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

At a glance: The STEP trials

A round-up of the STEP phase 3 clinical trials evaluating semaglutide for weight loss in people with overweight or obesity.
ADVANCED SEARCH
Log in
Top
Journal of Experimental & Clinical Cancer Research
Published in: Journal of Experimental & Clinical Cancer Research 1/2017

Open Access 01-12-2017 | Research

Cooperative antitumor activities of carnosic acid and Trastuzumab in ERBB2+ breast cancer cells

Authors: Carolina D’Alesio, Grazia Bellese, Maria Cristina Gagliani, Cinzia Aiello, Elena Grasselli, Gianluca Marcocci, Angela Bisio, Sara Tavella, Tiziana Daniele, Katia Cortese, Patrizio Castagnola

Published in: Journal of Experimental & Clinical Cancer Research | Issue 1/2017

Login to get access

Abstract

Background

ERBB2 is overexpressed in up to 20–30% of human breast cancers (BCs), and it is associated with aggressive disease. Trastuzumab (Tz), a humanized monoclonal antibody, improves the prognosis associated with ERBB2-amplified BCs. However, the development of resistance remains a significant challenge. Carnosic acid (CA) is a diterpene found in rosemary and sage, endowed with anticancer properties. In this in vitro study, we have investigated whether Tz and CA have cooperative effects on cell survival of ERBB2 overexpressing (ERBB2+) cells and whether CA might restore Tz sensitivity in Tz-resistant cells.

Methods

We have studied BC cell migration and survival upon CA and Tz treatment. In particular, migration ability was assessed by transwell assay while cell survival was assessed by MTT assay. In addition, we have performed cell cycle and apoptosis analysis by high-resolution DNA flow cytometry and annexin-V, resazurin and sytox blue staining by flow cytometry, respectively. The expression of proteins involved in cell cycle progression, ERBB2 signaling pathway, and autophagy was evaluated by immunoblot and immunofluorescence analysis. Cellular structures relevant to the endosome/lysosome and autophagy pathways have been studied by immunofluorescence and transmission electron microscopy.

Results

We report that, in ERBB2+ BC cells, CA reversibly enhances Tz inhibition of cell survival, cooperatively inhibits cell migration and induces cell cycle arrest in G0/G1. These events are accompanied by ERBB2 down-regulation, deregulation of the PI3K/AKT/mTOR signaling pathway and up-regulation of both CDKN1A/p21WAF1 and CDKN1B/p27KIP1. Furthermore, we have demonstrated that CA impairs late autophagy and causes derangement of the lysosomal compartment as shown by up-regulation of SQSTM1/p62 and ultrastructural analysis. Accordingly, we have found that CA restores, at least in part, sensitivity to Tz in SKBR-3 Tz-resistant cell line.

Conclusions

Our data demonstrate the cooperation between CA and Tz in inhibiting cell migration and survival of ERBB2+ BC cells that warrant further studies to establish if CA or CA derivatives may be useful in vivo in the treatment of ERBB2+ cancers.
Appendix
Available only for authorised users
Literature
1.
Hynes NE, MacDonald G. ErbB receptors and signaling pathways in cancer. Curr Opin Cell Biol. 2009;21(2):177–84.CrossRefPubMed
2.
3.
Kennecke H, Yerushalmi R, Woods R, Cheang MC, Voduc D, Speers CH, et al. Metastatic behavior of breast cancer subtypes. J Clin Oncol. 2010;28(20):3271–7.CrossRefPubMed
4.
Carter P, Presta L, Gorman CM, Ridgway JB, Henner D, Wong WL, et al. Humanization of an anti-p185HER2 antibody for human cancer therapy. Proc Natl Acad Sci U S A. 1992;89(10):4285–9.CrossRefPubMedPubMedCentral
5.
6.
Junttila TT, Akita RW, Parsons K, Fields C, Lewis Phillips GD, Friedman LS, et al. Ligand-independent HER2/HER3/PI3K complex is disrupted by trastuzumab and is effectively inhibited by the PI3K inhibitor GDC-0941. Cancer Cell. 2009;15(5):429–40.CrossRefPubMed
7.
Yakes FM, Chinratanalab W, Ritter CA, King W, Seelig S, Arteaga CL. Herceptin-induced inhibition of phosphatidylinositol-3 kinase and Akt is required for antibody-mediated effects on p27, cyclin D1, and antitumor action. Cancer Res. 2002;62(14):4132–41.PubMed
8.
Albanell J, Codony J, Rovira A, Mellado B, Gascon P. Mechanism of action of anti-HER2 monoclonal antibodies: scientific update on trastuzumab and 2C4. Adv Exp Med Biol. 2003;532:253–68.CrossRefPubMed
9.
Nagata Y, Lan KH, Zhou X, Tan M, Esteva FJ, Sahin AA, et al. PTEN activation contributes to tumor inhibition by trastuzumab, and loss of PTEN predicts trastuzumab resistance in patients. Cancer Cell. 2004;6(2):117–27.CrossRefPubMed
10.
Martinez MT, Perez-Fidalgo JA, Martin-Martorell P, Cejalvo JM, Pons V, Bermejo B, et al. Treatment of HER2 positive advanced breast cancer with T-DM1: a review of the literature. Crit Rev Oncol Hematol. 2016;97:96–106.CrossRefPubMed
11.
Uppal H, Doudement E, Mahapatra K, Darbonne WC, Bumbaca D, Shen BQ, et al. Potential mechanisms for thrombocytopenia development with trastuzumab emtansine (T-DM1). Clin Cancer Res. 2015;21(1):123–33.CrossRefPubMed
12.
Baron JM, Boster BL, Barnett CM. Ado-trastuzumab emtansine (T-DM1): a novel antibody-drug conjugate for the treatment of HER2-positive metastatic breast cancer. J Oncol Pharm Pract. 2015;21(2):132–42.CrossRefPubMed
13.
Amaravadi RK, Lippincott-Schwartz J, Yin XM, Weiss WA, Takebe N, Timmer W, et al. Principles and current strategies for targeting autophagy for cancer treatment. Clin Cancer Res. 2011;17(4):654–66.CrossRefPubMedPubMedCentral
14.
Chen S, Zhu X, Qiao H, Ye M, Lai X, Yu S, et al. Protective autophagy promotes the resistance of HER2-positive breast cancer cells to lapatinib. Tumour Biol. 2016;37(2):2321–31.CrossRefPubMed
15.
Vazquez-Martin A, Oliveras-Ferraros C, Menendez JA. Autophagy facilitates the development of breast cancer resistance to the anti-HER2 monoclonal antibody trastuzumab. PLoS One. 2009;4(7):e6251.CrossRefPubMedPubMedCentral
16.
Cufi S, Vazquez-Martin A, Oliveras-Ferraros C, Corominas-Faja B, Cuyas E, Lopez-Bonet E, et al. The anti-malarial chloroquine overcomes primary resistance and restores sensitivity to trastuzumab in HER2-positive breast cancer. Sci Rep. 2013;3:2469.CrossRefPubMedPubMedCentral
17.
Moore J, Yousef M, Tsiani E. Anticancer Effects of Rosemary (Rosmarinus officinalis L.) Extract and Rosemary Extract Polyphenols. Nutrients. 2016;8(11):731.
18.
19.
Baselga-Escudero L, Souza-Mello V, Pascual-Serrano A, Rachid T, Voci A, Demori I, Grasselli E. Beneficial effects of the Mediterranean spices and aromas on non-alcoholic fatty liver disease. Trends Food Sci Technol. 2017;61:141–59.CrossRef
20.
Gao Q, Liu H, Yao Y, Geng L, Zhang X, Jiang L, et al. Carnosic acid induces autophagic cell death through inhibition of the Akt/mTOR pathway in human hepatoma cells. J Appl Toxicol. 2015;35(5):485–92.CrossRefPubMed
21.
Kim YJ, Kim JS, Seo YR, Park JH, Choi MS, Sung MK. Carnosic acid suppresses colon tumor formation in association with antiadipogenic activity. Mol Nutr Food Res. 2014;58(12):2274–85.CrossRefPubMed
22.
Kar S, Palit S, Ball WB, Das PK. Carnosic acid modulates Akt/IKK/NF-kappaB signaling by PP2A and induces intrinsic and extrinsic pathway mediated apoptosis in human prostate carcinoma PC-3 cells. Apoptosis. 2012;17(7):735–47.CrossRefPubMed
23.
Cortese K, Daga A, Monticone M, Tavella S, Stefanelli A, Aiello C, et al. Carnosic acid induces proteasomal degradation of Cyclin B1, RB and SOX2 along with cell growth arrest and apoptosis in GBM cells. Phytomedicine. 2016;23(7):679–85.CrossRefPubMed
24.
Einbond LS, Wu HA, Kashiwazaki R, He K, Roller M, Su T, et al. Carnosic acid inhibits the growth of ER-negative human breast cancer cells and synergizes with curcumin. Fitoterapia. 2012;83(7):1160–8.CrossRefPubMed
25.
Gonzalez-Vallinas M, Molina S, Vicente G, Sanchez-Martinez R, Vargas T, Garcia-Risco MR, et al. Modulation of estrogen and epidermal growth factor receptors by rosemary extract in breast cancer cells. Electrophoresis. 2014;35(11):1719–27.CrossRefPubMed
26.
Bisio A, De Mieri M, Milella L, Schito AM, Parricchi A, Russo D, et al. Antibacterial and hypoglycemic Diterpenoids from salvia Chamaedryoides. J Nat Prod. 2017;80(2):503–14.CrossRefPubMed
27.
Richheimer SL, Bernart MW, King GA, Kent MC, Bailey DT. Antioxidant activity of lipid-soluble phenolic diterpenes from rosemary. J Am Oil Chem Soc. 1996;73(4):507–14.CrossRef
28.
Denizot F, Lang R. Rapid colorimetric assay for cell growth and survival. Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability. J Immunol Methods. 1986;89(2):271–7.CrossRefPubMed
29.
Schindelin J, Rueden CT, Hiner MC, Eliceiri KW. The ImageJ ecosystem: an open platform for biomedical image analysis. Mol Reprod Dev. 2015;82(7–8):518–29.CrossRefPubMedPubMedCentral
30.
Limame R, Wouters A, Pauwels B, Fransen E, Peeters M, Lardon F, et al. Comparative analysis of dynamic cell viability, migration and invasion assessments by novel real-time technology and classic endpoint assays. PLoS One. 2012;7(10):e46536.CrossRefPubMedPubMedCentral
31.
Monticone M, Biollo E, Fabiano A, Fabbi M, Daga A, Romeo F, et al. Z-Leucinyl-leucinyl-norleucinal induces apoptosis of human glioblastoma tumor-initiating cells by proteasome inhibition and mitotic arrest response. Mol Cancer Res. 2009;7(11):1822–34.CrossRefPubMed
32.
Cortese K, Sahores M, Madsen CD, Tacchetti C, Blasi F. Clathrin and LRP-1-independent constitutive endocytosis and recycling of uPAR. PLoS One. 2008;3(11):e3730.CrossRefPubMedPubMedCentral
33.
34.
Neve RM, Chin K, Fridlyand J, Yeh J, Baehner FL, Fevr T, et al. A collection of breast cancer cell lines for the study of functionally distinct cancer subtypes. Cancer Cell. 2006;10(6):515–27.CrossRefPubMedPubMedCentral
35.
Soule HD, Maloney TM, Wolman SR, Peterson WD Jr, Brenz R, CM MG, et al. Isolation and characterization of a spontaneously immortalized human breast epithelial cell line, MCF-10. Cancer Res. 1990;50(18):6075–86.PubMed
36.
Qu Y, Han B, Yu Y, Yao W, Bose S, Karlan BY, et al. Evaluation of MCF10A as a reliable model for normal human mammary epithelial cells. PLoS One. 2015;10(7):e0131285.CrossRefPubMedPubMedCentral
37.
Scholzen T, Gerdes J. The Ki-67 protein: from the known and the unknown. J Cell Physiol. 2000;182(3):311–22.CrossRefPubMed
38.
Baselga J, Albanell J, Molina MA, Arribas J. Mechanism of action of trastuzumab and scientific update. Semin Oncol. 2001;28(5 Suppl 16):4–11.CrossRefPubMed
39.
Cuello M, Ettenberg SA, Clark AS, Keane MM, Posner RH, Nau MM, et al. Down-regulation of the erbB-2 receptor by trastuzumab (herceptin) enhances tumor necrosis factor-related apoptosis-inducing ligand-mediated apoptosis in breast and ovarian cancer cell lines that overexpress erbB-2. Cancer Res. 2001;61(12):4892–900.PubMed
40.
Neve RM, Sutterluty H, Pullen N, Lane HA, Daly JM, Krek W, et al. Effects of oncogenic ErbB2 on G1 cell cycle regulators in breast tumour cells. Oncogene. 2000;19(13):1647–56.CrossRefPubMed
41.
Le XF, Claret FX, Lammayot A, Tian L, Deshpande D, LaPushin R, et al. The role of cyclin-dependent kinase inhibitor p27Kip1 in anti-HER2 antibody-induced G1 cell cycle arrest and tumor growth inhibition. J Biol Chem. 2003;278(26):23441–50.CrossRefPubMed
42.
Alkarain A, Jordan R, Slingerland J. p27 deregulation in breast cancer: prognostic significance and implications for therapy. J Mammary Gland Biol Neoplasia. 2004;9(1):67–80.CrossRefPubMed
43.
Bjorkoy G, Lamark T, Pankiv S, Overvatn A, Brech A, Johansen T. Monitoring autophagic degradation of p62/SQSTM1. Methods Enzymol. 2009;452:181–97.CrossRefPubMed
44.
Mehrpour M, Esclatine A, Beau I, Codogno P. Overview of macroautophagy regulation in mammalian cells. Cell Res. 2010;20(7):748–62.CrossRefPubMed
45.
Koay DC, Zerillo C, Narayan M, Harris LN, DiGiovanna MP. Anti-tumor effects of retinoids combined with trastuzumab or tamoxifen in breast cancer cells: induction of apoptosis by retinoid/trastuzumab combinations. Breast Cancer Res. 2010;12(4):R62.CrossRefPubMedPubMedCentral
46.
Bickel PE, Tansey JT, Welte MA. PAT proteins, an ancient family of lipid droplet proteins that regulate cellular lipid stores. Biochim Biophys Acta. 2009;1791(6):419–40.CrossRefPubMedPubMedCentral
47.
Visanji JM, Thompson DG, Padfield PJ. Induction of G2/M phase cell cycle arrest by carnosol and carnosic acid is associated with alteration of cyclin a and cyclin B1 levels. Cancer Lett. 2006;237(1):130–6.CrossRefPubMed
48.
Rodriguez CE, Reidel SI, Bal de Kier Joffe ED, Jasnis MA, Fiszman GL. Autophagy protects from Trastuzumab-induced Cytotoxicity in HER2 Overexpressing breast tumor spheroids. PLoS One. 2015;10(9):e0137920.CrossRefPubMedPubMedCentral
49.
Zhang MH, Man HT, Zhao XD, Dong N, Ma SL. Estrogen receptor-positive breast cancer molecular signatures and therapeutic potentials (review). Biomed Rep. 2014;2(1):41–52.CrossRefPubMed
50.
Gaya M, Repetto V, Toneatto J, Anesini C, Piwien-Pilipuk G, Moreno S. Antiadipogenic effect of carnosic acid, a natural compound present in Rosmarinus Officinalis, is exerted through the C/EBPs and PPARgamma pathways at the onset of the differentiation program. Biochim Biophys Acta. 2013;1830(6):3796–806.CrossRefPubMed
51.
Rau O, Wurglics M, Paulke A, Zitzkowski J, Meindl N, Bock A, et al. Carnosic acid and carnosol, phenolic diterpene compounds of the labiate herbs rosemary and sage, are activators of the human peroxisome proliferator-activated receptor gamma. Planta Med. 2006;72(10):881–7.CrossRefPubMed
52.
Koppen A, Kalkhoven E. Brown vs white adipocytes: the PPARgamma coregulator story. FEBS Lett. 2010;584(15):3250–9.CrossRefPubMed
53.
Larigauderie G, Furman C, Jaye M, Lasselin C, Copin C, Fruchart JC, et al. Adipophilin enhances lipid accumulation and prevents lipid efflux from THP-1 macrophages: potential role in atherogenesis. Arterioscler Thromb Vasc Biol. 2004;24(3):504–10.CrossRefPubMed
54.
Kimmel AR, Brasaemle DL, McAndrews-Hill M, Sztalryd C, Londos C. Adoption of PERILIPIN as a unifying nomenclature for the mammalian PAT-family of intracellular lipid storage droplet proteins. J Lipid Res. 2010;51(3):468–71.CrossRefPubMedPubMedCentral
55.
Tansey JT, Sztalryd C, Gruia-Gray J, Roush DL, Zee JV, Gavrilova O, et al. Perilipin ablation results in a lean mouse with aberrant adipocyte lipolysis, enhanced leptin production, and resistance to diet-induced obesity. Proc Natl Acad Sci U S A. 2001;98(11):6494–9.CrossRefPubMedPubMedCentral
56.
Martinez-Botas J, Anderson JB, Tessier D, Lapillonne A, Chang BH, Quast MJ, et al. Absence of perilipin results in leanness and reverses obesity in Lepr(db/db) mice. Nat Genet. 2000;26(4):474–9.CrossRefPubMed
57.
Ito S, Murphy CG, Doubrovina E, Jasin M, Moynahan ME. PARP inhibitors in clinical use induce genomic instability in normal human cells. PLoS One. 2016;11(7):e0159341.CrossRefPubMedPubMedCentral
58.
Risinger AL, Dybdal-Hargreaves NF, Mooberry SL. Breast cancer cell lines exhibit differential sensitivities to microtubule-targeting drugs independent of doubling time. Anticancer Res. 2015;35(11):5845–50.PubMedPubMedCentral
59.
Stavik B, Tinholt M, Sletten M, Skretting G, Sandset PM, Iversen N. TFPIalpha and TFPIbeta are expressed at the surface of breast cancer cells and inhibit TF-FVIIa activity. J Hematol Oncol. 2013;6:5.CrossRefPubMedPubMedCentral
Metadata
Title
Cooperative antitumor activities of carnosic acid and Trastuzumab in ERBB2+ breast cancer cells
Authors
Carolina D’Alesio
Grazia Bellese
Maria Cristina Gagliani
Cinzia Aiello
Elena Grasselli
Gianluca Marcocci
Angela Bisio
Sara Tavella
Tiziana Daniele
Katia Cortese
Patrizio Castagnola
Publication date
01-12-2017
Publisher
BioMed Central
Published in
Journal of Experimental & Clinical Cancer Research / Issue 1/2017
Electronic ISSN: 1756-9966
DOI
https://doi.org/10.1186/s13046-017-0615-0

Other articles of this Issue 1/2017

Journal of Experimental & Clinical Cancer Research 1/2017 Go to the issue

Research

Blockage of glycolysis by targeting PFKFB3 suppresses tumor growth and metastasis in head and neck squamous cell carcinoma

Research

Pro-inflammation NF-κB signaling triggers a positive feedback via enhancing cholesterol accumulation in liver cancer cells

Research

miR-500a-3p promotes cancer stem cells properties via STAT3 pathway in human hepatocellular carcinoma

Research

Synthetic regulatory RNAs selectively suppress the progression of bladder cancer

Research

Musashi-2, a novel oncoprotein promoting cervical cancer cell growth and invasion, is negatively regulated by p53-induced miR-143 and miR-107 activation

Research

The oncogenic Golgi phosphoprotein 3 like overexpression is associated with cisplatin resistance in ovarian carcinoma and activating the NF-κB signaling pathway
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