Poptosis or Peptide-Induced Transmembrane Pore Formation: A Novel Way to Kill Cancer Cells without Affecting Normal Cells.

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.

Anti-Cancer Peptide PNC-27 Kills Cancer Cells by Unique Interactions with Plasma Membrane-Bound hdm-2 and with Mitochondrial Membranes Causing Mitochondrial Disruption.

OBJECTIVE: We have previously shown that the anti-cancer peptide PNC-27 kills cancer cells by co-localizing with membrane-expressed HDM-2, resulting in transmembrane pore formation causing extrusion of intracellular contents. We have also observed cancer cell mitochondrial disruption in PNC-27-treated cancer cells. Our objectives are to determine: 1. if PNC-27 binds to the p53 binding site of HDM-2 (residues 1-109) in the cancer cell membrane and 2. if this peptide causes selective disruption of cancer cell mitochondria. METHODS: For aim 1, we incubated MIA-PaCa-2 human pancreatic carcinoma cells with PNC-27 in the presence of a monoclonal antibody against the amino terminal p53 binding site of HDM-2 to determine if it, but not negative control immune serum, blocks PNC-27-induced tumor cell necrosis. For the second aim, we incubated these cells with PNC-27 in the presence of two specific dyes that highlight normal organelle function: mitotracker for mitochondria and lysotracker for lysosomes. We also performed immuno-electron microscopy (IEM) with gold-labeled anti-PNC-27 antibody on the mitochondria of these cells treated with PNC-27. RESULTS: Monoclonal antibody to the p53 binding site of HDM-2 blocks PNC-27-induced cancer cell necrosis, whereas negative control immune serum does not. The mitochondria of PNC-27-treated cancer cells fail to retain mitotracker dye while their lysosomes retain lysotracker dye. IEM of the mitochondria cancer cells reveals gold particles present on the mitochondrial membranes. CONCLUSIONS: PNC-27 binds to the p53 binding site of HDM-2 (residues 1-109) inducing transmembrane pore formation and cancer cell necrosis. Furthermore, this peptide enters cancer cells and binds to the membranes of mitochondria, resulting in their disruption.

Ketone Bodies Induce Unique Inhibition of Tumor Cell Proliferation and Enhance the Efficacy of Anti-Cancer Agents.

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.

Peptides That Block RAS-p21 Protein-Induced Cell Transformation.

This is a review of approaches to the design of peptides and small molecules that selectively block the oncogenic RAS-p21 protein in ras-induced cancers. Single amino acid substitutions in this protein, at critical positions such as at Gly 12 and Gln 61, cause the protein to become oncogenic. These mutant proteins cause over 90 percent of pancreatic cancers, 40-50 percent of colon cancers and about one third of non-small cell cancers of the lung (NSCCL). RAS-p21 is a G-protein that becomes activated when it exchanges GDP for GTP. Several promising approaches have been developed that target mutant (oncogenic) RAS-p21 proteins in these different cancers. These approaches comprise: molecular simulations of mutant and wild-type proteins to identify effector domains, for which peptides can be made that selectively inhibit the oncogenic protein that include PNC-1 (ras residues 115-126), PNC-2 (ras residues 96-110) and PNC7 (ras residues 35-47); the use of contiguous RAS-p21 peptide sequences that can block ras signaling; cyclic peptides from large peptide libraries and small molecule libraries that can be identified in high throughput assays that can selectively stabilize inactive forms of RAS-p21; informatic approaches to discover peptides and small molecules that dock to specific domains of RAS-p21 that can block mitogenic signal transduction by oncogenic RAS-p21; and the use of cell-penetrating peptides (CPPs) that are attached to the variable domains of the anti-RAS-p21 inactivating monoclonal antibody, Y13 259, that selectively enters oncogenic RAS-p21-containing cancer cells, causing these cells to undergo apoptosis. Several new anti-oncogenic RAS-p21 agents, i.e., Amgen's AMG510 and Mirati Therapeutics' MRTX849, polycyclic aromatic compounds, have recently been FDA-approved and are already being used clinically to treat RAS-p21-induced NSCCL and colorectal carcinomas. These new drugs target the inactive form of RAS-p21 bound to GDP with G12C substitution at the critical Gly 12 residue by binding to a groove bordered by specific domains in this mutant protein into which these compounds insert, resulting in the stabilization of the inactive GDP-bound form of RAS-p21. Other peptides and small molecules have been discovered that block the G12D-RAS-p21 oncogenic protein. These agents can treat specific mutant protein-induced cancers and are excellent examples of personalized medicine. However, many oncogenic RAS-p21-induced tumors are caused by other mutations at positions 12, 13 and 61, requiring other, more general anti-oncogenic agents that are being provided using alternate methods.

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.

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.

Molecular Targeting of H/MDM-2 Oncoprotein in Human Colon Cancer Cells and Stem-like Colonic Epithelial-derived Progenitor Cells.

BACKGROUND/AIM: We have tested whether the anticancer peptide, PNC-27, that kills cancer cells but not normal cells by binding to cancer cell membrane HDM-2 forming pores, kills CD44+ colon cancer stem cells. MATERIALS AND METHODS: Flow cytometry determined the CD44 and HDM-2 expression on six-colon cancer cell lines and one normal cell line (CCD-18Co). MTT, LDH release, annexin V binding and caspase 3 assays were used to assess PNC-27-induced cell death. Bioluminescence imaging measured PNC-27 effects on in vivo tumor growth. RESULTS: High percentages of cells in all six tumor lines expressed CD44. PNC-27 co-localized with membrane HDM-2 only in the cancer cells and caused total cell death (tumor cell necrosis, high LDH release, negative annexin V and caspase 3). In vivo, PNC-27 caused necrosis of tumor nodules but not of normal tissue. CONCLUSION: PNC-27 selectively kills colon cancer stem cells by binding of this peptide to membrane H/MDM-2.

Anti-Cancer Tumor Cell Necrosis of Epithelial Ovarian Cancer Cell Lines Depends on High Expression of HDM-2 Protein in Their Membranes.

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.

Targeting Membrane HDM-2 by PNC-27 Induces Necrosis in Leukemia Cells But Not in Normal Hematopoietic Cells.

BACKGROUND/AIM: Anticancer peptide PNC-27 binds to HDM-2 protein on cancer cell membranes inducing the formation of cytotoxic transmembrane pores. Herein, we investigated HDM-2 membrane expression and the effect of PNC-27 treatment on human non-stem cell acute myelogenous leukemia cell lines: U937, acute monocytic leukemia; OCI-AML3, acute myelomonocytic leukemia and HL60, acute promyelocytic leukemia. MATERIALS AND METHODS: We measured cell surface membrane expression of HDM-2 using flow cytometry. Cell viability was assessed using MTT assay while direct cytotoxicity was measured by lactate dehydrogenase (LDH) release and induction of apoptotic markers annexin V and caspase-3. RESULTS: HDM-2 is expressed at high levels in membranes of U937, OCI-AML3 and HL-60 cells. PNC-27 can bind to membrane HDM-2 to induce cell necrosis and LDH release within 4 h. CONCLUSION: Targeting membrane HDM-2 can be a potential strategy to treat leukemia. PNC-27 targeting membrane HDM-2 demonstrated significant anti-leukemia activity in a variety of leukemic cell lines.

Unique Features of Prostate Cancer in African American and West Indian Patients Including Diagnosis of High Grade Cancers Using Only Elevated Serum Levels of Prostate Specific Antigen.

OBJECTIVE: We have studied the occurrence and nature of prostate cancer in 330 African American patients with respect to its frequency of occurrence, the prevalence of high grade cancers (Gleason score ≥7), distribution of prostate specific antigen (PSA) levels, whether serum PSA levels correlate with Gleason scores, and whether tumor grade correlates with tumor extension and/or metastasis. METHODS: We reviewed the medical charts of patients at the University Hospital of SUNY Downstate Medical Center for whom prostate biopsies or excisions were performed (2015-2019). We then computed the prevalence of prostate cancer and high grade tumors. To determine if there was a quantitative relationship between PSA and Gleason score, we used linear regression analysis. We further used the Fisher exact test to determine if there exists a serum PSA level beyond which the diagnosis of high-grade prostate cancer is definitive. RESULTS: The prevalence of prostate cancer was high at 75.8%; of these cancers, 70% were found to be high grade. Ninety two percent of PSA values were ≥ 4.0ng/mL; the sensitivity was 94%; the positive predictive value was 80%. There was a poor correlation between PSA and Gleason score (R2=0.1), but almost 30 percent of PSA values were >20 ng/mL, and almost all of these corresponded to high grade tumors (Fisher exact test, p<0.00001, α=0.05). Fifteen cancers extended beyond the capsule or metastasized; all were high grade tumors. CONCLUSIONS: These patients presented with a high frequency of high-grade prostate cancer and elevated PSA values, such that PSA> 20ng/mL are virtually diagnostic of high-grade prostate cancer. Since the average age, 65, of screening for biopsy in this population is the same as for other demographic groups, screening should be performed at younger ages in this population.

Low Energy Conformations for S100 Binding Peptide from the Negative Regulatory Domain of p53.

We have computed the low energy conformations of the negative regulatory domain of p53, residues 374-388 using Empirical Conformational Energies of Peptides Program including solvation and computed the statistical weights of distinct conformational states. We find that there are two high probability conformations, one an α-helix from Lys 374-Lys 381, followed by another helical structure involving Lys 382-Glu 388 (statistical weight of 0.48) and an all-α-helix for the entire sequence (statistical weight of 0.23). Both structure are superimposable on the NMR structure of this sequence bound to the S100 protein. The global minimum structure (statistical weight of 0.014) is a beta structure from Gly 374-Arg 379 followed by an α-helix from His 380-Glu 388. Based on these results, we propose a possible strategy for enhancement of p53 anti-tumor activity in cancer cells. Since the structure of this sequence bound to the sirtuin protein, Sir2, is a ß-sheet, we further propose that the global minimum may be an intermediate on the α-ß structure transition.

Mitochondrial Iron Accumulation in Parietal and Chief Cells in Iron Pill Gastritis Following Billroth II Gastrectomy: Case Report Including Electron Microscopic Examination.

Iron pill gastritis has been shown to be associated with superficial gastric erosion and deposition of iron in lamina propria and gastric antral glands. However, iron absorption in gastric parietal and chief cells is rare. We present a case of a 62-year-old man with iron deficiency anemia. His past medical history is significant for Billroth II surgery. His medications include ferrous sulphate 325mg. Esophagogastroduodenoscopy showed diffuse circumferential abnormal mucosa at the gastro-jejunal anastomosis. The mucosa was erythematous and violaceous. Biopsy showed reactive gastropathy with iron deposits predominantly in macrophages, parietal cells, and chief cells. These findings were confirmed by iron stain and later by electron micrography of the gastric mucosa that showed iron deposits in mitochondria and cytoplasm of the parietal and chief cells.

Emerging Role of MDM2 as Target for Anti-Cancer Therapy: A Review.

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.

Papillary urothelial carcinoma with squamous differentiation in association with human papilloma virus: case report and literature review.

BACKGROUND: The human papilloma virus (HPV) is a carcinogen known for its strong association with cervical cancers and cervical lesions. It is also known to be associated with a variety of squamous cell carcinomas in other areas, such as the penis, vulva, anus and head and neck. However, the association with urothelial carcinoma remains controversial. Here, we report a case of urothelial carcinoma with squamous differentiation associated with HPV-6/HPV-11. CASE PRESENTATION: This is a case of a 70 year old man who presented with nocturia and pressure during urination. During the TURP procedure for what was clinically thought to be benign prostate hyperplasia with pathologic diagnosis as prostate carcinoma, a 2 cm papillary mass was found in the distal penile urethra. The papillary mass was found to be a high grade urothelial carcinoma positive for GATA 3 expression, with focal areas of squamous differentiation. The areas with squamous differentiation demonstrated koilocytic differentiation, which were positive for strong p16 expression. The tumor was found to harbor low risk HPV 6/11 by in situ hybridization. CONCLUSIONS: This study case demonstrates HPV infection with a low risk subtype (HPV 6/11) associated with an urothelial carcinoma with squamous differentiation and condylomatous features.

Metastatic Small Cell Carcinoma of the Lung: An Unusual Cause of Acute Fulminant Hepatic Failure.

For patients with acute fulminant liver failure, imaging and histopathologic studies are indicated to reveal the underlying etiology, and metastatic small cell carcinoma should be included in the clinical differential diagnosis when appropriate.

Ex vivo Efficacy of Anti-Cancer Drug PNC-27 in the Treatment of Patient-Derived Epithelial Ovarian Cancer.

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.

The low-energy conformations of gonadotropin-releasing hormone in aqueous solution.

Using the chain-build-up method based on Empirical Conformational Energies of Peptides Program including solvation, we have computed, the low energy conformations of gonadotrpin-releasing hormone, GnRH, whose sequence is Pyro-Glu(PG)-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2. We have found 5,077 solvated conformations with conformational energies that were within 5 kcal/mole of that of the global minimum. These minima were found to occur in 802 distinct conformational classes of which 25 represented 70 % of the Boltzmann energy-weighted structures. Virtually all of these structures adopted bend conformations from Tyr 5-Leu 8, and 3,861 structures adopted bend conformations at residues 4-7. However, these structures differed significantly from one another, indicating that GnRH does not adopt a well-defined structure in aqueous solution consistent with the absence of a well-defined NMR structure of GnRH in water. A total of 300 of these structures were found to be superimposable on possible NMR structures for GnRH in DMSO with a combined statistical weight of 1.6 %. We found that Gly 6 adopts low energy "starred" states, e.g., C* and D*, that are energetically forbidden to L-amino acids but are low energy for D-amino acids, with a statistical weight of 43 %. This can explain why substitutions of L-amino acids for Gly 6 are known to inactivate GnRH while D-amino acid substitutions enhance its activity. Using these findings, in the accompanying manuscript, we compute the low energy conformations for the substituted GnRHs that enable inference of possible receptor-bound conformations.

Low energy conformations for gonadotropin-releasing hormone with D- and L-amino acid substitutions for Gly 6: possible receptor-bound conformations.

In the preceding paper, using ECEPP, including the effects of water, and the chain build-up procedure, we computed the low energy structures for GnRH and found that there were no distinct low energy structures or structures with high statistical weights. To attempt to deduce possible structures of GnRH that may bind to the GnRH receptor, we computed the low energy structures for GnRH peptides that have L- and D-amino acids substituting for Gly 6. The L-amino acid-substituted peptides (L-Ala and L-Val) have very low or no affinity for the receptor and on activity (release of FSH and LH) while the D-Ala-, D-Leu-, D-Trp- and D-Phe-substituted peptides have significantly higher relative affinities and activities than those for native GnRH; the D-Val-substituted peptide has about one-third of the affinity and activity as native GnRH. Unlike native GnRH, our computations suggest that both sets of peptides form well-defined structures in water: the L-amino acid-substituted peptides are predominantly α-helical while the D-amino acid-substituted peptides adopted E*A A A E D*(C*) A E C A(C*) and minor variants of these structures. By eliminating structures that lay in common to the D-Ala and L-Val peptides and further eliminating structures that differed between the D-Ala and D-Leu peptides, we reduced the number of possible distinct binding conformations to 254. Searching for structures among these 254 conformations that had relative statistical weights that paralleled their relative affinities, we found two candidate structures: D*E A A E C*A E C A and D*G A A E D*A E C G*, both of which have conformations for residues 3-9 that are similar to the computed most probable structures for the D-amino acid-substituted GnRH peptides in water.

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.

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.

Imbalanced expression of Tif1γ inhibits pancreatic ductal epithelial cell growth.

Transcriptional intermediary factor 1 gamma (Tif1γ) (Ectodermin/PTC7/RFG7/TRIM33) is a transcriptional cofactor with an important role in the regulation of the TGFß pathway. It has been suggested that it competes with Smad2/Smad3 for binding to Smad4, or alternatively that it may target Smad4 for degradation, although its role in carcinogenesis is unclear. In this study, we showed that Tif1γ interacts with Smad1/Smad4 complex in vivo, using both yeast two-hybrid and coimmunoprecipitation assays. We demonstrated that Tif1γ inhibits transcriptional activity of the Smad1/Smad4 complex through its PHD domain or bromo-domainin pancreatic cells by luciferase assay. Additionally, there is a dynamic inverse relationship between the levels of Tif1γ and Smad4 in benign and malignant pancreatic cell lines. Overexpression of Tif1γ resulted in decreased level of Smad4. Both overexpression and knockdown of Tif1γ resulted in growth inhibition in both benign and cancerous pancreatic cell lines, attributable to a G2-phase cell cycle arrest, but only knockdown of Tif1γ reduces tumor cell invasiveness in vitro. Our study demonstrated that imbalanced expression of Tif1γ results in inhibition of pancreatic ductal epithelial cell growth. In addition, knockdown of Tif1γ may inhibit tumor invasion. These data suggest that Tif1γ might serve as a potential therapeutic target for pancreatic cancer.