• 대한한방부인과학회지
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• 제19권4호
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• pp.47-60
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• 2006

Purpose : This study was conducted to investigate the inhibitory effects of Scutellaria barbata D. D on on the cell proliferation of HeLa Cells. Methods : Human uterine cervical carcinoma HeLa cells were cultured in the 1%, 5% and 10% concentration of Scutellaria barbata D. D on solution for 24, 48 and 72 hours for the direct inhibitory effects of Scutellaria barbata D. D on. Then we examined the effect of Scutellaria barbata D. D on solution on the cell proliferation inhibition by XTT assay. DNA fragmentation, MAP kinase activity and caspase activity by FACS analysis in HeLa cells. Results : We found that the proliferation of HeLa cells was significantly decreased in Scutellaria barbata D. D on solution containing groups comparing with a control group in a concentration-dependant manner. When HeLa cells were cultivated for 24 hours with 5% Scutellaria barbata D. D on solution containing group, the percentage of HeLa cells with activated caspase was the highest. Scutellaria barbata D. D on solution reduced the MAP kinase activity of HeLa cells comparing with the control group. By the XTT assay, the cell's activity was decreased in 5% and 10% Scutellaria barbata D. D on solution containing groups in 24 and 72 hours cultivation and 10% group in 48 hours. DNA fragmentation and caspase-3 activity of HeLa cells, however, were changed insignificantly. Conclusion : From this study we could suggest that Scutellaria barbata D. D on is available to the inhibition and apoptosis of human cervical carcinoma cell line, HeLa cells in vitro.

• 한방안이비인후피부과학회지
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• 제20권2호통권33호
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• pp.10-35
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• 2007

Objectives : To investigate the effects of Ohbaesangami (OBSGM) on mucosa and skin diseases, anti-microbial and anti-inflammatory tests were performed using several in vitro test models. Results : In anti-microbial test, OBSGM showed the slight inhibitory effect against Propionibacterium acnes (P. acnes) and Staphylococcus aureus (S. aureus). In anti-oxidant test, OBSGM showed the potent radical scavenging activity. In anti-inflammatory test, OBSGM weakly inhibited the lipopolysaccharide (LPS)-induced nitric oxide(NO) release from the RAW 264.7 macrophage cells. OBSGM also inhibited the LPS-induced $interleukin-1{\beta}(IL-1{\beta})$ and cyclooxygenase-2 (COX-2) expressions. The inhibitory effects of OBSGM on macrophage activation was via the inhibition of $NF-{\kappa}B$, evidenced by transient transfection assay. Furthermore, OBSGM markedly inhibited the activation of Jun-N-terminal kinase (JNK) and p38 MAP kinase in RAW 264.7 cells. In skin wrinkle formation assay, OBSGM strongly inhibited collagnease and elastase, whose activities are tightly related with the wrinkle formation. In addition, OBSGM inhibited the activities of MMP-1, MMP-2 on the mRNA levels in RAW 264.7 cells. However, OBSGM did not show an inhibitory potential on tyrosinase activity and melanin synthesis, indicating that it could not be applicable for skin whitening. Conclusion : These results suggest that the anti-inflammatory effect of OBSGM may be due to its inhibitory potentials on the macrophage activation. And, the anti-wrinkle effects of OBSGM may be due to its inhibitory potential on the collagnease and elastase activities. Therefore, OBSGM could be applicable for the treatment of mucosa and skin diseases.

• 방사선산업학회지
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• 제5권1호
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• pp.15-20
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• 2011

Protein kinases are the most important drug targets for the treatment of numerous diseases. The involvement of protein kinase C (PKC) in many biological processes such as development, memory, cell differentiation, and proliferation has been demonstrated. PKC is recognized as an important player in carcinogenesis. Thus, a variety of PKC inhibitors have been investigated. Among them, flavonoids have been demonstrated to affect the activity of many mammalian in vitro enzyme systems. The recent investigation was performed to evaluate the inhibitory effects of hesperidin, which is a flavonoid, on the proliferation and carcinogenesis of many cancers. In this study, an efficient kinase assay based on a biochip using radio-phosphorylation was established and performed for an examination of the inhibitory effects of hesperidin on PKC activity at different concentrations of 50, 200, 500 nM. It was found that hesperidin shows inhibitory potency on PKC, and that the biochip can be used to rapidly screen kinase inhibitors resulting in the therapeutic agents.

• Biomolecules & Therapeutics
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• 제27권2호
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• pp.216-221
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• 2019

The c-Met protein is a receptor tyrosine kinase involved in cell growth, proliferation, survival, and angiogenesis of several human tumors. Overexpression of c-Met has been found in gastric cancers and correlated with a poor prognosis. Indirubin is the active component of Danggui Longhui Wan, which is a traditional Chinese antileukemic recipe. In the present study, we tested the anti-cancer effects of an indirubin derivative, LDD-1937, on human gastric cancer cells SNU-638. When we performed the in vitro kinase assay against the c-Met activity, LDD-1937 inhibited the activity of c-Met. This result was confirmed by immunoblot and immunofluorescence of phosphorylated c-Met. Immunoblot analysis showed that LDD-1937 decreased the expression of the Erk1/2, STAT3, STAT5, and Akt, downstream proteins of c-Met. In addition, LDD-1937 reduced the cell viability and suppressed colony formation and migration of SNU-638 cells. Furthermore, LDD-1937 induced $G_2/M$ phase arrest in the SNU-638 cells by decreasing the expression levels of cyclin B1 and CDC2. Cleaved-PARP, an apoptosis-related protein, was up-regulated in cells treated with LDD-1937. Overall, this study suggests that LDD-1937 may be a novel small-molecule with therapeutic potential for selectively inhibiting c-Met and c-Met downstream pathways in human gastric cancers overexpressing c-Met.

• Journal of Microbiology and Biotechnology
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• 제22권2호
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• pp.170-175
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• 2012

Clostridium difficile toxin A glucosylates Rho family proteins, resulting in actin filament disaggregation and cell rounding in cultured colonocytes. Given that the cellular toxicity of toxin A is dependent on its receptor binding and subsequent entry into the cell, we herein sought to identify additional colonocyte proteins that might bind to toxin A following its internalization. Our results revealed that toxin A interacted with ERK1 and ERK2 in two human colonocyte cell lines (NCM460 and HT29). A GST-pulldown assay also showed that toxin A can directly bind to ERK1 and ERK2. In NCM460 cells exposed to PMA (an ERK1/2 activator), the phosphorylation of ERK1/2 did not affect the interaction between toxin A and ERK1/2. However, an in vitro kinase assay showed that the direct binding of toxin A to ERK1 or ERK2 inhibited their kinase activities. These results suggest a new molecular mechanism for the cellular toxicity seen in cells exposed to toxin A.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• 제28권6호
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• pp.434-439
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• 2002

Oral squamous cell carcinoma (OSCC) is one of the most common head and neck cancers. OSCC generally has a poor prognosis due to its tendency towards a local invasion and subsequent metastasis, which is mediated by multiple proteolytic enzymes and angiogenesis. Soy products contain high levels of isoflavonoids, including the tyrosine kinase inhibitor, genistein, which has been identified as a potent inhibitor of cell proliferation and in vitro angiogenesis. The purpose of this in vitro study is to evaluate the anti-cancer effect of genistein with respect to the angiogenesis and basement membrane invasion in OSCC. The highly invasive OSCC cell line, HSC-3 cells were cultured in the presence of $10{\mu}M$ genistein for 24h. To evaluate the effects of genistein on the invasiveness and the gelatinolytic activity, in vitro invasion assay and zymography were performed. In order to evaluate the effect on the VEGF and bFGF mRNA expression, RT-PCR and northern hybridization reaction, and chemiluminescence detection were applied. The in vitro invasion assay showed that the genistein treatment reduced the cellular invasion through the artificial basement membrane and significant difference between the control group and the genistein treated group was shown in MMP-2 activity. Especially, the 62 kDa activated form of MMP-2 in the control group was 1.8 times higher than that in the genistein treated group. The results of the northern blot analyses indicated that VEGF mRNA expression in the genistein treated group was significantly down regulated. This study showed that genistein inhibits angiogenesis and reduces basement membrane invasion in OSCC. It seems to support the possibility of genistein as an anti-cancer agent.

• 한국미생물·생명공학회지
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• 제33권4호
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• pp.260-266
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• 2005

본 실험에서는 MRF가 다량 존재하는 RBL-2H3 세포주에 다양한 PKC isotype별 억제제를 처리하여 in vitro상에서 Na, K-ATPase $\alpha$1L3를 이용한 pull-down assay와 RBL-2H3 세포를 이용한 membrane fractionation을 실시하였다. 그 결과 HRF는 in vitro에서 $\alpha$1L3와 결합한다는 사실을 재확인 할 수 있었고 실제 세포주 내에서 Na,K-ATPase와 결합한다는 것을 알 수 있었다. 사용한 약물로부터 PKC $\alpha,\;\beta,\;\delta$뿐 아니라 protein phosphatase 2B(PP2B)도 HRF와 $\alpha$1L3의 결합에 관여한다는 사실을 알 수 있었다. 또한 이들 PKC, PP2B에 의해 인산화된 HRF 분자는 cytosolic fraction으로 이행할 수 있으며 이러한 결과는 탈인산화된 HRF가 Na,K-ATPase와 결합하여 Na, K-ATPase의 기능을 조절한다고 추정할 수 있다. 그러나 약물자체가 histamine 분비에 영향을 미칠 수 있으며 cytosolic HRF보다 exocytosis된 HRF가 histamine를 더 분비하도록 할 수 있으므로, 약물을 전처리한 세포에 외부에서 HRF를 첨가하여 histamine이 유리되는 정도가 어떻게 변화하는지를 HRF를 가하지 않은 결과와 비교해야 할 것이다.

• BMB Reports
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• 제42권12호
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• pp.840-845
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• 2009

Mitogen- and stress-activated protein kinase (MSK1) palys a crucial role in the regulation of transcription downstream of extracellular-signal-regulated kinase1/2 (ERK1/2) and mitogen-activated protein kinase p38. MSK1 can be phosphorylated and activated in cells by both ERK1/2 and p38$\alpha$. In this study, Casein Kinase 2 (CK2) was identified as a binding and regulatory partner for MSK1. Using the yeast two-hybrid system, MSK1 was found to interact with the CK2$\beta$ regulatory subunit of CK2. Interactions between MSK1 and the CK2$\alpha$ catalytic subunit and CK2$\beta$ subunit were demonstrated in vitro and in vivo. We further found that CK2$\alpha$ can only interact with the C-terminal kinase domain of MSK1. Using site-directed mutagenesis assay and mass spectrometry, we identified five sites in the MSK1 C-terminus that could be phosphorylated by CK2 in vitro: Ser757, Ser758, Ser759, Ser760 and Thr793. Of these, Ser757, Ser759, Ser760 and Thr793 were previously unknown.

• Molecular & Cellular Toxicology
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• 제2권3호
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• pp.151-158
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• 2006

Khal was a synthetic congener of halocidin, a heterodimeric peptide consisting of 19 and 15 amino acid residues detected in Halocynthia aurantium. This compound was considered a candidate for the development of a novel peptide antibiotic. The genotoxicity of Khal was subjected to high throughput toxicity screening (HTTS) because they revealed strong antibacterial effects. Mouse lymphoma thymidine kinase ($tk^{+/-}$) gene assay (MOLY), single cell gel electrophoresis (Comet) assay and chromosomal aberration assay in mammalian cells and Ames reverse mutation assay in bacterial system were used as simplified, inexpensive, short-term in vitro screening tests in our laboratory. These compounds are not mutagenic in S. typhimurium TA98 and TA100 strains both in the presence and absence of metabolic activation. Before performing the comet assay, $IC_{20}$ of Khal was determined the concentration of $25.51\;{\mu}/mL\;and\;21.99\;{\mu}g/mL$ with and without S-9, respectively. In the comet assay, Khal was not induced DNA damage in mouse lymphoma cell line. Also, the mutation frequencies in the Khal-treated cultures were similar to the vehicle controls. It is suggests that Khal is non-mutagenic in MOLY assay. And no clastogenicity was observed in Khal-treated Chinese hamster lung cells. The results of this battery of assays indicate that Khal has no genotoxic potential in bacterial or mammalian cell systems. Therefore, we suggest that Khal, as the optimal candidates with both no genotoxic potential and antibacterial effects must be chosen.

• 생명과학회지
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• 제33권9호
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• pp.703-712
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• 2023

기존 혈관에서 새로운 혈관을 형성하는 혈관 신생은 혈관 신생 조절인자에 의해 조절되는 다단계 과정이며 배아 발달, 만성 염증 및 상처 복구를 포함한 다양한 생리학적 과정에 필수적이다. 혈관 신생의 조절장애는 암, 자가 면역 질환, 류마티스 관절염, 심혈관 질환 및 상처 치유 지연과 같은 많은 질병을 유발한다. 그러나 효과적인 혈관신생 억제 약물은 제한되어 있으며, 최근 연구에서는 천연 자원에서 잠재적인 약물후보를 식별하는 데 중점을 두고 있다. 예를 들어, 해양 천연물은 항암, 항산화, 항염증, 항바이러스 및 상처 치유 효과를 입증했다. 따라서 본 연구에서는 톳(갈조류) 추출물의 혈관 신생 억제 효과를 확인했습니다. H. fusiformis 추출물은 인간 제대 정맥 내피 세포(HUVECs)에서 세포 이동, 침윤 및 관 형성을 억제하며, 동시에 Matrigel 겔 플러그 분석을 통해 생체 내 혈관 신생을 억제를 확인했다. 또한, 톳 추출물 처리 후 VEGF, Erk, Akt의 활성이 감소하는 것을 확인했다. 이 결과를 토대로 H. fusiformis 추출물이 in vitro 및 in vivo 혈관 신생을 억제함을 시사한다.