Invivo Antitumor Activity Research Articles

Anti-Tumor and Anti-Oxidant Activity of Ethanolic Extract of Epipremnum Aureum Linn. Leaves against DAL Induced Tumor in Swiss Albino Mice

Neoplasia literally means the process of “new growth,” and a new growth is called a neoplasm. The term tumor was originally applied to the swelling caused by inflammation. Neoplasms also may induce swellings, but by long precedent, the non-euplastic usage of tumor has passed into limbo; thus, the term is now equated with neoplasm. Oncology (Greek oncos = tumor) is the study of tumors or neoplasm’s. Cancer is the common term for all malignant tumors. The present study was designed to investigate the anti-tumor activity of ethanolic leaves extract of Epipremnum aureum Linn. and evaluated by in-vitro and in-vivo experimental models. To achieve objectives, EEEA was subjected to phytochemical screening and tested for oral toxicity test. The in-vitro study was carried out by means of MTT assay and Trypan blue dye exclusion assay using DAL cell lines. The in-vivo anti-tumor activity was evaluated against DAL tumour bearing mice by liquid tumour models. Preliminary phytochemical screening was confirmed the presence of flavonoids, glycosides, tannins, phenolic, steroids, and triterpeniods etc. EEEA showed good cytotoxic effect on DAL cell line in MTT assay and Trypan blue dye exclusion assay. Oral administration of EEEA in tumour bearing mice for 14 days, showed significant reduction in the percent increase in body weight, tumour volume, tumour weight, viable cell count when compared to the untreated mice of the DAL control group. The restoration of the haematological parameters towards the normal control was also observed. The results suggested that the EEEA exhibits significant anti-tumor activity towards both methods. The DAL-bearing mice orally administered leaves of Epipremnum aureum Linn., at 200 and 400 mg/kg body weight showed significant change in the average life span compared to animals of the tumor control group. The percentage increase in body weight, tumor cell volume, and number of viable tumor cells were found to be significantly less than the tumor control animals, indicating the anti-tumor nature of the extract.

Abstract 1970: Preclinical candidate TQB3824, a small molecule inhibitor of CDC7, shows strong antitumor efficacy in colorectal and pancreatic tumor models

Abstract Objective: Cell division cycle 7 (CDC7), a receptor serine/threonine kinase which features high-affinity with protein DBF4, plays an important role in DNA replication and DNA damage response via MCM2 phosphorylation. CDC7 has been reported to be up-regulated by about 50% in various human cancers and is being investigated as a therapeutic target in clinical trials. The purpose of this study is to investigate the in-vitro and in-vivo antitumor activities of TQB3824, a small molecule of CDC7 kinase inhibitor, in preclinical models with CDC7 overexpression. Method: Kinase inhibition activities of TQB3824 were determined with CDC7/DBF4 kinase assays. Cellular anti-proliferative activities were evaluated in COLO205 cell line and NCI-H226 cell line, which features CDC7 overexpression and no CDC7 expression, respectively. Three CDX models, colorectal cancer COLO205/SW620 and pancreatic cancer CAPAN-1 with KRAS mutation, were used to test the efficacy of TQB-3824 in vivo. Result: TQB3824 showed potent activities in both kinase inhibition and COLO205 cell anti-proliferation, with IC50 of 2.9 nM and 14.5 nM, separately. In contrast, TQB3824 showed much weaker anti-proliferative activity in NCI H226 cells with IC50 of 8154 nM. TQB3824 displayed strong antitumor efficacy in CDC7 highly expressed colorectal cancer CDX model COLO205 (TGI = 77% @5 mpk, BID), SW620 CDX model (TGI = 100% @5 mpk, BID), and pancreatic cancer CAPAN-1 CDX model (TGI = 115% @6 mpk, BID). Conclusion: We have identified a novel potent CDC7 inhibitor TQB3824, which shows high antitumor efficacy in CDC7 overexpressed solid tumor models. TQB3824 represents a promising clinical candidate for treating solid tumors with high CDC7 expression. Citation Format: Gang Li, Xiquan Zhang, Ling Yang, Xin Tian, Zhong xiong, Lihong Hu, Yuanfeng xia, Chi-chung chan, Charles Z. Ding, Shuhui Chen. Preclinical candidate TQB3824, a small molecule inhibitor of CDC7, shows strong antitumor efficacy in colorectal and pancreatic tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1970.

Chemical modification of NSC12 leads to a specific FGF-trap with antitumor activity in multiple myeloma.

Inhibition of FGF/FGFR signaling is a promising strategy for the treatment of malignances dependent from FGF stimulation, including multiple myeloma (MM). The steroidal derivative NSC12 (compound 1) is a pan-FGF trap endowed with antitumor activity invivo. Chemical modifications of compound 1 were explored to investigate structure-activity relationships, focusing on the role of the bis(trifluoromethyl)1,3-propanediol chain, the stereochemistry at C20 and functionalization of C3 position. Our studies unveiled compound 25b, the pregnane 3-keto 20R derivative of compound 1 as an effective agent, blocking the proliferation of MM cells invitro by inhibiting FGF-dependent receptor activation and slowing MM growth invivo. Importantly, the absence of the hydroxyl group at C3 prevents binding to estrogen receptors, which might concur to the antitumor activity observed for compound 1, leading to a specific FGF/FGFR system inhibitor, and further supporting the role of FGFR in anticancer therapy in MM.

Metabolic flexibility determines human NK cell functional fate in the tumor microenvironment

NK cells are central to anti-tumor immunity and recently showed efficacy for treating hematologic malignancies. However, their dysfunction in the hostile tumor microenvironment remains a pivotal barrier for cancer immunotherapies against solid tumors. Using cancer patient samples and proteomics, we found that human NK cell dysfunction in the tumor microenvironment is due to suppression of glucose metabolism via lipid peroxidation-associated oxidative stress. Activation of the Nrf2 antioxidant pathway restored NK cell metabolism and function and resulted in greater anti-tumor activity invivo. Strikingly, expanded NK cells reprogrammed with complete metabolic substrate flexibility not only sustained metabolic fitness but paradoxically augmented their tumor killing in the tumor microenvironment and in response to nutrient deprivation. Our results uncover that metabolic flexibility enables a cytotoxic immune cell to exploit the metabolic hostility of tumors for their advantage, addressing a critical hurdle for cancer immunotherapy.

Prevalence of Homologous Recombination Pathway Gene Mutations in Melanoma: Rationale for a New Targeted Therapeutic Approach

Homologous recombination DNA damage repair (HR-DDR) deficient patients with various solid tumors have been treated with PARP inhibitors. However, the clinical characteristics of patients with melanoma who have HR-DDR gene mutations and the consequences of PARP inhibition are poorly understood. We compared the commercially available next-generation sequencing data from 84 patients with melanomas from our institution with a dataset of 1,986 patients as well as 1,088 patients profiled in cBioportal. In total, 21.4% of patients had ≥1 functional HR-DDR mutation, most commonly involving BRCA1, ARID1A, ATM, ATR, and FANCA. Concurrent NF1, BRAF, and NRAS mutations were found in 39%, 39%, and 22% of cases, respectively. HR-DDR gene mutation was associated with high tumor mutational burden and clinical response to checkpoint blockade. A higher prevalence of HR-DDR mutations was observed in the datasets from Foundation Medicine (Cambridge, CA) and those from the Cancer Genome Atlas. Treatment of HR-DDR‒mutated patient-derived xenograft models of melanoma with PARP inhibitor produced significant antitumor activity invivo and was associated with increased apoptotic activity. RNA sequencing analysis of PARP inhibitor-treated tumors indicated alterations in the pathways involving extracellular matrix remodeling, cell adhesion, and cell-cycle progression. Melanomas with HR-DDR mutations represent a unique subset, which is more likely to benefit from checkpoint blockade and may be targeted with PARP inhibitor.

Antitumor and Radiosensitizing Effects of Zinc Oxide-Caffeic Acid Nanoparticles against Solid Ehrlich Carcinoma in Female Mice.

This study aimed to evaluate the anticancer and radio-sensitizing efficacy of Zinc Oxide-Caffeic Acid Nanoparticles (ZnO-CA NPs). ZnO-CA NPs were formulated by the conjugation of Zinc Oxide nanoparticles (ZnO NPs) with caffeic acid (CA) that were characterized by Fourier Transform Infrared Spectra (FT-IR), X-ray Diffractometer (XRD), and Transmission Electron Microscopy (TEM). In vitro anticancer potential of ZnO-CA NPs was evaluated by assessing cell viability in the human breast (MCF-7) and hepatocellular (HepG2) carcinoma cell lines. In vivo anticancer and radio-sensitizing effects of ZnO-CA NPs in solid Ehrlich carcinoma-bearing mice (EC mice) were also assessed. Treatment of EC mice with ZnO-CA NPs resulted in a considerable decline in tumor size and weight, down-regulation of B-cell lymphoma 2 (BCL2) and nuclear factor kappa B (NF-κB) gene expressions, decreased vascular cell adhesion molecule 1 (VCAM-1) level, downregulation of phosphorylated-extracellular-regulated kinase 1 and 2 (p-ERK1/2) protein expression, DNA fragmentation and a recognizable peak at sub-G0/G1 indicating dead cells’ population in cancer tissues. Combined treatment of ZnO-CA NPs with γ-irradiation improved these effects. In conclusion: ZnO-CA NPs exhibit in-vitro as well as in-vivo antitumor activity, which is augmented by exposure of mice to γ-irradiation. Further explorations are warranted previous to clinical application of ZnO-CA NPs.

Antitumor Activity In Vivo and Vitro of New Chiral Derivatives of Baicalin and Induced Apoptosis via the PI3K/Akt Signaling Pathway

In this study, a pair of chiral baicalin (BA) derivatives were synthesized by combining BA with phenylalanine methyl ester based on molecular docking technology, namely BAD and BAL. Cell cytotoxicity trails showed that the cell growth inhibitory effects of both BAD and BAL were increased by 8- to 12-fold compared with BA on A549 cells. Flow cytometry showed that the apoptotic rates of 50 μg/mL BA, BAD, and BAL to A549 cells for 48 h were 17.94%, 24.32%, and 39.69%, respectively. Western blotting analysis showed that BAD and BAL could promote Bax, caspase-3, and caspase-9 expression and inhibit Bcl-2 expression by inhibiting the expression of p-Akt. The tumor inhibition rates of BA, BAD, and BAL in nude mice of tumor-bearing experiment lasting for 24 days were 35.01%, 53.30%, and 59.35%, respectively. These results in vitro and in vivo showed that BAL had higher antitumor activity than did BAD and BA, which were related to promotion of the apoptosis of tumor cells by inhibiting the expression of p-Akt on PI3K/Akt pathway. This study provides an experimental basis for the development of a new configuration of BA for the treatment of cancer.

Preclinical evaluation of CLX-155, a novel prodrug of 5-FU for the treatment of cancer.

e15614 Background: CLX-155 is a novel prodrug of 5-FU, developed by adding two acetyl groups (ester bond) and a caprylic acid moiety (amide bond) to 5'-DFCR ring structure. CLX-155 is designed to have a differentiated ADME profile, with improved safety and efficacy over capecitabine. Here, we present the preclinical data of CLX-155 in comparison with capecitabine. Methods: Solubility/stability of CLX-155 was studied in buffers, simulated gastrointestinal fluids and liver microsomes. In vitro metabolite profiling in hepatocytes was done following standard protocol. An oral (gavage) dose range finding (DRF) toxicity, comparative oral pharmacokinetic study and antitumor activity in HCT-116 human colon cancer cell xenograft model was conducted in mice. DRF study in mice evaluated the toxicity of CLX-155 when administered at once daily doses of 100 to 1000 mg/kg/day for 7-days. The oral pharmacokinetics CLX-155/metabolites (5'-DFCR, 5'-DFUR and 5-FU) was determined in mice at MTD dose of 500 mg/kg and for capecitabine at 1000 mg/kg. Antitumor activity of CLX-155 was evaluated in Foxn1 athymic mice at 125, 250 and 500 mg/kg/day and for capecitabine at 1000 mg/kg/day. Treatment was performed for three consecutive weeks (5 days on; 2 days off per week). Results: The results of in-vitro studies confirmed an acceptable profile for further development of CLX-155. Maximum tolerated dose of CLX-155 in mice was 500 mg/kg/day. The key toxicity findings were consistent with the mechanism of action and comparable with capecitabine. Oral PK study in mice showed a lower plasma Cmax for 5'-DFCR, 5'-DFUR and 5-FU. Plasma AUCs of the metabolites were close to or higher than that of capecitabine, indicating an extended absorption or altered metabolism in comparison with capecitabine. CLX-155 caused significant, tumor growth inhibition at all the dose levels tested. Complete tumor regression was seen in 2/10 animals at 500 mg/kg/day of CLX-155. CLX-155 was better tolerated in the mouse xenograft study, no mortality in CLX-155 versus 2/10 in capecitabine group. Conclusions: CLX-155 has an excellent safety and a differentiated ADME profile in relation to capecitabine. This translated into an improved in-vivo antitumor activity for CLX-155 in the HCT 116 xenograft model in relation to capecitabine. Overall data indicate that CLX-155 could offer significant improvements over the currently approved capecitabine in terms of dose size, frequency of administration, safety and interpatient variability in pharmacokinetics.

The Preclinical Activities of PTC596, a Novel Tubulin Binding Agent That Down-Regulates BMI1, Alone and in Combination with Bortezomib in Multiple Myeloma

Introduction: A novel tubulin binding agent PTC596, which is currently in clinical trials for solid tumors, was originally identified by its ability to kill cancer stem cells and to reduce BMI1 activity. PTC596 treatment results in hyperphosphorylation of the BMI1 protein and loss of BMI1 function as demonstrated by a reduction in H2A ubiquitination levels in a range of solid tumor lines. Subsequent studies have shown that the down-regulation of BMI1 protein is due to a G2/M arrest. In this study, we aimed to investigate the in-vitro and in-vivo anti-tumor activities of PTC596 and the combination with bortezomib in multiple myeloma (MM). Methods: For in-vitro evaluation, MTS and BrdU ELISA assays were performed using human MM cell lines. Approved by the Institutional Review Committee at Chiba University, primary myeloma cells and bone marrow stromal cells (BMSCs) were collected from the bone marrow of MM patients with informed consent. For in-vivo evaluation, the MM.1S subcutaneous xenograft model in NOG mice was used. To understand the mechanisms of action and target genes of the treatments, flow cytometry (FCM), western blotting, RNA-seq, and ChIP-seq were performed. Results: PTC596 induced significant cytotoxicity in all MM cell lines tested, including bortezomib-resistant OPM-2/BTZ and KMS-11/BTZ cells (CC50: 24-98 nM). PTC596 also suppressed cell proliferation when these cell lines were co-cultured with BMSCs. As expected, PTC596 reduced the levels of BMI1 protein and uH2A in a dose-dependent manner. Of note, PTC596 induced cell cycle arrest as detected by a BrdU FCM assay in MM cells and apoptosis as detected by annexin-V FCM in MM cell lines and primary myeloma cells. Moreover, oral administration of PTC596 twice a week for three weeks significantly inhibited the growth of MM.1S tumors implanted in immunodeficient mice and improved the survival of mice as compared with mice treated with vehicle only (p=0.0021). Of interest, bortezomib appeared to transcriptionally repress the expression of BMI1 and reduce the levels of uH2A. We then tested the efficacy of the combination of PTC596 with bortezomib in MM cells and found additive or synergistic effects when MM cell lines were co-cultured with BMSCs. Reductions in the levels of BMI1 protein and uH2A by PTC596 or bortezomib alone were significantly enhanced in the combination treatment. Furthermore, apoptosis induced by bortezomib was significantly enhanced by the combination with PTC596 as evidenced by increased annexin-positive cells detected in flow cytometric analysis and increased cleavage of caspases with reduction in MCL1 protein in western blotting. RNA-seq of MM.1S cells treated with PTC596 alone or in combination with bortezomib demonstrated repression of gene sets related to the cell cycle in either setting and enrichment of gene sets related to apoptosis in the combination. Ongoing analysis of our ChIP-seq data will reveal the direct targets of BMI1 in MM cells. Remarkably, oral administration of PTC596 combined with subcutaneous injection of bortezomib twice a week for five weeks significantly reduced MM.1S tumor growth in comparison to the control or either single treatment (p<0.0001 vs. control, p=0.0003 vs. PTC596, p=0.0104 vs. bortezomib). Severe body weight loss was not observed in the combination-treated mice except one mouse which needed washout period until recovery. White blood cell count and hemoglobin level in peripheral blood of mice were not significantly changed during the treatment with PTC596 or even the combination. Survival of the host mice treated with the combination was significantly prolonged as compared with the control or either single treatment (p<0.0001 vs. control, p=0.0002 vs. PTC596, p=0.0072 vs. bortezomib). Conclusions: Our results demonstrate that PTC596 alone and in combination with proteasome inhibition are potential novel therapeutic options in MM. The results of this study support the clinical evaluation of this promising therapeutic approach to improve the outcome of MM patients. Disclosures Sheedy: PTC Therapeutics: Employment. Weetall:PTC Therapeutics: Employment.

Dimeric c(RGD) peptide conjugated nanostructured lipid carriers for efficient delivery of Gambogic acid to breast cancer.

Background and purpose: Gambogic acid (GA) is a natural compound that exhibited a promising multi-target antitumor activity against several types of cancer. However, the clinical application of this drug is limited due to its poor solubility and low tumor cell-specific delivery. In this study, the monomeric and dimeric Cyclo (Arg-Gly-Asp) c(RGD) tumor targeting peptides (c(RGDfK) and E-[c(RGDfK)2]) were used to modify GA loaded nanostructured lipid carriers (NLC) to reduce the limitations associated with GA and improve its antitumor activity.Methods: GA-NLC was prepared by emulsification and solvent evaporation methods and the surface of the NLC was conjugated with the c(RGD) peptides via an amide bond. The formulations were characterized for particle size, morphology and zeta potential, encapsulation efficiency and drug loading. The in-vitro cytotoxicity and cell uptake studies were conducted using 4T1 cell. Furthermore, the in-vivo antitumor activity and bio-distribution study were performed on female BALB/c nude mice.Results: The c(RGD) peptides modified GA-NLC was successfully prepared with the particles size about 20 nm. The HPLC analysis, FT-IR and 1H-NMR spectra confirmed the successful conjugation of the peptides with the NLC. The in-vitro cytotoxicity study on 4T1 cells revealed that c(RGD) peptides modified GA-NLCs showed significantly higher cytotoxicity at 0.25 and 0.5 µg/mL as compared to unmodified GA-NLC. Furthermore, the cell uptake study demonstrated that better accumulation of E-[c(RGDfK)2] peptides modified NLC in 4T1 cell after 12 h incubation. Moreover, the in-vivo study showed that c(RGD)s functionalized GA-NLC exhibited better accumulation in tumor tissue and tumor growth inhibition. In contrast to the monomeric c(RGD) peptide, the dimeric c(RGD) peptide (E-[c(RGDfK)2]) conjugated GA-NLC showed the improved antitumor activity and tumor targeting ability of GA-NLC.Conclusion: These data provide further support for the potential clinical applications of E-[c(RGDfK)2]-GA-NLC in breast cancer therapy.

Overcoming genetically based resistance mechanisms to PD-1 blockade.

2584 Background: Mechanism-based strategies to overcome resistance to anti-PD1 therapy are urgently needed. Using CRISPR/Cas9 genome editing tools, we developed acquired resistant models through JAK1/2 and B2M loss of function (LoF) mutations in human melanoma cell lines and in the murine MC38 colon carcinoma, known for high mutational load and good response to anti-PD-1. We hypothesized that the downstream activation of the IFN-receptor pathway or the activation of natural killer (NK) cells would overcome this resistance. Methods: We studied signaling changes in four human cell lines (parental and LoFs) exposed to IFN-gamma using RNAseq. In addition, we analyzed the in-vivo antitumor activity in MC38 variants with anti-PD1 and characterized the tumor microenvironment using mass cytometry (CyTOF). Finally, we tested strategies to overcome resistance mechanisms with SD-101 (TLR-9 agonist) and bempegaldesleukin (NKTR-214, CD-122 biased agonist) with the extent of CD8 and NK1.1 depletion. Results: RNAseq differential gene expression analysis showed that the IFN-gamma induced increased expression of antigen presenting machinery, IFN-gamma signaling and chemokines (CXCL9/10) was lost in JAK1/2-LoF human melanoma cell lines. The significant antitumor activity of anti-PD-1 against MC38 parental cell line was lost in JAK1/2 and B2M LoF sublines, and CyTOF analysis revealed that anti-PD-1 therapy was unable to increase tumor CD8+ T-effectors in these LoF tumors. The intratumoral administration of SD-101 (50 μg/injection q4dx3wks) was able to overcome local resistance even in non-injected sites in JAK1/2 and IFNAR-type-I LoF tumors, and systemic administration of bempegaldesleukin (0.8 mg/kg, q9dx2, i.v.) was able to overcome resistance in B2M LoF with significantly increased survival (Table). Depletion studies showed complete abrogation of anti-tumor response with anti-NK1.1 in JAK1 LoF and B2M LoF, and partial abrogation with anti-NK1.1 or anti-CD8 in JAK2 LoF tumors. Conclusions: Even in the extreme setting of genetic resistance to PD-1 blockade by JAK1/2 LoF, resistance can be overcome by SD-101, a TLR9 agonist, while resistance of B2M LoF can be overcome by bempegaldesleukin (NKTR-214), a CD-122 biased agonist. Our findings support the testing of these rational mechanistic strategies in patients with a-PD1 resistance. [Table: see text]

In-vivo anti-tumor activity of a novel poloxamer-based thermosensitive in situ gel for sustained delivery of norcantharidin

In order to develop a novel norcantharidin (NCTD) delivery system with slow drug release and specific targeting characteristics, we have developed a Poloxamer-based NCTD thermosensitive in situ gel. The evaluation of the characteristics of this system using both in vitro and in vivo methods was previously reported. However, its anti-tumor activity in vivo is still not confirmed. Thus, the potential anti-tumor activity and relative mechanism were investigated in a murine H22 hepatoma model. Tumor-bearing mice were treated with different dose of NCTD thermosensitive in situ gel (3.3 mg/kg, 6.6 mg/kg, and 9.9 mg/kg, respectively by intra-tumor injection once every three days, totaling 5 injections per group. Control groups included untreated or NCTD injection (2.2 mg/kg, qd) or blank in situ gel. The expression of vascular endothelial growth factor (VEGF) and CD44 in tumor tissue was examined by immunohistochemistry (IHC) staining. Treatment with middle or high dose of NCTD thermosensitive in situ gel significantly induced tumor regression, inhibited VEGF and CD44 expression and improved survival of tumor-bearing mice. The efficacy of NCTD thermosensitive in situ gel is higher than that of free NCTD injection. Therefore, NCTD thermosensitive in situ gel is a novel NCTD delivery approach for chemotherapeutic treatment of cancer.

Assessment of antiproliferative activity of Capparis spinosa L extract against Ehrlich ascites carcinoma in Swiss albino mice

Background: Medicinal plants have been known as one of the most important therapeutic agents for cancer treatment and prevention. Capparis spinosa L is a multipurpose plant that contains different bioactive phytochemicals including phenols and flavonoids.Aims: In the present study we explore the invivo anti-tumor activity of Capparis spinosa L extract and most potent constituents rutin and hesperidin against Ehrlich ascites carcinoma (EAC). Also study the side effect of treatments on different organs (liver, kidney and heart). Material and method: The antitumor effect was assessed by evaluating tumor volume , tumor cell count , survival time and increase in life span of EAC bearing mice. We assessed the effect of Capparis spinosa L extract , rutin and hesperidin on the level of malonaldialdhyde (MDA) and Catalase activity. Also, we estimated their effect on Alanine aminotransferase (ALT), Aspartate aminotransferase (AST) , Albumin , urea , creatinine ,Lactate dehydrogenase activity (LDH), Creatine Kinase -MB activity (CK-MB). Apoptosis was assessed by BCL2 and caspase3 activity. Results : The Capparis spinosa L extract , rutin and hesperidin showed significantly decreased (p<0.001) in the volume of the EAC and in the count of EAC cells and increase the life span of EAC bearing mice. The treatment with Capparis spinosa L extract , rutin and hesperidin showed significantly decreased (p<0.001) the lipid peroxidation marker (MDA) and recovered catalase activity towards normal as compared to positive control. We founded that Capparis spinosa L extract , rutin and hesperidin treatment induced apoptosis demonstrated by an increased in Caspase 3 activity and decreased in BCL2 .The treatment of Capparis spinosa L extract , rutin and hesperidin significantly reduced the elevated levels of ALT , AST ,LDH ,CK-MB,Urea and Creatinine in positive control as compared with negative control. Also, they showed protection for both liver and kidney histopathologically. Conclusions: The present study demonstrated that Capparis spinosa L extract , rutin and hesperidin have potent antitumor activity against Ehrlich ascites carcinoma. The anti-tumor mechanism may be mediated by preventing oxidative damage and induction of apoptosis.

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.

Dual compartmental targeting of cell cycle and angiogenic kinases in colorectal cancer models.

Cancer is a disease caused by several factors characterized by uncontrolled cell division, growth, and survival. ENMD-2076, is a novel orally active small molecule multikinase inhibitor targeting angiogenesis, proliferation, and the cell cycle. It is selectively active against the mitotic kinases aurora A and B, and kinases responsible for angiogenesis including VEGFR2/KDR and FGFR1 and 2. ENMD-2076 has been shown to inhibit tumor growth and prevent angiogenesis in vitro and in vivo in preclinical cancer models. Moreover, in a phase I trial, ENMD-2076 was well tolerated, exhibited a linear pharmacokinetic profile, and showed a promising antitumor activity in a number of solid tumors. In this study, we show that ENMD-2076 has antiproliferative effects, causes cell cycle arrest, and has activity in preclinical models of colorectal cancer (CRC), including patient-derived xenograft (PDX) models. Forty-seven human CRC cell lines were exposed in vitro to ENMD-2076 and analyzed for effects on cell cycle, apoptosis, and downstream effector proteins. The drug was then tested against 20 human CRC PDX models to further evaluate in-vivo antitumor activity. We show that ENMD-2076 exhibits a broad range of activity against a large panel of CRC cell lines with varying molecular characteristics. Mechanistically, ENMD-2076 exposure resulted in a G2/M cell cycle arrest, an increase in aneuploidy, and cell death in responsive cell lines. In addition, ENMD-2076 treatment resulted in a promising antitumor activity in CRC PDX models. These results support the continued development of ENMD-2076 in CRC including further exploration of rational combinations.

Lobaplatin inhibits prostate cancer progression in part by impairing AR and ERG signal.

Lobaplatin is the third-generation platinum drug that has been shown promising antitumor activity in preclinical studies and clinical trials for multiple human cancers except prostate cancer. In this study, we investigated the role of lobaplatin on prostate cancer progression and the underlying molecular mechanism. We treated a variety of prostate cancer cell lines with different doses of lobaplatin and examined cell cycle progression, cell apoptosis, cell proliferation, and cell migration. Moreover, we also assessed the in-vivo antitumor activity of lobaplatin using xenograft mouse model. Importantly, we further dissected the underlying molecular mechanism by examining the expression of AR and ERG, as well as their downstream targets. We found that lobaplatin arrested cell cycle progression in G2/M phase and trigged cell apoptosis. Lobaplatin also dramatically inhibited cell proliferation and migration in vitro. Consistently, lobaplatin significantly suppressed tumor growth in xenograft mouse model. Mechanistically, the expression of AR and ERG, as well as their downstream targets, was markedly decreased upon treatment with lobaplatin in prostate cancer cells. Altogether, our results suggest that lobaplatin displayed favorable antitumor activity on prostate cancer both in vitro and in vivo, providing molecular basis and rational for using lobaplatin to combat human prostate cancer.

Anti‑cancer effects of a novel Pan‑RAF inhibitor in a hepatocellular carcinoma cell line.

The RAF/mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK (RAF/MEK/ERK) signaling cascade serves a prominent role in hepatocellular carcinoma (HCC) proliferation. Sorafenib (BAY 43‑9006) is a potent multikinase inhibitor of RAF kinases and a few receptor tyrosine kinases. Additionally, sorafenib causes apoptosis in a number of human tumor cell lines such as leukemia cell lines. Sorafenib is the first targeted drug to prolong the overall survival of patients with advanced HCC. However, sorafenib activity is less favorable in certain cancers, including sarcomas and melanomas, due to patient insensitivity and drug resistance. In the present study, a novel bi‑aryl urea, N‑(3‑trifluoromethylphenyl)‑N'-(2-methyl-4-(6‑cyclopropanecarboxamido-pyrimidin-4-yl) oxyphenyl) urea (CBI‑5725), is shown to be a potential candidate for the treatment of liver cancer. In the present study, the invitro activities of CBI‑5725 and sorafenib in PLC/PRF/5 HCC cells were examined and the corresponding invivo antitumor activities in PLC/PRF/5 human tumor xenografts. An alamar blue assay confirmed that CBI‑5725 was more cytotoxic than sorafenib to PLC/PRF/5 cells, suggesting that CBI‑5725 inhibited tumor cell proliferation more potently than sorafenib. CBI‑5725 inhibited the RAF/MEK/ERK signaling pathway to the same extent as sorafenib. In addition, CBI‑5725 elicited cell cycle arrest in the G2/M phase, while sorafenib did not markedly alter the cell cycle. Furthermore, CBI‑5725 induced apoptosis more strongly than sorafenib in a dose‑dependent manner, which may be attributed to greater caspase‑3 and poly(adenosine 5'‑diphosphate‑ribose) polymerase activation by CBI‑5725. In the PLC/PRF/5 xenograft model, 2mg/kg CBI‑5725 inhibited tumor growth by 73%. At doses ranging from 6 to 18mg/kg, CBI‑5725 nearly completely prevented tumor growth. These results imply that the antitumor efficacy of CBI‑5725 in HCC models may result from the suppression of the RAF/MEK/ERK signaling pathway, the induction of cell cycle arrest in the G2/M phase, and the initiation of caspase‑3‑dependent apoptosis. These observations suggested that CBI‑5725 may be a potent novel compound for the treatment of HCC.

Optimization of IL13Rα2-Targeted Chimeric Antigen Receptor T Cells for Improved Anti-tumor Efficacy against Glioblastoma.

T cell immunotherapy is emerging as a powerful strategy to treat cancer and may improve outcomes for patients with glioblastoma (GBM). We have developed a chimeric antigen receptor (CAR) Tcell immunotherapy targeting IL-13 receptor α2 (IL13Rα2) for the treatment of GBM. Here, we describe the optimization of IL13Rα2-targeted CAR Tcells, including the design of a 4-1BB (CD137) co-stimulatory CAR (IL13BBζ) and a manufacturing platform using enriched central memory Tcells. Utilizing orthotopic human GBM models with patient-derived tumor sphere lines in NSG mice, we found that IL13BBζ-CAR Tcells improved anti-tumor activity and Tcell persistence as compared to first-generation IL13ζ-CAR CD8+ Tcells that had shown evidence for bioactivity in patients. Investigating the impact of corticosteroids, given their frequent use in the clinical management of GBM, we demonstrate that low-dose dexamethasone does not diminish CAR Tcell anti-tumor activity invivo. Furthermore, we found that local intracranial delivery of CAR Tcells elicits superior anti-tumor efficacy as compared to intravenous administration, with intraventricular infusions exhibiting possible benefit over intracranial tumor infusions in a multifocal disease model. Overall, these findings help define parameters for the clinical translation of CAR Tcell therapy for the treatment of brain tumors.

Abstract 4140: Identification of a selective MKLP2/KIF20A inhibitor with high in-vivo antitumor activity

Abstract Mitotic kinesins are essential regulators of cancer cell replication and migration. The mitotic kinesin MKLP2/KIF20A, a member of the kinesin-6 family, plays an essential role during cytokinesis and was identified as a potential new target for cancer chemotherapy1. We have previously identified Paprotrain, a new synthetic compound, as the first selective MKLP2 inhibitor2. Recently, we obtained Paprotrain analogues with higher potency on MKLP23. Herein we describe the identification and characterization of BKS0349, a new potent analogue of Paprotrain. BKS0349 compound is 10 times more potent than paprotain on MKLP2 inhibition and has shown an even more restricted specificity profile when tested on a large set of kinases. In-vitro this compound is highly cytotoxic on a wide panel of human cancer cell lines (IC50 ranged 10-70 nM, which corresponds to 1000 fold improvement of paprotrain potency) while no toxicity is observed on human normal cells such as peripheral blood mononuclear cells (PBMC) and primary hepatocytes (IC50 &amp;gt;50µM). BKS0349 compound is well tolerated in mice using repeated intravenous administrations (200 mg/kg, twice a week for 4 weeks). In xenografted nude mice, in-vivo treatment with BKS0349 compound demonstrates a high antitumor activity against various human cancer cell models, either sensitive or resistant to some standard-of-care treatments. In addition, human cancer cells treated in-vitro as well as in-vivo with BSK0349 compound display Golgi scattering and a mitotic arrest leading to cell death, as hallmarks of BKS0349’ mode of action. These findings show that MKLP2 is a potential new target for cancer chemotherapy and BKS0349 a good candidate to be developed for cancer treatment. 1Paclitaxel targets FOXM1 to regulate KIF20A in mitotic catastrophe and breast cancer paclitaxel resistance. P. Khongkow et al. Oncogene, 2015, 1-13. 2Relocation of Aurora B and Survivin from centromeres to the central spindle impaired by a kinesin-specific MKLP-2 inhibitor. S. Tcherniuk et al. Angew. Chem. Int. Ed., 2010, 49:8228-8231. 3New MKLP-2 inhibitors in the paprotrain series: design, synthesis and biological evaluations. C. Labrière et al. Bioorg and Med Chem, 2016, 24:731-734. Note: This abstract was not presented at the meeting. Citation Format: Yves Collette, Stephanie Miserey-Lenkei, Catherine Guillou, Denis Carniato, Bernard Pau, Bruno Goud, Norbert Vey, Cecile E. Bougeret. Identification of a selective MKLP2/KIF20A inhibitor with high in-vivo antitumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4140. doi:10.1158/1538-7445.AM2017-4140

Interrogating resistance mechanisms to PD-1 blockade therapy with CRISPR.

3077 Background: We tested the biological significance of the loss of function (LOF) mutations in JAK1 or JAK2 within the IFN-receptor-pathway and in beta-2-microglobulin (B2M), which had been found in patient biopsies with resistance to anti-PD-1 therapy. Methods: We used CRISPR/Cas9 genome editing to generate JAK1, JAK2 and B2M knockout (KO) sublines of HLA-A*02:01 MART-1 or NY-ESO-1 positive human melanoma cell lines, tested using in-vitro T cell co-culture systems and in a syngeneic mouse model (MC38) to analyze the in-vivo antitumor activity with anti-PD1 therapy. Results: The JAK2-KO cell line was insensitive to IFN-gamma induced signaling and growth arrest (p &lt; 0.001 compared with IFN-alpha or beta), while the JAK1-KO cell line was insensitive to all three IFNs. Baseline MHC class I expression after JAK1-KO was unaffected (baseline-MFI 1230 JAK1-KO vs 1570 parental, p = 0.66), but the magnitude of change was lower upon IFN-gamma exposure compared to the parental (MFI change with IFN-gamma, 26% decrease for JAK1-KO vs 50% increase for parental). There was no difference in in-vitro cytotoxicity by NY-ESO-1-TCR transgenic T-cells against JAK1-KO-NY-ESO-1+ melanoma cells compared to the parental (78% vs 82% cytotoxicity at 10:1 E:T ratio, p NS). However, B2M-KO was resistant to killing by MART-1 specific T-cells (2% vs 96% cytotoxicity at 10:1 E:T ratio, p &lt; 0.0001). On the other hand, in the MC38 model the significant antitumor activity of anti-PD-1 against the wild type cells was lost in both JAK2-KO and B2M-KO. The percentage of CD8+ T cells has a trend of increase with anti-PD1 compared to untreated in the MC38 wild type (p = 0.1 d12), and a trend of decrease in MC38 B2M-KO (p = 0.2 d12), but no change in JAK2-KO tumors (p = 0.7 d12). Conclusions: JAK1/2 LOF mutations result in insensitivity to IFN induced antitumor effects, but does not impair T cell recognition and cytotoxicity, while B2M LOF results in lack of antigen presentation to T cells and loss of antitumor activity. However both lead to in-vivo resistance to anti-PD-1 therapy, suggesting they do so by independent mechanisms.