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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.