Effect And Mechanism Of 3MH-EGCG-β-LG Nanoparticles Against Tumor Growth

(-)-Epigallocatechin-3-gallate (EGCG), the major catechin in green tea, is primary compound responsible for the health benefits. EGCG possesses remarkable cancer chemopreventive and therapeutic potential in various cancers. However, the instability and low bioavailability of EGCG limit its applications.In the present study, EGCG and 3-mercapto-1-hexanol (3MH, a food additive) was loaded in heat treated β-lactoglobulin (β-LG) to prepare 3MH-EGCG-β-LG nanoparticles (MEP nanoparticles) since 3MH is an excellent antioxidant of EGCG, and nanotechnology can improve thermal stability and bioavailability of bioactive compounds. The investigation included (1) the screening of tumor cell lines which are sensitive to MEβ nanoparticles by cell proliferation inhibition assay, (2) the safety evaluation of MEP nanoparticles by acute toxicity test against mice, (3) investigation of the effect and mechanism of MEβ nanoparticles aganist tumor growth in vivo and in vitro. The main results were listed as follows:1. With a molar ratio of 3MH/EGCG/P-LG of 8:32:1, 3MH-EGCG-β-LG nanoparticles (MEβ) were prepared with size 31.19 nm, zeta potential-32.5 mV, PDI 0.083, entrapment efficiency of EGCG 61.22% and EGCG loading amount 1070.68 nmol/mg. In the simulated body fluid condition (37 ℃ and pH=7.4), the sustained release of EGCG was observed. The scavenging capacities of the MEβ nanoparticles on DPPH radicals in vitro were investigated. The results showed that the combination of EGCG and 3MH could synergistically increase the scavenging capacities of the MEP nanoparticles on DPPH radicals. The MEP nanoparticles are in a stable state in a storage duration of 6-8 d at 4 ℃ though the stability of the MEβ nanoparticles were influenced by its storage temperature and time.2. The effect of the MEβ nanoparticles on three cell lines of tumor cells was evaluated by MTT assay and cell morphological observation. The results showed that the MEP nanoparticles (60 μgEGCG/mL, 3MH/EGCG/β-LG=8:32:1) significantly induced cell morphological changes, decreased the percentage of viable cells, inhibited the growth of human melanoma A375 cells, human hepatoma HepG2 cells, human esophageal cancer TE-1 cells.3. The safety of the MEβ nanoparticles was evaluated by acute toxicity experiment in vivo. The results showed MEP nanoparticles with a dose of 100 mg EGCG/kg.BW (3MH/EGCG/β-LG=8:32:1) did not exhibit toxicity on the mice, which suggested that the MEβ nanoparticles were safe when it was used with a moderate dose.4. The tumor volume, inhibition rate, immunohistochemical indicators in the tumors and the cytokines in serum change in nude mice were detected by animal model with male nude mice bearing transplanted tumor from human melanoma A375 cells. The results showed that the MEP nanoparticles had significant inhibition effect on the transplanted tumor when the nude mice were treated with the MEβ nanoparticles at 10 mg EGCG/kg.BW (3MH/EGCG/β-LG=8:32:1) by tail intravenous injection lasted 14 days. The tumor growth inhibition rate of MEβ nanoparticles was 51.78%, which was double of those of EGCG alone. Enzyme-linked immunosorbent assay (ELISA) showed that the MEP nanoparticles could significantly increase the content of IL-2, TNF-a and IFN-y, and decrease the content of IL-6 in the mice serum. TUNEL and immunohistochemical detection showed that the MEP nanoparticles could significantly increase the apoptosis index and the activities of caspase-3, and decrease the content of PCNA and VEGF in the tumors. These results indicated that MEβ nanoparticles could inhibit the proliferation and induce the tumor cell apoptosis, and reduce tumor angiogenesis, simultaneously may adjust the immune system of the mice.5. After the treatment with the MEP nanoparticles for 48 h at 60 μg EGCG/mL (3MH/EGCG/β-LG=8:32:1), the formation of apoptotic bodies and the condensation of nuclear chromatin in A375, HepG2 and TE-1 were found by fluorescent microscope with DAPI staining. Single cell gel electrophoresis (SCGE) showed that MEβ nanoparticles could significantly increase the DNA damage in A375, HepG2 and TE-1. The effect of the MEp nanoparticles on cell cycle and cell apoptosis of A375, HepG2 and TE-1 was measured with flow cytometry. The results showed that the apoptotic cells significantly more than the control in A375, HepG2 and TE-1, respectively. Under the concentration of 60 ug EGCG/mL, the MEβ nanoparticles could arrest cell cycle in S phase, inhibit the proliferation and induce the apoptosis of the three tumor cells.6. The activities of caspase-3, caspase-8 and caspase-9 in A375 were determined by spectrophotometry. The results showed that MEβ nanoparticles could significantly increase the activities of caspase-3 and caspase-9, which indicated that the MEβ nanoparticles induced the apoptosis of A375 cells may via mitochondrial-mediated signaling pathways.7. Western blot analysis showed that the MEβ nanoparticles promoted the expression of p21 and inhibited the expression of cyclinE and CDK2, which lead to S phase arrest of tumor cells. Furthermore, the data also showed that the MEβ nanoparticles inhibited the level of phosphorylation of AKt and increased the activity of cleaved caspase-3, cleaved caspase-9 and Bax, and decreased the activity of Bcl-2, which lead to apoptosis of tumor cells. The results suggested that the apoptosis induced by the MEβ nanoparticles in A375 cells may via the mitochondrial-mediated pathway.