Induction Of Apoptotic Cell Death Research Articles

A Multi-Ingredient Nutritional Supplement Exerts Antiapoptotic and Proliferative Effects on Rat Neural Stem Cells: A Novel Therapeutic Option

Background: The importance of neural stem cell (NSC) survival in the appropriate brain function, synapsis formation, and neural cell communication is to the degree that any perturbation in this process leads to many neurodegenerative diseases. Numerous therapeutic approaches have recently been developed to prolong the survival of NSCs, so that these multipotent stem cells could differentiate into other neural cells and improve the function of the brain. Methods: In this study, we aimed to investigate whether the treatment of isolated NSCs from the Wistar rats with the nutrition bio-shield (NBS) supplement could boost the survival of NSCs. Results: Of note, we found that the extract had negative effects neither on the biochemical and hematological parameters nor on the morphology of hepatocytes of the rats, suggestive of the lack of toxicity of the extract. Our results also suggested that the extract robustly increased the survival and the proliferative capacity of NSCs by elevating the metabolic activity of the cells. The NBS supplement also prevented the induction of apoptotic cell death in NSCs, as revealed by the decrease in the number of terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)-positive cells as compared to the control group. Conclusion: Overall, this study highlighted the potent efficacy of the NBS supplement in prolonging the survival of NSCs and proposed the possibility of the clinical use of the extract in the treatment of different neural diseases. However, further investigations in this field are required to provide the extract’s mechanism of action.

Gemcitabine in combination with epibrassinolide enhanced the apoptotic response in an ER stress-dependent manner and reduced the epithelial-mesenchymal transition in pancreatic cancer cells.

Gemcitabine is a broad-spectrum antimetabolite and a deoxycytidine analog recognized as a standard therapy alone or in combination with other antineoplastic agents in the therapy of pancreas cancer. Drug resistance following gemcitabine treatment is a common phenomenon; therefore, combinational therapy models are usually preferred. Pancreatic ductal adenocarcinoma, or pancreas cancer, is the fourth leading cause of cancer-related deaths worldwide. With the increasing incidence of pancreatic cancer every year, the mortality rate is also rising significantly because of late diagnosis, and limited chemotherapy options. Adjuvant chemotherapy after surgical resection is the typical option for the treatment of early pancreatic cancer. Mostly, 5-fluorouracil/leucovorin with irinotecan and oxaliplatin (FOLFIRINOX) and gemcitabine/nab-paclitaxel is used for the prognosis of advanced pancreatic cancer; however, chemoresistance usually occurs limiting the effectiveness of the chemotherapy. Therefore, most of the studies are focused on gemcitabine combination with other drugs to overcome the situation. As an apoptotic agent and a member of brassinosteroids, epibrassinolide (EBR) induces endoplasmic reticulum (ER) stress-dependent cell death in different cancer cells, as shown by our group. In this study, we aimed to enhance the gemcitabine apoptotic effect by EBR combined treatment in pancreatic cancer cells. EBR treatment reduced cell viability and inhibited cell proliferation in PANC-1, MIA PaCa-2, and AsPC-1 cells. Each pancreatic cancer cell gave different responses to the EBR treatment because of different aggressiveness. However, EBR induced apoptosis through increasing ROS generation, which was associated with ER stress in PANC-1 and MIA PaCa-2 cells. Gemcitabine alone reduced the cell viability of each pancreatic cancer cell line; however, combination with EBR led to further induction of apoptotic cell death in each pancreatic cancer cell line. In addition, combined treatment of gemcitabine and EBR further decreased N-cadherin and vimentin expressions, suggesting that epithelial-mesenchymal transition of pancreatic cells is reduced. In conclusion, EBR had therapeutic potential to avoid the gemcitabine-induced side effects during the treatment of pancreatic cancer.

Induction of apoptotic cell death in human bladder cancer cells by ethanol extract of Zanthoxylum schinifolium leaf, through ROS-dependent inactivation of the PI3K/Akt signaling pathway

BACKGROUND/OBJECTIVESZanthoxylum schinifolium is traditionally used as a spice for cooking in East Asian countries. This study was undertaken to evaluate the anti-proliferative potential of ethanol extracts of Z. schinifolium leaves (EEZS) against human bladder cancer T24 cells.MATERIALS/METHODSSubsequent to measuring the cytotoxicity of EEZS, the anti-cancer activity was measured by assessing apoptosis induction, reactive oxygen species (ROS) generation, and mitochondrial membrane potential (MMP). In addition, we determined the underlying mechanism of EEZS-induced apoptosis through various assays, including Western blot analysis.RESULTSEEZS treatment concentration-dependently inhibited T24 cell survival, which is associated with apoptosis induction. Exposure to EEZS induced the expression of Fas and Fas-ligand, activated caspases, and subsequently resulted to cleavage of poly (ADP-ribose) polymerase. EEZS also enhanced the expression of cytochrome c in the cytoplasm by suppressing MMP, following increase in the ratio of Bax:Bcl-2 expression and truncation of Bid. However, EEZS-mediated growth inhibition and apoptosis were significantly diminished by a pan-caspase inhibitor. Moreover, EEZS inhibited activation of the phosphoinositide 3-kinase (PI3K)/Akt pathway, and the apoptosis-inducing potential of EEZS was promoted in the presence of PI3K/Akt inhibitor. In addition, EEZS enhanced the production of ROS, whereas N-acetyl cysteine (NAC), a ROS scavenger, markedly suppressed growth inhibition and inactivation of the PI3K/Akt signaling pathway induced by EEZS. Furthermore, NAC significantly attenuated the EEZS-induced apoptosis and reduction of cell viability.CONCLUSIONSTaken together, our results indicate that exposure to EEZS exhibits anti-cancer activity in T24 bladder cancer cells through ROS-dependent induction of apoptosis and inactivation of the PI3K/Akt signaling pathway.

Exploring the MEN1 dependent modulation of caspase 8 and caspase 3 in human pancreatic and murine embryo fibroblast cells

MEN1 mutation causes pancreatic neuroendocrine neoplasia and benign malignancies of the parathyroid, the adrenal cortex and pituitary gland. The transcriptional activity of its product menin promotes the expression of genes deputed to several cellular mechanism including cell death. Here, we focused on its implication in the activation of the initiator and executioner caspases after staurosporine mediated cell death in 2D and 3D human and murine cell models. The administration of staurosporine, a well-known inducer of apoptotic cell death, caused a significant reduction of BON1, QGP1 and HPSC2.2 cell viability. The transient knockdown of MEN1, performed by using a specific siRNA, caused a significant down-regulation of CDKN1A and TP53 transcripts. The treatment with 1 µM of staurosporine caused also a significant down-regulation of MEN1 and was able to restore the basal expression of TP53 only in QGP1 cells. Transient or permanent MEN1 inactivation caused a decrease of caspase 8 activity in BON1, HPSC2.2 cells and MEN1−/− MEFs treated with staurosporine. Caspase 3/7 activity was suppressed after administration of staurosporine in MEN1 knocked down HPSC2.2 and MEN1−/− MEFs as well. The cleaved caspase 8 and caspase 3 decreased in human cells after MEN1 knockdown and in MEN1−/− MEFs. The treatment with staurosporine caused a reduction of the size of MEN1+/+ MEFs spheroids. Instead, MEN1−/− MEFs spheroids did not show any significant reduction of their size. In conclusion, MEN1 controls the activity of the initiator caspase 8 and the executioner caspase 3 in human and murine cells. Restoring of a functional MEN1 and interfering with the apoptotic mechanism could represent a future strategy for the treatment of MEN1-related malignancies.

Phototoxicity of two positive-charged diaryl porphyrins in multicellular tumor spheroids.

Photodynamic therapy (PDT) is a clinically approved cancer treatment in which reactive oxygen species are formed only when three harmless components, a photosensitizer (PS), light and molecular oxygen, are present at the same time, leading to cell death. Most of the PSs were tested on monolayer cells, but differences between 2D cells and solid tumors significantly limit the value of in vitro PDT studies, whereas the use of 3D spheroid might be more suitable for drug development and preclinical drug testing for PDT. In a previous work we have shown that two positive-charged diaryl porphyrins (2 and 4) were more potent than the corresponding neutral molecules (1 and 3) on a panel of 2D-cultured cancer cell lines. In the present study the photodynamic effects of these molecules have been evaluated on HCT116 and MCF7 spheroids. Induction of apoptotic and necrotic cell death, and generation of reactive oxygen species (ROS) have been also evaluated, along with accumulation and localization of PSs into spheroids. Our findings indicate that 2 and 4 retained their phototoxic effects also in 3D spheroids; furthermore, they were more potent than 1 and 3 and as potent as Foscan (m-THPC), the most successful PS approved for clinical PDT of cancer, used as reference. Although further aspects of their mechanisms of action need to be addressed, our results strongly suggest a potential in vivo photodynamic application of 2 and 4, considering that spheroids represent a more realistic indicator of in vivo therapeutic efficacy than 2D cell lines.

Photoreactive Cytosine-Functionalized Self-Assembled Micelles with Enhanced Cellular Uptake Capability for Efficient Cancer Chemotherapy.

Design, fabrication, and control of photoreactive supramolecular macromers─which are composed of a thermoresponsive polymer backbone and photoreactive nucleobase end-groups─to achieve the desired physical-chemical performance and provide the high efficiency required for chemotherapy drug delivery purposes still present challenges. Herein, a difunctional cytosine-terminated supramolecular macromer was successfully obtained at high yield. UV-irradiation induces the formation of cytosine photodimers within the structure. The irradiated macromer can self-assemble into nanosized spherical micelles in water that possess a number of interesting and unique features, such as desired micellar size and morphology, tunable drug-loading capacity, and excellent structural stability in serum-containing medium, in addition to well-controlled drug-release behaviors in response to changes in environmental temperature and pH; these extremely desirable, rare features are required to augment the functions of polymeric nanocarriers for drug delivery. Importantly, a series of in vitro studies demonstrated that photodimerized cytosine moieties within the drug-loaded micelles substantially enhance their internalization and accumulation inside cells via endocytosis and subsequently lead to induction of massive apoptotic cell death compared with the corresponding nonirradiated micelles. Thus, this newly developed "photomodified" nanocarrier system could provide a potentially fruitful route to enhance the drug delivery performance of nanocages without the need to introduce targeting moieties or additional components.

Roles for ADAM17 in TNF-R1 Mediated Cell Death and Survival in Human U937 and Jurkat Cells.

Signaling via death receptor family members such as TNF-R1 mediates pleiotropic biological outcomes ranging from inflammation and proliferation to cell death. Pro-survival signaling is mediated via TNF-R1 complex I at the cellular plasma membrane. Cell death induction requires complex IIa/b or necrosome formation, which occurs in the cytoplasm. In many cell types, full apoptotic or necroptotic cell death induction requires the internalization of TNF-R1 and receptosome formation to properly relay the signal inside the cell. We interrogated the role of the enzyme A disintegrin and metalloprotease 17 (ADAM17)/TACE (TNF-α converting enzyme) in death receptor signaling in human hematopoietic cells, using pharmacological inhibition and genetic ablation. We show that in U937 and Jurkat cells the absence of ADAM17 does not abrogate, but rather increases TNF mediated cell death. Likewise, cell death triggered via DR3 is enhanced in U937 cells lacking ADAM17. We identified ADAM17 as the key molecule that fine-tunes death receptor signaling. A better understanding of cell fate decisions made via the receptors of the TNF-R1 superfamily may enable us, in the future, to more efficiently treat infectious and inflammatory diseases or cancer.

SLC5A3 Transports Myo-Inositol to Support the Growth of Acute Myeloid Leukemia

Metabolic reprogramming contributes to tumor development and sustains cancer cell proliferation. Like other cancers, acute myeloid leukemia (AML), a devastating hematologic malignancy with poor overall survival, has altered metabolic features, providing new possibilities for AML treatment. Since the niche can reshape the metabolic properties of cancer cells, it is critical to validate AML metabolic vulnerabilities in a proper microenvironment. To this end, we optimized a protocol for CRISPR screening in orthotopic xenograft AML models, including patient-derived-xenograft (PDX) models tractable for CRISPR-editing, to enable the systematic evaluation of the physiological relevance of top AML dependencies. We performed in vivo screens in MV4-11 and U937 cell lines and a PDX model, which converged to reveal the sodium/myo-inositol cotransporter SLC5A3 as a top-ranked in vivo gene target.We have validated the SLC5A3 dependency in additional AML cell line and PDX models; SLC5A3 deletion consistently induced apoptotic cell death in all AML models tested. We also observed cell-context dependent alterations of cell cycle and differentiation. In addition, using a PDX model with a doxycycline-inducible CRISPR system, we have confirmed that induction of SLC5A3 knockout post transplantation significantly represses AML progression and prolonged mouse survival. Next, we investigated whether the growth defect caused by SLC5A3 knockout results from the myo-inositol deficiency. Augmentation of myo-inositol concentration in the standard culture medium completely rescued the proliferation of SLC5A3-knockout cells. In accordance, depletion of myo-inositol from the culture medium largely impeded the growth of parental AML cells, causing similar phenotypes as SLC5A3-deletion, with cell-context dependent alterations of cell cycle and induction of apoptotic cell death. Together, these data reveal that myo-inositol is a critical metabolite for AML.Since a subset of AML cell lines were not dependent on SLC5A3 based on a genome-scale CRISPR screen dataset (DepMap), we explored the potential biomarkers associated with SLC5A3 essentiality in AML. Intriguingly, the low expression of Inositol-3-phosphate synthase 1 (ISYNA1) predicted a strong SLC5A3 dependency in AML cell lines. In addition to importing myo-inositol from the extracellular fluid, cells can also synthesize myo-inositol de novo from glucose 6-phosphate, and ISYNA1 encodes the rate-limiting enzyme in this myo-inositol biosynthesis pathway. We confirmed low expression of ISYNA1 protein in AML cells sensitive to SLC5A3 deletion, and importantly, overexpression of ISYNA1 can completely relieve the SLC5A3 dependency. Moreover, knockout of ISYNA1 in an ISYNA1-high cell line M07e exacerbated the growth defect associated with SLC5A3 deletion. Altogether, these results strongly demonstrate that SLC5A3 becomes essential in AML cells with insufficient myo-inositol biosynthesis capacity to support AML proliferation.Finally, we have investigated the clinical features associated with low ISYNA1 expression in genomic datasets of primary AML samples, including TCGA and Beat AML, to postulate the patient population that can benefit from a SLC5A3-directed therapy. Interestingly, low ISYNA1 expression is associated with FAB M4 and M5 AML subtypes. In accordance, a monocyte lineage gene signature is enriched in ISYNA1-low samples. In addition, AML samples with low ISYNA1 expression tend to have IDH2 or DNMT3 mutations. Collectively, our study demonstrated that SLC5A3 is a strong metabolic dependency in AML, and targeting SLC5A3 can provide a therapeutic opportunity for a subset of monocytic AML. DisclosuresDharia: Genentech: Current Employment. Piccioni: Merck Research Laboratories: Current Employment. Stegmaier: AstraZeneca: Consultancy; KronosBio: Consultancy; Bristol Myers Squibb: Consultancy; Auron Therapeutics: Consultancy, Current equity holder in publicly-traded company; Novartis: Research Funding.

Restoration of MiR-34a Expression by 5-Azacytidine Augments Alimta -Induced Cell Death in Non-Small Lung Cancer Cells by Downregulation of HMG B1, A2 and Bcl-2 Pathway.

Alimta (Pemetrexed) as an antifolate drug has been approved for the treatment of lung cancer. The aim of the present study was to investigate the combination effect of 5-Azacytidine (5-aza) and Alimta on the miR-34a and its target genes expression and induction of apoptotic cell death in non-small lung cancer A549 cells. In this experimental study, lung cancer A549 cells were treated with various concentrations of Alimta alone and combined with 5-Aza. Then, viability was assessed by trypan blue and MTT assays. mRNA expressions were performed by real time-polymerase chain reaction (PCR) and western blot. Flow cytometry used to detect apoptotic/ necrotic cells and cell cycle arrest. Alimta alone reduced viability of the cells in a dose dependent manner with the half-maximal inhibitory concentration (IC50) value of 12 μM. Pretreatment of the cells with 5-aza (5 μM) induced a synergistic cytotoxic effect with IC50 of 3 μM. Sequential exposure of the cells to 5-aza and Alimta enhanced miR-34a expression and significantly downregulated HMGB1, HMGA2 and BCL-2 expressions. Also, it was associated with reduction of nuclear HMGB1 and HMGA2 content. Caspase-3 activation, HMGB1 release into extracellular space and staining of the cells with annexine V/PI suggested that 5-aza reduced late apoptotic/necrotic cell death induced by Alimta. In addition, combination of 5-aza and Alimta arrested the cells at S and sub-G1 phases and inhibited colony formation. 5-aza synergistically enhances Alimta induced apoptotic cell death through HMG proteins regulation, MIR34A gene expression and intrinsic apoptosis mechanism, providing a promising combination therapy in clinical lung cancer therapy.

Restoration of MiR-34a Expression by 5-Azacytidine Augments Alimta -Induced Cell Death in Non-Small Lung Cancer Cells by Downregulation of HMG B1, A2 and Bcl-2 Pathway

Objective Alimta (Pemetrexed) as an antifolate drug has been approved for the treatment of lung cancer. The aim of the present study was to investigate the combination effect of 5-Azacytidine (5-aza) and Alimta on the miR-34a and its target genes expression and induction of apoptotic cell death in non-small lung cancer A549 cells. Materials and Methods In this experimental study, lung cancer A549 cells were treated with various concentrations of Alimta alone and combined with 5-Aza. Then, viability was assessed by trypan blue and MTT assays. mRNA expressions were performed by real time-polymerase chain reaction (PCR) and western blot. Flow cytometry used to detect apoptotic/ necrotic cells and cell cycle arrest. Results Alimta alone reduced viability of the cells in a dose dependent manner with the half-maximal inhibitory concentration (IC50) value of 12 µM. Pretreatment of the cells with 5-aza (5 µM) induced a synergistic cytotoxic effect with IC50of 3 µM. Sequential exposure of the cells to 5-aza and Alimta enhanced miR-34a expression and significantly downregulated HMGB1, HMGA2 and BCL-2 expressions. Also, it was associated with reduction of nuclear HMGB1 and HMGA2 content. Caspase-3 activation, HMGB1 release into extracellular space and staining of the cells with annexine V/PI suggested that 5-aza reduced late apoptotic/necrotic cell death induced by Alimta. In addition, combination of 5-aza and Alimta arrested the cells at S and sub-G1 phases and inhibited colony formation. Conclusion5-aza synergistically enhances Alimta induced apoptotic cell death through HMG proteins regulation, MIR34A gene expression and intrinsic apoptosis mechanism, providing a promising combination therapy in clinical lung cancer therapy.

TCTP protein degradation by targeting mTORC1 and signaling through S6K, Akt, and Plk1 sensitizes lung cancer cells to DNA-damaging drugs

Translationally controlled tumor protein (TCTP) is expressed in many tissues, particularly in human tumors. It plays a role in malignant transformation, apoptosis prevention, and DNA damage repair. The signaling mechanisms underlying TCTP regulation in cancer are only partially understood. Here, we investigated the role of mTORC1 in regulating TCTP protein levels, thereby modulating chemosensitivity, in human lung cancer cells and an A549 lung cancer xenograft model. The inhibition of mTORC1, but not mTORC2, induced ubiquitin/proteasome-dependent TCTP degradation without a decrease in the mRNA level. PLK1 activity was required for TCTP ubiquitination and degradation and for its phosphorylation at Ser46 upon mTORC1 inhibition. Akt phosphorylation and activation was indispensable for rapamycin-induced TCTP degradation and PLK1 activation, and depended on S6K inhibition, but not mTORC2 activation. Furthermore, the minimal dose of rapamycin required to induce TCTP proteolysis enhanced the efficacy of DNA-damaging drugs, such as cisplatin and doxorubicin, through the induction of apoptotic cell death in vitro and in vivo. This synergistic cytotoxicity of these drugs was induced irrespective of the functional status of p53. These results demonstrate a new mechanism of TCTP regulation in which the mTORC1/S6K pathway inhibits a novel Akt/PLK1 signaling axis and thereby induces TCTP protein stabilization and confers resistance to DNA-damaging agents. The results of this study suggest a new therapeutic strategy for enhancing chemosensitivity in lung cancers regardless of the functional status of p53.

Induction of Apoptotic Cell Death by Oral Streptococci in Human Periodontal Ligament Cells.

Initiation and progression of oral infectious diseases are associated with streptococcal species. Bacterial infection induces inflammatory responses together with reactive oxygen species (ROS), often causing cell death and tissue damage in the host. In the present study, we investigated the effects of oral streptococci on cytotoxicity and ROS production in human periodontal ligament (PDL) cells. Streptococcus gordonii showed cell cytotoxicity in a dose- and time-dependent manner. The cytotoxicity might be due to apoptosis since S. gordonii increased annexin V-positive cells, and the cytotoxicity was reduced by an apoptosis inhibitor, Z-VAD-FMK. Other oral streptococci such as Streptococcus mitis, Streptococcus sanguinis, and Streptococcus sobrinus also induced apoptosis, whereas Streptococcus mutans did not. All streptococci tested except S. mutans triggered ROS production in human PDL cells. Interestingly, however, streptococci-induced apoptosis appears to be ROS-independent, as the cell death induced by S. gordonii was not recovered by the ROS inhibitor, resveratrol or n-acetylcysteine. Instead, hydrogen peroxide (H2O2) appears to be important for the cytotoxic effects of streptococci since most oral streptococci except S. mutans generated H2O2, and the cytotoxicity was dramatically reduced by catalase. Furthermore, streptococcal lipoproteins are involved in cytotoxicity, as we observed that cytotoxicity induced by the lipoprotein-deficient S. gordonii mutant was less potent than that by the wild-type and was attenuated by anti-TLR2-neutralizing antibody. Indeed, lipoproteins purified from S. gordonii alone were sufficient to induce cytotoxicity. Notably, S. gordonii lipoproteins did not induce H2O2 or ROS but cooperatively induced cell death when co-treated with H2O2. Taken together, these results suggest that most oral streptococci except S. mutans efficiently induce damage to human PDL cells by inducing apoptotic cell death with bacterial H2O2 and lipoproteins, which might contribute to the progression of oral infectious diseases such as apical periodontitis.

The Natural Pigment Violacein Potentially Suppresses the Proliferation and Stemness of Hepatocellular Carcinoma Cells In Vitro.

Hepatocellular carcinoma (HCC) is a malignant type of primary liver cancer with high incidence and mortality, worldwide. A major challenge in the treatment of HCC is chemotherapeutic resistance. It is therefore necessary to develop novel anticancer drugs for suppressing the growth of HCC cells and overcoming drug resistance for improving the treatment of HCC. Violacein is a deep violet-colored indole derivative that is produced by several bacterial strains, including Chromobacterium violaceum, and it possesses numerous pharmacological properties, including antitumor activity. However, the therapeutic effects of violacein and the mechanism underlying its antitumor effect against HCC remain to be elucidated. This study is the first to demonstrate that violacein inhibits the proliferation and stemness of Huh7 and Hep3B HCC cells. The antiproliferative effect of violacein was attributed to cell cycle arrest at the sub-G1 phase and the induction of apoptotic cell death. Violacein induced nuclear condensation, dissipated mitochondrial membrane potential (MMP), increased generation of reactive oxygen species (ROS), activated the caspase cascade, and upregulated p53 and p21. The anticancer effect of violacein on HCC cells was also associated with the downregulation of protein kinase B (AKT) and extracellular signal-regulated kinase (ERK)1/2 signaling. Violacein not only suppressed the proliferation and formation of tumorspheres of Huh7 and Hep3B cancer stem-like cells but also reduced the expression of key markers of cancer stemness, including CD133, Sox2, Oct4, and Nanog, by inhibiting the signal transducer and activator of transcription 3 (STAT3)/AKT/ERK pathways. These results suggest the therapeutic potential of violacein in effectively suppressing HCC by targeting the proliferation and stemness of HCC cells.

CCT245718, a dual FLT3/Aurora A inhibitor overcomes D835Y-mediated resistance to FLT3 inhibitors in acute myeloid leukaemia cells.

Activating mutations in the Fms-like tyrosine kinase 3 (FLT3) are among the most prevalent oncogenic mutations in acute myeloid leukaemia. Inhibitors selectively targeting FLT3 kinase have shown promising clinical activity; their success in the clinic, however, has been limited due to the emergence of acquired resistance. CCT245718 was identified and characterised as a dual Aurora A/FLT3 inhibitor through cell-based and biochemical assays. The ability of CCT245718 to overcome TKD-mediated resistance was evaluated in a cell line-based model of drug resistance to FLT3 inhibitors. CCT245718 exhibits potent antiproliferative activity towards FLT3-ITD + AML cell lines and strongly binds to FLT3-ITD and TKD (D835Y) mutants in vitro. Activities of both FLT3-ITD and Aurora A are also inhibited in cells. Inhibition of FLT3 results in reduced phosphorylation of STAT5, downregulation of survivin and induction of apoptotic cell death. Moreover, CCT245718 overcomes TKD-mediated resistance in a MOLM-13-derived cell line containing FLT3 with both ITD and D835Y mutations. It also inhibits FLT3 signalling in both parental and resistant cell lines compared to FLT3-specific inhibitor MLN518, which is only active in the parental cell line. Our results demonstrate that CCT245718 is a potent dual FLT3/Aurora A inhibitor that can overcome TKD-mediated acquired resistance.

Two parallel pathways connect glutamine metabolism and mTORC1 activity to regulate glutamoptosis

Glutamoptosis is the induction of apoptotic cell death as a consequence of the aberrant activation of glutaminolysis and mTORC1 signaling during nutritional imbalance in proliferating cells. The role of the bioenergetic sensor AMPK during glutamoptosis is not defined yet. Here, we show that AMPK reactivation blocks both the glutamine-dependent activation of mTORC1 and glutamoptosis in vitro and in vivo. We also show that glutamine is used for asparagine synthesis and the GABA shunt to produce ATP and to inhibit AMPK, independently of glutaminolysis. Overall, our results indicate that glutamine metabolism is connected with mTORC1 activation through two parallel pathways: an acute alpha-ketoglutarate-dependent pathway; and a secondary ATP/AMPK-dependent pathway. This dual metabolic connection between glutamine and mTORC1 must be considered for the future design of therapeutic strategies to prevent cell growth in diseases such as cancer.

Transcriptional CDK inhibitors, CYC065 and THZ1 promote Bim-dependent apoptosis in primary and recurrent GBM through cell cycle arrest and Mcl-1 downregulation

Activation of cyclin-dependent kinases (CDKs) contributes to the uncontrolled proliferation of tumour cells. Genomic alterations that lead to the constitutive activation or overexpression of CDKs can support tumourigenesis including glioblastoma (GBM), the most common and aggressive primary brain tumour in adults. The incurability of GBM highlights the need to discover novel and more effective treatment options. Since CDKs 2, 7 and 9 were found to be overexpressed in GBM, we tested the therapeutic efficacy of two CDK inhibitors (CKIs) (CYC065 and THZ1) in a heterogeneous panel of GBM patient-derived cell lines (PDCLs) cultured as gliomaspheres, as preclinically relevant models. CYC065 and THZ1 treatments suppressed invasion and induced viability loss in the majority of gliomaspheres, irrespective of the mutational background of the GBM cases, but spared primary cortical neurons. Viability loss arose from G2/M cell cycle arrest following treatment and subsequent induction of apoptotic cell death. Treatment efficacies and treatment durations required to induce cell death were associated with proliferation velocities, and apoptosis induction correlated with complete abolishment of Mcl-1 expression, a cell cycle-regulated antiapoptotic Bcl-2 family member. GBM models generally appeared highly dependent on Mcl-1 expression for cell survival, as demonstrated by pharmacological Mcl-1 inhibition or depletion of Mcl-1 expression. Further analyses identified CKI-induced Mcl-1 loss as a prerequisite to establish conditions at which the BH3-only protein Bim can efficiently induce apoptosis, with cellular Bim amounts strongly correlating with treatment efficacy. CKIs reduced proliferation and promoted apoptosis also in chick embryo xenograft models of primary and recurrent GBM. Collectively, these studies highlight the potential of these novel CKIs to suppress growth and induce cell death of patient-derived GBM cultures in vitro and in vivo, warranting further clinical investigation.

Synergistic Activity of the HSP90 Inhibitor Ganetespib With Lapatinib Reverses Acquired Lapatinib Resistance in HER2-Positive Breast Cancer Cells.

Lapatinib is an FDA-approved EGFR and HER2 tyrosine kinase inhibitor for the treatment of HER2-positive breast cancer patients. However, its therapeutic efficacy is limited by primary or acquired resistance. In the present study, we established breast cancers cells with acquired lapatinib resistance and investigated the antitumor activity of the second-generation HSP90 inhibitor ganetespib in association with lapatinib in lapatinib-sensitive and -resistant cells. The combination treatment showed synergistic inhibition of HER and the downstream PI3K/Akt and Ras/MEK/ERK pathways, in addition to enhancing induction of early apoptotic cell death and G1 arrest in both parent and lapatinib-resistant cells in vitro. The joint administration of ganetespib and lapatinib depleted the aberrant nuclear transcription factor STAT3, a mediator of the cell cycle and apoptosis-related pathways that is probably involved in the lapatinib resistance of HER2-positive breast cancer cells. In conjunctive with the augmented inhibition of tumor growth observed in both SKBR3 and SKBR3-L xenografts compared to monotherapy, our data provide a sound preclinical basis for combination treatment with lapatinib and ganetespib for refractory HER2-positive breast cancer.

Abstract 1752: Exploring the relation of eosinophilic esophagitis (EoE) with esophageal cancer

Abstract A negative correlation between allergic inflammation and cancer risk has been identified in a variety of organs via population-based studies; however, functional investigations are necessary to define the relationship between allergy and cancer and determine the feasibility of therapeutic approaches leveraging allergic inflammation for cancer prevention and therapy. Esophageal cancer is amongst the most aggressive forms of human cancer. Reflux esophagitis is a primary risk factor for esophageal adenocarcinoma. Eosinophilic esophagitis (EoE) represents a distinct, food allergen-mediated type of esophagitis characterized by esophageal eosinophilia. In contrast to patients with reflux esophagitis, epidemiological data indicates that EoE patients fail to develop esophageal cancer despite the presence of chronic esophageal inflammation. To investigate the functional relationship between EoE and esophageal cancer, we have paired the food allergen-driven MC903/Ovalbumin (OVA) model of EoE with esophageal squamous cell carcinoma induced by the carcinogen 4-nitroquinoline 1-oxide (4NQO). 10/10 mice treated with 4NQOalone developed ESCC as expected in this robust carcinogen-driven tumor model. In mice co-treated with MC903/OVA and 4NQO, esophageal tumors were detected in 4/5 mice with a significant decrease in tumor load as compared to mice treated with 4NQO alone. Notably, 6/6 mice treated withMC903/OVA alone and 4/4 mice treated with propylene glycol (vehicle for 4NQO) failed to display tumors. To examine the possibility that EoE may induce an anti-tumor inflammatory milieu, we exposed primary esophageal tumor cells to CD45positive immune cells isolated from the peripheral blood of strain- matched donor mice with or without food allergen-mediated EoE. These experiments revealed a robust induction of apoptotic cell death in tumor cells exposed to immune cells from EoE donor mice as compared to those exposed to immune cells from naïve controls. Finally, in order to investigate how EoE and cancer may differentially impact esophageal biology, single cell RNA-sequencing was performed and further identified differences in the esophageal epithelial and immune cell compartments in mice with EoE as compared to those with cancer. The innovative approaches employed here, including combining mouse models of EoE and esophageal cancer capability, establish a robust experimental platform to define the relationship between EoE and esophageal cancer which will be further used for future functional investigations to define the precise cellular and molecular mechanisms through which EoE inflammation limits carcinogenesis. As allergic disorders promote systemic inflammation, findings from the current study may be applicable to cancers occurring in organs other than the esophagus. Citation Format: Mohammad Faujul Kabir, Adam Karami, Anbin Mu, Kelly A. Whelan. Exploring the relation of eosinophilic esophagitis (EoE) with esophageal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1752.

Abstract 1946: Synergistic cell death induction in breast cancer cell lines by combining ABT-199 and cisplatin irrespective of estrogen receptor and p53 expression

Abstract 50 Years after the generation of the most studied human breast cancer cell line, MCF-7, many research results fundamentally changed patient outcomes for the better. The mortality of breast cancer in women has decreased significantly, especially in the last 30 years. But the total number of deaths is rising again in the last ten years. Considering that breast cancer is the most common cancer in women and the second most common cancer overall, with incidences of 24,2% and 11,6%, respectively, total numbers of new cases are in the millions every year. First line drugs for the treatment of breast cancer often target microtubules (e.g. Vinblastine and Paclitaxel) or induce DNA damage (e.g. Carboplatin and Cisplatin (CisPt)). In addition to these classic therapeutic drugs new compounds that specifically target cancer-driving molecular alterations have been developed. Among these is the group of BH3-mimetics. The efficacy of several BH3-mimetics is investigated in numerous clinical trials while the BCL-2 specific inhibitor ABT-199 (Venetoclax) is already approved for clinical application in chronic lymphatic leukemia (CLL), acute myeloid leukemia (AML) and small lymphocytic lymphoma (SLL). BH3-mimetics target the apoptosis machinery by interacting with anti-apoptotic Bcl-2 proteins, mimicking the function of pro-apoptotic BH3-only proteins. In turn, the pro-apoptotic potential of pore-forming BCL-2 effectors BAX, BAK and BOK is released inducing mitochondrial outer membrane permeabilization and cell death. Recently we reported synergistic cell death induction by ABT-199 in combination with the proteasome inhibitor Bortezomib (BOZ) in soft tissue sarcoma (STS) cells. ABT-199 and BOZ induce accumulation of BOK and the BH3-only protein NOXA (Muenchow et al. 2020). Here, we combined ABT-199, CisPt and Nutlin-3 in breast cancer cell lines that differ in expression of the estrogen receptor (ER) and p53 activity. Similar to the results in STS cells synergistic induction of apoptotic cell death was observed by FACS analysis of the mitochondrial membrane potential (TMRM) and exposure of phosphatidyl serine (Annexin-V). Western blot analysis revealed that ABT-199 induced accumulation of NOXA and MCL-1, irrespective of the ER status or p53 mutation. Simultaneous treatment with ABT-199 and CisPt or Nutlin-3 further increased expression of NOXA and MCL-1. Interestingly, qRT-PCR revealed enhanced expression of NOXA and MCL-1 mRNA in ABT-199 treated cells indicating transcriptional regulation. However, transcriptional induction of NOXA was also detected independent of p53 activity. Thus, combined therapeutic approaches using CisPt and ABT-199 should be effective irrespective of ER and p53 expression. Nevertheless, in p53 proficient tumors Nutlins might increase therapeutic efficacy. Citation Format: Benjamin Schaefer, Sandra Weller, Tobias Beigl, Kathrin Boepple, Reiner Hoppe, Klaus Schulze-Osthoff, Hans-Georg Kopp, Walter E. Aulitzky, Frank Essmann. Synergistic cell death induction in breast cancer cell lines by combining ABT-199 and cisplatin irrespective of estrogen receptor and p53 expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1946.

Pancracine, a Montanine-Type Amaryllidaceae Alkaloid, Inhibits Proliferation of A549 Lung Adenocarcinoma Cells and Induces Apoptotic Cell Death in MOLT-4 Leukemic Cells.

Pancracine, a montanine-type Amaryllidaceae alkaloid (AA), is one of the most potent compounds among natural isoquinolines. In previous studies, pancracine exhibited cytotoxic activity against diverse human cancer cell lines in vitro. However, further insight into the molecular mechanisms that underlie the cytotoxic effect of pancracine have not been reported and remain unknown. To fill this void, the cell proliferation and viability of cancer cells was explored using the Trypan Blue assay or by using the xCELLigence system. The impact on the cell cycle was determined by flow cytometry. Apoptosis was evaluated by Annexin V/PI and by quantifying the activity of caspases (-3/7, -8, and -9). Proteins triggering growth arrest or apoptosis were detected by Western blotting. Pancracine has strong antiproliferative activity on A549 cells, lasting up to 96 h, and antiproliferative and cytotoxic effects on MOLT-4 cells. The apoptosis-inducing activity of pancracine in MOLT-4 cells was evidenced by the significantly higher activity of caspases. This was transmitted through the upregulation of p53 phosphorylated on Ser392, p38 MAPK phosphorylated on Thr180/Tyr182, and upregulation of p27. The pancracine treatment negatively altered the proliferation of A549 cells as a consequence of an increase in G1-phase accumulation, associated with the downregulation of Rb phosphorylated on Ser807/811 and with the concomitant upregulation of p27 and downregulation of Akt phosphorylated on Thr308. This was the first study to glean a deeper mechanistic understanding of pancracine activity in vitro. Perturbation of the cell cycle and induction of apoptotic cell death were considered key mechanisms of pancracine action.