Decreased Tumor Weight Research Articles

Aerobic Exercise Training Attenuates Tumor Growth and Reduces Insulin Secretion in Walker 256 Tumor-Bearing Rats.

Aerobic exercise training can improve insulin sensitivity in many tissues; however, the relationship among exercise, insulin, and cancer cell growth is unclear. We tested the hypothesis that aerobic exercise training begun during adolescence can attenuate Walker 256 tumor growth in adult rats and alter insulin secretion. Thirty-day-old male Wistar rats engaged in treadmill running for 8 weeks, 3 days/week, 44 min/day, at 55–65% VO2max until they were 90 days old (TC, Trained Control). An equivalently aged group was kept inactive during the same period (SC, Sedentary Control). Then, half the animals of the SC and TC groups were reserved as the control condition and the other half were inoculated with Walker 256 cancer cells, yielding two additional groups (Sedentary Walker and Trained Walker). Zero mortalities were observed in tumor-bearing rats. Body weight (BW), food intake, plasma glucose, insulin levels, and peripheral insulin sensitivity were analyzed before and after tumor cell inoculation. We also evaluated tumor growth, metastasis and cachexia. Isolated pancreatic islets secretory activity was analyzed. In addition, we evaluated mechanic sensibility. Our results showed improved physical performance according to the final workload and VO2max and reduced BW in trained rats at the end of the running protocol. Chronic adaptation to the aerobic exercise training decreased tumor weight, cachexia and metastasis and were associated with low glucose and insulin levels and high insulin sensitivity before and after tumor cell inoculation. Aerobic exercise started at young age also reduced pancreatic islet insulin content and insulin secretion in response to a glucose stimulus, without impairing islet morphology in trained rats. Walker 256 tumor-bearing sedentary rats also presented reduced pancreatic islet insulin content, without changing insulin secretion through isolated pancreatic islets. The mechanical sensitivity test indicated that aerobic exercise training did not cause injury or trigger inflammatory processes prior to tumor cell inoculation. Taken together, the current study suggests that aerobic exercise training applied during adolescence may mitigate tumor growth and related disorders in Walker 256 tumor-bearing adult rats. Improved insulin sensibility, lower glucose and insulin levels and/or reduced insulin secretion stimulated by glucose may be implicated in this tumor attenuation.

MiR-103 Functions as a Tumor Suppressor by Directly Targeting Programmed Cell Death 10 in NSCLC.

Non-small cell lung cancer (NSCLC) accounts for about 85% of all lung cancer cases. Absence of miR-103 has recently been identified to be associated with metastatic capacity of primary lung tumors. However, the exact role of miR-103 in NSCLC and the molecular mechanism are unclear. In the present study, we showed that miR-103 expression was reduced in NSCLC tissues and cells. miR-103 expression was negatively correlated with tumor size and stage. The overall survival was longer in patients with higher miR-103 level than in those with lower miR-103 expression. miR-103 inhibited cell proliferation in A549 cells, decreased tumor weight and volume, and prolonged survival of tumor-implanted nude mice. miR-103 increased apoptotic cell death in A549 cells. Furthermore, miR-103 decreased the invasion and migration abilities in A549 cells, as evidenced by Transwell and wound healing results. Downregulation of miR-103 significantly reduced the level of programmed cell death 10 (PDCD10). We found a significant decrease in the relative luciferase activity of the reporter gene in A549 cells cotransfected with the miR-103 mimic and pGL3-PDCD10 WT 3′-UTR, but not pGL3-PDCD10 mut 3′-UTR. We showed that overexpression of PDCD10 significantly inhibited miR-103-induced inhibition of cell proliferation, increased apoptosis, and decreased invasion and migration in A549 cells. Moreover, we found that PDCD10 expression was increased in NSCLC tissues and cells. PDCD10 expression was positively correlated with tumor size and stage. Overexpression of PDCD10 increased cell proliferation and inhibited apoptosis in A549 cells. The data demonstrated that dysregulation of the miR-103/PDCD10 signal may be a novel therapeutic target for the treatment of NSCLC.

CUL4B promotes metastasis and proliferation in pancreatic cancer cells by inducing epithelial-mesenchymal transition via the Wnt/β-catenin signaling pathway.

This study determines whether cullin 4B (CUL4B) promotes pancreatic cancer (PC) metastasis by inducing epithelial-mesenchymal transition (EMT) via the Wnt/β-catenin signaling pathway. A total of 64 PC patients were enrolled in this study. Human PC cell lines were distributed into blank, negative control, shCUL4B, PLOC, PLOC-CUL4B, and PLOC-CUL4B + siRNA-β-catenin groups. The expressions of CUL4B, Wnt/β-catenin signaling pathway-related proteins, and EMT-related proteins were determined using RT-qPCR and Western blotting. The positive expressions of CUL4B and β-catenin protein in tissues were detected by immunohistochemistry. MTT assay and flow cytometry was performed for cell proliferation and cell cycle, scratch test, and transwell assay for cell migration and invasion ability. CUL4B and β-catenin were expressed at a higher level in PC tissues than in paracancerous tissues though paracancerous tissues had higher expressions of CUL4B and β-catenin than normal tissues. The PLOC-CUL4B group showed increased CUL4B, Wnt, β-catenin, LEF-1, c-Jun, Cyclin D1, N-cadherin, Vimentin, Snail, and ZEB1 expression; decreased E-cadherin expression; accelerated cell proliferation; increased S-phase cell percentages; increased cell migration ability; more liver metastases; and enlarged tumor than the PLOC and PLOC-CUL4B + siRNA-β-catenin groups. The shCUL4B group showed decreased CUL4B, Wnt, β-catenin, LEF-1, c-Jun, Cyclin D1, N-cadherin, Vimentin, Snail, and ZEB1 expression; increased E-cadherin expression; decelerated cell proliferation; decreased S-phase cell percentages; reduced cell migration ability; less liver metastases; and decreased tumor weight than the blank and negative control groups. We demonstrate that CUL4B promotes PC metastasis by inducing EMT via the Wnt/β-catenin signaling pathway. Therefore, CUL4B might be the clinical target for treating PC.

Endometrial Cancers Harboring Mutated Fibroblast Growth Factor Receptor 2 Protein Are Successfully Treated With a New Small Tyrosine Kinase Inhibitor in an Orthotopic Mouse Model.

AL3818 (anlotinib) is a receptor tyrosine kinase inhibitor targeting vascular endothelial growth factor receptors (VEGFR1, VEGFR2/KDR, and VEGFR3), stem cell factor receptor (C-kit), platelet-derived growth factor (PDGFβ), and fibroblast growth factor receptors (FGFR1, FGFR2, and FGFR3). This study evaluates the efficacy of AL3818 studying tumor regression in an orthotopic murine endometrial cancer model. We tested the cytotoxicity of AL3818 on a panel of 7 human endometrial cancer cell lines expressing either wild-type or mutant FGFR2 and also assessed the in vivo antitumor efficacy in a murine, orthotopic AN3CA endometrial cancer model. AL3818 was administered daily per os either alone or in combination with carboplatin and paclitaxel, which represent the current standard of adjuvant care for endometrial cancer. AL3818 significantly reduces AN3CA cell number in vitro, characterized by high expression of a mutated FGFR2 protein. Daily oral administration of AL3818 (5 mg/kg) resulted in a complete response in 55% of animals treated and in a reduced tumor volume, as well as decreased tumor weights of AN3CA tumors by 94% and 96%, respectively, following a 29-day treatment cycle. Whereas carboplatin and paclitaxel failed to alter tumor growth, the combination with AL3818 did not seem to exhibit a superior effect when compared with AL3818 treatment alone. AL3818 shows superior efficacy for the treatment of endometrial cancer irresponsive to conventional carboplatin and paclitaxel combination and warrants further investigation.

Possible anticancer effects of rutin and orlistat in Ehrlich-ascites carcinoma in mice.

Cancer is a broad term used to describe a large number of diseases characterized by uncontrolled cell proliferation that leads to tumor production. Cancer is associated with mutations in genes controlling proliferation, oxidative stress and other mechanisms. Currently, most antineoplastic drugs have severe adverse effects and new effective and safe drugs are needed. This study aims to investigate the possible anticancer activity of rutin and orlistat which are both safely used clinically in humans against an in vivo (Ehrlich ascites carcinoma; EAC) model of cancer.Our results have shown that both rutin and orlistat exerted an in vivo anticancer activity as evidenced by the decrease in tumor weight and the exerted antioxidant action (increased catalase content). In conclusion, the anticancer activity of rutin and orlistat makes them promising candidates for cancer treatment alone or in combination with other anticancer drugs specially that they are used clinically with acceptable safety profile.

Histone deacetylase 3 is associated with gastric cancer cell growth via the miR-454-mediated targeting of CHD5.

Gastric cancer (GC) is the third leading cause of cancer-related mortality in China and worlwide; hence, the identification of GC-related genes is necessary for the development of effective treatment strategies. In this study, histone deacetylase 3 (HDAC3) was identified as the most significantly upregulated cancer-related gene in GC tissues by microarray. In accordance with this, HDAC3 expression was found to be upregulated in GC cell lines/tissues. Further experiments indicated that the knockdown of HDAC3 decreased GC cell viability, reduced the colony formation number and decreased tumor weight. To explore the underlying mechanisms, the overexpression of HDAC3 was induced by transfection with an overexpression plasmid, followed by miRNA microarray, and we identified miR-454 as the most markedly upregulated miRNA. Accordingly, miR-454 expression was upregulated in GC cell lines/tissues and a high level of miR-454 indicated a high HDAC3 expression in GC tissues, and miR-454 knockdown reduced cell viability. In addition, a high level of miR-454 was significantly associated with an advanced clinical stage, lymph node metastases and a poor prognosis of patients with GC. Furthermore, CHD5 was identified as a direct target of miR-454. CHD5 was downregulated in GC tissues/cell lines and the expresssion of CHD5 inversely correlated with the level of miR-454 in GC tissues. Taken together, these observations indicate that HDAC3 is associated with GC cell growth via the miR-454-mediated targeting of CHD5.

The anti-cancer effects of itraconazole in epithelial ovarian cancer

We assessed the anti-proliferative activity of itraconazole using an EOC cell line (SKOV3ip1) and endothelial cell lines (HUVEC & SVEC4-10). We also examined angiogenesis (VEGFR2, p-ERK, p-PLCr1/2), hedgehog (Gli1, Ptch1, SMO), and mTOR (pS6K1) signaling pathways to determine the mechanism of action of itraconazole. Furthermore, we evaluated the synergistic effects of itraconazole and paclitaxel using orthotopic mouse models with established EOC cells (SKOV3ip1 or HeyA8) as well as patient-derived xenografts (PDXs). Itraconazole treatment inhibited proliferation of endothelial cells in a dose-dependent manner, but had no effect on EOC cells. The endothelial cell antiproliferative effect was associated with inhibition of hedgehog, and mTOR pathways and angiogenesis. In xenograft models of EOC using SKOV3ip1 or HeyA8, mice treated with the combination of itraconazole and paclitaxel had significantly decreased tumor weight than the control, paclitaxel-alone, or itraconazole-alone groups. Tissue derived from these tumors had significantly lower microvessel density than tissue from the other groups as well as hedgehog and mTOR pathway inhibition. We confirmed those effects in two EOC PDX models. These results suggest that itraconazole selectively inhibits endothelial cells rather than cancer cells by targeting multiple pathways including hedgehog, and mTOR pathways and angiogenesis.

Ultrasound-Responsive Polymeric Micelles for Sonoporation-Assisted Site-Specific Therapeutic Action.

Targeting drug delivery remains a challenge in various disease treatment including cancer. The local drug deposit could be greatly enhanced by some external stimuli-responsive systems. Here we develop pluronic P123/F127 polymeric micelles (M) encapsulating curcumin (Cur) that are permeabilized directly by focused ultrasound, in which ultrasound triggers drug release. Tumor preferential accumulation and site-specific sonochemotherapy were then evaluated. Cur-loaded P123/F127 mixed micelles (Cur-M) exhibited longer circulating time and increased cellular uptake compared to free Cur. With the assistance of focused ultrasound treatment, Cur-M showed tumor-targeting deposition in a time-dependent manner following systemic administration. This was due to enhanced permeabilization of tumor regions and increased penetration of Cur-M in irradiated tumor cells by ultrasound sonoporation. Furthermore, Cur-M self-assembly could be regulated by ultrasound irradiation. In vitro Cur release from mixed micelles was greatly dependent on ultrasound intensity but not on duration, suggesting the cavitational threshold was necessary to initiate subsequent sonochemotherapy. In vivo site-specific drug release was demonstrated in dual-tumor models, which showed spatial-temporal release of entrapped drugs following intratumoral injection. The sonoporation-assisted site-specific chemotherapy significantly inhibited tumor growth and the decrease in tumor weight was approximately 6.5-fold more than without exposure to ultrasound irradiation. In conclusion, the established ultrasound-guided nanomedicine targeting deposit and local release may represent a new strategy to improve chemotherapy efficiency.

Anticancer activity of salicin and fenofibrate.

Cancer refers to a disorder of cell proliferation that leads to tumor production. Cancer is usually treated by surgery, chemotherapeutic drugs, and radiation. Despite the presence of many anticancer drugs, cancer is still an uncontrolled disease and is a major cause of death worldwide. In addition, most anticancer drugs have severe side effects that can limit their use in some patients. This study aims to investigate the possible anticancer activity of two clinically used drugs: a natural antioxidant agent (salicin) and an antihyperlipidemic agent (fenofibrate) against two breast cancer models (in vivo EAC and in vitro MCF7) and the pancreatic cancer cell line (Panc-1).Our results have shown that both salicin and fenofibrate exerted an in vivo anticancer activity as evidenced by the decrease in tumor weight, tumor volume, CEA level, and reduced tumor cholesterol content through an antioxidant (reduced MDA level and increased GSH and catalase content) and an antiinflammatory activity (reduced TNF-∝ level). In addition, both salicin and fenofibrate were shown to be cytotoxic to MCF-7 and Panc-1 cell lines through activation of the caspase 3/7 apoptotic pathway.In conclusion, salicin and fenofibrate are promising anticancer drugs that are already used clinically with acceptable safety profile which can be incorporated into clinical trials to determine their possible application in cancer treatment.

Abstract 2083: Development of novel targeted adjuvant therapy for triple negative breast cancer

Abstract There is an unmet clinical need for targeted adjuvant therapy in Triple Negative Breast Cancer (TNBC) to overcome its poor prognosis, short disease-free interval and metastatic dissemination. We previously reported that blocking interactions between the PAH2 domain of chromatin regulator Sin3 and Sin3 interaction domain (SID) containing proteins like PF1 and TGIF1 by SID decoys (peptides and small molecule, C16) decreased the cancer stem cell population, invasion, EMT, and metastases. This, programmed upregulation of retinoid signaling that sensitized TNBC cells to AM80, a novel clinically available RARα-specific agonist. Here we report preclinical investigations on effects of SID decoys and AM80 treatments on cellular heterogeneity, primary tumors, metastatic dissemination, minimum residual disease (MRD) and host microenvironment. Using CyTOF2, we made single cell measurements of markers of differentiation, proliferation and stemness in CSC-enriched 4T1 tumorspheres. Treatment with SID peptide decreased cell populations expressing nanog, sox2, vimentin and β-catenin with increase in γH2AX. Addition of AM80 in combination with C16, resulted in populations with increased expression of differentiation marker CD24 with decrease in vimentin, β-catenin and Ki-67. To interrogate the neo-adjuvant effects of C16-AM80 treatments, primary 4T1 tumors in Balb/c mice were treated with C16 and AM80 alone or in combination. Compared to DMSO, ~40 % decrease in tumor weight, 60% decrease in ALDH activity and 60% decrease in lung metastasis was seen in mice treated with C16-AM80 combination. In post-surgical adjuvant settings, in both 4T1 and MMTV-myc xenografts, we observed 100% disease-free survival and absence of macrometastasis in mice receiving minimally toxic adjuvant therapy with the C16-AM80 combination for 90 days. However, MRD was found consisting of a small number of single CK8+ cells which failed to form colonies when recovered from the bone marrow and selectively cultured in vitro. Upon stopping the treatments, 20-40% animals developed macrometastases within the first three months. To test the influence of C16-AM80 to condition the host microenvironment to prevent macrometastases mice received only pretreatment with C16 and AM80 followed by 4T1 cells injected in the tail vein. The percentage of parenchyma occupied by metastatic nodules were: DMSO = 50%; AM80 = 25%; C16 >10% and C16-AM = <<10%; the mitotic features were greatly reduce from 0-5 in DMSO to 0-1 per 400x/field in the C16-AM80 combination. The predominant effects were observed with C16 treatment, which was enhanced to a small degree in combination with AM80. These results suggest that C16 can potentially induce changes in the host microenvironment to prevent the colonization and metastatic growth of cancer cells in TNBC. Altogether, our preclinical studies merit expansion of a pre-clinical program for development of C16-AM80 combination as a targeted adjuvant therapy to treat TNBC. Citation Format: Nidhi Bansal, Eduardo Farias, Veronica Gonzalez, Garry Nolan, Samuel Waxman. Development of novel targeted adjuvant therapy for triple negative breast cancer [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 2083. doi:10.1158/1538-7445.AM2017-2083

Abstract 2676: Role of the RON kinase in the regulation of the antitumor immune response in pancreatic cancer

Abstract RON is a cell-surface receptor for the macrophage-stimulating protein expressed in tissue-resident macrophages and overexpressed in multiple epithelial neoplasms including pancreatic cancer. Our laboratory recently found that RON serves to mediate pancreatic duct carcinogenesis initiated by KRAS. We demonstrated that RON overexpression in the pancreas results in more rapid local and metastatic progression ultimately associated with decreased survival. We also discovered profound changes in the immunophenotype within the tumor microenvironment associated with RON expression. We notably established that RON expression drives the polarization of tumor-associated macrophages into an alternative, pro-tumorigenic state. We hypothesize that these unique activities help to shape the immunosuppressive milieu of the pancreatic cancer microenvironment and that RON directed therapy has the potential to serve as a novel immunomodulatory strategy in this disease. The aim of this work is to define how RON signaling contributes to shaping the pancreatic cancer microenvironment. In order to further interrogate the role of RON during pancreatic carcinogenesis, we bred kinase-dead RON mice (KD) to Pdx1-cre/LSL-KRASG12D mice. We observed a significant delay in the onset of pancreatic neoplasia and a significant decrease in tumor weight. The absence of functional RON kinase markedly reduced trafficking of CD11b+ cells to the pancreas and immunophenotyping in tumors showed a clear reduction of alternatively polarized macrophages when compared to RON overexpression or KRAS mutation alone. Because RON is expressed in both epithelial and stromal cell populations, we further sought to dissect the relevance of RON function to tumor growth within these cellular compartments. We performed orthotopic injections of organoïd-3D cells derived from tumors of KPC mice (LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre). These cells were injected in wild type (WT) or RON KD mice. Preliminary results show that tumor growth is impaired in KD mice, suggesting that RON activity in the host microenvironment influence tumor growth. In order to identify all immune populations regulated by RON, we will conduct an exhaustive immunophenotyping of murine pancreatic tumors. A transcriptomic analysis will then be conducted on selected cell populations to better define the molecular mechanisms associated with the RON regulation. Finally, given the effect of RON signaling on macrophage polarization, we will also dissect the effects of RON signaling from bone marrow derived myeloid cells versus resident tissue macrophages. Preliminary results and future experiments will significantly narrow the gap in our understanding of how RON signaling contributes to shaping the pancreatic cancer chimera and therefore how RON-directed therapies could be most successfully utilized to the benefit of those afflicted with this most deadly disease Citation Format: Alex Cazes, Michele L. Babicky, Jeffery Chakedis, Divya Sood, Dawn Jaquish, Andrew M. Lowy. Role of the RON kinase in the regulation of the antitumor immune response in pancreatic cancer [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 2676. doi:10.1158/1538-7445.AM2017-2676

Abstract 4075: Effect of PEG chain length on antibody-drug conjugate tumor and tissue distribution in tumor bearing xenograft mice

Abstract Antibody-drug conjugates (ADCs) are a promising new therapeutic modality having demonstrated clinical efficacy in the treatment of cancer. While ADCs have better exposures than their constitutive chemotypes, it is important that they maintain the favorable pharmacokinetics of their antibody backbones. Previous work incorporating PEG into the drug-linker as a sidechain helped to mask the hydrophobicity of the chemotypes, resulting in improved ADC exposures in rats compared to ADCs with non-PEGylated drug-linkers. The current studies were aimed at understanding the effect of PEG chain length on ADC efficacy, tumor and tissue distribution in tumor bearing mice. Efficacy and biodistribution of radiolabeled ADCs with 2, 4, 8, 12 and 24 PEG units in the drug-linker as a side chain were investigated in SCID mice bearing a subcutaneous xenograft of L540cy tumor cells. A non-PEGylated ADC was used as control. Antibody based radioactivity in plasma, tumor, liver, lung, kidney, spleen and brain were measured over 14 days. MMAE, the free drug released from the ADC, was measured by mass spectrometry. Reduction in tumor weight was determined as a measure of efficacy. Compartmental analysis of the plasma exposure data was conducted to understand the change in rate constants with change in PEG chain length. ADCs with side chain PEGylated drug-linkers had greater plasma and tumor exposures than the non-PEGylated control ADC. Increasing PEG chain length in the linker led to increased plasma and tumor exposures, and lower plasma clearances. Tumor distribution was binary with ADCs with 2 and 4 PEG units in the linker showing similar tumor exposures while the ADCs with 8, 12 and 24 PEG units providing similar but significantly higher tumor exposures. ADCs with 8, 12 and 24 PEG units in the linker had significantly higher tumor to plasma exposure ratios than ADCs with 2 and 4-PEG units in the drug-linker. The reduction in tumor weights was also binary. ADCs with 2 and 4 PEG units provided a 35-45% decrease in tumor weights while ADCs with 8, 12 and 24 PEG units in the linker provided 75-85% reduction in tumor weights. The non-PEGylated control ADC decreased the tumor weights by 11%. In general, PEGylated ADCs increased the extent of tissue distribution, but the tissue to plasma ratios remained the same indicating that the increased tissue exposures was a function of increased plasma exposures. However, PEGylated ADC tumor exposure increased beyond that expected based solely on the increase in plasma exposure. Incorporation of PEG as a side chain into the drug-linker improved the tumor disposition of ADCs with little alteration in tissue disposition. ADCs with 8, 12 and 24 PEG units in the drug- linker showed preferential uptake into tumor, but not the tissues as evidenced by tumor (or tissue) to plasma exposure ratios. Over all, the ADCs with 8, 12 and 24 PEG units in the linker provided the optimal combination of tumor distribution, efficacy and tissue distribution. Citation Format: Nagendra V. Chemuturi, Martha Anderson, Julia Cochran, Thomas Pires, Josh Hunter, David Meyer, Jason Neale, Alice Chin, Paul Pittman, Cindy Balasubramanian, Francisco Zapata, Jessica Simmons, Robert Lyon, Steve Alley. Effect of PEG chain length on antibody-drug conjugate tumor and tissue distribution in tumor bearing xenograft mice [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 4075. doi:10.1158/1538-7445.AM2017-4075

EVALUATION OF IN VIVO ANTICANCER AND IMMUNOSTIMULATORY ACTIVITY OF FLOWERS OF MIMOSA PUDICA LINN. (FABACEAE)

Objective: To investigate the in vivo anticancer and immunostimulatory activity of dichloromethane (DCM) extract of flowers of Mimosa pudica and its isolated compound 11β hydroxy-3 methoxy 1,2 dehydro crinane.Methods: The anticancer activity was performed on Ehrlich ascites carcinoma (EAC) cell line in Swiss albino mice. The activity was assessed by evaluating tumor volume, viable and nonviable tumor cell count, tumor weight, hematological, and histopathological parameters of EAC-bearing animals. The immunostimulatory activity was performed through carbon clearance, delayed type hypersensitivity (DTH), neutrophil adhesion and humoral antibody (HA) titer methods.Results: At the dose of 500 and 1000 mg/kg/day p.o for the extracts and 2.5 mg/kg/day p.o. for the isolated compound, significantly decrease the tumor volume (3.46±0.135 ml, 2.25±0.153 ml, and 1.84±0.012), increased the life span (59.32%, 76.39%, and 82.43%) and significantly (p<0.05) decreased tumor weight as compared with control. Hematological profiles were found to be nearly normal level in extract treated mice compared with tumor bearing control mice. The immunostimulatory activity was also found to be effective in the above dosage regimen. The results revealed that animals treated with above doses show a significant increase in the rate of carbon clearance from blood, increase in HA titer value, increase in neutrophil adhesion and significant (p<0.05) increase in mean paw edema in DTH reactions in dose-dependent manner.Conclusion: The results demonstrated that the extract is possessing dose-dependent anticancer activity and immunostimulatory activity attributed to the presence of crinane alkaloid.

Abstract 231: The polyphenolic plant lignan secoisolariciresinol diglycoside reduces mammary tumor growth, possibly via inhibition of local inflammatory signaling

Abstract Background: Secoisolariciresinol diglycoside (SDG) is a polyphenolic plant lignan found in flax and sesame seeds as well as legumes, whole grains, fruits and vegetables. It is metabolized by the gut bacteria into two major enterolignans: enterolactone (ENL) and enterodiol (END). These enterolignans have been associated with reduced breast cancer risk and progression in population studies as well as decreased tumor growth in preclinical models of breast cancer. Methods: The impact of SDG supplementation on tumor growth in a mouse model of basal-like breast cancer was examined. C57BL/6 mice were fed a control diet (10% kcal from fat) or control diet with SDG supplementation (100 mg/kg food) for eight weeks, then both groups were orthotopically injected with E0771 mammary tumor cells. An inflammatory signaling qPCR array (Qiagen) was performed on mammary tissue distal to tumor. Tumors were stained by immunohistochemistry (IHC) for Ki67 to measure proliferation levels and phospho-p65 to determine inflammatory signaling pathway activation. Tumors and mammary tissue were also stained for F4/80 to quantify macrophage infiltration. Serum level of hormones, adipokines, and cytokines were measured via luminex assay (Bio-Rad). Results: SDG supplementation significantly decreased tumor weight (p<0.05). SDG did not affect body weight or body fat percentage but did significantly decrease expression of F4/80, CRP, and other pro-inflammatory markers in the mammary tissue. IHC staining revealed no difference in tumor proliferation; however, SDG supplementation did reduce inflammatory signaling in the tumors, indicated by a significant decrease in phospho-p65 staining. However, serum cytokine levels were not significantly different between the groups. Tumors are currently being stained by IHC for cleaved caspase 3 to measure levels of apoptosis. In addition, cell culture experiments will be conducted to define the impact of ENL treatment on protumorigenic cross-talk between tumor cells, adipocytes, and macrophages. Specifically, the effects of conditioned media from adipocyte/macrophage co-cultures (with +/- ENL treatment) on mouse E0771 and human MDA-MB-231 tumor cell proliferation, migration and invasion will be examined. Conclusions: SDG supplementation reduced mammary tumor growth in association with SDG’s effects on local, but not systemic, inflammatory signaling. Citation Format: Claire G. Lineberger, Laura W. Bowers, Nikki A. Ford, Emily L. Rossi, Bruce K. Kimler, Carol J. Fabian, Stephen D. Hursting. The polyphenolic plant lignan secoisolariciresinol diglycoside reduces mammary tumor growth, possibly via inhibition of local inflammatory signaling [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 231. doi:10.1158/1538-7445.AM2017-231

Abstract 3229: Drug repurposing of topiramate in obesity-driven endometrial cancer

Abstract Introduction: Topiramate (TPM) is a B-D-fructopyranose sulfamate that acts by inhibiting carbonic anhydrases (CAs) and has been implicated as a novel inhibitor of angiogenesis. TPM is commonly used for the treatment of epilepsy and migraine headaches; however, when combined with phentermine, TPM can induce weight loss. Due to its weight loss and anti-angiogenic properties, we assessed TPM’s potential as an anti-tumorigenic agent in endometrial cancer (EC), a highly obesity-driven disease. Methods: Cell proliferation was assessed by MTT assay after exposure to TPM for 72 hours in the HEC-1A, KLE, Ishikawa and ECC-1 EC cell lines. Two representative cell lines, ECC-1 and Ishikawa, were used to analyze apoptosis, cell cycle progression, cell adhesion and invasion. Apoptosis was analyzed by Annexin V-FITC assay. Inhibition of adhesion and invasion by TPM were assessed by ELISA and transwell assay, respectively. Cell cycle progression was evaluated by Cellometer. Western immunoblotting was performed to assess downstream targets of the MAPK and mTOR pathways. The LKB1fl/flp53fl/fl EC mouse model was utilized to assess the in vivo effects of TPM. AdCre was injected at six weeks of age to induce invasive EC. Eight wks following AdCre injection, mice (N=10 per group) were treated with placebo or TPM (200 mg/kg/day, oral) for four weeks. The expression of phosphorylated-S6 and Ki-67 was assessed by IHC. Results: TPM inhibited cell proliferation in a dose dependent manner in all four EC cell lines (IC50 range=1500-3000 mM). Treatment with TPM resulted in G1 arrest and induction of apoptosis (p<0.05). TPM also reduced adhesion and invasion in the EC cell lines (p=0.05), with a corresponding decrease in VEGF expression. In addition, TPM significantly inhibited phosphorylation of p42/44 and S6 in the EC cell lines. Lastly, TPM decreased tumor weight in the LKB1fl/flp53fl/fl mice by 68% compared to those treated with placebo (p<0.05), accompanied by a reduction in Ki-67 and phosphorylated-S6 expression (p<0.05). Conclusion: TPM inhibited cell proliferation and tumor growth in EC cell lines and an EC mouse model. Therefore, TPM may be worthy of drug repurposing as an anti-tumorigenic agent in EC, with the potential added benefit of weight loss in this obesity-linked disease. Citation Format: Arthur-Quan Tran, Stephanie A. Sullivan, Chunxiao Zhou, David Kaufman, Victoria Bae-Jump. Drug repurposing of topiramate in obesity-driven endometrial cancer [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 3229. doi:10.1158/1538-7445.AM2017-3229

Modulation of the anti-tumor activity of VEGF blockade by metformin.

e23009 Background: The payoff from first-generation angiogenesis inhibitors was commonly temporary, due to the development of intrinsic as well as acquired resistance. Apart from the vascular resistance due to circumventing VEGF blockade by proangiogenic factors, resistance has also been associated with assortment of resistant clones. Accordingly, a combination strategy was proposed to overcome such resistance. Metformin, as a potential candidate, has received considerable concern as an anticancer agent. Methods: IN-VITRO WORK;Cells were divided into normoxic and hypoxic groups. For both, four subgroups were developed; control, metformin, glucose deprivation and a subgroup in which cells were subjected to both glucose deprivation and metformin treatment. Cells were then subjected to analysis for Apoptosis, Cell cycle progression and western blot. IN-VIVO WORK; Tumor growth was assessed IGROV-1 subcutaneous xenografts. Animal were divided into four groups; control, Metformin, Bevacizumab and a fourth group receiving both drugs. Tumor growth was assessed along treatments and at time of sacrifice.After sacrificing animals, tumors were further analyzed for; necrosis, apoptosis, proliferative capacity, activation of AMPK and its downstream effectors. Results: Our in-vitro results suggested an anti-proliferative effect of metformin in terms of apoptosis, cell cycle arrest, AMPK activation and mTOR inhibition at various time points. In-vivo experiments revealed a significant decrease of tumor weight and proliferative capacity in tumors treated with both drugs and a trend toward a decrease in tumor growth along the treatment. Yet, there was no difference between the bevacizumab and the combination groups regarding AMPK activation suggesting another mechanism to exert the combination effect. The effect of Metformin on CSC percentage and viability was tested. Glucose deprivation plus hypoxia lead to increase in CSC population, while adding metformin to bevacizumab reverse this augmentation in CSC numbers. Conclusions: Study represents a proof of concept study demonstrating a positive modulatory effect of metformin addition to bevacizumab. Additional experimental work on a wider scale is running to ascertain the current results.

MP73-01 IMMUNE CHECKPOINT INHIBITOR COMBINED WITH THE GROWTH INHIBITOR LYCORINE SYNERGISTICALLY FUNCTION TO MEDIATE ANTI-TUMOR EFFECTS IN A MOUSE MODEL OF RENAL CELL CARCINOMA

You have accessJournal of UrologyKidney Cancer: Basic Research & Pathophysiology III1 Apr 2017MP73-01 IMMUNE CHECKPOINT INHIBITOR COMBINED WITH THE GROWTH INHIBITOR LYCORINE SYNERGISTICALLY FUNCTION TO MEDIATE ANTI-TUMOR EFFECTS IN A MOUSE MODEL OF RENAL CELL CARCINOMA Xiezhao Li, Naijin Xu, Aibai Xu, Masami Watanabe, Peng Huang, Yasutomo Nasu, and Chunxiao Liu Xiezhao LiXiezhao Li More articles by this author , Naijin XuNaijin Xu More articles by this author , Aibai XuAibai Xu More articles by this author , Masami WatanabeMasami Watanabe More articles by this author , Peng HuangPeng Huang More articles by this author , Yasutomo NasuYasutomo Nasu More articles by this author , and Chunxiao LiuChunxiao Liu More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.3346AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Immune checkpoint inhibitors-based therapy is rapidly developing into an effective treatment option for a surprising range of cancers. Despite the autoimmune or inflammatory immune-mediated adverse effects which have been seen, the responses and overall survival benefits exhibited thus far warrant further clinical development. Lycorine, an alkaloid extracted from plants in the Amaryllidaceae family, which showed a strong anticancer effect by inducing cell lysis of various carcinomas. This study investigated the effectiveness of a combination therapy of lycorine and anti-CTLA-4 in a mouse model of renal cell carcinoma (RCC). METHODS We investigated the anti-tumor efficacy of lycorine in various RCC cell lines including Caki-1, ACHN, KPK-1 and Renca through XTT proliferation, scratch motility, migration and invasion assays. We also discuss that mechanism underlying this anticancer potential with flow cytometry and western blot. Furthermore, luciferase-expressing Renca cells were implanted in the left kidney and the lung of BALB/c mice to develop a RCC metastatic mouse model for investigated the combination therapy effect of lycorine and anti-CTLA-4. RESULTS We confirmed the anticancer potential of lycorine in vitro as observed time-dependent inhibition in several RCC cell lines. Moreover, lycorine suppressed the migratory and invasive abilities of Renca cells. The possible mechanism underlying this anticancer potential was cell cycle profile arrest. Lycorine and anti-CTLA-4 additively decreased tumor weight, lung metastasis, and luciferin-stained tumor images. Importantly, these effects were dependent on significantly suppressing regulatory T-cells while upregulating effector T-cells. CONCLUSIONS Herein, as we know, it's the first report that lycorine in combination with anti-CTLA-4 inhibited orthotopic and metastatic tumors by downregulating Treg, which was accompanied with upregulation of effector T-cells. Our findings suggest and indicate lycorine as a potent candidate for treating RCC and will serve as an excellent aid for developing a better treatment strategy for the use of immune checkpoint inhibitors in RCC. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e963 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Xiezhao Li More articles by this author Naijin Xu More articles by this author Aibai Xu More articles by this author Masami Watanabe More articles by this author Peng Huang More articles by this author Yasutomo Nasu More articles by this author Chunxiao Liu More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Knockdown of KRT17 by siRNA induces antitumoral effects on gastric cancer cells.

Keratin 17 (KRT17) was shown to be an important molecular marker for predicting the carcinogenesis, progression, and prognosis of various cancer types. Our previous studies identified KRT17 as a possible biomarker for gastric cancer by gene microarray, with an elevated expression that occurred early during tumorigenesis and increased during tumor progression. Based on these findings, we aimed to investigate KRT17 biological functions in gastric adenocarcinoma and its possible use as a rational molecular target for anticancer therapy. We used RNA interference-mediated knockdown of KRT17 expression and analyzed the effects on cell proliferation, cell migration, and signal transduction in two gastric cell lines (AGS and NCI-N87) in vitro and on xenograft growth in vivo. The functional analysis of KRT17 knockdown cell lines showed a decreased cell proliferation (with 42.36%±3.2%) and migration ability (with 37.2%±6.2%) relative to scrambled siRNA control. The in vivo tumorigenicity on nude mice exhibited a significant decrease in tumor weight with 69.14% in xenografts obtained from AGS cells and 84.43% in xeno-NCI-N87 tumors. The analysis on KRT17 knockdown outcome on intracellular signaling identifies AKT/mTOR as the main affected pathway that sustains proliferation and survival, and also the AMPKα1/CREB pathway that was recently shown to induce organ protection and antiinflammatory response. Our results highlight KRT17 as a possible biomarker in gastric cancer promoting tumor growth, motility, and invasion, and suggest that KRT17 can be a valuable molecular target for development of anti-gastric cancer-specific therapies.

Synergistic effects of the immune checkpoint inhibitor CTLA-4 combined with the growth inhibitor lycorine in a mouse model of renal cell carcinoma.

Renal cell carcinoma (RCC) management has undergone a major transformation over the past decade; immune checkpoint inhibitors are currently undergoing clinical trials and show promising results. However, the effectiveness of immune checkpoint inhibitors in patients with metastatic RCC (mRCC) is still limited. Lycorine, an alkaloid extracted from plants of the Amaryllidaceae family, is touted as a potential anti-cancer drug because of its demonstrative growth inhibition capacity (induction of cell cycle arrest and inhibition of vasculogenic mimicry formation). Moreover, T cell checkpoint blockade therapy with antibodies targeting cytotoxic T-lymphocyte associated protein 4 (CTLA-4) has improved outcomes in cancer patients. However, the anti-tumor efficacy of combined lycorine and anti-CTLA-4 therapy remains unknown. Thus, we investigated a combination therapy of lycorine hydrochloride and anti-CTLA-4 using a murine RCC model. As a means of in vitro confirmation, we found that lycorine hydrochloride inhibited the viability of various RCC cell lines. Furthermore, luciferase-expressing Renca cells were implanted in the left kidney and the lung of BALB/c mice to develop a RCC metastatic mouse model. Lycorine hydrochloride and anti-CTLA-4 synergistically decreased tumor weight, lung metastasis, and luciferin-staining in tumor images. Importantly, the observed anti-tumor effects of this combination were dependent on significantly suppressing regulatory T cells while upregulating effector T cells; a decrease in regulatory T cells by 31.43% but an increase in effector T cells by 31.59% were observed in the combination group compared with those in the control group). We suggest that a combination of lycorine hydrochloride and anti-CTLA-4 is a viable therapeutic option for RCC patients.

High Glucose-Mediated STAT3 Activation in Endometrial Cancer Is Inhibited by Metformin: Therapeutic Implications for Endometrial Cancer.

ObjectivesSTAT3 is over-expressed in endometrial cancer, and diabetes is a risk factor for the development of type 1 endometrial cancer. We therefore investigated whether glucose concentrations influence STAT3 expression in type 1 endometrial cancer, and whether such STAT3 expression might be inhibited by metformin.MethodsIn Ishikawa (grade 1) endometrial cancer cells subjected to media with low, normal, or high concentrations of glucose, expression of STAT3 and its target proteins was evaluated by real-time quantitative PCR (qPCR). Ishikawa cells were treated with metformin and assessed with cell proliferation, survival, migration, and ubiquitin assays, as well as Western blot and qPCR. Expression of apoptosis proteins was evaluated with Western blot in Ishikawa cells transfected with a STAT3 overexpression plasmid and treated with metformin. A xenograft tumor model was used for studying the in vivo efficacy of metformin.ResultsExpression of STAT3 and its target proteins was increased in Ishikawa cells cultured in high glucose media. In vitro, metformin inhibited cell proliferation, survival and migration but induced apoptosis. Metformin reduced expression levels of pSTAT3 ser727, total STAT3, and its associated cell survival and anti-apoptotic proteins. Additionally, metformin treatment was associated with increased degradation of pSTAT3 ser727. No change in apoptotic protein expression was noticed with STAT3 overexpression in Ishikawa cells. In vivo, metformin treatment led to a decrease in tumor weight as well as reductions of STAT3, pSTAT3 ser727, its target proteins.ConclusionsThese results suggest that STAT3 expression in type 1 endometrial cancer is stimulated by a high glucose environment and inhibited by metformin.