Reduced Tumor Weight Research Articles

Abstract 2366: HDAC inhibition improves immune checkpoint inhibitor efficacy in renal cell carcinoma

Abstract Background: Immune checkpoint inhibitors have shown clinical benefit in solid tumors, including renal cell carcinoma (RCC); however, the rate of clinical response remains modest and improved therapeutic approaches need to be tested. Growing evidence suggests that epigenetic modifying agents may have an immunomodulatory effect that improves the efficacy of immune checkpoint inhibitors. Our group has previously demonstrated that entinostat, a histone deacetylase (HDAC) inhibitor, decreases the function of regulatory T cells (Treg) and myeloid derived suppressor cells (MDSC), synergizing with PD-1 blockade. Here we assessed the combination of PD-1 blockade with pan-HDAC inhibition in a RCC model. Methods: To test the efficacy of combined PD-1 inhibition, mDX-400 (10 and 20 mg/kg I.P) (Merck & Co, Inc) with pan-HDAC inhibition, vorinostat (100 and 150 mg/kg I.P) (Merck & Co, Inc), we utilized a syngeneic mouse model of metastatic RCC following orthotopic implantation of RENCA cells in immunocompetent mice. Antitumor activity was assessed by measuring bioluminescence, end point tumor weights, and survival times. Immune profiling of tumor infiltrating lymphocytes (TILs) was performed by flow cytometry, immunohistochemistry, and immunofluorescence. Peripheral blood mononuclear cells (PBMC) were assessed for differential chromatin accessibility by Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq). Results: Significant reductions in tumor weight and lung metastases were observed in mice treated with the combination of vorinostat and mDX-400. Combination therapy significantly increased the survival of the mice (median survival = 60) compared to treatment with mDX-400 alone (median survival=42 days). Immune landscape profiling demonstrated an increase in natural killer cell infiltration (P=0.048) and decrease of exhausted T cells (P=0.049, CD8+ PD1+) in the combination group. Furthermore, decreased immunosuppressive Treg (CD4+ FOXP4+) and MDSC (CD11b+ Gr1+) populations were identified in the combination group. Analysis of the mouse PBMC ATAC-seq data in the combination and mDX400 alone conditions demonstrated numerous regions of differential chromatin accessibility. Pathway analysis of genes associated with increased accessibility in the combination treatment identified enrichment of cell cycle and immune activation pathways. Conclusions: Our results demonstrate that pan-HDAC inhibition augments the antitumor effect of immune checkpoint inhibitors, prolonging survival in our preclinical mouse model. This antitumor effect was achieved by changing the immune landscape in TILs and was associated with higher chromatin accessibility near genes involved in cell cycle progression and immune cell activation. Taken together, our results support the clinical testing of pan-HDAC inhibitors in combination with anti-PD-1. Citation Format: Justin Budka, Nur Damayanti, Roberto Pili. HDAC inhibition improves immune checkpoint inhibitor efficacy in renal cell carcinoma [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 2366.

Abstract 4804: Pre-clinical development of ISOX; A novel therapy for pancreatic cancer identified utilizing system biology approach(s)

Abstract Background: Pancreatic Cancer (PC) remains one of the most common causes of cancer-related deaths. The low survival percentage in patients can be attributed primarily to ineffective therapeutic targeting and lack of new potent therapies. Over the years the conventional one target at a time approach for drug development has failed to achieve promising survival and better approaches are needed. Large-scale genomic database connectivity map (CMAP), a collection of perturbagen profiles from >2800 drugs is a potential tool to identify effective therapies for PC by identifying novel drugs that essentially reverse the global gene signature originating due to malignant PC. Considering this information, we hypothesize that “a big data approach using CMAP will lead to the identification of novel drugs for highly lethal PC.” Methods and Results: To generate a meta-gene signature for PC, GEO data for PC cases (N=106) and normal samples (N=68) was extracted, normalized & assessed for differential gene expression using R Bioconductor. Drugs specific to this gene signature across various datasets were identified using CMAP and the top hit ISOX chosen for further assessment. Considering the high mutational heterogeneity of PC, a five-cell line panel with diverse mutations was chosen for the validation studies. Cell line-based assessment for changes to proliferation using MTT and motility using matrigel assisted invasion showed the high potency of ISOX with an IC50 of 2.4nM-1.4µM & up to 90% reduction in invasion supporting initial hypothesis. It was further supported by the 48% induction of apoptosis. Molecular mechanism of ISOX was further assessed using an RNA-seq on ISOX treated PC cell lines followed by ingenuity pathway analyses. Gene ontology pathway analysis indicated SHH-WNT, PI3K-mTOR-AKT, and EGFR signaling as key pathways affected by ISOX. Further, 60% loss of viability was observed in pancreatic tumor organoids at 500nM of ISOX. Furthermore, about 10 fold statistically significant (p-value = 0.014) reduction in tumor weight at 50 mg/kg of ISOX both alone and in combination with 50 mg/kg 5FU (p-value=0.02) was observed in orthotopic mice models. Conclusion: Our data suggest that ISOX is a potential new therapeutic for PC. In future, we aim to evaluate the potency of newly identified drug in both transgenic mouse models of PC as well as clinical phase I studies. Citation Format: Pranita Atri, Parthasarathy Seshacharyulu, Satyanaryana Rachagani, Palanisamy Nallasamy, Sanchita Rauth, Garima Kaushik, Koelina Ganguly, Dario Ghersi, Moorthy P. Ponnusamy, Sukhwinder Kaur, Surinder K. Batra. Pre-clinical development of ISOX; A novel therapy for pancreatic cancer identified utilizing system biology approach(s) [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 4804.

Abstract 2762: Development of a novel peptide-formed nanoparticle for pancreatic cancer treatment

Abstract Background and objective: Pancreatic ductal adenocarcinoma (PDAC) is the most common malignant tumor of pancreas with an overall 5-year survival rate of less than 5%. Surgical operation is an curable treatment, however, only 10-20% of patients with PDAC are respectable at diagnosis. Taking advantage of the progression in nanoparticle study, we developed a novel peptide-formed nanoparticle (PF-Nano) and investigate its therapeutic effect in PDAC treatment. Methods: Phage display peptide library was used to screen a high-affinity tumor-homing peptide (THP) for pancreatic cancer (PC) cells. Ligation of THP with cell penetrating peptide was used to prepare a tandem peptide, which automatically formed a nanoparticle. We used this PF-Nano as a vehicle to load and deliver siRNA.Dynamic light scattering (DLS) was employed to define nanoparticle size; and fluorescence imaging was applied to assess PF-Nano siRNA delivery efficiency. In vitro, we used MTT assay and colony formation to evaluate cell growth; wound healing assay to detect cell migration, and flow cytometry to evaluate cell cycle and apoptosis. Therapeutic effect was evaluated by measuring final tumor weight in the mice receiving different treatments. qPCR and Western Blot (WB) were used to detect gene expression in mRNA and protein level. Immunoprecipitation in combination with mass spectrometry and immunofluorescence were used to identify THP’s ligands. Results: The screened peptide has a high-affinity with PC cells. DLS identified evenly distributed PF-Nano with a diameter of approximately 230nm. Detection of fluorescence signal from labeled siRNAs in PC cells and tumor tissue suggested that PF-Nano owned the capacity to successfully deliver siRNA in vitro and in vivo. In vitro treatment with PF-Nano significantly suppressed PC cell proliferation, colony formation, and cell migration. Also it caused cycle arrest in S phase (DNA synthesis) and increase in cell apoptosis. Treatment of tumor-bearing mice with PF-Nano by intraperitoneal injection markedly suppressed tumor growth, evidenced by the reduction of tumor weight to 25% relevant to that in control mice without treatment. Immunoprecipitation and mass spectrometry revealed that protein phosphatase 1 catalytic subunit gamma (PP1C) was a potential binding target of the specific THP. Mechanistically, PF-Nano treatment strongly suppressed RAC-alpha serine/threonine-protein kinase (AKT) phosphorylation, suggesting PP1-AKT signal pathway may be responsible for peptide effect in pancreatic tumor. Conclusion: The established PF-Nano is able to specifically deliver siRNAs to PC tumors and therapeutically suppresses PC tumor growth. Citation Format: Qiongling Wang, Kevin F. Staveley-O’Carroll, Guangfu Li. Development of a novel peptide-formed nanoparticle for pancreatic cancer treatment [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 2762.

Role of docosahexaenoic acid in enhancement of docetaxel action in patient-derived breast cancer xenografts.

The objective of this study was to investigate if DHA dietary supplementation enhances the anticancer actions of docetaxel (TXT) in two different drug resistant triple negative breast cancer (TNBC) patient-derived xenografts (PDX)s. In two experiments, female NSG mice bearing TNBC PDXs were randomized to one of two nutritionally adequate diets (20% w/w): control (0% DHA), or DHA (3.9% w/w of total fat) and injected with 0 or 5mg/kg TXT, twice weekly for 6weeks (n = 8 per group). Treatment response was determined by significant differences in tumor weight, and apoptotic, proliferation and cell cycle markers at endpoint. Mice bearing MAXF574 xenografts fed DHA diet and treated with TXT had a 57% reduction in tumor weight compared to mice fed control diet (P < 0.004), a 64% reduction compared to control + TXT (P < 0.01) and a 34% reduction compared to DHA with no TXT (P < 0.04). DHA + TXT reduced MAXF401 xenografts growth compared to control and control + TXT (by 43% and 34%, respectively, P < 0.05). In both xenografts, DHA + TXT resulted in a higher expression of proapoptotic proteins Ripk1 and Bid, lower expression of proliferation marker Ki67 and anti-apoptotic proteins Bcl-2 and Parp, and a greater increase in cell cycle arrest as measured by decreased Survivin expression when compared to control + TXT mice (P < 0.05). This work is the first to confirm that DHA supplementation during chemotherapy treatment improves TXT action in two PDX models of TNBC. The results suggest that decreases in tumor size occurred via changes in apoptosis, cell proliferation, and cell cycle pathways.

Antitumor effects and hematotoxicity of C60-Cis-Pt nanocomplex in mice with Lewis lung carcinoma.

Cisplatin (Cis-Pt) is a widely used anticancer drug but its therapeutic efficiency is limited by hemato-, cardio-, hepato-, nephro- and neurotoxicity. Complexation of Cis-Pt with C60 fullerene nanoparticle will allow to enhance the antitumor activity of the drug and to reduce its side toxic effects. To estimate the antitumor effects of С60-Cis-Pt nanocomplex in Lewis lung carcinoma (LLC) and analyze hematological toxicity in tumor-bearing mice. Complexation of C60 fullerene and Cis-Pt molecule was studied by computer simulation. С60-Cis-Pt nanocomplex was i.p. injected to LLC-bearing mice in a total dose of 7.5mg/kg (C60:Cis-Pt as 3.75:3.75mg/kg). The survival of tumor-bearing mice and the relative reduction of tumor weight was recorded. Blood indices were determined using the Particle Counter PCE-210automatic hematology analyzer. Computer simulation demonstrated the formation of С60-Cis-Pt nanocomplex in physiological medium and its stability due to the hydrophobic interactions. Treatment with C60-Cis-Pt nanocomplex increased survival time of LLC-bearing mice by 32%, normalized hemoglobin content (up to 100g/l), erythrocyte and platelet count as compared to the untreated LLC-bearing mice. Tumor weight decreased by 35.5%; the mitotic index of tumor cells decreased by 78%, and apoptotic index increased by 75%. The revealed effects of the C60-Cis-Pt nanocomplex were more pronounced than the effects of Cis-Pt or C60 fullerene alone in equivalent dose. Treatment with C60-Cis-Pt nanocomplex prolonged the survival of LLC-bearing mice and reduced anemia in LLC-bearing mice.

Cold atmospheric plasma and silymarin nanoemulsion synergistically inhibits human melanoma tumorigenesis via targeting HGF/c-MET downstream pathway

BackgroundRecent studies claimed the important role of cold atmospheric plasma (CAP) with nanotechnology in cancer treatments. In this study, silymarin nanoemulsion (SN) was used along with air CAP as therapeutic agent to counter human melanoma.MethodsIn this study, we examined the combined treatment of CAP and SN on G-361 human melanoma cells by evaluating cellular toxicity levels, reactive oxygen and nitrogen species (RONS) levels, DNA damage, melanoma-specific markers, apoptosis, caspases and poly ADP-ribose polymerase-1 (PARP-1) levels using flow cytometer. Dual-treatment effects on the epithelial–mesenchymal transition (EMT), Hepatocyte growth factor (HGF/c-MET) pathway, sphere formation and the reversal of EMT were also assessed using western blotting and microscopy respectively. SN and plasma-activated medium (PAM) were applied on tumor growth and body weight and melanoma-specific markers and the mesenchymal markers in the tumor xenograft nude mice model were checked.ResultsCo-treatment of SN and air CAP increased the cellular toxicity in a time-dependent manner and shows maximum toxicity at 200 nM in 24 h. Intracellular RONS showed significant generation of ROS (< 3 times) and RNS (< 2.5 times) in dual-treated samples compared to control. DNA damage studies were assessed by estimating the level of γ-H2AX (1.8 times), PD-1 (> 2 times) and DNMT and showed damage in G-361 cells. Increase in Caspase 8,9,3/7 (> 1.5 times), PARP level (2.5 times) and apoptotic genes level were also observed in dual treated group and hence blocking HGF/c-MET pathway. Decrease in EMT markers (E-cadherin, YKL-40, N-cadherin, SNAI1) were seen with simultaneously decline in melanoma cells (BRAF, NAMPT) and stem cells (CD133, ABCB5) markers. In vivo results showed significant reduction in SN with PAM with reduction in tumor weight and size.ConclusionsThe use of air CAP using μ-DBD and the SN can minimize the malignancy effects of melanoma cells by describing HGF/c-MET molecular mechanism of acting on G-361 human melanoma cells and in mice xenografts, possibly leading to suitable targets for innovative anti-melanoma approaches in the future.

Cationic lipoplexes for treatment of cancer stem cell-derived murine lung tumors

Side population (SP) cells with stem-like properties, also known as cancer stem cells (CSC) have been recognized as drivers of the resistance phenotype in many cancers. Central to the characteristic stem-like phenotype of CSCs in cancer is the activity of the SOX2 transcription factor whose upregulation has been associated with enrichment of many oncogenes. This study outlines the fabrication of a lipoplex of SOX2 small interfering RNA (CL-siSOX2) for targeted treatment of SOX2-enriched, CSC-derived orthotopic and xenograft lung tumors in CB-17 SCID mice. CL-siSOX2 induced tumor contraction in cisplatin-naïve and cisplatin-treated groups by 85% and 94% respectively. Reduction in tumor weight and volume following treatment with CL-siSOX2 was associated with reduced protein expression of SOX2 and markers of tumor initiation, inflammation, invasion and metastasis in mice tumor xenografts. In addition, histological staining of lung tumor sections showed reduction in SOX2 expression was associated with inhibition markers of epithelial-to-mesenchymal transition.

Inhibition of tumor growth and angiogenesis of tamoxifen-resistant breast cancer cells by ruxolitinib, a selective JAK2 inhibitor.

Tamoxifen (TAM) is the most widely used treatment for estrogen receptor-positive breast cancer patients. Unfortunately, the majority of these patients exhibit TAM resistance following treatment. We previously reported that proliferation and migration were greater in TAM-resistant MCF-7 (TAMR-MCF-7) cells than in parental MCF-7 cells. Janus kinases (JAKs) are cytosolic tyrosine kinases that transduce signals from plasma membrane cytokines and growth factor receptors. JAK2 selectively phosphorylates signal transducer and activator of transcription (STAT)-3, and the JAK2-STAT3 signaling pathway is known as a crucial signaling pathway for the regulation of cancer progression and metastasis. In the present study, basal phosphorylation of STAT3 was revealed to be greater in TAMR-MCF-7 cells than in control MCF-7 cells. Ruxolitinib, a potent JAK2 inhibitor, was demonstrated to attenuate STAT3 phosphorylation and the proliferation of TAMR-MCF-7 cells. Ruxolitinib also suppressed the enhanced cell migration of TAMR-MCF-7 cells through the inhibition of epithelial mesenchymal transition. Vascular endothelial growth factor (VEGF), a representative target gene of the JAK2-STAT3 pathway, functions as a key regulator of invasion and angiogenesis. Ruxolitinib significantly inhibited VEGF mRNA expression and transcriptional activity. The present study also performed a chick embryo chorioallantoic membrane assay to assess tumor growth and angiogenesis in TAMR-MCF-7 cells. Ruxolitinib reduced tumor weight and the number of blood vessels produced by TAMR-MCF-7 cells in a concentration-dependent manner. These results indicated that JAK2 could be a new therapeutic target for TAM-resistant breast cancer.

The ROMP: A Powerful Approach to Synthesize Novel pH-Sensitive Nanoparticles for Tumor Therapy.

Fast clearance, metabolism, and systemic toxicity are major limits for the clinical use of anti-cancer drugs. Histone deacetylase inhibitors (HDACi) present these defects, despite displaying promising anti-tumor properties on tumor cells in vitro and in in vivo models of cancer. The specific delivery of anti-cancer drugs into the tumor should improve their clinical benefit by limiting systemic toxicity and by increasing the anti-tumor effect. This paper deals with the synthesis of the polymeric nanoparticle platform, which was produced by Ring-Opening Metathesis Polymerization (ROMP), able to release anti-cancer drugs in dispersion, such as histone deacetylase inhibitors, into mesothelioma tumors. The core-shell nanoparticles (NPs) have stealth properties due to their poly(ethylene oxide) shell and can be viewed as universal nano-carriers on which any alkyne-modified anti-cancer molecule can be grafted by click chemistry. A cleavage reaction of the chemical bond between NPs and drugs through the contact of NPs with a medium presenting an acidic pH, which is typically a cancer tumor environment or an acidic intracellular compartment, induces a controlled release of the bioactive molecule in its native form. In our in vivo syngeneic model of mesothelioma, a highly selective accumulation of the particles in the tumor was obtained. The release of the drugs led to an 80% reduction of tumor weight for the best compound without toxicity. Our work demonstrates that the use of theranostic nanovectors leads to an optimized delivery of epigenetic inhibitors in tumors, which improves their anti-tumor properties in vivo.

Long non-coding RNA CRNDE regulates cell proliferation, migration, invasion, epithelial-mesenchymal transition and apoptosis in oral squamous cell carcinoma.

The present study aimed to investigate whether the long non-coding RNA (lncRNA) colorectal neoplasia differentially expressed (CRNDE) can promote the migration and invasion of human oral squamous cell carcinoma (OSCC) cells via the regulation of epithelial-mesenchymal transition (EMT). CAL-27 and SCC-15 cells were classified into a control group, a small interfering negative control (si-NC) group (cells transfected with control siRNA) and an si-CRNDE group (cells transfected with CRNDE siRNA). The expression of CRNDE in OSCC tissues and cell lines was detected by in situ hybridization (ISH) and reverse transcription-quantitative polymerase chain reaction. An MTT assay was used to detect cell proliferation, flow cytometry was performed to determine cell apoptosis, wound-healing and Transwell assays were conducted to evaluate cell metastasis, and immunofluorescence staining and western blotting were performed to measure the expression of proteins associated with EMT. Tumor-bearing mouse models were established, and the tumor volumes were recorded. An immunohistochemical assay was performed to determine the expression of EMT-related proteins. CRNDE expression was increased in OSCC tissues and cell lines compared with that in normal tissues and cell lines. Compared with the control group, the si-CRNDE group displayed a reduction in the expression of CRNDE, in the proliferation, migration and invasion of cells, in the protein expression of N-cadherin, vimentin and Snail, and in the expression of proteins in the Wnt/β-catenin pathway. However, an increase was displayed in the apoptosis of cells and the expression of E-cadherin. Compared with the control group of tumor-bearing nude mice, the sh-CRNDE group demonstrated slowed tumor growth, reduced tumor weight and elevated E-cadherin, as well as reduced expression of N-cadherin, vimentin and Snail. In conclusion, silencing CRNDE may inhibit EMT, thus decreasing the migration and invasion of human OSCC cells by repressing the activation of the Wnt/β-catenin signaling pathway, thereby restricting cell growth and promoting cell apoptosis.

Momelotinib sensitizes glioblastoma cells to temozolomide by enhancement of autophagy via JAK2/STAT3 inhibition.

Temozolomide (TMZ) is a widely used chemotherapeutic agent for glioblastoma multiforme (GBM). However, chemoresistance to TMZ is still a major obstacle for GBM patients. An abundance of candidates has been reported to improve the chemotherapeutic sensitization of TMZ. In the present study, it was demonstrated that momelotinib (MTB) enhanced the sensitivity of glioma cells to TMZ in vitro, as evidenced by a noticeable decrease in cell growth and a significant increase in apoptosis and autophagy following treatment with the combination of TMZ and MTB compared to TMZ alone. Mechanistically, MTB and TMZ combination treatment reduced U251 cell growth by activating both apoptosis and autophagy pathways. MTB potentiated TMZ to inhibit the phosphorylation of JAK2 and STAT3 in U251 cells, resulting in the inactivation of JAK2/STAT3 signaling pathways. Moreover, we investigated the effect of MTB in xenograft tumor model mice. MTB and TMZ combination reduced tumor weight, decreased the expression of Ki‑67, P62, p‑STAT3 and p‑JAK2, while increased the ratio of LC3‑II/I and the expression of caspase‑3 and Beclin1 in vivo. Importantly, this combination was well tolerated, and caused significant tumor growth inhibition in the GBM xenografts. In summary, the present study provides pharmacological evidence that MTB has potential value in the treatment of GBM.

Evaluation of antitumor activity and toxicity of Schinus terebinthifolia leaf extract and lectin (SteLL) in sarcoma 180-bearing mice

Ethnopharmacological relevanceSchinus terebinthifolia Raddi is a plant broadly used in folk medicine and the use of its leaf extract as an antitumor agent has been reported. Aim of the studyTo evaluate the antitumor potential and the toxicity of saline extract (SE) and lectin (SteLL) from S. terebinthifolia leaves in sarcoma 180-bearing mice. Materials and methodsCytotoxicity to sarcoma 180 cells was tested in vitro, and antitumor assay was performed using Swiss female mice. The treatments (0.15 M NaCl, negative control; methotrexate 1.5 mg/kg, positive control; SE 100 mg/kg; SteLL 1 and 5 mg/kg) by intraperitoneal injections started on the 8th day after tumor inoculation and lasted 7 days. It was analyzed: tumor weight; number and gauge of tumor vessels; hematological and biochemical parameters; histopathological changes; and occurrence of micronuclei in bone marrow cells. ResultsSE and SteLL showed IC50 values (concentrations that reduced cell viability to 50%) of 301.65 and 8.30 μg/mL, respectively. The lectin was able to induce apoptosis. Treatments with the extract and lectin caused a 57.6–73.6% reduction in tumor weight, which was not significantly different from the reduction in the methotrexate group. Tumors of animals treated with SteLL at 5 mg/kg showed reduced number of secondary vessels while the gauge was lower in all treated groups. In the groups treated with SteLL, tumors showed reduced and slightly vascularized parenchyma, with necrosis in the center and at the periphery. No alterations in the blood levels of urea, creatine, and glucose were detected while serum AST level was moderately increased in the SE group. Histopathological analysis revealed vacuolization and steatosis in the liver of animals treated with the extract and lectin. In addition, the treatments with SE and SteLL resulted in the reduction of filtration space and alterations in tubular architecture in kidneys. In respect to hematological parameters, it was only detected increase in the number of monocytes in SE group. The extract and lectin did not induce the formation of micronuclei in the bone marrow cells. ConclusionsSE and SteLL had antitumor effect against sarcoma 180 without inducing hematological changes and genotoxic effects in mice; however, some degree of hepatic and renal toxicity was observed, suggesting the evaluation of drug delivery strategies in the future.

The Novel Combination of Nitroxoline and PD-1 Blockade, Exerts a Potent Antitumor Effect in a Mouse Model of Prostate Cancer.

Programmed cell death protein 1 (PD-1) blockade is a promising therapeutic strategy against prostate cancer. Nitroxoline has been found to have effective anticancer properties in several cancer types. We investigated the efficacy of a combination therapy involving nitroxoline and PD-1 blockade in a prostate cancer mouse model. In our in vitro analysis, we found that nitroxoline inhibited the viability and proliferation of the mouse prostate cancer cell line RM9-Luc-PSA. Additionally, nitroxoline downregulated the expressions of phospho-PI3 kinase, phospho-Akt (Thr308), phospho-Akt (Ser473), phospho-GSK-3β, Bcl-2, and Bcl-xL. Nitroxoline also downregulated programmed death-ligand 1 (PD-L1) expression levels in prostate cancer cell line and tumor tissue. In our murine prostate cancer orthotopic model, nitroxoline plus PD-1 blockade synergistically suppressed tumor growth when compared with nitroxoline or PD-1 blockade alone, leading to reductions in tumor weight, bioluminescence tumor signals, and serum prostate-specific antigen levels. Furthermore, fluorescence-activated cell sorting analysis showed that the combination strategy significantly enhanced antitumor immunity by increasing CD44+CD62L+CD8+ memory T cell numbers and reducing myeloid-derived suppressor cell numbers in peripheral blood. In conclusion, our findings suggest that nitroxoline plus PD-1 blockade may be a promising treatment strategy in patients with prostate cancer.

Anti-tumor effect of volatile oil from Houttuynia cordata Thunb. on HepG2 cells and HepG2 tumor-bearing mice.

The aim of this paper is to study the anti-tumor mechanism of volatile oil from Houttuynia cordata Thunb. (sodium new houttuyfonate, SNH). In vitro, SNH exhibited a concentration-dependent cytotoxic effect against four human cancer lines (HepG2, A2780, MCF-7, SKOV-3). SNH treatment with different concentrations induced HepG2 cells to exhibit varying degrees of morphological changes in apoptotic features, such as round shape, cell shrinkage and formation of apoptotic body. It was observed that SNH caused the decrease in Bcl-2 mRNA expression and triggered the apoptosis of HepG2 cells. Wound healing assay and RT-PCR results showed that the decrease in the expression level of MMP9 and VEGF was observed in HepG2 cells after treatment with SNH for 48 h, suggesting that the extracellular matrix pathway degradation was involved in the HepG2 cells migration. Moreover, we got an insight into the binding mode of SNH into the MMP9 active site through 3D pharmacophore models. Docking study and molecular dynamics (MD) simulation analysis sheds light on that SNH was completely embedded into the MMP9 active site and formed hydrogen bonds with key catalytic residues of MMP9, including Ala191, His190, Ala189 and Glu227. The prediction of SNH binding interaction energies in the MMP9 was almost in good agreement with the original inhibitor EN140. In vivo experiments, both SNH and cyclophosphamide significantly reduced tumor weights and their tumor inhibitory rates were 50.78% and 82.61% respectively. This study demonstrated that SNH was an apoptosis inducer in HepG2 cells. SNH has four possible functions, that it could induce apoptosis by mitochondria pathway in HepG2 cells, inhibit the tumor growth, regulate Bcl-2 family mRNA expression and effectively subdue migration of hepatocellular carcinoma cells by decreasing the expression of MMP9 and VEGF. Therefore, SNH might be a potential candidate drug for the treatment of hepatocellular carcinoma, which could provide a reference for further clinical research.

Antineoplastic Properties of Phyto-synthesized Silver Nanoparticles from Hibiscus Sabdariffa Linn. Bark Extract

Objectives: In recent years, green synthesis of silver nanoparticles (AgNPs) has gained much interest from researchers as biomedical treatments. The purpose of this study was to determine if AgNPs synthesized from Hibiscus Sabdariffa L. bark extract are an effective stabilizing agent and inhibit Ehrlich ascites carcinoma (EAC) cell growth. Methods: AgNPs were prepared by the reaction of 0.6mM silver nitrate and 5% bark extract. Synthesis of AgNPs were confirmed by UV-visible spectroscopy as well as characterized by scanning electron microscopy, x-ray diffraction, and fourier transform infrared spectroscopy. AgNPs was applied on 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) experiments to determine its antioxidant activity as well as its cytotoxic effect against EAC cells. In vivo, the antineoplastic effect of AgNPs was tested against EAC cells. Results: The synthesized AgNPs have a surface plasmon resonance band centered at 475 nm. Fourier transform infrared spectroscopy (FTIR) spectra showed major peaks of phytochemicals and proteins involved in AgNPs biosynthesis by identifying different functional groups involved in effective capping of AgNPs. The scanning electron microscope (SEM) analysis of AgNPs showed spherical nanoparticles with mean size 90.82 nm. The DPPH and ABTS assays revealed the antioxidant activity of AgNPs with IC50 31.74±2.06 and 15.45±2.72 μg/ml that was comparable to catechin. The synthesized AgNPs exhibited effective cytotoxic activity against EAC cells and the inhibitory concentration (IC50) was recorded at 32.00 µg/ml. AgNPs showed effective cell growth inhibition, increased life span, reduced tumor weight, restored all hematological characteristics to normal level and also induced apoptosis of EAC cells. Conclusion: This study proves that the phyto-synthesized AgNPs show effective anticancer properties against EAC cells.

The FAK inhibitor BI 853520 inhibits spheroid formation and orthotopic tumor growth in malignant pleural mesothelioma

No tyrosine kinase inhibitors are approved for malignant pleural mesothelioma (MPM). Preclinical studies identified focal adhesion kinase (FAK) as a target in MPM. Accordingly, we assessed the novel, highly selective FAK inhibitor (BI 853520) in 2D and 3D cultures and in vivo. IC50 values were measured by adherent cell viability assay. Cell migration and 3D growth were quantified by video microscopy and spheroid formation, respectively. Phosphorylation of FAK, Akt, S6, and Erk was measured by immunoblot. The mRNA expression of the putative tumor stem cell markers SOX2, Nanog, CD44, ALDH1, c-myc, and Oct4 was analyzed by qPCR. Cell proliferation, apoptosis, and tumor tissue microvessel density (MVD) were investigated in orthotopic MPM xenografts. In all 12 MPM cell lines, IC50 exceeded 5 μM and loss of NF2 did not correlate with sensitivity. No synergism was found with cisplatin in adherent cells. BI 853520 decreased migration in 3 out of 4 cell lines. FAK phosphorylation was reduced upon treatment but activation of Erk, Akt, or S6 remained unaffected. Nevertheless, BI 853520 inhibited spheroid growth and significantly reduced tumor weight, cell proliferation, and MVD in vivo. BI 853520 has limited effect in adherent cultures but demonstrates potent activity in spheroids and in orthotopic tumors in vivo. Based on our findings, further studies are warranted to explore the clinical utility of BI 853520 in human MPM.Key messagesResponse to FAK inhibition in MPM is independent of NF2 expression or histotype.FAK inhibition strongly interfered with MPM spheroid formation.BI 853520 has been shown to exert anti-tumor effect in MPM.

Anti-lung cancer mechanisms of diterpenoid tanshinone via endoplasmic reticulum stress-mediated apoptosis signal pathway

At present, lung cancer ranks second and first respectively in the incidence and the mortality among malignant tumors. It is urgent to find new effective anti-lung cancer drugs with less side effects and relatively defined mechanisms. Endoplasmic reticulum stress (ERS)-mediated apoptosis pathway is an effective way to promote tumor cell apoptosis; diterpenoid tanshinone (DT), an effective part separated from Salviae Miltiorrhizae Radix et Rhizoma, was found to have an anti-lung cancer effect in previous studies via ERS-induced PERK-EIF2α pathway. In this paper, human lung adenocarcinoma PC9 cell line and nude mouse transplantation tumor model were applied to verify the anti-lung cancer effect of DT in vivo and in vitro, and illuminate the potential mechanism via ERS induced IRE1α/caspase 12 apoptosis pathway. The results showed that in vivo, DT could promote PC9 cell apoptosis in a concentration-dependent manner, up-regulate Bip, IRE1 and TRAF2 protein expressions in tumor tissue, reduce tumor weight and alleviate bodyweight loss. In vitro, DT inhibited the proliferation of PC9 cell line in a concentration-dependent manner, and destroyed the structure of mitochondria in PC9 cell, promoted Bax, IRE1α, Bip, TRAF2 and caspase 12 protein expressions, lower Bcl-2 protein expression in a time-dependent manner. DT shows a good effect on anti-lung cancer both in vivo and in vitro. The mechanism is related to the activation of ERS-induced IRE1α/caspase 12 apoptosis pathway and the promotion of cell apoptosis. ERS-mediated apoptosis pathway may be an important target of DT on anti-lung cancer.

Therapeutic Potential of Axl Blockade in BCR-ABL Negative Myeloproliferative Neoplasms (MPN)

Axl, a member of the TAM family of receptor tyrosine kinases, mediates survival and therapy resistance of different cancer cells. The Axl ligand growth-arrest specific gene 6 (Gas6) was discovered to promote proliferation of leukemia cells in acute and chronic myeloid leukemia and Axl was identified as a potential therapeutic target in these diseases. Based on these data we investigated the role of Axl in BCR-ABL negative myeloproliferative neoplasms (MPN) and the therapeutic potential of Axl blockade in this group of diseases.We studied the effects of Axl blockade using the small molecule Axl inhibitor BGB324 and performing a lentivirus shRNA mediated knockdown of Axl in human SET-2 and murine BaF3-Jak2V617F MPN cell lines. Pharmacologic Axl blockade resulted in a significant dose dependent decrease in viability of MPN cell lines as measured by WST-1 cell viability assay. Annexin+ staining revealed an increased rate of apoptotic cells upon BGB324 treatment for SET-2 (increase by 15% at 1µM, p<0.001) and BaF3-Jak2V617F cells (increase by 54% at 2µM, p<0.05). Moreover, Western Blot analysis showed higher levels of cleaved caspase 3 in BGB324 treated SET-2 cells and decreased levels of anti-apoptotic bcl-2 in BGB324 treated BaF3-Jak2V617F cells. Additionally, BrdU incorporation assays showed a dose dependent decrease in proliferating cells upon treatment with BGB324 in MPN cell lines (p<0.05). Genetic knockdown of Axl in SET-2 cells decreased cell viability by 75% (p<0.01), increased apoptosis levels as measured by Annexin+ staining by 61% (p<0.05) and decreased proliferation as measured by BrdU incorporation by 35% (p<0.001) compared to control-transduced cells. Furthermore, Western Blot analysis revealed that genetic knockdown of Axl resulted in decreased phosphorylation of Stat3 and Stat5 compared to control-transduced cells. Combined Axl and Jak2 blockade, using BGB324 and the Jak2-inhibitor ruxolitinib, showed additive effects on reducing cell viability in SET-2 and BaF3-Jak2V617F cells (p<0.01 and p<0.001, respectively). Western Blot analysis identified inhibition of Stat5 by BGB324 single treatment in SET-2 cells whereas additive effects of combined Axl and Jak2 blockade resulted from additional inhibition of Stat3. In BaF3-Jak2V617F cells, BGB324 single treatment resulted in downstream inhibition of Akt signaling whereas additive effects of combined Axl and Jak2 blockade were exerted via additional inhibition of Stat5, Stat3 and Erk.The finding that BGB324 inhibits growth of MPN cells was further corroborated in vivo. A xenograft tumor model with SET-2 cells was set up in vivo. SET-2 tumor bearing mice treated with BGB324 50mg/kg showed a slower tumor growth (n=8, p<0.01), with a 60% reduction of tumor weight compared to vehicle treated mice (n=8/8, p<0.01). As a second in vivo model, a systemic model of Jak2V617F driven disease was used. After intravenous injection of BaF3-Jak2V617F cells, mice were treated with 50mg/kg BGB324 or vehicle starting the day after inoculation. BGB324 treated mice had a longer overall survival compared to vehicle treated mice (n=10/11, p*<0.05).Furthermore, to evaluate the potential of BGB324 in primary MPN cells, peripheral blood mononuclear cells (PBMC) were isolated from MPN patients and healthy donors. Western Blot analysis showed higher levels of Axl expression by PBMC from MPN patients compared to PBMC from healthy donors. Moreover, colony-forming assays with PBMC were performed in the presence of different concentrations of BGB324. Here, a higher reduction in the number of colony forming units (BFU-E and CFU-GEMM) was observed in samples from MPN patients compared to healthy donors upon treatment with 1µM (77% vs. 5%, respectively; p<0.001) or 2µM (100% vs. 60%, respectively; p<0.01) of BGB324 (n=5/5).In conclusion, these data indicate therapeutic potential of Axl blockade in BCR-ABL negative MPN as monotherapy and in combination with Jak2-inhibition, supporting the need for clinical investigation. Disclosuresvon Amsberg:Novartis: Honoraria, Speakers Bureau; Ipson: Honoraria, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Speakers Bureau; Sanofi: Honoraria, Speakers Bureau; Astellas: Honoraria, Speakers Bureau; MSD: Honoraria, Speakers Bureau. Loges:BerGenBio: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Icotinib inhibits the proliferation of hepatocellular carcinoma cells in vitro and in vivo dependently on EGFR activation and PDL1 expression.

PurposeHepatocellular carcinoma (HCC) accounts for one of the most prevalent tumor types in the world and still lacks an effective treatment regimen. The EGFR tyrosine-kinase inhibitor icotinib is capable of inhibiting proliferation of several kinds of cancer cells, but its anticancer effect in HCC is still not verified.MethodsIn the current study, ten HCC cell lines were selected to test their original EGFR-activation status and PDL1 protein level, and in vitro antiproliferation assays were also conducted to analyze the IC50 and further investigate the correlation between IC50 and protein level of phosphorylated EGFR and PDL1. A in vivo nude mouse xenograft animal model was used as well to analyze its anticancer effect.ResultsIcotinib showed significant inhibitory effects only on HCC cell lines that had both higher p-EGFR and PDL1 protein level. This specific HCC cell line was subcutaneously injected to establish the in vivo xenograft tumor model, and icotinib reduced tumor weight remarkably and growth dose dependently. Molecular mechanism study revealed that icotinib inhibited the phosphorylation of EGFR and PDL1 expression in cancer cells and activated apoptosis. Knocking down PDL1 significantly reduced the inhibitory effect of icotinib on HCC, and knocking in PDL1 increased the sensitivity of icotinib in HCC.ConclusionThe current research suggests that icotinib has an inhibitory effect on a subgroup of HCC cells that have both higher p-EGFR and PDL1. This hints at the potential clinical usage of icotinib in HCC based on PDL1-biomarker examination.

Therapeutic targeting of PFKFB3 with a novel glycolytic inhibitor PFK158 promotes lipophagy and chemosensitivity in gynecologic cancers.

Metabolic alterations are increasingly recognized as important novel anti‐cancer targets. Among several regulators of metabolic alterations, fructose 2,6 bisphosphate (F2,6BP) is a critical glycolytic regulator. Inhibition of the active form of PFKFB3ser461 using a novel inhibitor, PFK158 resulted in reduced glucose uptake, ATP production, lactate release as well as induction of apoptosis in gynecologic cancer cells. Moreover, we found that PFK158 synergizes with carboplatin (CBPt) and paclitaxel (PTX) in the chemoresistant cell lines, C13 and HeyA8MDR but not in their chemosensitive counterparts, OV2008 and HeyA8, respectively. We determined that PFK158‐induced autophagic flux leads to lipophagy resulting in the downregulation of cPLA2, a lipid droplet (LD) associated protein. Immunofluorescence and co‐immunoprecipitation revealed colocalization of p62/SQSTM1 with cPLA2 in HeyA8MDR cells uncovering a novel pathway for the breakdown of LDs promoted by PFK158. Interestingly, treating the cells with the autophagic inhibitor bafilomycin A reversed the PFK158‐mediated synergy and lipophagy in chemoresistant cells. Finally, in a highly metastatic PTX‐resistant in vivo ovarian mouse model, a combination of PFK158 with CBPt significantly reduced tumor weight and ascites and reduced LDs in tumor tissue as seen by immunofluorescence and transmission electron microscopy compared to untreated mice. Since the majority of cancer patients will eventually recur and develop chemoresistance, our results suggest that PFK158 in combination with standard chemotherapy may have a direct clinical role in the treatment of recurrent cancer.