AMPK Activation of Apoptotic Markers in Human Breast Cancer Cell Lines with Different p53 Backgrounds: MCF-7, MDA-MB-231 and T47D Cells.

BACKGROUND: Downregulation of AMPK has been established as a major contributor to carcinogenesis in many types of human cancer. We sought to investigate the influence of activated AMPK on apoptotic markers in human breast cancer cells differing in their p53 status, as well as estrogen receptor status (MCF-7 (p53+ and ER+), MDA-MB-231 (p53 mutant and ER-) and T47D (p53 mutant and ER+)). METHODS: We examined the effect of AICAR-activated AMPK on PARP cleavage, Bax redistribution, the involvement of intrinsic and extrinsic pathways of apoptosis using selective caspase inhibitors and cell cycle progression and p21 levels. RESULTS: PARP cleavage occurred to a greater extent in MCF-7 and MDA-MB-231 cells, whereas Bax translocation was slower in MDA-MB-231 cells. Although there were quantitative differences in the effect of caspase inhibitors, it was clear that AMPK activation predominately affected the intrinsic pathway of apoptosis. Although, p21 was increased in all 3 cell types, there were quantitative and time differences. Apoptosis, as measured by fluorimetry, was increased in all three cell types. CONCLUSION: The impact of AMPK activation was cell type dependent resulting in differential activation of apoptotic markers, confirming that the genetic background of breast cancer may have an influence on the mode of action of AMPK. Thus, different anti-tumour mechanisms may be elicited depending on the cellular genotype.

Differential crosstalk between the AMPK and PI3K/Akt pathways in breast cancer cells of differing genotypes: Leptin inhibits the effectiveness of AMPK activation.

AMP-activated protein kinase (AMPK), a sensor of cellular energy, is widely reported as a potential therapeutic target in treatment of breast and other cancers. The activated enzyme has been shown to be a promising anti-proliferative agent in breast cancer cell lines. However, little data exist on crosstalk between AMPK and the cellular survival axis of PI3K/Akt/mTOR pathway and the impact of microenvironment on cellular responses to AMPK activation. We present results which show differential crosstalk between AMPK and Akt, dependent on the cellular genetics of each breast cancer cell type. We also show that leptin blocks activation of AMPK and partially or completely attenuates the anti-proliferative effect of AMPK activation depending on the cell type. This suggests that leptin within the local environment might impose limitations on therapeutic usage of AMPK activators in cancer, thereby attenuating their effective use in many obese subjects.

Effects of activation of AMPK on human breast cancer cell lines with different genetic backgrounds.

Breast cancer remains a therapeutic challenge, and this has intensified the search for new drug targets. The AMP-activated protein kinase (AMPK) signalling pathway is emerging as having potential for intervention. We assessed the possible different effects of AMPK action on breast cancer cells by studying their impact on proliferation, apoptosis and the mitochondrial membrane potential in three breast cancer cell lines (MCF-7, MDA-MB-231 and T47D) differing in their p53 and estrogen receptor (ER) status. Activation of AMPK by 5-aminoimidazole carboxamide ribonucleotide (AICAR) and phenformin elicited clear anti-proliferative effects in all breast cancer cell lines, but with differences in sensitivity. However, the anti-proliferative effects were accompanied by varying responses between the different cells, with a marked cell cycle arrest effect in T47D cells and an apoptotic effect in MCF-7 and MDA-MB-231 cells. The mitochondrial apoptotic pathway was potentially involved in all cell lines. These results suggest that AMPK potentially serves as a therapeutic target in breast cancer, but one which may be dependent on the genetic background of the cancer cells.

Differential enhancement of the anti-cancer effect of doxorubicin by Akt inhibitors on human breast cancer cells with differing genetic backgrounds.

The phosphatidylinositol 3 OH-kinase (PI3K) pathway is a key intracellular signalling cascade in cellular survival. Our previous studies indicated that specific blockade of this enzyme led to sensitisation of human breast carcinoma cells to killing by doxorubicin through induction of both G2 arrest and apoptosis in some, but not all, breast cancer cells. In the present study, we report that inhibition of a down-stream component of this pathway, Akt, is an effective means of enhancing doxorubicin killing in some breast cell types. Doxorubicin (Dox) and six Akt inhibitors were used individually or in combination on MDA-MB-231 (p53 mutant, ER-), T47D (p53 mutant, ER+), and MCF-7 (p53 wt, ER+) human breast cancer cell lines. In MDA-MB-231 breast cancer cells, all six Akt inhibitors, which have differing mechanisms of action to inhibit Akt, synergised with the growth inhibitory effects of doxorubicin. Two Akt inhibitors also enhanced the effect of Dox in T47D cells but the other inhibitors induced additive effects in these cells. None of the inhibitors used elicited enhanced effects in MCF-7 cells. These results support the notion that combination therapies of doxorubicin (and possibly other chemotherapeutics) with inhibitors of elements of the PI3K pathway are a realistic possibility for future breast cancer therapy, which could lead to reduced side-effects, but that this could be dependent on the genetic background of each breast cancer.

Prognostic significance of Akt, phospho-Akt and BAD expression in primary breast cancer.

Apoptotic markers in breast cancer are reported to have prognostic significance. The aim of this study was to assess the prognostic value of Akt, phospho-Akt and BAD expression in primary tumours from breast cancer patients. Expression of phospho-Akt did not correlate with menopausal status, nodal involvement or tumour size, although there was a significant correlation between phospho-Akt and oestrogen receptor status and tumour grade. No association was found between phospho-Akt and BAD. However, a significant correlation was found between Akt and BAD. Akt and phospho-Akt expression did not correlate with either disease-free survival (DFS) or overall survival (OS). Conversely, BAD immunostaining correlated significantly with increasing tumour size and with oestrogen receptor (ER) immunostaining in both frozen and paraffin sections. Expression of BAD appeared to be nucleolar in addition to its cytoplasmic and nuclear staining. Comparison of immunohistochemical staining on frozen sections and paraffin sections showed a reasonable concordance in Akt and BAD immunoreactivity. However, the results showed for the first time that strong BAD expression is related to a favourable prognosis but is not an independent prognostic factor. In conclusion, these results could provide the basis for understanding how Akt, phospho-Akt and BAD expression contributes to the prognosis of invasive breast cancer.

Enhanced anti-cancer effect of a phosphatidylinositol-3 kinase inhibitor and doxorubicin on human breast epithelial cell lines with different p53 and oestrogen receptor status.

New efforts are being focused on signalling pathways as targets for cancer therapy. This particular study was designed to investigate whether blockade of the phosphatidylinositol 3OH-kinase (PI3K) pathway (a survival/anti-apoptosis pathway, overexpressed in various tumours) could sensitise human breast cancer cells to the effect of chemotherapeutics. Doxorubicin (Dox) and LY294002 (LY, a PI3K inhibitor) were used individually or in combination on MDA-MB-231 (p53 mutant, ER-), T47D (p53 mutant, ER+), and MCF-7 (p53 wildtype, ER+) human breast cancer cell lines, and on 184A1, a nonmalignant human breast epithelial cell line (p53 wildtype, ER-). Each drug showed time- and dose-dependent growth inhibition of cell proliferation on all 4 cell lines. The combination of Dox+LY resulted in enhanced cell growth inhibition in MDA-MB-231 and T47D cells, and additive inhibition in MCF-7 and 184A1 cells. Cell cycle analysis showed that Dox+LY enhanced the arrest of MDA-MB-231 and T47D cells in G2 with the appearance of a sub-G1 peak indicating apoptosis/necrosis, a notion supported by enhanced depolarisation of mitochondrial membrane potential in these cell types. The combination also caused a greater additive increase in Cyclin B1. Thus, the synergistic effect of the combination on cell proliferation in some, but not all, breast cancer cells may be through enhanced induction of both G2 arrest and apoptosis, in which p53 may play a role. Substantially lower doses of doxorubicin could be used with low doses of inhibitors of the PI3K pathway, without compromising the anti-cancer effect, but also lowering detrimental side-effects of doxorubicin. This study supports the notion that survival signalling pathways offer special targets for chemotherapy in cancer.

The survival effect of prolactin on PC3 prostate cancer cells.

OBJECTIVES: Recent studies suggest a paracrine/autocrine loop involving prolactin (PRL) within the human prostate. The aims of this study were to determine the effects of PRL on the growth and survival of prostate cancer cells and the intracellular signalling mechanisms underlying such effects. METHODS: The effect of PRL on proliferation of LNCaP, PC3 and DU145 was assessed by Coulter counting. The effect of PRL on TRAIL-, staurosporine- and flavopiridol-induced apoptosis was assessed by Timelapse microscopy and Annexin V binding. The status of the PRL receptor (PRL-R) and Akt/PKB (protein kinase B) activity were assessed by Western blotting. RESULTS: All three cell lines expressed both the short and long forms of the PRL receptor. Although, no significant effect of PRL on the proliferation of these cells was found, PRL partially inhibited TRAIL-induced apoptosis in PC3 cells. PRL also enhanced the phosphorylation of Akt/PKB in these cells. CONCLUSIONS: PRL had no significant effect on the proliferation of PC3, DU145 and LNCaP, but inhibited TRAIL-induced apoptosis in PC3 cells, possibly via enhanced Akt/PKB phosphorylation in PC3 cells. Further investigations are underway to determine the survival effect of PRL on the other two prostate cancer cell line.

Protein kinase C-eta (PKC-eta) is required for the development of inducible nitric oxide synthase (iNOS) positive phenotype in human monocytic cells.

Several murine and human monocytic cell lines and monocyte-derived macrophages (MDM) from healthy volunteers were studied to compare their production of nitric oxide (NO) and induction of iNOS following endotoxin treatment. Although the human cells were sensitive to endotoxin and responded well by producing TNF-alpha and matrix metalloproteases (MMP), there was no induction of iNOS expression or NO production by any of these cells. Murine cells, however, produced large amounts of NO and expressed iNOS following similar endotoxin stimulation. We investigated the expression of PKC isotypes in all human and murine cell lines as well as in MDM, and found that the human cells lacked PKC-eta while the murine counterparts lacked PKC-beta1. Subsequently, human cells that were transfected with PKC-eta were found to make large quantities of NO following endotoxin exposure, an observation not seen in untransfected cells. We propose that PKC-eta is essential for the development of the iNOS positive phenotype in human monocytic cells, and may be responsible for the development of a number of inflammatory related conditions. As such it may be a suitable target for therapeutic intervention.