Effects Of Liposome Nanoparticles Of Hydrazinocurcumin On Cell Proliferation,Apoptosis, Invasion, And Migration In Breast Cancer Cells

Objective:Although the synthetic derivative of curcumin, hydrazinocurcumin (HC) has an evident of anti-cancer progression and metastasis, its relative poor bioavailability has been highlighted as one of the major problem in therapeutic application. To investigate the anti-cancer effects of liposome nanoparticles of hydrazinocurcumin (HC-NPs) on breast cancer cells, further explore its mechanism in vitro and establish an experimental basis for research in vivo, the study introduced liposome nanoparticles (NPs) as a carrier encapsulating HC to treat4T1cells.In addition, Stat3is an important signaling molecules regulating tumorigenesis and tumor development. In order to investigate the feasibility and availability of PR39delivering Stat3siRNA into breast cancer cells and further explore the mechanism of PR39/Stat3siRNA on breast cancer cells invasion and migration, we utilized antimicrobial peptide PR39to deliver Stat3siRNA into mouse breast cancer cells in vitro.Methods:1. HC-NPs were prepared by film-sonication method; the particles size distribution and morphology were observed by transmission electron microscope; internalization of nanoparticles was observed by fluorescence microscopy.2. Inhibited proliferation in breast cancer cells treated with HC-NPs was observed, and the probable mechanisms of anti-tumor effects was discussed in vitro from the following aspects:the inhibitory rate of cell proliferation were detected by MTT assay; morphological changes were analyzed by Liu staining; the cell cycle and apoptosis were investigated by FCM assay; then invasion and migration of the cells were analyzed by Transwell. Moreover, the expression of proteins correlated to cell proliferation, cell cycle arrest, apoptosis, invasion and migration, such as CyclinD1, Bcl-2, Bax, Survivin, MMP-9were detected by Western blotting.3. Inhibited invasion and migration in4T1cells treated with PR39/Stat3siRNA or single PR39was observed, then the probable mechanism of anti-tumor effects was discussed in vitro from the following aspects:cell internalization of FITC-PR39/Cy3-siRNA complexes were observed by fluorescence microscope, the growth inhibition rate of4T1cells was measured by MTT; then invasion and migration of the4T1cells were analyzed by Transwell. Moreover, the expression of Stat3and proteins correlated to cell invasion and migration were assayed by RT-PCR and Western blotting.Results:1. HC-NPs and NPs were successfully prepared and the nanoparticles size was about150nm by transmission electron microscope, size distribution was (158±11) nm. The nanoparticles were internalized by4T1cells after incubation for1h, and the peak time was at4h.The cell proliferation of MDA-MB-231and4T1cells was inhibited by HC-NPs compared with NPs group in vitro; many cells became round in shape and a few cells contained vacuole; the cells were arrested in G2/M phase after treated with HC-NPs; the ratio of apoptosis was increased (P<0.01), the cells invasion and migration were restrained. The expression of p-Stat3, CyclinD1, Survivin, Bcl-2, and MMP-9were down-regulated, while the expression of Bax was up-regulated.2. The optimal ratio of Stat3siRNA with PR39was1:90, and demonstrated maximum gene silencing effect; Stat3and MMP-9expression was significantly inhibited by PR39/Stat3siRNA compared with control group, but Lipo2000/Stat3siRNA displayed more potent than PR39/Stat3siRNA in silenced Stat3expression; proliferation and cell cycle of4T1cells were not affected by Stat3knockdown by MTT and FCM. Transwell assay suggested that PR39and Stat3siRNA could have synergistic effect in invasion and migration of4T1cells, and the effects of single PR39on cells invasion and migration were also investigated.Conclusion:1. The effects of HC-NPs on suppression of cell proliferation, invasion, and migraion, and induction of cells apoptosis may be related to the inhibition of Stat3activation, down regulation downstream proteins of JAK/STAT signalling pathway related to cell proliferation, apoptosis, invasion and migration; Therefore, HC-NPs is a novel promising delivery system in future.2. PR39could successfully deliver Stat3siRNA into4T1cells and Stat3expression was significantly inhibited by PR39/siRNA complex; then cells invasion and migration were inhibited significantly by PR39/Stat3siRNA; Surprisingly, although Lipo2000/Stat3siRNA displayed more potent than PR39/Stat3siRNA in silenced Stat3expression, single PR39could synergistically inhibit4T1cells invasion and migration with Stat3siRNA, and the cells invasive and migration capacity after treated with Lipo2000/Stat3siRNA was not less than PR39/siRNA group. This study provides new idea for vector in tumor gene therapy of RNAi strategy.