The number of women in the United States who are obese has doubled in the past 25 years. As the average body mass index climbs, the overall levels of Acrp30 will decline making this area of research progressively more important (Smigal et al, 2006
; Wyatt et al, 2006
). We have been able to show that five different breast cancer cell lines express either AdipoR1 and/or AdipoR2 protein. Previously, mRNA for AdipoR2 and protein for AdipoR1 had been detected in MCF-7, T47D and MDA-MB-231 cells (Dieudonne et al, 2006
; Arditi et al, 2007
). We confirmed the protein expression and found that AdipoR1 protein was also expressed by SK-BR-3 cells. Our results also indicate AdipoR2 protein was expressed by MCF-7, T47D, MDA-MB-231, and MDA-MB-361 breast cancer cells. Additionally, these receptors appear to be functional since in some breast cancer cell lines growth was inhibited by the addition of Acrp30 (). We did not find large changes in the cell cycle indicators, PCNA or cyclin D1, possibly due to the use of serum-free media, which may have caused the cells to enter a G0
state in which PCNA and cyclin D levels were already relatively low. We did find that caspase-8 and PARP appeared to be activated by Acrp30 in ER+ cell lines (). These findings suggest that apoptosis was initiated in ER+ but not ER− cell lines by Acrp30 in this serum-free experiment.
To better understand why some women would be more adversely affected by obesity, we initially compared both Her2/neu status and ERα
status in conjunction with the response of the cell lines we examined for decreased proliferation in response to Acrp30. shows that two of the lines that did not express Her2/neu, the MCF-7 and T47D, did decrease their proliferation in response to Acrp30 but that the MDA-MB-231 line which also lacks Her2/neu did not decrease its proliferation in response to Acrp30. Of the two lines that expressed Her2/neu, the SK-BR-3 cells did have a decrease in proliferation but the MDA-MB-361 cells did not. When we examined the status of ERα
in these cell lines as compared to decreased proliferation in response to Acrp30, we found that the MCF-7 and T47D cells which express ERα
had a decrease in proliferation but that the MDA-MB-361 cells which also express ERα
did not exhibit the same decrease. Of the cells that did not express ERα
, SK-BR-3 had a decreased proliferation rate at higher Acrp30 levels as compared to MCF-7 and T47D cells but the MDA-MB-231 cells did not. Because there appeared to be a relationship between expression of ERα
and decreased sensitivity of the cells' antiproliferation response to Acrp30, we chose to further investigate the interaction of Acrp30 and ERα
by developing a new cell line designated MDA-ERα
7. This cell line expresses ERα
and is oestrogen responsive in vivo
since it exhibited increased growth in response to exogenous oestradiol (). Other investigators have transfected ERα
into MDA-MB-231 cells and have found no change in growth (Touitou et al, 1991
; Bandyopadhyay et al, 2007
) or even a decrease in the presence of oestradiol (Jiang et al, 1992
). The difference may be attributable to clonal variation or the fact that we first tested the MDA-ERα
7 cells soon after selection. Interestingly, while initial experiments with very early passage MDA-ERα
7 cells suggested it was oestrogen responsive, in vitro
testing with later passages of MDA-ERα
7 cells showed little or no increase in growth in response to oestradiol (data not shown) despite the fact that similar passages were responsive to oestradiol in vivo
. This may be due to interactions between oestradiol and other growth factors that would be possible in vivo
but that did not occur in our in vitro
system due to the fact that we used serum-free media to perform our oestradiol growth experiments. These results illustrate that in vivo
growth and in vitro
growth can be very different.
ERα and Her2/neu status of breast cancer cell lines and response to Acrp30
When we tested the effects of Acrp30 and gAcrp30 using in vitro assays we found that MDA-ERα7 cells have decreased cell growth and were more sensitive to gAcrp30 as compared to the parental MDA-wt cells (). These decreases in proliferation may be attributable to decreased signalling through JNK2 since phosphorylation of JNK2 by FCS is inhibited by adiponectin, particularly gAcrp30 (). The concentration levels of the receptors, AdipoR1 and AdipoR2, did not seem to be involved in cellular response since they were very similar (). Interestingly, the ERα+ MDA-ERα7 cells did not show phosphorylation of Akt following serum stimulation even though the parental MDA-wt cells did ().
We did not find any indication of growth inhibition in relationship to the cell cycle as no changes in levels of cyclin D1 or PCNA in the absence of serum were detected. However, we did find that the ER+ cells treated with low doses of Acrp30 appear to have an increase in apoptosis. This was suggested by the increase in cleaved caspase-8 and cleaved PARP in these cells. Other reports have found that apoptosis is involved in growth inhibition by Acrp30 by the use of TUNEL or annexin V assays (Kang et al, 2005
; Dieudonne et al, 2006
; Wang et al, 2006
). We have extended those earlier observations by identifying increased cleavage of caspase-8 and PARP following treatment with Acrp30. We also looked for cleavage of caspase-3, caspase-6, and caspase-9 but did not find any changes due to Acrp30 treatment suggesting that under these conditions the parts of the apoptosis pathway controlled by these proteins was not being utilised.
Several groups reported previously that MDA-MB-231 cells were growth inhibited by Acrp30 (Kang et al, 2005
; Wang et al, 2007
). However, in our hands proliferation of this cell line was not statistically inhibited by the addition of Acrp30. This lack of agreement may be attributable to different culture conditions, subclone variation of the cells and/or the use of different sources of Acrp30. We purchased Acrp30 while the other groups made theirs using at least three different methods.
Our study is one of the first to investigate the function of gAcrp30 vs
Acrp30. Very little work has been done in this area with only one previous mention that gAcrp30 did not affect MDA-MB-231 cells growth but the data were not shown (Wang et al, 2006
). In our hands the addition of gAcrp30 to MDA-MB-231 cells resulted in fewer cells after 48 hours but the reduction was not statistically significant. Previous work has shown that AdipoR1 has a higher affinity for gAcrp30 (Yamauchi et al, 2003a
). Our study was not able to address which of the Acrp30 receptors was most important for the reduction in cell number since our cell lines expressed both receptors. Additional work using shRNA to knockout AdipoR1, AdipoR2 or both receptors may help to clarify the roles of the two receptors with regards to cell growth and/or death.
Here, cell growth inhibition by Acrp30 was examined independently of any other serum factors. However, the interplay between Acrp30 and other growth factors has been shown to be important for Acrp30's ability to inhibit cell proliferation (Wang et al, 2005
). With respect to breast cancer, and obesity, leptin has been an adipokine implicated in mammary tumorigenesis (Hu et al, 2002
; Cleary et al, 2003
; Frankenberry et al, 2006
; Garofalo et al, 2006
). Overweight or obese individuals usually have elevated levels of the growth factor leptin, which is positively correlated to body mass index while Acrp30 is negatively correlated with body mass index. We and others are now showing that Acrp30 may be an important negative regulator of breast cancer cell growth (Miyoshi et al, 2003
; Kang et al, 2005
; Dieudonne et al, 2006
). In particular, we are trying to assess the interrelationship of these two adipokines on tumour growth and development. It is likely that a balance between the two adipokines will determine if a tumour increases or decreases in size.
Our current study has illustrated that Acrp30 may be involved in apoptosis as well as inhibition of growth factors found in serum which initiate proliferation through the JNK and Akt pathways. This is consistent with a prior study showing that pretreatment of human aortic smooth muscle cells with Acrp30 inhibited the function of growth factors in serum (Wang et al, 2005
). Previously it had been anticipated that there would be increased levels of growth factors released during refeeding following calorie restriction which would enhance tumour development; however, our previous in vivo
findings indicated that intermittent calorie restriction resulted in increased mammary tumour latency and decreased tumour incidence (Cleary et al, 2002
). Human findings on calorie restriction indicate that serum Acrp30 levels increased following weight loss either through diet (Weiss et al, 2006
), MacLean vertical banded gastroplasty or biliopancreatic diversion with duodenal switch (Kotidis et al, 2006
). We are currently investigating the levels of Acrp30 during calorie restriction in vivo
with ongoing mouse mammary and prostate tumorigenesis studies. We feel that factors in the serum of the mice, specifically adipokines have the ability to function as both growth stimulators such as has been shown for leptin or as growth inhibitors such as seems to be the case with Acrp30 for breast cancer. Additional experiments will confirm and define the roles of adipokines such as Acrp30 and leptin and determine how the balance of the two impact breast cancer cell growth and death.