Mitochondrial levels determine variability in cell death by modulating apoptotic gene expression

Background: Ovarian cancer is the leading cause of death caused by genital cancers. One of the most common treatments for this type of cancer is chemotherapy by cisplatin, which induces apoptosis in cancer cells. Apoptosis is a type of physiological cell death. Cisplatin chemotherapy usually has several side effects and cellular resistance to cisplatin is a common incidence. In order to overcome these problems, the use of combination therapies using natural substances has been considered. Fisetin is a flavonoid with anti-cancer activity which induces apoptosis. In this study, the apoptosis induced by cisplatin along with Fisetin in cisplatin-resistant ovarian cancer cell line (A2780) was investigated. Methods: In the present experimental study, the effect of combined use of Fisetin and cisplatin on ovarian cancer cell lines (A2780) was investigated by using MTT assay. Cell death was also determined by DAPI, acridine orange/propidium iodide, and Annexin/PI assay. Apoptotic gene expression of Bax, BCL-2, caspase 3, and caspase 9 was also assessed by real time PCR. Results: The results of MTT assay indicated that the combined treatment of Fisetin and cisplatin effectively inhibits proliferation of A2780 cells. The results of DAPI staining showed that fragmentation of chromatin in cells occurred in the combined treatment. Acridine orange-propidium iodide staining and Annexin/PI staining showed an increase in the rate of apoptotic cells in cells under combined treatment. The results of the study regarding changes in gene expression also indicated that Bax pro-apoptotic gene expression and BCL-2 anti-apoptotic gene expression increased in cells under treatment; moreover, gene expression of caspases 3 and 9 significantly increased as well. Conclusion: According to the findings of this study, the combined use of cisplatin and Fisetin increases the induction of apoptosis in cisplatin-resistant ovarian cancer cells (A2780); therefore, the combined use of cisplatin and Fisetin can be considered a promising strategy in the treatment of ovarian cancer.

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4-1BB-Costimulated CAR-Mediated Non-Canonical NF-Kb Signaling Enhances CAR T Cell Survival and Suppresses Bim Expression

Blood ◽

10.1182/blood-2018-99-119522 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3713-3713

Author(s):

Michael C. Milone ◽

Roddy OConnor ◽

Michael May ◽

Steven Albelda ◽

Benjamin Philipson

Keyword(s):

Gene Expression ◽

T Cells ◽

Cell Death ◽

T Cell ◽

Western Blot ◽

Fold Increase ◽

Apoptotic Gene ◽

Car T Cell ◽

Apoptotic Protein ◽

Car T

Abstract Chimeric Antigen Receptor (CAR) T cell therapy induces deep and durable responses in a large percentage of patients with B-cell malignancies. These responses often correlate with CAR T cell persistence in patients. The first two FDA-approved CAR-T cell therapies employ 2nd generation CARs that use different costimulatory domains derived from either CD28 or 4-1BB. Data from pre-clinical studies as well as clinical trials suggest that 4-1BB CAR (BBz) T cells persist longer than CD28 CAR (28z) T cells. One signal associated with cellular survival and activated by endogenous 4-1BB, but not CD28, is the non-canonical NF-kB (ncNF-kB) pathway. NcNF-kB is required for memory T cell persistence and enhances tumor cell survival by suppressing pro-apoptotic gene transcription. Therefore, we hypothesize that the BBz, but not the 28z CAR, activates ncNF-kB signaling, which promotes CAR T cell persistence by suppressing pro-apoptotic gene expression. Using primary human T cells isolated from the apheresis products of anonymous healthy donors, we generated 28z or BBz T cells by lentiviral transduction following activation by CD3 and CD28. Induction of ncNF-kB signaling following CAR activation was assessed by western blot. NcNF-kB function was evaluated by concomitant expression of a dominant-negative mutant of NF-kB-inducing Kinase (dnNIK) to block the ncNF-kB pathway. In addition to pathway analysis by western blotting, T cell proliferation, immunophenotype and survival were assessed by bead-based counting via flow cytometry. To explore mechanisms affecting survival, pro- and anti-apoptotic gene expression was analyzed by qPCR and western blot over three weeks following restimulation through the CAR. Following CAR activation, ncNF-kB signaling was detected in BBz but not 28z T cell lysates. Control mRFP BBz T cells expanded approximately 10 fold more than BBz T cells coexpressing dnNIK. DnNIK-expressing BBz T cells exhibited higher rates of cell death contributing to the observed differences apparent in ex vivo expansion. This cell death was associated with a 2 fold increase in the message and nearly 3 fold increase in the expression of the pro-apoptotic protein, Bim, in the dnNIK BBz T cells relative to control. In contrast, dnNIK 28z T cells had no appreciable difference in proliferation or survival compared with control. Therefore, BBz, but not 28z signaling activates the ncNF-kB pathway, which protects BBz T cells from cell death likely by restricting the expression of the pro-apoptotic protein, Bim. Disclosures Milone: Novartis: Patents & Royalties.

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Regulation of Pro-Apoptotic Bax and Trail Gene Expressions by H5N1 in Madin-Darby Canine Kidney (Mdck) Cell Line

Pakistan Veterinary Journal ◽

10.29261/pakvetj/2020.055 ◽

2020 ◽

Vol 40 (04) ◽

pp. 509-513

Author(s):

Wei Boon Yap

Keyword(s):

Gene Expression ◽

Cell Death ◽

Fas Ligand ◽

Mdck Cells ◽

Influenza Viruses ◽

High Morbidity ◽

Gene Expressions ◽

Apoptotic Gene ◽

Dna Laddering ◽

Cellular Apoptosis

Avian influenza virus (AIV) A H5N1 is able to cause zoonosis with high morbidity and mortality. Several studies suggested the regulation of cellular apoptosis for virus survival by influenza viruses. This sheds light on the development of antivirals that can improve cellular apoptosis to fight influenza infections. This study aimed to investigate the regulation of pro-apoptotic gene expressions by H5N1 in MDCK cells. The H5N1 infection (102.67TCID50/ml) was performed for 8, 24, 48, 72, 96 and 120 hours. Upon incubation, the percentage of cell death, DNA fragmentation and pro-apoptotic gene expressions i.e. Bax, TRAIL, Caspase 3 (Cas3) and 8 (Cas8), and Fas-ligand (FasL) were determined. The degree of DNA laddering and pro-apoptotic gene expressions were determined on agarose gels. The results indicated that severe cytopathic effects (CPE) caused by H5N1 appeared as early as 24 hour post-infection (hpi) meanwhile a significant cell death was observed starting at 48 hpi. However, DNA laddering was not observed in both of the control and infected DNA samples. Downregulation of Bax gene expression was recorded at 24 -72 hpi and a total suppression was observed at 96 and 120 hpi. Similarly, a significant decrease in the TRAIL gene expression was observed at 48-120 hpi. On the contrary, the expressions of Cas3, Cas8 and FasL genes were not detected in the mock and infected samples. Conclusively, this study suggested that H5N1 might downregulate cellular apoptosis prematurely for viral survival which can be a target cellular mechanism for newly developed antivirals to combat H5N1 infections

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Reverse Screening Bioinformatics Approach to Identify Potential Anti Breast Cancer Targets Using Thymoquinone from Neutraceuticals Black Cumin Oil

Anti-Cancer Agents in Medicinal Chemistry ◽

10.2174/1871520619666190124155359 ◽

2019 ◽

Vol 19 (5) ◽

pp. 599-609 ◽

Cited By ~ 1

Author(s):

Sumathi Sundaravadivelu ◽

Sonia K. Raj ◽

Banupriya S. Kumar ◽

Poornima Arumugamand ◽

Padma P. Ragunathan

Keyword(s):

Breast Cancer ◽

Gene Expression ◽

Cell Cycle ◽

Cell Death ◽

In Silico ◽

Chemotherapeutic Agents ◽

Normal Cells ◽

Black Cumin ◽

In Silico Docking ◽

Wide Range

Background: Functional foods, neutraceuticals and natural antioxidants have established their potential roles in the protection of human health and diseases. Thymoquinone (TQ), the main bioactive component of Nigella sativa seeds (black cumin seeds), a plant derived neutraceutical was used by ancient Egyptians because of their ability to cure a variety of health conditions and used as a dietary food supplement. Owing to its multi targeting nature, TQ interferes with a wide range of tumorigenic processes and counteracts carcinogenesis, malignant growth, invasion, migration, and angiogenesis. Additionally, TQ can specifically sensitize tumor cells towards conventional cancer treatments (e.g., radiotherapy, chemotherapy, and immunotherapy) and simultaneously minimize therapy-associated toxic effects in normal cells besides being cost effective and safe. TQ was found to play a protective role when given along with chemotherapeutic agents to normal cells. Methods: In the present study, reverse in silico docking approach was used to search for potential molecular targets for cancer therapy. Various metastatic and apoptotic targets were docked with the target ligand. TQ was also tested for its anticancer activities for its ability to cause cell death, arrest cell cycle and ability to inhibit PARP gene expression. Results: In silico docking studies showed that TQ effectively docked metastatic targets MMPs and other apoptotic and cell proliferation targets EGFR. They were able to bring about cell death mediated by apoptosis, cell cycle arrest in the late apoptotic stage and induce DNA damage too. TQ effectively down regulated PARP gene expression which can lead to enhanced cancer cell death. Conclusion: Thymoquinone a neutraceutical can be employed as a new therapeutic agent to target triple negative breast cancer which is otherwise difficult to treat as there are no receptors on them. Can be employed along with standard chemotherapeutic drugs to treat breast cancer as a combinatorial therapy.

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Programmed cell death 4 and BCR-ABL fusion gene expression are negatively correlated in chronic myeloid leukemia

Oncology Letters ◽

10.3892/ol.2016.4942 ◽

2016 ◽

Vol 12 (4) ◽

pp. 2976-2981 ◽

Cited By ~ 2

Author(s):

Xia Zhang ◽

Riming Liu ◽

Baohua Huang ◽

Xiaolu Zhang ◽

Weijuan Yu ◽

...

Keyword(s):

Gene Expression ◽

Cell Death ◽

Chronic Myeloid Leukemia ◽

Programmed Cell Death ◽

Myeloid Leukemia ◽

Fusion Gene ◽

Programmed Cell Death 4

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Molecular pathology associated with altered synaptic transcriptome in the dorsolateral prefrontal cortex of depressed subjects

Translational Psychiatry ◽

10.1038/s41398-020-01159-9 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Yuta Yoshino ◽

Bhaskar Roy ◽

Nilesh Kumar ◽

M. Shahid Mukhtar ◽

Yogesh Dwivedi

Keyword(s):

Gene Expression ◽

Cell Death ◽

Prefrontal Cortex ◽

Dorsolateral Prefrontal Cortex ◽

Gene Network ◽

Nervous System Development ◽

Network Analyses ◽

Dorsolateral Prefrontal ◽

Total Fraction ◽

Transcriptomic Changes

AbstractDisrupted synaptic plasticity is the hallmark of major depressive disorder (MDD), with accompanying changes at the molecular and cellular levels. Often, the maladaptive molecular changes at the synapse are the result of global transcriptional reprogramming dictated by activity-dependent synaptic modulation. Thus far, no study has directly studied the transcriptome-wide expression changes locally at the synapse in MDD brain. Here, we have examined altered synaptic transcriptomics and their functional relevance in MDD with a focus on the dorsolateral prefrontal cortex (dlPFC). RNA was isolated from total fraction and purified synaptosomes of dlPFC from well-matched 15 non-psychiatric controls and 15 MDD subjects. Transcriptomic changes in synaptic and total fractions were detected by next-generation RNA-sequencing (NGS) and analyzed independently. The ratio of synaptic/total fraction was estimated to evaluate a shift in gene expression ratio in MDD subjects. Bioinformatics and network analyses were used to determine the biological relevance of transcriptomic changes in both total and synaptic fractions based on gene–gene network, gene ontology (GO), and pathway prediction algorithms. A total of 14,005 genes were detected in total fraction. A total of 104 genes were differentially regulated (73 upregulated and 31 downregulated) in MDD group based on 1.3-fold change threshold and p < 0.05 criteria. In synaptosomes, out of 13,236 detectable genes, 234 were upregulated and 60 were downregulated (>1.3-fold, p < 0.05). Several of these altered genes were validated independently by a quantitative polymerase chain reaction (qPCR). GO revealed an association with immune system processes and cell death. Moreover, a cluster of genes belonged to the nervous system development, and psychological disorders were discovered using gene–gene network analysis. The ratio of synaptic/total fraction showed a shift in expression of 119 genes in MDD subjects, which were primarily associated with neuroinflammation, interleukin signaling, and cell death. Our results suggest not only large-scale gene expression changes in synaptosomes, but also a shift in the expression of genes from total to synaptic fractions of dlPFC of MDD subjects with their potential role in immunomodulation and cell death. Our findings provide new insights into the understanding of transcriptomic regulation at the synapse and their possible role in MDD pathogenesis.

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