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The efficacy of Paclitaxel on solid tumour analysed by ATP bioluminescence assay and VEGF expression: a translational research study
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SCIENCE ~____IDIRECT e
ELSEVIER
& BIOMEDICINE PHARMACOTHERAPY
Biomedicine & Pharmacotherapy 59 (2005) $337-$339 http://france.elsevier.com/direct/BIOPHA/
The efficacy of Paclitaxel on solid tumour analysed by ATP bioluminescence assay and VEGF expression: a translational research study W.T.Y. Loo, J.H.M. Fong, M.N.B. Cheung, L.W.C. Chow* Department of Surgery, The University of Hong Kong Medical Centre, PokfulamRoad, QueenMary Hospital, Hong Kong, China
Abstract Background. - Paclitaxel (Taxol), is known to induce mitotic arrest and apoptosis by inhibiting the depolymerisation of microtubules. Tumour growth and metastasis are affected by the metabolic rate and angiogenesis. We investigated the effect of Paclitaxel on tumour metabolism and markers of angiogenesis, vascular endothelial growth factor (VEGF). M a t e r i a l s a n d m e t h o d s . - Tissue samples of 39 patients diagnosed with invasive carcinoma were obtained. The solid tumours were cultured with Paclitaxel at a concentration of 4.27 ug/ml for 24 h. The metabolic rate of the samples was measured by ATP Bioluminescence assay and the levels of VEGF in culture medium were measured by ELISA. R e s u l t . - The mean ATP concentration of control and test groups were 7.169 and 5.004 rlu/ml, respectively, suggesting that the metabolic rate was inhibited by Paclitaxel. The mean VEGF levels in the control and test groups were 5.335 and 4.567 pg/ml, respectively. All data analysed were statistically significant (P < 0.005). The finding of the study showed that Paclitaxel could inhibit metabolic rate in solid tumour. It could also downregulate VEGF. Conclusion. - Our result suggested that Paclitaxel is an effective cytotoxic possibly with anti-angiogenic effects. © 2005 Elsevier SAS. All rights reserved.
Keywords: Paclitaxel; Metastasis; ATP bioluminescence assay
1. Introduction
Breast cancer is the most common primary cancer with poor prognosis and high mortality rate while primary malignant breast tumours are among those human neoplasms that exhibit the greatest angiogenic activity [1]. Vascular endothelial growth factor (VEGF), the most potent regulator of angiogenesis, is one of the factors leading to frequent metastasis and tumour development. As a result it has been acting as a prognostic indicator for various kinds of human tumours [2-4]. Tumour angiogenesis is a complex mechanism consisting of multi-step events including secretion or activation of angiogenic factors by tumour cells, activation of proteolytic enzymes, and proliferation, migration, and differentiation of endothelial cells [5].
* Corresponding authors. E-mail address: [email protected] (Louis W.C. Chow). © 2005 Elsevier SAS. All rights reserved.
The detection of the expression of soluble and insoluble VEGF in vitro and in vivo is an established method in determining the efficacy of treatment performed [6]. Paclitaxel (Taxol), an anti-neoplastic agent, is known to induce mitotic arrest and apoptosis by inhibiting the depolymerisation of microtubules [7-12]. It has been widely single or combined used in clinical to treated various cancer, such as neuroblastoma, non-Hodgkin lymphoma, oral squamous cell carcinoma, stomach, ovarian, breast, prostate and lung cancer [13-15]. ATP bioluminescence assay in solid tumour has been well established in our previous studies as one of the most sophisticated assays available, and early clinical trials show an enhanced response rate in patients undergoing ATP assay-directed preoperative chemotherapy compared to empirical chemotherapy [16]. By combining the ATP bioluminescence assay and ELISA assay to test for the metabolic rate and expression of VEGF, respectively, tumours' response may be predicted in vitro.
$338
W.T.Y. Loo et al. / Biomedicine & Pharmacotherapy 59 (2005) $337-$339
Therefore, more information w ill become available for clinicians in making decisions for treating malignant diseases. We would like to investigate the effect of Paclitaxel on breast solid tumour by means of the ATP assay and ELISA assay tested on level of VEGF. 2. Material and methods
Breast tumour tissues were obtained from 39 patients diagnosed with invasive ductal carcinoma. The tissue was minced into 1 mm 3 and cultured in DMEM (Invitrogen Corporation, California, USA) with or without 4.27 ug/ml (PPC) Paclitaxel for 24 h. After 24 h, the tissues were lysed by a sonicator (Sonics and Materials Inc., Danbury, CT. USA). The ATP Bioluminescence assay employed TD-20/ 20 Luminometer (Turner Designs, CA. USA) for measuring the metabolic rate. Apoptosis was determined by DNA fragmentation whereas the level of VEGF secretion in culture medium was by ELISA Kit (Pierice Endogen, USA). All data collected were analysed by Independent T-test (SPSS 11.0, USA). 3. Results
Natural logarithm was applied to the ATP readings due to the large range. Statistical significance was observed between the metabolic rate of control and test groups (Fig. 1). The DNA fragmentation test with 1% agarose gel revealed DNA ladder for different cases treated with Paclitaxel 4.27 ug/ml (Fig. 2). The mean level VEGF in culture media of the control and test groups was 5.335 pg/ml and 4.567 pg/ml. 4. Discussion
tumour chemosensitivity assay (ATP-TCA) have been implemented in evaluating cell viability, cell lines were chosen for most studies [17,18]. This analysis had been employed by our former studies o;n the determination of cell viability though not specifically to chemodrugs [16]. There are multiple advantages with using the chemosensitivity testing system we proposed. Firstly, by using a tissue-based three-dimensional histoculture, only a small amount of tissue is needed [19]. Moreover, the in vivo effects due to the stromal elements can be simulated. Currently, the ATPTCA can be regarded as the most sophisticated assay to investigate both solid samples and effusions derived from patients with various organ tumours [20]. Our results were analysed by comparing the inhibitory rate of the chemodrugs with the magnitude of tumour area reduced. Statistically significant positive correlation was observed between the chemosensitivity response and clinical response. The clinicians in our parallel study made use of the in vitro ATP results after tested with Paclitaxel on breast cancer patients, the pathological and clinical response rate reached 50%. Other researchers reported that Paclitaxel arrests tumour cell growth through impairing of the cytoskeletal system by forming microtubules in the malignant cells, giving rise to programmed cell death [21,22] which coincides with our finding (Fig. 2). Solid tumours were cultured with and without the anticancer agent, the level of soluble VEGF measured was definitely lower than the control group. Zhong et al. [6] also accounted for an effective agent in cancer treatment through the inhibition of VEGF expression, and the activation of signalling pathway in their study. Clinical evidence has been provided that HER-2/neu overexpression is associated with expression of two of the most abundantly expressed VEGF isoforms in breast cancer, suggesting that VEGF may in part mediate the aggressive phenotype of
We described an innovative chemosensitivity test by measuring cell metabolism of tumour tissues with an ATP bioluminescence assay. Although some other ATP-based 8.0
¸
0.. I.- 7.0' <
~5 W
"~ t~
6.0'
O'J
g ¢)
5.0,
4.0 Control
Paclitaxel 4.27ug/ml
Group
Fig. 1. The comparison of metabolic rate between control and test groups.
Fig. 2. The DNA ladder produced by Paclitaxel-treated solid tumour of various patients. From left, Lane 1: Control; Lanes 2-6: Different cases treated with Paclitaxel 4.27 ug/ml and Lane 7:100 bp DNA marker.
W. T. Y. Loo et al. / Biomedicine & Pharmacotherapy 59 (2005) $337-$339
breast cancers that overexpress HER-2/neu [23]. Recognising which are the more biologically active positive and negative angiogenic factors is the key for the identification not only of new prognostic markers but also of targets for anti-angiogenic therapy in human breast cancer [24]. This translational assay by means of the application of ATP assay as well as measurement of secretion of VEGF in tumour cells is proved to be practically responsive for patients given that the clinicians make use of the data provided by the laboratory tests. The pathological and clinical response rate with this combined analysis approach attained more than 50% in clinical use. References [1]
Rahman MA, Toi M. Anti-angiogenic therapy in breast cancer. Biomed Pharmacother 2003;57:463-70. [2] Weidner N, Folkman J, Pozza F, Bevilacqua P, Alfred EN, Moore DH, Meli S, Gasparin G. Tumor angiogenesis: a new significant and independent prognostic indicator in early-stage breast carcinoma. J Natl Cancer Inst 1992;84:1875-87. [3] Toi M, Kashitani J, Tominaga T. Tumor angiogenesis is an independent prognostic indicator in primary breast carcinoma. Int J Cancer 1993;55:371-4. [4] Niethammer AG. A DNA vaccine against VEGF receptor 2 prevents effective angiogenesis and inhibits tumor growth. Nat Med 2002;8:1369-75. [5] Folkman J. What is the evidence that tumors are angiogenesis dependent? J Natl Cancer Inst 1990;82:4-6. [6] Zhong XS, Zheng JZ, Reed E, Jiang BH. SU5416 inhibited VEGF and HIF-lalpha expression through the PI3K/AKT/p70S6K1 signaling pathway. Biochem Biophys Res Commun 2004;324:471-80. [7] Horwitz SB. Mechanism of action of taxol. Trends Pharmacol Sci. 1992;13:134-6. [8] Spencer CM, Faulds D. Paclitaxel. A review of its pharmacodynamic and pharmacokinetic properties and therapeutic potential in the treatment of cancer. Drugs 1994;48:794-847. [9] Rowinsky EK, Donehower RC. Paclitaxel (taxol) N Engl J Med 1995;332:1004-14. [10] Jordan MA, Wilson L. Microtubules and actin filaments: dynamic targets for cancer chemotherapy. Curr Opin Cell Biol 1998; 10:123-30. [11] Blagosklonny MV, Fojo T. Molecular effects of paclitaxel: myths and reality (a critical review). Int J Cancer 1999;83:151-6.
$339
[12] Giannakakou P, Robey R, Fojo T, Blagosklonny MV. Low concentrations of paclitaxel induce cell type-dependent p53, p21 and G1/G2 arrest instead of mitotic arrest: molecular determinants of paclitaxel-induced cytotoxicity. Oncogene 2001 ;20:3806-13. [13] Huang GC, Liu SY, Lin MH, Kuo YY, Liu YC. The synergistic cytotoxicity of cisplatin and taxol in killing oral squamous cell carcinoma. Jpn J Clin Oncol 2004;34:499-504. [14] Perol M. First-line treatment of metastatic non-small cell lung cancers. Rev Pneumol Clin 2004;60:51-6. [15] Sit AS, Kelley JL, Gallion HH, Kunschner AJ, Edwards RP. Paclitaxel and carboplatin for recurrent or persistent cancer of the cervix. Cancer Invest 2004;22:368-73. [16] Tse EY, Loo WT, Cheung MN, Chow LW, Cheng CW. Afimidex inhibition on proliferation of human breast solid tumors measured by ATP bioluminescence. Life Sci 2004, Dec 31.76(7):827-34. [17] Boabang P, Kurbacher CM, Kohlhagen H, Waida A, Amo-Takyi B K. Anti-neoplastic activity of topotecan versus cisplatin, etoposide and paclitaxel in four squamous cell cancer cell lines of the female genital tract using an ATP-Tumor Chemosensitivity Assay. Anticancer Drugs 2000; 1:843-8. [18] Kochli OR, Sevin BU, Schar G, Haller M. Value of the adenosine triphosphate chemosensitivity assay for pre-therapeutic determination of the chemosensitivity of breast cancer cells in cell culture. Geburtshilfe Frauenheilkd 1995;55:7-16. [19] Furukawa T, Kubota T, Tanino H, Oura S, Yuasa S, Murate H, Morita K, Kozakai K, Yano T, Hoffman RM. Chemosensitivity of breast cancer lymph node metastasis compared to the primary tumor from individual patients tested in the histoculture drug response assay. Anticancer Res 2000;20:3657-8. [20] Kurbacher CM, Grecu OM, Stier U, Gibster TJ, Janat MM, Untch M, Konecny G, Bruckner HW, Cree IA. ATP chemosensitivity testing in ovarian and breast cancer: early clinical trials. Recent results. Cancer Res 2003;161:221-30. [21] Zhou J, Liu M, Aneja R, Chandra R, Joshi HC. Enhancement of paclitaxel-induced microtubule stabilization, mitotic arrest, and apoptosis by the microtubule-targeting agent EM012. Biochem Pharmaco12004;68:2435-41. [22] Sui M, Dziadyk JM, Zhu X, Fan W. Cell cycle-dependent antagonistic interactions between paclitaxel and gamma-radiation in combination therapy. Clin Cancer Res 2004; 10:4848-57. [23] Konecny GE, Meng YG, Untch M, et al. Association between HER2/neu and vascular endothelial growth factor expression predicts clinical outcome in primary breast cancer patients. Clin Cancer Res 2004; 10:1706-16. [24] Locopo N, Fanelli M, Gasparini G. Clinical significance of angiogenic factors in breast cancer. Breast Cancer Res Treat 1998;52(1-3): 159-73.
SCIENCE ~____IDIRECT e
ELSEVIER
& BIOMEDICINE PHARMACOTHERAPY
Biomedicine & Pharmacotherapy 59 (2005) $337-$339 http://france.elsevier.com/direct/BIOPHA/
The efficacy of Paclitaxel on solid tumour analysed by ATP bioluminescence assay and VEGF expression: a translational research study W.T.Y. Loo, J.H.M. Fong, M.N.B. Cheung, L.W.C. Chow* Department of Surgery, The University of Hong Kong Medical Centre, PokfulamRoad, QueenMary Hospital, Hong Kong, China
Abstract Background. - Paclitaxel (Taxol), is known to induce mitotic arrest and apoptosis by inhibiting the depolymerisation of microtubules. Tumour growth and metastasis are affected by the metabolic rate and angiogenesis. We investigated the effect of Paclitaxel on tumour metabolism and markers of angiogenesis, vascular endothelial growth factor (VEGF). M a t e r i a l s a n d m e t h o d s . - Tissue samples of 39 patients diagnosed with invasive carcinoma were obtained. The solid tumours were cultured with Paclitaxel at a concentration of 4.27 ug/ml for 24 h. The metabolic rate of the samples was measured by ATP Bioluminescence assay and the levels of VEGF in culture medium were measured by ELISA. R e s u l t . - The mean ATP concentration of control and test groups were 7.169 and 5.004 rlu/ml, respectively, suggesting that the metabolic rate was inhibited by Paclitaxel. The mean VEGF levels in the control and test groups were 5.335 and 4.567 pg/ml, respectively. All data analysed were statistically significant (P < 0.005). The finding of the study showed that Paclitaxel could inhibit metabolic rate in solid tumour. It could also downregulate VEGF. Conclusion. - Our result suggested that Paclitaxel is an effective cytotoxic possibly with anti-angiogenic effects. © 2005 Elsevier SAS. All rights reserved.
Keywords: Paclitaxel; Metastasis; ATP bioluminescence assay
1. Introduction
Breast cancer is the most common primary cancer with poor prognosis and high mortality rate while primary malignant breast tumours are among those human neoplasms that exhibit the greatest angiogenic activity [1]. Vascular endothelial growth factor (VEGF), the most potent regulator of angiogenesis, is one of the factors leading to frequent metastasis and tumour development. As a result it has been acting as a prognostic indicator for various kinds of human tumours [2-4]. Tumour angiogenesis is a complex mechanism consisting of multi-step events including secretion or activation of angiogenic factors by tumour cells, activation of proteolytic enzymes, and proliferation, migration, and differentiation of endothelial cells [5].
* Corresponding authors. E-mail address: [email protected] (Louis W.C. Chow). © 2005 Elsevier SAS. All rights reserved.
The detection of the expression of soluble and insoluble VEGF in vitro and in vivo is an established method in determining the efficacy of treatment performed [6]. Paclitaxel (Taxol), an anti-neoplastic agent, is known to induce mitotic arrest and apoptosis by inhibiting the depolymerisation of microtubules [7-12]. It has been widely single or combined used in clinical to treated various cancer, such as neuroblastoma, non-Hodgkin lymphoma, oral squamous cell carcinoma, stomach, ovarian, breast, prostate and lung cancer [13-15]. ATP bioluminescence assay in solid tumour has been well established in our previous studies as one of the most sophisticated assays available, and early clinical trials show an enhanced response rate in patients undergoing ATP assay-directed preoperative chemotherapy compared to empirical chemotherapy [16]. By combining the ATP bioluminescence assay and ELISA assay to test for the metabolic rate and expression of VEGF, respectively, tumours' response may be predicted in vitro.
$338
W.T.Y. Loo et al. / Biomedicine & Pharmacotherapy 59 (2005) $337-$339
Therefore, more information w ill become available for clinicians in making decisions for treating malignant diseases. We would like to investigate the effect of Paclitaxel on breast solid tumour by means of the ATP assay and ELISA assay tested on level of VEGF. 2. Material and methods
Breast tumour tissues were obtained from 39 patients diagnosed with invasive ductal carcinoma. The tissue was minced into 1 mm 3 and cultured in DMEM (Invitrogen Corporation, California, USA) with or without 4.27 ug/ml (PPC) Paclitaxel for 24 h. After 24 h, the tissues were lysed by a sonicator (Sonics and Materials Inc., Danbury, CT. USA). The ATP Bioluminescence assay employed TD-20/ 20 Luminometer (Turner Designs, CA. USA) for measuring the metabolic rate. Apoptosis was determined by DNA fragmentation whereas the level of VEGF secretion in culture medium was by ELISA Kit (Pierice Endogen, USA). All data collected were analysed by Independent T-test (SPSS 11.0, USA). 3. Results
Natural logarithm was applied to the ATP readings due to the large range. Statistical significance was observed between the metabolic rate of control and test groups (Fig. 1). The DNA fragmentation test with 1% agarose gel revealed DNA ladder for different cases treated with Paclitaxel 4.27 ug/ml (Fig. 2). The mean level VEGF in culture media of the control and test groups was 5.335 pg/ml and 4.567 pg/ml. 4. Discussion
tumour chemosensitivity assay (ATP-TCA) have been implemented in evaluating cell viability, cell lines were chosen for most studies [17,18]. This analysis had been employed by our former studies o;n the determination of cell viability though not specifically to chemodrugs [16]. There are multiple advantages with using the chemosensitivity testing system we proposed. Firstly, by using a tissue-based three-dimensional histoculture, only a small amount of tissue is needed [19]. Moreover, the in vivo effects due to the stromal elements can be simulated. Currently, the ATPTCA can be regarded as the most sophisticated assay to investigate both solid samples and effusions derived from patients with various organ tumours [20]. Our results were analysed by comparing the inhibitory rate of the chemodrugs with the magnitude of tumour area reduced. Statistically significant positive correlation was observed between the chemosensitivity response and clinical response. The clinicians in our parallel study made use of the in vitro ATP results after tested with Paclitaxel on breast cancer patients, the pathological and clinical response rate reached 50%. Other researchers reported that Paclitaxel arrests tumour cell growth through impairing of the cytoskeletal system by forming microtubules in the malignant cells, giving rise to programmed cell death [21,22] which coincides with our finding (Fig. 2). Solid tumours were cultured with and without the anticancer agent, the level of soluble VEGF measured was definitely lower than the control group. Zhong et al. [6] also accounted for an effective agent in cancer treatment through the inhibition of VEGF expression, and the activation of signalling pathway in their study. Clinical evidence has been provided that HER-2/neu overexpression is associated with expression of two of the most abundantly expressed VEGF isoforms in breast cancer, suggesting that VEGF may in part mediate the aggressive phenotype of
We described an innovative chemosensitivity test by measuring cell metabolism of tumour tissues with an ATP bioluminescence assay. Although some other ATP-based 8.0
¸
0.. I.- 7.0' <
~5 W
"~ t~
6.0'
O'J
g ¢)
5.0,
4.0 Control
Paclitaxel 4.27ug/ml
Group
Fig. 1. The comparison of metabolic rate between control and test groups.
Fig. 2. The DNA ladder produced by Paclitaxel-treated solid tumour of various patients. From left, Lane 1: Control; Lanes 2-6: Different cases treated with Paclitaxel 4.27 ug/ml and Lane 7:100 bp DNA marker.
W. T. Y. Loo et al. / Biomedicine & Pharmacotherapy 59 (2005) $337-$339
breast cancers that overexpress HER-2/neu [23]. Recognising which are the more biologically active positive and negative angiogenic factors is the key for the identification not only of new prognostic markers but also of targets for anti-angiogenic therapy in human breast cancer [24]. This translational assay by means of the application of ATP assay as well as measurement of secretion of VEGF in tumour cells is proved to be practically responsive for patients given that the clinicians make use of the data provided by the laboratory tests. The pathological and clinical response rate with this combined analysis approach attained more than 50% in clinical use. References [1]
Rahman MA, Toi M. Anti-angiogenic therapy in breast cancer. Biomed Pharmacother 2003;57:463-70. [2] Weidner N, Folkman J, Pozza F, Bevilacqua P, Alfred EN, Moore DH, Meli S, Gasparin G. Tumor angiogenesis: a new significant and independent prognostic indicator in early-stage breast carcinoma. J Natl Cancer Inst 1992;84:1875-87. [3] Toi M, Kashitani J, Tominaga T. Tumor angiogenesis is an independent prognostic indicator in primary breast carcinoma. Int J Cancer 1993;55:371-4. [4] Niethammer AG. A DNA vaccine against VEGF receptor 2 prevents effective angiogenesis and inhibits tumor growth. Nat Med 2002;8:1369-75. [5] Folkman J. What is the evidence that tumors are angiogenesis dependent? J Natl Cancer Inst 1990;82:4-6. [6] Zhong XS, Zheng JZ, Reed E, Jiang BH. SU5416 inhibited VEGF and HIF-lalpha expression through the PI3K/AKT/p70S6K1 signaling pathway. Biochem Biophys Res Commun 2004;324:471-80. [7] Horwitz SB. Mechanism of action of taxol. Trends Pharmacol Sci. 1992;13:134-6. [8] Spencer CM, Faulds D. Paclitaxel. A review of its pharmacodynamic and pharmacokinetic properties and therapeutic potential in the treatment of cancer. Drugs 1994;48:794-847. [9] Rowinsky EK, Donehower RC. Paclitaxel (taxol) N Engl J Med 1995;332:1004-14. [10] Jordan MA, Wilson L. Microtubules and actin filaments: dynamic targets for cancer chemotherapy. Curr Opin Cell Biol 1998; 10:123-30. [11] Blagosklonny MV, Fojo T. Molecular effects of paclitaxel: myths and reality (a critical review). Int J Cancer 1999;83:151-6.
$339
[12] Giannakakou P, Robey R, Fojo T, Blagosklonny MV. Low concentrations of paclitaxel induce cell type-dependent p53, p21 and G1/G2 arrest instead of mitotic arrest: molecular determinants of paclitaxel-induced cytotoxicity. Oncogene 2001 ;20:3806-13. [13] Huang GC, Liu SY, Lin MH, Kuo YY, Liu YC. The synergistic cytotoxicity of cisplatin and taxol in killing oral squamous cell carcinoma. Jpn J Clin Oncol 2004;34:499-504. [14] Perol M. First-line treatment of metastatic non-small cell lung cancers. Rev Pneumol Clin 2004;60:51-6. [15] Sit AS, Kelley JL, Gallion HH, Kunschner AJ, Edwards RP. Paclitaxel and carboplatin for recurrent or persistent cancer of the cervix. Cancer Invest 2004;22:368-73. [16] Tse EY, Loo WT, Cheung MN, Chow LW, Cheng CW. Afimidex inhibition on proliferation of human breast solid tumors measured by ATP bioluminescence. Life Sci 2004, Dec 31.76(7):827-34. [17] Boabang P, Kurbacher CM, Kohlhagen H, Waida A, Amo-Takyi B K. Anti-neoplastic activity of topotecan versus cisplatin, etoposide and paclitaxel in four squamous cell cancer cell lines of the female genital tract using an ATP-Tumor Chemosensitivity Assay. Anticancer Drugs 2000; 1:843-8. [18] Kochli OR, Sevin BU, Schar G, Haller M. Value of the adenosine triphosphate chemosensitivity assay for pre-therapeutic determination of the chemosensitivity of breast cancer cells in cell culture. Geburtshilfe Frauenheilkd 1995;55:7-16. [19] Furukawa T, Kubota T, Tanino H, Oura S, Yuasa S, Murate H, Morita K, Kozakai K, Yano T, Hoffman RM. Chemosensitivity of breast cancer lymph node metastasis compared to the primary tumor from individual patients tested in the histoculture drug response assay. Anticancer Res 2000;20:3657-8. [20] Kurbacher CM, Grecu OM, Stier U, Gibster TJ, Janat MM, Untch M, Konecny G, Bruckner HW, Cree IA. ATP chemosensitivity testing in ovarian and breast cancer: early clinical trials. Recent results. Cancer Res 2003;161:221-30. [21] Zhou J, Liu M, Aneja R, Chandra R, Joshi HC. Enhancement of paclitaxel-induced microtubule stabilization, mitotic arrest, and apoptosis by the microtubule-targeting agent EM012. Biochem Pharmaco12004;68:2435-41. [22] Sui M, Dziadyk JM, Zhu X, Fan W. Cell cycle-dependent antagonistic interactions between paclitaxel and gamma-radiation in combination therapy. Clin Cancer Res 2004; 10:4848-57. [23] Konecny GE, Meng YG, Untch M, et al. Association between HER2/neu and vascular endothelial growth factor expression predicts clinical outcome in primary breast cancer patients. Clin Cancer Res 2004; 10:1706-16. [24] Locopo N, Fanelli M, Gasparini G. Clinical significance of angiogenic factors in breast cancer. Breast Cancer Res Treat 1998;52(1-3): 159-73.