The prohibitin-binding compound fluorizoline induces the pro-inflammatory cytokines interleukin-8 and interleukin-6 through the activation of JNK and p38 MAP kinases.

Fluorizoline is a prohibitin (PHB)-binding compound that induces apoptosis in several cancer cell lines as well as in primary cells from hematologic malignancies. In this study, we show that fluorizoline treatment triggers the activation of the stress-activated kinases c-Jun N-terminal kinase (JNK) and p38 prior to caspase activation in human cell lines. However, the blockage of p38 and JNK activity with chemical inhibitors or siRNA-mediated downregulation of MAPK14 (p38) does not prevent fluorizoline-induced apoptosis, suggesting that the activation of these kinases plays an alternative role in the cell response to fluorizoline treatment. Here, we describe that fluorizoline treatment leads to the secretion of pro-inflammatory cytokines interleukin-8 (IL-8) and interleukin-6 (IL-6). Importantly, we demonstrate that the activation of the stress-activated kinases JNK and p38 mediates the secretion of both IL-8 and IL-6. This study shows novel insights into the pro-inflammatory role exhibited by a compound that binds to PHB, thus supporting the potential of PHBs as anti-inflammatory proteins.

Purine Nucleotide Alterations in Tumoral Cell Lines Maintained with Physiological Levels of Folic Acid.

Most cancer cells have an increased synthesis of purine nucleotides to fulfil their enhanced division rate. The de novo synthesis of purines requires folic acid in the form of N10-formyltetrahydrofolate (10-formyl-THF). However, regular cell culture media contain very high, non-physiological concentrations of folic acid, which may have an impact on cell metabolism. Using cell culture media with physiological levels of folic acid (25 nM), we uncover purine alterations in several human cell lines. HEK293T, Jurkat, and A549 cells accumulate 5'-aminoimidazole-4-carboxamide ribonucleotide (ZMP), an intermediary of the de novo biosynthetic pathway, at physiological levels of folic acid, but not with the artificially high levels (2200 nM) present in regular media. Interestingly, HEK293T and Jurkat cells do not accumulate high levels of ZMP when AICAr, the precursor of ZMP, is added to medium containing 2200 nM folate; instead, ATP levels are increased, suggesting an enhanced de novo synthesis. On the other hand, HeLa and EHEB cells do not accumulate ZMP at physiological levels of folic acid, but they do accumulate in medium containing AICAr plus 2200 nM folate. Expression of SLC19A1, which encodes the reduced folate carrier (RFC), is increased in HEK293T and Jurkat cells compared with HeLa and EHEB, and it is correlated with the total purine nucleotide content at high levels of folic acid or with ZMP accumulation at physiological levels of folic acid. In conclusion, tumoral cell lines show a heterogenous response to folate changes in the media, some of them accumulating ZMP at physiological levels of folic acid. Further research is needed to clarify the ZMP downstream targets and their impact on cell function.

The Prohibitin-Binding Compound Fluorizoline Activates the Integrated Stress Response through the eIF2α Kinase HRI.

Fluorizoline is a synthetic molecule that induces apoptosis, by selectively targeting prohibitins (PHBs), through induction of the BH3-only protein NOXA. This induction is transcriptionally regulated by the integrated stress response (ISR)-related transcription factors ATF3 and ATF4. Here, we evaluate the role of the four eIF2α kinases, to decipher which is responsible for the mechanism of ISR activation triggered by fluorizoline in HeLa and HAP1 cells. First, we demonstrated the involvement of the eIF2α kinases using ISR inhibitor (ISRIB) and by simultaneous downregulation of all four eIF2α kinases, as both approaches were able to increase cell resistance to fluorizoline-induced apoptosis. Furthermore, we confirmed that fluorizoline treatment results in endoplasmic reticulum (ER) stress, as evidenced by PERK activation. Despite PERK activation, this kinase was not directly involved in the ISR activation by fluorizoline. In this regard, we found that the eIF2α kinases are capable of compensating for each other's loss of function. Importantly, we demonstrated that the mitochondrial-stress-related eIF2α kinase HRI mediates ISR activation after fluorizoline treatment.

The prohibitin-binding compound fluorizoline inhibits mitophagy in cancer cells.

Fluorizoline is a prohibitin-binding compound that triggers apoptosis in several cell lines from murine and human origin, as well as in primary cells from hematologic malignancies by inducing the integrated stress response and ER stress. Recently, it was described that PHB (Prohibitin) 1 and 2 are crucial mitophagy receptors involved in mediating the autophagic degradation of mitochondria. We measured mitophagy in HeLa cells expressing Parkin and in A549, a lung cancer cell line that can undergo mitophagy in a Parkin-independent manner, and we demonstrated that both fluorizoline and rocaglamide A, another PHB-binding molecule, inhibit CCCP- and OA-induced mitophagy. Moreover, we demonstrated that PHBs are mediating Parkin-dependent mitophagy. In conclusion, besides being a potent pro-apoptotic compound, we present fluorizoline as a promising new mitophagy modulator that could be used as anticancer agent.

Activation of the Integrated Stress Response and ER Stress Protect from Fluorizoline-Induced Apoptosis in HEK293T and U2OS Cell Lines.

The prohibitin (PHB)-binding compound fluorizoline as well as PHB-downregulation activate the integrated stress response (ISR) in HEK293T and U2OS human cell lines. This activation is denoted by phosphorylation of eIF2α and increases in ATF4, ATF3, and CHOP protein levels. The blockage of the activation of the ISR by overexpression of GRP78, as well as an increase in IRE1 activity, indicate the presence of ER stress after fluorizoline treatment. The inhibition of the ER stress response in HEK293T and U2OS led to increased sensitivity to fluorizoline-induced apoptosis, indicating a pro-survival role of this pathway after fluorizoline treatment in these cell lines. Fluorizoline induced an increase in calcium concentration in the cytosol and the mitochondria. Finally, two different calcium chelators reduced fluorizoline-induced apoptosis in U2OS cells. Thus, we have found that fluorizoline causes increased ER stress and activation of the integrated stress response, which in HEK293T and U2OS cells are protective against fluorizoline-induced apoptosis.

Fluorizoline-induced apoptosis requires prohibitins in nematodes and human cells.

We previously showed that fluorizoline, a fluorinated thiazoline compound, binds to both subunits of the mitochondrial prohibitin (PHB) complex, PHB1 and PHB2, being the expression of these proteins required for fluorizoline-induced apoptosis in mouse embryonic fibroblasts. To investigate the conservation of this apoptotic mechanism, we studied the effect of PHB downregulation on fluorizoline activity on two human cell lines, HEK293T and U2OS. Then, we asked whether PHBs mediate the effect of fluorizoline in a multicellular organism. Interestingly, reduced levels of PHBs in the human cells impaired the induction of apoptosis by fluorizoline. We observed that fluorizoline has a detrimental dose-dependent effect on the development and survival of the nematode model Caenorhabditis elegans. Besides, such effects of fluorizoline treatment in living nematodes were absent in PHB mutants. Finally, we further explored the apoptotic pathway triggered by fluorizoline in human cell lines. We found that the BH3-only proteins NOXA, BIM and PUMA participate in fluorizoline-induced apoptosis and that the induction of NOXA and PUMA is dependent on PHB expression.

NOXA upregulation by the prohibitin-binding compound fluorizoline is transcriptionally regulated by integrated stress response-induced ATF3 and ATF4.

Fluorizoline is a new synthetic molecule that induces p53-independent apoptosis, in several tumor cell lines and in primary leukemia cells, by selectively targeting prohibitins (PHBs). In this study, we describe how fluorizoline induces BCL-2 homology 3-only protein NOXA, without modulating the protein levels of anti-apoptotic B-cell lymphoma-2 (BCL-2) family members prior to caspase activation, as well as how it synergizes with the BCL-2 and BCL-XL inhibitor ABT-737 to induce apoptosis. Interestingly, fluorizolinetreatment triggers the activation of the integrated stress response (ISR) in HeLa and HAP1 cells, with increased eukaryotic translation initiation factor 2α phosphorylation, and induction of ATF3, ATF4, and CHOP. Moreover, PHB downregulation induces similar ISR activation and apoptosis as with fluorizoline treatment. In addition, we studied the essential role of the pro-apoptotic protein NOXA in fluorizoline-induced apoptosis and we describe its mechanism of induction in HeLa and HAP1 cells. Moreover, we identified ATF3 and ATF4 as the transcription factors that bind to NOXA promoter upon fluorizoline treatment. Furthermore, using ATF3 and ATF4 CRISPR HeLa and HAP1 cells, we confirmed that both factors mediate the induction of NOXA and apoptosis by fluorizoline. In conclusion, fluorizoline treatment triggers the activation of the ISR that results in the induction of ATF3 and ATF4, important regulators of NOXA transcription in fluorizoline-induced apoptosis.

The BCL-2 family members NOXA and BIM mediate fluorizoline-induced apoptosis in multiple myeloma cells.

Fluorizoline is a new synthetic molecule that induces apoptosis by selectively targeting prohibitins. In this study, we have assessed the pro-apoptotic effect of fluorizoline in 3 different multiple myeloma cell lines and 12 primary samples obtained from treatment-naïve multiple myeloma patients. Fluorizoline induced apoptosis in both multiple myeloma cell lines and primary samples at concentrations in the low micromolar range. All primary samples were sensitive to fluorizoline. Moreover, fluorizoline increased the mRNA and protein levels of the pro-apoptotic BCL-2 family member NOXA both in cell lines and primary samples analyzed. Finally, NOXA-depletion by CRISPR/Cas9 in cells that do not express BIM conferred resistance to fluorizoline-induced apoptosis in multiple myeloma cells. These results suggest that targeting prohibitins could be a new therapeutic strategy for myeloma multiple.

Involvement of both caspase-8 and Noxa-activated pathways in endoplasmic reticulum stress-induced apoptosis in triple-negative breast tumor cells.

Recent evidences indicate that triple-negative breast cancer (TNBC) cells with a mesenchymal phenotype show a basal activation of the unfolded protein response (UPR) that increases their sensitivity to endoplasmic reticulum (ER) stress although the underlying cell death mechanism remains largely unexplored. Here we show that both caspase-8-dependent and -independent apoptotic mechanisms are activated in TNBC cells undergoing sustained ER stress. Activation of the extrinsic apoptotic pathway by ER stress involves ATF4-dependent upregulation of tumor necrosis factor-related apoptosis-inducing ligand receptor 2 (TRAIL-R2/DR5). In addition, accumulation of BH3-only protein Noxa at the mitochondria further contributes to apoptosis following ER stress in TNBC cells. Accordingly, simultaneous abrogation of both extrinsic and intrinsic apoptotic pathways is required to inhibit ER stress-induced apoptosis in these cells. Importantly, persistent FLICE-inhibitory protein (FLIP) expression plays an adaptive role to prevent early activation of the extrinsic pathway of apoptosis upon ER stress. Overall, our data show that ER stress induces cell death through a pleiotropic mechanism in TNBC cells and suggest that targeting FLIP expression may be an effective approach to sensitize these tumor cells to ER stress-inducing agents.

The prohibitin-binding compound fluorizoline induces apoptosis in chronic lymphocytic leukemia cells through the upregulation of NOXA and synergizes with ibrutinib, 5-aminoimidazole-4-carboxamide riboside or venetoclax.

Fluorizoline is a new synthetic molecule that induces apoptosis by selectively targeting prohibitins. In the study herein, the pro-apoptotic effect of fluorizoline was assessed in 34 primary samples from patients with chronic lymphocytic leukemia. Fluorizoline induced apoptosis in chronic lymphocytic leukemia cells at concentrations in the low micromolar range. All primary samples were sensitive to fluorizoline irrespective of patients' clinical or genetic features, whereas normal T lymphocytes were less sensitive. Fluorizoline increased the protein levels of the pro-apoptotic B-cell lymphoma 2 family member NOXA in chronic lymphocytic leukemia cells. Furthermore, fluorizoline synergized with ibrutinib, 5-aminoimidazole-4-carboxamide riboside or venetoclax to induce apoptosis. These results suggest that targeting prohibitins could be a new therapeutic strategy for chronic lymphocytic leukemia.

Targeting prohibitins induces apoptosis in acute myeloid leukemia cells.

Fluorizoline is a new synthetic molecule that induces apoptosis by selectively targeting prohibitins (PHBs). In this study, the pro-apoptotic effect of fluorizoline was assessed in two cell lines and 21 primary samples from patients with debut of acute myeloid leukemia (AML). Fluorizoline induced apoptosis in AML cells at concentrations in the low micromolar range. All primary samples were sensitive to fluorizoline irrespectively of patients' clinical or genetic features. In addition, fluorizoline inhibited the clonogenic capacity and induced differentiation of AML cells. Fluorizoline increased the mRNA and protein levels of the pro-apoptotic BCL-2 family member NOXA both in cell lines and primary samples analyzed. These results suggest that targeting PHBs could be a new therapeutic strategy for AML.

A novel prohibitin-binding compound induces the mitochondrial apoptotic pathway through NOXA and BIM upregulation.

We previously described diaryl trifluorothiazoline compound 1a (hereafter referred to as fluorizoline) as a first-in-class small molecule that induces p53-independent apoptosis in a wide range of tumor cell lines. Fluorizoline directly binds to prohibitin 1 and 2 (PHBs), two proteins involved in the regulation of several cellular processes, including apoptosis. Here we demonstrate that fluorizoline-induced apoptosis is mediated by PHBs, as cells depleted of these proteins are highly resistant to fluorizoline treatment. In addition, BAX and BAK are necessary for fluorizoline-induced cytotoxic effects, thereby proving that apoptosis occurs through the intrinsic pathway. Expression analysis revealed that fluorizoline induced the upregulation of Noxa and Bim mRNA levels, which was not observed in PHB-depleted MEFs. Finally, Noxa(-/-)/Bim(-/-) MEFs and NOXA-downregulated HeLa cells were resistant to fluorizoline-induced apoptosis. All together, these findings show that fluorizoline requires PHBs to execute the mitochondrial apoptotic pathway.

A trifluorinated thiazoline scaffold leading to pro-apoptotic agents targeting prohibitins.

A new class of small molecules, with an unprecedented trifluorothiazoline scaffold, were synthesized and their pro-apoptotic activity was evaluated. With an EC50 in the low micromolar range, these compounds proved to be potent inducers of apoptosis in a broad spectrum of tumor cell lines, regardless of the functional status of p53. Fast structure-activity relationship studies allowed the preparation of the strongest apoptosis-inducing candidate. Using a high performance affinity purification approach, we identified prohibitins 1 and 2, key proteins involved in the maintenance of cell viability, as the targets for these compounds.

Activated ERBB2/HER2 licenses sensitivity to apoptosis upon endoplasmic reticulum stress through a PERK-dependent pathway.

HER2/Neu/ERBB2 is a receptor tyrosine kinase overexpressed in approximately 20% of human breast tumors. Truncated or mutant isoforms that show increased oncogenicity compared with the wild-type receptor are found in many breast tumors. Here, we report that constitutively active ERBB2 sensitizes human breast epithelial cells to agents that induce endoplasmic reticulum stress, altering the unfolded protein response (UPR) of these cells. Deregulation of the ERK, AKT, and mTOR activities elicited by mutant ERBB2 was involved in mediating this differential UPR response, elevating the response to endoplasmic reticulum stress, and apoptotic cell death. Mechanistic investigations revealed that the increased sensitivity of mutant ERBB2-expressing cells to endoplasmic reticulum stress relied upon a UPR effector signaling involving the PERK-ATF4-CHOP pathway, upregulation of the proapoptotic cell surface receptor TRAIL-R2, and activation of proapoptotic caspase-8. Collectively, our results offer a rationale for the therapeutic exploration of treatments inducing endoplasmic reticulum stress against mutant ERBB2-expressing breast tumor cells.

AICAR induces Bax/Bak-dependent apoptosis through upregulation of the BH3-only proteins Bim and Noxa in mouse embryonic fibroblasts.

5-Aminoimidazole-4-carboxamide (AICA) riboside (AICAR) is a nucleoside analogue that is phosphorylated to 5-amino-4-imidazolecarboxamide ribotide (ZMP), which acts as an AMP mimetic and activates AMP-activated protein kinase (AMPK). It has been recently described that AICAR triggers apoptosis in chronic lymphocytic leukemia (CLL) cells, and its mechanism of action is independent of AMPK as well as p53. AICAR-mediated upregulation of the BH3-only proteins BIM and NOXA correlates with apoptosis induction in CLL cells. Here we propose mouse embryonic fibroblasts (MEFs) as a useful model to analyze the mechanism of AICAR-induced apoptosis. ZMP formation was required for AICAR-induced apoptosis, though direct Ampk activation with A-769662 failed to induce apoptosis in MEFs. AICAR potently induced apoptosis in Ampkα1 (-/-) /α2 (-/-) MEFs, demonstrating an Ampk-independent mechanism of cell death activation. In addition, AICAR acts independently of p53, as MEFs lacking p53 also underwent apoptosis normally. Notably, MEFs lacking Bax and Bak were completely resistant to AICAR-induced apoptosis, confirming the involvement of the mitochondrial pathway in its mechanism of action. Apoptosis was preceded by ZMP-dependent but Ampk-independent modulation of the mRNA levels of different Bcl-2 family members, including Noxa, Bim and Bcl-2. Bim protein levels were accumulated upon AICAR treatment of MEFs, suggesting its role in the apoptotic process. Strikingly, MEFs lacking both Bim and Noxa displayed high resistance to AICAR. These findings support the notion that MEFs are a useful system to further dissect the mechanism of AICAR-induced apoptosis.

Epigenetic profile in chronic lymphocytic leukemia using methylation-specific multiplex ligation-dependent probe amplification.

AIM: To analyze the methylation status of 35 tumor suppressor genes using methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) in chronic lymphocytic leukemia (CLL). MATERIALS & METHODS: The DNA of 37 samples from patients with CLL, six healthy donors, and Jurkat and Ramos cell lines was analyzed by MS-MLPA. RESULTS: Our results confirm that hypermethylation is a common and not randomly distributed event in CLL, and some genes, such as WT1, CDH13, IGSF4/TSLC1, GATA5, DAPK1 and RARB, are hypermethylated in more than 25% of the analyzed samples. Importantly, MS-MLPA also detected hypermethylation of some genes not reported previously in CLL, and their methylation status was confirmed by bisulfite sequencing. CONCLUSION: These results indicate that MS-MLPA is a useful technique for the detection of methylation in CLL samples. Selecting CLL-specific methylation targets in order to generate a CLL-specific MS-MLPA probe set could enhance its usefulness as a tool in studies of risk stratification and guiding the best therapeutic decision.

Glycogen synthase kinase-3ß is involved in ligand-dependent activation of transcription and cellular localization of the glucocorticoid receptor.

Glucocorticoids (GC) induce cell cycle arrest and apoptosis in different cell types and therefore are widely used to treat a variety of diseases including autoimmune disorders and cancer. This effect is mediated by the GC receptor (GR), a ligand-activated transcription factor that translocates into the nucleus where it modulates transcription of target genes in a promoter-specific manner. Glycogen synthase kinase-3 (GSK3) regulates GR response by genomic and nongenomic mechanisms, although the specific role of each isoform is not well defined. We used GSK3 pharmacological inhibitors and isoform-specific small interfering RNA to evaluate the role of GSK3 in the genomic regulation induced by GC. GSK3 inhibition resulted in the reduction of GC-induced mRNA expression of GC-induced genes such as BIM, HIAP1, and GILZ. Knockdown of GSK3ß but not GSK3α reduced endogenous GILZ induction in response to dexamethasone and GR-dependent reporter gene activity. Chromatin immunoprecipitation experiments revealed that GSK3 inhibition impaired the dexamethasone-mediated binding of GR and RNA polymerase II to endogenous GILZ promoter. These results indicate that GSK3ß is important for GR transactivation activity and that GSK3ß inhibition suppresses GC-stimulated gene expression. Furthermore, we show that genomic regulation by the GR is independent of known GSK3ß phosphorylation sites. We propose that GC-dependent transcriptional activation requires functional GSK3ß signaling and that altered GSK3ß activity influences cell response to GC.

Analysis of apoptosis regulatory genes altered by histone deacetylase inhibitors in chronic lymphocytic leukemia cells.

Histone deacetylases (HDACs) play a key role in the regulation of acetylation status not only of histones but also of many other non-histone proteins involved in cell cycle regulation, differentiation or apoptosis. Therefore, histone deacetylase inhibitors (HDACi) have emerged as promising anticancer agents. Herein, we report the characterization of apoptosis in B-cell chronic lymphocytic leukemia (CLL) induced by two HDACi, Kendine 92 and SAHA. Both inhibitors induce dose-, time- and caspase-dependent apoptosis through the mitochondrial pathway. Interestingly, Kendine 92 and SAHA show a selective cytotoxicity for B lymphocytes and induce apoptosis in CLL cells with mutated or deleted TP53 as effectively as in tumor cells harboring wild-type TP53. The pattern of apoptosis-related gene and protein expression profile has been characterized. It has shown to be irrespective of TP53 status and highly similar between SAHA and Kendine 92 exposure. The balance between the increased BAD, BNIP3L, BNIP3, BIM, PUMA and AIF mRNA expression levels, and decreased expression of BCL-W, BCL-2, BFL-1, XIAP and FLIP indicates global changes in the apoptosis mRNA expression profile consistent with the apoptotic outcome. Protein expression analysis shows increased levels of NOXA, BIM and PUMA proteins upon Kendine 92 and SAHA treatment. Our results highlight the capability of these molecules to induce apoptosis not only in a selective manner but also in those cells frequently resistant to standard treatments. Thus, Kendine 92 is a novel HDACi with anticancer efficacy for non-proliferating CLL cells.

AICAR induces apoptosis independently of AMPK and p53 through up-regulation of the BH3-only proteins BIM and NOXA in chronic lymphocytic leukemia cells.

5-Aminoimidazole-4-carboxamide riboside or acadesine (AICAR) induces apoptosis in chronic lymphocytic leukemia (CLL) cells. A clinical study of AICAR is currently being performed in patients with this disease. Here, we have analyzed the mechanisms involved in AICAR-induced apoptosis in CLL cells in which it activates its only well-known molecular target, adenosine monophosphate-activated protein kinase (AMPK). However, AMPK activation with phenformin or A-769662 failed to induce apoptosis in CLL cells and AICAR also potently induced apoptosis in B lymphocytes from Ampkα1(-/-) mice, demonstrating an AMPK-independent mechanism of cell death. Importantly, AICAR induced apoptosis irrespective of the tumor suppressor TP53 or ataxia telangiectasia mutated (ATM) status via induction of the mitochondrial pathway. Apoptosis was preceded by an increase in mRNA and protein levels of proapoptotic BCL-2 family proteins of the BH3-only subgroup, including BIM, NOXA, and PUMA in CLL cells. Strikingly, B lymphocytes from Noxa(-/-) or Bim(-/-) mice were partially protected from the cytotoxic effects of AICAR. Consistently, B cells from Noxa(-/-)/Bim(-/-) mice resisted induction of apoptosis by AICAR as potently as B lymphocytes overexpressing transgenic BCL-2. These findings support the notion that AICAR is an interesting alternative therapeutic option for CLL patients with impaired p53 function and resistance to conventional chemotherapy.