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Clinical Cancer Research Vol. 10, 794-801, January 2004
© 2004 American Association for Cancer Research

Experimental Therapeutics, Preclinical Pharmacology

ZD1839 Modulates Paclitaxel Response in Renal Cancer by Blocking Paclitaxel-Induced Activation of the Epidermal Growth Factor Receptor–Extracellular Signal-Regulated Kinase Pathway

Makoto Sumitomo, Tomohiko Asano, Junichi Asakuma, Takako Asano, Akio Horiguchi and Masamichi Hayakawa

Department of Urology, National Defense Medical College, Tokorozawa, Saitama, Japan


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Purpose: We evaluated the antitumor activity of ZD1839, a selective epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor, in combination with paclitaxel in human renal cell carcinomas (RCCs).

Experimental Design: Eight human RCC lines and the surgical specimens obtained from 10 RCC patients were used. The protein expression was detected by Western blotting, immunohistochemistry and/or flow cytometry. Apoptosis was evaluated by flow cytometry and fragmented DNA ELISA. SKRC-49 tumor xenografts in athymic nude mice were treated with ZD1839 and/or paclitaxel, and tumor volume was determined

Results: EGFR protein was expressed and phosphorylated in eight RCC lines and EGFR expression was markedly increased in RCC specimens compared with adjacent normal renal tissues. Treatment of SKRC-49 with 1 µM ZD1839 resulted in a marked decrease in the phosphorylation of EGFR but not of HER-2. Treatment of SKRC-49 with ZD1839 in combination with 5 nM paclitaxel resulted in a significant increase in apoptotic cell number compared with paclitaxel alone, whereas ZD1839 alone failed to induce apoptosis. Although administration of ZD1839 or paclitaxel resulted in a transient growth inhibition in SKRC-49 xenografts, significant tumor regrowth delay was observed when paclitaxel was combined with ZD1839. Paclitaxel phosphorylated extracellular signal-regulated kinase through EGFR activation predominantly in cancer cells. ZD1839 promoted paclitaxel-induced Bcl-2 down-regulation resulting in promoting apoptosis by blocking paclitaxel-induced activation of the EGFR—extracellular signal-regulated kinase antiapoptotic pathway independent of Akt activity in SKRC-49.

Conclusions: Our findings support the idea that the significant clinical benefit is obtained from ZD1839 in combination with paclitaxel for the treatment of RCC.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Renal cell carcinoma (RCC) is the most common malignancy of the kidney and accounts for about 3% of all adult neoplasms (1) . The number of new cases with RCC in the United States in 2003 is projected to be 31,900 with an estimated 11,900 deaths (1) . In disseminated RCCs, conventional chemotherapy, radiation therapy, and immunotherapy have been tried with limited efficacy (2 , 3) . Moreover, chemotherapeutic agents such as cisplatin are toxic to normal renal tubular cells, which might limit the clinical use of chemotherapeutic agents because most patients with metastatic RCCs usually undergo radical nephrectomy. Thus, the novel therapeutic strategies should be sought to improve the efficacy in treating disseminated RCCs.

Epidermal growth factor (EGF) receptor (EGFR, ErbB1) is overexpressed in a wide variety of epithelial malignancies including RCC (4, 5, 6) . The known downstream effectors of EGFR such as extracellular signal-regulated kinase (ERK) in the mitogen-activated protein kinase pathway play a crucial role in the apoptosis inhibition in cancer cells (7 , 8) . Importantly, it has recently been demonstrated that a variety of chemotherapeutic agents by themselves activate the EGFR pathway, which has been demonstrated to contribute to the resistance to chemotherapy in many tumor types (9, 10, 11, 12) .

ZD1839 is the development of quinazoline-derived agents that are specific ATP-competitors of EGFR tyrosine kinase. ZD1839 shows antiproliferative activity in various human cancer cell types (13) . The antitumor activity of chemotherapeutic agents such as a microtubule-stabilizing agent paclitaxel is potentiated by combination with ZD1839 (14 , 15) . In the present study, we evaluated the antitumor activity of ZD1839 in combination with low concentrations of paclitaxel in human RCCs because of the following reasons. First of all, there are few reports describing the efficacy of ZD1839 with or without chemotherapeutic agents in treating EGFR-overexpressing RCCs. Second, low concentrations of paclitaxel may be promising for the treatment of RCC with minimal toxicity in normal tissues because we have recently reported that, at low concentrations, paclitaxel is able to induce mitotic cell death, especially in RCC cells (16) . Third, the precise mechanism by which ZD1839 potentiates paclitaxel response synergistically, not additively, in cancer cells remains unclear. We report here that ZD1839 is able to modulate paclitaxel response in RCC both in vitro and in vivo. Furthermore, we present a finding that explains why ZD1839 is able to promote chemotherapeutic agents-induced apoptosis synergistically in cancer cells.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Reagents.
RCC lines and a prostate cancer cell line PC-3 were maintained in DMEM or MEM each supplemented with 2 mM glutamine, 1% nonessential amino acids, 100 units/ml streptomycin and penicillin, and 10% FCS. The normal human renal proximal tubular epithelial cells and prostate epithelial cells PrEC (Clonetics, Walkersville, MD) were maintained in PrEBM medium (Clonetics) supplemented with the PrEGM BulletKit (Clonetics). ZD1839 was generously provided by AstraZeneca (Macclesfield, United Kingdom) and was dissolved with Tween 80. Paclitaxel was supplied by Bristol-Myers Squibb KK (Tokyo, Japan). The mitogen-activated protein kinase or ERK (mitogen-activated protein kinase/ERK kinase) inhibitor U0126 and the phosphatidylinositol 3-kinase-Akt inhibitor LY294002 were purchased from Calbiochem-Novabiochem Ltd. (La Jolla, CA).

Western Blot Analysis.
Total cell lysates were prepared with radioimmunoprecipitation assay buffer and immunoblotting were performed as described previously (17 , 18) using anti-EGFR antibody (Ab), anti-phospho Tyr1068-EGFR Ab, anti-HER-2 (ErbB2) Ab, anti-phospho Tyr1248-HER-2 Ab, anti-ERK1/2 Ab, anti-phospho Thr202/Tyr204-ERK1/2 Ab, Ser473-Akt, (Cell Signaling Technology, Inc., Beverly, MA, 1:500–1000), and anti-Akt and anti-Bcl-2 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA; 1:500–1000).

Flow Cytometry and Immunohistochemistry for EGFR Expression.
For detecting EGFR expression in surgical specimens by flow cytometry, RCC surgical specimens and adjacent normal renal tissues from 10 patients with RCC were gently pressed and ground in a course glass grinder with chilled medium. The cells were counted in a hemacytometer by the trypan blue dye exclusion method. Cells were then incubated in 1% BSA in PBS for 30 min and then were stained with the FITC-conjugated anti-EGFR monoclonal Ab (PharMingen, San Diego, CA; 1:50) for 1 h on ice. As a negative control, an aliquot of the cells was stained with the FITC-conjugated monoclonal Ab of the same phenotype (PharMingen; 1:50). Flow cytometry was performed on a FAC-Scan analyzer (Becton Dickinson, Mountain View, CA), and median EGFR-positive values in RCC specimens and adjacent normal renal tissues were calculated by using the CellQuest program (Becton Dickinson). Immunohistochemical analyses of paraffin sections obtained from the same RCC patients were performed as described previously (19) using anti-EGFR Ab (Cell Signaling Technology; 1:100).

Assays for Cell Cycle Progression and Apoptosis.
Cell cycle analyses and apoptosis assays were performed by flow cytometry and/or fragmented DNA ELISA as described previously (16 , 18) . In cell cycle analyses, fragmented apoptotic nuclei are recognized by their subdiploid (sub-G1) DNA content. All of the experiments were performed at least three times in triplicate.

Assay of Tumor Growth in Athymic Nude Mice.
Athymic nude mice (3–4-week-old) were maintained in a laminar air-flow cabinet under aseptic conditions. The care and treatment of experimental animals were in accordance with institutional guidelines. Human RCC cells (1 x 106) were injected s.c. into the right flank area of the mice. When tumor volume reached about 5 mm in diameter (day 0), mice were randomized into four groups: (a) control, 200 µl of Tween 80 on days 1–15 by mouth; (b) ZD1839 (100 mg/kg on days 1–15) by mouth; (c) paclitaxel i.v. (2 mg/kg on days 1, 8, and 15); and (d) paclitaxel in combination with ZD1839. Tumor volume was determined by direct measurement with calipers and was calculated as follows: ({pi}/6) x (large diameter) x (small diameter)2.

Statistical Analysis.
The statistical analysis was performed using a paired t test or an unpaired t test. Ps of less than 0.05 were regarded as statistically significant.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
EGFR Is Overexpressed in RCC.
We first evaluated EGFR expression in eight RCC lines. Western blot analyses showed that EGFR protein was expressed and phosphorylated at Tyr1068 in all RCC lines at a level equivalent to that in a lung cancer cell line, A549, used as the positive control (Fig. 1A)Citation Citation . We also confirmed by flow cytometry that EGFR was highly expressed at the cell surface of RCC lines and A549 cells (Fig. 1B)Citation Citation . We next investigated the difference of EGFR expression between RCC tissues and adjacent normal renal tissues in 10 patients with RCC who underwent radical nephrectomy. Immunohistochemical analyses revealed that EGFR expression in RCC tissues was much higher than that in adjacent normal renal tissues, and similar results were obtained by flow cytometry (Fig. 1C)Citation Citation . Flow cytometry for evaluating EGFR expression in surgical specimens showed that the mean value of median fluorescent intensities in 10 RCC tissues was much higher than that in normal renal tissues (P = 0.0007; Fig, 1DCitation Citation ). These results suggest that EGFR is overexpressed in RCC.



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  		Fig. 1. Epidermal growth factor receptor (EGFR) is overexpressed in renal cell carcinoma (RCC). A, RCC cell lysates were analyzed by Western blot analysis using specific antibodies (Abs) as described in the "Materials and Methods"; kDa, Mr in thousands. B, EGFR expression in RCC lines and A549 cells was detected by flow cytometry.

 


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  		Fig. 1A. Continued. C and D, EGFR expression in RCC surgical specimens was detected by immunohistochemistry and flow cytometry. Representative immunostaining of RCC (T) and adjacent normal renal tissues (N) using H&E (HE, reduced from x100) and EGFR Abs (reduced from x100) and a higher magnification of N and T using EGFR Ab (reduced from x400) are shown in C. Note that EGFR is overexpressed in RCC tissues compared with normal renal tissues (right top panel). Higher magnification (middle panels) and flow cytometry data (bottom panels) show that EGFR is overexpressed at the cell surface of RCC. The mean ± SD of median fluorescent intensities from 10 RCC tissue samples and adjacent normal renal tissue stained with the FITC-conjugated anti-EGFR Ab were calculated and compared. The data are shown as values relative to those from samples stained with the control Ab set to 10 (D). Experiments were repeated three times with similar results. kDa, Mr in thousands.

 
ZD1839 Specifically Blocks Phosphorylation of EGFR in SKRC-49 Cells.
Recent studies suggest that ZD1839 blocks the phosphorylation of HER-2, one of the EGFR families, as well as of EGFR (20, 21, 22) . As shown in Fig. 2ACitation , treatment of SKRC-49 grown in serum-free medium overnight with increasing amounts of EGF for 20 min resulted in a marked increase in phosphorylation of both EGFR and HER-2. We, therefore, sought to examine the specific effects of ZD1839 on the phosphorylation of EGFR in SKRC-49 cells. As shown in Fig. 2BCitation , the addition of 1 µM ZD1839 markedly inhibited EGFR phosphorylation, whereas >5 µM was required to block HER-2 phosphorylation. These results suggest that 1 µM ZD1839 is specific for EGFR in SKRC-49 cells.



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  		Fig. 2. ZD1839 specifically blocks phosphorylation of epidermal growth factor receptor (EGFR; ErbB1) in SKRC-49 cells. A, SKRC-49 cells in serum-free medium were treated with indicated concentrations of epidermal growth factor (EGF) for 20 min, and cell lysates were analyzed by Western blot analysis using specific antibodies; kDa, Mr in thousands. B, SKRC-49 cells in regular medium were treated with indicated concentrations of ZD1839 for 20 min, and cell lysates were analyzed by Western blot analysis.

 
ZD1839 Fails to Induce Apoptosis by Itself but Promotes Paclitaxel-Induced Apoptosis in RCC Cells in Vitro.
Recent studies suggest that ZD1839 is able to induce apoptosis in a variety of cancer cells (14 , 23) and that it synergistically promotes cytotoxic effects induced by conventional chemotherapeutic agents such as paclitaxel in various cancers (14 , 15) . We, therefore, examined the effect of ZD1839 with or without paclitaxel on apoptosis induction in RCC lines. Flow cytometric analyses showed that treatment of SKRC-49 cells with 1 µM ZD1839 in combination with 5 nM paclitaxel for 48 h resulted in a significant increase in sub-G1 DNA content compared with paclitaxel alone (P < 0.05), whereas treatment with ZD1839 alone failed to produce an increase in sub-G1 DNA content (Fig. 3A)Citation . These results correlated well with those obtained by fragmented DNA ELISA (Fig. 3B)Citation . Similar results were obtained using 769P and ACHN cells, whereas the combination therapy had no synergistic apoptosis-inducing effect on the normal renal proximal tubular epithelial cells (Fig. 3B)Citation . These results suggest that ZD1839 fails to induce apoptosis by itself but promotes paclitaxel-induced apoptosis predominantly in RCC cells.



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  		Fig. 3. ZD1839 fails to induce apoptosis by itself but promotes paclitaxel-induced apoptosis in renal cell carcinoma (RCC) cells in vitro. RCC cells were treated with the complex (ZD1839, paclitaxel) indicated for 48 h, and cell cycle progression and apoptotic cell death were analyzed by flow cytometry (A) and/or fragmented DNA ELISA (B) as described in the "Materials and Methods." Experiments were repeated three times with similar results. Bars, ±SD. The data of DNA flow cytometry histograms are representative of three independent experiments using SKRC-49 cells and percentage apoptosis (sub-G1 DNA content; M1) is indicated for each sample.

 
ZD1839 in Combination with Paclitaxel Blocks SKRC-49 Tumor Regrowth in Vivo.
Wakeling et al. (24) recently have provided evidence that the continuous administration of ZD1839 is required for blocking tumor regrowth in vivo. Unlike recent reports (14 , 23) , we have shown that ZD1839 alone fails to promote apoptosis in RCC lines in vitro. These findings suggest that ZD1839 alone is cytostatic but not lethal to RCC. To confirm this in vivo, we studied the effect of ZD1839 with or without paclitaxel on RCC tumor proliferation using SKRC-49 tumor xenografts. As shown in Fig. 4Citation , treatment with ZD1839 or paclitaxel alone produced a measurable growth delay in SKRC-49 xenografts, compared with the untreated control, within 4 weeks after treatment (P < 0.05), but these tumors subsequently regrew on the withdrawal of each agent. In contrast, synergistic antitumor efficacy was observed when paclitaxel was combined with ZD1839, resulting in a significant tumor regrowth delay (P < 0.01), compared with each single treatment in SKRC-49 xenografts, within 7 weeks after treatment. Treatment of ZD1839 with or without paclitaxel was well tolerated, as determined by the maintenance of body weight (data not shown). These results suggest that ZD1839 in combination with paclitaxel is able to block RCC regrowth in vivo.



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  		Fig. 4. ZD1839 (Z) in combination with paclitaxel (P) blocks SKRC-49 tumor regrowth in vivo. SKRC-49 (1 x 106) was injected s.c. into the right flank of athymic mice, and mice were treated with ZD1839 p.o. and/or paclitaxel i.v. as described in the "Materials and Methods." A, representative of each group 4 weeks and 7 weeks after treatment. Arrows, SKRC-49 xenografts. Tumor volume was measured as described in the "Materials and Methods." B, values represent mean tumor size (mm3; n = 8 or 10/group); bars, ±SD. Experiments were repeated twice with similar results.

 
ZD1839 Blocks Paclitaxel-Induced Activation of the EGFR-ERK Antiapoptotic Pathway.
It has recently been reported that various chemotherapeutic agents activate the ERK antiapoptotic pathway, which blocks apoptosis induced by chemotherapeutic agents (9, 10, 11) . Furthermore, Benhar et al. (12) reported that chemotherapeutic agents were capable of inducing EGFR activation. These findings suggest that EGFR-mediated activation of the ERK pathway in response to chemotherapy may be responsible for the antagonistic effect observed on the administration of chemotherapy in cancer cells. We, therefore, studied the effect of paclitaxel on the activation of EGFR and ERK in various cell lines. As shown in Fig. 5ACitation , treatment with 5 nM paclitaxel for 12 h resulted in a marked increase in the phosphorylation of EGFR and ERK in SKRC-49 but not in normal renal proximal tubular epithelial cells. We obtained similar results using prostate cell lines PC-3 and PrEC (Fig. 5A)Citation , suggesting that paclitaxel-induced phosphorylation of EGFR and ERK occurs predominantly in cancer cells. Next, we sought to investigate whether paclitaxel-induced ERK activation influenced the antiapoptosis against paclitaxel. Fragmented DNA ELISA showed that the specific MAPK/ERK kinase inhibitor U0126 (10 µM) significantly promoted paclitaxel-induced apoptosis in SKRC-49 and in PC-3 cells (Fig. 5B)Citation . These results suggest that paclitaxel-induced ERK activation contributes to the inhibition of apoptosis induced by paclitaxel in cancer cells. To exclude the possibility that ERK might be activated independent of EGFR activation, we further evaluated whether paclitaxel-induced ERK activation resulted from paclitaxel-induced EGFR activation. As shown in Fig. 5CCitation , treatment of SKRC-49 cells with 1 µM ZD1839 resulted in a marked decrease in the basal and paclitaxel-induced phosphorylation of both EGFR and ERK, suggesting that paclitaxel-induced ERK activation is caused by paclitaxel-induced EGFR activation in SKRC-49 cells. In contrast, ZD1839 failed to decrease the phosphorylation of Akt, which is also well known as the downstream effector of EGFR and as one of the major kinases related to chemoresistance (25 , 26) . We found that ZD1839 promoted paclitaxel-induced phosphorylation and down-regulation of Bcl-2 protein (Fig. 5C)Citation , which plays a crucial role in the control of sensitivity of ZD1839 and/or paclitaxel by regulating mitochondrial signaling (27 , 28) , whereas ZD1839 alone had no effect on Bcl-2 protein expression. We finally studied whether paclitaxel-induced activation of the ERK pathway influenced the Bcl-2 signaling in SKRC-49 cells. Western blot analysis showed that U0126 (1–10 µM) significantly decreased paclitaxel-induced ERK phosphorylation concomitant with a decrease in Bcl-2 protein expression, whereas the specific phosphatidylinositol 3-kinase-Akt inhibitor LY294002 (1–10 µM) failed to promote Bcl-2 down-regulation induced by paclitaxel. Taken together, these results strongly suggest that ZD1839 promotes paclitaxel-induced Bcl-2 down-regulation and apoptosis by blocking paclitaxel-induced activation of the EGFR-ERK antiapoptotic pathway in RCC, which is independent of the Akt antiapoptotic pathway.



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  		Fig. 5. ZD1839 blocks paclitaxel-induced activation of the epidermal growth factor receptor (EGFR)–extracellular signal-regulated kinase (ERK) antiapoptotic pathway. A. renal and prostate cell lines were treated with or without 5 nM paclitaxel for 12 h, and cell lysates were analyzed by Western blot analysis using specific antibodies (Abs). B, renal and prostate cell lines were treated with the complex (paclitaxel, ZD1839, U0126, LY294002) indicated for 48 h and apoptotic cell death was analyzed by fragmented DNA ELISA. Bars, ±SD. Experiments were repeated three times with similar results. C and D, SKRC-49 cells were treated with the complex indicated for 12 h, and cell lysates were analyzed by Western blot analysis using specific Abs. Experiments were repeated twice with similar results.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Consistent with previous reports (4 , 29) , we demonstrated that EGFR was overexpressed in RCC surgical specimens compared with adjacent normal renal tissues. We showed that EGFR protein was expressed and phosphorylated in RCC lines as in the equivalent level in A549, one of the cell lines derived from non-small cell lung cancer patients for whom ZD1839 has been widely used with significant efficacy. Numerous groups reported that EGFR was expressed and activated in A549 (15 , 30 , 31) . We found in our preliminary study that EGFR was more than 10-fold phosphorylated after the addition of 10 ng/ml EGF, both in A549 and SKRC-49 cells (data not shown). EGFR overexpression has been implicated in rapid tumor growth and metastatic behavior in RCC (4 , 29) . These findings suggest that EGFR is one of the hopeful targets for the treatment of RCC as well as non-small cell lung cancer.

Recent studies reported that tumor cells expressing high levels of HER-2 and other EGFR-homologs were highly sensitive to ZD1839 (20, 21, 22) , suggesting the possibility that ZD1839 cross-reacts nonspecifically with HER2 and other EGFR-homologous kinases. In general, EGFR phosphorylation is specifically blocked by lower concentrations of ZD1839 compared with HER-2 phosphorylation (20) . We have also shown that more than 5 µM ZD1839 is required to block HER-2 phosphorylation in SKRC-49 cells, whereas 1 µM ZD1839 markedly inhibited EGFR phosphorylation. According to a review of ZD1839 by the Pharmaceuticals and Medical Evaluations Center (32) , maximal steady-state (trough) plasma levels of 5 µM ZD1839 have been achieved in nude mice when >200 mg/kg ZD1839 was administered p.o. once a day for 14 days. It is, therefore, likely that the antiproliferative effects achieved by treatment with 100 mg/kg ZD1839 results from the specific EGFR blockade in RCC. However, several reports have shown that tumor cells expressing high levels of HER-2 are highly sensitive to ZD1839 (20 , 21) . We, therefore, consider the possibility that the effect of ZD1839 on HER-2 might be minimal because SKRC-49 cells do not express high levels of HER-2. Our present interest is whether the coexpression of HER receptors and their functional interaction with EGFR in cancer cells might dictate the tumor progression as well as the true antitumor effect of ZD1839 in RCC.

We have recently reported (16) that the induction of aneuploid G1 cells, which has been implicated in drug sensitivity for microtubule-stabilizing agents like paclitaxel, is observed in SKRC-49 but not in renal proximal tubular epithelial cells treated with 5 nM paclitaxel. However, our study in vivo demonstrated that SKRC-49 tumors regrew on the withdrawal of paclitaxel, which had a transient antitumor effect, suggesting that low concentrations of paclitaxel as a single agent may not be lethal to RCC. We obtained similar results using ZD1839 alone both in vitro and in vivo, which supports a recent study by Wakeling et al. (24) showing that the continuous administration of ZD1839 is required for blocking tumor regrowth. In contrast, the combination therapy presented here has the great advantage of augmenting the lethal killing of RCC because synergistic and continuous antitumor effects were observed even after the withdrawal of these agents.

We have shown that paclitaxel-induced activation of the EGFR-ERK antiapoptotic pathway occurs predominantly in cancer cells. We observed in the preliminary study that at least 6–8 h incubation with paclitaxel was required for the activation of EGFR and ERK, which was not influenced by treatment with EGFR blocking Ab (data not shown), suggesting that paclitaxel-induced EGFR activation does not require its ligand as EGF does, as several researchers reported (10 , 11 , 33) . We speculate that cancer cells but not normal cells possess unique mechanisms for activating EGFR in a ligand-independent fashion in response to paclitaxel, which may contribute to the drug-resistance in cancer cells, although further study will be needed to clarify the details.

Recent studies suggest that Akt protects cancer cells from apoptosis induced by anticancer therapies (25 , 26) . However, our results presented here suggest that ERK but not Akt is involved in the mechanism by which ZD1839 exerts its chemosensitizing effects. The implication of ERK activation in the response to cytotoxic drugs such as paclitaxel has been well documented by many groups (9, 10, 11) . However, most of their reports lack the mechanical approach needed to investigate whether ERK activation is mainly mediated by EGFR activation, to exclude the possibility that ERK is activated by factors other than the EGFR pathway; those reports also fail to connect their findings with the mechanism by which ZD1839 exerts its chemosensitizing effects. In the present study, we demonstrated that ERK activation in response to paclitaxel was predominantly mediated by EGFR activation induced by paclitaxel itself as Benhar et al. (12) showed in their recent report that chemotherapeutic agents such as cisplatin activated EGFR. The inhibition of ERK and the potentiation of cytotoxic drugs that are produced by ZD1839 have also been well documented by other groups (27 , 34) . They have reported the implication of the basal EGFR-ERK activation in the inhibition of apoptosis induced by cytotoxic drugs. However, we showed that ZD1389 alone failed to down-regulate Bcl-2 protein expression and that neither ZD1389 nor U0126 promoted apoptosis despite their inhibitory effect on constitutively active ERK. Our findings provide the insight that paclitaxel-induced EGFR-ERK activation, rather than the basal EGFR-ERK activation, contributes to the chemoresistance in cancer cells. We believe that our findings are more reasonable than similar results by others for explaining why ZD1839 synergistically, not additively, exerts its chemosensitizing effects.

In summary, we have shown that ZD1839 is able to modulate paclitaxel response in RCC both in vitro and in vivo. Furthermore, we have clarified a part of the mechanism by which ZD1839 exerts its chemosensitizing effects in cancer cells. ZD1839 is able to promote paclitaxel-induced cell death in cancer cells predominantly by blocking paclitaxel-induced activation of the EGFR-ERK antiapoptotic pathway. In our ongoing study, similar results were obtained using cisplatin instead of paclitaxel.1 Our findings may provide the clue to clarify one of the mechanisms for the drug-resistance in cancer cells and support the idea that the significant clinical benefit is obtained from ZD1839 in combination with a variety of widely used chemotherapeutic agents.


   ACKNOWLEDGMENTS
 
We thank AstraZeneca for kindly providing us with ZD1839 for experimental studies.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Makoto Sumitomo, Department of Urology, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama 359-8513, Japan. Phone: 81-(42)-995-1676; Fax: 81-(42)-996-5210; E-mail: mh712{at}me.ndmc.ac.jp

1 Unpublished data. Back

Received 6/17/03; revised 9/18/03; accepted 9/24/03.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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