OBJECTIVE: This study was designed to investigate the therapeutic and anti-metastatic potential of the two newly obtained anti-nNav1.5 antibodies, polyclonal anti-nNav1.5 (pAb-nNav1.5) and monoclonal anti-nNav1.5 (mAb-nNav1.5), on breast cancer invasion and metastasis.
METHODS: MDA-MB-231 and 4T1 cells were used as in vitro models to study the effect of pAb-nNav1.5 (59.2 µg/ml) and mAb-nNav1.5 (10 µg/ml) (24 hours treatment) on cell invasion. 4T1-induced mammary tumours in BALB/c female mice were used as an in vivo model to study the effect of a single dose of intravenous pAb-nNav1.5 (1 mg/ml) and mAb-nNav1.5 (1 mg/ml) on the occurrence of metastasis. Real-time PCR and immunofluorescence staining were conducted to assess the effect of antibody treatment on nNav1.5 mRNA and protein expression, respectively. The animals' body weight, organs, lesions, and tumour mass were also measured and compared.
RESULTS: pAb-nNav1.5 and mAb-nNav1.5 treatments effectively suppressed the invasion of MDA-MB-231 and 4T1 cells in the 3D spheroid invasion assay. Both antibodies significantly reduced nNav1.5 gene and protein expression in these cell lines. Treatment with pAb-nNav1.5 and mAb-nNav1.5 successfully reduced mammary tumour tissue size and mass and prevented lesions in vital organs of the mammary tumour animal model whilst maintaining the animal's healthy weight. mRNA expression of nNav1.5 in mammary tumour tissues was only reduced by mAb-nNav1.5.
CONCLUSION: Overall, this work verifies the uniqueness of targeting nNav1.5 in breast cancer invasion and metastasis prevention, but more importantly, humanised versions of mAb-nNav1.5 may be valuable passive immunotherapeutic agents to target nNav1.5 in breast cancer.
METHODS: Potential CYP inhibition by F3 was first determined using fluorescence assays, using known CYP inhibitors as reference. To further ascertain the inhibitory potency and mode of inhibition, high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analysis of specific metabolites of a CYP probe substrate was conducted.
RESULTS: The half-maximal inhibitory concentration (IC50) values indicate that F3 exhibited relatively weak inhibition on CYP2B6, CYP2C19, CYP2D6, and CYP3A4. Highest susceptibility to inhibition by F3 was observed for CYP2C9, where the IC50 value from fluorescence-based assay was 35-fold higher than control. Further analysis by HPLC-MS/MS revealed relatively weak mixed-type inhibition of F3 on CYP2C9, as indicated by IC50 and inhibition constant (KI) values. The risk of clinically significant CYP2C9 inhibition by F3 was then predicted based on the attained KI value and the presumed amount of F3 absorbed from S. crispus leaves following consumption. The calculated maximum plasma concentration to inhibition constant Cmax/KI) ratio suggests that F3 consumption could potentially result in clinically significant drug interactions with medications metabolized by CYP2C9.
CONCLUSION: Taken together, the results revealed a low probability of inhibition by F3 on CYP enzymes involved in tamoxifen metabolism. However, further in vivo investigation is necessary for potential F3 interaction with CYP2C9. The utility of a preliminary in vitro approach in the assessment of potential HDI was demonstrated in this study.
OBJECTIVE: This study aims to determine the effect of S. crispus active fraction (F3) and its bioactive components on glycolysis in triple-negative breast cancer cells (MDA-MB-231).
METHODS: This study utilizes F3, lutein, β-sitosterol, and stigmasterol to be administered in MDA-MB-231 cells for measurement of antiglycolytic activities through cell poliferation, glucose uptake, and lactate concentration assays. Cell proliferation was assessed by MTT assay of MDA-MB-231 cells after treatment with F3 and its bioactive components lutein, β-sitosterol, and stigmasterol. The IC50 value in each compound was determined by MTT assay to be used in subsequent assays. The determination of glucose uptake activity and lactate concentration were quantified using fluorescence spectrophotometry.
RESULTS: Antiproliferative activities were observed for F3 and its bioactive components, with IC50 values of 100 µg/mL (F3), 20 µM (lutein), 25 µM (β-sitosterol), and 90 μM (stigmasterol) in MDA-MB-231 cells at 48 h. The percentage of glucose uptake and lactate concentration in MDA-MB-231 cells treated with F3, lutein, or β sitosterol were significantly lower than those observed in the untreated cells in a time-dependent manner. However, treatment with stigmasterol decreased the concentration of lactate without affecting the glucose uptake in MDA-MB-231 cells.
CONCLUSION: The antiglycolytic activities of F3 on MDA-MB-231 cells are attributed to its bioactive components.
AIM OF THE STUDY: In this study, the effects of F3, lutein and β-sitosterol on tumor development and metastasis were investigated in 4T1-induced mouse mammary carcinoma model.
MATERIALS AND METHODS: Tumor-bearing mice were fed with F3 (100 mg/kg/day), lutein (50 mg/kg/day) and β-sitosterol (50 mg/kg/day) for 30 days (n = 5 each group). Tumor physical growth parameters, animal body weight and development of secondary tumors were investigated. The safety profile of F3 was assessed using hematological and histomorphological changes on the major organs in normal control mice (NM).
RESULTS: Our findings revealed significant reduction of physical tumor growth parameters in all tumor-bearing mice treated with F3 (TM-F3), lutein (TM-L) or β-sitosterol (TM-β) as compared with the untreated group (TM). Statistically significant reduction in body weight was observed in TM compared to the NM or treated (TM-F3, TM-L and TM-β) groups. Histomorphological examination of tissue sections from the F3-treated group showed normal features of the vital organs (i.e., liver, kidneys, lungs and spleen) which were similar to those of NM. Administration of F3 to NM mice (NM-F3) did not cause significant changes in full blood count values.
CONCLUSION: F3 significantly reduced the total tumor burden and prevented secondary tumor development in metastatic breast cancer without significant toxicities in 4T1-induced mouse mammary carcinoma model. The current study provides further support for therapeutic development of F3 with further pharmacokinetics studies.
METHODS: Initially, MTT proliferation assay was used to test the cell viability with various doses of MNQ (5-100 µM). As the half maximal inhibitory concentration (IC50) was obtained, glucose uptake and lactate assays of the cells were tested with IC50 dose of MNQ. The treated cells were also subjected to gene and protein analysis of glycolysis-related molecules (GLUT1 and Akt).
RESULTS: The results showed that MNQ decreased the percentage of MDA-MB-231 cell viability in a dose-dependent manner with the IC50 value of 29 µM. The percentage of glucose uptake into the cells and lactate production decreased significantly after treatment with MNQ as compared to untreated cells. Remarkably, the expressions of GLUT1 and Akt molecules decreased in MNQ-treated cells, suggesting that the inhibition of glycolysis by MNQ is GLUT1-dependent and possibly mediated by the Akt signaling pathway.
CONCLUSION: Our findings indicate the ability of MNQ to inhibit the glycolytic activities as well as glycolysis-related molecules in MDA-MB-231 cells, suggesting the potential of MNQ to be further developed as an effective anticancer agent against TNBC cells.