Hep3B 인간 간암세포에서 caspase 의존적이며 PI3K/Akt 신호전달의 불활성화와 관련된 β-lapachone의 세포사멸 유도

권재임,최영현,황보현 한국생명과학회 2024 생명과학회지 Vol.34 No.2

β-Lapachone is a natural quinone compound originally obtained from the bark of the lapacho tree (Tabebuia vellanedae), which has been used in traditional medicine in several South and Central American countries for treating various diseases. Although β-lapachone has been reported to have potent anticancer activity in many types of cancer cells, its effect on the proliferation of hepatocellular carcinoma (HCC) cells is still unclear. Therefore, in this study, we investigated the effect of β-lapachone on the proliferation of human HCC Hep3B cells. According to our results, the decrease in cell viability of Hep3B cells caused by β-lapachone was closely related to the induction of apoptosis, which was confirmed through changes in nuclear morphology and flow cytometry. In addition, in Hep3B cells treated with β-lapachone, the expression of Bcl-2, an anti-apoptotic factor, was decreased, while the expression of Bax, an apoptosis inducer, was increased, and the activity of the caspase cascade was also increased. However, in the presence of a pan-caspase inhibitor, β-lapachone-induced apoptosis was weakened, indicating that the induction of apoptosis by β-lapachone was caspase-dependent. Moreover, β-lapachone treatment activated extracellular-regulated kinase (ERK) signaling while inhibiting activation of the phosphoinositide 3 kinase (PI3K)/Akt pathway. Furthermore, the effect of the ERK inhibitor on suppressing the induction of apoptosis by β-lapachone was minimal, and the PI3K inhibitor significantly increased β-lapachone-induced apoptosis. The findings from this study will contribute to a better understanding of the anticancer activity of β-lapachone in HCC cells. β-lapachone은 다양한 유형의 질병을 치료하기 위해 남미 및 중미 지역의 전통 의학에서 널리 사용되어 온 Tabebuia vellanedae의 껍질에서 분리된 천연 퀴논 화합물의 일종이다. β-lapachone은 여러 유형의 암세포에서 강력한 항암 활성을 갖는 것으로 보고되었지만, 간세포암종 세포의 증식에 대한 효과는 아직 불분명하다. 따라서 본 연구에서는 β-lapachone 인간 간세포암종 Hep3B 세포의 증식에 미치는 영향을 조사하였으며, 본 연구의 결과에 의하면, β-lapachone 처리에 의한 Hep3B 세포의 세포생존율 감소는 세포사멸 유도와 밀접한 관련이 있었다. 또한, β-lapachone이 처리된 Hep3B 세포에서는 항세포사멸 인자인 Bcl-2의 발현이 감소한 반면, 세포사멸 유도 인자인 Bax의 발현은 증가하였으며, 이는 caspase cascade의 활성 증가와 연관성이 있었다. 그러나 pan-caspase 억제제가 존재하는 경우 β-lapachone에 의해 유발된 세포사멸은 약화되었으며, 이는 β-lapachone에 의한 세포사멸 유도가 caspase 의존적인 현상임을 의미한다. 아울러, β-lapachone의 처리는 ERK 경로를 활성화시키면서 PI3K/Akt 경로의 활성을 억제하였으며, β-lapachone 유도 세포사멸에 ERK 억제제의 효과는 미미했지만, PI3K 억제제는 β-lapachone에 의해 유도된 세포사멸을 유의하게 증가시켰다. 비록 생체 내 동물 모델에서의 확인이 필요하지만, 본 연구의 결과는 간세포암종 세포에서 β-lapachone의 항암 활성을 이해하는 데 유용한 자료로 활용될 것이다.

β-Lapachone-Induced Apoptosis is Associated with Inhibition of Cyclooxygenase-2 Activity in Human Lung Cancer A549 Cells

최영현(Yung Hyun Choi) 한국생명과학회 2011 생명과학회지 Vol.21 No.10

β-lapachone은 남미에 자생하는 lapacho 나무(Tabeuia avellanedae)의 수액에 함유된 quinone계열의 일종으로 많은 인체암세포에서apoptosis를 유발하는 것으로 알려져 있다. 본 연구는 A549 인체폐암세포를 대상으로 β-lapachone에 의한 apoptosis 유발과정에서 나타나는 또 다른 현상들을 조사하기 위하여 실시되었다. β-lapachone이 처리된 A549 세포는 처리 농도의 증가에 따라 생존율이 감소되었으며, 이는 apoptosis 유발과 연관이 있음을 MTT assay와 flow cytometry 분석을 통하여 확인하였다. β-lapachone에 의한 A549 세포의 증식억제는 종양억제유전자 p53과 cyclin-dependent kinase 저해제인 p21의 발현을 전사 및 번역 수준에서 증가시켰으며, p53 단백질의 인산화 증가와 연관성이 있었다. 또한 β-lapachone은 caspase-3과 -9를 활성화시켰으며, 활성화된 caspase-3의 기질 단백질들[poly(ADP-ribose) polymerase, β-catenin 및 phospholipase C-γ1]의 단편화를 유도하였다. 아울러 β-lapachone은 cyclooxygenase (COX)-2의 mRNA 및 단백질의 발현을 억제하였으나 COX-1의 발현에는 영향을 미치지 않았으며, β-lapachone에 의한 COX-2의 발현억제는 prostaglandin E₂의 생성 저하에 관련이 있었다. 본 연구의 결과는 β-lapachone의 항암활성 기전의 이해와 더불어 β-lapachone이 폐암세포에서 강력한 항암활성이 있음을 보여주는 것이다. β-lapachone, a quinone of lapachol extracted from the bark of the lapacho tree, has been found to induce apoptosis in various human cancer cells. In the present study, we investigated further possible mechanisms by which β-lapachone exerts its pro-apoptotic action in cultured human lung cancer A549 cells. β-lapachone treatment resulted in inhibition of growth and induction of apoptosis in a concentration-dependent manner, as determined by MTT assay and flow cytometry analysis. The induction of apoptosis by β-lapachone was associated with up-regulation of the expression of p53 and p21 in both transcriptional and translational levels, and the phosphorylation of p53. In addition, β-lapachone activated caspase-3 and -9, and induced degradation of caspase-3 target proteins such as poly (ADP-ribose) polymerase (PARP) and β-catenin. Furthermore, β-lapachone treatment caused a progressive decrease in the expression levels of cyclooxygenase (COX)-2 without significant changes in the levels of COX-1, which was correlated with a decrease in prostaglandin E₂ synthesis. Taken together, these results indicated that β-lapachone may have therapeutic potential in human lung cancer treatment.

Preclinical Pharmacokinetic Evaluation of β-Lapachone: Characteristics of Oral Bioavailability and First-Pass Metabolism in Rats

( Ik Soo Kim ),( Hyeong Min Kim ),( Jie Un Ro ),( Kang Hee Jo ),( San Deep Karki ),( Prakash Khadka ),( Gyi Ae Yun ),( Jae Hwi Lee ) 한국응용약물학회 2015 Biomolecules & Therapeutics(구 응용약물학회지) Vol.23 No.3

β-Lapachone has drawn increasing attention as an anti-inflammatory and anti-cancer drug. However, its oral bioavailability has not been yet assessed, which might be useful to develop efficient dosage forms possibly required for non-clinical and clinical studies and future market. The aim of the present study was thus to investigate pharmacokinetic properties of β-lapachone as well as its first-pass metabolism in the liver, and small and large intestines after oral administration to measure the absolute bioavailability in rats. A sensitive HPLC method was developed to evaluate levels of β-lapachone in plasma and organ homogenates. The drug degradation profiles were examined in plasma to assess the stability of the drug and in liver and intestinal homogenates to evaluate first-pass metabolism. Pharmacokinetic profiles were obtained after oral and intravenous administration of β-lapachone at doses of 40 mg/kg and 1.5 mg/kg, respectively. The measured oral bioavailability of β-lapachone was 15.5%. The considerable degradation of β-lapachone was seen in the organ homogenates but the drug was quite stable in plasma. In conclusion, we suggest that the fairly low oral bioavailability of β-lapachone may be resulted from the first-pass metabolic degradation of β-lapachone in the liver, small and large intestinal tracts and its low aqueous solubility.

β-lapachone-Induced Apoptosis of Human Gastric Carcinoma AGS Cells Is Caspase-Dependent and Regulated by the PI3K/Akt Pathway

( Hai Yang Yu ),( Sung Ok Kim ),( Cheng Yun Jin ),( Gi Young Kim ),( Wun Jae Kim ),( Young Hyun Yoo ),( Yung Hyun Choi ) 한국응용약물학회 2014 Biomolecules & Therapeutics(구 응용약물학회지) Vol.22 No.3

β-lapachone is a naturally occurring quinone that selectively induces apoptotic cell death in a variety of human cancer cells in vitro and in vivo; however, its mechanism of action needs to be further elaborated. In this study, we investigated the effects of β-lapachone on the induction of apoptosis in human gastric carcinoma AGS cells. β-lapachone significantly inhibited cellular proliferation, and some typical apoptotic characteristics such as chromatin condensation and an increase in the population of sub-G1 hypodiploid cells were observed in β-lapachone-treated AGS cells. Treatment with β-lapachone caused mitochondrial transmembrane potential dissipation, stimulated the mitochondria-mediated intrinsic apoptotic pathway, as indicated by caspase-9 activation, cytochrome c release, Bcl-2 downregulation and Bax upregulation, as well as death receptor mediated extrinsic apoptotic pathway, as indicated by activation of caspase-8 and truncation of Bid. This process was accompanied by activation of caspase- 3 and concomitant with cleavage of poly(ADP-ribose) polymerase. The general caspase inhibitor, z-VAD fmk, significantly abolished β-lapachone-induced cell death and inhibited growth. Further analysis demonstrated that the induction of apoptosis by β-lapachone was accompanied by inactivation of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. The PI3K inhibitor LY29004 significantly increased β-lapachone-induced apoptosis and growth inhibition. Taken together, these findings indicate that the apoptotic activity of β-lapachone is probably regulated by a caspase-dependent cascade through activation of both intrinsic and extrinsic signaling pathways, and that inhibition of the PI3K/Akt signaling may contribute to β lapachone mediated AGS cell growth inhibition and apoptosis induction.

β-Lapachone Inhibits Lung Metastasis of Colorectal Cancer by Inducing Apoptosis of CT26 Cells

Kee, Ji-Ye,Han, Yo-Han,Park, Jinbong,Kim, Dae-Seung,Mun, Jeong-Geon,Ahn, Kwang Seok,Kim, Hyun-Jung,Um, Jae-Young,Hong, Seung-Heon SAGE Publications 2017 Integrative cancer therapies Vol.16 No.4

<P><B>Background:</B> β-Lapachone is a quinone-containing compound found in red lapacho (<I>Tabebuia impetiginosa</I>, syn. <I>T avellanedae</I>) trees. Lapacho has been used in traditional medicine by several South and Central American indigenous people to treat various types of cancer. The purpose of this study was to investigate the antimetastatic properties of β-lapachone and the underlying mechanisms using colon cancer cells. <B>Methods:</B> This research used metastatic murine colon cancer cell lines, colon 26 (CT26) and colon 38 (MC38). A WST assay, annexin V assay, cell cycle analysis, wound healing assay, invasion assay, western blot analysis, and real-time reverse transcription–polymerase chain reaction were performed to examine the effects of β-lapachone on metastatic phenotypes and molecular mechanisms. The effect of β-lapachone on lung metastasis was assessed in a mouse experimental metastasis model. <B>Results:</B> We found that the inhibition of proliferation of the colon cancer cell lines by β-lapachone was due to the induction of apoptosis and cell cycle arrest. β-Lapachone induced the apoptosis of CT26 cells through caspase-3, -8, and -9 activation; poly(ADP-ribose) polymerase cleavage; and downregulation of the Bcl-2 family in a dose- and time-dependent manner. In addition, a low concentration of β-lapachone decreased the cell migration and invasion by decreasing the expression of matrix metalloproteinases-2 and -9, and increased the expression of tissue inhibitors of metalloproteinases-1 and -2. Moreover, β-lapachone treatment regulated the expression of epithelial-mesenchymal transition markers such as E- and N-cadherin, vimentin, β-catenin, and Snail in CT26 cells. In the mouse experimental metastasis model, β-lapachone significantly inhibited the lung metastasis of CT26 cells. <B>Conclusions:</B> Our results demonstrated the inhibitory effect of β-lapachone on colorectal lung metastasis. This compound may be useful for developing therapeutic agents to treat metastatic cancer.</P>

Suppression of Human Prostate Cancer Cell Growth by β-Lapachone via Down-regulation of pRB Phosphorylation and Induction of Cdk Inhibitor p21WAF1/CIP1

( Yung Hyun Choi ),( Ho Sung Kang ),( Mi Ae Yoo ) 생화학분자생물학회 2003 BMB Reports Vol.36 No.2

The product of a tree (Tabebuia avellanedae) from South America, β-lapachone, is known to exhibit various pharmacological properties, the mechanisms of which are poorly understood. The aim of the present study was to further elucidate the possible mechanisms by which p- lapachone exerts its anti-proliferative action in cultured human prostate cancer cells. We observed that the prolieration-inhibitory effect of β-lapachone was due to the induction of apoptosis, which was confirmed by observing the morphological changes and cleavage of the poly(ADP-ribose) polymerase protein. A DNA flow cytometric analysis also revealed that βlapachone arrested the cell cycle progression at the G1 phase. The effects were associated with the down-regulation of the phosphorylation of the retinoblastoma protein (pRB) as well as the enhanced binding of pRB and the transcription factor E2F-1. Also, β-lapachone suppressed the cyclin- dependent kinases(Cdks) and cyclin E-associated kinase activity without changing their expressions. Furthermore, this compound induced the levels of the Cdk inhibitor p21^(WAFI/CIPI) expression in a p-530independent manner, and the p21 proteins that were induced by β-lapachone were associated with Cdk2. β-lapachone also activated the reporter construct of a p21 promoter. Overall, our results demonstrate a combined mechanism that involves the inhibition of pRB phosphorylation and induction of p21 as targets for βlapachone. This may explain some of its anti-cancer effects.

Effects of β-Lapachone on Gastric Secretion

박동선,조인근,양윤희,경장빈,김다정,최은경,곽태환,유상구,김윤배 충북대학교 동물의학연구소 2011 Journal of Biomedical and Translational Research Vol.12 No.3

The effects of β-lapachone on gastric secretion were investigated. The pylorus of male Sprague-Dawley rats were ligated and intraduodenally injected with β-lapachone, and the volume, pH, free HCl and total acidity of gastric fluid were measured 6 hours after the operation. The gastric secretion was inhibited by treatment with β-lapachone, in a dose-dependent manner. The gastric fluid was reduced to 42.9% of control level by 100 mg/kg of β-lapachone, leading to an increase of pH to 6.70 from 1.85 in control group. In parallel with the increase of pH, at this dosage, free HCl and total acidity decreased to 16.7% and 12.0%, respectively, of control levels. β-Lapachone exhibited ED50 values of 72, 46 and 47 mg/kg for the inhibition of gastric volume, free HCl and total acidity, respectively, implying a superior efficacy on gastric acid to volume. In comparison, pantoprazole (30 mg/kg) reduced the volume, free HCl and total acidity of gastric fluid to 53.0%, 26.0% and 25.0%, respectively, of control levels, resulting in the increase in pH to 6.36. In the present study, it was confirmed that β-lapachone at an appropriate dose (100 mg/kg) exerted a higher inhibitory effect on gastric secretion than pantoprazole (30 mg/kg), a well-known proton-pump inhibitor. Therefore, it is suggested that β-lapachone could be a candidate compound for the prevention or treatment of gastric ulcers induced by diverse psychological and physical stimuli.

Solubility evaluation and thermodynamic modeling of β-lapachone in water and ten organic solvents at different temperatures

Kim, Ki Hyun,Oh, Hee Kyung,Heo, Bora,Kim, Nam Ah,Lim, Dae Gon,Jeong, Seong Hoon Elsevier 2018 Fluid phase equilibria Vol.472 No.-

<P><B>Abstract</B></P> <P>Although β-lapachone is a promising drug with pharmacological activity, issues concerning its low aqueous solubility are known. The objective of this study was to measure the solubility of β-lapachone in water and ten organic solvents at temperatures ranging from 298.15 K to 318.15 K under atmospheric pressure. The modified Apelblat model, the Buchowski-Ksiazaczak <I>λh</I> model, and the ideal model were used to correlate experimentally obtained solubility values. Moreover, thermodynamic analysis of β-lapachone dissolution was performed based on experimental solubility data using the van't Hoff equation. The highest mole fraction solubility of β-lapachone at 298.15 K was found in acetone (2.05 × 10<SUP>−2</SUP>), followed by acetonitrile (1.80 × 10<SUP>−2</SUP>), ethyl acetate (8.53 × 10<SUP>−3</SUP>), 1-butanol (7.43 × 10<SUP>−3</SUP>), 1-propanol (6.69 × 10<SUP>−3</SUP>), 2-butanol (5.65 × 10<SUP>−3</SUP>), methanol (5.40 × 10<SUP>−3</SUP>), ethanol (4.99 × 10<SUP>−3</SUP>), 2-propanol (3.76 × 10<SUP>−3</SUP>), propylene glycol (3.06 × 10<SUP>−3</SUP>), and water (2.85 × 10<SUP>−6</SUP>). Correlation results showed that the modified Apelblat model was more accurate than the Buchowski-Ksiazaczak <I>λh</I> model and the ideal model. Thermodynamic analysis indicated that β-lapachone dissolution was endothermic and entropy-driven process in all solvents studied. Data on solubility and thermodynamic properties in various solvents obtained in this study could be helpful in formulation development, purification, and crystallization of β-lapachone.</P>

Induction of Egr-1 Is Associated with Anti-Metastatic and Anti-Invasive Ability of β-Lapachone in Human Hepatocarcinoma Cells

KIM, Sung Ok,KWON, Jae Im,JEONG, Yong Kee,KIM, Gi Young,KIM, Nam Deuk,CHOI, Yung Hyun Japan Society for Bioscience, Biotechnology, and A 2007 Bioscience, Biotechnology, and Biochemistry Vol.71 No.9

<P>β-lapachone, a quinone compound obtained from the bark of the lapacho tree (<I>Tabebuia avellanedae</I>), was reported to have anti-inflammatory and anti-cancer activities. In this study, we investigated novel functions of β-lapachone in terms of anti-metastasis and anti-invasion abilities using human hepatocarcinoma cell lines, HepG2 and Hep3B. β-lapachone dose-dependently inhibited cell viability and migration of both HepG2 and Hep3B cells, as determined by methylthiazoletetrazolium (MTT) assay and wound healing assay. RT-PCR and Western blot data revealed that β-lapachone dramatically increased the levels of protein, as well as mRNA expression of early growth response gene-1 (Egr-1) and throbospondin-1 (TSP-1) at an early point in time, and then decreased in a time-dependent manner. In addition, down-regulation of Snail and up-regulation of E-cadherin expression were observed in β-lapachone-treated HepG2 and Hep3B cells, and this the associated with decreased invasive ability as measured by matrigel invasion assay. Taken together, our results strongly suggest that β-lapachone may be expected to inhibit the progression and metastasis of hepatoma cells, at least in part by inhibiting the invasive ability of the cells <I>via</I> up-regulation of the expression of the Egr-1, TSP-1, and E-cadherin.</P>

피부 항노화제로써 라파콘에 관한 연구

김성우(Sung-Woo Kim),강혜리(Hye-Ri Kang),이소연(So-Yeon Lee),박기현(Gi-Hyeon Park),박소현(So-Hyeon Park),김명래(Myeoung-Rae Kim),한현탁(Hyeon-Tak Han) 한국화장품미용학회 2017 한국화장품미용학회지 Vol.7 No.1

The action mechanism of β-lapachone on fibrillogenesis in human dermal fibroblasts was studied to develop a potential skin anti-aging agent. In a prior study, we found that β-lapachone stimulated type I collagen expression in human dermal fibroblasts. In cultured human dermal fibroblasts, both Smad 2 and Smad 3 (Smad 2/3) were phosphorylated by β-lapachone treatment, suggesting that β-lapachone stimulates type I collagen synthesis via the transforming growth factor-β receptor I kinase-dependent pathway. Secreted transforming growth factor-β I levels were not elevated after β-lapachone treatment, suggesting that the molecular mechanism of β-lapachone for the upregulation of collagen synthesis is due to the extracellular regulation of availability and activities of TGF-β. Our findings provide new insights into the role of β-lapachone in collagen synthesis in human dermal fibroblasts. These findings could be used for developing novel therapeutic strategies for treating aging skin.