Long Term Effect of Curcumin in Restoration of Tumour Suppressor p53 and Phase-II Antioxidant Enzymes via Activation of Nrf2 Signalling and Modulation of Inflammation in Prevention of Cancer

<div><p>Inhibition of carcinogenesis may be a consequence of attenuation of oxidative stress via activation of antioxidant defence system, restoration and stabilization of tumour suppressor proteins along with modulation of inflammatory mediators. Previously we have delineated significant role of curcumin during its long term effect in regulation of glycolytic pathway and angiogenesis, which in turn results in prevention of cancer via modulation of stress activated genes. Present study was designed to investigate long term effect of curcumin in regulation of Nrf2 mediated phase-II antioxidant enzymes, tumour suppressor p53 and inflammation under oxidative tumour microenvironment in liver of T-cell lymphoma bearing mice. Inhibition of Nrf2 signalling observed during lymphoma progression, resulted in down regulation of phase II antioxidant enzymes, p53 as well as activation of inflammatory signals. Curcumin potentiated significant increase in Nrf2 activation. It restored activity of phase-II antioxidant enzymes like GST, GR, NQO1, and tumour suppressor p53 level. In addition, curcumin modulated inflammation via upregulation of TGF-β and reciprocal regulation of iNOS and COX2. The study suggests that during long term effect, curcumin leads to prevention of cancer by inducing phase-II antioxidant enzymes via activation of Nrf2 signalling, restoration of tumour suppressor p53 and modulation of inflammatory mediators like iNOS and COX2 in liver of lymphoma bearing mice.</p></div

Förder- und Sortiertechniken

<p>Effect of curcumin on mRNA expression and protease activity of MMP 9 and 2 in liver of lymphoma bearing mice (A) RT-PCR of MMP9, MMP2 and β-actin genes, (B) Densitometric scanning of MMP9 and MMP2 genes after normalization with β-actin, (C) Gelatin Zymography showing protease activity of MMP9 and MMP2, (D) Densitometric scanning of the activity band of MMP9 and MMP2. Livers of all six animals of each group were pooled separately and used for extraction of total RNA and proteins at non denaturing condition. Data represent mean ± S.E.M. #p<0.05 and ##<0.01 compared to N group, *p<0.05 and **p<0.01 compared to DL+DMSO group respectively. Cur is curcumin, M is 100 bp marker and bw is body weight. N, DL, DL+DMSO, DLT50, DLT100 and DLT150 represents normal, Dalton's lymphoma bearing, Dalton's lymphoma bearing mice treated with DMSO and Dalton's lymphoma bearing mice treated with 50, 100 and 150 mg curcumin/kg body weight dissolved in DMSO respectively.</p

Expression and activities of SOD isozymes.

<p>Effect of curcumin on mRNA expression and enzymatic activity of SOD isozymes in liver of lymphoma bearing mice (A) RT-PCR of Mn-SOD, Cu/Zn-SOD and β-actin gene, (B) Densitometric scanning of Mn-SOD and Cu/Zn-SOD genes after normalization with β-actin, (C) Specific staining showing activity of SOD isozymes, (D) Densitometric scanning of the activity band of SOD isozymes. Livers of all six animals of each group was pooled separately and used for extraction of total RNA and proteins at non denaturing condition. Data represent mean ± S.E.M. #p<0.05 compared to N group and *p<0.05 compared to DL+DMSO group respectively. Cur is curcumin, M is 100 bp marker and bw is body weight. N, DL, DL+DMSO, DLT50, DLT100 and DLT150 represents normal, Dalton's lymphoma bearing, Dalton's lymphoma bearing mice treated with DMSO and Dalton's lymphoma bearing mice treated with 50, 100 and 150 mg curcumin/kg body weight dissolved in DMSO respectively.</p

Expression and activity of GR.

<p>Effect of curcumin on expression & enzymatic activity of GR (A) RT-PCR of GR and β-actin (B) Densitometric scanning of GR after normalization with β-actin. (C) Specific staining showing activity of GR. (D) Densitometric scanning of the activity band of GR. Liver of all six animals of each group was pooled separately and used for extraction of total RNA and proteins. Data represent mean ± S.E.M. # and * denotes significant difference compared with N and DL+DMSO group respectively. Cur is curcumin and bw is body weight. N, DL, DL+DMSO, DLT50, DLT100 and DLT150 represents normal, Dalton’s lymphoma bearing, Dalton’s lymphoma bearing mice treated with DMSO and Dalton’s lymphoma bearing mice treated with 50, 100 and 150 mg curcumin/kg body weight dissolved in DMSO respectively.</p

Nrf2 activation (ARE binding).

<p>Effect of curcumin on DNA binding activity of Nrf2 with ARE consensus sequences. (A) Titration with unlabelled probe as specific competitor. (B) Titration with poly-dI/dC as non-specific competitor. (C) Autoradiogram showing Nrf2-ARE complex (D) Densitometric scanning of Nrf2-ARE complex. Liver of all six animals of each group was pooled separately and used for extraction of nuclear protein. Data represent mean ± S.E.M. # and * denotes significant difference compared with N and DL+DMSO group respectively. Cur is curcumin and bw is body weight. N, DL, DL+DMSO, DLT50, DLT100 and DLT150 represents normal, Dalton’s lymphoma bearing, Dalton’s lymphoma bearing mice treated with DMSO and Dalton’s lymphoma bearing mice treated with 50, 100 and 150 mg curcumin/kg body weight dissolved in DMSO respectively.</p

Nrf2 activation (NFE2 binding).

<p>Effect of curcumin on DNA binding activity of Nrf2 with NFE2 consensus sequences. (A) Titration with unlabelled probe as specific competitor. (B) Titration with poly-dI/dC as non-specific competitor. (C) Autoradiogram showing Nrf2-NFE2 complex (D) Densitometric scanning of Nrf2-NFE2 complex. Liver of all six animals of each group was pooled separately and used for extraction of nuclear proteins. Data represent mean ± S.E.M. # and * denotes significant difference compared with N and DL+DMSO group respectively. Cur is curcumin and bw is body weight. N, DL, DL+DMSO, DLT50, DLT100 and DLT150 represents normal, Dalton’s lymphoma bearing, Dalton’s lymphoma bearing mice treated with DMSO and Dalton’s lymphoma bearing mice treated with 50, 100 and 150 mg curcumin/kg body weight dissolved in DMSO respectively.</p

Nrf2 activation (NFE2 binding).

<p>Effect of curcumin on DNA binding activity of Nrf2 with NFE2 consensus sequences. (A) Titration with unlabelled probe as specific competitor. (B) Titration with poly-dI/dC as non-specific competitor. (C) Autoradiogram showing Nrf2-NFE2 complex (D) Densitometric scanning of Nrf2-NFE2 complex. Liver of all six animals of each group was pooled separately and used for extraction of nuclear proteins. Data represent mean ± S.E.M. # and * denotes significant difference compared with N and DL+DMSO group respectively. Cur is curcumin and bw is body weight. N, DL, DL+DMSO, DLT50, DLT100 and DLT150 represents normal, Dalton’s lymphoma bearing, Dalton’s lymphoma bearing mice treated with DMSO and Dalton’s lymphoma bearing mice treated with 50, 100 and 150 mg curcumin/kg body weight dissolved in DMSO respectively.</p

Expression of COX2.

<p>Effect of curcumin on mRNA expression and protein level of COX2 (A) RT-PCR of COX2 and β-actin (B) Densitometric scanning of COX2 mRNA after normalization with β-actin. (C) Western analysis of COX2 and β-actin (D) Densitometric scanning of COX2 after normalization with β-actin. Liver of all six animals of each group was pooled separately and used for extraction of total RNA and proteins. Data represent mean ± S.E.M. # and * denotes significant difference compared with N and DL+DMSO group respectively. Cur is curcumin and bw is body weight. N, DL, DL+DMSO, DLT50, DLT100 and DLT150 represents normal, Dalton’s lymphoma bearing, Dalton’s lymphoma bearing mice treated with DMSO and Dalton’s lymphoma bearing mice treated with 50, 100 and 150 mg curcumin/kg body weight dissolved in DMSO respectively.</p

<i>In vivo</i> angiogenesis assay.

<p>Effect of curcumin on in vivo angiogenesis studied by using Matrigel plug (A) Photographs showing angiogenesis pattern on peritoneal membrane of mouse of different groups, (B) Photographs showing appearance of colour of Matrigels due to vascularisation, removed from different groups, (C) Histogram showing haemoglobin content in Matrigel due to vascularisation (D) Histogram showing weight of Matrigel plug weighed just after removing from different groups. Data represent mean ± S.E.M. #p<0.05 and ##<0.01 compared to N group, *p<0.05 and **p<0.01 compared to DL+DMSO group respectively. N, DL, DL+DMSO, DLT50, DLT100 and DLT150 represents normal, Dalton's lymphoma bearing, Dalton's lymphoma bearing mice treated with DMSO and Dalton's lymphoma bearing mice treated with 50, 100 and 150 mg curcumin/kg body weight dissolved in DMSO respectively.</p

Total H₂O₂ and ROS level.

<p>Effect of curcumin on oxidative stress in terms of total H2O2 level and total ROS level in liver of lymphoma bearing mice (A) Histogram shows total H2O2 level in different groups, (B) Histogram shows total ROS level in different groups. Livers of all six animals of each group were pooled separately and homogenate was prepared for determination of H2O2 and ROS level. Data represent mean ± S.E.M. #p<0.05 compared to N group, *p<0.05 compared to DL+DMSO group respectively. N, DL, DL+DMSO, DLT50, DLT100 and DLT150 represents normal, Dalton's lymphoma bearing, Dalton's lymphoma bearing mice treated with DMSO and Dalton's lymphoma bearing mice treated with 50, 100 and 150 mg curcumin/kg body weight dissolved in DMSO respectively.</p