Figure 1.
Structure of andrographolide and its derivatives.
Table 1.
High Resolution Mass Spectroscopic (HRMS) data of different andrographolide derivatives.
Figure 2.
Viability of three cancer lines HCT116, MiaPaCa-2, HepG2 and a non–cancer cell line Chang liver cells was observed in response to andrographolide and its derivatives.
Experimental cells (2×105) were treated with andrographolide and its derivatives. MTT assay was performed. O. D. at 595 nm reflects the viability of cells. (A) Concentration dependent cell viability of potent derivatives from proline series viz. CY2, CY14, CY15 and andrographolide. (B) Time dependent cell viability of potent derivatives from proline series viz. CY2, CY14, CY15 and andrographolide. Percent viability of cells calculated from MTT assay after treatment with 20 µM of each of the potent derivatives and andrographolide for indicated time points. Values are mean ± S.D. and represent one of the 3 representative experiments (P<0.001).
Table 2.
Inhibition of HepG2 cell growth by andrographolide and its derivatives.
Figure 3.
Morphological and nuclear changes seen in HCT116, MiaPaCa2 and HepG2 cells after treatment with CY2.
Cells, treated with 20 µM of CY2 for 36 h were seen under light microscope and/or under fluorescence microscope following nuclear staining with DAPI or acridine orange/ethidium bromide (AO/EtBr). Results in the upper panel shows bright field, middle panel shows acridine orange/ethidium bromide (AO/EtBr) and lower panel shows DAPI stained nuclei of HCT116, MiaPaCa-2 and HepG2 cells respectively. Scale Bar = 15 µm.
Figure 4.
Analysis of apoptosis by flow cytometry in HCT116 cells.
Cells were treated with 20 µM of CY2 (left panel) and andrographolide (right panel) for 24 h and 36 h. Binding of annexin-V/FITC to phosphatidyl serine was determined by flow cytometry. Percentages of apoptotic cells determined by the number of annexin V(+)/propidium iodide cells are shown in the scattered plot.
Figure 5.
Study of cell cycle arrest in HCT116 cells by propidium iodide.
Percentage of G0–G1 cell population increases after treatment of 20 µM of CY2 and andrographolide for 24 h and 36 h, indicating G1/S phase cell cycle arrest.
Figure 6.
Analysis of caspase-3 and caspase-9 status, cytochrome c level, DNA fragmentation and mitochondrial membrane potential in treated and untreated HCT116 cell.
Cells (2×105) after treated with different concentrations (0–40 µM) of CY2 for 24 h were put to ELISA based colorimetric assay using kits for determining the levels of activated caspase-3, caspase-9, and DNA fragmentation. Enhancement of O.D. represents activation of (A) caspase-3, (B) caspase-9. (C) Cell viability in presence or absence of caspase-3 and caspase-9 inhibitors (Z-DEVD-FMK, and Z-LEHD-FMK respectively). (D) Cytochrome c level alteration determined by spectrofuorometric analysis. (E) DNA fragmentation represented through change of O.D. (F) Mitochondrial membrane potential measured by spectrofluorometry using rhodamine123 is reflected through change of O.D. Values are mean ± S.D. and represent one of the 3 representative experiments (P<0.001).
Figure 7.
ROS generation in HCT116 cells upon treatment with 20 µM of CY2 and the scavenging action by NAC.
(A) Time and dose response of ROS generation by different concentrations (0–40 µM) of CY2 incubated for 0–36 h. (B) Percentage cell viability after 20 µM CY2 and andrographolide treatment and alteration in ROS level by NAC treatment, indirectly indicating decrease in apoptotic cell death by NAC treatment. (C) Fluorometric study of ROS generation by different concentrations (0–40 µM) of CY2 and andrographolide in presence and absence of 5 mM and 10 mM NAC (D)Flow cytometric study for ROS generation by CY2 and andrographolide at 24 h on HCT116 and different degrees of scavenging effect by two concentraions (5 mM and 10 mM) of NAC. Values are mean ± S.D. and represent one of the 3 representative experiments (P<0.001).
Figure 8.
Western Blot analyses of some important pro- and anti-apoptotic proteins in treated and untreated HCT116 cells.
Cells were treated with 10 µM or 20 µM each of CY2, CY14, CY15 for 24 h or left as such. Cells were lysed and cell lysate were used for Western Blot analysis. (A) Panel represents western blot analysis of different apoptotic proteins. Panel indicates Bcl2, Bad, Bax, caspase 3 and 9, cytochrome c, P53,PARP, NF-κB p65 (cytosolic), PI3K, Akt, p-Akt and β-Actin expression after 10 µM or 20 µM of andrographolide and CY2 treatment respectively for 24 h. (B) Bar graph shows semiquantified densitometry from western blot analysis for lower dose (10 µM) of both andrographolide and CY2 treatment. (C) Bar graph shows semiquantified densitometry from Western blot analysis for higher dose (20 µM) of both andrographolide and CY2 treatment (P<0.001).
Figure 9.
Immunocytochemistry showing localization of P53, P21, NF-κB p50, NF-κB p65 and MMP-9 in presence and absence of CY2 in HCT116 cells.
DAPI was used for genomic DNA counterstaining (A) Translocation status of P53 and P21 in control cells (upper panel) and 20 µM CY2 treated cells (lower panel). HCT116 cells coincubated with primary antibodies for P53 and P21 and developed with FITC (λEm∼525 nm, green) and TRITC (λEm∼576 nm, red) labeled secondary antibodies respectively, cotranslocation (left composite panel) in the nuclei was observed for both of these proteins with respect to the untreated cells, where they were found to reside mainly in the cytoplasm. Scale Bar = 20 µm. (B) Translocation status of NF-κB p50 and NF-κB p65 in control cells (upper panel) and 20 µ M CY2 treated cells (lower panel). The nuclear localization of the two main subunits of the NF-κB family viz. p65 and p50 (developed with FITC and TRITC -labeled secondary antibodies respectively) was found in untreated control cells but upon treatment, these two subunits tends to localize into the cytoplasm with respect to the untreated control as shown in the composite confocal micrograph. Scale Bar = 15 µm. (C) Localization of MMP-9 in control cells (upper panel) and 20 µM CY2 treated cells (lower panel). Confocal microscopy revealed that immunocytochemistry of MMP-9 protein (developed with TRITC-labelled secondary antibody) expression decreased following treatment with CY2 with respect to the untreated control. Scale Bar = 15 µm.