Role of Renin-Angiotensin-System in Human Breast Cancer Cells: Is There a Difference in Regulation of Angiogenesis between Hormone-Receptor Positive and Negative Breast Cancer Cells?

Abstract

Objective This study examined the role of the RAS in human breast cancer cells to question if there are differences between HR-positive and HR-negative cells with regard to regulation of VEGF.

Methods Expression of different RAS components in hormone receptor (HR)-positive and HR-negative breast cancer cells was investigated using RT-PCR. Different stimulation protocols with different RAS inhibitors were used to investigate the effect on VEGF expression. Angiotensin II-dependent expression of VEGF was quantified by real time PCR. In addition, the effect of intrinsic RAS was studied performing siRNA knockdown of angiotensinogen (AGT). Statistical analysis were calculated using IBM SPSS Statistics Version 21.

Results Expression of AT₁R, AT₂R, AGT and ACE was shown in HR-positive and HR-negative breast cancer cell lines. Extrinsic stimulation with angiotensin II increased VEGF significantly. After treatment with captopril or AT₁R-inhibitor candesartan, VEGF-expression decreased significantly in HR-positive and HR-negative cell lines. However, inhibition of AT₂R using PD 123,319 did not show any significant changes of VEGF. After prevention of intrinsic angiotensin II, extrinsic angiotensin II as well as the combination with inhibitors of the receptors caused a significant reduction of VEGF. Surprisingly, the overall effect of the RAS after knockdown of AGT revealed a significant increase of VEGF in HR-positive cells at any time while a significant decrease was observed in HR-negative cells after 144 hours incubation.

Conclusion The RAS-dependent regulation of VEGF between HR-positive and HR-negative breast cancer cells seems do be different. These findings provide evidence for a possible future therapeutic strategy.

Zusammenfassung

Zielsetzung Im Rahmen dieser Studie wurde die Bedeutung des RAS für die Regulation von VEGF in humanen Mammakarzinomzellen im Hinblick auf mögliche Unterscheide zwischen HR-positiven und HR-negativen Zellen untersucht.

Methoden Die Expression verschiedener Komponenten des RAS wurde in hormonrezeptorpositiven und -negativen Mammakarzinom-Zelllinien durch RT-PCR nachgewiesen und die Angiotensin-II-abhängige Expression von VEGF mittels Real-Time-PCR quantifiziert. Außerdem wurde die Wirkung von intrinsischem Angiotensin II durch siRNA-Knockdown von AGT ausgeschaltet. Die Statistik wurde mittels IBM SPSS Statistics Version 21 berechnet.

Ergebnisse Die Expression von AT₁R, AT₂R, AGT und ACE wurde in hormonrezeptorpositiven und -negativen Mammakarzinomzellen gezeigt. Extrinsische Stimulation mit Angiotensin II erhöhte dabei die VEGF-Expression signifikant. Im Gegensatz dazu war letztere nach Behandlung mit Captopril oder dem AT₁R-Inhibitor Candesartan in HR-positiven und -negativen Zellen signifikant reduziert. Dagegen führte die Blockade des AT₂R mit PD 123,319 zu keiner signifikanten Veränderung von VEGF. Nach Ausschalten von intrinsischem Angiotensin II wurde VEGF durch extrinsisches Angiotensin II oder durch die Kombination mit den Inhibitoren der Rezeptoren signifikant verringert. Überaschenderweise zeigte sich als Nettoeffekt des RAS nach Ausschalten von AGT eine signifikante Zunahme von VEGF in HR-positiven Zellen zu allen Zeitpunkten. Dagegen war in den HR-negativen Zellen eine Abnahme von VEGF nur nach 144 Stunden zu beobachten.

Schlussfolgerung Die RAS-abhängige Regulation von VEGF scheint zwischen hormonrezeptorpositiven und -negativen Mammakarzinomzellen unterschiedlich zu sein. Diese Ergebnisse könnten auf eine mögliche zukünftige therapeutische Option hinweisen.

Key words

breast cancer - RAS - renin - angiotensin - angiogenesis - VEGF

Schlüsselwörter

Mammakarzinom - RAS - Renin - Angiogenese - VEGF

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