Inducing cell death in vitro in cancer cells by targeted delivery of cytochrome c via a transferrin conjugate

<div><p>One of the major drawbacks of many of the currently used cancer drugs are off-target effects. Targeted delivery is one method to minimize such unwanted and detrimental events. To actively target lung cancer cells, we have developed a conjugate of the apoptosis inducing protein cytochrome c with transferrin because the transferrin receptor is overexpressed by many rapidly dividing cancer cells. Cytochrome c and transferrin were cross-linked with a redox sensitive disulfide bond for the intra-cellular release of the protein upon endocytosis by the transferrin receptor. Confocal results demonstrated the cellular uptake of the cytochrome c-transferrin conjugate by transferrin receptor overexpressing A549 lung cancer cells. Localization studies further validated that this conjugate escaped the endosome. Additionally, an in vitro assay showed that the conjugate could induce apoptosis by activating caspase-3. The neo-conjugate not only maintained an IC₅₀ value similar to the well known drug cisplatin (50 μM) in A549 cancer cells but also was nontoxic to the normal lung (MRC5) cells. Our neo-conjugate holds promise for future development to target cancers with enhanced transferrin receptor expression.</p></div

Thiol-maleimide poly(ethylene glycol) crosslinking of L-asparaginase subunits at recombinant cysteine residues introduced by mutagenesis

<div><p>L-Asparaginase is an enzyme successfully being used in the treatment of acute lymphoblastic leukemia, acute myeloid leukemia, and non-Hodgkin’s lymphoma. However, some disadvantages still limit its full application potential, e.g., allergic reactions, pancreatitis, and blood clotting impairment. Therefore, much effort has been directed at improving its performance. A popular strategy is to randomly conjugate L-asparaginase with mono-methoxy polyethylene glycol, which became a commercial FDA approved formulation widely used in recent years. To improve this formulation by PEGylation, herein we performed cysteine-directed conjugation of the L-asparaginase subunits to prevent dissociation-induced loss of activity. The recombinant cysteine conjugation sites were introduced by mutagenesis at surface-exposed positions on the protein to avoid affecting the catalytic activity. Three conjugates were obtained using different linear PEGs of 1000, 2000, and 5000 g/mol, with physical properties ranging from a semi-solid gel to a fully soluble state. The soluble-conjugate exhibited higher catalytic activity than the non-conjugated mutant, and the same activity than the native enzyme. The cysteine-directed crosslinking of the L-asparaginase subunits produced a higher molecular weight conjugate compared to the native tetrameric enzyme. This strategy might improve L-asparaginase efficiency for leukemia treatment by reducing glomerular filtration due to the increase in hydrodynamic size thus extending half-live, while at the same time retaining full catalytic activity.</p></div

Endosomal localization of Cytc-Tf conjugate in A549 cells using confocal imaging.

<p>(A) A549 cells were treated with the Cyt c-Tf conjugate for 12 h and endosome localization was probed with an endosomal marker antibody (primary antibody against EEA1 and a secondary antibody conjugated with Alexafluor-555) and Cyt c was localized with an anti-Cyt c FITC labeled antibody. The upper panel shows untreated cells and the lower panel the cells treated with Cyt c-Tf. (B) Merged image of A549 cells treated with conjugate enlarged to show nonover lapping green (Cyt c) and red (endosomes) signal.</p

Size determination (hydrodynamic radii) using dynamic light scattering measurements.

<p>(A) Tf (B) Cyt c-Tf conjugate (C) Reduced Cyt c-Tf conjugate after incubation with 10 mM of TCEP.</p

Purification of the Cyt c-Tf conjugate on a Superdex 200 increase gel filtration column.

<p>(A) Elution chromatogram from the Sephadex 200 column, peak 1 indicates the peak containing the conjugate. (B) Coomassie stained SDS PAGE 4–20% gradient gel loaded with unmodified transferrin and a protein molecular weight marker (C) Coomassie stained SDS PAGE 4–20% gradient gel of the fractions from the purification A; lanes 1–6 from the peak 1 in the chromatogram contain the conjugate (transferrin and cytochrome c co-eluting); lanes 7–10 corresponding to peak 2 in the chromatogram contain unconjugated free Cyt c. Lane 11 contains the concentrated conjugate before purification and lane 12 molecular weight markers.</p

Cell viability assays in a lung cancer cell line (A549), in normal (MRC5) lung cells, a cervical cancer cells (HeLa) and in a chronic myleloid leukemia (K562) cells upon incubation with the Cyt c-Tf conjugate.

<p>(A) Cell viability assay (MTS) in A549 cells to determine the IC₅₀ value of Cyt c-Tf conjugate. Cells were treated with an increasing amount of Cyt c-TF. (B) Equimolar concentrations (50 μM) of cisplatin, Cyt c-Tf, Cyt c, and Tf were incubated with MRC5 cells (C) HeLa and (D) K562 cells and subsequently cell viability was tested by MTS assay. * Indicates P<0.05, significant difference from control cells as assessed by two tailed unpaired t-test with n = 4.</p

Activation of caspase-3 and caspase-9 using cell-free caspase assay.

<p>(A) Compared to the cells alone control, the Cyt c-Tf conjugate activates caspase-3 to a significantly greater extent indicating that the conjugate can induce apoptosis. (B) Cells treated with the Cyt c-Tf conjugate were able to activate the caspase-9 to a significantly greater extent compared to untreated cells or cells treated with Tf alone. * Indicates P<0.05, significant difference from control cells as assessed by two tailed unpaired t-test with n = 4.</p

Three step synthesis of the Cyt c-Tf conjugate.

<p>In the first step, the proteins (molar ratio of 6 Cyt c:Tf) were reacted with the crosslinker (Sulfo-LC SPDP). In the second step, the LC-SPDP bound Cyt c was reduced to expose the–SH group. In the last step reduced Cyt c-LC-SPDP and Tf-LC-SPDP were incubated to obtain the final conjugate. The final protein conjugate was purified on a Superdex-200 10/300 column. The final conjugate contained a population of Cyt c-Tf conjugates with molar ratios of Cyt c to Tf varying from 1 to 5 Cyt c molecules attached to one Tf molecule.</p

Subunit mass of recombinant native and mutant A38C-T263C L-asparaginase.

<p>The <i>m/z</i> peak for the native L-asparaginase subunit (in green) was observed at 34605 g/mol, while the <i>m/z</i> peak for the mutant A38C-T263C subunit (in black) was observed at 34634 g/mol.</p

Catalytic activity of L-asparaginase C77-105S mutant.

<p>Catalytic activity of L-asparaginase C77-105S mutant.</p