Increase In Caspase Research Articles

Panobinostat Synergizes with Chemotherapeutic Agents and Improves Efficacy of Standard-of-Care Chemotherapy Combinations in Ewing Sarcoma Cells

Background: The survival rate of patients with Ewing sarcoma (EWS) has seen very little improvement over the past several decades and remains dismal for those with recurrent or metastatic disease. HDAC2, ALK, JAK1, and CDK4 were identified as potential targets using RNA sequencing performed on EWS patient tumors with the bioinformatic analysis of gene expression. Methods/Results: The pan-HDAC inhibitor Panobinostat was cytotoxic to all the Ewing sarcoma cell lines tested. Mechanistically, Panobinostat decreases the expression of proteins involved in the cell cycle, including Cyclin D1 and phospho-Rb, and DNA damage repair, including CHK1. Further, Panobinostat induces a G1 cell cycle arrest. The combination of Panobinostat with Doxorubicin or Etoposide, both of which are used as standard of care in upfront treatment, leads to a synergistic effect in EWS cells. The combination of Panobinostat and Doxorubicin induces an accumulation of DNA damage, a decrease in the expression of DNA damage repair proteins CHK1 and CHK2, and an increase in caspase 3 cleavage. The addition of Panobinostat to standard-of-care chemotherapy combinations significantly reduces cell viability compared to that of chemotherapy alone. Conclusions: Overall, our data indicate that HDAC2 is overexpressed in many EWS tumor samples and HDAC inhibition is effective in targeting EWS cells, alone and in combination with standard-of-care chemotherapy agents. This work suggests that the addition of an HDAC inhibitor to upfront treatment may improve response.

Prooxidant and anti-inflammatory potential of Garcinia xanthochymus fruit and its phytochemical characterisation by UHPLC-Q-Orbitrap HRMS

Pro-oxidants play a crucial role in cancer by causing oxidative stress that leads to apoptosis. The present study demonstrates the prooxidant and anti-inflammatory potential of ethyl acetate and methanolic extracts of Garcinia xanthochymus fruit. Oxidation of Trolox and NADH activity indicated the pro-oxidant capacity of the extracts. Significant decrease in cell viability in B16F10 and MDA-MB-231 cancer cell lines and significant increase in caspase 3 activity after treatment with extracts indicated pro-oxidant induced apoptosis. Pre-treatment with the extracts significantly inhibited ROS, reduced NO production, inhibited LPS-induced COX-2 and suppressed IL-6 and TNF-α expression. HRMS analysis showed the presence of compounds like biflavonoids, xanthones, phloroglucinols, benzophenones, etc. The fruit is rich in total phenolic and flavonoid contents, and have DPPH radical scavenging, ferric reducing antioxidant and metal chelating potential. This study report for the first time about the anticancer and anti-inflammatory properties of G. xanthochymus whole fruit.

Turkish coffee has an antitumor effect on breast cancer cells in vitro and in vivo

BackgroundBreast cancer is the most diagnosed cancer in women. Its pathogenesis includes several pathways in cancer proliferation, apoptosis, and metastasis. Some clinical data have indicated the association between coffee consumption and decreased cancer risk. However, little data is available on the effect of coffee on breast cancer cells in vitro and in vivo.MethodsIn our study, we assessed the effect of Turkish coffee and Fridamycin-H on different pathways in breast cancer, including apoptosis, proliferation, and oxidative stress. A human breast cancer cell line (MCF-7) was treated for 48 h with either coffee extract (5% or 10 v/v) or Fridamycin-H (10 ng/ml). Ehrlich solid tumors were induced in mice for in vivo modeling of breast cancer. Mice with Ehrlich solid tumors were treated orally with coffee extract in drinking water at a final concentration (v/v) of either 3%, 5%, or 10% daily for 21 days. Protein expression levels of Caspase-8 were determined in both in vitro and in vivo models using ELISA assay. Moreover, P-glycoprotein and peroxisome proliferator-activated receptor gamma (PPAR-γ) protein expression levels were analyzed in the in vitro model. β-catenin protein expression was analyzed in tumor sections using immunohistochemical analysis. In addition, malondialdehyde (MDA) serum levels were analyzed using colorimetry.ResultsBoth coffee extract and Fridamycin-H significantly increased Caspase-8, P-glycoprotein, and PPAR-γ protein levels in MCF-7 cells. Consistently, all doses of in vivo coffee treatment induced a significant increase in Caspase-8 and necrotic zones and a significant decrease in β- catenin, MDA, tumor volume, tumor weight, and viable tumor cell density.ConclusionThese findings suggest that coffee extract and Fridamycin-H warrant further exploration as potential therapies for breast cancer.

Comprehensive analysis of butyric acid impact on immunology, histopathology, gene expression, and metabolomic responses in pacific shrimp experiencing cold stress

In this study, our objective was to investigate the impact of dietary butyric acid (BA) on the homeostasis mechanism of Pacific shrimp under cold stress. Specifically, we analyzed its effects on immunity, antioxidant capacity, gene expression, and metabolomics response. To carry out this research, Litopenaeus vannamei were fed a diet supplemented with BA for 8 weeks. Following this feeding period, a total of 180 shrimp, with an average weight of 12.76 ± 0.38 g, were exposed to cold conditions, with the temperature decreasing from 28 °C to 14 °C within an hour. The results of our study revealed survival rates ranging from 90 % to 100 %. Shrimp that were fed a diet containing 1.5 % BA exhibited a significant increase in acid phosphatase (ACP) and alkaline phosphatase (AKP) activity. Conversely, the control groups showed an increase in aspartate aminotransferase (AST) and alanine transaminase (ALT) activity. Shrimp that consumed diets containing 1.5 % BA displayed the lowest malondialdehyde (MDA) levels with the highest superoxide dismutase (SOD) content. The shrimp fed the BA diet exhibited tightly organized hepatic tubules with a star-shaped lumen filled with numerous B and R cells. Furthermore, shrimp fed the BA diet demonstrated a significant increase in caspase 3 (CASP) expression. There were no significant variations in the expression levels of prophenoloxidase (ProPO), manganese superoxide dismutase (MnSOD), and glutathione S-transferase (GST) The metabolites of Dl-carnitine, acetyl-l-carnitine, propionylcarnitine, hexanoylcarnitine, palmitoylcarnitine, decanoylcarnitine, and Dl-carnitine exhibited significantly increased expression in shrimp that were fed BA, suggesting their role in the lipolysis process. Based on the findings, adding 2 % BA to the diet of Pacific shrimp helps reduce inflammation and oxidative stress when they are under cold stress.

WITHDRAWN: Assessment of the therapeutic potential of a novel phosphoramidate acyclic nucleoside on induced hepatocellular carcinoma in rat model

AimsHepatocellular Carcinoma (HCC) is renowned as a deadly primary cancer of hepatic origin. Sorafenib is the drug-of-choice for targeted treatment of unresectable end-stage HCC. Unfortunately, great proportion of HCC patients showed intolerance or unresponsiveness to treatment. This study assesses potency of novel ProTide; SH-PAN-19 against N-Nitrosodiethylamine (DEN)-induced HCC in male Wistar rats, compared to Sorafenib. Main methodsStructural entity of the synthesized compound was substantiated via FT-IR, UV–Vis, 1H NMR and 13C NMR spectroscopic analysis. In vitro, SH-PAN-19 cytotoxicity was tested against 3 human cell lines; hepatocellular carcinoma; HepG-2, colorectal carcinoma; HCT-116 and normal fibroblasts; MRC-5. In vivo, therapeutic efficacy of SH-PAN-19 (300 mg/kg b.w./day) against HCC could be revealed and compared to that of Sorafenib (15 mg/kg b.w./day) by evaluating the morphometric, biochemical, histopathological, immunohistochemical and molecular key markers. Key findingsSH-PAN-19 was relatively safe toward MRC-5 cells (IC50 = 307.6 μg/mL), highly cytotoxic to HepG-2 cells (IC50 = 24.9 μg/mL) and prominently hepato-selective (TSI = 12.35). Oral LD50 of SH-PAN-19 was >3000 mg/kg b.w. DEN-injected rats suffered hepatomegaly, oxidative stress, elevated liver enzymes, hypoalbuminemia, bilirubinemia and skyrocketed AFP plasma titre. SH-PAN-19 alleviated the DEN-induced alterations in apoptotic, angiogenic and inflammatory markers. SH-PAN-19 produced a 2.5-folds increase in Caspase-9 and downregulated VEGFR-2, IL-6, TNF-α, TGFβ-1, MMP-9 and CcnD-1 to levels comparable to that elicited by Sorafenib. SH-PAN-19 resulted in near-complete pathological response versus partial response achieved by Sorafenib. SignificanceThis research illustrated that SH-PAN-19 is a promising chemotherapeutic agent capable of restoring cellular plasticity and could stop HCC progression.

Abstract 5930: Measuring apoptotic effects of EP4A1 and EP4A2 on Kuramochi with a high-throughput multiplex image cytometric method

Abstract Ovarian cancer accounts for approximately 6% of all cancer death in women and has one of the highest mortality rates out of all gynecologic malignancies in the United States. Ovarian cancer can exhibit innate or acquired chemoresistance behavior, thus it is critical to identify novel therapeutic drug candidates. One of important characteristics of identifying potential chemotherapy drug candidate is the ability to induce apoptosis or programmed cell death in the target cancer cells. In order to measure apoptosis, various biomarkers are commonly employed such as Caspase 3, 7, 8 or 9, as well as Annexin V, which are all part of the cascade in apoptotic intrinsic pathway. However, typical fluorescent staining for image cytometry is not multiplexed that required multiple steps in order to determine the different type of apoptotic populations. In this work, we demonstrate a multiplexing apoptosis detection method to investigate the apoptotic effects of EP4A1, EP4A2, Paclitaxel, and Carboplatin on Kuramochi ovarian cancer cells. We employed the use of a high-throughput plate-based image cytometer (Celigo, Revvity Health Sciences Inc.) to image and analyze drug-treated Kuramochi cells stained with Annexin V-APC, Caspase 3/7 (488), and propidium iodide (PI) to assess the early- and late-stage apoptosis, as well as necrosis. We expected to see an increase in dual staining with the addition of EP4A to paclitaxel and carboplatin; however, the more interesting data was observed by separating single positive cell populations (Caspase, Annexin or PI only) from dual positive cell populations (Annexin + PI, Caspase + PI, Annexin +PI). The results show that treatment with EP4A lead to a time dependent increase in Caspase 3 cleavage but only in the single positive cell population. This trend was also observed in Annexin V single positive cell populations which is an indication of early apoptosis. The additive effect observed with the addition of EP4A was not seen in the dual positive cell populations. The high throughput nature of the image cytometry also allowed us to easily and quickly determine the optimal treatment conditions for this experiment. We were able to record multiple time points and not only capture proliferation, but also simultaneously collect data on caspase cleavage, Annexin-V staining and PI staining on multiple concentrations of compounds in monotherapy, dual therapy, and triple therapy combinations. Typically, it may be difficult to test multiple conditions in duplicate and triplicate using flow cytometry, while Celigo Image Cytometry may be used to obtain more robust data, and simultaneously acquire data of proliferation and multiplex staining with Annexin-V, PI and Caspase 3/7. Citation Format: James McDonald, Leo Li-Ying Chan, Jocelyn Reader, Sergio Mojica, Mackenzie Pierce. Measuring apoptotic effects of EP4A1 and EP4A2 on Kuramochi with a high-throughput multiplex image cytometric method [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 5930.

Abstract 6444: Assessing cytotoxic T cell responses to immune checkpoint inhibitors in murine and human tumor samples using metabolic imaging

Abstract Background Immune checkpoint inhibitors (ICI) have revolutionized cancer treatment. Unfortunately, existing FDA approved biomarkers do not accurately predict which patients will respond. The Elephas Cybrid™ platform aims to improve ICI response prediction by imaging a patient’s biopsy treated with ICI ex vivo. Multiphoton microscopy (MPM) is a powerful imaging technique to interrogate the biological changes in live tumor fragments (LTFs) following ICI treatment. MPM allows rapid visualization of biological processes in 3D living tissues in a non-destructive fashion allowing for longitudinal assessment of a sample. We and others have used label-free metabolic imaging to demonstrate that changes in T cell metabolism and activation status are correlated. Using MPM imaging we successfully demonstrated T cell activation and T cell mediated cytotoxicity in response to ICI in LTFs and tumor biopsies. Methods Tumors were cut into LTFs, sorted, and cultured using the Cybrid platform. LTF structure and metabolic status were assessed using intrinsically fluorescent NAD(P)H and FAD. Anti-CD8 nanobodies and cleaved caspase 3/7 dye were used to characterize T cell cytotoxicity. Multi-channel fluorescence intensity and lifetime were obtained over 48 hours using MPM. Results Murine CT26 LTFs generated with the Cybrid platform were treated with ICI and cytotoxicity was assessed in the intact LTFs by monitoring caspase 3/7 activity using MPM over 48 hours. We found that ICI treatment resulted in a significant increase in caspase 3/7 activity compared to control treated LTFs. By blocking the interaction of T cells with tumor cells through antibody blockade, we demonstrated that the cytotoxicity observed was T cell mediated. As additional confirmation, we measured changes in cytotoxicity in the ICI resistant LLC1 tumor model. As expected, we did not observe an increase in cytotoxicity in LLC1 LTFs treated with ICI. MPM allows for the tracking of labeled T cells and characterization of their metabolism as a means of assessing changes in their activation state. We used a CD8 nanobody to label T cells in CT26 LTFs and then characterized the shift in metabolism following ICI treatment. Finally, we applied the Cybrid platform to human biopsies treated with ICI and characterized the cytotoxicity and shifts in T cell metabolism in living tissue. These data show that the Cybrid platform can measure changes in T cell metabolism and T cell cytotoxicity in response to ICI in 3D living tissue. Conclusions The methods presented herein will advance the field of cancer diagnostics by progressing from fixed tissue imaging and genetic characterization to the assessment of clinically relevant ICI-treated biopsies. Understanding the response of biopsies to immunotherapies will allow clinicians to make more informed treatment decisions and improve the lives of those with cancer. Citation Format: Jonathan Ouellette, Janey Degnan, Anna Kellner, Evan Flietner, Lindsay Doebert, Zachary Swider, Eric Wait, Victoria Pope, Laura Hrycyniak, Sean Caenepeel, Kevin Eliceiri, T S. Ramasubramanian, Michael J. Korrer. Assessing cytotoxic T cell responses to immune checkpoint inhibitors in murine and human tumor samples using metabolic imaging [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 6444.

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.

Abstract 351: Quaratusugene ozeplasmid mediated TUSC2 upregulation in EML4-ALK bearing non-small cell lung carcinoma can induce cellular apoptosis

Abstract Anaplastic Lymphoma Kinase (ALK), a potent oncogenic driver in Non-Small Cell Lung Carcinoma (NSCLC), is found to be rearranged and fused to Echinoderm microtubule-associated protein-like 4 (EML4), contributing to approximately 5% of NSCLC as a distinct clinicopathological subset. Tumors bearing the EML4-ALK fusion are sensitive to ALK Tyrosine Kinase Inhibitors (TKIs), that form the first and second line of treatment for these patients. However, ALK+ lung cancers develop resistance to ALK inhibitors, creating the need for newer treatment strategies in ALK+ NSCLC. Tumor Suppressor Candidate 2 (TUSC2) is a tumor suppressor gene that is known to have low endogenous expression in NSCLC in general but data on TUSC2 in ALK+ lung cancers are not available. Quaratusugene ozeplasmid, developed by Genprex, is an immunogene therapy that upregulates TUSC2 expression in cancer cells by delivering the functional TUSC2 gene. It consists of the TUSC2 gene expressing plasmid encapsulated in non viral lipid nanoparticles. We evaluated TUSC2 expression in 3 ALK+ cell lines, both before and after exposure to quaratusugene ozeplasmid and to a TUSC2-containing plasmid. Controls were non-ALK+ cell lines and transfection with a plasmid containing no insert. Our studies in ALK+ lung cancer reveal that overexpressing TUSC2 using quaratusugene ozeplasmid treatment of ALK+ lung cancer cell lines is able to suppress colony formation by 50%, the effect being more significant than using a TUSC2-containing plasmid. Furthermore, we have observed a robust pro-apoptotic response to TUSC2 expression in ALK+ NSCLC, as both quaratusugene ozeplasmid and TUSC2-containing plasmid induced an increase in caspase 3/7 activity in the cancer cells, accompanied by an increase in cleaved PARP expression. Taken together, our data indicate that overexpression of TUSC2 in ALK+ NSCLC cell lines with quaratusugene ozeplasmid or with TUSC2-containing plasmid is effective in decreasing growth and proliferation through the activation of apoptotic pathways and warrants further investigation as an anti-ALK NSCLC strategy. Citation Format: Ananya Banerjee, Neeke Busette, Mark S. Berger, Matthew B. Soellner, Angel Qin, Sofia D. Merajver, Nathan M. Merrill. Quaratusugene ozeplasmid mediated TUSC2 upregulation in EML4-ALK bearing non-small cell lung carcinoma can induce cellular apoptosis [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 351.

Metabolic profiling, antimicrobial, anticancer, and in vitro and in silico immunomodulatory investigation of Aspergillus niger OR730979 isolated from the Western Desert, Egypt.

Ten fungal species were isolated from soil in the Western Desert and Wadi El-Natron in Egypt. All fungal isolates were morphologically recognized down to the species level. Methanol extracts of fungal mycelia and ethyl acetate extracts of culture filtrate from the isolated fungi were evaluated for antimicrobial activity against six pathogenic bacteria and one pathogenic yeast (Candida albicans ATCC20231). Only ethyl acetate extracts of Fusarium circinatum, Aspergillus niger, and Aspergillus terreus culture filtrates showed significant antimicrobial activity against the majority of the investigated pathogens. The culture filtrate extract of Aspergillus niger exhibited notable cytotoxicity towards the breast cancer (MCF-7) cell line, with the lowest detected IC50 recorded at 8 μg/μl. Whereas Fusarium circinatum and Aspergillus terreus had IC50s of 15.91 μg/μl and 18 μg/μl, respectively. A gas chromatography-mass spectroscopy (GC-MS) investigation of A. niger's potent extract revealed 23 compounds with different biological activities. Glycidyleoleate was found to be the main extract component. Aspergillus niger extract was chosen to study its possible cytotoxic mechanism. The extract was found to induce apoptosis and cell cycle arrest at the < 2n stage. Despite a significant increase in caspases 8 and 9, the production levels of tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6) have shown a significant decrease. The high interaction of glycidyleoleate against the studied cytokines' binding receptors was demonstrated via docking studies. In conclusion, the available data revealed that the culture filtrate extract of A. niger possesses promising antimicrobial, cytotoxic, and immunomodulatory properties.

Synthesis and biological activity of 11-Oxygenated and heterocyclic estrone analogs in pancreatic cancer monolayers and 3D spheroids

Pancreatic Ductal Adenocarcinoma (PDAC), representing over 90 % of pancreatic cancer diagnoses, is an aggressive disease with survivability among the worst of all cancers due to its difficulty in detection and its high metastatic properties. Current therapies for PDAC show limited success at extending life expectancies, primarily due to cancer resistance and lack of patient-specific targeted therapies. This work highlights the design and evaluation of estrone-derived analogs with both heterocyclic side-chain functionality and 11-oxygenated functionality for use in pancreatic cancer. First-round heterocyclic analogs show preliminary promise in AsPC-1 and Panc-1 cell lines, with IC50 values as low as 10.16 ± 0.83 µM. Their success, coupled with design choices from other studies, led to the synthesis of novel 11-hydroxyl and 11-keto estrone analogs that show potent in-vitro toxicity against various pancreatic cancer models. The three most cytotoxic analogs, KA1, KA2, and KA9 demonstrated low micromolar activities in both MTT and CellTiter assays in three pancreatic cancer cell lines: AsPC-1, Panc-1, and BxPC-3, as well as in a co-culture of Panc-1 and pancreatic stellate cells. IC50 values for KA9 (4.17 ± 0.90, 5.28 ± 1.87, and 5.70 ± 0.65 µM respectively) shows consistency in all cell lines tested. KA9 is also able to cause an increase in caspases 3 and 7 activity, key markers for apoptosis, at non-cytotoxic concentrations. Additional work was performed by generating 3D pancreatic cancer spheroids to better modulate the pancreatic tumor microenvironment, and KA9 continued to show the best IC50 values (21.0 and 24.3 µM) in both cell types tested. KA9 was also able to prevent the growth of spheroids whereas the standard chemotherapy, Gemcitabine, could not, suggesting that it may be a potent analog for future development of treatments. Molecular dynamic simulations were also performed to confirm biological findings and uncovered that KA9′s preferential binding location is in the active site pocket of key proteins involved in cytotoxicity.

New modified thieno[2,3-d]pyrimidine derivatives as VEGFR-2 inhibitors: Design, synthesis, in vitro anti-cancer evaluation and divers in silico studies

Angiogenesis is a critical regulatory mechanism for tumor development and metastasis. Tumor growth is closely related to the production of the vascular endothelial growth factor (VEGF), which is mediated by the cell-surface kinase VEGFR-2. Accordingly, blocking of VEGFR-2 is an important approach to find new treatments for malignancies. The aim of this research is the synthesis of new thieno[2,3-d]pyrimidine derivatives that could be potential anticancer leads inhibiting VEGFR-2. The prepared thieno[2,3-d]pyrimidine derivatives were tested in vitro for their inhibitory effects against the kinase activity of VEGFR-2. In addition, the synthesized compounds were assessed for their anti-proliferative activities against MCF-7 and HepG2 cell lines. Compound 4c displayed the strongest anti-VEGFR-2 potentiality with an IC50 value of 0.15 µM and exhibited good anti-proliferative effects against HepG2 and MCF-7 cells with IC50 of 17.14 and 11.56 µM, respectively. Compound 4c induced cell cycle arrest at the S phase and boosted early and late apoptosis in MCF-7 cells. Additionally, compound 4c increased BAX (4.5-fold), decreased Bcl-2 (3.5-fold), and demonstrated a notable increase in caspase-8 (2.9-fold) and caspase-9 (3.3-fold) levels. Furthermore, compound 4c significantly reduced TNF-α (3.6-fold) and IL-6 (3.2-fold) levels in MCF-7 cells. Molecular docking studies indicated good binding affinities as well as energies of the thieno[2,3-d]pyrimidine derivatives against the VEGFR-2. Molecular dynamics (MD) simulations were utilized to study the structural, energetic, and conformational changes of the VEGFR-2–4c complex, and the results indicated its stability. The MM-GBSA study of the VEGFR-2–4c complex showed a stable thermodynamic behavior with a binding free energy of -44 kcal/mol. The PLIP analysis identified the 3D interactions and binding conformation through the VEGFR-2–4c complex. Moreover, the DFT studies have been performed to study, Mullikan atomic charge distribution, FMO, ESP, the total density of state, and the QTAIM maps of compound 4c to theoretically verify its reactivity. Finally, computational ADMET and toxicity studies were conducted to assess the drug development potential of the thieno[2,3-d]pyrimidine derivatives.

Targeting apoptosis; design, synthesis and biological evaluation of new benzoxazole and thiazole based derivatives

Several novel approaches to target Bcl-2 proteins and apoptotic pathways have been identified in recent years for the treatment of different types of cancer including colorectal cancer. However, no effective treatments were yet developed for colorectal cancer. Twenty two novel benzoxazole and thiazole−based compounds were designed, synthesized, and evaluated as potential Bcl-2 inhibitors with anti−proliferative activity. Compounds 8g, 12e and 13d showed good to moderate anti−proliferative activity against most of the NCI 60 cell line panel with mean growth inhibition percent of 45.13, 42.29 and 29.25%, respectively. They showed the greatest cell growth inhibition percent to HCT-116 cell line with the values of 68.0, 59.11 and 43.44%, respectively. The aforementioned compounds were furtherly investigated for their effect on HCT-116 cell cycle, and they showed increase in the total apoptosis with 17, 22, and 5%, respectively. Also, the apoptotic effect of compounds 8g, 12e and 13d, were tested by their effect on altering caspase-3 expression level in HCT-116 human cell line. The three compounds showed an increase in the caspase-3 levels by 6, 8 and 3 folds, respectively in comparison with the same untreated ones. Moreover, they were evaluated for their in–vitro Bcl-2 inhibitory activity and they showed percent inhibition of 60.2, 69.2 and 50.0%, respectively. Finally, the most potent compounds 8g and 12e showed 3.864 and 2.834 folds increase in Bax level compared to the control respectively. On the other hand, Bcl-2 was down−regulated to 0.31 and 0.415 folds compared to the control. The induction of apoptosis through increase in caspase 3 expression and down−regulation of Bcl-2 is the suggested mechanism of action.

Exposure to subthreshold dose of UVR‐B does not induce increase in caspase 3 mRNA expression in the rat lens in vivo within first 24 hours

Aims/Purpose: The aim of this study is to investigate the time evolution of caspase 3 mRNA expression within first 24 h in the rat lens after in vivo exposure to subthreshold dose of UVR‐B.Methods: Twelve six‐week‐old female albino Sprague–Dawley rats were exposed to subthreshold dose (1 kJ/m2) of UVR‐B unilaterally and sacrificed at 1, 8, 16 and 24 h after exposure. Lenses were enucleated and caspase 3 mRNA expression was detected by qRT‐PCR. The time evolution of caspase 3 mRNA expression was then plotted as a function of mean ratios in caspase 3 mRNA expression between exposed and contralateral nonexposed lenses.Results: There is no difference in mean ratios in caspase 3 mRNA expression between exposed and nonexposed lenses in all four postexposure groups.Conclusions: Exposure to subthreshold dose of UVR‐B does not induce increase in caspase 3 mRNA expression in the rat lens in vivo within first 24 h.

The Extract of Angelica sinensis Inhibits Hypoxia-Reoxygenation and Copper-Induced Oxidative Lesions and Apoptosis in Branchiae and Red Blood Corpuscles of Fish.

The study explored the effects of Angelica sinensis extract (AsE) on oxidative lesions and apoptosis in branchiae and red blood corpuscles in hypoxia-reoxygenation (HR) and Cu-treated carp (Cyprinus carpio var. Jian). After feeding trial for 30 days, the carp were exposed to HR and CuSO4. The results indicated that dietary AsE increased the durative time, decreased the oxygen consumption rate, suppressed ROS generation and cellular component oxidation, decreased enzymatic antioxidant activity and reduced glutathione (GSH) levels in red blood corpuscles and branchiae in carp under hypoxia. Moreover, dietary AsE avoided the loss of Na+,K+-ATPase, metabolic and antioxidant enzyme activities, ROS generation and cellular component oxidation, as well as the increase in caspase-8, 9, and 3 activities in the branchiae of the carp and inhibited ROS generation. It furthermore avoided the loss of Na+,K+-ATPase and metabolic enzyme activities, the decrease in GSH levels and hemoglobin content, the increase in the activities of caspase-8, 9, and 3 and the increase in the levels of cytochrome c and phosphatidylserine exposure in the red blood corpuscles of Cu-exposed carp. The present results suggested that dietary AsE improved hypoxia tolerance and inhibited HR or Cu-triggered oxidative lesions and apoptosis. Therefore, AsE can be utilized as a natural inhibitor of Cu and HR stress in fish.

Elovl2 and Elovl4 reduction in Adipor1-/- and Mfrprd6 Leads to Neuroinflammation and Neurodegeneration

The omega-3 fatty acid docosahexaenoic acid (DHA) is enriched in the central nervous system, including the retina, where it preserves vision by maintaining photoreceptor disc integrity. DHA plays a central role in neural cell homeostasis, having antioxidative, anti- inflammatory, and antiapoptotic properties through derived lipid mediators. We have discovered that mutant Adipor1 and Mfrp mice have a selective reduction of DHA and very long- chain polyunsaturated fatty acids (VLC-PUFAs; &gt;28 carbons) formed notably by elongases ELOVL2 and 4 (ELOngation of Very Long chain fatty acids). These mice have impaired retinal function and progressive loss of photoreceptor cells. We have now determined the underlying mechanism leading to inflammaging in these models for retinal degeneration. We harvested retinas and RPE-eyecups from Adipor1-/-, Mfrprd6 and C57BL/6J (wild- type; WT) male and female mice and performed capillary Jess (Protein Simple) Western blot for Elovl2 and Elovl4. FAM-FLICA Caspase-1 Assay was performed on eye sections to determine caspase-1 activation. Immunohistochemistry was performed with Iba-1 antibody to determine microglia invasion in mutant mice. Protein expressions of Elovl2 and 4 were reduced in the mutants. FAM-FLICA showed Adipor1-/- and Mfrprd6 had a 47% and 189% increase in caspase 1 activity. Immunostaining demonstrated greater Iba-1 positive cells in both mutant retinas. Adipor1-/- and Mfrprd6 are susceptible to uncontrolled neuroinflammation, leading to microglia invasion and caspase 1-dependent retina damage, suggesting pyroptotic immune response and targeting of the inflammasome pathway.

Abstract A022: CDK2 inhibition demonstrates synthetic lethality in SCLC through apoptotic induction

Abstract Small cell lung cancer (SCLC), a malignant thoracic neoplasm genetically defined by dysregulation of the Retinoblastoma (RB1) gene and tumor suppressor protein 53 (TP53), remains one of the world’s deadliest tumors with a 5-year overall survival rate ranging from 5% to 10%. Despite numerous clinical trials and recent FDA approval of immune checkpoint inhibitors, the prognosis for SCLC has not significantly improved in decades. There remains a strong need for new therapeutic approaches. In this study, we found that the combined effect of dysregulation or deletion of RB1 and TP53, as is found in the majority of SCLC, results in sensitivity to CDK2 inhibition or ablation. This relationship is reflected in the DepMap database, with cell lines harboring deleterious mutations in RB1 demonstrating markedly higher susceptibility to CDK2 inhibition. This relationship is surprising given CDK2’s established role in suppressing the growth inhibition activity of RB and CDK2’s ability to thus facilitate G1/S transition. To validate this discovery, we evaluated the effects of CDK2 inhibition on a panel of SCLC cell lines. ARTS-021 is a potent and highly selective orally administered inhibitor of CDK2 currently in clinical development. In contrast to CCNE1 amplified cells, which undergo G1 arrest upon ARTS-021 treatment, SCLC cells treated with ARTS-021 exhibited minimal G1 accumulation. Instead, ARTS-021 treatment induced apoptosis in SCLC cell lines with loss of functional RB1 and TP53, as demonstrated by a dose-dependent increase in Caspase 3/7 activity and the accumulation of cleaved PARP. This phenotype was further demonstrated to be CDK2 dependent though generation of stable cell lines expressing a dox-inducible shRNA targeting CDK2. Combination studies further demonstrated that this apoptotic phenotype can be enhanced synergistically through inhibition of the anti-apoptotic proteins BCL-2 and BCL-xL with clinical stage inhibitor Navitoclax. In addition, combinations with currently approved standard of care chemotherapies were tested both in vitro and in vivo, and demonstrated potent combinatorial activity. These findings suggest that the clinical development of CDK2 inhibitors for SCLC may represent a novel therapeutic option for this difficult to treat malignancy. Citation Format: Nathan Schomer, Jinhong Chen, Wei Pang, Tingru Zhou, Xipan Liu, Wenhen Liang, Yali Guo, Dai Cheng, Fang Li, Jiaqi Liang, Yaoyu Chen, Qing Sheng. CDK2 inhibition demonstrates synthetic lethality in SCLC through apoptotic induction [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A022.

Folic acid antioxidant supplementation to binge drinking adolescent rats improves hydric-saline balance and blood pressure, but fails to increase renal NO availability and glomerular filtration rate.

Binge drinking (BD) is an especially pro-oxidant pattern of alcohol consumption, particularly widespread in the adolescent population. In the kidneys, it affects the glomerular filtration rate (GFR), leading to high blood pressure. BD exposure also disrupts folic acid (FA) homeostasis and its antioxidant properties. The aim of this study is to test a FA supplementation as an effective therapy against the oxidative, nitrosative, and apoptotic damage as well as the renal function alteration occurred after BD in adolescence. Four groups of adolescent rats were used: control, BD (exposed to intraperitoneal alcohol), control FA-supplemented group and BD FA-supplemented group. Dietary FA content in control groups was 2 ppm, and 8 ppm in supplemented groups. BD provoked an oxidative imbalance in the kidneys by dysregulating antioxidant enzymes and increasing the enzyme NADPH oxidase 4 (NOX4), which led to an increase in caspase-9. BD also altered the renal nitrosative status affecting the expression of the three nitric oxide (NO) synthase (NOS) isoforms, leading to a decrease in NO levels. Functionally, BD produced a hydric-electrolytic imbalance, a low GFR and an increase in blood pressure. FA supplementation to BD adolescent rats improved the oxidative, nitrosative, and apoptotic balance, recovering the hydric-electrolytic equilibrium and blood pressure. However, neither NO levels nor GFR were recovered, showing in this study for the first time that NO availability in the kidneys plays a crucial role in GFR regulation that the antioxidant effects of FA cannot repair.

CXCR4 S338X Promotes Resistance to Second Generation BTK Inhibitor and BCL-2 Inhibitor in WM Cells

Introduction: Waldenström macroglobulinemia (WM) is an IgM-secreting lymphoplasmacytic lymphoma. CXCR4 somatic mutations are common in WM and are associated with clinical resistance to Bruton's tyrosine kinase (BTK) inhibitors and other therapeutic agents. CXCR4 mutations occur nearly exclusively with MYD88 L265Pmutation, with CXCR4 S338X constituting the most common in WM patients. The aim of this study was to further investigate the effect of CXCR4 S338X on BTK inhibitors (ibrutinib and zanubrutinib) and a BCL-2 inhibitor (venetoclax) resistance. Methods: MWCL-1 cells were transfected with scramble, CXCR4 or CXCR4 S338Xplasmids using Lipofectamine 3000 followed by neomycin selection. CXCR4 expression was confirmed using qPCR and flow cytometry. Transfected cells were cultivated in presence of ibrutinib (5μΜ), zanubrutinib (5μΜ) and venetoclax (1μΜ) for 48 hours. Proliferation was estimated using CCK-8 colorimetric assay while cell cycle was evaluated by propidium iodide staining and flow cytometry. Apoptosis was estimated by Annexin V/7-AAD staining and flow cytometry while the expression of apoptotic genes was determined by qPCR. The statistical analysis was performed using Student's t-test and Graphpad Prism. Results: Drug-induced cytotoxicity was observed only in the case of venetoclax in CXCR4 S338X transfectedcells (p= 0.0007) while increased cytotoxicity was noted in the presence of ibrutinib, zanubrutinib and venetoclax in the case of scramble-transfected cells (p=0.038, p=0.006 and p=0.0006, respectively). Moreover, no significant difference in proliferation rate was noted between CXCR4 S338Xtransfectedcells treated with the therapeutic agents and CXCR4 S338X untreated cells, underlining the S338X role to promote cell proliferation. Interestingly, CXCR4 S338X transfectedcells exhibited apoptosis resistance not only compared to scramble-transfected cells but also in comparison with CXCR4-transfected cells, associated with resistance to all three drug agents (p=0.004, p=0.003 and p=0.034, respectively). In the case of scramble-transfected cells, an increase in apoptosis was demonstrated in parallel with an increase in caspases 8, 9 and 3 in presence of ibrutinib (p=0.006, p=0.0001 and p=0.025, respectively) and zanubrutinib (p=0.027, p=0.007 and p=0.052, respectively), whereas no statistically significant increase in caspase gene expression was observed in the case of CXCR4 S338Xtransfectedcells, highlighting S338X role in the inhibition of intrinsic and extrinsic apoptosis pathways. Lastly, the presence of S338X mutation acted as a mitotic promoting factor when cells treated with the aforementioned drugs showed a high proportion of cells in G2/M phases of cell cycle compared to scramble and CXCR4 controls. Conclusions: The above data demonstrate the role of CXCR4 S338X mutation in drug resistance and suggest the necessity to develop therapeutic strategies to address this issue and overcome this therapeutic barrier in WM. Data on more cell lines and downstream signaling will be presented at the meeting.

Pyrrolidinedione-thiazolidinone hybrid molecules with potent cytotoxic effect in squamous cell carcinoma SCC-15 cells

The hybrid heterocyclic molecules are perspective materials in the development of anticancer drugs. Here, the pyrrolidinedione-thiazolidinone hybrid molecules were designed as potent anticancer agents. This study aimed to investigate the cytotoxic effect of three derivatives 1-(4-hydroxyphenyl)-, 1-(4-chlorophenyl)- and 1-(4-bromophenyl)-3-[5-[2-chloro-3-(4-nitrophenyl)prop-2-enylidene]-4-oxo-2-thioxothiazolidine-3-yl]pyrrolidine-2,5-diones (Les-6287, Les-6294, and Les-6328, respectively), their effect on the production of the reactive oxygen species (ROS), apoptosis induction, and expression of genes - PPARγ, AHR, and NRFL2 – whose products are important in metabolism in human tongue squamous cell carcinoma cells of SCC-15 line. The results of resazurin reduction and lactate dehydrogenase (LDH) release assays proved the toxicity of the tested derivatives for the SCC-15 cells. Les-6287, Les-6294, and Les-6328 inhibited the viability of SCC-15 cells with the half-maximal effective concentration (EC50) in the range of 10.18–32.75 µM at 24 and 48 h treatment. These derivatives reduced the metabolism of SCC-15 cells with the half-maximal inhibitory concentration (IC50) of 6.72–39.85 µM at 24 and 48 h treatment. Les-6287, Les-6294, and Les-6328 reduced the metabolism of normal human keratinocytes of HaCaT line murine fibroblasts of Balb/c 3T3 line to a lesser extent. The compounds used in a range from 50 to 100 µM concentrations decreased ROS production in the SCC-15 cells. The derivatives Les-6287 and Les-6328 decreased the level of expression of mRNA of PPARγ, AHR, and NRFL2 genes in these cells at PPARγ siRNA knockdown and without it. Thus, the anticancer effect of studied hybrid pyrrolidinedione-thiazolidinones in the SCC-15 carcinoma cells is accompanied by a reduction of their metabolic activity and ROS level, and increase in caspase 3 activity. However, these changes are not the result of direct interaction of Les-6287, Les-6294, and Les-6328 with the PPARγ molecule.