Co-Chemotherapy Effect of Glycosylated Nanoalbumin Genitri Seed Extract Targeting Induced Apoptotic on Overexpressed HER2+ Breast Cancer

Event Abstract Back to Event Multiple siRNA delivery against cell cycle and anti-apoptosis proteins in breast cancer and normal cells Manoj Parmar1*, Bárbara E. Arteaga Ballesteros2*, Timothy Fu2*, Remant Bahadur Kc2*, Hamidreza Montazeri Aliabadi3*, Judith C. Hugh4* and Hasan Uludag1, 2, 5* 1 University of Alberta, Faculty of Pharmacy and Pharmaceutical Sciences, Canada 2 University of Alberta, Department of Chemical and Materials Engineering, Canada 3 Chapman University, School of Pharmacy, United States 4 University of Alberta, Department of Laboratory Medicine and Pathology, Canada 5 University of Alberta, Department of Biomedical Engineering, Canada Introduction: Current therapies for breast cancer induce severe side effects that warrant a search for alternative therapies. Short-interfering RNA (siRNA)-based therapeutics directly target and silence specific genes critical for cancer cells, thus holding promise as an effective therapy with less side effects. Given the unregulated cell cycle progression in cancer cells, proteins involved in cell cycle regulation such as spindle assembly checkpoint kinase TTK and cell-division cycle protein 20 (CDC20) are promising targets to stop uncontrolled cell growth. Other targets that might act in conjunction with cell cycle proteins are anti-apoptosis proteins such as survivin [1], which are often up-regulated in transformed cells. We hypothesize that the dual silencing of cell cycle and anti-apoptosis proteins might be exceptionally effective and specific for treatment of breast cancer. Here, we explored the combinational siRNA therapy in breast cancer as well as effects on normal cells in vitro. Methods & Results: Since siRNA is highly labile and anionic, we synthesized a library of cationic and hydrophobic (lipid-modified) polymers from 0.6, 1.2 and 2.0 kDa polyethylenimines (PEI). We screened this library for delivery of CDC20 siRNA in MDA-MB-231 cells, where 1.2 kDa PEI substituted with linoleic acid (PEI-LA) was found to be the most effective [2,][3]. We additionally determined efficacy of 6 siRNAs targeting at different locations of TTK gene; all TTK siRNAs inhibited growth of MDA-MB-231 and MCF7 breast cancer cells (as assessed by MTT Assay), which was further confirmed with the down-regulation of TTK transcripts by qPCR. We then explored the feasibility of combinational siRNA therapy by mixing specific siRNAs with polymers at different weight ratios to form complexes. TTK/CDC20 and CDC20/Survivin siRNA combinations decreased the growth of MDA-MB-231 cells significantly, while only TTK/CDC20 combination inhibited MCF7 cell growth significantly at a relatively low (15 nM each) siRNA concentration (Figure 1). The transcripts of each of these targets were significantly down-regulated in specific siRNA treated cells compared to scrambled siRNA treated cells. The combinational siRNA delivery was also performed in normal breast epithelial MCF10A cells, human umbilical vein endothelial cells (HUVEC) and human bone marrow stromal cells (HBMSC) to determine the side effects of siRNA treatments using 15 nM each siRNA at different siRNA:polymer ratios. The combinational siRNA therapy at lower ratio (1:2) showed higher side effects of siRNA treatment in these cells compared to higher ratios (1:4, 1:8; Figure 2). Based on relative effect of combinational siRNAs in breast cancer and normal cells, TTK/CDC20 siRNA combination inhibited MDA-MB-231 (33%) and MCF7 (24%) cell growth at 1:4 siRNA:polymer ratio without showing much side effects in MCF10A and HUVEC cells (Figure 2D). However, this combination showed significant side effects in HBMSC cells (33%). Conclusions: This study has indicated improved efficacy of combinational siRNA therapy against cell cycle and anti-apoptosis proteins in breast cancer therapy. The lipid-substituted polymers could serve as a viable platform for delivery of multiple siRNAs against critical targets. However, the siRNA therapy has also showed side effects on non-malignant (normal) cells in vitro and more selective therapies might be needed to target cancer cells solely. Manoj Parmar is a recipient of Women and Children's Health Research Institute (WCHRI) Graduate Studentship Grant and Alberta Innovates: Health Solutions (AIHS) Graduate Studentship.; This study was supported by operating grants from Canadian Breast Cancer Foundation (CBCF) and Natural Sciences and Engineering Council of Canada (NSERC).

World Health Organization articulated 9.8 million casualties globally in 2018 due to cancer. Cancer, as the world's second most fatal disease, can be recuperated well if diagnosed at an early stage. In this work, a gradient-based impedance synthesis of normal and cancerous cells of breast and lungs, is demonstrated numerically for early-stage cancer detection. Low-voltage single-cell level examination is employed for indomitable diagnosis. MCF-7 and MCF-10A are utilized as breast cancer and breastnormal cells, respectively; likewise, SK-MES and NL-20 are utilized as lung cancer and lung normal cell. Pre-examination numerical setup validity ensured with multiple test regimes. Micro-scaled planar and nano-structured electrodes are employed individually to witness the effect of the electrode's structure during electrical impedance examination of cancer and non-cancer cell. Frequency range, at which differential impedance effect is found detectable, for breast and lung cancer cell pairs is determined to be 107Hz and 108Hz, respectively. By surpassing the conventional impedance spectroscopy with tedious data fitting formalities, the gradient synthesis technique for cancer detection is introduced. The gradient synthesis for cancer detection is found independent of electrode shape effect. Gradient for breast cancer cell is found to be 2 times greater than the normal breast cell while for lung cancer cell it is found to be 1.5 times greater than the normal lung cell. Our results suggest that as the frequency of applied electrical stimulus increases, impedance of cancerous cell falls at the rate almost double than its counterpart normal cell. This work provides a theoretical basis for further experimental exploration of gradient-based impedance synthesis in cancer therapy and serves as a design tool for performance optimization. Figure 1 (a) Represents electrical Impedance analysis of breast normal cell MCF-10A and breast cancer cell MCF-7 using micro-scaled planar and nano-structured electrodes. (b) Gradient impedance synthesis performed, for breast normal cell (MCF-10A) and breast cancer cell (MCF-7) likewise for lungs normal cell (NL-20) and lungs cancer cell (SK-MES), which assures clear differential effect for cancer screening. Surpassing the conventional and tedious data fitting impedance spectroscopies, a novel gradient-based impedance spectroscopy for early cancer detection is introduced. It clearly detects cancer without any data fitting formalities to find parameter of identification. Planar and nano structure electrodes are used to witness the impact of electrode shape on cell impedance. Breast normal MCF-10A and cancer cell MCF-7 as well as lungs normal NL-20 and cancer cell SK-MES are examined to reflect the efficacy of our work. Single cell level examination is performed for authenticated results.

Breast cancer is one of the most lethal types of cancer in women worldwide. The human epidermal growth factor receptor 2 (HER2) is considered as a validated target in breast cancer therapy. Previously, we have used quantitative structure activity relationship QSAR equations and their associated pharmacophore models to screen for new promising HER2 structurally diverse inhibitory leads which were tested against HER2-overexpressing SKOV3 ovarian cancer cell line. In this study, we sought to explore the effect of most active ligands against different normal and breast cancer cell lines that represent different breast cancer subtypes with distinguished expression levels in HER2 and HER1. We have tested the promising compounds against SKBR3, MDA-MB-231, MCF7, human fibroblast, and MCF10 cell lines. To understand the inhibitory effects of the active ligands against HER2 over expressed breast cancer cell lines, all inhibitors and the control compound, lapatinib, were docked into the active site of HER2 enzyme performed using Ligand Fit docking engine and PMF scoring function. Five ligands exhibited promising results with relatively low IC50 values on cells that amplify HER2 and high IC50 on those that do not express such a receptor. The most potent compound (compound 13) showed an IC50 of 0.046µM. To test their toxicity against normal cells, the active compounds were tested against both normal fibroblast and normal breast cancer cell MCF-10 and relatively high IC50 values were scored. The IC50 values on HER2 over-expressed breast cancer and normal fibroblast cells provided a promising safety index. Docking results showed the highest similarity in the binding site between the most active ligand and the lapatinib. Our pharmacophore model resulted in a high potent ligand that shows high potency against HER2 positive breast cancer and relatively low toxicity towards the normal human cells.

Breast cancer is one of the most prevalent woman cancers. Genomic instability, accumulative mutations, and subsequent changes in intracellular signaling cascades play key roles in the development of human breast cancers. Activation of nuclear factor-kappaB (NF-kappaB) has been implicated in oncogenesis of breast cancers and is known to be associated with resistance to anticancer agents and apoptosis. Blocking NF-kappaB signaling may represent a therapeutic strategy in breast cancer therapy. The objective of this study is to investigate the in vitro effects of epoxypseudoisoeugenol-2-methyl butyrate (EPB), a phenylpropranoid isolated from Pimpinella corymbosa, on the activation of NF-kappaB, cell growth, cell cycle progression and apoptosis in MCF-7 (estrogen-dependent) and BT-549 (estrogen-independent) breast cancer cells. Transcriptional activity of NF-kappaB was measured by cell based reporter gene assay. Cell proliferation was determined by MTT assay. Cell cycle analysis was carried out by flow cytometry and apoptosis was observed by DAPI staining assy. EPB inhibited the NF-kappaB-mediated transcription activity induced by tumor necrosis factor-alpha (TNF-alpha) and phorbol myristate acetate (PMA) in MCF-7 cells. EPB also inhibited constitutive NF-kappaB transcriptional activity in BT-549 cells. EPB inhibited the proliferation of both MCF-7 and BT-549 cells in a concentration- and time-dependent manner. EPB induced cell cycle arrest in G(1)/G(0) phase and apoptosis in both MCF-7 and BT 549 cells. These in vitro results indicated that EPB has a potential for use against both hormone-dependent and hormone-independent breast cancers and its effects seem to be mediated by inhibiting the NF-kappaB activity.

Differential Allele-Specific Expression Uncovers Breast Cancer Genes Dysregulated by Cis Noncoding Mutations.

Anastasis is a phenomenon described as a cellular escape from ethanol-induced cell death. Although the relevant mechanism has not yet been fully elucidated, anastasis is thought to play a role in drug resistance in cancer cells. To date, the regulation of anastasis in normal and cancerous cells has not been clarified. The current cancer treatment strategies are expected to selectively attack cancer cells without negatively affecting normal cell proliferation. Inspired by the anti-cancer potential of bee venom, this study is the first to evaluate whether bee venom has similar selectivity in producing an anastatic effect. The results indicated that bee venom induces anastasis in normal cells (Michigan Cancer Foundation-10A (MCF10A), Adult Retinal Pigment Epithelium cell line-19 (ARPE-19), and National Institutes of Health 3T3 cell line (NIH3T3)) but causes irreversible cell death in breast cancer cells (M.D. Anderson-Metastatic Breast-231 (MDA-MB-231) and Michigan Cancer Foundation-7 (MCF7)). Liver cancer (HepG2) cells were moderately more resistant to permanent cell death after bee venom treatment compared to breast cancer cells. However, cisplatin caused permanent non-selective cell death in both normal and cancerous cells. The selectivity indices after bee venom treatment were higher compared to cisplatin. Taken together, bee venom was shown to induce selective anastasis only in normal cells, not in cancer cells, which suggests that bee venom has significant potential in selective cancer therapy, especially for breast cancer, via promoting the recovery and maintenance of viability of normal cells.

Selective Cancer Targeting via Aberrant Behavior of Cancer Cell-associated Glucocorticoid Receptor

Artemisinin (ART), a common anti‐malarial drug, and its analogs are also useful as repurposed anti‐cancer drugs, but their properties are not fully elucidated. We previously synthesized novel trioxane (DMR) and dioxazanine (HSM) ART analogs and showed that: a. at low doses (5–10 μM), HSM > than DMR in inducing apoptosis in human colon and lung cancer cells, but not in normal lung cells; b. The actions involved reactive oxygen species (ROS) (Faseb J, ′18:32, 616.2). TfR expression is increased in cancer vs. normal cells. Thus, we hypothesize that, in cancer cells, TfR increases [Fe2+]i which acts on the endoperoxide to produce ROS and induce apoptosis. We examined the mechanism of action of DMR/HSM in inducing apoptosis in human breast, lung, and colon cancer cell lines as compared to normal cell lines. To increase the efficacy of the drug, we also designed and synthesized a 3rd novel analog with dual dioxazanine pharmacophores, PMW.Confluent normal (Breast: MCF10A, Lung: A549) and cancer (Breast: MCF7, Colon: T84, Lung: A549) cells were treated (18 H) with 1–50μM of DMR, HSM or PMW±Deferoxamine (DFO, iron chelator; 1–25 μM). Cells were stained with FITC‐Annexin V (apoptosis), propidium iodide (cell death) or CellRox Green (mitochondrial/nuclear ROS), imaged and quantified by flow cytometry and/or microscopy (Image J). Normal and cancer cell lysates (30 μg) were subjected to SDS‐PAGE and immunoblotting with polyclonal TfR antibody or anti‐GAPDH (control).As in other cancer cells, both DMR and HSM (5 – 50μM) induced apoptosis in breast cancer, but not normal, cells. In contrast to A549 lung cells, where HSM was more effective than DMR, in MCF7 breast cells, 50μM DMR was more efficient (~40%) in inducing apoptosis than HSM (10%). This dictates the development of novel tissue‐specific analogs. The new analog, PMW (1 – 50μM, 18H) did not induce apoptosis in the cancer cells. The larger size of PMW may have slowed entry, suggesting longer incubations.Image J analysis (mean pixel intensity; 18 H) of CellRox+ lung cells (n=3) showed that HSM dose‐dependently increased ROS in cancer A549: (DMSO: 1.5±0.2; HSM, 5 μM: 19±2; 10 μM: 48±4; 50 μM: 59±2); but not normal BEAS2B: (DMSO: 1.0±0.3; HSM, 5 μM: 1.8±1; 10 μM: 2.2±0.4; 50 μM: 2.9±2). Similarly, in breast cancer cells (n=3), DMR dose‐dependently increased ROS in MCF7 (DMSO: 1.0±0.3; DMR, 5 μM: 12±2; 10 μM: 51±5; 50 μM: 42±3); but not in MCF10A (DMSO: 1.0±0.2; DMR, 5 μM: 1.6±1; 10 μM: 3.1±1; 50 μM: 2.5±1).Immunoblotting detected a distinct 94 kDa TfR protein in colon, lung, and breast cancer, but not normal cells. The effect of DMR/HSM±DFO on apoptosis was examined to study the role of TfR. Albeit showing an inhibitory trend, DFO did not significantly alter ART analogs actions (% Annexin V+ cells,18 H: A549: HSM,10μM: 46±4; HSM+5μM DFO: 38±6; HSM +10μM DFO: 33±9; MCF7: DMR,10μM: 36±4; DMR+5μM DFO: 31±7; DMR+10μM DFO: 29±5; n≥3).Synthesizing novel ART‐analogs with improved pharmacokinetics to specifically target cancer cells and elucidating their mechanism of action will help develop new alternatives to treat cancer.Support or Funding InformationNSF ‐ MRI: DBI‐1427937 to JS; Ben U Funds to JS and DMR; UIC Funds to MCR; APS‐STRIDE to UD; APS‐UGSRF to MHThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Tamoxifen therapy for the treatment of hormone responsive breast cancer has limitations due to acquired resistance in the case of recurrences. Embelin, a known inhibitor of X-linked inhibitor of apoptosis protein (XIAP) was also reported to exhibit strong antiestrogenic effects in animal models. Dual role of embelin as a proapoptotic and antiestrogenic agent may have potential benefits in the therapy of breast cancer. In this study, the effects of embelin treatment on estrogen receptor positive Human breast adenocarcinoma (MCF-7) cells was investigated to primarily understand if embelin being an antiestrogen and XIAP inhibitor could be a potential alternative to tamoxifen therapy. Results revealed that, embelin at a concentration of 65μg/ml attenuated proliferation, inhibited metastatic migration, modulated the expression of Bcl2, Caspases and induced apoptosis in MCF-7 cells which was found to be p53 mediated. Hence, chemotherapy with embelin could be a promising strategy to be experimented in hormone responsive breast cancers.

The role of radiation therapy in the management of primary breast cancer

Paclitaxel (PTX) is one of the first-line chemotherapeutic agents for treating breast cancer. However, PTX resistance remains a major hurdle in breast cancer therapy. Crocin, the main chemical constituent of saffron, shows anti-cancer activity against various types of cancer. However, the effect of crocin on the resistance of PTX in breast cancer is still unknown. CCK-8 and TUNEL assays were employed to detect cell viability and apoptosis, respectively. The targets of crocin were predicted using HERB database and the targets associated with breast cancer were acquired using GEPIA database. The Venn diagram was utilized to identify the common targets between crocin and breast cancer. Baculoviral inhibitor of apoptosis repeat containing 5 (BIRC5) expression was detected by qRT-PCR and western blot analysis. The correlation between BIRC5 expression and survival was analyzed by Kaplan-Meier plotter and PrognoScan databases. Our data suggested that crocin aggravated PTX-induced decrease of viability and increase of apoptosis in MCF-7 and MCF-7/PTX cells. BIRC5 was identified as the target of crocin against breast cancer. Crocin inhibited BIRC5 expression in MCF-7 and MCF-7/PTX cells. BIRC5 is overexpressed in breast cancer tissues, as well as PTX-sensitive and PTX-resistant breast cancer cells. BIRC5 expression is related to the poor survival of patients with breast cancer. Depletion of BIRC5 strengthened PTX-induced viability reduction and promotion of apoptosis in MCF-7 and MCF-7/PTX cells. Moreover, BIRC5 overexpression reversed the inhibitory effect of crocin on PTX resistance in breast cancer cells. In conclusion, crocin enhanced the sensitivity of PTX in breast cancer cells partially through inhibiting BIRC5 expression.

Adjuvant therapy of primary breast cancer. 4th International Conference on Adjuvant Therapy of Primary Breast Cancer St. Gallen, Switzerland.

Cancer remains the leading cause of death worldwide, with breast cancer being the most prevalent disease diagnosed globally. Liver cancer is the fourth most common cause of death globally, while prostate cancer accounts for the second most frequent malignancy among males worldwide. The main treatment options for cancer include surgery, radiation therapy, and chemotherapy. The stem bark of Tieghemella heckelii has been exploited for its medicinal properties. On a broader scale, research on the anticancer properties of T. heckelii has not been explored. It is therefore important to investigate the anticancer potential of the leaves of T. heckelii. The goal of the study was to evaluate the in vitro anticancer activities of the leaves of T. heckelii on breast, prostate, and liver cancer cell lines. The leaves of T. heckelii were collected from Asantemanso, Akim Oda, in the eastern region of Ghana. Authenticity of the leaves was performed at the Department of Plant and Environmental Biology, University of Ghana. Aqueous and ethanol extraction were performed on the leaves of T. heckelii after grinding into fine particles, followed by low-temperature drying. Breast cancer (MDA-MB-468), liver cancer (HepG2), and normal kidney (Vero E6) cell lines were cultured in DMEM media, while prostate cancer (PC3) cell lines were cultured in RPMI medium. Anticancer activity of the leaves of T. heckelii was conducted on breast (MDA-MB-468), liver (HepG2), and prostate (PC3) cancer cell lines. Cell viability was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. Both ethanolic and aqueous extracts demonstrated similar cytotoxicity for the HepG2 cell line, with IC50 values of about 200 μg/mL. selectivity index of > 2 was recorded by all cell lines. When compared to other cell lines, the aqueous extract for prostate cancer showed the lowest IC50 value with a selectivity of 8. In addition, the extracts showed less cytotoxic activity against the normal (Vero) cell line. Leaves of T. heckelii possess cytotoxic properties with notable selectivity against prostate (PC3) and liver (HepG2) cancer cell lines. However, findings should be evaluated in in vivo studies because biological processes can be more complex in living organisms. Further investigation should be conducted to ascertain the bioactive compounds responsible for the anticancer activity.

Precise Detection and Visualization of Cyclooxygenase-2 for Golgi Imaging by a Light-Up Aggregation-Induced Emission-Based Probe

The skin mucus in lower vertebrates such as fish with strong innate immune system has many unique and valuable bioactive compounds that can be used for inducing apoptosis in cancer cells. This study was looking for the cytotoxic potential of mucus from the two fish species, including round goby (Neogobius melanostomus) and common carp (Cyprinus carpio), and inducing apoptosis in MCF7 and LNCaP cancer cell lines via influencing P53 gene expression and cell cycle arrest. Results showed that the both mucus types have cytotoxic effects on the both cancer cell lines whereas they have no severe effect on normal primary fibroblast cells. In addition, round goby mucus and common carp mucus selectively induced apoptosis in the LNCaP and MCF7 cells, respectively, through up-regulating P53 gene and arresting cell cycle at the G1 phase. Taken together, this study suggested that the both mucus types can selectively influence P53 pathway and induce apoptosis in especial cancer cells. The skin mucus derived from round Goby and common Carp can be a promising candidate for investigation about apoptosis and molecular targeting therapy in cancer.