Am J Clin Oncol (CCT) 26(1): 50–54, 2003.
`
`© 2003 Lippincott Williams & Wilkins, Inc., Philadelphia
`
`Decreased Response to Paclitaxel Versus Docetaxel
`in HER-2/neu Transfected Human Breast
`Cancer Cells
`
`Lois M. Witters, B.S., Samuel M. Santala, B.S., Linda Engle, B.S.,
`Vernon Chinchilli, Ph.D., and Allan Lipton, M.D.
`
`Taxanes are effective in the treatment of metastatic breast
`cancer. Docetaxel has been shown to be more potent than
`paclitaxel in inducing bcl-2 phosphorylation and apoptosis and
`is clinically active in some paclitaxel-resistant breast tumors.
`HER-2/neu overexpression has been shown to correlate with
`resistance to hormonal therapy as well as chemotherapy. Using
`a HER-2/neu transfected MCF-7 human breast cancer cell line,
`we investigated the role of HER-2/neu overexpression on
`resistance to paclitaxel and docetaxel treatment. A control
`vector transfected MCF-7 human breast cancer cell line (MCF/
`neo) and a HER-2/neu transfected MCF-7 line (MCF/18) were
`treated with various concentrations of docetaxel or paclitaxel.
`Cell number was assessed using the MTT tetrazolium dye
`assay. In the control vector transfected MCF/neo cell line,
`paclitaxel and docetaxel gave similar dose-dependent growth
`inhibition (p ⫽ 0.175). In HER-2/neu transfected MCF/18
`cells, docetaxel treatment resulted in a dose-dependent inhibi-
`tion similar to that seen in MCF/neo cells. Paclitaxel, however,
`gave significantly less growth inhibition than docetaxel in the
`HER-2/neu overexpressing MCF/18 cells (p ⫽ 0.0003). These
`data suggest that HER-2/neu overexpression may contribute to
`paclitaxel resistance. In contrast, the cytotoxic effects of do-
`cetaxel in these breast carcinoma cells are not affected by
`HER-2/neu expression. Therefore, docetaxel may be the pre-
`ferred taxane therapy in HER-2/neu overexpressing breast
`tumors.
`Key Words:
`cancer.
`
`Paclitaxel—Docetaxel—HER-2/neu—Breast
`
`Activation of the HER-2/neu receptor tyrosine kinase
`triggers a cascade of events leading to cell prolifera-
`tion.1,2 HER-2/neu is overexpressed in 20% to 30% of
`
`From the Departments of Medicine (L.M.W., S.M.S., A.L.) and
`Biostatistics & Epidemiology (L.E., V.C.), Penn State College of
`Medicine, Hershey, Pennsylvania, U.S.A.
`Presented in poster form at the San Antonio Breast Cancer Confer-
`ence, San Antonio, Texas, 2000.
`Supported by a grant from Aventis Pharmaceuticals, Parsippany, NJ.
`Address correspondence and reprint requests to Dr. Allan Lipton,
`Department of Medicine, H046, The Milton S. Hershey Medical
`Center, Penn State College of Medicine, 500 University Drive, Her-
`shey, PA 17033, U.S.A. E-mail: alipton@psu.edu
`
`breast tumors3,4 and is associated with a more aggressive
`disease and worse prognosis.3– 6
`Docetaxel and paclitaxel are members of the taxane
`family. They are promoters of tubulin polymerization
`and microtubule stabilization.7–9 They also induce Bcl-2
`phosphorylation and apoptosis. Docetaxel has been
`shown to induce Bcl-2 phosphorylation and apoptosis at
`100-fold lower concentration than paclitaxel.10,11 It has
`also been reported that docetaxel produces greater anti-
`tumor activity than paclitaxel at equal doses both in
`vitro12,13 and in vivo.14,15 Docetaxel is 2 to 12 times
`more potent than paclitaxel in causing cell death.13,16 In
`addition, the cellular uptake of docetaxel is greater than
`that of paclitaxel, and the efflux of docetaxel from the
`tumor cell is slower, meaning longer tumor retention
`time.7,17
`Both drugs are effective as first-line treatment of
`metastatic breast cancer (MBC). Docetaxel, however,
`has proven to be more effective in second-line treatment
`of MBC. In a phase II clinical study in anthracycline-
`resistant breast cancer, the response rate (RR) with do-
`cetaxel was greater than that with paclitaxel (RR ⫽
`32–51%, 6–30%, respectively).18 –21 Docetaxel has also
`been active in patients with paclitaxel-resistant breast
`cancer, suggesting that
`there is only a partial cross
`resistance between paclitaxel and docetaxel.7,22 In a
`phase III study in MBC, Chan showed that docetaxel was
`superior to the previously accepted treatment, doxorubi-
`cin.23,24 In contrast, paclitaxel was not better
`than
`doxorubicin.25,26
`Many reports have suggested a relationship between
`HER-2/neu expression and resistance to paclitaxel. Yu27
`has shown that HER-2/neu transfected MDA-MB435
`human breast cancer cells are more resistant to paclitaxel
`than the lower HER-2/neu expressing parental
`line.
`Perez-Soler28 has reported that in non–small-cell lung
`cancer (NSCLC) heterotransplants, lack of HER-2/neu
`expression was associated with response to paclitaxel
`(0% of
`responding tumors expressed HER-2/neu,
`whereas 48% of the nonresponders expressed HER-2/
`neu). Use of trastuzumab, the humanized monoclonal
`
`50
`
`Genentech 2054
`Hospira v. Genentech
`IPR2017-00737
`
`

`

`PACLITAXEL VERSUS DOCETAXEL IN CANCER
`
`51
`
`FIG. 1. Percentage of growth in the
`control vector
`transfected MCF/neo
`cell line after a 3-day exposure to var-
`ious concentrations of paclitaxel or
`docetaxel.
`
`HER-2/neu antibody, to block HER-2/neu activity en-
`hances the cytotoxic effect of paclitaxel in vitro and in
`HER-2/neu overexpressing human breast cancer xeno-
`grafts.29 Zhang30 has reported that the tyrosine kinase
`inhibitor, emodin, which inhibits growth of HER-2/neu
`overexpressing tumors in mice, sensitizes the tumors to
`paclitaxel. Konecny has reported preclinical data show-
`ing synergy with the combination of docetaxel and
`trastuzumab.31
`The observation that docetaxel is effective in paclitax-
`el-resistant breast cancer suggests that these drugs are
`not cross-resistant. The purpose of this study was to
`explore the sensitivity of HER-2/neu transfected breast
`cancer cells to paclitaxel and docetaxel.
`
`MATERIALS AND METHODS
`
`Materials
`The MCF-7 human breast cancer cell line transfected with a
`control vector, MCF/neo, and the HER-2/neu transfected
`MCF-7 line, MCF/18, were supplied by Genentech (South San
`Francisco, CA, U.S.A.). Paclitaxel was a gift from Bristol-
`Myers Squibb (Princeton, NJ, U.S.A.), and docetaxel was a gift
`from Aventis Pharmaceuticals (Bridgewater, NJ, U.S.A.). The
`tetrazolium dye (MTT), Cremophor EL, and polyoxyethylene-
`sorbitan monooleate were purchased from Sigma Chemical
`Company (St. Louis, MO, U.S.A.).
`
`Cell Growth Experiments
`The MCF/neo and MCF/18 cells were maintained in a 50:50
`mix of Ham’s media and high glucose Dulbecco’s Modified
`Eagle Medium supplemented with 10% fetal bovine serum,
`L-glutamine, and G418 in 5% CO2 at 37°C. Cells were plated
`in 24-well plates at 20,000 cells/well and were incubated
`overnight. Cells were treated with various concentrations of
`docetaxel or paclitaxel (1–5 nmol/l) or their corresponding
`soluent (polyoxyethylene-sorbitan monooleate or Cremophor
`EL, respectively) and incubated for an additional 3 days. Cell
`number was assessed using the colorimetric MTT tetrazolium
`
`dye assay. Percent of growth was normalized to control (un-
`treated) cells (100% growth).
`Statistical Analyses
`Analysis of variance was applied using PROC MIXED of
`SAS to model treatment and dose (together as an interaction
`term) on growth. A separate analysis for the MCF/neo cell line
`(n ⫽ 26 with nonmissing growth) and a separate analysis for
`the MCF/18 cell line (n ⫽ 41 with nonmissing growth) was
`performed. We also modeled cell line and dose (together as an
`interaction term) with separate analysis for paclitaxel (n ⫽ 31
`with nonmissing growth) and separate analysis for docetaxel
`(n ⫽ 36 with nonmissing growth).
`
`RESULTS
`Control vector transfected MCF-7 cells (MCF/neo)
`and HER-2/neu transfected MCF-7 cells (MCF/18) were
`treated with similar concentrations of either paclitaxel or
`docetaxel (1–5 nmol/l). In the MCF/neo cell line, both
`drugs inhibited cell growth in a dose-dependent manner.
`Treatment with paclitaxel resulted in slightly less growth
`inhibition overall (2.5 nmol/l; 31% ⫾ 12.5 inhibition)
`compared with docetaxel (2.5 nmol/l: 43% ⫾ 12.5 inhi-
`bition) (Fig. 1). This difference was not statistically
`significant (p ⫽ 0.179). There was no significant differ-
`ence in the mean for percent growth between the treat-
`ments either overall or separately by dose.
`In the HER-2/neu transfected MCF/18 cells, docetaxel
`gave dose-dependent growth inhibition similar to that
`seen in the MCF/neo line. Treatment with paclitaxel,
`however, resulted in significantly (p ⬍ 0.0001) less
`inhibition at the 2.5 nmol/l dose than that seen with
`docetaxel (17% ⫾ 8.7 versus 44% ⫾ 7.7, respectively)
`(Fig. 2). Also, overall, the mean for percent growth with
`paclitaxel treatment was significantly more than for the
`mean with docetaxel treatment (p ⫽ 0.0003). The other
`doses had no significant difference between the
`treatments.
`
`Am J Clin Oncol (CCT), Vol. 26, No. 1, 2003
`
`

`

`52
`
`L. M. WITTERS ET AL.
`
`FIG. 2. Percentage of growth in the
`HER-2/neu transfected MCF/18 cell line
`after a 3-day exposure to various con-
`centrations of paclitaxel or docetaxel.
`
`Figure 3 illustrates that docetaxel treatment resulted in
`an identical growth inhibitory effect on both cell lines
`regardless of levels of HER-2/neu expression. There was
`no significant difference between the cell lines, either
`overall or separately by dose. Paclitaxel over the same
`dose range inhibited growth in a manner similar to
`docetaxel in the MCF/neo cell line. In contrast, paclitaxel
`had significantly less of an inhibitory effect on the
`HER-2/neu transfected MCF/18 cells.
`
`DISCUSSION
`In phase II trials, paclitaxel as a single agent at
`first-line or second-line doses without a high toxicity
`profile (175–200 mg/m2) for 3 hours results in a 25% to
`30% objective response rate in the treatment of
`MBC.32,33 In contrast, docetaxel as a single agent in
`
`phase II trials results in response rates of 38% to 68% (as
`first-line), 34% to 58% (as second-line), and 29% to 50%
`(in patients previously exposed to anthracyclines).33 In
`vitro studies have shown that docetaxel induces Bcl-2
`phosphorylation and apoptosis at 100-fold lower concen-
`tration than paclitaxel.10 Clinically, docetaxel is active in
`patients with breast cancer who are paclitaxel resistant,7
`suggesting that these agents are not entirely identical in
`their mechanisms of action.
`HER-2/neu overexpression has been suggested as a
`mechanism for resistance to both hormonal therapy as
`well as chemotherapy.28,30,34 HER-2/neu expression may
`also predict response to certain chemotherapy. The pur-
`pose of our study was to assess whether HER-2/neu
`overexpression plays a role in resistance to taxane anti-
`tumor activity.
`We have shown that paclitaxel is indeed less effective
`
`FIG. 3. Percentage of growth in the
`control vector
`transfected MCF/neo
`and HER-2/neu transfected MCF/18
`human breast cancer cells after a
`3-day exposure to various concentra-
`tions of paclitaxel or docetaxel.
`
`Am J Clin Oncol (CCT), Vol. 26, No. 1, 2003
`
`

`

`PACLITAXEL VERSUS DOCETAXEL IN CANCER
`
`53
`
`as an antiproliferative agent in HER-2/neu transfected
`MCF/18 breast cancer cells (2.5 nmol/l: 17% ⫾ 7.9
`inhibition) than in the control transfected MCF/neo cells
`(2.5 nmol/l: 31% ⫾ 9.3 inhibition). Docetaxel, however,
`resulted in the same percent inhibition at 2.5 nmol/l in
`both cell lines (MCF/neo: 43% ⫾ 12.4; MCF/18: 44% ⫾
`9.3). Docetaxel inhibited growth regardless of HER-2/
`neu expression.
`In clinical trials, tumors that overexpress HER-2/neu
`appear to be less responsive to paclitaxel therapy. As
`first-line therapy for patients with MBC after failure
`of adjuvant anthracycline therapy, paclitaxel induced a
`clinical response in only 17% of HER-2/neu-positive
`patients.35 In earlier studies with unselected patients
`(HER-2/neu positive and negative) with MBC receiving
`first-line paclitaxel, the response rates were up to 60%.33
`Perhaps the low response rate in the anthracycline fail-
`ures was because the patients all had HER-2/neu-positive
`tumors.
`It has been reported that the cellular uptake of do-
`cetaxel is greater than that of paclitaxel and that the
`efflux rate is three times slower than with paclitaxel.7,17
`This might result in a higher intracellular concentration,
`longer exposure, and greater cytotoxicity with do-
`cetaxel.7 Significant difference in growth inhibition pro-
`duced by treatment with paclitaxel or docetaxel was only
`seen in the MCF/18 cell line (p ⫽ 0.0003). The only
`difference between this line and the MCF/neo cell line
`where a significant difference was not seen between the
`two drugs (p ⫽ 0.175) is the level of HER-2/neu expres-
`sion. These data would suggest, therefore, that the results
`were not caused by only greater intracellular concentra-
`tions with docetaxel. If it were just a concentration
`phenomenon, the inhibitory activity of paclitaxel would
`be similar in both cell
`lines, as was observed with
`docetaxel.
`Paclitaxel and docetaxel are both members of the
`taxane family and inhibit microtubule depolymerization.
`However, they do not appear to inhibit cell proliferation
`by identical mechanisms, with HER-2/neu signaling
`pathways potentially inhibiting the paclitaxel antitumor
`response. The cytotoxic effects of docetaxel, however,
`are not inhibited by HER-2/neu overexpression. Further
`investigation is warranted to determine the molecular
`pathways involved in the interaction between taxanes
`and HER-2/neu. This study should further alert clinicians
`that
`these two agents may have different effects in
`similar types of carcinomas, suggesting that docetaxel
`may be the preferred taxane therapy for patients with
`HER-2/neu-positive breast cancer.
`
`REFERENCES
`1. Burris AB. Docetaxel (Taxotere) in HER-2-positive patients and in
`combination with trastuzumab (Herceptin). Semin Oncol 2000;27:
`19–23.
`2. Scott GK, Dodson JM, Montgomery PA, et al. p185HER2 signal
`transduction in breast cancer cells. J Biol Chem 1991;266:14300–
`14305.
`3. Slamon DJ, Clark GM, Wong SG, et al. Human breast cancer:
`
`correlation of relapse and survival with amplification of HER-2/
`neu oncogene. Science 1987;235:177–182.
`4. Van de Vijver MJ, Peterse JL, Mooi WJ, et al. Neu protein
`overexpression in breast cancer. Association with comedo-type
`ductal carcinoma in situ and limited prognostic value in stage II
`breast cancer. N Engl J Med 1988;319:1239–1245.
`5. Slamon DJ, Godolphin W, Jones LA, et al. Studies of HER-2/neu
`proto-oncogene in human breast and ovarian cancer. Science 1989;
`244:707–712.
`6. Press MF, Jones LA, Godolphin W, et al. HER-2/neu oncogene
`amplification and expression in breast and ovarian cancers. Prog
`Clin Biol Res 1990;354A:209–221.
`7. Valero V, Jones SE, Von Hoff DD, et al. A phase II study of
`docetaxel in patients with paclitaxel-resistant metastatic breast
`cancer. J Clin Oncol 1998;16:3362–3368.
`8. Ringel I, Horwitz SB. Studies with RPR 56976 (Taxotere): a
`semisynthetic analogue of Taxol. J Natl Cancer Inst 1991;83:288–
`291.
`9. Diaz JF, Andreau JM. Assembly of purified GDP-tubulin into
`microtubules induced by Taxol and Taxotere: reversibility, ligand
`stoichiometry, and competition. Biochemistry 1993;32:2747–2755.
`10. Haldar S, Basu A, Croce CM. Bcl2 is the guardian of microtubule
`integrity. Cancer Res 1997;57:229–233.
`11. Gumerlock PH, Mack PC, Manorek GH, et al. p27 induction as a
`potential p53-independent mechanism of apoptotic response to
`taxanes in non-small cell lung cancer (NSCLC) [Abstract 723].
`Proc Am Soc Clin Oncol 1999.
`12. Keland LR, Abel G. Comparative in vitro cytotoxicity of Taxol and
`Taxotere against cisplatin-sensitive and -resistant human ovarian
`carcinoma cell lines. Cancer Chemother Pharmacol 1992;30:444–
`450.
`13. Riou JF, Naudin A, Lavelle F. Effects of Taxotere on murine and
`human tumor cell lines. Biochem Biophys Res Commun 1992;187:
`164–170.
`14. Nicoletti MI, Lucchini V, D’Incalci M, et al. Comparison of
`paclitaxel and docetaxel activity on human ovarian carcinoma
`xenografts. Eur J Cancer 1994;30A:691–696.
`15. Vogel M, Hilsenbeck SG, Depenbrock H, et al. Preclinical activity
`of Taxotere (RP56976, NSC 628503) against freshly explanted
`clonogenic human tumour cells: comparison with Taxol and con-
`ventional antineoplastic agents. Eur J Cancer 1993;29A:2009–
`2014.
`16. Hanauske AR, Degen D, Hilsenbeck SG, et al. Effects Taxotere
`and Taxol on in vitro colony formation of freshly explanted human
`tumor cells. Anticancer Drugs 1992;3:121–124.
`17. Riou JF, Petitgenet O, Combeau C, et al. Cellular uptake and efflux
`of docetaxel (Taxotere) and paclitaxel (Taxol) in P388 cell line
`[Abstract 2292]. Proc Am Assoc Cancer Res 1994;35.
`18. Valero V, Holmes FA, Walters RS, et al. Phase II trial of do-
`cetaxel: a new highly effective antineoplastic agent in the manage-
`ment of patients with anthracycline-resistant breast cancer. J Clin
`Oncol 1995;13:2886–2894.
`19. Ravdin PM, Burris HA, Cook G, et al. Phase II of docetaxel in
`advanced anthracycline-resistant or
`anthracenedione-resistant
`breast cancer. J Clin Oncol 1995;13:2879–2885.
`20. Seidman AD, Hudis CA, Raptis G, et al. Paclitaxel for breast
`cancer: the Memorial Sloan-Kettering Cancer Center experience.
`Oncology 1997;11:20–28.
`21. Vermorken JB, ten Bokkel Huinink WW, Mandjes IAM, et al.
`High-dose paclitaxel with granulocyte colony-stimulating factor in
`patients with advanced breast cancer refractory to anthracycline
`therapy: a European Cancer Center trial. Semin Oncol 1995;22:
`16–22.
`22. Verweij J, Clavel M, Chevallier B. Paclitaxel (Taxol) and do-
`cetaxel (Taxotere): not simply two of a kind. Ann Oncol 1994;5:
`495–505.
`23. Chan S, Friedrichs K, Noel D, et al. Prospective randomized trial
`of docetaxel versus doxorubicin in patients with metastatic breast
`cancer. The 303 Study Group. J Clin Oncol 1999;17:2341–2354.
`24. Nabholtz JM, Senn HJ, Bezwoda WR, et al. Prospective random-
`ized trial of docetaxel versus mitomycin plus vinblastine in patients
`with metastatic breast cancer progressing despite previous anthra-
`
`Am J Clin Oncol (CCT), Vol. 26, No. 1, 2003
`
`

`

`54
`
`L. M. WITTERS ET AL.
`
`cycline-containing chemotherapy. 304 Study Group. J Clin Oncol
`1999;17:1413–1424.
`25. Paridaens R, Biganzoli L, Bruning P, et al. Paclitaxel versus
`doxorubicin as first-line single agent chemotherapy for metastatic
`breast cancer: a European Organization for Research and Treat-
`ment of Cancer randomized study with cross-over. J Clin Oncol
`2000;18:724–733.
`26. Sledge GW, Neuberg D, Ingle J, et al. Phase III trial of doxorubicin
`(A) vs. paclitaxel (T) vs. doxorubicin ⫹ paclitaxel (A ⫹ T) as
`first-line therapy for metastatic breast cancer (MBC): an intergroup
`trial [Abstract 2]. Proc Am Soc Clin Oncol 1997.
`27. Yu D, Liu B, Tan M, et al. Overexpression of c-erbB-2/neu in
`breast cancer cells confers increased resistance to Taxol via mdr-
`1-independent mechanisms. Oncogene 1996;13:1359–1365.
`28. Perez-Soler R, Kemp B, Wu QP, et al. Response and determinants
`of sensitivity to paclitaxel in human non-small cell lung cancer
`tumors heterotransplanted in nude mice. Clin Cancer Res 2000;6:
`4932–4938.
`29. Baselga J, Norton L, Albanell J, et al. Recombinant humanized
`anti-HER2 antibody (Herceptin) enhances the antitumor activity of
`paclitaxel and doxorubicin against HER2/neu overexpressing hu-
`man breast cancer xenografts. Cancer Res 1998;58:2825–2831.
`
`30. Zhang L, Lau Y-K, Xia W, et al. Tyrosine kinase inhibitor emodin
`suppresses growth of HER-2/neu-overexpressing breast cancer
`cells in athymic mice and sensitizes these cells to the inhibitory
`effect of paclitaxel. Clin Cancer Res 1999;5:343–353.
`31. Konecny G, Pegram MD, Beryt M, et al. Therapeutic advantage of
`chemotherapy drugs in combination with Herceptin against human
`breast cancer cells with HER-2/neu overexpression. San Antonio
`Breast Cancer Conference, 1999: Abstract 467.
`32. Wiseman LR, Spencer CM. Paclitaxel: an update of its use in the
`treatment of metastatic breast cancer and ovarian and other gynae-
`cological cancers. Drugs Aging 1998;12:305–334.
`33. Nabholtz JM, Tonkin K, Smiylie M, et al. Chemotherapy of breast
`cancer: are the taxanes going to change the natural history of breast
`cancer? Expert Opin Pharmacother 2000;1:187–206.
`34. Leitzel K, Teramoto Y, Konrad K, et al. Elevated serum c-erbB-2
`antigen levels and decreased response to hormone therapy of breast
`cancer. J Clin Oncol 1995;13:1129–1135.
`35. Slamon DJ, Leyland-Jones B, Shak S, et al. Use of chemotherapy
`plus a monoclonal antibody against HER2 for metastatic breast
`cancer that overexpresses HER2. N Engl J Med 2001;344:783–
`792.
`
`Am J Clin Oncol (CCT), Vol. 26, No. 1, 2003
`
`