Apoptosis-inducing Effects Research Articles

Resistance to the Non-Covalent BTK Inhibitor Pirtobrutinib

Pirtobrutinib is a non-covalent BTK inhibitor (BTKi) designed to maintain activity despite the most common resistance mutation to covalent inhibitors, at BTK C481. To investigate mechanisms of disease progression on pirtobrutinib, we evaluated the effect of pirtobrutinib in vitro on the BCR pathway in pre- and post-treatment patient samples (n=5), and on 2 of these patients, we performed whole exome sequencing longitudinally including prior to and at relapse on pirtobrutinib. Phylogenetics and subclonal dynamics associated with resistance were evaluated using the PhylogicNDT and Concerti tools. To investigate the impact of identified BTK mutations on BCR activation, we generated 6 single and 3 double mutants using site directed mutagenesis and expressed them in the BTK null DT40 B cell line. We demonstrate that primary CLL cells from responding patients on the pirtobrutinib trial show reduced BCR signaling, reduced CCL3/CCL4 chemokine secretion, as well as effective induction of apoptosis and inhibition of proliferation, in response to pirtobrutinib. At time of progression, these primary CLL cells show increasing resistance to pirtobrutinib in signaling inhibition and cytokine production, with reduced inhibition of proliferation and induction of apoptosis. In WES analysis, patient #1 had 16 samples evaluated prior to, during, and at relapse on acalabrutinib, vecabrutinib and pirtobrutinib. Clonal analysis of samples collected during acalabrutinib shows steady selection of a clone harboring BTK p.C481S mutation with CCF 92% at relapse but then steadily decreases during pirtobrutinib treatment. Concerti's time-scaled phylogenetic tree shows the birth of a new clone containing the BTK gatekeeper mutation, p.T474I, during acalabrutinib treatment, which then grows rapidly under pirtobrutinib treatment, taking over nearly the entire cancer cell population and replacing the prior p.C481S clone. This complete clonal shift during pirtobrutinib treatment suggests that pirtobrutinib effectively inhibits the p.C481S clone, while the p.T474I gatekeeper clone is likely driving resistance in this patient. We also observed an additional gatekeeper clone BTK p.T474L develop at low levels, as well as another previously undescribed BTK mutation at p.M477I. Manual inspection showed that BTK mutations p.M477I and p.T474I are in cis and therefore in the same clone. Patient #2 had 10 samples evaluated prior to, during and at relapse on ibrutinib and pirtobrutinib. During ibrutinib therapy, we observed a steady increase in a clone with TP53 p.S240G and SF3B1 p.K666N mutations, reaching CCFs >40% at relapse on ibrutinib. We also noted a significant increase in BTK p.C481R (CCF 33%), BTK p.C481S (CCF 11%) and TP53 p.R196* (CCF 5%) at progression on ibrutinib. Under pirtobrutinib treatment the clone carrying BTK p.C481R decreased to CCF 20%, while BTK p.C481S (28%) and TP53 p.R197* (35%) both increased. Concerti's phylogenetic tree captures the birth of a resistant clone, harboring BTK p.L528W which significantly increases to CCF 30% at progression on pirtobrutinib. Functional characterization of the identified BTK mutations demonstrated that only T474I, T474L and C481S mutants showed adequate kinase activity, while all the other mutants including M477I, C48IR and L528W essentially lacked kinase activity as judged by phosphorylation at BTK Y223 and at PLCG2 Y753. As expected, the C481S variant was resistant to ibrutinib, but not to pirtobrutinib, while the T474I/L variants were sensitive to ibrutinib but resistant to pirtobrutinib. Interestingly, phosphorylation of AKT and ERK were retained downstream, even with mutations that failed to activate proximal BCR signaling. Furthermore, phosphorylation of AKT and ERK was also observed in the B7.10 cell line which lacks endogenous BTK, demonstrating significant activation of these pathways independent of BTK. In this study, we demonstrate that ex vivo efficacy of pirtobrutinib declines as patients’ CLL starts to progress, in concert with the development of gatekeeper and alternative site BTK mutations that lead to resistance to pirtobrutinib. Interestingly, many of the second-site BTK mutations fail to activate BTK phosphorylation but are still associated with downstream activation of phospho-AKT and phospho-ERK; the mechanism of this activation remains to be elucidated.

Escape from Type II JAK2 Inhibition in MPN Depends on AXL/MAPK Activation and Is Targetable

Myeloproliferative neoplasms (MPN) are characterized by dysregulated JAK2 signaling. Clinical type I JAK2 inhibitors as ruxolitinib are hampered by limited disease-modifying effects and occurrence of resistance. Based on pre-clinical data, novel type II JAK2 inhibitors as CHZ868 effectively stabilize inactive JAK2, reduce MPN clones and overcome resistance to ruxolitinib. We study whether MPN cells could escape from type ll JAK2 inhibition, and characterize therapeutic approaches. To assess potential escape from type II JAK inhibition, JAK2V617F MPN cells incl. SET2, UKE-1 and Ba/F3 EPOR Jak2V617F were exposed long-term to CHZ868. Outgrowing cells showed significantly increased IC50 to CHZ868 and reduced susceptibility for apoptosis induction. No second-site mutations in JAK2 were detected. Phosphoproteomic analysis identified >2500 differentially phosphorylated sites with most prominent activation of the MAPK pathway in JAK inhibitor resistant cells. Type II JAK inhibition persistently suppressed JAK2 - STAT3/5 signaling, while MAPK target activation incl. MEK and ERK was prominent also by phospho-immunoblot suggesting MAPK pathway signaling as a driver of escape from type II JAK inhibition. To characterize the molecular basis, we profiled differential gene expression and chromatin accessibility by RNA-sequencing and ATAC-sequencing in MPN cells escaping JAK inhibition. Paired analysis of RNA-seq/ATAC-seq revealed enrichment of H3K27me3 and PRC2 signatures with overall more compacted chromatin. Expression of AXL tyrosine kinase known to signal through the MAPK pathway, was significantly increased along with enriched RAS signaling target gene patterns by GSEA. Motif analysis suggested differential transcriptional programs with loss of ETS and increased accessibility of GATA transcription factor family binding sites. Enhanced AXL expression was confirmed by immunoblotting and depletion of AXL by AXL-specific shRNA knock-down resensitized resistant cells to CHZ868 with decreased IC50. Since these findings pointed towards a role of AXL activation for escape of MPN cells from type II JAK inhibition, we targeted AXL activation using respective inhibitors. AXL inhibitor bemcentinib and AXL/FLT3 inhibitor gilteritinib reduced proliferation of resistant cells with up to 5.6-fold and 3.4-fold decreased IC50, respectively. Given that type II JAK inhibition with CHZ868 maintained suppression of JAK2-STAT3/5 activation in resistant cells, we combined JAK2 and AXL inhibition. Notably, dual JAK2 / AXL inhibition with CHZ868 / bemcentinib or CHZ868 / gilteritinib showed most pronounced inhibitory effects with IC50 similar to values seen in JAK inhibitor sensitive cells and effective induction of apoptosis. Resistant MPN cells engrafted subcutaneously into immunocompromised NSG mice and resulted in robust tumor growth despite JAK2 inhibitor treatment. However, AXL inhibition with bemcentinib reduced progression of type II JAK inhibitor resistant tumors. Combined JAK2 and AXL inhibition showed most pronounced suppression of subcutaneous tumor growth with minimal progression. Given that inhibitors of MAPK signaling targeting e.g. MEK-ERK activation, are in clinical use for other malignancies with favorable tolerability profiles, we then characterized combined JAK2 / MAPK inhibition to overcome AXL-MAPK driven escape of MPN cells from type II JAK inhibitors. Combination of CHZ868 and MEK inhibitor trametinib significantly reduced subcutaneous tumor growth in NSG mice similarly to JAK2 / AXL inhibition. Expression of MAPK pathway effectors as DUSP6 was substantially reduced. Furthermore, intravenous engraftment of JAK inhibitor resistant cells into NSG mice led to substantial bone marrow (BM) infiltration shown by luciferase imaging and hCD45 immunohistochemistry despite treatment with CHZ868. Combined JAK2 / MAPK inhibition with CHZ868 and trametinib significantly reduced BM infiltration by luciferase imaging and histology supporting that escape from type II JAK inhibition is dependent on AXL-MAPK activation. In summary, we report on a novel mechanism of AXL-MAPK driven escape from type II JAK2 inhibition which is targetable in vitro and in vivo. Our findings highlight that rationally designed combination therapy approaches can restore sensitivity to JAK inhibitor therapy in MPN, which may have potential implications for other malignant settings. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Anti-proliferative and Apoptosis Inducing Effects of Gallic Acid on Human Acute Myeloid Leukaemia Cell Lines (HL-60)

Gallic acid (GA) comes from benzoic acid or, more specifically, 3,4,5-tryhydroxybenzoic, which derives from the phenolic acid of the non-flavonoid part of the polyphenol compound. It is found ubiquitously in various plants and fruits, such as grapes, gallnuts, pomegranates, and tea leaves [1]. Many scientific journals and articles reported on the pharmacological properties of the photochemical like GA, which has antioxidant properties, antimicrobial, anti-inflammatory, anticancer, cardioprotective, gastroprotective, and neuroprotective activity [2]. Moreover, the anticancer properties of GA have been recognised in several cancers, such as lung cancers, oesophageal cancer cells and leukaemia [3].
 
 The objective of this study is to examine the anti-proliferative and apoptosis inducing effects of GA on HL-60 cell lines. Six concentrations of GA were made using the stock solution of GA compound dissolved in dimethyl sulfide (DMSO). HL-60 cells were treated with concentrations of 100, 50, 25, 12.5, 6.25, and 3.125 µg/mL and was incubated at three incubation period which were 24, 48, and 72 hours. The quantitative measure was determined with cytotoxicity assay of 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and was read by microplate reader at 570 nm. For qualitative measure was through staining with acridine orang (AO) and propidium iodide (PI) and observed the morphological changes of the treated cells.
 
 Results from the MTT assays show that GA has cytotoxicity effect on HL-60 cells especially for 72 hours incubation period. The maximal half inhibitory concentrations (IC50) value of GA decreases as incubation period increases. The IC50 of GA were 9.03, 6.76, and 3.65 µg/ml for 24-, 48-, and 72-hours incubation, respectively. The IC50 value of GA (p<0.05) was significantly different for different incubation periods. The morphological changes were seen through the AO/PI staining with the appearance of the cell blebbing, early apoptosis, and late apoptosis.
 
 Figure 1 shows early apoptosis (EA), late apoptosis (LA), cell blebbing (CB), membrane loose (MB), nuclear fragmentation (NF), and apoptotic bodies (AB).
 
 These findings show that GA has potential as anti-proliferative and apoptosis inducing effects on HL-60 cells line. More research is needed to determine the pathways of apoptosis in HL-60 treated with GA.

Prodigiosin inhibits the proliferation of glioblastoma by regulating the KIAA1524/PP2A signaling pathway

Prodigiosin (PG), a member of a family of natural red pigments produced by a variety of bacteria, was first discovered in Serratia marcescens. PG has been reported to have an apoptosis-inducing effect in many cancers, such as lymphoma, colon cancer and nasopharyngeal carcinoma. For this study, we used three glioblastoma (GBM) cell lines (LN229, U251 and A172) to explore the effect of prodigiosin on GBM cells. A CCK8 assay was used to evaluate cell viability. We determinedthe cell cycle distribution by flow cytometry and measured proliferation by an EdU incorporation assay. The expression of different molecules was investigated by western blotting and RT-PCR. We further confirmed our results by plasmid transfection and lentiviral transduction. The LN229 xenograft model was used to study the effect of prodigiosin in vivo. We confirmed that prodigiosin played an anticancer role in several GBM cell lines through the KIAA1524/PP2A/Akt signalling pathway. Prodigiosin inhibited the protein expression of KIAA1524 by suppressing its transcription, which led to activation of PP2A. Afterward, PP2A inhibited the phosphorylation of Akt, thereby inducing increased expression of p53/p21. Furthermore, it was verified that prodigiosin inhibited the KIAA1524/PP2A/Akt axis in vivo in the LN229 xenograft model. These data improve the understanding of the anticancer effects of prodigiosin and further highlight the potential of prodigiosin for the development of anti-glioma drugs.

Brucein D imparts a growth inhibitory effect in multiple myeloma cells by abrogating the Akt-driven signaling pathway.

The Akt signaling pathway is an oncogenic cascade activated in the bone marrow microenvironment of multiple myeloma (MM) cells and contributes to their uncontrolled proliferation. Abrogation of Akt signaling has been presentedas one of the prime therapeutic targets in the treatment of MM. In the present report, we have investigated the effect of Brucein D (BD) on Akt-driven signaling events in MM cells. BD (300 nM) substantially inhibited cell viability and imparted growth-inhibitory effects in U266 cells as evidenced by cell viability assays and flow cytometric analysis. Effect of BD on cell viability was evaluated by MTT assay. Apoptotic cells and cell cycle arrest by BD were analyzed by flow cytometer. The results of the TUNEL assay and western blotting showed that BD induces apoptosis of MM cells by activating caspase-8 and 9 with subsequent reduction in the expression of antiapoptotic proteins (Bcl-2, Bcl-xl, survivin, cyclin D1, COX-2, VEGF, MMP-9). Analysis of activated kinases by Phospho-Kinase Array Kit revealed that Akt, p70S6K, HSP60, p53, and WNK1 were strongly expressed in untreated cells and BD treatment reversed this effect. Using transfection experiments, AKT depletion led to a decrease in phosphorylation of Akt, mTOR, p70S6K, and WNK. However, Akt overexpression led to increase in phosphorylation of these proteins. Depletion of Akt potentiated the apoptosis-inducing effect of BD whereas overexpression displayed resistance to BD-induced apoptosis suggesting the role of Akt in chemoresistance. Taken together, BD mitigates Akt-dependent signaling pathways in MM cells to impart its anticancer activity.

Interleukin 24 promotes cell death in renal epithelial cells and is associated with acute renal injury

Ischemia-reperfusion injury is a major cause of acute kidney injury. Many cytokines are involved in the pathogenesis of renal ischemia-reperfusion injury. IL24 is a member of the IL10 family and has gained importance because of its apoptosis-inducing effects in tumor disease besides its immunoregulative function. Littles is known about the role of IL24 in kidney disease. Using a mouse model, we found that IL24 is upregulated in the kidney after renal ischemia-reperfusion injury and that tubular epithelial cells and infiltrating inflammatory cells are the source of IL24. Mice lacking IL24 are protected from renal injury and inflammation. Cell culture studies showed that IL24 induces apoptosis in renal tubular epithelial cells, which is accompanied by an increased endoplasmatic reticulum stress response. Moreover, IL24 induces robust expression of endogenous IL24 in tubular cells, fostering ER-stress and apoptosis. In kidney transplant recipients with delayed graft function and patients at high risk to develop acute kidney injury after cardiac surgery IL24 is upregulated in the kidney and serum. Taken together, IL24 can serve as a biomarker, plays an important mechanistic role involving both extracellular and intracellular targets, and is a promising therapeutic target in patients at risk of or with ischemia-induced acute kidney injury.

Antineoplastic Properties by Proapoptotic Mechanisms Induction of Inula viscosa and Its Sesquiterpene Lactones Tomentosin and Inuviscolide.

Cancer is a complex disease including approximately 200 different entities that can potentially affect all body tissues. Among the conventional treatments, radiotherapy and chemotherapy are most often applied to different types of cancers. Despite substantial advances in the development of innovative antineoplastic drugs, cancer remains one of the most significant causes of death, worldwide. The principal pitfall of successful cancer treatment is the intrinsic or acquired resistance to therapeutic agents. The development of more effective or synergistic therapeutic approaches to improve patient outcomes and minimize toxicity has become an urgent issue. Inula viscosa is widely distributed throughout Europe, Africa, and Asia. Used as a medicinal plant in different countries, I. viscosa has been characterized for its complex chemical composition in order to identify the bioactive compounds responsible for its biological activities, including anticancer effects. Sesquiterpene lactones (SLs) are natural, biologically active products that have attracted considerable attention due to their biological activities. SLs are alkylating agents that form covalent adducts with free cysteine residues within enzymes and key proteins favoring cancer cell cytotoxicity. They are effective inducers of apoptosis in several cancer cell types through different molecular mechanisms. This review focuses on recent advances in the cytotoxic effects of I. viscosa and SLs in the treatment of neoplastic diseases, with a special emphasis on their proapoptotic molecular mechanisms.

Red Blood Cell Membrane-Camouflaged Gold Nanoparticles for Treatment of Melanoma.

Background Patients with melanoma have poor response and low long-term survival to conventional cisplatin (CP). Recently, biomimetic nanoparticles have played a significant role in tumor therapy. The purpose of this study was to mechanistically evaluate the effect of red blood cell membrane camouflaged gold nanoparticles loaded with CP (RBCm@AuNPs-CP) on enhancing chemotherapy in melanoma. Methods Treated B16-F10 cells with RBCm@AuNPs-CP, the antimelanoma effect in vitro was explored by detecting cell viability, apoptosis rate, level of reactive oxygen species (ROS), and singlet oxygen. RBCm@AuNPs-CP was injected into the melanoma-bearing mice via tail vein, and the target-ability, therapeutic effect, and toxicity were detected in melanoma tumor-bearing mice. Results RBCm@AuNPs-CP had an antiproliferation and apoptosis-inducing effect on B16-F10 cells, which might be mediated by oxidative stress of ROS, and its effect was significantly enhanced compared with the CP treatment group. In vivo experiments suggested the same outcome, with better target-ability of RBCm@AuNPs-CP. Conclusion The erythrocyte camouflage nanosystem RBCm@AuNPs-CP exhibited well passive tumor target-ability and promoted apoptosis of melanocytes by inducing ROS. RBCm@AuNPs-CP as a novel safe and effective targeted drug delivery system may provide a promising choice for the treatment of melanoma.

In Vitro Cytotoxicity and Spectral Analysis-Based Phytochemical Profiling of Methanol Extract of Barleria hochstetteri, and Molecular Mechanisms Underlying Its Apoptosis-Inducing Effect on Breast and Lung Cancer Cell Lines

The objectives of this research were to carry out GC–MS and LC–MS-based phytochemical profiling of Barleria hochstetteri, as well as flow cytometry-based mechanistic investigations of the cytotoxic effect of its extracts against breast and lung cancer cell lines. This preclinical in vitro study was carried out in Saudi Arabia and India, from 11 August to 15 January 2022. Barleria hochstetteri was sequentially extracted using the Soxhlet extraction technique. Utilizing LC–MS and GC–MS methods, the phytochemical profiling was performed. Additionally, the total phenolic compounds and flavonoids were quantified in the plant extract using spectrophotometric techniques. In this study, we first examined the cytotoxicity of the plant extract on non-malignant L929 cells and on the carcinogenic MCF-7 and A549 cell lines. Then, we studied the underlying molecular pathways by means of Anti-Bcl-2, caspase-3, and DNA fragmentation (TUNEL) assays, using flow cytometry. The results revealed phenolic compounds and flavonoids to be the two major components in the methanolic extract of B. hochstetteri, with concentrations of 3210 µg GAE/g dwt and 1863 µg QE/g dwt, respectively. Results from GC–MS and LC–MS analyses revealed the presence of bioactive phytochemicals with known cytotoxicity. From the MTT assay on cell viability, the IC50 of the methanol extract for the MCF-7 and A549 cell lines were 219.67 and 144.30 µg/mL, respectively. With IC50 values of 324.24 and 266.66 µg/mL, respectively, the aqueous and methanol extracts were less toxic when tested against the non-cancerous L929 cell line. The extract caused early and late apoptosis in the tested breast and lung cancer cells by activating caspase-3 and inhibiting Bcl-2 protein, and it also caused cell death via DNA damage, based on flow cytometric and molecular marker analyses. These findings indicate that the methanol extract of B. hochstetteri was cytotoxic on breast cancer and lung cancer cell lines. To uncover cancer-fighting chemicals, there is a need for further research on B. hochstetteri, as it is a promising source of anti-cancer chemotherapeutic drugs.

Development and characterization of Spodoptera mauritia ovarian primary cell culture and evaluation of fenoxycarb toxicity.

The rice swarming caterpillar, Spodoptera mauritia (Biosduval, 1833) (Lepidoptera: Noctuidae), has been considered a severe pest and has caused the extreme loss of rice paddies in the nursery stages. The massive reproductive efficiency of pests results from balanced endocrine functioning. Insect treatments with exogenous either juvenile hormones (JH) or juvenile hormone analogues (JHAs) during low endogenous JH titers disrupt metamorphosis andovarian development. Hence, it was thought worthwhile to develop a primary ovarian cell culture of Spodoptera mauritia to study the biological efficiency of JHA, fenoxycarb, at the cellular level. The study envisioned the cell characteristics and growth properties and found that most cells were spherical. Spindle-shaped cells were also present during the initial stage of active cell division. The majority of the cells grew attached to the bottom of the culture plates, and a few grew in suspension. The cell doubling time was 144 ± 14h. The growth curve exhibited a sigmoid pattern of cell proliferation at first, followed by a declining phase. These properties indicate that the primary ovarian culture is a suitable candidate for developing a novel cell line of S. mauritia. The preliminary study investigated the cytotoxicity of the insect growth regulator fenoxycarb in ovarian, primary cell culture. Incubated cells showed noticeable morphological changes, including cell shrinkage and apoptotic body formation; inhibition of cellular proliferation; and apoptosis-inducing effects in ovarian, primary cell culture of S. mauritia. Evidently, from these results, it is clear that juvenile hormone mimics can react in a meaningful way to control the rice pest S. mauritia by inducing cellular apoptosis in ovarian cells, which may cause adult females to become sterile.

Yeast Smy2 and its human homologs GIGYF1 and -2 regulate Cdc48/VCP function during transcription stress

The "last resort" pathway results in ubiquitylation and degradation of RNA polymerase II in response to transcription stress and is governed by factors such as Def1 in yeast. Here, we show that the SMY2 gene acts as a multi-copy suppressor of DEF1 deletion and functions at multiple steps of the last resort pathway. We also provide genetic and biochemical evidence from disparate cellular processes that Smy2 works more broadly as a hitherto overlooked regulator of Cdc48 function. Similarly, the Smy2 homologs GIGYF1 and -2 affect the transcription stress response in human cells and regulate the function of the Cdc48 homolog VCP/p97, presently being explored as a target for cancer therapy. Indeed, we show that the apoptosis-inducing effect of VCP inhibitors NMS-873 and CB-5083 is GIGYF1/2 dependent.

Investigating the effects of quercetin fatty acid esters on apoptosis, mechanical properties, and expression of ERK in melanoma cell line (A375)

AimsMalignant melanoma (MM) is the most fatal skin cancer with a critical increase in the number of cases in the last decades. Recent studies have shown the antitumor potential of active biological phytochemical structures of flavonoids for the prevention and treatment of cancerous cells. In this study, two quercetin fatty acid esters (α-linolenic acid (ALA) and linoleic acid (LA)) compounds were evaluated in terms of inducing apoptotic human melanoma cells (A375) death in vitro. Main methodsThe MTT assay was utilized for comparing the effects of quercetin, ALA, and LA on A375 cell viability concentrations of 5, 25, 35, 50, and 100μg/mL for 24 and 48 h to obtain IC50. To detect the effects on apoptosis and to determine p-ERK/ERK apoptosis-related signaling pathway proteins level, flow-cytometry and western blot were used. Finally, the nano-mechanical properties of the melanoma A375 membrane structure containing elastic modulus value and cell-cell adhesion forces were investigated using Atomic Force Microscopy (AFM). Statistical data was analyzed in GraphPad v.8.0.0. Key findingsThe most significant A375 cell viability amplified effect of Q-LA was observed with a half-maximal inhibitory concentration (IC50 = 35 μg/mL, 48 h), proportional to dose. Ester compounds, especially Q-LA, showed the highest cell proliferation inhibition with improved elastic modulus, cell-cell adhesion forces (253 ± 11.2), and elevated apoptosis-inducing effect (p < 0.01**). Moreover, Q-LA significantly decreased the mean levels of p-ERK phosphorylation (0.1439) and, subsequently, apoptosis in A375 cells. SignificanceThe data presented in this study confirmed the antitumor activity of ester compounds against A375 cells, high-lighting the ability of the tested compounds to induce apoptosis.

Targeting apoptosis to manage acquired resistance to third generation EGFR inhibitors.

A significant clinical challenge in lung cancer treatment is management of the inevitable acquired resistance to third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (EGFR-TKIs), such as osimertinib, which have shown remarkable success in the treatment of advanced NSCLC with EGFR activating mutations, in order to achieve maximal response duration or treatment remission. Apoptosis is a major type of programmed cell death tightly associated with cancer development and treatment. Evasion of apoptosis is considered a key hallmark of cancer and acquisition of apoptosis resistance is accordingly a key mechanism of drug acquired resistance in cancer therapy. It has been clearly shown that effective induction of apoptosis is a key mechanism for third generation EGFR-TKIs, particularly osimertinib, to exert their therapeutic efficacies and the development of resistance to apoptosis is tightly associated with the emergence of acquired resistance. Hence, restoration of cell sensitivity to undergo apoptosis using various means promises an effective strategy for the management of acquired resistance to third generation EGFR-TKIs.

Design, Synthesis, and Biological Evaluation of Novel Dihydropyridine and Pyridine Analogs as Potent Human Tissue Nonspecific Alkaline Phosphatase Inhibitors with Anticancer Activity: ROS and DNA Damage-Induced Apoptosis.

Small molecules with nitrogen-containing scaffolds have gained much attention due to their biological importance in the development of new anticancer agents. The present paper reports the synthesis of a library of new dihydropyridine and pyridine analogs with diverse pharmacophores. All compounds were tested against the human tissue nonspecific alkaline phosphatase (h-TNAP) enzyme. Most of the compounds showed excellent enzyme inhibition against h-TNAP, having IC50 values ranging from 0.49 ± 0.025 to 8.8 ± 0.53 µM, which is multi-fold higher than that of the standard inhibitor (levamisole = 22.65 ± 1.60 µM) of the h-TNAP enzyme. Furthermore, an MTT assay was carried out to evaluate cytotoxicity against the HeLa and MCF-7 cancer cell lines. Among the analogs, the most potent dihydropyridine-based compound 4d was selected to investigate pro-apoptotic behavior. The further analysis demonstrated that compound 4d played a significant role in inducing apoptosis through multiple mechanisms, including overproduction of reactive oxygen species, mitochondrial dysfunction, DNA damaging, and arrest of the cell cycle at the G1 phase by inhibiting CDK4/6. The apoptosis-inducing effect of compound 4d was studied through staining agents, microscopic, and flow cytometry techniques. Detailed structure–activity relationship (SAR) and molecular docking studies were carried out to identify the core structural features responsible for inhibiting the enzymatic activity of the h-TNAP enzyme. Moreover, fluorescence emission studies corroborated the binding interaction of compound 4d with DNA through a fluorescence titration experiment.

Isolation and Characterization of a Discrete Genetically Homogeneous Viral Subpopulation of Mumps Virus RS-12 Strain with Superior Oncolytic Potency Compared to Its Progenitor Virus

Background: Oncolytic virotherapy can serve as a novel therapeutic strategy in oncology. In this study, we aimed to evaluate the oncolytic activity of the mumps virus RS-12 strain after its adaptation to cancer cells via serial passaging. Methods: To adapt the RS-12 strain-based vaccine to cancer cells, it was passaged eight times in the HT1080 cell line and was isolated via two terminal endpoint dilutions. The genetic homogeneity of isolated cancer cell-adapted RS-12 variant was confirmed by direct sequencing of regions, encompassing four known heterogeneous genomic positions. The in vitro cytotoxic effects of viruses was assessed in two different cancer cell lines using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The apoptosis-inducing effects of the cancer cell-adapted variant and its parental virus on cancer cells were quantified by flow cytometry. Results: According to the chromatograms, the RS-12 strain vaccine seed exhibited two peaks at the genomic nucleotide positions 1591, 2417, 3774, and 12977. On the contrary, cancer cell-adapted RS-12, isolated by terminal endpoint serial dilutions, contained no viral subpopulations in these positions. A significant improvement was observed in the oncolytic potency of our cancer cell-adapted variant compared to its parental virus vaccine seed in vitro. Besides, the variant efficiently induced apoptosis in the human fibrosarcoma and adenocarcinoma cell lines. Conclusions: Considering the increased oncolytic potency and apoptosis-inducing capacity of this variant in cancer cells, it can be a promising option for future experiments.

Synergistic anticancer effects of metformin and Achillea vermicularis Trin-loaded nanofibers on human pancreatic cancer cell line: An in vitro study

Treatment options for pancreatic cancer are limited and not effective enough despite recent advances in chemotherapy. In this study, Metformin (Met)/Achillea vermicularis Trin. (AV)-loaded polylactic acid (PLA)/polycaprolactone (PCL) nanofibers (NFs) were fabricated by electrospinning and their chemical, mechanical, degradation, thermal, drug release behaviour and kinetic, and morphological properties were characterized. Met/AV-loaded NFs were found more effective against insulinoma cells in vitro than Met-loaded and AV-loaded NFs. Lactate dehydrogenase (LDH) leakage was remarkably higher in Met/AV-loaded NF-treated cells (on day 1 is 50.14% and on day 7 is 46.59%) than in pure NF-treated cells. The density of live cells was lower on the surface of Met/AV-loaded NF compared to Met-loaded and AV-loaded NFs. Met/AV-loaded NF demonstrated a higher apoptosis-inducing effect on insulinoma cells than single drug-loaded NFs according to DAPI nuclear staining. The combination of Met and AV in PLA/PCL NFs could be a novel and effective therapy against pancreatic cancer via a local application.

Design, Synthesis and Cytotoxicity Screening of New Thiazole Derivatives as Potential Anticancer Agents through VEGFR-2 Inhibition

Z-configurated isomers are kinetically preferred molecules. Compounds with Z-configuration are contained in many natural products, biologically active compounds and as synthons for organic synthesis. Two series of new thiazole-based analogs were synthesized from appropriate starting materials hydrazinecarbothioamide derivatives (Z)-2a,b to be evaluated for their inhibitory activity towards VEGFR-2. The prepared thiazole compounds 3a-5b were screened for their cytotoxic potency against the MDA-MB-231 breast cancer cell line and their percentage inhibition against VEGFR-2. Compound 4d exhibited good VEGFR-2 inhibitory activity. A DNA flow cytometry analysis was conducted, and compound 4d demonstrated cell cycle arrest at the G1 and G2/M phases of the cell cycle profile and an apoptosis-inducing effect by increasing the percentage of pre-G1 phase. Compound 4d was further evaluated for its apoptosis-inducing effect by studying the effect on mitochondrial membrane potential (MMP) and p53 activation. It was found to boost the level of p53 and reduce the level of MMP compared with the untreated control cells.

Dehydroabietic acid chemosensitizes drug-resistant acute lymphoblastic leukemia cells by downregulating survivin expression

Objective: To explore the mechanism of drug resistance in acute lymphoblastic leukemia and the anti-tumor effect of combination therapy of dehydroabietic acid and vincristine against acute lymphoblastic leukemia cells. Methods: Acute lymphoblastic leukemia cells REH and CCRF- CEM were employed to detect the anti-tumor effect of vincristine and doxorubicin on proliferation and apoptosis using EdU assay, human active caspase-3 Quantikine ELISA kit, and flow cytometry. Vincristine-resistant REH cells (REH-R), survivin knockdown and overexpressing REH cells were established to verify the role of survivin in drug resistance. Additionally, in vitro and in vivo assays were performed to determine the effect of dehydroabietic acid on the cytotoxicity of vincristine. Results: Vincristine and doxorubicin markedly suppressed proliferation and induced apoptosis of REH and CCRF-CEM cells. Survivin expression was upregulated in REH-R cells compared with REH cells. Knockdown of survivin expression obviously restored the sensitivity of REH-R cells to vincristine. Akt phosphorylation was also increased in REH-R cells compared to REH cells. In addition, LY294002, a PI3k/Akt pathway blocker, inhibited survivin expression and enhanced cytotoxicity of vincristine to REH-R cells. Dehydroabietic acid effectively reduced survivin expression in REH-R cells, thereby enhancing the therapeutic effect of vincristine on drug-resistant cells. Survivin overexpression markedly reduced the effect of dehydroabietic acid on enhancing the anti-proliferation and inducing apoptosis effect of vincristine. Moreover, the combination of dehydroabietic acid with vincristine significantly extended the survival rate in a mouse xenograft model of acute lymphoblastic leukemia, compared with vincristine treatment alone. Conclusions: Dehydroabietic acid may be used as a potential candidate for the treatment of acute lymphoblastic leukemia in combination with vincristine.

Pharmacological Small Molecules against Prostate Cancer by Enhancing Function of Death Receptor 5.

Death receptor 5 (DR5) is a membrane protein that mediates exogenous apoptosis. Based on its function, it is considered to be a target for the treatment of cancers including prostate cancer. It is encouraging to note that a number of drugs targeting DR5 are now progressing to different stages of clinical trial studies. We collected 38 active compounds that could produce anti-prostate-cancer effects by modulating DR5, 28 of which were natural compounds and 10 of which were synthetic compounds. In addition, 6 clinically used chemotherapeutic agents have also been shown to promote DR5 expression and thus exert apoptosis-inducing effects in prostate cancer cells. These compounds promote the expression of DR5, thereby enhancing its function in inducing apoptosis. When these compounds were used in combination with the natural ligand of DR5, the number of apoptotic cells was significantly increased. These compounds are all promising for development as anti-prostate-cancer drugs, while most of these compounds are currently being evaluated for their anti-prostate-cancer effects at the cellular level and in animal studies. A great deal of more in-depth research is needed to evaluate whether they can be developed as drugs. We collected literature reports on small molecules against prostate cancer through modulation of DR5 to understand the current dynamics in this field and to evaluate the prospects of small molecules against prostate cancer through modulation of DR5.

Aspiletrein A Induces Apoptosis Cell Death via Increasing Reactive Oxygen Species Generation and AMPK Activation in Non-Small-Cell Lung Cancer Cells.

Lung cancer remains a leading cause of death in cancer patients, and deregulation of apoptosis is a serious concern in clinical practice, even though therapeutic intervention has been greatly improved. Plants are a versatile source of biologically active compounds for anticancer drug discovery, and aspiletrein A (AA) is a steroidal saponin isolated from Aspidistra letreae that has a potent cytotoxic effect on various cancer cell lines. In this study, we investigated and determined the underlying molecular mechanism by which AA induces apoptosis. AA strongly induced apoptosis in NSCLC cells by mediating ROS generation and thereby activating AMP-activated protein kinase (AMPK) signaling. Consequently, downstream signaling and levels of phosphorylated mTOR and Bcl-2 were significantly decreased. Pretreatment with either an antioxidant, N-acetylcysteine, or an AMPK inhibitor, compound C, could reverse the apoptosis-inducing effect and counteract the effect of AA on the AMPK signaling pathway. Decreased levels of Bcl-2 were due to AA-mediating Bcl-2 degradation via a ROS/AMPK/mTOR axis-dependent proteasomal mechanism. Consistently, the apoptotic-inducing effect of AA was also observed in patient-derived malignant lung cancer cells, and it suppressed an in vitro 3D-tumorigenesis. This study identified the underlying mechanism of AA on lung cancer apoptosis, thereby facilitating potential research and development of this compound for further clinical implications.