Benzylidene derivatives of andrographolide inhibit growth of breast and colon cancer cells in vitro by inducing G₁ arrest and apoptosis

Abstract

Background and purpose: Andrographolide, the major phytoconstituent of Andrographis paniculata, was previously shown by us to have activity against breast cancer. This led to synthesis of new andrographolide analogues to find compounds with better activity than the parent compound. Selected benzylidene derivatives were investigated for their mechanisms of action by studying their effects on the cell cycle progression and cell death. Experimental approach: Microculture tetrazolium, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and sulphorhodamine B (SRB) assays were utilized in assessing the in vitro growth inhibition and cytotoxicity of compounds. Flow cytometry was used to analyse the cell cycle distribution of control and treated cells. CDK1 and CDK4 levels were determined by western blotting. Apoptotic cell death was assessed by fluorescence microscopy and flow cytometry. Key results: Compounds, in nanomolar to micromolar concentrations, exhibited growth inhibition and cytotoxicity in MCF-7 (breast) and HCT-116 (colon) cancer cells. In the NCI screen, 3,19-(2-bromobenzylidene) andrographolide (SRJ09) and 3,19-(3-chloro-4-fluorobenzylidene) andrographolide (SRJ23) showed greater cytotoxic potency and selectivity than andrographolide. SRJ09 and SRJ23 induced G 1 arrest and apoptosis in MCF-7 and HCT-116 cells, respectively. SRJ09 downregulated CDK4 but not CDK1 level in MCF-7 cells. Apoptosis induced by SRJ09 and SRJ23 in HCT-116 cells was confirmed by annexin V-FITC/PI flow cytometry analysis. Conclusion and implications:The new benzylidene derivatives of andrographolide are potential anticancer agents. SRJ09 emerged as the lead compound in this study, exhibiting anticancer activity by downregulating CDK4 to promote a G 1 phase cell cycle arrest, coupled with induction of apoptosis.

Andrographolide derivatives inhibit guanine nucleotide exchange and abrogate oncogenic Ras function

“…1) exhibit an enhanced ability to induce apoptosis and G1 cell-cycle arrest in breast and colon cancer cells (25,27). Other studies have shown that AGP interferes with MAPK activation, increases sensitivity of Ras-transformed cells to radiation treatment in vitro and in vivo (27)(28)(29)(30), and is not toxic (31).…”

“…5F). Moreover, the ability of AGP, SRJ09, and SRJ23 to induce growth inhibition against PC-3 (prostate), HCT-116 (colon), and MDA-231 (breast) cancer cells was assessed as described previously (25). PC-3 cancer cells harbor wild-type Ras for all of the three different isoforms (H-, K-, and N-Ras), whereas HCT-116 and MDA-231 cancer cells express G13D mutant K-Ras and wild-type H-and N-Ras.…”

“…Based on initial work that formed the basis of this study (25,48), AGP, SRJ09, and SRJ23 were prepared as reported earlier (48) A B C D E F Fig. 5.…”

“…The potential of AGP, SRJ09, and SRJ23 to induce growth inhibition against PC-3 (prostate), HCT-116 (colon), and MDA-231 (breast) cancer cells was assessed according to Jada et al (25). Briefly, exponentially growing cells were seeded in flat-bottom 96-well plates at a density of 2,000 cells in 0.18 mL per well and incubated overnight for cell attachment.…”

Anti-tumor Initiating Potential of Andrographolide in 7,12-dimethylbenz[a]anthracene Induced Hamster Buccal Pouch Carcinogenesis

“…Andrographolide protected normal cells against chemical induced toxicity (Ye et al, 2011). Andrographolide induced G0/G1 cell cycle arrest in various types of cancer cells (Jada et al, 2008). The cytotoxic activity of andrographolide is probably due to intact U-butyrolactone ring, the double bonds between C-12 and C-13, C-8 and C-17 and hydroxyl group at C-14 present in the chemical structure of andrographolide (Varma et al, 2009;Sirion et al, 2012).…”