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Table 1 Overview of in vitro studies in breast cancer cell lines on the mitogenic potential of insulin analogues

From: Treatment with insulin (analogues) and breast cancer risk in diabetics; a systematic review and meta-analysis of in vitro, animal and human evidence

Author, year Cell line INSR/IGF1R Method Starvation Stimulation time Refreshment of medium Type of stimulation medium Presence phenol red Analogues tested Concentrations tested nM Mitogenic response Sig. PI3K pathway* MAPK pathway*
Milazzo et al., 1997 [26] MCF7A 1:4 [3H]Thymidine incorporation Yes 24 hrs stim 2 hrs measure Yes MEM DME/F12 + 0.1 % BSA Yes AspB10 10 A,B Yes   
1:0.8 DNA measurement Yes 3−5 days Yes, every two days MEM DME/F12 + 0.1 % BSA Yes AspB10 0.01−10 A,B yes   
MCF10B   Colony forming assay No 2 weeks Yes, every two days MEM DME/F12 + 2 % BSA Yes AspB10 100 A -B Yes   
Staiger et al., 2007 [32] MCF7A - [3H]Thymidine incorporation 48hA 20 hrs stim 4 hrs measure Yes DME/F12 SFM No Glargine 10, 50, 100 A No   
MCF10B - MTT 24hB 4 days Yes, every two days DME/F12 SFM No Glargine 1, 5, 10, 25 A,B No   
No
Liefvendahl et al., 2008 [24] MCF7 SKBR-3 1:20 [3H]Thymidine incorporation 24 hrs 21 hrs stim 3 hrs measure No DMEM SFM No Glargine 0.01−100 -    
1:1.8
Mayer et al., 2008 [25] MCF7A 1:3 Cristal violet cell staining No 4 days No DMEM + 1 % SD-FBS No Aspart 1.5A,B     
Lispro 15A,B   
MCF10AB 1:1.2 Glargine 1500C A YesA
Glulisine
T47DC 1:2 Detemir
Shukla et al., 2009 [31] MCF7A - Cristal violet cell staining 24 hrs 3 daysA Yes, every 24 hrs DMEM + 2 % CDFBS No Aspart 1.5, 15, 150, A No   
Lispro 1500 -  
2 daysB MEGMB Glargine A yes
Detemir A No
MCF10AB - WB 24 hrs 10 min - DMEM + 2 % CDFBS No Aspart     - -
Lispro     - -
MEGMB Glargine    Yes A,B A
Detemir    Yes A -
Shukla et al., 2009 [30] MCF7A - Cristal violet cell staining 24 hrs 3 daysA Yes, every 24 hrs DMEM + 2 % CDFBS No Glulisine 1.5, 15, 150, 1500 AB No   
MCF10AB - 2 daysB MEGM
MMOC/ki67 nuclei count No 3 days No Waymouth medium SFM   Glulisine 750 No   
WB 24 hrs 10 min - DMEM + 2 % CDFBSA No Glulisine    Yes AB AB
MEGMB
Weinstein et al., 2010 [35] MCF7 - Cell counting No 72 hrs Yes every day DMEM/SFM   Glargine 100 No   
Detemir No
Oleksiewicz et al., 2011 [27] MCF7 - FACS 72 hrs 24−30 hrs No DMEM + 0.1 % FCS No X10 0.074−2 Yes   
WB 72 hrs 20−40 min No DMEM + 0.1 % FCS No X10 0.67, 2   Yes
Teng et al., 2011 [33] MCF7A - MTT 24 hours 2 days Yes, every two days RPMI + 0.5 % CS-FBS No Glargine 20−200 A Yes   
Yes
WB No 0, 30, 60, 120, 240 min No RPMI + 0.5 % CS-FBS No Glargine 100nM A    
FACS anti-apoptotic No 48 hrs No RPMI + 0.5 % CS-FBS   Glargine  A anti-Apoptotic response Yes   
Glendorf et al., 2012 [21] HMEC 1:20 [3H]Thymidine incorporation No 70 hrs stim 2 hrs measure No MEGM ? B10A, 0.0001−1000    
B10R,
X10,
B10Q,
B10E,
B10H,
B10I,
B10F,
B10W,
B10V
Hansen et al., 2012 [22] HMECA 1:21 [3H]Thymidine incorporation 24 hrs 70 hrs stim 2 hrs measure No MEGM No Detemir 0.001−1000 A Yes   
Glargine A Yes
X10 A Yes
Knudsen et al., 2012 [23] MCF7A - [3H]Thymidine incorporation 2 hrs 24 hrs stim 2 hrs measure No DMEM + 0.1 % serum No S961 0.0001−100 A    
Pierre-Eugene et al., 2012 [28] MCF7A - BRET-PIP3 No 45 min No DMEM/F12 + 5 % FBS ? Aspart     -  
MDA-MB-231B -        Lispro     -
Glargine    Yes A
M1    A
M2    A
Glulisine    Yes A
Detemir    Yes B
WB 12 5 or 20 min No DMEM/F12 ? Glargine    A A
SFM   M1     - -
M2     - -
[14C]Thymidine incorporation 4 hrs 19 hrs stim 6 hrs measure No DMEM/F12 SFM ? Glargine 0.01−1000 A    
M1   -
M2   -
Gallagher et al., 2013 [20] MET1   WB 1 hr 10 min No DMEM + 0.1 % BSA   X10 10 Yes   
MVT1
Ter Braak et al., 2014 [34] MCF7 IGF1RA 1:25 WB   30 min No RPMI + 5 % CDFBS No Aspart 10, 33, 100    - -
MCF7 INSRB 1:0.02 Lispro    Yes A -
MCF7 INSRC 1:0.07 Glargine   A ABC  
M1     - -
M2     - -
Glulisine    - -  
Detemir    Yes AB C ABC
X10    Yes A ABC
SRB 24 hrs 4 days Yes RPMI + 5 % CDFBS No Aspart 0.01−100 -    
Lispro   -  
Glargine   Yes
M1   -  
M2   -  
Glulisine   -  
Detemir   Yes
X10   Yes
Sciacca et al., 2014 [29] MCF7A 1:6 BRDU incorporation 24 hrs 12 hrs, 6 hrs measure No MEM SFM ? Aspart 5 nM AB,C,D    
MDA-MB- 1:2 Lispro (only detemir -A,C,DB YesB
157B   Glargine at 19 nM) -A,C,DB YesB
MDA-MB-468C 1:0.2 M1   -A,B,DC  
T47DD 1:8 M2  A -B,DC  
Glulisine   -A,C,DB YesB
Detemir   -A,C,DB YesB
X10  A,Bc,D YesB
Collagen invasion assay (Boyden chamber technique) No 18 hrs No MEM SFM ? Aspart   -A,D,B,C    
Lispro   -A,DB,C
Glargine  A,B,CD
M1  A,CB,D
M2   -A,DB,C
Glulisine  A,DB,C
Detemir  A,B,C,D
X10  A,B,C,D
  1. A/BOften studies used multiple cell lines. A, B, C, D Specific cell line for cell-line-specific conclusions. *Some studies used a specific experimental setup that allowed discrimination between the involvement of different pathways. For all these studies the p-ERK and p-AKT served as biomarker for activation of mitogen-activated protein kinase (MAPK) or phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K), respectively. IGF1R insulin-like growth factor-1 receptor, BRDU 5-Bromo-2’-deoxyuridine, RPMI Roswell Park Memorial Institute medium, MTT Microculture Tetrazolium proliferation Assay, WB Western Blot, BRET-PIP Bioluminescence Resonance Energy Transfer assay in which the phophatidylinositol-3 phosphate (PIP(3)) production was monitored, SRB SulfoRhodamine B proliferation assay, MEGM Mammary Epithelial Cell Growth Medium, MEM Minimum Essential Medium, SFM Serum Free Medium, CDFBS Charcoal-Dextran-Treated Fetal Bovine Serum, Sig Significant.