Histone Deacetylase Inhibitor (HDACi) PCI-24781 and Bortezomib Result in Synergistic Apoptosis in Hodgkin Lymphoma (HL) and Non-Hodgkin’s Lymphoma (NHL) Cell Lines: Investigation of Cell Death and NFKB-Mediated Pathways.

Abstract HDACi block cancer cell proliferation by mechanisms that involve epigenetic gene regulation leading to cell growth arrest, differentiation, and apoptosis. Bortezomib inhibits NFKB signaling and induces apoptosis. Furthermore, anti-tumor activity of HDACi and bortezomib both depend in part on reactive oxygen species (ROS)-mediated pathways. Both have activity in NHL. We reasoned that these agents may be synergistic in part due to their dependence on overlapping pathways. We investigated the biology of PCI-24781, a pan-HDACi currently in clinical trials, and bortezomib both alone, and in combination, in HL (L428) and NHL cell lines (HF1, Ramos, & SUDHL4). Cells were incubated with increasing concentrations of PCI-24781 and bortezomib (0.25–2.0μM and 2.5–20nM, respectively) for 24–72 hour (hr). Apoptosis was determined by fluorescence-activated cell sorting (FACS) using AnnexinV-FITC/propidium iodide (AnnexinV+/PI+) staining. Reactive oxygen species (ROS) were measured by oxidation of 2′7′dichlorofluorescein diacetate (H2DCFDA) to DCF and detected by FACS. Downstream targets of NFKB such as NFKB1, Myc and IL-8 were measured in Ramos using quantitative real time polymerase chain reaction (RT-PCR) following 24 hr incubation of cells with PCI-24781 and bortezomib alone, and in combination. Dose-dependent apoptosis was seen with PCI-24781 and bortezomib alone in all HL and NHL cell lines. IC70 (dose to achieve 70% AnnexinV+/PI+) was 1μM for PCI-24781 and 2μM for L428. With bortezomib, the IC50 was 10nM in Ramos, HF1, and SUDHL4 and 20 nM in L428. The combination of PCI-24781 and bortezomib resulted in synergistic apoptosis (combination index <0.2) in all 3 NHL cell lines (IC80=0.25μM PCI-24781/5nM bortezomib) and L428 (IC80=0.5μM PCI-24781/10nM bortezomib) compared with minimal cell death using each agent alone at those concentrations. Furthermore, immunoblots of L428 and Ramos showed enhanced caspase 3 and caspase 8 cleavage with the combination of PCI-24781 and bortezomib compared to either agent alone, suggesting that the synergy seen was in part caspase-dependent. HL and NHL cell lines showed a 3- to 4-fold increase in ROS with PCI-24781 or bortezomib alone and in combination at 24hr. Moreover, we found that hyperacetylation of histone-3 and histone-4 on immunoblots of cells treated with combination PCI-2478/bortezomib was significantly increased compared to PCI-24781 alone. Finally, we found that in Ramos cells PC-24781/bortezomib together resulted in downregulation of NFKB targets NFKB1 and Myc, but not IL-8. We conclude that PCI-24781 and bortezomib are active in lymphoma cell lines and that the combination results in synergistic apoptosis. Apoptosis was accompanied by caspase activation and synergistic downregulation of the NFkB pathway. These data have important clinical implications for NHL and HL.

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Promotion of Ferroptosis in Oral Cancer Cell Lines by Chrysophanol

Current Topics in Nutraceutical Research ◽

10.37290/ctnr2641-452x.18:273-276 ◽

2019 ◽

Vol 18 (3) ◽

pp. 273-276

Author(s):

Lin Ya-Hsuan ◽

Chiu Valeria ◽

Huang Chun-Yen ◽

Tzeng I-Shiang ◽

Hsieh Po-Chun ◽

...

Keyword(s):

Reactive Oxygen Species ◽

Squamous Cell Carcinoma ◽

Cell Death ◽

Oral Cancer ◽

Cell Carcinoma ◽

Cell Lines ◽

Cancer Cell ◽

Squamous Cell ◽

Reactive Oxygen ◽

Oxygen Species

Oral cancer is a type of head and neck cancer that can be life threatening if not diagnosed and treated early. Ferroptosis is a type of programmed or regulated cell death dependent on iron and reactive oxygen species but is a caspase-independent form of non-apoptotic cell death. Therefore, there is a need to identify candidate natural compound that may attenuate carcinogenesis through ferroptosis. To this end, we determined the pharmacological effects of chrysophanol on ferroptosis in two different oral cancer cell lines—FaDu, a hypopharyngeal squamous cell carcinoma and SAS, a poorly differentiated squamous cell carcinoma cell line from human tongue primary lesion. Results indicated that chrysophanol caused overproduction of lipid reactive oxygen species, decreased the level of glutathione peroxidase 4, and increased the level of lipocalin-2 and CCAAT-enhancer-binding protein homologous protein. These findings suggest that chrysophanol has the therapeutic potential to alleviate the progression of oral carcinogenesis through activation of ferroptosis.

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The Regulation of Reactive Oxygen Species Production during Programmed Cell Death

The Journal of Cell Biology ◽

10.1083/jcb.141.6.1423 ◽

1998 ◽

Vol 141 (6) ◽

pp. 1423-1432 ◽

Cited By ~ 505

Author(s):

Shirlee Tan ◽

Yutaka Sagara ◽

Yuanbin Liu ◽

Pamela Maher ◽

David Schubert

Keyword(s):

Gene Expression ◽

Reactive Oxygen Species ◽

Cell Death ◽

Programmed Cell Death ◽

Reactive Oxygen ◽

Fold Increase ◽

Glutamate Toxicity ◽

Ros Production ◽

Oxygen Species ◽

Ht22 Cells

Reactive oxygen species (ROS) are thought to be involved in many forms of programmed cell death. The role of ROS in cell death caused by oxidative glutamate toxicity was studied in an immortalized mouse hippocampal cell line (HT22). The causal relationship between ROS production and glutathione (GSH) levels, gene expression, caspase activity, and cytosolic Ca2+ concentration was examined. An initial 5–10-fold increase in ROS after glutamate addition is temporally correlated with GSH depletion. This early increase is followed by an explosive burst of ROS production to 200–400-fold above control values. The source of this burst is the mitochondrial electron transport chain, while only 5–10% of the maximum ROS production is caused by GSH depletion. Macromolecular synthesis inhibitors as well as Ac-YVAD-cmk, an interleukin 1β–converting enzyme protease inhibitor, block the late burst of ROS production and protect HT22 cells from glutamate toxicity when added early in the death program. Inhibition of intracellular Ca2+ cycling and the influx of extracellular Ca2+ also blocks maximum ROS production and protects the cells. The conclusion is that GSH depletion is not sufficient to cause the maximal mitochondrial ROS production, and that there is an early requirement for protease activation, changes in gene expression, and a late requirement for Ca2+ mobilization.

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Chrysophanol Regulates Cell Death, Metastasis, and Reactive Oxygen Species Production in Oral Cancer Cell Lines

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2020/5867064 ◽

2020 ◽

Vol 2020 ◽

pp. 1-10

Author(s):

Po-Chih Hsu ◽

Ching-Feng Cheng ◽

Po-Chun Hsieh ◽

Yi-Hsuan Chen ◽

Chan-Yen Kuo ◽

...

Keyword(s):

Reactive Oxygen Species ◽

Cell Cycle ◽

Cell Death ◽

Oral Cancer ◽

Cell Lines ◽

Cancer Cell ◽

Cell Cycle Progression ◽

Reactive Oxygen ◽

Oxygen Species ◽

Cycle Progression

Background. Oral cancer belongs to the class of head and neck cancers and can be life threatening if not diagnosed and treated early. Activation of cell death via apoptosis or reactive oxygen species (ROS) accumulation and inhibition of cell cycle progression, migration, and epithelial-to-mesenchymal transition (EMT) may be a good strategy to arrest the development of oral cancer. In this study, we analyzed the possible action of chrysophanol isolated from the rhizomes of Rheum palmatum on the oral cancer cell lines FaDu (human pharynx squamous cell carcinoma) and SAS (human tongue squamous carcinoma) by investigating whether chrysophanol could influence cell death. Method. Cell viability was measured by using the MTT assay. For the detection of apoptosis, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining and subG1 population analysis were used. We also examined cell cycle progression and ROS levels by flow cytometry. Additionally, the expression of p53, p21, procaspase 3, cyclin D1, CDK4, cdc2, CDK2, E-cadherin, vimentin, and PCNA was evaluated by western blotting. Conclusion. Chrysophanol has an anticancer effect on FaDu and SAS cell lines. There is an increase in subG1 accumulation, ROS production, and cell cycle G1 arrest after treatment with chrysophanol. On the other hand, chrysophanol inhibited cell migration/metastasis and EMT. We proposed that chrysophanol may be a good candidate compound on oral cancer treatment in the further.

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Antioxidants Threaten Multikinase Inhibitor Efficacy against Liver Cancer by Blocking Mitochondrial Reactive Oxygen Species

Antioxidants ◽

10.3390/antiox10091336 ◽

2021 ◽

Vol 10 (9) ◽

pp. 1336

Author(s):

Blanca Cucarull ◽

Anna Tutusaus ◽

Tania Hernáez-Alsina ◽

Pablo García de Frutos ◽

María Reig ◽

...

Keyword(s):

Reactive Oxygen Species ◽

Cell Death ◽

Liver Cancer ◽

Cell Lines ◽

Reactive Oxygen ◽

Chemotherapeutic Agents ◽

Glutathione Depletion ◽

Ros Production ◽

Oxygen Species ◽

Tumor Spheroids

Sorafenib and regorafenib, multikinase inhibitors (MKIs) used as standard chemotherapeutic agents for hepatocellular carcinoma (HCC), generate reactive oxygen species (ROS) during cancer treatment. Antioxidant supplements are becoming popular additions to our diet, particularly glutathione derivatives and mitochondrial-directed compounds. To address their possible interference during HCC chemotherapy, we analyzed the effect of common antioxidants using hepatoma cell lines and tumor spheroids. In liver cancer cell lines, sorafenib and regorafenib induced mitochondrial ROS production and potent cell death after glutathione depletion. In contrast, cabozantinib only exhibited oxidative cell death in specific HCC cell lines. After sorafenib and regorafenib administration, antioxidants such as glutathione methyl ester and the superoxide scavenger MnTBAP decreased cell death and ROS production, precluding the MKI activity against hepatoma cells. Interestingly, sorafenib-induced mitochondrial damage caused PINK/Parkin-dependent mitophagy stimulation, altered by increased ROS production. Finally, in sorafenib-treated tumor spheroids, while ROS induction reduced tumor growth, antioxidant treatments favored tumor development. In conclusion, the anti-tumor activity of specific MKIs, such as regorafenib and sorafenib, is altered by the cellular redox status, suggesting that uncontrolled antioxidant intake during HCC treatment should be avoided or only endorsed to diminish chemotherapy-induced side effects, always under medical scrutiny.

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Parthenolide and DMAPT Induce Cell Death in Chronic Myeloid Leukemia Cells Via Generation of Reactive Oxygen Species

Blood ◽

10.1182/blood.v128.22.4242.4242 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4242-4242

Author(s):

Gabriela Flores-López ◽

Hector Mayani ◽

María Antonieta Chávez-Gonzalez

Keyword(s):

Reactive Oxygen Species ◽

Cell Death ◽

Chronic Myeloid Leukemia ◽

Tyrosine Kinase ◽

Cell Lines ◽

Myeloid Leukemia ◽

Reactive Oxygen ◽

Hl60 Cell ◽

Oxygen Species ◽

Induce Cell Death

Abstract Chronic Myeloid Leukemia (CML) is a malignant hematologic disease characterized by the presence of a chromosomal alteration known as the Philadelphia Chromosome (Ph). Ph originates in Leukemia Stem Cells (LSC) and is caused by the reciprocal translocation between chromosome 9 and 22, giving rise to the Bcr-Abl oncogene, which produces a constitutively active tyrosine kinase -the BCR-ABL oncoprotein. This CML feature has made it possible the development of Tyrosine Kinase Inhibitors (TKI's) Imatinib, Dasatinib, and Nilotinib, which are able to inhibit BCR-ABL and take patients into molecular remission. However TKI's are not able to eliminate the population of LSC, which cause patients to relapse if treatment is discontinued. This has made it necessary search for alternative ways to eliminate LSC in CML patients. Parthenolide (PTL), a sesquiterpene lactone extracted from the feverfew plant (Thanacethum partenium), which has been used in folk medicine to treat inflammatory symptoms, has shown activity against LSC in Acute Myeloid Leukemia (AML). Recently Guzman and collaborators have developed an analog of PTL, named DMAPT, which is orally bioavailable. Both molecules have shown to be active inhibitors of NFkB and induce high levels of Reactive Oxygen Species (ROS) in LSC of AML. In the present study we asked whether CML cells (LSC, Progenitors and CML cell lines) are sensitive to PTL and DMAPT. Cell lines K562, Kcl-22 and HL60 (as an AML control) were cultured in liquid suspension cultures with increasing concentrations of PTL or DMAPT at a cell density of 300,000 cells per ml. At 24 hours of exposure, PTL and DMAPT showed significant induction of apoptosis at 7.5 µM and 10 µM, respectively. To determine whether NFkB activity is inhibited by PTL and DMAPT, K562, Kcl-22, HL60 cell lines, as well as CD34+ cells from a CML patient in Chronic phase where exposed to 7.5 µM PTL or 10 µM DMAPT. After 6 hours, protein lysis was induced and Western blots were performed with protein extracts from 250,000 cells per lane. Membranes where probed for Phosphorilated-p65, total p65 and b-actin as a loading control. We observed a decrease in the levels of phosphorylated p65 in cells treated with PTL and DMAPT, compared with untreated cells, while levels of total p65 remained constant. We had previosly used DCFDA stain and HMOX expression, to observe ROS induction in CML cells exposed to PTL. To asses if ROS induction is necessary for CML cell death we pretreated cells with N-acetyl cysteine (NAC), precursor of glutathione, for 1 hour before treating cells with 7.5 µM PTL or 10 µM DMAPT, and evaluated ROS levels by staining with Cell rox deep red, Mitosox-PE, and also assessing cell viability with YoPro-1 and 7AAD stain. After 6 hours of PTL treatment, we found that NAC pretreatment diminished ROS levels in cytoplasm and mitochondrial dysfunction when cells were treated with PTL. Pretreatment with NAC was able to rescue the majority of cells from apoptosis induced by PTL. Similar results were observed in cultures treated with DMAPT. Taken together, our results indicate that PTL and DMAPT were able to induce cell death via apoptosis in CML cells. This process seems to involve inhibition of p65 phosphorylation and an increase in ROS levels, which we found to be necessary for cell death to occur. Disclosures No relevant conflicts of interest to declare.

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Gadd153 Sensitizes Cells to Endoplasmic Reticulum Stress by Down-Regulating Bcl2 and Perturbing the Cellular Redox State

Molecular and Cellular Biology ◽

10.1128/mcb.21.4.1249-1259.2001 ◽

2001 ◽

Vol 21 (4) ◽

pp. 1249-1259 ◽

Cited By ~ 1288

Author(s):

Karen D. McCullough ◽

Jennifer L. Martindale ◽

Lars-Oliver Klotz ◽

Tak-Yee Aw ◽

Nikki J. Holbrook

Keyword(s):

Reactive Oxygen Species ◽

Endoplasmic Reticulum ◽

Cell Death ◽

Er Stress ◽

Cell Lines ◽

Reactive Oxygen ◽

Oxygen Species ◽

Down Regulation ◽

Gadd153 Expression ◽

Bcl2 Expression

ABSTRACT gadd153, also known as chop, is a highly stress-inducible gene that is robustly expressed following disruption of homeostasis in the endoplasmic reticulum (ER) (so-called ER stress). Although all reported types of ER stress induce expression of Gadd153, its role in the stress response has remained largely undefined. Several studies have correlated Gadd153 expression with cell death, but a mechanistic link between Gadd153 and apoptosis has never been demonstrated. To address this issue we employed a cell model system in which Gadd153 is constitutively overexpressed, as well as two cell lines in which Gadd153 expression is conditional. In all cell lines, overexpression of Gadd153 sensitized cells to ER stress. Investigation of the mechanisms contributing to this effect revealed that elevated Gadd153 expression results in the down-regulation of Bcl2 expression, depletion of cellular glutathione, and exaggerated production of reactive oxygen species. Restoration of Bcl2 expression in Gadd153-overexpressing cells led to replenishment of glutathione and a reduction in levels of reactive oxygen species, and it protected cells from ER stress-induced cell death. We conclude that Gadd153 sensitizes cells to ER stress through mechanisms that involve down-regulation of Bcl2 and enhanced oxidant injury.

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