A novel synthetic C-1 analogue of 7-deoxypancratistatin induces apoptosis in p53 positive and negative human colorectal cancer cells by targeting the mitochondria: enhancement of activity by tamoxifen

Abstract

The natural compound pancratistatin (PST), isolated from the Hymenocallis littoralis plant, specifically induces apoptosis in many cancer cell lines. Unlike many other chemotherapeutics, PST is not genotoxic and has minimal adverse effects on non-cancerous cells. However, its availability for preclinical and clinical work is limited due to its low availability in its natural source and difficulties in its chemical synthesis. Several synthetic analogues of 7-deoxypancratistatin with different modifications at C-1 were synthesized and screened for apoptosis inducing activity in human colorectal cancer (CRC) cells. We found that a C-1 acetoxymethyl derivative of 7-deoxypancratistatin, JC-TH-acetate-4 (JCTH-4), was effective in inducing apoptosis in both p53 positive (HCT 116) and p53 negative (HT-29) human CRC cell lines, demonstrating similar efficacy to that of natural PST. JCTH-4 was able to decrease mitochondrial membrane potential (MMP), increase levels of reactive oxygen species in isolated mitochondria, cause release of the apoptogenic factor cytochrome c (Cyto c) from isolated mitochondria, and induce autophagy in HCT 116 and HT-29 cells. Interestingly, when JCTH-4 was administered with tamoxifen (TAM), there was an enhanced effect in apoptosis induction, reactive oxygen species (ROS) production and Cyto c release by isolated mitochondria, and autophagic induction by CRC cells. Minimal toxicity was exhibited by a normal human fetal fibroblast (NFF) and a normal colon fibroblast (CCD-18Co) cell line. Hence, JCTH-4 is a novel compound capable of selectively inducing apoptosis and autophagy in CRC cells alone and in combination with TAM and may serve as a safer and more effective alternative to current cancer therapies.

Selective Induction of Apoptosis and Autophagy Through Treatment With Dandelion Root Extract in Human Pancreatic Cancer Cells

“…This conjugation results in the lipidated protein LC3-II that is recruited to autophagosomal membranes and can be used as another marker for autophagy detection. 22 To confirm the results of autophagy seen by MDC staining, immunocytochemical analysis was used to detect the conversion of LC3-I to LC3-II. BxPC-3 treated with DRE at 2.5 mg/mL had a similar effect on the conversion of LC3-I to LC3-II, compared to the tamoxifen-treated cells.…”

Phenanthridone Alkaloids of the Amaryllidaceae as Activators of the Apoptosis-related Proteolytic Enzymes, Caspases

“…As observed in the Saos-2, BxPC-3 and PANC-1 cells above [23,26], the caspase inhibitor Z-VAD-FMK (at both 25 and 50 μM) could not protect HCT116 cells from the apoptotic onslaught of compound 2 (1 μM) [27]. This confirmed the action of this alkaloid to be at the stage of mitochondrial destabilization, upstream of executioner caspase activation, which in turn caused the release of various apoptogenic factors involved in both caspase dependent and independent pathways of apoptotic induction [27]. Similar effects were observed for alkaloid 2 in human neuroblastoma (SHSY-5Y) and breast adenocarcinoma (MCF-7) cells [28].…”

“…To this extent, compound 2 was able to decrease MMP, increase levels of ROS and cytochrome c in isolated mitochondria and induce autophagic effects in both cell lines [27]. As observed in the Saos-2, BxPC-3 and PANC-1 cells above [23,26], the caspase inhibitor Z-VAD-FMK (at both 25 and 50 μM) could not protect HCT116 cells from the apoptotic onslaught of compound 2 (1 μM) [27]. This confirmed the action of this alkaloid to be at the stage of mitochondrial destabilization, upstream of executioner caspase activation, which in turn caused the release of various apoptogenic factors involved in both caspase dependent and independent pathways of apoptotic induction [27].…”

“…Given that phenanthridone (2) proved to be a potent growth inhibitor of Saos-2, U-2 OS, HCT116, HT-29, BxPC-3 and PANC-1 cells [23,[26][27][28], a series of C-1 homologated analogs (4-6) were targeted for further screening against various cancer cells [29]. Structurally these compounds were distinguishable by the presence of a C-7 phenolic hydroxyl group, similar to the parent compound pancratistatin (1) [29].…”

Exploiting mitochondrial and oxidative vulnerabilities with a synthetic analog of pancratistatin in combination with piperlongumine for cancer therapy

“…Although the precise mechanism of its affinity for cancerous mitochondria remains elusive to date, we have found that the natural compound pancratistatin (PST), isolated from the Hymenocallis littoralis plant, specifically induces apoptosis in numerous cancer cell types with minimal effect on noncancerous cells by mitochondrial targeting (16)(17)(18)(19)(20). We have previously synthesized JCTH-4, a C-1 acetoxymethyl analog of 7-deoxypancratistatin, and found it to possess comparable anticancer activity to ABBREVIATIONS: AIF, anti-apoptosis-inducing factor; AV, annexin V; BMX, basement membrane extract; DCF, 29,79-dicholorofluorescein; DIC, differential interference contrast; FBS, fetal bovine serum; H 2 DCFDA, 29,79-dicholorofluorescin diacetate; MMP, mitochondrial membrane potential; NAC, N-acetyl cysteine; PBMC, peripheral blood mononuclear cell; PBMCs V1/2, peripheral blood mononuclear cells from healthy volunteer 1/2; PI, propidium iodide; PL, piperlongumine; PST, pancratistatin; ROS, reactive oxygen species; RPMI, Roswell Park Memorial Institute; TBST, Tris-buffered saline-Tween 20; TMRM, tetramethylrhodamine methyl ester that of PST, inducing apoptosis in numerous cancer cell types by way of mitochondrial targeting (21)(22)(23)(24). Recently we created a C-7 hydroxylated version of JCTH-4, SVTH-6, a C-1 acetoxymethyl analog of PST, by de novo synthesis; to our knowledge, this is the first time that chemical synthesis of a PST analog with the full pharmacophore for anticancer activity has been achieved (24).…”