The binding activity with MDA-MB231 increased with fusion protein concentration (from 0.5 to 10 μM). When the protein concentration reached 10 μM, the binding activity was found to be at max capacity (Figure 1I). Real-time Q-PCR and translational analysis Transcription of RGD- core-IFN-α2a
was examined by RT-PCR, using total RNA isolated from Sf9 cells infected with the recombinant virus vAcH1, vAcH2, vAcH3, and vAcH4. The transcriptional levels of vAcH1 and vAcH2 are higher than the vAcH3 and vAcH4 (Figure 2C). At the same time, the Western blotting results show that RGD-core-IFN-α2a expression levels in vAcH1 and vAcH2 are higher than click here levels from vAcH3 and vAcH4 (Figure 2D). From the results of binding, transcription, and translation analysis, we concluded that vAcH1 and vAcH2 are more effective on cancer cells. We then used vAcH1 and vAcH2 to analyze the VLP functions. Figure 2 Transcription
and expression of HCV core-IFN-α2a recombinant viruses. (A) Identification of pFBD-H1 and pFBD-H2. M: 1Kb Plus DNA ladder; pFBD-H1 and pFBD-H2 samples were digested by BamHI and EcoRI. (B) Identification of pFBD-H3 and pFBD-H4. M: 1Kb Plus DNA ladder; pFBD-H3 and pFBD-H4 samples were digested by BamHI and EcoRI. (C) RT-PCR results of HCV core gene in recombination viruses infect cells. Total RNA was isolated from Sf9 infected with vAcH1, vAcH2, vAcH3, or vAcH4. cDNA was synthesized with SuperScript First Strand Synthesis kit (Invitrogen) with 0.5 to 1.0 μg RNA according to the manufacturer’s instructions. Quantitative RT-PCR reactions were carried
out using SYBR Green PCR master mix reagents on an ABI 7500 Fast Real-Time BI 10773 PCR System (Applied Biosystems). (D) Expression of HCV core-IFN-α2a fusion protein in recombinant virus infected cells. M: protein marker. Cells were harvested at 72 h post-infection (hpi) and lysed in SDS-PAGE loading buffer. Twenty micrograms of total protein was separated by SDS-PAGE and subjected to Western blot assay. vAcH1 and vAcH2 inhibit breast cancer cells MDA-MD-231 Necrostatin-1 migration and invasion IFN-α has an established role in cancer therapy in some cancer types [19–21]. We set out to examine the role of VLP H1 and VLP H2 in breast cancer cell migration and invasion. MDA-MB-231 cells were plated on glass-bottomed dishes coated with 5 μg/ml fibronectin; we then add 10 μM purified VLP H1, VLP H2, or Oxymatrine PBS (as control) for 2 h. The migration was determined using time-lapse cell migration assays. VLP H1 and VLP H2 significantly reduced the total distance and directionality of cell migration and strongly inhibited the net distance of cell migration (Figure 3B,C,D,E,F). Figure 3 VLP H1 and VLP H2 inhibit breast cancer cell migration and invasion. (A) VLP H1 and VLP H2 inhibited the invasion of MDA-MB-231 cells. Data are presented as mean ± SEM, n = 5. Ctrl vs VLP H1; Ctrl vs VLP H2, p < 0.01. (B) Statistic results of net distance of the cells that treated with PBS, 10 μM VLP H1 or VLP H2.