Fig 1: Cholesterol promoted temozolomide (TMZ) resistance of U251 cells. (a) Inhibition rate of 200 µM TMZ or DMSO on U251 wild-type (U251-WT) and TMZ-resistant U251 (U251-R) cells evaluated by CCK-8. (b) Inhibition rate of 200 µM TMZ on U251-R cells treated with 5 µg/ml cholesterol (Chol) or DMSO. (c, d) The apoptosis proportion of U251-WT and U251-R cells treated with DMSO, 200 µM TMZ alone, or combined with 5 µg/ml Chol. (e, f) The cell cycle of U251-WT and U251-R cells treated with DMSO, 200 µM TMZ alone, or combined with 5 µg/ml Chol. (g, h) The protein expression of MGMT and MDR1 (integrated density referred to GAPDH) in DMSO, 200 µM TMZ, or additional 5 µg/ml Chol-treated U251-WT and U251-R cells. The histograms show the mean, and the error bars indicate SD. The percentage of cells (%) is displayed in the flow cytometry chart. P* < 0.05, P** < 0.01, P*** < 0.001, P**** < 0.0001; ns, no significant.
Fig 2: Ginsenosides Rg1 and compound K (CK) regulated cholesterol metabolism and lipid raft distribution and inhibited the temozolomide (TMZ) resistance of U251 cells by upregulating LXRa. (a, b) The protein expression of LXRa (integrated density referred to GAPDH) in TMZ-resistant U251 (U251-R) cells. (c) NaTC-induced cholesterol efflux rate in U251-R cells incubated with 10 µg/mL 25-NBD cholesterol. (d) Confocal laser scanning of the distribution of lipid rafts labeled with cholera toxin subunit B (CT-B) in U251-R cells. Alexa Fluor 594-conjugated CT-B emits red fluorescence, and the blue fluorescence shows 4', 6-diamidino-2-phenylindole (DAPI)-labeled nucleus. The arrows point to the area where lipid rafts accumulated on the cell membrane (magnification, ×600; scale bar, 10 µm). (e) Inhibition rate on U251-R cells assessed by CCK-8. (f, g) The proportion of apoptotic U251-R cells at each stage. (h, i) The protein expression of MDR1 (integrated density referred to GAPDH) in U251-R cells. Cells in all groups were pretreated with 5 µg/ml cholesterol and dosed with 200 µM TMZ, and cells were additionally supplemented with 100 µM Rg1 or CK in the presence or absence of 100 nM GSK2033. The histogram and dots represent the mean, and the error bars indicate SD. P* < 0.05, P* < 0.05, P** < 0.01, P*** < 0.001, P**** < 0.0001; ns, no significant.
Fig 3: Expression of P-glycoprotein in tumor tissues and tumor cells after various treatments. (a). The expression of P-gp in tumor tissues by IHC staining. (b). The expression of P-gp in tumor cells by western blot assay. (c). Gray value of P-gp bands in western blot assay. *** P < 0.001.
Fig 4: Schematic illustration of albumin nanoparticles preparation and their targeted delivery to breast cancer cells and metastatic lymph node. (a). The synthesis process of BSA-CYC-DOX NPs and BSA NPs. (b). The dual-drug-loaded NPs were delivered to breast cancer via the active targeting mediated by BSA-binding proteins gp60 and SPARC. CYC increased DOX accumulation by inhibiting HH signaling pathway and reducing P-gp expression. BSA-CYC-DOX NPs were drained into the metastatic lymph node through lymphatic vessels and retained in macrophages.
Fig 5: Ginsenosides Rg1 and compound K (CK) increased the sensitivity of resistant glioblastoma cells to temozolomide (TMZ) by governing cholesterol metabolism and lipid raft distribution. (a) Cholesterol in the brain tumor microenvironment is taken up in the form of cholesterol esters by low-density lipoprotein receptor (LDLR). Restricted expression of LXRa in TMZ-resistant glioblastoma cells leads to attenuated ABCA1-mediated cholesterol efflux, which retains a higher concentration of intracellular cholesterol to maintain a stable distribution of lipid rafts and increases the resistance to TMZ. The amplified TMZ resistance is potentially associated with the upregulation of MDR1 in lipid rafts. (b) Ginsenosides Rg1 and CK facilitated cholesterol efflux by stimulating the expression of LXRa. The decreased intracellular cholesterol redistributed lipid rafts in an uneven and aggregated state and reserved the cytotoxicity of TMZ on glioblastoma cells. Following the regulation of cholesterol metabolism, Rg1 treatment also diminished MDR1 expression.
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