Enhancement of Apoptotic and Autophagic Induction by a Novel Synthetic C-1 Analogue of 7-deoxypancratistatin in Human Breast Adenocarcinoma and Neuroblastoma Cells with Tamoxifen

Summary

We have synthesized a novel analogue of pancratistatin with comparable anti-cancer activity as native pancratistatin; interestingly, combinatory treatment with tamoxifen yielded a drastic enhancement in apoptotic and autophagic induction by mitochondrial targeting with minimal effect on noncancerous fibroblasts. Thus, JCTH-4 in combination with tamoxifen could provide a safe anti-cancer therapy.

Abstract

Breast cancer is one of the most common cancers amongst women in North America. Many current anti-cancer treatments, including ionizing radiation, induce apoptosis via DNA damage. Unfortunately, such treatments are non-selective to cancer cells and produce similar toxicity in normal cells. We have reported selective induction of apoptosis in cancer cells by the natural compound pancratistatin (PST). Recently, a novel PST analogue, a C-1 acetoxymethyl derivative of 7-deoxypancratistatin (JCTH-4), was produced by de novo synthesis and it exhibits comparable selective apoptosis inducing activity in several cancer cell lines. Recently, autophagy has been implicated in malignancies as both pro-survival and pro-death mechanisms in response to chemotherapy. Tamoxifen (TAM) has invariably demonstrated induction of pro-survival autophagy in numerous cancers. In this study, the efficacy of JCTH-4 alone and in combination with TAM to induce cell death in human breast cancer (MCF7) and neuroblastoma (SH-SY5Y) cells was evaluated. TAM alone induced autophagy, but insignificant cell death whereas JCTH-4 alone caused significant induction of apoptosis with some induction of autophagy. Interestingly, the combinatory treatment yielded a drastic increase in apoptotic and autophagic induction. We monitored time-dependent morphological changes in MCF7 cells undergoing TAM-induced autophagy, JCTH-4-induced apoptosis and autophagy, and accelerated cell death with combinatorial treatment using time-lapse microscopy. We have demonstrated these compounds to induce apoptosis/autophagy by mitochondrial targeting in these cancer cells. Importantly, these treatments did not affect the survival of noncancerous human fibroblasts. Thus, these results indicate that JCTH-4 in combination with TAM could be used as a safe and very potent anti-cancer therapy against breast cancer and neuroblastoma cells.

Protocol

Introduction

Apoptosis, or type I programmed cell death, is a physiological process that can operate extrinsically, via binding of a death ligand to a death receptor, or intrinsically. The intrinsic pathway of apoptosis is initiated by intracellular stress such as DNA damage and mitochondrial dysfunction; this ultimately leads to the permeabilization of the mitochondria, dissipation of mitochondrial membrane potential (MMP), release of apoptogenic factors from the mitochondrial intermembrane space, and subsequent execution of apoptosis¹.

Autophagy is a process in which a cell breaks, degrades, and recycle....

Discussion

PST and similar compounds have been shown to have anti-cancer properties^11-15,21. We have previously reported natural PST to destabilize the mitochondria selectively in cancer cells, which thereby induces apoptosis by the release of apoptogenic factors^12,14. It is most likely that JCTH-4 acts through the same mechanism; JCTH-4 caused MMP collapse in MCF7 cells as seen with TMRM staining (Fig. 5a), and increased generation of ROS in isolated mitochondria from SH-SY5Y cells (F.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
SH-SY5Y cell line	ATCC	CRL-2266
Dulbecco’s Modified Eagles Medium F-12 HAM	Sigma-Aldrich	51448C
Fetal bovine serum	GIBCO, by Life Technologies	16000-044
MCF7 cell line	ATCC	HTB-22
RPMI-1640 medium	Sigma-Aldrich	R 0883
Apparently normal human fetal fibroblast cell line (NFF)	Coriell Institute for Medical Research	AG04431B
Dulbecco’s Modified Eagle’s Medium, High Glucose medium	Thermo Fisher Scientific, Inc.	SH30022.01
Tamoxifen citrate salt	Sigma-Aldrich	T9262
35 mm glass bottom culture dishes	MatTek Corp.	P35G-014-C
Leica DMI6000 B inverted microscope	Leica Microsystems	N/A
H–chst 33342 dye	Molecular Probes, Life Technologies	H3570
Leica DM IRB inverted fluorescence microscope	Leica Microsystems	N/A
Annexin V AlexaFluor-488	Invitrogen	A13201
Trypan Blue solution	Sigma-Aldrich	T8154-20ML
Haemocytometer	Fisher Scientific	267110
WST-1 reagent	Roche Group	11644807001
Wallac Victor3 1420 Multilabel Counter	PerkinElmer, Inc.	1420-011
Tetramethylrhodamine methyl ester (TMRM)	GIBCO, by Life Technologies	T-668
Glass tissue grinder	Fisher Scientific	K8885300-0002
BioRad protein assay	Bio-Rad	500-0001Bottom of Form
Amplex Red	Invitrogen	A12222
Horseradish peroxidase (HRP)	Sigma-Aldrich	P8125
SpectraMax Gemini XPS	Molecular Devices	3126666
Anti-LC3 antibody raised in rabbit	Novus Biologicals	NB100-2220
Anti-mouse HRP-conjugated secondary antibody	Abcam	ab6728
Anti-rabbit HRP-conjugated secondary antibody	Abcam	ab6802
Chemiluminescence peroxidase substrate	Sigma-Aldrich	CPS160
Monodansylcadaverine	Sigma-Aldrich	30432