Reduced Cancer Cell Viability Research Articles

BRC-mediated RNAi targeting of USE1 inhibits tumor growth in vitro and in vivo

USE1 has been demonstrated to play crucial roles in the development and progression of human lung cancer. However, the antitumor efficacy of RNA interference (RNAi) targeting of USE1 has not yet been evaluated as a possible clinical application. We here synthesized USE1 targeting bubbled RNA-based cargo (BRC) composed of densely packed multimeric pre-siRNAs with specific Dicer cleavage sites to enable efficient siRNA release upon entry to target cells. The physical entanglement and continuous networking of RNAs via hybridization during enzymatic replication serve as a driving force for the self-assembly of BRCs. These molecules effectively suppressed the transcription of their target genes, leading to tumor growth suppression in vitro and in vivo. Moreover, their repeated intravenous administration efficiently inhibited the growth of A549 tumor xenografts. Based on these findings of a reduced cancer cell viability following a USE1 knockdown, we further explored cell cycle arrest and apoptosis pathways. The observed tumor cell growth suppression was found to be controlled by cell cycle arrest and apoptosis signals induced by the USE1 reduction. These results suggest that USE1 BRCs may have future clinical applications as an RNAi-based cancer therapy.

Next-Generation Multimodality of Nanomedicine Therapy: Size and Structure Dependence of Folic Acid Conjugated Copolymers Actively Target Cancer Cells in Disabling Cell Division and Inducing Apoptosis.

Nanomedicine as a multimodality treatment of cancer utilizes the advantages of nanodelivery systems of drugs. They are superior to the clinical administration of different therapeutic agents in several aspects, including simultaneous delivery of drugs to the active site, precise ratio control of the loading drugs and overcoming multidrug resistance. The role of nanopolymer size and structural shape on the internalization process and subsequent intracellular toxicity is limited. Here, the size and shape dependent mechanism of a functionalized copolymer was investigated using folic acid (FA) covalently bonded to the copolymer poly (styrene-alt-maleic anhydride) (SMA) on its hydrophilic exterior via a biological linker 2,4-diaminobutyric acid (DABA) to target folic acid receptors (FR) overly expressed on cancer cells actively. We recently reported that unloaded FA-DABA-SMA copolymers significantly reduced cancer cell viability, suggesting a secondary therapeutic mechanism of action of the copolymer carrier post-internalization. Here, we investigated the size and shape dependent secondary mechanism of unloaded 350 kDa and 20 kDa FA-DABA-SMA. The 350 kDa and 20 kDa copolymers actively target folic acid receptors (FR) to initialize internationalization, but only the large size and sheet shaped copolymer disables cell division by intracellular disruptions of essential oncogenic proteins including p53, STAT-3 and c-Myc. Furthermore, the 350 kDa FA-DABA-SMA activates early and late apoptotic events in both PANC-1 and MDA-MB-231 cancer cells. These findings indicate that the large size and structural sheet shape of the 350 kDa FA-DABA-SMA copolymer facilitate multimodal tumor targeting mechanisms together with the ability to internalize hydrophobic chemotherapeutics to disable critical oncogenic proteins controlling cell division and to induce apoptosis. The significance of these novel findings reveals copolymer secondary cellular targets and therapeutic actions that extend beyond the direct delivery of chemotherapeutics. This report offers novel therapeutic insight into the intracellular activity of copolymers critically dependent on the size and structural shape of the nanopolymers.

A high-throughput drug combination screen of targeted small molecule inhibitors in cancer cell lines

While there is a high interest in drug combinations in cancer therapy, openly accessible datasets for drug combination responses are sparse. Here we present a dataset comprising 171 pairwise combinations of 19 individual drugs targeting signal transduction mechanisms across eight cancer cell lines, where the effect of each drug and drug combination is reported as cell viability assessed by metabolic activity. Drugs are chosen by their capacity to specifically interfere with well-known signal transduction mechanisms. Signalling processes targeted by the drugs include PI3K/AKT, NFkB, JAK/STAT, CTNNB1/TCF, and MAPK pathways. Drug combinations are classified as synergistic based on the Bliss independence synergy metrics. The data identifies combinations that synergistically reduce cancer cell viability and that can be of interest for further pre-clinical investigations.

Antiproliferative effect of MoS2 in human prostate cancer cell lines

In this investigation, the anti-proliferative activity of a novel molybdenum complex was distinguished on LNCaP (as an androgen-dependent), PC3 (as an androgen-independent) cancer cells, and normal PBD2-fib cells (as a control) using MTT assay, flow cytometry, RT-qPCR, and western blotting. The MoS2 was prepared by the hydrothermal method, and the synthetic MoS2 characterized using XRD, EDX, FESEM, HRTEM, and Raman spectroscopies to confirm the success of the synthesis and the unique crystal structure. The cells were treated with different concentrations of MoS2 (0, 5, 10, 20, 35 and 50 μg ml−1) for 24, 48 and 72 h. The obtained results showed that the IC50 values for LNCaP (21.02 ± 0.09 μg ml−1) and PC3 (23.03 ± 0.07 μg ml−1) were significantly lower than that recorded for normal fibroblast cells (41.56 ± 0.012 μg ml−1). Flow cytometry findings demonstrated that the complex is effective in reducing cancer cell viability via apoptosis. RT-qPCR data showed a decrease in BCL2 expression and increases in BAX and P53 gene expression, which were also correlated with the synthetic complex response. The expression of P53 protein increased in LNCaP and PC3 cells after treating with MoS2. Also, these data show the anti-tumor properties of synthetic molybdenum complexes in prostate cancer cells. To conclude, the results indicated that the novel design of nanoparticles can be created a new generation of nano-therapeutics strategies in different types of cancer.

Co-Treatment with Sulforaphane and Nano-Metformin Molecules Accelerates Apoptosis in HER2+ Breast Cancer Cells by Inhibiting Key Molecules

Breast cancer cell lines MCF-10, MCF-7 and BT-474 expressing various levels of HER2 were examined for their response to treatment with sulforaphane (SLFN), metformin (MTFN), Nano-MTFN or combinations. Direct correlation was found between SLFN effect on cell death and HER2 levels. Bioinformatic studies suggested the possibility of additive co-effects on cell fate by SLFN-MTFN co-treatment. This co-treatment specially with SLFN + Nano-MTFN significantly affected the survival of the cells and killed more BT-474 cells than the other two. Cell sensitivity to SLFN-MTFN combination correlated with HER2 expression levels. RT-PCR showed that parallel with cell death, expression of BCL-2, SRC, WNT1, β-catenin and CD44 are diminished, whereas BAX levels are elevated significantly. Cell co-staining indicated that apoptosis percent correlates with cell death following different treatments. We also found that cell death induced by SLFN-MTFN co-treatment is in direct correlation with HER2 levels and increased cell death correlates directly with BAX levels but inversely with levels of cancer stem cell (CSC) signaling genes and CD44. In conclusion, our data indicate that SLFN and MTFN can reduce cancer cell viability via both collaborative and differential effects and suggest that MTFN increases SLFN effectiveness by targeting common molecules/pathways downstream of HER2 and key for CSC signaling.

(-)-Oleocanthal and (-)-oleocanthal-rich olive oils induce lysosomal membrane permeabilization in cancer cells.

(-)-Oleocanthal (oleocanthal) is a phenolic compound found in varying concentrations in extra virgin olive oil oleocanthal has been shown to be active physiologically, benefiting several diseased states by conferring anti-inflammatory and neuroprotective benefits. Recently, we and other groups have demonstrated its specific and selective toxicity toward cancer cells; however, the mechanism leading to cancer cell death is still disputed. The current study demonstrates that oleocanthal, as well as naturally oleocanthal-rich extra virgin olive oils, induced damage to cancer cells’ lysosomes leading to cellular toxicity in vitro and in vivo. Lysosomal membrane permeabilization following oleocanthal treatment in various cell lines was assayed via three complementary methods. Additionally, we found oleocanthal treatment reduced tumor burden and extended lifespan of mice engineered to develop pancreatic neuroendocrine tumors. Finally, following-up on numerous correlative studies demonstrating consumption of olive oil reduces cancer incidence and morbidity, we observed that extra virgin olive oils naturally rich in oleocanthal sharply reduced cancer cell viability and induced lysosomal membrane permeabilization while oleocanthal-poor oils did not. Our results are especially encouraging since tumor cells often have larger and more numerous lysosomes, making them especially vulnerable to lysosomotropic agents such as oleocanthal.

PEGylated gold nanoparticles-ribonuclease induced oxidative stress and apoptosis in colorectal cancer cells.

Introduction: Currently, drug-induced reactive oxygen species (ROS) mediating apoptosis pathway have extensively been investigated in designing effective strategies for colorectal cancer (CRC) chemotherapy. Bovine pancreatic ribonuclease A (RNase A) represents a new class of cytotoxic and non-mutagenic enzymes, and has gained more attention as a potential anticancer modality; however, the cytosolic ribonuclease inhibitors (RIs) restrict the clinical application of this enzyme. Nowadays, nanotechnology-based diagnostic and therapeutic systems have provided potential solutions for cancer treatments. Methods: In this study, the gold nanoparticles (AuNPs) were synthesized, stabilized by polyethylene glycol (PEG), functionalized, and covalently conjugated with RNase A. The physicochemical properties of engineered nanobiomedicine (AuNPs-PEG-RNase A) were characterized by scanning electron microscope (SEM), dynamic light scattering (DLS), and UV-vis spectrum. Then, its biological impacts including cell viability, apoptosis, and ROS production were evaluated in the SW-480 cells. Results: The engineered nanobiomedicine, AuNPs-PEG-RNase A, was found to effectively induce apoptosis in SW-480 cells and result in a significant reduction in cancer cell viability. Besides, the maximum production of ROS was obtained after the treatment of cells with an IC50 dose of AuNPs-PEG-RNase A. Conclusion: Based on the efficient ROS-responsiveness and the anticancer activity of RNase A of the engineered nanomedicine, this nanoscaled biologics may be considered as a potential candidate for the treatment of CRC.

Abstract LB-149: Genome-wide prediction of synthetic rescue mediators of resistance to targeted and immunotherapy

Abstract Background: Most patients with advanced cancer eventually acquire resistance to targeted therapies, spurring extensive efforts to identify molecular events mediating therapy resistance. Here we demonstrate that many of these molecular events involve synthetic rescue (SR) genetic interactions, where the reduction in cancer cell viability caused by targeted gene inactivation is rescued by an adaptive alteration of another gene (the rescuer). Methods: Analyzing recently published large scale in vitro functional screens we identify 100,000s candidate SR interactions that show evidence of rescue events in cancer cell lines. We then analyze tumor transcriptomic, genomic, and genetic profiles together with survival and clinical characteristics of 10,000 TCGA cancer patients to identify the few thousand SR interactions that have strong evidence that they are mediators of emerging resistance in patient tumors. Results: We identify the first rescue network, composed of SR interactions common to many cancer types. The identified SRs successfully match the resistance mediators identified in recently published clinical studies. We perform multiple in vitro analyses in head and neck and lung cancer, showing that targeting predicted rescuer genes successfully re-sensitizes resistant cancer cells, providing specific leads for targeting resistance proactively. We further demonstrate that SR-based combination therapy can improve the progression free survival in mouse xenografts models. Notably, we show that SR interactions successfully predict cancer patients' response in the TCGA compendium, showing performance superior to existing machine learning based predictive models. Finally, we show that SR analysis of melanoma patients successfully identifies known mediators of resistance to checkpoint immunotherapy (reported previously in mice) and suggests new combination therapies that counteract the resistance. Conclusions: This work presents a new paradigm identifying and harnessing synthetic rescue interactions to counteract resistance to both targeted- and immuno-therapies. Future implementations of this approach will have two broad implications in the precision oncology era: first for determining the most effective treatment regimen based on the molecular characteristics of individual patient’s tumor; second for identifying conjunct drugs to counteract resistance to existing primary therapies, for both targeted and immune checkpoint therapies. Citation Format: Avinash Das, Joo Sang Lee, Gao Zhang, Zhiyong Wang, Arnaud Amzallag, Genevieve Boland, Sridhar Hannenhalli, Meenhard Herlyn, Cyril Benes, J. Silvio Gutkind, Keity Flaherty, Eytan Ruppin. Genome-wide prediction of synthetic rescue mediators of resistance to targeted and immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-149.

Abstract 509: Acarbose, but not metformin, reduces tumor burden and improves intra-tumoral immune responses in a pre-clinical breast cancer model

Abstract Finding ways to enhance T cell infiltration into tumors and boost effector function remains a critical knowledge gap that must be addressed to improve responses to immune-stimulatory therapies. Acarbose and metformin are FDA-approved drugs for the treatment of type two diabetes that display potent anti-hyperglycemic activity. A treatment strategy that decreases intra-tumoral glucose could reduce cancer cell viability and render tumors more susceptible to immunotherapy-mediated clearance. The goal of the current study was to determine if acarbose and/or metformin reduce tumor burdens and promote anti-tumor immunity in a preclinical model of breast cancer. Eight-week-old female C57BL/6 mice were orthotopically injected with EO771-Fluc mammary tumor cells then randomized to control (CTRL), CTRL+acarbose (ACA), CTRL+metformin (MET), or acarbose+metformin (ACA+MET) for four weeks. Compared to CTRL, single-agent ACA reduced primary tumor growth (50% decrease; p<0.001) and spontaneous lung metastases (48% decrease; p=0.035) without altering body weight. No effects of single-agent MET or the combination of ACA+MET on EO771 tumor growth or spontaneous lung metastases were observed. nanoString analysis of whole-tumors revealed increased dendritic cell-, T cell-, T helper 1 cell-, CD8+ T cell-, and exhausted CD8+ T cell-based gene signature scores in ACA compared to CTRL-fed mice. Cellular analysis revealed an ACA-induced increase in the percent of tumor-infiltrating PD-1+CD8+ T cells (p=0.042) and a trend toward decreased tumor-infiltrating myeloid-derived suppressor cells, resulting in improved CD8:MDSC ratios. The beneficial effects of ACA on intra-tumoral immunity, specifically promoting T cell immunity within the tumor microenvironment, illustrate the potential for future combined use of ACA with immunotherapies, such as PD-1 or CTLA-4 blockade. Citation Format: William J. Turbitt, Rachael M. Orlandella, Justin T. Gibson, Lyse A. Norian. Acarbose, but not metformin, reduces tumor burden and improves intra-tumoral immune responses in a pre-clinical breast cancer model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 509.

Preparation, structural characterization and bioactivity of 4-O-Methylglucuronoxylan from Artemisia sphaerocephala Krasch

We obtained four soluble acid xylan fractions AGP-III-A, AGP-III-B, AGP-III-C and AGP-III-D from the insoluble Artemisia sphaerocephala Krasch gum (ASKG) polysaccharide by weak alkali treatment combined with H2O2-Vc oxidative degradation. Activity studies showed that the degradation components could reduce the cell viability of several cancer cell lines in a concentration-dependent manner, especially 4-O-Methylglucuronoxylan AGP-III-C with specific molecular weight and branching degree significantly reduced cancer cells viability and induced HepG2 apoptosis, also caused mitochondrial membrane dysfunction upregulated ROS levels, and induced G0/G1 arrest in HepG2 cells by cell cycle assay. Further, AGP-III-C mediates apoptosis in HepG2 cells by upregulating MAPK phosphorylation. The structure of AGP-III-C was characterized by uronic acid reduction, permethylation with GC-MS, and 2D-NMR analysis. The structure of AGP-III-C had a linear (1→4)-linked β-Xylf residue backbone with one branched 4-O-Me-α-GlcAp attached to the main chain by a (1→2)-glycosidic bond at every two β-(1→4)-Xylf units.

Comparison of ionic polymers in the targeted drug delivery applications as the coating materials on the Fe3O4 nanoparticles

The objective of the study was to obtain multifunctional core shell nanostructures of superparamagnetic iron oxide nanoparticles (Fe3O4) coated with various ionic biopolymers that can optimize toxicity to healthy cells, colloidal instabilities and drug loading capacities. These nanostructures can also allow drug delivery to tumor tissue because of their magnetic properties, accumulation and drug release at tumor site could be controlled by means of an external magnetic field.The impact of the biopolymers with different ionic properties to final core shell structures were investigated and compared in terms of their colloidal properties, cytotoxicities, drug adsorption and drug delivery capacities. Besides, the effect of the surface charges on the healthy cells and cancer cells is very important factor affecting toxicity and drug delivery. The results showed that the drug delivery agents coated with cationic biopolymers with cationic surface properties significantly reduced cancer cell viability compared to the anionic and nonionic polymer coatings even though their drug loading capacities were found to be the lowest.

PH‐Responsive Nanoparticles Loaded with Graphene Quantum Dots and Doxorubicin for Intracellular Imaging, Drug Delivery and Efficient Cancer Therapy

AbstractMultifunctional core‐shell structural nanoparticles (NPs) integrating of therapeutic and imaging modalities have presented superior prospects as drug delivery systems in the treatment of cancer. In this work, graphene quantum dots (GQDs) and doxorubicin (DOX) were encapsulated into the cores of PLGA (Poly (D, L‐lactide‐co‐glycolide)) nanoparticles coated with bovine serum albumin via a double emulsion‐solvent evaporation method to construct pH‐responsive nanoparticles (DOX/PB and GQDs@DOX/PB NPs) with spherical core‐shell structures. Subsequently, the cellular uptake, cytotoxicity and biocompatibility of the NPs were studied via in vitro experiments. The results demonstrated that DOX/PB NPs and GQDs@DOX/PB NPs exhibit high drug‐loading efficiency and promote the release of DOX in a mild acidic microenvironment, which was expected to result in intelligent intracellular drug carriers. In vitro cytotoxicity measurements showed that all the NPs exhibit dose‐ and time‐dependent cytotoxicity to the HeLa cells and PLGA NPs loaded with GQDs contribute to reducing cancer cell viability. Moreover, no significant difference in ROS level was determined during the endocytosis. In addition, all the NPs displayed excellent blood compatibility by hemolysis assay. These results demonstrate that pH‐responsive NPs may be excellent candidates for efficient cancer therapy and in vivo biological imaging probes.

Telmisartan Influences the Antiproliferative Activity of Linoleic Acid in Human Colon Cancer Cells

Aim: Linoleic acid (LA) and telmisartan as PPARgamma agonists exhibit anticancer activity. The LA effect is observed for high non-achievable in vivo concentrations and in short treatment period, therefore we evaluate the effect of supplemental LA and pharmacological telmisartan plasma concentrations on human primary (SW480) and metastatic (SW620) colon cancer cells and immortal keratinocytes (HaCaT) cells in long-term treatment. Methods: Cell viability and proliferation were determined by TB and MTT and pro-apoptotic effect was measured by Annexin V binding assays, respectively.Results: LA decreased cancer cell viability and proliferation in a concentration-dependent manner, whereas no significant effect was found for HaCaT cells. Telmisartan (0.2 µM) suppresses antiproliferative effect of 60 µM LA on cancer cells in short-term treatment. Long-term administration of 60 µM LA reduced cancer cells viability after one week, while telmisartan delayed this effect by two weeks. Growth of all cell lines with 20 µM LA was unchanged during all treatment time. Telmisartan decreased late apoptosis of cancer and normal cells with 60 and 120 µM LA. Conclusion: The cytotoxic LA action depends not only on its concentration but also duration of treatment. Telmisartan exhibits biphasic but not synergistic effect on LA cytotoxicity in cancer cells.

Genome‐wide prediction of synthetic rescue mediators of resistance to targeted and immunotherapy

Most patients with advanced cancer eventually acquire resistance to targeted therapies, spurring extensive efforts to identify molecular events mediating therapy resistance. Many of these events involve synthetic rescue (SR) interactions, where the reduction in cancer cell viability caused by targeted gene inactivation is rescued by an adaptive alteration of another gene (the rescuer). Here, we perform a genome‐wide in silico prediction of SR rescuer genes by analyzing tumor transcriptomics and survival data of 10,000 TCGA cancer patients. Predicted SR interactions are validated in new experimental screens. We show that SR interactions can successfully predict cancer patients’ response and emerging resistance. Inhibiting predicted rescuer genes sensitizes resistant cancer cells to therapies synergistically, providing initial leads for developing combinatorial approaches to overcome resistance proactively. Finally, we show that the SR analysis of melanoma patients successfully identifies known mediators of resistance to immunotherapy and predicts novel rescuers.

Mathematical modeling and control for cancer treatment with cold atmospheric plasma jet

Cold atmospheric plasma (CAP) jet exhibits remarkable properties that trigger cell death in cancer cells. The effect of CAP on cancer cells is influenced by several factors including plasma jet discharge voltages, gas composition and cancer cell type. Consequently in clinics it becomes challenging to plan plasma cancer treatments for a particular cancer types. To address this, we present preliminary results for an in vitro model which includes an optimal feedback control scheme that can adjust treatment conditions based on the actual cancer cell response. Translation to an in vivo model will be the next objective of the presented project. First, a mathematical model is presented for the dynamic response of cancer cells to CAP jets based on experimental data that provide temporal measurements of cancer cell viability after CAP treatments. A differential equation is developed to model the influence of CAP on the viability of two cancer cell lines, U-87 MG and MDA-MB-231, under varying treatment duration and plasma discharge voltages. Subsequently, a control scheme is presented to determine CAP treatment conditions in an optimal fashion by reducing cancer cell viability less than a prescribed goal while minimizing a weighted sum of the treatment duration and the discharge voltage. This is further extended to a model predictive control framework such that a pre-planned CAP treatment schedule is revised according to the actual cancer cell response. The efficacy of the proposed approach is illustrated by numerical simulations based on experimental data.

Abstract P2-06-21: Endogenous expression of ERβ variants contributes towards chemotherapy-resistance in the triple negative breast cancer cell line HCC-1806

Abstract Triple negative breast cancer (TNBC) still remains a challenge to treat in the clinic due to a lack of good targets for treatment. Although TNBC lacks expression of ERα, the expression of ERβ and its variants are detected quite frequently in this cancer type and can represent an avenue for treatment. We show that the variants of ERβ, namely ERβ1, ERβ2, ERβ4, and ERβ5, contributes to aggressiveness of the TNBC cell line HCC1806 by affecting E-Cadherin expression and chemotherapy sensitivity. We have previously shown that the HCC1806 cell line expresses all the ERβ variants. To investigate their function we used the recently developed dCas9-KRAB system with Guide RNA for both ERβ promoters 0N and 0K, using 3 guide RNA's for each promoter and we found that expression of panERβ decreased from a ct. of 28 to 33 indicating loss of total ERβ. Treatment of HCC1806 cells with chemotherapy in combination with an ERβ agonist, LY500307, is more effective in reducing cancer cell viability. Knockdown of total ERβ in HCC1806 cells using CRISPR-Cas9 technology, decreased the proliferation and increased chemo-sensitivity to docetaxel agent in cells with the 0K promoter knock down, while cells with the 0N promoter knock down were less affected. Furthermore, after ERβ knockdown, the cell morphology of HCC-1806 changed to a more epithelial phenotype and there was increase in the expression of the classical epithelial marker, E-cadherin. N-Cadherin was unchanged in the cells with the 0K promoter knock down, while E-Cadherin was slightly decreased in cells with the 0N promoter knock down. We also found that cells with the 0K promoter knock down migrated less than control cells in a migration assay, while cells with the 0N promoter knock down were less affected. These findings are in agreement with reports showing that the 0K promoter is responsible for expression of the variants ERβ2 and ERβ5, while the 0N promoter expresses more ERβ1. Furthermore, we have earlier shown that ERβ2 and ERβ5 increase aggressiveness of TNBC via several oncogenic signaling pathways. In conclusion, knockdown of ERβ variants in HCC-1806 indicated that the variants may be responsible for conferring oncogenic properties to TNBC such as EMT, cancer cell proliferation and chemo-resistance. Therefore, treating TNBC patients with combination therapy including ERβ agonists, may prove to be extremely beneficial for securing better treatment responses and future pre-clinical studies should design appropriate experiments to confirm these findings in vivo. Citation Format: Faria M, Gustafsson J-A, Strom A. Endogenous expression of ERβ variants contributes towards chemotherapy-resistance in the triple negative breast cancer cell line HCC-1806 [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-06-21.

Differential anti-tumour effects of MTH1 inhibitors in patient-derived 3D colorectal cancer cultures

Reactive oxygen species (ROS) function as second messengers in signal transduction, but high ROS levels can also cause cell death. MTH1 dephosphorylates oxidized nucleotides, thereby preventing their incorporation into DNA and protecting tumour cells from oxidative DNA damage. Inhibitors of MTH1 (TH588 and (S)-crizotinib) were shown to reduce cancer cell viability. However, the MTH1-dependency of the anti-cancer effects of these drugs has recently been questioned. Here, we have assessed anti-tumour effects of TH588 and (S)-crizotinib in patient-derived 3D colorectal cancer cultures. Hypoxia and reoxygenation – conditions that increase intracellular ROS levels – increased sensitivity to (S)-crizotinib, but not to TH588. (S)-crizotinib reduced tyrosine phosphorylation of c-MET and ErbB3 whereas TH588 induced a mitotic cell cycle arrest, which was not affected by adding ROS-modulating compounds. Furthermore, we show that both compounds induced DNA damage that could not be prevented by adding the ROS inhibitor N-acetyl-L-cysteine. Moreover, adding ROS-modulating compounds did not alter the reduction in viability in response to TH588 and (S)-crizotinib. We conclude that TH588 and (S)-crizotinib have very clear and distinct anti-tumour effects in 3D colorectal cancer cultures, but that these effects most likely occur through distinct and ROS-independent mechanisms.

Anti-EGFR anchored paclitaxel loaded PLGA nanoparticles for the treatment of triple negative breast cancer. In-vitro and in-vivo anticancer activities.

The aim of the present study is to analyze the viability of anti-EGFR anchored immunonanoparticle (INP) bearing Paclitaxel (PTX) to specifically bind the EGFR protein on the TNBC cells. The NP was prepared by nanoprecipitation and characterized the particle size, charge, entrapment of drug and release of it. The anti-EGFR anchored and the integrity was confirmed by SDS-PAGE. Cytotoxicity and NPs cellular uptake was analyzed with MDA-MB-468 type cancer cells and the EGFR expression was confirmed by PCR, qualitatively and quantitatively. The in-vivo antitumor activity of INP was determined by using athymic mice model and targeting efficiency was measured by calculating the PTX accumulation in the tumor plasma. The prepared INP with the size of 336.3 nm and the charge of -3.48 mV showed sustained drug release upto 48 h. The INP showed significant reduction of cancer cell viability of 10.6% for 48 h with 93 fold higher PTX accumulation in the tumor plasma compared with NPs. Based on these reports, we recommend that anti-EGFR anchored PTX loaded NP may have the ability to target the TNBC cells and improve the therapeutic action and subsidize the side effects of PTX for the treatment of TNBC.

Studies examining the synergy between Dihydrotanshinone and Temozolomide against MGMT+ glioblastoma cells in vitro: Predicting interactions with the blood-brain barrier

We showed previously that Dihydrotanshinone (DHT) augments temozolomide (TMZ) efficacy by inducing reactive oxygen species production in an in vitro model. Here, the underlying basis of the synergistic effect and the ability of DHT to potentially pass the blood brain barrier (BBB) is investigated using an in vitro model. Trypan blue exclusion assays were used to determine effects of DHT/TMZ combinatorial treatment on GBM cell viability. ELISA was utilized to determine effects on NFkB levels after singular and combinatorial treatment. An in vitro model of the BBB was constructed to predict the potential of DHT to penetrate the BBB in vivo. DHT and TMZ synergistically reduce cancer cell viability, NFkB activity, and markedly halt cell cycle progression. This regimen was also shown to exert minimal effects on astrocytes. Finally, DHT was shown to have the potential of passing through the BBB to a similar extent as TMZ and that paclitaxel’s oncolytic effects are completely ablated in the presence of our in vitro BBB. Our data confirms the synergistic interaction between DHT and TMZ and also highlights the potential of combination treatment to sequester NFkB activity and inhibit cell cycle progression. The encouraging data with the BBB model show that the DHT/TMZ combination may be clinically useful and warrants future in vivo testing.

Engineering methionine γ-lyase from Citrobacter freundii for anticancer activity

Methionine deprivation of cancer cells, which are deficient in methionine biosynthesis, has been envisioned as a therapeutic strategy to reduce cancer cell viability. Methionine γ-lyase (MGL), an enzyme that degrades methionine, has been exploited to selectively remove the amino acid from cancer cell environment. In order to increase MGL catalytic activity, we performed sequence and structure conservation analysis of MGLs from various microorganisms. Whereas most of the residues in the active site and at the dimer interface were found to be conserved, residues located in the C-terminal flexible loop, forming a wall of the active site entry channel, were found to be variable. Therefore, we carried out site-saturation mutagenesis at four independent positions of the C-terminal flexible loop, P357, V358, P360 and A366 of MGL from Citrobacter freundii, generating libraries that were screened for activity. Among the active variants, V358Y exhibits a 1.9-fold increase in the catalytic rate and a 3-fold increase in KM, resulting in a catalytic efficiency similar to wild type MGL. V358Y cytotoxic activity was assessed towards a panel of cancer and nonmalignant cell lines and found to exhibit IC50 lower than the wild type. The comparison of the 3D-structure of V358Y MGL with other MGL available structures indicates that the C-terminal loop is either in an open or closed conformation that does not depend on the amino acid at position 358. Nevertheless, mutations at this position allosterically affects catalysis.