Tumor Necrosis Factor-α (TNF-α) Sensitize Breast Cancer Cells To Natural Products With Proteasome Inhibitory Activities Leading To Apoptosis | Posted on:2015-07-31 | Degree:Master | Type:Thesis | Country:China | Candidate:W L Shi | Full Text:PDF | GTID:2284330422988148 | Subject:Pathology and pathophysiology | Abstract/Summary: | PDF Full Text Request | Inflammatory microenvironment plays important role in the process of tumor development. TNF-α, akey pro-inflammatory cytokine, has significant role in this process. Natural products such asWithaferin A (WA) and celastrol have shown anti-cancer and anti-inflammatory properties that canbe attributed to multiple mechanisms including, but not limited to apoptosis induction due to theinhibition of proteasomal activities. The objective of this study was to investigate the effects ofTNF-α in combination with these two natural products in vitro in cancer cells. TNF-α, whencombined with WA or celastrol, activated caspase-3and9with downregulation of XIAP in a dose-dependent manner leading to induction of apoptosis in MDA-MB-231breast cancer cells. Thecombination also caused accumulation of the proteasomal target protein IκBα, resulting in inhibitionof the nuclear translocation of nuclear factor-κB (NF-κB). Taken together, these results suggest thatTNF-α could sensitize breast cancer cells to WA and celastrol, at least in part, through inhibiting theactivation of NF-κB signaling leading to XIAP inhibition with subsequent upregulation of caspase-3and9activities. Thus, the anti-cancer activities of TNF-α are enhanced when combined with thenatural proteasome inhibitors, WA and celastrol.Methods1MTT assayã€Colony formation assaysã€Cell gap closure migration assays (scratch wound-healingassay) to detect activity, proliferation and migration of MDA-MB-231breast cancer cells2Flow cytometry technique to detect apoptosis of MDA-MB-231breast cancer cells3Caspase-3activity, chymotrypsin (CT)-like activity assay and Western blotting analysis to detectthe expression of caspase-3/7,-94Reverse transcription and RT-qPCR to detect the genic level of XIAP and cIAP1/25Immunofluorescence microscopy to detect the nuclear translocation of NF-κB-p65Results1. TNF-α sensitized breast cancer cells to WA and celastrol resulting in induction of cell death2. TNF-α sensitized breast cancer cells to WA and celastrol resulting in induction of apoptosis3. TNF-α combination with WA and celastrol induced apoptosis in breast cancer cells by affectingcaspase-9and caspase-3expression levels4. TNF-α+WA/celastrol has somewhat effect on proteasomal activities5. TNF-α combination with WA or celastrol affect XIAP levels at later time point6. WA or celastrol decrease XIAP, cIAP2mRNA levels and TNF-α mediated nuclear translocationof NF-κBConclusionsIn this study, we demonstrated that TNF-α could sensitize MDA-MB-231cells to WA and celastrolleading to apoptosis. One of the major mechanisms by which WA and celastrol mediate these effectson breast cancer cell appears to be through suppression of proteasomal activities, resulting ininhibition of IκBα degradation leading to suppression of NF-κB signaling. | Keywords/Search Tags: | proteasome inhibitors, inflammatory microenvironment, TNF-α, cancer, withaferin A/celastrol | PDF Full Text Request | Related items |
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