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SRJ23, a new semisynthetic andrographolide derivative: in vitro growth inhibition and mechanisms of cell cycle arrest and apoptosis in prostate cancer cells

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Abstract

Purpose

3,19-(3-Chloro-4-fluorobenzylidene)andrographolide (SRJ23), a new semisynthetic derivative of andrographolide (AGP), exhibited selectivity against prostate cancer cells in the US National Cancer Institute (NCI) in vitro anti-cancer screen. Herein, we report the in vitro growth inhibition and mechanisms of cell cycle arrest and apoptosis induced by SRJ23.

Methods

3-(4,5-Dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used in assessing in vitro growth inhibition of compounds against prostate cancer (PC-3, DU-145 and LNCaP) and mouse macrophage (RAW 264.7) cell lines. Flow cytometry was utilised to analyse cell cycle distribution, whereas fluorescence microscopy was performed to determine morphological cell death. DNA fragmentation and annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) flow cytometry were done to confirm apoptosis induced by SRJ23. Quantitation of cell cycle and apoptotic regulatory proteins were determined by immunoblotting.

Results

AGP and SRJ23 selectively inhibited the growth of prostate cancer cells compared with RAW 264.7 cells at low micromolar concentrations; however, SRJ23 was more potent. Mechanistically, SRJ23-treated PC-3 cells displayed down-regulation of cyclin-dependent kinase (CDK) 1 without affecting levels of CDK4 and cyclin D1. However, SRJ23 induced down-regulation of CDK4 and cyclin D1 but without affecting CDK1 in DU145 and LNCaP cell lines. DNA histogram analysis revealed that the SRJ23 induced G2/M in PC-3 cells but G1 arrest in DU-145 and LNCaP cells. Morphologically, both compounds induced predominantly apoptosis, which was further confirmed by DNA fragmentation and annexin V-FITC staining. The DNA fragmentation was inhibited in the presence of caspase 8 inhibitor (Z-IETD-FMK). Apoptosis was associated with an increase in caspase 8 expression and activation. This thought to have induced cleavage of Bid into t-Bid. Additionally, increased expression and activation of caspase 9 and Bax proteins were apparent, with a concomitant down-regulation of Bcl-2 protein. Similar apoptosis cascade of events was observed in SRJ23-treated DU145 and LNCaP cell lines.

Conclusion

SRJ23 inhibited the growth of prostate cancer cells by inducing G2/M and G1 arrest via down-regulation of CDK1, and CDK4 and cyclin, respectively, and initiated caspase-8-mediated mitochondrial apoptosis. Taken together, these data support the potential of this compound as a new anti-prostate cancer agent.

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Abbreviations

AGP:

Andrographolide

AO:

Acridine orange

CDK:

Cyclin-dependent kinase

ECL:

Enzyme chemiluminescence

ERK:

Extracellular signal-regulated kinase

FITC:

Fluorescein isothiocyanate

PAGE:

Polyacrylamide gel electrophoresis

PI:

Propidium iodide

RNase A:

Ribonuclease A

SDS:

Sodium dodecyl sulphate

SOM:

Self-organising map

SRJ23:

3,19-(3-Chloro-4-fluorobenzylidene)andrographolide

TEMED:

N,N,N′,N′-tetramethylethylenediamine

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Acknowledgments

The Ministry of Higher Education is thanked for funding this project through the Research University Grant Scheme (RUGS) (Grant 04-02-12-2017RU). Hui Chyn Wong is a recipient of the National Science Fellowship (NSF) awarded by the Ministry of Science, Technology and Innovation of Malaysia (MOSTI). We are grateful to Mr. Lim Siang Hui from Cancer Research Initiatives Foundation (CARIF), Malaysia, for assistance in the flow cytometry work. We are thankful to Mr. Teh Yuan Han (Pharmacotherapeutics Unit, Department of Medicine, Faculty of Medicine and Health Sciences, UPM) for his editorial assistance.

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Correspondence to Johnson Stanslas.

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Wong, H.C., Wong, C.C., Sagineedu, S.R. et al. SRJ23, a new semisynthetic andrographolide derivative: in vitro growth inhibition and mechanisms of cell cycle arrest and apoptosis in prostate cancer cells. Cell Biol Toxicol 30, 269–288 (2014). https://doi.org/10.1007/s10565-014-9282-5

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