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

Abstract

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.

A Cytochrome c‐Chlorotoxin Hybrid Protein as a Possible Antiglioma Drug

“…It is important to note that we have previously FITC-labeled Cytc and it was not endocytosed as expected because of the absence of CTX. [25] These observations support that FITC-Cytc-CTX is taken up by the cells via receptormediated endocytosis and is released from the endosome into the cytosol. Similar observations have been seen in cellular delivery of Cytc via different formulations by our laboratory.…”

“…Similar observations have been seen in cellular delivery of Cytc via different formulations by our laboratory. [21,[23][24][25] However, this was the first time that we have successfully achieved this result using a facile, cost-effective, and reproducible recombinant biology approach, yielding a highly pure and homogenous product that is potently cytotoxic.…”

“…[19][20][21][22] A major limitation that needs to be overcome is that Cytc is unable to cross the cytoplasmic membrane. [19][20] Numerous strategies have been taken to address this issue including encapsulating the protein inside nanoparticles, [19][20][21] nanoparticle formulation of the protein with conjugation to receptor recognition moieties, [23][24] and conjugation of Cytc with the serum proteins transferrin [25] and albumin. [26] While effective in improving cellular uptake of Cytc, many of these strategies involve complex chemical conjugation steps and can result in nonhomogenous products.…”

<p>Smart Targeting To Improve Cancer Therapeutics</p>

“…The results suggest that this conjugate design would be effective in cancers with enhanced expression of TfR due to its nontoxic pattern to lung normal cells. 105 These outcomes make TfR an important molecular target in the study of more effective cancer therapies.…”

Optimization and Characterization of Protein Nanoparticles for the Targeted and Smart Delivery of Cytochrome c to Non-Small Cell Lung Carcinoma

“…Several studies have shown that the direct external delivery of Cyt c to the cell cytosol can activate the caspase cascade and induce apoptosis in a dose-dependent manner [9,10]. Our group and others have developed several drug delivery systems for Cyt c which can induce apoptosis selectively in different cancer types by targeting the mitochondrial apoptotic pathway [11][12][13][14][15][16][17]. These systems specifically release the drugs under reducing conditions like the ones found in the cellular cytosol, but not outside the cell, increasing their effective dose and lowering off-targets and premature drug release [18].…”