In Vitro Investigations of miR-33a Expression in Estrogen Receptor-Targeting Therapies in Breast Cancer Cells.

(1) Background: Increased fatty acid synthesis leads to the aggressive phenotype of breast cancer and renders efficiency of therapeutics. Regulatory microRNAs (miRNAs) on lipid biosynthesis pathways as miR-33a have potential to clarify the exact mechanism. (2) Methods: We determined miR-33a expression levels following exposure of MCF-7 and MDA-MB-231 breast cancer cells to estrogen receptor (ER) activator (estradiol-17ß, E2) or anti-estrogens (ICI 182,780, Fulvestrant, FUL) at non-cytotoxic concentrations. We related miR-33a expression levels in the cells to cellular lipid biosynthesis-related pathways through immunoblotting. (3) Results: miR-33a mimic treatment led to significantly downregulation of fatty acid synthase (FASN) in MCF-7 cells but not in MDA-MB-231 cells in the presence of estradiol-17ß (E2) or Fulvestrant (FUL). In contrast to the miR-33a inhibitor effect, miR-33a mimic co-transfection with E2 or FUL led to diminished AMP-activated protein kinase α (AMPKα) activity in MCF-7 cells. E2 increases FASN levels in MDA-MB-231 cells regardless of miR-33a cellular levels. miR-33a inhibitor co-treatment suppressed E2-mediated AMPKα activity in MDA-MB-231 cells. (4) Conclusions: The cellular expression levels of miR-33a are critical to understanding differential responses which include cellular energy sensors such as AMPKα activation status in breast cancer cells.

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.

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.

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.

The molecular targets of diclofenac differs from ibuprofen to induce apoptosis and epithelial mesenchymal transition due to alternation on oxidative stress management p53 independently in PC3 prostate cancer cells.

BACKGROUND: Prostate cancer is the most common type of cancer among men. Studies showed that the regular use of nonsteroidal antiinflammatory drugs might reduce disease progression risk for prostate cancer patients with prostate cancer. We evaluated the effects of ectopic expression of p53 on the biological functions of ibuprofen and diclofenac. MATERIALS AND METHODS: For this purpose, We investigated cell death decision pathways related to survival and aggressive cellular phenotypes such as extrinsic/intrinsic apoptosis decision, Protein Kinase B/ Forkhead box O (AKT/FoxO) axis, mitogen-activated protein kinases (MAPKs), reactive oxygen species (ROS) generation, and EMT (epithelial mesenchymal transition) in wild type and p53 + PC3 prostate cancer cells. RESULTS AND CONCLUSIONS: Ibuprofen (1 mM) and diclofenac (250 µM) effectively induced cell cycle arrest and led to apoptosis via modulating both extrinsic and intrinsic pathways. However, diclofenac was the only drug to generate ROS intermediates. Diclofenac triggered a typical EMT process with downregulated E-cadherin and upregulated N-cadherin, vimentin, and Snail in PC3 cells, regardless of p53 expression. In conclusion, although both drugs are effective on cell death mechanism, only diclofenac caused EMT because of increased ROS generation independent of p53. On the other hand, ibuprofen could inhibit metastasis via upregulating E-cadherin. The biological targets of both nonsteroidal antiinflammatory drugs are different to highlight their role in cell survival and death axis.

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.

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.

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.

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.

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.

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.

DENSpm overcame Bcl-2 mediated resistance against Paclitaxel treatment in MCF-7 breast cancer cells via activating polyamine catabolic machinery.

PURPOSE: The Bcl-2 mediated resistance is one of the most critical obstacle in cancer therapy. Conventional chemotherapeutics such as Paclitaxel, a commonly used in the treatment of metastatic breast cancer, is not sufficient to overcome Bcl-2 mediated drug resistance mechanism. Thus, combinational drug regimes are favored by researchers to overcome resistance phenotype against drugs. N1,N11-diethylnorspermine (DENSpm), a polyamine analogue, which is a promising drug candidate induced-cell cycle arrest and apoptosis in various cancer cells such as prostate, melanoma, colon and breast cancer cells via activated polyamine catabolism and reactive oxygen generation. Recent studies indicated the potential therapeutic role of DENSpm in phase I and II trials in breast cancer cases. Although the molecular targets of Paclitaxel in apoptotic cell death mechanism is well documented, the therapeutic effect of DENSpm and Paclitaxel in breast cancer cells has not been investigated yet. In this study, our aim was to determine the time dependent effect of DENSpm and Paclitaxel on apoptotic cell death via determination of polyamine metabolism related targets in wt and Bcl-2 overexpressing MCF-7 breast cancer cells. RESULTS: In our experimental study, Paclitaxel decreased cell viability in dose-dependent manner within 24h. Co-treatment of Paclitaxel (30nM) with DENSpm (20µM) further increased the cytoxicity of Paclitaxel (30nM) compared to alone Paclitaxel (30nM) treatment in MCF-7 Bcl-2+ breast cancer cells. In addition, we determined that resistance against Paclitaxel-induced apoptotic cell death in Bcl-2 overexpressed MCF-7 cells was overcome due to activation of polyamine catabolic pathway, which caused depletion of polyamines. CONCLUSIONS: DENSpm combinational treatment might increase the effect of low cytotoxic paclitaxel in drug-resistant breast cancer cases.

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.

The inhibition of PI3K and NFκB promoted curcumin-induced cell cycle arrest at G2/M via altering polyamine metabolism in Bcl-2 overexpressing MCF-7 breast cancer cells.

Bcl-2 protein has been contributed with number of genes which are involved in oncogenesis. Among the many targets of Bcl-2, NFκB have potential role in induction of cell cycle arrest. Curcumin has potential therapeutic effects against breast cancer through multiple signaling pathways. In this study, we investigated the role of curcumin in induction of cell cycle arrest via regulating of NFκB and polyamine biosynthesis in wt and Bcl-2+ MCF-7 cells. To examine the effect of curcumin on cell cycle regulatory proteins, PI3K/Akt, NFκB pathways and polyamine catabolism, we performed immunoblotting assay. In addition, cell cycle analysis was performed by flow cytometry. The results indicated that curcumin induced cell cycle arrest at G2/M phase by downregulation of cyclin B1 and Cdc2 and inhibited colony formation in MCF-7wt cells. However, Bcl-2 overexpression prevented the inhibition of cell cycle associated proteins after curcumin treatment. The combination of LY294002, PI3K inhibitor, and curcumin induced cell cycle arrest by decreasing CDK4, CDK2 and cyclin E2 in Bcl-2+ MCF-7 cells. Moreover, LY294002 further inhibited the phosphorylation of Akt in Bcl-2+ MCF-7 cells. Curcumin could suppress the nuclear transport of NFκB through decreasing the interaction of P-IκB-NFκB. The combination of wedelolactone, NFκB inhibitor, and curcumin acted different on SSAT expression in wt MCF-7 and Bcl-2+ MCF-7 cells. NFκB inhibition increased the SSAT after curcumin treatment in Bcl-2 overexpressed MCF-7 cells. Inhibition of NFκB activity as well as suppression of ROS generation with NAC resulted in the partial relief of cells from G2/M checkpoint after curcumin treatment in wt MCF-7 cells. In conclusion, the potential role of curcumin in induction of cell cycle arrest is related with NFκB-regulated polyamine biosynthesis.

SILAC-Based Mass Spectrometry Analysis Reveals That Epibrassinolide Induces Apoptosis via Activating Endoplasmic Reticulum Stress in Prostate Cancer Cells.

Epibrassinolide (EBR) is a polyhydroxylated sterol derivative and biologically active compound of the brassinosteroids. In addition to well-described roles in plant growth, EBR induces apoptosis in the LNCaP prostate cancer cells expressing functional androgen receptor (AR). Therefore, it is suggested that EBR might have an inhibitory potential on androgen receptor signaling pathway. However, the mechanism by which EBR exerts its effects on LNCaP is poorly understood. To address this gap in knowledge, we used an unbiased global proteomics approach, i.e., stable-isotope labeling by amino acids in cell culture (SILAC). In total, 964 unique proteins were identified, 160 of which were differentially expressed after 12 h of EBR treatment. The quantification of the differentially expressed proteins revealed that the expression of the unfolded protein response (UPR) chaperone protein, calreticulin (CALR), was dramatically downregulated. The decrease in CALR expression was also validated by immunoblotting. Because our data revealed the involvement of the UPR in response to EBR exposure, we evaluated the expression of the other UPR proteins. We demonstrated that EBR treatment downregulated calnexin and upregulated BiP and IRE1α expression levels and induced CHOP translocation from the cytoplasm to nucleus. The translocation of CHOP was associated with caspase-9 and caspase-3 activation after a 12 h EBR treatment. Co-treatment of EBR with rapamycin, an upstream mTOR pathway inhibitor, prevented EBR-induced cell viability loss and PARP cleavage in LNCaP prostate cancer cells, suggesting that EBR could induce ER stress in these cells. In addition, we observed similar results in DU145 cells with nonfunctional androgen receptor. When proteasomal degradation of proteins was blocked by MG132 co-treatment, EBR treatment further induced PARP cleavage relative to drug treatment alone. EBR also induced Ca2+ sequestration, which confirmed the alteration of the ER pathway due to drug treatment. Therefore, we suggest that EBR promotes ER stress and induces apoptosis.

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.

Bag-1 promotes cell survival through c-Myc-mediated ODC upregulation that is not preferred under apoptotic stimuli in MCF-7 cells.

Bag-1, Bcl-2 associated athanogene-1, is a multifunctional protein that can regulate a wide variety of cellular processes: proliferation, cell survival, transcription, apoptosis and motility. Bag-1 interacts with various targets in the modulation of these pathways; yet molecular details of Bag-1's involvement in each cellular event are still unclear. We first showed that forced Bag-1 expression promotes cell survival and prevents drug-induced apoptosis in MCF-7 breast cancer cells. Increased mRNA expressions of c-myc protooncogene and ornithine decarboxylase (ODC), biosynthetic enzyme of polyamines, were detected in Bag-1L+ cells, and western blots against the protein product of c-Myc and ODC confirmed these findings. Once ODC, a c-Myc target, gets activated, polyamine biosynthesis increases. We observed enhanced polyamine content in the Bag-1L+ cells. On the contrary, when polyamine catabolic mechanisms were investigated, Bag-1 silencing suppressed biosynthesis of polyamines because of the downregulation of ODC and upregulation of PAO. Exposure of cells to apoptotic inducers enhances the cell death mechanism by producing toxic products such as H2 O2 and aldehydes. Bag-1L+ cells prevented drug-induced PAO activation leading to a decrease in H2 O2 production following cisplatin or paclitaxel treatment. In this line, our results suggested that Bag-1 indirectly affects cell survival through c-Myc activated signalling that causes elevation of ODC levels, leading to an increase of the polyamine content.

Inhibition of PI3K signaling triggered apoptotic potential of curcumin which is hindered by Bcl-2 through activation of autophagy in MCF-7 cells.

Curcumin is a natural anti-cancer agent derived from turmeric (Curcuma longa). Curcumin triggers intrinsic apoptotic cell death by activating mitochondrial permeabilization due to the altered expression of pro- and anti-apoptotic Bcl-2 family members. Phosphoinositol-3-kinase (PI3K) and Akt, key molecular players in the survival mechanism, have been shown to be associated with the Bcl-2 signaling cascade; therefore, evaluating the therapeutic efficiency of drugs that target both survival and the apoptosis mechanism has gained importance in cancer therapy. We found that Bcl-2 overexpression is a limiting factor for curcumin-induced apoptosis in highly metastatic MCF-7 breast cancer cells. Forced overexpression of Bcl-2 also blocked curcumin-induced autophagy in MCF-7 cells, through its inhibitory interactions with Beclin-1. Pre-treatment of PI3K inhibitor LY294002 enhanced curcumin-induced cell death, apoptosis, and autophagy via modulating the expression of Bcl-2 family members and autophagosome formation in MCF-7 breast cancer cells. Atg7 silencing further increased apoptotic potential of curcumin in the presence or absence of LY294002 in wt and Bcl-2+ MCF-7 cells. The findings of this study support the hypothesis that blocking the PI3K/Akt pathway may further increased curcumin-induced apoptosis and overcome forced Bcl-2 expression level mediated autophagic responses against curcumin treatment in MCF-7 cells.

Purvalanol induces endoplasmic reticulum stress-mediated apoptosis and autophagy in a time-dependent manner in HCT116 colon cancer cells.

Purvalanol, a novel cyclin-dependent kinase inhibitor, is referred to as a strong apoptotic inducer which causes cell cycle arrest in various cancer cells such as prostate, breast and colon cancer cell lines. Various physiological and pathological conditions such as glucose starvation, inhibition of protein glycosylation and oxidative stress may cause an accumulation of unfolded proteins in the endoplasmic reticulum (ER), leading to the unfolded protein response (UPR) and autophagy. Lacking proteosomal function on aggregates of unfolded proteins, ER stress may induce autophagic machinery. Autophagy, an evolutionarily conserved process, is characterized by massive degradation of cytosolic contents. In the present study, our aim was to determine the time-dependent, ER-mediated apoptotic and autophagy induction of purvalanol in HCT 116 colon cancer cells. Fifteen micromoles of purvalanol induced a reduction in cell viability by 20 and 35% within 24 and 48 h, respectively. HCT 116 colon cancer cells were exposed to purvalanol, which activated ER stress via upregulation of PERK, IRE1α gene expression, eIF-2α phosphorylation and ATF-6 cleavage at early time-points in the HCT 116 colon cancer cells. Moreover, we determined that during purvalanol-mediated ER stress, autophagic machinery was also activated prior to apoptotic cell death finalization. Beclin-1 and Atg-5 expression levels were upregulated and LC3 was cleaved after a 6 h purvalanol treatment. Purvalanol induced mitochondrial membrane potential loss, caspase-7 and caspase-3 activation and PARP cleavage following a 48 h treatment. Thus, we conclude that the anticancer effect of purvalanol in HCT 116 cells was due to ER stress-mediated apoptosis; however, purvalanol triggered autophagy, which functions as a cell survival mechanism at early time-points.

Polyamines modulate the roscovitine-induced cell death switch decision autophagy vs. apoptosis in MCF-7 and MDA-MB-231 breast cancer cells.

Current clinical strategies against breast cancer mainly involve the use of anti­hormonal agents to decrease estrogen production; however, development of resistance is a major problem. The resistance phenotype depends on the modulation of cell­cycle regulatory proteins, cyclins and cyclin­dependent kinases. Roscovitine, a selective inhibitor of cyclin­dependent kinases, shows high therapeutic potential by causing cell­cycle arrest in various cancer types. Autophagy is a type of cell death characterized by the enzymatic degradation of macromolecules and organelles in double­ or multi­membrane autophagic vesicles. This process has important physiological functions, including the degradation of misfolded proteins and organelle turnover. Recently, the switch between autophagy and apoptosis has been proposed to constitute an important regulator of cell death in response to chemotherapeutic drugs. The process is regulated by several proteins, such as the proteins of the Atg family, essential for the initial formation of the autophagosome, and PI3K, important at the early stages of autophagic vesicle formation. Polyamines (PAs) are small aliphatic amines that play major roles in a number of eukaryotic processes, including cell proliferation. The PA levels are regulated by ornithine decarboxylase (ODC), the rate­limiting enzyme in PA biosynthesis. In this study, we aimed to investigate the role of PAs in roscovitine­induced autophagic/apoptotic cell death in estrogen receptor­positive MCF­7 and estrogen receptor­negative MDA­MB­231 breast cancer cells. We show that MDA­MB­231 cells are more resistant to roscovitine than MCF­7 cells. This difference was related to the regulation of autophagic key molecules in MDA­MB­231 cells. In addition, we found that exogenous PAs have a role in the cell death decision between roscovitine­induced apoptosis or autophagy in MCF­7 and MDA­MB­231 breast cancer cells.