The curves adopted a hyperbolic pattern achieving a hundred% inhibition with S-(1,2-Dichlorovinyl)-L-cysteineall the proteasomes (Determine 1A-C) and CLA isomers analyzed, althought t10,c12- and c9,t11-CLA were the very best effectors (IC50=fourteen.eight. M and 31.two.8 M on 20S isoform and 1.one.two M and 6.four. M on 20Si isoform for t10,c12- and c9,t11-CLA, respectively). Similar experiments ended up carried out by making use of the proteasome inhibitor bortezomib (BTZ) (Figure 1D) simply because of its identified anti-proliferative action on most cancers cells. As anticipated, BTZ appeared to target each 20S (IC50=one.4.3 nM) and 20Si (IC50=2.1.five nM) isoforms indiscriminately, reaching about sixty% of inhibition in both cases, although octanoic acid (information not proven), utilised as a unfavorable manage, gave only negligible outcomes. Following, prior to to examine the feasible mechanisms underlying the CLA-lowered viability of most cancers cells, the likely contribution of APEH was explored. When the capacity of these compounds to modulate APEH in cell-totally free assays was evaluated (Figure 1E), only t10,c12-CLA was exposed to influence the enzyme activity in a dose-dependent way, reaching a greatest inhibition of about forty one% (IC50=110.eleven.7 M) as calculated by SigmaPlot ten. application. Therefore, a stereoselective binding in the interaction with APEH and proteasome isoforms of the CLA isomers can be proposed collectively with a specific potential of t10,c12-CLA to inhibit all these enzymes mobile applicant for additional investigations. As revealed in Determine 2A, when basal certain APEH exercise was plotted against the corresponding proteasomal chymotrypsin-like (CT-like) activity, a significant optimistic correlation was located (r2=.988, P=<0.01), supporting the idea of a functional relationship between these two enzymes which could act in cooperation for degradation of damaged proteins [15,16]. Moreover, on the basis of gene expression analysis and intracellular protein levels (Figure 2B and 2C), the different cancer cells could be divided into two groups displaying low (U87, HeLa, MDA-MB and MCF7: Group I) or high (A375, A375M, HepG2 and Caco2: Group II) protein, activity and transcript levels of APEH and proteasome (-5 subunit). Data on the immunoproteasome subunit (-5i subunit) were not reported due to the low detectable levels in all the cancer cells investigated. These findings suggested that cells exhibiting high basal activity and expression levels of APEH and proteasome could be highly dependent on these enzyme functions and therefore more sensitive to their down-regulation.The susceptibility of cell lines belonging to Group I and Group II to the growth inhibitory effects of CLA isomers was estimated upon 24h exposure at concentrations ranging from 50 to 200 M. Data indicated that only A375, A375M and MDAMB cells exhibited a moderate reduction (<40%) of cell viability by c9,t11-CLA treatment (Figure 3A), while a more marked anti-proliferative effect was observed following cell exposure with t10,c12-CLA on HeLa, A375M and A375 (40, 51 and 63% respectively) (Figure 3B). A375 cell viability was also greatly influenced by t9,t11-CLA (about 50%) (Figure 3C), whereas no significant results were obtained on all cancer cells by octanoic acid (up to 200 M) treatment, which was used as a negative control (data not shown). To assess the cytotoxicity and antiproliferative activity of the most abundant CLA isomers on the cancer cells considered, LDH activity was measured in spent media following 24h exposure to 200 M c9,t11-, t10,c12-CLA or to 10 nM BTZ, using octanoic acid as negative control. As expected, substantial cell death resulted from BTZ supplementation while the LDH activity in cultures exposed to CLA isomers was comparable to that of control (Figure 4A). Moreover, to examine the contribution of an apoptotic event in CLA-induced decline of cancer cells viability, caspase 3 activation was measured. Interestingly, results revealed that while caspase 3 activation varied slightly between the different tumor cell lines upon exposure with c9,t11-CLA, a more marked variation was observed by t10,c12-CLA treatment (Figure 4B), leading to inversely correlated measures of cell viability and caspase 3 activation (r2=0.78 P<0.01) (Figure 4C). It's worth to note that, although CLA reduced cell viability in the considered cell lines with no cytotoxicity (LDH release), nevertheless its proapoptotic activity couldn't be accounted for the observed cell death, therefore a cytostatic effect cannot be excluded. In addition, proteasome activity was differently downregulated by CLA isomers (Figure S1) but it was not significantly correlated with cell viability decrease in evaluating the involvement of APEH and proteasome in the anti-cancer effects of CLA isomers, we decided to examine the basal expression/activity levels of these enzymes in eight cancer cell lines (at their pre-confluent stage) to select the best fatty acids and CLA isomers exhibit dissimilar inhibitory ability towards chymotrypsin-like (CT-like) proteasome and APEH activities. The inhibitory effect of different CLA isomers, namely c9,t11- (A), t9,t11- (B), t10,c12-CLA (C), bortezomib, BTZ (D), was evaluated on commercially available pure 20S (black circles), 20Si (white circles) and 26S (black triangles) proteasomes. The synthetic fluorescent substrate N-Suc-LLVT-AMC (0.080 mM) was used for the measurement of the CT-like activity of the proteasomes. The hyperbolic curves indicate the best fits for the data obtained, with IC50 values calculated from the graphs by SigmaPlot 10.0 software. Mixtures treated with DMSO alone were used as blank. The dose-dependent inhibitory effect of c9,t11-, t9,t11-, t10,c12-CLA isomers, octanoic acid or bortezomib on APEH activity was shown (E). Results are presented as the mean standard deviation (SD) of triplicate analyses from three independent experiments. SD values lower than 5% were not shown suggesting that proteasome inhibition alone was not liable for the observed anti-proliferative activity of CLAs. Hence, it appears reasonable to hypothesize that an enzyme machinery, such as APEH/proteasome system, could be involved in the marked anti-proliferative and pro-apoptotic activity exerted by t10,c12-CLA through its specific capacity to down-regulate both enzymes (Figure 1).Human cancer cell lines may be grouped according to the basal enzyme activities and expression levels of APEH and proteasome. Cells from eight human cancer lines (U87, MCF7, MDA-MB, HeLa, Caco2, HepG2, A375M, A375) and non-cancerous cells (BHk21, fibroblasts) were harvested at the pre-confluent stage and used for cytoplasmic or mRNA extract preparation. Basal APEH and proteasomal CT-like activities were measured in cytoplasmic extracts (A). The mRNA levels of APEH and -5 subunit were evaluated by qRT-PCR and expressed as fold change in comparison to expressed levels in human fibroblast (B). Intracellular levels of -5 and APEH were detected by immunoblotting (C upper panel). Typical Western blot was shown and data from three different analyses were normalized to the density of control protein (-actin) and expressed as ratio over control (C lower panel). Results are presented as the mean values D of triplicate analyses from at least three different experiments. Significantly different (P < 0.01) from respective controls.On the basis of the marked cell viability reduction (Figure 3B) induced by t10,c12-CLA on A375 melanoma cell line, we decided to use this model system for investigations on the different cellular factors (redox status, caspase 3, APEH and proteasome) involved in the apoptotic pathway. In order to define the dose accountable for 50% decrease of cell viability (IC50), A375 cells were exposed for 24h to a concentration range of t10,c12-CLA or BTZ (from 10 nM to 400 M), using human fibroblasts as control. The resulting isobologram revealed that the IC50 values were 1.0.02 M or 10.0.02 nM for t10,c12-CLA or BTZ, respectively. Moreover, proliferation data obtained from fibroblasts, even at higher concentration of t10,c12-CLA, further supported the lack of toxic effects (Figure 5A). Next, cultures were incubated with increasing t10,c12-CLA doses (50, 100 or 200 M) and the possible additive effect elicited by sub-toxic amount of BTZ (5 nM) was evaluated in cells co-incubated with t10,c12-CLA for 24h. The results obtained (Figure 5B) demonstrated that the dose-dependent activation of caspase 3 was triggered by t10,c12-CLA, reaching an eightfold increase compared to the control culture. Notably, pro-apoptotic induction, associated with a significant decline in intracellular GSH, was not further improved by BTZ supplementation (Figure 5B). Similarly, APEH and proteasome mRNA levels were strongly down-regulated by 200 M t10,c12CLA treatment (Figure 5C, right panel) and only minor alterations were produced by the addition of BTZ (data not shown). Interestingly, while a dose-dependent inhibition of APEH activity was observed, the proteasomal CT-like activity was inhibited to 46 and 50% by 50 and 100 M t10,c12-CLA, respectively and a less marked effect resulted from cells exposed to 200 M CLA (25%) (Figure 5C, left panel). Moreover, the decline of APEH and -5 protein expression only occurred at the higher CLA dose (p<0.05) (Figure 5D). In addition, the noticeable decrease of the anti-apoptotic protein Bcl-2 expression, reaching the maximum reduction of 80%, further supported the role of apoptosis in the anti-proliferative effect of t10,c12-CLA (Figure 5D). Finally, we showed that cell exposure to high t10,c12-CLA doses markedly down-regulated the Nrf2 pathway, as evidenced by the declined mRNA levels of some target genes (NQO1 and GCL), expressed as fold change in comparison to untreated cells (Figure S2). These findings support the hypothesis that the combined down-regulation of antioxidant/detoxifying defences, APEH/ proteasome system and Bcl-2 levels, may play an important role in apoptosis induction triggered by t10,c12-CLA in A375 cells.Time-dependent monitoring of ROS production, APEH and proteasome (-5) at mRNA and enzyme activity level, was performed to evaluate the effects produced by the exposure to lower (50 M) or higher (200 M) t10,c12-CLA concentrations, on pre-confluent A375 cells. Sudden decrease (2h) of APEH and proteasomal CT-like activities in cells exposed to low doses, correlated with a transient reduction of their mRNA expression. Upon this early response, enzyme activities recovered, reaching a plateau after 8h with values corresponding to 80 or 70% of their starting values, respectively (Figure 6A). Similarly, mRNA profiles showed a short-lived gene repression, which quickly recovered towards the stable final values, being approximately one-fold lower than their initial expression level (Figure 6B). Conversely, the higher concentration of t10,c12-CLA produced a downshift of APEH activity reaching a plateau with average values of 70% compared to its starting level, whereas a long-term downregulation of proteasomal activity persisted up to 16h (Figure 6C). Interestingly, two transient minima of mRNA levels were observed after 2 and 6h, followed by a significant increase until 16h. After 24h of incubation, APEH and -5 expression decreased again reaching the corresponding lowest values (Figure 6D). The time-dependent ROS production indicated that the early down-regulation of APEH /proteasome enzyme activities could be induced by ROS yield (Figure 6A), while a direct modulation of the CLA isomer on both enzymes can possibly contribute to the following decrease of the activity/mRNA levels observed at 200 M (Figure 6C,D). Cell pre-incubation with the antioxidant N-acetyl cysteine (NAC, 5 mM) before the 200 M CLA exposure (2 or 24h) resulted in a marked cytotoxic effect (data not shown). Finally, time-dependent effects elicited by 200 M t10,c12CLA on GSH concentration and caspase 3 activity, together with GCL mRNA levels, were measured. As shown in Figure 7A, the decline of intracellular GSH was followed by caspase 3 activation (after 6-8h). To investigate the mechanism underlying the pro-oxidant activity of t10,c12-CLA, the mRNA expression of the rate-limiting enzyme responsible for cellular GSH synthesis (namely GCL) was monitored. As expected, the early activation triggered by CLA isomer (after 2h) was followed by a transient decrease in mRNA (peaking after 4h),human cancer cells exhibit differential sensitivity to the anti-proliferative activity of CLA isomers. The effects of c9,t11 (A), t10,c12- (B) or t9,t11-CLA isomers (C) on cell viability were assessed in eight cancer cell lines exposed for 24h to increasing concentrations of the CLA isomers. Data are expressed as means D values of triplicate data from three independent experiments. SD values lower than 5% were not shown.Anti-proliferative ability of t10,c12-CLA correlates with caspase 3 activation. LDH release (A) and caspase 3 activity (B) were measured to study the cytotoxic and pro-apoptotic ability of 200 of t10,c12- (dark grey bars) or c9,t11-CLA (light grey bars). Cell cultures exposed to octanoic acid (200 , black bars) or to BTZ (10 nM, white bars) were used as negative or positive controls, respectively. Average caspase 3 activity values (fold increase) in cancer cells exposed for 24h to 200 t10,c12(C upper panel) or to c9,t11- CLA (C lower panel) were plotted against cell viability (%)which temporarily recovered before leading to the downregulation (1.5 fold) of mRNA levels (Figure 7B).19561098Owing to their enhanced metabolic activity, cancer cells require elevated levels of energy to maintain a high rate of cell growth and proliferation. This is also guaranteed by an improved activity of the Ubiquitin-Proteasome System, which is the major pathway for protein turnover in eukaryotes [29], providing a secondary antioxidant defence mechanism, in combination with APEH [15,16]. Indeed, protein homeostasis is critically involved in cancer cell survival thus, one of the major focus in cancer research is targeting the balance between the production and destruction of proteins mediating cell proliferation. In this context, proteasome inhibition represents a novel strategy against many tumoral diseases, triggering an increase in apoptosis and decrease in cellular growth. Accordingly, in the last decade, research and development of new compounds able to down-regulate proteasome functions have attracted growing attention. It is known that the pro-apoptotic ability of CLA mixture (c9,t11- and t10,c12-CLA 50:50) or its individual isomers, affects tumor cell proliferation via different biochemical pathways involving apoptotic or survival genes (Bcl-2, p21, p53). The efficacy of these isomers in inhibiting the cancer cell viability was highly influenced by the model system used, within a concentration range of 1-200 mol/L and treatment lasting 1-11 days [18]. Specifically, t10,c12-CLA has revealed a more efficient activity, respect to c9,t11-CLA isomer, in modulating apoptosis or cell cycle. In human prostatic carcinoma cells, t10,c12-CLA anticancer effect associates to decreased Bcl-2 and increased p21(WAF1/Cip1) mRNA levels [30] while in human colon or bladder cancer cells it was accompanied by the activation of ATF/NAG-1 [31] or Insulin Growth Factor signaling [32]. Moreover, it was reported that t10,c12-CLA was able to down-regulate Fatty Acid Synthase [33] or antioxidant defence systems [21,22,34] in different human cancer cells.
