Limits...
Growth inhibition and apoptosis induction by (+)-Cyanidan-3-ol in hepatocellular carcinoma.

Monga J, Pandit S, Chauhan RS, Chauhan CS, Chauhan SS, Sharma M - PLoS ONE (2013)

Bottom Line: The expression levels of apoptosis-related genes that belong to bcl-2 and caspase family were increased and AP-1 and NF-κB activities were significantly suppressed by CD-3.Immunohistochemistry data revealed less localization of p53, p65 and c-jun in CD-3 treated tumors as compared to localization in NDEA/CCl4 treated tumors.Taken together, our data demonstrated that CD-3 could significantly inhibit the proliferation of HepG2 cells in-vitro and suppress HCC tumor growth in-vivo by apoptosis induction.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacy, Jaypee University of Information Technology, Waknaghat, Himachal Pradesh, India.

ABSTRACT
The objective of this study was to evaluate the cytotoxicity of (+)-cyanidan-3-ol (CD-3) in human hepatocellular carcinoma cell line (HepG2) and chemopreventive potential against hepatocellular carcinoma (HCC) in Balb/c mice. The HepG2 cell line was treated with CD-3 at various concentrations and the proliferation of the HepG2 cells was measure by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT), sulforhodamine B (SRB) and lactate dehydrogenase (LDH) assays. Cell apoptosis was detected by Hoechst 33258 (HO), Acridine orange/ethylene dibromide (AO/EB) staining, DNA fragmentation analysis and the apoptosis rate was detected by flow cytometry. The HCC tumor model was established in mice by injecting N-nitrosodiethylamine/carbon tetrachloride (NDEA/CCl4) and the effect of CD-3 on tumor growth in-vivo was studied. The levels of liver injury markers, tumor markers, and oxidative stress were measured. The expression levels of apoptosis-related genes in in-vitro and in vivo models were determined by RT-PCR and ELISA. The CD-3 induced cell death was considered to be apoptotic by observing the typical apoptotic morphological changes under fluorescent microscopy and DNA fragmentation analysis. Annexin V/PI assay demonstrated that apoptosis increased with increase in the concentration of CD-3. The expression levels of apoptosis-related genes that belong to bcl-2 and caspase family were increased and AP-1 and NF-κB activities were significantly suppressed by CD-3. Immunohistochemistry data revealed less localization of p53, p65 and c-jun in CD-3 treated tumors as compared to localization in NDEA/CCl4 treated tumors. Taken together, our data demonstrated that CD-3 could significantly inhibit the proliferation of HepG2 cells in-vitro and suppress HCC tumor growth in-vivo by apoptosis induction.

Show MeSH

Related in: MedlinePlus

Expression of apoptosis-related genes in liver after CD-3 treatment.mRNA expressions of β-actin, p53 (mut) (A), p53 (wild) (B), mdm2 (C), p65 (D), c-jun (E), c-fos (F), bcl-2 (G), bax (H), caspase-3 (I), -7 (J), -8 (K), and -9 (L), and cytochrome-c (M) and their densitometric analysis in hepatocarcinoma. Values are mean ± SEM of four independent observations. aP < 0.0001 w.r.t. control, bP < 0.001 w.r.t. control, cP < 0.01 w.r.t. control, dP < 0.0001 w.r.t. control, eP < 0.001 w.r.t. control, fP < 0.01 w.r.t. control, gP < 0.05 w.r.t. control, hP < 0.0001 w.r.t. NDEA/CCl4 (group 2), iP < 0.001 w.r.t. NDEA/CCl4 (group 2).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3722203&req=5

pone-0068710-g010: Expression of apoptosis-related genes in liver after CD-3 treatment.mRNA expressions of β-actin, p53 (mut) (A), p53 (wild) (B), mdm2 (C), p65 (D), c-jun (E), c-fos (F), bcl-2 (G), bax (H), caspase-3 (I), -7 (J), -8 (K), and -9 (L), and cytochrome-c (M) and their densitometric analysis in hepatocarcinoma. Values are mean ± SEM of four independent observations. aP < 0.0001 w.r.t. control, bP < 0.001 w.r.t. control, cP < 0.01 w.r.t. control, dP < 0.0001 w.r.t. control, eP < 0.001 w.r.t. control, fP < 0.01 w.r.t. control, gP < 0.05 w.r.t. control, hP < 0.0001 w.r.t. NDEA/CCl4 (group 2), iP < 0.001 w.r.t. NDEA/CCl4 (group 2).

Mentions: To elucidate cell signaling pathway activated by CD-3 in NDEA-induced hepatocarcinoma in Balb/c mice, RT-PCR analyses was used to measure the mRNA expression of p53, mdm2, p65, c-jun, c-fos, bcl-2, bax, caspase-3, -7, -8 and -9 and cytochrome-c as well as the expression of the β-actin (an internal standard). In the present study, two different primers from different exonic regions of p53 gene were used for mRNA expression analysis. One primer was designed from exon 4-7 (Primer A) and the second primer was from exon 10-11 (Primer B). The densitometric analysis of mRNA expression in each group was also carried and statistically compared (Figure 10).


Growth inhibition and apoptosis induction by (+)-Cyanidan-3-ol in hepatocellular carcinoma.

Monga J, Pandit S, Chauhan RS, Chauhan CS, Chauhan SS, Sharma M - PLoS ONE (2013)

Expression of apoptosis-related genes in liver after CD-3 treatment.mRNA expressions of β-actin, p53 (mut) (A), p53 (wild) (B), mdm2 (C), p65 (D), c-jun (E), c-fos (F), bcl-2 (G), bax (H), caspase-3 (I), -7 (J), -8 (K), and -9 (L), and cytochrome-c (M) and their densitometric analysis in hepatocarcinoma. Values are mean ± SEM of four independent observations. aP < 0.0001 w.r.t. control, bP < 0.001 w.r.t. control, cP < 0.01 w.r.t. control, dP < 0.0001 w.r.t. control, eP < 0.001 w.r.t. control, fP < 0.01 w.r.t. control, gP < 0.05 w.r.t. control, hP < 0.0001 w.r.t. NDEA/CCl4 (group 2), iP < 0.001 w.r.t. NDEA/CCl4 (group 2).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3722203&req=5

pone-0068710-g010: Expression of apoptosis-related genes in liver after CD-3 treatment.mRNA expressions of β-actin, p53 (mut) (A), p53 (wild) (B), mdm2 (C), p65 (D), c-jun (E), c-fos (F), bcl-2 (G), bax (H), caspase-3 (I), -7 (J), -8 (K), and -9 (L), and cytochrome-c (M) and their densitometric analysis in hepatocarcinoma. Values are mean ± SEM of four independent observations. aP < 0.0001 w.r.t. control, bP < 0.001 w.r.t. control, cP < 0.01 w.r.t. control, dP < 0.0001 w.r.t. control, eP < 0.001 w.r.t. control, fP < 0.01 w.r.t. control, gP < 0.05 w.r.t. control, hP < 0.0001 w.r.t. NDEA/CCl4 (group 2), iP < 0.001 w.r.t. NDEA/CCl4 (group 2).
Mentions: To elucidate cell signaling pathway activated by CD-3 in NDEA-induced hepatocarcinoma in Balb/c mice, RT-PCR analyses was used to measure the mRNA expression of p53, mdm2, p65, c-jun, c-fos, bcl-2, bax, caspase-3, -7, -8 and -9 and cytochrome-c as well as the expression of the β-actin (an internal standard). In the present study, two different primers from different exonic regions of p53 gene were used for mRNA expression analysis. One primer was designed from exon 4-7 (Primer A) and the second primer was from exon 10-11 (Primer B). The densitometric analysis of mRNA expression in each group was also carried and statistically compared (Figure 10).

Bottom Line: The expression levels of apoptosis-related genes that belong to bcl-2 and caspase family were increased and AP-1 and NF-κB activities were significantly suppressed by CD-3.Immunohistochemistry data revealed less localization of p53, p65 and c-jun in CD-3 treated tumors as compared to localization in NDEA/CCl4 treated tumors.Taken together, our data demonstrated that CD-3 could significantly inhibit the proliferation of HepG2 cells in-vitro and suppress HCC tumor growth in-vivo by apoptosis induction.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacy, Jaypee University of Information Technology, Waknaghat, Himachal Pradesh, India.

ABSTRACT
The objective of this study was to evaluate the cytotoxicity of (+)-cyanidan-3-ol (CD-3) in human hepatocellular carcinoma cell line (HepG2) and chemopreventive potential against hepatocellular carcinoma (HCC) in Balb/c mice. The HepG2 cell line was treated with CD-3 at various concentrations and the proliferation of the HepG2 cells was measure by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT), sulforhodamine B (SRB) and lactate dehydrogenase (LDH) assays. Cell apoptosis was detected by Hoechst 33258 (HO), Acridine orange/ethylene dibromide (AO/EB) staining, DNA fragmentation analysis and the apoptosis rate was detected by flow cytometry. The HCC tumor model was established in mice by injecting N-nitrosodiethylamine/carbon tetrachloride (NDEA/CCl4) and the effect of CD-3 on tumor growth in-vivo was studied. The levels of liver injury markers, tumor markers, and oxidative stress were measured. The expression levels of apoptosis-related genes in in-vitro and in vivo models were determined by RT-PCR and ELISA. The CD-3 induced cell death was considered to be apoptotic by observing the typical apoptotic morphological changes under fluorescent microscopy and DNA fragmentation analysis. Annexin V/PI assay demonstrated that apoptosis increased with increase in the concentration of CD-3. The expression levels of apoptosis-related genes that belong to bcl-2 and caspase family were increased and AP-1 and NF-κB activities were significantly suppressed by CD-3. Immunohistochemistry data revealed less localization of p53, p65 and c-jun in CD-3 treated tumors as compared to localization in NDEA/CCl4 treated tumors. Taken together, our data demonstrated that CD-3 could significantly inhibit the proliferation of HepG2 cells in-vitro and suppress HCC tumor growth in-vivo by apoptosis induction.

Show MeSH
Related in: MedlinePlus