Cytotoxic activity and apoptosis-inducing potential of di-spiropyrrolidino and di-spiropyrrolizidino oxindole andrographolide derivatives.

Anticancer role of andrographolide is well documented. To find novel potent derivatives with improved cytotoxicity than andrographolide on cancer cells, two series of di-spiropyrrolidino- and di-spiropyrrolizidino oxindole andrographolide derivatives prepared by cyclo-addition of azomethine ylide along with sarcosine or proline (viz. sarcosine and proline series respectively) and substitution of different functional groups (-CH3, -OCH3 and halogens) were examined for their cytotoxic effect on a panel of six human cancer cell lines (colorectal carcinoma HCT116 cells, pancreatic carcinoma MiaPaCa-2 cells, hepatocarcinoma HepG2 cells, cervical carcinoma HeLa cells, lung carcinoma A549 and melanoma A375 cells). Except halogen substituted derivatives of proline series (viz. CY2, CY14 and CY15 for Br, Cl and I substitution respectively), none of the other derivatives showed improved cytotoxicity than andrographolide in the cancer cell lines examined. Order of cytotoxicity of the potent compounds is CY2>CY14>CY15>andrographolide. Higher toxicity was observed in HCT116, MiaPaCa-2 and HepG2 cells. CY2, induced death of HCT116 (GI50 10.5), MiaPaCa-2 (GI50 11.2) and HepG2 (GI50 16.6) cells were associated with cell rounding, nuclear fragmentation and increased percentage of apoptotic cells, cell cycle arrest at G1 phase, ROS generation, and involvement of mitochondrial pathway. Upregulation of Bax, Bad, p53, caspases-3,-9 and cleaved PARP; downregulation of Bcl-2, cytosolic NF-κB p65, PI3K and p-Akt; translocation of P53/P21, NF-κB p65 were seen in CY2 treated HCT116 cells. Thus, three halogenated di-spiropyrrolizidino oxindole derivatives of andrographolide are found to be more cytotoxic than andrographolide in some cancer cells. The most potent derivative, CY2 induced death of the cancer cells involves ROS dependent mitochondrial pathway like andrographolide

Analysis of apoptosis by flow cytometry in HCT116 cells.

<p>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.</p

Western Blot analyses of some important pro- and anti-apoptotic proteins in treated and untreated HCT116 cells.

<p>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 (<i>P</i><0.001).</p

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.

<p>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 (<i>P</i><0.001).</p

Inhibition of HepG2 cell growth by andrographolide and its derivatives.

<p>Cells were treated with different concentration of compounds ranging from 0–40 µM for 36 h. GI₅₀ values were calculated from MTT assay.</p>*<p>Derivative which is most potent;</p>**<p>derivative(s) intermediately potent but more potent than andrographolide.</p><p>Values are mean ± S.D (ranging between 3–7%) and represent one of the 3 representative experiments (<i>P</i><0.001).</p

Immunocytochemistry showing localization of P53, P21, NF-κB p50, NF-κB p65 and MMP-9 in presence and absence of CY2 in HCT116 cells.

<p>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 (λ_Em∼525 nm, green) and TRITC (λ_Em∼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.</p

ROS generation in HCT116 cells upon treatment with 20 µM of CY2 and the scavenging action by NAC.

<p>(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 (<i>P</i><0.001).</p

Structure of andrographolide and its derivatives.

<p>Structure of andrographolide and its derivatives.</p

High Resolution Mass Spectroscopic (HRMS) data of different andrographolide derivatives.

<p><b>m/z</b> = mass/charge; <b>calcd</b>. = calculated data of the compound; <b>MF</b> = Molecular Formula.</p

Study of cell cycle arrest in HCT116 cells by propidium iodide.

<p>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.</p