• 생명과학회지
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• 제25권10호
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• pp.1184-1195
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• 2015

새로운 실험 동물로 대두되고 있는 제브라피쉬는 척추동물 생식생물학 연구에서도 중요한 역할을 한다. 제브라피쉬의 난자 성숙은 maturation inducing hormone (MIH, 17α,20β-Dihydroxy-4-pregnen-3-one)에 의해 촉발된다. 대부분의 동물의 난자성숙에는 cdc2 kinase와 cyclinB 단백질 복합체인 MPF의 활성화가 필요하다. 발톱개구리와 생쥐에서는 MPF 활성이 두 가지 기작에 의해 조절되는데, 하나는 cyclinB 결합이고 또 다른 하나는 Wee1과 Cdc25에 의한 T14/Y15 잔기의 억제성인산화와 탈인산화이다. 발톱개구리나 생쥐와 달리 제브라피쉬를 포함한 대부분의 진골어류(teleost)는 GV 난자에 pre-MPF complex가 존재하지 않으므로 MPF 활성화는 전적으로 cyclinB 단백질의 de novo synthesis에 의존한다. 다른 종과 마찬가지로 제브라피쉬의 모계유래 mRNA도 CPEB, Dazl, Pum1/Pum2, insulin-like growth factor2 mRNA-binding protein 3 등 다양한 RNA binding protein (RBP)의 결합에 의해 번역이 조절된다. 그러나 제브라피쉬 난자에서 단백질 번역 조절에 관여하는 자세한 작용 기작은 확실하게 규명되지 않았다. 그러므로 제브라피쉬 난자의 성숙과정을 연구하는 것은 척추동물 난자 초기 성숙과정에서 단백질 번역 조절의 역할을 규명할 수 있는 새로운 정보를 제공할 것이다.

• The Journal of Korean Physical Therapy
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• 제19권4호
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• pp.1-14
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• 2007

Background: It is generally accepted that skeletal muscle contraction is triggered by nerve impulse and intracellular $Ca^{2+}\;([Ca^{2+}]_i)$ released from intracellular $Ca^{2+}$ stores such as sarcoplasmic reticulum (SR). Specifically, this process, called excitation-contraction (E-C) coupling, takes place at intracellular junctions between the plasma membrane, the transverse (T) tubule L-type $Ca^{2+}$ channel (dihydropyridine-sensitive L-rype $Ca^{2+}$ channel, DHPR, also called tetrads), and the SR $Ca^{2+}$ release channel (ryanodine-sensitive $Ca^{2+}$ release channel, RyR, also called feet) of internal $Ca^{2+}$ stores in skeletal muscle cells. Furthermore, it has been reported that the $Ca^{2+-}$ dependent and -independent contraction determine the expression of skeletal muscle genes, thus providing a mechanism for tightly coupling the extent of muscle contraction to regulation of muscle plasticity-related excitation-transcription (E-T) coupling. Purpose: Expression and activity of plasticity-associated enzymes in gastrocnemius muscle strips have not been well studied, however. Methods: Therefore, in this study the expression and phosphorylation of E-C and E-T coupling-related mediators such as protein kinases, ROS(reactive oxygen species)- and apoptosis-related substances, and others in gastrocnemius muscles from rats was examined. Results: I found that expression and activity of MAPKs (mitogen-activated protein kinases, ERK1/2, p38MAPK, and SAPK/JNK), apoptotic proteins (cleaved caspase-3, cytochrome c, Ref-1, Bad), small GTP-binding proteins (RhoA and Cdc42), actin-binding protein (cofilin), PKC (protein kinase C) and $Ca^{2+}$ channel (transient receptor potential channel 6, TRPC6) was observed in rat gastrocnemius muscle strips. Conclusion: These results suggest that MAPKs, ROS- and apoptosis-related enzymes, cytoskeleton-regulated proteins, and $Ca^{2+}$ channel may in part functionally import in E-C and E-T coupling from rat skeletal muscles.

• Journal of Microbiology and Biotechnology
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• 제10권1호
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• pp.27-34
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• 2000

The chloroform and methanol (2;1, v/v) extract from an edible plant, Actinidia arguta Planchon, appeared to possess antitumor activity against human leukemias Jurkat T and U937 cells through inducing apoptosis. The substance in the solvent extract was purified by silica gel column chromatography, preparative TLC, and Sephadex LH-20 column chromatography. Characteristics of the substance analyzed by UV scanning analysis, $^1H$ and $^{13}C$ NMR spectra suggested that the substance belongs to the chlorophyll derivatives-like group. The $IC_{50}$ value of the chlorophyll derivative (Cp-D) determined by MTT assay was $15\mu\textrm{g}/ml$ for Jurkat, $10\mu\textrm{g}/ml$ for U937, and $11.4\mu\textrm{g}/ml$ for HL-60m and was more toxic to these leukemias than to solid tumors or normal fibroblast. In order to elucidate cellular mechanisms underlying the cytotoxicity, the effect of the Cp-D on Jurkat T cells was investigated. When cells were treated with the Cp-D at a concentration of $15\mu\textrm{g}/ml$, [3H]thymidine incorporation declined rapidly and wa undetectable in 1h. However, no significant changes were made in the cell cycle distribution of the cells by 24h. The sub-Gl peak representing apoptotic cells began to be detectable in 36h, at which time apoptotic DNA fragmentation was also detected on agarose gel electrophoresis, demonstrating that the cytotoxic effect of the Cp-D is attributable to the induced apoptosis. Under the same conditions, although the protein level of cyclin-dependent kinases such as cdc4, csk6, cdk2, and cdc2 was not significantly changed until 24h, the kinase activity of all c안 rapidly declined and reached a minimum level within 1-6h and then recovered to the initial level by 12h and sustained until 24h. These results suggest that inactivation of cdks at an inappropriate time during the cell cycle progression in jurkat T cells following a treatment with the Cp-D leads to induction of apoptotic cell death.

• 대한한의학방제학회지
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• 제23권2호
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• pp.199-208
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• 2015

Objectives : In the present study, we investigated the effects of ethanol extract of Scutellaria baicalensis (EESB) on the progression of cell cycle in human renal cell carcinoma Caki-1 cells. Methods : The effects of EESB on cell growth and apoptosis induction were evaluated by trypan blue dye exclusion assay and flow cytometry, respectively. The mRNA and protein levels were determined by Western blot analysis and reverse transcription-polymerase chain reaction, respectively. Results : It was found that EESB treatment on Caki-1 cells resulted in a dose-dependent inhibition of cell growth and induced apoptotic cell death as detected by Annexin V-FITC staining. The flow cytometric analysis indicated that EESB resulted in G2/M arrest in cell cycle progression which was associated with the down-regulation of cyclin A expression. Our results also revealed that treatment with EESB increased the mRNA and proteins expression of tumor suppressor p53 and cyclin-dependent kinase (Cdk) inhibitor p21(WAF1/CIP1), without any noticeable changes in cyclin B1, Cdk2 and Cdc2. In addition, the incubation of cells with EESB resulted in a significant increase in the binding of p21 and Cdk2 and Cdc2. These findings suggest that EESB-induced G2/M arrest and apoptosis in Caki-1 cells is mediated through the p53-mediated upregulation of Cdk inhibitor p21. Conclusions : Taken together, these findings suggest that EESB may be a potential chemotherapeutic agent and further studies will be needed to identify the biological active compounds that confer the anti-cancer activity of S. baicalensis.

• Biomolecules & Therapeutics
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• 제32권1호
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• pp.104-114
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• 2024

Licochalcone C (LCC; PubChem CID:9840805), a chalcone compound originating from the root of Glycyrrhiza inflata, has shown anticancer activity against skin cancer, esophageal squamous cell carcinoma, and oral squamous cell carcinoma. However, the therapeutic potential of LCC in treating colorectal cancer (CRC) and its underlying molecular mechanisms remain unclear. Chemotherapy for CRC is challenging because of the development of drug resistance. In this study, we examined the antiproliferative activity of LCC in human colorectal carcinoma HCT116 cells, oxaliplatin (Ox) sensitive and Ox-resistant HCT116 cells (HCT116-OxR). LCC significantly and selectively inhibited the growth of HCT116 and HCT116-OxR cells. An in vitro kinase assay showed that LCC inhibited the kinase activities of EGFR and AKT. Molecular docking simulations using AutoDock Vina indicated that LCC could be in ATP-binding pockets. Decreased phosphorylation of EGFR and AKT was observed in the LCC-treated cells. In addition, LCC induced cell cycle arrest by modulating the expression of cell cycle regulators p21, p27, cyclin B1, and cdc2. LCC treatment induced ROS generation in CRC cells, and the ROS induction was accompanied by the phosphorylation of JNK and p38 kinases. Moreover, LCC dysregulated mitochondrial membrane potential (MMP), and the disruption of MMP resulted in the release of cytochrome c into the cytoplasm and activation of caspases to execute apoptosis. Overall, LCC showed anticancer activity against both Ox-sensitive and Ox-resistant CRC cells by targeting EGFR and AKT, inducing ROS generation and disrupting MMP. Thus, LCC may be potential therapeutic agents for the treatment of Ox-resistant CRC cells.

• Biomolecules & Therapeutics
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• 제30권6호
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• pp.553-561
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• 2022

Chronic myeloid leukemia (CML) is a slowly progressing hematopoietic cell disorder. Sphingosine kinase 1 (SPHK1) plays established roles in tumor initiation, progression, and chemotherapy resistance in a wide range of cancers, including leukemia. However, small-molecule inhibitors targeting SPHK1 in CML still need to be developed. This study revealed the role of SPHK1 in CML and investigated the potential anti-leukemic activity of hirsuteine (HST), an indole alkaloid obtained from the oriental plant Uncaria rhynchophylla, in CML cells. These results suggest that SPHK1 is highly expressed in CML cells and that overexpression of SPHK1 represents poor clinical outcomes in CML patients. HST exposure led to G2/M phase arrest, cellular apoptosis, and downregulation of Cyclin B1 and CDC2 and cleavage of Caspase 3 and PARP in CML cells. HST shifted sphingolipid rheostat from sphingosine 1-phosphate (S1P) towards the ceramide coupled with a marked inhibition of SPHK1. Mechanistically, HST significantly blocked SPHK1/S1P/S1PR1 and BCR-ABL/PI3K/Akt pathways. In addition, HST can be docked with residues of SPHK1 and shifts the SPHK1 melting curve, indicating the potential protein-ligand interactions between SPHK1 and HST in both CML cells. SPHK1 overexpression impaired apoptosis and proliferation of CML cells induced by HST alone. These results suggest that HST, which may serve as a novel and specific SPHK1 inhibitor, exerts anti-leukemic activity by inhibiting the SPHK1/S1P/S1PR1 and BCR-ABL/PI3K/Akt pathways in CML cells, thus conferring HST as a promising anti-leukemic drug for CML therapy in the future.

• Asian Pacific Journal of Cancer Prevention
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• 제15권7호
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• pp.3293-3297
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• 2014

Background: Pine needle oil from crude extract of pine needles has anti-tumor effects, but the effective component is not known. Methods: In the present study, compounds from a steam distillation extract of pine needles were isolated and characterized. Alpha-pinene was identified as an active anti-proliferative compound on hepatoma carcinoma BEL-7402 cells using the MTT assay. Results: Further experiments showed that ${\alpha}$-pinene inhibited BEL-7402 cells by arresting cell growth in the G2/M phase of the cell cycle, downregulating Cdc25C mRNA and protein expression, and reducing cycle dependence on kinase 1(CDK1) activity. Conclusion: Taken together, these findings indicate that ${\alpha}$-pinene may be useful as a potential anti-tumor drug.

• 한국수정란이식학회지
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• 제32권3호
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• pp.131-138
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• 2017

본 연구에서는 체외성숙 배양액 내 caffeine 첨가가 돼지 난자의 성숙과 단위발생 및 체세포 핵이식 후 배 발육에 미치는 영향을 조사하였다. 난세포질 및 난구세포 부착정도의 형태학적 특징에 따라 MGCOCs와 MPCOCs로 구분된 미성숙난자를 각각 무처리군(대조군)과 2.5 mM caffeine이 첨가된 배양액에서 체외성숙 22-42(20시간), 34-42(8시간), 38-42(4시간)동안 처리하는 군으로 나누어 체외성숙을 유도하였다. 또한 체외성숙 난자를 단위발생 및 체세포 핵이식에 공여하여 배아를 생산한 후 7일 동안 체외배양하여 체외성숙 동안 caffeine 처리가 분할률, 배반포 형성률 및 배반포의 세포수에 미치는 영향을 조사하였다. 연구 결과, 분할률 및 배반포 세포 수는 caffeine의 처리 시간에 따라 유의적인 영향을 받지 않았다. 그러나 MPCOCs 유래 난자에서 체외성숙 후기 4시간 동안 caffeine 처리는 체세포 핵이식 배아의 배반포 형성률을 유의적으로 증가시켰다. 이 결과는 caffeine 처리가 체외성숙 동안 난자의 MPF 수준의 감소를 억제시킴으로써 체세포 핵의 리모델링이나 리프로그래밍에 영향을 미쳐 핵이식 배아의 발육능에 영향을 미친 것으로 사료된다.

• 한국미생물·생명공학회지
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• 제29권3호
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• pp.181-185
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• 2001

유방암 세포주에서는 우수한 항암활성을 가진 것으로 알려진 indole-3-carbinol을 HepG2세포주에 시간과 농도별로 처리한 결과 cell growth inhibition을 확인하였으며, $IC_{50}$ 값은 48시간배양에서 $446\mu$M 72시간 배양에서 444$\mu$M로 나타났다. $400\mu$M의 I3C을 투여하고, 24, 48, 72시간에 HePG2 세포주의 cell cycle pattern을 분석한 결과, G1 phase에서 P21의증가와 함께 Cdk 6와 cyclin D의 확연한 감소와 Pb protein의 hypo-phosphorylation을 확인하였다. 반면 G2 phase에서는 I3C의 직접적인 억제로 인해 24시간 후부터 Cdc2와 cyclin B1가 급격히 감소하는것을 확인하였다. Flow cytomery 분석결과 I3C 처리 24시간 뒤 G2 arrest (25%)가 발생하였으며, 72시간이 지난후 G1 arrest (53%)가 발생하였다. 이러한 I3C의 간암세포주인 HePG2 cell의 cell cycle arrest가 apoptosis를 유발하는지를 알고자 caspase 3 Bcl2 Bax protein의 발현양상을 확인한 결과 아무런 변화가 보이지 않았다. 즉 I3C은 간암세포주인 HepG2 cell에서 apoptosis를 유도하지 못한다는것을 확인하였따. 결론적으로 I3C은 HepG2 세포주에서 G1와 G2 phase에서 cell cycle arrest는 발생시키나, 특이적으로 apoptosis 와는 연관되지 않는다는 사실을 확인하였다.

• Journal of Microbiology and Biotechnology
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• 제26권2호
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• pp.287-294
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• 2016

The effect of kaempferol (3,5,7,4-tetrahydroxyflavone), a flavonoid compound that was identified in barnyard millet (Echinochloa crus-galli var. frumentacea) grains, on G₂-checkpoint and apoptotic pathways was investigated in human acute leukemia Jurkat T cell clones stably transfected with an empty vector (J/Neo) or a Bcl-xL expression vector (J/Bcl-xL). Exposure of J/Neo cells to kaempeferol caused cytotoxicity and activation of the ATM/ATR-Chk1/Chk2 pathway, activating the phosphorylation of p53 (Ser-15), inhibitory phosphorylation of Cdc25C (Ser-216), and inactivation of cyclin-dependent kinase 1 (Cdk1), with resultant G₂-arrest of the cell cycle. Under these conditions, apoptotic events, including upregulation of Bak and PUMA levels, Bak activation, mitochondrial membrane potential (Δψm) loss, activation of caspase-9, -8, and -3, anti-poly (ADP-ribose) polymerase (PARP) cleavage, and accumulation of apoptotic sub-G₁ cells, were induced without accompanying necrosis. However, these apoptotic events, except for upregulation of Bak and PUMA levels, were completely abrogated in J/Bcl-xL cells overexpressing Bcl-xL, suggesting that the G₂-arrest and the Bcl-xL-sensitive mitochondrial apoptotic events were induced, in parallel, as downstream events of the DNA-damage-mediated G₂-checkpoint activation. Together these results demonstrate that kaempferol-mediated antitumor activity toward Jurkat T cells was attributable to G₂-checkpoint activation, which caused not only G₂-arrest of the cell cycle but also activating phosphorylation of p53 (Ser-15) and subsequent induction of mitochondria-dependent apoptotic events, including Bak and PUMA upregulation, Bak activation, Δψm loss, and caspase cascade activation.