The authors have declared that no competing interests exist.
Conceived and designed the experiments: LD MV. Performed the experiments: LD. Analyzed the data: LD MV. Contributed reagents/materials/analysis tools: MV. Wrote the paper: LD MV.
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.
Nuclear factor E2-related factor 2 (Nrf2) is a critical transcription factor that binds to promotor region of a number of genes encoding phase-II antioxidant enzymes [
Wild-type p53 and TGF-β are key tumour suppressors which regulate an array of cellular responses. p53 physically interacts with Smads and co-ordinately induces transcription of a number of key tumour suppressive genes [
The use of whole extract of herbal source or single-isolated constituent or a metabolite of an isolated constituent, to achieve desirable health benefits has been an issue of the past several years. Curcumin, a polyphenolic phytochemical is reported to inhibit chemically induced carcinogenesis at multiple organ sites in various animal models. Rapid metabolism and elimination are major factors of low bioavailability of curcumin at tissue levels, irrespective of the route of administration, though metabolites of curcumin remain for longer time in different tissues. However, long term effect of curcumin, after withdrawal of administration is still to be reported. It is not clear whether anti-carcinogenic action of curcumin is due to cumulative effect of its metabolites or due to curcumin itself. Further, it is evidenced that the consumption of whole or partially purified food extracts is more beneficial over single-isolated constituent due to the existence of synergistic interactions among phytochemicals in whole foods [
All chemicals were of analytical and molecular biology grade as well as endotoxin free, and used without further purification. Curcumin, reagents for RNA isolation, Taq polymerase, PMSF, menadione, Sephadex G50 and HRP conjugated anti-β actin antibody were purchased from Sigma Aldrich. Reverse Transcriptase, Ribonuclease Inhibitor, random primers (hexamers), 100 bp Plus DNA ladder and Klenow enzyme from Fermentase Life Science and TURBO DNA-FreeTM Kit I were purchased from Ambion. Gene specific primers for RT-PCR were synthesized from Metabion. Anti-mouse p53 antibody from Imgenix, anti-mouse iNOS and COX2 antibody from Cayman and HRP conjugated goat anti-rabbit secondary antibody from Bangalore Genie. Radiolabelled α32P-dCTP from Board of Radiation and Isotope Technology (BRIT) and ECL (Super signal Kit) was purchased from PIERCE Biotechnology HYSEL India Pvt. Ltd.
Mice (
Development of DL was confirmed by abdominal swelling and increased body weight, which were visible clearly on 10–11 day post transplantation and DL mice survived for 20±2 days. Growth of Dalton’s lymphoma did not show any major change in body weight and ascite fluid accumulation during first 7–9 days starting from next day of DL transplantation (
Six groups of mice were taken with 6 mice in each group (n = 6) and curcumin treatment to DL mice was scheduled as described previously [
Nuclear protein was extracted and binding affinity to Nrf2 sequences was determined by electrophoretic mobility shift assay (EMSA) as described previously [
RNA isolation, cDNA synthesis and amplification were done as described earlier [
Activity of GR and NQO1 as well as activity and isozyme patterns of GST was measured by in gel activity staining. Non-denaturing PAGE analysis of antioxidant enzymes was preferred over immunodetection, because change in activity of an enzyme is associated with metabolic changes and the method utilizes substrate specificity based detection of only active part of enzymes in the same gel. It is considered highly relevant for correlating a change in level of a specific isozyme with that of metabolic alterations at cellular level [
The activity gel was performed by non-denaturing PAGE, as per the method of Ricci et al. with minor modification [
The activity staining of GR was performed according to the method of Hou et al. [
In-gel activity staining of NQO1 was conducted as described by Wrobel et al. [
Total glutathione and reduced glutathione were determined as total sulfhydryl (T-SH) content and non-protein sulfhydryl (NP-SH) content respectively using molar absorption coefficient of 13100 M-1 cm-1, and were expressed in micro moles per mg protein [
Equal amount of protein from each sample was separated in 10% SDS-PAGE and transferred to the PVDF membrane overnight at 4°C. Membrane was blocked in 5% non-fat milk in PBS (pH 7.4) for 2h at RT. Blot was incubated overnight at 4°C with rabbit anti-mouse p53 (1:500 dilution, Imgenix) or rabbit anti-mouse iNOS (1:200 dilution, Cayman) or rabbit anti-mouse COX2 (1:500 dilution, Cayman) in 1% BSA and 0.05% Tween-20 in PBS (pH 7.4). Blot was washed and incubated with HRP-conjugated goat anti-rabbit IgG (1:2500 dilution, Bangalore Genei) in PBS (pH 7.4) containing 1% BSA and 0.05% Tween-20 for 2 h at RT. Immunoreactive proteins were detected with ECL super signal kit (Pierce Biotechnology) in X-ray film. Band density values were normalized with β-actin.
NO level in liver was estimated by using Griess reagent; 100μl of liver homogenate was mixed with 100μl of Griess reagent (0.1% naphthylethylenediamine dihydrochloride, and 1% sulfanilamide in 5% phosphoric acid), incubated for 10 min at RT and absorbance was measured at 540 nm with a plate reader (ECL ELISA reader). The concentration of NO was determined using a standard curve, generated by taking known quantities of sodium nitrite and values were expressed in terms of μg equivalence of NaNO2/mg protein
Statistical analysis was performed by SPSS software using one-way ANOVA followed by Tucky’s test. Values were expressed as mean ± S.E.M. obtained from three different sets of experiments, p<0.05 was taken as statistically significant (95% confidence interval) compared to N group (#) and DL+DMSO group (*).
Specificity and binding affinity of synthesized oligonucleotide corresponding to antioxidant response element (ARE consensus sequence) was validated. Titration with unlabelled probe (cold probe) as specific competitor and with non-specific competitor (poly-dI/dC) confirmed specificity and affinity respectively [
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.
Titration with unlabelled probe as specific competitor and poly-dI/dC as non-specific competitor confirmed specificity and affinity respectively [
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.
The expression of Nrf2 was down regulated in DL and DL+DMSO mice upto approximately 63% and 73% of normal mice respectively, which was significantly up regulated towards normal level by curcumin treatment. The expression of Nrf2 was 107%, 127% and 108% of DL+DMSO with 50, 100 and 150 mg curcumin/kg bw respectively [
Effect of curcumin on mRNA expression of Nrf2. (A) RT-PCR of Nrf2 and β-actin. (B) Densitometric scanning of Nrf2 after normalization with β-actin. Liver of all six animals of each group was pooled separately and used for extraction of total RNA. 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.
The expression and activity of five isozymes of GST namely GSTa, GSTm, GSTp, GSTt and GSTo were observed in mouse liver, where GSTa is found to be the most abundant isozyme. As compared to normal mice, expression of GSTa, GSTm, GSTp, GSTt and GSTo was down regulated approximately upto 30%, 71%, 80%, 50% and 71% respectively in DL mice and approximately 35%, 61%, 82%, 68%, and 76% respectively in DL+DMSO mice. Curcumin treatment induced expression of five isozymes with 50, 100 and 150 mg curcumin/kg bw as compared to DL+DMSO, which was as follows—GSTa: 137%, 180%, 150%; GSTm: 117%, 110%, 125%; GSTp: 113%, 107%, 108%; GSTt: 102%, 122%, 115%; GSTo: 107%, 108% and 102% respectively [
Effect of curcumin on expression & enzymatic activity of GST isozymes (A) RT-PCR of GST isozymes and β-actin (B) Densitometric scanning of GST isozymes after normalization with β-actin. (C) Specific staining showing activity of GST isozymes. (D) Densitometric scanning of the activity band of GST isozymes. 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.
The activities of five isozymes of GST like GSTa, GSTm, GSTp, GSTt and GSTo did not follow similar variation pattern as that of expression. The activity of GSTa, GSTm and GSTp was reduced in DL and DL+DMSO mice, where as GSTt and GSTo isozymes showed induced activity in DL and DL+DMSO mice as compared to normal mice. The decrease in activity of GSTa: 46%, 41%; GSTm: 59%, 67% and GSTp: 35%, 21% in DL and DL+DMSO mice respectively as compared to normal mice. However, GSTt and GSTo isozymes showed induced activity upto 172% and 123% in case of GSTt and 145% and 126% in case of GSTo respectively in DL and DL+DMSO mice as compared to normal mice. Curcumin modulated activity of isozymes of GST towards normal level. GSTa, GSTm and GSTp were stimulated differentially with different doses of curcumin treatment. All the doses of curcumin elevated the activity of GSTa and GSTp. GSTa was elevated upto approximately 132%, 155%, 143% and GSTp upto approximately 247%, 366%, 420% of DL+DMSO mice with 50, 100 and 150 mg curcumin/kg bw respectively. However, the activity of GSTm was stimulated significantly upto 156%, 106% with 100 and 150 mg curcumin/kg bw respectively. Decrease in activity of GSTt was upto approximately 94%, 81% with 50 and 100 mg curcumin/kg bw respectively and GSTo upto approximately 75% with 50 mg curcumin/kg bw as compared to DL+DMSO mice [
The expression of GR in liver of DL and DL+DMSO mice was down regulated upto approximately 58% and 72% of normal mice respectively. Curcumin treatment induced expression of GR in a dose dependent manner. Up regulation of expression of GR by curcumin is approximately 118%, 129%, 134% of DL+DMSO mice with 50, 100 and 150 mg curcumin/kg bw respectively [
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.
Redox status in liver of lymphoma bearing mice was measured in terms of reduced versus oxidized form of glutathione (NP-SH/P-SH), as redox status is also an important indicator of oxidative stress. The status was observed to be 51% and 58% of normal mice in DL and DL+DMSO mice respectively. All the three doses elevated redox status significantly, which was upto approximately 131%, 159% and 124% of DL+DMSO mice with 50, 100 and 150 mg curcumin/kg bw respectively [
Effect of curcumin on cellular redox status in terms of NP-SH/P-SH. Liver of all six animals of each group was pooled separately and used for extraction of total 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.
Lymphoma bearing mice showed reduced expression of phase II antioxidant enzyme NQO1. The expression was down regulated in DL and DL+DMSO mice upto approximately 66% and 77% of normal mice. Curcumin treatment significantly up regulated expression towards normal level (95% of normal) with the dose of 100 mg curcumin/kg bw. The increased expression of NQO1 by curcumin was approximately 104% and 123% of DL+DMSO mice with 50 and 100 mg curcumin/kg bw respectively [
Effect of curcumin on mRNA expression & enzymatic activity of NQO1 (A) RT-PCR of NQO1 and β-actin (B) Densitometric scanning of NQO1 mRNA after normalization with β-actin. (C) Specific staining showing activity of NQO1. (D) Densitometric scanning of the activity band of NQO1. 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.
The p53 being a labile protein, is degraded fast under oxidative stress. The mRNA expression of Trp53 (p53 gene) was found to be down regulated in DL and DL+DMSO mice upto approximately 43% and 40% of normal mice. All the three doses of curcumin induced expression of Trp53 significantly. The up regulation of Trp53 expression was approximately upto 200%, 240% and 223% of DL+DMSO mice with 50, 100 and 150 mg curcumin/kg bw respectively [
Effect of curcumin on mRNA expression and protein level of p53 (A) RT-PCR of p53 and β-actin (B) Densitometric scanning of p53 mRNA, after normalization with β-actin. (C) Western analysis of p53 and β-actin. (D) Densitometric scanning of p53 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.
The expression of iNOS mRNA was observed to be up regulated upto 2.03-fold and 1.86-fold in DL and DL+DMSO mice respectively, as compared to normal mice. All the doses of curcumin significantly induced the expression of iNOS, which was approximately upto 122%, 166% and 148% of DL+DMSO mice with 50, 100 and 150mg/kg bw respectively [
Effect of curcumin on mRNA expression and protein level of iNOS (A) RT-PCR of iNOS and β-actin (B) Densitometric scanning of iNOS mRNA after normalization with β-actin. (C) Western analysis of iNOS and β-actin (D) Densitometric scanning of iNOS 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.
Activity of iNOS was measured in terms of NO level, as iNOS is the major contributor of NO production in liver. In comparison to normal mice, NO concentration in DL and DL+DMSO mice was observed to be 165% and 166% higher respectively. All the doses of curcumin treatment further induced the production of NO level significantly upto 150%, 211% and 198% of DL+DMSO mice with the dose of 50, 100 and 150mg/kg bw respectively [
Effect of curcumin on NO level in liver tissue. NO level was observed by using Griess reagent and expressed in μg equivalence of NaNO2/mg protein. Liver of all six animals of each group was pooled separately and used for extraction of total 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.
Expression of TGF-β1 mRNA was found to be down regulated approximately upto 29% and 32% in DL and DL+DMSO mice respectively as compared to normal mice. Curcumin modulated expression of TGF-β1 mRNA towards normal level. TGF-β1 mRNA expression was elevated approximately upto 150%, 300% and 265% of DL+DMSO mice with the dose of 50, 100 and 150mg/kg bw respectively [
Effect of curcumin on mRNA expression of TGF-β1 (A) RT-PCR of TGF-β1 and β-actin (B) Densitometric scanning of TGF-β1 after normalization with β-actin. Liver of all six animals of each group was pooled separately and used for extraction of total RNA. 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.
DL mice showed higher level of COX2 expression. It was upregulated in DL and DL+DMSO mice upto approximately 508% and 478% of normal mice respectively. Curcumin treatment modulated it towards normal level by down regulating the expression in a dose dependent manner, which was approximately 59%, 38% and 27% of DL+DMSO mice with 50, 100 and 150 mg curcumin/kg bw respectively [
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.
Being a vital organ, involvement of liver is a major determinant of survival from cancer. Most of the patients die due to hepatic metastases, irrespective of the origin of cancer; although true prevalence of metastatic liver disease is yet to be studied. Metastatic cancer cells trigger a proinflammatory response and reactive oxygen intermediates released by cancer cells in hepatic sinusoid microenvironment, leading to development of oxidative tumour microenvironment [
Our earlier findings show induced activity of NF-κB under oxidative tumour microenvironment in liver of DL mice, which is correlated with impaired antioxidant defence system and higher level of proinflammatory cytokines [
Therefore, we evaluated Nrf2 signalling in DL mice. Our finding of reduced Nrf2 signalling is correlated with high oxidative stress in DL mice in terms of redox status. Elevated binding of nuclear Nrf2 to ARE and NFE2 by curcumin treatment is capable of restoring Nrf2 expression with modulation of redox status towards normal level. Expression of GST and NQO1 are reported to be decreased abruptly in Nrf2 knockout mice as compared to wild-type counterpart [
Long term effect of curcumin is likely to inhibit the ability of Cullin3-Rbx1 E3 ubiquitin ligase to target Nrf2, needed for its ubiquitinization, facilitating activation and nuclear translocation of Nrf2 [
Further, Nrf2 plays an essential role in regulation of nonprotein thiol GSH level in oxidative tumour microenvironment through antioxidant genes [
Reduced oxidative stress in curcumin treated mice might be due to superoxide scavenging role of NQO1. Further, NQO1 regulates stability of p53 by inhibiting its ubiquitin-independent 20S proteasomal degradation [
Curcumin is reported to inhibit activity of NQO1
Modulation of level of NO production by tumour cells at the site of metastasis could be crucial [
Tumour progression is regulated by TGF-β via simultaneous activation of Smad-mediated mitoinhibition and decreased PGE2 production [
Thus, curcumin restored phase-II antioxidant enzyme activities and tumour suppressor p53 via activation of Nrf2 signalling which leads to upregulation of TGF-β and inhibition of carcinogenesis via reciprocal regulation of iNOS and COX2 in liver of lymphoma bearing mice.
Low bioavailability of curcumin at tissue level is due to rapid metabolism and elimination, irrespective of the route of administration. However, metabolites of curcumin remain for longer time in different tissues. Therefore the effect of curcumin, even after withdrawal of treatment might be due to synergistic action of its various metabolites. However, this needs further verification using different isolated metabolites. The treatment with 100 mg curcumin/kg body weight was found to be optimal dose. 50 mg/kg body weight may be insufficient to produce the maximum effect and 150 mg/kg body weight may lead to negative feedback regulation.
In summary, long term effect of curcumin potentiates its tumour preventing action by inducing phase-II antioxidant enzymes via activation of Nrf2 signalling, restoration of tumour suppressor p53 and modulation of inflammatory mediators like TGF-β and COX2 in liver of lymphoma bearing mice. Thus the observations suggest antioxidant and anti-inflammatory property of the metabolites of curcumin.
Body weights of DL mice were measured every day, starting from one day after DL transplantation till 18th day, to observe the growth pattern of Dalton’s lymphoma. Body weight verses time duration curve follows sigmoid curve of cell population growth (here ascite cell population growth).
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Primer pairs of corresponding gene with annealing and elongation condition as well as optimum cycle for amplification.
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