**2.3 Celecoxib (a selective inhibitor of COX-2) and ABCG2 expression and function**

Numerous studies showed that COX-2 inhibitors (coxibs) enhance the efficacy of different anticancer therapy methods. Different mechanisms have been suggested to contribute to the antitumor activity of coxibs such as the inhibition of cell cycle progression, induction of apoptosis, inhibition of angiogenesis and decreased invasive potential of tumor cells (Fife et al., 2004; Gasparini et al., 2003; Hashitani et al., 2003; Masferrer et al., 2000). Another mechanism by which COX-inhibitors could sensitize cells to chemotherapeutic drugs is functional blockade of membrane transporter proteins of the ABC-transporter family (Patel et al., 2002; Zatelli et al., 2005).

Kalalinia et. al. investigated the relationship between the inhibition of COX-2 and expression of ABCG2 in parental and resistance breast cancer cell lines. They reported that treatment of MCF-7 and MCF-7/MX cells with celecoxib up-regulates ABCG2 expression at mRNA levels. The results also indicated that, celecoxib reversed the inhibitory effects of TPA on ABCG2 protein expression and increased its expression to the basal level in MCF-7/MX, while co-treatment of MCF-7 cells with TPA and celecoxib caused increased ABCG2 protein expression to a small amount more than TPA lonely (Fig. 4 ). In the same way , Zrieki et al. showed that treatment of human colorectal Caco-2 cell line with COX-1/ COX-2 inhibitor naproxen led to an stimulation of ABCG2 expression which corresponded to the significant decrease of Rho123 retention achieved in activity study. In contrast, treatment with selective COX-2 inhibitors nimesulide did not influence the expression of ABCG2 at protein level (Zrieki et al., 2008). Several studies have shown that specific COX-2 inhibitors could prevent or reduce the development of chemoresistance phenotype by downregulation of the expression and function of P-glycoprotein (MDR1) (Huang et al., 2007; Kim et al., 2004; Roy et al., 2010; Zatelli et al., 2005; Zatelli et al., 2007). Xia et al. found that celecoxib significantly inhibited MDR1 expression without any effects on pump function of P-gp.

MCF-7 Breast Cancer Cell Line, a Model for the Study of the Association

effects are cell and efflux transporter specific (Nozaki et al., 2007).

of ABCG2 substrates (Pavek et al., 2005).

genes (Garcia-Gras et al., 2000; Lasa et al., 2002).

**2.5 The influence of dexamethasone on ABCG2 expression and function** 

Glucocorticoides are efficacious in the reducing of chemotherapy adverse side effects and show their intrinsic anticancer activity (Vee et al., 2009) (Pavek et al., 2005). Some glucocorticoids, such as beclomethasone, 6α-methylprednisolone, dexamethasone, and triamcinolone, at micromolar concentrations, are shown to efficiently decrease the transport

Glucocorticoide receptor agonists regulate gene expression in various ways, at the transcriptional (Adcock, 2001), posttranscriptional (Korhonen et al., 2002), and posttranslational levels (Kritsch et al., 2002). Direct interaction of ligand-activated GR with control elements of target genes could regulate gene transcription in a positive or negative way. However, there are different mechanisms for the negative regulation of gene transcription by glucocorticoides. They could interfere with general transcription factors such as activator protein-1 (AP-1) (Herrlich, 2001) and nuclear factor-κB (NF-κB) (Almawi et al., 2002), resulting in decreased transcription of AP-1- and NF-κB -responsive genes. Genomic organization of the ABCG2 gene revealed the presence of several AP-1 sites in the ABCG2 promoter (Bailey-Dell et al., 2001). So it could be a direct target of transcriptional repression in a similar way. On the other hand, it has been reported that dexamethasone mediates negative regulation of gene expression by destabilizing the mRNA of some target

Investigating the effects of dexamethasone on ABCG2 expression in MCF-7 cells showed that dexamethasone decreased the mRNA level of ABCG2 gene in comparison with control in MCF-7 and MCF-7/MX cell lines. Flow cytometry analysis indicated that a decrease in the level of ABCG2 protein was observed in dexamethasone treated MCF-7/MX cells. While the level of ABCG2 protein expressed as a ratio of the corresponding control was unchanged in

examined in detail.

Between Inflammation and ABCG2-Mediated Multi Drug Resistance 351

enzyme and subsequent reduction in prostaglandin production (Kismet et al., 2004; Zatelli et al., 2005). Modulation of the efficacy of cancer chemotherapy by NSAIDs has not been

Elahian et. al. investigated the pharmacological silencing of ABCG2 in MCF-7 cells through the use of indomethacin, in the hopes of opening a novel way in management of breast cancer. MTT assay showed that indomethacin did not significantly change the survival of MCF-7 and MCF-7/MX cells, but cotreatment of mitoxantrone with indomethacin increased the mitoxantrone cytotoxicity and reduced the IC50 of mitoxantrone in these cells. Altough indomethacin sensitized MCF-7 cells to mitoxantrone, but it did not alter mitoxantrone accumulation in MCF-7 cells, compared to the control (Elahian et al., 2010). It might suggest that indomethacin exerts the sensitising effects through a mechanism not involving the inhibition of ABCG2, but possibly reducing the synthesis of COX and its end-products (Spugnini et al., 2006; Verdina et al., 2008). Indeed, further studies would be necessary to clarify the molecular mechanisms involved in the potentiation of mitoxantrone cytotoxicity by indomethacin in MCF-7 and MCF-7/MX cells. Real-time PCR results showed that indomethacin-treated MCF-7 cells indicated no significant change in the amount of ABCG2 mRNA expression. This observation has been also confirmed on the level of ABCG2 protein expression (Elahian et al., 2009). As a result, expression of a MDR phenotype in human malignant cells may not always be sensitive to potentiation of drug cytotoxicity by NSAIDs (Roller et al., 1999). The present results also confirmed other studies that show NSAIDs'

They demonstrated that the inhibitory effect of celecoxib on MDR1 was COX-2-independent but directly correlated to hypermethylation of MDR1 gene promoter (Xia et al., 2009). In addition, it is shown that COX-2 inhibitors induced PGH2 generation and NF-κB activation, which result in inhibition of P-gp expression and function in breast cancer cells (Zatelli et al., 2009).

Fig. 4. Effects of celecoxib on the expression of ABCG2 at protein levels in MCF-7 (A) and MCF-7/MX (B) cell lines were studied by flow cytometry.

Cells were fixed and permeabilized by formaldehyde and methanol, blocked with BSA and then incubated with primary monoclonal antibody BXP-21. After washing, cells were incubated with a FITC-conjugated goat anti-mouse antibody. Each histogram shows the overlay of the TPA treated sample (black), TPA and celecoxib treated sample (dark gray), untreated sample (ligh gray) and secondary antibody as negative control (broken light gray).

In MCF-7 cell line, celecoxib in presence of TPA 10 nM caused reduction of ABCG2 function in a dose- and time-dependent manner (Kalalinia et al., 2010). Another study provides evidence that NS-398, selective COX-2 inhibitor, sensitizes chemoresistant breast cancer cells to the cytotoxic effects of doxorubicin and notably enhances intracellular DOX accumulation and retention in vitro. It was shown that these effects depended on the inhibition of P-gp expression and function in both native and chemoresistant MCF-7 cells (Zatelli et al., 2007).

#### **2.4 The influence of indomethacin on ABCG2 expression and function**

Several preclinical and clinical trials have shown that nonsteroidal anti-inflammatory drugs (NSAIDs), used as classical COX inhibitors, could reduce the incidence of cancers (Cha et al., 2007; Kang et al., 2005; Lin et al., 2005). Although the exact anticancer mechanisms of NSAIDs are not fully understood, it seems to be related closely to their suppression of COX

They demonstrated that the inhibitory effect of celecoxib on MDR1 was COX-2-independent but directly correlated to hypermethylation of MDR1 gene promoter (Xia et al., 2009). In addition, it is shown that COX-2 inhibitors induced PGH2 generation and NF-κB activation, which result in inhibition of P-gp expression and function in breast cancer cells (Zatelli et al.,

Fig. 4. Effects of celecoxib on the expression of ABCG2 at protein levels in MCF-7 (A) and

Cells were fixed and permeabilized by formaldehyde and methanol, blocked with BSA and then incubated with primary monoclonal antibody BXP-21. After washing, cells were incubated with a FITC-conjugated goat anti-mouse antibody. Each histogram shows the overlay of the TPA treated sample (black), TPA and celecoxib treated sample (dark gray), untreated sample (ligh gray) and secondary antibody as negative control (broken light gray). In MCF-7 cell line, celecoxib in presence of TPA 10 nM caused reduction of ABCG2 function in a dose- and time-dependent manner (Kalalinia et al., 2010). Another study provides evidence that NS-398, selective COX-2 inhibitor, sensitizes chemoresistant breast cancer cells to the cytotoxic effects of doxorubicin and notably enhances intracellular DOX accumulation and retention in vitro. It was shown that these effects depended on the inhibition of P-gp expression and function in both native and chemoresistant MCF-7 cells (Zatelli et al., 2007).

**2.4 The influence of indomethacin on ABCG2 expression and function** 

Several preclinical and clinical trials have shown that nonsteroidal anti-inflammatory drugs (NSAIDs), used as classical COX inhibitors, could reduce the incidence of cancers (Cha et al., 2007; Kang et al., 2005; Lin et al., 2005). Although the exact anticancer mechanisms of NSAIDs are not fully understood, it seems to be related closely to their suppression of COX

MCF-7/MX (B) cell lines were studied by flow cytometry.

(A)

(B)

2009).

enzyme and subsequent reduction in prostaglandin production (Kismet et al., 2004; Zatelli et al., 2005). Modulation of the efficacy of cancer chemotherapy by NSAIDs has not been examined in detail.

Elahian et. al. investigated the pharmacological silencing of ABCG2 in MCF-7 cells through the use of indomethacin, in the hopes of opening a novel way in management of breast cancer. MTT assay showed that indomethacin did not significantly change the survival of MCF-7 and MCF-7/MX cells, but cotreatment of mitoxantrone with indomethacin increased the mitoxantrone cytotoxicity and reduced the IC50 of mitoxantrone in these cells. Altough indomethacin sensitized MCF-7 cells to mitoxantrone, but it did not alter mitoxantrone accumulation in MCF-7 cells, compared to the control (Elahian et al., 2010). It might suggest that indomethacin exerts the sensitising effects through a mechanism not involving the inhibition of ABCG2, but possibly reducing the synthesis of COX and its end-products (Spugnini et al., 2006; Verdina et al., 2008). Indeed, further studies would be necessary to clarify the molecular mechanisms involved in the potentiation of mitoxantrone cytotoxicity by indomethacin in MCF-7 and MCF-7/MX cells. Real-time PCR results showed that indomethacin-treated MCF-7 cells indicated no significant change in the amount of ABCG2 mRNA expression. This observation has been also confirmed on the level of ABCG2 protein expression (Elahian et al., 2009). As a result, expression of a MDR phenotype in human malignant cells may not always be sensitive to potentiation of drug cytotoxicity by NSAIDs (Roller et al., 1999). The present results also confirmed other studies that show NSAIDs' effects are cell and efflux transporter specific (Nozaki et al., 2007).
