MCO Molecular and Clinical Oncology 2049-9450 2049-9469 D.A. Spandidos MCO-23-5-02896 10.3892/mco.2025.2896 Articles Prolonged exposure to axitinib alters the molecular profile of Caki-2 renal cell carcinoma cells NakayamaYuko 1 InoAya 2 YamamotoKazuhiro 3 TakaraKohji 2 Department of Clinical Pharmaceutics, Faculty of Pharmaceutical Sciences, Himeji Dokkyo University, Himeji, Hyogo 670-8524, Japan Department of Pharmaceutics and Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, Hyogo Medical University, Kobe, Hyogo 650-8530, Japan Department of Integrated Clinical and Basic Pharmaceutical Sciences, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama 700-8558, Japan Correspondence to: Professor Kohji Takara, Department of Pharmaceutics and Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, Hyogo Medical University, 1-3-6 Minatojima, Chuo, Kobe, Hyogo 650-8530, Japan ko-takara@hyo-med.ac.jp 112025 22092025 23 5 101 13 05 2025 05 09 2025 Copyright: © 2025 Nakayama et al. 2025 This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

Axitinib, an oral second-generation multitargeted tyrosine kinase inhibitor, is used as a second-line treatment for metastatic renal cell carcinoma (RCC). However, patients often develop resistance after initial responsiveness, necessitating the elucidation of the underlying resistance mechanisms. Therefore, the present study aimed to investigate the mechanisms underlying axitinib resistance using the Caki-2 human papillary RCC model cells. Cells tolerating 0.1 µM axitinib were designated as Caki/AX cells. Cell viability was assessed using the water-soluble tetrazolium salt assay. Notably, the 50% inhibitory concentration (IC50) values of axitinib and sunitinib were significantly higher in Caki/AX cells than those in Caki-2 cells, indicating 2.83- and 1.2-fold resistance, respectively. By contrast, the IC50 values of sorafenib and erlotinib were decreased in Caki/AX cells. Moreover, Caki/AX cells showed resistance to everolimus, temsirolimus and rapamycin, and decreased sensitivity to vinblastine, vincristine, paclitaxel, doxorubicin and SN-38 compared with Caki-2 cells. Notably, etoposide, 5-fluorouracil, cisplatin and carboplatin sensitivities were comparable in both cell types. Reverse transcription-quantitative polymerase chain reaction (PCR) analysis revealed that the mRNA levels of the ATP-binding cassette subfamily B member 1 and subfamily G member 2 were significantly higher in Caki/AX cells than those in Caki-2 cells. A PCR array related to vascular endothelial growth factor signalling showed that the mRNA levels of FIGF (also known as vascular endothelial growth factor D) and sphingosine kinase 1 were upregulated, whereas those of Rac family small GTPase 2 were downregulated in Caki/AX cells. Overall, these findings suggested that the upregulation of the ATP-binding cassette subfamily B member 1, FIGF and sphingosine kinase 1 mRNA levels, and downregulation of the Rac family small GTPase 2 mRNA levels may contribute to acquired resistance in Caki/AX cells.

 axitinib renal cell carcinoma drug resistance ABC transporter Funding: No funding was received. Introduction

Renal cell carcinoma (RCC) is among the most resistant variety of cancer showing resistance to conventional cytotoxic chemotherapy. Cytokine therapy was the standard treatment for RCC until 2006. However, development of various molecular-targeted drugs, such as vascular endothelial growth factor (VEGF) receptor (VEGFR) tyrosine kinase inhibitors (TKIs) and mammalian target of rapamycin (mTOR) inhibitors, significantly improved metastatic RCC treatment (1-3).

Axitinib, an oral second-generation multitargeted TKI targeting VEGFR-1, -2, and -3 was approved by the US Food and Drug Administration in 2012 (4-6). Currently, it is used as a second-line treatment for metastatic RCC (7-9); however, information on its third-line or later treatment use remains scarce. Approximately one-third of patients with RCC exhibit TKI resistance in clinical trials (10). Drug resistance can develop in initially responsive patients typically one year after treatment, thereby complicating advanced RCC management with TKIs (11). Therefore, understanding the mechanism underlying axitinib in the second-line and later settings is important for effective treatment.

Targeted therapy resistance is of two types: intrinsic and acquired. Intrinsic resistance refers to the immediate ineffectiveness of therapeutic agents, often due to pre-existing resistant tumour clones formed via inherited resistance or evolutionary clonal selection. In contrast, acquired resistance is observed when tumours regrow following initial regression despite continued therapy. Although the precise mechanisms of resistance to targeted therapies remain unclear, both laboratory and clinical studies have identified several factors contributing to intrinsic and acquired resistance (12).

We previously established everolimus-resistant papillary RCC (PRCC) cells exhibiting cross-resistance to other mTOR inhibitors, decreased mTOR activity, and downregulated mRNA levels of DNA damage-inducible transcript 4 (DDIT4), DEP domain-containing mTOR-interacting protein (DEPTOR), hypoxia-inducible factor 1 subunit alpha (HIF1A), and phospholipase D1 (PLD1), which possibly contributed to everolimus resistance (13). mRNA levels of ATP-binding cassette (ABC) transporter ABCB1 mRNA are downregulated in everolimus-resistant PRCC cells after long-term everolimus exposure (14). However, effects of long-term exposure to axitinib remain unknown. Therefore, in this study, we aimed to elucidate axitinib resistance mechanisms using PRCC cells by generating axitinib-resistant PRCC cells and comparing their molecular characteristics with those of parental PRCC cells.

 Materials and methods <sec> <title>Chemicals

Axitinib, sunitinib, temsirolimus, and 5-fluorouracil were purchased from Sigma-Aldrich; Merck KGaA. Everolimus and rapamycin were obtained from Selleck Chemicals, LLC and LC Laboratories, respectively. Erlotinib, sorafenib, and SN-38 (active metabolite of irinotecan hydrochloride) were purchased from LKT Laboratories Inc. Carboplatin, cisplatin, doxorubicin hydrochloride, etoposide, paclitaxel, vinblastine sulphate, and vincristine sulphate were purchased from FUJIFILM Wako Pure Chemical Corp. Water-soluble tetrazolium salt (WST)-1 and 1-methoxy phenazinium methylsulphate (PMS) were obtained from Dojindo Laboratories.

 Cells and cell culture

Caki-2 cells (RRID:CVCL_0235; DS Pharma Biomedical) were used as human PRCC model cells (13-15). Short tandem repeat-polymerase chain reaction-PCR profiling using the PowerPlex 16 System (Promega Corp.) confirmed that the cell used in this study was the same as the cell registered in DSMZ (ACC-54 CAKI-2), and the cell registered in ATCC (HTB-47 Caki-2), by the comparison with the database of JCRB Cell Bank. The cells were subsequently cultured in the Roswell Park Memorial Institute (RPMI)-1640 medium (Invitrogen, Life Technologies) supplemented with 10% heat-inactivated foetal bovine serum (Invitrogen, Life Technologies) and 100 IU/ml penicillin + 100 µg/ml streptomycin (Invitrogen, Life Technologies) in a humidified atmosphere containing 95% air and 5% CO2 at 37˚C. The cells were sub-cultured every 3-4 d using 0.05% trypsin-0.02% ethylenediaminetetraacetic acid (Invitrogen, Life Technologies).

 Establishment of axitinib-resistant sublines

Clinically achievable plasma concentration of axitinib at 10 mg is approximately 30 ng/ml (equivalent to approximately 0.08 µM) (16-18). Caki-2 cells were cultured in RPMI-1640 medium supplemented with 0.1 µM axitinib. After three months, the cells tolerating 0.1 µM axitinib were isolated and cloned, and the selected clones were named as Caki/AX cells. Caki/AX cells were maintained under conditions similar to those used for Caki-2 cells, except that the medium contained 0.1 µM axitinib.

 Cell growth assay

Caki-2 and Caki/AX cell growth was evaluated using growth curves. On day 0, the cells (1,000 cells/well) were seeded in a 96-well plate in a culture medium without axitinib and counted from day 0 to 12. The cell counts were determined via WST-1 colorimetric assay based on the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, as previously described (13,19,20). Three hours after the addition of the WST-1 reagent solution, absorbance at 450 nm and a reference wavelength of 630 nm was determined using the Spectra Fluor microplate reader (Tecan Group, Ltd.), according to the manufacturer's instructions. Preliminary experiments revealed a proportional relationship between the absorbance and cell number. Log phase doubling time of Caki-2 and Caki/AX cells were calculated as previously described (13,19,20).

 Growth inhibition assay

Effects of molecular targeted and cytotoxic anticancer drugs on Caki-2 and Caki/AX cell growth were evaluated using the WST-1 assay as previously described (13,14,19-22). To examine the effects of molecular targeted drugs, the cells were initially seeded at a density of 500 cells/well in a 96-well plate without any drugs. After 24 h of pre-culture, the medium was replaced with a fresh medium containing various concentrations of the tested molecular targeted drugs. After 168 h of incubation, cell counts were determined via WST-1 assay.

To examine the effects of cytotoxic anticancer drugs, the cells were seeded at a density of 1,000 cells/well, and drug exposure time was 72 h. All other experimental conditions were identical to those described above. Subsequently, 50% inhibitory concentration (IC50) values of the tested drugs were estimated using the sigmoid inhibitory effect model, as previously described (13,14,19-22).

 Reverse transcription-quantitative PCR (RT-qPCR)

Next, mRNA expression levels of the ABC transporters, ABCB1 and ABCG2, were measured via RT-qPCR. Total RNA was extracted from Caki-2 and Caki/AX cells using the GenElute Mammalian Total RNA Miniprep kit (Sigma-Aldrich; Merck KGaA), and an aliquot (500 ng) was used for reverse transcription using the PrimeScript RT reagent kit (Takara Bio Inc.). Reverse transcription reaction was performed at 37˚C for 15 min and terminated via heating at 85˚C for 5 sec, followed by cooling at 4˚C.

Real-time PCR was performed using the 7500 Fast Real-Time PCR system (Applied Biosystems) and SYBR Premix Ex Taq (Takara Bio). PCR cycling conditions were as follows: 95˚C for 30 sec, followed by 40 cycles of 95˚C for 3 sec and 60˚C for 30 sec. Dissociation curve analysis was performed via heating at 95˚C for 15 sec followed by 60˚C for 1 min, and 95˚C for 15 sec. All PCR primers used in this study are listed in Table SI (13,14,20). Primers were synthesised by GeneDesign, Inc. β-actin was used as an internal standard. The comparative Cq method was used to determine the relative target mRNA levels (13,14,20,23,24).

 PCR array

PCR array was performed using the RT2 Profiler PCR Array (catalogue No. PAHS-091Z; Qiagen), as previously described (13). Total RNA was extracted as described above, and an aliquot (500 ng of total RNA) was used for reverse transcription with the RT2 First Strand kit (Qiagen), according to the manufacturer's instructions. Real-time PCR was performed using the 7500 Fast Real-Time PCR System (Applied Biosystems) and RT2 SYBR-Green Master Mix (Qiagen). PCR conditions were as follows: 95˚C for 10 min, followed by 40 cycles of 95˚C for 15 sec, and 60˚C for 1 min. Dissociation was initiated at 95˚C for 15 sec, followed by 60˚C for 1 min, and 95˚C for 15 sec. The data were analysed using the 2-ΔΔCq method (25).

 Statistical analyses

Two groups were compared using an unpaired Student's t-test with the JMP Pro 15.2.0. software (SAS Institute Japan Ltd.). P<0.05 (two-tailed) was considered to indicate a statistically significant difference.

 Results <sec> <title>Caki-2 and Caki/AX cell growth curves

Growth curves of Caki-2 and Caki/AX cells revealed a logarithmic phase that continued for at least six days after cell seeding (Fig. 1). The cell doubling time were approximately 24.2 and 24.4 h for Caki-2 and Caki/AX cells, respectively. Notably, growth rates were comparable in both cell types.

 Cell sensitivities to TKIs and mTOR inhibitors

Table I shows the IC50 values of the tested TKIs in Caki-2 and Caki/AX cells. IC50 value of axitinib was significantly high in Caki/AX cells, showing 2.83-fold resistance. Similarly, IC50 value of sunitinib was significantly higher in Caki/AX cells than in Caki-2 cells, with Caki/AX cells showing a 1.2-fold higher resistance than Caki-2 cells. In contrast, IC50 values of sorafenib and erlotinib were lower in Caki/AX cells than in Caki-2 cells; however, the difference was not significant.

IC50 values of the tested mTOR inhibitors in Caki-2 and Caki/AX cells are presented in Table II. Their IC50 values were higher in Caki/AX cells than in Caki-2 cells, with the relative resistance to mTOR inhibitors being approximately 10-fold.

 Cell sensitivities to cytotoxic anticancer drugs

IC50 values of the tested cytotoxic anticancer drugs in Caki-2 and Caki/AX cells are presented in Table III. Vinblastine, vincristine, paclitaxel, and doxorubicin sensitivities were lower in Caki/AX cells than in Caki-2 cells. SN-38 sensitivity was decreased, but not significantly, in Caki/AX cells. Notably, etoposide, 5-fluorouracil, cisplatin, and carboplatin sensitivities were comparable between Caki-2 and Caki/AX cells.

 ABCB1 and ABCG2 mRNA expression levels

ABCB1 and ABCG2 mRNA levels were significantly higher in Caki/AX cells than in Caki-2 cells (Fig. 2).

 PCR array

Next, mRNA levels of molecules associated with VEGF-related signalling pathways were analysed using a PCR array (Fig. 3). Volcano plot showed the mRNAs up- or downregulated in Caki/AX cells compared to those in Caki-2 cells. Notably, mRNA levels of FIGF (also known as VEGFD) and sphingosine kinase 1 (SPHK1) were upregulated ≥ 2-fold, whereas those of Rac family small GTPase 2 (RAC2) were downregulated >2-fold in Caki/AX cells (Table IV).

Additionally, mRNA levels of VEGFA (Fig. S1) and cadherin 1 (Fig. S2), which encodes the calcium-dependent cell-cell adhesion protein E-cadherin, were significantly lower in Caki/AX cells than in Caki-2 cells.

 Discussion

Target gene mutations reducing the drug affinity for target molecules are involved in TKI resistance mechanisms. Activation of bypass signalling pathways also contributes to TKI resistance. Other resistance mechanisms include lysosomal sequestration of TKIs, activation of angiogenic switches, and involvement of ABC transporters (12). However, the specific mechanisms underlying axitinib resistance remain unclear. Therefore, in this study, axitinib-resistant PRCC cells were generated and molecularly compared with their parental cells to elucidate the underlying resistance mechanisms.

Axitinib-resistant Caki/AX cells exhibited cell growth comparable to that of their parental Caki-2 cells, with equivalent doubling time of 24.2 and 24.4 h for Caki-2 and Caki/AX cells, respectively. Moreover, continuous exposure to axitinib did not affect cell growth, suggesting that changes in cell growth do not contribute to the development of drug resistance in Caki/AX cells.

Caki/AX cells exhibited significantly lower sensitivity to axitinib than Caki-2 cells, with approximately 3-fold resistance. This suggests that long-term exposure to 0.1 µM axitinib, equivalent to the plasma concentrations achieved with clinical dosing, induces resistance in these cells. Additionally, Caki/AX cells showed cross-resistance to sunitinib, but not sorafenib and erlotinib. They exhibited altered sensitivity to TKIs; however, the specific factors responsible for this could not be identified in this study. Sensitivity to mTOR inhibitors was also reduced in Caki/AX cells, indicating the development of cross-resistance to mTOR inhibitors.

Caki/AX cells were resistant to vinblastine, vincristine, paclitaxel, and doxorubicin. These cytotoxic anticancer drugs are substrates of ABCB1, also known as P-glycoprotein (19,21). ABCB1 mRNA levels were higher in Caki/AX cells than in Caki-2 cells, suggesting that drug resistance is induced by ABCB1 mRNA upregulation. Axitinib and sunitinib also act as ABCB1 substrates (26-29); therefore, resistance to these drugs was partially due to the upregulation of ABCB1 mRNA levels in this study. Furthermore, moderate resistance to SN-38, an active irinotecan metabolite, was observed, possibly due to ABCG2 mRNA upregulation because SN-38 is a substrate of ABCG2(22).

Volcano plot revealed increased FIGF and SPHK1 mRNA levels and decreased RAC2 mRNA levels in Caki/AX cells. FIGF encodes a c-fos-induced growth factor, also known as VEGFD. Lieu et al (30) reported increased VEGFD levels after bevacizumab chemotherapy, suggesting their association with bevacizumab chemotherapy resistance. Therefore, axitinib resistance may be partially due to the upregulation of FIGF levels, despite bevacizumab being a human monoclonal VEGFA-neutralising antibody. Here, mRNA levels of VEGFA and VEGFC were decreased in Caki/AX cells, indicating an association between reduced VEGF levels and axitinib resistance in Caki/AX cells.

SPHK1, encoded by SPHK1, acts as a proto-oncogenic factor synthesizing sphingosine-1 phosphate (S1P). Tumour cells often exhibit elevated levels of S1P and its receptor, S1PR1, which promotes drug resistance. Signalling through S1P via its receptor, S1PR1, facilitates cancer cell survival by activating anti-apoptotic pathways (31). Therefore, targeting S1P and its receptors can potentially inhibit cancer cell proliferation and overcome drug resistance (31). Bao et al (32) reported that SPHK1 overexpression is associated with RCC development and resistance to antiangiogenic agents. Elevated SPHK1 levels predicts poor outcomes and resistance to angiogenic agents in patients with RCC. These findings indicate the potential role of SPHK1 in axitinib resistance in Caki/AX cells.

RAC2 is a small GTPase contributing to B-cell receptor (BCR)-activated calcium mobilization via phospholipase Cγ2 (33-35). It acts as a regulator of cell adhesion, linking BCR signalling pathways to cellular adhesion processes (33-35). Its diverse functions are crucial for fundamental cellular physiological processes and immune responses (33,35). Shaffer et al (35) reported the involvement of RAC2 in the resistance to the Bruton tyrosine kinase inhibitor, ibrutinib. Wu et al (36) revealed that the reduction of RAC2 expression using RNA interference and clustered regularly interspaced palindromic repeat technology impairs cell adhesion and that its overexpression reverses ibrutinib-induced cell adhesion impairment. RNA-sequencing analysis has shown that ibrutinib-resistant cells exhibit higher RAC2 levels than their parental cells (35). RAC2 knockdown significantly reduces the levels of the signatures associated with the activated B-cell-diffuse large B-cell lymphoma identity, B-cell-specific genes repressed by B-lymphocyte-induced maturation protein-1, and genes induced by nuclear factor-κB, signal transducer and activator of transcription 3, and interferon regulatory factor 4. Although the direct involvement of RAC2 in axitinib resistance remains unclear, its downregulation possibly contributes to axitinib resistance.

Overall, this study showed that Caki/AX cells develop drug resistance via various mechanisms, including ABCB1 mRNA upregulation. Our findings suggest that the upregulation of FIGF and SPHK1 mRNA levels and downregulation of RAC2 mRNA levels contribute to the acquired axitinib resistance of Caki/AX cells. These changes align with mechanisms previously reported in the literature, and particularly, the involvement of FIGF and SPHK1 suggests the possibility of resistance acquisition via angiogenic and sphingolipid-related pathways. Specifically, the decreased RAC2 expression is a novel finding of this study with limited prior documentation, indicating a potential cell line-specific molecular alteration. This investigation is based on a single cell line and remains a preliminary exploration. However, other factors may also be involved in axitinib resistance, warranting further gene expression analyses via next-generation sequencing. Additionally, mRNA levels of cadherin 1 were significantly lower in Caki/AX cells than in Caki-2 cells, suggesting the induction of epithelial-mesenchymal transition, consistent with a previous report (37). This study used only a few cell lines in vitro, which possibly limits the generalisability and warrants the cautious interpretation of our findings. Therefore, further research incorporating additional models, including animal and clinical specimens, is necessary to fully validate and extend our observations.

 Supplementary Material Vascular endothelial growth factor mRNA levels in Caki-2 and Caki/AX cells. Relative target gene level is expressed as 2<sup> ΔCq</sup>. ΔCq was calculated by subtracting the Cq of the internal standard (β-actin) from that of the target gene. Open (□) and closed (■) bars indicate the Caki-2 and Caki/AX cells, respectively. Each bar represents the mean ± standard deviation (n=4). <sup>**</sup>P<0.01 (unpaired Student's t-test). Cadherin 1 mRNA levels in Caki-2 and Caki/AX cells. Relative target gene expression is expressed as 2<sup>-ΔCq</sup>. ΔCq was calculated by subtracting the Cq of the internal standard (β-actin) from that of the target gene. Open (□) and closed (■) bars indicate the Caki-2 and Caki/AX cells, respectively. Each bar represents the mean ± standard deviation (n=4). <sup>**</sup>P<0.01 (unpaired Student's t-test). Primer sequences for reverse transcription-quantitative polymerase chain reaction. Acknowledgements

Not applicable.

 Availability of data and materials

The data generated in the present study may be requested from the corresponding author.

 Authors' contributions

YN conceptualized the study, performed the investigation, compiled the data, generated the figures, and wrote the original draft. AI and KY generated the figures, collected and organized the data, and reviewed and edited the manuscript. KT conceptualized the study, curated all of the data, reviewed and edited the manuscript, and supervised the study. All authors read and approved the final manuscript.

 Ethics approval and consent to participate

Not applicable.

 Patient consent for publication

Not applicable.

 Competing interest

The authors declare that they have no competing interests.

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(A) Growth curves of Caki-2 and Caki/AX cells. Each cell line was seeded in a 96-well plate on day 0. Cell counts were determined using the water-soluble tetrazolium salt-1 assay from day 0 to 12. Symbols indicate the growth curves of Caki-2 (○) and Caki/AX (●) cells. Each point represents the mean ± standard deviation of values from 12 independent experiments, and error bars are included in the symbols. (B) Microscopic images of Caki-2 and Caki/AX cells.

ABCB1 (left) and ABCG2 (right) mRNA levels in Caki-2 and Caki/AX cells. Relative target gene expression is expressed as 2-ΔCq. ΔCq was calculated by subtracting the Cq of the internal standard (β-actin) from that of the target gene. Open (□) and closed (■) bars indicate the Caki-2 and Caki/AX cells, respectively. Each bar represents the mean ± standard deviation (n=4). **P<0.01 (unpaired Student's t-test). ABC, ATP-binding cassette.

Volcano plot of the vascular endothelial growth factor-related signalling polymerase chain reaction array results in Caki-2 and Caki/AX cells. Graph shows Caki-2 vs. Caki/AX cells, and fold-difference indicates the normalized gene expression (2-ΔCq) in Caki/AX cells divided by that (2-ΔCq) in Caki-2 cells. Black line indicates a fold-difference of 1 in gene expression. Dotted line indicates a two-fold change in gene expression. Dashed-dotted line indicates a P-value threshold of 0.05, determined via the Student's t-test.

Sensitivities of Caki-2 and Caki/AX cells to tyrosine kinase inhibitors.

  IC50 value, µM  
Drug Caki-2 Caki/AX R.R.
Axitinib 3.92±1.39 11.1±4.27a 2.83
Sorafenib 3.65±0.24 3.34±0.07a 0.92
Sunitinib 2.84±0.22 3.33±0.35a 1.17
Erlotinib 0.44±0.30 0.36±0.11 0.81

R.R. was obtained by dividing the IC50 value of Caki/AX cells by that of Caki-2 cells. Each value represents the mean ± standard deviation (n=8).

aP<0.01 significantly different from Caki-2 cells (unpaired Student's t-test). R.R., relative resistance; IC50, 50% inhibitory concentration.

Sensitivities of Caki-2 and Caki/AX cells to mammalian target of rapamycin inhibitors.

  IC50 value, nM  
Drug Caki-2 Caki/AX R.R.
Everolimus 52.6±51.0 653±620a 12.4
Temsirolimus 6.20±11.0 92.2±84.3a 14.9
Rapamycin 62.5±55.6 814±487b 13.0

R.R. was obtained by dividing the IC50 value of Caki/AX cells by that of Caki-2 cells. Each value represents the mean ± standard deviation (n=6-8).

aP<0.05;

bP<0.01 significantly different from Caki-2 cells (unpaired Student's t-test). R.R., relative resistance; IC50, 50% inhibitory concentration.

Sensitivities of Caki-2 and Caki/AX cells to cytotoxic anticancer drugs.

  IC50 value  
Drug Caki-2 Caki/AX R.R.
Vinblastine, nM 9.16±2.86 29.1±9.00a 3.18
Vincristine, nM 13.5±2.06 44.7±3.54a 3.31
Paclitaxel, nM 6.34±2.09 26.6±8.00a 4.20
Doxorubicin, nM 149±110 349±188a 2.34
Etoposide, µM 5.00±3.65 4.75±2.85 0.95
SN-38, nM 36.3±26.0 72.2±47.9 1.99
5-fluorouracil, µM 20.4±12.1 19.4±12.9 0.95
Cisplatin, µM 1.99±0.63 2.52±0.53 1.27
Carboplatin, µM 25.2±14.1 17.4±12.1 0.69

R.R. was obtained by dividing the IC50 value of Caki/AX cells by that of Caki-2 cells. Each value represents the mean ± standard deviation (n=6-8).

aP<0.01 significantly different from Caki-2 cells (unpaired Student's t-test). R.R., relative resistance; IC50, 50% inhibitory concentration.

Changes in the expression levels of vascular endothelial growth factor signalling pathway-related mRNAs.

A, Increased in Caki/AX cells
Gene Description Log2 (fold-difference)a
FIGF C-fos-induced growth factor (vascular endothelial growth factor D) 2.21
NFATC3 Nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 3 1.21
NFATC4 Nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 4 0.82
PDGFC Mitogen-activated protein kinase 3 1.13
PIK3CA Mitogen-activated protein kinase-activated protein kinase 3 1.15
PIK3CB Nuclear factor of activated T-cells 5, tonicity-responsive 1.24
PIK3R1 Nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 3 1.58
PLCG2 Phospholipase C, gamma 2 (phosphatidylinositol-specific) 1.40
PPP3R2 Protein phosphatase 3, regulatory subunit B, beta 1.82
SH2D2A SH2 domain containing 2A 1.66
SPHK1 Sphingosine kinase 1 3.35
B, Decreased in Caki/AX cells
Gene Description Log2 (fold-difference)a
RAC2 Ras-related C3 botulinum toxin substrate 2 (rho family, small GTP binding protein Rac2) -2.86
VEGFC Vascular endothelial growth factor C -1.39

aFold-difference is the normalized gene expression (2-ΔCq) in Caki/AX cells divided by the normalized gene expression (2-ΔCq) in Caki-2 cells.