miR27a, a fine-tuning molecule, interacts with growth hormone (GH) signaling and ornithine decarboxylase (ODC) via targeting STAT5.

Autocrine growth hormone (GH) expression triggers cell proliferation, invasion-metastasis in vitro and in vivo models, but GH gene mutations inhibit postnatal growth. Natural polyamines (PA); putrescine, spermidine, spermine trigger cell growth and differentiation. The importance of miR27a has shown to exert a suppressive effect on ornithine decarboxylase (ODC) expression in dwarf mice models. We aimed to modulate the role of A13S, F166Δ, T24 GH gene mutations' impact on PA metabolism and epithelial-mesencyhmal transition (EMT) pathway through miR27a. Biologically active GH signaling triggered cell viability, growth, and colony formation, but T24A alteration significantly decreases aggressive profiles due to inactive GH signaling through a decline in STAT5 activity and expressions of STAT5, c-myc and ODC. Although statistically significant increase in intracellular PA levels in wt GH signaling HEK293 cells compared to HEK293 cells with a lack of GH signaling, a sharp decline in PA levels measured in each mutant GH expressing HEK293 cells. When we inhibited miR27a, proliferation and colony formation accelerated through a significant increase in putrescine levels and upregulation of ODC, STAT5 expression. In contrast, a substantial decline in GH-mediated colony enlargement observed via ODC, STAT5 downregulation, and PA depletion in both wt and mutant GH expressing HEK293 cell lines by miR27a mimic transfection. In conclusion, T24A mutant GH expression declines the GH signaling through STAT5 activity, and mutant GH signaling decreased cell proliferation, division, and colony formation via EMT inhibition. The autocrine GH-mediated proliferative profiles were under the control of miR27a that depletes intracellular putrescine levels via targeting ODC.

Palbociclib negatively regulates fatty acid synthesis due to upregulation of AMPKα and miR-33a levels to increase apoptosis in Panc-1 and MiaPaCa-2 cells.

Fatty acids (FAs) synthesis mechanism has various regulators such as fatty acid synthase (FASN), AMP-regulated protein kinase (AMPK), or mammalian target of rapamycin (mTOR), which are aberrantly dysregulated in various pancreatic cancer cells. In this study, we aim to understand the regulatory role of palbociclib, a CDK4/6 inhibitor, on the cellular energy metabolism through regulation of AMPK/mTOR signaling by modulation of intracellular miR-33a levels in Panc-1 and MiaPaCa-2 cells. Palbociclib downregulated FAs metabolism more effectively in MiaPaCa-2 cells than Panc-1 cells. Moreover, palbociclib treatment increased the levels of miR-33a in each cell line albeit a higher increase was evident in MiaPaCa-2 cells. Stress-mediated activation of mTOR signaling axis was found associated with palbociclib-mediated AMPKα activation and miR33a upregulation. These findings provided that a deeper understanding about possible interactions of cell cycle activity and reduction of FAs synthesis may facilitate the enhancement of cell death mechanisms in pancreatic cancer cells.

microRNA-21 Regulates Stemness in Pancreatic Ductal Adenocarcinoma Cells.

Pancreatic ductal adenocarcinoma (PDAC) is the most common and aggressive type of pancreatic cancer (PCa) with a low survival rate. microRNAs (miRs) are endogenous, non-coding RNAs that moderate numerous biological processes. miRs have been associated with the chemoresistance and metastasis of PDAC and the presence of a subpopulation of highly plastic "stem"-like cells within the tumor, known as cancer stem cells (CSCs). In this study, we investigated the role of miR-21, which is highly expressed in Panc-1 and MiaPaCa-2 PDAC cells in association with CSCs. Following miR-21 knockouts (KO) from both MiaPaCa-2 and Panc-1 cell lines, reversed expressions of epithelial-mesenchymal transition (EMT) and CSCs markers were observed. The expression patterns of key CSC markers, including CD44, CD133, CX-C chemokine receptor type 4 (CXCR4), and aldehyde dehydrogenase-1 (ALDH1), were changed depending on miR-21 status. miR-21 (KO) suppressed cellular invasion of Panc-1 and MiaPaCa-2 cells, as well as the cellular proliferation of MiaPaCa-2 cells. Our data suggest that miR-21 is involved in the stemness of PDAC cells, may play roles in mesenchymal transition, and that miR-21 poses as a novel, functional biomarker for PDAC aggressiveness.

Epibrassinolide prevents tau hyperphosphorylation via GSK3ß inhibition in vitro and improves Caenorhabditis elegans lifespan and motor deficits in combination with roscovitine.

Glycogen synthase kinase 3ß (GSK3ß) is considered an important element of glycogen metabolism; however, it has many other regulatory roles. Changes in the GSK3ß signaling mechanism have been associated with various disorders, such as Alzheimer's disease (AD), type II diabetes, and cancer. Although the effects of GSK3ß inhibitors on reducing the pathological effects of AD have been described, an effective inhibitor has not yet been developed. Epibrassinolide (EBR), a brassinosteroid (BR), is structurally similar to mammalian steroid hormones. Our studies have shown that EBR has an inhibitory effect on GSK3ß in different cell lines. Roscovitine (ROSC), a cyclin-dependent kinase (CDK) inhibitor, has also been identified as a potential GSK3 inhibitor. Within the scope of this study, we propose that EBR and/or ROSC might have mechanistic action in AD models. To test this hypothesis, we used in vitro models and Caenorhabditis elegans (C. elegans) AD strains. Finally, EBR treatment successfully protected cells from apoptosis and increased the inhibitory phosphorylation of GSK3ß. In addition, EBR and/or ROSC treatment had a positive effect on the survival rates of C. elegans strains. More interestingly, the paralysis phenotype of the C. elegans AD model due to Aß42 toxicity was prevented by EBR and/or ROSC. Our findings suggest that EBR and ROSC administration have neuroprotective effects on both in vitro and C. elegans models via inhibitory GSK3ß phosphorylation at Ser9.

Atiprimod triggered apoptotic cell death via acting on PERK/eIF2α/ATF4/CHOP and STAT3/NF-ΚB axis in MDA-MB-231 and MDA-MB-468 breast cancer cells.

PURPOSE: The constitutive activation of STAT3 through receptor tyrosine kinases triggered breast cancer cell growth and invasion-metastasis. Atiprimod impacts anti-proliferative, anti-carcinogenic effects in hepatocellular carcinoma, lymphoma, multiple myeloma via hindering the biological activity of STAT3. Dose-dependent atiprimod evokes first autophagy as a survival mechanism and then apoptosis due to prolonged ER stress in pituitary adenoma cells. The therapeutic efficiency and mechanistic action of atiprimod in breast cancer cells have not been investigated yet. Thus, we aimed to modulate the pivotal role of ER stress in atiprimod-triggered apoptosis in MDA-MB-231 and MDA-MB-468 breast cancer cells. RESULTS: Dose- and time-dependent atiprimod treatment inhibits cell viability and colony formation in MDA-MB-468 and MDA-MB-231 breast cancer cells. A moderate dose of atiprimod (2 µM) inhibited STAT3 phosphorylation at Tyr705 residue and also suppressed the total expression level of p65. In addition, nuclear localization of STAT1, 3, and NF-κB was prevented by atiprimod exposure in MDA-MB-231 and MDA-MB-468 cells. Atiprimod evokes PERK, BiP, ATF-4, CHOP upregulation, and PERK (Thr980), eIF2α (Ser51) phosphorylation's. However, atiprimod suppressed IRE1α-mediated Atg-3, 5, 7, 12 protein expressions and no alteration was observed on Beclin-1, p62 expression levels. PERK/eIF2α/ATF4/CHOP axis pivotal role in atiprimod-mediated G1/S arrest and apoptosis via Bak, Bax, Bim, and PUMA upregulation in MDA-MB-468 cells. Moreover, atiprimod renders MDA-MB-231 more vulnerable to type I programmed cell death by plasmid-mediated increased STAT3 expression. CONCLUSION: Atiprimod induced prolonged ER stress-mediated apoptosis via both activating PERK/eIF2α/ATF4/CHOP axis and suppressing STAT3/NF-κB transcription factors nuclear migration in TBNC cells.

MiR-21 Is Required for the Epithelial-Mesenchymal Transition in MDA-MB-231 Breast Cancer Cells.

Breast cancer (BCa) is one of the leading health problems among women. Although significant achievements have led to advanced therapeutic success with targeted therapy options, more efforts are required for different subtypes of tumors and according to genomic, transcriptomic, and proteomic alterations. This study underlines the role of microRNA-21 (miR-21) in metastatic MDA-MB-231 breast cancer cells. Following the knockout of miR-21 from MDA-MB-231 cells, which have the highest miR-21 expression levels compared to MCF-7 and SK-BR-3 BCa cells, a decrease in epithelial-mesenchymal transition (EMT) via downregulation of mesenchymal markers was observed. Wnt-11 was a critical target for miR-21, and the Wnt-11 related signaling axis was altered in the stable miR-21 knockout cells. miR-21 expression was associated with a significant increase in mesenchymal markers in MDA-MB-231 BCa cells. Furthermore, the release of extracellular vesicles (EVs) was significantly reduced in the miR-21 KO cells, alongside a significant reduction in relative miR-21 export in EV cargo, compared with control cells. We conclude that miR-21 is a leading factor involved in mesenchymal transition in MDA-MB-231 BCa. Future therapeutic strategies could focus on its role in the treatment of metastatic breast cancer.

Palbociclib, a selective CDK4/6 inhibitor, restricts cell survival and epithelial-mesenchymal transition in Panc-1 and MiaPaCa-2 pancreatic cancer cells.

The mortality rate of pancreatic cancer has close parallels to its incidence rate because of limited therapeutics and lack of effective prognosis. Despite various novel chemotherapeutics combinations, the 5-year survival rate is still under 5%. In the current study, we aimed to modulate the aberrantly activated PI3K/AKT pathway and epithelial-mesenchymal transition (EMT) signaling with the treatment of CDK4/6 inhibitor PD-0332991 (palbociclib) in Panc-1 and MiaPaCa-2 pancreatic cancer cells. It was found that PD-0332991 effectively reduced cell viability and proliferation dose-dependently within 24 hours. In addition, PD-0332991 induced cell cycle arrest at the G1 phase by downregulation of aberrant expression of CDK4/6 through the dephosphorylation of Rb in each cell lines. Although PD-0332991 treatment increased epithelial markers and decreased mesenchymal markers, the nuclear translocation of ß-catenin was not prevented by PD-0332991 treatment, especially in MiaPaCa-2 cells. Effects of PD-0332991 on the regulation of PI3K/AKT signaling and its downstream targets such as GSK-3 were cell type-dependent. Although the activity of AKT was inhibited in both cell lines, the phosphorylation of GSK-3ß at Ser9 increased only in Panc-1. In conclusion, PD-0332991 induced cell cycle arrest and reduced the cell viability of Panc-1 and MiaPaCa-2 cells. However, PD-0332991 differentially affects the regulation of the PI3K/AKT pathway and EMT process in cells due to its distinct influence on Rb and GSK-3/ß-catenin signaling. Understanding the effect of PD-0332991 on the aberrantly activated signaling axis may put forward a new therapeutic strategy to reduce the cell viability and metastatic process of pancreatic cancer.

Epibrassinolide-induced autophagy occurs in an Atg5-independent manner due to endoplasmic stress induction in MEF cells.

Epibrassinolide (EBR), a polyhydroxysteroid belongs to plant growth regulator family, brassinosteroids and has been shown to have a similar chemical structure to mammalian steroid hormones. Our findings indicated that EBR could trigger apoptosis in cancer cells via induction of endoplasmic reticulum (ER) stress, caused by protein folding disturbance in the ER. Normal cells exhibited a remarkable resistance to EBR treatment and avoid from apoptotic cell death. The unfolded protein response clears un/misfolded proteins and restore ER functions. When stress is chronic, cells tend to die due to improper cellular functions. To understand the effect of EBR in non-malign cells, mouse embryonic fibroblast (MEF) cells were investigated in detail for ER stress biomarkers, autophagy, and polyamine metabolism in this study. Evolutionary conserved autophagy mechanism is a crucial cellular process to clean damaged organelles and protein aggregates through lysosome under the control of autophagy-related genes (ATGs). Cells tend to activate autophagy to promote cell survival under stress conditions. Polyamines are polycationic molecules playing a role in the homeostasis of important cellular events such as cell survival, growth, and, proliferation. The administration of PAs has been markedly extended the lifespan of various organisms via inducing autophagy and inhibiting oxidative stress. Our data indicated that ER stress is induced following EBR treatment in MEF cells as well as MEF Atg5-/- cells. In addition, autophagy is activated following EBR treatment by targeting PI3K/Akt/mTOR in wildtype (wt) cells. However, EBR-induced autophagy targets ULK1 in MEF cells lacking Atg5 expression. Besides, EBR treatment depleted the PA pool in MEF cells through the alterations of metabolic enzymes. The administration of Spd with EBR further increased autophagic vacuole formation. In conclusion, EBR is an anticancer drug candidate with selective cytotoxicity for cancer cells, in addition the induction of autophagy and PA metabolism are critical for responses of normal cells against EBR.

Proinflammatory cytokine profile is critical in autocrine GH-triggered curcumin resistance engulf by atiprimod cotreatment in MCF-7 and MDA-MB-231 breast cancer cells.

Active growth hormone (GH) signaling triggers cellular growth and invasion-metastasis in colon, breast, and prostate cancer. Curcumin, an inhibitor of NF-Ò¡B pathway, is assumed to be a promising anti-carcinogenic agent. Atiprimod is also an anti-inflammatory, anti-carcinogenic agent that induces apoptotic cell death in hepatocellular carcinoma, multiple myeloma, and pituitary adenoma. We aimed to demonstrate the potential additional effect of atiprimod on curcumin-induced apoptotic cell death via cytokine expression profiles in MCF-7 and MDA-MB-231 cells with active GH signaling. The effect of curcumin and/or atiprimod on IL-2, IL-4, and IL-17A levels were measured by ELISA assay. MTT cell viability, trypan blue exclusion, and colony formation assays were performed to determine the effect of combined drug exposure on cell viability, growth, and colony formation, respectively. Alteration of the NF-Ò¡B signaling pathway protein expression profile was determined following curcumin and/or atiprimod exposure by RT-PCR and immunoblotting. Finally, the effect of curcumin with/without atiprimod treatment on Reactive Oxygen Species (ROS) generation and apoptotic cell death was examined by DCFH-DA and Annexin V/PI FACS flow analysis, respectively. Autocrine GH-mediated IL-6, IL-8, IL-10 expressions were downregulated by curcumin treatment. Atiprimod co-treatment increased the inhibitory effect of curcumin on cell viability, proliferation and also increased the curcumin-triggered ROS generation in each GH+ breast cancer cells. Combined drug exposure increased apoptotic cell death through acting on IL-2, IL-4, and IL-17A secretion. Forced GH-triggered curcumin resistance might be overwhelmed by atiprimod and curcumin co-treatment via modulating NF-Ò¡B-mediated inflammatory cytokine expression in MCF-7 and MDA-MB-231 cells.

Putative Roles for Peptidylarginine Deiminases in COVID-19.

Peptidylarginine deiminases (PADs) are a family of calcium-regulated enzymes that are phylogenetically conserved and cause post-translational deimination/citrullination, contributing to protein moonlighting in health and disease. PADs are implicated in a range of inflammatory and autoimmune conditions, in the regulation of extracellular vesicle (EV) release, and their roles in infection and immunomodulation are known to some extent, including in viral infections. In the current study we describe putative roles for PADs in COVID-19, based on in silico analysis of BioProject transcriptome data (PRJNA615032 BioProject), including lung biopsies from healthy volunteers and SARS-CoV-2-infected patients, as well as SARS-CoV-2-infected, and mock human bronchial epithelial NHBE and adenocarcinoma alveolar basal epithelial A549 cell lines. In addition, BioProject Data PRJNA631753, analysing patients tissue biopsy data (n = 5), was utilised. We report a high individual variation observed for all PADI isozymes in the patients' tissue biopsies, including lung, in response to SARS-CoV-2 infection, while PADI2 and PADI4 mRNA showed most variability in lung tissue specifically. The other tissues assessed were heart, kidney, marrow, bowel, jejunum, skin and fat, which all varied with respect to mRNA levels for the different PADI isozymes. In vitro lung epithelial and adenocarcinoma alveolar cell models revealed that PADI1, PADI2 and PADI4 mRNA levels were elevated, but PADI3 and PADI6 mRNA levels were reduced in SARS-CoV-2-infected NHBE cells. In A549 cells, PADI2 mRNA was elevated, PADI3 and PADI6 mRNA was downregulated, and no effect was observed on the PADI4 or PADI6 mRNA levels in infected cells, compared with control mock cells. Our findings indicate a link between PADI expression changes, including modulation of PADI2 and PADI4, particularly in lung tissue, in response to SARS-CoV-2 infection. PADI isozyme 1-6 expression in other organ biopsies also reveals putative links to COVID-19 symptoms, including vascular, cardiac and cutaneous responses, kidney injury and stroke. KEGG and GO pathway analysis furthermore identified links between PADs and inflammatory pathways, in particular between PAD4 and viral infections, as well as identifying links for PADs with a range of comorbidities. The analysis presented here highlights roles for PADs in-host responses to SARS-CoV-2, and their potential as therapeutic targets in COVID-19.

Inhibition of extracellular signal-regulated kinase potentiates the apoptotic and antimetastatic effects of cyclin-dependent kinase inhibitors on metastatic DU145 and PC3 prostate cancer cells.

Purvalanol and roscovitine are specific cyclin-dependent kinase (CDK) inhibitors, which have antiproliferative and apoptotic effects on various types of cancer. Although, the apoptotic accomplishment of purvalanol and roscovitine was elucidated at the molecular level, the underlying exact of drug-induced apoptosis through mitogen-activated protein kinase (MAPK) signaling still speculative. In addition, the role of CDK inhibitors in the downregulation of extracellular signal-regulated kinase 1/2 (ERK1/2)-mediated epithelial-mesenchymal transition (EMT) remains unclear. Here, we investigated the potential effect of each CDK inhibitors on cell proliferation, migration, and generation of reactive oxygen species due to the inhibition of MAPKs in metastatic DU145 and PC3 prostate cancer cells. We reported that purvalanol and roscovitine induced mitochondria membrane potential loss-dependent apoptotic cell death, which was also characterized by activation of several caspases, cleavage of poly (ADP-ribose) polymerase-1 in DU145 and PC3 cells. Cotreatment of either purvalanol or roscovitine with ERK1/2 inhibitor, U0126, synergistically suppressed cell proliferation, and induced apoptotic action. Also, ERK1/2 inhibition potentiated the effect of each CDK inhibitor on the downregulation of EMT processes via increasing the epithelial marker and decreasing mesenchymal markers through reduction of Wnt signaling regulators in DU145 cells. This study provides biological evidence about purvalanol and roscovitine have apoptotic and antimetastatic effects via MAPK signaling on prostate cancer cell by activation of GSK3ß signaling and inhibition of phosphoinositide-3-kinase/AKT (PI3K/AKT) pathways involved in the EMT process.

Atiprimod induce apoptosis in pituitary adenoma: Endoplasmic reticulum stress and autophagy pathways.

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 atiprimod-mediated 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 Bcl2 family members. Moreover, by N-acetyl 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 addition, overexpression of STAT3 increased atiprimod-mediated cell viability loss and apoptotic cell death through suppressing autophagy and ER stress key molecules expression profile. In conclusion, a low dose of atiprimod exposure triggers autophagy and mild-ER stress as a survival mechanism, but increased atiprimod dose induced caspase-dependent apoptotic cell death by targeting STAT3 in GH3 pituitary adenoma cells.

Bag-1 silencing enhanced chemotherapeutic drug-induced apoptosis in MCF-7 breast cancer cells affecting PI3K/Akt/mTOR and MAPK signaling pathways.

The multifunctional anti-apoptotic Bag-1 protein has important roles in apoptosis, proteasome-mediated degradation, transcriptional regulation, and intracellular signaling. Bag-1 promotes cell survival and proliferation, and is overexpressed in breast cancer. Therefore, Bag-1-targeted therapy might be a promising strategy to treat breast cancer. However, the effects of Bag-1 silencing in combination with conventional chemotherapeutic drugs on cell viability and major signaling pathways have not yet been fully investigated in breast cancer cells. In this study, we investigated the cytotoxic effects of Bag-1 silencing, alone and in combination with cisplatin or paclitaxel treatment, in MCF-7 breast cancer cells. Bag-1 knockdown by shRNA or siRNA transfection sensitized MCF-7 cells to apoptosis induced by cisplatin or paclitaxel. Combination of Bag-1 silencing and drug treatment more potently downregulated the pro-survival PI3K/Akt/mTOR and p44/42 mitogen activated protein kinase (MAPK) pathways, and more potently upregulated the stress-activated p38 and SAPK/JNK MAPK pathways. Bag-1-silenced drug-treated cells had also highly reduced proliferative capacity, downregulated cyclin-cyclin dependent kinase complexes and upregulated tumor suppressors p21 and Rb. These results overall indicated that Bag-1 silencing enhanced cisplatin- or paclitaxel-induced cytotoxicity through multiple pathways. In conclusion, Bag-1 targeted therapy might enhance the therapeutic potential of conventional anti-cancer drugs in the treatment of breast cancer.

Curcumin prevented human autocrine growth hormone (GH) signaling mediated NF-κB activation and miR-183-96-182 cluster stimulated epithelial mesenchymal transition in T47D breast cancer cells.

Autocrine growth hormone (GH) signaling is a promoting factor for breast cancer via triggering abnormal cell growth, proliferation, and metastasis, drug resistance. Curcumin (diferuloylmethane), a polyphenol derived from turmeric (Curcuma longa), has anti-proliferative, anti-carcinogenic, anti-hormonal effect via acting on PI3K/Akt, NF-κB and JAK/STAT signaling. Forced GH expression induced epithelial mesenchymal transition (EMT) through stimulation of miR-182-96-183 cluster expression in breast cancer cells. This study aimed to investigate the role of NF-κB signaling and miR-182-96-183 cluster expression profile on autocrine GH-mediated curcumin resistance, which was prevented by time-dependent curcumin treatment in T47D breast cancer cells. Dose- and time-dependent effect of curcumin on T47D wt and GH+ breast cancer cells were evaluated by MTT cell viability and trypan blue assay. Apoptotic effect of curcumin was determined by PI and Annexin V/PI FACS flow analysis. Immunoblotting performed to investigate the effect of curcumin on PI3K/Akt/MAPK, NF-κB signaling. miR182-96-183 cluster expression profile was observed by qRT-PCR. Overexpression of GH triggered resistant profile against curcumin (20 µM) treatment for 24 h, but this resistance was accomplished following 48 h curcumin exposure. Concomitantly, forced GH induced invasion and metastasis through EMT and NF-κB activation were prevented by long-term curcumin exposure in T47D cells. Moreover, 48 h curcumin treatment prevented the autocrine GH-mediated miR-182-96-183 cluster expression stimulation in T47D cells. In consequence, curcumin treatment for 48 h, prevented autocrine GH-triggered invasion-metastasis, EMT activation through inhibiting NF-κB signaling and miR-182-96-183 cluster expression and induced apoptotic cell death by modulating Bcl-2 family members in T47D breast cancer cells.

Curcumin inhibits autocrine growth hormone-mediated invasion and metastasis by targeting NF-κB signaling and polyamine metabolism in breast cancer cells.

Curcumin is assumed to be a plant-derived therapeutic drug that triggers apoptotic cell death in vitro and in vivo by affecting different molecular targets such as NF-κB. Phase I/II trial of curcumin alone or with chemotherapeutic drugs has been accomplished in pancreatic, colon, prostate and breast cancer cases. Recently, autocrine growth hormone (GH) signaling-induced cell growth, metastasis and drug resistance have been demonstrated in breast cancer. In this study, our aim was to investigate the potential therapeutic effect of curcumin by evaluating the molecular machinery of curcumin-triggered apoptotic cell death via focusing on NF-κB signaling and polyamine (PA) metabolism in autocrine GH-expressing MCF-7, MDA-MB-453 and MDA-MB-231 breast cancer cells. For this purpose, a pcDNA3.1 (+) vector with a GH gene insert was transfected by a liposomal agent in all breast cancer cells and then selection was conducted in neomycin (G418) included media. Autocrine GH-induced curcumin resistance was overcome in a dose-dependent manner and curcumin inhibited cell proliferation, invasion-metastasis and phosphorylation of p65 (Ser536), and thereby partly prevented its DNA binding activity in breast cancer cells. Moreover, curcumin induced caspase-mediated apoptotic cell death by activating the PA catabolic enzyme expressions, which led to generation of toxic by-products such as H2O2 in MCF-7, MDA-MB-453 and MDA-MB-231 GH+ breast cancer cells. In addition, transient silencing of SSAT prevented curcumin-induced cell viability loss and apoptotic cell death in each breast cancer cells. In conclusion, curcumin could overcome the GH-mediated resistant phenotype via modulating cell survival, death-related signaling routes and activating PA catabolic pathway.

Cyclin-dependent kinase inhibitors, roscovitine and purvalanol, induce apoptosis and autophagy related to unfolded protein response in HeLa cervical cancer cells.

Roscovitine (Rosc) and purvalanol (Pur) are competitive inhibitors of cyclin-dependent kinases (CDKs) by targeting their ATP-binding pockets. Both drugs are shown to be effective to decrease cell viability and dysregulate the ratio of pro- and anti-apoptotic Bcl-2 family members, which finally led to apoptotic cell death in different cancer cell lines in vitro. It was well established that Bcl-2 family members have distinct roles in the regulation of other cellular processes such as endoplasmic reticulum (ER) stress. The induction of ER stress has been shown to play critical role in cell death/survival decision via autophagy or apoptosis. In this study, our aim was to investigate the molecular targets of CDK inhibitors on ER stress mechanism related to distinct cell death types in time-dependent manner in HeLa cervical cancer cells. Our results showed that Rosc and Pur decreased the cell viability, cell growth and colony formation, induced ER stress-mediated autophagy or apoptosis in time-dependent manner. Thus, we conclude that exposure of cells to CDK inhibitors induces unfolded protein response and ER stress leading to autophagy and apoptosis processes in HeLa cervical cancer cells.

Diclofenac induced apoptosis via altering PI3K/Akt/MAPK signaling axis in HCT 116 more efficiently compared to SW480 colon cancer cells.

Diclofenac is a preferential cyclooxygenase 2 inhibitor (COX-2) and member of non-steroidal anti-inflammatory drugs (NSAIDs). Inflammation is one of the main reason of poor prognosis of colon cancer cases; thereby NSAIDs are potential therapeutic agents in colon cancer therapy. In this study, our aim to understand the potential molecular targets of diclofenac, which may propose new therapeutic targets in HCT 116 (wt p53) and SW480 (mutant p53R273H) colon cancer cells. For this purpose, we identified different response against diclofenac treatment through expression profiles of PI3K/Akt/MAPK signaling axis. Our hypothesis was diclofenac-mediated apoptosis is associated with inhibition of PI3K/Akt/MAPK signaling axis. We found that sub-cytotoxic concentration of diclofenac (400 µM) promoted further apoptosis in HCT 116 cells compared to SW480 colon cancer cells. Diclofenac triggered dephosphorylation of PTEN, PDK, Akt, which led to inhibition of PI3K/Akt survival axis in HCT 116 colon cancer cells. However, diclofenac showed lesser effect in SW480 colon cancer cells. In addition, diclofenac further activated p44/42, p38 and SAPK/JNK in HCT 116 cells compared to SW480 cells.

Calreticulin is a fine tuning molecule in epibrassinolide-induced apoptosis through activating endoplasmic reticulum stress in colon cancer cells.

Epibrassinolide (EBR), a member of brassinostreoids plant hormones with cell proliferation promoting role in plants, is a natural polyhydroxysteroid with structural similarity to steroid hormones of vertebrates. EBR has antiproliferative and apoptosis-inducing effect in various cancer cells. Although EBR has been shown to affect survival and mitochondria-mediated apoptosis pathways in a p53-independent manner, the exact molecular targets of EBR are still under investigation. Our recent SILAC (Stable Isotope Labeling by Amino Acids in Cell Culture) data showed that the most significantly altered protein after EBR treatment was calreticulin (CALR). CALR, a chaperone localized in endoplasmic reticulum (ER) lumen, plays role in protein folding and buffering Ca2+ ions. The alteration of CALR may cause ER stress and unfolded protein response correspondingly the induction of apoptosis. Unfolded proteins are conducted to 26S proteasomal degradation following ubiquitination. Our study revealed that EBR treatment caused ER stress and UPR by altering CALR expression causing caspase-dependent apoptosis in HCT 116, HT29, DLD-1, and SW480 colon cancer cells. Furthermore, 48 h EBR treatment did not caused UPR in Fetal Human Colon cells (FHC) and Mouse Embryonic Fibroblast cells (MEF). In addition our findings showed that HCT 116 colon cancer cells lacking Bax and Puma expression still undergo UPR and related apoptosis. CALR silencing and rapamycin co-treatment prevented EBR-induced UPR and apoptosis, whereas 26S proteasome inhibition further increased the effect of EBR in colon cancer cells. All these findings showed that EBR is an ER stress and apoptotic inducer in colon cancer cells without affecting non-malignant cells.

mTOR is a fine tuning molecule in CDK inhibitors-induced distinct cell death mechanisms via PI3K/AKT/mTOR signaling axis in prostate cancer cells.

Purvalanol and roscovitine are cyclin dependent kinase (CDK) inhibitors that induce cell cycle arrest and apoptosis in various cancer cells. We further hypothesized that co-treatment of CDK inhibitors with rapamycin, an mTOR inhibitor, would be an effective combinatory strategy for the inhibition of prostate cancer regard to androgen receptor (AR) status due to inhibition of proliferative pathway, PI3K/AKT/mTOR, and induction of cell death mechanisms. Androgen responsive (AR+), PTEN(-/-) LNCaP and androgen independent (AR-), PTEN(+/-) DU145 prostate cancer cells were exposed to purvalanol (20 µM) and roscovitine (30 µM) with or without rapamycin for 24 h. Cell viability assay, immunoblotting, flow cytometry and fluorescence microscopy was used to define the effect of CDK inhibitors with or without rapamycin on proliferative pathway and cell death mechanisms in LNCaP and DU145 prostate cancer cells. Co-treatment of rapamycin modulated CDK inhibitors-induced cytotoxicity and apoptosis that CDK inhibitors were more potent to induce cell death in AR (+) LNCaP cells than AR (-) DU145 cells. CDK inhibitors in the presence or absence of rapamycin induced cell death via modulating upstream PI3K/AKT/mTOR signaling pathway in LNCaP cells, exclusively only treatment of purvalanol have strong potential to inhibit both upstream and downstream targets of mTOR in LNCaP and DU145 cells. However, co-treatment of rapamycin with CDK inhibitors protects DU145 cells from apoptosis via induction of autophagy mechanism. We confirmed that purvalanol and roscovitine were strong apoptotic and autophagy inducers that based on regulation of PI3K/AKT/mTOR signaling pathway. Co-treatment of rapamycin with purvalanol and roscovitine exerted different effects on cell survival and death mechanisms in LNCaP and DU145 cell due to their AR receptor status. Our studies show that co-treatment of rapamycin with CDK inhibitors inhibit prostate cancer cell viability more effectively than either agent alone, in part, by targeting the mTOR signaling cascade in AR (+) LNCaP cells. In this point, mTOR is a fine-tuning player in purvalanol and roscovitine-induced apoptosis and autophagy via regulation of PI3K/AKT and the downstream targets, which related with cell proliferation.

Epibrassinolide alters PI3K/MAPK signaling axis via activating Foxo3a-induced mitochondria-mediated apoptosis in colon cancer cells.

Epibrassinolide (EBR), a steroid-derived plant growth regulator, has been recently suggested as an apoptotic inducer in different cancer cells. In this experimental study, we investigated the potential apoptotic effect of EBR on stress-related and survival signaling molecules in colon carcinoma cells. EBR decreased cell viability and colony formation in HCT 116 and HT-29 colon carcinoma cells. The inactivation of PI3K/AKT by EBR treatment led to upregulation of Foxo3a, which in turn induced apoptosis in HCT 116 and HT-29 cells. In addition, the upstream non-receptor protein tyrosine kinase Src was found elevated allowing to the upregulation of p38, stress-activated protein kinase/Jun amino-terminal kinase and extracellular signal-regulated kinase 1/2 and their target genes c-jun, c-fos and c-myc in a time-dependent manner in HCT 116 cells within 48h. The alterations in PA metabolism caused intracellular PA pool decrease. The upregulation of pro-apoptotic Bak, Bax, Puma and Bim were accompanied with the decrease in Mcl-1 in HCT 116 and Bcl-xL expression profiles in HT-29 following 48h EBR treatment. We suggest that the upregulation of Bim expression levels might be related with one of the PI3K/AKT target transcription factor Foxo3a, which was dephosphorylated by EBR treatment in HCT 116 and HT-29 cells.