Skip to main content
Key Specialties
Cardiology Diabetology Endocrinology Gastroenterology Geriatrics and Gerontology Gynecology and Obstetrics Hematology Infectious Disease Internal Medicine Nephrology Neurology Oncology Pediatrics Respiratory Medicine Rheumatology Surgery
Congress Coverage
2024 ACC Congress 2023 ESMO Congress 2023 EASD Congress 2023 ERS Congress 2023 ESC Congress
Clinical Collections
Advances in Alzheimer’s

Keynote webinar | Spotlight on medication adherence

LIVE: Thursday 27th June 2024, 18:00-19:30 (CEST)
Join our expert panel to discover why you need to understand the drivers of non-adherence in your patients, and how you can optimize medication adherence in your clinics to drastically improve patient outcomes.
ADVANCED SEARCH
Log in
Top
BMC Cancer
Published in: BMC Cancer 1/2011

Open Access 01-12-2011 | Research article

A human ribonuclease induces apoptosis associated with p21WAF1/CIP1induction and JNK inactivation

Authors: Jessica Castro, Marc Ribó, Susanna Navarro, Maria Victòria Nogués, Maria Vilanova, Antoni Benito

Published in: BMC Cancer | Issue 1/2011

Login to get access

Abstract

Background

Ribonucleases are promising agents for use in anticancer therapy. Among the different ribonucleases described to be cytotoxic, a paradigmatic example is onconase which manifests cytotoxic and cytostatic effects, presents synergism with several kinds of anticancer drugs and is currently in phase II/III of its clinical trial as an anticancer drug against different types of cancer. The mechanism of cytotoxicity of PE5, a variant of human pancreatic ribonuclease carrying a nuclear localization signal, has been investigated and compared to that of onconase.

Methods

Cytotoxicity was measured by the MTT method and by the tripan blue exclusion assay. Apoptosis was assessed by flow cytometry, caspase enzymatic detection and confocal microscopy. Cell cycle phase analysis was performed by flow cytometry. The expression of different proteins was analyzed by western blot.

Results

We show that the cytotoxicity of PE5 is produced through apoptosis, that it does not require the proapoptotic activity of p53 and is not prevented by the multiple drug resistance phenotype. We also show that PE5 and onconase induce cell death at the same extent although the latter is also able to arrest the cell growth. We have compared the cytotoxic effects of both ribonucleases in the NCI/ADR-RES cell line by measuring their effects on the cell cycle, on the activation of different caspases and on the expression of different apoptosis- and cell cycle-related proteins. PE5 increases the number of cells in S and G2/M cell cycle phases, which is accompanied by the increased expression of cyclin E and p21WAF1/CIP1 together with the underphosphorylation of p46 forms of JNK. Citotoxicity of onconase in this cell line does not alter the cell cycle phase distribution and it is accompanied by a decreased expression of XIAP

Conclusions

We conclude that PE5 kills the cells through apoptosis associated with the p21WAF1/CIP1 induction and the inactivation of JNK. This mechanism is significantly different from that found for onconase.
Appendix
Available only for authorised users
Literature
1.
Rybak SM, Newton DL: Natural and engineered cytotoxic ribonucleases: therapeutic potential. Exp Cell Res. 1999, 253 (2): 325-335. 10.1006/excr.1999.4718.CrossRefPubMed
2.
Darzynkiewicz Z, Carter SP, Mikulski SM, Ardelt WJ, Shogen K: Cytostatic and cytotoxic effects of Pannon (P-30 Protein), a novel anticancer agent. Cell Tissue Kinet. 1988, 21 (3): 169-182.PubMed
3.
Ardelt W, Shogen K, Darzynkiewicz Z: Onconase and amphinase, the antitumor ribonucleases from Rana pipiens oocytes. Curr Pharm Biotechnol. 2008, 9 (3): 215-225. 10.2174/138920108784567245.CrossRefPubMedPubMedCentral
4.
Costanzi J, Sidransky D, Navon A, Goldsweig H: Ribonucleases as a novel pro-apoptotic anticancer strategy: review of the preclinical and clinical data for ranpirnase. Cancer Invest. 2005, 23 (7): 643-650. 10.1080/07357900500283143.CrossRefPubMed
5.
Wu Y, Mikulski SM, Ardelt W, Rybak SM, Youle RJ: A cytotoxic ribonuclease. Study of the mechanism of onconase cytotoxicity. J Biol Chem. 1993, 268 (14): 10686-10693.PubMed
6.
Ita M, Halicka HD, Tanaka T, Kurose A, Ardelt B, Shogen K, Darzynkiewicz Z: Remarkable enhancement of cytotoxicity of onconase and cepharanthine when used in combination on various tumor cell lines. Cancer Biol Ther. 2008, 7 (7): 1104-1108. 10.4161/cbt.7.7.6172.CrossRefPubMedPubMedCentral
7.
Vasandani VM, Wu YN, Mikulski SM, Youle RJ, Sung C: Molecular determinants in the plasma clearance and tissue distribution of ribonucleases of the ribonuclease A superfamily. Cancer Res. 1996, 56 (18): 4180-4186.PubMed
8.
Vasandani VM, Burris JA, Sung C: Reversible nephrotoxicity of onconase and effect of lysine pH on renal onconase uptake. Cancer Chemother Pharmacol. 1999, 44 (2): 164-169. 10.1007/s002800050962.CrossRefPubMed
9.
Leland PA, Staniszewski KE, Kim BM, Raines RT: Endowing human pancreatic ribonuclease with toxicity for cancer cells. J Biol Chem. 2001, 276 (46): 43095-43102. 10.1074/jbc.M106636200.CrossRefPubMed
10.
Bosch M, Benito A, Ribo M, Puig T, Beaumelle B, Vilanova M: A nuclear localization sequence endows human pancreatic ribonuclease with cytotoxic activity. Biochemistry. 2004, 43 (8): 2167-2177. 10.1021/bi035729+.CrossRefPubMed
11.
Rodriguez M, Benito A, Tubert P, Castro J, Ribo M, Beaumelle B, Vilanova M: A cytotoxic ribonuclease variant with a discontinuous nuclear localization signal constituted by basic residues scattered over three areas of the molecule. J Mol Biol. 2006, 360 (3): 548-557. 10.1016/j.jmb.2006.05.048.CrossRefPubMed
12.
Tubert P, Rodriguez M, Ribo M, Benito A, Vilanova M: The nuclear transport capacity of a human-pancreatic ribonuclease variant is critical for its cytotoxicity. Invest New Drugs. 2010
13.
Jemal A, Siegel R, Ward E, Hao Y, Xu J, Thun MJ: Cancer statistics, 2009. CA Cancer J Clin. 2009, 59 (4): 225-249. 10.3322/caac.20006.CrossRefPubMed
14.
Ozols RF, Bundy BN, Greer BE, Fowler JM, Clarke-Pearson D, Burger RA, Mannel RS, DeGeest K, Hartenbach EM, Baergen R: Phase III trial of carboplatin and paclitaxel compared with cisplatin and paclitaxel in patients with optimally resected stage III ovarian cancer: a Gynecologic Oncology Group study. J Clin Oncol. 2003, 21 (17): 3194-3200. 10.1200/JCO.2003.02.153.CrossRefPubMed
15.
Kelley SK, Ashkenazi A: Targeting death receptors in cancer with Apo2L/TRAIL. Curr Opin Pharmacol. 2004, 4 (4): 333-339. 10.1016/j.coph.2004.02.006.CrossRefPubMed
16.
Leland PA, Schultz LW, Kim BM, Raines RT: Ribonuclease A variants with potent cytotoxic activity. Proc Natl Acad Sci USA. 1998, 95 (18): 10407-10412. 10.1073/pnas.95.18.10407.CrossRefPubMedPubMedCentral
17.
Canals A, Ribo M, Benito A, Bosch M, Mombelli E, Vilanova M: Production of engineered human pancreatic ribonucleases, solving expression and purification problems, and enhancing thermostability. Protein Expr Purif. 1999, 17 (1): 169-181. 10.1006/prep.1999.1112.CrossRefPubMed
18.
Ribo M, Benito A, Canals A, Nogues MV, Cuchillo CM, Vilanova M: Purification of engineered human pancreatic ribonuclease. Methods Enzymol. 2001, 341: 221-234. 10.1016/S0076-6879(01)41154-2.CrossRefPubMed
19.
Ribo M, Bosch M, Torrent G, Benito A, Beaumelle B, Vilanova M: Quantitative analysis, using MALDI-TOF mass spectrometry, of the N-terminal hydrolysis and cyclization reactions of the activation process of onconase. Eur J Biochem. 2004, 271 (6): 1163-1171. 10.1111/j.1432-1033.2004.04020.x.CrossRefPubMed
20.
Pace CN, Vajdos F, Fee L, Grimsley G, Gray T: How to measure and predict the molar absorption coefficient of a protein. Protein Sci. 1995, 4 (11): 2411-2423. 10.1002/pro.5560041120.CrossRefPubMedPubMedCentral
21.
Benito A, Ribo M, Vilanova M: On the track of antitumour ribonucleases. Mol Biosyst. 2005, 1 (4): 294-302. 10.1039/b502847g.CrossRefPubMed
22.
Liu J, Lin A: Role of JNK activation in apoptosis: a double-edged sword. Cell Res. 2005, 15 (1): 36-42. 10.1038/sj.cr.7290262.CrossRefPubMed
23.
Bennett BL, Sasaki DT, Murray BW, O'Leary EC, Sakata ST, Xu W, Leisten JC, Motiwala A, Pierce S, Satoh Y, et al: SP600125, an anthrapyrazolone inhibitor of Jun N-terminal kinase. Proc Natl Acad Sci USA. 2001, 98 (24): 13681-13686. 10.1073/pnas.251194298.CrossRefPubMedPubMedCentral
24.
Matlashewski G, Banks L, Pim D, Crawford L: Analysis of human p53 proteins and mRNA levels in normal and transformed cells. Eur J Biochem. 1986, 154 (3): 665-672. 10.1111/j.1432-1033.1986.tb09449.x.CrossRefPubMed
25.
Ogretmen B, Safa AR: Expression of the mutated p53 tumor suppressor protein and its molecular and biochemical characterization in multidrug resistant MCF-7/Adr human breast cancer cells. Oncogene. 1997, 14 (4): 499-506. 10.1038/sj.onc.1200855.CrossRefPubMed
26.
Batist G, Tulpule A, Sinha BK, Katki AG, Myers CE, Cowan KH: Overexpression of a novel anionic glutathione transferase in multidrug-resistant human breast cancer cells. J Biol Chem. 1986, 261 (33): 15544-15549.PubMed
27.
Gehrmann ML, Fenselau C, Hathout Y: Highly altered protein expression profile in the adriamycin resistant MCF-7 cell line. J Proteome Res. 2004, 3 (3): 403-409. 10.1021/pr0340577.CrossRefPubMed
28.
Shi Z, Liang YJ, Chen ZS, Wang XH, Ding Y, Chen LM, Fu LW: Overexpression of Survivin and XIAP in MDR cancer cells unrelated to P-glycoprotein. Oncol Rep. 2007, 17 (4): 969-976.PubMed
29.
Iordanov MS, Ryabinina OP, Wong J, Dinh TH, Newton DL, Rybak SM, Magun BE: Molecular determinants of apoptosis induced by the cytotoxic ribonuclease onconase: evidence for cytotoxic mechanisms different from inhibition of protein synthesis. Cancer Res. 2000, 60 (7): 1983-1994.PubMed
30.
31.
Grabarek J, Ardelt B, Du L, Darzynkiewicz Z: Activation of caspases and serine proteases during apoptosis induced by onconase (Ranpirnase). Exp Cell Res. 2002, 278 (1): 61-71. 10.1006/excr.2002.5568.CrossRefPubMed
32.
Michaelis M, Cinatl J, Anand P, Rothweiler F, Kotchetkov R, von Deimling A, Doerr HW, Shogen K, Cinatl J: Onconase induces caspase-independent cell death in chemoresistant neuroblastoma cells. Cancer Lett. 2007, 250 (1): 107-116. 10.1016/j.canlet.2006.09.018.CrossRefPubMed
33.
Ardelt B, Juan G, Burfeind P, Salomon T, Wu JM, Hsieh TC, Li X, Sperry R, Pozarowski P, Shogen K, et al: Onconase, an anti-tumor ribonuclease suppresses intracellular oxidative stress. Int J Oncol. 2007, 31 (3): 663-669.PubMed
34.
Jacotot E, Ferri KF, Kroemer G: Apoptosis and cell cycle: distinct checkpoints with overlapping upstream control. Pathol Biol (Paris). 2000, 48 (3): 271-279.
35.
Ye F, Che Y, McMillen E, Gorski J, Brodman D, Saw D, Jiang B, Zhang DY: The effect of Scutellaria baicalensis on the signaling network in hepatocellular carcinoma cells. Nutr Cancer. 2009, 61 (4): 530-537. 10.1080/01635580902803719.CrossRefPubMed
36.
Takezawa K, Okamoto I, Tsukioka S, Uchida J, Kiniwa M, Fukuoka M, Nakagawa K: Identification of thymidylate synthase as a potential therapeutic target for lung cancer. Br J Cancer. 2010, 103 (3): 354-361. 10.1038/sj.bjc.6605793.CrossRefPubMedPubMedCentral
37.
Harper JW, Adami GR, Wei N, Keyomarsi K, Elledge SJ: The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell. 1993, 75 (4): 805-816. 10.1016/0092-8674(93)90499-G.CrossRefPubMed
38.
Waga S, Hannon GJ, Beach D, Stillman B: The p21 inhibitor of cyclin-dependent kinases controls DNA replication by interaction with PCNA. Nature. 1994, 369 (6481): 574-578. 10.1038/369574a0.CrossRefPubMed
39.
Niculescu AB, Chen X, Smeets M, Hengst L, Prives C, Reed SI: Effects of p21(Cip1/Waf1) at both the G1/S and the G2/M cell cycle transitions: pRb is a critical determinant in blocking DNA replication and in preventing endoreduplication. Mol Cell Biol. 1998, 18 (1): 629-643.CrossRefPubMedPubMedCentral
40.
Ogryzko VV, Wong P, Howard BH: WAF1 retards S-phase progression primarily by inhibition of cyclin-dependent kinases. Mol Cell Biol. 1997, 17 (8): 4877-4882.CrossRefPubMedPubMedCentral
41.
Ameyar M, Shatrov V, Bouquet C, Capoulade C, Cai Z, Stancou R, Badie C, Haddada H, Chouaib S: Adenovirus-mediated transfer of wild-type p53 gene sensitizes TNF resistant MCF7 derivatives to the cytotoxic effect of this cytokine: relationship with c-myc and Rb. Oncogene. 1999, 18 (39): 5464-5472. 10.1038/sj.onc.1202919.CrossRefPubMed
42.
Dvory-Sobol H, Cohen-Noyman E, Kazanov D, Figer A, Birkenfeld S, Madar-Shapiro L, Benamouzig R, Arber N: Celecoxib leads to G2/M arrest by induction of p21 and down-regulation of cyclin B1 expression in a p53-independent manner. Eur J Cancer. 2006, 42 (3): 422-426. 10.1016/j.ejca.2005.11.009.CrossRefPubMed
43.
Suzuki T, Yokozaki H, Kuniyasu H, Hayashi K, Naka K, Ono S, Ishikawa T, Tahara E, Yasui W: Effect of trichostatin A on cell growth and expression of cell cycle- and apoptosis-related molecules in human gastric and oral carcinoma cell lines. Int J Cancer. 2000, 88 (6): 992-997. 10.1002/1097-0215(20001215)88:6<992::AID-IJC24>3.0.CO;2-9.CrossRefPubMed
44.
Goke R, Goke A, Goke B, El-Deiry WS, Chen Y: Pioglitazone inhibits growth of carcinoid cells and promotes TRAIL-induced apoptosis by induction of p21waf1/cip1. Digestion. 2001, 64 (2): 75-80. 10.1159/000048843.CrossRefPubMed
45.
Lincet H, Poulain L, Remy JS, Deslandes E, Duigou F, Gauduchon P, Staedel C: The p21(cip1/waf1) cyclin-dependent kinase inhibitor enhances the cytotoxic effect of cisplatin in human ovarian carcinoma cells. Cancer Lett. 2000, 161 (1): 17-26. 10.1016/S0304-3835(00)00586-3.CrossRefPubMed
46.
Shim J, Lee H, Park J, Kim H, Choi EJ: A non-enzymatic p21 protein inhibitor of stress-activated protein kinases. Nature. 1996, 381 (6585): 804-806. 10.1038/381804a0.CrossRefPubMed
47.
Potapova O, Gorospe M, Dougherty RH, Dean NM, Gaarde WA, Holbrook NJ: Inhibition of c-Jun N-terminal kinase 2 expression suppresses growth and induces apoptosis of human tumor cells in a p53-dependent manner. Mol Cell Biol. 2000, 20 (5): 1713-1722. 10.1128/MCB.20.5.1713-1722.2000.CrossRefPubMedPubMedCentral
48.
Du L, Lyle CS, Obey TB, Gaarde WA, Muir JA, Bennett BL, Chambers TC: Inhibition of cell proliferation and cell cycle progression by specific inhibition of basal JNK activity: evidence that mitotic Bcl-2 phosphorylation is JNK-independent. J Biol Chem. 2004, 279 (12): 11957-11966. 10.1074/jbc.M304935200.CrossRefPubMed
49.
Ennis BW, Fultz KE, Smith KA, Westwick JK, Zhu D, Boluro-Ajayi M, Bilter GK, Stein B: Inhibition of tumor growth, angiogenesis, and tumor cell proliferation by a small molecule inhibitor of c-Jun N-terminal kinase. J Pharmacol Exp Ther. 2005, 313 (1): 325-332. 10.1124/jpet.104.078873.CrossRefPubMed
50.
Iordanov MS, Wong J, Newton DL, Rybak SM, Bright RK, Flavell RA, Davis RJ, Magun BE: Differential requirement for the stress-activated protein kinase/c-Jun NH(2)-terminal kinase in RNAdamage-induced apoptosis in primary and in immortalized fibroblasts. Mol Cell Biol Res Commun. 2000, 4 (2): 122-128. 10.1006/mcbr.2000.0266.CrossRefPubMed
51.
Smith MR, Newton DL, Mikulski SM, Rybak SM: Cell cycle-related differences in susceptibility of NIH/3T3 cells to ribonucleases. Exp Cell Res. 1999, 247 (1): 220-232. 10.1006/excr.1998.4317.CrossRefPubMed
52.
Tsai SY, Ardelt B, Hsieh TC, Darzynkiewicz Z, Shogen K, Wu JM: Treatment of Jurkat acute T-lymphocytic leukemia cells by onconase (Ranpirnase) is accompanied by an altered nucleocytoplasmic distribution and reduced expression of transcription factor NF-kappaB. Int J Oncol. 2004, 25 (6): 1745-1752.PubMed
53.
Zhao H, Ardelt B, Ardelt W, Shogen K, Darzynkiewicz Z: The cytotoxic ribonuclease onconase targets RNA interference (siRNA). Cell Cycle. 2008, 7 (20): 3258-3261.CrossRefPubMedPubMedCentral
54.
Metadata
Title
A human ribonuclease induces apoptosis associated with p21WAF1/CIP1induction and JNK inactivation
Authors
Jessica Castro
Marc Ribó
Susanna Navarro
Maria Victòria Nogués
Maria Vilanova
Antoni Benito
Publication date
01-12-2011
Publisher
BioMed Central
Published in
BMC Cancer / Issue 1/2011
Electronic ISSN: 1471-2407
DOI
https://doi.org/10.1186/1471-2407-11-9

Other articles of this Issue 1/2011

BMC Cancer 1/2011 Go to the issue

Research article

Societal Costs and Benefits of Treatment with Trastuzumab in Patients with Early HER2neu-Overexpressing Breast Cancer in Singapore

Research article

Ki67 proliferation in core biopsies versus surgical samples - a model for neo-adjuvant breast cancer studies

Case report

Paraneoplastic syndrome mimicking adult-onset Still's disease caused by advanced lung cancer: a case report

Research article

Increased expression of transcription factor TFAP2α correlates with chemosensitivity in advanced bladder cancer

Research article

Downregulation of TFPI in breast cancer cells induces tyrosine phosphorylation signaling and increases metastatic growth by stimulating cell motility

Research article

5-allyl-7-gen-difluoromethoxychrysin enhances TRAIL-induced apoptosis in human lung carcinoma A549 cells
Webinar | 19-02-2024 | 17:30 (CET)

Keynote webinar | Spotlight on antibody–drug conjugates in cancer

Watch now

Antibody–drug conjugates (ADCs) are novel agents that have shown promise across multiple tumor types. Explore the current landscape of ADCs in breast and lung cancer with our experts, and gain insights into the mechanism of action, key clinical trials data, existing challenges, and future directions.

Dr. Véronique Diéras
Prof. Fabrice Barlesi
Developed by: Springer Medicine
Image Credits