The effect of second-line antiestrogen therapy on breast tumor growth after first-line treatment with the aromatase inhibitor letrozole: long-term studies using the intratumoral aromatase postmenopausal breast cancer model.

PURPOSE: The aromatase inhibitors letrozole and anastrozole have been approvedrecently as first-line treatment options for hormone-dependent advanced breast cancer. Although it is established that a proportion of patients who relapse on first-line tamoxifen therapy show additional responses to aromatase inhibitors, it has not been determined whether tumors that acquire resistance to aromatase inhibitors in the first line remain sensitive to second-line therapy with antiestrogens. The aim of this study was to determine whether aromatase-transfected and hormone-dependent MCF-7Ca human breast cancer cells remain sensitive to antiestrogens after: (a) long-term growth in steroid-depleted medium in vitro; and (b) long-term treatment with the aromatase inhibitor letrozole in vivo. METHODS: In the first approach, a variant of the MCF-7Ca human breast cancer cell line was selected that had acquired the ability to grow in estrogen-depleted medium after 6-8 months of culture. Steroid-deprived UMB-1Ca cells were analyzed for aromatase activity levels, hormone receptor levels, and sensitivity to estrogens and antiestrogens in vitro and in vivo. In the second approach, established MCF-7Ca breast tumor xenografts were treated with letrozole 10 microg/day for 12 weeks followed by 100 microg/day for 25 weeks until tumors acquired the ability to proliferate in the presence of the drug. Long-term letrozole-treated tumors were then transplanted into new mice, and the effects of antiestrogens and aromatase inhibitors on tumor growth were determined. RESULTS: Steroid-deprived UMB-1Ca breast cancer cells continued to express aromatase activity at levels comparable with the parental cell line. However, compared with MCF-7Ca cells, UMB-1Ca cells expressed elevated levels of functionally active estrogen receptor. The growth of UMB-1Ca cells in vitro was inhibited by the antiestrogens tamoxifen and faslodex and tumor growth in vivo was inhibited by tamoxifen. In the second approach, the time for MCF-7Ca tumor xenografts to approximately double in volume after being treated sequentially with the increasing doses of letrozole was thirty-seven weeks. Long-term letrozole-treated tumors continued to express functionally active aromatase. When transplanted into new mice, growth of the long-term letrozole-treated tumors was slowed by tamoxifen and inhibited more effectively by faslodex. Tumor growth was refractory to the aromatase inhibitors anastrozole and formestane but, surprisingly, showed sensitivity to letrozole. CONCLUSIONS: Steroid-deprived UMB-1Ca human breast cancer cells selected in vitro and long-term letrozole-treated MCF-7Ca breast tumor xenografts remain sensitive to second-line therapy with antiestrogens and, in particular, to faslodex. This finding is associated with increased expression of functionally active estrogen receptor after steroid-deprivation of MCF-7Ca human breast cancer cells in vitro.     

The pure antiestrogen ICI 182780 is more effective in the induction of apoptosis and down regulation of BCL‐2 than tamoxifen in MCF‐7 cells

There is increasing evidence that induction of apoptosis by antihormones is an important mechanism in regard to their growth inhibitory action on hormone dependent tumors. In this report we have compared the efficiency of tamoxifen (Tam) and the pure antiestrogen ICI 182780 (ZM) to induce apoptosis in the estrogen dependent breast cancer cell line MCF7. Clear evidence for induction of apoptosis could be demonstrated after treatment with both antiestrogens. Application of the pure antiestrogen ZM led to a significantly higher induction of apoptosis compared to the partial agonistic compound Tam. The ability of the two compounds to induce apoptosis correlated with their growth inhibitory action. On the molecular level administration of ZM led to a time dependent steady decrease of BCL2 mRNA and protein. Administration of Tam also initially decreased the expression of BCL2. In contrast to ZM treatment, BCL2 expression increased again after 8h of incubation with Tam. After 96h Tam treated cells expressed BCL2 levels nearly as high as untreated cells. In general, ZM decreased BCL2 levels more effectively than Tam. Our results demonstrate that ZM and Tam possess quantitative and qualitative differences in their ability to down regulate BCL2 expression. The higher ability of the pure antiestrogen to down regulate BCL2 expression may explain the superiority of the pure antiestrogen to induce apoptosis and to inhibit the growth of MCF7 cells.     

Regulation of BAX and BCL‐2 expression in breast cancer cells by chemotherapy

Optimizing chemotherapeutic drug delivery strategies relies, in part, on identification of the most clinically effective sequence, dose, and duration of drug exposure. The combination of dose intensive etoposide (VP16) followed by cyclophosphamide has clinical efficacy in the treatment of advanced breast cancer. However, molecular mechanisms that underlie the effectiveness of this combination of chemotherapeutic agents have not been investigated. In this study we investigated regulation of BAX and BCL2 expression by VP16 and cyclophosphamide as a potential mechanism for the induction of breast cancer cell death induced by this regimen.There was a dose and time dependent increase in BAX expression in the breast cancer cell lines MCF7, MDAMB435S, and MDAMBA231 following in vitro treatment with 50–100M VP16. Elevation of BAX protein expression in the presence of VP16 alone did not correlate with reduced viability or induction of apoptosis in MCF7, MDAMB435S, or MDAMBA231. VP16 did effectively block the breast cancer cell lines evaluated (MCF7 and MDAMB435S) at G₂/M phase of the cell cycle, confirming activity of the drug in vitro. MCF7 and MDAMB435S cells that were pretreated with VP16 and subsequently exposed to 1.0–12.0g/m1 4hydroperoxycyclophosphamide (4HC), an active metabolite of cyclophosphamide, had markedly reduced viability when compared to matched controls treated with either VP16 or 4HC individually. Consistent with this loss of viability, exposure of all three cell lines to the combination of VP16 and 4HC resulted in higher BAX protein levels than those observed following treatment with either single agent. This combination of chemotherapeutic agents also resulted in reduced BCL2 expression.These observations suggest that combination chemotherapy may derive its efficacy, in part, through coordinated regulation of specific gene products associated with apoptosis. Characterization of molecular events that underlie susceptibility of specific tumor cells to combination chemotherapeutic regimens may lead to additional improvements in treatment strategies for this disease.     

The effects of aromatase inhibitors and antiestrogens in the nude mouse model

The effects of antiestrogens, tamoxifen and ICI 182,780, and aromatase inhibitors, arimidex (anastrozole ZD1033) and letrozole (CGS 20,267), on the growth of tumors were studied in nude mice. In this model, estrogen dependent MCF-7 human breast cancer cells stably transfected with the aromatase gene were inoculated in four sites per mouse. Sufficient estrogen is produced from aromatization of androstenedione supplement (0.1 mg/mouse/day) by the cells to stimulate their proliferation, tumor formation, and maintain the uterus similar to that of the intact mouse. Once the tumors reached a measurable size, the mice were injected with antiestrogen or inhibitor for 35–56 days. Tumor volumes were measured weekly. At autopsy, the tumors were removed, cleaned, and weighed. Statistical data was determined from tumor weights. Both antiestrogens were effective in reducing tumor growth in these mice. Tamoxifen appears to be more effective than ICI 182,780, although the former stimulated the uterine weight whereas the pure antiestrogen did not. However, both aromatase inhibitors were more effective than the antiestrogens. Tumor regression was observed with letrozole. Thus, after-treatment tumor weights were less than those of a group of mice at the start of treatment. The aromatase inhibitors also reduced the weight of the uterus, suggesting that these compounds, as well as the pure antiestrogen, may not cause endometrial proliferation, unlike tamoxifen. These aromatase inhibitors may not only benefit patients who have relapsed from tamoxifen, but may be more effective in patients as first line agents for suppressing the effects of estrogen.     

Mechanism of captopril toxicity to a human mammary ductal carcinoma cell line in the presence of copper

Captopril (D3mercapto2methylpropanoylLproli ne) is an angiotensin converting enzyme (ACE) inhibitor, used widely in the treatment of hypertension and congestive heart failure. Captopril also inhibits proliferation of a variety of cell types, including several lacking ACE and renin acitvity. We have previously demonstrated that human mammary ductal carcinoma cells are among the cell types whose mitotic activity is inhibited by captopril. In those cells, captopril also reduces estrogen receptor (ER) and increases progesterone receptor (PR) concentrations. The present study evaluated the mechanism of captopril's antiproliferative action in an ER/PRnegative human mammary ductal carcinoma cell line, Hs578T. Cells grown in a 10% serum medium showed negligible changes in the presence of captopril alone. However, in the presence of subphysiologic concentrations of copper salts or copperloaded ceruloplasmin, captopril caused a dosedependent reduction in cell number, thymidine incorporation and mitochondrial dehydrogenase activity. In contrast, iron salts and ironsaturated transferrin had no effect on captopril activity. Catalase and horseradish peroxidase nullified the cytotoxic effects of captopril/Cu⁺⁺, whereas H₂O₂ mimicked those effects. These data are consistent with the notion of a coppercatalyzed oxidation of captopril, leading to the generation of H₂O₂ as the cytotoxin to this clinically important cell type.     

Signaling pathways of apoptosis activated by aromatase inhibitors and antiestrogens

Aromatase inhibitors have recently been reported to be more effective than the antiestrogen tamoxifen (Tam) in treating breast cancer. Here, we studied the mechanisms and signaling pathways of cell growth, cell cycle progression, and apoptosis induced by three aromatase inhibitors: letrozole (Let), anastrozole, and 4-hydroxyandrostenedione in comparison with estrogen withdrawal (E2W) and antiestrogens Tam and faslodex. Estrogen-dependent human breast cancer cells stably transfected with aromatase (MCF-7Ca) were used. All treatments induced growth suppression and cell cycle arrest at the G(0)-G(1) phase that was associated with up-regulation of p53 and p21 protein and mRNA levels and down-regulation of cyclin D1 and c-myc mRNA. The apoptotic index was increased 4-7 fold, Bcl-2 protein expression decreased, Bax increased, and caspase-9, caspase-6, and caspase-7 were activated but not caspase-3 and caspase-8. Let and E2W caused regression of tumors of MCF-7Ca cells grown in nude mice and increased the number of cells undergoing apoptosis. In contrast, Tam and faslodex did not induce tumor regression and a lower number of apoptotic cells was detected. Cleavage of poly(ADP-ribose) polymerase was detected. Treatment with Let, Tam, or E2W resulted in a dose- and time-dependent increase in active caspase-7 and up-regulation of p53 and p21 protein. Although the mechanisms involved appeared to be similar for antiestrogens and aromatase inhibitors, the most significant effects occurred with Let, which were significantly greater than with E2W and consistent with marked effects of Let on tumor and cell growth.     

Association between endometrial cancer and tamoxifen treatment of breast cancer

A retrospective cohortstudy in 4109 breast cancer patients was undertaken to determine how tamoxifen affected the risk of endometrial cancer. Data on 1701 tamoxifentreated women were analysed. Two thousand four hundred and eight nontamoxifen users served as control group. The occurrence of new primary uterine cancers was assessed by computerized linkage to the Austrian Cancer Registry. Twentyfive women who subsequently developed endometrial cancer were identified. Eight uterine cancers occurred in the tamoxifen group, whereas 17 uterine cancers were found in the control group. The estimate of the relative risk (RR) showed an increased risk to develop endometrial cancer for the tamoxifen group RR 1.136 (95% CI 0.71; 1.80). Analysis of relevant confounding variables did not show any differences in the two groups.In conclusion, this retrospective study demonstrated a nonsignificant increased risk of endometrial cancer in women receiving tamoxifen as treatment for breast cancer. However, the magnitude of RR and the absolute number of endometrial cancer cases in this long term observation demonstrate clearly that the clinical benefit of tamoxifen therapy greatly outweighs the risk.     

Differential regulation of normal and tumoral breast epithelial cell growth by fibroblasts and 1,25‐dihydroxyvitamin D3

Mesenchymalepithelial interactions are of paramount importance during normal and tumoral breast developments. We have investigated the paracrine growth regulation of normal and tumoral breast epithelial cells by fibroblasts derived from normal or pathological breast tissues. In some cases, breast cancer MCF7 cells or normal epithelial cells in primary culture were cocultured with fibroblasts in a Transwell system allowing diffusible factor exchanges. Alternatively, conditioned medium produced by fibroblast cultures was added to epithelial cell cultures. Fibroblasts were shown to stimulate the proliferation of normal and carcinoma cells through paracrine mechanisms. However, the paracrine exchanges appeared to be different in normal versus tumoral breast epithelial cell growth regulation. Moreover, vitamin Drelated compounds that have been proposed as antitumoral drugs were studied for their ability to affect normal and tumoral mammary epithelial cell proliferation and to interfere with the growthregulatory activity of fibroblasts. Whereas vitamin D compounds inhibited MCF7 cell growth, they led to a marked stimulation of the proliferation of normal mammary epithelial cells. Moreover, it was shown that the vitamin D analog EB 1089 can block the mitogenic effect of fibroblastconditioned medium on tumoral but not normal breast epithelial cells. The differential effects of vitamin D compounds on cell proliferation provide further data in favor of the different behaviours of normal and tumoral mammary epithelial cells. The potential therapeutic use of vitamin D derivatives in the treatment of breast cancer is supported by these results but their growthstimulatory properties on normal epithelial cells cannot be overlooked.     

Synergistic action of apoptosis induced by eicosapentaenoic acid and TNP‐470 on human breast cancer cells

The effects of eicosapentaenoic acid (EPA) and an angiogenesis inhibitor (TNP470) on the suppression of breast cancer cell growth were examined in five human breast cancer cell lines (MDAMB231, T47D, MCF7, KPL1, and MKLF). In all five cell lines, EPA and TNP470 alone both showed tumor growth inhibition in a time and dosedependent manner, and in combination, a synergistic effect was seen at high concentrations. EPA plus TNP470 treatment evoked apoptosis as confirmed by the appearance of sub G1 populations, by DNA fragmentation, and by cell morphology. With the combination, the expression of Bax and Bc1x_S, the apoptosisenhancing proteins, was more upregulated and that of Bcl2 and Bclx_L, the apoptosissuppressing proteins, was more downregulated compared to the use of EPA or TNP470 alone, suggesting that their synergistic effect was due to an acceleration of apoptosis.     

Successful co‐treatment with LHRH‐agonist for ovarian over‐stimulation and cystic formation in premenopausal tamoxifen exposure

The present study evaluates the potential beneficial effect of cotreatment with LHRHagonist in resolving premenopausal tamoxifen's induced supraphysiological serum 17 estradiol levels and persistent ovarian cysts. Ultrasonographic and serum hormonal evaluations were performed before, during, and following three consecutive injections of long acting LHRHagonist administered to 14 premenopausal breast cancer patients treated with tamoxifen, who had supraphysiological serum 17 estradiol levels and simultaneous persistent ovarian cysts. Within 3 weeks of the first LHRHagonist injection, all patients had menopausal serum estradiol levels. Ovarian cysts completely disappeared within 2 months following the first injection. Following the discontinuation of LHRHagonist cotreatment, serum estradiol levels remained in physiological levels and the ovaries remained a normal size in 64.3% of the patients for 13.3 ± 11.5 months. 28.6% of the patients had a gradual reappearance of high serum estradiol levels and of ovarian cysts, and were, therefore, treated with a second course of LHRHagonist. Following the second course, serum estradiol levels remained in physiological levels and the ovaries remained a normal size for 8–15 months. It is concluded that short duration of cotreatment with long acting LHRHagonist administered to premenopausal breast cancer patients treated with tamoxifen, successfully resolved the tamoxifeninduced supraphysiological serum 17 estradiol levels and the ovarian cysts.affiliated with Sackler Faculty of Medicine, Tel Aviv University     

Involvement of nuclear steroid/thyroid/retinoid receptors and of protein kinases in the regulation of growth and of c‐erbB and retinoic acid receptor expression in MCF‐7 breast cancer cells

Nuclear steroid/thyroid/retinoid receptors and cerbB membrane receptor tyrosine kinases control epithelial growth and differentiation. Retinoid receptors can dimerize with the vitamin D receptor, the glucocorticoid receptor or the thyroid receptor. Furthermore, multiple cerbB receptor dimers have been identified. It has been shown that some of these receptor pathways communicate with each other via crossconnected regulatory networks. Molecular interactions between retinoid receptors or estrogen receptors (ER) and cerbB2, and between ER and retinoic acid receptor(RAR) have been reported. Here, we demonstrate the effects of steroids/thyroids/retinoids and of activators of protein kinase A (forskolin, Forsk) and C (12Otetradecanoylphorbol13acetate, TPA), on growth and expression of cerbB and RARs in MCF7 breast cancer cells, which contain high levels of RAR and , and which express significant amounts of cerbB2 and 3. All transretinoic acid (tRA), the antiestrogen ICI 182 780 (ICI), Forsk and TPA reduced, whereas triiodothyronine and 17estradiol (E₂) stimulated cell growth. Flow cytometry revealed that tRA and E₂ reduced cerbB2 and 3, whereas tamoxifen, Forsk and TPA upregulatedcerbB2. cerbB3 was coregulated with cerbB2. Northern analysis demonstrated that RAR was downregulated by dexamethasone, ICI, and TPA, whereas vitamin D₃ and E₂ upregulated RAR. RAR expression was less responsive to such treatment, being reduced only by ICI and Forsk. These data indicate that nuclear receptor and protein kinase signaling communicate with each other and control the expression of RARs and cerbB receptors. Efficient growth control requires the coordinated interplay of both receptor systems.     

Prognostic significance of Ki‐67 and topoisomerase IIα expression in infiltrating ductal carcinoma of the breast

To evaluate the prognostic relevance of Ki67 and topoisomerase II expression in relation to tumor stage, grade, and hormone receptor content, 942 ductal infiltrating carcinomas of the breast were examined by means of the monoclonal antibodies KiS11 (Ki67) and KiS4 (topoisomerase II). pS2, cerbB2, and p53 were additionally considered as prognostic variables. The median followup time was 149 months. Eighthundredandsixtythree tumors reacted with KiS11 and KiS the labeling indices of the two antigens were closely associated (r=0.93). Both correlated positively with the tumor size, cerbB2, and p53 expression, and negatively with patient age, hormone receptor content, and pS2 immunostaining. In the univariate analysis, KiS11 and KiS4 scores, nodal status, tumor size, tumor grade, and progesterone receptor content strongly predicted both overall and metastasisfree survival (p <0.00001). Estrogen receptor status, p53, and cerbB2 were of minor significance. Concerning overall survival, multivariate Cox regression analysis selected a KiS4 score >25% (p < 0.00001) next to the nodal status, and before tumor size, progesterone receptor content, and patient age. Independent predictors of the occurrence of distant metastases were nodal status, KiS4, tumor size, grade 1, and progesterone receptor negativity, in that order. The KiS11 score was of independent prognostic significance only if examined as a continuous variable. We conclude that topoisomerase II expression as assessed by monoclonal antibody KiS4 may add valuable information to current prognostic models for breast cancer. Its predictive value appears to be essentially related to the proliferative activity of tumor cells.     

Invasion marker PAI‐1 remains a strong prognostic factor after long‐term follow‐up both for primary breast cancer and following first relapse

In 1991, our group was the first to report the prognostic strength of plasminogen activator inhibitor type 1 (PAI1) in primary breast cancer. The prognostic impact of invasion markers PAI1 and urokinasetype plasminogen activator (uPA) on diseasefree survival (DFS) and overall survival (OS) in breast cancer has since been independently confirmed. We now report on the prognostic impact of PAI1 and uPA after longterm median followup of 77 months for our cohort (n=316).Levels of uPA, PAI1, and cathepsin D were determined in tumor tissue extracts by immunoenzymatic methods. Sphase fraction (SPF) was measured flowcytometrically in paraffin sections. Using logrank statistics, optimized cutoffs were found for PAI1 (14ng/mg), uPA (3ng/mg), cathepsin D (41pmol/mg), and SPF (6%). In all patients, various factors (PAI1, uPA, nodal status, SPF, cathepsin D, grading, tumor size, hormone receptor status) showed significant univariate impact on DFS. In Cox analysis, only nodal status (p < 0.001, RR: 3.1) and PAI1 (p < 0.001, RR: 2.7) remained significant. In nodenegative patients (n = 147), PAI1, uPA, and SPF had significant univariate impact on DFS, whereas in Cox analysis, only PAI1 was significant. PAI1 was also significant for DFS within subgroups defined by established factors. In CART analysis, uPA enhanced the prognostic value of PAI1 and nodal status for determination of a verylowrisk subgroup. For OS, only lymph node status and PAI1 were significant in multivariate analysis. PAI1 levels in the primary tumor were also a significant prognostic marker for survival after first relapse in both univariate and multivariate analysis.     

p53 expression in breast and endometrium during estrogen and tamoxifen treatment of surgically postmenopausal cynomolgus macaques

Estrogens are important for both normal cell growth and malignant proliferation in the mammary gland as well as in the endometrium. Tamoxifen is a nonsteroidal antiestrogen widely used in breast cancer treatment. In recent years reports have been made of an increased risk of endometrial carcinoma during tamoxifen treatment. We used surgically menopausal cynomolgus macaques to study proliferation and p53 expression during hormonal replacement therapy (HRT) and tamoxifen treatment. Animals were treated continuously for 35 months with either conjugated equine estrogens (CEE; n = 20); medroxyprogesterone acetate (MPA; n =17); the combination of CEE + MPA (n = 13) or tamoxifen (n = 17) for 35 months. We found an increased expression of p53 in normal breast and endometrial tissue linked to CEE but not tamoxifen treatment. In the breast alveoli there was an association between proliferation measured by morphometry and p53 expression in all groups. However, in the endometrium CEE induced significantly more p53 positivity than tamoxifen, 9/20 vs. 3/17 in glands and 9/19 vs. 0/17 in stroma, respectively. If indeed longterm treatment with tamoxifen as in the present study could inactivate the tumorsuppressive function of p53, endometrial cells might thereby become more susceptible to genetic lesions associated with carcinogenesis.     

Tissue inhibitor of metalloprotease (TIMP)‐1 and proliferative behaviour of clonal breast cancer cells

In the present paper, we have examined whether human tissue inhibitor of metalloprotease1 (hTIMP1) is able to exert a growth factorlike effect on two clonal cell lines (BC3A and BC61), isolated from a parental line of human breast carcinoma cells (8701BC), and endowed with different growth and invasive behaviour in vitro and in nude mouse. The data obtained indicate that only the more tumorigenic clonal cell line (BC61) is responsive to hTIMP1 treatment by increasing its proliferative rate in a dosedependent manner. It was also found that BC61 cells selectively express a transmembrane protein of about 80kDa able to bind hTIMP1 in vitro and in vivo with high affinity (K_d of 0.07 ± 0.004 nM), and that treatment of BC61 cells with a proliferationpromoting concentration of hTIMP1 is able to stimulate tyrosinetargeted phosphorylation. The cumulative results obtained strongly support the hypothesis that hTIMP1, classically regarded as a collagenase inhibitor, may be a crucial element of the extracellular signalling network during breast cancer development by controlling cell growth phenotype in autocrine and paracrine manner, and that intratumoural heterogeneity for the biological response to TIMP1 may exist within the composite cell population of the primary tumour site.     

Induction of insulin‐like growth factor binding protein expression by ICI 182,780 in a tamoxifen‐resistant human breast cancer cell line

Earlier studies in our laboratory demonstrated that the steroidal antiestrogen ICI 182,780 is very effective in abolishing the tamoxifenresistant proliferation of MCF 7/523 cells [1]. In addition, preliminary binding studies showed that ICI 182,780 increased the binding of insulinlike growth factor (IGF)I to the MCF 7/523 cells, although this finding was not the result of an increase in the expression of the insulinlike growth factorI receptor (IGFIR). Hence, we reasoned that the inhibition of tamoxifenresistant cell growth by ICI 182,780 might have been due to increased expression of insulinlike growth factor binding proteins (IGFBPs).We observed the upregulation of noninsulinsuppressible IGFI binding in both the tamoxifensensitive MCF 7/521 cell line (1.5fold) and the tamoxifenresistant MCF 7/523 cell line (2.5fold) after 5 days of treatment with ICI 182,780 (10^–7 M) in serumfree medium, suggesting a role for cellassociated IGFBPs. Affinity crosslinking experiments confirmed the presence of an IGFI:IGFBP complex of approximately 38kDa in tamoxifen or ICI 182,780treated cells. Western ligand blots showed higher levels of a soluble 30kDa IGFBP in media conditioned by either of the subclones that had been treated with ICI 182,780, an effect consistently opposed by estrogen (E₂:10^–9 M). RTPCR showed higher levels of IGFBP5 mRNA than any of the other known IGFBPs, suggesting that this was the major IGFBP subtype. The protein was subsequently identified by Western immunoblotting as IGFBP5. In conclusion, we postulate that this may be a mechanism contributing to the greater potency of ICI 182,780 in the growth inhibition of the MCF 7/523, tamoxifenresistant cell line.     

Coexpression of c-kit and stem cell factor in breast cancer results in enhanced sensitivity to members of the EGF family of growth factors

Insulinlike growth factor (IGF)I protects many cell types from apoptosis. As a result, it is possible that IGFIresponsive cancer cells may be resistant to apoptosisinducing chemotherapies. Therefore, we examined the effects of IGFI on paclitaxel and doxorubicininduced apoptosis in the IGFIresponsive breast cancer cell line MCF7. Both drugs caused DNA laddering in a dosedependent fashion, and IGFI reduced the formation of ladders. We next examined the effects of IGFI and estradiol on cell survival following drug treatment in monolayer culture. IGFI, but not estradiol, increased survival of MCF7 cells in the presence of either drug. Cell cycle progression and counting of trypanblue stained cells showed that IGFI was inducing proliferation in paclitaxeltreated but not doxorubicintreated cells. However, IGFI decreased the fraction of apoptotic cells in doxorubicin but not paclitaxeltreated cells. Recent work has shown that mitogenactivated protein kinase (MAPK) and phosphotidylinositol3 (PI3) kinase are activated by IGFI in these cells. PI3 kinase activation has been linked to antiapoptotic functions while MAPK activation is associated with proliferation. We found that IGFI rescue of doxorubicininduced apoptosis required PI3 kinase but not MAPK function, suggesting that IGFI inhibited apoptosis. In contrast, IGFI rescue of paclitaxelinduced apoptosis required both PI3 kinase and MAPK, suggesting that IGFImediated protection was due to enhancement of proliferation. Therefore, IGFI attenuated the response of breast cancer cells to doxorubicin and paclitaxel by at least two mechanisms: induction of proliferation and inhibition of apoptosis. Thus, inhibition of IGFI action could be a useful adjuvant to cytotoxic chemotherapy in breast cancer.     

Quantitative changes in cytological molecular markers during primary medical treatment of breast cancer: A pilot study

Aim: To quantify the changes in biological molecular markers during primary medical treatment in patients with operable breast cancer and to assess their possible relationship with response to treatment.Methods: The treatment group consisted of 31 patients with operable breast carcinomas, median age 57 years (range 41–67), treated with four 3weekly cycles of chemotherapy with Mitoxantrone, methotrexate (± mitomycin C), and tamoxifen before surgery. Fine needle aspiration (FNA) was used to obtain samples from patients prior to and at 10 or 21 days posttreatment. The following molecular markers were assessed: estrogen receptor (ER), progesterone receptor (PgR), p53, Bcl2, and Ki67 measured by immunocytochemistry, and ploidy and Sphase fraction (SPF) by flow cytometry. To evaluate the reproducibility of the technique, repeat FNA was performed in a separate nontreatment control group of 20 patients and the same molecular markers assessed, two weeks after the first sample with no intervening treatment.Results: The nontreatment control group showed a high reproducibility for the measurement of molecular markers from repeat FNA. In the treatment group there was a nonsignificant reduction in SPF and a significant reduction (p = 0.005) in Ki67. Patients who responded to neoadjuvant therapy were more likely to have a reduction in these two markers than those who failed to respond. Similarly, a reduction in ER scores was observed between the first and second samples (p = 0.04). For PgR, the change between the first and second samples was not significant although there was a significant difference between responders and nonresponders (p = 0.03). All nine patients with an increase in PgR were responders. No significant changes in p53 or Bcl2 were observed during treatment.Conclusion: Molecular markers can be adequately measured from FNA samples prior to and during neoadjuvant therapy. Changes in cellular proliferation and hormone receptors have been shown that may be related to tumour response. These relationships should be assessed in a larger cohort of patients.     

Differential effects of chemotherapeutic agents on the Bcl‐2/Bax apoptosis pathway in human breast cancer cell line MCF‐7

The present study explored the effects of three commonly used chemotherapeutic agents on the Bcl2/Bax apoptosis pathway and the interaction of these chemotherapeutic drugs with the estradiolmediated regulation of this pathway. Our results showed that: (1) Treatment of MCF7 cells with Adriamycin resulted in time and concentrationdependent decreases in Bcl2 and increases in Bax mRNA and protein levels. (2) Camptothecin elicited similar trends on Bcl2 and Bax as Adriamycin, while etoposide, at 50–100 fold (1–5M) the effective concentration of Adriamycin and camptothecin, only resulted in an increase in Bax mRNA levels. (3) Adriamycin and camptothecin, but not etoposide, were effective in suppressing estradiolstimulated increases in Bcl2 mRNA levels. Our study provides evidence that the Bcl2/Bax apoptosis pathway may be differentially regulated by chemotherapeutic agents. In addition, interaction between these agents and estradiol on the Bcl2/Bax apoptosis pathway may also exist.     

Tamoxifen and fenretinide in women with metastatic breast cancer

Background: Tamoxifen and fenretinide combination therapy has been shown to be an active treatment regimen in metastatic breast cancer patients. This pilot study sought to determine whether the addition of fenretinide to tamoxifen would be associated with antitumor activity in metastatic breast cancer patients who had been previously treated with tamoxifen or who had hormone receptor negative disease. The effect of this therapy on circulating plasma transforming growth factorbeta (TGF) levels and serum lipids was also examined.Patientsand Methods: Thirtyone patients were treated with tamoxifen (20mg po daily), and fenretinide (400mg po daily with a 3day drug holiday each month). Plasma TGF testing was performed using isoform specific sandwich ELISA.Results: Twenty four of the 31 patients were evaluable for an antitumor response including 14 estrogen receptor (ER) positive patients who had failed prior tamoxifen therapy, seven ERnegative patients, and three hormone therapy naive ERpositive patients. There were no objective antitumor responses; three patients had stable disease for 8, 8, and 24 months. Five patients (16%) discontinued therapy for toxicity (one for grade 3 skin rash and four for abnormal dark adaptation). There was a statistically significant decrease in total cholesterol (median change per patient of –13.5mg/dl; p=0.049, a 6.5% decrease), and an increase in HDL levels (median change per patient of +18mg/dl, p=0.0001, a 35% increase) with tamoxifen and fenretinide therapy.TGF-1 plasma levels were normal in 26 of 28 patients, and no changes in these levels post-treatment were demonstrated.Conclusions: Tamoxifen and fenretinide therapy is not an active combination in ER negative metastatic breast cancer or in patients whose disease has progressed on tamoxifen. This combination had a beneficial effect on total serum cholesterol and HDL levels with no associated rise in serum triglyceride levels. The 400mg dose of fenretinide was associated with symptomatic nyctalopia in one-third of patients making it an unsuitable dose for use in breast cancer prevention studies.