Induction Of Apoptotic Cell Death Research Articles

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

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

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

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

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

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

Comparative Evaluation of Apoptosis Induction Using Needles, Bark, and Pollen Extracts and Essential Oils of Pinus eldarica in Lung Cancer Cells

Lung cancer is one of the leading causes of cancer-related mortality worldwide. Although effective clinical drugs for treating advanced stages are available, interest in alternative herbal medicines has gained momentum. Herbal extracts are potent antioxidants that reportedly inhibit the growth of various cancer cell lines. In the present study, we investigated the effects of essential oils and hexane, methanolic, and aqueous extracts, obtained from various parts (bark, needles, and pollen) of Pinus eldarica against human lung cancer (A549) cells. First, the DPPH radical scavenging activities of P. eldarica extracts and essential oils were examined, which revealed that methanolic extracts presented higher antioxidant activity than the other extracts and essential oils. Next, A549 cells were exposed to various concentrations of the extracts and essential oils for 48 h. P. eldarica extracts/essential oil-treated lung cancer cells demonstrated a significant decrease in cell proliferation, along with an induction of apoptotic cell death, particularly, the pollen hexane extract, bark essential oil, and methanolic needle extract showed superior results, with IC50 values of 31.7, 17.9, and 0.3 μg/mL, respectively. In the cell cycle analysis, treatment of A549 cells with the methanolic needle and pollen hexane extracts led to apoptosis and accumulation of cells in the sub-G1 phase. Further, exposure to the bark essential oil and methanolic needle extract decreased the cell population in the G2/M phase. Notably, treatment with the pollen hexane extract, bark essential oil, and methanolic needle extract resulted in caspase-3 activation, poly (ADP-ribose) polymerase cleavage, Bcl-2 downregulation, and Bax and p53 regulation in A549 cells. Furthermore, these extracts and essential oils decreased the migration, and colony formation of A549 cells. These findings provide experimental evidence for a new therapeutic effect of P. eldarica against human lung cancer and suggest P. eldarica as a potential chemopreventive natural resource for developing novel cancer therapeutics.

Mercury-containing supramolecular micelles with highly sensitive pH-responsiveness for selective cancer therapy

Construction and manipulation of metal-based supramolecular polymers—which are based on a combination of nucleobase hydrogen bonding interactions and functional metal ions—to obtain the desired physicochemical properties and achieve the efficacy and safety required for biomedical applications remain extremely challenging. We successfully designed and synthesized a new mercury-based supramolecular polymer, Hg-BU-PPG, containing an oligomeric polypropylene glycol backbone and pH-sensitive uracil-mercury-uracil (U-Hg-U) linkages. This multifunctional metallo-supramolecular material spontaneously self-organizes into nanosized spherical micelles in aqueous solution. The micelles possess several attractive properties, including desired long-term structural stability in serum-rich conditions, unique fluorescence behavior and highly sensitive, well-controlled pH-responsiveness. Interestingly, Hg-BU-PPG micelles exhibited strong, selective cytotoxic effects towards cancer cells in vitro, without harming normal cells. The highly selective cytotoxicity can be attributed to rapid dissociation of the U-Hg-U complexes within the micelles in the mildly acidic intracellular pH of cancer cells, followed by release of inherently toxic mercury ions. Importantly, fluorescence microscopy and flow cytometry clearly demonstrated that Hg-BU-PPG selectively entered the cancer cells via endocytosis and rapidly promoted massive apoptotic cell death. In contrast, internalization of Hg-BU-PPG by normal cells was limited, resulting in high biocompatibility and no cytotoxic effects. Thus, this newly discovered ‘cytotoxicity-concealing’ supramolecular system could represent a viable route to enhance the safety and efficacy of cancer therapy and bioimaging via a strategy that does not require incorporation of anticancer drugs and fluorescent probes. Statement of significanceWe report a significant breakthrough in the construction of mercury-containing supramolecular polymers, namely the creation of multifunctional micelles with unique chemical and physical properties conferred by pH-sensitive uracil-mercury-uracil (U-Hg-U) linkages and tunable structural and dynamical features due to the presence of hydrogen-bonded uracil moieties. Importantly, in vitro experiments clearly demonstrated that introduction of the U-Hg-U complexes into the micelles not only improved the efficiency of selective uptake via endocytosis into cancer cells, but also accelerated the induction of massive apoptotic cell death. Thus, this work provides crucial new insight for the development of metallo-supramolecular polymeric micelles that may substantially enhance the safety and efficacy of cancer therapy and bioimaging without requiring incorporation of anticancer drugs or fluorescent probes.

Vitamin D3 suppresses intestinal epithelial stemness via ER stress induction in intestinal organoids

BackgroundVitamin D3 is important for normal function of the intestinal epithelial cells (IECs). In this study, we aimed to investigate the effects of vitamin D3 on the differentiation, stemness, and viability of healthy IECs in intestinal organoids.MethodsIntestinal organoids derived from mouse small intestine were treated with vitamin D3, and the effects on intestinal stemness and differentiation were evaluated using real-time PCR and immunofluorescence staining of the distinct lineage markers. Cell viability was analyzed using viability and apoptosis assays.ResultsVitamin D3 enhanced IEC differentiation into the distinct lineages of specialized IECs, including Paneth, goblet, and enteroendocrine cells and absorptive enterocytes. Decreased expression levels of leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5) and the presence of several LGR5-green fluorescent protein (GFP)-positive cells were observed in vitamin D3-treated organoids derived from LGR5-GFP mice. The formation of the crypt-villus structure was also inhibited by vitamin D3, suggesting that vitamin D3 suppresses intestinal cell stemness. Furthermore, the expression levels of unfolded protein response genes, C/EBP homologous protein (CHOP), and activating transcription factor 6 (ATF6) were upregulated in vitamin D3-treated organoids. Moreover, vitamin D3 promoted apoptotic cell death in intestinal cells, which may be associated with the decrease in intestinal stemness. LGR5 gene expression, ISC number, and apoptotic cell death were partially recovered in the presence of the ER stress inhibitor tauroursodeoxycholic acid (TUDCA), suggesting that intestinal stemness suppression and intestinal apoptosis occurred via ER stress activation.ConclusionsOur study provides important insights into the effects of vitamin D3 on the induction of IEC differentiation and apoptotic cell death, and inhibition of intestinal stemness accompanied by ER stress augmentation.

Retracted] Anticancer activity of taraxerol acetate in human glioblastoma cells and a mouse xenograft model via induction of autophagy and apoptotic cell death, cell cycle arrest and inhibition of cell migration.

Following the publication of the above paper, a concerned reader drew to the Editor's attention that several figures (Figs.3, 4, 7 and 10) contained apparent anomalies, including repeated patternings of data within the same figure panels. Furthermore, Fig.3 contained data that bore striking similarities to data published in Fig.6 in another paper published in Molecular Medicine Reports, which has now been retracted [Zhu Y‑Y, Huang H‑Y and WuY‑L: Anticancer and apoptotic activities of oleanolic acid are mediated through cell cycle arrest and disruption of mitochondrial membrane potential in HepG2human hepatocellular carcinoma cells. Mol Med Rep12: 5012‑5018, 2015]. After having conducted an independent investigation in the Editorial Office, the Editor of Molecular Medicine Reports has determined that the above paper should be retracted from the Journal on account of a lack of confidence concerning the originality and the authenticity of the data. The authors were asked for an explanation to account for these concerns, but the Editorial Office never received any reply. The Editor regrets any inconvenience that has been caused to the readership of the Journal. [the original article was published in Molecular Medicine Reports 13: 4541‑4548, 2016; DOI: 10.3892/mmr.2016.5105].

Identification of a novel Bax-Cdk1 signalling complex that links activation of the mitotic checkpoint to apoptosis.

In eukaryotes, entry into and exit from mitosis is regulated, respectively, by the transient activation and inactivation of Cdk1. Taxol, an anti-microtubule anti-cancer drug, prevents microtubule-kinetochore attachments to induce spindle assembly checkpoint (SAC; also known as the mitotic checkpoint)-activated mitotic arrest. SAC activation causes mitotic arrest by chronically activating Cdk1. One consequence of prolonged Cdk1 activation is cell death. However, the cytoplasmic signal(s) that link SAC activation to the initiation of cell death remain unknown. We show here that activated Cdk1 forms a complex with the pro-apoptotic proteins Bax and Bak (also known as BAK1) during SAC-induced apoptosis. Bax- and Bak-mediated delivery of activated Cdk1 to the mitochondrion is essential for the phosphorylation of the anti-apoptotic proteins Bcl-2 and Bcl-xL (encoded by BCL2L1) and the induction of cell death. The interactions between a key cell cycle control protein and key pro-apoptotic proteins identify the Cdk1-Bax and Cdk1-Bak complexes as the long-sought-after cytoplasmic signal that couples SAC activation to the induction of apoptotic cell death.

Initial defensive secretory compounds emitted from the live millipede and the induction of apoptotic cell death

The initial defensive secretory compounds emitted from a live millipede have not yet been clarified. This study focused on elucidating the initial secretory compounds emitted from a live millipede. Pre-concentration of the defensive secretory volatile organic compounds (VOC) from the live Polidesmida millipedes, Chamberlinius hualienensis and Oxidus gracilis, was performed using a three-stage VOC concentration technique by an on-line GC/MS system. As a result, the monoterpenes derived from the plant metabolite; i.e., α-pinene, α-thujene, β-pinene, 3-carene, β-myrcene, β-phellandrene, γ-terpinene, o,m,p-cymenes, limonene and camphene were first detected as the initial secretory substances. It was elucidated that some plant monoterpenes have a repellent effect and antifungal and antibacterial actions which are used as defensive substances. In addition, this study also confirmed that these monoterpenes induced apoptotic cell death involved in the induction of the caspase 3/7 activity. The millipede feeds on fallen or withered leaves containing the monoterpenes. Thus, the millipede accumulates the plant defensive secretions in the exocrine defense glands of the body somites, which would be used as against predators.

Induction of Apoptosis in Hepatocellular Carcinoma Cell Lines by Novel Indolylacrylamide Derivatives: Synthesis and Biological Evaluation.

Hepatocellular carcinoma (HCC) is the most prevalent primary liver cancer and one of the leading causes of cancer associated death worldwide. This is due to the highly resistant nature of this malignancy and the lack of effective treatment options for advanced stage HCC patients. The hyperactivity of PI3K/Akt and Ras/Raf/MEK/ERK signaling pathways contribute to the cancer progression, survival, motility, and resistance mechanisms, and the interaction of these two pathways are responsible for the regulation of cancer cell growth and development. Therefore, it is vital to design and develop novel therapeutic options for HCC treatment targeting these hyperactive pathways. For this purpose, novel series of trans-indole-3-ylacrylamide derivatives originated from the lead compound, 3-(1H-indole-3-yl)-N-(3,4,5-trimethoxyphenyl)acrylamide, have been synthesized and analyzed for their bioactivity on cancer cells along with the lead compound. Based on the initial screening, the most potent compounds were selected to elucidate their effects on cellular signaling activity of HCC cell lines. Cell cycle analysis, immunofluorescence, and Western blot analysis revealed that lead compound and (E)-N-(4-tert-butylphenyl)-3-(1H-indole-3-yl)acrylamide induced cell cycle arrest at the G2/M phase, enhanced chromatin condensation and PARP-cleavage, addressing induction of apoptotic cell death. Additionally, these compounds decreased the activity of ERK signaling pathway, where phosphorylated ERK1/2 and c-Jun protein levels diminished significantly. Relevant to these findings, the lead compound was able to inhibit tubulin polymerization as well. To conclude, the novel trans-indole-3-ylacrylamide derivatives inhibit one of the critical pathways associated with HCC which results in cell cycle arrest and apoptosis in HCC cell lines.

The Superior Cytotoxicity of Dual Targeting of BCR/ABL and PI3K in K562 Cells: Proposing a Novel Therapeutic Potential for the Treatment of CML.

Apart from BCR/ABL which is the main player in the pathogenesis of chronic myeloid leukemia (CML), the role of other signaling cascades should not be underestimated especially for the maintenance of leukemic cells survival. The results of the present study indicate that either an isoform-specific or a pan-PI3K inhibitor could potently reduce the survival of CML-derived K562 cells, shedding more light on the involvement of the PI3K axis in the pathogenesis of CML. Of particular interest, the importance of the PI3K pathway in this disease became more evident when we found that there was a more remarkable reduction in the viability of K562 cells when BKM120 was used in combination with imatinib. Moreover, BKM120 robustly enhanced the growth-suppressive effect of imatinib through p21-mediated induction of G2/M cell cycle arrest and induction of apoptotic cell death. Despite the favorable anti-survival effects of the drug combination, these agents failed to induce inhibitory effects on the expression of c-Myc and NF-κB anti-apoptotic target genes. However, the ability of combinational therapy in diminishing K562 cell survival was potentiated either in the presence of 10058-F4 (c-Myc inhibitor) or Bortezomib (proteasome inhibitor), suggestive of the role of both NF-κB and c-Myc in overshadowing the therapeutic value of drugs combination. Taken together, the results of this study showed that inhibition of the PI3K pathway is a suitable approach to enhance the therapeutic value of imatinib in the treatment of CML.

Auranofin reveals therapeutic anticancer potential by triggering distinct molecular cell death mechanisms and innate immunity in mutant p53 non-small cell lung cancer

Auranofin (AF) is an FDA-approved antirheumatic drug with anticancer properties that acts as a thioredoxin reductase 1 (TrxR) inhibitor. The exact mechanisms through which AF targets cancer cells remain elusive. To shed light on the mode of action, this study provides an in-depth analysis on the molecular mechanisms and immunogenicity of AF-mediated cytotoxicity in the non-small cell lung cancer (NSCLC) cell line NCI–H1299 (p53 Null) and its two isogenic derivates with mutant p53 R175H or R273H accumulation.TrxR is highly expressed in a panel of 72 NSCLC patients, making it a valid druggable target in NSCLC for AF. The presence of mutant p53 overexpression was identified as an important sensitizer for AF in (isogenic) NSCLC cells as it was correlated with reduced thioredoxin (Trx) levels in vitro. Transcriptome analysis revealed dysregulation of genes involved in oxidative stress response, DNA damage, granzyme A (GZMA) signaling and ferroptosis. Although functionally AF appeared a potent inhibitor of GPX4 in all NCI–H1299 cell lines, the induction of lipid peroxidation and consequently ferroptosis was limited to the p53 R273H expressing cells. In the p53 R175H cells, AF mainly induced large-scale DNA damage and replication stress, leading to the induction of apoptotic cell death rather than ferroptosis. Importantly, all cell death types were immunogenic since the release of danger signals (ecto-calreticulin, ATP and HMGB1) and dendritic cell maturation occurred irrespective of (mutant) p53 expression. Finally, we show that AF sensitized cancer cells to caspase-independent natural killer cell-mediated killing by downregulation of several key targets of GZMA.Our data provides novel insights on AF as a potent, clinically available, off-patent cancer drug by targeting mutant p53 cancer cells through distinct cell death mechanisms (apoptosis and ferroptosis). In addition, AF improves the innate immune response at both cytostatic (natural killer cell-mediated killing) and cytotoxic concentrations (dendritic cell maturation).

Long non-coding RNA PTCSC3 inhibits human oral cancer cell proliferation by inducing apoptosis and autophagy.

IntroductionLong non-coding RNAs (lncRNAs) have gained increased attention due to the discovery of their roles in cancer-related processes. LncRNA PTCSC3 has been shown to have tumour-suppressive effects in thyroid cancer and glioblastoma. This study investigated the role of lncRNA PTSC3 in human oral cancer.Material and methodsCell viability was determined by MTT assay. The induction of apoptosis was confirmed by 4′,6-diamidino-2-phenylindole (DAPI) and Annexin V/PI assays. Ultrastructural analysis was performed by electron microscopy. Transwell assay was used to monitor the invasion of oral cancer cells.ResultsThe results revealed significant (p < 0.05) suppression of PTCSC3 expression in human oral cancer tissues and cell lines. The overexpression of PTCSC3 caused a significant (p < 0.05) decline in the proliferation of the human oral cancer cells via induction of apoptotic cell death which was accompanied by remarkable enhancement of Bax and suppression of Bcl-2. The electron microscopic analysis showed the development of autophagic vesicles in both the SCC-1 and SCC-9 cells indicative of autophagy. The western blotting analysis showed that PTCSC3 overexpression caused a remarkable increase in LC3B-I and Beclin 1 expression. PTCSC3 overexpression caused a significant (p < 0.05) decrease in invasion of the human SCC-1 and SCC-9 oral cancer cells. The invasion of the SCC-1 and SCC-9 cells was inhibited by 62% and 69% respectively.ConclusionsOverall, the evidence suggests that lncRNA PTCSC3 acts as a tumour suppressor in human oral cancer and suppresses oral cancer proliferation via induction of apoptosis and autophagy.

Correction: Hseu, Y.-C., et al. The In Vitro and In Vivo Anticancer Properties of Chalcone Flavokawain B through Induction of ROS-Mediated Apoptotic and Autophagic Cell Death in Human Melanoma Cells. Cancers 2020, 12, 2936

In the original article [...].

Homoharringtonine inhibits fibroblasts proliferation, extracellular matrix production and reduces surgery-induced knee arthrofibrosis via PI3K/AKT/mTOR pathway-mediated apoptosis

BackgroundThe prevention of surgery-induced intraarticular fibrosis remains a challenge following orthopedic surgery. Homoharringtonine (HHT) has been reported to have positive effects in preventing various kinds of fibrosis. However, little is known regarding its effect as well as the potential mechanism of HHT in preventing surgery-induced intraarticular fibrosis.MethodsVarious concentrations of HHTs were locally applied in vivo to reduce knee intraarticular fibrosis in rabbits. Histological macroscopic assessments such as hematoxylin and eosin (HE) staining, Masson’s trichrome staining, and Picric-sirius red polarized light were used to evaluate the effect of HHT in reducing intraarticular fibrosis. CCK-8, cell cycle assay, and EdU incorporation assay were used in vitro to detect HHT’s effect on inhibiting fibroblast viability and proliferation. The effect of HHT on fibroblast differentiation, extracellular matrix production, and apoptosis were evaluated by western blot, flow cytometry, immunofluorescent staining, and TUNEL analysis. Moreover, the expressions of PI3K/AKT/mTOR signaling pathway were detected.ResultsThe results demonstrated that HHT could reduce the formation of intraarticular fibrosis. HHT was also found to induce fibroblast apoptotic cell death in a dose- and time-dependent manner in vitro. Moreover, HHT could effectively inhibit the production of the extracellular matrix secreted by fibroblasts and inhibited the expression of p-PI3K, p-AKT, and p-mTOR in a dose-dependent manner. After treating with insulin-like growth factor-1 (IGF-1), an activator of the PI3K/AKT axis, the expressions of pro-apoptosis-related proteins were decreased, and the fibroblast apoptosis rate was also inhibited.ConclusionsIn conclusion, this study demonstrated that HHT could reduce the formation of intraarticular fibrosis through the inhibition of fibroblast proliferation, extracellular matrix production, and the induction of fibroblast apoptotic cell death. Furthermore, its potential mechanism may be through the suppression of the PI3K/AKT/mTOR signaling pathway.

Pre-Treatment of Pterostilbene Enhances H2O2-induced Cell Apoptosis Through Caspase-dependent Pathway in Human Keratinocyte Cells

Hydrogen peroxide (H2O2) is one of the reactive oxygen species (ROS), which can induce apoptotic cell death in numerous cancer cells. Pterostilbene (PTE), a natural polyphenolic compound, induces cell apoptosis in many human cancer cells. We investigated whether PTE could enhance H2O2-induced cell apoptosis in human keratinocyte HaCaT cells in vitro. The morphological change of HaCaT cells was observed and photographed under a contrast-phase microscope. The percentage of cell viability was measured by propidium iodide exclusion assay. Cell apoptosis was performed by Annexin V/PI double staining and assayed by flow cytometer. DNA condensation was measured by DAPI staining. The protein expression was determined by western blotting. ROS production-associated proteins were also assayed by confocal laser scanning microscopy. PTE pre-treatment enhanced H2O2 (600 μM)-induced cell morphological changes and reduced the total cell number (cell viability). The decreased cell viability in HaCaT cells was through induction of apoptotic cell death, which was confirmed by Annexin V/PI double staining and DAPI staining. Western blotting studies indicated that HaCaT cells which were pre-treated with PTE (100 μM) and then co-treated with H2O2 (600 μM) for 12 h showed significantly increased levels of SOD (Cu/Zn), SOD (Mn), Bax, caspase-3, caspase-8, caspase-9, PARP, p53, p-p53, and p-H2A.X but decreased levels Bcl-2 and catalase. Results also showed that HaCaT cells pre-treated with PTE and then co-treated with H2O2 had increased expression of SOD (Cu/Zn) and glutathione but decreased catalase. These observations suggest that PTE pre-treatment can enhance the H2O2-induced apoptotic cell death in keratinocyte cells and may be an effective candidate for the treatment of proliferative keratinocytes.

Induction of ROS-dependent apoptotic cell death by platycodin D in human prostate cancer PC3 cells

Background and objective: Platycodin D (PD), a triterpenoid saponin isolated from an edible and medicinal plant Platycodon grandiflorum, possesses multiple pharmacological properties. The purpose of this study is to investigate the effect of PD on the growth of PC3 human prostate cancer cells and the underlying molecular mechanisms.Materials and methods: Cell viability, apoptosis and mitochondrial membrane potential (MMP) were measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, nuclear staining and flow cytometry analysis. To investigate the mechanism of anti-cancer activity of PD, expression of apoptosis regulatory protein, caspase activity, and generation of intracellular reactive oxygen species (ROS) were determined.Results: PD treatment reduced PC3 cell proliferation, which was associated with induction of apoptosis, and accompanied by increased expression of Fas, Fas-ligand (FasL) and pro-apoptotic Bax, and decreased expression of anti-apoptotic Bcl-2 and truncation of Bid. PD also inhibited expression of c-FLIP and members of inhibitor of apoptosis protein family, and activated caspases, resulting in an increase in poly (ADP-ribose) polymerase cleavage. However, in the presence of a pan-caspase inhibitor, PD-mediated growth inhibition and apoptosis were significantly protected. PD also destroyed the integrity of mitochondria due to the loss of MMP, leading to cytosolic release of cytochrome c. Moreover, the levels of ROS were markedly increased by PD treatment, which was significantly attenuated by the ROS scavenger N-acetyl-L-cysteine (NAC). Furthermore, NAC fully suppressed PD-induced apoptotic events and cytotoxicity.Conclusions: The results of this study show that PD had chemopreventive potential through the induction of ROS-dependent apoptosis in PC3 cells, and that this compound could be useful for developing an effective and selective natural source to inhibit cancer cell proliferation.

Dual drug delivery of trapoxin A and methotrexate from biocompatible PLGA-PEG polymeric nanoparticles enhanced antitumor activity in breast cancer cell line

Nanoparticle (NP) mediated systems have been considered for chemotherapeutical delivery because of their ability to control drug release and reduce side effects These systems can also be used to deliver two or more drugs at the same time and prevent the growth of resistant cancer cells. Trapoxin A (TPX) and methotrexate (MTX) have shown promising results in treatment or controlling the progression of cancer. The present study examines the synergistic anticancer efficiency of TPX and MTX co-loaded PLGA-PEG NPs against MCF-7 breast cancer cells. The resultant drug-loaded PLGA-PEG NPs displayed a spherical morphology with the uniform nanosize distribution. Besides, the possible mechanisms for the inhibition of cell growth, distribution of cell cycle, and induction of apoptotic cell death were analyzed by DAPI staining, cell cycle analysis, and qRT-PCR. Interestingly, the obvious synergistic anticancer effects and the activation of the mitochondrial apoptosis pathway were observed on the breast cancer cell line MCF-7 by treating with TPX/MTX-co-loaded PLGA-PEG NPs. Also, it was revealed that the dual drug-loaded NPs could considerably change the expression levels of cyclin D1, and Bcl-2 compared to the free combination of the drugs as well as the single drug-loaded NPs. Taken together, this work revealed that TPX/MTX co-loaded PLGA-PEG NP based combination therapy might have an important potential to increase the efficiency of breast cancer therapy.

Suppression of proteasome induces apoptosis in APL cells and increases chemo-sensitivity to arsenic trioxide: Proposing a perception in APL treatment

In the last three decades, the pathogenesis of acute promyelocytic leukemia (APL) has been mostly studied with regard to the oncogenic role of PML/RAR fusion protein; however, the latest discoveries have stated that the concerns with the treatment of APL patients would not be resolved until the role of aberrant networks is overlooked. The present study was designed to evaluate the anti-cancer property of second-generation of the proteasome inhibitors carfilzomib (CFZ) on APL-derived NB4 cells. Our results showed that pharmacologic targeting of proteasome in NB4 reduced the proliferative rate of malignant cells through a c-Myc-mediated G2/M cell cycle arrest. Moreover, we found that the suppression of proteasome was coupled with the induction of apoptotic NB4 cell death, which is probably mediated through down-regulation of anti-apoptotic target genes. Interestingly, our results suggested that the suppression of the autophagy system using chloroquine could serve as a mechanism through which the cytotoxicity of CFZ in APL cells was ameliorated. Finally, and consistent with the favorable efficacy of single agent of CFZ, we also noted an intensifying effect of the inhibitor on the anti-leukemic activity of arsenic trioxide (ATO) when it was used in combination. Overall, this study suggests that pharmaceutical targeting of proteasome using CFZ, either as a single agent or in combination with ATO, could be a promising mechanism through which the obstacle on the way of APL would be tackled; however, further investigations are needed to determine the advantages of the inhibitor in clinical applications.

Efficacy of Tubulin Polymerization Inhibitor in Myelodysplastic Syndrome

Background: Myelodysplastic Syndrome (MDS) is a clonal bone marrow disorder characterized by ineffective and clonal hematopoiesis accompanied by morphological dysplasia and variable cytopenia. There are few treatment options for MDS, and allogenic hematopoietic stem cell transplantation is the only curative option. Tubulin belongs to protein superfamily of globular proteins. Monomeric tubulin can polymerize into microtubules, which play an important role in the attachment and segregation of chromosomes in various phases of cell division. Therefore, the targeting of microtubules represents a therapeutic strategy against both solid and hematological cancers. The first approved tubulin binding agent by the FDA was vincristine, which has been clinically used to treat multiple types of cancers, particularly hematological malignancies. Over the past few decades, additional tubulin binding agents have been developed and received FDA approval, mostly for applications to cancer therapy. These agents have been classified by their binding sites on tubulin, which influences their roles in the inhibition or stabilization of polymerized microtubules. We herein investigated the efficacy of PTC-028, a novel microtubule polymerization inhibitor for MDS. Method: Anti-MDS efficacy of PTC-028 was studied using human MDS cell lines and primary MDS cells in vitro. The efficacy of PTC-028 was also assessed in a xenograft mouse model of MDS using an MDS cell lines. PTC-028 was synthesized at PTC Therapeutics Inc. Mechanistic studies were conducted via flow cytometry and RNA sequencing. Results: A previous study reported that PTC596 suppressed cell proliferation and induced apoptosis in AML cell lines. Since MDS is regarded as a pre-leukemic stage, we examined the effects of PTC-028, another novel microtubule polymerization inhibitor, on MDS cells. PTC-028 induced a dose-dependent inhibition of cell proliferation on MDS cell lines, such as MDS-L and SKM-1 cells. Caspase 3/7 activity was also significantly induced in MDS-L and SKM-1 cells in the presence of PTC-028, suggesting the induction of apoptotic cell death by PTC-028. We then isolated CD34+ cells from primary MDS BM samples and investigated the efficacy of PTC-028 on primary MDS cells. The efficacy of PTC-028 in CD34+ MDS cells was also confirmed by cell proliferation assays. To enhance the therapeutic benefit of PTC-028 on MDS cells, we investigated synergism between PTC-028 and DNA hypomethylating agents. We treated MDS-L and SKM-1 cells with PTC-028 in combination with DNA hypomethylating agents. After 3 days of culture, cell growth was analyzed by MTS and Annexin V assays. PTC-028 synergized with hypomethylating agents, such as decitabine and azacitidine, to inhibit the growth and induce apoptosis of MDS cells. We assessed the efficacy of PTC-028 in a xenograft mouse model of MDS using an MDS cell line. Recipient mice were treated with PTC-028 for 7 weeks. PTC-028 significantly inhibited the growth of MDS-L cells and prolonged the overall survival of recipient mice. Furthermore, a significant synergistic effect was observed between PTC-028 and decitabine. Mice that received combination therapy showed moderate weight loss 11 days after the initiation of the treatment, but subsequently recovered. Mechanistically, a treatment with PTC-028 induced G2/M arrest followed by apoptotic cell death. We then investigated the effect of PTC-028 on the levels of soluble (unpolymerized) versus polymerized tubulin in MDS-L cells. Cells were treated with PTC-028 and paclitaxel for 4 hours, and cell lysates were then separated into soluble and polymerized fractions by centrifugation. The visualization of tubulin fractions by Western blotting demonstrated that the PTC-028 treatment for 4 hours resulted in the near-complete loss of polymerized microtubules. In contrast, polymerized microtubules increased in cells treated with paclitaxel, which stabilizes microtubules against depolymerization. These result indicates that PTC-028 also acts as a microtubule polymerization inhibitor. Conclusion: Our data reveal a possible chemotherapeutic strategy for PTC-028 and PTC-596 in MDS by disruption of microtubule dynamics as a single agent and in combination with hypomethylating agents. The present study provides a preclinical framework for the clinical evaluation of this promising therapeutic approach to improve outcomes in MDS patients. Disclosures Lennox: PTC therapeutics: Current Employment. Sheedy:PTC therapeutics: Current Employment. Weetall:PTC Therapeutic: Current Employment.