Evaluation of Radiation and Ammonium Lactate Effects on Hyaluronic Acid Expression as a Pro‑cancerous Factor in Supernatant and Exosome Isolated from Supernatant of Primary Mouse Fibroblast Cell Culture

Nasrin Zare, Amirhosein Kefayat, Shaghayegh Haghjooy Javanmard

Abstract


Background: Previous studies show that aberrant synthesis of Hyaluronan accelerates tumor
growth, angiogenesis, and metastasis. The fibroblasts are probably responsible for most of the
hyaluronic acid (HA) accumulation in tumor microenvironment after radiotherapy. Our goal is to
investigate and compare radiation and lactate effects on HA levels in supernatant and exosome
isolated from supernatant of primary mouse fibroblast cell culture. Methods: Fibroblast cells
were prepared from skin of C57BL6 mouse. These cells were divided into three groups (no
treatment, cells treated with 10 mM ammonium lactate, and irradiated cells). Then supernatant
was harvested from FBS‑free culture media after 48 h. Exosomes were purified by differential
centrifugation (300 × g for 10 min, 2000 × g for 30 min, 16500 g for 30 min) and were pelleted
by ultracentrifugation (150,000 × g for 180 min). Size of exosomes was determined using
a Zetasizer. HA concentration measured using a HA ELISA Kit. Data were analyzed using
one‑way ANOVA.

Results: There was a significant increase in HA‑coated exosomes isolated
from supernatants of irradiated cells compared to untreated cell and cells treated with 10 mM
ammonium lactate (P < 0.001). As well, there was a significant increase in the HA concentration
in the supernatants of cells treated with 10 mM ammonium lactate relative to untreated cells and
irradiated cells (P < 0.05).

Conclusions: It seems that routine radiation therapy leads to massive
shedding of HA‑coated exosomes by normal fibroblast cells and thus exosomes‑HA may contribute
to tumor promotion and induce of the premetastatic niche.


Keywords


Exosomes; hyaluronic acid; radiation

Full Text:

PDF

References


Östman A, Augsten M. Cancer‑associated fibroblasts and tumor

growth ‑ bystanders turning into key players. Curr Opin Genet

Dev 2009;1967‑73.

Pietras K, Östman A. Hallmarks of cancer: Interactions with the

tumor stroma. Exp Cell Res 2010;316:1324‑31.

Strell C, Rundqvist H, Östman A. Fibroblasts‑a key host cell

type in tumor initiation, progression, and metastasis. Ups J Med

Sci 2012;117:187‑95.

Slevin M, Krupinski J, Kumar S, Gaffney J. Angiogenic

oligosaccharides of hyaluronan induce protein tyrosine kinase

activity in endothelial cells and activate a cytoplasmic signal

transduction pathway resulting in proliferation. Lab Invest

;78:987‑1003.

Constant S, Huang S, Wisniewski L, Mas C. Advanced human

in vitro models for the discovery and development of lung cancer

therapies. In: Drug Discovery and Development‑From Molecules

to Medicine. In Tech; 2015.

Itano N, Zhuo L, Kimata K. Impact of the hyaluronan‑rich tumor

microenvironment on cancer initiation and progression. Cancer

Sci 2008;99:1720‑5.

Bourguignon LYW, Hongbo Z, Shao L, Chen YW. CD44

interaction with Tiam1 promotes Rac1 signaling and hyaluronic

acid‑ mediated breast tumor cell migration. J Biol Chem

;275:1829‑38.

Lopez JI, Camenisch TD, Stevens MV, Sands BJ, McDonald J,

Schroeder JA. CD44 attenuates metastatic invasion during breast

cancer progression. Cancer Res 2005;65:6755‑63.

Golshani R, Lopez L, Estrella V, Kramer M, Iida N,

Lokeshwar VB. Hyaluronic acid synthase‑1 expression regulates

bladder cancer growth, invasion, and angiogenesis through

CD44. Cancer Res 2008;68:483‑91.

Knudson W, Biswas C, Li XQ, Nemec RE, Toole BP. The role

and regulation of tumour‑associated hyaluronan. Ciba Found

Symp 1989;143:150‑9.

György B, Szabó TG, Pásztói M, Pál Z, Misják P,

Aradi B, et al. Membrane vesicles, current state‑of‑the‑art:

Emerging role of extracellular vesicles. Cell Mol Life Sci

;68:2667‑88.

Yáñez‑Mó M, Siljander PR, Andreu Z, Zavec AB, Borràs FE,

Buzas EI, et al. Biological properties of extracellular vesicles

and their physiological functions. J Extracell Vesicles

;4:1‑60.

Van DerPol E, Böing AN, Harrison P, Sturk A, Nieuwland R.

Classification, functions, and clinical relevance of extracellular

vesicles. Pharmacol Rev 2012;64:676‑705.

Rilla K, Siiskonen H, Tammi M, Tammi R. Hyaluronan‑coated

extracellular vesicles‑ A novel link between hyaluronan and

cancer. Adv Cancer Res 2014;123:121‑48.

Odintsova E, Van Niel G, Conjeaud H, Raposo G, Iwamoto R,

Mekada E, et al. Metastasis suppressor tetraspanin CD82/KAI1

regulates ubiquitylation of epidermal growth factor receptor.

J Biol Chem 2013;288:26323‑34.

Stern R, Shuster S, Neudecker BA, Formby B. Lactate stimulates

fibroblast expression of hyaluronan and CD44: The Warburg

effect revisited. Exp Cell Res 2002;276:24‑31.

Baskar R, Lee KA, Yeo R, Yeoh KW. Cancer and radiation

therapy: Current advances and future directions. Int J Med Sci

;9:193‑9.

Auvinen P, Tammi R, Parkkinen J, Tammi M, Ågren U,

Johansson R, et al. Hyaluronan in peritumoral stroma and

malignant cells associates with breast cancer spreading and

predicts survival. Am J Pathol 2000;156:529‑36.

Ponting J, Kumar S, Pye D. Co‑localisation of hyaluronan and

hyaluronectin in normal and neoplastic breast tissues. Int J Oncol

;2:889‑93.

Prasanna PGS, Stone HB, Wong RS, Capala J, Bernhard EJ,

Vikram B, et al. Normal tissue protection for improving

radiotherapy: Where are the Gaps? Transl Cancer Res

;1:35‑48.

Buch K, Peters T, Nawroth T, Sänger M, Schmidberger H,

Langguth P. Determination of cell survival after irradiation via

clonogenic assay versus multiple MTT Assay ‑ A comparative

study. Radiat Oncol 2012;7:1.

Ni J, Bucci J, Malouf D, Knox M, Graham P, Li Y. Exosomes

in cancer radioresistance. Front Oncol 2019. doi: 10.3389/fonc.

00869.

Anttila MA, Tammi RH, Tammi MI, Syrjänen KJ, Saarikoski SV,

Kosma VM. High levels of stromal hyaluronan predict poor

disease outcome in epithelial ovarian cancer. Cancer Res

;60:150‑5.

Josefsson A, Adamo H, Hammarsten P, Granfors T, Stattin P,

Egevad L, et al. Prostate cancer increases hyaluronan in

surrounding nonmalignant stroma, and this response is associated

with tumor growth and an unfavorable outcome. Am J Pathol

;179:1961‑8.

Lipponen P, Aaltomaa S, Tammi R, Tammi M, Ågren U,

Kosma VM. High stromal hyaluronan level is associated with

poor differentiation and metastasis in prostate cancer. Eur J

Cancer 2001;37:849‑56.

Ricciardelli C, Ween MP, Lokman NA, Tan IA, Pyragius CE,

Oehler MK. Chemotherapy‑induced hyaluronan production:

A novel chemoresistance mechanism in ovarian cancer. BMC

Cancer 2013;13:476.

Toole BP, Slomiany MG. Hyaluronan: A constitutive regulator of

chemoresistance and malignancy in cancer cells. Semin Cancer

Biol 2008;18:244‑50.

(PDF) Increased hyaluronan at sites of attachment to mesentery

by CD44‑positive mouse ovarian and breast tumor cells.

Jung T, Castellana D, Klingbeil P, Hernández IC,

Vitacolonna M, Orlicky DJ, et al. CD44v6 dependence of

premetastatic niche preparation by exosomes. Neoplasia

;11:1093‑105.

Semenza GL. Tumor metabolism: Cancer cells give and take

lactate. J Clin Investig 2008;118;3835‑7.

Auvinen P, Rilla K, Tumelius R, Tammi M, Sironen R, Soini Y,

et al. Hyaluronan synthases (HAS1‑3) in stromal and malignant

cells correlate with breast cancer grade and predict patient

survival. Breast Cancer Res Treat 2014;143:277‑86.

Friedl P, Wolf K. Tumour‑cell invasion and migration: Diversity

and escape mechanisms. Nat Rev Cancer 2003;3:362‑74.

Miletti‑González KE, Chen S, Muthukumaran N, Saglimbeni GN,

Wu X, Yang J, et al. The CD44 receptor interacts with

P‑glycoprotein to promote cell migration and invasion in cancer.

Cancer Res 2005;65:6660‑7.




ijpm_12_448