- Email: [email protected]
Arimidex inhibition on proliferation of human breast solid tumors measured by ATP bioluminescence
Life Sciences 76 (2004) 827 – 834 www.elsevier.com/locate/lifescie
Arimidex inhibition on proliferation of human breast solid tumors measured by ATP bioluminescence Edith Y.T. Tse, Wings T.Y. Loo, Mary N.B. Cheung, Louis W.C. Chow*, Carter W. Cheng Department of Surgery, University of Hong Kong Medical Centre, Pokfulam Road, Queen Mary Hospital, Hong Kong Received 10 February 2004; accepted 7 September 2004
Abstract To determine the degree of Arimidex (Anastrozole) inhibition on proliferation of human breast solid tumors in vitro by ATP bioluminescence assay. Breast cancer solid tumors with different hormone receptors and grading were collected from 38 Chinese women with invasive breast cancers. Tumors were treated with three concentrations of Arimidex (1.5 mM, 15 mM and 150 mM). ATP bioluminescence assay was used to measure the metabolic rate in order to determine the degree of inhibition of Arimidex on the breast cancer tumors by comparing to the untreated tumors. 15 mM Arimidex shows greatest inhibitory effect on the proliferation of solid tumors with ER-postive/PR-positive. It can also inhibit the growth of metastatic tumors and tumors with HER-2/neu expression. It shows greater inhibitory effect in lower grading of tumors then higher. Arimidex may effectively inhibit the growth of breast tumors in in vitro system by inhibiting aromatase and block estrogen dependent tumor growth. D 2004 Elsevier Inc. All rights reserved. Keywords: Arimidex; ATP bioluminescence; Estrogen receptor; Progesterone receptor; Human epidermal growth factor receptor-2
* Corresponding author. Tel.: +852 2855 4773; fax: +852 2817 2291. E-mail address: [email protected] (L.W.C. Chow). 0024-3205/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2004.09.016
828
E.Y.T. Tse et al. / Life Sciences 76 (2004) 827–834
Introduction In postmenopausal women, the major mechanism for estrogen synthesis is the conversion of androstenedion into estrone by aromatase (estrogen synthetase). Aromatase is a cytochrome P-450 enzyme which catalyzes the rate-limiting step, in the peripheral adipose tissues. (Harvey, 1998; Lake and Hudis, 2002; Spigel and Burstein, 2002) It provides a significant source of estrogen mediating tumor proliferation in postmenopausal women. Since intratumoral aromatase produces estrogen in situ, it is the target for successful inhibitor treatment (Tamulevicius and Streffer, 1995). Arimidex is a third-generation, non-steroidal competitive aromatase inhibitor, (Brodie et al., 1999) being both potent and selective for aromatase. (Suzuki et al., 2002; Bajetta et al., 2002; Naundorf et al., 1996) It inhibits the cytochrome P-450 component of the aromatase enzyme complex by interfering with the electron transfer from NADPH. (Lake and Hudis, 2002) Estradiol concentrations can be reduced to below detectable levels by inhibiting the terminal step of the estrogen synthesis. (Bhatnagar et al., 2001;) Arimidex has been shown to inhibit in vivo aromatization by 96–97% and to suppress plasma estrogen levels by 84–94%. Arimidex (1 and 10 mg daily) can profoundly decrease the in situ aromatase activity, and therefore the endogenous levels of estrone and estradiol of the estrogen-receptor rich breast cancer tumors. It is selective as it does not modify serum levels of androgens. (Geisler et al., 2001; Miller and Dixon, 2001) ATAC (Arimidex, Tamoxifen, Alone or in Combination) trial suggests Arimidex is superior to Tamoxifen for disease-free survival and reducing the occurrence of contralateral breast cancer in receptor-positive patients. (Lake and Hudis, 2002; Campos and Winer, 2003) Arimidex associates with fewer cardiovascular and thromboembolic side effects, less weight gain and episodes of vaginal bleeding than Tamoxifen (Brodie et al., 1999; Suzuki et al., 2002). Arimidex can be used in adjuvant setting in early breast cancer, as well as first-and second-line treatments of locally advanced and metastatic breast cancer. (Pietras et al., 1995) It is the only aromatase inhibitor with published clinical trial data and U.S. Food and Drug Administration approval for adjuvant therapy of postmenopausal women with early breast cancer (Buzdar et al., 1996). In this study, the ATP bioluminescence assay was used to examine the inhibitory effect of Arimidex on breast solid tumor in in vitro system.
Materials and methods Subject selection 38 patients with histology-confirmed ductal breast cancers, ages ranging from 29 to 85 (median age was 59.1) were recruited for this study in our department. Written consent was obtained from all patients. The breast cancer tissues were collected in sterilized bottle containing 0.9% normal saline. Pathological results of patients Patients’ tissues are obtained from Operation Theater, they were stained for estrogen receptor (ER), progesterone receptor (PR) and HER-2, the lymphovascular permeation and histological grading of tumor differentiation were also identified by Department of Pathology.
E.Y.T. Tse et al. / Life Sciences 76 (2004) 827–834
829
Preparation of Arimidex We obtained powdered Arimidex from Zenaca Limited (Pennsylvania, USA). Even though the drug concentration for Arimidex is 1 mg or 10 mg in clinical setting, the concentration used in experiments would no doubt be different as they were done in vitro. Therefore, before testing on the solid tumors, serial dilution was conducted in breast cancer cell to find out the optimum concentration which was 15 mM. Serial dilution is not shown in this paper. ATP bioluminescence assay applied on human solid tumor The tissue was minced into 1 mm3 and they were cultured in a 24-well culture plates with D-MEM (Dulbecco’s modified eagle medium with 15% fetal bovine serum (FBS), 15 mM HEPES buffer, Lglutamine, pyridoxine hydrochloride, penicillin (100 U/ml), streptomycin (100 Ag/ml) and insulin (4 Ag/ ml) (Invitrogen Corporation, California, USA) for 24 hours. The study consists of four groups, the control and Arimidex of concentrations 150 mM, 15 mM and 1.5 mM. The tissues were submerged in their respective concentrations with above medium for another 24 hours. At harvest day, the tissues were transferred to eppendorf tubes and 0.3 ml cell lysis reagent was added. In preparation for the luciferase reagent of the ATP Bioluminescence assay, 10 ml dilution buffer was added to the luciferase reagent supplied by the kit. Cell lysis was accelerated by a sonicator (Sonics and Materials Inc. Danbury, CT. USA) at a pulse of 30 per minute with 5% amplitude. A 10-second binding period was proceded by amalgamation of 50 ul of both samples and luciferase reagent. The absorbance values of the samples were read by TD-20/ 20 Luminometer (Turner Designs, CA. USA) at 420 nm wavelength. This kit provides a standard ATP for conversion of samplesT optical densities to bioluminescence (RLU, Relative Light Units). Determination of percentage inhibition of Arimidex on tumors The percentage inhibition (%INH) was calculated as: CL Test 100% %INH ¼ CL CL= control without Arimidex, Test= samples with Arimidex Correlation of ATP bioluminescence assay and standardized cell proliferation assay In order to confirm the reliability of ATP bioluminescence assay, cell proliferation reagent WST-1 (Roche, Germany) was employed for confirmation. Doxorubicin at concentrations of 0.1 ug/ml and 0.05 ug/ml were tested against the breast cancer cell line, BT-483 (ATCC, USA). The plates were incubated for 24 and 48 hours. At each harvest day, cell count, amount of ATP and cell proliferation by WST-1 were recorded.
Results We integrated 38 patients pathological report and among them, around 90% are aged over 40 years old. More than 50% are positive in ER, PR and lymphovascular permeation status. Less than 50% are positive
830
E.Y.T. Tse et al. / Life Sciences 76 (2004) 827–834
Table 1 Evaluation criteria for breast cancer marker study Staining intensity
Tumor cell positivity
+ ++ +++
b50% 51–75% N75%
in the HER-2 expression. The evaluation criteria for receptor status was based upon the pathological reports (Table 1). Most of the cases included in the current study belong to tumor grade II and III (Table 3). Results of ATP bioluminescence have been analysed by SPSS 11.0 (Chicago, USA) (Table 2 and Fig. 1), all three concentrations of Arimidex showed statistically significant different, after converted optical density into bioluminescence (rlu) unit. Both ATP bioluminescence assay and WST-1 showed significant results meaning that the former is proper for both primary culture cells from clinical tissues and established cultured cell line (Table 4). The ER, PR, HER-2 and tumor grades have been compared with the results of ATP test, most of them demonstrated greater than 50% inhibition (Table 3).
Discussion ATP bioluminescence assay is a reproducible, practicable and promising method for predicting and assessing the response of breast cancer tumor cells to cytotoxic drugs that can also be used for pretherapeutic drug testing. It is able to overcome major technical limitations of the older assays like the human tumor clonogenic assay (HTCA). Testing with drug combination chemotherapy and fresh human breast cancer tumor testing are warranted and ongoing. (Cree et al., 1995; Hunter et al., 1993a,b) The number of viable cells is quantified by using an enzyme cocktail to link the ATP with luciferase, so that the light emission is proportional to the ATP concentration. (Kurbacher et al., 1996) Sonication is performed in order to lyse the cells rapidly and release the ATP. ATP rapidly degrades in dead cells upon the action of ATPases and therefore is suitable for the sensitive measurement of biomass by luminescence. (Buzdar, 2003) Cell proliferation and metabolism were investigated with standardized cell proliferation reagent WST-1 in parallel with the results analyzed in Table 4 (Chow et al., 2003). Estrogen is important for the development and growth of hormone-dependent breast cancer tumors. (Lake and Hudis, 2002) It initiates and promotes the process of breast carcinogenesis by enhancing the cell division rate and reducing the time for DNA repair. (Harvey, 1998) Four fold to six fold higher estrogen levels are observed in tumors than in plasma of postmenopausal patients with breast cancer. (Tamulevicius and Streffer, 1995). Table 2 Mean of the relative light units(ATP) measured in different concentrations of Arimidex Group
Mean of ATP (RLU)
Control Arim150 mM Arim15 mM Arim1.5 mM
53.7 20.51* 21.65* 23.29*
* the mean difference is significant at the .05 level.
E.Y.T. Tse et al. / Life Sciences 76 (2004) 827–834
831
Fig. 1. Arimidex Inhibition in breast cancer solid tumor.
Progesterone is an important regulator of growth in breast tissues as it controls cell division and differentiation. PR binds to progesterone responsive elements in promoters and stimulates gene transcription after being activated by progesterone. Proliferation and differentiation of normal breast epithelium is stimulated. Progesterone has been reported to either potentiate growth, have no effect on, or inhibit proliferation in breast cancer cells. There is also in vitro evidence that progesterone could induce cellular differentiation in breast cancer. Progesterone has strong effect on human hormone-responsive breast cancers where it inhibits estrogen-mediated growth (Kester et al., 1997; Kurbacher et al., 2003). In carcinoma tissue, estrogen is metabolized to catecholestrogens and form quinones which damage DNA directly. (Harvey, 1998) A higher state of differentiation is associated with a lower metastasizing capacity generally. Estrogen’s dedifferentiation action therefore stimulates the invasion of cancer cells. (Kurbacher et al., 2003) Arimidex, being an aromatase inhibitor, is able to inhibit both intratumoral and peripheral aromatase activity by eliminating the external supply of estrogen to tumor cells so as to block the pathway of estrogen synthesis (Bhatnagar et al., 2001). From our data, 15 mM Arimidex showed an inhibition rate of 55.2% in inhibiting the growth of tumors with ER-positive and PR-positive. Arimidex’s inhibitory effect was effective in endocrine dependent breast cancers. It could inhibit estrogen synthesis within metastatic breast cancer tissue (Brodie et al., 1999). Our data showed that 15 mM Arimidex displays a an inhibition rate of 75% in ERpositive/PR-positive/LP-positive tumors. The inhibition rate of 15 mM Arimidex was higher on tumors with ER-positive/PR-positive than those being positive in both hormonal receptors and LP-positive. Though Arimidex was found to inhibit the estrogen synthesis in postmenopausal and therefore older women, there was a 75% and 72% inhibition rate on pre-and post-menopausal patients, respectively when 15 mM Arimidex was used. The inhibition rate of 15 mM Arimidex was at its highest in grade I tumors, higher than in both grade II and III tumors. The effect of Arimidex caused no significant difference in tumors with high grading. Human epidermal growth factor receptor-2 (HER-2) belongs to a family of 4 transmembrance receptors involved in signal transduction pathways that regulate cell growth and differentiation and causes the cancer cells to proliferate. (Yarden, 2001) HER-2 proto-oncogene encodes a 185-kd transmembrane glycoprotein growth factor receptor that contains an extracellular ligand-binding domain. Amplification of the gene and over-expression of its protein receptor was found in human breast cancer cell lines. (Lipton et al., 2002) This disrupts the normal control mechanisms, potentially leading to the
832
E.Y.T. Tse et al. / Life Sciences 76 (2004) 827–834
Table 3 Percentage inhibition of different groups of patients to different concentrations of Arimidex Group
Patients
ER+ ERPR+ PRHER-2/neu+ HER-2/neuER+, PR+ ER-, PR+ ER+, PRER-, PRER+, HER-2/neu+ ER-, HER-2/neu+ ER+, HER-2/neuER-, HER-2/neuPR+, HER-2/neu+ PR-, HER-2/neu+ PR+, HER-2/neuPR-, HER-2/neuER+, PR+, HER-2/neu+ ER-, PR-, HER-2/neu+ ER+, PR+, HER-2/neuER-, PR-, HER-2/neuER+, PR+, LP+ Tumor Grading I Tumor Grading II Tumor Grading III Pre-menopause Post-menopause
Percentage Inhibition (%INH)
81.25% (26/32) 18.75% (6/32) 56.25% (18/32) 56.25% (18/32) 43.75% (14/32) 43.75% (14/32) 56.25% (18/32) – 25% (8/32) 18.75% (6/32) 31.25%(10/32) 12.5% (4/32) 50% (16/32) 6.25% (2/32) 18.75% (6/32) 25% (8/32) 37.5% (12/32) 18.75% (6/32) 18.75% (6/32) 12.5% (4/32) 37.5% (12/32) 6.25% (2/32) 32.26% (10/31) 6.67% (2/30) 43.33% (13/30) 50% (15/30) 10.5% (4/38) 89.5% (34/38)
1.5 mM
15 mM
150 mM
52.0 36.3 51.1 66.7 21.4 71.9 54.1 – 66.3 36.3 52.47 29.9 50.9 49.2 51.2 42.2 55.6 56.6 51.2 29.9 55.6 49.15 80 100 46.15 53.85 50 61.76
52.6 47.8 55.2 85.7 70.0 69.9 55.2 – 46.7 47.78 68.72 48.4 55.3 46.5 54.5 36.0 50.6 62.0 64.4 48.4 50.6 46.54 75 100 55.56 76.9 75 72
48.3 54.5 52.6 66.7 71.4 56.4 52.6 – 38.6 54.5 55.64 59.5 43.7 44.6 73.3 44.3 42.3 46.9 73.3 59.5 42.3 44.6 80 50 66.67 66.67 75 58.82
ER: estrogen receptor status; PR: progesterone receptor status; LP: Lymphovascular permeation status.
formation of aggressive tumor cells. (Hunter et al., 1993a,b) Estrogen independence and antiestrogen Tamoxifen resistance in ER-positive human breast cancer cells are resulted. ER-positive/HER-2-positive primary breast carcinomas also show an impeded antiproliferative response and shorter duration of response to endocrine therapy (Pietras et al., 1995). Table 4 The effects of Doxorubicin on breast cancer cell line BT-483 on day 1 and 2 at different concentrations in terms of cell number, ATP and cell proliferation Group
Hours
Mean cell no.
Mean ATP (rlu)
WST-1
Control 0.1 ug/ml 0.05 ug/ml Control 0.1 ug/ml 0.05 ug/ml
24 24 24 48 48 48
660,000 306,667 313,333 1,100,000 386,667 273,333
23,383.89 11,794.55 15,515.88 161,790.7 31,452.68 38,842.68
1.33425 0.88525 0.891 1.357 0.78925 1.02675
E.Y.T. Tse et al. / Life Sciences 76 (2004) 827–834
833
Our data showed that tumors that were both positive in ER and HER-2 had a lower inhibition percentage with treatment of 15 mM Arimidex, when compared to those positive in hormone receptors and negative in HER-2. Though Arimidex’s inhibitory effect on ER-positive/HER-2-positive was not as great as that on ER-positive/PR-positive tumors, its inhibition rate was still more than 50%. 15 mM Arimidex showed a pleasing result as it displayed. The inhibition rate over 50% in all categories. 150 mM Arimidex could also achieve this results, but the high inhibition rate may due to the high concentrations of chemical. Arimidex may effectively inhibit the growth of breast tumors in in vitro system by inhibiting aromatase from converting androgen to estrogen, thereby blocking estrogen dependent tumor growth. It displayed its greatest inhibitory effect on ER-positive/PR-positive tumors. It also appeared to inhibit the HER-2-positive and metastatic tumors. Investigation of the effect of Arimidex on the molecular level and protein expression of the solid tumors could be the subject of further study.
References Bajetta, E., Martinetti, A., Zilembo, N., Pozzi, P., La Torre, I., Ferrari, L., Seregni, E., Longarini, R., Salvucci, G., Bombardieri, E., 2002. Biological activity of anastrozole in postmenopausal patients with advanced breast cancer: effects on estrogens and bone metabolism. Annals of Oncology 13, 1059 – 1066. Bhatnagar, A.S., Brodie, A.M., Long, B.J., Evans, D.B., Miller, W.R., 2001. Intracellular aromatase and its relevance to the pharmacological efficacy of aromatase inhibitors. Journal of Steroid Biochemistry and Molecular Biology 76, 199 – 202. Brodie, A., Lu, Q., Liu, Y., Long, B., 1999. Aromatase inhibitors and their antitumor effects in model systems. EndocrineRelated Cancer 6, 205 – 210. Buzdar, A.U., 2003. Advances in endocrine treatments for postmenopausal women with metastatic and early breast cancer. Oncologist 8, 335 – 341. Buzdar, A.U., Plourde, P.V., Hortobagyi, G.N., 1996. Aromatase inhibitors in metastatic breast cancer. Seminars in Oncology 23 (Suppl 9), 28 – 32. Campos, S.M., Winer, E.P., 2003. Hormonal therapy in postmenopausal women with breast cancer. Oncology 64, 289 – 299. Chow, L.W.C., Cheung, M.N.B., Loo, W.T.Y., Guan, X.Y., 2003. A rat cell line derived from DMBA-induced mammary carcinoma. Life Sciences 73, 27 – 40. Cree, I.A., Pazzagli, M., Mini, E., Mazzei, T., Hunter, E.M., Sutherland, L.A., Pinzani, P., Gerli, A., Andreotti, P.E., 1995. Methotrexate chemosensitivity by ATP luminescence in human leukemia cell lines and in breast cancer primary cultures: comparison of the TCA-100 assay with a clonogenic assay. Anticancer Drugs 6, 398 – 404. Lake, Diana E., Hudis, Clifford, 2002. Aromatase Inhibitors in Breast Cancer: An Update. Cancer Control 9, 490 – 497. Geisler, J., Detre, S., Berntsen, H., Ottestad, L., Lindtjorn, B., Dowsett, M., Einstein Lonning, P., 2001. Influence of neoadjuvant anastrozole (Arimidex) on intratumoral estrogen levels and proliferation markers in patients with locally advanced breast cancer. Clinical Cancer Research 7, 1230 – 1236. Harvey, H.A., 1998. Emerging role of aromatase inhibitors in the treatment of breast cancer. Oncology (Huntingt) 12 (Suppl 5), 32 – 35. Hunter, E.M., Sutherland, L.A., Cree, I.A., Dewar, J.A., Preece, P.E., Wood, R.A., Linder, D., Andreotti, P.E., 1993a. Heterogeneity of chemosensitivity in human breast carcinoma: use of an adenosine triphosphate (ATP) chemiluminescence assay. European Journal of Surgical Oncology 19, 242 – 249. Hunter, E.M., Sutherland, L.A., Cree, I.A., Dewar, J.A., Preece, P.E., Wood, R.A., Linder, D., Andreotti, P.E., 1993b. Heterogeneity of chemosensitivity in human breast carcinoma: use of an adenosine triphosphate (ATP) chemiluminescence assay. European Journal of Surgical Oncology 19, 242 – 249. Kester, H.A., van der Leede, B.M., van der Saag, P.T., van der Burg, B., 1997. Novel progesterone target genes identified by an improved differential display technique suggest that progestin-induced growth inhibition of breast cancer cells coincides with enhancement of differentiation. Journal of Biological Chemistry 272, 16637 – 16643.
834
E.Y.T. Tse et al. / Life Sciences 76 (2004) 827–834
Kurbacher, C.M., Grecu, O.M., Stier, U., Gilster, T.J., Janat, M.M., Untch, M., Konecny, G., Bruckner, H.W., Cree, I.A., 2003. ATP chemosensitivity testing in ovarian and breast cancer: early clinical trials. Recent Results in Cancer Research 161, 221 – 230. Kurbacher, C.M., Mallmann, P., Kurbacher, J.A., Hubner, H., Krebs, D., 1996. Chemosensitivity testing in gynecologic oncology. Experiences with an ATP bioluminescence assay. Geburtshilfe Frauenheilkd 56, 70 – 78. Lipton, A., Ali, S.M., Leitzel, K., Demers, L., Chinchilli, V., Engle, L., Harvey, H.A., Brady, C., Nalin, C.M., Dugan, M., Carney, W., Allard, J., 2002. Elevated serum Her-2/neu level predicts decreased response to hormone therapy in metastatic breast cancer. Journal of Clinical Oncology 20, 1467 – 1472. Miller, W.R., Dixon, J.M., 2001. Local endocrine effects of aromatase inhibitors within the breast. Journal of Steroid Biochemistry Molecular Biology 79, 93 – 102. Naundorf, H., Parczyk, K., Zschiesche, W., Reinecke, S., Buttner, B., Saul, G.J., Sinn, B., Fichtner, I., 1996. Relation of oestradiol-mediated growth stimulation with the expression of c-erbB-2 protein in xenotransplanted oestradiol-receptorpositive and-negative breast carcinomas. Journal of Cancer Research and Clinical Oncology 122, 14 – 20. Pietras, R.J., Arboleda, J., Reese, D.M., Wongvipat, N., Pegram, M.D., Ramos, L., Gorman, C.M., Parker, M.G., Sliwkowski, M.X., Slamon, D.J., 1995. HER-2 tyrosine kinase pathway targets estrogen receptor and promotes hormone-independent growth in human breast cancer cells. Oncogene 10, 2435 – 2446. Spigel, D.R., Burstein, H.J., 2002. HER2 overexpressing metastatic breast cancer. Current Treatment Options in Oncology 3, 163 – 174. Suzuki, T., Moriya, T., Ishida, T., Kimura, M., Ohuchi, N., Sasano, H., 2002. In situ production of estrogens in human breast carcinoma. Breast Cancer 9, 296 – 302. Tamulevicius, P., Streffer, C., 1995. Metabolic imaging in tumours by means of bioluminescence. British Journal of Cancer 72, 1102 – 1112. Yarden, Y., 2001. Biology of HER2 and its importance in breast cancer. Oncology 61 (Suppl 2), 1 – 13.
Arimidex inhibition on proliferation of human breast solid tumors measured by ATP bioluminescence Edith Y.T. Tse, Wings T.Y. Loo, Mary N.B. Cheung, Louis W.C. Chow*, Carter W. Cheng Department of Surgery, University of Hong Kong Medical Centre, Pokfulam Road, Queen Mary Hospital, Hong Kong Received 10 February 2004; accepted 7 September 2004
Abstract To determine the degree of Arimidex (Anastrozole) inhibition on proliferation of human breast solid tumors in vitro by ATP bioluminescence assay. Breast cancer solid tumors with different hormone receptors and grading were collected from 38 Chinese women with invasive breast cancers. Tumors were treated with three concentrations of Arimidex (1.5 mM, 15 mM and 150 mM). ATP bioluminescence assay was used to measure the metabolic rate in order to determine the degree of inhibition of Arimidex on the breast cancer tumors by comparing to the untreated tumors. 15 mM Arimidex shows greatest inhibitory effect on the proliferation of solid tumors with ER-postive/PR-positive. It can also inhibit the growth of metastatic tumors and tumors with HER-2/neu expression. It shows greater inhibitory effect in lower grading of tumors then higher. Arimidex may effectively inhibit the growth of breast tumors in in vitro system by inhibiting aromatase and block estrogen dependent tumor growth. D 2004 Elsevier Inc. All rights reserved. Keywords: Arimidex; ATP bioluminescence; Estrogen receptor; Progesterone receptor; Human epidermal growth factor receptor-2
* Corresponding author. Tel.: +852 2855 4773; fax: +852 2817 2291. E-mail address: [email protected] (L.W.C. Chow). 0024-3205/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2004.09.016
828
E.Y.T. Tse et al. / Life Sciences 76 (2004) 827–834
Introduction In postmenopausal women, the major mechanism for estrogen synthesis is the conversion of androstenedion into estrone by aromatase (estrogen synthetase). Aromatase is a cytochrome P-450 enzyme which catalyzes the rate-limiting step, in the peripheral adipose tissues. (Harvey, 1998; Lake and Hudis, 2002; Spigel and Burstein, 2002) It provides a significant source of estrogen mediating tumor proliferation in postmenopausal women. Since intratumoral aromatase produces estrogen in situ, it is the target for successful inhibitor treatment (Tamulevicius and Streffer, 1995). Arimidex is a third-generation, non-steroidal competitive aromatase inhibitor, (Brodie et al., 1999) being both potent and selective for aromatase. (Suzuki et al., 2002; Bajetta et al., 2002; Naundorf et al., 1996) It inhibits the cytochrome P-450 component of the aromatase enzyme complex by interfering with the electron transfer from NADPH. (Lake and Hudis, 2002) Estradiol concentrations can be reduced to below detectable levels by inhibiting the terminal step of the estrogen synthesis. (Bhatnagar et al., 2001;) Arimidex has been shown to inhibit in vivo aromatization by 96–97% and to suppress plasma estrogen levels by 84–94%. Arimidex (1 and 10 mg daily) can profoundly decrease the in situ aromatase activity, and therefore the endogenous levels of estrone and estradiol of the estrogen-receptor rich breast cancer tumors. It is selective as it does not modify serum levels of androgens. (Geisler et al., 2001; Miller and Dixon, 2001) ATAC (Arimidex, Tamoxifen, Alone or in Combination) trial suggests Arimidex is superior to Tamoxifen for disease-free survival and reducing the occurrence of contralateral breast cancer in receptor-positive patients. (Lake and Hudis, 2002; Campos and Winer, 2003) Arimidex associates with fewer cardiovascular and thromboembolic side effects, less weight gain and episodes of vaginal bleeding than Tamoxifen (Brodie et al., 1999; Suzuki et al., 2002). Arimidex can be used in adjuvant setting in early breast cancer, as well as first-and second-line treatments of locally advanced and metastatic breast cancer. (Pietras et al., 1995) It is the only aromatase inhibitor with published clinical trial data and U.S. Food and Drug Administration approval for adjuvant therapy of postmenopausal women with early breast cancer (Buzdar et al., 1996). In this study, the ATP bioluminescence assay was used to examine the inhibitory effect of Arimidex on breast solid tumor in in vitro system.
Materials and methods Subject selection 38 patients with histology-confirmed ductal breast cancers, ages ranging from 29 to 85 (median age was 59.1) were recruited for this study in our department. Written consent was obtained from all patients. The breast cancer tissues were collected in sterilized bottle containing 0.9% normal saline. Pathological results of patients Patients’ tissues are obtained from Operation Theater, they were stained for estrogen receptor (ER), progesterone receptor (PR) and HER-2, the lymphovascular permeation and histological grading of tumor differentiation were also identified by Department of Pathology.
E.Y.T. Tse et al. / Life Sciences 76 (2004) 827–834
829
Preparation of Arimidex We obtained powdered Arimidex from Zenaca Limited (Pennsylvania, USA). Even though the drug concentration for Arimidex is 1 mg or 10 mg in clinical setting, the concentration used in experiments would no doubt be different as they were done in vitro. Therefore, before testing on the solid tumors, serial dilution was conducted in breast cancer cell to find out the optimum concentration which was 15 mM. Serial dilution is not shown in this paper. ATP bioluminescence assay applied on human solid tumor The tissue was minced into 1 mm3 and they were cultured in a 24-well culture plates with D-MEM (Dulbecco’s modified eagle medium with 15% fetal bovine serum (FBS), 15 mM HEPES buffer, Lglutamine, pyridoxine hydrochloride, penicillin (100 U/ml), streptomycin (100 Ag/ml) and insulin (4 Ag/ ml) (Invitrogen Corporation, California, USA) for 24 hours. The study consists of four groups, the control and Arimidex of concentrations 150 mM, 15 mM and 1.5 mM. The tissues were submerged in their respective concentrations with above medium for another 24 hours. At harvest day, the tissues were transferred to eppendorf tubes and 0.3 ml cell lysis reagent was added. In preparation for the luciferase reagent of the ATP Bioluminescence assay, 10 ml dilution buffer was added to the luciferase reagent supplied by the kit. Cell lysis was accelerated by a sonicator (Sonics and Materials Inc. Danbury, CT. USA) at a pulse of 30 per minute with 5% amplitude. A 10-second binding period was proceded by amalgamation of 50 ul of both samples and luciferase reagent. The absorbance values of the samples were read by TD-20/ 20 Luminometer (Turner Designs, CA. USA) at 420 nm wavelength. This kit provides a standard ATP for conversion of samplesT optical densities to bioluminescence (RLU, Relative Light Units). Determination of percentage inhibition of Arimidex on tumors The percentage inhibition (%INH) was calculated as: CL Test 100% %INH ¼ CL CL= control without Arimidex, Test= samples with Arimidex Correlation of ATP bioluminescence assay and standardized cell proliferation assay In order to confirm the reliability of ATP bioluminescence assay, cell proliferation reagent WST-1 (Roche, Germany) was employed for confirmation. Doxorubicin at concentrations of 0.1 ug/ml and 0.05 ug/ml were tested against the breast cancer cell line, BT-483 (ATCC, USA). The plates were incubated for 24 and 48 hours. At each harvest day, cell count, amount of ATP and cell proliferation by WST-1 were recorded.
Results We integrated 38 patients pathological report and among them, around 90% are aged over 40 years old. More than 50% are positive in ER, PR and lymphovascular permeation status. Less than 50% are positive
830
E.Y.T. Tse et al. / Life Sciences 76 (2004) 827–834
Table 1 Evaluation criteria for breast cancer marker study Staining intensity
Tumor cell positivity
+ ++ +++
b50% 51–75% N75%
in the HER-2 expression. The evaluation criteria for receptor status was based upon the pathological reports (Table 1). Most of the cases included in the current study belong to tumor grade II and III (Table 3). Results of ATP bioluminescence have been analysed by SPSS 11.0 (Chicago, USA) (Table 2 and Fig. 1), all three concentrations of Arimidex showed statistically significant different, after converted optical density into bioluminescence (rlu) unit. Both ATP bioluminescence assay and WST-1 showed significant results meaning that the former is proper for both primary culture cells from clinical tissues and established cultured cell line (Table 4). The ER, PR, HER-2 and tumor grades have been compared with the results of ATP test, most of them demonstrated greater than 50% inhibition (Table 3).
Discussion ATP bioluminescence assay is a reproducible, practicable and promising method for predicting and assessing the response of breast cancer tumor cells to cytotoxic drugs that can also be used for pretherapeutic drug testing. It is able to overcome major technical limitations of the older assays like the human tumor clonogenic assay (HTCA). Testing with drug combination chemotherapy and fresh human breast cancer tumor testing are warranted and ongoing. (Cree et al., 1995; Hunter et al., 1993a,b) The number of viable cells is quantified by using an enzyme cocktail to link the ATP with luciferase, so that the light emission is proportional to the ATP concentration. (Kurbacher et al., 1996) Sonication is performed in order to lyse the cells rapidly and release the ATP. ATP rapidly degrades in dead cells upon the action of ATPases and therefore is suitable for the sensitive measurement of biomass by luminescence. (Buzdar, 2003) Cell proliferation and metabolism were investigated with standardized cell proliferation reagent WST-1 in parallel with the results analyzed in Table 4 (Chow et al., 2003). Estrogen is important for the development and growth of hormone-dependent breast cancer tumors. (Lake and Hudis, 2002) It initiates and promotes the process of breast carcinogenesis by enhancing the cell division rate and reducing the time for DNA repair. (Harvey, 1998) Four fold to six fold higher estrogen levels are observed in tumors than in plasma of postmenopausal patients with breast cancer. (Tamulevicius and Streffer, 1995). Table 2 Mean of the relative light units(ATP) measured in different concentrations of Arimidex Group
Mean of ATP (RLU)
Control Arim150 mM Arim15 mM Arim1.5 mM
53.7 20.51* 21.65* 23.29*
* the mean difference is significant at the .05 level.
E.Y.T. Tse et al. / Life Sciences 76 (2004) 827–834
831
Fig. 1. Arimidex Inhibition in breast cancer solid tumor.
Progesterone is an important regulator of growth in breast tissues as it controls cell division and differentiation. PR binds to progesterone responsive elements in promoters and stimulates gene transcription after being activated by progesterone. Proliferation and differentiation of normal breast epithelium is stimulated. Progesterone has been reported to either potentiate growth, have no effect on, or inhibit proliferation in breast cancer cells. There is also in vitro evidence that progesterone could induce cellular differentiation in breast cancer. Progesterone has strong effect on human hormone-responsive breast cancers where it inhibits estrogen-mediated growth (Kester et al., 1997; Kurbacher et al., 2003). In carcinoma tissue, estrogen is metabolized to catecholestrogens and form quinones which damage DNA directly. (Harvey, 1998) A higher state of differentiation is associated with a lower metastasizing capacity generally. Estrogen’s dedifferentiation action therefore stimulates the invasion of cancer cells. (Kurbacher et al., 2003) Arimidex, being an aromatase inhibitor, is able to inhibit both intratumoral and peripheral aromatase activity by eliminating the external supply of estrogen to tumor cells so as to block the pathway of estrogen synthesis (Bhatnagar et al., 2001). From our data, 15 mM Arimidex showed an inhibition rate of 55.2% in inhibiting the growth of tumors with ER-positive and PR-positive. Arimidex’s inhibitory effect was effective in endocrine dependent breast cancers. It could inhibit estrogen synthesis within metastatic breast cancer tissue (Brodie et al., 1999). Our data showed that 15 mM Arimidex displays a an inhibition rate of 75% in ERpositive/PR-positive/LP-positive tumors. The inhibition rate of 15 mM Arimidex was higher on tumors with ER-positive/PR-positive than those being positive in both hormonal receptors and LP-positive. Though Arimidex was found to inhibit the estrogen synthesis in postmenopausal and therefore older women, there was a 75% and 72% inhibition rate on pre-and post-menopausal patients, respectively when 15 mM Arimidex was used. The inhibition rate of 15 mM Arimidex was at its highest in grade I tumors, higher than in both grade II and III tumors. The effect of Arimidex caused no significant difference in tumors with high grading. Human epidermal growth factor receptor-2 (HER-2) belongs to a family of 4 transmembrance receptors involved in signal transduction pathways that regulate cell growth and differentiation and causes the cancer cells to proliferate. (Yarden, 2001) HER-2 proto-oncogene encodes a 185-kd transmembrane glycoprotein growth factor receptor that contains an extracellular ligand-binding domain. Amplification of the gene and over-expression of its protein receptor was found in human breast cancer cell lines. (Lipton et al., 2002) This disrupts the normal control mechanisms, potentially leading to the
832
E.Y.T. Tse et al. / Life Sciences 76 (2004) 827–834
Table 3 Percentage inhibition of different groups of patients to different concentrations of Arimidex Group
Patients
ER+ ERPR+ PRHER-2/neu+ HER-2/neuER+, PR+ ER-, PR+ ER+, PRER-, PRER+, HER-2/neu+ ER-, HER-2/neu+ ER+, HER-2/neuER-, HER-2/neuPR+, HER-2/neu+ PR-, HER-2/neu+ PR+, HER-2/neuPR-, HER-2/neuER+, PR+, HER-2/neu+ ER-, PR-, HER-2/neu+ ER+, PR+, HER-2/neuER-, PR-, HER-2/neuER+, PR+, LP+ Tumor Grading I Tumor Grading II Tumor Grading III Pre-menopause Post-menopause
Percentage Inhibition (%INH)
81.25% (26/32) 18.75% (6/32) 56.25% (18/32) 56.25% (18/32) 43.75% (14/32) 43.75% (14/32) 56.25% (18/32) – 25% (8/32) 18.75% (6/32) 31.25%(10/32) 12.5% (4/32) 50% (16/32) 6.25% (2/32) 18.75% (6/32) 25% (8/32) 37.5% (12/32) 18.75% (6/32) 18.75% (6/32) 12.5% (4/32) 37.5% (12/32) 6.25% (2/32) 32.26% (10/31) 6.67% (2/30) 43.33% (13/30) 50% (15/30) 10.5% (4/38) 89.5% (34/38)
1.5 mM
15 mM
150 mM
52.0 36.3 51.1 66.7 21.4 71.9 54.1 – 66.3 36.3 52.47 29.9 50.9 49.2 51.2 42.2 55.6 56.6 51.2 29.9 55.6 49.15 80 100 46.15 53.85 50 61.76
52.6 47.8 55.2 85.7 70.0 69.9 55.2 – 46.7 47.78 68.72 48.4 55.3 46.5 54.5 36.0 50.6 62.0 64.4 48.4 50.6 46.54 75 100 55.56 76.9 75 72
48.3 54.5 52.6 66.7 71.4 56.4 52.6 – 38.6 54.5 55.64 59.5 43.7 44.6 73.3 44.3 42.3 46.9 73.3 59.5 42.3 44.6 80 50 66.67 66.67 75 58.82
ER: estrogen receptor status; PR: progesterone receptor status; LP: Lymphovascular permeation status.
formation of aggressive tumor cells. (Hunter et al., 1993a,b) Estrogen independence and antiestrogen Tamoxifen resistance in ER-positive human breast cancer cells are resulted. ER-positive/HER-2-positive primary breast carcinomas also show an impeded antiproliferative response and shorter duration of response to endocrine therapy (Pietras et al., 1995). Table 4 The effects of Doxorubicin on breast cancer cell line BT-483 on day 1 and 2 at different concentrations in terms of cell number, ATP and cell proliferation Group
Hours
Mean cell no.
Mean ATP (rlu)
WST-1
Control 0.1 ug/ml 0.05 ug/ml Control 0.1 ug/ml 0.05 ug/ml
24 24 24 48 48 48
660,000 306,667 313,333 1,100,000 386,667 273,333
23,383.89 11,794.55 15,515.88 161,790.7 31,452.68 38,842.68
1.33425 0.88525 0.891 1.357 0.78925 1.02675
E.Y.T. Tse et al. / Life Sciences 76 (2004) 827–834
833
Our data showed that tumors that were both positive in ER and HER-2 had a lower inhibition percentage with treatment of 15 mM Arimidex, when compared to those positive in hormone receptors and negative in HER-2. Though Arimidex’s inhibitory effect on ER-positive/HER-2-positive was not as great as that on ER-positive/PR-positive tumors, its inhibition rate was still more than 50%. 15 mM Arimidex showed a pleasing result as it displayed. The inhibition rate over 50% in all categories. 150 mM Arimidex could also achieve this results, but the high inhibition rate may due to the high concentrations of chemical. Arimidex may effectively inhibit the growth of breast tumors in in vitro system by inhibiting aromatase from converting androgen to estrogen, thereby blocking estrogen dependent tumor growth. It displayed its greatest inhibitory effect on ER-positive/PR-positive tumors. It also appeared to inhibit the HER-2-positive and metastatic tumors. Investigation of the effect of Arimidex on the molecular level and protein expression of the solid tumors could be the subject of further study.
References Bajetta, E., Martinetti, A., Zilembo, N., Pozzi, P., La Torre, I., Ferrari, L., Seregni, E., Longarini, R., Salvucci, G., Bombardieri, E., 2002. Biological activity of anastrozole in postmenopausal patients with advanced breast cancer: effects on estrogens and bone metabolism. Annals of Oncology 13, 1059 – 1066. Bhatnagar, A.S., Brodie, A.M., Long, B.J., Evans, D.B., Miller, W.R., 2001. Intracellular aromatase and its relevance to the pharmacological efficacy of aromatase inhibitors. Journal of Steroid Biochemistry and Molecular Biology 76, 199 – 202. Brodie, A., Lu, Q., Liu, Y., Long, B., 1999. Aromatase inhibitors and their antitumor effects in model systems. EndocrineRelated Cancer 6, 205 – 210. Buzdar, A.U., 2003. Advances in endocrine treatments for postmenopausal women with metastatic and early breast cancer. Oncologist 8, 335 – 341. Buzdar, A.U., Plourde, P.V., Hortobagyi, G.N., 1996. Aromatase inhibitors in metastatic breast cancer. Seminars in Oncology 23 (Suppl 9), 28 – 32. Campos, S.M., Winer, E.P., 2003. Hormonal therapy in postmenopausal women with breast cancer. Oncology 64, 289 – 299. Chow, L.W.C., Cheung, M.N.B., Loo, W.T.Y., Guan, X.Y., 2003. A rat cell line derived from DMBA-induced mammary carcinoma. Life Sciences 73, 27 – 40. Cree, I.A., Pazzagli, M., Mini, E., Mazzei, T., Hunter, E.M., Sutherland, L.A., Pinzani, P., Gerli, A., Andreotti, P.E., 1995. Methotrexate chemosensitivity by ATP luminescence in human leukemia cell lines and in breast cancer primary cultures: comparison of the TCA-100 assay with a clonogenic assay. Anticancer Drugs 6, 398 – 404. Lake, Diana E., Hudis, Clifford, 2002. Aromatase Inhibitors in Breast Cancer: An Update. Cancer Control 9, 490 – 497. Geisler, J., Detre, S., Berntsen, H., Ottestad, L., Lindtjorn, B., Dowsett, M., Einstein Lonning, P., 2001. Influence of neoadjuvant anastrozole (Arimidex) on intratumoral estrogen levels and proliferation markers in patients with locally advanced breast cancer. Clinical Cancer Research 7, 1230 – 1236. Harvey, H.A., 1998. Emerging role of aromatase inhibitors in the treatment of breast cancer. Oncology (Huntingt) 12 (Suppl 5), 32 – 35. Hunter, E.M., Sutherland, L.A., Cree, I.A., Dewar, J.A., Preece, P.E., Wood, R.A., Linder, D., Andreotti, P.E., 1993a. Heterogeneity of chemosensitivity in human breast carcinoma: use of an adenosine triphosphate (ATP) chemiluminescence assay. European Journal of Surgical Oncology 19, 242 – 249. Hunter, E.M., Sutherland, L.A., Cree, I.A., Dewar, J.A., Preece, P.E., Wood, R.A., Linder, D., Andreotti, P.E., 1993b. Heterogeneity of chemosensitivity in human breast carcinoma: use of an adenosine triphosphate (ATP) chemiluminescence assay. European Journal of Surgical Oncology 19, 242 – 249. Kester, H.A., van der Leede, B.M., van der Saag, P.T., van der Burg, B., 1997. Novel progesterone target genes identified by an improved differential display technique suggest that progestin-induced growth inhibition of breast cancer cells coincides with enhancement of differentiation. Journal of Biological Chemistry 272, 16637 – 16643.
834
E.Y.T. Tse et al. / Life Sciences 76 (2004) 827–834
Kurbacher, C.M., Grecu, O.M., Stier, U., Gilster, T.J., Janat, M.M., Untch, M., Konecny, G., Bruckner, H.W., Cree, I.A., 2003. ATP chemosensitivity testing in ovarian and breast cancer: early clinical trials. Recent Results in Cancer Research 161, 221 – 230. Kurbacher, C.M., Mallmann, P., Kurbacher, J.A., Hubner, H., Krebs, D., 1996. Chemosensitivity testing in gynecologic oncology. Experiences with an ATP bioluminescence assay. Geburtshilfe Frauenheilkd 56, 70 – 78. Lipton, A., Ali, S.M., Leitzel, K., Demers, L., Chinchilli, V., Engle, L., Harvey, H.A., Brady, C., Nalin, C.M., Dugan, M., Carney, W., Allard, J., 2002. Elevated serum Her-2/neu level predicts decreased response to hormone therapy in metastatic breast cancer. Journal of Clinical Oncology 20, 1467 – 1472. Miller, W.R., Dixon, J.M., 2001. Local endocrine effects of aromatase inhibitors within the breast. Journal of Steroid Biochemistry Molecular Biology 79, 93 – 102. Naundorf, H., Parczyk, K., Zschiesche, W., Reinecke, S., Buttner, B., Saul, G.J., Sinn, B., Fichtner, I., 1996. Relation of oestradiol-mediated growth stimulation with the expression of c-erbB-2 protein in xenotransplanted oestradiol-receptorpositive and-negative breast carcinomas. Journal of Cancer Research and Clinical Oncology 122, 14 – 20. Pietras, R.J., Arboleda, J., Reese, D.M., Wongvipat, N., Pegram, M.D., Ramos, L., Gorman, C.M., Parker, M.G., Sliwkowski, M.X., Slamon, D.J., 1995. HER-2 tyrosine kinase pathway targets estrogen receptor and promotes hormone-independent growth in human breast cancer cells. Oncogene 10, 2435 – 2446. Spigel, D.R., Burstein, H.J., 2002. HER2 overexpressing metastatic breast cancer. Current Treatment Options in Oncology 3, 163 – 174. Suzuki, T., Moriya, T., Ishida, T., Kimura, M., Ohuchi, N., Sasano, H., 2002. In situ production of estrogens in human breast carcinoma. Breast Cancer 9, 296 – 302. Tamulevicius, P., Streffer, C., 1995. Metabolic imaging in tumours by means of bioluminescence. British Journal of Cancer 72, 1102 – 1112. Yarden, Y., 2001. Biology of HER2 and its importance in breast cancer. Oncology 61 (Suppl 2), 1 – 13.