Inhibition of MCF-7 breast cancer cell proliferation by MCF-10A breast epithelial cells in coculture.

A coculture system was developed to investigate the interactions between MCF-10A breast epithelial cells and MCF-7 breast cancer cells stably expressing the green fluorescent protein (MCF-7-GFP). Studies with this MCF-10A/MCF-7-GFP coculture system on microtiter plates and on reconstituted basement membrane (Matrigel), revealed paracrine inhibition of MCF-7-GFP cell proliferation. Epidermal growth factor, which in monocultures modestly enhanced MCF-7-GFP and markedly increased MCF-10A cell proliferation, greatly inhibited MCF-7-GFP cell proliferation in MCF-10A/MCF-7-GFP cocultures. 17beta-Estradiol, which stimulated MCF-7-GFP but not MCF-10A cell proliferation in monoculture, inhibited MCF-7-GFP cell proliferation in MCF-10A/MCF-7-GFP cocultures, an effect that was blocked by the antiestrogen, ICI 182,780. On Matrigel, complex MCF-10A/MCF-7-GFP cellular interactions were observed in real time that resulted in the formation of acinus-like structures. These results indicate a role of normal epithelial cells in inhibiting tumor-cell proliferation and demonstrate the utility of this coculture system as a model of early paracrine control of breast cancer.

Transient induction of cytochromes P450 1A1 and 1B1 in MCF-7 human breast cancer cells by indirubin.

The aryl hydrocarbon receptor (AhR), when activated by exogenous ligands such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), regulates expression of several phase I and phase II enzymes and is also involved in the regulation of cell proliferation. Several studies suggest that endogenous AhR ligand(s) may exist. One putative endogenous ligand is indirubin, which was recently identified in human urine and bovine serum. We determined the effect of indirubin in MCF-7 breast cancer cells on induction of the activities of cytochromes P450 (CYP) 1A1 and 1B1, as measured by estradiol and ethoxyresorufin metabolism, and on induction of the CYP1A1 and CYP1B1 mRNAs. With 4-hr exposure, the effects of indirubin and TCDD at 10nM on CYP activity were comparable, but the effects of indirubin, unlike those of TCDD, were transitory. Indirubin-induced ethoxyresorufin-O-deethylase activity was maximal by 6-9 hr post-exposure and had disappeared by 24 hr, whereas TCDD-induced activities remained elevated for at least 72 hr. The effects of indirubin on CYP mRNA induction were maximal at 3 hr. Indirubin was metabolized by microsomes containing cDNA-expressed human CYP1A1 or CYP1B1. The potency of indirubin was comparable to that of TCDD in a CYP1B1-promoter-driven luciferase assay, when MCF-7 cells were co-exposed to the AhR ligands together with the CYP inhibitor, ellipticine. Thus, if indirubin is an endogenous AhR ligand, then AhR-mediated signaling by indirubin is likely to be transient and tightly controlled by the ability of indirubin to induce CYP1A1 and CYP1B1, and hence its own metabolism.

Estrogen regulates Ah responsiveness in MCF-7 breast cancer cells.

Cytochrome P450 (CYP)1A1 and CYP1B1, which are under the regulatory control of the aryl hydrocarbon (Ah) receptor (AhR), catalyze the metabolic activation of numerous procarcinogens and the hydroxylation of 17beta-estradiol (E2) at the C-2 and C-4 positions, respectively. There is evidence of cross-talk between estrogen receptor alpha (ERalpha)- and AhR-mediated signaling in breast and endometrial cells. To further examine these interactions, we investigated the short- and long-term effects of E2 exposure on Ah responsiveness in MCF-7 human breast cancer cells. Short-term exposure to 1 nM E2 elevated the ratio of the 4- to 2-hydroxylation pathways of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced E2 metabolism and the ratio of the induced CYP1B1 to CYP1A1 mRNA levels, as determined by real-time PCR. Cells maintained long-term (9-12 months) in low-E2 medium progressively lost Ah responsiveness, as indicated by diminished rates of TCDD-induced E2 metabolism and ethoxyresorufin O-deethylase activity, and the reduced expression of the CYP1A1 and CYP1B1 mRNAs and proteins levels. These E2-deprived cells showed elevated levels of ERalpha mRNA, depressed levels of AhR mRNA, and unchanged levels of the AhR nuclear translocator mRNA. Transient transfection studies using a CYP1B1-promoter-luciferase reporter construct showed that reduced CYP1B1 promoter activity in E2-deprived cells could be restored by co-transfection with an AhR expression construct, indicating that AhR expression was limiting in these cells. The reduced Ah responsiveness of E2-deprived cells was reversed by culture for four passages in medium supplemented with 1 nM E2; ERalpha and AhR mRNAs returned to near-normal levels and the inducibility of the CYP1A1 and CYP1B1 mRNAs, proteins, and E2 metabolic activities by TCDD was restored. These studies indicate that the continued presence of estrogen is required to maintain high levels of AhR expression and inducibility of the procarcinogen-bioactivating enzymes, CYP1A1 and CYP1B1, in MCF-7 cells.

Induction of CYP1A1 and CYP1B1 in T-47D human breast cancer cells by benzo[a]pyrene is diminished by arsenite.

Polycyclic aromatic hydrocarbons (PAHs) and metals are often environmental cocontaminants, yet there have been relatively few studies of combined effects of PAHs and metals on cytochrome P450 (P450)-catalyzed metabolism. We examined the effects of NaAsO(2) in combination with benzo[a]pyrene (BAP) on CYP1A1 and CYP1B1 in T-47D human breast cancer cells by using estrogen metabolism as a probe of their activities. Exposure to BAP caused elevated rates of the 2- and 4-hydroxylation pathways of estrogen metabolism, indicating induction of both CYP1A1, an estradiol 2-hydroxylase, and CYP1B1, an estradiol 4-hydroxylase. BAP-induced metabolism peaked 9 to 16 h after exposure and returned to near-basal levels by 48 h. Concentration-response studies showed maximal induction of the 2- and 4-hydroxylation pathways at 3 microM BAP; higher levels caused reduced rates of metabolism due to inhibition of CYP1A1 and CYP1B1. NaAsO(2) caused pronounced decreases in the induction of CYP1A1 and CYP1B1 by 3 microM BAP because cotreatment with 10 microM NaAsO(2) inhibited the rates of the 2- and 4-hydroxylation pathways by 86 and 92%, respectively. Western immunoblots showed diminished levels of BAP-induced CYP1A1 by coexposure to NaAsO(2). The levels of the CYP1A1 and CYP1B1 mRNAs induced by BAP were not significantly affected by coexposure to NaAsO(2); however, heme oxygenase 1 mRNA levels were markedly induced by coexposure to BAP and NaAsO(2). These results indicate a post-transcriptional inhibitory effect of arsenite on the expression of CYP1A1 and CYP1B1 in T-47D cells, possibly resulting from reduced heme availability.