Ehsan Sarafraz-Yazdi

Anticancer peptide PNC-27 adopts an HDM-2-binding conformation and kills cancer cells by binding to HDM-2 in their membranes

The anticancer peptide PNC-27, which contains an HDM-2-binding domain corresponding to residues 12-26 of p53 and a transmembrane-penetrating domain, has been found to kill cancer cells (but not normal cells) by inducing membranolysis. We find that our previously determined 3D structure of the p53 residues of PNC-27 is directly superimposable on the structure for the same residues bound to HDM-2, suggesting that the peptide may target HDM-2 in the membranes of cancer cells. We now find significant levels of HDM-2 in the membranes of a variety of cancer cells but not in the membranes of several untransformed cell lines. In colocalization experiments, we find that PNC-27 binds to cell membrane-bound HDM-2. We further transfected a plasmid expressing full-length HDM-2 with a membrane-localization signal into untransformed MCF-10-2A cells not susceptible to PNC-27 and found that these cells expressing full-length HDM-2 on their cell surface became susceptible to PNC-27. We conclude that PNC-27 targets HDM-2 in the membranes of cancer cells, allowing it to induce membranolysis of these cells selectively.

The Penetratin Sequence in the Anticancer PNC-28 Peptide Causes Tumor Cell Necrosis Rather Than Apoptosis of Human Pancreatic Cancer Cells

BackgroundPNC-27 and PNC-28 are p53-derived peptides from the human double minute (hdm-2) binding domain attached to penetratin. These peptides induce tumor cell necrosis of cancer cells, but not normal cells. The anticancer activity and mechanism of PNC-28 (p53 aa17–26-penetratin) was specifically studied against human pancreatic cancer.MethodsMiaPaCa-2 cells were treated with PNC-28. Necrosis was determined by measuring lactate dehydrogenase (LDH) and apoptosis as assayed for measuring elevation of proapoptotic proteins. PNC-29, an unrelated peptide, and hdm-2-binding domain p53 aa12-26 without penetratin (PNC-26) were used as controls. Since there is evidence that penetratin is required for tumor cell necrosis, we tested “naked” p53 peptide without penetratin by transfecting a plasmid that encodes p53 aa17–26 segment of PNC-28 into MiaPaCa-2 and an untransformed rat pancreatic acinar cell line, BMRPA1. Time-lapse electron microscopy was employed to further elucidate anticancer mechanism.ResultsTreatment with PNC-28 does not result in the elevation of proapoptotic proteins found in p53-induced apoptosis, but elicits rapid release of LDH, indicative of tumor cell necrosis. Accordingly, we observed membrane pore formation and dose-dependent killing. In direct contrast, transfected MiaPaCa-2 cells underwent apoptosis, and not necrosis, as evidenced by expression of high levels of caspases-3 and 7 and annexin V with background levels of LDH.ConclusionThese results suggest that PNC-28 may be effective in treating human pancreatic cancer. The penetratin sequence appears to be responsible for the fundamental change in the mechanism of action, inducing rapid necrosis initiated by membrane pore formation. Cancer cell death by apoptosis was observed in the absence of penetratin.

Tumor-Targeting Peptides and Small Molecules as Anti-Cancer Agents to Overcome Drug Resistance

Since the introduction of chemotherapy in cancer therapy, development of resistance to every new therapeutic has been the universal experience. The growing understanding of cancer genomics, cancer-associated signal transduction pathways, and key protein drivers of cancer has enabled cancer biologists and medicinal chemists to develop targeted molecules to interfere with these pathways to tackle drug resistant cancers. However, to the dismay of oncologists, the clinical use of many of these tools has once again brought to the forefront the inevitable challenge of drug resistance. It is now understood that cancer resistance to different therapies involves multiple challenges that encompass the cancer cell itself as well as host physiology. This review presents small molecule inhibitors and peptides as two therapeutic approaches in anti-cancer drug development. Resistance to selected samples of these novel therapies is described in the context of cell autonomous resistance, the contributions of the tumor microenvironment, and germ line factors. For each approach, advantages and disadvantages are discussed on how to better overcome the inevitable challenge of resistance in cancer treatment.

Immunization with a peptide of Semliki Forest virus promotes remyelination in experimental autoimmune encephalomyelitis

Remyelination is one of the elusive topics in treatment of multiple sclerosis (MS). Our previous studies have shown that Semliki Forest virus (SFV)-infected δ-knock-out (KO) mice did not exhibit the extensive remyelination, seen in wild type (WT) B6 mice, after viral clearance and demyelination. The Remyelination in SFV-infected WT mice started on day 15 and was completed by day 35 post-infection (pi), whereas the KO mice remained partially demyelinated through day 42 pi. Treatment with E2 peptide2 in incomplete Freunds adjuvant (IFA), resulted in higher antibody production and earlier remyelination in SFV-infected KO (day 28 pi), than WT mice. This finding suggested that anti-E2 peptide2 antibody could play a part in remyelination. In the current study, the effect of E2 peptide2 treatment was evaluated in the experimental autoimmune encephalomyelitis (EAE) model. Mice with established EAE were treated with E2 peptide2 in IFA to develop antibody. Treated EAE mice made significantly higher anti-E2 peptide2 antibody than untreated EAE group. Average clinical disease scores were significantly lower in peptide treated compared to untreated EAE mice. Furthermore, histopathological and immunohistochemical studies demonstrated increased remyelinating areas and higher number of activated oligodendrocytes and astrocytes, in treated compared to untreated EAE groups. Moreover, the anti-E2 peptide2 antibody showed higher binding to the myelinated areas of treated than untreated EAE mice. We conclude that treatment with, or antibody to, SFV E2 peptide2 triggers some mechanism that promotes remyelination.

Anti-cancer peptides from ras-p21 and p53 proteins.

We have employed computer-based molecular modeling approaches to design peptides from the ras-p21 and p53 proteins that either induce tumor cell reversion to the untransformed phenotype or induce tumor cell necrosis without affecting normal cells. For rasp21, we have computed and superimposed the average low energy structures for the wild-type protein and oncogenic forms of this protein and found that specific domains change conformation in the oncogenic proteins. We have synthesized peptides corresponding to these and found that ras peptides, 35-47 (PNC-7) and 96-110 (PNC-2), block oncogenic ras-p21-induced oocyte maturation but have no effect on insulin-induced oocyte maturation that requires activation of endogenous wild-type ras-p21. These results show signal transduction pathway differences between oncogenic and activated wild-type ras-p21. Both peptides, attached to a membrane-penetrating peptide (membrane residency peptide or MRP), either induce phenotypic reversion to the untransformed phenotype or tumor cell necrosis of several ras-transformed cell lines, but have no effect on the growth of normal cells. Using other computational methods, we have designed two peptides, PNC-27 and 28, containing HDM-2-protein-binding domain sequences from p53 linked on their C-termini to the MRP that induce pore formation in the membranes of a wide range of cancer cells but not any normal cells tested. This is due to the expression of HDM-2 in the cancer cell membrane that does not occur in normal cells. These peptides eradicate a highly malignant tumor in nude mice with no apparent side effects. Both ras and p53 peptides show promise as anti-tumor agents in humans.

Abstract 2765A: Co-treatment of human pancreatic cancer MiaPaCa-2 Cells with PNC-27 and Gemzar

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Novel anti-cancer PNC-27 peptide, derived from the p53 binding domain of MDM2, induces necrosis of human pancreatic MiaPaCa-2 cancer cells but not normal cells in vitro, in a dose dependent fashion. Recent studies support a membranolytic mechanism of action for the peptide, initiated by binding of peptide monomers to the cells’ plasma membrane that subsequently aggregate to form transmembrane pores. Gemzar, on the other hand, is an established, non-selective chemotherapeutic used to treat pancreatic cancer. As a nucleoside analog, Gemzar inhibits DNA replication, resulting in cell cycle arrest, and ultimately apoptosis. Speculation regarding whether the two drugs, with different mechanisms, could produce a combined killing effect if administered simultaneously is investigated. Cell proliferation assays (MTT), cell cytotoxicity assays (LDH) and confocal microscopy were used to study the susceptibility of the MiaPaCa-2 cells to the co-treatment of PNC-27 and Gemzar. To determine the working concentration of PNC-27 that would result in pore formation but would not be cytotoxic to the cells, a dose response assay with serial dilutions was conducted. Results revealed that while MiaPaCa-2 cells treated with PNC-27 in DMEM have an LD50 = 150 ug/mL, the working concentration that would enable minimal pore formation without cell death was determined to be ∼10 ug/mL. Through confocal microscopy, it was then determined that propidium iodide (PI) (668.4 Da), a marker for dead cells and that is impermeable for intact cells, would nevertheless be able to enter the cell cytoplasm via PNC-27 pores. The results found PI staining in the cytoplasm but complete absence of nuclear staining. This observation not only indicates the sustained viability of the cells treated with 10 ug/mL PNC-27, but also supports the hypothesis that PI entered the cells through the PNC-27 induced pores. To explore a combined cytotoxic effect of Gemzar and PNC-27, MTT assays were carried out with Gemzar concentrations between 0 uM-100 uM for either 24 or 48 hours, in presence or absence of PNC-27. Our results demonstrate that co-treatment of MiaPaCa-2 cells with PNC-27 and Gemzar leads to a greater cytotoxic effect than with either Gemzar or PNC-27 alone. Additionally, the 48hr treatment resulted in a greater cytotoxic effect than did the 24hr condition. Our results indicate that the increased permeability of the cancer cells to Gemzar via the PNC-27-induced pores may allow for a more rapid mechanism of entry for Gemzar. Due to the selectivity of PNC-27, more Gemzar is able to target the cancer cells which have compromised membrane integrity caused by PNC-27. Therefore, co-treatment with Gemzar may allow for a dose reduction of Gemzar due to increased accessibility of the chemotherapeutic to its site of action. By reducing the dose of Gemzar required to achieve a significant cytotoxic effect, the severity of the side-effects of the drug experienced by patients may also be reduced. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2765A. doi:1538-7445.AM2012-2765A

Abstract C44: Ex vivo cytotoxicity of PNC-27 on primary human ovarian and endometrial cancers

We have developed a series of anti-cancer peptide drugs, PNC-27 and PNC-28 from the MDM2- binding domain of the p53 protein (AA12–26 and 17–26, respectively) and a newly designed membrane residency peptide (MRP) that allows the drug to insert itself into cellular plasma membrane. To date, we have treated in vitro cells from over 20 different human epithelial cancer cell lines, including pancreatic, colon, lung, breast and ovarian cancer with PNC-27 and PNC-28. In all cases both peptides induce tumor cell necrosis within minutes to hours. Recent biophysical and ultrastructural studies have shown that cancer cell necrosis is due to the formation of transmembrane pores by PNC-peptide inducing direct cell lysis. Furthermore, we have found that PNC-28 destroys a highly metatstatic pancreatic cancer (BMRPA1.TUC-3) xenotransplanted into Nu/Nu mice. Remarkably, the PNC-peptides have no effect on the growth or viability of normal cells in culture such as human fibroblasts, keratinocytes and normal pancreatic acinar cells. Most importantly, the PNC-peptides have shown no effect on human umbilical-cord derived human hematopoetic stem cells in vitro to differentiate into mature blood cells and in vivo on blood cell differentials from tumor bearing and PNC-peptide treated animals. These results strongly indicate that PNC-27 and PNC-28 are potent anti-cancer drugs. The results strongly suggest the drugs9 considerable potential against different epithelial cancers without exerting the limiting side effects of suppressing a patient9s bone marrow, hematopoesis, normal cell growth and differentiation. Clearly, the cytotoxic efficacy of the PNC-peptides against long-established cancer cell lines is not directly transferable to a primary cancer in a future patient. Thus, to study the effect of PNC-peptide on a primary human cancer as close as possible to the in vivo (the tumor) situation, we freshly established in culture, under an IRB-approved protocol, cancer cells from the tissues of two primary human ovarian cancers and human uterine cancers. The cells released from the tumor tissue placed into a tissue culture dish were collected and their derivation as a homogenous cell population and from their respective tumors confirmed by immune-cytopathological comparative analysis. The cells9 cancerous properties were confirmed in vitro within the first four passages (p) of the newly established primary cells when the cells9 genomic makeup is still virtually identical (>99%) to that present in the tumor tissue per se. Cell growth and cell death in cells from p1–p4 and treated with PNC-27 or a control peptide PNC-29 were measured using MTT cell proliferation and LDH cytotoxicity assays. The results clearly demonstrate that PNC-27 effectively kills these primary human ovarian and uterine cancer cells in a dose-dependant manner with an LD50 of 100 and 150μg/ml, respectively. Throughout these experiments the effect of PNC-27 on the cancer cells9 morphology was recorded, demonstrating the cells9 complete disruption during the course of the treatment whereas the cancer cells treated with PNC-29 (up to 500μg/ml) remained morphologically indistinguishable from untreated cancer cells. These findings demonstrate for the first time the potentials of PNC-27 anti-cancer peptide as an efficient drug against freshly established and thus primary cells from these rather frequent human gynecological malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr C44.