Forbedring av apoptotiske og autophagic Induksjon av en roman syntetisk C-1 Analog av 7-deoxypancratistatin i Human Breast adenokarsinom og neuroblastom celler med Tamoxifen
Summary
Vi har syntetisert en roman analog pancratistatin med sammenlignbare anti-kreft aktivitet som innfødt pancratistatin, interessant, combinatory behandling med tamoxifen ga en drastisk forbedring i apoptotisk og autophagic induksjon av mitokondrie målretting med minimal effekt på noncancerous fibroblaster. Dermed kunne JCTH-4 i kombinasjon med tamoxifen gi en trygg anti-kreft behandling.
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
Discussion
PST og lignende forbindelser har vist seg å ha anti-kreft egenskaper 11-15,21. Vi har tidligere rapportert naturlig PST å destabilisere mitokondriene selektivt i kreftceller, som derved induserer apoptose ved utgivelsen av apoptogenic faktorer 12,14. Det er mest sannsynlig at JCTH-4 handlinger gjennom den samme mekanismen, JCTH-4 forårsaket MMP kollaps i MCF7 celler som sett med TMRM farging (Fig. 5a), og økt produksjon av ros i isolert mitokondriene fra SH-SY5Y celler …
Disclosures
The authors have nothing to disclose.
Acknowledgements
Dette arbeidet har vært støttet av Knights of Columbus kapittel 9671 (Windsor, Ontario), og en CIHR Frederick Banting og Charles Best Canada Graduate Scholarship tildelt Dennis Ma. Takk til Robert Hodge og Elizabeth Fidalgo da Silva for deres hjelp med time-lapse mikroskopi. Takk til Katie Facecchia for redigering av time-lapse mikroskopi videoer. Vi ønsker også å takke Sudipa juni Chatterjee og Phillip Tremblay for kritisk gjennomgang av dette manuskriptet. Dette arbeidet er dedikert til minne om Kevin Couvillon som mistet sin kamp mot kreft i 2010.
Materials
| Material Name | Company | Catalogue number |
| SH-SY5Y cell line | ATCC | CRL-2266 |
| Dulbecco’s Modified Eagles Medium F-12 HAM | Sigma-Aldrich | 51448C |
| Fetal bovine serum | Gibco BRL | 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 Scientific | SH30022.01 |
| Tamoxifen citrate salt | Sigma-Aldrich | T9262 |
| 35 mm glass bottom culture dishes | MatTek | P35G-014-C |
| Leica DMI6000 B inverted microscope | Leica Microsystems | N/A |
| Hoechst 33342 dye | Molecular Probes | 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 Applied Science | 11644807001 |
| Wallac Victor3 1420 Multilabel Counter | PerkinElmer | 1420-011 |
| Tetramethylrhodamine methyl ester (TMRM) | Gibco BRL | T-668 |
| Glass tissue grinder | Fisher Scientific | K8885300-0002 |
| BioRad protein assay | Bio-Rad Laboratories | 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 |
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