Main Research Findings

1) PNC-27 has the potential to induce tumor cell necrosis of a non-solid, tissue human leukemia cell line dependent on the expression of HDM-2 throughout the plasma membrane of tumor cells.

2) The peptide PNC-27 successfully binds to HDM-2 resulting in the induction of selective membrane-pore formation and cancer cell lysis.

 

Selected Data

1) The research team of Davitt et. Al examined how the anti-cancer peptide, PNC-27, is capable of inducing tumor cell necrosis in non-solid tissue tumor cells in a manner similar to the interaction between the peptide and HDM-2. First, PNC-27 was synthesized by solid phase methods. The purity of the compound was confirmed by HPLC and mass spectrographic analysis. The peptide contains residues from the HDM-2 binding domain of p53 and attached to the membrane residency peptide (MRP) from the antennapedia sequence at the carboxyl terminal end of the compound.

Additionally, the American Type Culture Collection provided the human chronic myeloid K652 leukemia cell line that lacks p53 expression. The cell cultures were maintained in RPMI-1640 media and supplemented with 10% FBS and 1% penicillin/streptomycin at 5% CO2 in an incubator. The p53-positive control cancer cell line of the study was defined as human pancreatic cancer cells (MiaPaCa-2), cultured in DMEM and supplemented with 10% FBS and 1% penicillin/streptomycin [1].

The mouse peritoneal macrophages were isolated by a standard procedure using wild-type (WT) and SphK-1 knockout (KO) mice. The mice were injected with 4% thioglycollate medium intraperitoneally in order to isolate the peritoneal macrophages; peritoneal lavage fluid was collected 3 days after the initial injection of medium. The next step included the CD11b+ macrophages in peritoneal lavage followed by purification with MACS based on the positive selection method using CD11b+ magnetic micro beads. Each well of peritoneal macrophages were cultured in RPMI-1640 medium and incubated in 5% CO2.

After the desired confluence was reached, the K562 cells were incubated in tissue culture dishes for 24 hours with concentrations of PNC-27 ranging from 25. 50, 75, or 100 uM. In the cells treated for 48 hours, the peptide treatment was added on the second day as well. The cells were thoroughly examined on a daily basis in order to monitor changes in cell growth, viability, or morphology. After 48 hours an MTT assay was performed in order to determine cell viability. Furthermore, the K562 cells were incubated with PNC-27 over the course of 24 hours. Caspase Activity was assessed by Clontech’s assy while 10 ug/ml of TNF-alpha was used to induce apoptosis. A CytoTox96 assay was used to detect necrosis by evaluating the rate of release of lactate dehydrogenase (LDH) from the cells after 24 hours of peptide treatment [1] .

Confocal microscopy of the K562 cells was performed by initially maintain the cells in RPMI-1640 media and supplemented with 10% FBS and 1% penicillin/streptomycin, followed by a one-hour treatment with 100 ug/ml of PNC-27 in a 5% CO2 humidified incubator. The samples were repeatedly washed with PBS and the cells were then fixed with 4% formaldehyde in PBS for 5 minutes. The cell samples were then blocked with PBS containing 5% bovine serum albumin (BSA) and 0.3% Triton x-100 for 1 hour on a shaker.

Next, an antibody mixture of DO1 anti-p53 that recognized p53 11-25 sequences that overlap the p53 12-26 sequences of PNC-27, as well as the anti-HDM-2 polyclonal antibody in 1% BSA and 0.3% Triton x-100 containing PBS that was added to cells overnight on a rotator. The cells were then washed with PBS and treated with secondary antibodies, the polyclonal goat to mouse green-fluorophore and polyclonal goal to rabbit red fluorophore in PBS containing 1% BSA and 0.3% Triton x-100 overnight on a rotator. Following incubation, the cells were washed with PBS and visualized on glass slides under the confocal microscope. Sequential image recording was applied to avoid spectral overlap and all image analysis was performed using Olympus FV10-ASW 1.7 View Software [1].

After the cells were treated with the peptides for 48 hours, the cells were then harvested from culture plates and lysed in cell lysis buffer composed of 1% Triton X-100 in 0.05 M Tris-HCl, 0.15 mM NaCL, 0.02% sodium azide, 0.1 mg/ml PMSF< and 0.001 mg/ml Aprotinin. The lysates were subjected to SDS-PAGE technology and electrotransfer to PVDF membrane followed by immunoblotting with DO-1 monoclonal anti-p53 and anti-actin antibodies. Chemiluminescence using Super Signal West Pico Chemiluminescent Substrate identified all antibody-labeled proteins while protein concentration was determined by Bradford Dye Reagent [1].

2) The research team of Sarafraz-Yazdi et. Al examined how PNC-27 was capable of lysing the membranes of cancer cells causing necrosis of cancer cell lines and human tumors. The PNC-27 peptide was synthesized by Shaanxi Zhongbang Pharma-Tech Corp through the use of solid-phase methods. Streptolysin O was purchased from Sigma while the human pancreatic carcinoma cells, MIA-PaCa-2, as well as human melanoma cells and untransformed human fibroblasts were purchased from the American Type Culture Collection and sustained in the appropriate media.

The researchers were able to superimpose the atoms of the 2D-NMR-determined structure for PNC-27 for residues 17-26 of the p53 HDM-2-binding segment of the peptide on the corresponding atom coordinates, from the X-ray crystal structure of the p53 peptide residues 17-29. This was further complexed with residues 25-109 of HDM-2 as deposited in the Protein Data Bank. The 17-29 peptides were removed following the supposition of the PNC-27 structure on the corresponding p53 peptide structure in the HDM-2 binding site. This results in the complex of the HDM-2 structure with the least-squares best-fit structure for PNC-27 was considered the starting structure for energy minimization. After energy convergence, the coordinates for residues 17-26 of the resulting energy-minimized structures were superimposed with the corresponding residues of the H-ray structure for the 17-29 residue peptide bound to HDM-2. This procedure allowed the researchers to determine if the structures of PNC-27 residues were still superimposable [2].

Transmission electron microscopy (TEM) initially began with growing cells in 6-well dishes overnight. The medium was removed and cells were washed and treated with PNC-27 and varying, pre-determined concentrations. After 10 minutes of treatment with PNC-27 the cells were washed and fixed in 3% buffered paraformaldehyde supplemented with 1% glutaraldehyde for 2 hours, followed by scraping into PBS and centrifugation. The resulting pellet was embedded in 6% agar in PBS followed by osmication and dehydration in increasing concentration of ethanol. The pellet was then embedded in 50% resin for 1 hour followed by overnight embedment in 100% resin. The samples were then processed for TEM by preparing 60 nm sections staining with uranyl acetate and ultimately examined in a Zeiss EM10 TEM.

Immunogold TEM was then conducted by the research team, beginning with cell growth in a 6-well TVD overnight. The medium was removed followed by cell washing with PBSand treatment with PNC-27 for 10 minutes. The cells were washed and fixed in 3% buffered paraformaldehyde supplemented with 0.1% glutaraldehyde for 1.5 hours. After washing and quenching the cells with glycine and NaBH4, they were incubated overnight with anti-p53 mAB clone DO-1 that recognized the p53 residues 11-25 and overlaps with the PNC-27 p53 residues 12-26, or monoclonal rabbit anti-HDM-2. After removing all unbound Ab and washing with PVS, the cells were incubated for 6 hours with 6 nm of G-alpha-M F(ab’)2 or 15 nm G-alpha-R F(ab’)2. After washing, the cells were post-fixed in 1% glutaraldehyde in the cacodylate buffer overnight. This was followed by scraping the fixed cells into PBS, centrifuged into a pellet, washed in a cacodylate buffer, and finally post-fixed in 1% osmium tetroxide. The thin sections were stained with uranyl acetate and examined in a Zeiss EM10 TEM [2].

Scanning Electron Microscopy was performed by initially growing MIA-PaCa-2 cells on glass cover slips, followed by treatment with PNC-27 for 3-5 minutes. Control cells were left untreated under the same buffer conditions. At the end of the incubation period, the cell samples were fixed in a water bath in 3% glutaraldehyde in 0.113 m cacodylate buffer for approximately 15 to 30 minutes. This procedure was followed by overnight fixation of the cells and immuno-gold staining with antibodies against PNC-27 and HDM-2. All cell samples were dehydrated by moving the cover slip through alcohol concentrations, increasing from 50 to 100%. The cover slips were then mounted on metal stubs, platinum sputter-coated, and observed by the research team in a LEO 1550 SEM, equipped with a digital camera capable of inserting scale bars that are used during the photographic recording of the results. These same procedures were followed when the researchers evaluated the untransformed A-13145 human fibroblasts [2].

The next portion of the study examined whether tumor cell cytotoxicity induced by PNC-27 was dependent on temperature. Individual 96-well tissue culture dishes (TCDs) were prepared and 5000 cells were seeded into each well. The TCDs were then floated in water baths set to 17 and 37 degrees Celsius in order to precisely control incubation temperatures which were then continuously monitored by thermometers placed into a buffer-containing well. Once the medium was stable the original culture medium was removed and PBS containing the peptide was added and the samples were incubated for 30 minutes at the predetermined temperature. The amount of LDH released in the wells and the amount of LDH remaining in the cells was used to calculate the cytotoxic effect of PNC-27. All experiments were conducted three times to ensure validity. The three primary cell lines used in this study were: rat pancreatic cancer BMRPA1 TUC-3 cells, Hu-melanoma (A2058) cells, and Hu-pancreatic cancer MIA PaCa-2 cells [2].

 

Discussion

1. 
   Figure 1: The results of the confocal micrographs captured by the research team of Davitt et. Al reported that PNC-27 was shown to localize to the cell membrane, however, a small amount of peptide enters the cell (Panel A). Next, Panel B demonstrates prominent membrane fluorescence from the anti-HDM-2 antibody system, suggesting to the researchers that the HDM-2 proteins were primarily expressed on the membrane of the cells. Panel C shows the merged image that confirms the co-localization of the two proteins through the complete overlap of red and green fluorescent emission, indicating that PNC-27 and HDM-2 are co-localized in the cell membrane. Panels D-F represent the magnified image of the lowermost cell generated as superimposed cross sections in order to properly demonstrate the details of the labeled cell surface. Panel D displays the abundant cell surface presence of PNC-27 while Panel E shows the abundant cell surface presence of HMD-2 [1].

The MTT assay that was performed on the K562 cells incubated with varying concentrations of PNC-27 reported that the peptide causes a dose-dependent decrease in K562 cell viability over a 48 hour period of incubation. When administered at a concentration of 75 uM, almost all cancer cells were killed. PNC-27 was not shown to have any significant effects on cell viability of normal murine lymphocytes, however, the peptide was capable of selectively targeting human leukemia cells without affecting normal lymphoid control cells.

Figure 2: Results of the MTT assay following 48 hour incubation with the peptide

Furthermore, PNC-27 led to leakage of LDH after the 24 hour treatment period with the peptide, in a dose-dependent manner. The untransformed murine primary lymphocytes were not affected by the treatment. 48 hours of incubating the K562 cells with 75 uM of PNC-27 was shown to kill almost all cancer cells, however, there was insignificant activation of both Caspase 3 and Caspase 7 past the baseline levels. These results were compared to the control group treated with TNF-alpha, an activator of caspase-dependent apoptosis, thus inducing strong activation of both caspases and indicating the induction of apoptosis in the cells. These results allowed the research team to conclude that PNC-27 induces cellular toxicity in K562 cells through a mechanism of necrosis rather than apoptosis in the cells [1].

Figure 3: Dose-dependent LDH release from cells treated by PNC-27, PNC-29, and IGF-alpha

Figure 4: Caspase 3 and Caspase 7 activity in K562 cells incubated with PNC-27, PNC-29, and TNF-alpha

2) The results of the study reported that PNC-27 leds to the induction of transmembrane pore formation, lined at the outer membrane surface with PNC-27-HDM-2 complexes. The research team thought it was important to note that TEM experiment highlighted “bulky heads” protruding beyond the membrane into the extracellular environment. Additionally the SEM experiment identified spherically shaped particles surrounding the membrane pores. These protrusions and particles were identified as the segments of the complexes that overflow into the extracellular space. The exact stoichometry of the complexes could not be established due to the variation of cell samples that were dissected. The research team also developed several high-resolution images that revealed pores induced by PNC-27 in the solid tissue cancer cells are lined with PNC-27-HDM-2 complexes essential to the structure of the pore [2].

The researchers then proposed a model for transmembrane pore formation induced by amphipathic peptides, hypothesiizing that the amphipathic peptide forms pores in three different stages. The first stage of pore formation includes lying the peptides along the outer cell membrane so the positively charged peptide residues can interact with the phosphate negative charges present in the phospholipid membrane. During the second stage the peptide inserts into the membrane in order for the hydrophobic residues to interract with the lipid bilayer while the charged and polar residues form inntermolecular complexes that line the interior of the pore. In the final step, whole peptides or segments of the peptides a able to undergo a helix-to-extended conformation to span the membrane [2].

PNC-27 was also shown to traverse the membrane and localize to the nucleus in normal pancreatic acinar (BMRPA1) cells, leaving them untransformed. Treating the untransformed cells with PNC-27 did not result in pore formation, indicating that transmembrane pore formation induced by PNC-27 is dependent on complexing with membrane-bound HDM-2. Furthermore, the results reported that most of the hydrophobic face of the PNC-27 peptide, is composed of residues 6-15 in the HDM-2-binding domain. The structure calculations performed by the research team suggests that the hydrophobic face of the peptide is bound deep within the binding pocket of HDM-2 and is not able to interact with the lipids present in the membrane bilayer.

Furthermore, when examining the role of the MRP in PNC-27, the research team theoorized that if HDM-2 is expressed on the cell surface without traversing the cell membrane then PNC-27-HDM-2 complexes can combine in a way that the MRP would span the membrane and pore linings. However, MRP was not shown to be involved in binding to HDM-2, and was instead projecting away from the complex without interacting with any other molecules. After subjecting the p53-binding domain of HMD-2 to X-ray crystallographic analysis, 491 amino acids were identified in HDM-2, and while the location of the MRP in the full-length compound could not be determined the research team believes it may be in position primed to line the pores. Due to the alpha-helical structure of the MRP, the pores were lined by multiple MRP domains that extended down through the membrane from individual PNC-27-HDM-2 complexes at the surface of the extracellular membrane [2].

Finally, when examining the temperature dependence of PNC-27’s ability to induce tumor cell death, the results showed that the peptide induces pore formation in pore-forming proteins. The first step of the process includes forming a complex between HDM-2 and PNC-27 in the cancer cell membrane, followed by a diffusion step where the complex then fuses together in the cell membrane to form pores. However, the research team found that the pore formation seems to be primarily driven my a general affinity for the lipid and cholesterol present in the lipid bilayer of the cellular membrane. On the other hand, PNC-27-induced pore formation in the cancer cell membrane was driven by its affinity for membrane-bound HDM-2 rather than the lipids and cholesterols in the membrane [2].

Figure 5: Changes in cytotoxcity in response to different doses of PNC-27

PNC-27 is different from other anti-cancer agents as it induced tumor cell death by promoting transmembrane pore formation. This mechanism of action is optimal as is it does not depended on signal transduction pathways, the action of apoptopic agents, or the avoidance of MDR gene products. Because PNC-27 only requires the expression of cancer cell membrane-bound HDM-2, the peptide is able to kill various types of cancers regardless of the pro-mitotic intracellular processes that occur. PNC-27 also differs from traditional anti-cancer treatments as it kills cancer cell lines that have had p53 homozygously deleted, in comparison to other agents that block the binding of p53 to HDM-2 in order activate apoptosis. Additionally, there is no evidence suggesting that PNC-27 blocks cell growth and viability in normal or untransformed cell lines. These findings allowed the research team to conclude that the PNC-27 peptide has the potential to act as an effective anti-cancer agent [2].

 

Disclaimer

**LAB USE ONLY**
*This information is for educational purposes only and does not constitute medical advice. THE PRODUCTS DESCRIBED HEREIN ARE FOR RESEARCH USE ONLY. All clinical research must be conducted with oversight from the appropriate Institutional Review Board (IRB). All preclinical research must be conducted with oversight from the appropriate Institutional Animal Care and Use Committee (IACUC) following the guidelines of the Animal Welfare Act (AWA).

 

Citations

[1] Davitt K, Babcock BD, Fenelus M, Poon CK, Sarkar A, Trivigno V, Zolkind PA, Matthew SM, Grin’kina N, Orynbayeva Z, Shaikh MF, Adler V, Michl J, Sarafraz-Yazdi E, Pincus MR, Bowne WB. The anti-cancer peptide, PNC-27, induces tumor cell necrosis of a poorly differentiated non-solid tissue human leukemia cell line that depends on expression of HDM-2 in the plasma membrane of these cells. Ann Clin Lab Sci. 2014 Summer;44(3):241-8. PMID: 25117093.

[2] Sarafraz-Yazdi E, Mumin S, Cheung D, Fridman D, Lin B, Wong L, Rosal R, Rudolph R, Frenkel M, Thadi A, Morano WF, Bowne WB, Pincus MR, Michl J. PNC-27, a Chimeric p53-Penetratin Peptide Binds to HDM-2 in a p53 Peptide-like Structure, Induces Selective Membrane-Pore Formation and Leads to Cancer Cell Lysis. Biomedicines. 2022 Apr 20;10(5):945. doi: 10.3390/biomedicines10050945. PMID: 35625682; PMCID: PMC9138867.

 

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