Auranofin accelerates spermidine-induced apoptosis via reactive oxygen species generation and suppression of PI3K/Akt signaling pathway in hepatocellular carcinoma

황보현,김다혜,김민영,지선영,EunJin Bang,홍수현,최영현,정재헌 한국수산과학회 2023 Fisheries and Aquatic Sciences Vol.26 No.2

Auranofin is a US Food and Drug Administration (FDA)-approved anti-arthritis medication that functions as a thioredoxin reductase inhibitor. Spermidine, a polyamine present in marine algae, can exert various physiological functions. Herein, we examined the synergistic anticancer activity of auranofin and spermidine in hepatocellular carcinoma (HCC). Combined treatment with auranofin and spermidine suppressed cell viability more efficiently than either treatment alone in HCC Hep3B cells. The isobologram plotted by calculating the half maximal inhibitory concentration (IC50) values of each drug indicated that the two drugs exhibited a synergistic effect. Based on the analysis of annexin V and cell cycle distribution, auranofin and spermidine markedly induced apoptosis in Hep3B cells. Moreover, auranofin and spermidine increased mitochondria-mediated apoptosis by promoting mitochondrial membrane potential (Δψm) loss. Auranofin and spermidine significantly increased reactive oxygen species (ROS) production in Hep3B cells, and the blocking ROS suppressed apoptosis induced by spermidine and auranofin. In addition, auranofin and spermidine reduced the expression of phosphorylated phosphatidylinositol-3 kinase (PI3K) and protein kinase B (Akt), and PI3K inhibitor accelerated auranofin- and spermidine-induced apoptosis. Using ROS scavenger and PI3K inhibitor, we revealed that ROS acts upstream of auranofin- and spermidine-induced apoptosis. Collectively, our study suggests that combination treatment with auranofin and spermidine could afford synergistic anticancer activity via ROS overproduction and reduced PI3K/Akt signaling pathway.

Anti-fibrotic effect of aurocyanide, the active metabolite of auranofin

Hyun Young Kim,Undarmaa Otgontenger,Jun‑Woo Kim,Young Joo Lee,Sang‑Bum Kim,Sung Chul Lim,Young‑Mi Kim,Keon Wook Kang 대한약학회 2023 Archives of Pharmacal Research Vol.46 No.3

Drug repositioning has gained significant attention over the past several years. The anti-rheumatoid arthritis drug auranofin has been investigated for the treatment of other diseases, including liver fibrosis. Because auranofin is rapidly metabolized, it is necessary to identify the active metabolites of auranofin that have detectable levels in the blood and reflect its therapeutic effects. In the present study, we investigated whether aurocyanide as an active metabolite of auranofin, can be used to evaluate the anti-fibrotic effects of auranofin. Incubation of auranofin with liver microsomes showed that auranofin was susceptible to hepatic metabolism. Previously, we found that the anti-fibrotic effects of auranofin are mediated via system xc–-dependent inhibition of the NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome. Therefore, we tried to identify active metabolites of auranofin based on their inhibitory effects on system xc– and NLRP3 inflammasome in bone marrow-derived macrophages. Among the seven candidate metabolites, 1-thio-β-D-glycopyrano-sato-S-(triethyl-phosphine)-gold(I) and aurocyanide potently inhibited system xc– and NLRP3 inflammasome. A pharmacokinetics study on mice detected significant plasma levels of aurocyanide after auranofin administration. Oral administration of aurocyanide significantly prevented thioacetamide-induced liver fibrosis in mice. Moreover, the in vitro anti-fibrotic effects of aurocyanide were assessed in LX-2 cells, where aurocyanide significantly decreased the migratory ability of the cells. In conclusion, aurocyanide is metabolically stable and detectable in plasma, and has inhibitory effects on liver fibrosis, suggesting that it is a potential marker of the therapeutic effects of auranofin.

Auranofin Suppresses Plasminogen Activator Inhibitor-2 Expression through Annexin A5 Induction in Human Prostate Cancer Cells

( Dong-won Shin ),( Yeo-jung Kwon ),( Dong-jin Ye ),( Hyoung-seok Baek ),( Joo-eun Lee ),( Young-jin Chun ) 한국응용약물학회 2017 Biomolecules & Therapeutics(구 응용약물학회지) Vol.25 No.2

Auranofin has been developed as antirheumatic drugs, which is currently under clinical development for the treatment of chronic lymphocytic leukemia. Previous report showed that auranofin induced apoptosis by enhancement of annexin A5 expression in PC-3 cells. To understand the role of annexin A5 in auranofin-mediated apoptosis, we performed microarray data analysis to study annexin A5-controlled gene expression in annexin A5 knockdown PC-3 cells. Of differentially expressed genes, plasminogen activator inhibitor (PAI)-2 was increased by annexin A5 siRNA confirmed by qRT-PCR and western blot. Treatment with auranofin decreased PAI-2 and increased annexin A5 expression as well as promoting apoptosis. Furthermore, auranofin-induced apoptosis was recovered by annexin A5 siRNA but it was promoted by PAI-2 siRNA. Interestingly, knockdown of annexin A5 rescued PAI-2 expression suppressed by auranofin. Taken together, our study suggests that induction of annexin A5 by auranofin may enhance apoptosis through suppression of PAI-2 expression in PC-3 cells.

Auranofin Downregulates Nuclear Factor-κB Activation via Nrf2-Independent Mechanism

Nam-Hoon Kim(김남훈),Hyo Jung Park(박효정),In-Sook Kim(김인숙) 한국생명과학회 2010 생명과학회지 Vol.20 No.12

내재면역반응의 중요한 조절자인 Nrf2와 NF-κB는 염증시에 교차 작용을 통하여 서로의 전사활성을 조절할 수 있다고 보고된 바 있으나 상반된 결과도 제시되고 있어서 아직까지 확실하게 규명되어 있지 않다. 저자들은 선행연구에서 NF-κB 저해제인 금(I)-화합물 오라노핀이 인간 관절활막세포와 단핵구성 세포에서 Nrf2를 활성화시킴을 확인한 바 있기 때문에, 본 연구에서는 Nrf2를 knockdown 시킨 류마티스성 활막세포를 사용하여 오라노핀에 의해 저해되는 NF-κB 신호전달 과정에 Nrf2가 관여하는지를 조사하였다. 세포를 Nrf2 siRNA로 transfection 시켰을 때 Nrf2 발현은 대부분 차단됨을 확인하였다. 하지만 Nrf2 knockdown은 TNF-α에 의해 유도되는 IκB-α 분해를 막는 오라노핀의 작용에는 영향을 주지 않았다. Nrf2 target 단백질로서 항염 작용에 관여하는 HO-1을 knockdown 시켰을 경우에도 IκB-α 분해를 저해하는 오라노핀의 작용에 영향을 미치지 않았다. 또한, Nrf2 knockdown은 오라노핀에 의해 저해된 ICAM-1 발현을 다시 복원시키지 못했다. 이러한 결과들은 염증성 싸이토킨에 의해 유도되는 NF-κB 활성화를 오라노핀이 저해하는 기전에 Nrf2 및 HO-1이 관련되어 있지 않음을 시사한다. 따라서 류마티스성 관절활막세포에서 오라노핀의 항염작용 기전으로 알려진 Nrf2/HO-1 활성유도와 NF-κB 활성저해는 교차작용 없이 각각 독립적인 기전을 통해 나타나는 것으로 생각된다. Transcription factors Nrf2 and NF-κB are important regulators of the innate immune response, and their cross-talks in inflammation have been reported. Previously, we demonstrated that gold(I)-compound auranofin, an inhibitor of NF-κB signal, induced Nrf2 activation in human synovial cells and monocytic cells. To investigate whether the Nrf2 activation is involved in the mechanism of the auranofin- attenuated NF-κB signaling, we examined the effects of Nrf2 knockdown on NF-κB activation using rheumatic synovial cells. When the cells were transfected with a specific siRNA for Nrf2, the gene expression was perfectly blocked. However, the Nrf2 knockdown did not cancel the suppressive effect of auranofin on TNF-α-induced IκB-α degradation. Treatment with a specific siRNA for HO-1, which is a target of Nrf2 and plays a role in anti-inflammation, also did not affect the blocking activity of auranofin on IκB-α degradation. In addition, auranofin-inhibited ICAM-1 expression was not restored by Nrf2 knockdown. These findings indicate that the activated Nrf2 and HO-1 are not associated with the suppressive action of auranofin on the pro-inflammatory cytokines-stimulated NF-κB activation. This suggests that Nrf2/HO-1 and NF-κB signals, which are regulated by auranofin, participate in the anti-inflammatory action of auranofin via independent pathways in rheumatic synovial cells.

TBK1-Targeted Suppression of TRIF-Dependent Signaling Pathway of Toll-like Receptor 3 by Auranofin

Se-Jeong Park,A-Neum Lee,윤형선 대한약학회 2010 Archives of Pharmacal Research Vol.33 No.6

Toll-like receptors (TLRs) play an important role in induction of innate immune responses. The stimulation of TLRs by microbial components triggers two branches of downstream signaling pathways: myeloid differential factor 88 (MyD88)- and toll-interleukin-1 receptor domain-containing adapter inducing interferon-β (TRIF)-dependent signaling pathways. Auranofin, a sulfur-containing gold compound (Au[I]), has been widely used for the treatment of rheumatoid arthritis. Since dysregulation of TLRs can lead to severe systemic inflammatory and joint destructive process in rheumatoid arthritis, auranofin-mediated modulation of TLR activation may have therapeutic potential against such diseases. Previously, we demonstrated that auranofin suppressed TLR4 signaling pathway by inhibiting TLR4 dimerization induced by LPS. Here, we examined the effect of auranofin on signal transduction via the TRIFdependent pathway induced by a TLR3 agonist. Auranofin inhibited nuclear factor-κB and interferon (IFN) regulatory factor 3 (IRF3) activation induced by polyinosinic-polycytidylic acid (poly[I:C]). Auranofin inhibited poly[I:C]-induced phosphorylation of IRF3 as well as IFNinducible genes such as IFN inducible protein-10. Furthermore, auranofin inhibited TBK1 kinase activity in vitro. All the results suggest that auranofin suppress TLR signaling at multiple steps.

Auranofin Suppresses Plasminogen Activator Inhibitor-2 Expression through Annexin A5 Induction in Human Prostate Cancer Cells

Shin, Dong-Won,Kwon, Yeo-Jung,Ye, Dong-Jin,Baek, Hyoung-Seok,Lee, Joo-Eun,Chun, Young-Jin The Korean Society of Applied Pharmacology 2017 Biomolecules & Therapeutics(구 응용약물학회지) Vol.25 No.2

Auranofin has been developed as antirheumatic drugs, which is currently under clinical development for the treatment of chronic lymphocytic leukemia. Previous report showed that auranofin induced apoptosis by enhancement of annexin A5 expression in PC-3 cells. To understand the role of annexin A5 in auranofin-mediated apoptosis, we performed microarray data analysis to study annexin A5-controlled gene expression in annexin A5 knockdown PC-3 cells. Of differentially expressed genes, plasminogen activator inhibitor (PAI)-2 was increased by annexin A5 siRNA confirmed by qRT-PCR and western blot. Treatment with auranofin decreased PAI-2 and increased annexin A5 expression as well as promoting apoptosis. Furthermore, auranofin-induced apoptosis was recovered by annexin A5 siRNA but it was promoted by PAI-2 siRNA. Interestingly, knockdown of annexin A5 rescued PAI-2 expression suppressed by auranofin. Taken together, our study suggests that induction of annexin A5 by auranofin may enhance apoptosis through suppression of PAI-2 expression in PC-3 cells.

Auranofin Suppresses Plasminogen Activator Inhibitor-2 Expression through Annexin A5 Induction in Human Prostate Cancer Cells

신동원,권여정,예동진,백형석,이주은,천영진 한국응용약물학회 2017 Biomolecules & Therapeutics(구 응용약물학회지) Vol.25 No.2

Auranofin has been developed as antirheumatic drugs, which is currently under clinical development for the treatment of chronic lymphocytic leukemia. Previous report showed that auranofin induced apoptosis by enhancement of annexin A5 expression in PC-3 cells. To understand the role of annexin A5 in auranofin-mediated apoptosis, we performed microarray data analysis to study annexin A5-controlled gene expression in annexin A5 knockdown PC-3 cells. Of differentially expressed genes, plasminogen activator inhibitor (PAI)-2 was increased by annexin A5 siRNA confirmed by qRT-PCR and western blot. Treatment with auranofin decreased PAI-2 and increased annexin A5 expression as well as promoting apoptosis. Furthermore, auranofin-induced apoptosis was recovered by annexin A5 siRNA but it was promoted by PAI-2 siRNA. Interestingly, knockdown of annexin A5 rescued PAI-2 expression suppressed by auranofin. Taken together, our study suggests that induction of annexin A5 by auranofin may enhance apoptosis through suppression of PAI-2 expression in PC-3 cells.

Auranofin Enhances Sulforaphane-Mediated Apoptosis in Hepatocellular Carcinoma Hep3B Cells through Inactivation of the PI3K/Akt Signaling Pathway

( Hyun Hwangbo ),( So Young Kim ),( Hyesook Lee ),( Shin-hyung Park ),( Su Hyun Hong ),( Cheol Park ),( Gi-young Kim ),( Sun-hee Leem ),( Jin Won Hyun ),( Jaehun Cheong ),( Yung Hyun Choi ) 한국응용약물학회 2020 Biomolecules & Therapeutics(구 응용약물학회지) Vol.28 No.5

The thioredoxin (Trx) system plays critical roles in regulating intracellular redox levels and defending organisms against oxidative stress. Recent studies indicated that Trx reductase (TrxR) was overexpressed in various types of human cancer cells indicating that the Trx-TrxR system may be a potential target for anti-cancer drug development. This study investigated the synergistic effect of auranofin, a TrxR-specific inhibitor, on sulforaphane-mediated apoptotic cell death using Hep3B cells. The results showed that sulforaphane significantly enhanced auranofin-induced apoptosis by inhibiting TrxR activity and cell proliferation compared to either single treatment. The synergistic effect of sulforaphane and auranofin on apoptosis was evidenced by an increased annexin-V-positive cells and Sub-G1 cells. The induction of apoptosis by the combined treatment caused the loss of mitochondrial membrane potential (ΔΨm) and upregulation of Bax. In addition, the proteolytic activities of caspases (-3, -8, and -9) and the degradation of poly (ADP-ribose) polymerase, a substrate protein of activated caspase-3, were also higher in the combined treatment. Moreover, combined treatment induced excessive generation of reactive oxygen species (ROS). However, treatment with N-acetyl-L-cysteine, a ROS scavenger, reduced combined treatment-induced ROS production and apoptosis. Thereby, these results deduce that ROS played a pivotal role in apoptosis induced by auranofin and sulforaphane. Furthermore, apoptosis induced by auranofin and sulforaphane was significantly increased through inhibition of the phosphoinositide 3-kinase (PI3K)/Akt pathway. Taken together, the present study demonstrated that down-regulation of TrxR activity contributed to the synergistic effect of auranofin and sulforaphane on apoptosis through ROS production and inhibition of PI3K/Akt signaling pathway.

급성전골수성백혈병 세포의 Thromnomoduline 발현에 미치는 Auranofin의 효과

이일하,전종률,김명신,김인숙 대한혈액학회 2005 Blood Research Vol.40 No.3

배경: 급성전골수성백혈병(acute promyelocytic leukemia, APL)에서는 혈액응고장애가 특징적으로 나타난다. 효과적인 APL 치료제로 사용되고 있는 all- trans retinoic acid (ATRA)와 arsenic trioxide (As2O3)는 혈액응고와 관련된 인자인 thrombomodulin (TM)과 tissue factor (TF)의 발현을 조절함으로써 혈액응고장애를 신속하게 개선시킨다고 알려져 있다. 최근에 저자들은 auranofin (AF)이 NB4 세포의 사멸과 분화유도에 관여함을 보고한 바 있으며 AF의 작용이 As2O3의 작용과 유사하였기 때문에 AF도 항응고기능이 있는지를 알아보고자 본 연구에서는 TM과 TF의 발현조절에 대한 AF의 효과를 조사하였다. 방법: APL 세포주인 NB4 세포에 AF을 1μM 농도로 가하고 12, 24 및 48시간 동안 배양하였다. 배양 후, AF에 의해 조절되는 TM 및 TF 발현양상은 RT-PCR, Northern blot과 Western blot 방법으로 확인하였다. 또한 유동세포분석법을 사용하여 세포표면에 존재하는 TM의 변화도 동시에 측정하였다. 결과: TM mRNA 발현량은 AF 처치 후 12시간부터 증가하였으나 TF mRNA 발현량은 변화를 보이지 않았다. 또한 AF에 의해 TM 단백질량도 함께 증가함을 알 수 있었으며 세포표면에서도 TM이 48시간까지 지속적으로 증가되어 있음을 확인하였다.

Synergistic induction of apoptosis by combination treatment with mesupron and auranofin in human breast cancer cells

이주은,권여정,백형석,예동진,조은아,최형균,오경수,천영진 대한약학회 2017 Archives of Pharmacal Research Vol.40 No.6

Urokinase-type plasminogen activator (uPA) hasbeen validated as a predictive or prognostic biomarkerprotein, and mesupron is considered the first-in-class anticanceragent to inhibit uPA activity in human breast cancer. In the present study, we showed that the synergismbetween mesupron and auranofin, a thioredoxin reductaseinhibitor, for inducing of apoptosis in MCF-7 human breastcancer cells. Our results demonstrated that mesupron andauranofin significantly lead to inhibition of the cancer cellsproliferation; cell cycle arrest at the G1/S phase of the cellcycle, and apoptosis as indicated by caspase 3 activation,poly(ADP-ribose) polymerase cleavage, and annexin Vstaining. Isobologram analyses of MCF-7 cells showed aclear synergism between mesupron and auranofin. Thiscombined treatment decreased the levels of mitochondrialanti-apoptotic factors, such as BCL-2, BCL-xL, and MCL-1 and caused nuclear translocation of apoptosis-inducingfactor. Mitochondrial membrane potential (Dwm) wasfound to be strongly disrupted in combination-treated cells. In addition, combination treatment significantly enhancedthe overproduction of reactive oxygen species, which wasrescued by N-acetylcysteine treatment. The combinationtreatment suppressed phosphorylation of Akt, thus contributingto apoptosis. Taken together, our data suggest thatthe use of mesupron in combination with auranofin may beimportant in achieving high anticancer synergy.