Abstract
Background/Aim: Advanced renal cell carcinoma is treated with mammalian target of rapamycin (mTOR) inhibitors or tyrosine kinase inhibitors (TKIs). The effects of these drugs are, however, limited and novel treatment strategies are required. Clear-cell type renal cell carcinoma (ccRCC) is chemo-resistant, in part, due to expression of multidrug resistance proteins such as p-glycoprotein. Cabazitaxel, a tubulin-binding taxane drug used for castration-resistant prostate cancer, has less affinity for p-glycoprotein compared to docetaxel. In the current study, the effects of docetaxel and cabazitaxel on ccRCC cells were investigated. Materials and Methods: The expression of p-glycoprotein was evaluated in the ccRCC cell lines, Caki-1, KMRC-1 and OS-RC-2 by western blotting. Cells were treated with cabazitaxel or docetaxel, and growth kinetics and tubulin polymerization were determined by the WST-1 assay and cell-based tubulin polymerization assay, respectively. Intracellular drug concentrations were measured by chromatography. AKT activation after treatment was examined by western blotting. Results: All ccRCC cell lines expressed p-glycoprotein. Cabazitaxel inhibited cell growth and induced tubulin polymerization more potently than docetaxel. The intracellular concentration of cabazitaxel was much higher than docetaxel in all cell lines. Both docetaxel and cabazitaxel inhibit AKT phosphorylation at 5 min among three cells. Conclusion: Cabazitaxel inhibits growth of ccRCC cells expressing p-glycoprotein and could thus be possibly used for advanced ccRCC patients in combination with targeted-therapy enhancing their effects.
Renal cell carcinoma is one of the most lethal malignant tumors, whose most common pathological type is clear-cell type renal cell carcinoma (ccRCC). Metastatic and advanced renal cancer remains largely incurable. Molecular targeted therapies such as mammalian target of rapamycin (mTOR) inhibitors and tyrosine kinase inhibitors (TKIs) have been used to inhibit the PI3K/ AKT /mTOR pathway and improve outcomes for metastatic ccRCC (1, 2). However, currently approved therapies have limited success and the cure remains rare because of multiple genetic heterogeneity and/or emergence of resistant cells (3, 4, 5). Thus, novel therapeutic approaches to the treatment of metastatic and advanced ccRCC are required (6, 7).
ccRCC has been thought to be a chemotherapy-resistant cancer (8). One of the mechanisms responsible for resistance of ccRCC is increased expression of p-glycoprotein (also known as multidrug resistance protein 1, MDR1) (8-14). P-glycoprotein is one of the drug efflux pumps to transport pharmacological compounds from inside to outside of the cells and its overproduction results in resistance to a wide range of structurally unrelated anticancer agents. Among 60 cell lines of the National Cancer Institute, RCC cell lines were found to express the highest levels of MDR1 mRNA (10). Dan et al. also showed that renal cancer cells were resistant to taxanes such as paclitaxel and docetaxel (11).
Paclitaxel, the first taxane available for clinical use, has been used to treat ovarian, breast, lung cancer, Kaposi's sarcoma etc. Previous studies have revealed that paclitaxel is one of the major p-glycoprotein substrates (10, 12). Despite significant responses in cell-based experiments, clinical trials of chemotherapy in combination with p-glycoprotein-blockers that enhance intracellular accumulation of the drugs did not show significant antitumor effects for RCC patients (8, 10, 15).
Cabazitaxel, a dimethoxy derivative of docetaxel, was shown to exert cytotoxic effects in docetaxel-resistant cell lines due to its lower affinity for p-glycoprotein (16, 17). Recent studies have demonstrated that treatment with cabazitaxel, but not with mitoxantrone, produced survival benefits to metastatic castration-resistant prostate cancer patients who previously received docetaxel chemotherapy (18). In the present study, we hypothesized that cabazitaxel could be an effective treatment for ccRCC expressing p-glycoprotein because of its intracellular accumulation due to its low affinity for p-glycoprotein. We investigated the effects of cabazitaxel and docetaxel on ccRCC cell lines and also examined if AKT inactivation is involved in their effects.
Multiple reaction monitoring (MRM) chromatograms of blank cell lysate spiked with cabazitaxel, docetaxel and docetaxel-d9 (IS), each at 5 ng/mL
Materials and Methods
Tumor cell lines. Human ccRCC cell lines Caki-1 (JCRB0801) established by Fogh, J. and KMRC-1 (JCRB1010) established by Ashida, S. obtained from the JCRB Cell Bank (Osaka, Japan) were maintained in D-MEM (Wako Pure Chemical Industries, Osaka Japan) containing penicillin, streptomycin and 10% fetal bovine serum (Equitech-Bio, Kerrville, TX). Human OS-RC-2 (RCB0735) cell line that was established by Kinouchi, T. was provided by the RIKEN BRC through the National Bio-Resource Project of the MEXT, Japan and maintained in RPMI1640 containing the aforementioned antibiotics and serum.
Collision energy and MRM transitions for the three analytes and internal standard.
Chemicals. Docetaxel and cabazitaxel were purchased from Selleckchem (Houston, TX, USA). Both docetaxel and cabazitaxel for cell-based assay was dissolved in DMSO to obtain 1 mg/ml stock solutions and stored at −20°C. Stock solutions were diluted in medium for each experiment. Docetaxel-d9 was purchased from Toronto Research Chemicals (North York, ON, Canada).
Cytotoxicity assay. WST-1 assay was performed as previously described (19). Briefly, cells were seeded at a density of 5×102 cells in 96-well plates in each complete medium. Twenty-four hours after seeding, cells were treated with docetaxel or cabazitaxel. Cell growth inhibition was determined at 72 h (Caki-1 and KMRC-1) or 96 h (OS-RC-2) after treatment using the WST-1 assay kit (Roche Diagnostics, Mannheim, Germany).
IC50 for docetaxel and cabazitaxel in ccRCC cell lines.
Western blotting. Cells (3×104) were seeded onto 6 well-plates and treated with drugs. Cells were prepared as previously reported (20). Membranes were incubated overnight at 4°C with following primary antibodies: mouse anti-p-glycoprotein antibody (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-β-actin antibody, rabbit anti-AKT antibody and rabbit anti-phospho-AKT antibody (Cell Signaling Technology, Danvers, MA). The immunoreactive bands were detected with horseradish peroxidase-conjugated anti-rabbit and -mouse IgG (GE Healthcare, Buckinghamshire, UK) and the ECL detection system (GE Healthcare).
Cell-based tubulin polymerization assay. Cell-based tubulin polymerization assay was performed according to the method described in our previous report with minor modifications (21). Briefly, cells (3×104) were seeded onto 6 well-plates and treated with 1 nM docetaxel or cabazitaxel for 90 min. Cell lysates prepared in a low salt buffer (20 mM Tris-HCl, pH 6.8, 1 mM MgCl2, 2 mM EGTA, 0.5% NP-40, protease inhibitor cocktail) were centrifuged at 20,000×g for 30 min. The supernatant was used as a soluble tubulin fraction. The pellet was sonicated in RIPA buffer and used as a polymerized tubulin fraction. The soluble and polymerized fractions (1.5 and 3.0 μg respectively) from the same aliquot of cell lysates were used for Western blotting with rabbit anti-α-tubulin antibody (Cell Signaling Technology).
Chromatographic analysis. Intracellular and extracellular drug concentration was measured following a previous report with minor modifications (22). Briefly, cells (5×105) were seeded onto 55 cm2 dish and treated with 5 nM of docetaxel or cabazitaxel for 24 hrs. At 24 h, cells were collected with distilled water and sonicated. Drug concentration of both docetaxel and cabaxitaxel were measured by validated liquid chromatography/tandem mass spectrometry (LC-MS/MS) methods using the multiple reaction monitoring mode. Standard stock solutions of the two drugs and internal standard (IS) were individually prepared at 100 μg/mL in methanol in polypropylene tubes. Docetaxel-d9, deuterated docetaxel, was used as an IS for chromatography. IS (20 μL; 125 ng / mL) was added to cell lysate (10 μL) or culture medium (500 μL) that had been spiked with calibration standards or experiment samples in polypropylene tubes. After mixing samples, tert-butyl methyl ether (2 mL) was added, and mixed vigorously for 60 sec on a vortex mixer following centrifugation at 2,300 g for 10 min at ambient temperature. Supernatants (1.8 mL) were transferred to polypropylene tubes and dried in nitrogen gas. Samples were reconstituted by mixing with mobile phase (50 μL). Then samples were filtrated with 0.2-μm pore-membrane filter (Millex-LG, 4 mm i.d. disk, Merck-Millipore, Darmstadt, Germany) and transferred to 200-μL polypropylene autosampler vials. Samples (10 μL) were injected onto the LC instrument for quantitative analysis using an autosampler operating at 4°C. XBridge Shield RP18 3.5 μm 2.1×50 mm (Waters, Milford, MA, USA) and XBridge Guard columns Shield RP18 2.1×10 mm (Waters) were used for the chromatographic separation of the drugs using a Nexera X2 UHPLC system (Shimadzu, Kyoto, Japan). The column was heated to 40°C, and the autosampler temperature was set at 4°C. The injection volume for all samples was 10 μL. The mobile phase was a 70:30 (v/v) mixture of 1 mM ammonium hydroxide in methanol and 10 mM aqueous ammonium hydroxide (pH 10.5), and the flow rate was 0.2 mL/min. MS was performed using the electrospray ionization positive ion mode on an LCMS8040 system (Shimadzu). The ion spray needle was maintained at 4.5 kV. The turbo gas temperature was 400°C. The collision energy and multiple reaction monitoring (MRM) transitions for the two analytes and IS are shown in Table I. The dwell time was 100 msec. The representative spiked chromatogram was shown in Figure 1.
The concentration of docetaxel and cabazitaxel within cells (nmol/g) of ccRCC cell lines and in the medium.
Results
ccRCC cells express p-glycoprotein. Expression of p-glycoprotein in both human tissues and cell lines of renal cell carcinoma and correlation of chemo-resistance with expression of p-glycoprotein has been previously reported (9, 11, 12). To examine p-glycoprotein expression in ccRCC cell lines, we performed western blot analysis. All three cell lines expressed p-glycoprotein with a molecular weight of approximately 150 kDa (Figure 2A). The expression level of p-glycoprotein was much higher in Caki-1 and KMRC-1 cells than in OS-RC-2 cells (Figure 2A).
Cabazitaxel inhibits ccRCC cell growth. To examine the cytotoxic effect of docetaxel and cabazitaxel on ccRCC cells, cells were treated under various concentrations (0-10 nM) of each agent for 72 h (Caki-1 and KMRC-1) and 96 h (OS-RC-2) and then WST-1 assay was performed. Cell viability was lower in cells treated with cabazitaxel than in those treated with docetaxel (Figure 2B-D). The half-maximal inhibitory concentrations (IC50) for docetaxel and cabazitaxel were 4.48 nM and 0.48 nM in Caki-1 cells, 3.46 nM and 0.43 nM in KMRC-1 cells and 3.55 nM and 0.50 nM in OS-RC-2 cells, respectively (Table II). In OS-RC-2 cells, 96 h treatment was required to determine the IC50. These results indicate that cabazitaxel inhibits ccRCC cell growth more potently than docetaxel.
(A) The p-glycoprotein expression levels in ccRCC cells. Total protein was extracted from Caki-1, KMRC-1 and OCRC-2 cells and subjected to western blotting with anti-p-glycoprotein antibody. (B-D) Effects of docetaxel and cabazitaxel on ccRCC cells growth. Caki-1, KMRC-1 and OS-RC-2 were treated with various concentration of docetaxel and cabazitaxel. Cell viability was determined by WST-1 assay at 72 h (Caki-1 and KMRC-1) and 96 hrs (OS-RC-2) after treatment. Values represent means±SD (bars), n=5.
Tubulin polymerization induced by docetaxel and cabazitaxel. Caki-1, KMRC-1 and OS-RC-2 cells were treated with 1 nM of docetaxel and cabazitaxel for 90 min. The soluble and polymerized tubulin fractions were subjected to Western blotting for β-tubulin. Data are representative of two independent experiments.
Cabazitaxel induces stabilization of tubulin. Since both docetaxel and cabazitaxel are tubulin-stabilizing agents, we examined their effects on the level of polymerized tubulin. As shown in Figure 3, cabazitaxel (1 nM) induced more polymerized tubulin than docetaxel in all ccRCC cell lines. The level of soluble tubulin was decreased in KMRC-1 cells, concomitant with an increase in polymerized tubulin. These results suggest that the anti-proliferative effect of cabazitaxel was accompanied by an accumulation of polymerized tubulin.
Intracellular concentration of cabazitaxel is higher than docetaxel. Under the assumption that the difference in substrate affinity for p-glycoprotein may affect intracellular drug concentration, the concentrations of cabazitaxel and docetaxel in conditioned media and cell lysates were analyzed (Table III). Cells were treated with 5 nM of each drug because 1 nM was too low to determine intracellular concentrations by chromatography. Intracellular concentrations of cabazitaxel were higher than those of docetaxel in all ccRCC cell lines (Table III). In KMRC-1 cells, docetaxel was not detectable within cells and the concentration in conditioned media was highest among all three cell lines, suggesting that docetaxel was transported to the outside of cells effectively. The ratio of intracellular to extracellular concentration of docetaxel and cabazitaxel were 1.86 and 31.82 in Caki-1 cells, 0 and 61.10 in KMRC-1 cells and 22.08 and 166.43 in OS-RC-2 cells, respectively (Figure 4). Although variable among cell lines, the ratio of intracellular to extracellular concentration of cabazitaxel was much higher than that of docetaxel in all ccRCC cell lines.
Intracellular drug concentrations. Caki-1, KMRC-1 and OS-RC-2 cells were treated with 5 nM of docetaxel and cabazitaxel for 24 h. Cells were collected in distilled water. Drug concentrations within cells and conditioned media were analyzed as described in Materials and Methods.
Docetaxel and cabazitaxel inhibit AKT phosphorylation. Since mTOR inhibitors and TKIs target the PI3K/AKT/mTOR pathway, we examined the effects of docetaxel and cabazitaxel on AKT phosphorylation. Cells were treated with 10 nM docetaxel and cabazitaxel, at which concentration maximum cell growth inhibition occurred. The level of phosphorylated AKT significantly decreased 5 min after treatment with docetaxel and cabazitaxel, but returned to normal and remained unchanged 24 h after, in all ccRCC cell lines (Figure 5). Ten nanomolar of docetaxel and cabazitaxel inhibit cell growth strongly, suggesting that the AKT pathway, at least in part, was involved in taxane-induced ccRCC growth inhibition.
Discussion
P-glycoprotein correlates with chemo-resistance in various human cancer types including ccRCC. Alvarez et al. have reported that increased expression of MDR-1 gene encoding p-glycoprotein in human renal cell lines resulting in a high correlation with resistance to a large number of compounds (10). We previously reported that p-glycoprotein was increased in cells and exosomes derived from a docetaxel-resistant prostate cancer cell line and also in exosomes isolated from serum of patients with docetaxel-resistant prostate cancer. We concluded that up-regulation of p-glycoprotein in exosomes could be a diagnostic marker for docetaxel-resistance (17). Similarly to previous reports, our current experiment showed that p-glycoprotein was strongly expressed in ccRCC cell lines, Caki-1, KMRC-1 and OS-RC-2, but the expression levels varied among cell lines.
AKT phosphorylation after docetaxel and cabazitaxel treatment. Cell lysates were harvested at indicated times after treatment with 10 nM docetaxel or cabazitaxel and subjected to western blotting with anti-p-AKT and anti-AKT antibodies. Data are representative of two independent experiments.
In the present study, the IC50 for docetaxel in ccRCC cell lines ranged from 3.46 to 4.48 nM. Although OS-RC-2 cells expressed the lowest level of p-glycoprotein in those three cell lines, 96 h incubation were required to determine the IC50, suggesting its lowest sensitivity to both drugs among them. This discrepancy may originate from cancer cell types or other factors. In addition to p-glycoprotein expression, several mechanisms for resistance to docetaxel have been proposed including mutated p53 and Bax, overexpressed class βIII-tubulin, clusterin and protease-activated receptor 1. The results presented here suggest that p-glycoprotein is not the sole determinant for docetaxel-resistance and thus further investigation will be required to dissect the underlying mechanisms.
Cabazitaxel, a dimethoxy derivative of docetaxel, is a third-generation taxane, which was developed to overcome resistance to docetaxel-resistant cancers. Nowadays, cabazitaxel is used for patients previously treated with a docetaxel containing regimen (18). Docetaxel binds to the β-tubulin subunit of the α/β-tubulin heterodimer, which inhibits tubulin depolymerization and blocks mitosis, resulting in apoptosis. Our results suggest that cabazitaxel induces tubulin polymerization and inhibits cell proliferation. This is consistent with previous reports for other cancer cell lines.
It has been reported that cabazitaxel has less affinity to p-glycoprotein compared to docetaxel. Intracellular drug concentration analysis confirmed a striking difference of drug accumulation in ccRCC cells. Although cells were treated with 5 nM of each agent, their concentrations in conditioned media were much lower than expected. This difference may be ascribed to metabolism or adsorption to flasks of drugs. Nevertheless, it is of a great interest to note that a significant amount of cabazitaxel was accumulated within ccRCC cells that exhibited growth inhibition.
There exists accumulating evidence suggesting that tyrosine kinases involving the AKT signaling pathway play an important role in cancer progression. Several drugs that target and inhibit the function succeeded in ccRCC treatment. The AKT signaling pathway also plays a key role in cancer cell survival via modulating extracellular growth factors (23, 24). McCarroll et al. reported that βIII-tubulin that reduces taxane binding promotes non-small lung cancer growth via PTEN/AKT signaling activation (25). In order to study the involvement of AKT signal transduction in taxane treatment of ccRCC cells, AKT phosphorylation after treatment with docetaxel and cabazitaxel was analyzed. Our study revealed that the level of AKT phosphorylation decreased 5 min after treatment with both cabazitaxel and docetaxel, but returned to normal and remained unchanged until 24 h. This result indicates that AKT inactivation may be involved in docetaxel and cabazitaxel-induced growth inhibition in ccRCC cells. Previous reports have also demonstrated that the relationship of AKT activation and docetaxel efficacy in other cancer cell lines (26-29), that reminds us that the combination use of targeted therapy and cabazitaxel potentially represents more effective regimen for advanced ccRCC. A limitation of the present study is the lack of animal and clinical studies. Nevertheless, our result demonstrated that cabazitaxel inhibits cell growth of ccRCC cell lines. A clinical investigation will be necessary to determine the efficacy of cabazitaxel for advanced ccRCC patients with or without targeted therapy.
- Received August 17, 2015.
- Revision received September 18, 2015.
- Accepted September 23, 2015.
- Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
