Anticancer effects of Curcuma C20-dialdehyde against colon and cervical cancer cell lines

Background: Recent attention on chemotherapeutic intervention against cancer has been focused on discovering and developing phytochemicals as anticancer agents with improved efficacy, low drug resistance and toxicity, low cost and limited adverse side effects. In this study, we investigated the effects of Curcuma C20-dialdehyde on growth, apoptosis and cell cycle arrest in colon and cervical cancer cell lines. Materials and Methods: Antiproliferative, apoptosis induction, and cell cycle arrest activities of Curcuma C20-dialdehyde were determined by WST cell proliferation assay, flow cytometric Alexa fluor 488-annexin V/propidium iodide (PI) staining and PI staining, respectively. Results: Curcuma C20 dialdehyde suppressed the proliferation of HCT116, HT29 and HeLa cells, with IC50 values of 65.4±1.74 μg/ml, 58.4±5.20 μg/ml and 72.0±0.03 μg/ml, respectively, with 72 h exposure. Flow cytometric analysis revealed that percentages of early apoptotic cells increased in a dose-dependent manner upon exposure to Curcuma C20-dialdehyde. Furthermore, exposure to lower concentrations of this compound significantly induced cell cycle arrest at G1 phase for both HCT116 and HT29 cells, while higher concentrations increased sub-G1 populations. However, the concentrations used in this study could not induce cell cycle arrest but rather induced apoptotic cell death in HeLa cells. Conclusions: Our findings suggest that the phytochemical Curcuma C20-dialdehyde may be a potential antineoplastic agent for colon and cervical cancer chemotherapy and/or chemoprevention. Further studies are needed to characterize the drug target or mode of action of the Curcuma C20-dialdehyde as an anticancer agent.

Defining the Role of the Histone Methyltransferase, PR-Set7, in Maintaining the Genome Integrity of Drosophila Melanogaster

The complete and faithful duplication of the genome is essential to ensure normal cell division and organismal development. Eukaryotic DNA replication is initiated at multiple sites termed origins of replication that are activated at different time through S phase. The replication timing program is regulated by the S-phase checkpoint, which signals and repairs replicative stress. Eukaryotic DNA is packaged with histones into chromatin, thus DNA-templated processes including replication are modulated by the local chromatin environment such as post-translational modifications (PTMs) of histones.

One such epigenetic mark, methylation of lysine 20 on histone H4 (H4K20), has been linked to chromatin compaction, transcription, DNA repair and DNA replication. H4K20 can be mono-, di- and tri-methylated. Monomethylation of H4K20 (H4K20me1) is mediated by the cell cycle-regulated histone methyltransferase PR-Set7 and subsequent di-/tri- methylation is catalyzed by Suv4-20. Prior studies have shown that PR-Set7 depletion in mammalian cells results in defective S phase progression and the accumulation of DNA damage, which may be partially attributed to defects in origin selection and activation. Meanwhile, overexpression of mammalian PR-Set7 recruits components of pre-Replication Complex (pre-RC) onto chromatin and licenses replication origins for re-replication. However, these studies were limited to only a handful of mammalian origins, and it remains unclear how PR-Set7 impacts the replication program on a genomic scale. Finally, the methylation substrates of PR-Set7 include both histone (H4K20) and non-histone targets, therefore it is necessary to directly test the role of H4K20 methylation in PR-Set7 regulated phenotypes.

I employed genetic, cytological, and genomic approaches to better understand the role of H4K20 methylation in regulating DNA replication and genome stability in Drosophila melanogaster cells. Depletion of Drosophila PR-Set7 by RNAi in cultured Kc167 cells led to an ATR-dependent cell cycle arrest with near 4N DNA content and the accumulation of DNA damage, indicating a defect in completing S phase. The cells were arrested at the second S phase following PR-Set7 downregulation, suggesting that it was an epigenetic effect that coupled to the dilution of histone modification over multiple cell cycles. To directly test the role of H4K20 methylation in regulating genome integrity, I collaborated with the Duronio Lab and observed spontaneous DNA damage on the imaginal wing discs of third instar mutant larvae that had an alanine substitution on H4K20 (H4K20A) thus unable to be methylated, confirming that H4K20 is a bona fide target of PR-Set7 in maintaining genome integrity.

One possible source of DNA damage due to loss of PR-Set7 is reduced origin activity. I used BrdU-seq to profile the genome-wide origin activation pattern. However, I found that deregulation of H4K20 methylation states by manipulating the H4K20 methyltransferases PR-Set7 and Suv4-20 had no impact on origin activation throughout the genome. I then mapped the genomic distribution of DNA damage upon PR-Set7 depletion. Surprisingly, ChIP-seq of the DNA damage marker γ-H2A.v located the DNA damage to late replicating euchromatic regions of the Drosophila genome, and the strength of γ-H2A.v signal was uniformly distributed and spanned the entire late replication domain, implying stochastic replication fork collapse within late replicating regions. Together these data suggest that PR-Set7-mediated monomethylation of H4K20 is critical for maintaining the genomic integrity of late replicating domains, presumably via stabilization of late replicating forks.

In addition to investigating the function of H4K20me, I also used immunofluorescence to characterize the cell cycle regulated chromatin loading of Mcm2-7 complex, the DNA helicase that licenses replication origins, using H4K20me1 level as a proxy for cell cycle stages. In parallel with chromatin spindown data by Powell et al. (Powell et al. 2015), we showed a continuous loading of Mcm2-7 during G1 and a progressive removal from chromatin through S phase.

Synthetic Lethal Targeting of ARID1A-Mutant Ovarian Clear Cell Tumors with Dasatinib

New targeted approaches to ovarian clear cell carcinomas (OCCC) are needed, given the limited treatment options in this disease and the poor response to standard chemotherapy. Using a series of high-throughput cell-based drug screens in OCCC tumor cell models, we have identified a synthetic lethal (SL) interaction between the kinase inhibitor dasatinib and a key driver in OCCC, ARID1A mutation. Imposing ARID1A deficiency upon a variety of human or mouse cells induced dasatinib sensitivity, both in vitro and in vivo, suggesting that this is a robust synthetic lethal interaction. The sensitivity of ARID1A-deficient cells to dasatinib was associated with G₁ -S cell-cycle arrest and was dependent upon both p21 and Rb. Using focused siRNA screens and kinase profiling, we showed that ARID1A-mutant OCCC tumor cells are addicted to the dasatinib target YES1. This suggests that dasatinib merits investigation for the treatment of patients with ARID1Amutant OCCC. Mol Cancer Ther; 15(7); 1472-84. Ó2016 AACR.

Recurrent CDKN1B (p27) mutations in hairy cell leukemia

Hairy cell leukemia (HCL) is marked by near 100% mutational frequency of BRAFV600E mutations. Recurrent cooperating genetic events that may contribute to HCL pathogenesis or affect the clinical course of HCL are currently not described. Therefore, we performed whole exome sequencing to explore the mutational landscape of purine analog refractory HCL. In addition to the disease-defining BRAFV600E mutations, we identified mutations in EZH2, ARID1A, and recurrent inactivating mutations of the cell cycle inhibitor CDKN1B (p27). Targeted deep sequencing of CDKN1B in a larger cohort of HCL patients identify deleterious CDKN1B mutations in 16% of patients with HCL (n = 13 of 81). In 11 of 13 patients the CDKN1B mutation was clonal, implying an early role of CDKN1B mutations in the pathogenesis of HCL. CDKN1B mutations were not found to impact clinical characteristics or outcome in this cohort. These data identify HCL as having the highest frequency of CDKN1B mutations among cancers and identify CDNK1B as the second most common mutated gene in HCL. Moreover, given the known function of CDNK1B, these data suggest a novel role for alterations in regulation of cell cycle and senescence in HCL with CDKN1B mutations.

Activation of STING-Dependent Innate Immune Signaling By S-Phase-Specific DNA Damage in Breast Cancer

BACKGROUND: Previously we identified a DNA damage response-deficient (DDRD) molecular subtype within breast cancer. A 44-gene assay identifying this subtype was validated as predicting benefit from DNA-damaging chemotherapy. This subtype was defined by interferon signaling. In this study, we address the mechanism of this immune response and its possible clinical significance.

METHODS: We used immunohistochemistry (IHC) to characterize immune infiltration in 184 breast cancer samples, of which 65 were within the DDRD subtype. Isogenic cell lines, which represent DDRD-positive and -negative, were used to study the effects of chemokine release on peripheral blood mononuclear cell (PBMC) migration and the mechanism of immune signaling activation. Finally, we studied the association between the DDRD subtype and expression of the immune-checkpoint protein PD-L1 as detected by IHC. All statistical tests were two-sided.

RESULTS: We found that DDRD breast tumors were associated with CD4+ and CD8+ lymphocytic infiltration (Fisher's exact test P < .001) and that DDRD cells expressed the chemokines CXCL10 and CCL5 3.5- to 11.9-fold more than DNA damage response-proficient cells (P < .01). Conditioned medium from DDRD cells statistically significantly attracted PBMCs when compared with medium from DNA damage response-proficient cells (P < .05), and this was dependent on CXCL10 and CCL5. DDRD cells demonstrated increased cytosolic DNA and constitutive activation of the viral response cGAS/STING/TBK1/IRF3 pathway. Importantly, this pathway was activated in a cell cycle-specific manner. Finally, we demonstrated that S-phase DNA damage activated expression of PD-L1 in a STING-dependent manner.

CONCLUSIONS: We propose a novel mechanism of immune infiltration in DDRD tumors, independent of neoantigen production. Activation of this pathway and associated PD-L1 expression may explain the paradoxical lack of T-cell-mediated cytotoxicity observed in DDRD tumors. We provide a rationale for exploration of DDRD in the stratification of patients for immune checkpoint-based therapies.

Sonic hedgehog signaling regulates mode of cell division of early cerebral cortex progenitors and increases

The morphogen Sonic Hedgehog (SHH) plays a critical role in the development of different tissues. In the central nervous system, SHH is well known to contribute to the patterning of the spinal cord and separation of the brain hemispheres. In addition, it has recently been shown that SHH signaling also contributes to the patterning of the telencephalon and establishment of adult neurogenic niches. In this work, we investigated whether SHH signaling influences the behavior of neural progenitors isolated from the dorsal telencephalon, which generate excitatory neurons and macroglial cells in vitro. We observed that SHH increases proliferation of cortical progenitors and generation of astrocytes, whereas blocking SHH signaling with cyclopamine has opposite effects. In both cases, generation of neurons did not seem to be affected. However, cell survival was broadly affected by blockade of SHH signaling. SHH effects were related to three different cell phenomena: mode of cell division, cell cycle length and cell growth. Together, our data in vitro demonstrate that SHH signaling controls cell behaviors that are important for proliferation of cerebral cortex progenitors, as well as differentiation and survival of neurons and astroglial cells.

Development of A Flow Cytometry Assay for Measurement of CD4+ T Cell Proliferation

Thesis (Master's)--University of Washington, 2016-06

Role for cell-to-cell communication in stem cell specification toward pancreatic progenitors: relevance to the design of novel therapies for diabetes.

Thesis (Ph.D.)--University of Washington, 2016-08

Sonic hedgehog signaling regulates mode of cell division of early cerebral cortex progenitors and increases

The morphogen Sonic Hedgehog (SHH) plays a critical role in the development of different tissues. In the central nervous system, SHH is well known to contribute to the patterning of the spinal cord and separation of the brain hemispheres. In addition, it has recently been shown that SHH signaling also contributes to the patterning of the telencephalon and establishment of adult neurogenic niches. In this work, we investigated whether SHH signaling influences the behavior of neural progenitors isolated from the dorsal telencephalon, which generate excitatory neurons and macroglial cells in vitro. We observed that SHH increases proliferation of cortical progenitors and generation of astrocytes, whereas blocking SHH signaling with cyclopamine has opposite effects. In both cases, generation of neurons did not seem to be affected. However, cell survival was broadly affected by blockade of SHH signaling. SHH effects were related to three different cell phenomena: mode of cell division, cell cycle length and cell growth. Together, our data in vitro demonstrate that SHH signaling controls cell behaviors that are important for proliferation of cerebral cortex progenitors, as well as differentiation and survival of neurons and astroglial cells.

From genes to radioresistance in head and neck squamous cell carcinoma

Head and Neck Cancers (HNC) are a group of tumours located in the upper aero-digestive tract. Head and Neck Squamous Cell Carcinoma (HNSCC) represent about 90% of all HNC cases. It has been considered the sixth most malignant tumour worldwide and, despite clinical and technological advances, the five-year survival rate has not improved much in the last years. Nowadays, HNSCC is well established as a heterogeneous disease and that its development is due to accumulation of genetic events. Apart from the majority of the patients being diagnosed in an advanced stage, HNSCC is also a disease with poor therapeutic outcome. One of the therapeutic approaches is radiotherapy. However, this approach has different drawbacks like the radioresistance acquired by some tumour cells, leading to a worse prognosis. A major knowledge in radiation biology is imperative to improve this type of treatment and avoid late toxicities, maintaining patient quality of life in the subsequent years after treatment. Then, identification of genetic markers associated to radiotherapy response in patients and possible alterations in cells after radiotherapy are essential steps towards an improved diagnosis, higher survival rate and a better life quality. Not much is known about the radiation effects on cells, so, the principal aim of this study was to contribute to a more extensive knowledge about radiation treatment in HNSCC. For this, two commercial cell lines, HSC-3 and BICR-10, were used and characterized resorting to karyotyping, aCGH and MS-MLPA. These cell lines were submitted to different doses of irradiation and the resulting genetic and methylation alterations were evaluated. Our results showed a great difference in radiation response between the two cell lines, allowing the conclusion that HSC-3 was much more radiosensitive than BICR-10. Bearing this in mind, analysis of cell death, cell cycle and DNA damages was performed to try to elucidate the motifs behind this difference. The characterization of both cell lines allowed the confirmation that HSC-3 was derived from a metastatic tumour and the hypothesis that BICR-10 was derived from a dysplasia. Furthermore, this pilot study enabled the suggestion of some genetic and epigenetic alterations that cells suffer after radiation treatment. Additionally, it also allowed the association of some genetic characteristics that could be related to the differences in radiation response observable in this two cell lines. Taken together all of our results contribute to a better understanding of radiation effects on HNSCC allowing one further step towards the prediction of patients’ outcome, better choice of treatment approaches and ultimately a better quality of life.

Molecular and biochemical mechanisms involved in the toxicity of a marine cyanobacteria compound on cancer cell lines

In recent years marine biotechnology has revealed a crucial role in the future of bioindustry. Among the many marine resources, cyanobacteria have shown great potential in the production of bioactive compounds with diverse applicability. The pharmacological potential of these organisms has been one of the most explored areas in particular its antibacterial, antifungal and anticancer potential. This work was based on the assessment of potential anticancer compound E13010 F 5.4 isolated from marine cyanobacteria strain Synechocystis salina LEGE 06099. Thus the aim of this work was to explore molecular and biochemical mechanisms underlying the bioactivity detected in human cancer cells, specifically in lines RKO colon carcinoma and HT-29. The isolation of the compound was performed from biomass obtained by large-scale culture. To obtain the compound fractionation was carried and confirmation and isolation performed by Nuclear Magnetic Resonance (NMR), Thin Layer Chromatography (TLC) and High-Performance Liquid Chromatography (HPLC). Cell viability assays were performed based on reduction of 3- (4,5-dimetiltiaziol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) to assess the cytotoxic potential of the compound. From the battery of cell lines RKO (colon carcinoma), HT-29 (colorectal adenocarcinoma), MG-63 (osteosarcoma) and T47D (breast carcinoma) the cell lines RKO and HT-29 were selected for elucidation of mechanisms of cytotoxicity. For the elucidation of the mechanisms involved in cytotoxicity the cell lines RKO and HT29 were exposed to the compound. A genomic approach based in the mRNA expression of genes involved in apoptosis and cell cycle by Real-Time PCR and a proteomic approach based on the separation of proteins by two-dimensional electrophoresis (2DGE) was performed. For mRNA expression were selected the genes RPL8, HPRT1, VDAC, SHMT2, CCNE, CCNB1, P21CIP, BCL-2 and BAD and for proteomics isoelectric focussing between 3 – 10 and molecular weight of 19 – 117 kDa separated by polyacrylamide gels (2DGE). The MTT results confirmed the reduction of the cell viability. The RT-PCR results for the expression of genes studied were not yet fully elucidative. For the cell line RKO there was a significant reduction in the expression of the gene P21CIP, and a tendency for reduction in the BAD gene expression and for increased expression of gene CCNB1, pointing to an effort for cell proliferation. In HT-29 cell line, there was a tendency for increase in the expression of P21CIP and BAD, which may explain the reduction in cell viability. The 2DGE results indicate proteomic patterns with differentially altered spots in the treated and control cells with both qualitative and quantitative differences, and differences in response between the RKO and HT-29 cell lines.

Inhibition of proliferation, migration and invasion of human non-small cell lung cancer cell line A549 by phlomisoside F from Phlomis younghusbandii Mukerjee

Purpose: To determine the effect of phlomisoside F (PMF) on the proliferation, migration and invasion of human non-small cell lung cancer cell line A549 and explore the possible mechanisms. Methods: The anti-proliferative effect of PMF on A549 cells was determined by CCK-8. Subsequently, migration and invasion were evaluated by Transwell and Transwell with matrigel assays, respectively. Furthermore, cell cycle and apoptosis were assessed by flow cytometry, while the mechanisms of action were determined by Western blotting. Results: PMF exhibited significant anti-proliferative effect on A549 cells in concentration-dependent and time-dependent manners, with half maximal inhibitory concentration (IC50) of 54.51 μM. Treatment with PMF (10, 20 and 40 μM) for 48 h resulted in significantly decreased migration and invasion in A549 cells. In addition, PMF at concentrations of 25, 50 and 75 μM induced cell cycle arrest in G0/G1phase and enhanced cell apoptosis in A549 cells. Furthermore, caspase-3, caspase-9 and Bax protein expressions were up-regulated while Bacl-2 and COX-2 protein expressions were significantly downregulated at 10, 20 and 40 μM concentrations of PMF. Conclusion: PMF suppresses A549 cell growth, migration and invasion. The mechanism may be related to the induction of mitochondria-mediated apoptosis pathway via regulation of caspase-3, caspase-9, Bcl-2 and Bax expressions, and inhibition of PGE2 synthesis by reducing COX-2 expression.

6-(Methylsulfonyl) hexyl isothiocyanate as potential chemopreventive agent: molecular and cellular profile in leukaemia cell lines

Despite numerous therapeutic interventions cancer is still today the second leading cause of death. A growing interest has been addressed to isothiocytanates and more recently, the 6- (methylsulfonyl) hexyl isothiocyanate (6-MITC), the main constituent of the rhizome of Wasabia Japonica, has stimulated the interest of researchers. Aim of the research was to study if 6-MITC is able to modulate the main mechanisms underlying chemopreventive process in leukemic cells lines, verify the selectivity of action and the safety of use in terms of mutagenicity. The study was conducted on different cell types. In particular, Jurkat and HL-60 cells were treated with increasing concentrations of 6-MITC and cell viability, induction of apoptosis, cell cycle analysis, autophagy modulation and stimulation of differentiation were evaluated by flow cytometry. PBL, the non-transformed counterparty of leukemia cells, was used to analyse the selectivity of action by studying the same mechanisms previously indicated. Finally, safety of use and antimutagenicity were studied in TK6 cells adopting an automated protocol in flow cytometry. The achieved results have demonstrated that isothiocyanate modulates many signaling pathways involved in chemopreventive mechanism. In fact, 6-MITC induces apoptosis of both transformed cells, limits tumor growth by slowing down the cell cycle of Jurkat cells and blocks HL-60 cell cycle, increases the autophagic flux and induces cytodifferentiation of promyelocytic HL-60 into macrophage and granulocytic phenotypes. Furthermore, the results obtained with 6-MITC on PBL from healthy donors suggest that the isothiocyante is a good selective cytotoxic agent. Essential feature of a good chemopreventive agent is selectivity toward cancer cells and low toxicity towards non-transformed cells. Finally, the analysis of the micronuclei revealed that 6-MITC is not mutagenic, ensuring safe use, and that instead, it is able to counteract the mutagenic activity of the aneuploidogen Vinblastine, demonstrating another important and interesting chemopreventive activity.

Pipkiiα Is Widely Expressed In Hematopoietic-derived Cells And May Play A Role In The Expression Of Alpha- And Gamma-globins In K562 Cells.

Characterized for the first time in erythrocytes, phosphatidylinositol phosphate kinases (PIP kinases) belong to a family of enzymes that generate various lipid messengers and participate in several cellular processes, including gene expression regulation. Recently, the PIPKIIα gene was found to be differentially expressed in reticulocytes from two siblings with hemoglobin H disease, suggesting a possible relationship between PIPKIIα and the production of globins. Here, we investigated PIPKIIα gene and protein expression and protein localization in hematopoietic-derived cells during their differentiation, and the effects of PIPKIIα silencing on K562 cells. PIPKIIα silencing resulted in an increase in α and γ globins and a decrease in the proliferation of K562 cells without affecting cell cycle progression and apoptosis. In conclusion, using a cell line model, we showed that PIPKIIα is widely expressed in hematopoietic-derived cells, is localized in their cytoplasm and nucleus, and is upregulated during erythroid differentiation. We also showed that PIPKIIα silencing can induce α and γ globin expression and decrease cell proliferation in K562 cells.

Obesity And Inflammation And The Effect On The Hematopoietic System.

Bone marrow is organized in specialized microenvironments known as 'marrow niches'. These are important for the maintenance of stem cells and their hematopoietic progenitors whose homeostasis also depends on other cell types present in the tissue. Extrinsic factors, such as infection and inflammatory states, may affect this system by causing cytokine dysregulation (imbalance in cytokine production) and changes in cell proliferation and self-renewal rates, and may also induce changes in the metabolism and cell cycle. Known to relate to chronic inflammation, obesity is responsible for systemic changes that are best studied in the cardiovascular system. Little is known regarding the changes in the hematopoietic system induced by the inflammatory state carried by obesity or the cell and molecular mechanisms involved. The understanding of the biological behavior of hematopoietic stem cells under obesity-induced chronic inflammation could help elucidate the pathophysiological mechanisms involved in other inflammatory processes, such as neoplastic diseases and bone marrow failure syndromes.