Evaluation of Anticancer and Anti-Mitotic Properties of Quinazoline and Quinazolino-Benzothiadiazine Derivatives

Background: Cancer is one of the major health and social-economic problems despite considerable progress in its early diagnosis and treatment. Owing to the emergence and increase of multidrug resistance to various conventional drugs, and the continuing importance of health-care expenditure, many researchers have focused on developing novel and effective anticancer compounds. Objective: Chemical repositories provide a good platform to evaluate and exploit known chemical entities for the identification of other biological activities. In the present study, we have selected an in-house library of synthesized compounds based on two different pharmacophoric scaffolds to evaluate their cytotoxic potency on various cancer cell lines and mechanisms of action. Methods: A series of in-house synthesized quinazoline and quinazolino-benzothiadiazine derivatives were investigated for their anticancer efficacy against a panel of five cancer (DU145, MCF7, HepG2, SKOV3 and MDA-MB-231) and one normal (MRC5) cell lines. Furthermore, the active compound of the study was investigated to elucidate the mechanism of cytotoxicity by performing series of experiments such as cell cycle analysis, inhibition of tubulin polymerization, alteration of mitochondrial membrane potential, determination of endocytic pathway for drug uptake pathway and combination drug treatment. Results: Among all the tested compounds, fifteen of them exhibited promising growth-inhibitory effect (0.15- 5.0μM) and induced cell cycle arrest in the G2/M phase. In addition, the selected compounds inhibited the microtubule assembly; altered mitochondrial membrane potential and enhanced the levels of caspase-9 in MCF-7 cells. Furthermore, the active compound with a combination of drugs showed a synergistic effect at lower concentrations, and the drug uptake was mediated through clathrin-mediated endocytic pathway. Conclusion: Our results indicated that quinazoline and quinazolino-benzothiadiazine conjugates could serve as potential leads in the development of new anticancer agents.

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Itraconazole, an Oral Antifungal Drug, Is Active in Chemotherapy Resistant B-Cell Non-Hodgkin Lymphoma and Enhances the Anti-Tumor Activity of Chemotherapy Agents

Blood ◽

10.1182/blood.v128.22.5138.5138 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5138-5138

Author(s):

Juan J Gu ◽

Lianjuan Yang ◽

Cory Mavis ◽

Matthew J. Barth ◽

Francisco J. Hernandez-Ilizaliturri

Keyword(s):

Cell Cycle ◽

Membrane Potential ◽

B Cell ◽

Mitochondrial Membrane Potential ◽

Cell Lines ◽

Mitochondrial Membrane ◽

In Vitro Exposure ◽

Chemotherapy Agents ◽

Anti Tumor Activity

Abstract Background: Relapsed/refractory diffuse large B-cell lymphoma (DLBCL) patients previously treated with rituximab-based therapy have poor clinical outcome, according to the results from collaborative trial in relapsed aggressive lymphoma (CORAL) study. It stresses the need to identify and/or optimize novel targeted agents. To better understand the molecular mechanisms underlining the acquired resistance to rituximab, we generated and characterized several rituximab-resistant DLBCL cell lines (RRCLs). Itraconazole, an oral antifungal agent, was reported had novel anticancer activity in basal cell carcinoma, non-small cell lung cancer and prostate cancer. In our current work, we define and characterize the anticancer activity of itraconazole in preclinical rituximab-sensitive or -resistant lymphoma models. Methods: A panel of rituximab-sensitive (RSCL) and rituximab-resistant (RRCL) cell lines were exposed to escalating doses of itraconazole (0-20μM) for 24, 48 and 72h. Changes in cell viability and cell cycle distribution were evaluated using the Presto Blue assay and flow cytometry respectively. IC50 was calculated by Graphpad Prism6 software. Loss of mitochondrial membrane potential (∆ψm) following itraconazole exposure was assessed by DiOC6 and flow cytometry. Subsequently lymphoma cells were exposed to itraconazole or vehicle and various chemotherapy agents such as doxorubicin (1µM), dexamethasone (1µM), cDDP (20μg/ml), bortezomib (20nM), carfilzomib (20nM) or MLN2238 (20nM) for 48 hours. Coefficient of synergy was calculated using the CalcuSyn software. Changes in hexokinase II (HKII), Voltage dependent anion channel protein (VDAC), LC3 and BCL-xL expression levels were determined by western blotting after exposure cells to itraconazole. VDAC-HKII interactions following in vitro exposure to itraconazole were determined by immunoprecipitation of VDAC and probing for HKII in RSCL and RRCLs. Result:Itraconazole consistently showed potent, specific, dose-and time- dependent inhibition of all our sensitive and resistant lymphoma cell lines. In vitro exposure cells to itraconazole resulted in a loss of mitochondrial membrane potential and caused G2 cell cycle arrest. Itraconazole significantly had a synergistic anti-tumor effect combined with various chemotherapeutic agents, including doxorubicin, dexamethasone, cisplatin and different generations of proteasome inhibitors (bortezomib, carfilzomib or ixazomib) in both RSCL and RRCL. Western blot and immunoprecipitation studies demonstrated that following exposure to itraconazole, HKII bound less to mitochondrial specific protein VDAC. Complete silencing of HKII (using HKII siRNA interference) resulted in a rescue of loss in the mitochondrial membrane potential induced by intraconazole. Conclusion: Taking together, our data suggest that itraconazole had a potent anti-tumor activity against rituximab-sensitive or resistant pre-clinical models. The disruption of HKII from mitochondria following itraconazole exposure may contribute to lower the mitochondrial membrane potential and enhance the chemotherapeutic efficacy. Our finding highlights itraconazole as a potential therapeutic agent in the treatment of B-cell malignancies, and strongly supports clinical translation of its use. Disclosures No relevant conflicts of interest to declare.

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Dihydroorotate Dehydrogenase Inhibitors Promote Cell Cycle Arrest and Disrupt Mitochondria Bioenergetics in Ramos Cells

Current Pharmaceutical Biotechnology ◽

10.2174/1389201021666200611113734 ◽

2020 ◽

Vol 21 (15) ◽

pp. 1654-1665

Author(s):

Mohamad F.A. Kadir ◽

Shatrah Othman ◽

Kavitha Nellore

Keyword(s):

Cell Cycle ◽

Membrane Potential ◽

Mitochondrial Membrane Potential ◽

Cell Lines ◽

Mitochondrial Membrane ◽

Myelogenous Leukemia ◽

Therapeutic Window ◽

Proliferation Inhibition ◽

Dihydroorotate Dehydrogenase ◽

Cancerous Cells

Background: The re-emerging of targeting Dihydroorotate Dehydrogenase (DHODH) in cancer treatment particularly Acute Myelogenous Leukemia (AML) has corroborated the substantial role of DHODH in cancer and received the attention of many pharmaceutical industries. Objective: The effects of Brequinar Sodium (BQR) and 4SC-101 on lymphoblastoid cell lines were investigated. Methods: DHODH expression and cell proliferation inhibition of lymphoblastoid and lymphoma cell lines were analyzed using Western blot analysis and XTT assay, respectively. JC-1 probe and ATP biochemiluminescence kit were used to evaluate the mitochondrial membrane potential and ATP generation in these cell lines. Furthermore, we explored the cell cycle progression using Muse™ Cell Cycle Kit. Results: Ramos, SUDHL-1 and RPMI-1788 cells are fast-growing cells with equal expression of DHODH enzyme and sensitivity to DHODH inhibitors that showed that the inhibition of DHODH was not cancer-specific. In ATP depletion assay, the non-cancerous RPMI-1788 cells showed only a minor ATP reduction compared to Ramos and SUDHL-1 (cancer) cells. In the mechanistic impact of DHODH inhibitors on non-cancerous vs cancerous cells, the mitochondrial membrane potential assay revealed that significant depolarization and cytochrome c release occurred with DHODH inhibitors treatment in Ramos but not in the RPMI-1788 cells, indicating a different mechanism of proliferation inhibition in normal cells. Conclusion: The findings of this study provide evidence that DHODH inhibitors perturb the proliferation of non-cancerous cells via a distinct mechanism compared to cancerous cells. These results may lead to strategies for overcoming the impact on non-cancerous cells during treatment with DHODH inhibitors, leading to a better therapeutic window in patients.

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Reserpine Induces Apoptosis and Cell Cycle Arrest in Hormone Independent Prostate Cancer Cells through Mitochondrial Membrane Potential Failure

Anti-Cancer Agents in Medicinal Chemistry ◽

10.2174/1871520618666180209152215 ◽

2019 ◽

Vol 18 (9) ◽

pp. 1313-1322 ◽

Cited By ~ 5

Author(s):

Manjula Devi Ramamoorthy ◽

Ashok Kumar ◽

Mahesh Ayyavu ◽

Kannan Narayanan Dhiraviam

Keyword(s):

Prostate Cancer ◽

Reactive Oxygen Species ◽

Cell Cycle ◽

Membrane Potential ◽

Cancer Cells ◽

Mitochondrial Membrane Potential ◽

Mitochondrial Membrane ◽

Reactive Oxygen ◽

Oxygen Species ◽

Prostate Cancer Cells

Background: Reserpine, an indole alkaloid commonly used for hypertension, is found in the roots of Rauwolfia serpentina. Although the root extract has been used for the treatment of cancer, the molecular mechanism of its anti-cancer activity on hormonal independent prostate cancer remains elusive. Methods: we evaluated the cytotoxicity of reserpine and other indole alkaloids, yohimbine and ajmaline on Prostate Cancer cells (PC3) using MTT assay. We investigated the mechanism of apoptosis using a combination of techniques including acridine orange/ethidium bromide staining, high content imaging of Annexin V-FITC staining, flow cytometric quantification of the mitochondrial membrane potential and Reactive Oxygen Species (ROS) and cell cycle analysis. Results: Our results indicate that reserpine inhibits DNA synthesis by arresting the cells at the G2 phase and showed all standard sequential features of apoptosis including, destabilization of mitochondrial membrane potential, reduced production of reactive oxygen species and DNA ladder formation. Our in silico analysis further confirmed that indeed reserpine docks to the catalytic cleft of anti-apoptotic proteins substantiating our results. Conclusion: Collectively, our findings suggest that reserpine can be a novel therapeutic agent for the treatment of androgen-independent prostate cancer.

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Synthesis and Biological Evaluation of Novel Heterocyclic Imines Linked Coumarin- Thiazole Hybrids as Anticancer Agents

Anti-Cancer Agents in Medicinal Chemistry ◽

10.2174/1871520619666190207140120 ◽

2019 ◽

Vol 19 (4) ◽

pp. 557-566 ◽

Cited By ~ 7

Author(s):

Nerella S. Goud ◽

Mahammad S. Ghouse ◽

Jatoth Vishnu ◽

Jakkula Pranay ◽

Ravi Alvala ◽

...

Keyword(s):

Cell Cycle ◽

Membrane Potential ◽

Fluorescence Spectroscopy ◽

Mitochondrial Membrane Potential ◽

Mitochondrial Membrane ◽

Annexin V ◽

Biological Evaluation ◽

Dependent Manner ◽

Dapi Staining ◽

Dose Dependent

Background: Human Galectin-1, a protein of lectin family showing affinity towards β-galactosides has emerged as a critical regulator of tumor progression and metastasis, by modulating diverse biological events including homotypic cell aggregation, migration, apoptosis, angiogenesis and immune escape. Therefore, galectin-1 inhibitors might represent novel therapeutic agents for cancer. Methods: A new series of heterocyclic imines linked coumarin-thiazole hybrids (6a-6r) was synthesized and evaluated for its cytotoxic potential against a panel of six human cancer cell lines namely, lung (A549), prostate (DU-145), breast (MCF-7 & MDA-MB-231), colon (HCT-15 & HT-29) using MTT assay. Characteristic apoptotic assays like DAPI staining, cell cycle, annexin V and Mitochondrial membrane potential studies were performed for the most active compound. Furthermore, Gal-1 inhibition was confirmed by ELISA and fluorescence spectroscopy. Results: Among all, compound 6g 3-(2-(2-(pyridin-2-ylmethylene) hydrazineyl) thiazol-4-yl)-2H-chromen-2- one exhibited promising growth inhibition against HCT-15 colorectal cancer cells with an IC50 value of 1.28 ± 0.14 µM. The characteristic apoptotic morphological features like chromatin condensation, membrane blebbing and apoptotic body formation were clearly observed with compound 6g on HCT-15 cells using DAPI staining studies. Further, annexin V-FITC/PI assay confirmed effective early apoptosis induction by treatment with compound 6g. Loss of mitochondrial membrane potential and enhanced ROS generation were confirmed with JC-1 and DCFDA staining method, respectively by treatment with compound 6g, suggesting a possible mechanism for inducing apoptosis. Moreover, flow cytometric analysis revealed that compound 6g blocked G0/G1 phase of the cell cycle in a dose-dependent manner. Compound 6g effectively reduced the levels of Gal-1 protein in a dose-dependent manner. The binding constant (Ka) of 6g with Gal-1 was calculated from the intercept value which was observed as 1.9 x 107 M-1 by Fluorescence spectroscopy. Molecular docking studies showed strong interactions of compound 6g with Gal-1 protein. Conclusion: Our studies demonstrate the anticancer potential and Gal-1 inhibition of heterocyclic imines linked coumarin-thiazole hybrids.

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Sulofenur cytotoxicity and changes in cytosolic calcium and mitochondrial membrane potential in human colon adenocarcinoma cell lines

Cancer Letters ◽

10.1016/0304-3835(94)03607-k ◽

1995 ◽

Vol 88 (1) ◽

pp. 27-35 ◽

Cited By ~ 9

Author(s):

Patricia C. Phelps ◽

Pramod T. Jain ◽

Irene K. Berezesky ◽

George B. Boder ◽

Benjamin F. Trump

Keyword(s):

Membrane Potential ◽

Mitochondrial Membrane Potential ◽

Cell Lines ◽

Mitochondrial Membrane ◽

Colon Adenocarcinoma ◽

Human Colon ◽

Cytosolic Calcium ◽

Adenocarcinoma Cell ◽

Human Colon Adenocarcinoma

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Metabolic Profile Of Leukemia Cells Influences Treatment Efficacy Of L‑Asparaginase

10.21203/rs.2.17519/v1 ◽

2019 ◽

Author(s):

Katerina Hlozkova ◽

Alena Pecinova ◽

David Pajuelo Reguera ◽

Marketa Simcikova ◽

Lenka Hovorkova ◽

...

Keyword(s):

Membrane Potential ◽

Mitochondrial Membrane Potential ◽

Cell Lines ◽

Mitochondrial Membrane ◽

Metabolic Profile ◽

Lymphoblastic Leukemia ◽

Leukemia Cell ◽

Leukemia Cells ◽

Lower Sensitivity ◽

Leukemia Cell Lines

Abstract Background Effectiveness of L-asparaginase administration in acute lymphoblastic leukemia treatment is mirrored in overall outcome of patients. Generally, leukemia patients differ in their sensitivity to L-asparaginase; however, the mechanism underlying their inter-individual differences is still not fully understood. We have previously shown that L-asparaginase rewires the biosynthetic and bioenergetic pathways of leukemia cells to activate both anti-leukemic and pro-survival processes. Herein, we investigated the relationship between the metabolic profile of leukemia cells and their sensitivity to currently used cytostatic drugs.Methods Altogether, 19 leukemia cell lines and primary leukemia cells from 11 patients were used. Glycolytic function and mitochondrial respiration were measured using Seahorse bioanalyzer. Sensitivity to cytostatics was measured using MTS assay and/or absolute count and flow cytometry. Mitochondrial membrane potential was determined as TMRE fluorescence.Results We characterized the basal metabolic state of the cells derived from different leukemia subtypes using cell lines and primary samples and assessed their sensitivity to cytostatic drugs. We found that leukemia cells cluster into distinct groups according to their metabolic profile, which is mainly driven by their hematopoietic lineage of origin from which they derived. However, majority of lymphoid leukemia cell lines and patients with lower sensitivity to L-asparaginase clustered regardless their hematopoietic phenotype together with myeloid leukemias. Furthermore, we observed a correlation of specific metabolic parameters with sensitivity to L-asparaginase. Greater ATP-linked respiration and lower basal mitochondrial membrane potential in cells significantly correlated with higher sensitivity to L-asparaginase. No such correlation was found in other tested cytostatic drugs.Conclusions These data support the prominent role of the cell metabolism in the treatment effect of L-asparaginase. Based on these findings metabolic profile could identify leukemia patients with lower sensitivity to L-asparaginase with no specific genetic characterization.

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Differential Effects of Silibinin A on Mitochondrial Function in Neuronal PC12 and HepG2 Liver Cells

Oxidative Medicine and Cellular Longevity ◽

10.1155/2019/1652609 ◽

2019 ◽

Vol 2019 ◽

pp. 1-10 ◽

Cited By ~ 4

Author(s):

Carsten Esselun ◽

Bastian Bruns ◽

Stephanie Hagl ◽

Rekha Grewal ◽

Gunter P. Eckert

Keyword(s):

Membrane Potential ◽

Oxidative Phosphorylation ◽

Mitochondrial Membrane Potential ◽

Cell Lines ◽

Mitochondrial Function ◽

Mitochondrial Membrane ◽

Nitrosative Stress ◽

Citrate Synthase ◽

Atp Levels ◽

Age Related

The Mediterranean plant Silybum marianum L., commonly known as milk thistle, has been used for centuries to treat liver disorders. The flavonolignan silibinin represents a natural antioxidant and the main bioactive ingredient of silymarin (silybin), a standard extract of its seeds. Mitochondrial dysfunction and the associated generation of reactive oxygen/nitrogen species (ROS/RNS) are involved in the development of chronic liver and age-related neurodegenerative diseases. Silibinin A (SIL A) is one of two diastereomers found in silymarin and was used to evaluate the effects of silymarin on mitochondrial parameters including mitochondrial membrane potential and ATP production with and without sodium nitroprusside- (SNP-) induced nitrosative stress, oxidative phosphorylation, and citrate synthase activity in HepG2 and PC12 cells. Both cell lines were influenced by SIL A, but at different concentrations. SIL A significantly weakened nitrosative stress in both cell lines. Low concentrations not only maintained protective properties but also increased basal mitochondrial membrane potential (MMP) and adenosine triphosphate (ATP) levels. However, these effects could not be associated with oxidative phosphorylation. On the other side, high concentrations of SIL A significantly decreased MMP and ATP levels. Although SIL A did not provide a general improvement of the mitochondrial function, our findings show that SIL A protects against SNP-induced nitrosative stress at the level of mitochondria making it potentially beneficial against neurological disorders.

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Effects of Berberine on Cell Cycle, DNA, Reactive Oxygen Species, and Apoptosis in L929 Murine Fibroblast Cells

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2015/796306 ◽

2015 ◽

Vol 2015 ◽

pp. 1-13 ◽

Cited By ~ 7

Author(s):

Manman Gu ◽

Jing Xu ◽

Chunyang Han ◽

Youxi Kang ◽

Tengfei Liu ◽

...

Keyword(s):

Reactive Oxygen Species ◽

Cell Cycle ◽

Dna Damage ◽

Membrane Potential ◽

Mitochondrial Membrane Potential ◽

Cell Apoptosis ◽

Mitochondrial Membrane ◽

Oxygen Species ◽

Cycle Arrest ◽

Murine Fibroblast

Berberine, an isoquinoline alkaloid isolated from several traditional Chinese herbal medicines (TCM), exhibits a strong antimicrobial activity in the treatment of diarrhea. However, it causes human as well as animal toxicity from heavy dosage. The present study was conducted to investigate the cytotoxicity of berberine and its possible trigger mechanisms resulting in cell cycle arrest, DNA damage, ROS (reactive oxygen species) level, mitochondrial membrane potential change, and cell apoptosis in L929 murine fibroblast (L929) cells. The cells were culturedin vitroand treated with different concentrations of berberine for 24 h. The results showed that cell viability was significantly decreased in a subjected dose-dependent state; berberine concentrations were higher than 0.05 mg/mL. Berberine at a concentration above 0.1 mg/mL altered the morphology of L929 cells. Cells at G2/M phase were clear that the level of ROS and cell apoptosis rates increased in 0.1 mg/mL group. Each DNA damage indicator score (DIS) increased in groups where concentration of berberine was above 0.025 mg/mL. The mitochondrial membrane potential counteractive balance mechanics were significantly altered when concentrations of berberine were above 0.005 mg/mL. In all, the present study suggested that berberine at high dosage exhibited cytotoxicity on L929 which was related to resultant: cell cycle arrest; DNA damage; accumulation of intracellular ROS; reduction of mitochondrial membrane potential; and cell apoptosis.

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