Pituitary adenoma is the most common tumor with a high recurrence rate due to a hormone-dependent JAK/signal transducer and activator of transcriptions (STAT) signaling. Atiprimod, a novel compound belonging to the azaspirane class of cationic amphiphilic drugs, has antiproliferative, anticarcinogenic effects in multiple myeloma, breast, and hepatocellular carcinoma by blocking STAT3 activation. Therapeutic agents' efficiency depends on endoplasmic reticulum (ER) stress-autophagy regulation during drug-mediated apoptotic cell death decision. However, the molecular machinery of dose-dependent atiprimod treatment regarding ER stress-autophagy has not been investigated yet. Thus, our aim is to investigate the ER stress-autophagy axis in atiprimodmediated apoptotic cell death in GH-secreting rat cell line (GH3) pituitary adenoma cells. Dose-dependent atiprimod treatment decreased GH3 cell viability, inhibited cell growth, and colony formation. Upregulation of Atg5, Atg12, Beclin-1 expressions, cleavage of LC-3II and formation of autophagy vacuoles were determined only after 1 µM atiprimod exposure. In addition, atiprimod-triggered ER stress was evaluated by BiP, C/EBP-homologous protein (CHOP), p-PERK upregulation, and Ca +2 release after 1 µM atiprimod exposure.Concomitantly, increasing concentration of atiprimod induced caspase-dependent apoptotic cell death via modulating Bcl 2 family members. Moreover, by Nacetyl cycteinc pretreatment, atiprimod triggered reactive oxygen species generation and prevented apoptotic induction. Concomitantly, dose-dependent atiprimod treatment decreased both GH and STAT3 expression in GH3 cells. In Abbreviations: AMPK, AMP-activated protein kinase; AO, acridine orange; ATF6, activating transcription factor 6; CHOP, C/EBP-homologous protein; DiOC6, 3,3′-dihexyloxacarbocyanine iodide; DMSO, dimethyl sulfoxide; EDTA, ethylenediaminetetraacetic acid; eIF-2 α, eukaryotic initiation factor 2; HRP, horseradish peroxidase; IRE-1α, inositol-requiring protein-1α; mTOR, mammalian target of rapamycin; MTT, 3-(4,5-
Background Growth Hormone Releasing Hormone (GHRH), 44 amino acids containing hypothalamic hormone, retains the biological activity by its first 29 amino acids. GHRH (NH2 1–29) peptide antagonists inhibit the growth of prostate, breast, ovarian, renal, gastric, pancreatic cancer in vitro and in vivo. Aptamers, single-strand RNA, or DNA oligonucleotides are capable of binding to target molecules with high affinity. Our aim in this study is to synthesize and select X-aptamers against both GHRH NH2 (1–29) and GHRH NH2 (1–44) and demonstrate synthesized aptamers’ target binding activity as well as serum stability. Methods and results Aptamers against GHRH NH2 (1–44) and NH2 (1–29) peptides were synthesized, and binding affinity (Kd) of 24 putative X-aptamers was determined by the dot-blot method, co-immunofluorescence staining and, SPR analysis. The serum stability of TKY.T1.08, TKY1.T1.13, TKY.T2.08, TKY.T2.09 X-aptamers was 90–120 h, respectively. The dose-dependent binding of TKY1.T1.13, TKY.T2.08, TKY.T2.09 X-aptamers on GHRHR in MIA PaCa-2 was approved by co-IF assay results. Moreover, SPR analysis indicated the Kd (4.75, 1.21, and 4.0 nM) levels of TKY2.T1.13, TKY.T2.08, TKY.T2.09 putative X-aptamers, respectively. Conclusion Our results illustrate the synthesis of 24 putative X-aptamers against both GHRH NH2 (1–44) and NH2 (1–29) peptides and TKY1.T1.13, TKY.T2.08, TKY.T2.09 X-aptamers have high serum stability, high target binding potential with low Kd levels.
Pancreatic cancer is the fourth most common cause of cancer death worldwide with limited therapeutic potential and low survival rate. A natural agent; triptolide (diterpenoid triepoxide), induce cell viability loss, growth inhibition and apoptotic cell death in various cancer cells such as breast, prostate and pancreatic cancer. Metformin, a common therapeutic agent for type II Diabetes mellitus, has been shown to induce apoptotic cell death in pancreatic cancer via modulating various pathways such as PI3K/Akt pathways. In this study, our aim was to investigate the potential additional effect of metformin on triptolide-induced apoptotic cell death in MiaPaCa-2 cells. Drugs-mediated cell viability loss, growth inhibition and colony formation potential were examined by MTT, trypan blue exclusion, colony formation, hanging drop assays. Drugs triggered apoptotic cell death was determined by PI FACS flow analysis. According to MTT cell viability assay, 20 nM triptolide decreased cell viability by 78%, but triptolide and metformin co-treatment decreased cell viability by 57%. Metformin co-treatment increased the triptolide-mediated cell growth inhibition, and prevention of colony formation in MiaPaCa-2 cells. In addition, co-treatment increased triptolide-triggered mitochondrial membrane potential loss, cell death and ROS generation. In conclusion, metformin co-treatment accelerated triptolide-triggered apoptotic cell death in MiaPaCa-2 cells.
Background: Cycline-dependent kinase inhibitors (CDKi); roscovitine and purvalanol, are promising anti-cancer drugs due to their strong anti-proliferative effectiveness due to activation of PA catabolism. Besides transforming acetylated spermine and spermidine into spermidine and putrescine, respectively, polyamine oxidase (PAO) also generates hydrogen peroxide in high concentrations as a by-product. PAO was assumed as a pivotal key molecule during drug-induced apoptosis in cancer cells. Our aim is to reveal the role of PAO action in CDKi-triggered apoptosis in Puma knock-out HCT116 colon cancer cells. Methods: HCT116 wt and HCT116 Puma-/- cells were treated with Roscovitine and Purvalanol and cell viability and apoptosis were determined. Protein was isolated from treated and untreated cells and key molecules of cell cycle control and polyamine pathways were investigated at translational level. Polyamine content was determined by HPLC for all conditions. MDL-72527 was used as a PAO inhibitor and apoptotic cell death was analysed. Results: Roscovitine and purvalanol induced apoptosis and increased the cytotoxic responses in HCT116 wt and HCT116 Puma-/- colon carcinoma cell lines by modulating CDK1, 4, cyclin-B1, D3. Both, CDKi altered intrinsic apoptotic pathways in HCT116 wt. Whereas, drug-induced apoptosis occurred caspase-independent in Puma-/- colon cancer cells. Roscovitine and purvalanol up-regulated polyamine catabolic enzymes, whereas CDK inhibitors decreased the polyamine levels in HCT116 wt and HCT116 Puma-/- colon cancer cells. In addition, PAO inhibitor MDL72527 prevented drug-induced apoptosis. Conclusion: PAO expression profile might be a critical target in CDK inhibitors-triggered apoptosis in HCT116 colorectal cancer cells. Thus, MAPK signaling pathway relations with cell cycle and polyamine catabolic pathway investigations are in progress.
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