Anti-KRAS siRNA nanoparticles for targeted colorectal cancer therapy.

2017 ◽  
Vol 35 (4_suppl) ◽  
pp. 636-636 ◽  
Author(s):  
Bradley Krasnick ◽  
Matthew S. Strand ◽  
Ye Bi ◽  
Peter S. Goedegebuure ◽  
Timothy Fleming ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cell Viability ◽  
Caspase 3 ◽  
Fluorescent Microscopy ◽  
Mutant Type ◽  
Viability Assay ◽  
Mutational Status ◽  
Promising Target ◽  
Folfox Chemotherapy

636 Background: Standard treatment for metastatic colorectal cancer (mCRC) is systemic chemotherapy with anti-EGFR treatment, depending on KRAS mutational status. However, tumors harboring a KRAS mutation do not respond to existing targeted therapy. Moreover, targeting mutant KRAS has, to date, not been possible. Herein, we explore using a KRAS inhibitory nanoparticle (NP), to directly knock down mutant KRAS. Methods: Utilizing fluorescent-labeled small interfering RNA (siRNA) NPs, uptake was assessed via fluorescent microscopy. KRAS mutant CT26 and wild-type MC38 CRC cell lines were incubated with either scramble (Sc) sequence siRNA NP, KRAS siRNA NP, or FOLFOX (fluorouracil + oxaliplatin) chemotherapy ± KRAS siRNA NP. Cell viability was assessed via a luminescent viability assay. KRAS and cleaved caspase 3 protein expression were assessed using western blotting. Results: Fluorescent NP uptake was demonstrated in CT26 cells as early as 260 minutes post treatment, with increased uptake through 780 minutes. Decreased cellular viability was seen with KRAS siRNA NP treated CT26 cells, as compared to both Sc siRNA NP and non-treated CT26 cells (both p < 0.0001). Cell viability was significantly diminished with FOLFOX combined with KRAS siRNA NP as compared to FOLFOX alone for CT26 cells ( p = 0.0003), but not MC38 cells (p = 0.2259). Western blot demonstrated decreased KRAS and increased cleaved caspase 3 expression in CT26 cells treated with KRAS siRNA NP. Conclusions: A KRAS siRNA tagged NP was internalized by the CRC cells in vitro, and induced cellular death via apoptosis in mutant type KRAS CRC. In addition, KRAS siRNA NP acted synergistically with FOLFOX chemotherapy to enhance cell death. We believe KRAS inhibition based NP treatment is a promising target for mutant type KRAS CRC. [Table: see text]

scholarly journals β-Adrenergic Receptor Inhibitor and Oncolytic Herpesvirus Combination Therapy Shows Enhanced Antitumoral and Antiangiogenic Effects on Colorectal Cancer

2021 ◽  
Vol 12 ◽  
Author(s):  
Jiali Hu ◽  
Cuiyu Chen ◽  
Ruitao Lu ◽  
Yu Zhang ◽  
Yang Wang ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Oncolytic Virus ◽  
Caspase 3 ◽  
Therapeutic Potential ◽  
Tumor Regression ◽  
Oncolytic Viruses ◽  
Cell Viability Assay ◽  
Viability Assay

Oncolytic viruses (OVs) are considered a promising therapeutic alternative for cancer. However, despite the development of novel OVs with improved efficacy and tumor selectivity, their limited efficacy as monotherapeutic agents remains a significant challenge. This study extended our previously observed combination effects of propranolol, a nonselective β-blocker, and the T1012G oncolytic virus into colorectal cancer models. A cell viability assay showed that cotreatment could induce synergistic killing effects on human and murine colorectal cell lines. Moreover, cotreatment caused sustained tumor regression compared with T1012G monotherapy or propranolol monotherapy in human HCT116 and murine MC38 tumor models. The propranolol activity was not via a direct effect on viral replication in vitro or in vivo. Western blotting showed that cotreatment significantly enhanced the expression of cleaved caspase-3 in HCT116 and MC38 cells compared with the propranolol or T1012G alone. In addition, propranolol or T1012G treatment induced a 35.06% ± 0.53% or 35.49% ± 2.68% reduction in VEGF secretion in HUVECs (p &lt; 0.01/p &lt; 0.01). Cotreatment further inhibited VEGF secretion compared with the monotherapies (compared with propranolol treatment: 75.06% ± 1.50% decrease, compared with T1012G treatment: 74.91% ± 0.68%; p＜0.001, p &lt; 0.001). Consistent with the in vitro results, in vivo data showed that cotreatment could reduce Ki67 and enhance cleaved caspase 3 and CD31 expression in human HCT116 and murine MC38 xenografts. In summary, β-blockers could improve the therapeutic potential of OVs by enhancing oncolytic virus-mediated killing of colorectal cancer cells and colorectal tumors.

scholarly journals Direct Effect of Septic Plasma in Human Cell Lines Viability

2018 ◽  
Vol 47 (1-3) ◽  
pp. 270-276
Author(s):  
Grazia Maria Virzì ◽  
Chiara Borga ◽  
Chiara Pasqualin ◽  
Silvia Pastori ◽  
Alessandra Brocca ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Cell Viability ◽  
Cell Lines ◽  
Caspase 3 ◽  
Test Group ◽  
Organ Injury ◽  
Enzyme Linked Immunosorbent Assay ◽  
Caspase 9 ◽  
Renal Tubular Cells

Background: Sepsis is a life-threatening condition often associated with a high incidence of multiple organs injury. Several papers suggested the immune response by itself, with the production of humoral inflammatory mediators, is crucial in determining organ injury. However, little is known of how sepsis directly induces organ injury at the cellular levels. To assess this point, we set up an in vitro study to investigate the response of renal tubular cells (RTCs), monocytes (U937) and hepatocytes (HepG2) after 24 h-incubation with septic patients’ plasma. Methods: We enrolled 26 septic patients (“test” group). We evaluated cell viability, apoptosis and necrosis by flow cytometer. Caspase-3,-8,-9 and cytochrome-c concentrations have been analyzed using the Human enzyme-linked immunosorbent assay kit. Results: We found that a decrease of cell viability in all cell lines tested was associated to the increase of apoptosis in RTCs and U937 (p < 0.0001) and increase of necrosis in HepG2 (p < 0.5). The increase of apoptosis in RTCs and U937 cells was confirmed by higher levels of caspase-3 (p < 0.0001). We showed that apoptosis in both RTCs and U937 was triggered by the activation of the intrinsic pathway, as caspase-9 and cytochrome-c levels significantly increased (p < 0.0001), while caspase-8 did not change. This assumption was strengthened by the significant correlation of caspase-9 with both cytochrome-c (r = 0.73 for RTCs and r = 0.69 for U937) and caspase-3 (r = 0.69 for RTCs and r = 0.63 for U937). Conclusion: Humoral mediators in septic patients’ plasma induce apoptosis. This fact suggests that apoptosis inhibitors should be investigated as future strategy to reduce sepsis-induced organ damages.

Evaluation of Paclitaxel (Taxol), Cisplatin, and the Combination Paclitaxel-Cisplatin in Ovarian Cancer in Vitro with the ATP Cell Viability Assay

1994 ◽  
Vol 53 (1) ◽  
pp. 44-49 ◽  
Author(s):  
Michael Untch ◽  
Bernd-Uwe Sevin ◽  
James P. Perras ◽  
Roberto Angioli ◽  
Andrea Untch ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Cell Viability ◽  
Cell Viability Assay ◽  
Viability Assay

scholarly journals Overexpression of IRS-4 Correlates with Procaspase 3 Levels in Tumoural Tissue of Patients with Colorectal Cancer

2018 ◽  
Vol 2018 ◽  
pp. 1-14
Author(s):  
Patricia Sanmartín-Salinas ◽  
Luis G. Guijarro
Keyword(s):  
Colorectal Cancer ◽  
Caspase 3 ◽  
Cultured Cells ◽  
Cyclin Dependent Kinase ◽  
Kinase Activation ◽  
Tnm System ◽  
Protein Levels ◽  
Receptor Signalling ◽  
T Values

We reported that insulin receptor substrate 4 (IRS-4) levels increased in tissue from colorectal cancer (CRC) patients and promoted retinoblastoma-cyclin-dependent kinase activation. The aim of the present study was to evaluate the effect of IRS-4 on IGF-1 receptor pathway and its impact on procaspase 3 and PARP expression in RKO and HepG2 cancer cell lines. The results obtained in vitro were compared with those obtained from biopsies of patients with CRC (n = 18), tubulovillous adenomas (TA) (n = 2) and in matched adjacent normal colorectal (MANC) tissue (n = 20). IRS-4 overexpression in cultured cells induced the overactivation of IGF-1/BRK/AKT/GSK-3/β-catenin/cyclin D1 pathways, which led to increased expression of procaspase 3 and PARP protein levels. Studies carried out on CRC and TA tissues revealed the overactivation of the IGF-1 receptor signalling pathway, as well as the overexpression of procaspase 3 and PARP in tumoural tissue with respect to MANC tissue. The upregulation of IRS-4 in tumoural samples correlated significantly with the increase in pIGF-1 receptor (Tyr 1165/1166) (r = 0.84; p < 0.0001), procaspase 3 (r = 0. 77; p < 0. 0005) and PARP (r = 0. 89; p < 0. 0005). Similarly, we observed an increase in the proteolysis of procaspase 3 in tumoural tissue with respect to MANC tissue, which correlated significantly with the degradation of PARP (r = 0.86; p < 0.0001), p53 (r = 0.84; p < 0.0001), and GSK-3 (r = 0.78; p < 0.0001). The stratification of patient samples using the TNM system revealed that procaspase 3 and caspase 3 increased gradually with T values, which suggests their involvement in the size and local invasion of primary tumours. Taken together, our findings suggest that IRS-4 overexpression promotes the activation of the IGF-1 receptor pathway, which leads to the increase in procaspase 3 levels in CRC.

scholarly journals N6-Isopentenyladenosine Inhibits Colorectal Cancer and Improves Sensitivity to 5-Fluorouracil Targeting FBXW7 Tumor Suppressor

Cancers ◽  
2019 ◽  
Vol 11 (10) ◽  
pp. 1456 ◽  
Author(s):  
Donatella Fiore ◽  
Chiara Piscopo ◽  
Maria Proto ◽  
Michele Vasaturo ◽  
Fabrizio Dal Piaz ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Tumor Suppressor ◽  
In Vitro Models ◽  
Antitumor Action ◽  
Cancer Proliferation ◽  
Ubiquitinated Proteins ◽  
Mutational Status ◽  
Ubiquitin Ligase Complex

N6-isopentenyladenosine has been shown to exert potent in vitro antitumor activity on different human cancers, including colorectal cancer. Although some potential biochemical targets have been identified, its precise mechanism of action remains unclear. We found that N6-isopentenyladenosine affects colorectal cancer proliferation in in vitro models carrying different mutational status of FBXW7 and TP53 genes, and in HCT116 xenografts in SCID mice, by increasing the expression of the well-established tumor suppressor FBXW7, a component of the SCF-E3 ubiquitin ligase complex that promotes degradation of various oncoproteins and transcription factors, such as c-Myc, SREBP and Mcl1. Corroborating our previous studies, we identified for the first time the FBXW7/SREBP/FDPS axis as a target of the compound. Pull down of ubiquitinated proteins, immunoprecipitation and luciferase assays, reveal that through the increase of FBXW7/c-Myc binding, N6-isopentenyladenosine induces the ubiquitination of c-Myc, inhibiting its transcriptional activity. Moreover, in FBXW7- and TP53-wild type cells, N6-isopentenyladenosine strongly synergizes with 5-Fluorouracil to inhibit colon cancer growth in vitro. Our results provide novel insights into the molecular mechanism of N6-isopentenyladenosine, revealing its multi-targeting antitumor action, in vitro and in vivo. Restoring of FBXW7 tumor-suppressor represents a valid therapeutic tool, enabling N6-isopentenyladenosine as optimizable compound for patient-personalized therapies in colorectal cancer.

scholarly journals Targeting REV7 effectively reverses 5-FU and oxaliplatin resistance in colorectal cancer

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Xianjun Sun ◽  
Wenhou Hou ◽  
Xin Liu ◽  
Jie Chai ◽  
Hongliang Guo ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cell Viability ◽  
Research Effort ◽  
Translesion Synthesis ◽  
Xenograft Model ◽  
Mice Model ◽  
Cell Viability Assay ◽  
Viability Assay ◽  
Xenograft Mice Model

Abstract Background Despite an enormous research effort, patients diagnosed with advanced colorectal cancer (CRC) still have low prognosis after surgical resection and chemotherapy. The major obstacle for CRC treatment is chemoresistance to front line anti-cancer drugs, such as 5-fluorouracil (5-FU) and oxaliplatin. However, the mechanism of chemoresistance to these drugs remains unclear. Methods Cell viability to 5-FU and oxaliplatin was measured by the CellTiter-Glo® 2.0 Cell Viability Assay. The endogenous REV7 protein in CRC cells was detected by western blotting. The translesion synthesis (TLS) events were measured by plasmid-based TLS efficiency assay. Cell apoptosis was evaluated by caspase3/7 activity assay. The in vivo tumor progression was analyzed by HT29 xenograft mice model. Results In this study, we found that expression of REV7, which is a key component of translesion synthesis (TLS) polymerase ζ (POL ζ), is significantly increased in both 5-FU and oxaliplatin resistant CRC cells. TLS efficiency analysis revealed that upregulated REV7 protein level results in enhanced TLS in response to 5-FU and oxaliplatin. Importantly, inhibition of REV7 by CRISPR/Cas9 knockout exhibited significant synergy with 5-FU and oxaliplatin in cell culture and murine xenograft model. Conclusion These results suggest that combination of REV7 deficiency and 5-FU or oxaliplatin has strong inhibitory effects on CRC cells and identified REV7 as a promising target for chemoresistant CRC treatment.

Lipid emulsion attenuates apoptosis induced by a toxic dose of bupivacaine in H9c2 rat cardiomyoblast cells

2016 ◽  
Vol 35 (9) ◽  
pp. 929-937 ◽  
Author(s):  
S-H Ok ◽  
J Yu ◽  
Y Lee ◽  
H Cho ◽  
I-W Shin ◽  
...  
Keyword(s):  
Cell Viability ◽  
Lipid Emulsion ◽  
Caspase 3 ◽  
In Vitro Study ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Caspase 8 ◽  
Lipid Solubility ◽  
H9c2 Cells ◽  
Toxic Dose

The goal of this in vitro study was to investigate the effect of lipid emulsion on apoptosis induced by a toxic dose of bupivacaine (BPV) in H9c2 rat cardiomyoblast cell lines. The effect of lipid emulsion on the decreased cell viability and count induced by BPV or mepivacaine (MPV) in the H9c2 cells was assessed using an 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide assay or a cell count assay. The effect of BPV or lipid emulsion combined with BPV on cleaved caspase 3, caspase 8, and Bax in H9c2 cells was investigated using Western blotting. A terminal deoxynucleotidyl transferase dUTP2′-deoxyuridine 5′-triphosphate nick end-labeling (TUNEL) assay was performed to detect apoptosis of H9c2 cells treated with BPV alone or lipid emulsion combined with BPV. The magnitude of lipid emulsion-mediated attenuation of decreased cell viability induced by BPV was higher than that of lipid emulsion-mediated attenuation of decreased cell viability induced by MPV. Lipid emulsion attenuated the increases in cleaved caspase 3, caspase 8 and Bax induced by BPV. Lipid emulsion attenuated the increases in TUNEL-positive cells induced by BPV. These results suggest that lipid emulsion attenuates a toxic dose of BPV-induced apoptosis via inhibition of the extrinsic and intrinsic apoptotic pathways. The protective effect of lipid emulsion may be partially associated with the relatively high lipid solubility of BPV.

scholarly journals Apoptosis Induced by Ziziphora tenuior Essential Oil in Human Colorectal Cancer Cells

2021 ◽  
Vol 2021 ◽  
pp. 1-9
Author(s):  
Mohammadreza Azimi ◽  
Jalil Mehrzad ◽  
Armita Ahmadi ◽  
Elnaz Ahmadi ◽  
Ali Ghorbani Ranjbary
Keyword(s):  
Colorectal Cancer ◽  
Essential Oil ◽  
Mtt Assay ◽  
Caspase 3 ◽  
Rna Extraction ◽  
Gastrointestinal Diseases ◽  
Control Group ◽  
Gene Expressions ◽  
Ht 29

Ziziphora (Cacotti in Persian) belongs to the Lamiaceae family (mint group) and is vastly found in Iran and Asia. This traditional medicinal plant is normally used as analgesic and for treatment of particular gastrointestinal diseases. Since colorectal cancer is one of the most common causes of death in the world and the second leading cause of cancer death among adults, there is a pressing need to inhibit this malignancy by using methods with minimal side effects. One of these methods is the use of natural resources such as medical plants. This study is aimed at investigating the expression of apoptosis-related genes in the adjacent culture of colorectal cancer epithelial cells (HT-29) with Ziziphora essential oil (ZEO). The essential oil was extracted from Ziziphora leaves, and its compounds were determined and then added to the HT-29 culture medium at different concentrations. After 24 hours, the HT-29 cells were harvested from the medium and cytotoxicity was analyzed by MTT assay. After MTT assay and determination of the percentage of apoptosis by flow cytometry, RNA extraction was performed and the expression levels of Bax, Bcl-2, caspase 3 (C3), and caspase 9 (C9) were analyzed using newly designed primers by reverse transcription (RT) qPCR method and GeniX6 software. Also, specific antibodies were used for western blot analyses of those molecules. GC analysis revealed 42 different compounds in the ZEO, including pulegone (26.65%), menthone (5.74%), thymol (5.51%), and menthol (1.02%). MTT assay showed that the concentration of 200 μg/ml of ZEO had the highest HT-29 cell death during 24 hours. After incubation with the concentration of 50 μg/ml of ZEO for 24 and 48 hours, caspase 3 and 9 gene expressions in the treated group increased compared to those in the control group ( P < 0.001 ), while the Bcl-2 expression decreased. The results showed that having anticancer compounds, ZEO can increase C3 and C9 and decrease Bcl-2 expressions, causing apoptosis in HT-29 cells in vitro. This can lead to the use of ZEO as a factor for colorectal cancer treatment.

scholarly journals Anlotinib Overcomes Multiple Drug Resistant Colorectal Cancer Cells via Inactivating PI3K/AKT Pathway

2021 ◽  
Vol 21 ◽  
Author(s):  
Weilan Lan ◽  
Jinyan Zhao ◽  
Wujin Chen ◽  
Haixia Shang ◽  
Jun Peng ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Drug Resistance ◽  
Cell Viability ◽  
Cancer Cells ◽  
Cell Apoptosis ◽  
Caspase 3 ◽  
Akt Pathway ◽  
Inhibition Of Proliferation ◽  
Colorectal Cancer Cells ◽  
Induced Apoptosis

Background: Anlotinib is a multi-target tyrosine kinase inhibitor that has been reported to have activity against colorectal cancer. However, the mechanisms of how anlotinib mediates drug-resistance of colorectal cancer have not been fully described. Particularly the potential mechanisms regarding to the inhibition of proliferation and induction of apoptosis remain unknown. Objective: In this study, we intended to study the effect and related-mechanism of the proliferation, migration, invasion and induced apoptosis of anlotinib overcoming multidrug resistant colorectal cancer cells through in vitro experiments. Methods: Cell viability was determined by MTT assays and the resistant index was calculated. Colony formation and PI/RNase Staining were used for testing the proliferation of resistant cells. DAPI staining and Annexin V-FITC/PI staining were used to detect cell apoptosis. Migration and invasion were examined by transwell. Protein expression and activation of PI3K/AKT pathway were detected by western blot. LY294002 was used to verify whether anlotinib overcomes the drug-resistance of CRC cells by inactivating the PI3K/AKT pathway. Results: The results showed that the HCT-8/5-FU cells were resistant to multiple chemotherapy drugs (5-FU, ADM and DDP). Anlotinib significantly inhibited the cell viability, proliferation, migration, invasion and induced the cell apoptosis. Moreover, anlotinib downregulated the expression of survivin, cyclin D1, CDK4, caspase-3, Bcl-2, MMP-2, MMP-9, vimentin and N-cadherin, but up-regulated cleaved-caspase-3, Bax and E-cadherin and blocked the activity of the PI3K/AKT in HCT-8/5-FU cells. We found anlotinib and LY294002 overcame the drug resistance of HCT-8/5-FU cells by reducing the expression of PI3K/p-AKT. Conclusions: Anlotinib inhibited the proliferation, migration, invasion and induced apoptosis of HCT-8/5-FU cells, and the mechanisms may be that anlotinib conquered multidrug resistance of colorectal cancer cells via inactivating of PI3K/AKT pathway.

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