RESUMEN
Recent advances in cancer treatment like personalized chemotherapy and immunotherapy are aimed at tumors that meet certain specifications. In this review, we describe a new approach to general cancer treatment, termed peptide-induced poptosis, in which specific peptides, e.g., PNC-27 and its shorter analogue, PNC-28, that contain the segment of the p53 transactivating 12-26 domain that bind to HDM-2 in its 1-109 domain, bind to HDM-2 in the membranes of cancer cells, resulting in transmembrane pore formation and the rapid extrusion of cancer cell contents, i.e., tumor cell necrosis. These peptides cause tumor cell necrosis of a wide variety of solid tissue and hematopoietic tumors but have no effect on the viability and growth of normal cells since they express at most low levels of membrane-bound HDM-2. They have been found to successfully treat a highly metastatic pancreatic tumor as well as stem-cell-enriched human acute myelogenous leukemias in nude mice, with no evidence of off-target effects. These peptides also are cytotoxic to chemotherapy-resistant cancers and to primary tumors. We performed high-resolution scanning immuno-electron microscopy and visualized the pores in cancer cells induced by PNC-27. This peptide forms 1:1 complexes with HDM-2 in a temperature-independent step, followed by dimerization of these complexes to form transmembrane channels in a highly temperature-dependent step parallel to the mode of action of other membranolytic but less specific agents like streptolysin. These peptides therefore may be effective as general anti-cancer agents.
RESUMEN
The ketone bodies, sodium and lithium salts of acetoacetate (AcAc) and sodium 3-hydroxybutyrate (3-HB; commonly called beta-hydroxybutyrate) have been found to inhibit the proliferation of cancer cells. Previous studies have suggested that lithium itself may be an inhibiting agent but may be additive or synergistic with the effect of AcAc. We previously found that sodium acetoacetate (NaAcAc) inhibits the growth of human colon cancer cell line SW480. We report here similar results for several other cancer cell lines including ovarian, cervical and breast cancers. We found that NaAcAc does not kill cancer cells but rather blocks their proliferation. Similar inhibition of growth was seen in the effect of lithium ion alone (as LiCl). The effect of LiAcAc appears to be due to the combined effects of acetoacetate and the lithium ion. The ketone bodies, when given together with chemotherapeutic agents, rapamycin, methotrexate and the new peptide anti-cancer agent, PNC-27, substantially lowers their IC50 values for cancer cell, killing suggesting that ketone bodies and ketogenic diets may be powerful adjunct agents in treating human cancers.
RESUMEN
PNC-27, a 32-residue peptide that contains an HDM-2 binding domain and a cell-penetrating peptide (CPP) leader sequence kills cancer, but not normal, cells by binding to HDM-2 associated with the plasma membrane and induces the formation of pores causing tumor cell lysis and necrosis. Conformational energy calculations on the structure of PNC-27 bound to HDM-2 suggest that 1:1 complexes form between PNC-27 and HDM-2 with the leader sequence pointing away from the complex. Immuno-scanning electron microscopy was carried out with cancer cells treated with PNC-27 and decorated with an anti-PNC-27 antibody coupled to 6 nm gold particles and an anti-HDM-2 antibody linked to 15 nm gold particles. We found multiple 6 nm- and 15 nm-labeled gold particles in approximately 1:1 ratios in layered ring-shaped structures in the pores near the cell surface suggesting that these complexes are important to the pore structure. No pores formed in the control, PNC-27-treated untransformed fibroblasts. Based on the theoretical and immuno-EM studies, we propose that the pores are lined by PNC-27 bound to HDM-2 at the membrane surface with the PNC-27 leader sequence lining the pores or by PNC-27 bound to HDM-2.
RESUMEN
Multimodal analysis of circulating tumour cells (CTCs) has the potential to provide remarkable insight for cancer development and metastasis. CTCs and CTC clusters investigation using single-cell analysis, enables researchers to gain crucial information on metastatic mechanisms and the genomic alterations responsible for drug resistance, empowering treatment, and management of cancer. Despite a plethora of CTC isolation technologies, careful attention to the strengths and weaknesses of each method should be considered in order to isolate these rare cells. Here, we provide an overview of cutting-edge technologies used for single-cell isolation and analysis of CTCs. Additionally, we highlight the biological features, clinical application, and the therapeutic potential of CTCs and CTC clusters using single-cell analysis platforms for cancer management.
Asunto(s)
Células Neoplásicas Circulantes , Separación Celular/métodos , Humanos , Análisis de la Célula IndividualRESUMEN
The aim of this study was to assess the effects of pirfenidone (PFD) on promoting epithelial-mesenchymal-transition (EMT) and stemness features in breast carcinoma cells through targeting cancer-associated-fibroblasts (CAFs). Using The Cancer Genome Atlas (TCGA) database, we analyzed the association between stromal index, EMT, and stemness-related genes across 1084 breast cancer patients, identifying positive correlation between YAP1, EMT, and stemness genes in samples with a high-stromal index. We monitored carcinoma cell invasion and spheroid formation co-cultured with CAFs in a 3D microfluidic device, followed by exposing carcinoma cells, spheroids, and CAFs with PFD. We depicted a positive association between the high-stromal index and the expression of EMT and stemness genes. High YAP1 expression in samples correlated with more advanced EMT status and stromal index. Additionally, we found that CAFs promoted spheroid formation and induced the expression of YAP1, VIM, and CD44 in spheroids. Treatment with PFD reduced carcinoma cell migration and decreased the expression of these genes at the protein level. The cytokine profiling showed significant depletion of various EMT- and stemness-regulated cytokines, particularly IL8, CCL17, and TNF-beta. These data highlight the potential application of PFD on inhibiting EMT and stemness in carcinoma cells through the targeting of critical cytokines.
RESUMEN
OBJECTIVE: Patients with epithelial ovarian cancers experience the highest fatality rates among all gynecological malignancies which require development of novel treatment strategies. Tumor cell necrosis was previously reported in a number of cancer cell lines following treatment with a p53-derived anti-cancer peptide called PNC-27. This peptide induces necrosis by transmembrane pore formation with HDM-2 protein that is expressed in the cancer cell membrane. We aimed to extend these studies further by investigating expression of membrane HDM-2 protein in ovarian cancer as it relates to susceptibility to PNC-27. PROCEDURES: Herein, we measured HDM-2 membrane expression in two ovarian cancer cell lines (SKOV-3 and OVCAR-3) and a non-transformed control cell line (HUVEC) by flow cytometric and western blot analysis. Immunofluorescence was used to visualize colocalization of PNC-27 with membrane HDM-2. Treatment effects with PNC-27 and control peptide were assessed using a MTT cell proliferation assay while direct cytotoxicity was measured by lactate dehydrogenase (LDH) release and induction of apoptotic markers; annexin V and caspase-3. RESULTS: HDM-2 protein was highly expressed and frequently detected in the membranes of SKOV-3 and OVCAR-3 cells; a prominent 47.6 kDa HDM-2 plasma membrane isoform was present in both cell lines whereas 25, 29, and 30 kDa isoforms were preferentially expressed in OVCAR-3. Notably, PNC-27 colocalized with HDM-2 in the membranes of both cancer cell lines that resulted in rapid cellular necrosis. In contrast, no PNC-27 colocalization and cytotoxicity was observed with non-transformed HUVEC demonstrating minimal expression of membrane HDM-2. CONCLUSIONS: Our results suggest that HDM-2 is highly expressed in the membranes of these ovarian cancer cell lines and colocalizes with PNC-27. We therefore conclude that the association of PNC-27 with preferentially expressed membrane HDM-2 isoforms results in the proposed model for the formation of transmembrane pores and epithelial ovarian cancer tumor cell necrosis, as previously described in a number of solid tissue and hematologic malignancies.
Asunto(s)
Neoplasias Ováricas/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteína p53 Supresora de Tumor/farmacología , Anexina A5/análisis , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Carcinoma Epitelial de Ovario/metabolismo , Caspasa 3/análisis , Línea Celular Tumoral , Membrana Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Femenino , Humanos , L-Lactato Deshidrogenasa/análisis , Necrosis/metabolismo , Neoplasias Ováricas/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
OBJECTIVES: Paclitaxel is widely used in the treatment of gynecologic malignancies. It targets tumor cells in the M phase of the cell cycle. Cells in other phases survive the insult and repopulate the tumor. PNC-27 is a peptide synthesized of amino acids of the p53-MDM-2 binding domain. It kills various cancer cell lines in a dose-dependent manner. The goal of this study is to assess ovarian cancer cells' sensitivity to PNC-27 after surviving exposure to paclitaxel and to investigate the potential for synergy between PNC-27 and paclitaxel in the treatment of ovarian cancer. METHODS: The impact of exposure to paclitaxel on the surface expression of MDM-2 was assessed with the use of flow cytometry. For measurement of cytotoxicity in vitro, ID8 cells were exposed to paclitaxel for 12 hours in various concentrations. At 12 hours, the drug containing media was removed and the cells were cultured in media containing various concentrations of PNC-27 for 24 hours. Viability was assessed with the use of an MTT assay. Survival fractions were plotted against drug concentrations and the data were fit to logistic dose-response curves. Isoeffective combinations were used to create isobolograms. The combined treatment with weekly paclitaxel and PNC-27 was tested in an intraperitoneal mouse model of ovarian cancer (ID8). RESULTS: Exposure to paclitaxel rendered incomplete time-dependent killing, while PNC-27 mediated comprehensive, dose-dependent killing of ID8 cells. The cytotoxic effect of PNC-27 was dependent on its binding to MDM-2. Blocking MDM-2 inhibited the killing by PNC-27. ID8 cells surviving paclitaxel demonstrated increased expression of MDM-2 and increased susceptibility to PNC-27. Isobologram for dose combinations that were isoeffective indicates synergistic effect between the 2 agents (Combination index <1). In an in vivo model of ovarian cancer (ID8), the addition of PNC-27 to weekly paclitaxel administration significantly reduces tumor growth. CONCLUSIONS: These data demonstrate synergism between PNC-27 and paclitaxel. PNC-27 could target cells surviving paclitaxel and improve its antitumor effect.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Neoplasias Ováricas/tratamiento farmacológico , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Femenino , Humanos , Neoplasias Ováricas/patología , Paclitaxel/administración & dosificación , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Estudios Retrospectivos , Proteína p53 Supresora de Tumor/administración & dosificación , Proteína p53 Supresora de Tumor/farmacocinéticaRESUMEN
The mouse/murine protein, MDM2, and its human homolog, HDM2, are important negative regulators of the p53 tumor suppressor protein. In normal, untransformed cells, MDM2 levels are tightly regulated to control expression of p53 and apoptosis. Conversely, MDM2 expression appears inherently higher in multiple types of cancer cells, thereby supporting its role as a suppressor of p53 pro-apoptotic activity. MDM2 amplification ranges between two- and ten-fold as reported in brain, breast, lung, and soft tissue tumors. MDM2 regulates p53 by two mechanisms: acting as a physical blockade of the transcriptional activation domain and E3 ubiquitin ligase. In addition to its relationship with p53, MDM2 behaves as an independent oncogene. These inherent characteristics make MDM2 a promising target for developing anti-cancer therapies. Investigators are now exploring both p53- dependent and independent cancer cell death pathways by targeting MDM2. Disrupting MDM2-p53 interaction with resultant increase in p53 induces cancer cell cycle arrest and apoptosis. Targeting over-expressed MDM2 on cancer cell membranes disrupts membrane integrity by pore formation, causing membrane destabilization and rapid cancer cell-specific necrosis. In this review, evidence supporting the evolving role of MDM2 as an anti-cancer target and a molecular-based tumor biomarker will be discussed.
Asunto(s)
Terapia Molecular Dirigida , Neoplasias/terapia , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , Animales , Apoptosis , Genómica , Humanos , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
OBJECTIVE: Despite an 80% response rate to chemotherapy, epithelial ovarian cancer has the highest case fatality rate of all gynecologic malignancies. Several studies have shown the efficiency of anticancer peptides PNC-27 and PNC-28 in killing a variety of cancer cells selectively in vitro and in vivo. The purpose of this study was to evaluate the efficacy of PNC-27 against human primary epithelial ovarian cancer. METHODS: We established primary cultures of freshly isolated epithelial ovarian cancer cells from patients with newly diagnosed ovarian cystadenocarcinomas. Two cell lines were obtained, one from mucinous cystadenocarcinoma, and the other from high-grade papillary serous carcinoma. The cancerous properties of these cells were characterized in vitro morphologically, by their growth requirements and serum independence. Treatment effects with PNC-27 were followed qualitatively by light microscopy, and quantitatively by measuring inhibition of cell growth using the MTT cell proliferation assay and direct cytotoxicity by measuring lactate dehydrogenase (LDH). RESULTS: PNC-27 inhibits in a dose-dependent manner the growth of and is cytotoxic to human primary cancer cells that had been freshly isolated from two ovarian epithelial cancers. The results further show that the control peptide PNC-29 has no effect on the primary cancer cells. Our results also show that PNC-27 is cytotoxic to cells from long-established and chemotherapy-resistant human ovarian cancer cell lines. CONCLUSION: These findings show, for the first time, the efficacy of PNC-27 on freshly isolated, primary human cancer cells. Our results indicate the potential of PNC-27 peptide as an efficient alternative treatment of previously untreated ovarian cancer as well as for ovarian cancers that have become resistant to present chemotherapies.
Asunto(s)
Antineoplásicos/farmacología , Cistadenocarcinoma Seroso/tratamiento farmacológico , Neoplasias Glandulares y Epiteliales/tratamiento farmacológico , Neoplasias Ováricas/tratamiento farmacológico , Proteína p53 Supresora de Tumor/farmacología , Antineoplásicos/administración & dosificación , Carcinoma Epitelial de Ovario , Cistadenocarcinoma Seroso/patología , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Neoplasias Glandulares y Epiteliales/patología , Neoplasias Ováricas/patología , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/administración & dosificaciónRESUMEN
GOALS: We have developed the anti-cancer peptide, PNC-27, which is a membrane-active peptide that binds to the HDM-2 protein expressed in the cancer cell membranes of solid tissue tumor cells and induces transmembrane pore formation in cancer, but not in normal cells, resulting in tumor cell necrosis that is independent of p53 activity in these cells. We now extend our study to non-solid tissue tumor cells, in this case, a primitive, possible stem cell human leukemia cell line (K562) that is also p53-homozygously deleted. Our purpose was twofold: to investigate if these cells likewise express HDM-2 in their plasma membranes and to determine if our anti-cancer peptide induces tumor cell necrosis in these non-solid tissue tumor cells in a manner that depends on the interaction between the peptide and membrane-bound HDM-2. PROCEDURES: The anti-cancer activity and mechanism of PNC-27, which carries a p53 aa12-26-leader sequence connected on its carboxyl terminal end to a trans-membrane-penetrating sequence or membrane residency peptide (MRP), was studied against p53-null K562 leukemia cells. Murine leukocytes were used as a non-cancer cell control. Necrosis was determined by measuring the lactate dehydrogenase (LDH) release and apoptosis was determined by the detection of Caspases 3 and 7. Membrane colocalization of PNC-27 with HDM-2 was analyzed microscopically using fluorescently labeled antibodies against HDM-2 and PNC-27 peptides. RESULTS: We found that K562 cells strongly express HDM-2 protein in their membranes and that PNC-27 co-localizes with this protein in the membranes of these cells. PNC-27, but not the negative control peptide PNC-29, is selectively cytotoxic to K562 cells, inducing nearly 100 percent cell killing with LDH release. In contrast, this peptide had no effect on the lymphocyte control cells. CONCLUSIONS: The results suggest that HDM-2 is expressed in the membranes of non-solid tissue tumor cells in addition to the membranes of solid tissue tumor cells. Since K-562 cells appear to be in the stem cell family, the results suggest that early developing tumor cells also express HDM-2 protein in their membranes. Since PNC-27 induces necrosis of K-562 leukemia cells and co-localizes with HDM-2 in the tumor cell membrane as an early event, we conclude that the association of PNC-27 with HDM-2 in the cancer cell membrane results in trans-membrane pore formation which results in cancer cell death, as previously discovered in a number of different solid tissue tumor cells. Since K562 cells lack p53 expression, these effects of PNC-27 on this leukemia cell line occur by a p53-independent pathway.
Asunto(s)
Membrana Celular/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteína p53 Supresora de Tumor/farmacología , Animales , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Diferenciación Celular , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Humanos , Células K562 , Ratones , Microscopía Confocal , Unión Proteica/efectos de los fármacosAsunto(s)
MicroARNs/fisiología , Nanotubos , Osteosarcoma/metabolismo , Neoplasias Ováricas/metabolismo , Animales , Femenino , HumanosRESUMEN
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.
Asunto(s)
Antineoplásicos/farmacología , Resistencia a Antineoplásicos , Neoplasias/tratamiento farmacológico , Péptidos/farmacología , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Humanos , Neoplasias/patología , Proteínas Tirosina Quinasas/antagonistas & inhibidoresRESUMEN
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 Freund's 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.
Asunto(s)
Anticuerpos Antivirales/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/terapia , Regeneración Nerviosa/efectos de los fármacos , Péptidos/inmunología , Virus de los Bosques Semliki/inmunología , Animales , Astrocitos/inmunología , Astrocitos/patología , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/patología , Femenino , Inmunización , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Esclerosis Múltiple/inmunología , Esclerosis Múltiple/patología , Esclerosis Múltiple/terapia , Vaina de Mielina/inmunología , Vaina de Mielina/patología , Glicoproteína Mielina-Oligodendrócito/inmunología , Regeneración Nerviosa/inmunología , Oligodendroglía/inmunología , Oligodendroglía/patología , Péptidos/farmacología , Médula Espinal/inmunología , Médula Espinal/patologíaRESUMEN
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.
Asunto(s)
Antineoplásicos/farmacología , Diseño de Fármacos , Péptidos/farmacología , Animales , Antineoplásicos/efectos adversos , Antineoplásicos/síntesis química , Diseño Asistido por Computadora , Humanos , Ratones , Ratones Desnudos , Modelos Moleculares , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Proteína Oncogénica p21(ras)/química , Péptidos/efectos adversos , Péptidos/síntesis química , Estructura Terciaria de Proteína , Transducción de Señal , Proteína p53 Supresora de Tumor/químicaRESUMEN
PURPOSE: PNC-27, a peptide that contains an HDM-2-binding domain from p53 attached to a membrane-penetrating peptide on its carboxyl terminal end, is cytotoxic to cancer, but not normal, cells. It forms transmembrane pores in the cancer cell membrane. Our purpose is to determine if the whole peptide or critical fragments induce pore formation in cancer cells. METHODS: We have prepared PNC-27 with a green fluorescent label on its amino terminus and a red fluorescent label on its carboxyl terminus and treated MCF-7 breast cancer cells and untransformed MCF-10-2A breast epithelial cells with this double-labeled peptide to determine if combined yellow fluorescence occurs in the membrane of the cancer cells during cancer cell killing. RESULTS: At 30 min, there is significant combined punctate yellow fluorescence, indicative of intact peptide, in the cell membrane of cancer cells that increases during cancer cell lysis. MCF-10-2A cells show initial (30 min) uniform combined yellow membrane fluorescence that subsequently disappears. Unlike the cancer cells, these untransformed cells remain viable. CONCLUSIONS: PNC-27 induces cancer cell membrane lysis by acting as the whole peptide, not fragments. The punctate yellow fluorescence is due to interaction of PNC-27 with intramembrane targets of MCF-7 cells that do not exist in the membrane of the untransformed cell line. This interaction increases the lifetime of PNC-27. Absence of these targets in the membranes of the untransformed MCF-10-2A cells results in initial uniform fluorescence of the double-labeled peptide in their membranes after which the peptide is degraded.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias/tratamiento farmacológico , Proteína p53 Supresora de Tumor/farmacología , Apoptosis/efectos de los fármacos , Caspasas/metabolismo , Línea Celular Tumoral , Membrana Celular/patología , Membrana Celular/ultraestructura , Colorantes , Fluoresceína-5-Isotiocianato , Colorantes Fluorescentes , Proteínas Fluorescentes Verdes , Humanos , L-Lactato Deshidrogenasa/metabolismo , Microscopía Confocal , Necrosis/patología , Neoplasias/patología , Rodaminas , Sales de Tetrazolio , TiazolesRESUMEN
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.
Asunto(s)
Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteína p53 Supresora de Tumor/farmacología , Secuencia de Aminoácidos , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacología , Secuencia de Bases , Sitios de Unión , Muerte Celular/efectos de los fármacos , Línea Celular , Línea Celular Tumoral , Membrana Celular/metabolismo , Cristalografía por Rayos X , Femenino , Colorantes Fluorescentes , Humanos , Masculino , Microscopía Confocal , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/farmacología , Plásmidos/genética , Conformación Proteica , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transfección , Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
BACKGROUND: PNC-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. METHODS: MiaPaCa-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. RESULTS: Treatment 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. CONCLUSION: These 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.