The association between rat mammary tumor weight and AA levels, p-Akt/Akt ratio, IkB, nuclear p65, c-Myc or VEGF expression
Our lab has previous found that AA levels are 10 times higher in rat mammary tumor tissue than in the normal mammary gland, and are positively correlated with the tumor weight [1]. Akt signaling and NF-kB activation play important roles in cancer proliferation and angiogenesis [7]. Constitutive NF-kB activation has been detected in DMBA-induced rat mammary tumors [6, 8]. Therefore, we examined AA levels, p-Akt/Akt ratio, IkB, nuclear p65, c-Myc and VEGF expression in the DMBA-induced rat mammary tumors, weighting between 0.3 g and 19.2 g (Fig. 1). We observed that the weight of the rat mammary tumors was significantly positively associated with AA levels (Pearson correlation coefficient r = 0.5671, p = 0.002, n = 27) (Fig. 1A), p-Akt/Akt ratio (r = 0.5881, p = 0.0006, n = 30) (Fig. 1B), nuclear p65 (r = 0.6289, p = 0.0002, n = 30) (Fig. 1D), c-Myc (r = 0.7107, p < 0.0001, n = 30) (Fig. 1E), and VEGF (r = 0.8272, p < 0.0001, n = 30) (Fig. 1F). Further, tumor weights were significantly negatively corrected with IkBa expression (r = -0.6894, p < 0.0001, n = 28) (Fig. 1C). Owing to the limitation of tumor weight being less than 0.5 g, tumor tissue was insufficient for fatty acid analysis and therefore, the sample size for AA level analysis was smaller than that taken for the analyses of the other parameters. The similar results were obtained for the association between AA levels in the rat mammary tumor and above parameters, AA levels in the rat mammary tumor showed a significantly positive correlation with p-Akt/Akt ratio (r = 0.5521, p = 0.0028, n = 27), nuclear p65 (r = 0.4853, p = 0.012, n =27), c-Myc (r = 0.5053, p = 0.0085, n = 27) and VEGF (r = 0.6372, p = 0.0004, n = 27). Further, a significantly negative correlation with IkBa (r = -0.6324, p = 0.0005, n = 27) expression was observed. These results indicated that AA plays an important role in mammary tumor development, and that, AA levels in rat mammary tumor tissue were positively correlated with the tumor weight. Furthermore, the tumor weights were positivity associated with p-Akt signaling, NF-kB activation, c-Myc and VEGF expression, for tumor development. Moreover, we examined the role of AA without or with TNF-α stimulation on proliferation in MCF-7 human breast cancer cells.
AA incorporation into MCF-7
We first examined whether the supplemented AA was incorporated into MCF-7 cells (Fig. 2 and Additional file 1: Table S1). After 2 days of culturing the cells with 0-100 mM AA, the AA levels were found to increase from 5.3% of total fatty acids at 0 mM AA to 17.2%, 24.8% or 28.8% of total fatty acids with AA supplementation of 10, 50 and 100 mM, respectively. Docosatetraenoic acid (22:4n-6) levels were also found to increase from 0.4% to 10.9%, with increasing AA supplementation. The increase in AA and 22:4n-6 levels was accompanied by a decrease in the total monounsaturated fatty acids from 53% to 22%. The monounsaturated fatty acids that were found to be reduced were oleic acid (18:1n-9) from 34% to 14%, 18:1n-7 from 7.2% to 3.2% and 16:1n-7 from 7.6 to 2.9% of the total fatty acids, others being changed by less than 1%. In contrast, the saturated fatty acids and n-3 fatty acids showed little change, ranging from 38% to 35% and from 2.5% to 0.9%, of the total fatty acids.
AA and TNF-α on p-Akt signaling and NF-kB activation in MCF-7
About two-thirds of breast cancers in women are hormone-dependent cancers that contain estrogen receptors. NF-kB activation is found to be constitutive in human breast cancers, often showing higher expression in the more aggressive human breast cancer cell lines such as estrogen receptor-negative MDA-MB-461, but less frequently in estrogen receptor-positive MCF-7, where NF-kB activity can be induced by TNF-α stimulation [18-21]. In order to distinguish the role of AA on NF-kB activity, we further examined the effect of pretreatment of AA, without and with TNF-α stimulation on p-Akt/Akt, p-IkB, IkB, nuclear p65, NF-kB luciferase gene reporter activity, nuclear c-Myc expression, and 3H-thymidine incorporation in MCF-7 cells. We incubated the cells for 48 h in culture medium supplemented with BSA or 10, 50 and 100 mM AA, after which the same medium was added alone or with 10 ng/mL TNF-α.
Western blotting was performed to study the effect of AA treatment, with or without TNF-α stimulation on the p-Akt/Akt ratio (Fig 3). Two-way ANOVA revealed a main effect of both AA and TNF-α without a AA x TNF-α interaction on p-Akt/Akt ratio. The p-Akt/Akt ratio was significantly increased in response to treatment with 10-100 mM AA for 48 h, and was significantly induced by treatment with TNF-α for 20 min. Moreover, p-Akt/Akt ratios induced by TNF-α were significantly enhanced after pretreatment with 10-100 mM AA for 48 h, compared to controls (0 mM).
Furthermore, the effect of AA and TNF-α interaction on p-IkBa, IkBa and nuclear p65 expression was revealed by two-way ANOVA in the cells that received treatment with both, AA and TNF-α (Fig. 4). AA treatment alone had no effect on p-IkB (Fig 4B), IkBa (Fig 4C) and nuclear p65 (Fig 4D) expression. Significant effects were observed in TNF-α stimulated cells. Compared to the 0 mM AA with TNF-a stimulation, pretreatment with 50 or 100 mM AA, significantly enhanced TNF-α stimulated increase in p-IkBa, with a subsequent decrease in IkBa, and increase in nuclear p65 expression. These data suggest that 50 or 100 mM AA enhances the TNF-α-induced NF-kB activation.
To further evaluate NF-kB reporter luciferase activity after AA and TNF-α stimulation, we transfected MCF-7 cells with a NF-kB-driven luciferase reporter plasmid and pretreated it with 50 mM AA before, and TNF-α stimulation after, transfection. The NF-kB reporter luciferase activity was significantly increased by TNF-α stimulation and AA significantly enhanced the TNF-α stimulated luciferase activity (Fig 5). AA alone, without TNF-α stimulation, had no effect on the NF-kB reporter luciferase activity. Further, we used a PI3K/Akt inhibitor, LY294002, to study the effect of LY294002 and AA interaction on the NF-kB reporter luciferase activity. Two-way ANOVA revealed that LY294002, significantly decreased TNF-α stimulated NF-kB luciferase activity and AA significantly inhibited the effect of LY294002 on NF-kB reporter luciferase activity. These results suggest that AA alone has no effect on NF-kB activation, while AA pretreatment enhances TNF-α stimulation, with subsequent increase in p-Akt signaling, decrease in IkB expression and increase in NF-kB activation in MCF-7.
AA and TNF-α on nuclear c-Myc expression and 3H-thymidine incorporation assay in MCF-7
Western blotting was performed to study the effect of AA treatment with or without TNF-α stimulation, two-way ANOVA revealed a main effect of both AA and TNF-α without a AA x TNF-α interaction on nuclear c-Myc expression (Fig. 6A). Nuclear c-Myc expression was significantly increased by treatment with 10-100 mM AA for 48 h and was significantly induced by TNF-α treatment for 24 h. It is noteworthy that nuclear c-Myc expression induced by TNF-α was significantly enhanced after pretreatment for 48 h with 10-100 mM AA, compared to untreated controls (0 mM AA). The data suggests that AA can effectively increase and enhance TNF-α-induced nuclear c-Myc expression. 3H-thymidine incorporation assay (Fig. 6B), two-way ANOVA revealed main effect of AA but no effect of TNF-α and no AA x TNF-α interaction, indicating that treatment with AA significantly augmented MCF-7 cell proliferation.
These results indicates that AA supplemented in the culture medium was incorporated by the cells, which significantly increased the p-Akt/Akt ratio, nuclear c-Myc expression and 3H-thymidine incorporation for cell proliferation, but had no effect on NF-kB activation. However, stimulation with TNF-α significantly increased the NF-kB activation including p-IkB, nuclear p65 expression and NF-kB transcriptional activity, and nuclear c-Myc expression. Moreover, these effects were significantly enhanced after pretreatment with 50-100 mM AA. Interestingly, supplementation with 50 mM AA had no effect on NF-kB transcriptional activity, which was significantly increased in response to TNF-α stimulation, but was inhibited by Ly294002. Nevertheless, AA significantly enhanced TNF-α induction and reversed Ly294002 inhibition on NF-kB reporter activity. These results suggest that AA enhances TNF-α-induction, even though, it is unable to influence NF-kB activation, independently.