Cyclooxygenase-2 inhibition: effects on tumour growth, cell cycling and lymphangiogenesis in a xenograft model of breast cancer

Abstract

Cyclooxygenase-2 (COX-2) is associated with poor-prognosis breast cancer. We used a nude mouse xenograft model to determine the effects of COX-2 inhibition in breast cancer. Oestrogen receptor (ER)-positive MCF7/HER2-18 and ER-negative MDAMB231 breast cancer cell lines were injected into nude mice and allowed to form tumours. Mice then received either chow containing Celecoxib (a COX-2 inhibitor) or control and tumour growth measured. Tumour proliferation, apoptosis, COX-2, lymphangiogenesis and angiogenesis were assessed by immunohistochemistry (IHC), Western blotting or Q-PCR. Celecoxib inhibited median tumour growth in MCF7/HER2-18 (58.7%, P=0.029) and MDAMB231 (46.3%, P=0.0002) cell lines compared to control. Cyclooxygenase-2 expression decreased following Celecoxib treatment (MCF7/HER2-18 median control 65.3% vs treated 22.5%, P=0.0001). Celecoxib increased apoptosis in MCF7/HER2-18 tumours (TUNEL 0.52% control vs 0.73% treated, P=0.0004) via inactivation of AKT (median pAKT(ser473) 57.3% control vs 35.5% treated, P=0.0001--confirmed at Western blotting). Q-PCR demonstrated decreased podoplanin RNA (lymphangiogenesis marker) in the MCF7/HER2-18 - median 2.9 copies treated vs 66.6 control (P=0.05) and MDAMB231-treated groups--median 160.7 copies vs 0.05 control copies (P=0.015), confirmed at IHC. Cyclooxygenase-2 is associated with high levels of activated AKT(ser473) and lymphangiogenesis in breast cancer. Cyclooxygenase-2 inhibition decreases tumour growth, and may potentially decrease recurrence, by inactivating AKT and decreasing lymphangiogenesis.

Figure 1

Western blot MCF7/HER2-18 pAkt^ser473, total Akt. (A) Representative Western blots from MCF7/MCF7/HER2-18 control and Celecoxib-treated tumours. The top blot shows a decrease in pAKT^ser473 in the treated samples (60 kDa). At the bottom, no change was seen in total AKT (60 kDa) as a loading control. The median arbitrary quantity from the control to treated tumours showed a decrease of 28.2% (IQR −1.5–89.1%) at densitometry over all of the tumours studied. (B) A representative Western blot using lysates from the MDAMB231 cell line. There was no overall pattern seen. Total AKT was used as a loading control (60 kDa), bottom blot.

Figure 2

The MCF7/HER2-18 control tumours showed expression of pAKT^ser473 in both the cell nucleus and the cell cytoplasm (A). Following Celecoxib treatment both the nuclear and cytoplasmic components of pAKT^ser473 decreased, this was predominantly seen as a decrease in nuclear pPAKT^ser473 (B), which decreased from a median of 57.3% (IQR 54.1–61.6) in the control tumours to 35.7% (IQR 24.4–47.6) in the treated tumours, P=0.0001. The MDAMB231 cell line expressed predominantly cytoplasmic pAKT^ser473 (C), which did not significantly decrease following Celecoxib treatment. Decreases in lymphatic vessel density were seen in the MCF7/HER2-18 xenografts (D and E) from a median of 12 Chalkley counts to 9 Chalkley counts (P=0.0001). The MDAMB231 xenografts (F and G) also showed a decrease in podoplanin expression from a median of 7 Chalkley counts to 4 Chalkley counts, P=0.0001) following COX-2 inhibition. Representative IHC staining for pAKT^ser473 and podoplanin in the harvested xenografts: (A) pAKT^ser473 staining in MCF7/HER2-18 control xenografts (× 400); (B) pAKT^ser473 staining in MCF7/HER2-18 treated xenografts (× 400); (C) pAKT^ser473 staining in MDAMB231 control xenografts (× 400); (D) Podoplanin staining in MCF7/HER2-18 control xenografts (× 400); (E) Podoplanin staining in MCF7/HER2-18 treated xenografts (× 400); (F) Podoplanin staining in MDAMB231 control xenografts (× 100); (G) Podoplanin staining in MDAMB231 treated xenografts (× 100).

Figure 3

Cyclooxygenase-2 immunoscore in control and Celecoxib-treated MCF7/HER2-18 tumours. Cyclooxygenase-2 immunoscore was calculated as the sum of the intensity score 0–4 (0=none, 1=mild, 2=moderate, 3=strong, 4=very strong) and the percent positivity score (0=nil, 1⩽10%, 2=10–50%, 3⩾50%). No intensity score and therefore immunoscore could be calculated for the MDAMB231 cell line as the staining was confined in cytoplasmic vesicles.