIM ; Jang Hyun CHOI ; Pann Ghill SUH ; Jong Soo CHANG
Experimental & Molecular Medicine 2006;38(3):265-272
Phosphoinositide-specific phospholipase C-gamma1 (PLC-gamma1) has two pleckstrin homology (PH) domains: an amino-terminal domain (PH1) and a split PH domain (PH2). Here, we show that overlay assay of bovine brain tubulin pool with glutathione-S-transferase (GST)-PLC-gamma1 PH domain fusion proteins, followed by matrix-assisted laser-desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), identified 68-kDa neurofilament light chain (NF-L) as a binding protein of amino-terminal PH domain of PLC-gamma1. NF-L is known as a component of neuronal intermediate filaments, which are responsible for supporting the structure of myelinated axons in neuron. PLC-gamma1 and NF-L colocalized in the neurite in PC12 cells upon nerve growth factor stimulation. In vitro binding assay and immunoprecipitation analysis also showed a specific interaction of both proteins in differentiated PC12 cells. The phosphatidylinositol 4, 5-bisphosphate [PI(4,5)P2] hydrolyzing activity of PLC-gamma1 was slightly decreased in the presence of purified NF-L in vitro, suggesting that NF-L inhibits PLC-gamma1. Our results suggest that PLC-gamma1-associated NF-L sequesters the phospholipid from the PH domain of PLC-gamma1.
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
;
Rats
;
Protein Interaction Mapping
;
Protein Biosynthesis/drug effects
;
Protein Binding/drug effects
;
Phosphoproteins/chemistry/*metabolism
;
Phospholipase C gamma/antagonists & inhibitors/chemistry/*metabolism
;
Phosphatidylinositol 4,5-Diphosphate/metabolism
;
Peptides/chemistry/metabolism
;
PC12 Cells
;
Neurofilament Proteins/chemistry/*metabolism
;
Nerve Growth Factor/pharmacology
;
Molecular Weight
;
Molecular Sequence Data
;
Microtubules/metabolism
;
Microscopy, Fluorescence
;
Isoenzymes/metabolism/pharmacology/physiology
;
Glutathione Transferase/metabolism
;
Blotting, Far-Western
;
Blood Proteins/chemistry/*metabolism
;
Binding Sites
;
Animals
;
Amino Acid Sequence