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High dose human insulin and insulin glargine promote T24 bladder cancer cell proliferation via PI3K-independent activation of Akt
diabetes research and clinical practice 91 (2011) 177–182
Contents lists available at ScienceDirect
Diabetes Research and Clinical Practice journ al h omepage: www .elsevier.co m/lo cate/diabres
High dose human insulin and insulin glargine promote T24 bladder cancer cell proliferation via PI3K-independent activation of Akt S. Liu a, Y. Li b,*, T. Lin a, X. Fan a, Y. Liang b, U. Heemann c a
Research Center of Medicine, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou 510120, People’s Republic of China Department of Endocrinology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, 107th Yanjiang Western Road, Guangzhou, Guangdong 510120, People’s Republic of China c Department of Nephrology, Technical University Munich, 81675 Munich, Germany b
article info
abstract
Article history:
Background: This study was to investigate the effects of human insulin and insulin glargine
Received 10 October 2010
on proliferation of T24 human bladder cancer cells and the implication of the PI3K/Akt and
Received in revised form
MEK/ERK1/2 pathways.
27 October 2010
Methods: After exposure to insulin or glargine at the indicated concentrations for certain
Accepted 4 November 2010
time courses, in the absence or presence of inhibitor for MEK (PD98059) or PI3K (LY294002),
Published on line 3 December 2010
T24 cell proliferation was evaluated by CCK-8 assay. Phosphorylation of Akt and ERK1/2 was analyzed by Western blot.
Keywords:
Results: Insulin and glargine similarly induced phosphorylation of Akt and slight increases
Insulin
in T24 cell proliferation at 10–100 IU/L. LY294002 remarkably reduced T24 cell proliferation
Glargine
in all groups. However, in the presence of LY294002, cell growth was still promoted by
Proliferation
insulin and glargine relative to LY294002-treated group. Accordingly, LY294002 profoundly
Bladder cancer
reduced protein levels of pAkt, while insulin and glargine increased pAkt in T24 cells pretreated with LY294002 as compared with cells treated with LY294002 alone. PD98059 reduced pERK while enhanced T24 cell proliferation. Insulin and glargine increased pERK at 15, 30, 60 min, not at 24 h. Conclusions: High dose human insulin and insulin glargine similarly promoted T24 bladder cancer cell proliferation via PI3K-independent activation of Akt. # 2010 Elsevier Ireland Ltd. All rights reserved.
1.
Introduction
In recent years, the association between increased carcinogenesis and diabetes mellitus has been revealed by epidemiologic studies [1–5]. The underlying mechanisms remain unclear. Early in vitro studies demonstrated that insulin stimulates DNA synthesis at physiologically relevant concentrations in breast cancer cells [6], and insulin deficiency has been associated with less aggressive cancer proliferation in rats [7]. Insulin promotes
the induction of genes involved in cell proliferation and differentiation by interacting with insulin receptor (IR) [8]. Type 1 insulin-like growth factor receptor (IGF-1R) is a member of the tyrosine kinase receptor family and is structurally similar to IR [9]. Activation of both phosphatidylinositol 3-kinases (PI3Ks)/ Akt and mitogen-activated protein/extracellular signal-regulated kinase (MEK)/extracellular signal-regulated kinase (ERK)1/ 2, downstream to IGF-1R as well as IR, has been associated with increase in cell number caused by insulin [5,10].
* Corresponding author. Tel.: +86 20 81332415; fax: +86 20 81332405. E-mail addresses: [email protected], [email protected] (Y. Li). 0168-8227/$ – see front matter # 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.diabres.2010.11.009
178
diabetes research and clinical practice 91 (2011) 177–182
[()TD$FIG]
Concerns on the mitogenicity of insulin analogues, especially in cancer incidence and development, due to their varied affinity for IR or IGF-1R, have been raised again as insulin glargine has been recently reported to be associated with increased cancer occurrence [11]. Insulin glargine, a long-term insulin analogue, is produced by recombinant DNA techniques by adding two arginine residues to the B chain at positions 31 and 32 and substituting asparagine with glycine in the A chain at position 21. Structural modification resulted in a modified affinity for IR and IGF-1R. In vitro studies demonstrated that insulin glargine exhibited 1.5-fold and 6.4-fold higher affinity for IR and IGF-1R than human insulin, respectively [12]. Bladder cancer has been reported to be one of the ten leading malignancies in the USA, and it has been estimated that there will be 70,980 new cases of bladder cancer and that 14,330 people will die from the disease in 2009 [13]. An increased risk for bladder cancer has also been reported in diabetes [1]. Both IR and IGF-1R have been detected on bladder cancer cells [14,15]. Whether insulin therapy is involved in bladder cancer occurrence and whether structural modification associated changes in receptor affinity of glargine contributes to its mitogenic potential remains to be clarified. In this study, we investigated the effects of glargine in comparison to insulin on proliferation of T24 bladder cancer cell line and the involvement of PI3K/Akt and MEK/ERK1/2 pathways in the process.
2.
Materials and methods
2.1.
Materials
Cell Counting Kit 8 (cck-8) was purchased from Da-jindo (Da-jindo Laboratories, Shanghai, China). Human insulin and insulin glargine preparations used were commercially available as used in clinical practice. PD98059 and LY294002 from Beyotime (Beyotime Institute of Biotechnology, Jiangsu, China); IR, IGF1R, pERK, ERK, pAkt, Akt antibodies were purchased from Cell Signaling (Cell Signaling Technology, Inc. Danvers, MA, USA), tubulin antibody from Novus Biologicals (Novus Biologicals, LLC, Littleton, Colorado, USA), secondary goat-anti-rabbit IgGHRP polyclonal antibody from Chemicon (Chemicon International, Temecula, CA, USA). Enzymatic chemiluminescence: SuperSignal1West Pico was purchased from Thermo Scientific (Pierce Biotechnology, Rockford, IL, USA).
2.2.
2 103 cells/well and cultured in 96-well flat-bottomed microplates. After exposure to human insulin or insulin glargine at the indicated concentrations for the indicated time courses, in the absence or presence of inhibitor of MEK (PD98059) or PI3K (LY294002), CCK-8 reagent (10 ml) was added to each well of a 96-well flat-bottomed microplate containing 100 ml of culture medium and the plate was incubated for 1 h at 37 8C. Viable cells were evaluated by absorbance measurements at 450 nm using auto microplate reader (Wellscan MK3, Labsystems Dragon Inc., Helsinki, Finland). The OD450 value was proportional to the degree of cell proliferation. All experiments were performed in quintuplicate on three separate occasions.
2.4.
Western blotting
Cells were harvested and incubated for 30 min on ice with lysis buffer (Cell Signaling Technology, Inc., Danver, MA, USA). The lysates were then centrifuged at 16,000 g for 30 min at 4 8C and the supernatants were collected. Protein concentration in the lysates was measured by PIERCE BCATM Protein Assay kit (Thermo Fisher Scientific Inc., Rockford, IL, USA). Protein (10 mg) samples were mixed with sample loading buffer, heated for 5 min at 95 8C and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were then transferred to BioTrace NT nitrocellulose membrane (Pall Corporation, Beijing, China).
Cell culture
Human bladder cancer T24 cell line was purchased from American Type Culture Collection (Manassas, VA, USA). The cells were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (Gibco, Invitrogen, Guangzhou, China) in a humidified atmosphere of 5% CO2 at 37 8C. Cells were subcultured every 2–3 days by total medium replacement using 0.25% (w/v) trypsin–0.53 mM EDTA solution (Gibco, Invitrogen, Guangzhou, China).
2.3.
Fig. 1 – Insulin and glargine induced dose-dependent proliferation of T24 bladder cancer cells. T24 cells were seeded in a 96-well plate at a density of 2000/well for 24 h followed by incubation with human insulin or insulin glargine at 0.1, 1, 10, and 100 IU/L, respectively, for 72 h. Cell proliferation was evaluated by CCK-8 assay. Results were calculated as folds increase of the A450 absorbance in the treated group over that in the controls at the respective concentrations. Data represent means W SD. *p < 0.05 vs. control; n = 5.
[()TD$FIG]
Cell proliferation assay
Cell proliferation was determined with the Cell Counting Kit-8 (CCK-8) assay. Cells were suspended at a final concentration of
Fig. 2 – IR and IGF1R protein expression in T24 cells.
diabetes research and clinical practice 91 (2011) 177–182
[()TD$FIG]
179
Fig. 3 – Insulin- and glargine-induced intracellular signaling in T24 bladder cancer cells. T24 cells at subconfluence were challenged with human insulin or insulin glargine at 100 IU/L for 0, 15, 30, 60 min, and 24 h, respectively. Protein levels of total and phosphorylated Akt and ERK1/2 were detected by Western blotting: Akt phosphorylation at 0, 15, 30, 60 min by insulin (A) or glargine (C); ERK1/2 phosphorylation by insulin (B) or glargine (D); results are given as optical density of the pAkt or pERK1/2 normalized to that of total protein of Akt or ERK1/2, respectively. Data are means W SD. *p < 0.05 vs. amounts of protein phosphorylation at 0 min; +p < 0.05 vs. phosphorylation at 15 min; #p < 0.05 vs. phosphorylation at 30 min. (E) Akt phosphorylation and (F) ERK1/2 phosphorylation by insulin and glargine at 24 h. Results are means W SD. *p < 0.05 vs. protein phosphorylation of control group.
Nonspecific binding sites were blocked in 5% nonfat milk and 0.1% Tween-20 in TBS for 1 h at room temperature, after which the membranes were incubated in primary antibody buffer overnight shaking gently at 4 8C, followed by being washed three times in 0.1% TBST after which they were incubated with peroxidase-labeled secondary antibodies at
room temperature for 1 h and again washed three times. The blots were then detected using SuperSignal1West Pico Chemiluminescent Substrate Kit (Thermo Fisher Scientific Inc., Rockford, IL, USA). Densitometric analysis of the bands corresponding to detected protein was done using Adobe Photoshop 7.0 software.
180
2.5.
diabetes research and clinical practice 91 (2011) 177–182
Statistical analysis
Data were analyzed using ANOVA as appropriate. All statistics and data analysis were performed using SPSS 10.0 software. Data represent means SD, p < 0.05 was considered significant.
T24 cells were pretreated with LY294002 (40 mM) for 30 min followed by addition of either insulin or glargine at 100 IU/L for 24 h. LY294002 profoundly reduced the protein level of pAkt. Pretreatment with LY294002 followed by adding insulin (Fig. 5B) or glargine (Fig. 5C) induced a remarkable increase in pAkt as compared with cells treated with LY294002 alone.
3.
4.
Results
3.1. Human insulin and insulin glargine promote T24 cell proliferation
In this study we demonstrated that insulin and glargine similarly promoted an increase in proliferation of T24 bladder cancer cells at 10 and 100 IU/L that are equivalent to 60 and 600 nM, respectively. Such doses are much higher than physiological concentration of insulin (0.1–1 nM). Similar comparable does-dependent growth response to insulin and glargine has been observed in other cell lines such as MCF-7 breast cancer cells [16] and vascular smooth muscle cells [17]. Both IR and IGF1R were detected in T24 cells. Although the expression of IGF1R was in a higher abundance than that of IR, and glargine has been demonstrated of much higher affinity to IGF-1R than insulin [12], no significant increase in the proliferative effect was detected with glargine as compared with insulin. This result suggested that glargine-IGF1R pathway might not be more mitogenic for T24 cells. The baseline level of phosphorylated ERK1/2 was found higher as compared with that of Akt in T24 cells. Insulin and glargine similarly induced phosphorylation of ERK1/2 for a short phase. However, no increase in pERK was observed upon a prolonged exposure to insulin or glargine for 24 h. Furthermore, we found that PD98059-enhanced T24 cell
Human insulin and insulin glargine comparably promoted T24 cell proliferation at concentrations of 10 IU/L and 100 IU/L after incubation for 72 h. No significant increase in cell proliferation was observed with insulin or glargine at 0.1 IU/ L and 1 IU/L. No significant difference in cell growth was detected between insulin and glargine treatments at concentrations from 0.1 to 100 IU/L (Fig. 1).
3.2. Expression of IR and IGF1R and time course of insulin or glargine-induced phosphorylation of Akt and ERK1/2 in T24 cells Protein expression of IRb and IGF1Rb was detected (Fig. 2), with IGF1Rb in a relatively higher abundance in T24 cells. The phosphorylation of Akt and ERK1/2 induced by 100 IU/L of human insulin (Fig. 3A and B) or glargine (Fig. 3C and D) occurred in a similar time frame at 0, 15, 30, and 60 min. Prolonged exposure to either drug for 24 h resulted in increased phosphorylation of Akt (Fig. 3E); while the level of pERK1/2 was not increased in treated groups as compared with controls (Fig. 3F). A tendency of reduction in pERK1/2 was found in glargine group, the difference is not of statistical significance.
Discussion
[()TD$FIG]
3.3. Effect of LY294002 and PD98059 on proliferation of T24 cells, and phosphorylation of Akt or ERK1/2 Treatment with LY294002, an inhibitor of PI3K, reduced T24 cell proliferation in a dose-dependent manner. The maximal reduction of 89.4% was observed at a concentration of 40 mM of LY294002 after 72 h (Fig. 4A). Phosphorylation of Akt was inhibited by LY294002 after incubation at 10, 20, and 40 mM for 24 h (Fig. 4B). By contrast, an inhibitor of MEK, PD98059, led to a significant increase in T24 cell proliferation at concentrations from 5 to 40 mM (Fig. 4A). Phosphorylation of ERK1/2 was inhibited by PD98059 after incubation at 10, 20, and 40 mM for 24 h (Fig. 4C).
3.4. Insulin and glargine promote proliferation and Akt phosphorylation in T24 cell treated with LY294002 In the presence of LY294002, T24 cell growth was reduced in each group as compared to non-LY294002 treated controls. However, combination with insulin or glargine still similarly increased cell growth as compared with cells only treated with LY294002 (Fig. 5A).
Fig. 4 – Effects of LY294002 or PD98059 on proliferation and phosphorylation of Akt and ERK1/2 in T24 cells. T24 cells were treated with LY294002 or PD98059 at 5, 10, 20, and 40 mM or vehicle, respectively, for 72 h. (A) Cell proliferation was measured by CCK-8 assay. Results were calculated as folds increase of the A450 absorbance of the treated group over that of the control at the respective concentrations. Data represent means W SD. *p < 0.05 vs. control; n = 5. Protein phosphorylation of Akt (B) and ERK1/ 2 (C) was measured by Western blotting after T24 cells were treated with LY294002 or PD98059 at 0, 10, 20, and 40 mM, respectively, for 24 h. Data are representatives of three separate experiments.
diabetes research and clinical practice 91 (2011) 177–182
[()TD$FIG]
Fig. 5 – Effect of LY294002 on human insulin and glargineinduced T24 cell proliferation and Akt phosphorylation. (A) T24 cells were pre-treated with LY294002 at 10, 20, and 40 mM or vehicle, respectively, for 30 min followed by adding 100 IU/L of human insulin and glargine for 72 h. CCK-8 assay was used to measure cell proliferation. Results were values of A450 absorbance. Data represent means W SD. *p < 0.05 vs. the group only treated with the respective concentration of LY294002; n = 5. T24 cells were pre-treated with LY294002 at 40 mM or vehicle for 30 min followed by adding 100 IU/L of insulin (B) and glargine (C) for 24 h. Phosphorylation of Akt was evaluated by Western blot as described above. Data are representatives of three separate experiments. con: control, ins: insulin, gla: glargine, and LY: LY294002.
181
alone, which suggested that PI3K-independent pathways might contribute to insulin- or glargine-induced T24 cell proliferation. We further found that when PI3K was blocked by LY294002, insulin and glargine still increased pAkt. These results suggested that alternative pathways to the activation of Akt might be involved in insulin- or glargine-induced T24 cell proliferation. In cultured chicken embryo retinal cells, it has been found that ATP-induced proliferation was associated with increased phosphorylation of Akt that was not blocked by LY294002 [27]. In freshly isolated B Cell chronic lymphocytic leukemia cells, PMA induced phosphorylation of Akt was independent of PI3K, although basal Akt phosphorylation was reduced by PI3K inhibition [28]. Therefore, insulin- or glargineinduced T24 cell proliferation might be associated with PI3Kindependent activation of Akt. In summary, our data demonstrated that human insulin and insulin glargine similarly promoted T24 cell proliferation at high doses. The effect was slight. PI3K-independent activation of Akt might contribute to the mitogenic action of insulin and glargine on T24 cells. MEK/ERK1/2 pathway might act as a negative regulator for T24 cell growth. Further study on the detailed mechanisms of Akt activation induced by insulin or its analogues and the behavior of effectors downstream to Akt may help to find potential targets for the prevention of bladder cancer in diabetes mellitus.
Conflict of interest There is no conflict of interest.
Acknowledgements proliferation was associated with a reduction in pERK1/2. These results suggested that MEK/ERK1/2 pathway might negatively regulate T24 cell growth. ERK activation has typically been associated with cell survival, proliferation and differentiation in response to mitogens and growth factors [18–20]. Inhibition of ERK activation represents a primary approach for treatment of human malignancies. However, activation of ERK has recently been demonstrated to contribute to cell death, depending on stimuli as well as cell types involved [21–24]. Weinstein et al. [25] reported that glargine promoted proliferation in HCT-116 colorectal cancer cells as compared with insulin. However, glargine-induced activation of ERK1/2 was less than that of insulin. In another human bladder cancer cell line, 5673 cells, ERK1/2 activation was involved in the inhibition of cell growth induced by naringin due to induction of p21WAF1 [26]. Taken together, these data suggested that insulin- or glargine-induced proliferation of bladder cancer cells was not mediated by activation of MEK/ERK1/2. In our study, insulin and glargine similarly induced phosphorylation of pAkt. LY294002, an inhibitor of PI3K, reduced phosphorylation of Akt and inhibited proliferation of T24 cells in all groups, which indicated the essential role of PI3K/Akt activity in T24 cell proliferation. However, when PI3K was blocked by LY294002, insulin and glargine still promoted cell growth as compared to the cells treated with LY294002
This work was supported by grants from Natural Science Foundation of China (30671974) and Guangdong Provincial Natural Science Foundation (04009408, 06021329).
references
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Contents lists available at ScienceDirect
Diabetes Research and Clinical Practice journ al h omepage: www .elsevier.co m/lo cate/diabres
High dose human insulin and insulin glargine promote T24 bladder cancer cell proliferation via PI3K-independent activation of Akt S. Liu a, Y. Li b,*, T. Lin a, X. Fan a, Y. Liang b, U. Heemann c a
Research Center of Medicine, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou 510120, People’s Republic of China Department of Endocrinology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, 107th Yanjiang Western Road, Guangzhou, Guangdong 510120, People’s Republic of China c Department of Nephrology, Technical University Munich, 81675 Munich, Germany b
article info
abstract
Article history:
Background: This study was to investigate the effects of human insulin and insulin glargine
Received 10 October 2010
on proliferation of T24 human bladder cancer cells and the implication of the PI3K/Akt and
Received in revised form
MEK/ERK1/2 pathways.
27 October 2010
Methods: After exposure to insulin or glargine at the indicated concentrations for certain
Accepted 4 November 2010
time courses, in the absence or presence of inhibitor for MEK (PD98059) or PI3K (LY294002),
Published on line 3 December 2010
T24 cell proliferation was evaluated by CCK-8 assay. Phosphorylation of Akt and ERK1/2 was analyzed by Western blot.
Keywords:
Results: Insulin and glargine similarly induced phosphorylation of Akt and slight increases
Insulin
in T24 cell proliferation at 10–100 IU/L. LY294002 remarkably reduced T24 cell proliferation
Glargine
in all groups. However, in the presence of LY294002, cell growth was still promoted by
Proliferation
insulin and glargine relative to LY294002-treated group. Accordingly, LY294002 profoundly
Bladder cancer
reduced protein levels of pAkt, while insulin and glargine increased pAkt in T24 cells pretreated with LY294002 as compared with cells treated with LY294002 alone. PD98059 reduced pERK while enhanced T24 cell proliferation. Insulin and glargine increased pERK at 15, 30, 60 min, not at 24 h. Conclusions: High dose human insulin and insulin glargine similarly promoted T24 bladder cancer cell proliferation via PI3K-independent activation of Akt. # 2010 Elsevier Ireland Ltd. All rights reserved.
1.
Introduction
In recent years, the association between increased carcinogenesis and diabetes mellitus has been revealed by epidemiologic studies [1–5]. The underlying mechanisms remain unclear. Early in vitro studies demonstrated that insulin stimulates DNA synthesis at physiologically relevant concentrations in breast cancer cells [6], and insulin deficiency has been associated with less aggressive cancer proliferation in rats [7]. Insulin promotes
the induction of genes involved in cell proliferation and differentiation by interacting with insulin receptor (IR) [8]. Type 1 insulin-like growth factor receptor (IGF-1R) is a member of the tyrosine kinase receptor family and is structurally similar to IR [9]. Activation of both phosphatidylinositol 3-kinases (PI3Ks)/ Akt and mitogen-activated protein/extracellular signal-regulated kinase (MEK)/extracellular signal-regulated kinase (ERK)1/ 2, downstream to IGF-1R as well as IR, has been associated with increase in cell number caused by insulin [5,10].
* Corresponding author. Tel.: +86 20 81332415; fax: +86 20 81332405. E-mail addresses: [email protected], [email protected] (Y. Li). 0168-8227/$ – see front matter # 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.diabres.2010.11.009
178
diabetes research and clinical practice 91 (2011) 177–182
[()TD$FIG]
Concerns on the mitogenicity of insulin analogues, especially in cancer incidence and development, due to their varied affinity for IR or IGF-1R, have been raised again as insulin glargine has been recently reported to be associated with increased cancer occurrence [11]. Insulin glargine, a long-term insulin analogue, is produced by recombinant DNA techniques by adding two arginine residues to the B chain at positions 31 and 32 and substituting asparagine with glycine in the A chain at position 21. Structural modification resulted in a modified affinity for IR and IGF-1R. In vitro studies demonstrated that insulin glargine exhibited 1.5-fold and 6.4-fold higher affinity for IR and IGF-1R than human insulin, respectively [12]. Bladder cancer has been reported to be one of the ten leading malignancies in the USA, and it has been estimated that there will be 70,980 new cases of bladder cancer and that 14,330 people will die from the disease in 2009 [13]. An increased risk for bladder cancer has also been reported in diabetes [1]. Both IR and IGF-1R have been detected on bladder cancer cells [14,15]. Whether insulin therapy is involved in bladder cancer occurrence and whether structural modification associated changes in receptor affinity of glargine contributes to its mitogenic potential remains to be clarified. In this study, we investigated the effects of glargine in comparison to insulin on proliferation of T24 bladder cancer cell line and the involvement of PI3K/Akt and MEK/ERK1/2 pathways in the process.
2.
Materials and methods
2.1.
Materials
Cell Counting Kit 8 (cck-8) was purchased from Da-jindo (Da-jindo Laboratories, Shanghai, China). Human insulin and insulin glargine preparations used were commercially available as used in clinical practice. PD98059 and LY294002 from Beyotime (Beyotime Institute of Biotechnology, Jiangsu, China); IR, IGF1R, pERK, ERK, pAkt, Akt antibodies were purchased from Cell Signaling (Cell Signaling Technology, Inc. Danvers, MA, USA), tubulin antibody from Novus Biologicals (Novus Biologicals, LLC, Littleton, Colorado, USA), secondary goat-anti-rabbit IgGHRP polyclonal antibody from Chemicon (Chemicon International, Temecula, CA, USA). Enzymatic chemiluminescence: SuperSignal1West Pico was purchased from Thermo Scientific (Pierce Biotechnology, Rockford, IL, USA).
2.2.
2 103 cells/well and cultured in 96-well flat-bottomed microplates. After exposure to human insulin or insulin glargine at the indicated concentrations for the indicated time courses, in the absence or presence of inhibitor of MEK (PD98059) or PI3K (LY294002), CCK-8 reagent (10 ml) was added to each well of a 96-well flat-bottomed microplate containing 100 ml of culture medium and the plate was incubated for 1 h at 37 8C. Viable cells were evaluated by absorbance measurements at 450 nm using auto microplate reader (Wellscan MK3, Labsystems Dragon Inc., Helsinki, Finland). The OD450 value was proportional to the degree of cell proliferation. All experiments were performed in quintuplicate on three separate occasions.
2.4.
Western blotting
Cells were harvested and incubated for 30 min on ice with lysis buffer (Cell Signaling Technology, Inc., Danver, MA, USA). The lysates were then centrifuged at 16,000 g for 30 min at 4 8C and the supernatants were collected. Protein concentration in the lysates was measured by PIERCE BCATM Protein Assay kit (Thermo Fisher Scientific Inc., Rockford, IL, USA). Protein (10 mg) samples were mixed with sample loading buffer, heated for 5 min at 95 8C and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were then transferred to BioTrace NT nitrocellulose membrane (Pall Corporation, Beijing, China).
Cell culture
Human bladder cancer T24 cell line was purchased from American Type Culture Collection (Manassas, VA, USA). The cells were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (Gibco, Invitrogen, Guangzhou, China) in a humidified atmosphere of 5% CO2 at 37 8C. Cells were subcultured every 2–3 days by total medium replacement using 0.25% (w/v) trypsin–0.53 mM EDTA solution (Gibco, Invitrogen, Guangzhou, China).
2.3.
Fig. 1 – Insulin and glargine induced dose-dependent proliferation of T24 bladder cancer cells. T24 cells were seeded in a 96-well plate at a density of 2000/well for 24 h followed by incubation with human insulin or insulin glargine at 0.1, 1, 10, and 100 IU/L, respectively, for 72 h. Cell proliferation was evaluated by CCK-8 assay. Results were calculated as folds increase of the A450 absorbance in the treated group over that in the controls at the respective concentrations. Data represent means W SD. *p < 0.05 vs. control; n = 5.
[()TD$FIG]
Cell proliferation assay
Cell proliferation was determined with the Cell Counting Kit-8 (CCK-8) assay. Cells were suspended at a final concentration of
Fig. 2 – IR and IGF1R protein expression in T24 cells.
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[()TD$FIG]
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Fig. 3 – Insulin- and glargine-induced intracellular signaling in T24 bladder cancer cells. T24 cells at subconfluence were challenged with human insulin or insulin glargine at 100 IU/L for 0, 15, 30, 60 min, and 24 h, respectively. Protein levels of total and phosphorylated Akt and ERK1/2 were detected by Western blotting: Akt phosphorylation at 0, 15, 30, 60 min by insulin (A) or glargine (C); ERK1/2 phosphorylation by insulin (B) or glargine (D); results are given as optical density of the pAkt or pERK1/2 normalized to that of total protein of Akt or ERK1/2, respectively. Data are means W SD. *p < 0.05 vs. amounts of protein phosphorylation at 0 min; +p < 0.05 vs. phosphorylation at 15 min; #p < 0.05 vs. phosphorylation at 30 min. (E) Akt phosphorylation and (F) ERK1/2 phosphorylation by insulin and glargine at 24 h. Results are means W SD. *p < 0.05 vs. protein phosphorylation of control group.
Nonspecific binding sites were blocked in 5% nonfat milk and 0.1% Tween-20 in TBS for 1 h at room temperature, after which the membranes were incubated in primary antibody buffer overnight shaking gently at 4 8C, followed by being washed three times in 0.1% TBST after which they were incubated with peroxidase-labeled secondary antibodies at
room temperature for 1 h and again washed three times. The blots were then detected using SuperSignal1West Pico Chemiluminescent Substrate Kit (Thermo Fisher Scientific Inc., Rockford, IL, USA). Densitometric analysis of the bands corresponding to detected protein was done using Adobe Photoshop 7.0 software.
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2.5.
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Statistical analysis
Data were analyzed using ANOVA as appropriate. All statistics and data analysis were performed using SPSS 10.0 software. Data represent means SD, p < 0.05 was considered significant.
T24 cells were pretreated with LY294002 (40 mM) for 30 min followed by addition of either insulin or glargine at 100 IU/L for 24 h. LY294002 profoundly reduced the protein level of pAkt. Pretreatment with LY294002 followed by adding insulin (Fig. 5B) or glargine (Fig. 5C) induced a remarkable increase in pAkt as compared with cells treated with LY294002 alone.
3.
4.
Results
3.1. Human insulin and insulin glargine promote T24 cell proliferation
In this study we demonstrated that insulin and glargine similarly promoted an increase in proliferation of T24 bladder cancer cells at 10 and 100 IU/L that are equivalent to 60 and 600 nM, respectively. Such doses are much higher than physiological concentration of insulin (0.1–1 nM). Similar comparable does-dependent growth response to insulin and glargine has been observed in other cell lines such as MCF-7 breast cancer cells [16] and vascular smooth muscle cells [17]. Both IR and IGF1R were detected in T24 cells. Although the expression of IGF1R was in a higher abundance than that of IR, and glargine has been demonstrated of much higher affinity to IGF-1R than insulin [12], no significant increase in the proliferative effect was detected with glargine as compared with insulin. This result suggested that glargine-IGF1R pathway might not be more mitogenic for T24 cells. The baseline level of phosphorylated ERK1/2 was found higher as compared with that of Akt in T24 cells. Insulin and glargine similarly induced phosphorylation of ERK1/2 for a short phase. However, no increase in pERK was observed upon a prolonged exposure to insulin or glargine for 24 h. Furthermore, we found that PD98059-enhanced T24 cell
Human insulin and insulin glargine comparably promoted T24 cell proliferation at concentrations of 10 IU/L and 100 IU/L after incubation for 72 h. No significant increase in cell proliferation was observed with insulin or glargine at 0.1 IU/ L and 1 IU/L. No significant difference in cell growth was detected between insulin and glargine treatments at concentrations from 0.1 to 100 IU/L (Fig. 1).
3.2. Expression of IR and IGF1R and time course of insulin or glargine-induced phosphorylation of Akt and ERK1/2 in T24 cells Protein expression of IRb and IGF1Rb was detected (Fig. 2), with IGF1Rb in a relatively higher abundance in T24 cells. The phosphorylation of Akt and ERK1/2 induced by 100 IU/L of human insulin (Fig. 3A and B) or glargine (Fig. 3C and D) occurred in a similar time frame at 0, 15, 30, and 60 min. Prolonged exposure to either drug for 24 h resulted in increased phosphorylation of Akt (Fig. 3E); while the level of pERK1/2 was not increased in treated groups as compared with controls (Fig. 3F). A tendency of reduction in pERK1/2 was found in glargine group, the difference is not of statistical significance.
Discussion
[()TD$FIG]
3.3. Effect of LY294002 and PD98059 on proliferation of T24 cells, and phosphorylation of Akt or ERK1/2 Treatment with LY294002, an inhibitor of PI3K, reduced T24 cell proliferation in a dose-dependent manner. The maximal reduction of 89.4% was observed at a concentration of 40 mM of LY294002 after 72 h (Fig. 4A). Phosphorylation of Akt was inhibited by LY294002 after incubation at 10, 20, and 40 mM for 24 h (Fig. 4B). By contrast, an inhibitor of MEK, PD98059, led to a significant increase in T24 cell proliferation at concentrations from 5 to 40 mM (Fig. 4A). Phosphorylation of ERK1/2 was inhibited by PD98059 after incubation at 10, 20, and 40 mM for 24 h (Fig. 4C).
3.4. Insulin and glargine promote proliferation and Akt phosphorylation in T24 cell treated with LY294002 In the presence of LY294002, T24 cell growth was reduced in each group as compared to non-LY294002 treated controls. However, combination with insulin or glargine still similarly increased cell growth as compared with cells only treated with LY294002 (Fig. 5A).
Fig. 4 – Effects of LY294002 or PD98059 on proliferation and phosphorylation of Akt and ERK1/2 in T24 cells. T24 cells were treated with LY294002 or PD98059 at 5, 10, 20, and 40 mM or vehicle, respectively, for 72 h. (A) Cell proliferation was measured by CCK-8 assay. Results were calculated as folds increase of the A450 absorbance of the treated group over that of the control at the respective concentrations. Data represent means W SD. *p < 0.05 vs. control; n = 5. Protein phosphorylation of Akt (B) and ERK1/ 2 (C) was measured by Western blotting after T24 cells were treated with LY294002 or PD98059 at 0, 10, 20, and 40 mM, respectively, for 24 h. Data are representatives of three separate experiments.
diabetes research and clinical practice 91 (2011) 177–182
[()TD$FIG]
Fig. 5 – Effect of LY294002 on human insulin and glargineinduced T24 cell proliferation and Akt phosphorylation. (A) T24 cells were pre-treated with LY294002 at 10, 20, and 40 mM or vehicle, respectively, for 30 min followed by adding 100 IU/L of human insulin and glargine for 72 h. CCK-8 assay was used to measure cell proliferation. Results were values of A450 absorbance. Data represent means W SD. *p < 0.05 vs. the group only treated with the respective concentration of LY294002; n = 5. T24 cells were pre-treated with LY294002 at 40 mM or vehicle for 30 min followed by adding 100 IU/L of insulin (B) and glargine (C) for 24 h. Phosphorylation of Akt was evaluated by Western blot as described above. Data are representatives of three separate experiments. con: control, ins: insulin, gla: glargine, and LY: LY294002.
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alone, which suggested that PI3K-independent pathways might contribute to insulin- or glargine-induced T24 cell proliferation. We further found that when PI3K was blocked by LY294002, insulin and glargine still increased pAkt. These results suggested that alternative pathways to the activation of Akt might be involved in insulin- or glargine-induced T24 cell proliferation. In cultured chicken embryo retinal cells, it has been found that ATP-induced proliferation was associated with increased phosphorylation of Akt that was not blocked by LY294002 [27]. In freshly isolated B Cell chronic lymphocytic leukemia cells, PMA induced phosphorylation of Akt was independent of PI3K, although basal Akt phosphorylation was reduced by PI3K inhibition [28]. Therefore, insulin- or glargineinduced T24 cell proliferation might be associated with PI3Kindependent activation of Akt. In summary, our data demonstrated that human insulin and insulin glargine similarly promoted T24 cell proliferation at high doses. The effect was slight. PI3K-independent activation of Akt might contribute to the mitogenic action of insulin and glargine on T24 cells. MEK/ERK1/2 pathway might act as a negative regulator for T24 cell growth. Further study on the detailed mechanisms of Akt activation induced by insulin or its analogues and the behavior of effectors downstream to Akt may help to find potential targets for the prevention of bladder cancer in diabetes mellitus.
Conflict of interest There is no conflict of interest.
Acknowledgements proliferation was associated with a reduction in pERK1/2. These results suggested that MEK/ERK1/2 pathway might negatively regulate T24 cell growth. ERK activation has typically been associated with cell survival, proliferation and differentiation in response to mitogens and growth factors [18–20]. Inhibition of ERK activation represents a primary approach for treatment of human malignancies. However, activation of ERK has recently been demonstrated to contribute to cell death, depending on stimuli as well as cell types involved [21–24]. Weinstein et al. [25] reported that glargine promoted proliferation in HCT-116 colorectal cancer cells as compared with insulin. However, glargine-induced activation of ERK1/2 was less than that of insulin. In another human bladder cancer cell line, 5673 cells, ERK1/2 activation was involved in the inhibition of cell growth induced by naringin due to induction of p21WAF1 [26]. Taken together, these data suggested that insulin- or glargine-induced proliferation of bladder cancer cells was not mediated by activation of MEK/ERK1/2. In our study, insulin and glargine similarly induced phosphorylation of pAkt. LY294002, an inhibitor of PI3K, reduced phosphorylation of Akt and inhibited proliferation of T24 cells in all groups, which indicated the essential role of PI3K/Akt activity in T24 cell proliferation. However, when PI3K was blocked by LY294002, insulin and glargine still promoted cell growth as compared to the cells treated with LY294002
This work was supported by grants from Natural Science Foundation of China (30671974) and Guangdong Provincial Natural Science Foundation (04009408, 06021329).
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