Reduced Tumor Weight Research Articles

LncRNA GA-Binding Protein Transcription Factor Subunit Beta-1 Antisense RNA 1 Inhibits Renal Carcinoma Growth Through an MiR-1246/Phosphoenolpyruvate Carboxykinase 1 Pathway.

ObjectiveTo clarify the role and mechanism of GABPB1-AS1 in renal cell carcinoma.MethodsWe collected 48 pairs of tumor and adjacent normal tissues from patients with clear cell renal cell carcinoma (ccRCC). Both 786-o and caki-1 ccRCC cell lines were transfected with GA-binding protein transcription factor subunit beta-1 antisense RNA 1 (GABPB1-AS1), miR-1246, or small interfering RNA phosphoenolpyruvate carboxykinase 1 (siPCK1) vectors. RNA expression was examined by quantitative reverse transcription-PCR and protein expression by Western blot. Cell proliferation was measured by Cell Counting Kit-8 assays. Cell migration and invasion were measured by transwell assays. Targeting relationships between genes were tested by luciferase reporter gene assays.ResultsLower GABPB1-AS1 expression was found in ccRCC cells and tissues. GABPB1-AS1 expression was inversely associated with tumor size, TNM stage, and Furhman stage. High GABPB1-AS1 expression was associated with a better prognosis. GABPB1-AS1 overexpression significantly inhibited proliferation, migration, and invasion by 786-o and caki-1 cells. GABPB1-AS1 overexpression reduced tumor weights in xenograft experiments. Luciferase reporter assays showed that miR-1246 overexpression significantly inhibited the luciferase activity of 786-o and caki-1 cells transfected with wild-type (WT)-GABPB1-AS1 or WT-PCK1. Knockdown of PCK1 weakened the inhibition of proliferation, migration, and invasion induced by GABPB1-AS1 overexpression in 786-o and caki-1 cells.ConclusionGABPB1-AS1 inhibits ccRCC growth and plays a tumor suppressor role through an miR-1246/PCK1 axis.

Antitumor Actions of Intratumoral Delivery of Membrane-Fused Mitochondria in a Mouse Model of Triple-Negative Breast Cancers.

BackgroundThe transfer of whole mitochondria has been demonstrated to be beneficial for treating breast cancer because it induces apoptosis and drug sensitivity; however, in vivo evidence of this benefit remains scant. The present study compared the transplantation of mitochondria with instinctive (Mito) and membrane-fused morphologies induced by Pep-1 conjugation (P-Mito) using a mouse model of triple-negative breast cancers.Materials and MethodsMice with advanced severe immunodeficiency received orthotopic implantation of MDA-MB-231 human breast cancer cells followed by transplants of 5-bromo-2ʹ-deoxyuridine (BrdU)-labeled Mito or P-Mito (200 μg [10 μg/μL]) through intratumoral injection at multiple points once a week for 4 weeks.ResultsAfter 1 month of consecutive treatment, 8.2% and 14.2% of the BrdU-labeled mitochondria were preserved in tumors of the Mito and P-Mito groups, respectively. Both Pep-1 and P-Mito treatments reduced tumor weight (21.7% ± 2.43% vs 40.6% ± 2.28%) and led to marked inhibition of Ki67 staining and angiogenesis. However, only the P-Mito group exhibited obvious necrosis and DNA fragmentation accompanied by an altered tumor microenvironment, which included reduced oxidative stress and size of cancer-associated fibroblast populations and enhanced immune cell infiltration. Transmission electron microscopy images further revealed an elongated network of perinuclear mitochondria fused with a few peripheral mitochondria in the nonnecrotic area in the P-Mito group as well as increases in mitochondrial fusion proteins and parkin compared with mitochondrial fission proteins.ConclusionIn this study, the results of mitochondrial transplantation emphasized that the facilitation of mitochondrial fusion is a critical regulator in breast cancer therapy.

Abstract B15: Gut microbiome populations modulate neoadjuvant chemotherapy responsiveness in preclinical triple-negative breast cancer murine model

Abstract Triple-negative breast cancer (TNBC) is a highly aggressive subtype with a 5-year survival rate significantly worse when compared to other breast cancer types. There are no targeted therapy options, limiting these patients to cytotoxic chemotherapy regimens. Our group demonstrated increased tumoral Pseudomonas abundance in breast cancer patients receiving neoadjuvant chemotherapy, suggesting that systemic anticancer therapy administration may modulate microbiota populations. The purpose of our study was to determine whether gut microbiota populations correlate with chemotherapeutic responsiveness and may be a predictive biomarker outcome. We wanted to determine the impact of shifting the microbiome on chemotherapy efficacy. To do so, 8-week old female BALB/c mice were injected with 4T1-luciferase cells in the mammary fat pad. Once tumors reached 100 mm3, mice were either untreated (control group), treated with 1x weekly 2.5 mg/kg doxorubicin (DOX) for 4 weeks, or treated with doxorubicin +antibiotics (mixture of streptomycin, ampicillin, and colistin in the drinking water to ablate the microbiome). Tumor size was monitored. Tumors and lungs were collected after the study. Fecal samples were collected at T0 (before treatment) and T4 (after treatment). Mice receiving DOX were stratified into DOX-responders or DOX-nonresponders based upon tumor size. Mice from DOX-responders and DOX +antibiotics groups displayed reduced tumor weight and decreased lung metastatic burden. 16S-bacterial sequencing indicates elevated fecal Ruminococcus correlates with DOX-nonresponsiveness and increased abundance of Oscillospira and Bacteroidales is associated with better therapeutic outcome. Protein analysis of tumor tissue indicates a significant increase in apoptosis in DOX-responders and DOX +antibiotic groups. To determine whether modulating the gut microbiome influences drug responsiveness, BALB/c mice were stratified into a control group or group receiving lard diet-derived fecal transplant (LDFT) by oral gavage. Mice were injected with 4T1-luciferase cells in the mammary fat pad. Once tumors reached 100 mm3, mice were treated with 1x weekly 2.5 mg/kg DOX for 4 weeks. Tumors and lungs were collected at the end of the study. Fecal samples were collected at T0 (before gavage), T3 (after 3 weeks of LDFT and before DOX-treatment), and T7 (at end of study). All tumors from LDFT-group were larger and displayed reduced chemotherapy responsiveness when compared with control animals, suggesting gut microbiome populations can modulate chemotherapy resistance. Taken together, our data demonstrate that chemotherapy efficacy is modulated by gut microbiome and suggest that modulation of the gut microbiome through dietary or probiotic interventions may affect therapeutic outcomes. Moreover, fecal microbiota populations could be used as a predictive biomarker of chemotherapeutic responsiveness. Citation Format: Kenysha Clear, Adam Wilson, Akiko Chiba, Katherine L. Cook, Alaa Bawaneh. Gut microbiome populations modulate neoadjuvant chemotherapy responsiveness in preclinical triple-negative breast cancer murine model [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr B15.

Synergistic inhibitory effect of resveratrol and TK/GCV therapy on melanoma cells.

To investigate the synergistic effect of resveratrol on the bystander effect of TK/GCV suicide gene system in melanoma cells. The effect of resveratrol on the growth of B16 cells and the synergistic effect of resveratrol with or without GCV were detected by MTT assay and high content screening assay. The effect of resveratrol on GJIC function was detected by flow cytometry combined with fluorescence tracer and fluorescence microscope, and the expression of gap junction protein was detected by western blotting. Synergistic killing effect of resveratrol plus TK/GCV was tested in vivo using transplanted melanoma model. In vitro, resveratrol can enhanced GJ function and upregulated Cx32 and Cx43 protein expression in B16 cells. Resveratrol synergized with GCV to kill mixed B16 melanoma cells (20% TK+ cells and 80% TK- cells) and to improve apoptosis rate of TK- cells (the bystander effect of TK system), and the synergistic action was reversed by the GJ inhibitor AGA. In vivo, when B16 cells were mixed with 30% TK+ B16 cells, significantly reduced tumor weight and volume were observed after combinational treatment with resveratrol plus GCV as compared with GCV or resveratrol treatment alone. Resveratrol could synergistically enhance the killing effect of TK/GCV suicide gene system in melanoma B16 cells and transplanted melanoma. It might be a promising adjuvant of TK/GCV therapy.

Doxorubicin Cardiotoxicity in Young Tumor‐Bearing Mice

BackgroundChildhood cancer survivors have about 15‐times higher risk of heart failure than their siblings mainly due to the cardiotoxicity of chemotherapy. Nearly 50% of pediatric cancer patients receive doxorubicin (DOX) which is known to be cardiotoxic. DOX‐induced cardiotoxicity had been extensively studied in tumor‐free mouse models. The objective of the current study is to characterize a juvenile tumor‐bearing immunocompetent mouse model of DOX‐induced cardiotoxicity.MethodsJuvenile five‐week old male C57Bl/6 mice were injected subcutaneously with EL4 lymphoma cells (5X104 cells/mouse) in the flank region suspended in sterile PBS, while tumorfree mice were only injected with sterile PBS. One week after tumor implantation, both tumorfree and tumor‐bearing mice were injected intraperitoneally with either a clinically relevant dose of DOX (4 mg/kg/week) or saline for 3 weeks. One week after the last DOX injection, mice were euthanized and the hearts and tumors were harvested and weighed. Gene expression of molecular markers of cardiotoxicity and inflammation was quantified using real‐time PCR.ResultsDOX 4 mg/kg/week for 3 weeks significantly reduced tumor weights in EL4 tumor‐bearing mice. DOX administration caused significant cardiac atrophy in tumor‐free and tumor‐bearing mice. Moreover, tumor itself caused a significant reduction in heart weight compared to tumor‐free mice. Additionally, DOX administration induced the expression of markers of cardiotoxicity, ANP and BNP, in both tumor‐free and tumor‐bearing mice. Saline‐treated tumor‐bearing mice demonstrated higher expression of TNF‐alpha, an inflammatory marker, compared to tumor‐free mice.ConclusionWe have established a juvenile tumor‐bearing immunocompetent mouse model to investigate DOX‐induced cardiotoxicity. Our tumor‐bearing mouse model will optimize our understanding of the interplay between tumor and DOX‐induced cardiotoxicity. Additionally, this clinically relevant model will be used for investigation of cardioprotective strategies that mitigate DOX cardiotoxicity without compromising its chemotherapeutic efficacy before translating to the clinical settings.Support or Funding InformationResearch was supported by the National Institutes of Health’s National Center for Advancing Translational Sciences, grant UL1TR002494, and by a grant from the St. Baldrick’s Foundation [B.N.Z.]. B.N.Z. is a Masonic Cancer Center Women’s Health Scholar, sponsored by the Masonic Cancer Center, and administered by the University of Minnesota Women’s Health Research Program.

Antitumor activity of methoxylated flavonoids separated from Achillea fragrantissima extract in Ehrlich’s ascites carcinoma model in mice

Introduction: Achillea fragrantissima extract has been used in folk medicine as an anticancer. The present study describes the antitumor activities of the extract and the flavonoid compounds and attempts to explain the mechanisms underlying these activities. Methods: The whole plant of A. fragrantissima was collected and ground to produce a fine powder which subjected to the maceration process. Step gradient elution was used using silica gel vacuum liquid chromatography (VLC). The fractions were compared by thin layer chromatography (TLC) and concentrated. Final purification was performed using Sephadex LH-20 to give three compounds cirsiliol, chrysosplenol D and cirsimaritin from F4 and one compound, eupatilin-7-methyl ether from F6. In this study, Ehrlich’s ascites carcinoma (EAC) model was used as the model of cancer. Results: Achillea fragrantissima extract and its isolated methoxylated flavonoids significantly reduced the weight of tumor discs compared to EAC-control group. In addition, cirsimaritin and eupatilin 7-methyl ether treatments produced a dose-dependent reduction in tumor weight. Serum tumor necrosis factor-alpha (TNF-ɑ) level showed that A. fragrantissima extract and its isolated methoxylated flavonoid compounds significantly reduced its serum level compared to the EAC-control group. Furthermore, A. fragrantissima extract and the flavonoids significantly increased the Total antioxidant capacity (TAC) compared to EAC-control. A. fragrantissima extract and its isolated methoxylated flavonoids produced a better differentiation of tumor cells, with reduced nuclear pleomorphism and better formed tubular structures especially with high dose indicating the induction of apoptotic mechanism. Conclusion: Achillea fragrantissima extract and its isolated methoxylated flavonoids exhibit antitumor activities that may be attributed to the antioxidant properties and the induction of the apoptotic process.

B4GalT1 Regulates Apoptosis and Autophagy of Glioblastoma In Vitro and In Vivo.

Our study was designed to investigate the role of B4GalT1 in glioblastoma, in vitro and in vivo, to detect whether B4GalT1 knockdown could regulate the development of glioblastoma, and further observe the relationship between B4GalT1 knockdown and the apoptosis and autophagy of glioblastoma. To begin, we looked at TCGA and GEPIA systems to predict the potential function of B4GalT1. Western blot and RT-PCR were used to analyze the expression, or mRNA level, of B4GalT1 at different tissue or cell lines. Next, the occurrence and development of glioblastoma, in vitro and in vivo, was observed by using B4GalT1 knocked down by lentivirus. Finally, the apoptosis and autophagy of glioblastoma was observed in vitro and in vivo. Results show that B4GalT1 was a highly variable gene, and GEPIA and TCGA systems show B4GalT1 expression in GBM tumor tissue was higher than in normal tissue. Pair-wise gene correlation analysis revealed a probable relationship between B4GalT1 and autophagy related proteins. The B4GalT1 expression and mRNA level were increased in tumor cells, or U87 cells. B4GalT1 knocked down by lentivirus could inhibit glioblastoma development, in vitro and in vivo, by reducing tumor weight and volume, increasing survival, and weakening tumor cells proliferation, migration, invasion. B4GalT1 knockdown could increase apoptosis and autophagy of glioblastoma in vitro and in vivo. Our study demonstrates that B4GalT1 may be able to regulate apoptosis and autophagy of glioblastoma. Bax, Bcl-2, cleaved caspase-3, Beclin-1, and LC3 s may be the downstream target factors of B4GalT1 in apoptosis and autophagy, which may provide a new strategy to reduce glioblastoma development by regulating apoptosis and autophagy.

Arnicolide D Inhibits Triple Negative Breast Cancer Cell Proliferation by Suppression of Akt/mTOR and STAT3 Signaling Pathways.

Triple-Negative Breast Cancer (TNBC) is a most dangerous breast cancer subtype. The naturally occurring sesquiterpene lactone, arnicolide D (AD), has proven effective against a variety of tumors, however, the inhibitory effects of AD against TNBC and the underlying mechanisms remain unclear. In the present study, two TNBC cell lines (MDA-MB-231 and MDA-MB-468) and an MDA-MB-231 xenograft mouse model were employed to investigate the anti-TNBC effects of AD in vitro and in vivo. Cell viability was assessed by MTT assay. Cell cycle arrest and apoptosis were analyzed by flow cytometry. Protein levels were determined by immunoblotting. In vitro studies demonstrated that AD significantly decreased cell viability, and induced G2/M cell cycle arrest and apoptosis. In vivo assays showed that oral administration of 25 or 50 mg/kg AD for 22 days led to a reduction of tumor weights by 24.7% or 41.0%, without appreciable side effects. Mechanistically, AD inhibited the activation of Akt/mTOR and STAT3 signaling pathways. Based on our findings, AD is a promising candidate for development as an adjunctive therapeutic drug for TNBC.

Anticancer Effect of Mountain Ginseng on Human Breast Cancer: Comparison with Farm‐Cultivated Ginseng

Mountain ginseng has been used generally as a pharmacopuncture for cancer therapy in clinical practice in Northeast Asia. Nonetheless, there have been few scientific reports for the anticancer action of mountain ginseng. In this study, we investigated whether mountain ginseng extract (MGE) could inhibit the growth of breast cancer in in vitro and in vivo models. MGE showed stronger cytotoxicity than farm-cultivated ginseng extract (FGE) through promoting ROS generation. Also MGE dose-dependently brought about mitochondrial dysfunction in MCF-7 cells. In addition, MGE induced apoptosis through enhancing the activities of caspase-3/7 by regulation of expression of Bcl-2, Bax, cytochrome c, and cleaved caspase-3 in the MCF-7 cells. Consistent with the in vitro results, MGE significantly reduced tumor weights compared with FGE in mice transplanted with MCF-7 cells, and it regulated the expression of apoptosis-related proteins, such as Bcl-2, Bax, cytochrome c, cleaved caspase-3, and cleaved PARP, in the tumor tissues. Additionally, MGE included higher total ginsenoside contents than FGE. In conclusion, MGE, which is richer in ginsenosides, exerts a stronger anticancer action than FGE in breast cancer. The anticancer action of MGE may be closely correlated with caspase-mediated apoptosis through upregulating ROS generation. Therefore, these findings may be helpful for a clinical understanding of the anticancer mechanism of MGE for breast cancer patients.

Demethoxycurcumin Inhibits In Vivo Growth of Xenograft Tumors of Human Cervical Cancer Cells

Demethoxycurcumin (DMC), a derivate of curcumin from natural plants, exerts antitumor effects on various human cancer cells in vitro and in vivo. Nevertheless, no reports have disclosed whether DMC can affect the growth of human cervical cancer cells in vivo. Therefore we investigated the antitumor effects of DMC on a HeLa cell xenograft model in nude mice in this study. Twenty-four nude mice were subcutaneously injected with HeLa cells. All mice were randomly divided into control, low-dose DMC (30 mg/kg), and high-dose DMC (50 mg/kg) groups and individual mice were treated intraperitoneally accordingly every 2 days. DMC significantly reduced tumor weights and volumes of HeLa cell xenografts in mice, indicating the suppression of growth of xenograft tumors. These effects and findings might provide evidence for investigating the potential use of DMC as an anti-cervical cancer drug in the future.

Anti-proliferative effect of ZnO NPs against the growth of Ehrlich Solid carcinoma

Background: Zinc oxide nanoparticle (ZnO NPs) has been widely used in biomedical applications and cancer therapy and has been reported to induce a selective cytotoxic effect on cancer cell proliferation. Methods: Female Swiss Albino mice were inoculated subcutaneously with Ehrlich ascites carcinoma (EAC) cells. ZnO NPs or MTX were injected intraperitoneally. Tumor growth inhibition rate (TIR %), increase in life span (ILS%), zinc concentration, lipid peroxidation marker (MDA), glutathione (GSH) contents, and the activity of the antioxidant scavenger enzymes SOD and catalase CAT was examined. Also, DNA fragmentation and histopathological studies of all groups were carried out. Results: Treatment of tumor-bearing mice with ZnO NPs significantly increased (MST), (TIR %), (ILS %) and reduced tumor weight, compared to tumor-bearing controls. Additionally, Zn concentration and DNA fragmentation were significantly increased in tumor tissues. ZnO nanoparticles were also found to induce oxidative stress, evidenced by the generation of reactive oxygen species and depletion of the antioxidants in tumor tissues, with no changes in the liver. Our data demonstrate that ZnO NPs exert distinct effects via the killing of cancer cells. Conclusion: The study findings demonstrated that the ZnO NPs can induce selective cytotoxicity through reactive oxygen spe­cies generation and oxidative stress.

CYTOTOXIC AND ANTITUMOR EFFECT OF THE ETHANOLIC EXTRACT OF THE BARK OF HIMATANTHUS BRACTEATUS (A. DC.) WOODSON

Introduction Himatanthus bracteatus has been popularly used in the treatment of cancer, but its antitumor properties have not been scientifically reported yet. Objectives The purpose of this study was to assess the antitumor effect of Himatanthus bracteatus ethanolic extract (HBEE) in mice. Design The HBEE was obtained under high pressure conditions (100 bar, 2 mL/min, 25°C) and its cytotoxic activity was assessed in vitro against MCF-7 and MCF-10 A cells. For the biologic assay, 180 sarcoma cells were transplanted into 40 mice assigned into 5 groups: CTR, 5-fluorouracil (5-FU), and HBEE10, 30, and 100 (treated with saline, 5-FU, and HBEE at 10, 30, and 100 mg/Kg, respectively). Hematologic and biochemical analyses, as well as gross and morphologic analyses of the tumors, were performed in order to evaluate the toxicologic aspects. Results The IC50 of HBEE for MCF-7 tumor cells (36.28 µg/mL) was significantly higher than for MCF-10 A nontumor cells (145.13 µg/mL). In vivo assay demonstrated that HBEE at 30 mg/Kg significantly reduced tumor weight (P Conclusions These data suggest that the HBEE exerted selective cytotoxic for tumor cells and antitumor activity comparable to 5-FU.

The effect of low-dose naltrexone on solid Ehrlich carcinoma in mice: The role of OGFr, BCL2, and immune response

AimsCancer is a major worldwide health problem. Cancer cells express opioid growth factor (OGF) which controls their growth. Naltrexone in low dose (LDN) blocks opioid receptors intermittently and controls the replication of cancer cells. The aim of this study was to investigate the effect of LDN and its chemotherapeutic additive effect on the growth of solid Ehrlich carcinoma in mice with focus on the OGFr and immune responses. Main methodsSixty female Swiss albino mice were assigned into 5 groups (n: 12 mice each): (i): normal control, (ii): Solid Ehrlich carcinoma (SEC), (iii): SEC treated with LDN, (iv): SEC treated with 5-fluorouracil (5-FU), (v): SEC treated with LDN + 5-FU. All drugs were started when the tumor became palpable on 9th day. At the end of the study animals were sacrificed, blood and tissue samples were collected. Tumor weight and volume were measured. Splenocytes and myeloid derived suppressor cells (MDSC) were counted. Tumor expression of opioid growth factor receptors (OGFr), serum level of IFN-γ, tumor histopathology (H&E) and immunohistochemistry staining of p21, p53, Bcl2 were assessed. Key findingsAll drug-treated groups showed reduction in tumor weight and volume, significant increase of splenocyte with tendency to reduce MDSC cell counts. LDN led to significant increase in OGFr both in solo and in combination with 5FU. Serum IFN-γ is significantly increased by LDN but decreased by 5-FU. Also, LDN and 5FU increased immunehistochemical staining of p21 while decreased immunostaining of Bcl2. In animals treated with a combination of LDN and 5FU a maximal downregulation of the antiapoptotic mediator BCL2 was observed. SignificanceThe current study suggested that LDN may play a role in inhibiting cancer cell growth and highlights the possibility of promising combination with cancer chemotherapeutics, which guarantee further clinical studies for approval.

MiR-182 regulates cell proliferation and apoptosis in laryngeal squamous cell carcinoma by targeting the CRR9.

Background: The effect of miR-182 on the expressions of CRR9 in laryngeal squamous cell carcinoma (LSCC) cells, and the impact on invasion and metastasis of LSCC were investigated in the present paper.Methods: The expressions of miR-182 in LSCC tissue and cell line were detected by RT-qPCR. MTT assay and Annexin V staining were used to detect the effects of miR-182 on tumor cells proliferation. Target gene prediction and screening, and luciferase reporter assay were designed to verify downstream target genes of miR-182. The mRNA and protein expressions of CRR9 were detected by qRT-PCR and Western blot. Finally, the expressions of CRR9 were measured by transfecting cells with miR-182 in mice.Results: Compared with normal tissue and cell, the expressions of miR-182 in tumor tissues and cells were much lower. Over-expressions of miR-182 can increase apoptosis rate. Luciferase reporter assay revealed that CRR9 was a downstream gene of miR-182. Reintroduction of CRR9 abolished miR-182-induced LSCC cell growth inhibition. In animal models, over-expressions of miR-182 can reduce tumor weight and promote apoptosis.Conclusion: miR-182 can inhibit the proliferation of LSCC cells by directly inhibiting the expressions of CRR9, thereby suppressing the occurrences and developments of LSCC.

LncRNA MAGI2-AS3 Prevents the Development of HCC via Recruiting KDM1A and Promoting H3K4me2 Demethylation of the RACGAP1 Promoter

Accumulating studies have implicated the role of long non-coding RNAs (lncRNAs) in the pathogenesis of hepatocellular carcinoma (HCC) through the regulating transcription and mRNA stability. A recent report has linked Rac GTPase-activating protein 1 (RACGAP1) to the early recurrence of HCC. The current study aimed to ascertain whether MAGI2 antisense RNA 3 (MAGI2-AS3) influences the development of HCC by regulating RACGAP1. MAGI2-AS3 expression was initially quantified in both the HCC tissues and cell lines. In order to elucidate the role of MAGI2-AS3 in the development of HCC, MAGI2-AS3 was overexpressed or silenced in HCC cells after which cell proliferation, apoptosis, invasion, and migration were evaluated. Chromatin immunoprecipitation (ChIP), RNA immunoprecipitation (RIP), and biotin-labeled RNA pull-down assays were conducted to determine the interactions among MAGI2-AS3, KDM1A, and RACGAP1. Finally, the effects of MAGI2-AS3 and RACGAP1 on the tumorigenesis of transplanted HCC cells in nude mice were evaluated. MAGI2-AS3 was found to be under-expressed in HCC tissues and cell lines. The restoration of MAGI2-AS3 was identified to markedly inhibit HCC cell growth, migrating ability, and invasiveness, and promote cell apoptosis. Interaction between MAGI2-AS3 and KDM1A was identified. KDM1A recruited by MAGI2-AS3 was found to promote H3K4me2 demethylation at the RACGAP1 promoter, which ultimately decreased the expression of RACGAP1. We also identified that RACGAP1 knockdown eliminated the stimulatory effects of MAGI2-AS3 silencing on the malignant phenotypes of HCC cells. Additionally, the expression of MAGI2-AS3 reduced tumor weight and size in HCC transplanted nude mice. Taken together, the key observations of the current study demonstrate the potential of MAGI2-AS3 as a tumor suppressor and a promising target for HCC treatment.

MRTF-A controls myofibroblastic differentiation of human multipotent stromal cells and their tumour-supporting function in xenograft models

Tumour growth and metastatic colonization is strongly influenced by the tumour stroma, including cancer-associated fibroblasts (CAF). Multipotent mesenchymal stromal cells (MSC) are a possible source of CAF following myofibroblastic differentiation, and we have previously shown that MSC support tumour growth. Triggered by tumour cell-derived factors like transforming growth factor β1 (TGF-β1), myofibroblastic MSC differentiation is associated with the increased expression of markers including alpha smooth muscle actin (α-SMA). Here we show that myocardin-related transcription factor A (MRTF-A) plays an important role in myofibroblastic differentiation of primary human MSC in vitro and their tumour-supporting function in vivo. Recombinant TGF-β1 or tumour cell conditioned medium (TCM) elevated α-SMA, calponin 1 and collagen 1 A1 (COL1A1) amount on mRNA and protein level in MSC. This correlated with increased MRTF-A activity during MSC differentiation. MRTF-A knockdown by siRNA or shRNA impaired TGF-β1 and TCM induction of α-SMA and calponin 1, but not of COL1A1. Mixed xenograft experiments using HCT8 colorectal carcinoma cells and primary MSC of different donors revealed a significant reduction in tumour weight and volume upon MRTF-A knockdown in MSC. Our study suggests that MRTF-A is involved in the functional differentiation of MSC towards a tumour-promoting CAF phenotype in vivo.

Apoptosis and metastasis inhibitory potential of pineapple vinegar against mouse mammary gland cells in vitro and in vivo

BackgroundPlant-based food medicine and functional foods have been consumed extensively due to their bioactive substances and health-beneficial effects. Vinegar is one of them due to its bioactivities, which confers benefits on human body. Our previous study has produced pineapple vinegar that is rich in gallic acid and caffeic acid via 2 steps fermentation. There are many evidences that show the effectiveness of these resources in inhibiting the proliferation and metastasis of the cancer cells through several mechanisms.MethodsFreeze-dried pineapple vinegar was evaluated for its in vitro apoptosis and metastasis inhibitory potential using MTT, cell cycle, Annexin V and scratch assays. The in vivo test using BALB/c mice challenged with 4 T1 cells was further investigated by pre-treating the mice with 0.08 or 2 ml/kg body weight of freshly-prepared pineapple vinegar for 28 days. The tumor weight, apoptotic state of cells in tumor, metastasis and immune response of the untreated and pineapple vinegar treatment group were evaluated and compared.ResultsFrom the in vitro study, an IC50 value of 0.25 mg/mL after 48 h of treatment was established. Annexin V/PI and scratch closure assays showed that pineapple vinegar induced 70% of cell population to undergo apoptosis and inhibited 30% of wound closure of 4 T1 cells. High concentration of pineapple vinegar (2 ml/kg body weight) led to the reduction of tumor weight and volume by 45%as compared to the untreated 4 T1-challenged mice. This effect might have been contributed by the increase of T cell and NK cells population associated with the overexpression of IL-2 andIFN-γ cytokines and splenocyte cytotoxicity. Furthermore, fewer instances of metastasis events were recorded in the pineapple vinegar treatment group and this could be explained by the downregulation of inflammation related genes (iNOS, NF-kB and COX2), metastasis related genes (iCAM, VEGF and MMP9) and angeogenesis related genes (CD26, TIMP1, HGF, MMP3, IGFBP-1 and IGFBP-2).ConclusionThe ability of pineapple vinegar to delay cancer progression portrayed its potential as chemopreventive dietry intervention for cancer therapy.

LncRNA ANRIL affects the sensitivity of ovarian cancer to cisplatin via regulation of let-7a/HMGA2 axis.

This paper tried to explore ANRIL expression in ovarian cancer and how it affects cisplatin-sensitivity of ovarian cancer cells via regulation of let-7a/high-mobility group protein A2 (HMGA2) axis. qRT-PCR was used to detect ANRIL and let-7a levels in ovarian cancer tissues and cell lines (SKOV3 and SKOV3/DDP). Then cells were randomly assigned into Blank, negative control siRNA, ANRIL siRNA, let-7a inhibitor, and ANRIL siRNA+let-7a-inhibitor groups. CCK-8 assay was applied for assessing cell viability of cells treated with different concentrations of cisplatin. Flow cytometry was employed to test cell apoptosis rate. qRT-PCR and Western blot were performed for related molecules detection. Nude mice transplanted with SKOV3/DDP cells were used to confirm the effects of ANRIL siRNA on the cisplatin-sensitivity. Ovarian cancer tissues and cisplatin-resistant cells had increased ANRIL expression and decreased let-7a expression, and those patients with higher clinical stage and pathological grade showed higher ANRIL and lower let-7a. Dual-luciferase reporter-gene assay confirmed the targeting relationship between ANRIL and let-7a, and between let-7a and HMGA2. The cell viability and cisplatin IC50 were decreased in ANRIL siRNA group exposed to different concentrations of cisplatin, with enhanced apoptosis, as well as elevated let-7a and declined HMGA2, which would be reversed by let-7a inhibitor. Meanwhile, ANRIL down-regulation enhanced the inhibitory effect of cisplatin on tumor growth of nude mice and reduced tumor weight. Silencing ANRIL expression reduced HMGA2 expression to promote the apoptosis and improve cisplatin-sensitivity of ovarian cancer cells via up-regulating let-7a expression.

Abstract LB-186: The rexinoid LG100268 decreases immunosuppressive immune cells and activates T cells in two preclinical models of breast cancer

Abstract Breast cancer (BC) was considered an immunologically silent tumor, however recent findings suggest that immune cells present in the tumor microenvironment play important roles in tumor growth. Retinoid X receptors (RXRs) are a subclass of nuclear receptors that act as ligand-dependent transcription factors that regulate a variety of cellular processes including proliferation and differentiation. Rexinoids are synthetic molecules that bind and activate RXRs. Bexarotene (Targretin®) is the only rexinoid approved by the FDA for the treatment of refractory cutaneous T-cell lymphoma. Other more potent rexinoids have been synthesized, such as LG100268 (LG268) and LG101506. Because recent studies have suggested that RXRs are involved in immune regulation, we studied the effects of LG268 as an immune modulator in a preclinical model of breast cancer. MMTV-Neu mice with tumors approximately 32 mm3 in volume were treated with control diet or LG268 (100 mg/kg diet) for 5 days. Rexinoid treatment significantly (p=0.0047) reduced tumor weight from 3.2% of body weight in the control group to 1.1% in the treated group. Bexarotene (100 mg/kg diet), however, did not reduce tumor weight even after 10 days of treatment. Analysis of the tumor by flow cytometry showed a significant (p=0.0013) reduction in the percentage of myeloid derived suppressor cells (MDSCs) in the treated mice (3.4% vs 1.6%). MDSCs are associated with a lack of immune surveillance against tumor cells, and in BC, are correlated with higher metastatic burden and poor survival. The reduction in MDSCs was accompanied by a 80% reduction in p-STAT1 (p=0.02). The percentage of activated CD4 T cells (CD45, CD3, CD4, CD25) also decreased when mice were treated with LG268 (27.5% vs 13%; p=0.012). Moreover, the ratio of CD8/CD4, CD25 cells was increased in the tumors of treated mice (1.58 vs 3.07; p=0.04), suggesting increased activation of cytotoxic T cells. Higher CD8/CD4, CD25 ratios correlate with better outcomes in patients with several solid tumors. To further elucidate the effects of LG268 on immunosuppressive T cells, CD4 cells were isolated from spleen and stimulated with CD28, IL-2 and TGFβ, skewing the cells towards regulatory T cells. CD4 Treg FOXP3 expressing cells contribute to the immunosuppressive milieu in tumors. Treatment with LG268 at 100 nM reduced the mRNA expression of FOXP3 by 40% (p=0.03) when compared with untreated CD4 T cells. In the MMTV-PyMT model of triple negative BC, LG268 (100 mg/kg diet) significantly (p=0.018) extended survival by an average of 29 days when compared with the controls. Histopathological analysis revealed that tumors treated with LG268 had higher infiltration of CD8 cytotoxic T cells, which correlated with increased cleaved caspase 3. These results show that RXR agonists can be used to modulate immune cell populations present in the tumor microenvironment, and reduce immunosuppressive MDSC cells and CD4 T regulatory cell populations in breast cancer models. We thank the Breast Cancer Research Foundation for support. Citation Format: Ana Sofia Leal, Sarah Carapellucci, Kayla ZydecK, Michael B. Sporn, Karen T. Liby. The rexinoid LG100268 decreases immunosuppressive immune cells and activates T cells in two preclinical models of breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-186.

Abstract 2658: PFKFB3 inhibition reprograms malignant pleural mesothelioma to glycolytic stress-induced macropinocytosis and ER stress as independent binary adaptive responses

Abstract The metabolic signatures of cancer cells are often associated with elevated glycolysis which promotes alteration in cellular bioenergetics. 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), a critical control point in the glycolytic pathway has emerged as a potential anti-cancer target associated with many aspects of neoplastic progression. Pharmacological (PFK158 treatment) and genetic inhibition of PFKFB3 activity, decreases glucose uptake, ATP production, and lactate dehydrogenase activity and inhibits cell proliferation in vitro by arresting the cells in the G0/G1 phase induced cell death in malignant pleural mesothelioma (MPM). It induces an early response of increased macropinocytosis and drastic endosomal vacuolization to overcome the nutrient stress both in sarcomatoid (H28) and epithelioid (EMMeso) MPM cells. Inhibition of PFKFB3 leads to the activation of the endocytic Rac1-Rab5-Rab7 pathway resulting in a unique form of cell death called “methuosis”. Transmission Electron Microscopy analysis showed that pharmacological and genetic inhibition of PFKFB3 activity leads to the formation of nascent macropinocytotic vesicles, which rapidly coalesced to form large vacuoles with compromised lysosomal function. Confocal microscopy revealed F-actin and α-tubulin disassembly leading to the disruption of cell structure integrity. In parallel, PFKFB3 expression inversely modulates endoplasmic reticulum (ER) stress in MPM. Pharmacological and genetic inhibition of PFKFB3 led to an escalation in ER activity and aggravated ER stress mostly by upregulating BiP and GADD153 expression levels. However, EHT1864, a Rac1 inhibitor, could not rescue MPM cells from ER stress, suggesting that the activation of macropinocytosis and ER stress are a result of two independent PFKFB3 modulated adaptive responses. Both immunofluorescence microscopy and co-immunoprecipitation analyses revealed that the two crucial biomolecules of each pathway, Rac1 and Calnexin, interact with each other only at the later stages of PFKFB3 inhibition. Finally, PFK158, in a combination of with CBPt, demonstrated a significant reduction in tumor weight in mesothelin overexpressed EMMeso xenograft model. Since most cancer cells exhibit an increased glycolytic rate, these results provide evidence for PFK158, in combination with standard chemotherapy, may have a potential in the treatment of MPM. Citation Format: Sayantani Sarkar Bhattacharya, Ling Jin, Debarshi Roy, Deokbeom Jung, Prabhu Thirusangu, Yinan Xiao, Julie Staub, Julian Molina, Viji Shridhar. PFKFB3 inhibition reprograms malignant pleural mesothelioma to glycolytic stress-induced macropinocytosis and ER stress as independent binary adaptive responses [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 2658.