Raptor mediates the selective inhibitory effect of cardamonin on RRAGC-mutant B cell lymphoma.

BACKGROUND: mTORC1 (mechanistic target of rapamycin complex 1) is associated with lymphoma progression. Oncogenic RRAGC (Rag guanosine triphosphatase C) mutations identified in patients with follicular lymphoma facilitate the interaction between Raptor (regulatory protein associated with mTOR) and Rag GTPase. It promotes the activation of mTORC1 and accelerates lymphomagenesis. Cardamonin inhibits mTORC1 by decreasing the protein level of Raptor. In the present study, we investigated the inhibitory effect and possible mechanism of action of cardamonin in RRAGC-mutant lymphoma. This could provide a precise targeted therapy for lymphoma with RRAGC mutations. METHODS: Cell viability was measured using a cell counting kit-8 (CCK-8) assay. Protein expression and phosphorylation levels were determined using western blotting. The interactions of mTOR and Raptor with RagC were determined by co-immunoprecipitation. Cells overexpressing RagC wild-type (RagCWT) and RagC Thr90Asn (RagCT90N) were generated by lentiviral infection. Raptor knockdown was performed by lentivirus-mediated shRNA transduction. The in vivo anti-tumour effect of cardamonin was assessed in a xenograft model. RESULTS: Cardamonin disrupted mTOR complex interactions by decreasing Raptor protein levels. RagCT90N overexpression via lentiviral infection increased cell proliferation and mTORC1 activation. The viability and tumour growth rate of RagCT90N-mutant cells were more sensitive to cardamonin treatment than those of normal and RagCWT cells. Cardamonin also exhibited a stronger inhibitory effect on the phosphorylation of mTOR and p70 S6 kinase 1 in RagCT90N-mutant cells. Raptor knockdown abolishes the inhibitory effects of cardamonin on mTOR. An in vivo xenograft model demonstrated that the RagCT90N-mutant showed significantly higher sensitivity to cardamonin treatment. CONCLUSIONS: Cardamonin exerts selective therapeutic effects on RagCT90N-mutant cells. Cardamonin can serve as a drug for individualised therapy for follicular lymphoma with RRAGC mutations.

Raptor couples mTORC1 and ERK1/2 inhibition by cardamonin with oxidative stress induction in ovarian cancer cells.

Background: A balance on nutrient supply and redox homeostasis is required for cell survival, and increased antioxidant capacity of cancer cells may lead to chemotherapy failure. Objective: To investigate the mechanism of anti-proliferation of cardamonin by inducing oxidative stress in ovarian cancer cells. Methods: After 24 h of drug treatment, CCK8 kit and wound healing test were used to detect cell viability and migration ability, respectively, and the ROS levels were detected by flow cytometry. The differential protein expression after cardamonin administration was analyzed by proteomics, and the protein level was detected by Western blotting. Results: Cardamonin inhibited the cell growth, which was related to ROS accumulation. Proteomic analysis suggested that MAPK pathway might be involved in cardamonin-induced oxidative stress. Western blotting showed that cardamonin decreased Raptor expression and the activity of mTORC1 and ERK1/2. Same results were observed in Raptor KO cells. Notably, in Raptor KO cells, the effect of cardamonin was weakened. Conclusion: Raptor mediated the function of cardamonin on cellular redox homeostasis and cell proliferation through mTORC1 and ERK1/2 pathways.

Cardamonin suppresses pro-tumor function of macrophages by decreasing M2 polarization on ovarian cancer cells via mTOR inhibition.

Ovarian cancer is the most fatal tumor characterized by an abundance of tumor-associated macrophage (TAM) infiltrations in women. Functional TAMs, which mainly present M2-like phenotypes and perform key functions on tumor progress, have been considered an attractive target for ovarian cancer therapy. Cardamonin showed an excellent antitumor activity in multiple tumor cells. This study aimed to investigate the role of cardamonin on TAMs. With the conditioned medium of ovarian cancer cells, macrophages were induced to TAMs and, accordingly, promoted the proliferation, migration, and invasion of ovarian cancer cells. Cardamonin suppressed alternatively activated (M2) polarization of TAMs and downregulated TAM-secreted tumorigenic factors, thereby hindering the pro-tumor function of TAMs on ovarian cancer cells. Moreover, cardamonin inhibited tumor growth in xenograft nude mice and lowered the expression of CD163 and CD206. Mechanistically, cardamonin inhibited the phosphorylation of mammalian target of rapamycin (mTOR) and signal transducer and activator of transcription 3 (STAT3), resulting in the suppression of M2 polarization. Furthermore, STAT3 is tightly related with mTOR activity. Altogether, these findings implied that cardamonin suppresses the pro-tumor function of TAMs by decreasing M2 polarization via mTOR inhibition, and cardamonin may be a potential therapeutic agent for ovarian cancer.

Cardamonin inhibits the expression of P-glycoprotein and enhances the anti-proliferation of paclitaxel on SKOV3-Taxol cells.

Paclitaxel is widely used in the first-line treatment of ovarian cancer. Nevertheless, the development of acquired resistance to paclitaxel is a major obstacle for the therapy in clinic. Cardamonin is a novel anticancer chalcone which exhibits a wide range of pharmacological activities. However, the effect of cardamonin on paclitaxel-resistant ovarian cancer cells and its underlying molecular mechanisms are unknown. Here, we revealed whether cardamonin had a resensitivity for paclitaxel and furtherly explored the underlying mechanisms on SKOV3-Taxol cells. Our results showed that cardamonin combined with paclitaxel had a synergistic effect of anti-proliferation in SKOV3-Taxol cells, and CI was less than one. Cells apoptosis and G2/M phase arrest were enhanced by cardamonin with paclitaxel in a concentration-dependent way on SKOV3-Taxol cells (P < 0.05). Cardamonin significantly increased drug accumulation in SKOV3-Taxol cells (P < 0.05). Similar to verapamil, cardamonin decreased MDR1 mRNA and P-gp expression (P < 0.05). Cardamonin restrained NF-κB activation in SKOV3-Taxol cells (P < 0.05). Inhibitory effect of P-gp and NF-κB p65 (nuclear protein) expression was enhanced by cardamonin combined with PDTC, a NF-κB inhibitor. Cardamonin significantly inhibited the upregulation of NF-κB p65 (nuclear protein) and P-gp expression induced by TNF-α (P < 0.05). Taken together, cardamonin enhanced the effect of paclitaxel on inhibiting cell proliferation, inducing apoptosis and G2/M phase arrest, and then strengthened the cytotoxic effect of paclitaxel in SKOV3-Taxol cells. The mechanism might be involved in inhibition of P-gp efflux pump, reducing MDR1 mRNA and P-gp expression by cardamonin via suppression of NF-κB activation in SKOV3-Taxol cells.

Establishment and application of population pharmacokinetics model of vancomycin in infants with meningitis.

BACKGROUND: To establish a population pharmacokinetics (PPK) model of vancomycin (VCM) for dose individualization in Chinese infants with meningitis. METHODS: We collected the data of 82 children with meningitis in hospital from July 2014 to June 2016. The initial vancomycin dosage regimen for children was 10 or 15 mg/kg for q12 h, q8 h or q6 h. Serum concentrations were determined by Viva-E Analyzer before and after the fifth administration. The PPK model was developed by nonlinear mixed-effect model software, assessed by the bootstrap method and then tested in 20 infant patients. RESULTS: The VCM clearance (CL) was increased by body weight (WT) and decreased by blood urea nitrogen (BUN). Pharmacokinetic parameters of VCM were not influenced by co-administered drugs. The trough concentrations of VCM were accurately predicted by the PPK model, with the prediction errors less than 32%. CONCLUSION: A new individual strategy for VCM regimens was proposed and validated by the PPK model.

A meta-analysis of the efficacy of progestin-primed ovarian stimulation with medroxyprogesterone acetate in ovulation induction in poor ovarian responders.

PURPOSE: To systematically evaluate the effect of progestin-primed ovarian stimulation (PPOS) in in vitro fertilization (IVF)/oocyte intracytoplasmic sperm injection-embryo transfer (ICSI-ET) in patients with poor ovarian response and to find an optimal ovulation induction protocol for such patients. METHOD: A literature search of PubMed, Medline, EBSCO, Cochrane Library, Vip.com, CNKI, and the Wanfang database was conducted to find case-control studies of PPOS with medroxyprogesterone acetate and other traditional stimulation regimens for ovulation induction in patients with poor ovarian response. The period of time searched was from the database establishment to August 2020. Patients in the experimental group underwent PPOS and those in the control group underwent another program (e.g., the gonadotropin-releasing hormone antagonist protocol). RevMan 5.3 software was used for meta-analysis. RESULTS: A total of sixteen case-control studies (one of them is randomized controlled trial), with 4422 induction cycles, were included. All the included patients met the 2011 Bologna diagnostic criteria for poor ovarian response. The numbers of mature eggs, available embryos, optimal embryos, and the rate of cumulative pregnancies in the PPOS group were all better than those in the control group (P<0.05). There was a lower Serum luteinizing hormone on the day of human chorionic gonadotropin (HCG) injection and a lower rate of cycle cancellation in the PPOS group (P<0.05). No other differences between PPOS and other treatments were statistically significant. CONCLUSION: PPOS can reduce the need for cycle cancellation, improve the follicles and embryos, and improve the pregnancy rate and thus, can present an effective choice for IVF/ICSI-ET in patients with poor ovarian response.

Cardamonin inhibits cell proliferation by caspase-mediated cleavage of Raptor.

The antiproliferative effect of cardamonin on mTORC1 is related with downregulation of Raptor. We investigated the mechanism that cardamonin decreases Raptor expression through caspase-mediated protein degradation. SKOV3 cells and HeLa cells were pretreated with caspase inhibitor z-VAD-fmk for 30 min and then exposed to different doses of cardamonin and cisplatin, respectively. We analyzed the gene expression of caspases based on TCGA and GTEx gene expression data in serous cystadenocarcinoma and normal tissues, monitored caspase activity by caspase colorimetric assay kit, detected expression of mTORC1-associated proteins and apoptosis-associated proteins by western blotting, and finally detected cell viability by methyl thiazolyl tetrazolium (MTT) assay. A different expression of caspases except caspase-1 was found between serous cystadenocarcinoma and normal tissues. Raptor was cleaved when caspases were activated by cisplatin and caspase-6/caspase-8 was activated by cardamonin in SKOV3 cells. We further used a monoclonal antibody recognizing the N-terminal part of Raptor to find that Raptor was cleaved into a smaller fragment of about 70 kDa by cardamonin and was rescued by z-VAD-fmk treatment. As a result of Raptor cleavage, mTORC1 activity was decreased and cell viability was inhibited, while cell apoptosis was induced in SKOV3 cells. Notably, similar results are only observed in HeLa cells with a high dose of cardamonin. We concluded that caspase-mediated cleavage of Raptor might be an important mechanism in that cardamonin regulated Raptor and mTORC1 activity.

High PRAS40 mRNA expression and its role in prognosis of clear cell renal cell carcinoma.

BACKGROUND: Clear cell renal cell carcinoma (ccRCC) is one of the most common type of kidney malignancy. The proline-rich Akt substrate of 40 kDa (PRAS40) plays an important role in tumor growth. The present study aimed to analysis the prognostic value of PRAS40 mRNA expression in ccRCC. METHODS: We analyzed the PRAS40 mRNA expression using the data from TCGA-KIRC cohort. A receiver operating characteristic (ROC) curve was performed to assessed the diagnostic value of PRAS40 mRNA expression in ccRCC. Chi-square test was used to analyzed the correlation between clinical characteristics and PRAS40 mRNA expression. Kaplan-Meier analysis and Cox analysis were performed to determine the prognostic value of PRAS40 mRNA expression in ccRCC. Gene set enrichment analysis (GSEA) was conducted using TCGA database. RESULTS: Our results revealed that PRAS40 mRNA expression was higher in ccRCC tissues than in normal tissues. PRAS40 presented a moderate diagnostic value in ccRCC. High PRAS40 mRNA expression was correlated with histological grade, clinical stage, T classification, distant metastasis and vital status of ccRCC. High PRAS40 mRNA expression was associated with poor overall survival. Furthermore, Multivariate analysis revealed that PRAS40 was an independent risk factor for ccRCC patients. Myc targets, DNA repair, oxidative phosphorylation, glycolysis, adipogenesis, p53 pathway, reactive oxygen species pathway, myogenesis were differentially enriched in the phenotype that positively correlated with PRAS40. CONCLUSIONS: In conclusion, our results suggest that PRAS40 was a promising diagnostic and prognostic biomarker for ccRCC.

DAP1 negatively regulates autophagy induced by cardamonin in SKOV3 cells.

Autophagy is closely related to the formation and development of multiple human tumors including ovarian cancer. As a major regulator of this process, the role of mTOR (mammalian target of rapamycin) has been well proven. Cardamonin, a kind of flavonoid from plants, has effects on induction of autophagy and thus antiproliferation of cancer cells. However, the detailed mechanism remains unclear. DAP1 (death-associated protein 1) is a proline-rich protein, which is involved in the regulation of cellular growth and programmed cell death including autophagy and apoptosis. The aim of this study was to investigate whether DAP1 is involved in proliferation inhibition and autophagy induced by cardamonin in tumor cells. Using online bioinformatics tools, we found that DAP1 expression is closely related to the survival of patients with ovarian cancer. Our study showed that autophagy induced by cardamonin was associated with mTOR inhibition, and DAP1 was involved in this process. Silence of DAP1 decreased cell proliferation but enhanced the antiproliferative effect of cardamonin in SKOV3 cells. The level of autophagy was elevated by DAP1 silencing in SKOV3 cells. Notably, cardamonin showed higher autophagy flux in the DAP1 small interfering RNA group. Taken together, our results implied that DAP1 negatively regulates autophagy induced by cardamonin, and it may be a potential target for ovarian cancer therapy.

Anti­proliferative effect of cardamonin on mTOR inhibitor­resistant cancer cells.

A number of mammalian target of rapamycin (mTOR) inhibitors have been approved for the treatment of certain types of cancer or are currently undergoing clinical trials. However, mTOR targeted therapy exerts selective pressure on tumour cells, which leads to the preferential growth of resistant subpopulations. There are two classes of mTOR inhibitors: i) The rapalogs, such as rapamycin, which bind to the 12­kDa FK506­binding protein/rapamycin­binding domain of mTOR; and ii) the ATP­competitive inhibitors, such as AZD8055, which block the mTOR kinase domain. Cardamonin inhibits mTOR by decreasing the expression of regulatory­associated protein of mTOR (Raptor), a mechanism of action which differs from the currently available mTOR inhibitors. The present study investigated the inhibitory effects of cardamonin on mTOR inhibitor­resistant cancer cells. HeLa cervical cancer cells and MCF­7 breast cancer cells were exposed to high concentrations of mTOR inhibitors, until resistant clones emerged. Cytotoxicity was measured using the MTT and colony forming assays. The inhibitory effect of cardamonin on mTOR signalling was assessed by western blotting. The resistant cells were less sensitive to mTOR inhibitors compared with the parental cells. Consistent with the anti­proliferation effect, rapamycin and AZD8055 had no effect on the phosphorylation of rapamycin­sensitive sites on ribosomal protein S6 kinase B1 (S6K1) and AZD8055­sensitive sites on protein kinase B and eukaryotic translation initiation factor 4E binding protein 1 (Thr 37/46), respectively, in rapamycin­ and AZD8055­resistant cells. Cardamonin inhibited cell proliferation and decreased the phosphorylation of mTOR and S6K1, as well as the protein level of raptor, in the mTOR inhibitor­resistant cells. Therefore, cardamonin may serve as a therapeutic agent for patients with cervical and breast cancer resistant to mTOR inhibitors.

Contribution of genetic polymorphism of methylene tetrahydrofolate reductase on the effect of methotrexate in ectopic pregnancy patients.

BACKGROUND: Methotrexate (MTX) is the prior drug in ectopic pregnancy (EP). However, approximately 10% of patients suffer from failure by MTX therapy. Reduced folate carrier 1 (RFC1), methylene tetrahydrofolate reductase (MTHFR), and dihydrofolate reductase (DHFR) are involved in the transport and effects of MTX in vivo. In the present study, we aim to investigate the relationship between the genetic polymorphisms of RFC1, MTHFR, and DHFR and the clinical efficacy of MTX in tubal pregnancies. METHODS: 100 patients of EP were enrolled in this study. Polymorphisms of RFC1 G80A, MTHFR C677T, and DHFR A-317G were genotyped. ß-hCG level was detected in day 0, 4, and 7 after MTX injection. Association of MTX efficacy and genetic polymorphisms was analyzed. RESULTS: Methylene tetrahydrofolate reductase C677T was associated with MTX treatment (P = .017). The success rate of first MTX injection was superior in patients with harboring mutation allele of MTHFR gene than that in patients with wild-type gene (P = .001). However, there was no significant association between the polymorphisms of RFC1 G80A, DHFR A-317G, and surgical treatment (P = .709 and .476, respectively). In addition, ß-hCG level decrement was not significantly changed by MTX injection with different polymorphisms of RFC1, MTHFR, and DHFR on either day 4 (P = .214, 0.197 and 0.270, respectively) or day 7 (P = .172, .554, and .726, respectively). CONCLUSION: Our results suggested that the reliable indicator was polymorphism of MTHFR C677T in failure by MTX injection.

Glycolysis inhibition via mTOR suppression is a key step in cardamonin-induced autophagy in SKOV3 cells.

BACKGROUND: Autophagy occurs in cells that undergoing nutrient deprivation. Glycolysis rapidly supplies energy for the proliferation of cancer cells. Cardamonin inhibits proliferation and enhances autophagy by mTORC1 suppression in ovarian cancer cells. Here, we investigate the relationship between cardamonin-triggered autophagy and glycolysis inhibition via mTORC1 suppression. METHODS: Treated with indicated compounds, ATP content and the activity of hexokinase (HK) and lactate dehydrogenase (LDH) were analyzed by the assay kits. Autophagy was detected by monodansylcadaverin (MDC) staining. The relationship between cardamonin-triggered autophagy and glycolysis inhibition via mTORC1 suppression was analyzed by Western blot. RESULTS: We found that cardamonin inhibited the lactate secretion, ATP production, and the activity of HK and LDH. The results demonstrated that cardamonin enhanced autophagy in SKOV3 cells, as indicated by acidic compartments accumulation, microtubule-associated protein 1 Light Chain 3-II (LC3-II) and lysosome associated membrane protein 1 up-regulation. Our results showed that the activation of mTORC1 signaling and the expression HK2 were reduced by cardamonin; whereas the phosphorylation of AMPK (AMP-activated protein kinase) was increased. We also confirmed that the AMPK inhibitor, Compound C, reversed cardamonin-induced upregulation of LC3-II. CONCLUSION: These results suggest that cardamonin-induced autophagy is associated with inhibition on glycolysis by down-regulating the activity of mTORC1 in ovarian cancer cells.

Autophagy induced by cardamonin is associated with mTORC1 inhibition in SKOV3 cells.

BACKGROUND: The mammalian target of rapamycin (mTOR) integrates energy level to modulate cell proliferation and autophagy. Cardamonin exhibits anti-proliferative activity through inhibiting mTOR. In this study, the effect of cardamonin on autophagy and its mechanism on mTOR inhibition were investigated. METHODS: Cell viability and proliferation were measured by MTT assay and BrdU incorporation, respectively. Cell apoptosis was assayed by flow cytometry and cell autophagy was detected by electron microscopy and GFP-LC3 fluorescence. The mechanism of cardamonin on mTORC1 inhibition was investigated by Raptor siRNA and Raptor over-expression. RESULTS: The cell viability and proliferation were inhibited by cardamonin. The autophagosomes and the protein level of LC3-II were increased by cardamonin. Cell apoptosis and the levels of cleaved PARP and Caspase-3 were increased by cardamonin. Cardamonin inhibited the phosphorylation of mTOR and ribosome S6 protein kinase 1 (S6K1) as well as the protein level of regulatory associated protein of mTOR (Raptor). However, cardamonin had no effect on the component of mTORC2 and its downstream substrate Akt. The inhibitory effect of cardamonin on the phosphorylation of mTOR and S6K1 was eliminated by Raptor knockdown with siRNA, whereas this effect of cardamonin was stronger than that of rapamycin and AZD8055 in Raptor over-expression cells. Cell viability was inhibited by cardamonin in both Raptor knockdown and Raptor over-expression cells, which was consistent with the inhibitory effect of cardamonin on mTOR. CONCLUSION: These findings demonstrated that the autophagy induced by cardamonin was associated with mTORC1 inhibition through decreasing the protein level of Raptor in SKOV3 cells.

Anti-inflammatory Effects of Cardamonin in Ovarian Cancer Cells Are Mediated via mTOR Suppression.

Cardamonin exhibits a variety of pharmacological activities including anti-inflammatory and antitumor, which are correlated with the inhibition of nuclear factor-kappaB and the mammalian target of rapamycin, respectively. However, whether the anti-inflammatory effects of cardamonin are mediated by the mammalian target of rapamycin remains unknown. In this study, ovarian cancer SKOV3 cells were cultured with lipopolysaccharide to induce inflammation, and the inhibitory effects and underlying molecular mechanisms of cardamonin were investigated using specific inhibitors of the mammalian target of rapamycin and the nuclear factor-kappaB pathway (rapamycin and pyrrolidine dithiocarbamate, respectively). Our results indicated that cardamonin inhibited the viability of normal and lipopolysaccharide-pretreated SKOV3 cells in a concentration-dependent manner. In accordance with rapamycin, the activation of the mammalian target of rapamycin and its downstream target, ribosomal protein S6 kinase 1, was inhibited by cardamonin, while pyrrolidine dithiocarbamate substantially blocked nuclear factor-kappaB activation and mildly inhibited the phosphorylation of the mammalian target of rapamycin and ribosomal protein S6 kinase 1. Pretreated with pyrrolidine dithiocarbamate, the effect of cardamonin on the mammalian target of rapamycin signalling was not affected, but the expression of inflammatory factors was further reduced. In cells pretreated with rapamycin, the inhibitory effects of cardamonin were completely suppressed with regards to the phosphorylation of the mammalian target of rapamycin, ribosomal protein S6 kinase 1, TNF-α, and interleukin-6, and nuclear factor-kappaB p65 protein expression was decreased. In conclusion, our findings indicate that the anti-inflammatory effects of cardamonin are correlated with mammalian target of rapamycin inhibition.

CYP3A4 and GCK genetic polymorphisms are the risk factors of tacrolimus-induced new-onset diabetes after transplantation in renal transplant recipients.

PURPOSE: We intend to investigate the association between tacrolimus-induced new-onset diabetes after transplantation (NODAT) and polymorphisms of CYP3A4, CYP3A5, ATP-binding cassette transporter sub-family C member 8 (ABCC8), and glucokinase (GCK) in renal transplant recipients. METHODS: Polymorphisms of CYP3A4 *18B, CYP3A5 *3, ABCC8 T-3C, and GCK G-30A were genotyped in 169 renal transplant recipients. Trough concentrations of tacrolimus were detected by an ELISA kit. The relative materials were collected in all patients and volunteers. The association of NODAT and polymorphisms was analyzed. RESULTS: CYP3A4 *18B and GCK G-30A were related to NODAT (p < 0.05). The lower concentration/dose or fasting serum glucose was in CYP3A4 *1/*1 carriers than that in *18B/*18B carriers in all the renal transplant recipients (p < 0.05), respectively. Genotype of ABCC8 T-3C was associated with fasting serum glucose in both NODAT and non-NODAT patients (p < 0.05). Furthermore, family history of DM (OR = 3.734, p = 0.002), concentration/dose above 70 ([ng/mL]/[mg/kg]) (OR = 2.154, p = 0.034) and GCK G-30A (OR = 2.272, p = 0.026) were independently correlated with the incidence of NODAT in logistic regression. CONCLUSIONS: The polymorphisms of CYP3A4 *18B and GCK G-30A were related to NODAT induced by tacrolimus.

Cardamonin enhances the anti-proliferative effect of cisplatin on ovarian cancer.

The mammalian target of rapamycin (mTOR) is well-known as a promising therapeutic target in various cancer cells. mTOR activation decreases the sensitivity of ovarian cancer to cisplatin. Cardamonin inhibits the proliferation of various cancer cells by mTOR suppression. The present study examined whether cardamonin combined with cisplatin is efficacious for the anti-proliferation of ovarian cancer cells. The anti-proliferative effect was determined by MTT and cell cycle assays. Activation of the mTOR signal pathway and the expression of anti-apoptotic proteins were evaluated by western blot analysis. Cardamonin significantly enhanced the effects of cisplatin on cell proliferation and cell cycle progression. The expression of B cell lymphoma-2, X-linked inhibitor of apoptosis protein and Survivin was significantly decreased following combination treatment. Furthermore, the activation of mTOR and its downstream 70 kDa ribosomal protein S6 kinase was inhibited by cardamonin. These results demonstrated that the combinatorial effects of cardamonin and cisplatin on anti-proliferation were enhanced by suppressing the expression of anti-apoptotic proteins and activation of mTOR in ovarian cancer cells.

Raptor mediates the antiproliferation of cardamonin by mTORC1 inhibition in SKOV3 cells.

PURPOSE: Cardamonin inhibits the proliferation of SKOV3 cells by suppressing the mammalian target of rapamycin complex 1 (mTORC1). However, the mechanism of cardamonin on mTORC1 inhibition has not been well demonstrated. The regulatory-associated protein of TOR (Raptor) is an essential component of mTORC1. Here, we investigated the role of Raptor in the mTORC1 inhibition effect of cardamonin in SKOV3 cells. METHODS: The expression of Raptor was knockdown by small interfering RNA (siRNA). The expressions of specific binding proteins of mTORC1 were analyzed by Western blotting, and the cell proliferation was detected by methyl thiazolyl tetrazolium (MTT) assay. RESULTS: Rapamycin, AZD8055, and cardamonin inhibited the activity of mammalian target of rapamycin (mTOR). Different from rapamycin and AZD8055, cardamonin suppressed the phosphorylation and protein expression of Raptor. Transfected with Raptor siRNA, the mTOR activation and proliferation of SKOV3 cells were decreased, and these effects were strengthened by cardamonin in Raptor siRNA SKOV3 cells. Cardamonin interfered with the lysosomal colocalization of mTOR with lysosomal associated membrane protein 2 (LAMP2), which was also hindered by Raptor siRNA. Furthermore, cardamonin strengthened the inhibitory effect on the lysosomal localization of mTOR in Raptor siRNA cells. CONCLUSION: Our results suggested that Raptor mainly mediated the inhibition of cardamonin on mTORC1 in SKOV3 cells.

Cardamonin Inhibits Angiogenesis by mTOR Downregulation in SKOV3 Cells.

The mammalian target of rapamycin is critical in hypoxia-triggered angiogenesis. Cardamonin inhibits proliferation of various cancer cells through suppressing the mammalian target of rapamycin. In this study, the antiangiogenic effect of cardamonin on CoCl2-mimicked hypoxic SKOV3 cells was investigated. Cardamonin exhibited an antiproliferative effect on normal and CoCl2-mimicked hypoxic SKOV3 cells. Messenger RNA expression of vascular endothelial growth factor was inhibited with cardamonin and rapamycin in SKOV3 cells under both conditions. However, cardamonin had little effect on the messenger RNA expression of hypoxia-inducible factor-α. Cardamonin inhibited the protein expression of hypoxia-inducible factor-1α, hypoxia inducible factor-2α, vascular endothelial growth factor, and the phosphorylation of mammalian target of rapamycin and ribosomal S6 kinase 1. Furthermore, angiogenesis induced by a medium of SKOV3 cells was reduced by cardamonin in a chicken embryo allantois membrane model. These findings suggest that cardamonin inhibits protein expression of hypoxia-inducible factor-α, and vascular endothelial growth factor, which was induced by CoCl2-mimicked hypoxia and this effect partially correlates with the mammalian target of rapamycin inhibition. Cardamonin might be a potential angiogenesis inhibitor for ovarian cancer therapy.

Cardamonin Inhibits Metastasis of Lewis Lung Carcinoma Cells by Decreasing mTOR Activity.

The mammalian target of rapamycin (mTOR) regulates the motility and invasion of cancer cells. Cardamonin is a chalcone that exhibits anti-tumor activity. The previous study had proved that the anti-tumor effect of cardamonin was associated with mTOR inhibition. In the present study, the anti-metastatic effect of cardamonin and its underlying molecule mechanisms were investigated on the highly metastatic Lewis lung carcinoma (LLC) cells. The proliferation, invasion and migration of LLC cells were measured by MTT, transwell and wound healing assays, respectively. The expression and activation of mTOR- and adhesion-related proteins were assessed by Western blotting. The in vivo effect of cardamonin on the metastasis of the LLC cells was investigated by a mouse model. Treated with cardamonin, the proliferation, invasion and migration of LLC cells were significantly inhibited. The expression of Snail was decreased by cardamonin, while that of E-cadherin was increased. In addition, cardamonin inhibited the activation of mTOR and its downstream target ribosomal S6 kinase 1 (S6K1). Furthermore, the tumor growth and its lung metastasis were inhibited by cardamonin in C57BL/6 mice. It indicated that cardamonin inhibited the invasion and metastasis of LLC cells through inhibiting mTOR. The metastasis inhibitory effect of cardamonin was correlated with down-regulation of Snail and up-regulation of E-cadherin.

mTOR inhibition of cardamonin on antiproliferation of A549 cells is involved in a FKBP12 independent fashion.

AIMS: Cardamonin has previously demonstrated that it had an antiproliferative effect on vascular smooth muscle cells by inhibiting the activity of mammalian target of rapamycin (mTOR). The antiproliferative effect and the possible mechanism of combining with mTOR of cardamonin were investigated on A549 cells. MAIN METHODS: Cell proliferation, cell cycle and apoptosis were measured by methyl thiazolyl tetrazolium (MTT) and flow cytometry, respectively. mTOR and 12 kDa FK506 binding protein (FKBP12) were transfected into A549 cells by Lipofectamine. Western blots were used to examine the mTOR expressions and its activities, and the expressions of 70 kDa ribosomal S6 kinase (p70S6K), FKBP12 and Interleukin-2 (IL-2), respectively. KEY FINDINGS: Treated with cardamonin, the proliferation of A549 cells was inhibited. Meanwhile, cell cycle was blocked and DNA synthesis was decreased whereas cell apoptosis was promoted, and the activation of mTOR and p70S6K was decreased by cardamonin. Transfected with mTOR or FKBP12, proliferation of A549 cells was increased. Rapamycin had a similar degree of effect on antiproliferation of both transfected cells. However, the antiproliferative effect of cardamonin on mTOR transfected cells was stronger than that on FKBP12 transfected cells. Both rapamycin and cardamonin decreased the phosphorylation of mTOR and p70S6K in two kinds of transfected cells. Cardamonin had no effect on the expression of FKBP12 and IL-2, whereas the expressions were decreased by rapamycin. SIGNIFICANCE: Cardamonin inhibited proliferation and induced apoptosis of A549 cells via mTOR. It might directly interact with mTOR independently of binding with FKBP12.