Evaluation of the effect of intensity-modulated radiotherapy (IMRT) and volumetric-modulated arc radiotherapy (VMAT) techniques on survival response in cell lines with a new radiobiological modeling.

BACKGROUND: The optimal radiobiological model, which assesses the biological effects of novel radiotherapy techniques that concurrently modify multiple physical factors, has not yet been defined. This study aimed to investigate the impact of intensity-modulated radiotherapy (IMRT) and volumetric-modulated arc therapy (VMAT) on cellular response in head and neck cancer and melanoma models. METHODS: Clonogenic analysis, DNA double-strand break analysis, apoptosis, and cell cycle analysis were performed on cancer stem cell models, cancer models, and normal tissue cell models to assess radiation sensitivity. RESULTS: The segmented radiation approach used in IMRT applications enhanced radiosensitivity and cytotoxicity in the cancer models, while changes in dose rate had varying effects on cytotoxicity depending on the tumor cell type. VMAT increased cellular resistance, favoring treatment outcomes. CONCLUSIONS: The biological processes were influenced differently by dose rate, IMRT, and VMAT depending on the tumor cell type. The selection of the most appropriate technique is crucial in representing new radiotherapy approaches. The obtained data can serve as a model to address clinical questions in daily practice. The integration of non-standard outcomes with standard applications should be considered in clinical settings.

The effects of PKI-402 on breast tumor models' radiosensitivity via dual inhibition of PI3K/mTOR.

PURPOSE: PI3K/Akt/mTOR pathway activation causes relapse and resistance after radiotherapy in breast cancer (BC). We aimed to radiosensitize BC cell lines to irradiation (IR) by PKI-402, a dual PI3K/mTOR inhibitor. METHODS: We performed cytotoxicity, clonogenicity, hanging drop, apoptosis and double-strand break detection, and phosphorylation of 16 essential proteins involved in the PI3K/mTOR pathway. RESULTS: Our findings showed that PKI-402 has cytotoxic efficiency in all cell lines. Clonogenic assay results showed that PKI-402 plus IR inhibited the colony formation ability of MCF-7 and breast cancer stem cell lines. Results showed that PKI-402 plus IR causes more apoptotic cell death than IR alone in the MCF-7 cells but did not cause significant changes in the MDA-MB-231. γ-H2AX levels were increased in MDA-MB-231 in PKI-402 plus IR groups, whereas we did not observe any apoptotic and γ-H2AX induction in BCSCs and MCF-10A cells in all treatment groups. Some pivotal phosphorylated proteins of the PI3K/AKT pathway decreased, several proteins increased and others did not change. CONCLUSION: In conclusion, if the combined use of PKI-402 with radiation is supported by in vivo studies, it can contribute to the treatment options and the course of the disease.

Origanum Sipyleum Methanol Extract in Combination with Ponatinib Shows Synergistic anti-Leukemic Activities on Chronic Myeloid Leukemia Cells.

Origanum sipyleum is used in folk medicine due to its anti-inflammatory, antimicrobial, and antioxidant properties. Ponatinib, an effective tyrosine kinase inhibitor in the treatment of chronic myeloid leukemia (CML), has severe side effects. Thus, we aimed to determine a novel herbal combination therapy that might not only increase the anti-leukemic efficacy but also reduce the dose of ponatinib in targeting CML cells. Origanum sipyleum was extracted with methanol (OSM), and secondary metabolites were determined by phytochemical screening tests. The cytotoxic effects of OSM on K562 cells were measured by WST-1 assay. Median-effect equation was used to analyze the combination of ponatinib and OSM (p-OSM). Apoptosis, proliferation, and cell-cycle were investigated by flow-cytometry. Cell-cycle-related gene expressions were evaluated by qRT-PCR. OSM that contains terpenoids, flavonoids, tannins, and anthracenes exhibited cytotoxic effects on K562 cells. The median-effect of p-OSM was found as synergistic; OSM reduced the ponatinib dose ∼5-fold. p-OSM elevated the apoptotic and anti-proliferative activity of ponatinib. Consistently, p-OSM blocked cell-cycle progression in G0/G1, S phases accompanied by regulations in TGFB2, ATR, PP2A, p18, CCND1, CCND2, and CCNA1 expressions. OSM enhanced the anti-leukemic activity of ponatinib synergistically via inducing apoptosis, suppressing proliferation, and cell-cycle. As a result, OSM might offer a potential strategy for treating patients with CML.

Comparative expression analysis of dasatinib and ponatinib-regulated lncRNAs in chronic myeloid leukemia and their network analysis.

LncRNAs are associated with malignancies with their tumor suppressor/oncogenic properties. Although many studies are conducted related to the mechanism of action for dasatinib and ponatinib in chronic myeloid leukemia (CML), their comparative effects on lncRNA expressions are largely unknown. Hence, we aimed to define the lncRNAs involved in the treatment of CML with dasatinib and ponatinib. We measured the cytotoxicities of dasatinib/ponatinib with CCK-8 assay and identified differentially expressed lncRNAs (DEL) by qRT-PCR. We determined the principal functions of DELs by Ingenuity Pathway Analysis (IPA) and performed gene ontology (GO) analysis for apoptosis and anti-proliferation-related lncRNAs. Apoptotic and anti-proliferative activities of dasatinib/ponatinib were confirmed by flow-cytometry. In K562 cells, dasatinib/ponatinib re-regulated lncRNAs which were dysregulated in leukemia. DELs after treatment (forty with dasatinib, thirty-seven with ponatinib) were related to increased cell death; decreased cell viability, proliferation, tumor growth, invasion, migration. Dasatinib-mediated network was related to cancer, hematological disease while ponatinib-mediated network was associated with cancer, cell death/survival, cell-to-cell signaling/interaction. Both treatments predicted activation of IFNγ, IL1ß, TNF as upstream regulators, specially this effect was higher in dasatinib. Comparison analysis showed that ponatinib was predicted more effective in cell death of tumor cell line than dasatinib. We confirmed that ponatinib was more potent than dasatinib to induce apoptosis and inhibit proliferation of CML cells, in consensus with IPA and GO analysis results. LncRNAs are specifically involved in anti-leukemic activities of dasatinib and ponatinib. Our findings will contribute to understanding signalization occurring in CML cells after standard treatments.

The effect of ICRT-3 on Wnt signaling pathway in head and neck cancer.

The effect of Wnt pathway in head and neck cancer could not be elucidated, even though the aberrant Wnt signaling plays a key role in the development of many types of cancer. The inhibitor of ß-catenin responsive transcription (ICRT-3) blocks the Wnt signaling pathway by binding to ß-catenin, which is a coactivator of the Wnt signaling pathway and a promising agent for inhibiting aberrant signaling. In our study, we aimed to evaluate the effect of ICRT-3 on the cytotoxicity, apoptosis, cell cycle progression, migration, and gene expressions in head and neck cancer stem cell (HNCSC) and hypopharynx cancer. The effect of this compound on cytotoxicity and cell viability in FaDu and HNCSC line was assessed by using the water-soluble tetrazolium salt-1 method. The effect of ICRT-3 on apoptosis was detected by using Annexin V and caspase-3, caspase-9 kit, on cell cycle progression by cycle test plus DNA reagent kit, on gene expression by dual luciferase reporter assay, and on migration activity by wound healing assay in both cell lines. ICRT-3 was determined to have cytotoxic and apoptotic effect in both cell lines. In addition, it was also found that the administration of ICRT-3 caused cell cycle arrest and significant decrease in gene expression level and migration ability of the cells.

Investigation of the synergistic effects of paclitaxel and herbal substances and endemic plant extracts on cell cycle and apoptosis signal pathways in prostate cancer cell lines.

Paclitaxel, which isolated from Taxus brevifolia, is recently started to be used against prostate cancer treatment and it is a very effective compound against cancer. In this study, we aimed to test the synergistic effect of two plant active compounds (sulphoraphane (SFN) and silymarin (SILY)) and several endemic plant species from Turkey (such as Phlomis leucophracta, Rubia davisiana, Alkanna tinctoria), which are known to have anticarcinogenic effect on androgen-independent PC3 and DU145, and androgen-dependent VCaP prostate cancer cell lines, with paclitaxel on the expression of cell cycle signaling and apoptosis regulator genes. Herbal substances and endemic herbal extracts were combined with Paclitaxel drug. IC50 doses were identified as real-time online. The most effective synergistic doses were determined according to isobologram analysis. The apoptotic effects of effective combined doses were evaluated by TUNEL, Annexin V, and JC-1 methods. Apoptotic and/or cell cycle arrest effects of confirmed combined doses on the expression of genes in these pathways were assessed by real-time online. Endemic plant extracts (Alkanna tinctoria, Phlomis leucophracta and Rubia davisiana, IC50 < 220 µg/ml) and herbal substances (SILY, and SFN IC50 < 130 µM) indicated antiproliferative and apoptotic effects in prostate cancer cell lines. They testified to the synergistic effect of paclitaxel with endemic plant extracts (Combination Index CI, ED50 < 0.41). The combinations, which indicate the synergistic effect was increased to the Bax/Bcl­2 ratio by suppressing Bcl­2 gene expression into the prostate cancer cell lines. Besides, they increased the expression of TNFRSF10A, TNFRSF1A, CHEK1, CDKN1A, CDKN2B, CDK8, CDKN3 and CASP14 and decreased BAD, CDK5RAP1, CDC20, cyclin H, CDK5RAP1, CDC20. The effective doses of paclitaxel were reduced and G2/M arrest was induced by the endemic plant extracts and herbal substances that indicate a synergistic effect with paclitaxel. By using different combination of herbal extracts or active substances with paclitaxel, more economical and efficient treatment strategies can be developed.

Analysis of long non-coding RNA (lncRNA) expression in hepatitis B patients.

Long non-coding RNAs (lncRNAs) have been implicated in numerous biological processes, including epigenetic regulation, cell-cycle control, and transcriptional/translational regulation of gene expression. Differential expression of lncRNAs and disruption of the regulatory processes are recognized as critical steps in cancer development. The role of lncRNAs in hepatitis B virus (HBV) infection is not well understood. Here we analyzed the expression of 135 lncRNAs in plasma samples of 82 HBV patients (classified as chronic patients, inactive carriers, or resolved patients) at diagnosis and at 12 months of treatment in relation to control group (81 healthy volunteers). We also investigated the effect of small interfering RNA (siRNA)-mediated silencing of lincRNA-SFMBT2 on HBV-positive human liver cancer cell line. lncRNA expression was analyzed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Chemically synthesized siRNAs were transfected into the cell lines using Lipofectamine 2000 Reagent (Thermo Fisher Scientific). HBV DNA and HBsAg and HBeAg were detected in transfected cultures by real-time PCR and ELISA, respectively, using commercial kits. We observed changes in lncRNA expression in all three HBV groups, compared to control group. Most notably, the expression of anti-NOS2A, lincRNA-SFMBT2, and Zfhx2as was significantly increased and expression of Y5 lncRNA was decreased in chronic HBV patients. A decreased Y5 expression and increased lincRNA-SFMBT2 expression were observed in inactive HBsAg carriers. The expression of HOTTIP, MEG9, and PCAT-32 was increased in resolved HBV patients, and no significant change in the expression of Y5 was observed, compared to control group. siRNA-mediated inhibition of lincRNA-SFMBT2 decreased the level of HBV DNA in human liver cancer cells. Further research is needed to confirm the prognostic as well as therapeutic role of these lncRNAs in HBV patients.

Antileukemic effect of paclitaxel in combination with metformin in HL-60 cell line.

Acute promyelocytic leukemia (APL) is a subtype of AML that is a mixture of hematological malignancy, characterized by a specific translocation t(15;17). The using of all-trans retinoic acid (ATRA) with arsenic trioxide (ATO) or chemotherapeutic agents or both of these agents, composes main treatment strategy of APL. While it is possible to achieve success in treatment of low-risk APL with current treatment regimens, such success is not mentioned in high-risk APL. So, it may develop new approaches for treatment regimens for high-risk APL. In the present study, we aimed to investigate the effects of combinational of a classic anticancer agent paclitaxel and antidiabetic agent metformin on HL-60 APL cell line. The combination dose of paclitaxel and metformin was determined by WST-1 analysis. The effect of combinational dose on apoptosis was assessed in fluorescence microscope after using AnnexinV-EGFP Apoptosis and JC-1 Assay Kit. The effect of combinational dose on cell cycle, apoptosis and differentiation, and signaling pathways were determined investigating gene expression changes by using real time qRT-PCR. The combinational dose of paclitaxel and metformin was determined as 4.8nM and 398.7µM for 72h, respectively. The combination dose significantly increased apoptosis for 48h. In expression changes of genes associated cell cycle, apoptosis, cytokines, co-stimulator molecules, NF-kB and MAP/MAPK pathways, TLRs (Toll-like receptors) were found to be decreased or increased to provide apoptosis or differentiation. Consequently, we suggest that the combination of paclitaxel and metformin can be used as an option assessable for development of new treatment strategies for APL.

Expression profiling of RE1-silencing transcription factor (REST), REST corepressor 1 (RCOR1), and Synapsin 1 (SYN1) genes in human gliomas.

PURPOSE: The repressor element 1 (RE-1) silencing transcription factor (REST) is a transcription factor which represses the expression of neuronal differentiation-related genes including SYN1 gene. CoREST, encoded by RCOR1 gene, binds to the REST protein for remodeling of chromatin structure. Although there is a relation among REST, RCOR1, and SYN1 genes, the role of these genes in glioma tumors is still unclear. In this study, expressions of REST, RCOR1, and SYN1 genes were detected in primary cultures derived from tumor samples of diffuse astrocytoma (DA), anaplastic oligodendroglioma (AO), and glioblastoma multiforme (GBM) cases. METHODS: Expression profiles were analysed by RT-qPCR and the copy number variations were examined with qPCR in primary cultures. ChIP assay was performed to show binding characteristics of REST and CoREST proteins on promoter region of SYN1 gene. RESULTS: Means of relative expression for REST were as follows: 0.7898, 0.7606, and 0.7318 in DA, AO, and GBM groups, respectively. For RCOR1, expression means in DA, AO, and GBM groups were 0.7203, 0.7334, and 0.7230, respectively. SYN1 expression means were as follows: 0.3936, 0.3192, and 0.3197 in DA, AO, and GBM groups, respectively. Neither gain nor loss of copy numbers were detected for REST and RCOR1 genes in all groups. Copy loss for SYN1 was detected in primary culture of a DA case. REST and CoREST presented positive precipitation pattern on promoter region of SYN1 gene. CONCLUSIONS: Expressions of REST and RCOR1 genes may downregulate SYN1 expression in gliomas. Low expression pattern of SYN1 may maintain cancer stem-like phenotype which contributes to development of gliomas.

Analysis of dysregulated long non-coding RNA expressions in glioblastoma cells.

Long non coding RNAs (lncRNAs) are associated with various biological roles such as embryogenesis, stem cell biology, cellular development and present specific tissue expression profiles. Aberrant expression of lncRNAs are thought to play a critical role in the progression and development of various cancer types, including gliomas. Glioblastomas (GBM) are common and malignant primary brain tumours. Brain cancer stem cells (BCSC) are isolated from both low and high-grade tumours in adults and children, by cell fraction which express neuronal stem cell surface marker CD133. The purpose of this study was to investigate the expression profiles of lncRNAs in brain tumour cells and determine its potential biological function. For this purpose, U118MG-U87MG; GBM stem cell series were used. Human parental brain cancer cells were included as the control group; the expressions of disease related human lncRNA profiles were studied by LightCycler 480 real-time PCR. Expression profiles of 83 lncRNA genes were analyzed for a significant dysregulation, compared to the control cells. Among lncRNAs, 51 lncRNA genes down-regulated, while 8 lncRNA genes were up-regulated. PCAT-1 (-2.36), MEG3 (-5.34), HOTAIR (-2.48) lncRNAs showed low expression in glioblastoma compared to the human (parental) brain cancer stem cells, indicating their role as tumour suppressor genes on gliomas. As a result, significant changes for anti-cancer gene expressions were detected with disease-related human lncRNA array plates. Identification of novel target genes may lead to promising developments in human brain cancer treatment.

Evaluation of cancer imaging potential and photodynamic therapy efficacy of copper (II) benzyloxypheophorbide-a.

The biological potential of a synthetic copper chlorophyll derivative was investigated via in vivo and in vitro experiments. The Cu-chlorophyll derivative photosensitizer (Cu-PH-A) was labeled with (131)I with high efficiency (92.9 ± 4.2%) using the iodogen method. Cell culture studies were performed with the MCF-7 and MDAH-2774 cell lines after radiolabeling. The photosensitizing activity of Cu-PH-A was more effective in MDAH-2774 cells than in MCF-7 cells at a concentration of 50 µM. When the biodistribution in female Albino Wistar rats was examined, uptake of the radiolabeled photosensitizer was maximal in the liver and ovaries after 60 min. It is concluded that radiolabeled Cu-chlorophyll derivative photosensitizer has high uptake in ovaries in normal rats. In addition, the intercellular uptake and PDT efficacy of the Cu-PH-A in MDAH-2774 were good compared with MCF-7 cells. This photosensitizer could be useful for both ovary tumour imaging and PDT.

Synergistic effect of ponatinib and epigallocatechin-3-gallate induces apoptosis in chronic myeloid leukemia cells through altering expressions of cell cycle regulatory genes.

PURPOSE: Ponatinib (P) has been used for the treatment of chronic myeloid leukemia (CML) and it is known that inhibition of BCR-ABL fusion protein by ponatinib induces apoptosis of CML cells. Epigallocatechin-3-gallate (EGCG), which is a polyphenol in green tea, induces apoptosis in different types of cancer cells. The purpose of this study was to determine the cytotoxic and apoptotic effects of ponatinib and EGCG combination in K562 CML cell line. This study also aimed to detect alterations of the expression levels of cell cycle-regulation related genes after ponatinib and EGCG combination in K562 CML cell line. METHODS: The cytotoxic effects of the compounds on K562 cells were determined in a time-and dose-dependent manner by using WST-1 analysis. The combination index (CI) isobologram was used to analyze the data. Apoptotic effects of P-EGCG were defined by flow cytometry and gene expressions were detected by RT-qPCR. RESULTS: IC50values of ponatinib and EGCG were 87.13 nM and 50µM, respectively. CI value of the P-EGCG was 0.658 and the combination showed synergistic effect (ED90 value: 28.39 nM ponatinib, 117.12 µg/ml EGCG). Ponatinib, EGCG and P-EGCG induced apoptosis compared to control cells. CyclinD1 and CDC25A were downregulated by P-EGCG by 2.49 and 2.63-fold, respectively. TGF-ß2 was upregulated by 4.57-fold. CONCLUSION: EGCG possesses cytotoxic and apoptotic properties and may cooperate with the growth inhibiting activity of ponatinib synergistically against CML cells. P-EGCG mediated apoptosis might be associated with upregulation of TGF-ß2 gene and downregulation of cyclinD1 and CDC25A genes.