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Published on: March 8, 2018
Characterization of a novel cytotoxic cell-penetrating peptide derived from p14ARF protein
Henrik J Johansson1, Samir El-Andaloussi, Tina Holm
1Department of Neurochemistry, Stockholm University, Stockholm, Sweden. henrik@neurochem.su.se
Abstract:
The tumor suppressor p14ARF is widely deregulated in many types of cancers and is believed to function as a failsafe mechanism, inhibiting proliferation and inducing apoptosis as cellular response to a high oncogene load. We have found that a 22-amino-acid-long peptide derived from the N-terminal part of p14ARF, denoted ARF(1-22), which has previously been shown to mimic the function of p14ARF, has cell-penetrating properties. This peptide is internalized to the same extent as the cell-penetrating peptide (CPP) TP10 and dose-dependently decreases proliferation in MCF-7 and MDA MB 231 cells. Uptake of the ARF(1-22) peptide is associated with low membrane disturbance, measured by deoxyglucose and lactate dehydrogenase (LDH) leakage, as compared to its scrambled peptide. Also, flow cytometric analysis of annexin V/propidium iodide (PI) binding and Hoechst staining of nuclei suggest that ARF(1-22) induces apoptosis, whereas scrambled or inverted peptide sequences have no effect. The ARF(1-22) peptide mainly translocates cells through endocytosis, and is found intact inside cells for at least 3 hours. To our knowledge, this is the first time a CPP having pro-apoptopic activity has been designed from a protein.
Insights
A novel cell-penetrating peptide (CPP) derived from the tumor suppressor p14ARF, named ARF(1-22), effectively reduces cancer cell proliferation and induces apoptosis. This peptide shows promise as a targeted cancer therapy by mimicking p14ARF's tumor-suppressing functions.
Area of Science:
- Molecular Biology
- Cancer Research
- Biochemistry
Background:
- The tumor suppressor p14ARF is crucial in preventing cancer by halting cell proliferation and triggering apoptosis in response to oncogene activation.
- Dysregulation of p14ARF is common in various cancers, highlighting its significance as a tumor suppressor.
- Cell-penetrating peptides (CPPs) offer a promising method for delivering therapeutic molecules into cells.
Purpose of the Study:
- To investigate the cell-penetrating and anti-cancer properties of a novel peptide derived from p14ARF, designated ARF(1-22).
- To determine if ARF(1-22) can mimic the tumor-suppressive functions of p14ARF, including inhibiting proliferation and inducing apoptosis.
- To characterize the mechanism of cellular uptake and intracellular stability of the ARF(1-22) peptide.
Main Methods:
- Synthesized a 22-amino-acid peptide (ARF(1-22)) from the N-terminal region of p14ARF.
- Assessed peptide internalization in MCF-7 and MDA MB 231 cancer cells using flow cytometry.
- Measured cell proliferation and apoptosis using assays like deoxyglucose uptake, lactate dehydrogenase (LDH) leakage, annexin V/propidium iodide (PI) staining, and Hoechst nuclear staining.
- Investigated cellular uptake mechanisms via endocytosis and determined intracellular peptide stability.
Main Results:
- The ARF(1-22) peptide demonstrated significant cell-penetrating properties, comparable to the known CPP TP10.
- ARF(1-22) exhibited dose-dependent inhibition of proliferation in both MCF-7 and MDA MB 231 cell lines.
- Cellular uptake of ARF(1-22) resulted in minimal membrane disturbance, unlike its scrambled counterpart.
- Flow cytometry and nuclear staining confirmed that ARF(1-22) induces apoptosis, while scrambled or inverted sequences did not.
- The peptide was primarily internalized through endocytosis and remained intact within cells for at least 3 hours.
Conclusions:
- The ARF(1-22) peptide effectively penetrates cancer cells and induces apoptosis, mimicking the tumor-suppressive activity of p14ARF.
- This study presents the first instance of a cell-penetrating peptide with pro-apoptotic activity designed from a protein.
- ARF(1-22) holds potential as a novel therapeutic agent for cancer treatment due to its targeted delivery and apoptosis-inducing capabilities.
