7-Geranyloxcycoumarin enhances radio sensitivity in human prostate cancer cells.

BACKGROUND: Prostate cancer is the second most prevalent and the fifth deadliest cancer among men worldwide. To improve radiotherapy outcome, we investigated the effects of 7-geranyloxycoumarin, also known as auraptene (AUR), on radiation response of prostate cancer cells. METHODS AND RESULTS: PC3 cells were pretreated with 20 and 40 µM AUR for 24, 48 and 72 h, followed by X-ray exposure (2, 4 and 6 Gy). After 72 h recovery, cell viability was determined by alamar Blue assay. Flow cytometric analysis was performed to assess apoptosis induction, clonogenic assay was carried out to investigate clonogenic survival, and the expression of P53, BAX, BCL2, CCND1 and GATA6 was analyzed by quantitative polymerase chain reaction (qPCR). Cell viability assay indicated that toxic effects of radiation was enhanced by AUR, which was also confirmed by increased numbers of apoptotic cells and reduced amount of survival fraction. The qPCR results demonstrated significant induction of P53 and BAX, while the expression of BCL2, GATA6, and CCND1 was significantly downregulated. CONCLUSION: The findings of the present study indicated, for the first time, that AUR improved radio sensitivity in prostate cancer cells, and thus, has the potential to be used in future clinical trials.

Comparing toxicity of galbanic acid, auraptene and umbelliprenin on adult T-cell leukaemia-lymphoma in normoxia and hypoxia.

Natural coumarins are valuable agents that induce anticancer effects and/or enhance sensitivity to therapeutic modalities. Galbanic acid (GBA), auraptene (AUR) and umbelliprenin (UMB) are coumarins derived from Ferula species with various pharmaceutical activities. The aim of the current research was to compare toxic effects of GBA, AUR, and UMB on human lymphoma cells in normoxia and hypoxia. In this regard, GBA and AUR were extracted from the roots of F. szowitsiana and UMB was derived from the roots of F. persica, all by thin-layer chromatography. MT-2 cells were treated with each agent for 3 consequent periods, while exposed to different O2 contents (21% and 2%). By the end of each treatment, the viability of MT-2 cells was determined by resazurin dye-based colorimetric assay. Obtained results revealed that low doses of GBA (10 and 20 µM) induced significant (p < 0.0001) toxic effects in hypoxia. However, similar toxicity was observed when cells were treated with 40 µM AUR in normoxia and hypoxia. Notably, UMB was the only coumarin that exerted cytotoxic effects in all time points (48, 72 and 96 h) in normoxia and hypoxia, although its concentration was highest (80 µM). In conclusion, this is the first report indicating GBA was the most toxic coumarin against ATL cells in hypoxia, AUR induced similar effects in normoxia and hypoxia, and low toxicity of UMB was stable during the time and different O2 contents. Future studies on other ATL cell lines are recommended to better evaluate the toxic effects of GBA, AUR and UMB in vitro.

Efficacy of hyperthermia in human colon adenocarcinoma cells is improved by auraptene.

Colon adenocarcinoma is one of the most common cancers worldwide, and resistance to current therapeutic modalities is a serious drawback in its treatment. Auraptene is a natural coumarin with considerable anticancer effects. The goal of this study was to introduce a novel combinatorial approach for treatment against colon adenocarcinoma cells. To do so, HT29 cells were pretreated with nontoxic auraptene and then hyperthermia was induced. Afterwards, the viability of the cells was assessed, changes induced in the cell cycle were analyzed, and the expression patterns of candidate genes were studied. Results from the MTT assay demonstrated significant (p < 0.01) decreases in cell viability when 20 µg/mL auraptene was used for 72 h, heat shock was induced, and cells were allowed to recover for 24 h. Flow cytometry analysis also indicated considerable changes in the distribution of cells between the sub-G1/G1 and G2/M phases of cell cycle after the combinatorial treatment. Real-time RT-PCR studies revealed significant (p < 0.01) up-regulation of P21 in the cells pretreated with auraptene after heat shock, whereas no significant change was observed in HSP27 expression. Our findings not only indicate, for the first time, that the efficacy of hyperthermia was improved by auraptene pretreatment, but also suggest that this coumarin could be used in the future to achieve more effective therapeutic outcomes.

Synergy between Auraptene, Ionizing Radiation, and Anticancer Drugs in Colon Adenocarcinoma Cells.

Colorectal cancer is a growing health concern with increasing mortality rates, and resistance to anticancer drugs and radiotherapy is a serious drawback in its treatment. Auraptene is a natural prenyloxycoumarin with valuable anticancer effects. The aim of current study was to determine the synergy between auraptene, ionizing radiation, and chemotherapeutic drugs in colon adenocarcinoma cells for the first time. To do so, HT29 cells were treated with combination of auraptene + cisplatin, + doxorubicin, or + vincristine. Furthermore, cells were pretreated with nontoxic auraptene and then exposed to various doses of X-radiation. Assessment of cell viability not only indicated significant (p < 0.05) synergic effects of auraptene and anticancer agents, also revealed more significant (p < 0.01) increase in the toxicity of applied radiations in auraptene pretreated cells. Interesting synergy between auraptene and radiotherapy was then confirmed by morphological alterations, DAPI staining, and flow cytometric analysis of the cell cycle. Moreover, real-time reverse transcription polymerase chain reaction analysis indicated significant (p < 0.01) overexpression of p21, but not GATA6, in auraptene pretreated cells after radiotherapy, and also significant (p < 0.01) down regulation of CD44 and ALDH1 by auraptene. According to present results, auraptene could be considered as an effective natural coumarin to improve the outcome of current chemoradiotherapy options. Copyright © 2017 John Wiley & Sons, Ltd.

Cancer stem cells in human digestive tract malignancies.

Digestive tract malignancies, including oral, pharyngeal, esophageal, gastric, and colorectal cancers, are among the top 10 most common cancers worldwide. In spite of using various treatment modalities, cancer patients still suffer from recurrence and metastasis of malignant cells. Cancer stem cells (CSCs) are undifferentiated and highly proliferative malignant cells with unique properties mediated by overexpression of stemness markers, metastasis-related proteins, drug transporters, and DNA repair machinery. Due to their salient characteristics, it has been suggested that CSCs are responsible for tumor initiation, progression, invasion, recurrence, and therapy resistance. Exploring different aspects of CSC biology has fueled a great enthusiasm in designing novel therapeutic strategies to help patients. For instance, identification of markers associated with digestive tract CSCs, such as CD44, CD133, CD24, EpCAM, LGR5, ALDH1, and BMI1, has made it possible to develop more accurate diagnosis approaches. In addition, specifically targeting CSCs by their markers imposes fewer side effects and improves therapeutic outcomes. Here, we focus on the current status of CSC biology in digestive tract cancers, with emphasis on CSC markers, and review achieved progress in eradication of digestive tract CSC cells.

The cytotoxic activities of 7-isopentenyloxycoumarin on 5637 cells via induction of apoptosis and cell cycle arrest in G2/M stage.

BACKGROUND: Bladder cancer is the second common malignancy of genitourinary tract, and transitional cell carcinomas (TCCs) account for 90% of all bladder cancers. Due to acquired resistance of TCC cells to a wide range of chemotherapeutic agents, there is always a need for search on new compounds for treatment of these cancers. Coumarins represent a group of natural compounds, which some of them have exerted valuable anti-tumor activities. The current study was designed to evaluate anti-tumor properties and mechanism of action of 7-isopentenyloxycoumarin, a prenyloxycoumarin, on 5637 cells (a TCC cell line). RESULTS: MTT results revealed that the cytotoxic effects of 7-isopentenyloxycoumarin on 5637 cancerous cells were more prominent in comparison to HDF-1 normal cells. This coumarin increased the amount of chromatin condensation and DNA damage in 5637 cells by 58 and 33%, respectively. The results also indicated that it can induce apoptosis most probably via activation of caspase-3 in these cells. Moreover, propidium iodide staining revealed that 7-isopentenyloxycoumarin induced cell cycle arrest at G2/M stage, after 24 h of treatment. CONCLUSION: Our results indicated that 7-isopentenyloxycoumarin had selective toxic effects on this bladder cancer cell line and promoted its effects by apoptosis induction and cell cycle arrest. This coumarin can be considered for further studies to reveal its exact mechanism of action and also its anti-cancer effects in vivo.

Natural Flavonoid Apigenin, an Effective Agent Against Nervous System Cancers.

Cancer is a serious public health concern worldwide, and nervous system (NS) cancers are among the most life-threatening malignancies. Efforts have been devoted to introduce natural anticancer agents with minimal side effects. Apigenin is an edible flavonoid that is abundantly found in many vegetables and fruits. Various pharmaceutical activities, including anti-inflammatory, antioxidative, antimicrobial, and anticancer effects have been reported for apigenin. This review provides insights into the therapeutic effects of apigenin and flavonoids with similar structure on glioblastoma and neuroblastoma. Current evidence indicates that apigenin has the unique ability to cross the blood-brain barrier, and its antioxidative, anti-inflammatory, neurogenic, and neuroprotective effects have made this flavonoid a great option for the treatment of neurodegenerative disorders. Meanwhile, apigenin has low toxicity on normal neuronal cells, while induces cytotoxicity on NS cancer cells via triggering several signal pathways and molecular targets. Anticancer effects of apigenin have been contributed to various mechanisms such as induction of cell cycle arrest and apoptosis, and inhibition of migration, invasion, and angiogenesis. Although apigenin is a promising pharmaceutical agent, its low bioavailability is an important issue that must be solved before introducing to clinic. Recently, nano-delivery of apigenin by liposomes and poly lactic-co-glycolide nanoparticles has greatly improved functionality of this agent. Hence, investigating pharmaceutical effects of apigenin-loaded nanocarriers on NS cancer cell lines and animal models is recommended for future studies.

Joining up the scattered anticancer knowledge on auraptene and umbelliprenin: a meta-analysis.

Auraptene (AUR) and umbelliprenin (UMB) are naturally occurring prenylated coumarins that have demonstrated promising anticancer effects across various human cancer cell lines. This meta-analysis aimed to systematically assess, compare, and quantify the anticancer efficacy of AUR and UMB by synthesizing evidence from in vitro studies. A comprehensive literature search identified 27 eligible studies investigating AUR or UMB against cancer cells. Mixed-effects models revealed significant negative associations between coumarin dose and viability for AUR (est. = - 2.27) and UMB (est. = - 3.990), underscoring their dose-dependent cytotoxicity. Meta-regression indicated slightly higher potency for UMB over AUR, potentially due to increased lipophilicity imparted by additional isoprenyl units. Machine learning approaches identified coumarin dose and cancer type as the most influential determinants of toxicity, while treatment duration and the specific coumarin displayed weaker effects. Moderate (AUR) to substantial (UMB) between-study heterogeneity was detected, although the findings proved robust. In summary, this meta-analysis establishes AUR and UMB as promising natural anticancer candidates with clear dose-toxicity relationships across diverse malignancies. The structural insights and quantifications of anticancer efficacy can inform forthcoming efforts assessing therapeutic potential in pre-clinical models and human trials.

Illuminating new possibilities: Effects of copper oxide nanoparticles on gastrointestinal adenocarcinoma cells in hypoxic condition.

Cancer remains a major global health concern, necessitating the development of novel therapeutic approaches. Hypoxia is a common characteristic of solid tumors that plays a critical role in tumor progression, making it a prime target for anticancer therapies. This study aimed to determine the effects of copper oxide nanoparticles (CuONPs) on human gastrointestinal cancer cells in hypoxic condition for the first time. Toxicity of CuONPs was evaluated on human colon and gastric adenocarcinoma cells and normal fibroblasts by alamarBlue assay. Real-time polymerase chain reaction (PCR) was performed to study the effects of CuONPs on genes involved in cell apoptosis. To elucidate the molecular mechanisms underlying the effects of CuONPs in hypoxic condition, molecular docking was conducted on HIF-1α. Results revealed dose- and cell-type-dependent toxic effects of CuONPs, as a more significant (p < 0.0001) decrease in viability of LoVo cells (23 %) was observed compared to MKN-45 and HDF cells. In addition, CuONPs significantly (p < 0.0001) reduced LoVo cell viability down to 30.2 % in hypoxic condition. Gene expression analysis revealed significant (p < 0.0001) overexpression of P53 and BAX but downregulation of BCL-2 and CCND1 after treatment with CuONPs. Molecular docking indicated the preferable binding of CuONPs to the HIF-1α PAS-B domain through interaction with 15 residues with -4.8 kcal/mol binding energy. Our findings open up new possibilities for modulating HIF-1 activity and inhibiting hypoxia-induced tumor progression.

Developing new drugs for adult T-cell leukemia/lymphoma by targeting hypoxia: insights from toxicity of MS-275 and its analogs.

The low survival rate of adult T-cell leukemia/lymphoma (ATL) underscores the critical need for innovative therapeutic agents. While the pharmacokinetics of HDACis have been documented in several hematological neoplasms, there is a notable gap in research regarding their activity against ATL. Given that hypoxia can induce unpredictable effects on lymphoma cells, this study aimed to evaluate the toxic effects of MS-275 and novel analogs on ATL cells in hypoxic condition for the first time. Protein-protein interaction and gene set enrichment analyses were performed, the expression of HIF1A and downstream targets were assessed, and molecular docking was conducted on MS-275 and novel analogs with HIF-1α. For in vitro studies, at first benzamide analogs of MS-275 were synthesized and then, viability of MT-2 cells was evaluated in hypoxic condition. Enrichment analyses confirmed the involvement of hub genes in HIF-1 signaling pathway and volcano plot revealed over expression of HIF1A, GAL3ST1 and CD274. Molecular docking indicated favorable interaction between MS-275 and analogs with HIF-1α PAS-B domain. Results of alamarBlue assay demonstrated that MS-275 and analogs significantly (p < 0.001) reduced viability of MT-2 cells in hypoxic condition. Findings of the present study hold promise for developing new drugs targeting hypoxia-induced changes in ATL.

Umbelliprenin improved anti-proliferative effects of ionizing radiation on adult T-cell leukemia/lymphoma cells via interaction with CDK6; an in vitro and in silico study.

Adult T-cell leukemia/lymphoma (ATL) is an aggressive malignancy with poor survival rates. The efficacy of radiotherapy in ATL needs enhancement with radiosensitizing agents. This study investigated whether umbelliprenin (UMB) could improve the therapeutic effects of ionizing radiation (IR) in ATL cells. UMB, a naturally occurring prenylated coumarin, exhibits anticancer properties and has shown synergistic effects when combined with chemotherapeutic drugs. Despite this promising profile, there is a notable lack of research on its potential combinatorial effects with IR, particularly for ATL treatment. UMB was extracted from Ferula persica using thin layer chromatography. MT-2 cells were treated with UMB alone and in combination with various doses of IR, and cell proliferation was assessed via alamarBlue assay. Flow cytometry with annexin V and PI staining was conducted, and candidate gene expression was analyzed by qPCR. In silico analysis involved identifying pathogenic targets of ATL, constructing protein-protein interaction (PPI) networks, and evaluating CDK6 expression in MT-2 cells. Molecular docking was used to determine the interaction between UMB and CDK6. The alamarBlue assay and flow cytometry showed that pretreating ATL cells with UMB significantly (p < .0001) enhanced anti-proliferative effects of IR. The combination index indicated a synergistic effect between UMB and IR. qPCR revealed significant (p < .0001) downregulation of CD44, CDK6, c-MYC, and cFLIPL, and overexpression of cFLIPS. Computational analysis identified CDK6 as a hub gene in the PPI network, and CDK6 overexpression was confirmed in MT-2 cells. Molecular docking revealed a favorable binding interaction between UMB and the ATP-binding site of CDK6, with a JAMDA score of -2.131, surpassing the control selonsertib. The current study provides evidence that UMB enhances the anti-proliferative effects of IR on ATL cells, and highlights the significance of targeting CDK6 in combinatorial approaches.

Galbanic acid suppresses melanoma cell migration and invasion by reducing MMP activity and downregulating N-cadherin and fibronectin.

High mortality rate of melanoma is due to the metastasis of malignant cells. Galbanic acid (GBA) is a natural sesquiterpene coumarin with valuable pharmaceutical activities. Our study aimed to investigate whether GBA can affect the migration, invasion, and adhesion of melanoma cells. The survival rate of B16F10 cells was measured using the alamarBlue assay. Scratch, adhesion, and invasion assays were performed to determine the effect of GBA on metastatic behavior of cells. Moreover, gelatin zymography was done to assess the activity of MMP-2 and MMP-9, and qRT-PCR was used to investigate the effect of GBA on the expression of candidate genes. Based on the results of alamarBlue assay, 40 µM GBA was chosen as the optimum concentration for all tests. Our findings indicated that GBA significantly decreased the invasion and migration of B16F10 cells while enhancing their adhesion ability. In addition, gelatin zymography demonstrated that GBA reduced the enzymatic activity of MMP-2 and MMP-9. Moreover, qRT-PCR revealed that GBA reduced the expression of N-cadherin and fibronectin. Current findings demonstrated, for the first time, that GBA inhibited the migration and invasion of melanoma cells via reducing the activity of MMP-2 and MMP-9 and downregulating N-cadherin and fibronectin expression. Accordingly, GBA could be suggested as a potential therapeutic agent for the treatment of melanoma.

Inhibition of melanoma cell migration and invasion by natural coumarin auraptene through regulating EMT markers and reducing MMP-2 and MMP-9 activity.

Melanoma, the most invasive form of skin cancer, shows a rising incidence trend in industrial countries. Since the main reason for the failure of current therapeutic approaches against melanoma is metastasis, there is a great interest in introducing effective natural agents to combat melanoma cell migration and invasion. Auraptene (AUR) is the most abundant coumarin derivative in nature with valuable pharmaceutical effects. In this study, we aimed to investigate whether AUR could induce inhibitory effects on the migration and invasion of melanoma cells. B16F10 melanoma cells were treated with different concentrations of AUR and the viability of cells was evaluated by alamarBlue assay. Then, cells were treated with 20 µM AUR, and wound healing, invasion, and adhesion assays were carried out. In addition, the activity of matrix metalloproteinase-2 (MMP-2) and MMP-9 was assessed by gelatin zymography and the expression of genes related to epithelial-mesenchymal transition (EMT) was investigated by qPCR. Finally, the interactions between AUR and MMPs were stimulated by molecular docking. Findings revealed that AUR significantly reduced the migration and invasion of B16F10 cells while improved their adhesion. Furthermore, results of gelatin zymography indicated that AUR suppressed the activity of MMP-2 and MMP-9, and qPCR revealed negative regulatory effect of AUR on the expression of mesenchymal markers including fibronectin and N-cadherin. In addition, molecular docking verified the interactions between AUR and the active sites of wild-type and mutant MMP-2 and MMP-9. Accordingly, AUR could be considered as a potential natural agent with inhibitory effects on the migration and invasion of melanoma cells for future preclinical studies.

Evaluating stem and cancerous biomarkers in CD15+CD44+ KYSE30 cells.

Digestive system cancers are listed among the ten top causes of cancer-related death worldwide. Cancer stem cells (CSCs) are malignant cells that share some of their characteristics with normal stem cells, including self-renewal and multipotency, and also cancer cells, such as drug resistance and metastasis. Despite many reports on CSCs with digestive system origin, identification and characterization of esophageal CSCs have remained elusive. To examine the validity of routine SC, cancer cell and CSC markers in KYSE30 cells, derived from esophageal carcinoma, cells were first characterized by immunofluorescence and RT-PCR techniques, and then the significance of candidate biomarkers was evaluated in retinoic acid-treated cells by flow cytometry and/or real-time RT-PCR. Meanwhile, to study CD15 (a newly introduced CSC marker) expression in digestive tract cancers, human normal and tumoral tissues of esophagus, stomach, and colon were analyzed by immunohistochemistry. Using several experimental approaches, we show that CD44, but not CD15, could serve as a reliable marker for undifferentiated malignant squamous cells of esophagus. In conclusion, our study confirms the role of CD44 as a CSC marker in KYSE30 cells, an esophageal squamous cell carcinoma cell line, and for the first time indicates the expression of CD15 in non-neural stem-like cancer cells. Although the importance of CD15 was not indicated in diagnosis of digestive cancers, further studies are needed to better understand the biological identity and function of this molecule in non-neural malignancies.

Bacterial-mediated synthesis and characterization of copper oxide nanoparticles with antibacterial, antioxidant, and anticancer potentials.

The application of novel bacterial strains for effective biosynthesis of nanoparticles minimizes negative environmental impact and eliminates challenges of available approaches. In the present study, cell-free extract of Stenotrophomonas sp. BS95. was used for synthesis of copper oxide nanoparticles (CuONPs). Characterization of crude and calcined CuONPs was carried out by UV-vis spectroscopy, X-ray diffraction (XRD), fourier transform infrared (FTIR) spectroscopy, zeta potential, dynamic light scattering, field emission scanning electron microscopy, transmission electron microscopy, and atomic force microscopy. Afterward, biogenic CuONPs were evaluated for antibacterial, antioxidant, and cytotoxic effects using broth micro-dilution method, DPPH assay and alamarBlue assay, respectively. Finally, molecular mechanisms behind anticancer effects of CuONPs was ascertained by real time PCR. UV-vis absorbance spectra registered surface plasmon resonance peaks at 286 nm and 420 nm for crude and calcined CuONPs, respectively. FTIR spectra exhibited bands associated with organic functional groups of bacterial proteins, confirming capping and functionalization of CuONPs. The average crystallite size of crude and calcined CuONPs was determined as 18.24 and 21.3 nm by XRD, respectively. The average zeta potentials of crude and calcined CuONPs were as -28.57 ± 5.13 and -29.47 ± 4.78 mV, respectively, indicating their high stability. Electron microscopy revealed that crude and calcined CuONPs were roughly spherical particles with an average size of 35.24 ± 4.64 and 43.68 ± 2.31 nm, respectively. Biogenic CuONPs induced antibacterial effects with minimal inhibitory concentrations ranging from 62.5 to 1,000 µg/ml against Gram-negative and Gram-positive strains. The antioxidant activity of crude and calcined CuONPs was found to be 83% ± 2.64% and 78% ± 1.73%, respectively. More intriguingly, CuONPs exerted considerable cytotoxic effects on human colon and gastric adenocarcinoma cells, while induced low toxicity on normal cells. Anticancer effects of biogenic CuONPs were confirmed by significant changes induced in the expression of apoptosis-related genes, including P53, BAX, BCL2 and CCND1. Hence, biosynthesized CuONPs could be considered as potential antimicrobial, antioxidant and anticancer agents.

Conferone, a coumarin from Ferula flabelliloba, induced toxic effects on adult T-cell leukemia/lymphoma cells.

BACKGROUND: Adult T-cell leukemia/lymphoma (ATL) is a lymphoid malignancy caused by HTLV-1 infection, with distinct geographical distribution. Despite advances in cancer treatment, the average survival rate of ATL is low. Conferone is a natural coumarin extracted from Ferula species with a wide range of pharmaceutical effects. In search for a novel chemotherapeutic agent, we investigated the cytotoxicity of conferone on ATL cells. METHODS: To obtain conferone, the methanolic extract of the roots of F. flabelliloba was subjected to silica gel column chromatography, followed by 1H- and 13C-NMR to confirm its structure. For cytotoxicity assay, MT-2 cells were treated with different concentrations of conferone (2.5, 5, 10, 20, and 40 µM) for 24, 48, and 72 h, and viability was evaluated by a colorimetric assay using alamarBlue. Cell cycle was analyzed by PI staining and flow cytometry, and qPCR was used to study the expression of candidate genes. RESULTS AND CONCLUSION: Obtained findings indicated that conferone induced considerable cytotoxic effects on MT-2 cells in a time- and dose-dependent manner. In addition, accumulation of cells in the sub-G1 phase of the cell cycle was detected upon conferone administration. Moreover, conferone reduced the expression of CDK6, c-MYC, CFLIPL, and NF-κB (Rel-A) in MT-2 cells. Accordingly, conferone could be considered as a potent agent against ATL, although complementary investigations are required to define more precisely its mechanism of action.

7-geranyloxycoumarin enhanced radiotherapy effects on human gastric adenocarcinoma cells.

Background: Gastric adenocarcinoma (GA) is a serious malignancy with growing incidence and mortality rate worldwide. The objective of the present study was to determine whether 7-geranyloxycoumarin, a natural monoterpene coumarin, could induce anticancer effects, in single use and/or in combination with anticancer drugs and ionizing radiation, on GA cells. Materials and Methods: 7-geranyloxycoumarin was synthesized by a reaction between 7-hydroxycoumarin and transgeranyl bromide. MKN45 cells were treated with 7-geranyloxycoumarin, and the viability of cells was determined by resazurin. Apoptosis was then evaluated by flow cytometric analysis using annexin V and propidium iodide, and the expression of P53 and BCL2 was analyzed by quantitative polymerase chain reaction (qPCR). Combinatorial effects of 7-geranyloxycoumarin with 5-fluorouracil (5-FU), cisplatin (CDDP), and X radiation were also evaluated. Results: Assessment of cell viability indicated that 7-geranyloxycoumarin induced its toxic effects in a time- and dose-dependent manner. This was confirmed by the detection of apoptotic cells, and qPCR results revealed a significant downregulation in BCL2 expression. Although combinatorial use of 7-geranyloxycoumarin + 5-FU or + CDDP did not improve cytotoxicity of anticancer drugs, significant increase in the effectiveness of applied radiations was detected upon pretreatment with 7-geranyloxycoumarin. Conclusion: Our findings provide valuable insights into single and combinatorial effects of 7-geranyloxycoumarin on the GA cells.

Galbanic Acid Improves Accumulation and Toxicity of Arsenic Trioxide in MT-2 Cells.

BACKGROUND: Galbanic acid (GBA) is a sesquiterpene coumarin with valuable pharmacological effects. Adult T-cell lymphoma (ATL) is an aggressive lymphoid malignancy with a low survival rate. Although arsenic trioxide (ATO) is a standard therapeutic agent for ATL treatment, the efficacy of chemotherapy is limited due to the chemoresistance of cells. OBJECTIVE: The present study was carried out to investigate whether GBA in combination with ATO would improve cytotoxicity against ATL cells. METHODS: GBA was isolated from the roots of Ferula szowitsiana by column chromatography on silica gel. MT-2 cells were treated with 20 µM GBA + 4 µM ATO, and viability was evaluated by alamarBlue assay. The cell cycle was analyzed by PI staining, while the activity of P-glycoprotein (P-gp) was evaluated by mitoxantrone efflux assay. To understand the molecular mechanisms of GBA effects, the expression of NF-κB (RelA), P53, CDK4, c-MYC, c-FLIPL, and c-FLIPS was evaluated using real-time PCR. RESULTS: Combinatorial use of GBA + ATO significantly reduced the viability of MT-2 cells and induced cell cycle arrest in the sub-G1 phase. GBA improved mitoxantrone accumulation in cells, indicating that this agent has inhibitory effects on the functionality of the P-gp efflux pump. Moreover, real-time PCR analysis revealed that GBA + ATO negatively regulated the expression of P53, CDK4, c-FLIPL, and c-FLIPS. CONCLUSION: Due to the interesting effects of GBA on the accumulation and toxicity of ATO, combinatorial use of these agents could be considered a new therapeutic approach for ATL treatment.

Urolithins increased anticancer effects of chemical drugs, ionizing radiation and hyperthermia on human esophageal carcinoma cells in vitro.

Despite progress in diagnosis and treatment of esophageal cancer (EC), it is still considered as a serious malignancy with very poor prognosis. Urolithins are colonic microbiota metabolites with a wide range of pharmacological properties including chemopreventive, anti-inflammatory and anticancer activities. In this study, we hypothesized that urolithins might possess the potential to improve the efficacy of chemical drugs, ionizing radiation (IR) and/or hyperthermia on EC cells. After synthesis of urolithin A (UA), methylurolithin A (mUA) and urolithin B (UB), KYSE30 esophageal cancer cells were treated with urolithins + paclitaxel (PTX), + cisplatin (DDP), + different doses of IR or + heat-shock. Viability of cells was then determined by alamarBlue assay. To further elucidate the effects of UA, we used flow cytometry for investigation of induced apoptosis, and qRT-PCR for evaluating changes in the expression of HSP27, CCND1 and BCL2. Assessment of cell viability demonstrated that mUA increased the toxicity of PTX and DDP (up to 22.4 % and 20 %, respectively) and improved the effects of 6 Gy IR (26.5 %). Our main results achieved after UA treatment were improved toxicity of PTX and 6 Gy IR, beside enhanced effects of hyperthermia (37.3 %), which was confirmed by flow cytometry analysis and downregulation of HSP27, CCND1 and BCL2 expression. Taken together, our findings suggest that UA and mUA could be used as promising agents in combination with therapeutic modalities to improve the clinical outcomes of EC treatment.

Radiation Response of Human Leukemia/Lymphoma Cells was Improved by 7-Geranyloxycoumarin.

Objectives: Adult T-cell leukemia/lymphoma (ATLL) is a blood neoplasm with specific geographic distribution. Although radiotherapy is a palliative treatment that provides long-term local control, single use of radiation leads to complications for patients. To introduce a novel multimodal approach against ATLL, we investigated combinatorial effects of 7-geranyloxycoumarin and radiation in vitro. Methods: Viability of MT-2 cells was determined by resazurin assay upon administration of 7-geranyloxycoumarin alone and followed by radiation. Then, apoptosis was detected by annexin V and propidium iodide, and the expression of candidate genes was analyzed by qPCR. Results: Findings revealed significant (P<.0001) improvement in radiation effects upon 7-geranyloxycoumarin pretreatment, most notably when cells were pretreated with 5 µg/ml 7-geranyloxycoumarin for 96 h, exposed to 6 Gy radiation and recovered for 48 h. These results were confirmed by flow cytometry, as the percentage of early and late apoptotic cells was increased after combinatorial treatment. In addition, significant (P< .0001) changes in CD44, c-MYC, cFLIPL, BMI-1, NF-κB (Rel A), and P53 expression was induced by 7-geranyloxycoumarin and radiation. Conclusions: Current research indicated, for the first time, that combinatorial use of 7-geranyloxycoumarin and ionizing radiation could be considered as an effective therapeutic modality for ATLL.