Team:Korea HS/Description
KORHS
Cell-Penetrating Peptides (CPPs) and Cancer
Membrane-mediated mechanisms for internalization are classified as energy-dependent (i.e. phagocytosis, macropinocytosis, clathrin-mediated endocytosis, and caveolae-mediated endocytosis) and energy-independent (toroidal model, Barrel-Stave model, Carpet model, and inverted micelles) pathways. Cell-penetrating peptide (CPP, <30 aa) is a heterogeneous cargo conjugator characterized by an amphipathic receptor-independent infiltration. Conjugation of effector (i.e. peptide, protein, small drug, DNA, siRNA) to CPP via covalent (i.e. disulfide or thioester bonds) or non-covalent (i.e. hydrophobic interactions) strategies have been proposed. To date, CPP models (i.e. R7, PTD4, TAT, MTS) fused with cargoes (i.e. cyclosporine A, HSP20 phosphopeptide, JNK-1, botulinum toxin A) are in phase I/II/II clinical trials for the targeting of psoriasis, wound healing, intraocular inflammation, and cervical dystonia.
The literature classifies novel tissue growth into four touchstones of hyperplasia (normal, by size), hyperproliferation (normal, by quantity), dysplasia (pathogenic), and oncogenesis (pathogenic) by severity. Oncogenesis disrupts superior cellular movement from the basal cell layer (superior to epithelium) to squamous cells (inferior to incrust), thus satisfying proposed hallmarks - sustaining proliferative signaling, evading growth suppressors, activating invasion and metastasis, enabling replicative immortality, inducing angiogenesis, and resisting cell death. Reported cases of neoplasia presented discrepancies by gender cohort. In 2020, out of 10,065,305 total cases worldwide, lung carcinoma (1,435,943, 14.3%), prostatic carcinoma (1,414,259, 14.1%), and colorectal carcinoma (1,065,960, 10.6%) were most prevalently admitted in the male cohort. In the same year, out of 9,227,484 total cases worldwide, breast carcinoma (2,261,419, 24.5%), colorectal carcinoma (865,630, 9.4%), and lung carcinoma (770,828, 8.4%) were most prevalently admitted in the female cohort. Reported cases of neoplasia presented discrepancies by age cohort.
(Feasibility of cell line-specific cell penetration and flexible covalent/non-covalent conjugation of cargoes promise medicinal applications of CPP.)
(Defining hallmarks of mutagenesis and tumorigenesis require synthetic construct for coexpressed suppression.)
Modification and Assessment of Target CPP
A careful literature review was performed to select a cancer-specific cell-penetrating peptide. Cases of cationic antimicrobial cell-penetrating peptides (AMPs) displaying anti-cancer activity have been extensively reported in the literature. Studies of melittin, cecropins, and mainins - to ras oncogene overexpressed and neoplastic cells, respectively - have been investigated. To date, AMP is understood to induce cytolytic activity via the formation of ion-permeable membrane channels, but the precise anticancer mechanism is yet to be elucidated. Herein, buforin II (TRSSRAGLQFPVGRVHRRLLRK, Sphaenorhynchus lacteus), a histone H2A derived AMP with helix-hinge-helix structure and 𝝰-helix, was reviewed for its bacterial penetration selectivity and cytotoxicity.
Buforin IIb (RAGLQFPVGRLLRRLLRRLLR), synthetic analog of buforin II with proline hinge and 𝝰-helical C-terminus was selected for its selective anticancer activity hitherto described. AMP interacts with negatively charged phospholipids and polyanionic lipopolysaccharides of bacterial membranes to display remarkable selective toxicity. It has been explained that plasma membranes of neoplastic cancers, comparable to neutral zwitterionic phospholipids and sterols of normal mammalian cells (or 1-phenyl-2-palmitoylamino-3-morpholino-1-propanol-treated) are abundant with negatively charged gangliosides, phosphatidylserine, and heparan sulfate, inducing buforin IIb internalization. Subsequently, mitochondrial-dependent apoptosis via caspase-9 activation and cytosolic cytochrome c release ensues following ganglioside interaction in situ.
Buforin IIb displays cytotoxicity at high concentrations. Helicity and cytotoxicity of 𝝰-helical AMPs are understood to be directly related - complete non-polar face (or hydrophobicity) inducing hemolytic activity. Lim et al. in 2012 thereby demonstrated BR1 (RAGLQFPVGRLLRRLLR, MV1 in this research) and BR2 (RAGLQFPVGRLLR, MV2 in this research) via stepwise elimination of RLLR C-terminus, retaining cancer cell specificity with minimized alpha-helical cytotoxicity in vitro. Internalization of BR1 and BR2 is attributable to clathrin-independent lipid raft-mediated macropinocytosis not fusing with early endosomes and bypassing lysosomal pathways. Ergo, BR1 and BR2 are noteworthy cancer therapy candidates with the feasibility of covalent conjugation with bioactive cargoes in vivo.
Fluorescent dye fluorescein isothiocyanate (FITC) was conjugated to peptides of interest (PI: buforin IIb, MV1, and MV2) via primary amine. Hence, PI-FITC constructs were designed, which following covalent conjugation (incubation) were excited at 450nm (argon laser) and collected at 530nm via confocal microscopy for FITC cell specificity analysis. Colocalization of nuclei and gross cell morphology were performed via 4’,6-diamidino-2-phenylindole (DAPI) staining - merged with PI-FITC construct localization for the assessment of the intracellular distribution and transfection efficacy. Herein, all assays were performed on cell lines A549 (human lung adenocarcinoma) and MRC5 (human normal lung). Colorimetric MTT (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) metabolic assay was performed for the evaluation of PI construct (buforin IIb, MV1, and MV2) cell cytotoxicity to proposed cell lines (A549 and MRC5). Yellow MTT was reduced to purple-colored formazan via mitochondria-dependent pathway (reductase) of viable cells quantified under 550-600nm by ELISA reader. Both assays were performed in a dose-dependent manner.
(FITC specificity assay via PI-FITC construct and confocal microscopy allows assessment of transduction efficacy and intracellular distribution.)
(Colorimetric MTT metabolic assay provides analysis of PI cytotoxicity to cell lines A549 and MRC5 in dose-dependent manner.)
Identification and Assessment of Target siRNA
siRNA, or small interfering RNA, is a class of double-stranded RNA molecules. They are 20~25 nucleotides in length (usually 21-nucleotides) with two nucleotide 3’ overhangs on each strand. Introduction of siRNA to the cell causes RNA interference or RNAi, a sequence-specific target mRNA cleavage through siRNA winding and RNA-induced silencing complex (RISC) formation; that is, siRNA has the ability to specifically inhibit target gene expression. It is apt to identify the lead compound rapidly, synthesize easily, and target any protein. Yet, it is also prone to degradation and possesses a short half-life along with a high molecular size and a strong anionic charge - all of which can hinder intracellular delivery.
We have chosen to target the CYP1A1 gene which encodes cytochrome P450 1A1 with siRNA. Doxorubicin is a chemotherapy drug designed to inhibit the proliferation of cancer cells via DNA intercalation and topoisomerase II degradation. However, use over an elongated period of time can lead to the development of drug resistance in cancer cells through mutation. A study conducted by AbuHammad and Zihlif in 2012 aimed to find out which genes were associated with gene alterations leading to doxorubicin resistance in MCF7 breast cancer cells through results of an MTT assay and real-time based PCR array performed on doxorubicin-resistant MCF7 breast cancer cell line, displayed an up-regulation of CYP1A1 and CYP1A2 that were up-regulated by 206- and 96-fold respectively. From this prior research, we derived our choice to target CYP1A1 as we determined that if we are able to silence CYP1A1 with the appropriate siRNA, we will be able to most effectively decrease anticancer drug resistance.
Several peptides can absorb on the surface of siRNA leading to the complexation of siRNA with multiple peptides and subsequently a CPP-siRNA complex. Peptide-nucleic acid complexation is largely caused by electrostatic interaction where the negatively charged backbone of the polynucleotide is the primary interactor with the positively charged species. The dense complexes that form as a result have a net positive charge as multiple peptides can bind to one siRNA. However, the ideal ratio of CPP:siRNA for effective translocation must be determined experimentally. Then, these complexes can aggregate and translocate through the cell membrane to carry out desired intracellular function - in our case, target gene silencing and delivery of therapeutics.
To see that sequence-specific gene silencing and subsequent target mRNA cleavage have occurred, we will perform a reverse transcription-polymerase chain reaction also known as RT-PCR. RT-PCR is one of the most sensitive methods of mRNA detection and quantitation currently available. It catalyzes DNA synthesis using existing RNA as a template leading to a product known as complementary DNA (cDNA). With this product, normal PCR amplification proceeds to determine if the treated cell lines do, as predicted, present a statistically insignificant quantity of CYP1A1 phase I gene or don’t.
( siRNA is a mode of RNA interference that can target specific mRNA and induce inhibition of gene expression.)
(RT-PCR can detect presence and quantity of given mRNA by quantifying cDNA reversed-transcribed from the RNA.)
Overall Synthetic Construct Design
Doxorubicin (DOX) is a naturally occurring (Streptomyces peucetius variant caesius) anthracycline neoplastic investigated and approved for the treatment of lymphoblastic leukemia, Wilms’ tumor, Hodgkin’s disease, etc. It is reported with high antimitotic and cytotoxic activity - DNA intercalation and strand breakage, topoisomerase II degradation (ligation-religation action inhibition), and pharmacodynamics impact to nucleolar and coiled-body phosphoprotein 1/DNA polymerase - presumably cell type and cycle-nonspecific. Hence, in this study, PI-DOX conjugate was designed via covalent conjugation of primary amine with siRNA electrostatic attachment for three primary objectives: (1) A549-specific cell penetration, (2) downregulated DOX resistance via CYP1A1 silencing, and (3) A549-specific cell apoptosis. Cumulative cell death analysis of the synthetic construct (PI-CYP1A1 siRNA-DOX) was performed via trypan blue hemocytometer cell count.
(PI-CYP1A1 siRNA-DOX synthetic construct displays cancer cell-specific cell penetration, target gene silencing, and cell apoptosis.)
(Trypan blue hemocytometer cell count visualizes evaluation of synthetic construct cancer killing efficacy.)
References:
Lee, H. S., Park, C. B., Kim, J. M., Jang, S. A., Park, I. Y., Kim, M. S., Cho, J. H., & Kim, S. C. (2008). Mechanism of anticancer activity of buforin IIb, a histone H2A-derived peptide. Cancer letters, 271(1), 47–55. https://doi.org/10.1016/j.canlet.2008.05.041
Lim, K. J., Sung, B. H., Shin, J. R., Lee, Y. W., Kim, D. J., Yang, K. S., & Kim, S. C. (2013). A cancer specific cell-penetrating peptide, BR2, for the efficient delivery of an scFv into cancer cells. PloS one, 8(6), e66084. https://doi.org/10.1371/journal.pone.0066084.
AbuHammad, S., & Zihlif, M. (2013). Gene expression alterations in doxorubicin resistant MCF7 breast cancer cell line. Genomics, 101(4), 213–220. https://doi.org/10.1016/j.ygeno.2012.11.009