APIM-peptide targeting PCNA improves the efficacy of docetaxel treatment in the TRAMP mouse model of prostate cancer

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Mannlige vernepleiere og deres yrkesvalg

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Increased Anticancer Efficacy of Intravesical Mitomycin C Therapy when Combined with a PCNA Targeting Peptide

Non–muscle-invasive bladder cancers (NMIBCs) are tumors confined to the mucosa or the mucosa/submucosa. An important challenge in treatment of NMIBC is both high recurrence and high progression rates. Consequently, more efficacious intravesical treatment regimes are in demand. Inhibition of the cell’s DNA repair systems is a new promising strategy to improve cancer therapy, and proliferating cell nuclear antigen (PCNA) is a new promising target. PCNA is an essential scaffold protein in multiple cellular processes including DNA replication and repair. More than 200 proteins, many involved in stress responses, interact with PCNA through the AlkB homologue 2 PCNA-interacting motif (APIM), including several proteins directly or indirectly involved in repair of DNA interstrand crosslinks (ICLs). In this study, we targeted PCNA with a novel peptide drug containing the APIM sequence, ATX-101, to inhibit repair of the DNA damage introduced by the chemotherapeutics. A bladder cancer cell panel and two different orthotopic models of bladder cancer in rats, the AY-27 implantation model and the dietary BBN induction model, were applied. ATX-101 increased the anticancer efficacy of the ICL-inducing drug mitomycin C (MMC), as well as bleomycin and gemcitabine in all bladder cancer cell lines tested. Furthermore, we found that ATX-101 given intravesically in combination with MMC penetrated the bladder wall and further reduced the tumor growth in both the slow growing endogenously induced and the rapidly growing transplanted tumors. These results suggest that ATX-101 has the potential to improve the efficacy of current MMC treatment in NMIBC

Increased Anticancer Efficacy of Intravesical Mitomycin C Therapy when combined with a PCNA Targeting Peptide

Non–muscle-invasive bladder cancers (NMIBCs) are tumors confined to the mucosa or the mucosa/submucosa. An important challenge in treatment of NMIBC is both high recurrence and high progression rates. Consequently, more efficacious intravesical treatment regimes are in demand. Inhibition of the cell’s DNA repair systems is a new promising strategy to improve cancer therapy, and proliferating cell nuclear antigen (PCNA) is a new promising target. PCNA is an essential scaffold protein in multiple cellular processes including DNA replication and repair. More than 200 proteins, many involved in stress responses, interact with PCNA through the AlkB homologue 2 PCNA-interacting motif (APIM), including several proteins directly or indirectly involved in repair of DNA interstrand crosslinks (ICLs). In this study, we targeted PCNA with a novel peptide drug containing the APIM sequence, ATX- 101, to inhibit repair of the DNA damage introduced by the chemotherapeutics. A bladder cancer cell panel and two different orthotopic models of bladder cancer in rats, the AY-27 implantation model and the dietary BBN induction model, were applied. ATX-101 increased the anticancer efficacy of the ICL-inducing drug mitomycin C (MMC), as well as bleomycin and gemcitabine in all bladder cancer cell lines tested. Furthermore, we found that ATX-101 given intravesically in combination with MMC penetrated the bladder wall and further reduced the tumor growth in both the slow growing endogenously induced and the rapidly growing transplanted tumors. These results suggest that ATX-101 has the potential to improve the efficacy of current MMC treatment in NMIBC

Targeting proliferating cell nuclear antigen and its protein interactions induces apoptosis in multiple myeloma cells.

Multiple myeloma is a hematological cancer that is considered incurable despite advances in treatment strategy during the last decade. Therapies targeting single pathways are unlikely to succeed due to the heterogeneous nature of the malignancy. Proliferating cell nuclear antigen (PCNA) is a multifunctional protein essential for DNA replication and repair that is often overexpressed in cancer cells. Many proteins involved in the cellular stress response interact with PCNA through the five amino acid sequence AlkB homologue 2 PCNA-interacting motif (APIM). Thus inhibiting PCNA's protein interactions may be a good strategy to target multiple pathways simultaneously. We initially found that overexpression of peptides containing the APIM sequence increases the sensitivity of cancer cells to contemporary therapeutics. Here we have designed a cell-penetrating APIM-containing peptide, ATX-101, that targets PCNA and show that it has anti-myeloma activity. We found that ATX-101 induced apoptosis in multiple myeloma cell lines and primary cancer cells, while bone marrow stromal cells and primary healthy lymphocytes were much less sensitive. ATX-101-induced apoptosis was caspase-dependent and cell cycle phase-independent. ATX-101 also increased multiple myeloma cells' sensitivity against melphalan, a DNA damaging agent commonly used for treatment of multiple myeloma. In a xenograft mouse model, ATX-101 was well tolerated and increased the anti-tumor activity of melphalan. Therefore, targeting PCNA by ATX-101 may be a novel strategy in multiple myeloma treatment

"Two hits - one stone" increased efficacy of cisplatin-based therapies by targeting PCNA's role in both DNA repair and cellular signaling

Low response rate and rapid development of resistance against commonly used chemotherapeutic regimes demand new multi-targeting anti-cancer strategies. In this study, we target the stress-related roles of the scaffold protein PCNA with a cell-penetrating peptide containing the PCNA-interacting motif APIM. The APIM-peptide increased the efficacy of cisplatin-based therapies in a muscle-invasive bladder cancer (MIBC) solid tumor model in rat and in bladder cancer (BC) cell lines. By combining multiple omics-levels, from gene expression to proteome/kinome and metabolome, we revealed a unique downregulation of the EGFR/ERBB2 and PI3K/Akt/mTOR pathways in the APIM-peptide-cisplatin combination treated cells. Additionally, the combination treatment reduced the expression of anti-apoptotic proteins and proteins involved in development of resistance to cisplatin. Concurrently, we observed increased levels of DNA breaks in combination treated cells, suggesting that the APIM-peptide impaired PCNA - DNA repair protein interactions and reduced the efficacy of repair. This was also seen in cisplatin-resistant cells, which notably was re-sensitized to cisplatin by the APIM-peptide. Our data indicate that the increased efficacy of cisplatin treatment is mediated both via downregulation of known oncogenic signaling pathways and inhibition of DNA repair/translesion synthesis (TLS), thus the APIM-peptide hits both nuclear and cytosolic functions of PCNA. The novel multi-targeting strategy of the APIM-peptide could potentially improve the efficacy of chemotherapeutic regiments for treatment of MIBC, and likely other solid tumors.publishedVersionCopyright: Søgaard et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Anti-Cancer Potential of Homemade Fresh Garlic Extract Is Related to Increased Endoplasmic Reticulum Stress

The use of garlic and garlic-based extracts has been linked to decreased incidence of cancer in epidemiological studies. Here we examine the molecular and cellular activities of a simple homemade ethanol-based garlic extract (GE). We show that GE inhibits growth of several different cancer cells in vitro, as well as cancer growth in vivo in a syngeneic orthotopic breast cancer model. Multiple myeloma cells were found to be especially sensitive to GE. The GE was fractionated using solid-phase extractions, and we identified allicin in one GE fraction; however, growth inhibitory activities were found in several additional fractions. These activities were lost during freeze or vacuum drying, suggesting that the main anti-cancer compounds in GE are volatile. The anti-cancer activity was stable for more than six months in −20 °C. We found that GE enhanced the activities of chemotherapeutics, as well as MAPK and PI3K inhibitors. Furthermore, GE affected hundreds of proteins involved in cellular signalling, including changes in vital cell signalling cascades regulating proliferation, apoptosis, and the cellular redox balance. Our data indicate that the reduced proliferation of the cancer cells treated by GE is at least partly mediated by increased endoplasmic reticulum (ER) stress

APIM and PIP-box peptides have overlapping binding site on PCNA.

<p>(A) Protein sequence and structural model of PCNA (PDB entry 1vym) with M40 highlighted in red and the center loop (CL) in yellow (upper panel). Live cell (HeLa) confocal fluorescence images of CFP-PCNA wild type (WT) and CFP-PCNA M40 mutants. Bar, 5 µm (lower panel). (B) Normalized FRET (N_FRET) measurements between WT and mutated CFP-PCNA M40/APIM-YFP (light grey diamonds, PCNA WT−/PCNA M40A−/PCNA M40N−/PCNA M40R−/PCNA M40S- APIM) and WT and mutated CFP-PCNA M40/PIP-YFP (dark grey diamonds, PCNA WT−/PCNA M40A−/PCNA M40N−/PCNA M40R/PCNA M40S- PIP). CFP/YFP (vectors only) was used as background control (open diamonds). Data is from three independent experiments (mean ± SEM, n = 72–214). P-values were calculated by the unpaired Student’s t-test.</p

ATX-101 induces apoptosis in the MM cell line JJN-3.

<p>(A–C) Flow cytometric measurement of the apoptotic cell population by annexin V-Pacific Blue labeling. (A) JJN-3 cells treated with 6 µM ATX-101 and 0.5 µM melphalan alone or combined were incubated for 1, 2, and 3 days. Control cells were left unexposed. (B and C) JJN-3 cells treated with 6 and 10 µM ATX-101 were incubated for 1, 2, and 4 h. In addition to annexin V labeling, cells were stained with DRAQ5 for DNA profile. (C) The histograms show the cell cycle distribution of live (blue) and apoptotic (pink) cells after 1 h of ATX-101 treatments. (A–C) show data from representative experiments out of three. (D) Flow cytometric measurement of caspase 8, 9, and 3/7 activity by Fluorescent Labeled Inhibitor of Caspases (FLICA) assay. JJN-3 cells were left unexposed and exposed to 8 µM ATX-101 for 2 and 4 h before the FLICA probe was added for staining. The FLICA probe binds irreversible only to the activated caspase and labels apoptotic cells. Data is from four independent experiments for caspase 8 activity and three independent experiments for caspase 9 and 3/7 activity (mean ± SD, ** P < 0.01, Student’s t-test).</p

ATX-101, a cell-penetrating APIM-peptide, targets PCNA.

<p>(A) Confocal fluorescence image of live HeLa cells 2 minutes after addition of fluorescently tagged ATX-101. Bar, 5 µm. (B) Cell growth measured by MTT assay of HeLa cells stably expressing YFP and APIM-(hABH2 _1–7 F4W)-YFP unexposed (♦ and×, respectively) and after continuous exposure to 0.5 µM cisplatin (▴ and •, respectively) (left panel) and parental HeLa cells unexposed (♦) and after continuous exposure to 8 µM ATX-101 (×), 0.5 µM cisplatin (▴), and combination of ATX-101 and cisplatin (•) (right panel). Data is from one representative experiment out of at least three. (C) Normalized FRET (N_FRET) measurements in HeLa cells between CFP-PCNA and APIM-YFP without and in the presence of ATX-101. The cells were treated with 8 µM ATX-101 8 h after transient transfection and incubated for 16 h before the N_FRET measurements. CFP/YFP (vectors only) was used as background control. Data is from three independent experiments (mean ± SEM, n = 36–40). P-value was calculated by the unpaired Student’s t-test. (D) Cell growth measured by MTT assay of HeLa cells unexposed (♦) and after continuous exposure to 8 µM ATX-A (—), 8 µM ATX-101 (×), 0.5 µM cisplatin (▴), and combination of ATX-A or ATX-101 and cisplatin (▪ and •, respectively). The confocal image shows fluorescently tagged ATX-A in HeLa cells as in (A). Bar, 5 µm. Data is from one representative experiment out of three.</p