Novel Epigallocatechin-3-Gallate (EGCG) Derivative as a New Therapeutic Strategy for Reducing Neuropathic Pain after Chronic Constriction Nerve Injury in Mice

<div><p>Neuropathic pain is common in peripheral nerve injury and often fails to respond to ordinary medication. Here, we investigated whether the two novel epigallocatechin-3-gallate (EGCG) polyphenolic derivatives, compound <b>23</b> and <b>30</b>, reduce the neuropathic pain in mice chronic constriction nerve injury (CCI). First, we performed a dose-response study to evaluate nociceptive sensation after administration of EGCG and its derivatives <b>23</b> and <b>30</b>, using the Hargreaves test at 7 and 21 days after injury (dpi). We daily administered EGCG, <b>23</b> and <b>30</b> (10 to 100 mg/Kg; i.p.) during the first week post-CCI. None of the doses of compound <b>23</b> caused significant pain diminution, whereas 50mg/kg was optimal for both EGCG and <b>30</b> to delay the latency of paw withdrawal. With 50 mg/Kg, we showed that EGCC prevented the thermal hyperalgesia from 7 to 21 dpi and compound <b>30</b> from 14 to 56 dpi. To evaluate the molecular mechanisms underpinning why EGCG and compound <b>30</b> differentially prevented the thermal hyperalgesia, we studied several biochemical parameters in the dorsal horn of the spinal cord at 14 and 56 dpi. We showed that the effect observed with EGCG and compound <b>30</b> was related to the inhibition of fatty acid synthase (FASN), a known target of these polyphenolic compounds. Additionally, we observed that EGCG and compound <b>30</b> reduced the expression of CCI-mediated inflammatory proteins and the nuclear localization of nuclear factor-kappa B at 14 dpi, but not at 56 dpi. We also strongly detected a decrease of synaptic plasma membrane levels of N-methyl-D-asparte receptor 2B in CCI-mice treated with compound <b>30</b> at 56 dpi. Altogether, compound <b>30</b> reduced the chronic thermal hyperalgesia induced by CCI better than the natural compound EGCG. Thus, our findings provide a rationale for the preclinical development of compound <b>30</b> as an agent to treat neuropathic pain.</p></div

Decreased levels of NMDAR2B in the synaptic plasma membrane of spinal cord of CCI-mice treated with compound 30 at 56 dpi.

<p>(A) Protein extracts from the dorsal horn of the spinal cord of control mice (sham; sh) and CCI-mice treated with vehicle (VEH) and 50 mg/Kg of EGCG, compound <b>23</b> (c23) and compound <b>30</b> (c30) at 14 and 56 days post injury (dpi) were subjected to western blot to study the phospho-NMDAR2B and NMDAR2B protein levels. Data were expressed as a percentage with respect to sham-mice. Results are the mean± SEM (<i>n</i> = 5) and represent the ratio between phospho-NMDAR2B and NMDAR2B levels, obtained by densitometric analysis of western blot. Data were analyzed by one-way ANOVA with Bonferroni’s post-hoc test. **p<0.01 and ***p<0.001 compared to sham mice and ^<b>+</b>p<0.05 compared to vehicle-treated CCI-mice. Representative immuno-blots are presented. (B) The synaptic plasma membrane of the dorsal horn of the spinal cord at 56 days post injury (dpi) of all groups was obtained as indicated in materials and methods and then subjected to Western blot to study the levels of NMDAR2B. Data were expressed as a percentage with respect to sham-mice. Results are the mean± SEM (<i>n</i> = 5) and represent the ratio between NMDAR2B and N-cadherin levels, obtained by densitometric analysis of western blot. Data were analyzed by one-way ANOVA with Bonferroni’s post-hoc test. **p<0.01 compared to sham mice and ⁺p<0.05 compared to vehicle-treated CCI-mice. Representative immuno-blots are presented.</p

EGCG and compound 30 inhibit FASN activity in the spinal cord of CCI-mice.

<p>(A) The FASN activity was analyzed in the dorsal horn of the spinal cord of CCI-mice treated with vehicle, and 50 mg/Kg of EGCG, compound <b>23</b> (c23) and compound <b>30</b> (c30) as indicated in materials and methods at 14 and 56 days post injury (dpi). Data were expressed as a percentage with respect to vehicle-treated CCI-mice and represent the mean± SEM (<i>n</i> = 5). Data were analyzed by one-way ANOVA with Bonferroni’s post-hoc test. *p<0.05 and ***p<0.001 compared to vehicle-treated CCI-mice. (B) Protein extracts from the dorsal horn of the spinal cord of control mice (sham; Sh) and CCI-mice treated with vehicle (V), EGCG, compound <b>23</b> (c23) and compound <b>30</b> (c30) at 14 and 56 dpi were subjected to western blot to study the FASN protein levels. Representative immuno-blots showing no differences in FASN levels between all groups are presented. Actin levels were used as loading control.</p

Spinal cord of CCI-mice treated with EGCG and compound 30 at 14 dpi showed decreased levels of inflammatory cytokines

<p>(A) Total RNA of the dorsal horn of spinal cord of control mice (sham; Sh) and CCI-mice treated with vehicle (VEH) and 50 mg/Kg of EGCG, compound <b>23</b> (c23) and compound <b>30</b> (c30) was isolated at 14 and 56 days post injury (dpi) as indicated in materials and methods. Quantitative RT-PCR analysis of TNF-α, IL-1β and IL-6 was performed. Data were expressed as fold change normalized to 18S and represent the mean± SEM (<i>n</i> = 5). * p<0.05 and *** p<0.001 compared to vehicle-treated CCI-mice using a one-way ANOVA with Bonferroni’s post-hoc test. (B) Protein extracts from the dorsal horn of the spinal cord of control mice (sham; Sh) and CCI-mice treated with vehicle (VEH) and 50 mg/Kg of EGCG, compound <b>23</b> (c23) and compound <b>30</b> (c30) at 14 and 56 days post injury (dpi) were subjected to western blot to study the TNF-α, IL-1β and IL-6 protein levels. Data were expressed as a percentage with respect to sham-mice. Results are the mean± SEM (<i>n</i> = 5) and represent the ratio between each protein and actin levels, obtained by densitometric analysis of western blot. Data were analyzed by one-way ANOVA with Bonferroni’s post-hoc test. *** p<0.001 compared to sham mice and ⁺⁺⁺p<0.001 compared to vehicle-treated CCI-mice. (C) Representative immuno-blots are presented.</p

Administration of EGCG and compound 30 produce a time-dependent increase in CCI-mediated thermal paw withdrawal latency.

<p>The latency to paw withdrawal to a thermal stimulus was recorded in CCI-mice treated with vehicle and 50mg/Kg of EGCG (A), compound <b>23</b> (B) and compound <b>30</b> (C) from 7 to 56 days post injury. The results are expressed in seconds and data shown are the mean ± SEM (<i>n</i> = 15). Data were analyzed by two-way ANOVA with Bonferroni’s post-hoc test. *** p<0.001 compared to vehicle-treated CCI-mice.</p

EGCG, alone or combined with temsirolimus, inhibits tumor growth of sensitive and resistant HER2+ orthoxenopatients.

<p><b>(a)</b> Mice bearing HER2-PDX and resistant HER2-PDX (HER2-PDXR) were treated with control (C), EGCG (30 mg/kg, 3 days a week), temsirolimus (10 mg/kg, 1 day a week) or the combination (EGCG plus temsirolimus) for 21 days. Dots are mean of each experimental group and bars, SE. * (p ≤ 0.05), ** (p ≤ 0.01) and *** (p ≤ 0.001). <b>(b)</b> Apoptosis, by TUNEL fluorescent assay, was perfomed in control (C), EGCG (E), temsirolimus (T) and combination (T+E) treated as in (A) tumors. Tumors were collected at the end of the experiment and fixed in formalin. Pictures are representative of two samples of each group. <b>(c)</b> Body weight of the mice treated as in (A). Data are expressed as body weight at the end of the experiment and boxes show the 25th to 75th percentiles, whereas whiskers extend to the 5th and 95th percentiles.</p

FASN inhibitors improve pertuzumab and temsirolimus activity in parental and resistant cells.

<p><b>(a)</b> Cells were treated with pertuzumab (5 μg/ml) combined with EGCG (60 μM) or G28UCM (5 μM) for 5 days. Results were determined using an MTT assay and are expressed as ratio of inhibition of cell proliferation induced for each treatment alone versus inhibition induced for co- treatment from three independent experiments performed in triplicate. Dashed lines represent the effect of each drug alone, ratio 1. <b>(b)</b> Cells were treated with temsirolimus (0.05, 0.1, 0.5 and 1 μM) combined with EGCG (60 μM) or G28UCM (5 μM) for 2 days. Results were determined using an MTT assay and are expressed as ratio of inhibition of cell proliferation induced for each treatment alone versus inhibition induced for co- treatment from three independent experiments performed in triplicate and with several temsirolimus concentrations. Dashed lines represent the effect of each drug alone, ratio 1. * (p ≤ 0.05), ** (p ≤ 0.01) and *** (p ≤ 0.001) indicate levels of statistically significant difference compared with drugs administered alone.</p

EGCG, alone or combined with pertuzumab, inhibits tumor growth of sensitive and resistant HER2+ orthoxenopatients.

<p><b>(a)</b> Mice bearing HER2-PDX and resistant HER2-PDX (HER2-PDXR) were treated with control (C), EGCG (30 mg/kg, 3 days a week), pertuzumab (30 mg/kg, 1 day a week) or the combination (EGCG plus pertuzumab) for 24 days. Dots are mean of each experimental group and bars, SE. * (p ≤ 0.05), ** (p ≤ 0.01) and *** (p ≤ 0.001). <b>(b)</b> Apoptosis, by TUNEL fluorescent assay, was perfomed in control (C), EGCG (E), pertuzumab (P) and combination (P+E) treated as in (A) tumors. Tumors were collected at the end of the experiment and fixed in paraffin. Pictures are representative of two samples of each group. <b>(c)</b> Body weight of the mice treated as in (A). Data are expressed as body weight at the end of the experiment and boxes show the 25th to 75th percentiles, whereas whiskers extend to the 5th and 95th percentiles.</p

Pertuzumab plus trastuzumab combination improves effects in SK and SKLTR.

<p>Cells were treated with trastuzumab (20 μM), pertuzumab (5 μg/ml) and the combination of both for 5 days. Results were determined using an MTT assay and are expressed as the percentage of cell proliferation inhibition from three independent experiments performed in triplicate. Columns represent % of cell proliferation inhibition after trastuzumab or pertuzumab exposure and bars SE. * (p ≤ 0.05), ** (p ≤ 0.01) and *** (p ≤ 0.001) indicate levels of statistically significant difference compared with effect of the same drug in SKBr3 cells or compared with drugs administered alone (dashed line).</p

New Synthetic Inhibitors of Fatty Acid Synthase with Anticancer Activity

Fatty acid synthase (FASN) is a lipogenic enzyme that is highly expressed in different human cancers. Here we report the development of a new series of polyphenolic compounds <b>5</b>–<b>30</b> that have been evaluated for their cytotoxic capacity in SK-Br3 cells, a human breast cancer cell line with high FASN expression. The compounds with an IC₅₀ < 50 μM have been tested for their ability to inhibit FASN activity. Among them, derivative <b>30</b> blocks the 90% of FASN activity at low concentration (4 μM), is highly cytotoxic in a broad panel of tumor cells, induces apoptosis, and blocks the activation of HER2, AKT, and ERK pathways. Remarkably, <b>30</b> does not activate carnitine palmitoyltransferase-1 (CPT-1) nor induces in mice weight loss, which are the main drawbacks of other previously described FASN inhibitors. Thus, FASN inhibitor <b>30</b> may aid the validation of this enzyme as a therapeutic target for the treatment of cancer