Genistein improves 3-NPA-induced memory impairment in ovariectomized rats: impact of its antioxidant, anti-inflammatory and acetylcholinesterase modulatory properties.

Huntington's disease (HD) is a progressive neurodegenerative disorder. The pre-motor symptomatic stages of the disease are commonly characterized by cognitive problems including memory loss. 3-Nitropropionic acid (3-NPA) is a mitochondrial toxin that produces selective lesions in the brain similar to that of HD and was proven to cause memory impairment in rodents. Phytoestrogens have well-established neuroprotective and memory enhancing effects with fewer side effects in comparison to estrogens. This study investigated the potential neuroprotective and memory enhancing effect of genistein (5, 10 and 20 mg/kg), a phytoestrogen, in ovariectomized rats challenged with 3-NPA (20 mg/kg). These potential effects were compared to those of 17β-estradiol (2.5 mg/kg). Systemic administration of 3-NPA for 4 consecutive days impaired locomotor activity, decreased retention latencies in the passive avoidance task, decreased striatal, cortical and hippocampal ATP levels, increased oxidative stress, acetylcholinesterase (AChE) activity, cycloxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) expressions. Pretreatment with genistein and 17β-estradiol attenuated locomotor hypoactivity, increased retention latencies in the passive avoidance task, increased ATP levels, improved the oxidative stress profile, attenuated the increase in AChE activity and decreased the expression of COX-2 and iNOS. Overall, the higher genistein dose (20 mg/kg) was the most effective. In conclusion, this study suggests neuroprotective and memory enhancing effects for genistein in a rat model of HD. These effects might be attributed to its antioxidant, anti-inflammatory and cholinesterase inhibitory activities

The chemomodulatory effects of glufosfamide on docetaxel cytotoxicity in prostate cancer cells

Background. Glufosfamide (GLU) is a glucose conjugate of ifosfamide in which isophosphoramide mustard is glycosidically linked to the β-D-glucose molecule. Based on GLU structure, it is considered a targeted chemotherapy with fewer side effects. The main objective of the current study is to assess the cytotoxic potential of GLU for the first time in prostate cancer (PC) cells representing different stages of the tumor. Furthermore, this study examined the potential synergistic activity of GLU in combination with docetaxel (DOC). Methods. Two different cell lines were used, LNCaP and PC-3. Concentration-response curves were assessed. The tested groups per cell line were, control, GLU, DOC and combination. Treatment duration was 72 h. Cytotoxicity was assessed using sulforhodamine B (SRB) assay and half maximal inhibitory concentration (IC50) was calculated. Synergy analyses were performed using Calcusyn®software. Subsequent mechanistic studies included β-glucosidase activity assay, glucose uptake and apoptosis studies, namely annexin V-FITC assay and the protein expression of mitochondrial pathway signals including Bcl-2, Bax, Caspase 9 and 3 were assessed. Data are presented as mean ± SD; comparisons were carried out using one way analysis of variance (ANOVA) followed by Tukey-Kramer’s test for post hoc analysis. Results. GLU induced cytotoxicity in both cell lines in a concentration-dependent manner. The IC50 in PC-3 cells was significantly lower by 19% when compared to that of LNCaP cells. The IC50 of combining both drugs showed comparable effect to DOC in PC-3 but was tremendously lowered by 49% compared to the same group in LNCaP cell line. β-glucosidase activity was higher in LNCaP by about 67% compared to that determined in PC-3 cells while the glucose uptake in PC-3 cells was almost 2 folds that found in LNCaP cells. These results were directly correlated to the efficacy of GLU in each cell line. Treatment of PC cells with GLU as single agent or in combination with DOC induced significantly higher apoptosis as evidenced by Annexin V-staining. Apoptosis was significantly increased in combination group by 4.9 folds and by 2.1 Folds when compared to control in LNCaP cells and PC-3 cells; respectively. Similarly, the expression of Bcl-2 was significantly decreased while Bax, caspase 9 and 3 were significantly increased in the combined treatment groups compared to the control. Conclusion. GLU has a synergistic effect in combination with DOC as it increases the cell kill which can be attributed at least partially to apoptosis in both the tested cell lines and it is suggested as a new combination regimen to be considered in the treatment of the prostate cancer. Further experiments and clinical investigations are needed for assessment of that regimen

Effects of 3-NPA and/or genistein on striatal, cortical and hippocampal AChE activity of ovariectomized rats.

<p>3-NPA was administered i.p. (20 mg/kg) for 4 consecutive days. 17β-estradiol (2.5 mg/kg body weight, s.c) and genistein (10 and 20 mg/kg body weight, i.p) were administered for 8 days, beginning 4 days before and continued for 4 days one hour before 3-NPA injections. Data are presented as means ±S.E.M. (n = 6). ^x statistically significant compared to sham group at P< 0.05, * statistically significant compared to control group at P< 0.05, ^# statistically significant compared to 3-NPA-treated group at P< 0.05 (one-way ANOVA followed by Tukey test).</p

Effects of 3-NPA and/or genistein on striatal, cortical and hippocampal AChE activity of ovariectomized rats.

<p>3-NPA was administered i.p. (20 mg/kg) for 4 consecutive days. 17β-estradiol (2.5 mg/kg body weight, s.c) and genistein (10 and 20 mg/kg body weight, i.p) were administered for 8 days, beginning 4 days before and continued for 4 days one hour before 3-NPA injections. Data are presented as means ±S.E.M. (n = 6). ^x statistically significant compared to sham group at P< 0.05, * statistically significant compared to control group at P< 0.05, ^# statistically significant compared to 3-NPA-treated group at P< 0.05 (one-way ANOVA followed by Tukey test).</p

Effects of 3-NPA and/or genistein on step through passive avoidance in ovariectomized rats.

<p>3-NPA was administered i.p. (20 mg/kg) for 4 consecutive days. 17β-estradiol (2.5 mg/kg body weight, s.c) and genistein (5, 10 and 20 mg/kg body weight, i.p) were administered for 8 days, beginning 4 days before and continued for 4 days one hour before 3-NPA injections. Training was performed on day 5. First and second retention latencies were assessed at days 6 (A) and 8 (B). Data are presented as medians and quartiles (n = 8). ^x statistically significant compared to sham group at P< 0.05, * statistically significant compared to control group at P< 0.05, ^# statistically significant compared to 3-NPA-treated group at P< 0.05 (Kruskal-Wallis nonparameteric test followed by Dunn’s test).</p

Effects of 3-NPA and/or genistein on locomotor activity in ovariectomized rats.

<p>3-NPA was administered i.p. (20 mg/kg) for 4 consecutive days. 17β-estradiol (2.5 mg/kg body weight, s.c) and genistein (5, 10 and 20 mg/kg body weight, i.p) were administered for 8 days, beginning 4 days before and continued for 4 days one hour before 3-NPA injections. Data are presented as means ±S.E.M. (n = 8). ^x statistically significant compared to sham group at P< 0.05, * statistically significant compared to control group at P< 0.05, ^# statistically significant compared to 3-NPA-treated group at P< 0.05 (two-way ANOVA followed by Bonferroni test).</p

H & E staining of the hippocampi of rats belonging to the sham group (A), control group (B), 3-NPA-treated group (C), 17β-estradiol- and 3-NPA-treated group (D), genistein (5 mg/kg)- and 3-NPA-treated group (E), genistein (10 mg/kg)- and 3-NPA-treated group (F), genistein (20 mg/kg)- and 3-NPA-treated group (G) and genistein alone-treated group (H): A, B, D, F, G and H showed no histological alterations, C showed severe neurodegeneration (d) and hemorrhage (h) and E showed some degenerated hippocampal cells (d).

<p>H & E staining of the hippocampi of rats belonging to the sham group (A), control group (B), 3-NPA-treated group (C), 17β-estradiol- and 3-NPA-treated group (D), genistein (5 mg/kg)- and 3-NPA-treated group (E), genistein (10 mg/kg)- and 3-NPA-treated group (F), genistein (20 mg/kg)- and 3-NPA-treated group (G) and genistein alone-treated group (H): A, B, D, F, G and H showed no histological alterations, C showed severe neurodegeneration (d) and hemorrhage (h) and E showed some degenerated hippocampal cells (d).</p

Immunohistochemical staining of cortical COX-2-positive cells immunized with goat-anti-rabbit antibodies of the sham group (A), control group (B), 3-NPA-treated group (C), 17β-estradiol- and 3-NPA-treated group (D), genistein (10 mg/kg)- and 3-NPA-treated group (E), genistein (20 mg/kg)- and 3-NPA-treated group (F) and genistein alone-treated group (G).

<p>Immunohistochemical staining of cortical COX-2-positive cells immunized with goat-anti-rabbit antibodies of the sham group (A), control group (B), 3-NPA-treated group (C), 17β-estradiol- and 3-NPA-treated group (D), genistein (10 mg/kg)- and 3-NPA-treated group (E), genistein (20 mg/kg)- and 3-NPA-treated group (F) and genistein alone-treated group (G).</p

H & E staining of the cortices of rats belonging to the sham group (A), control group (B), 3-NPA-treated group (C), 17β-estradiol- and 3-NPA-treated group (D), genistein (5 mg/kg)- and 3-NPA-treated group (E), genistein (10 mg/kg)- and 3-NPA-treated group (F), genistein (20 mg/kg)- and 3-NPA-treated group (G) and genistein alone-treated group (H): A, B, D and H showed no histological alterations, C showed severe hemorrhage (h), E showed focal gliosis (g) F showed focal gliosis (g) and G showed congested blood vessel (C).

<p>H & E staining of the cortices of rats belonging to the sham group (A), control group (B), 3-NPA-treated group (C), 17β-estradiol- and 3-NPA-treated group (D), genistein (5 mg/kg)- and 3-NPA-treated group (E), genistein (10 mg/kg)- and 3-NPA-treated group (F), genistein (20 mg/kg)- and 3-NPA-treated group (G) and genistein alone-treated group (H): A, B, D and H showed no histological alterations, C showed severe hemorrhage (h), E showed focal gliosis (g) F showed focal gliosis (g) and G showed congested blood vessel (C).</p