Inhibitory Effect of Cardamonin on Human Osteosarcoma Cells and Its Underlying Mechanism / 中国药学杂志

OBJECTIVEP: To analyze and confirm the inhibitory effect of cardamonin(CAR) on human osteosarcoma(OS) cells and its related mechanism. METHODS: Human osteosarcoma cells were treated with 0, 4, 12, 16 μmol•L-1 CAR and 0.08% DMSO, respectively. Cell proliferation was detected by crystal violet staining and MTT assay. Cell apoptosis was detected by Hoechst staining and flow cytometry(FCM). Cell migration ability was detected by scratch healing test. Cell invasion ability was detected by Transwell method. Western blot detects changes in cell proliferation, apoptosis, migration and invasion-associated proteins and Wnt/β-catenin signaling pathway-related proteins. RESULTS: CAR inhibits proliferation, migration and invasion of osteosarcoma cells. CAR inhibits the expression of PCNA, MMP-7 and vimentin. CAR inhibits the expression of Bcl-2, promotes the expression of apoptotic markers caspase 3 and cleaved-caspase 3. CAR inhibits the expression of the key molecule β-catenin of Wnt/β-catenin signaling and its downstream target molecules cyclin D1 and c-Myc. CONCLUSION: CAR can inhibit the proliferation, invasion and migration of osteosarcoma cells, but promote its apoptosis. Its molecular mechanism may be related to the interference of Wnt/beta-catenin signaling pathway activation.

Cardamonin from a medicinal herb protects against LPS-induced septic shock by suppressing NLRP3 inflammasome

Aberrant activation of NLRP3 inflammasome has been implicated in the pathogenesis of diverse inflammation-related diseases, and pharmacological molecules targeting NLRP3 inflammasome are of considerable value to identifying potential therapeutic interventions. Cardamonin (CDN), the major active ingredient of the traditional Chinese medicinal herb , has exerted an excellent anti-inflammatory activity, but the mechanism underlying this role is not fully understood. Here, we show that CDN blocks canonical and noncanonical NLRP3 inflammasome activation triggered by multiple stimuli. Moreover, the suppression of CDN on inflammasome activation is specific to NLRP3, not to NLRC4 or AIM2 inflammasome. Besides, the inhibitory effect is not dependent on the expression of NF-B-mediated inflammasome precursor proteins. We also demonstrate that CDN suppresses the NLRP3 inflammasome through blocking ASC oligomerization and speckle formation in a dose-dependent manner. Importantly, CDN improves the survival of mice suffering from lethal septic shock and attenuates IL-1 production induced by LPS , which is shown to be NLRP3 dependent. In conclusion, our results identify CDN as a broad-spectrum and specific inhibitor of NLRP3 inflammasome and a candidate therapeutic drug for treating NLRP3 inflammasome-driven diseases.

Determination of the raw material feeding (Gentiana scabra, Katsumade Galangal Seed and dried tangerine peel) in compound gentian tincture by HPLC-MS/MS / 国际中医中药杂志

Objective To establish a HPLC-MS/MS method for comprehensive monitor and control of the raw material feeding (Gentiana scabra,Katsumade Galangal Seed and dried tangerine peel),and determination of Gentiopicroside,Alpinetin,Cardamonin and Hesperidin in Compound gentian tincture.Methods The separation was performed on an Inertsil ODS-3 (4.6 mm× 150 mm,5 μm) analytical column with the mobile phase consisting of acetonitrile-0.1％ formic acid solution by gradient elution program,and the column temperature was 40 ℃.Active ingredients were separated by HPLC.The Electrospray Ionization Mass (ESI) source was applied and operated in the negative ion mode,and reactions ion monitoring mode (MRM) for quantitative analysis were selected.Results Through the analysis of the samples with mixed extract the same characteristic peak in MS was found to determine the proprietary Chinese medicine according to the prescription feeding process.The calibration curve of Gentiopicroside,Alpinetin,Cardamonin and Hesperidin were linear:103.26-619.56 μg (r=0.999 0),109.50-657.00 μg (r=0.999 5),105.50-633.00 μg (r=0.996 9),105.02-630.12 μg (r=0.999 5).The precision was Gentiopicroside 0.81％,Alpinetin 0.48％,Cardamonin 0.61％ and Hesperidin 1.55％ respectively.The average recovery rate were Gentiopicroside 95.08％,Alpinetin 93.28％,Cardamonin 94.78％ and Hesperidin 95.04％ respectively.Conclusions The method was proved to be simple,accurate,reliable,high sensitivity and can be used for determination and control of the raw material feeding (Gentiana scabra,Katsumade Galangal Seed and dried tangerine peel).

The regulatory effect of cardamonin on TLR4/MyD88/NF-κB/iNOS pathway / 中国药理学通报

Aim Toassesstheregulatoryeffectsofcar-damonin (CDN ) on toll-like receptor (TLR )-4/MyD88/NF-κB/iNOS signaling pathway in lipopolysac-charide (LPS )-stimulated RAW264. 7 macrophage cells.Methods LPS-stimulatedRAW264.7cells were divided into three groups:vehicle-treated group, LPS-treated group and LPS +CDN-treated group.Cell viability was assessed by CCK-8 assay.The concentra-tion of nitric oxide (NO)in cell culture medium was measured by Griess reagent.The mRNA levels of iN-OS,COX-2,MCP-1 ,TNF-α,IL-6 and IL-1βwere de-termined by reverse transcription real-time quantitative PCR(RT-qPCR).The protein levels of inducible nitric oxide synthase(iNOS),TLR4,myeloid differentiation factor 88(MyD88),nuclear factor κB(NF-κB)phos-phorylated (p )-p65 ,inhibitor κBα(IκBα),and p-IκBαweredeterminedbyWesternblot.Results 1~50 μmol·L-1 CDN had no cytotoxicity in RAW264. 7 cells.However,CDN inhibited the LPS-induced secre-tion of nitric oxide(NO)and mRNA expressions of iN-OS,COX-2,MCP-1 ,TNF-α,IL-6 and IL-1βin a dose-dependent manner.Moreover,50 μmol · L-1 CDN inhibited the LPS-induced up-regulation of iNOS, TLR4,MyD88,NF-κB p-p65,p-IκBαand down-reg-ulationofIκBα.Conclusion Cardamonininhibitsthe production of NO via a mechanism associated with the inhibition of TLR4/MyD88/NF-κB/iNOS pathway.

Research progress in antitumor activity of cardamonin and its derivatives / 肿瘤

Cardamonin (2, 4-dihydroxy-6-methoxychalcone, CAR), the main flavonoid that derived from the seed of Alpinia katsumadai, has received growing attention due to a wide range of biological activities, especially for antitumor activities. A series of homologous compounds are synthesized. Previous studies have demonstrated that CAR can inhibit the growth of glioblastoma, Lewis lung cancer, breast cancer, colon cancer, prostate cancer cells and so on. The mechanism underlying the antitumor effects of CAR is involved in the pathways of mammalian target of rapamycin (mTOR), nuclear factor-kappa B (NF-κβ), transglutaminase-2 (tgase-2), endogenous apoptosis, signal transducer and activator of transcription 3 (STAT3), E-cadherin, autophagy, β-catenin, and cell cycle regulation etc. This review summarizes the recent progress in the antitumor activities and mechanisms of CAR and its derivatives.

Cardamonin inhibits agonist-induced vascular contractility via Rho-kinase and MEK inhibition

The present study was undertaken to investigate the influence of cardamonin on vascular smooth muscle contractility and to determine the mechanism(s) involved. Denuded aortic rings from male rats were used and isometric contractions were recorded and combined with molecular experiments. Cardamonin significantly relaxed fluoride-, phenylephrine-, and phorbol ester-induced vascular contractions, suggesting that it has an anti-hypertensive effect on agonist-induced vascular contraction regardless of endothelial nitric oxide synthesis. Furthermore, cardamonin significantly inhibited the fluoride-induced increase in pMYPT1 level and phenylephrine-induced increase in pERK1/2 level, suggesting inhibition of Rho-kinase and MEK activity and subsequent phosphorylation of MYPT1 and ERK1/2. This study provides evidence that the relaxing effect of cardamonin on agonist-induced vascular contraction regardless of endothelial function involves inhibition of Rho-kinase and MEK activity.

Dimethyl Cardamonin Exhibits Anti-inflammatory Effects via Interfering with the PI3K-PDK1-PKCalpha Signaling Pathway

Consumption of herbal tea [flower buds of Cleistocalyx operculatus (Roxb.) Merr. et Perry (Myrtaceae)] is associated with health beneficial effects against multiple diseases including diabetes, asthma, and inflammatory bowel disease. Emerging evidences have reported that High mobility group box 1 (HMGB1) is considered as a key "late" proinflammatory factor by its unique secretion pattern in aforementioned diseases. Dimethyl cardamonin (2',4'-dihydroxy-6'-methoxy-3',5'-dimethylchalcone, DMC) is a major ingredient of C. operculatus flower buds. In this study, the anti-inflammatory effects of DMC and its underlying molecular mechanisms were investigated on lipopolysaccharide (LPS)-induced macrophages. DMC notably suppressed the mRNA expressions of TNF-alpha, IL-1beta, IL-6, and HMGB1, and also markedly decreased their productions in a time- and dose-dependent manner. Intriguingly, DMC could notably reduce LPS-stimulated HMGB1 secretion and its nucleo-cytoplasmic translocation. Furthermore, DMC dose-dependently inhibited the activation of phosphatidylinositol 3-kinase (PI3K), phosphoinositide-dependent kinase 1 (PDK1), and protein kinase C alpha (PKCalpha). All these data demonstrated that DMC had anti-inflammatory effects through reducing both early (TNF-alpha, IL-1beta, and IL-6) and late (HMGB1) cytokines expressions via interfering with the PI3K-PDK1-PKCalpha signaling pathway.

Cardamonin Suppresses TGF-beta1-Induced Epithelial Mesenchymal Transition via Restoring Protein Phosphatase 2A Expression

Epithelial mesenchymal transition (EMT) is the first step in metastasis and implicated in the phenotype of cancer stem cells. Therefore, understanding and controlling EMT, are essential to the prevention and cure of metastasis. In the present study, we examined, by Western blot, reverse transcription polymerase chain reaction (RT-PCR), and confocal microscopy, the effects of cardamonin (CDN) on transforming growth factor-beta1 (TGF-beta1)-induced EMT of A549 lung adenocarcinoma cell lines. TGF-beta1 induced expression of N-cadherin and decreased expression of E-cadherin. CDN suppressed N-cadherin expression and restored E-cadherin expression. Further, TGF-beta1 induced migration and invasion of A549 cancer cells, which was suppressed by CDN. TGF-beta1 induced c-Jun N-terminal kinase (JNK) activation during EMT, but CDN blocked it. Protein serine/threonine phosphatase 2A (PP2A) expression in A549 cancer cells was reduced by TGF-beta1 but CDN restored it. The overall data suggested that CDN suppresses TGF-beta1-induced EMT via PP2A restoration, making it a potential new drug candidate that controls metastasis.

Cardamonin induces apoptosis and expression of apoptosis-related proteins in K562 cells / 中国比较医学杂志

ObjectiveTo observe the apoptotic effect of cardamonin on K562 cells and its relationship with the expressions of PTEN, p-Akt, NF-κB and Bcl-2.Methods K562 cells were treated with cardamonin for 48 h, and the following tests were performed:(1) The cell morphology was observed by light microscopy.(2)IC50 of the K562 cells was dtermined by MTT test.(3) The apoptosis rate was detected by flow cytometry.(4) The expressions of Bcl-2 and Bax mRNA were detected a by RT-PCR.(5) The expressions of PTEN, p-Akt, NF-κB and Bcl-2 proteins were detected by Western blot.Results Obvious apoptosis was observed in the K562 cells after treated with cardamonin for 48 h.MTT assay indicated that the proliferation of K562 cells was obviously inhibited in a dose-and time-dependent manner. Comparing with the blank group, the early apoptosis rate and expression of Bax mRNA were significantly increased.At the same time, the expression of Bcl-2 mRNA was significantly decreased.All of them presented a dose-dependent manner. The expression of PTEN obviously increased with the increasing dose of cardamonin and the expressions of p-Akt, NF-κB and Bcl-2 were decreased.Conclusions Cardamonin promotes the apoptosis in K562 cells in a dose-dependent manner by increasing the expression of PTEN and decreasing the expressions of p-Akt, NF-κB, and Bcl-2.