Significant Increase In Cell Death Research Articles

Doxorubicin induces phosphorylation of lamin A/C and loss of nuclear membrane integrity: A novel mechanism of cardiotoxicity

Lamin A/C, essential inner nuclear membrane proteins, have been linked to progeria, a disease of accelerated aging, and many other diseases, which include cardiac disorder. Lamin A/C mutation and its phosphorylation are associated with altering nuclear shape and size. The role of lamin A/C in regulating normal cardiac function was reported earlier. In the present study, we hypothesized that Doxorubicin (Dox) may alter total lamin A/C expression and phosphorylation, thereby taking part in cardiac injury. An in vitro cellular injury model was generated with Dox (0.1–10.0 μM) treatment on cardiomyoblast cells (H9c2) to prove our hypothesis. Increased size and irregular (ameboid) nucleus shape were observed in H9c2 cells after Dox treatment. Similarly, we have observed a significant increase in cell death on increasing the Dox concentration. The expression of lamin A/C and its phosphorylation at serine 22 significantly decreased and increased, respectively in H9c2 cells and rat hearts after Dox exposure. Phosphorylation led to depolymerization of the lamin A/C in the inner nuclear membrane and was evidenced by their presence throughout the nucleoplasm as observed by immunocytochemistry techniques. Thinning and perforation on the walls of the nuclear membrane were observed in Dox-treated H9c2 cells. LMNA-overexpression in H9c2 protected the cells from Dox-induced cell death, reversing all changes described above. Further, improvement of lamin A/C levels was observed in Dox-treated H9c2 cells when treated with Purvalanol A, a CDK1 inhibitor and N-acetylcysteine, an antioxidant. The study provides new insight regarding Dox-induced cardiac injury with the involvement of lamin A/C and alteration of inner nuclear membrane structure.

MhZAT10L positively modulates NaCl-induced cell death by triggering MhVPEγ transcription in Malus hupehensis

Excess sodium chloride (NaCl) in soil causes several secondary stresses for plants. The plant-specific vacuolar processing enzyme (VPE) plays a positive role in regulating stress-induced cell death. Here, we observed that NaCl treatment leads to a significant increase in cell death and upregulation of MhVPEγ expression in Malus hupehensis roots. Meanwhile, we also identified a C2H2-type transcription factor upstream of MhVPEγ, MhZAT10L, which positively regulated MhVPEγ transcription. The transcription of MhZAT10L was induced by NaCl. And genetic experiments using transgenic Malus hupehensis roots and apple calli showed that the suppression of MhZAT10L decreased their sensitivity to NaCl, as well as mitigated the generation of reactive oxygen species (ROS), cell death, and MhVPEγ expression induced by NaCl. Conversely, the overexpression of MhZAT10L enhanced their sensitivity to NaCl, accelerated ROS generation, and promoted cell death by directly activating MhVPEγ transcription. Overall, our findings indicate that MhZAT10L positively regulates NaCl-induced cell death by activating MhVPEγ transcription.

Monitoring mitochondrial calcium in cardiomyocytes during coverslip hypoxia using a fluorescent lifetime indicator

An increase in mitochondrial calcium via the mitochondrial calcium uniporter (MCU) has been implicated in initiating cell death in the heart during ischemia-reperfusion (I/R) injury. Measurement of calcium during I/R has been challenging due to the pH sensitivity of indicators coupled with the fall in pH during I/R. The development of a pH-insensitive indicator, mitochondrial localized Turquoise Calcium Fluorescence Lifetime Sensor (mito-TqFLITS), allows for quantifying mitochondrial calcium during I/R via fluorescent lifetime imaging. Mitochondrial calcium was monitored using mito-TqFLITS in neonatal mouse ventricular myocytes (NMVM) isolated from germline MCU-KO mice and MCUfl/fl treated with CRE-recombinase to acutely knockout MCU. To simulate ischemia, a coverslip was placed on a monolayer of NMVMs to prevent access to oxygen and nutrients. Reperfusion was induced by removing the coverslip. Mitochondrial calcium increases threefold during coverslip hypoxia in MCU-WT. There is a significant increase in mitochondrial calcium during coverslip hypoxia in germline MCU-KO, but it is significantly lower than in MCU-WT. We also found that compared to WT, acute MCU-KO resulted in no difference in mitochondrial calcium during coverslip hypoxia and reoxygenation. To determine the role of mitochondrial calcium uptake via MCU in initiating cell death, we used propidium iodide to measure cell death. We found a significant increase in cell death in both the germline MCU-KO and acute MCU-KO, but this was similar to their respective WTs. These data demonstrate the utility of mito-TqFLITS to monitor mitochondrial calcium during simulated I/R and further show that germline loss of MCU attenuates the rise in mitochondrial calcium during ischemia but does not reduce cell death.

Abstract 618: Preclinical assessment of APL-030, a selective and orally bioavailable inhibitor of the integrated stress response regulator GCN2 with activity against acute myeloid leukemia

Abstract GCN2 (EIF2AK4) is an evolutionarily conserved kinase and a pivotal regulator of the Integrated Stress Response (ISR), an adaptive cellular program triggered primarily by amino acid scarcity. Active GCN2 phosphorylates the translation initiation factor eIF2α, resulting in the attenuation of global protein synthesis and a largely ATF4-driven program of resource enhancement. GCN2 promotes cancer cell survival under conditions of microenvironmental or drug-induced intracellular nutrient scarcity. Here, we describe the preclinical development of APL-030, a novel and selective ATP-competitive inhibitor of GCN2. APL-030 was confirmed as a potent inhibitor of GCN2 in an on-target biochemical kinase assay with a Ki of 4.4nM. In a cell-based assay APL-030 decreased eIF2α phosphorylation in a dose-dependent manner via direct GCN2 inhibition with an IC50 of 50.8nM. In acute myeloid leukemia (AML) cell lines GCN2 inhibition resulted in a decrease in gene expression of effector genes CHAC1 and DDIT3. Moreover, treatment with APL-030 dose-dependently decreased cell viability in multiple AML cell lines, which was accompanied by an increase in caspase 3/7 activity. Bulk mRNA sequencing of MOLM-13 AML cells showed that APL-030 inhibited the extensive transcriptome changes triggered by glutamine depletion, in line with a high degree of selectivity of APL-030 for GCN2-mediated ISR signaling. In cells grown in rich medium, APL-030 triggered a largely ISR-independent transcriptome response that was dominated by the enrichment of pathways related to cell cycle as well as RNA and protein metabolism. In vivo, APL-030 treatment of an AML xenograft animal model completely inhibited tumor growth and prolonged survival time. Inhibition of AML tumor growth was shown to be a consequence of decreased cell viability and induction of caspase-dependent apoptosis. APL-030 was also tested on primary diagnostic AML cells, which were assessed for cell death by flow cytometry following staining for Annexin-V, 7-AAD, CD45, CD34, and CD38. Treatment with APL-030 caused a statistically significant increase in AML cell death in all six samples studied. Three samples exhibited a measurable CD34+/CD38- compartment, enabling evaluation of this leukemic stem cell-enriched population. APL-030 caused a statistically significant increase in cell death in the leukemic stem cell-enriched population in all three samples studied. APL-030 is a novel GCN2 inhibitor that that has shown encouraging efficacy in preclinical studies using both AML cell lines and patient-derived AML samples. Based on these preclinical results, a phase 1/2 study is planned in hematological tumors. Citation Format: Monica Roman-Trufero, Gavin Whitlock, Matthew Fuchter, Maxmila Jeyakumar, Panagiota Chaida, Sereina Annik Herzog, Armin Zebisch, Richard Butt, Holger W. Auner, Nadine Clemo. Preclinical assessment of APL-030, a selective and orally bioavailable inhibitor of the integrated stress response regulator GCN2 with activity against acute myeloid leukemia [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 618.

Influence of Different Photobiomodulation Parameters on Multi-Potent Adipose Tissue Mesenchymal Cells In Vitro.

Objective: Investigating the effect of different parameters of photobiomodulation (PBM) with low-power laser on multi-potent mesenchymal stem cells (MSCs) derived from adipose tissue in terms of proliferation and cell death. Methods: MSCs were submitted to PBM applications with combinations of the following physical parameters: control group (no intervention), wavelengths of 660 and 830 nm; energy of 0.5, 2, and 4 J; and power of 40 and 100 mW. MSC analysis was performed using MetaXpress® software at 24, 48, and 72 h. Results: Irradiation promoted a significant increase in cell proliferation (p < 0.05), with 830 nm laser, 100 mW, with energy of 0.5, 2, and 4 J in relation to the control group at all times. PBM with 660 nm, power of 40 mW, and energy of 0.5, 2, and 4 J produced greater cell death at 24 h compared with the control group. At the time of 72 h, there was no significant difference concerning cell death. Conclusions: According to the results found, we can conclude that both wavelengths were effective; however, the 830 nm laser was more effective in terms of cell proliferation compared with the 660 nm laser. The 660 nm wavelength showed a significant increase in cell death when compared with the 830 nm laser.

Fusariotoxin-Induced Toxicity in Mesenchymal Stem Cells and Fibroblasts: A Comparison Between Differentiated and Undifferentiated Cells.

Humans are unknowingly exposed to mycotoxins through the consumption of plant-derived foods and processed products contaminated with these toxic compounds. In addition to agricultural losses, Fusarium toxins pose a threat to human health. However, the effects of fusariotoxins on the viability and proliferation of stem cells have not been fully explored. We investigated the cytotoxic effects of deoxynivalenol (DON) and B-trichothecene mix (MIX) on mesenchymal stem cells (MSCs) and the L929 fibroblast cell line. MSCs were isolated from the dental pulp tissue. The doubling time and viability of dental pulp stem cells (DPSCs) and L929 cells were determined using the MTT assay. The following doses of B-trichothecenes (0.25-16 µg/mL; 24 hours and 48 hours) were used to evaluate cytotoxicity. In addition, changes in the confluency-dependent response of DPSCs to DON toxicity were determined. Moreover, we investigated the effect of DON on cell death via acridine orange/ethidium bromide (AO/EB) double staining. A DON and MIX showed a dose- and time-dependent inhibitory effect on the proliferation of both cells. DPSCs exposed to DON for 48 hours (IC50 = 0.5 μg/mL) were found to be 16-fold more sensitive than L929 cells (IC50 = 8 μg/mL). Compared with a culture with 80% confluency, DPSCs from a 50% confluent culture were more sensitive to varying doses of DON (0.25-4 µg/mL, 24-48 hours). Moreover, AO/EB staining showed that treatment of DPSCs with DON led to a significant increase in cell death (17% for 2.4 µg/mL; 50% for 4.8 µg/mL). This study reveals that undifferentiated MSCs are significantly more sensitive to DON than differentiated somatic cells (L929). Given that humans are frequently exposed to these mycotoxins, our findings imply that prolonged exposure to them may also have harmful effects on cellular differentiation and embryonic development.

Synergistic lethality in chronic myeloid leukemia – targeting oxidative phosphorylation and unfolded protein response effectively complements tyrosine kinase inhibitor treatment

Chronic myeloid leukemia (CML) is effectively treated with tyrosine kinase inhibitors (TKIs), targeting the BCR::ABL1 oncoprotein. Still, resistance to therapy, relapse after treatment discontinuation, and side effects remain significant issues of long-term TKI treatment. Preliminary studies have shown that targeting oxidative phosphorylation (oxPhos) and the unfolded protein response (UPR) are promising therapeutic approaches to complement CML treatment. Here, we tested the efficacy of different TKIs, combined with the ATP synthase inhibitor oligomycin and the ER stress inducer thapsigargin in the CML cell lines K562, BV173, and KU812 and found a significant increase in cell death. Both, oligomycin and thapsigargin, triggered the upregulation of the UPR proteins ATF4 and CHOP, which was inhibited by imatinib. We observed comparable effects on cell death when combining TKIs with the ATP synthase inhibitor 8-chloroadenosine (8-Cl-Ado) as a potentially clinically applicable therapeutic agent. Stress-related apoptosis was triggered via a caspase cascade including the cleavage of caspase 3 and the inactivation of poly ADP ribose polymerase 1 (PARP1). The inhibition of PARP by olaparib also increased CML death in combination with TKIs. Our findings suggest a rationale for combining TKIs with 8-Cl-Ado or olaparib for future clinical studies in CML.

Design, Synthesis, and Comparison of PLA-PEG-PLA and PEG-PLA-PEG Copolymers for Curcumin Delivery to Cancer Cells.

Curcumin (CUR) has potent anticancer activities, and its bioformulations, including biodegradable polymers, are increasingly able to improve CUR's solubility, stability, and delivery to cancer cells. In this study, copolymers comprising poly (L-lactide)-poly (ethylene glycol)-poly (L-lactide) (PLA-PEG-PLA) and poly (ethylene glycol)-poly (L-lactide)-poly (ethylene glycol) (PEG-PLA-PEG) were designed and synthesized to assess and compare their CUR-delivery capacity and inhibitory potency on MCF-7 breast cancer cells. Molecular dynamics simulations and free energy analysis indicated that PLA-PEG-PLA has a higher propensity to interact with the cell membrane and more negative free energy, suggesting it is the better carrier for cell membrane penetration. To characterize the copolymer synthesis, Fourier transform-infrared (FT-IR) and proton nuclear magnetic resonance (1H-NMR) were employed, copolymer size was measured using dynamic light scattering (DLS), and their surface charge was determined by zeta potential analysis. Characterization indicated that the ring-opening polymerization (ROP) reaction was optimal for synthesizing high-quality polymers. Microspheres comprising the copolymers were then synthesized successfully. Of the two formulations, PLA-PEG-PLA experimentally exhibited better results, with an initial burst release of 17.5%, followed by a slow, constant release of the encapsulated drug up to 80%. PLA-PEG-PLA-CUR showed a significant increase in cell death in MCF-7 cancer cells (IC50 = 23.01 ± 0.85 µM) based on the MTT assay. These data were consistent with gene expression studies of Bax, Bcl2, and hTERT, which showed that PLA-PEG-PLA-CUR induced apoptosis more efficiently in these cells. Through the integration of nano-informatics and in vitro approaches, our study determined that PLA-PEG-PLA-CUR is an optimal system for delivering curcumin to inhibit cancer cells.

Instabilities induced by mechanical loading determine the viability of chondrocytes grown on porous scaffolds

Tissue-engineered cartilage constructs have shown promise to treat focal cartilage defects in multiple clinical studies. Notably, products in clinical use or in late-stage clinical trials often utilize porous collagen scaffolds to provide mechanical support and attachment sites for chondrocytes. Under loading, both the local mechanical responses of collagen scaffolds and the corresponding cellular outcomes are poorly understood, despite their wide use. As such, the architecture of collagen scaffolds varies significantly among tissue-engineered cartilage products, but the effects of such architectures on construct mechanics and cell viability are not well understood. This study investigated the effects of local mechanical responses of collagen scaffolds on chondrocyte viability in tissue-engineered cartilage constructs. We utilized fast confocal microscopy combined with a strain mapping technique to analyze the architecture-dependent instabilities under quasi-static loading and subsequent chondrocyte death in honeycomb and sponge scaffolds. More specifically, we compared the isotropic and the orthotropic planes for each type of collagen scaffold. Under compression, both planes exhibited elastic, buckled, and densified deformation modes. In both loading directions, cell death was minimal in regions that experienced elastic deformation mode and a trend of increase in buckled mode. More interestingly, we saw a significant increase in cell death in densified mode. Overall, this study suggests that local instabilities are directly correlated to chondrocyte death in tissue-engineered cartilage constructs, highlighting the importance of understanding the architecture-dependent local mechanical responses under loading.

Effects of low-dose x-ray radiation at varying dose rates on human aortic smooth muscle in vitro.

Radiation has a variety of uses in the medical field such as diagnostic imaging and cancer treatment. However, we still know very little about radiation's effect on cells in the body particularly at low doses such as those used in x-ray and CT imaging. Our group has designed studies to determine the effects of low doses of x-rays on vascular cells. We helped to design a novel x-ray fluorescent experimental setup to irradiate human aortic smooth muscle cells with dosing rates varying from at the lower end of the medical imaging range. This novel setup allows us to expose the cells inside a standard incubator with x-rays that have very precisely controlled energy, total dose, and dose rate. Three specific protocols were conducted in 24 hour intervals over several days, ultimately showing clear and statistically significant differences between dose rates even when the total dose was kept constant. As dose rate increased, the total number of cells and the number of cells in a constricted state increased, while no significant increase in cell death was observed. We have shown, for the first time, that x-rays, at doses similar to what is given to patients during standard x-ray imaging, can cause changes in vascular cells in culture. Furthermore, RT-PCR was conducted at 12 hour intervals, showing along with the assays that radiation quantitatively changes the amount of collagen in vascular cells, thus supporting our previous assays. Future experiments will include determining a specific relationship between and mechanisms behind varying dose rate and the number of cells, as well as determining whether changing other characteristics of radiation has an impact on the cells being irradiated.

Protrusion of KCNJ13 Gene Knockout Retinal Pigment Epithelium Due to Oxidative Stress-Induced Cell Death.

This study was performed to elucidate the mechanisms of morphological abnormalities in a Leber congenital amaurosis 16 (LCA16) cell model using KCNJ13 knockout (KO) retinal pigment epithelial cells derived from human iPS cells (hiPSC-RPE). In KCNJ13 KO and wild-type hiPSC-RPE cells, ZO-1 immunofluorescence was performed, and confocal images were captured. The area and perimeter of each cell were measured. To detect cell death, ethidium homodimer III (EthD-III) staining and LDH assay were used. Scanning electron microscopy (SEM) was used to observe the cell surface. The expression levels of oxidative stress-related genes were examined by quantitative PCR. To explore the effects of oxidative stress, tert-butyl hydroperoxide (t-BHP) was administered to the hiPSC-RPE cells. Cell viability was tested by MTS assay, whereas oxidative damage was monitored by oxidized (GSSG) and reduced glutathione levels. The area and perimeter of KCNJ13-KO hiPSC-RPE cells were enlarged. EthD-III-positive cells were increased with more dead cells in the protruded region. The KO RPE had significantly higher LDH levels in the medium. SEM observations revealed aggregated cells having broken cell surfaces on the KO RPE sheet. The KCNJ13-deficient RPE showed increased expression levels of oxidative stress-related genes and total glutathione levels. Furthermore, t-BHP induced a significant increase in cell death and GSSG levels in the KO RPE. We suggest that in the absence of the Kir.7.1 potassium channel, human RPE cells are vulnerable to oxidative stress and ultimately die. The dying/dead cells form aggregates and protrude from the surviving KCNJ13-deficient RPE sheet.

Norcantharidin induces ferroptosis via the suppression of NRF2/HO-1 signaling in ovarian cancer cells.

Increasing evidence has indicated a crucial role of ferroptosis in ovarian cancer (OC). Norcantharidin (NCTD), a normethyl compound of cantharidin, is extensively used in clinical practice as an optional anticancer drug. However, whether NCTD leads to ferroptosis in OC has not been previously explored, at least to the best of our knowledge. In the present study, the effect of NCTD on SKOV3 and OVCAR-3 cells was evaluated. The experimental data of the present study revealed that NCTD significantly suppressed SKOV3 and OVCAR-3 cell viability in a concentration- and time-dependent manner. The results of Cell Counting Kit-8 assay revealed that NCTD treatment decreased SKOV3 and OVCAR-3 cell viability. In comparison, pre-incubation with ferrostatin-1 (Fer-1) significantly reversed the NCTD-induced reduction in SKOV3 and OVCAR-3 cell viability; however, no changes in cell viability were observed when the SKOV3 and OVCAR-3 cells were treated with NCTD, in combination with the apoptosis inhibitor, Z-VAD-FMK, the ferroptosis inhibitor, necrostatin-1, and the autophagy inhibitor, 3-methyladenine. Additionally, it was observed that NCTD markedly enhanced reactive oxygen species production and malondialdehyde and ferrous ion levels in the SKOV3 and OVCAR-3 cells; however, pre-incubation with Fer-1 abolished these effects. Flow cytometry also demonstrated a significant increase in cell death following treatment of the SKOV3 and OVCAR-3 cells with NCTD; however, pre-incubation with Fer-1 also reversed these effects. In vivo experiments demonstrated that NCTD significantly reduced tumor volume and weight. More importantly, it was revealed that nuclear factor erythroid 2-related factor 2 (NRF2), heme oxygenase 1 (HO-1), glutathione peroxidase 4 (GPX4) and solute carrier family 7 member 11 (xCT) expression levels were significantly decreased following NCTD treatment. Collectively, NCTD may represent a potent anticancer agent in OC cells, and NCTD-induced ferroptotic cell death may be achieved by inhibiting the NRF2/HO-1/GPX4/xCT axis.

NO-Dependent Mechanisms of p53 Expression and Cell Death in Rat's Dorsal Root Ganglia after Sciatic-Nerve Transection.

Peripheral-nerve injury is a frequent cause of disability. Presently, no clinically effective neuroprotectors have been found. We have studied the NO-dependent expression of p53 in the neurons and glial cells of the dorsal root ganglia (DRG) of a rat’s spinal cord, as well as the role of NO in the death of these cells under the conditions of axonal stress, using sciatic-nerve axotomy as a model. It was found out that axotomy led to the nuclear–cytoplasmic redistribution of p53 in neurons, 24 h after trauma. The NO donor led to a considerable increase in the level of p53 in nuclei and, to a smaller degree, in the cytoplasm of neurons and karyoplasm of glial cells 4 and 24 h after axotomy. Application of a selective inhibitor of inducible NO-synthase (iNOS) provided the opposite effect. Introduction of the NO donor resulted in a significant increase in cell death in the injured ipsilateral DRG, 24 h and 7 days after trauma. The selective inhibitor of iNOS demonstrated a neuroprotective effect. Axotomy was shown to upregulate the iNOS in nuclei and cytoplasm of DRG cells. The NO-dependent expression of p53, which is particularly achieved through iNOS activation, is believed to be a putative signaling mechanism of neural and glial-cell death after axotomy.

Abstract 2305: Centriolin depletion inhibits cellular proliferation of rhabdomyosarcoma cells

Abstract Rhabdomyosarcoma (RMS), the most common pediatric soft tissue sarcoma, accounts for approximately five percent of all childhood cancers. It develops from rhabdomyoblasts and can arise in a variety of anatomic locations. Recent findings have assisted the prediction of disease prognosis and treatment regimens based on genetic and cellular differences among the major types of RMS. One potential target is a cellular appendage called the primary cilium. Primary cilia are thought to play a critical role in the regulation of myogenesis, a process deregulated in RMS. During myogenesis, Gli protein accumulates at the primary cilia in myoblasts and induces proliferation. The centrosome protein, centriolin (CNTRL), is known to be a key structural component of primary cilia. Therefore, we hypothesized that disruption of CNTRL, using CRISPR/Cas9, may affect Gli protein function and, therefore, the proliferation of these cells. Two cell lines were used: A204, a rhabdoid cell line, and CCL-136, an embryonal RMS cell line. When CNTRL was depleted and analyzed 2 days post-transfection, a significant reduction in cell viability was observed in CCL-136 compared to control, whereas no significant increase in cell death in A204 was observed. Gli1 expression decreased in both cells lines when CNTRL was eliminated, while cell cycle progression was also reduced, as confirmed by immunofluorescence staining of Ki67. Unexpectedly, primary cilia formation, indicated by Arl13B expression, increased in A204 with CNTRL loss, with no perceptible change in CCL-136 cells. To assess differentiation potential, A204 and CCL-136 were cultured in differentiation media using 2% horse serum. Separately, cells were cultured in 0% FBS (serum) to test differentiation potential under starvation conditions. As expected, both cell lines expressed robust levels of MyoG, a muscle differentiation marker, with 2% horse serum. However, loss of CNTRL leads to a perceptible decrease in MyoG in the CCL-136, and a slight decrease in A204. Additionally, wound healing assays were conducted and showed CCL-136 had inherently greater ability to migrate 24 hours post-wound compared to A204., However, CCL-136 showed diminished migration under stressed conditions. Interestingly, cell migration was significantly diminished in A204 under starvation conditions when CNTRL was depleted (p&amp;lt;0.05). We show that CNTRL loss inhibits the cell cycle in two different forms of RMS. This arrest occurs with a corresponding reduction in Gli1 expression. This confirms previous studies that suggest Gli1 is a critical factor in maintaining the proliferative state. In addition, our results show that in cells normally producing primary cilia, like A204, CNTRL loss increases the number of ciliated cells. Taken together, our data support the possibility of inhibiting cellular proliferation of RMS cells through manipulation of CNTRL and suggest that CNTRL may be a viable future treatment target. Citation Format: Yoshinori Takeda, Ariel Cole, Caitlin Boyle, Adam Gromley. Centriolin depletion inhibits cellular proliferation of rhabdomyosarcoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2305.

Abstract LB204: Theclass Iselective, oral HDAC inhibitor OKI-179 increases tumor regressions when combined with the MEK inhibitor binimetinib inmodels of NRAS melanoma

Abstract Activating RAS pathway mutations are found in &amp;gt;30% of human tumors, and often associated with poor outcomes. While RAS-pathway targeted drugs have been approved, their activity as single-agents remains modest, supporting the need for rational targeted combinations to improve patient outcomes. Multiple studies have proposed a chemical synthetic lethality between Class I histone deacetylase inhibitors (HDACi), and RAS-pathway inhibitors in RAS-pathway mutated models. This synthetic lethality occurs due to a combined effect to inhibit double-stranded (ds) DNA repair and other survival pathways to drive apoptosis and tumor regressions. The use of HDACi in solid tumors and combinations has been challenged by poor potency, selectivity, safety and convenience, highlighting the need for a better HDACi in the clinic. OKI-179, a novel largazole derivative, is a potent, Class I-selective, oral HDACi that has completed Ph 1 clinical trials in patients with solid tumors. The clinical profile shows the potential to achieve exposure consistent with preclinical activity with strong pharmacodynamic activity at tolerated doses, supporting the development of OKI-179 in solid tumor combinations. Here we show synergistic activity of combining OKI-179 with the MEK inhibitor binimetinib (bini) or bini + RAF-inhibitor encorafenib (enco/bini) in both CDX and PDX models of NRAS-mutated melanoma and BRAF-mutated colorectal cancer (CRC). In vitro, combining OKI-179 with bini in SKMEL-2 NRASMT melanoma cells shows strong synergy, inducing a significant increase in cell death compared to single agents at clinically achievable drug concentrations. This activity was associated with a significant increase in gH2AX, a marker of ds DNA breaks, and apoptosis as measured by cleaved PARP. The activity of this combination was further investigated in vivo in established PDx models of NRAS-mutant melanoma, MEL278 and MM415. As single agents, both bini (3.5 mg/kg PO bid,21 d) and OKI-179 (80 mg/kg PO; 5/wk x 3 wk), doses that mimic the clinical exposure of these molecules, showed modest tumor growth inhibition (TGI). Combining bini and OKI-179 induced significantly greater TGI, including regression in 28-50% of the tumors, as compared to 0-6% for bini alone. A similar outcome was also observed in HT29 tumors (BRAFmt CRC) where the combination of OKI-179 and enco/bini showed regression in 50% of tumors, compared to 0% for OKI-179 or enco/bini alone. This supports the potential for additive efficacy of HDACi combined with RAS-pathway inhibitors across RAS-pathway mutations and tumor types. No additivity was observed in only one model (CRC563 - BRAFmt). These data show the potential for OKI-179 to synergize with RAS-pathway inhibitors across multiple indications. To investigate, a Phase1b/2 trial of OKI-179 with binimetinib in NRAS melanoma will initiate in early 2022. Citation Format: Richard Woessner, Patrice Lee, Eric Brown, Duncan H. Walker, James Winkler, Tony Piscopio. Theclass Iselective, oral HDAC inhibitor OKI-179 increases tumor regressions when combined with the MEK inhibitor binimetinib inmodels of NRAS melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB204.

In vitro assessment of neutrophil response to proteins and antibodies of SARS-CoV-2

Abstract The SARS-CoV-2 virus caused a worldwide epidemic of respiratory disease (COVID-19). Acute respiratory distress syndrome (ARDS) has been associated with severe COVID-19. Activation of neutrophils have been linked to ARDS in COVID-19. While emerging results indicate that SARS-CoV-2 leads to neutrophil activation, it remains unknown which components of the virus are responsible for this. We assessed in vitro how the spike (S) protein and anti-spike antibodies affect neutrophils. Human neutrophils were isolated from healthy donors. Different concentrations of recombinant S protein, a monoclonal antibody (mAb) produced against the S protein and their immunocomplex were used to stimulate neutrophils. Unstimulated and PMA-treated neutrophils were used as negative and positive controls. Reactive oxygen species (ROS) production was measured by Diogenes chemiluminescence, and cell death was measured by fluorescence. While no significant increase of cell death was observed, neutrophils responded with a dose-dependent increase in the production of ROS when stimulated with either the S protein or the mAb. Neutrophils produced significantly (p&amp;lt;0.05) more ROS when activated by 1,000 ng of the mAb. Immune complex stimulation (1,000 ng each component) of human neutrophils also lead to significantly (p&amp;lt;0.005) enhanced ROS production compared to stimulations by the protein or the mAb alone. Neutrophil cell death was also significantly enhanced when stimulated by the immune complex (500 ng and 1,000 ng each component) (p&amp;lt;0.05, p&amp;lt;0.005, respectively). Our work indicates that the S protein and its immune complex formed with mAb could stimulate neutrophils in vitro and contribute to neutrophil-mediated tissue damage in COVID-19 patients. Supported by grants from: Oswaldo Cruz Foundation, International Graduate Programme (PrInt FIOCRUZ-CAPES), University of Georgia and NIH.

Cytotoxicity and inflammation induced by Philodryas patagoniensis venom

The Green racer Philodryas patagoniensis is a snake species from South America and accidents with this genus are often neglected. Therefore, this study aimed to evaluate the toxicological, cytotoxic, and inflammatory potential of P. patagoniensis venom (PpV). The experimental model Artemia salina was used to determine toxicity through the median lethal dose (LD50). Cell viability and genotoxicity were evaluated in human mononuclear cells using the Trypan blue test and the Comet assay, respectively. To assess inflammation, mice had the ventral surface of the right hind paw injected with saline, formalin, and three different concentrations of venom (1, 1.5, and 2 μg. 50 μL−1). LD50 in A. salina was 461 μg. mL−1. PpV caused a significant increase in cell death and genotoxicity in human mononuclear cells at two concentrations (575 and 1150 μg. mL−1). PpV shown also to be a strong agent causing nociception in mice. Paw edema totaled four days at 1.5 μg. 50 μL−1. The hyperalgesia caused by the venom had a long duration in mice, lasting eight days at all concentrations evaluated. Thus, we evaluated for the first time the toxicological potential of PpV in A. salina model and in leukocytes. We concluded that systemic oxidative stress, which we infer to be in the genesis of cytotoxicity and genotoxicity observed in vitro, and the inflammatory process are part of the pathways that trigger the venom damage cascades. Relevant data for both scientific research and clinical medicine. Nonetheless, studies are needed to elucidate these mechanisms.

Effect of Different Intracanal Medicaments on the Viability and Survival of Dental Pulp Stem Cells

Background: Stem cells play an important role in the success of regenerative endodontic procedures. They are affected by the presence of medicaments that are used before the induction of bleeding or the creation of a scaffold for endodontic regeneration. This study examines the effects of different intracanal medicaments on the viability and survival of dental pulp stem cells at different doses and over different exposure times. Methods: Dental pulp stem cells were cultured from healthy third molar teeth using the long-term explant culture method and characterized using flow cytometry and exposed to different concentrations of calcium hydroxide, doxycycline, potassium iodide, triamcinolone, and glutaraldehyde, each ranging from 0 (control) to 1000 µg/mL. Exposure times were 6, 24, and 48 h. Cell viability was measured using the MTT assay, and apoptosis was measured using the Annexin V-binding assay. Results: All medicaments significantly reduced cell viability at different concentrations over different exposure times. Calcium hydroxide and triamcinolone favored cell viability at higher concentrations during all exposure times compared to other medicaments. The apoptosis assay showed a significant increase in cell death on exposure to doxycycline, potassium iodide, and glutaraldehyde. Conclusion: The intracanal medicaments examined in our study affected the viability of dental pulp stem cells in a time and dose-dependent manner. They also adversely affected the survival of dental pulp stem cells. Further studies are needed to better understand the effect of prolonged exposure to medicaments according to clinical protocols and their effect on the stemness of dental pulp stem cells.

Caryocar brasiliense Camb. fruit peel butanolic fraction induces antiproliferative effects against murine melanoma cell line

BackgroundCutaneous malignant melanoma is a skin cancer type highly resistant to standard cancer therapies. Natural compounds have been reported as important sources for the creation of new drugs for cancer treatment. Caryocar brasiliense Camb., popularly known as Pequi, is often used in Brazilian folk medicine, with anticancer effects reported. Objectives: The present study evaluated the antiproliferative activity of the crude extract butanolic fraction of the C. brasiliense Camb. peel on the B16F10 cell linage MethodC. brasiliense peel fraction was analyzed by Gas Chromatography coupled to massa spectra, and its biological effects were evaluated on the melanoma cell line B16F10. ResultsThe chromatography analysis of the butanolic fraction of the C. brasiliense peel fraction identified a majority presence of gallic acid and sarothrin compounds. These compounds might have been responsible for an antiproliferative effect on B16F10, with the inhibitory concentration (IC50) equal to 390.9 µg/mL (24 h) and 226.4 µg/mL (48 h) after treatments. Our results revealed that cell death assay via bromide and acridine orange tests indicated an increase in cell death observed after 24 h treatment with the pequi fraction at 250 μg/mL (p < 0.05) and 500 μg/mL (p < 0.01). In addition, a significant increase in cell death at 250 μg/mL (p < 0.01) and 500 μg/mL (p < 0.0001) occurred after 48 h. Furthermore, a significant reduction in migratory activity in cells treated at 250 μg/mL (p < 0.05) and 500 μg/mL (p < 0.01) occurred and was enhanced by the 48 h treatment (p < 0.001). ConclusionsThe present study is the first to demonstrate the use of "Pequi" residual by product as a potential reservoir of bioactive compounds with antiproliferative activity on B16F10 melanoma cells.

Focused Ultrasound Stimulation of Microbubbles in Combination With Radiotherapy for Acute Damage of Breast Cancer Xenograft Model.

Objective: Several studies have focused on the use of ultrasound-stimulated microbubbles (USMB) to induce vascular damage in order to enhance tumor response to radiation. Methods: In this study, power Doppler imaging was used along with immunohistochemistry to investigate the effects of combining radiation therapy (XRT) and USMB using an ultrasound-guided focused ultrasound (FUS) therapy system in a breast cancer xenograft model. Specifically, MDA-MB-231 breast cancer xenograft tumors were induced in severe combined immuno-deficient female mice. The mice were treated with FUS alone, ultrasound and microbubbles (FUS + MB) alone, 8 Gy XRT alone, or a combined treatment consisting of ultrasound, microbubbles, and XRT (FUS + MB + XRT). Power Doppler imaging was conducted before and 24 h after treatment, at which time mice were sacrificed and tumors assessed histologically. The immunohistochemical analysis included terminal deoxynucleotidyl transferase dUTP nick end labeling, hematoxylin and eosin, cluster of differentiation-31 (CD31), Ki-67, carbonic anhydrase (CA-9), and ceramide labeling. Results: Tumors receiving treatment of FUS + MB combined with XRT demonstrated significant increase in cell death (p = 0.0006) compared to control group. Furthermore, CD31 and Power Doppler analysis revealed reduced tumor vascularization with combined treatment indicating (P < .0001) and (P = .0001), respectively compared to the control group. Additionally, lesser number of proliferating cells with enhanced tumor hypoxia, and ceramide content were also reported in group receiving a treatment of FUS + MB + XRT. Conclusion: The study results demonstrate that the combination of USMB with XRT enhances treatment outcomes.