Theranostic Protein Targeting ErbB2 for Bioluminescence Imaging and Therapy for Cancer

<div><p>A combination of molecular-targeted cancer imaging and therapy is an emerging strategy to improve cancer diagnosis and minimize the side effects of conventional treatments. Here, we generated a recombinant protein, EC1-GLuc-p53C, by fusing EC1 peptide, an artificial ligand of ErbB2, with <i>Gaussia</i> luciferase (GLuc) and a p53-activating peptide, p53C. EC1-GLuc-p53C was expressed and purified from <i>E. coli</i> BL21. <i>In vitro</i> experiments showed that EC1-GLuc-p53c was stable in luminescent activity and selectively targeted ErbB2-overexpressing BT474 cells for bioluminescence imaging. Moreover, the internalized EC1-GLuc-p53C in BT474 cells exerted its function to reactivate p53 and significantly inhibited cellular proliferation. In tumor-bearing mice, the ErbB2-targeted bioluminescence imaging and therapeutic effect of EC1-GLuc-p53C were also observed specifically in BT474 tumors but not in MCF7 tumors, which does not overexpress ErbB2. Thus, the present study demonstrates EC1-GLuc-p53C to be an effective theranostic reagent targeting ErbB2 for bioluminescence imaging and cancer therapy.</p> </div

Stability and bioluminescent activity of the recombinant proteins in serum.

<p>Proteins incubated with an equal volume of mouse serum at 37°C were withdrawn at the indicated time points for bioluminescent activity evaluation (a) and Western blotting (c). b) The bioluminescent activity of EC1-GLuc-p53C and GLuc was examined 3 h after incubation in serum. The bioluminescent values of EC1-GLuc-p53C were normalized with GLuc. n = 6 each.</p

Purification and activity of the recombinant proteins.

<p>a) Schema of the purification of EC1-GLuc-p53C with the GST expression system. The GST tag was cleaved by PreScission protease after purification. b) Coomassie brilliant blue staining of purified proteins after 15% SDS-PAGE. Lane 1~4 are marker, GLuc, EC1-GLuc and EC1-GLuc-p53C, respectively. c) The purified proteins after 15% SDS-PAGE were detected using Western blotting with rabbit anti-<i>Gaussia</i> Luciferase serum. Lanes 1~3 are GLuc, EC1-GLuc and EC1-GLuc-p53C, respectively. d) Bioluminescent activities of the purified proteins. Each protein (2 µg) was evaluated using a plate luminometer. The bioluminescent values of EC1-GLuc and EC1-GLuc-p53C were normalized with GLuc. n=6 each, * p<0.01.</p

Bioluminescence imaging in living nude mice bearing MCF7 and BT474 tumors.

<p>MCF7 and BT474 tumors were established on the left and right flank of nude mice, respectively. At 6, 8, 12 and 24 h after the application of EC1-GLuc-p53C, the mice were imaged immediately after the intravenous injection of CTZ solution (3mg/Kg). The color scale represents p/sec/cm²/steradian. n = 3 in each group.</p

Effect of EC1-GLuc-p53C on tumor growth.

<p>30 µL of EC1-GLuc-p53C (0.5 µg/µL) or the same volume of PBS (control) were injected into MCF7 and BT474 tumors every day for 5 days. The mice were monitored daily and their tumor volume was measured twice a week to evaluate the effect of EC1-GLuc-p53C on tumor growth. n = 9 in each group.</p

Effect of EC1-GLuc-p53C on proliferation of MCF7 and BT474 cells.

<p>a) Time-dependent changes in proliferation of MCF7 (top panel) and BT474 (bottom panel) cells. Cells were supplemented with 1 µM of each recombinant protein or PBS (control) on day 0 and further cultured 96 h (day 4). Cellular proliferation was assessed by the WST-1 assay every 24 h. ♦, PBS (control); ■, GLuc; ▲, EC1-GLuc; ×, EC1-GLuc-p53C. n = 6 each. *p<0.05 and **p<0.01 vs control. b) Dose-dependent effect of EC1-GLuc-p53C on the proliferation of BT474 cells. BT474 cells were treated with EC1-GLuc-p53C at various concentrations or PBS (Cont.), and WST-1 assay was performed on day 4. n = 6 each. *, p < 0.05; **, p < 0.01. c) p53-driven transcriptional activity with the p21^<i>WAF1</i> luciferace reporter assay. BT474 transfected with the luciferase reporter vector were incubated with 1µM of GLuc, EC1-GLuc, EC1-GLuc-p53C or PBS for 24h. p21^<i>WAF1</i> luciferase reporter activities in cells were measured with Luciferase Assay System. Data are presented as the mean±S.E.M. <i>n</i> = 5 in each group; **P < 0.01.</p

Tumor site retention of EC1-GLuc-p53C after injection.

<p>At indicated time points after intratumoral injection of EC1-GLuc-p53C, mice were killed and tumors were excised. Tumors were then immediately fixed with 4% PFA. Paraffin-embedded slices of tumors were prepared at a thickness of 10 µm. Immunofluorescence staining with rabbit anti-<i>Gaussia</i> Luciferase serum was used to detect the retention of EC1-GLuc-p53C in tumors. Scale bars = 100 µm. n = 3 in each group.</p

ErbB2-targeted bioluminescence imaging of EC1-GLuc-p53C <i>in vitro</i>.

<p>a) Expression of ErbB2 in MCF7 and BT474 cells. Cell lysate of the two cell lines was subjected to 6% SDS-PAGE and immunoblotted with anti-ErbB2 antibody. β-actin was used as an endogenous control. b) Bioluminescence imaging <i>in </i><i>vitro</i>. Cells incubated with 1 µM of EC-GLuc-p53C were washed with culture medium without serum. Images were acquired with a bioluminescence microscope immediately after the addition of 1 µg/mL CTZ. I and III, bioluminescence images; II and IV, phase-contrast images. Bar = 100 µm. c) Internalization of EC1-fused proteins into ErbB2-overexpressing BT474 cells. Cells were incubated with 1 µM of protein for 24 h, lane 1: GLuc; lane 2: EC-GLuc-p53C; lane 3: EC-GLuc; lane 4: anti-ErbB2+EC1-GLuc-p53C; lane 5: anti-ErbB2+EC-GLuc. The internalization of fusion protein was immunoblotted with rabbit anti-<i>Gaussia</i> Luciferase serum. β-actin was used as an endogenous control.</p

(A) HeLa cells were transfected with control siRNA (siCont) and siRNA for Drp1 (siDrp1) before the transfection with GFP, YFP-Drp1, or YFP-Drp1-S600A and mito-DsRed as described above

HeLa cells were then either untreated or incubated with 20 mM K for 15 min. (B) Quantitative analysis of exogenous Drp1 foci. Data were obtained from the results from at least five independent experiments for each condition shown in A. Values were analyzed using one-way ANOVA followed by post-Turkey test. Control is siDrp1 without stimulation. Error bars indicate SD in each group. *, P < 0.01 versus siDrp1+YFP-Drp1+No stimulation. Bars, 10 μm.<p><b>Copyright information:</b></p><p>Taken from "CaM kinase Iα–induced phosphorylation of Drp1 regulates mitochondrial morphology"</p><p></p><p>The Journal of Cell Biology 2008;182(3):573-585.</p><p>Published online 11 Aug 2008</p><p>PMCID:PMC2500141.</p><p></p

(A) Recombinant Drp1 was incubated with either CaMKIα or CaMKII as indicated in the presence of PATP

Proteins were separated by SDS-PAGE and analyzed by autoradiography and Coomassie staining. (B) Wild-type Drp1 or mutant Drp1 in which Ser600 was replaced by alanine was incubated with CaMKIα and [P]ATP, and proteins were separated by SDS-PAGE and analyzed by autoradiography. (C) Recombinant Drp1 was incubated with CaMKIα in the absence or presence of ATP. Proteins were separated by SDS-PAGE and analyzed by immunoblotting using a phosphospecific antibody selective for phospho-Ser600. (D) HeLa cells were transfected without (control siRNA) or with siRNA selective for CaMKIα. HeLa cells were incubated without (−) or with 45 mM K for 15 min (+). Proteins were separated by SDS-PAGE and analyzed by immunoblotting with phospho-Ser600 or Drp1 antibodies. (E) Neurons were incubated for the indicated times with 45 mM K in the absence or presence of KN93 (20 μM added 30 min before high K). Proteins were separated by SDS-PAGE and analyzed by immunoblotting with phospho-Ser600 or Drp1 antibodies as indicated. (F) Neurons were untreated (control) or treated with 45 mM K for 15 min. Proteins were separated by SDS-PAGE and analyzed by immunoblotting with phospho-Ser600 or total Drp1 antibodies as indicated (bottom). Bar graphs show cumulative data from three independent experiments for each condition. Error bars indicate SEM in each group. *, P < 0.05.<p><b>Copyright information:</b></p><p>Taken from "CaM kinase Iα–induced phosphorylation of Drp1 regulates mitochondrial morphology"</p><p></p><p>The Journal of Cell Biology 2008;182(3):573-585.</p><p>Published online 11 Aug 2008</p><p>PMCID:PMC2500141.</p><p></p