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[Cancer Research 63, 1365-1370, March 15, 2003]
© 2003 American Association for Cancer Research

Molecular Biology and Genetics

Selective Akt Inactivation and Tumor Necrosis Factor-related Apoptosis-inducing Ligand Sensitization of Renal Cancer Cells by Low Concentrations of Paclitaxel

Junichi Asakuma, Makoto Sumitomo1, Takako Asano, Tomohiko Asano and Masamichi Hayakawa

Department of Urology, National Defense Medical College, Tokorozawa, Saitama 359-8513, Japan


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Recent studies demonstrated that the resistance of cancer cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) could be reversed by various chemotherapeutic agents. In the present study, we investigated the role of Akt in the apoptosis resistance to TRAIL and chemotherapeutic agents-induced TRAIL sensitization in human renal cell carcinoma cells. Apoptosis assays and Western blot analyses revealed that apoptosis resistance to TRAIL correlates well with the level of Akt phosphorylation at Ser 473 rather than protein expression levels of TRAIL receptors, DR4 and DR5. The apoptosis sensitivity to TRAIL in TRAIL-resistant SKRC-49 and TRAIL-sensitive Caki-1 cells was altered by modulation of Akt activity, which increased the protein expression of cellular FADD-like IL-1ß-converting enzyme-like inhibitory protein (cFLIP). Paclitaxel (5 and 100 nM) and cisplatin (10 µM) but not etoposide (1 and 10 µM) promoted TRAIL-induced apoptosis in SKRC-49 cells, which was not mediated by increased TRAIL receptor expression but by chemotherapeutic agents-induced Akt inactivation through ceramide formation derived from sphingomyelin hydrolysis. Of note, the low concentration (5 nM) of paclitaxel promoted ceramide formation and TRAIL-induced apoptosis predominantly in SKRC-49 cells but not in the normal renal proximal tubular epithelial cells. Our results may provide a novel therapeutic modality for selective killing of renal cell carcinoma with minimal toxicity on normal renal cells.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
TRAIL2 , also known as Apo-2 ligand, is a proapoptotic cytokine that is capable of inducing apoptosis in a wide variety of cancer cells but not in normal tissues (1 , 2) . However, despite the ubiquitous expression of TRAIL receptors, a significant proportion of cell lines originating from various cancer types demonstrate either partial or complete resistance to the proapoptotic effects of TRAIL. Recent studies demonstrated that the resistance of cancer cells to TRAIL could be reversed by various chemotherapeutic agents such as paclitaxel and CDDP, which increased the protein expression of two closely related TRAIL receptors, DR4 and DR5, which is able to decrease the threshold of TRAIL-sensitivity (3, 4, 5) . However, other recent reports in which chemotherapeutic agents failed to increase the expression of TRAIL receptors despite the augmentation of TRAIL sensitivity (6 , 7) suggest that alternative mechanisms may be involved in chemotherapeutic agents-enhanced TRAIL cytotoxicity.

Akt/protein kinase B is well known as a major serine-threonine kinase, which phosphorylates various signaling molecules, including the Bcl-2 family member Bad and caspase-9, and plays an important role in the cell survival in various cell types (8 , 9) . Recent studies reported that Akt protected cancer cells from apoptosis induced by anticancer therapies (10 , 11) . Akt activation has also been implicated in TRAIL resistance (12 , 13) . Importantly, quite recent studies demonstrated that chemotherapeutic agents such as topotecan and paclitaxel were capable of inactivating Akt (14 , 15) . These findings lead us to hypothesize that chemotherapeutic agents target Akt to augment TRAIL-induced apoptosis.

In disseminated RCC, novel therapeutic modalities need to be established, because conventional chemotherapy, radiation therapy, and immunotherapy have been tried with limited efficacy. Moreover, chemotherapeutic agents such as CDDP are toxic to normal renal tubular cells, which might limit the clinical use of the combination of them with TRAIL. In the present study, we first studied the role of Akt in the apoptosis resistance to TRAIL in seven established RCC lines. We found that the apoptosis-sensitivity to TRAIL in TRAIL-resistant SKRC-49 and TRAIL-sensitive Caki-1 cells was altered by modulation of Akt activity. We next demonstrated that paclitaxel and CDDP could promote TRAIL-induced apoptosis in SKRC-49 cells by Akt inactivation through Cer formation derived from SM hydrolysis. Furthermore, we present the novel finding that low concentrations of paclitaxel can induce Cer formation and TRAIL sensitization selectively in SKRC-49 cells but not in normal renal tubular cells.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Reagents.
Seven RCC lines were maintained in DMEM or MEM media supplemented with 2 mM glutamine, 1% nonessential amino acids, 100 units/ml streptomycin and penicillin, and 10% FCS. The human renal proximal tubular epithelial cells RPTEC (Clonetics, Walkersville, MD) were cultured as directed. Paclitaxel and etoposide were provided from Bristol-Myers Squibb KK (Tokyo, Japan), and CDDP was provided from Nippon Kayaku Co., LTD. (Tokyo, Japan). Various inhibitors were purchased from Calbiochem-Novabiochem Corp. (La Jolla, CA), and MeSM was from BIOMOL Research Laboratories, Inc. (Plymouth Meeting, PA).

Immunoblotting and Akt Kinase Assay.
Total cell lysates were prepared with radioimmunoprecipitation assay buffer, and immunoblotting was performed as described previously (16) using anti-Akt (Santa-Cruz Biotechnology Inc., Santa Cruz, CA; 1:500), anti-phospho Ser 473-Akt, anticaspase-8 (Cell Signaling Technology, Inc., Beverly, MA; 1:1000), anti-DR4 (BD Biosciences, San Jose, CA), anti-DR5, anti-DcR2 (Stressgene Biotechnologies, Victoria, British Columbia, Canada; 1:1000), anti-DcR1 (Chemicon International, Inc., Temecula, CA; 1:1000), anti-FLAG (M2; Sigma-Aldrich, St. Louis, MO; 1:100), and anti-cFLIP (R&D Systems, Inc., Minneapolis, MN; 1:1000). To monitor the loading equality of each lane, membranes were also immunoblotted with anti-actin (Chemicon International, Inc.; 1:3000) for total cell lysates. Akt kinase assays were performed using the manufacturer’s recommendations (Cell Signaling Technology, Inc.). Briefly, total cell lysates (300 µg) were allowed to immunoprecipitate for 3 h at 4°C with anti-Akt Ab. After washing immunoprecipitates, kinase reaction was performed for 30 min at 30°C in kinase buffer supplemented with 200 µM ATP and 1 µg GSK-3{alpha}/ß fusion protein. The samples were heated at 100°C for 5 min and loaded into a 12% acrylamide gel. Membranes were immunoblotted with anti-phospho Ser 21/9 GSK-3{alpha}/ß and detected using Phototope-horseradish peroxidase Western Detection kit (Cell Signaling Technology, Inc.).

Overexpression of Constitutively Active Akt and Dominant-negative (DN)-Type of Akt in RCC Cells.
Replication-deficient adenovirus vectors for constitutively active Akt (Ax-MyrAkt) and FLAG-tagged DN-type of Akt (Ax-AktAA), and a control vector for LacZ (Ax-LacZ) were constructed and transfected as described previously (17 , 18) . Briefly, subconfluent RCC cells were infected with adenovirus vectors at different MOIs. and the cells were incubated in the infection media (MEM with 2% L-glutamine and 100 µg/ml streptomycin) for 3 h. After removing the virus, cells were additionally incubated for specific time periods.

Cell Cycle Analyses, Apoptosis Assays, and Cell Growth Assays.
Cell cycle analyses and apoptosis assays were performed by flow cytometry and/or fragmented DNA ELISA as described previously (17 , 19) . 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 duplicate. The results obtained by fragmented DNA ELISA correlated well with those obtained by cell cycle analyses (data not shown). Cell growth assays were performed using a Coulter Counter ZM (Coulter Electronics, Hialeah, FL) as described previously (16 , 20) .

Cer Quantitation.
Intracellular Cer level was determined after metabolic labeling with [14C]serine as described previously (19) . Briefly, RCC cells (1 x 106/ml) were incubated for 24 h with [14C]serine (0.2 µCi/ml) in MEM medium containing 0.3% FCS, washed with PBS, and then treated for specific time periods. After cells were detached with trypsin, lipids were extracted and spotted on Silica Gel 60 TLC plates (Whatman KK, Tokyo, Japan), and developed. The intensity of each Cer band was measured relative to total radioactivity in phosphatidylethanolamine, which remained unaltered on stimulation and was expressed as a relative value to each control set to 1.

Statistical Analysis.
The statistical analysis was performed using an unpaired t test. Ps < 0.05 were regarded as statistically significant.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Correlation of Apoptosis Resistance to TRAIL with Akt Activation in RCC Cells.
We first examined the apoptosis sensitivity to TRAIL in seven RCC lines. Cell cycle analyses showed that 100 ng/ml TRAIL treatment for 24 h resulted in a significant increase in sub-G1 DNA content in ACHN, A498, and Caki-1 cells compared with untreated controls (P < 0.01), but not in other RCC cell lines (Fig. 1A)Citation . Western blot analyses showed little correlation between the protein expression levels of TRAIL receptors and TRAIL-induced apoptosis in RCC cells, although TRAIL-sensitive A498 cells expressed very high level of DR5 (Fig. 1B)Citation . None of the RCC lines expressed detectable levels of DcR1 as reported previously (data not shown; Ref. 5 ). On the other hand, a marked correlation was recognized between apoptosis resistance to TRAIL and the levels of Akt phosphorylation at Ser 473 in total cell lysates from RCC cell lines (Fig. 1CCitation , top and middle panels). The Akt phosphorylation level of each TRAIL-resistant cell line was nearly equal to that of the prostate cancer LNCaP cells in which Akt is constitutively activated because of PTEN mutation. We then performed Akt kinase assay using GSK-3{alpha}/ß as a substrate for Akt. The results showed that the level of Akt phosphorylation at Ser 473 correlated well with Akt kinase activity in RCC cells (Fig. 1CCitation , top and bottom panels). These results suggest that apoptosis resistance to TRAIL correlates well with the level of Akt phosphorylation at Ser 473, i.e., Akt activation rather than protein expression levels of TRAIL receptors.



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  		Fig. 1. Correlation of apoptosis resistance to TRAIL with Akt activation in RCC cells. A, RCC cells were treated with or without 100 ng/ml TRAIL for 24 h. Sub-G1 DNA content in each sample was measured by cell cycle analysis. Bars, ±SD. B and C, RCC cell lysates were analyzed by Western blot analysis and Akt kinase assay as described in "Materials and Methods." The prostate cancer LNCaP cells were used as positive controls. Experiments were repeated three times with similar results.

 
Activated Akt Is Required for Apoptosis Resistance to TRAIL in RCC Cells.
We next examined whether modulation of Akt activity could alter the sensitivity of RCC cells to TRAIL using Ax-AktAA, Ax-MyrAkt, and the specific inhibitor of phosphatidylinositol 3'-kinase-Akt pathway, Wortmannin. Western blot analysis using an anti-FLAG Ab revealed that DN-type Akt was highly expressed in SKRC-49 cells 24 h after Ax-AktAA transfection at concentrations of >=5 MOI, and treatment with Wortmannin decreased Akt phosphorylation in a dose-dependent fashion (Fig. 2A)Citation . After Ax-AktAA (10 MOI) transfection, culturing of TRAIL-resistant SKRC-49 cells in medium containing 100 ng/ml TRAIL for 24 h resulted in ~4-fold increase in sub-G1 DNA content compared with TRAIL treatment after Ax-LacZ transfection (P < 0.01; Fig. 2BCitation ). Similarly, pretreatment of SKRC-49 cells with Wortmannin augmented TRAIL-induced apoptosis in a dose-dependent fashion (Fig. 2B)Citation . We found that treatment with Wortmannin itself for 24 h failed to promote apoptosis in SKRC-49 cells, although Akt was markedly dephosphorylated (data not shown), suggesting that Akt inactivation itself has little effect on apoptosis in SKRC-49 cells at least within 24 h. We additionally studied whether constitutively active Akt overexpression by Ax-MyrAkt transfection could inhibit TRAIL-induced apoptosis in TRAIL-sensitive Caki-1 cells. Western blot analysis showed that Akt was highly expressed and phosphorylated in Caki-1 cells 24 h after Ax-MyrAkt transfection at concentrations of >=5 MOI (Fig. 2C)Citation . After Ax-MyrAkt (5 MOI) transfection, culturing of Caki-1 cells in medium containing 100 ng/ml of TRAIL for 48 h resulted in a significant decrease in sub-G1 DNA content compared with TRAIL treatment after Ax-LacZ transfection (P < 0.01; Fig. 2DCitation ). These results suggest that activated Akt is required for apoptosis resistance to TRAIL in RCC cells.



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  		Fig. 2. Activated Akt is required for apoptosis resistance to TRAIL in RCC cells. A, SKRC-49 cells, infected with Ax-LacZ or Ax-AktAA at different MOIs (top panel), or treated with increased concentrations of Wortmannin (bottom panels), are incubated for 24 h. Cell lysates were analyzed using anti-FLAG Ab (top panel) or anti-Akt Abs (bottom panels). B, SKRC-49 cells, infected with Ax-LacZ or Ax-AktAA at 10 MOI, or treated with increased concentrations of Wortmannin, were incubated with 100 ng/ml TRAIL for 24 h, and apoptosis was determined as described in Fig. 1. C and DCitation , Caki-1 cells, infected with Ax-MyrAkt or Ax-LacZ at different MOIs, were incubated for 24 h. Cell lysates were analyzed using anti-Akt Abs (C) and apoptosis was determined (D) as described above. Bars, ±SD. Experiments were repeated three times with similar results.

 
Increased cFLIP Expression by Akt Is One of the Major Mechanisms Underlying TRAIL Resistance.
Caspase-8 expression and its processing (activation) has been implicated in TRAIL-induced apoptosis (21) . Therefore, we studied whether caspase-8 protein expression correlated with TRAIL sensitivity in RCC cells. Western blot analysis revealed that the protein expression levels of caspase-8 were variable in RCC cells and did not correlate well with TRAIL sensitivity (Fig. 3A)Citation . However, we found that caspase-8 activation levels induced by TRAIL correlated well with TRAIL sensitivity in RCC cells (data not shown). Recent studies suggest that Akt up-regulates the caspase-8 inhibitory protein cFLIP, which is able to block TRAIL-induced caspase-8 activation and apoptosis (22 , 23) . Therefore, we examined whether Akt activation level and TRAIL resistance correlated with cFLIP protein expression. Western blot analysis revealed high levels of cFLIP protein expression in TRAIL-resistant 769P and SKRC-49 cells, and low levels in TRAIL-sensitive Caki-1 and A498 cells, although there was not a clear correlation recognized between Akt activation levels and cFLIP protein expression levels in other RCC cell lines (Fig. 3A)Citation . In addition, as shown in Fig. 3BCitation , cFLIP protein expression levels were altered by modulation of Akt activity in Caki-1 and SKRC-49 cells. These results suggest that increased cFLIP protein expression by Akt is one of the major mechanisms underlying TRAIL resistance in RCC cells, although other antiapoptotic factors regulated by Akt might be involved in TRAIL resistance.



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  		Fig. 3. Increased cFLIP expression by Akt is one of the major mechanisms underlying TRAIL resistance. A, RCC cell lysates were analyzed by Western blot analysis using specific Abs. B, Caki-1 cells infected with Ax-MyrAkt (top panels) and SKRC-49 cells infected with Ax-AktAA (bottom panels) at different MOIs were incubated for 24 h. Cell lysates were analyzed using specific Abs. Experiments were repeated three times with similar results.

 
Paclitaxel and CDDP Inactivate Akt and Augment TRAIL-induced Apoptosis in TRAIL-resistant SKRC-49 Cells.
We studied whether several chemotherapeutic agents could alter the TRAIL sensitivity of SKRC-49 cells. As shown in Fig. 4ACitation , synergistic increase in sub-G1 DNA content was recognized in SKRC-49 cells treated with 100 ng/ml of TRAIL combined with paclitaxel (5 and 100 nM) or CDDP (10 µM) for 24 h, whereas etoposide (1 and 10 µM) or 1 µM CDDP failed to augment TRAIL-induced apoptosis. Recent studies demonstrated that chemotherapeutic agents such as paclitaxel were capable of inactivating Akt (14 , 15) . Therefore, we investigated whether chemotherapeutic agents can modulate Akt activity in RCC cells. Western blot analysis showed that treatment of SKRC-49 cells in the medium including paclitaxel (5 and 100 nM) or CDDP (10 µM) for 24 h resulted in a marked decrease in Akt phosphorylation levels, whereas etoposide (1 and 10 µM) or 1 µM CDDP failed (Fig. 4B)Citation . The protein expression of DR4 or DR5 was not altered by treatment with each chemotherapeutic agent (Fig. 4B)Citation . In SKRC-49 cells, Akt phosphorylation was stably inhibited 12–24 h after treatment with 5 nM paclitaxel, whereas the protein expression of DR4 or DR5 was not altered (Fig. 4C)Citation . These results strongly suggest that chemotherapeutic agents-induced TRAIL sensitization is predominantly dependent on modulation of the Akt activity but not on the increase in the expression of TRAIL receptors in SKRC-49 cells.



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  		Fig. 4. Paclitaxel and CDDP inactivate Akt and augment TRAIL-induced apoptosis in TRAIL-resistant SKRC-49 cells. A, SKRC-49 cells were treated with the complex indicated for 24 h, and apoptosis was determined as described in Fig. 1Citation . Bars, ±SD. Experiments were repeated three times with similar results. B, SKRC-49 cells were treated with chemotherapeutic agents at indicated concentrations for 24 h, and cell lysates were analyzed using specific Abs. C, SKRC-49 cells were treated with 5 nM paclitaxel for various times periods, and cell lysates were analyzed using specific Abs. Experiments were repeated three times with similar results.

 
Cer Formation Mediates Chemotherapeutic Agents-induced Akt Inactivation and TRAIL Sensitization.
Cer formation has been implicated in the apoptosis signaling in response to chemotherapeutic agents (19 , 24) . Furthermore, Schubert et al. (25) reported that Cer could affect Akt signaling by dephosphorylation of Ser 473. To evaluate the importance of Cer formation in Akt inactivation and TRAIL sensitization induced by chemotherapeutic agents, we first measured intracellular Cer levels in SKRC-49 cells treated by chemotherapeutic agents. As shown in Fig. 5ACitation , treatment of SKRC-49 cells with paclitaxel (5 and 100 nM) or 10 µM CDDP for 12–24 h resulted in >1.5-fold increase in Cer formation (P < 0.05). In contrast, 10 µM etoposide or 1 µM CDDP failed to increase Cer formation. We next studied whether Cer could dephosphorylate Akt in SKRC-49 cells using a Cer analogue C2-Cer. As shown in Fig. 5BCitation , the level of Akt phosphorylation was decreased by C2-Cer in a dose-dependent fashion. It is well known that intracellular Cer formation by chemotherapeutic agents is derived mainly from de novo Cer synthesis pathway and/or nSMase (19 , 26) . Therefore, we investigated whether increased Cer by paclitaxel affects the Akt phosphorylation level and TRAIL sensitization using a de novo Cer synthesis inhibitor FB-1 and nSMase inhibitor MeSM. Western blot analysis revealed that 10 µM MeSM treatment of SKRC-49 cells for 24 h markedly reversed 5 nM paclitaxel-induced Akt dephosphorylation, whereas 10 µM FB-1 failed to reverse it (Fig. 5C)Citation . Consistent with these results, MeSM but not FB-1 significantly reversed paclitaxel-induced TRAIL sensitization in a dose-dependent fashion (Fig. 5D)Citation . Similar results were obtained by the treatment with 10 µM CDDP (data not shown). Together, these results suggest that Cer formation via SM hydrolysis mediates chemotherapeutic agents-induced Akt inactivation and TRAIL sensitization in SKRC-49 cells.



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  		Fig. 5. Cer formation mediates chemotherapeutic agents-induced Akt inactivation and TRAIL sensitization. A, SKRC-49 cells were treated with chemotherapeutic agents at indicated concentrations for 12 and 24 h, and Cer levels were determined as described in "Materials and Methods." The data are expressed as fold increase relative to each untreated control. Bars, ± SD. Experiments were repeated three times with similar results. B, SKRC-49 cells were treated with C2-Cer at various concentrations for 24 h, and cell lysates were analyzed using Akt Abs. Experiments were repeated three times with similar results. C and D, SKRC-49 cells were treated with the complex indicated for 24 h, and cell lysates were analyzed using Akt Abs (C) and apoptosis was determined (D) as described in Fig. 1Citation . Bars, ±SD. Experiments were repeated at least twice with similar results.

 
Low Concentrations of Paclitaxel Can Induce Selective Cer Formation, Akt Inactivation, and TRAIL Sensitization in SKRC-49 but not in Normal RPTEC.
Although numerous studies like ours have shown that chemotherapeutic agents augment TRAIL sensitivity, there seems to be a lack in evaluation on the cytotoxicity to normal cells by combined treatments. For example, CDDP is well known to induce nephrotoxicity. Therefore, we compared the Cer formation effect by paclitaxel and CDDP between SKRC-49 and normal RPTEC. Surprisingly, treatment with 5 nM paclitaxel for 24 h resulted in a significant increase in Cer formation in SKRC-49 cells compared with that in RPTEC where intracellular Cer was little increased (P < 0.05; Fig. 6ACitation ). On the other hand, treatment with 100 nM paclitaxel or 10 µM CDDP resulted in a significant increase in Cer formation compared with untreated controls in both cell lines (P < 0.05), and Cer increase levels between these two cell lines were not significantly different. Next, we studied the levels of Akt phosphorylation and TRAIL-receptor expression in SKRC-49 and RPTEC treated with paclitaxel or CDDP. Western blot analysis showed that treatment with 5 nM paclitaxel for 24 h had little effect on the levels of Akt phosphorylation in RPTEC cells where 100 nM paclitaxel or 10 µM CDDP markedly dephosphorylate Akt (Fig. 6B)Citation . The protein expression levels of DR4 and DR5 in RPTEC were nearly equal to those in SKRC-49, whereas RPTEC expressed higher levels of DcR2 protein than SKRC-49 cells, and the protein expressions of these TRAIL receptors were not altered by treatment with paclitaxel or CDDP (Fig. 6B)Citation . We next studied the effect of chemotherapeutic agents combined with TRAIL on the apoptosis induction in RPTEC by cell-cycle analyses. Twenty-four h after the treatment with 5 nM paclitaxel, a large population of aneuploid cells, close to the G1 peak, was observed in SKRC-49, whereas G2-M block was observed in RPTEC. In contrast, treatment with 100 nM paclitaxel or 10 µM CDDP resulted in a marked increase in sub-G1 and G2-M DNA content in both cell lines (Fig. 6C)Citation . The combination of 5 nM paclitaxel with 100 ng/ml TRAIL resulted in a significant increase in sub-G1 DNA content in SKRC-49 but not in RPTEC. On the other hand, 100 nM paclitaxel or 10 µM CDDP promoted TRAIL-induced apoptosis in RPTEC as well as SKRC-49 (Fig. 6C)Citation . We have obtained similar results using 769P cells instead of SKRC-49 cells (data not shown). Consistent with these findings, the cell growth in SKRC-49 cells treated with 5 nM paclitaxel was significantly inhibited compared with that in the untreated SKRC-49 or paclitaxel-treated RPTEC (P < 0.01; Fig. 6DCitation ), which was additionally promoted by the combination of TRAIL (P < 0.05 to paclitaxel-treated SKRC-49 and P < 0.01 to RPTEC treated with paclitaxel and TRAIL). On the other hand, the growth-inhibitory level was not significantly different between these cell lines treated with 100 nM paclitaxel or 10 µM CDDP with or without TRAIL. Taken together, these results strongly suggest the possibility that the combination of low concentrations of paclitaxel with TRAIL promotes apoptosis selectively in renal cancer cells with limited toxic effect in normal renal tubular cells.



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  		Fig. 6. Low concentrations of paclitaxel can induce selective Cer formation, Akt inactivation, and TRAIL sensitization in SKRC-49 but not in normal RPTEC. A and B, SKRC-49 and RPTEC were treated with paclitaxel or CDDP at indicated concentrations for 24 h, and Cer levels were determined (A) and cell lysates were analyzed using specific Abs (B). The Cer data are expressed as fold increase relative to each untreated control. C and D, SKRC-49 and RPTEC were treated with paclitaxel or CDDP at indicated concentrations with or without 100 ng/ml TRAIL for 24 h. Cell cycle analyses (C) and cell growth assays (D) were performed. The data of DNA flow cytometry histograms are representative of three experiments. Percentage apoptosis (sub-G1 DNA content; M1) is indicated for each sample. Bars, ±SD. Experiments were repeated twice with similar results.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Consistent with a previous study (5) , we found that the sensitivity to TRAIL was variable in RCC lines. Our investigation revealed that the sensitivity to TRAIL did not correlate well with TRAIL receptor expression. Instead, we found that Akt was highly phosphorylated in TRAIL-resistant RCC cells. Furthermore, we demonstrated that the TRAIL-resistant SKRC-49 could be converted to TRAIL-sensitive by overexpression of DN-type Akt, and TRAIL-sensitive Caki-1 could be converted to TRAIL-resistant by overexpression of constitutively active Akt, indicating that Akt activation plays a crucial role in protection against the apoptotic effects of TRAIL in RCC.

Several intracellular inhibitors of apoptosis have been identified that regulate TRAIL receptor-mediated cell death. For example, high expression of cFLIP has been implicated in TRAIL resistance (22 , 23) . Griffith et al. (5) observed high levels of the inhibitor of apoptosis protein (IAP) family member survivin in the TRAIL-resistant RCC line 786-O. Although we have demonstrated that modulation of Akt activity alters cFLIP protein expression in Caki-1 and SKRC-49 cells, the clear correlation between TRAIL resistance and cFLIP expression levels was not recognized in some RCC lines. Thus, it is likely that the mechanisms for TRAIL resistance are complicated. However, Akt locates upstream of several apoptosis-inhibitory proteins including cFLIP and survivin (22 , 27) . Therefore, it is reasonable to suppose that Akt plays a central role in TRAIL resistance by modulating the expression and/or activity of multiple TRAIL-resistant factors in RCC.

We have shown that the TRAIL-resistant SKRC-49 cells were converted to TRAIL-sensitive by treatment with paclitaxel and CDDP. In the present study, TRAIL sensitization by paclitaxel and CDDP was independent of the increase in the protein expression of DR4 or DR5 as reported previously (6 , 7) . We demonstrated that paclitaxel and CDDP markedly dephosphorylated Akt, and that the levels of Akt dephosphorylation induced by chemotherapeutic agents correlated well with those of TRAIL sensitization, strongly suggesting that TRAIL sensitization is predominantly caused by Akt inactivation. Furthermore, we have shown that Cer formation mediates Akt inactivation and TRAIL sensitization induced by chemotherapeutic agents in SKRC-49 cells. Our study is the first report to demonstrate that the Cer-Akt pathway is a key mediator for chemotherapeutic agents-induced TRAIL sensitization.

Notably, we demonstrated that low concentrations of paclitaxel induced Cer formation, Akt inactivation, and TRAIL sensitization in SKRC-49 but not in normal RPTEC. Unlike a recent study (5) , our study showed that the protein expression levels of DR4 and DR5 in RPTEC were similar to those in SKRC-49, and that 100 nM paclitaxel or 10 µM CDDP promoted TRAIL-induced apoptosis in RPTEC as well as SKRC-49. By contrast, our present and ongoing study have demonstrated that 5 nM paclitaxel treatment results in a decrease in Akt phosphorylation in 769P cells as well as SKRC-49 cells, but not in primary cultured normal renal tubular cells or normal small-airway epithelial cells.3 Thus, the combination of low concentrations of paclitaxel with TRAIL may be widely useful for the treatment of disseminated RCCs with pulmonary metastases without toxic effects on normal tissues.

The mechanism for the selective Cer formation followed by Akt inactivation in response to low concentrations of paclitaxel in SKRC-49 cells but not in RPTEC cells remains unclear. Although we have shown that Cer formation induced by 5 nM paclitaxel is derived from SM hydrolysis but is not dependent on de novo Cer synthesis pathway, we found in the preliminary study that the de novo Cer synthesis pathway is required for Cer formation induced by 100 nM paclitaxel (data not shown). These findings suggest that the mechanism for Cer formation induced by low concentrations of paclitaxel may be different from that by high concentrations of paclitaxel. In relation to this possibility, it is of interest that the induction of aneuploid G1 cells, which has been implicated in drug sensitivity for microtubule-stabilizing agents like paclitaxel (28 , 29) , was observed in SKRC-49 but not in RPTEC treated with low concentrations of paclitaxel. Unlike high concentrations of paclitaxel, low concentrations of paclitaxel stabilize microtubule dynamics without affecting the overall architecture of the microtubule cytoskeleton (30) , and increase aneuploid G1 cells resulted from aberrant mitosis in the absence of mitotic block (29 , 31 , 32) . Although additional study will be needed to clarify these mechanisms, Cer formation as well as aberrant mitosis by low concentrations of paclitaxel may be selectively induced in cancer cells lacking in G2-M checkpoint as reported recently (33) .

It is apparent from our findings that TRAIL resistance is overcome by adjuvant treatment with low concentrations of paclitaxel, which can inactivate Akt, a critical determinant for RCC sensitivity to TRAIL. Although one may think that low concentrations of paclitaxel alone may be enough for the treatment of RCC cells because they may selectively induce growth inhibition and cell death in RCC as presented, we put special emphasis on the observation that treatment with low concentrations of paclitaxel for longer periods causes SKRC-49 cells to change to the paclitaxel-resistant phenotype (data not shown). In this regard, the combination therapy presented here has a great advantage to additional augment the selective and lethal killing of RCC cells.


   ACKNOWLEDGMENTS
 
We thank Dr. Wataru Ogawa (the Second Department of Internal Medicine, Kobe University School of Medicine, Kobe, Japan) for kindly providing Ax-MyrAkt and Ax-AktAA.


   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.

1 To whom requests for reprints should be addressed, at 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 Back

2 The abbreviations used are: TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; RCC, renal cell carcinoma; CDDP, cisplatin; cFLIP, cellular FADD-like IL-1ß-converting enzyme-like inhibitory protein; Cer, ceramide; SM, sphingomyelin; FB-1, fumonisin B1; MeSM, 3-O-methyl-sphingomyelin; Ab, antibody; GSK-3{alpha}/ß, glycogen synthase kinase-3{alpha}/ß; DN, dominant-negative; MOI, multiplicity of infection; nSMase, neutral sphingomyelinase. Back

3 Unpublished observations. Back

Received 8/ 6/02. Accepted 1/17/03.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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