Mechanism Of Antitumor Effect Research Articles

Hydroalcoholic extract of Taraxacum officinale induces apoptosis and autophagy in 4T1 breast cancer cells.

Triple-negative breast cancer (TNBC) is an aggressive and deadly breast cancer sub-type with limited therapeutic options. Dandelion (Taraxacum officinale) exhibiting extensive anti-cancer activity is reported to be effective against TNBC; however, its anti-tumor effect mechanisms have not been fully elucidated. The purpose of this study was to determine the anti-cancer activity of hydroalcoholic extract of dandelion (HADE) on 4T1 cells, and the mechanism of HADE-induced cell death. The effect of HADE on cell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and lactate dehydrogenase assays. Apoptotic cell death was monitored by flow cytometry. The DNA fragmentation was evaluated by Acridine orange/Ethidium bromide (AO/EB) staining. Nitric oxide (NO) level was detected using Griess assay. The effects of HADE on Atg-7, Beclin-1, Bcl2, Bax and p53 genes were investigated by real-time reverse transcription-polymerase chain reaction. The results showed that HADE inhibited cell growth and proliferation in a dose- and time-dependent manner. The HADE induced 4T1 breast cancer cell death via apoptosis and autophagy. The DNA fragmentation was improved as the concentration of HADE increased. The NO secretion was declined with increasing concentration of HADE. Gene expression analysis confirmed HADE-induced apoptosis and autophagy in cancer cells. The Bax, Bax/Bcl-2 ratio, p53, Beclin-1 and Atg-7 over-expression as well as Bcl-2 down-regulation were also evident in treated cancer cells.

Abstract 3551: Oncolytic adenovirus serotype 35-mediated activation of NK cells leads to efficient anti-tumor effects

Abstract Oncolytic adenoviruses (OAds) are getting much attention as cancer immunotherapy agents. Almost all OAds are based on human adenovirus serotype 5 (Ad5) (OAd5). However, OAd5 has several drawbacks which possibly attenuate the anti-tumor effects of OAd5. More than 80% of adults have neutralizing antibodies against Ad5. The expression of coxsackievirus-adenovirus receptor (CAR), which is the primary infection receptor of Ad5, often declines on malignant tumor cells. Previously, we have developed a novel OAd fully composed of human adenovirus serotype 35 (Ad35) (OAd35) (Ono et al., Mol.Ther.Oncolytics, 2021). Only 20% or fewer adults have neutralizing antibodies against Ad35. Ad35 recognizes CD46, which is highly expressed on malignant tumor cells, as an infection receptor. OAd35 showed efficient tumor cell lysis activities in many types of tumor cells; however, the mechanism of anti-tumor effects of OAd35 remains to be fully evaluated. In this study, we examined the mechanism of anti-tumor effects of OAd35. Although OAd35 showed efficient tumor growth suppression at levels comparable to OAd5 following intratumoral administration in nude mice bearing H1299 human lung cancer xenografts, the genome copy numbers of OAd35 in the tumor were about 500-folds lower than those of OAd5 at 18 days after administration and were almost background levels. These data suggest that other mechanisms different from infection of tumor cells with OAd35 were involved in the anti-tumor effects of OAd35. Since previous studies reported that Ad35 efficiently activated innate immunity in natural killer (NK) cells, we examined whether OAd35-mediated activation of NK cells contributed to the anti-tumor effects of OAd35. Intratumoral administration of OAd35 resulted in efficient infiltration and activation of NK cells in H1299 tumors. In contrast, OAd5 did not apparently induce the infiltration and activation of NK cells in the tumors. OAd35-mediated activation and infiltration of NK cells were also found in B16 tumor-bearing immunocompetent mice. Next, in order to examine the roles of NK cells in the anti-tumor effects of OAd35, NK cells were depleted by intravenous injection of anti-GM1 antibody in nude mice, followed by intratumoral administration of OAd35. The depletion of NK cells significantly attenuated the anti-tumor effects of OAd35 on H1299 tumors. Furthermore, pretreatment with anti-GM1 antibody significantly increased the genome copy numbers of OAd35 in the tumor, suggesting that activated NK cells removed the OAd35-infected tumor cells. This study demonstrated that OAd35 significantly activated NK cells following intratumoral administration and that activated NK cells efficiently removed the tumor cells, leading to efficient tumor growth suppression. Citation Format: Ryosuke Ono, Kosuke Takayama, Fuminori Sakurai, Hiroyuki Mizuguchi. Oncolytic adenovirus serotype 35-mediated activation of NK cells leads to efficient anti-tumor effects [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3551.

Advantages of combined photodynamic therapy in the treatment of oncological diseases.

When conducting combined therapy of malignant neoplasms, treatment methods with various mechanisms of antitumor effects are used, while an additive or even synergistic effect can be realized. Combination treatment regimens are aimed at increasing the efficiency and, above all, at the complete eradication of the tumor, which can be achieved either by suppressing the survival mechanisms in PDT-resistant tumor cells or by pre-attenuation of tumor cells so that they become more susceptible to subsequent PDT. Photodynamic therapy is an approved medical technology for the treatment of various malignant neoplasms, and several precancerous and non-cancer diseases. To date, numerous data have been published on the combined use of PDT with traditional and innovative methods of treatment. This review considers research in this area in recent years.

Downregulation of ATP1A1 Expression by Panax notoginseng (Burk.) F.H. Chen Saponins: A Potential Mechanism of Antitumor Effects in HepG2 Cells and In Vivo.

The Na+/K+-ATPase α1 subunit (ATP1A1) is a potential target for hepatic carcinoma (HCC) treatment, which plays a key role in Na+/K+ exchange, metabolism, signal transduction, etc. In vivo, we found that Panax notoginseng saponins (PNS) could inhibit tumor growth and significantly downregulate the expression and phosphorylation of ATP1A1/AKT/ERK in tumor-bearing mice. Our study aims to explore the potential effects of PNS on the regulation of ATP1A1 and the possible mechanisms of antitumor activity. The effects of PNS on HepG2 cell viability, migration, and apoptosis were examined in vitro. Fluorescence, Western blot, and RT-PCR analyses were used to examine the protein and gene expression. Further analysis was assessed with a Na+/K+-ATPase inhibitor (digitonin) and sorafenib in vitro. We found that the ATP1A1 expression was markedly higher in HepG2 cells than in L02 cells and PNS exhibited a dose-dependent effect on the expression of ATP1A and the regulation of AKT/ERK signaling pathways. Digitonin did not affect the expression of ATP1A1 but attenuated the effects of PNS on the regulation of ATP1A1/AKT/ERK signaling pathways and enhanced the antitumor effect of PNS by promoting nuclear fragmentation. Taken together, PNS inhibited the proliferation of HepG2 cells via downregulation of ATP1A1 and signal transduction. Our findings will aid a data basis for the clinical use of PNS.

Abstract 1935: Antitumor effect mechanism of statin in PCa cells focusing on autophagy

Abstract Antitumor effect mechanism of statin in prostate cancer cells focusing on autophagy Background:Statins have recently been studied for their proapoptotic and antimetastatic effects. However, the exact mechanisms of their anticancer actions remain unclear. We have studied the novel castration resistant prostate cancer (CRPC) treatment with statins. Using microarrays, we discovered up-regulation of ATG4B (Autophagy related 4 homolog B) in PC-3 cells after simvastatin treatment. In this study, we evaluated the effects of simvastatin focusing on androgen-independent prostate cancer cells. Methods:PC-3, LNCaP-LA, 22RV-1-LA (taht were derived from LNCaP and 22RV1 cells and cultured in 10% charcoal-stripped fetal bovine serum for 3 months), and DU145 human prostate cancer cell lines were used. Microarrays were performed using PC-3 untreated or administered with simvastatin (0 uM versus 5 uM). Proliferation, migration, and invasion were evaluated using MTS assay, Migration assay, and Invasion assay, respectively. The increases of autophagy were detected by western blot using microtubule associated protein 1 light chain 3 (LC-3) antibody and confirmed by a fluorescence microscope with staining autophagosomes by autophagy detection kit (abcam, Cambridge, UK). Results:Using microarrays, we discovered up-regulation of ATG4B in PC-3 cells after simvastatin treatment. Simvastatin inhibited the proliferation, migration, and invasion of PC-3, LNCaP-LA, 22RV1-LA and DU145 cells. The expression level of LC-3 was up-regulated by simvastatin in PC-3, LNCaP-LA, 22RV1-LA and DU145 cells. In PC-3 cells treated with statin, a significant increase in autophagosomes was confirmed by fluorescence microscopy. The expression of LC-3 and autophagosomes were increased in a concentration-dependent manner. Conclusions:It was found that statin administration enhances autophagy induction in a prostate cell line. We plan to study the combined effect with other drugs that induce autophagy. We would like to develop CRPC treatment by inducing autophagy cell death. Citation Format: Yoshiyuki Miyazawa, Daisuke Oka, Hiroshi Nakayama, Yoshitaka Sekine, Seiji Arai, Kazuhiro Suzuki. Antitumor effect mechanism of statin in PCa cells focusing on autophagy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1935.

Abstract 6123: An indole compound MA-35 attenuates tumorigenesis in an inflammation-induced colon cancer model

Abstract Backgrounds: In inflammatory bowel disease (IBD), chronic inflammation results in the development of colon cancer known as colitis-associated cancer (CAC), that is associated with tumor necrosis factor-α (TNF-α) signaling. In addition, intestinal fibrosis in IBD patients is a common clinical complication, which is promoted by transforming growth factor β1 (TGF-β1). In our previous study, mitochonic acid (MA-35), derivative of indole-3-acetic acid known as a plant hormone auxin, showed anti-TNF-α effect by inhibiting IκB kinase (IKK) phosphorylation and anti-TGF-β1 effect by inhibiting Smad3 phosphorylation in a renal fibrosis mouse model. Aim: To identify the possible mechanism of anti-tumor effects and anti-fibrotic effects of MA-35 using azoxymethane (AOM)/ dextran sulfate sodium (DSS)-induced CAC mouse model. Methods and Results: MA-35 was orally administered every day for 70 days in an AOM/DSS model with a feeding tube. There was no difference in weight loss between the AOM/DSS group and the AOM/DSS+MA-35 group, however the disease activity index score and the survival rate were improved by MA-35. MA-35 also blocked progression of the anemia and shortening of the colon induced by AOM/DSS. MA-35 surprisingly reduced the formation of tumors in the colon. In the microscopic evaluation, MA-35 reduced inflammation and fibrosis in area with dysplasia. Furthermore, the TNF-α mRNA level in the colon tended to be reduced and the interleukin 6, TGF-β1 and fibronectin 1 mRNA levels in the colon were significant reduced by MA-35. In western blotting, MA-35 inhibited IKK phosphorylation, and both NF-κB p65 phosphorylation and TNF-α as the downstream signals for p-IKK in human colorectal cancer cell line HT-29. Similarly, MA-35 inhibited Smad2/3 phosphorylation, and both fibronectin 1 and TGF-β1 as the downstream signals for p-Smad2/3 in HT-29. Conclusions: MA-35 inhibited the development of CAC by reducing inflammation and fibrosis in an AOM/DSS mouse model by inhibiting both TNF-α signaling and TGF-β1 signaling. MA-35 may be a potent therapeutic agent for inhibiting the development of CAC in IBD patients. Citation Format: Keigo Kanehara, Shinobu Ohnuma, Takeshi Naitoh, Michiaki Uuno, Takaaki Abe. An indole compound MA-35 attenuates tumorigenesis in an inflammation-induced colon cancer model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6123.

Induction of different cellular arrest and molecular responses in low EGFR expressing A549 and high EGFR expressing A431 tumor cells treated with various doses of 177Lu-Nimotuzumab

Introduction Radioimmunotherapy (RIT) is a major anti-cancer therapy in cancer management multimodalities. 177Lu-Nimotuzumab has been in the use for radioimmunotherapy of EGFR expressing tumors. This study focuses on understanding the differential cellular and molecular mechanisms of anti-tumor effects of 177Lu-Nimotuzumab on low EGFR expressing A549 and high EGFR expressing A431 tumor cells. Materials and methods Nimotuzumab labeled with 177Lu was characterized by SE-HPLC. Specificity of 177Lu-Nimotuzumab to EGFR expressed on A549 and A431 cells was confirmed by competitive assay using increasing amounts of unlabeled Nimotuzumab. Cellular responses of A549 (low EGFR) and A431 (high EGFR) in response to different doses of 177Lu-Nimotuzumab were determined by Viable count assay for cellular viability, cell-cycle analysis by DNA staining, apoptotic assay for cell death, and CFSE dilution assay for cellular proliferation capacity. The number of DNA DSBs formed was determined using γ-H2AX assay with PI staining. Transcription of genes involved in DNA damage response and repair (DRR) pathways was monitored by RT-qPCR. Results 177Lu-Nimotuzumab characterized by SE-HPLC exhibited a radiochemical purity of 99.1 ± 0.6%. Cell binding competition studies with 177Lu-Nimotuzumab showed specific binding of 34.3 ± 1.7% with A431 cells and 18.4 ± 1.9% with A549 cells which decreased when co-incubated with unlabeled Nimotuzumab. Cytotoxicity and DNA damage (DNA DSBs) increased with an increase in the dose of 177Lu-Nimotuzumab. A549 displayed G2/M arrest while A431 showed G1 arrest. Apoptotic death was determined to be one of the modes of death of arrested A549 and A431 cells which increases with the increase in the dose of 177Lu-Nimotuzumab. Loss of proliferation capacity was higher in A431 showed by CFSE staining at different doses of 177Lu-Nimotuzumab. Transcription profile of most DRR genes in A431 and A549 showed a decrease in the transcription at 4 h followed by recovery at 16 h post-treatment. The degree of transcription of most DRR genes was similar, irrespective of 177Lu-Nimotuzumab dose. Conclusion 177Lu-Nimotuzumab induces different cellular arrest and molecular responses in low EGFR expressing A549 and high EGFR expressing A431 tumor cells. This study would enable the development of integrative novel treatment strategies for radioimmunotherapy in low and high EGFR expressing tumors by 177Lu-Nimotuzumab with therapeutic gains.

Abstract A32: Combination panobinostat and olaparib treatment promotes DNA damage and antitumor immunity in ovarian cancer

Abstract Background and Objective: High-grade serous ovarian cancers (HGSOC) retaining functional homologous recombination (HR) have poorer clinical outcomes and show relative resistance to DNA-damaging poly ADP ribose polymerase inhibitors (PARPi). We have shown that epigenetic drugs such as histone deacetylase inhibitors (HDACi) sensitize ovarian cancer cells to PARPi, in part through reduction of HR efficiency. However, these studies were conducted in immunocompromised mouse models, and drug effects on immune cell populations could not be fully examined. In this study, we will identify novel mechanisms of antitumor effects of combined HDACi/PARPi treatment by transcriptome analysis, and test drug effects in an established immune competent mouse model of ovarian cancer. Methods: HR-proficient SKOV-3 ovarian cancer cells were treated with vehicle (CON), PARPi olaparib (OLA; 10μM), HDACi panobinostat (PANO; 50nM) or OLA/PANO for 12h, and RNA-seq analysis performed. Differentially expressed genes between treatment groups were detected by DESeq2, based on fold change >1.5 and the corrected P value (FDR) <0.05. Functional group analysis of differentially expressed genes was performed using WebGestalt, with enrichment p-values generated by the Fisher’s exact test with Benjamini/Hochberg correction. C57BL/6 mice with intraperitoneal ID8-luc tumors were treated with vehicle, PANO (2.5 mg/kg), OLA (100mg/kg) and OLA/PANO for 4 weeks, starting 30 days after tumor injection. Tumor burden was measured by bioluminescence imaging of the constitutive luc reporter, and harvested tumors assayed for IHC markers of proliferation, apoptosis and DNA damage, and for total (CD3) and cytotoxic (CD8) T cells. Differences in group means were evaluated by Student’s t-test (p<0.05 is significant). Results: Functional group analysis of RNA-seq data showed significant enrichment of genes involved in cell proliferation, apoptosis, and DNA damage and repair with OLA/PANO treatment compared to CON or OLA alone. For a panel of 37 HR pathway genes, OLA/PANO treatment significantly reduced expression of 20 genes (e.g., BRCA1, BRCA2, WEE1). Examples of other significantly enriched functional pathways were cytokine secretion, inflammatory response genes, T cell activation (e.g., CD274/PD-L1, TNFSF9) and immune cell recruitment (e.g., IL-8, CXCL3). Consistent with these RNA-seq data, combined OLA/PANO treatment significantly reduced ID8-luc tumor burden and cell growth, and increased apoptosis and DNA damage, compared to CON or either drug alone. Moreover, there was increased infiltration of CD8+ T cells into OLA/PANO tumors, indicative of enhanced antitumor immunity. Conclusions: We have confirmed mechanisms such as reduced HR and identified a novel role for T-cell recruitment for OLA/PANO treatment of ovarian tumors. These findings support expanding the use of PARPi in HR-proficient HGSOC through rational combinations with HDACi and have the potential to benefit a substantial number of women with this devastating disease. Citation Format: Andrew J. Wilson, Xiaozhuan Dai, Qi Liu, Scott Hiebert, Marta Crispens, Dineo Khabele. Combination panobinostat and olaparib treatment promotes DNA damage and antitumor immunity in ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A32.

Efficacy of OR21, a Novel Oral Demethylating Agent in Chronic Myeloid Leukemia

Chronic myeloid leukemia (CML) is a clonal hematopoietic stem cell disease induced by t(9;22)(q34;q11) translocation. The prognosis of patients with CML has dramatically improved since tyrosine kinase inhibitors (TKIs) were introduced, and recent studies show that approximately 40 to 50 % of CML patients achieved deep molecular response (DASISION and ENESTnd trial) within several years. However, therapeutic options for patients with CML who are resistant for TKIs are limited. Besides BCR-ABL kinase domain mutation, somatic mutations associated with epigenetic gene alteration (e.g. TET, DNMT3A) are reportedly involved in TKI resistance and disease progression. Thus, we investigated the efficacy of DNA demethylating agents in CML. Azacitidine (AZA) and decitabine (DAC), currently available as DNA demethylating agents, have low bioavailability for oral administration because they are easily degraded by cytidine deaminase. Then, we are developing a novel demethylating agent, OR21, with possible oral absorbability as a prodrug of DAC. In vivo analysis using cynomolgus monkeys, the area under the curve of DAC after intraduodenally administration of DAC (1.5mg/kg, 6.6µmol/kg) or OR21 (2.25mg/kg, 6.6µmol/kg) was 0.01 µM·h, 0.298 µM·h respectively, indicating OR21 have high oral absorbability. To assess the demethylating activity of OR21 for CML, we performed western blot analysis and bisulfite pyrosequencing assay to measure LINE1 methylation, using CML cell-lines (K562, BV173). OR21 decreased DNMT1 protein level as a result of demethylating and LINE1 methylation in K562 and BV173 comparable to DAC. Next, we performed cell growth inhibition and cell apoptosis assay after 72 hours exposure of OR21 to assess anti-tumor effect in vitro. OR21 inhibited cell growth comparable to DAC in CML cell-lines (K562, BV173, KCL22, MYL) in a dose-dependent manner. Notably, OR21 inhibited the cell growth and apoptosis against BV173 with an extremely low concentration (IC50; 5nM) than that of AZA (IC50; 122nM) via significant accumulation of reactive oxygen species. Whereas, OR21 weakly induced cell apoptosis against K562. OR21 induced G2/M phase cell-cycle arrest in CML cell-lines except for BV173 via pRb downregulation. These results indicated mechanisms of anti-tumor effect in OR21 were induction of cell apoptosis (BV173) or cell cycle arrest (K562, KCL22, MYL). Because of the different mechanism of action, we assessed whether OR21 and TKIs combination can enhance the anti-tumor effect of CML. We investigated the combination effects of OR21 with imatinib (IM) or dasatinib (DAS) in K562. Combination index values at IC80 calculated by Calucusyn software showed 0.642±0.129 (with IM), 1.182±0.2 (with DAS), respectively. Accordingly, OR21 combined with TKIs showed at least additive or synergistic effect for K562. TKI resistance, which can be associated with somatic mutations leading to epigenetic gene alteration or loss of function of p53, is an obstacle for molecular remission in patients with CML, thus we examined the effects of OR21 in TKI resistant cell lines or efficacy on p53 mutational status. OR21 inhibited the cell growth in IM-resistant cell-line MYL-R, a derivative of MYL, which had overexpression of Lyn, and Ba/F3 BCR-ABLT315I, which exogenously expressed Bcr-Abl (T315I), indicating OR21 could overcome TKI resistance in CML. OR21 or cytarabine did not enhance cell apoptosis against K562, MYL and KCL22 (p53 deficient or mutant cell lines) combination with nutlin-3a (MDM-2 inhibitor), while increased cell apoptosis was observed in BV173 (p53 wild type) treated with cytarabine and nutlin-3a, but not with OR21 and nutlin-3a. These results suggested the effects of OR21 did not depend on p53 mutational status. Finally, we used a mouse xenograft model to evaluate anti-tumor effect of OR21 in vivo. BALB/c Rag-2/JAK3 double-deficient (BRJ) mice were injected intravenously via tail vein with 5 ×106 BV173 cells. OR21 were administered at a dose of 2.7mg/kg (equivalent to DAC 1.0mg/kg in AUC) and PBS (vehicle) twice weekly. OR21 significantly prolonged survival in a xenograft mice model (median 35 days vs not reached, P<0.01). In conclusion, a novel orally available demethylating agent OR21 is effective for CML cells including TKI resistant clones. The efficacy and safety of OR21 for CML is expected to be verified by early-phase clinical trials. Disclosures Kamachi: Ohara Pharmaceutical Co.: Research Funding. Ureshino:OHARA Pharmaceutical Co.: Research Funding. Yoshida:OHARA Pharmaceutical Co., Ltd.: Research Funding. Kurahashi:Ohara Pharmaceutical Co.: Employment. Watanabe:Ohara Pharmaceutical Co.: Research Funding. Okada:Japan Agency for Medical Research and Development: Research Funding; Bristol-Myers Squibb: Research Funding. Kimura:Ohara Pharmaceutical Co.: Research Funding; Novartis: Honoraria, Research Funding.

Abstract 1025: Mechanism of antitumor effect of Sorafenib in anaplastic thyroid cancer cell lines

Abstract Introduction: Anaplastic thyroid cancer (ATC) is a rare refractory disease, found in 1-3% of thyroid cancer. Because of its highly malignant potential, patients often become lethal within 6 months, nevertheless for intensive multimodal therapies. Recent observations indicated the strong efficacy of multi-kinase inhibitor to control disease progression of ATC. Frequent BRAF mutation (40-60%) in tumors of ATC was reported in Japan, indicating BRAF gene alteration as possible molecular target. Moreover, the secretions of VEGF were often found in ATC cell lines, suggesting the important role of VEGF-mediated tumor neo-vascularization in the aggressive progression of ATC. We, thus, examined the antitumor effect of Sorafenib, a multi-kinase inhibitor demonstrating its effect by inhibiting both BRAF and VEGF signal. Materials & Methods: Human ATC cell lines having four different gene mutations were used (OCUT-4; BRAF mutation, OCUT-6, ACT-1; NRAS mutation, OCUT-2; BRAS and PI3KCA mutation). The effect of Sorafenib on cellular viability was evaluated by MTT assay. In order to investigate the mechanism of cellular damage by Sorafenib, cell cycle was evaluated by flowcytometry, and the signal transduction of RAF/MEK pathway was examined by Western blotting. The amount of VEGF secretion from cells was measured by ELISA. Expression of VEGF-receptor in cells was evaluated by Western blotting. Furthermore, the effects of Sorafenib on human vascular endothelial cells were investigated by recruiting HUVEC (Kurabo, Japan) stimulated with VEGF and conditioned medium of the cancer cells. Results: The effect of Sorafenib appears most strongly in OCUT-4 cell with BRAF mutation compared with other cell lines. However, the impairment of cellular viability was shown at relatively high concentration (>100 nM). OCUT-4 showed G1 phase arrest by Sorafenib exposure. The specific effect of BRAF inhibition was confirmed by the suppression of MEK and ERK phosphorylation in OCUT-4. The suppression was not observed in OCUT-6, a cell line with NRAS mutation. The paradoxical increase of MEK phosphorylation was found in this cell line, instead. Every cell line secreted different amount of VEGF, and the proliferation of HUVEC was stimulated by their conditioned mediums similarly as by VEGF. The stimulation of proliferation was suppressed both by anti-VEGF antibody and Sorafenib. Discussion: In the present study, we clearly demonstrated that Sorafenib inhibits growth of human vascular endothelium mediated by VEGF secretion from cancer cells, as well as direct cell cycle arrest on ATC cells. A high concentration of Sorafenib may be needed to show inhibition on the RAF/MEK pathway. Furthermore, the inhibition of the pathway was not effective when additional growth stimulatory pathway than RAF was activated as in case of OCUT-2, or RAS gene were mutated. However, in any case, cancer cell growth mediated by autocrine system was inhibited clearly by Sorafenib. Citation Format: SAE ISHIHARA. Mechanism of antitumor effect of Sorafenib in anaplastic thyroid cancer cell lines [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 1025.

Administration of High-Dose Vitamin C and Irinotecan Ameliorates Colorectal Cancer Induced by Azoxymethane and Dextran Sodium Sulfate in Mice.

High-dose vitamin C administration has been reported to exhibit antitumor effect in various mouse models of cancer. However, the underlying mechanism of antitumor effect against colorectal cancer remains to be elucidated. In this study, we investigated the antitumor effect of high-dose vitamin C in a mouse model of chronic inflammation-associated colorectal cancer induced by azoxymethane (AOM) and dextran sodium sulfate (DSS). After cancer induction, the mice were administered vitamin C and/or irinotecan. Because irinotecan is a key drug in colorectal cancer treatment, it was used for comparison in this study. We examined reactive oxygen species (ROS) and interleukin-6 (IL-6) levels in the plasma of mice, as well as collagen type I and caspase-1 expression and neutrophil and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL)-positive cell counts in the colon tissue. Vitamin C and/or irinotecan administration decreased the plasma level of ROS and IL-6 and increased the expression of collagen type I and caspase-1. Furthermore, it increased neutrophil and TUNEL-positive cell counts. The most significant changes in the parameters analyzed were observed when both vitamin C and irinotecan were administered.

Abstract 3511: The mechanism of antitumor effect of metformin forgemcitabine-resistant pancreatic adenocarcinoma

Abstract Metformin (MET) is the first-line treatment for type 2 diabetes mellitus. Several epidemiological studies have reported anti-cancer effects of MET, including against pancreatic ductal adenocarcinoma (PDAC), which mainly acts through induction of AMP activated protein kinase (AMPK). Gemcitabine (GEM) has become the standard chemotherapy for PDAC but tolerance to GEM has become a burdensome issue. We evaluated the anti-tumor effects of MET for GEM-resistant PDAC in a xenograft mouse model. For this in vivo study, BxG30 (the cell line for GEM-resistant PDCA) was implanted into both flanks of female BALB/c nude mice. Mice were divided into four groups: (i) control (no treatment); (ii) the GEM-treated group (100 mg/kg); (iii) the MET-treated group (600 mg/kg); and (iv) the combined treatment group (G+M). Mice were fed for 4 weeks. Estimated tumor volumes and body weights were measured each week. Treatments were initiated 2 weeks after implantation. MET was administrated orally once per day. GEM was given by intraperitoneal injection once per week. Compared with the control, the final tumor volumes were decreased significantly only in the G+M group. [SCW1] The treated control ratio (T/C%) was calculated: GEM, 80.2%; MET, 54.0%; G+M, 47.2%. The anti-tumor effect of GEM for BxG30 was clearly limited[SCW2] . MET group showed satisfactory anti-tumor effects, but T/C% was <50% only in the G+M group. This result revealed that combination therapy had excellent anti-tumor effects even for GEM-resistant PDAC. The phosphorylation of ribosomal proteins S6 and 4E-BP, important targets of the mammalian target of rapamycin (mTOR) signaling pathway, was surveyed by western blot analysis. Western blot analysis showed inhibition of S6 and 4E-BP phosphorylation by co-incubation with MET, but not with GEM. Hypoxia-inducible factor 1 (HIF-1) is the one of the most important target of mTOR signaling pathway influencing tumor cell to progress, thus the expression level of HIF-1 was evaluated by western blot analysis as well. The results showed significant inhibition of HIF-1 expression by MET treatment, but not by GEM incubated under hypoxia (95%N2, 5%O2). Then the production of VEGF was evaluated by ELISA under hypoxia. The result showed suppression of VEGF production by MET treatment, but not by GEM. Our data showed that MET develops a different anti-tumor effect from GEM by suppression of mTOR-HIF-1 signaling. These results are of great clinical interest and reveal the potential of another anti-tumor agent for treatment of PDAC. Citation Format: Keiichi Suzuki, Osamu Takeuchi, Yukio Suzuki. The mechanism of antitumor effect of metformin forgemcitabine-resistant pancreatic adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3511.

LOW-DOSE SYSTEMIC RADIOTHERAPY AS AN ELEMENT OF GENERAL ANTITUMOR EFFECT IN TREATMENT FOR MALIGNANT TUMORS

The mechanisms of antitumor effect of polychemotherapy and systemic radiation therapy in non-tumorocidal doses are considered in the article, the advantages and disadvantages of each of them are noted as well as the common points connecting them with each other. Equivalence of the therapeutic effect mediated by myelodepression is substantiated. Methods of systemic radiation therapy and technical solutions for their implementation are given. It is concluded that it is well tolerated and effective.

Mechanism of anti-tumor effect of dihydroartemisinin

Dihydroartemisinin (DHA), the major active metabolite of artemisinin, participates in tumor progression through the following ways: forming free radicals to induce cancer cells death dependent on iron, inducing apoptosis, inhibiting angiogenesis, tumor cells invasion and metastasis, modulating multidrug resistance, controlling intracellular Ca2+ concentration, regulating cell cycles, cell autophagy and the immune system and so on. Generally, it is considered to be a potential anti-tumor drug. Key words: Artemisinins; Neoplasms; Apoptosis; Cell proliferation; Dihydroartemisinin

Inhibition of Yuyihe Powder on Tumor Growth in Mice Models of Sarcoma 180

ObjectiveTo explore the antitumor effect of Yuyihe Powder (Yu Yi He San, YYHS) and its antitumor mechanism. MethodsAfter treatment, tumor weight, immune apparatus weight, the life span of transplanted animals, spleen lymphocyte proliferation assays, and IL-2 concentration in mouse serum were recorded or detected. ResultsYYHS showed strong antitumor ability. Compared with control group, mid-dose YYHS (1.0 g/kg) could inhibit the tumor growth, prolong the life span of S180-bearing mice to some extent, significantly increase the thymic and splenic indices of S180 mice, and strongly promote the secretion of IL-2 in blood; The inhibitory rate on tumor growth and life prolongation rate were 37.1% and 38.37%, respectively. ConclusionYYHS could not only significantly inhibit the growth of S180 cells, but also markedly prolong the survival time of S180 bearing mice. The mechanism of antitumor effect could obviously enhance immunologic function of the S180 bearing mice to inhibit the growth of S180 cells.

Aspirin inhibits the proliferation of canine mammary gland tumor cells in vitro and in vivo

Background: Aspirin (acetylsalicylic acid), a non-steroidal anti-inflammatory drug (NSAID), is widely used to manage pain, arthritis, and cardiovascular diseases. Recent studies have shown that Aspirin could inhibit the growth of various human cancer cells in vitro and in vivo . The beneficial impact of Aspirin on human breast cancer has not been confirmed, and the precise mechanism of anti-tumor effect of Aspirin is largely unknown. Moreover, there is no evidence for the anti-tumor effect of Aspirin on canine mammary gland tumors (CMGT) cells. In this study, we investigated the effects of Aspirin on CMGT cells and the underlying mechanisms Methods: Cell proliferation was determined by cell viability assay and clone formation assay. Changes of apoptosis after Aspirin treatment were determined by Annexin-V-FITC apoptosis assay using flow-cytometry analysis, western blot assay for PARP, Bcl-2 and BAX expression. Effects of Aspirin on cell cycle arrest regulation were determined by flow-cytometry analysis, western blot assay for cyclin D1, CDK4, E2F1 and p21 expression. CMGT xenograft model was used to validate the anti-tumor effects of Aspirin in vivo . Results: We found that Aspirin not only effectively suppressed the proliferation and colony formation of CMGT cells in vitro , but also inhibited the tumor growth in vivo . Aspirin selectively induce apoptosis of metastatic CMGT cells, and our investigation showed that Aspirin could inhibit cell cycle progression at the G0/G1 checkpoint leading to decreased viability of the cells. Conclusions: Our results demonstrate that Aspirin efficiently inhibits the proliferation of CMGT cells and suggest that Aspirin might be a potential chemotherapy drug for CMGT.

RESEARCH OF SUBCHRONIC TOXICITY IN RATS OF GLYCOSIDE DERIVATIVE OF INDOLOCARBAZOLE LCS-1208

Background. In N.N. Blokhin Russian Cancer Research Center has been developed injectable dosage form based on the substance of indolocarbazole derivative LCS-1208, studied its antitumor activity and mechanism of antitumor effect, which was the basis for the pre-clinical study of the drug toxicity. Objective. The study of subchronic toxicity of formulation of glycoside derivative of indolocarbazole - LCS-1208 after intraperitoneal application to rats. Materials and methods. Dosage form of study drug: lyophilisate for preparation of solution for injection of 9,0 mg in vial. The study was conducted on 40 healthy non inbred male rats, which were obtained from the breeding of the N.N. Blokhin Russian Cancer Research Center. It was studied the action of the drug on the peripheral blood, the functional state of the liver, kidney and gastrointestinal tract of animals. Results. The drug LCS-1208 by daily intraperitoneal administration to rats for 15 days in the 3 studied doses (total dose 200 mg/kg, 100 mg/kg and 50 mg/kg) did not cause death of animals, had no effect on the general condition of the animals, did not cause external manifestations of toxicity, did not change behavioral responses of animals and had no effect on the functioning of the studied organs. Conclusion. The results obtained by the study of the subchronic toxicity in rats were the basis for further preclinical toxicology study of the drug LCS-1208.

Abstract 3273: The efficiency of anti-tumor effect of metformin for gemcitabine-resistant pancreatic adenocarcinoma

Abstract Metformin (MET) is the first-line treatment for type 2 diabetes mellitus. Several epidemiological studies have revealed the anti-cancer effects of metformin (MET), including against pancreatic ductal carcinoma (PDC). Gemcitabine (GEM) has become the standard chemotherapy for PDC but tolerance to GEM becomes an important issue. We evaluated the anti-tumor effects of MET for GEM-resistant PDC in a xenograft mouse model. For this in vivo study, wild-type BxPC-3 was implanted into both flanks of female BALB/c nude mice. Mice were divided into four groups: (i) control (without any treatment); (ii) GEM-treated group (100 mg/kg); (iii) MET-treated group (600 mg/kg); and (iv) combined treatment group (G+M). Mice were fed for 4 weeks. Estimated tumor volumes and body weights were measured each week. Treatments were initiated 2 weeks after implantation. MET was administrated orally every day. GEM was given by intraperitoneal injection every week. In the final tumor volumes, the two groups treated with GEM had significantly reduced tumors compared with control, but there were no differences between control and MET groups. The anti-tumor effect of MET for BxG30 (the cell line for GEM-resistant PDC) was evaluated using the method described above. Tumor volumes were decreased significantly in GEM+MET groups compared with control. The treated control ratio (T/C%) was calculated: GEM, 80.2%; MET, 54.0%; GEM+MET, 47.2%. The anti-tumor effect of GEM for BxG30 was limited. The MET group showed satisfactory anti-tumor effects, but T/C% was <50% only in the GEM+MET group. This result revealed that combination therapy had excellent anti-tumor effects even for GEM-resistant PDC. Western blot analysis was performed to confirm mammalian target of rapamycin (mTOR) expression level was surely inhibited. The results revealed that the expression level of mTOR treated with MET was significantly decreased, but no in GEM group. The expression level of hypoxia-inducible factor 1 (HIF-1) was evaluated by western blot analysis also. The results showed significant inhibition of HIF-1 expression by MET treatment, but not by GEM, again. Then the expression of VEGF mRNA, downstream of HIF-1 signaling pathway, was evaluated by qRT-PCR. The result showed inhibition of VEGF mRNA expression by MET treatment. Our results showed that MET has a partial anti-tumor effect for PDC. Combination therapy has an excellent effect not only for wild-type PDC but also for GEM-resistant PDC. The mechanism of anti-tumor effect of MET seems to be, at least partly, the inhibition of HIF-1/mTOR signaling pathway. These data suggest that MET could be used to treat PDC. Citation Format: Keiichi Suzuki, Osamu Takeuchi, Yoshiyuki Ishii, Masayoshi Osaku. The efficiency of anti-tumor effect of metformin for gemcitabine-resistant pancreatic adenocarcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3273.

Features of the Antitumor Effect of Vaccinia Virus Lister Strain.

Oncolytic abilities of vaccinia virus (VACV) served as a basis for the development of various recombinants for treating cancer; however, “natural” oncolytic properties of the virus are not examined in detail. Our study was conducted to know how the genetically unmodified L-IVP strain of VACV produces its antitumor effect. Human A431 carcinoma xenografts in nude mice and murine Ehrlich carcinoma in C57Bl mice were used as targets for VACV, which was injected intratumorally. A set of virological methods, immunohistochemistry, light and electron microscopy was used in the study. We found that in mice bearing A431 carcinoma, the L-IVP strain was observed in visceral organs within two weeks, but rapidly disappeared from the blood. The L-IVP strain caused decrease of sizes in both tumors, however, in different ways. Direct cell destruction by replicating virus plays a main role in regression of A431 carcinoma xenografts, while in Ehrlich carcinoma, which poorly supported VACV replication, the virus induced decrease of mitoses by pushing tumor cells into S-phase of cell cycle. Our study showed that genetically unmodified VACV possesses at least two mechanisms of antitumor effect: direct destruction of tumor cells and suppression of mitoses in tumor cells.

Abstract IA11: Monoclonal antibodies and in situ vaccination.

Abstract Traditional therapies for lymphoma have targeted features of the tumor, itself, such as cell surface molecules and intracellular signaling pathways. Increasingly, we are gaining therapeutic approaches that target the host microenvironment, and especially the host immune system. An exciting challenge is to combine these therapies in ways that are at least additive, and hopefully synergistic. We will present recent examples that combine therapies that target both the tumor and the host. Small molecule inhibitors of survival pathways in lymphoma cells, such as B cell receptor signaling, also have effects on the immune system. A case is point is Ibrutinib, a covalent inhibitor of Bruton's tyrosine kinase (Btk), recently approved for the treatment of CLL and MCL and with significant clinical activity in other lymphoid malignancies. Ibrutinib inhibits other members of the Tec kinase family, such as Itk, an important signaling molecule in subsets of T cells. By doing so, it can sculpt the T cell immune response and direct it against the tumor. Recent preclinical experiments indicate that Ibrutinib can enhance the T cell immune response induced by in situ vaccination as well as other immunotherapeutic maneuvers. These results imply a greater role for Ibrutinib than currently envisioned, one where it may be employed as an immune enhancer in addition to a targeted anti-tumor agent. Abscopal tumor responses can be induced in patients with lymphoma by local in situ vaccination. CpG ologonucleotide, a ligand for a Toll-like receptor, is injected into one site of tumor and low dose radiation is directed to the same site (Brody et. al. J Clin Oncol. 2010;28(28):4324). In preclinical animal models this treatment generates an anti-tumor T cell immune response. Such T cell responses can be enhanced by additional intra-tumoral injections of antibodies against immune checkpoints, such as CTLA4 and OX40 (Marabelle et al. J Clin Invest. 2013;123(6):2447). These antibodies work by depleting the negative T regulatory cells (Tregs) in the local tumor microenvironment, unleasing T effector cells to attack tumor at distant sites. Very low doses of checkpoint antibodies can be used to avoid autoimmune adverse effects. Such combinations of low dose radiation with immune enhancing agents may change the way we employ radiotherapy in the future- to help trigger immune responses rather than to sterilize the local tumor site. The therapeutic power of antibodies can be enhanced by stimulating ADCC, a primary mechanism of anti-tumor effect. One way to accomplish this is to target CD137, an activation molecule that appears on host NK cells after they encounter tumor cells coated by an anti-tumor antibody. In preclinical models the combination of an anti-CD20 with an anti-CD137 antibody is impressively synergistic (Kohrt et. al. Blood. 2011,117(8):2423). CD137 is also expressed on activated T cells and therefore the antibody against CD137 might also enhance an underlying host anti tumor T cell immune response. Two clinical trials are now under way to test the effect of an antibody against CD137 as a single agent and in combination with rituximab in patients with relapsed/refractory lymphoma. Citation Format: Ronald Levy. Monoclonal antibodies and in situ vaccination. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr IA11.