Dose-dependent Increase In Apoptosis Research Articles

Abstract 676: The integrated stress activator BTM-3566 overcomes therapeutic resistance in mantle cell lymphoma

Abstract Successive generations of BTK inhibitors and CAR T-cell therapy have transformed the treatment of mantle cell lymphoma (MCL). However, treatment resistance has inevitably emerged, creating significant medical need. Recently a novel, clinical stage small molecule, BTM-3566, has been described that targets a mitochondrial process essential for Diffuse Large B-cell lymphoma (DLBCL) survival. BTM-3566 activates the mitochondrial protease OMA1, triggering the ATF4 integrated stress response (ISR). BTM-3566 has robust in vitro efficacy in DLBCL cell lines irrespective of genomic background and elicits complete tumor regression in multiple CDX and PDX mouse models of DLBCL. Based on the activity observed in DLBCL, we further explored the effectiveness of BTM-3566 in MCL models in vitro and in xenograft mouse models in vivo. BTM-3566 inhibited cell proliferation across a panel of MCL cell lines, irrespective of resistance status to ibrutinib and venetoclax (IC50s of 142.5-687.6 nM). Following a 24-hour treatment with BTM-3566, a dose-dependent increase in apoptosis was observed in four tested MCL cell lines—JeKo-1, JeKo-IBN-R, Mino, and Mino-VEN-R, as determined by increased caspase-3 activation and PARP cleavage. Similarly, western blot analysis revealed dose-dependent reductions in MCL-1 and c-MYC, accompanied by an increase in the transcription factor ATF4, indicative of activation of the stress response. We evaluated the in vivo efficacy of BTM-3566 in the JeKo-CDX mouse model. Daily oral administration of BTM-3566 at 10, 20 and 30 mg/kg (n = 5 for each group) in a 5-day on and 2-day off schedule resulted in tumor regression at all three doses (Treatment vs. Vehicle, p < 0.01). We expanded our investigation to PDX mouse models, including those derived from patients resistant to multiple therapies. Four PDX models encompassing naive, rituximab-resistant, BTKi-CAR T dual-resistant and BTKi-VEN-CAR T triple-resistant models were evaluated. BTM-3566 treatment results in nearly complete tumor growth inhibition associated with robust reductions in tumor burden in treated mice across all four PDX models (reduction in tumor weight at the study endpoint, p < 0.001). Notably, no toxicity was observed during the treatment period. In summary, BTM-3566 is effective in treating MCL in vitro and in vivo in preclinical studies. Citation Format: Yang Liu, Heng-Huan Lee, Joseph McIntosh, Yijing Li, Tianci Zhang, Wei Wang, Matthew J. Kostura, Michael Wang. The integrated stress activator BTM-3566 overcomes therapeutic resistance in mantle cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 676.

Tumor-targeting engineered probiotic Escherichia coli Nissle 1917 inhibits colorectal tumorigenesis and modulates gut microbiota homeostasis in mice

AimsPreliminary studies have identified the use of probiotics as a potential treatment strategy against colorectal cancer (CRC). However, natural probiotics lack direct tumor-targeting and tumor-killing activity in the intestine. This study aimed to construct a tumor-targeting engineered probiotic to combat CRC. Main methodsStandard adhesion assay was performed to analyze the adherence ability of tumor-binding protein HlpA to CT26 cells. CCK-8 assay, Hoechst 33258 staining and flow cytometry analysis were used for examining cytotoxicity of tumoricidal protein azurin toward CT26 cells. An engineered probiotic Ep-AH harboring azurin and hlpA genes was developed using Escherichia coli Nissle 1917 (EcN) chassis. Antitumor effects of Ep-AH were evaluated in the azoxymethane (AOM) and dextran sodium sulfate salt (DSS)-induced CRC mice. Moreover, analysis of gut microbiota was conducted via fecal 16S rRNA gene sequencing and shotgun metagenomic sequencing. Key findingsAzurin caused a dose-dependent increase of apoptosis in CT26 cells. Ep-AH treatment reversed weight loss (p < 0.001), fecal occult blood (p < 0.01), and shortening of colon length (p < 0.001) than model group, as well as reducing tumorigenesis by 36 % (p < 0.001). Both Ep-H and Ep-A (EcN expressing HlpA or azurin) were less effective than Ep-AH. Furthermore, Ep-AH enriched the members of beneficial bacteria (e.g., Blautia and Bifidobacterium) and reversed abnormal changes of genes associated with several metabolic pathways (e.g., lipopolysaccharide biosynthesis). SignificanceThese results demonstrated that Ep-AH had excellent therapeutic benefits on cancer remission and gut microbiota modulation. Our study provides an effective strategy for anti-CRC treatment.

Abstract 4975: Anti-cancer effect of a probiotic bacteria-derived compound, 1,4-Dihydroxy-2-naphthoic acid in prostate cancer

Abstract Cancer incidence has been increasing globally and the major cause for mortality associated with this disease is due to metastasis development. Followed by skin and lung cancer, prostate cancer (PCa) is one of the most common cancers prevailing among older men in the United States. Although androgen deprivation therapy (ADT) is the mainstay treatment for PCa, most patients undergoing ADT eventually develop metastatic PCa. Moreover, other treatment options, such as chemotherapy and radiotherapy, have toxic side effects and are ineffective in controlling advanced PCa. Hence, there arises a need for identifying alternative agents with possibly fewer side effects. The gut biome plays a significant role in regulating many bodily functions like digestion, immunity, and including cancer prevention. Probiotics, commonly known as 'good bacteria,' are living microorganisms that provide health promoting benefits. A probiotic metabolite, 1,4-Dihydroxy-2-naphthoic acid (DHNA), is an anti-inflammatory aryl hydrocarbon receptor (Ahr) agonist which is yet to be explored for its anticancer activity. Inhibiting the inflammatory signaling axis and activating Ahr by DHNA could be a promising strategy to counteract metastatic PCa. Cell viability assays revealed that 1,4-DHNA is highly toxic in metastatic prostate cancer cell lines such as DU145 and PC3 compared to normal prostate epithelial cells like RWPE-1 and human prostate stromal myofibroblast cell line, WPMY-1, with an approximate IC50 value of 5 µM in DU145 and PC3 and about 20 µM and 75 µM in WPMY-1 and RWPE-1 respectively. DHNA reduced the colony forming abilities of BPH-Cd, PC3, and DU145 in clonogenic assay and suppressed the BPH-Cd spheroid growth in spheroid formation assay, revealing its anti-tumorigenic potential. Trans-well migration assay using BPH-Cd, PC3, and DU145 showed a decrease in cell migration, hinting at the drug's anti-metastatic ability. G2/M phase arrest in cell cycle analysis and dose-dependent increase in apoptosis of DHNA-treated DU145, PC3, and LNCaP cell lines reiterated its anti-cancer potential. Western blot results in PC3 and DU145 showed significant dose-dependent inhibition of protooncogene β-catenin expression. Downregulation of cell cycle proteins like cyclin D1 and increased expression of effector caspase, cleaved caspase 7 support cell death promoting effects of DHNA via an intrinsic apoptotic pathway. Our pre-clinical data suggest that DHNA can be a potential drug for inhibiting both prostate tumor growth and metastasis. Further research is warranted to ascertain the anti-metastatic and underlying anti-cancer mechanisms to help translate the findings to clinical use. Citation Format: Shreeja Srinivas Bitla, Gnanasekar Munirathinam. Anti-cancer effect of a probiotic bacteria-derived compound, 1,4-Dihydroxy-2-naphthoic acid in prostate cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4975.

Abstract 1558: Targeting CDK9 via the small-molecule inhibitor enitociclib as a therapeutic strategy to treat MYCN-amplified rhabdomyosarcoma and neuroblastoma in children

Abstract Introduction: MYCN amplification is the genetic aberration most consistently associated with poor outcomes in alveolar rhabdomyosarcomas (aRMS) and neuroblastoma (NBL). It regulates key oncogenic processes including tumor cell survival, proliferation and metastasis. In aRMS, MYCN transcription is driven by the PAX3-FOXO1 gene fusion. MYCN and other short half-life mRNA transcripts depend on CDK9 to drive transcription of target genes to initiate and maintain the oncogenic program, making CDK9 blockade an effective therapeutic route against MYC-driven cancers. We have previously shown the activity and tolerability of enitociclib in tumor xenograft models. In this study, we investigated the activity of the selective small-molecule CDK9 inhibitor enitociclib (VIP152/BAY1521152) in preclinical models of pediatric MYCN-amplified aRMS and NBL. Methods: Cell-based viability assays were performed in aRMS (Rh30 and Rh41) and NBL (LAN1, SK-N-AS, SK-N-BE(2) and SK-N-MC) cell lines. Enitociclib antitumor activity was measured by Alamar Blue cell viability assay after 96 h exposure at 9 dose levels (8 nM-2 μM) with DMSO used as control. We performed combination screens of enitociclib with several FDA-approved drugs (n=215). Selected agents with IC50 values &amp;lt;1 μM were analyzed at constant dilution ratios of two inhibitors. Drug synergy was calculated by established methods using SynergyFinder 3.0. Target modulation was determined for RNA polymerase II (RNAPII), MYCN and apoptosis inducers caspase-3 and PARP, in addition to PAX3-FOXO1 and PLK1 protein levels in aRMS cells, by western blotting after continuous exposure with enitociclib for 24 h. Results: Significant cytotoxic activity, with IC50 values ranging from 48-182 nM and 39-123 nM was seen for aRMS cells and NBL cells, respectively. SynergyFinder found synergistic effects of enitociclib with irinotecan, carfilzomib, etoposide, bortezomib, selinexor or topotecan tested at clinically relevant concentrations in the aRMS cell line Rh41. Enitociclib induced apoptosis with cleavage of caspase-3 and PARP by western blotting in a dose- and time-dependent manner in addition to the depletion of phosphorylated RNAPII (Ser2) and MYCN protein levels. In aRMS cells, PAX3-FOXO1 fusion and PLK1 proteins were downregulated. Annexin V/propidium iodide staining confirmed a dose-dependent increase in apoptosis after enitociclib exposure. Conclusions: Our results suggest that CDK9 inhibition is potentially clinically relevant for MYCN-amplified solid tumors such as aRMS and NBL due to its significant antitumor activity and pharmacologic targetability. The data provide essential information on the distinct targets, biomarkers of activity and clinically feasible drug combinations for the development of enitociclib in clinical trials addressing an unmet need in pediatric oncology. Citation Format: Son Tran, Patrick Sipila, Melanie M. Frigault, Amy J. Johnson, Joseph Birkett, Raquel Izumi, Ahmed Hamdy, Beatrix Stelte-Ludwig, David P. Douglass, Anne-Marie Langevin, Norman J. Lacayo, Aru Narendran. Targeting CDK9 via the small-molecule inhibitor enitociclib as a therapeutic strategy to treat MYCN-amplified rhabdomyosarcoma and neuroblastoma in children [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1558.

The PRMT5 inhibitor EPZ015666 is effective against HTLV-1-transformed T-cell lines in vitro and in vivo.

Human T-cell leukemia virus type 1 (HTLV-1) is the infectious cause of adult T-cell leukemia/lymphoma (ATL), an extremely aggressive and fatal malignancy of CD4+ T-cells. Due to the chemotherapy-resistance of ATL and the absence of long-term therapy regimens currently available for ATL patients, there is an urgent need to characterize novel therapeutic targets against this disease. Protein arginine methyltransferase 5 (PRMT5) is a type II PRMT enzyme that is directly involved in the pathogenesis of multiple different lymphomas through the transcriptional regulation of relevant oncogenes. Recently, our group identified that PRMT5 is overexpressed in HTLV-1-transformed T-cell lines, during the HTLV-1-mediated T-cell immortalization process, and in ATL patient samples. The objective of this study was to determine the importance of PRMT5 on HTLV-1 infected cell viability, T-cell transformation, and ultimately disease induction. Inhibition of PRMT5 enzymatic activity with a commercially available small molecule inhibitor (EPZ015666) resulted in selective in vitro toxicity of actively proliferating and transformed T-cells. EPZ015666-treatment resulted in a dose-dependent increase in apoptosis in HTLV-1-transformed and ATL-derived cell lines compared to uninfected Jurkat T-cells. Using a co-culture model of infection and immortalization, we found that EPZ015666 is capable of blocking HTLV-1-mediated T-cell immortalization in vitro, indicating that PRMT5 enzymatic activity is essential for the HTLV-1 T-cell transformation process. Administration of EPZ015666 in both NSG xenograft and HTLV-1-infected humanized immune system (HIS) mice significantly improved survival outcomes. The cumulative findings of this study demonstrate that the epigenetic regulator PRMT5 is critical for the survival, transformation, and pathogenesis of HTLV-1, illustrating the value of this cellular enzyme as a potential therapeutic target for the treatment of ATL.

Discovery of HCD3514 as a potent EGFR inhibitor against C797S mutation in vitro and in vivo.

Osimertinib (AZD9291), a third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI), has significantly improved the survival of non-small cell lung cancer (NSCLC) patients with EGFRT790M mutation, the major mechanism of acquired resistance to first-generation EGFR TKI. However, resistance to AZD9291 arises eventually and EGFRC797S mutation was reported to be a major resistance mechanism. Thus, it is highly valuable to develop novel EGFR fourth-generation inhibitors targeting C797S mutation to override the acquired resistance. In this study, we identified HCD3514 as a novel EGFR fourth-generation inhibitors targeting C797S triple mutation. It strongly inhibited EGFRL858R/T790M/C797S and EGFR19del/T790M/C797S mutations with IC50 values of 1.0 and 2.0 nM, respectively. HCD3514 dose-dependently inhibited the activation of EGFR in both engineered BaF3 cells and tumor cells harboring EGFRC797S triple mutant and thus effectively suppressed the proliferation of the cells. Moreover, HCD3514 caused a dose-dependent increase of apoptosis in C797S triple mutant cells accompanied by increased levels of cleaved caspase-3 and cleaved PARP. Furthermore, HCD3514 induced tumor growth inhibition in EGFR19del/T790M/C797S xenograft model as a single oral agent by decreasing the activation of EGFR. In addition to EGFRC797S triple mutations, HCD3514 also potently and selectively inhibited EGFRT790M double mutations (L858R/T790M and 19del/T790M). Collectively, HCD3514 is a highly selective and potent EGFR inhibitor against EGFRC797S triple mutations as well as EGFRT790M double mutations and is confirmed potently anti-tumor activity in preclinical models.

Prevention of carcinogen-induced oral cancers by polymeric black tea polyphenols via modulation of EGFR-Akt-mTOR pathway

The overexpression of Epidermal Growth Factor Receptor (EGFR) and dysregulation of its downstream effector pathways are important molecular hallmarks of oral cancers. Present study investigates the chemopreventive potential of polymeric black tea polyphenols (PBPs)/thearubigins (TRs) in the hamster model of oral carcinogenesis as well as determine the effect of PBPs on EGFR and the molecular players in the EGFR pathway. In dose-dependent manner, pre and concurrent treatment with PBPs (1.5%, 5%, 10%) decreased the number and volume of macroscopic tumors as well as the number and area of microscopic lesions. Interestingly, at 10% dose of PBPs, no macroscopic or microscopic tumors were observed. We observed PBPs mediated dose-dependent decrease in oxidative DNA damage (8OHdG); inflammation (COX-2); proliferation (PCNA, Cyclin D1); expression of EGFR, and its downstream signaling kinases (pAkt, Akt, and mTOR); hypoxia (HIF1α) and angiogenesis (VEGF). There was also a PBPs mediated dose-dependent increase in apoptosis (Bax). Thus, our data clearly indicate that the observed chemopreventive potential of PBPs was due to modulation in the EGFR pathway associated with cell proliferation, hypoxia, and angiogenesis. Taken together, our results demonstrate preclinical chemopreventive efficacy of PBPs and give an insight into its mechanistic role in the chemoprevention of experimental oral cancer.

Targeting the Non-Canonical NF-κB Pathway in Chronic Lymphocytic Leukemia and Multiple Myeloma.

Simple SummaryThis study was designed to investigate the potential for targeting the NF-κB-inducing kinase, NIK, in two common B-cell malignancies, chronic lymphocytic leukemia (CLL) and multiple myeloma (MM). Using a selective NIK inhibitor, CW15337, we were able to demonstrate that cell lines and primary tumor cells were sensitive to the effects of NIK inhibition, whilst normal lymphocytes were significantly more resistant to its cytotoxic effects. Sensitivity to CW15337 was associated with the nuclear expression of the NF-κB subunit, p52. Importantly, tumor samples from a subset of poor prognosis CLL patients, with mutations in a gene called BIRC3, showed elevated p52 expression and were particularly sensitive to NIK inhibition. Furthermore, the combination of CW15337 and ABT-199 (venetoclax) reversed the drug resistance observed when treating tumor cells with ABT-199 alone. Our study shows the potential for targeting NIK in both CLL and MM.In this study, we evaluated an NF-κB inducing kinase (NIK) inhibitor, CW15337, in primary chronic lymphocytic leukemia (CLL) cells, CLL and multiple myeloma (MM) cell lines and normal B- and T-lymphocytes. Basal NF-κB subunit activity was characterized using an enzyme linked immunosorbent assay (ELISA), and the effects of NIK inhibition were then assessed in terms of cytotoxicity and the expression of nuclear NF-κB subunits following monoculture and co-culture with CD40L-expressing fibroblasts, as a model of the lymphoid niche. CW15337 induced a dose-dependent increase in apoptosis, and nuclear expression of the non-canonical NF-κB subunit, p52, was correlated with sensitivity to CW15337 (p = 0.01; r2 = 0.39). Co-culture on CD40L-expressing cells induced both canonical and non-canonical subunit expression in nuclear extracts, which promoted in vitro resistance against fludarabine and ABT-199 (venetoclax) but not CW15337. Furthermore, the combination of CW15337 with fludarabine or ABT-199 showed cytotoxic synergy. Mechanistically, CW15337 caused the selective inhibition of non-canonical NF-κB subunits and the transcriptional repression of BCL2L1, BCL2A1 and MCL1 gene transcription. Taken together, these data suggest that the NIK inhibitor, CW15337, exerts its effects via suppression of the non-canonical NF-κB signaling pathway, which reverses BCL2 family-mediated resistance in the context of CD40L stimulation.

Features of the cytoprotective effect of selenium nanoparticles on primary cortical neurons and astrocytes during oxygen\u2013glucose deprivation and reoxygenation

The study is aimed at elucidating the effect of selenium nanoparticles (SeNPs) on the death of cells in the primary culture of mouse cerebral cortex during oxygen and glucose deprivation (OGD). A primary cell culture of the cerebral cortex containing neurons and astrocytes was subjected to OGD and reoxygenation to simulate cerebral ischemia-like conditions in vitro. To evaluate the neuroprotective effect of SeNPs, cortical astrocytes and neurons were incubated for 24 h with SeNPs, and then subjected to 2-h OGD, followed by 24-h reoxygenation. Vitality tests, fluorescence microscopy, and real-time PCR have shown that incubation of primary cultured neurons and astrocytes with SeNPs at concentrations of 2.5–10 µg/ml under physiological conditions has its own characteristics depending on the type of cells (astrocytes or neurons) and leads to a dose-dependent increase in apoptosis. At low concentration SeNPs (0.5 µg/ml), on the contrary, almost completely suppressed the processes of basic necrosis and apoptosis. Both high (5 µg/ml) and low (0.5 µg/ml) concentrations of SeNPs, added for 24 h to the cells of cerebral cortex, led to an increase in the expression level of genes Bcl-2, Bcl-xL, Socs3, while the expression of Bax was suppressed. Incubation of the cells with 0.5 µg/ml SeNPs led to a decrease in the expression of SelK and SelT. On the contrary, 5 µg/ml SeNPs caused an increase in the expression of SelK, SelN, SelT, SelP. In the ischemic model, after OGD/R, there was a significant death of brain cells by the type of necrosis and apoptosis. OGD/R also led to an increase in mRNA expression of the Bax, SelK, SelN, and SelT genes and suppression of the Bcl-2, Bcl-xL, Socs3, SelP genes. Pre-incubation of cell cultures with 0.5 and 2.5 µg/ml SeNPs led to almost complete inhibition of OGD/R-induced necrosis and greatly reduced apoptosis. Simultaneously with these processes we observed suppression of caspase-3 activation. We hypothesize that the mechanisms of the protective action of SeNPs involve the activation of signaling cascades recruiting nuclear factors Nrf2 and SOCS3/STAT3, as well as the activation of adaptive pathways of ESR signaling of stress arising during OGD and involving selenoproteins SelK and SelT, proteins of the Bcl-2 family ultimately leading to inactivation of caspase-3 and inhibition of apoptosis. Thus, our results demonstrate that SeNPs can act as neuroprotective agents in the treatment of ischemic brain injuries.

Zhoushi Qiling decoction induces apoptosis of human prostate cancer cells via miR-143/Bcl-2 axis.

A number of traditional Chinese medicines (TCMs) are widely used in prostate cancer treatment in China. The aim of this study was to test the efficacy of a TCM, Zhoushi Qiling Decoction (ZQD), in combination with androgen deprivation therapy (ADT) and explore its underlying mechanism. A total of 151 patients were recruited to receive ADT treatment or ADT+ZQD treatment. The survival of patients who received ADT+ZQD treatment was significantly higher than those who received ADT therapy only. DU145 prostate cancer cells were treated with ZQD (50 mg/mL) for 24 h in vitro and expression levels of an array of miRNAs were examined. Our results suggested that miR-143 demonstrated prominent upregulation in DU145 cells after treatment with ZQD. In patient serum samples, miR-143 expression was also significantly upregulated after ADT+ZQE treatment, which was however absent in patients treated with ADT only. In DU145 cells, ZQD treatment led to a dose-dependent increase in apoptosis, which could be reduced by anti-miR-143 treatment. There was a binding site between miR-143 and B cell CLL/lymphoma-2 (Bcl-2) and ZQD treatment reduced Bcl-2 expression. ZQD treatment led to increased caspase-3 and Bax expression. ZQD treatment could promote apoptosis of prostate cancer cells by promoting miR-143 upregulation, which could be a possible mechanism underlying the inhibitory effect of ZQD in prostate cancer in patient.

High Mobility Group Box 1 Protein (HMGB1) Mediates Anemia of Inflammation through Lineage Skewing and Direct Inhibition of EPO Signaling

Erythropoiesis is sensitive to inflammation and its associated mediators. We previously identified the damage-associated molecular pattern, high mobility group box 1 protein (HMGB1), as a causative factor involved in the development of anemia using a murine model of chronic inflammation, and recently, showed that HMGB1 negatively affects human erythropoiesis, in part, through altered erythropoietin (EPO) signaling and hematopoietic stem and progenitor cell (HSPC) lineage commitment. Despite these findings, a mechanistic understanding of erythroid dysfunction in the presence of HMGB1 is lacking.We first documented a dose-dependent logarithmic difference in erythroid cell growth at Days 7 and 11 of culture of CD34+ HSPCs during erythroid differentiation as well as a relatively smaller loss of cells at Day 14 between HMGB1-treated and vehicle-treated cultures. These results suggest that HMGB1 exerts its effects at distinct stages of erythroid differentiation: at both the progenitor and the precursor stages. Indeed, flow analysis of erythroblast differentiation showed that HMGB1 caused an accumulation of precursors at the basophilic erythroblast stage. The loss of poly- and orthochromatic erythroblasts resulted from increased apoptosis at these stages of erythroid differentiation as assessed by annexin-V staining. Induction of apoptosis was confirmed by a dose-dependent increase in apoptosis and 3-fold increase in cleaved caspase-3 in HUDEP-2 cells, an erythroid cell line. Furthermore, a mutant form of HMGB1 (3S-HMGB1) in which critical cysteine residues were mutated to abolish the activity of the protein failed to alter erythroid proliferation and survival.HMGB1 appears to deprive differentiating erythroid cells of growth and survival signals through attenuated phosphorylation of downstream EPO effectors including pJAK2 and pSTAT5, independent of SHP1/2 activity. As a consequence of perturbed EPO signaling, erythroid cells contained reduced levels of nuclear HSP70 and GATA1, which ultimately led to apoptosis. Importantly, EPO co-administrated with either tumor necrosis factor (TNF), interleukin-1β (IL-1β), or IL-6 led to phosphorylation levels of JAK2-STAT5 comparable to controls, suggesting that direct inhibition of EPO signaling is specific to HMGB1.To document the effect of HMGB1 on EPO-dependent and independent progenitors, erythroid progenitor growth and differentiation were monitored at 24hr intervals in culture. CFU-E but not BFU-E numbers were decreased following treatment with HMGB1. Moreover, HMGB1-treated cells expressed high levels of GATA2 indicating deregulation of GATA transcription factor switching. Myelo-erythroid culture systems demonstrated that HMGB1 strongly inhibited erythroid commitment of CD34+ HSPCs while it favored the generation of increased numbers of CD11b+ and CD13+ expressing myeloid cells. The induced switch in lineage commitment is consistent with increased GATA2 expression and decreased GATA1 expression. Further, we observed that erythroid and hematopoietic progenitor cultures became more acidic in the presence of HMGB1 suggesting that HMGB1 may also influence progenitor metabolic processes.Our data demonstrate that HMGB1 negatively affects erythropoiesis at EPO-dependent and -independent stages of erythroid differentiation through decreased CFU-E numbers and apoptosis of erythroblasts, and also favor increased myeloid differentiation of HSPCs. Contrary to classical inflammatory mediators, HMGB1 relies on a novel mechanism, involving downstream EPO effectors, to mediate erythroid dysfunction. Ongoing pharmacologic and genetic screens of EPO signaling axes, putative HMGB1 receptor(s), and progenitor metabolic profiling are currently underway to better understand how HMGB1 causes erythropoietin refractory anemia in the setting of inflammation. DisclosuresNo relevant conflicts of interest to declare.

Cytotoxic impacts of treated electroplating industrial effluent and the comparative effect of their metal components (Zn, Hg, and Zn+Hg) on Danio rerio gill (DrG) cell line

Effluent produced during the electroplating process can contain high concentrations of heavy metals that can enter the environment and induce toxicity to aquatic organisms. Relatively high concentrations of zinc (Zn) and mercury (Hg) have been detected in treated electroplating industrial effluent (TEPIE), though the cytotoxic potential of these compounds has not been well assessed in fish gills. A novel cell line, Danio rerio gill (DrG), were exposed to TEPIE and concentrations of Zn, Hg, and Zn + Hg previously measured in treated effluent to evaluate the use of the DrG cell line following exposure to environmental pollutants. Several cytotoxic assays were employed to assess the effect of TEPIE, Zn, and Hg on this cell line. The percent cell viability was significantly reduced in a concentration-dependent manner following exposure to TEPIE, Zn, Hg, and Zn + Hg (p < 0.05) for 24 h, with additional morphological changes observed in exposure treatments relative to controls. Additionally, there was a significant induction of DNA damage detected in all exposure treatments determined through comet assay tail length. An increase in intracellular ROS generation was also observed in cells exposed to TEPIE, Zn, Hg, and Zn + Hg, corresponding to dose-dependent increases in apoptosis. Our study confirmed that TEPIE and the metals present in it induced cytotoxicity in the DrG cell line, demonstrating its usefulness as a model to explore relationships between pollutants and fish gills.

Cyproheptadine causes apoptosis and decreases inflammation by disrupting thiol/disulfide balance and enhancing the levels of SIRT1 in C6 glioblastoma cells

Cyproheptadine is first-generation antihistamine drug, that is, H1 receptor antagonist, with a drug being anesthetic, anti-serotonergic and anti-cholinergic and started to be used clinically in the 1960s. As firstly utilized as an anti-allergic drug, usage of cyproheptadine was expanded to other cases including serotonin syndrome, appetite increasing, migraines and insomnia. However, there are almost few studies seeking to explore the association between cyproheptadine and cancer in general. In the present study, we sought to determine the impact of cyproheptadine on C6 glioblastoma cells by morphological, biochemical and cytotoxic analyzes. We searched the effective doses of cyproheptadine for C6 glioblastoma cells and examined the cells under an inverted microscope. Next, we determined the protein levels of SIRT1, NFκB and IL-6 protein. Then, we measured and calculated the levels of thiols, disulfide bonds and related parameters. After that, we evaluated apoptotic activity by Annexin V and caspase 3 assays. As a result, we detected a dose-dependent increase in apoptosis and SIRT 1 protein levels, and a decrease in inflammatory proteins. Furthermore, we have detected a drop in thiol and disulfide content. Our study suggests that Cyproheptadine causes apoptosis and decreases inflammation by disrupting thiol/disulfide balance and enhancing the levels of SIRT1, offering the potential for being an anti-cancer drug. Therefore, it might be further investigated in future studies.

Anticancer studies on methanolic extract of aerial parts of Uraria Picta (Jacq.) DC

BackgroundUraria picta (Jacq.) DC is a traditional Indian herb used in the Ayurvedic system of medicine. It is an ingredient of the popular Ayurvedic formulation “Dasamoola.” It is also traditionally used as an anti-inflammatory herb in Ayurveda. The plant also has excellent antioxidant potential. Different parts of Uraria picta are useful and have diverse medicinal properties. The present investigation was done to evaluate the anticancer activity of methanolic extract of aerial parts of Uraria picta (Jacq.).ResultsPreliminary phytochemical screening of MEUP confirmed the presence of flavonoids, phenolics, glycosides, and tannins. Total phenolic and flavonoid contents in MEUP were found to be 14.6 gram equivalent of gallic acid and 0.735 gram equivalent of catechin, respectively. IC50 values of cisplatin and MEUP were found to be 8.75 μg/ml and 436.92 μg/ml, respectively. Induction of apoptosis was evident from DAPI staining showing a dose-dependent increase in apoptosis. Also, a reduction in intracellular ROS was evident from the DCFH-da assay, where a dose-dependent decrease in intracellular ROS (fluorescence) was observed. Significant reduction (P < 0.05) in tumor volume was observed in mice receiving cisplatin and MEUP at both dose levels.ConclusionThe methanolic extract of aerial parts of Uraria picta (Jacq.) DC is effective in inhibiting tumor growth and has significant anticancer property.

CC-486 Mechanism Imparted By Extended Exposure of Azacitidine Upregulates Myeloid Differentiation Markers and Induces Cell Death in AML Cells

CC-486 Mechanism Imparted By Extended Exposure of Azacitidine Upregulates Myeloid Differentiation Markers and Induces Cell Death in AML Cells

Synthetic Makaluvamine Analogs Decrease c-Kit Expression and Are Cytotoxic to Neuroendocrine Tumor Cells.

In an effort to discover viable systemic chemotherapeutic agents for neuroendocrine tumors (NETs), we screened a small library of 18 drug-like compounds obtained from the Velu lab against pulmonary (H727) and thyroid (MZ-CRC-1 and TT) neuroendocrine tumor-derived cell lines. Two potent lead compounds (DHN-II-84 and DHN-III-14) identified from this screening were found to be analogs of the natural product makaluvamine. We further characterized the antitumor activities of these two compounds using pulmonary (H727), thyroid (MZ-CRC-1) and pancreatic (BON) neuroendocrine tumor cell lines. Flow cytometry showed a dose-dependent increase in apoptosis in all cell lines. Induction of apoptosis with these compounds was also supported by the decrease in myeloid cell leukemia-1 (MCL-1) and X-chromosome linked inhibitor of apoptosis (XIAP) detected by Western blot. Compound treatment decreased NET markers chromogranin A (CgA) and achaete-scute homolog 1 (ASCL1) in a dose-dependent manner. Moreover, the gene expression analysis showed that the compound treatment reduced c-Kit proto-oncogene expression in the NET cell lines. Induction of apoptosis could also have been caused by the inhibition of c-Kit expression, in addition to the known mechanisms such as damage of DNA by topoisomerase II inhibition for this class of compounds. In summary, makaluvamine analogs DHN-II-84 and DHN-III-14 induced apoptosis, decreased neuroendocrine tumor markers, and showed promising antitumor activity in pulmonary, thyroid, and pancreatic NET cell lines, and hold potential to be developed as an effective treatment to combat neuroendocrine tumors.

Antiproliferative effect of bupivacaine on patient-derived sarcoma cells.

Sarcomas are rare tumors with limited treatment options. Although chemotherapy is standard for certain subtypes, overall survival has not improved in several decades. Bupivacaine has been shown to induce apoptosis and prevent cell growth in multiple different types of malignancies but has not been studied in sarcoma. The current study evaluated the effects of bupivacaine on multiple patient-derived sarcoma cells and a commercial sarcoma cell line. Multiple patient-derived sarcoma cell subtypes and a commercial synovial cell sarcoma cell line were exposed to bupivacaine for different durations and at different concentrations. The patient-derived cells included a high-grade conventional osteosarcoma, a high-grade undifferentiated pleomorphic sarcoma of bone, and a high-grade synovial sarcoma. Flow cytometry and an MTT assay were used to evaluate whether a treatment effect was observed. Treatment of all the subtypes of sarcomas in this study with bupivacaine demonstrated a time- and dose-dependent increase in apoptosis and decrease in cell viability. A cell viability assay demonstrated that the IC50 was between 0.04 and 0.05% and that the treatment effect occurred at clinically relevant doses in vitro. Bupivacaine was toxic to both the patient-derived cells and the commercial cell line at doses commonly used in the clinical setting. These findings provide a foundation for further in vivo studies to evaluate whether these effects will translate to the clinical setting. Although further research is necessary, bupivacaine shows promise as not only an adjunct for pain management but as a treatment modality for sarcoma.

24R,25-Dihydroxyvitamin D3 regulates breast cancer cells in vitro and in vivo

BackgroundEpidemiological studies indicate high serum 25(OH)D3 is associated with increased survival in breast cancer patients. Pre-clinical studies attributed this to anti-tumorigenic properties of its metabolite 1α,25(OH)2D3. However, 1α,25(OH)2D3 is highly calcemic and thus has a narrow therapeutic window. Here we propose another metabolite, 24R,25(OH)2D3, as an alternative non-calcemic vitamin D3 supplement. MethodsNOD-SCID-IL2γR null female mice with MCF7 breast cancer xenografts in the mammary fat pad were treated with 24R,25(OH)2D3 and changes in tumor burden and metastases were assessed. ERα66+ MCF7 and T47D cells, and ERα66- HCC38 cells were treated with 24R,25(OH)2D3in vitro to assess effects on proliferation and apoptosis. Effects on migration and metastatic markers were assessed in MCF7. Results24R,25(OH)2D3 reduced MCF7 tumor growth and metastasis in vivo. In vitro results indicate that this was not due to an anti-proliferative effect; 24R,25(OH)2D3 stimulated DNA synthesis in MCF7 and T47D. In contrast, markers of invasion and metastasis were decreased. 24R,25(OH)2D3 caused dose-dependent increases in apoptosis in MCF7 and T47D, but not HCC38 cells. Inhibitors to palmitoylation, caveolae integrity, phospholipase-D, and estrogen receptors (ER) demonstrate that 24R,25(OH)2D3 acts on MCF7 cells through caveolae-associated, phospholipase D-dependent mechanisms via cross-talk with ERs. ConclusionThese results indicate that 24R,25(OH)2D3 shows promise in treatment of breast cancer by stimulating tumor apoptosis and reducing metastasis. General significance24R,25(OH)2D3 regulates breast cancer cell survival through ER-associated mechanisms similar to 24R,25(OH)2D3 effects on chondrocytes. Thus, 24R,25(OH)2D3 may modulate cell survival in other estrogen-responsive cell types, and its therapeutic potential should be investigated in ER-associated pathologies.

The potential toxic impact of different gadolinium-based contrast agents combined with 7-T MRI on isolated human lymphocytes

BackgroundTo investigate a potentially amplifying genotoxic or cytotoxic effect of different gadolinium-based contrast agents (GBCAs) in combination with ultra-high-field 7-T magnetic resonance imaging (MRI) exposure in separated human peripheral blood lymphocytes.MethodsThis in vitro study was approved by the local ethics committee and written informed consent was obtained from all participants. Isolated lymphocytes from twelve healthy donors were incubated with gadobutrol, gadoterate meglumine, gadodiamide, gadopentetate dimeglumine, or gadoxetate either alone or combined with 7-T MRI (1 h). Deoxyribonucleic acid (DNA) double-strand breaks were assessed 15 min after MRI exposure by automated γH2AX foci quantification. Cytotoxicity was determined at later endpoints by Annexin V/propidium iodide apoptosis assay (24 h) and [3H]-thymidine proliferation test (72 h). As a reference, lymphocytes from four different donors were exposed analogously to iodinated contrast agents (iomeprol, iopromide) in combination with computed tomography.ResultsBaseline γH2AX levels (0.08 ± 0.02 foci/cell) were not significantly (p between 0.135 and 1.000) enhanced after administration of GBCAs regardless of MRI exposure. In contrast to the two investigated macrocyclic GBCAs, lymphocytes exposed to the three linear GBCAs showed a dose-dependent increase in apoptosis (maximum 186% of unexposed control, p < 0.001) and reduced proliferation rate (minimum 0.7% of unexposed control, p < 0.001). However, additional 7-T MRI co-exposure did not alter GBCA-induced cytotoxicity.ConclusionsExposure of lymphocytes to different GBCAs did not reveal significant induction of γH2AX foci, and enhanced cytotoxicity was only observed in lymphocytes treated with the linear GBCAs used in this study, independent of additional 7-T MRI co-exposure.

Thermal photodynamic therapy increases apoptosis and reactive oxygen species generation in cutaneous and mucosal squamous cell carcinoma cells

Thermal photodynamic therapy (PDT) is an emerging modality to optimize treatment of pre-cancerous squamous cell carcinoma (SCC) lesions, known as actinic keratoses. Thermal PDT involves heating the tissue, skin, or mucosa above normal skin temperature during 5-aminolevulinic (5-ALA) incubation and irradiating with blue light, which leads to cell apoptosis and reactive oxygen species (ROS) generation. To our knowledge, thermal PDT has not been studied for the treatment of cutaneous or mucosal SCC. We incubated two SCC cell lines with 5-ALA for 30 minutes at temperatures between 21 °C and 42 °C and then irradiated cells with 1000 seconds of blue light. We measured changes in apoptosis, necrosis, and ROS. At 36 °C, there was a dose-dependent increase in apoptosis and ROS generation. Thermal incubation of 5-ALA at 39° and 42 °C followed by blue light increased cell apoptosis and ROS generation compared to untreated control samples incubated at the same temperatures. Thermal PDT may represent a new treatment option for cutaneous and mucosal SCC cancer. Thermal PDT is associated with an increase in SCC cellular apoptosis and is associated with an upregulation in ROS. Clinical trials are required to determine optimal thermal PDT treatment parameters and efficacy for cutaneous and mucosal SCC.