5FU Treatment Research Articles

The Acute Effects of 5 Fluorouracil on Skeletal Muscle Resident and Infiltrating Immune Cells in Mice.

5 fluorouracil (5FU) has been a first-choice chemotherapy drug for several cancer types (e.g., colon, breast, head, and neck); however, its efficacy is diminished by patient acquired resistance and pervasive side effects. Leukopenia is a hallmark of 5FU; however, the impact of 5FU-induced leukopenia on healthy tissue is only becoming unearthed. Recently, skeletal muscle has been shown to be impacted by 5FU in clinical and preclinical settings and weakness and fatigue remain among the most consistent complaints in cancer patients undergoing chemotherapy. Monocytes, or more specifically macrophages, are the predominate immune cell in skeletal muscle which regulate turnover and homeostasis through removal of damaged or old materials as well as coordinate skeletal muscle repair and remodeling. Whether 5FU-induced leukopenia extends beyond circulation to impact resident and infiltrating skeletal muscle immune cells has not been examined. The purpose of the study was to examine the acute effects of 5FU on resident and infiltrating skeletal muscle monocytes and inflammatory mediators. Male C57BL/6 mice were given a physiologically translatable dose (35 mg/kg) of 5FU, or PBS, i.p. once daily for 5 days to recapitulate 1 dosing cycle. Our results demonstrate that 5FU reduced circulating leukocytes, erythrocytes, and thrombocytes while inducing significant body weight loss (>5%). Flow cytometry analysis of the skeletal muscle indicated a reduction in total CD45+ immune cells with a corresponding decrease in total CD45+CD11b+ monocytes. There was a strong relationship between circulating leukocytes and skeletal muscle CD45+ immune cells. Skeletal muscle Ly6cHigh activated monocytes and M1-like macrophages were reduced with 5FU treatment while total M2-like CD206+CD11c- macrophages were unchanged. Interestingly, 5FU reduced bone marrow CD45+ immune cells and CD45+CD11b+ monocytes. Our results demonstrate that 5FU induced body weight loss and decreased skeletal muscle CD45+ immune cells in association with a reduction in infiltrating Ly6cHigh monocytes. Interestingly, the loss of skeletal muscle immune cells occurred with bone marrow cell cycle arrest. Together our results highlight that skeletal muscle is sensitive to 5FU’s off-target effects which disrupts both circulating and skeletal muscle immune cells.

Evaluation of metronomic chemotherapy response using diffusion and dynamic contrast-enhanced MRI.

To investigate the feasibility of using diffusion MRI (dMRI) and dynamic contrast-enhanced (DCE) MRI to evaluate the treatment response of metronomic chemotherapy (MCT) in the 4T1 mammary tumor model of locally advanced breast cancer. Twelve Balb/c mice with metastatic breast cancer were divided into treated and untreated (control) groups. The treated group (n = 6) received five treatments of anti-metabolite agent 5-Fluorouracil (5FU) in the span of two weeks. dMRI and DCE-MRI were acquired for both treated and control groups before and after MCT. Immunohistochemically staining and measurements were performed after the post-MRI measurements for comparison. The control mice had significantly (p<0.005) larger tumors than the MCT treated mice. The DCE-MRI analysis showed a decrease in contrast enhancement for the control group, whereas the MCT mice had a more stable enhancement between the pre-chemo and post-chemo time points. This confirms the antiangiogenic effects of 5FU treatment. Comparing amplitude of enhancement revealed a significantly (p<0.05) higher enhancement in the MCT tumors than in the controls. Moreover, the MCT uptake rate was significantly (p<0.001) slower than the controls. dMRI analysis showed the MCT ADC values were significantly larger than the control group at the post-scan time point. dMRI and DCE-MRI can be used as potential biomarkers for assessing the treatment response of MCT. The MRI and pathology observations suggested that in addition to the cytotoxic effect of cell kills, the MCT with a cytotoxic drug, 5FU, induced changes in the tumor vasculature similar to the anti-angiogenic effect.

A Novel Nomenclature for Repeat Motifs in the Thymidylate Synthase Enhancer Region and Its Relevance for Pharmacogenetic Studies.

Inhibition of thymidylate synthase (TS) is the primary mode of action for 5-fluorouracil (5FU) chemotherapy. TS expression is modulated by a variable number of tandem repeats in the TS enhancer region (TSER) located upstream of the TS gene (TYMS). Variability in the TSER has been suggested to contribute to 5FU-induced adverse events. However, the precise genetic associations remain largely undefined due to high polymorphism and ambiguity in defining genotypes. To assess toxicity associations, we sequenced the TSER in 629 cancer patients treated with 5FU. Of the 13 alleles identified, few could be unambiguously named using current TSER-nomenclature. We devised a concise and unambiguous systematic naming approach for TSER-alleles that encompasses all known variants. After applying this comprehensive naming system to our data, we demonstrated that the number of upstream stimulatory factor (USF1-)binding sites in the TSER was significantly associated with gastrointestinal toxicity in 5FU treatment.

Inhibitory effect of Ubenimex combined with fluorouracil on multiple drug resistance and P-glycoprotein expression level in non-small lung cancer.

Tumour drug resistance is one of the most urgent issues faced by anti‐tumour therapies. P‐glycoprotein (P‐gp) has been reported to be correlated with drug resistance. In this study, we aimed to study the synergistic effect of fluorouracil (5FU) and Ubenimex (UBE) on drug resistance in lung cancer. In this study, the tumour inhibitory role of 5FU and UBE was assessed in nude mice bearing A549 or A549/ADR. Real‐time polymerase chain reaction, Western blot and immunohistochemical were performed to analyse the mRNA and protein expression of P‐gp. TUNEL assay was used to evaluate the apoptosis of A549/ADR cells under 5FU and UBE treatment. MTT assay was performed to calculate the IC50 value of 5FU and UBE in A549 or A549/ADR. Combined administration of 5FU and UBE significantly inhibited the tumour growth of multidrug‐resistant cell lines A549/ADR in nude mice by down‐regulating the mRNA and protein expression of P‐gp. The apoptosis of A549/ADR was remarkably elevated in nude mice treated with 5FU and UBE. The IC50 value of 5FU and UBE was dramatically declined in A549/ADR cells compared with that of 5FU or UBE alone. Combined treatment of 5FU and UBE remarkably enhanced the apoptosis of A549/ADR cells by enhancing the intracellular accumulation of the drugs. The results of this study demonstrated that UBE combined with fluorouracil attenuated multiple drug resistance and inhibited the expression of P‐gp in lung cancer.

Abstract 3130: Evaluation of TK1 protein changes using the AroCell TK 210 ELISA in an acute myeloid leukemia (AML) xenograft model during 5-fluoruracil treatment

Abstract Background Thymidine kinase 1 (TK1) is a cytosolic enzyme involved in DNA precursor synthesis and it is up-regulated in proliferating cells. Serum TK1 is an established biomarker for prognosis and therapy monitoring of malignancies. However, commercial TK1 enzyme activity assays have limitations for their use in xenograft models as they cross-react with TK1 activity from the host. The AroCell TK 210 ELISA is specific for human TK1 and offers the opportunity to measure the release of TK1 from human xenografts free from the background of TK1 release from the host (mouse). Here we studied the changes in serum TK1 protein levels using the AroCell TK 210 ELISA in a xenograft model during 5-fluorouracil (5FU) treatment of Acute Myeloid Leukemia (AML). Experimental Procedures Human MV-4-11 leukemic cells from ATCC were cultured with 95% O2 and 5% CO2 at 37°C and in IMD medium (Sigma) supplemented with 10% FBS (HI media) containing 4mmol/L L-glutamine, 5 ng/mL GM-CSF and 1% penicillin and streptomycin. The cells were harvested during exponential growth and injected subcutaneously into the left flank of athymic BALB/c nude mice 8-10 weeks old. The mice were divided into controls that received no therapy (n=8) and an active group receiving 5FU (n=8). The treatment was initiated when the average tumor volume reached 50-100 mm3. Tumor bearing mice were treated with PBS (control) or 5-FU (active group 10 mg/kg, once daily) for three weeks. Serum samples (70-80 µL) were collected before treatment initiation, during treatment (once weekly for 3 weeks) and a final termination. Serum samples were stored at -80°C and TK1 protein levels were determined with the AroCell TK 210 ELISA. All the animal experiments were performed at TheraIndx life sciences Pvt Ltd, India. Results The tumor volumes were measured post-implantation of cells every 3 days throughout the study. There was a significant difference (p&amp;lt;0.05) in the mean tumor volume between 5-FU treated (5-FU) (108.75± 36.3 mm3) and untreated controls (294.63± 50.17 mm3) at the end of treatment. There was a significant reduction in the tumor volume from 2nd week onwards in 5-FU treated mice up to termination (4th week) compared to control mice. TK1 protein concentrations increased significantly from the 2nd week onwards in 5-FU treated mice (0.42± 0.19 µg/L) in comparison to controls (0.1± 0.02 µg/L) in spite of the decrease in tumor volume in response to treatment. No cross-reactivity of TK 210 ELISA with mouse TK1 was observed. Conclusions This study is the first evaluation of TK 210 ELISA as a biomarker for monitoring therapy with anticancer drugs in an AML xenograft model. The changes in TK1 protein levels were significant enough to monitor the response of the xenograft to 5FU therapy in this animal model. Furthermore, the use of the AroCell TK 210 ELISA in xenograft models may enable more accurate measurement of the anti-cancer drug effects on cell proliferation and these results encourage further applications of TK 210 ELISA in drug development projects. Citation Format: Kiran Kumar Jagarlamudi, Ramesh Jayaraman, Sundaresh Babu, Martin Shaw, Staffan Eriksson. Evaluation of TK1 protein changes using the AroCell TK 210 ELISA in an acute myeloid leukemia (AML) xenograft model during 5-fluoruracil treatment [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 3130.

Single-Cell Transcriptome Analysis of Colon Cancer Cell Response to 5-Fluorouracil-Induced DNA Damage

DNA damage often induces heterogeneous cell-fate responses, such as cell-cycle arrest and apoptosis. Through single-cell RNA sequencing (scRNA-seq), we characterize the transcriptome response of cultured colon cancer cell lines to 5-fluorouracil (5FU)-induced DNA damage. After 5FU treatment, a single population of colon cancer cells adopts three distinct transcriptome phenotypes, which correspond to diversified cell-fate responses: apoptosis, cell-cycle checkpoint, and stress resistance. Although some genes are regulated uniformly across all groups of cells, many genes showed group-specific expression patterns mediating DNA damage responses specific to the corresponding cell fate. Some of these observations are reproduced at the protein level by flow cytometry and are replicated in cells treated with other 5FU-unrelated genotoxic drugs, camptothecin and etoposide. This work provides a resource for understanding heterogeneous DNA damage responses involving fractional killing and chemoresistance, which are among the major challenges in current cancer chemotherapy.

Multidrug resistance gene expression response to cisplatin and 5FU treatment in hepatoma, prostate and colon cancer cells

Variable expression of drug transporters at the plasma membrane of tumor cells contributed to drug resistance. The aim of the present study was to explore the expression levels of MRP2 and MRP3genes in Cisplatin and 5 fluorouracil (5FU) treated Hep3B, PC3 and HT29 cell lines. MTT assay was used to determine the cytotoxic effects of cisplatin and 5FU. The results from the MTT assay revealed that PC3 cell line is the most sensitive for cisplatin treatment presenting the lowest IC50 value (0.5 µg/ml) for 48h. 5FU treatment altered the proliferation of PC3 cells with the IC50 values 6.0 µg/ml for 48 h and 8.2 µg/ml for 72h. The mRNA expression levels of MRP2 and MRP3 were determined by quantitative reverse transcription polymerase chain reaction. qRT-PCR results revealed that while the expression levels of MRP3 mRNA was generally down regulated in all cell lines treated with cisplatin and 5FU, MRP2 was upregulated in PC3 and Hep3B cells.

Natural Anthraquinone Emodin May Protect Immune and Gastrointestinal Health During Chemotherapy Treatment

The clinical utility of 5‐fluorouracil (5FU) chemotherapy remains hampered by hematopoietic and gastrointestinal toxicities. Emodin, a natural anthraquinone, has been shown to have anti‐inflammatory potential and may be an effective complementary therapeutic. The purpose of this study was to investigate the ability of emodin to reduce non‐specific toxicities, especially inflammation, associated with 5FU treatment. We evaluated acute and chronic exposure to 5FU utilizing tumor‐free mice. In the chronic phase, we randomized male C57BL/6 mice into four groups; Control + Vehicle (n=10), Control + Emodin (n=10), 5FU + Vehicle (n=15), 5FU + Emodin (n=15). 5FU was administered via I.P. for 3 cycles (5 consecutive days of 5FU at 35mg/kg followed by 9 days of recovery). Emodin was administered via oral gavage 3x/week at 40mg/kg throughout the entire 3 cycles of 5FU treatment. 5FU + Emodin treated mice showed improved grip strength (p&lt;0.05), run‐to‐fatigue time (p&lt;0.05), and reduced sensitivity to peripheral nociception (p&lt;0.05) compared to 5FU + Vehicle mice. Gene expression of the small intestine showed increased TNFα expression in 5FU + Vehicle treated mice which was decreased (p&lt;0.05) with emodin treatment. 5FU + Emodin treated mice show decreased colonic NOS2 expression (p&lt;0.05) but did not decrease MCP‐1, IL‐6, and TNFα expression compared to 5FU + Vehicle treated mice. To investigate the acute benefits of Emodin, male and female mice (n=125/sex) were randomized into four groups; 5FU + Vehicle, 5FU + 20mg/kg, 5FU + 40mg/kg, 5FU + 80mg/kg. Emodin treatment prevented body weight loss (p&lt;0.05) during one cycle of 5FU and was able to prevent performance loss during run‐to‐fatigue (p&lt;0.05). In male mice only, we observed a maintenance of the hematopoietic system, specifically in circulating lymphocytes, with increased emodin dosage (p&lt;0.05). Complementary to this trend, we found that bone marrow cells of 5FU + Emodin treated mice had greater percentage of cells in S (p&lt;0.05) and G2 (p&lt;0.05) phases, compared to 5FU + Vehicle mice. Flow cytometric analysis of spleen, mesenteric lymph node, and colon lamina propria did not show differences in total macrophages or neutrophils. Gene expression analysis of the colon did not show significant differences between all groups. Chronic exposure to 5FU causes increased production of Muc5ac, an indication of dysplasia in the colon, which is ameliorated with complementary emodin. Despite this, we did not see decreased Muc5ac from emodin treatment acutely. However, utilizing 16S rRNA FISH, we can see that 5FU can cause pockets of mucus disruption, gut barrier damage, and alterations in bacterial spatial arrangement, all of which was mitigated with increased dosage of emodin treatment. Taken together, emodin treatment may contribute to improved GI health by reducing damaging inflammation which may improve overall quality of life. Future analysis will expand on the inflammatory signaling pathways involved in emodin signaling and will investigate the potential role of the gut microbiome in reducing GI inflammation in this model of 5FU treatment.Support or Funding Information(AT009964) (F31AT009820)

Casein Kinase 2α Enhances 5-Fluorouracil Resistance in Colorectal Cancer Cells by Inhibiting Endoplasmic Reticulum Stress.

Anti-cancer drug resistance restricts the efficacy of chemotherapy in malignant tumors. Casein kinase 2α (CK2α) is highly expressed in 5-fluorouracil (5FU)-resistant colorectal cancer (CRC) cells. We hypothesized that inhibition of CK2α might reduce CRC resistance to 5FU. To investigate the role of CK2α in 5FU-resistant CRC cells, we assessed cell viability, apoptosis, cyclin-dependent kinase 4 (CDK4) activity, cell-cycle progression, invasion, and sphere formation in 5FU-resistant CRC cells. CK2α levels were significantly increased in 5FU-resistant CRC cells compared to those in wild-type CRC cells. During exposure to 5FU, viability, CDK4 activity, cell-cycle progression, invasion, and sphere formation were enhanced, while apoptosis was decreased in 5FU-resistant CRC cells. These effects were mediated by the inhibiting effects of CK2α on endoplasmic reticulum (ER) stress. Combination of CK2α knockdown with 5FU treatment promoted apoptosis of 5FU-resistant CRC cells by inducing ER stress. 5FU treatment in combination with a CK2α inhibitor may exert a synergistic effect against drug-resistant cancer cells.

Prostaglandin E2 accelerated recovery of chemotherapy-induced intestinal damage by increasing expression of cyclin D

Intestinal stem cells (ISCs) play a crucial role in maintaining intestinal homeostasis upon chemotherapy and radiotherapy. It has been documented that prostaglandin E2 (PGE2) treatment improved hematopoietic stem cell function in vitro and in vivo, while the relationship between PGE2 and intestinal stem cells remains unclear. Presently, mice were exposed to PGE1, dmPGE2 and indomethacin. Numbers and function of ISCs were assessed by analyzing Olfm4+ ISCs. Intestinal protection of dmPGE2 was investigated on a 5-fluorouracil (5FU)-induced intestinal damage mouse model. The results showed that dmPGE2 treatment, but not PGE1, increased numbers of Olfm4+ ISCs in dose- and time-dependent manners. Indomethacin treatment decreased numbers of Olfm4+ ISCs. The beneficial effects of short-term dmPGE2 treatment on intestine were supported in a 5FU-induced intestinal damage model. Our data showed that 5FU treatment significantly decreased numbers of Olfm4+ ISCs and goblet cells in intestine, which could be ameliorated by dmPGE2 treatment. dmPGE2 treatment accelerated the recovery of 5FU-induced ISC injury via increasing expression of cyclin D1 and D2 in intestine. Furthermore, dmPGE2 treatment-induced expression of cyclin D1 and D2 might be mediated by up-regulation of FOXM1 expression in intestine. These findings feature PGE2 as an effective protector against chemotherapy-induced intestinal damage.

Isoprenaline protects intestinal stem cells from chemotherapy-induced damage.

Damage to intestinal epithelial cells and mucosa limits the effectiveness of several anti-cancer chemotherapeutic agents but the underlying mechanism (s) remain unknown. Little is known of how enteric nervous system regulates proliferation, differentiation, impairment, and regeneration of intestinal stem cells. Here we have investigated the effects of isoprenaline on the damaged intestinal stem cells induced by chemotherapeutic treatments in mice. The effects of inhibiting sympathetic and parasympathetic nerves on intestinal stem cells were examined in male C57BL/6J mice. Protection by isoprenaline of intestinal stem cells was assessed in the presence or absence of 5-fluorouracil (5FU) or cisplatin. Cellular apoptosis, cell cycle, PI3K/Akt signalling, and NF-κB signalling in intestinal stem cells were mechanistically evaluated. The sympathetic nerve inhibitor 6-OHDA decreased the number and function of intestinal stem cells. 5FU or cisplatin treatment damaged both intestinal stem cells and sympathetic nerves. Notably, isoprenaline accelerated the recovery of intestinal stem cells after 5FU or cisplatin treatment. This protective effect of isoprenaline on damaged intestinal stem cells was mediated by β2 -adrenoceptors. The benefits of isoprenaline were mainly mediated by inhibiting cellular apoptosis induced by 5FU treatment, which might contribute to fine-tuning regulating NF-κB signalling pathway by isoprenaline administration. Treatment with isoprenaline is a new approach to ameliorate the damage to intestinal stem cells following exposure to cancer chemotherapeutic agents.

Abstract 305: Combination analysis for the potential chemomodulatory effects of mansorin-A and its naphthoquinone derivative (mansonone-G) to 5-fluorouracil against liver cancer cells

Abstract Mansorin-A and its naphthoquinone derivative, mansonone-G are naturally occurring compounds isolated from the trunk of Mansonia gagei, family Sterculariaceae. In a previous work, they showed potential anticancer effects besides their antioxidant properties. Herein, we investigated their potential chemomodulatory effects to 5-fluorouracil (5FU) against two liver cancer cell lines (Huh7 and HepG2). Despite the significant cell killing effect of 5FU to Huh7 and HepG2 cells (IC50’s of 2.6±0.2 µM and 0.82 ± 0.2 µM, respectively), the resistance fractions of both cell lines were considerably high (53.2±1.7% and 39.4±2.7%, respectively). Mansorin-A and mansonone-G induced moderate cytotoxicity against both liver cancer cell lines with IC50’s ranging from 25.8±3.2 µM to 36.3±2.7 µM. In Huh7, mansorin-A and mansonone-G synergized cell killing effect of 5FU and decreased its IC50’s to 0.9±0.1 and 1.0±2.1 µM, respectively. On the other hand, mansorin-A and mansonone-G showed apparent antagonism by combination with 5FU (CI values of 6.2 and 9.4, respectively) against HepG2 cells. However, both compounds significantly abolished the resistant fraction of HepG2 to 5FU (3.3±0.3% and 8.4 ± 2.6%, respectively). Further investigation showed that mansonone-G alone showed antiproliferative effect and arrested cells in G0/G1-phase. Both mansorin-A and mansonone-G enhanced 5FU induced cell cycle arrest at G0/G1-phase with reciprocal decrease in cells entering S-phase and G2/M-phase. In addition, apoptosis due to 5FU treatment alone and in combination with mansorin-A and mansonone-G was assessed using annexin-V/FITC staining coupled with flowcytometry. Yet, both mansorin-A and mansonone-G enhanced 5FU induced apoptotic effect against liver cancer cells. Mansonone-G alone induced significant apoptosis in both HepG2 and Huh7 cells. Besides apoptosis, autophagy induction properties of mansorin-A and mansonone-G were assessed using flowcytometry after acridine orange staining. Mansonone-G aborted autophagy in both HepG2 and Huh7 cell lines and indirectly forced liver cancer cells to undergo apoptotic cell death. On the other hand, both mansorin-A and mansonone-G significantly induced the production of reactive oxygen species within both HepG2 and Huh-7 cells which might directly induce apoptosis in both cell lines. In conclusion, mansonone-G and to a lesser extent mansorin-A, potentiated the anticancer properties of 5FU against liver cancer cells via potentiating its cell cycle arrest and potentiating its apoptotic influence due to ROS production. Citation Format: Hanadi G. Aljohani, Gehan A. Hegazy, Ali M. El-Halawany, Aliaa A. Alamoudi, Ghada M.A. Agabnoor, Ahmed M. Al-Abd. Combination analysis for the potential chemomodulatory effects of mansorin-A and its naphthoquinone derivative (mansonone-G) to 5-fluorouracil against liver cancer cells [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 305.

Emodin, a natural anthraquinone, may help protect gastrointestinal health during chemotherapy treatment by decreasing inflammation of the gastric mucosa and preserving gut morphology

The clinical utility of 5‐fluorouracil (5FU) chemotherapy remains hampered by hematopoietic and gastrointestinal toxicities. Emodin, a natural anthraquinone, has been shown to have anti‐inflammatory potential and may be effective as a complementary therapeutic used during chemotherapy treatments. The purpose of this study was to evaluate the efficacy of emodin on reducing the non‐specific toxicities associated with 5FU treatment. 50 C57BL/6 were randomized into four groups; Control + Vehicle (n=5), Control + Emodin (n=5), 5FU + Vehicle (n=20), 5FU + Emodin (n=20). 5FU was administered via I.P. over 3 cycles of the following regime: 5 consecutive days of 5FU at 35mg/kg followed by 9 days of recovery. Emodin was administered via oral gavage 3x/week at 40mg/kg through the entire 3 cycles of 5FU treatment. 5FU + Emodin treated mice showed improved muscular strength (p&lt;0.05), fun‐to‐fatigue time (P&lt;0.05), and peripheral nociception (p&lt;0.05) compared to 5FU + Vehicle treated mice. 5FU + Emodin treated mice did not show recovery of decreased circulating immune cells and red blood cells that is common with 5FU treatment. 5FU + Vehicle treated mice did show slightly increased splenic CD11b+ F4/80+ macrophages (6.8%) vs. control mice (4.3%), a phenotype that was not decreased in 5FU + Emodin treated mice (6.5%). 5FU treatment shows classic inflammatory damage to the gastric mucosa. 5FU + Vehicle treated mice show decreased villus length (p&lt;0.05) compared to control mice. 5FU + Emodin treated mice showed increased villus length (p&lt;0.05) compared to 5FU + Vehicle treated mice but remained shorter (p&lt;0.05) than control mice. Further, small intestine tissue shows increased TNFα expression in 5FU + Vehicle treated mice which was decreased (p&lt;0.05) in 5FU + Emodin treated mice. Similar increases in inflammatory gene expression are seen in the colon tissue of 5FU + Vehicle treated mice. 5FU + Emodin treated mice show decreased colonic TNFα expression (p&lt;0.05) and trending decrease in MCP‐1 (p=0.052) and NOS2 (p=0.065) expression compared to 5FU + Vehicle treated mice. Taken together, Emodin treatment may contribute to improved GI health by reducing damaging inflammation which may contribute to improved overall quality of life. Future analysis will expand on the inflammatory signaling pathways involved in emodin signaling and will investigate the potential role of the gut microbiome in reducing GI inflammation in this model of 5FU treatment.Support or Funding Information1F31AT009820 to ATS.1RO1CA218578 to EAM. 1R41AT009964 to EAM. 5R21CA191966 to EAM.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Impact of 5 fluorouracil chemotherapy on gut inflammation, functional parameters, and gut microbiota

Emerging evidence suggests that gut microbiota may influence the response to chemotherapy. We sought to characterize the effects of 5 fluorouracil (5FU) chemotherapy on colon inflammation and functional measures in colorectal cancer (CRC) and to further determine whether gut microbiota can influence this response. 50 C57BL/6 were randomized into four groups; Control + Vehicle (n = 10), Control + 5FU (n = 10), AOM/DSS + Vehicle (n = 15), and AOM/DSS + 5FU (n = 15). CRC was induced chemically by a single 10 mg/kg injection of azoxymethane (AOM) followed by two cycles (2% and 1%) of dextran sodium sulfate (DSS). Mice were then treated with 3 cycles of vehicle or 5FU (cycle 1: 40 mg/kg, cycle 2 + 3: 20 mg/kg). Functional tests (grip strength and run-to-fatigue) were performed prior to 5FU treatment (baseline) and at the completion of the second cycle of 5FU. Following the third 5FU cycle, mice were euthanized and the colon was evaluated for expression of inflammatory genes using RT-qPCR and stool samples were profiled using 16S rRNA sequencing. A second experiment used fecal microbiota transplantation from 5FU treated mice to control mice (n = 10–15/group) to determine whether 5FU associated changes in the microbiota could influence functional measures and colon inflammation. 5FU reduced grip strength (p < 0.05) and caused a trending decrease in run-to-fatigue performance in cancer mice (p = 0.06). Select intestinal inflammatory genes were significantly elevated with 5FU treatment and this was further exacerbated with cancer (p < 0.05). Microbiota analysis revealed increased dissimilarity and alterations in bacterial taxonomy in 5FU and AOM/DSS-treated mice (p < 0.05). Fecal transplant from 5FU treated mice reduced functional performance (p < 0.05) and altered select colon inflammatory markers (p < 0.05). This study provides evidence of an effect of 5FU on inflammatory responses and functional measures in a mouse model of CRC and suggests that gut microbes may play a role in some, but not all, 5FU related perturbations.

Multidose 5-Fluorouracil is Highly Toxic to Growing Ovarian Follicles in Mice.

With increasing improvements in cancer survival rates, it is critical to reduce the significant long-term side effects that afflict patients following treatment. For women, consequences of chemotherapy-induced damage to the reproductive system include infertility and premature menopause, which adversely effects cognition, mood, cardiovascular, bone, and sexual health, and increases the risk of early mortality. These long-term effects impact patient's life quality and highlight a significant and on-going burden on the health system after treatment. However, the precise mechanisms through which chemotherapeutic agents induce ovarian damage and primordial follicle depletion remain to be characterized. Hence, preventing the development of effective pharmacological methods to preserve fertility and improve quality of life after treatment. The chemotherapeutic agent 5-Fluorouracil (5FU) is not deemed cytotoxic to the ovary, however, risks to long-term fertility after multiple doses are not known. Therefore, we sought to evaluate the impact of 3, weekly doses of 5FU treatment on the ovary. Using a mouse model enabled accurate histomorphometric analysis of follicle numbers and ovarian structure and function, to accurately assess cumulative impact of 5FU on the ovary. This study clearly demonstrated that multidose 5FU treatment resulted in dramatic and progressive atresia of growing follicles and a profound decrease in ovarian volume due to reduced corpus luteum counts. However, primordial follicle numbers were unaffected. Thus, 5FU is unlikely to cause permanent infertility when administered to women of pre or reproductive age. Furthermore, this study suggests that depletion of the growing follicle population is insufficient to stimulate follicle activation and primordial follicle depletion.

MASTL induces Colon Cancer progression and Chemoresistance by promoting Wnt/\u03b2-catenin signaling

BackgroundChemotherapeutic agents that modulate cell cycle checkpoints and/or tumor-specific pathways have shown immense promise in preclinical and clinical studies aimed at anti-cancer therapy. MASTL (Greatwall in Xenopus and Drosophila), a serine/threonine kinase controls the final G2/M checkpoint and prevents premature entry of cells into mitosis. Recent studies suggest that MASTL expression is highly upregulated in cancer and confers resistance against chemotherapy. However, the role and mechanism/s of MASTL mediated regulation of tumorigenesis remains poorly understood.MethodsWe utilized a large patient cohort and mouse models of colon cancer as well as colon cancer cells to determine the role of Mastl and associated mechanism in colon cancer.ResultsHere, we show that MASTL expression increases in colon cancer across all cancer stages compared with normal colon tissue (P < 0.001). Also, increased levels of MASTL associated with high-risk of the disease and poor prognosis. Further, the shRNA silencing of MASTL expression in colon cancer cells induced cell cycle arrest and apoptosis in vitro and inhibited xenograft-tumor growth in vivo. Mechanistic analysis revealed that MASTL expression facilitates colon cancer progression by promoting the β-catenin/Wnt signaling, the key signaling pathway implicated in colon carcinogenesis, and up-regulating anti-apoptotic proteins, Bcl-xL and Survivin. Further studies where colorectal cancer (CRC) cells were subjected to 5-fluorouracil (5FU) treatment revealed a sharp increase in MASTL expression upon chemotherapy, along with increases in Bcl-xL and Survivin expression. Most notably, inhibition of MASTL in these cells induced chemosensitivity to 5FU with downregulation of Survivin and Bcl-xL expression.ConclusionOverall, our data shed light on the heretofore-undescribed mechanistic role of MASTL in key oncogenic signaling pathway/s to regulate colon cancer progression and chemo-resistance that would tremendously help to overcome drug resistance in colon cancer treatment.

Abstract LB-091: Hedgehog signaling induces drug resistance in gastric cancer cells via upregulation of ABCG2

Abstract Gastric cancer is the third leading cause of cancer-related mortality worldwide. Currently, there are no specific therapeutic options to cure gastric cancer, and chemotherapy is frequently used. Some patients respond initially to chemotherapy but all patients eventually succumb to the disease. Thus, identifying molecular mechanisms responsible for chemotherapy resistance may help design novel strategies to sensitize cancer cells to chemotherapy. In this study, we revealed that treatment with chemotherapeutic reagents CDDP and 5Fu induced expression of GLI1, GLI2 and PTCH1 in gastric cancer cell lines, suggestive of hedgehog (Hh) signaling activation. GLI1 knockdown sensitized gastric cancer cells to CDDP and 5Fu treatment whereas ectopic GLI1 expression desensitized cancer cells to these treatments. Further analyses indicate elevated Hh signaling is associated with an increase in putative cancer stem cell marker expression, such as ABCG2. We found that GLI1 protein binds to the promoter of ABCG2 through a Gli-binding consensus site. Disruption of ABCG2 function, through ectopic expression of an ABCG2 dominant negative construct or a specific ABCG2 inhibitor, resulted in an increase of drug sensitivity both in culture and in mice. The relevance of our studies to gastric cancer patient care is reflected by our discovery that high ABCG2 expression was associated with poor survival in the gastric cancer patients who underwent chemotherapy. Taken together, we have identified a molecular mechanism by which gastric cancer cells gain chemotherapy resistance. Citation Format: BEIQIN YU, DONGSHENG GU, XIAOLI ZHANG, BINGYA LIU, JINGWU XIE. Hedgehog signaling induces drug resistance in gastric cancer cells via upregulation of ABCG2 [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 LB-091.

PO-169 Involvement of neurotensin and neurotrophins pathways in human colorectal cancer cells 5-fluorouracil treatment resistance

IntroductionColorectal cancer (CRC) is one of the leading causes of cancer-related deaths. Surgical resection is the only curative treatment. Depending on disease stage, chemotherapies and targeted therapies are available. Despite considerable progress, recidive cases remain frequent. Studies lead in our laboratory on different cancer models, such as CRC, highlighted the importance of neuropeptides in cancer cells survival and progression: neurotrophines (NT) and neurotensin (NTS). NT is a family of four growth factors, BDNF, NT4/5, NT3 and NGF respectively binding a specific Tropomyosin Receptor Kinase (Trk A, B or C), tyrosin kinase receptors, with a strong affinity. NT can all bind another receptor, p75NTR, with low affinity, as well as the Sortilin, a TRK co-receptor, a NT and NTS intracellular trafficking regulator and also known as Neurotensin Receptor 3 (NTR3). Neurotensin can also bind two other receptors: NTR1 with strong affinity and 2 with low affinity; both are G protein coupled receptors. Very few is known about NT and NTS pathways in CRC cells resistance to chemotherapies, 5-Fluorouracil (5FU) being the backbone of any chemotreatments. The aim of my project is to understand if and how these pathways could be involved in 5FU CRC cell resistance and survival.Material and methodsTwo human CRC cell lines from different disease stages (WiDr, SW620) were treated with 8 µM of 5FU to obtain stably 5FU resistant cell lines. Both cell lines were also xenografted in Nude mice which were treated with 5FU. Protein expressions were assessed by western blot. Cell activation was assessed by flow cytometry. Stable knockdown cell lines (NTR3) were obtained by shRNA transfection. Exosomes were purified from culture supernatants by ultracentrifugation.Results and discussionsPreliminary results show that NTSR3 protein expression is increased after 5FU treatment both in whole cell lysates and exosomes especially in the most agressive cell line (SW620). The same results were obtained in vitro and in vivo. Moreover, 5FU treatment induces a decrease of tumour size only for the least agressive line (WiDr). Indeed, the 5FU induces a quiescence state of these cells.ConclusionIt is the first time that, in CRC, the NTSR3 seems to be overexpressed in 5FU-resistant tumour cells (from primary and advanced stages), in vitro and in vivo. This receptor could constitute a new potential therapeutic target.

Abstract 5026: Alcohol enriches therapy-resistant cancer cells with high autophagy, CD44 expression and tumor initiating capability

Abstract Introduction: Esophageal squamous cell carcinoma (ESCC) is the deadliest of all human cancers with alcohol (EtOH) as a major risk factor. Malignant properties of ESCC have been attributed to ESCC cells with high CD44 expression (CD44H) and autophagy; however, the pathogenic role of EtOH in ESCC remains elusive Methods: With a goal of translation in personalized medicine, we analyzed therapy-naïve ESCC tumor biopsies from patients and ESCC cell lines in three-dimensional (3D) organoids for functional characterization of ESCC cells following exposure to 5-fluorouracil (5FU) or EtOH ex vivo. Tumor growth was assessed in EtOH-fed mice carrying xenograft tumors with two independent ESCC cell lines in the presence or absence of 4-methylpyrazole (4MP), an alcohol dehydrogenase inhibitor. 3D organoids and tumors were analyzed by flow cytometry and immunostaining for CD44, Ki67, p53 and autophagic vesicle (AV) content. Results: ESCC patient biopsies contained CD44H cells with increased AV content. ESCC 3D organoids were grown successfully from 11 out of 16 tumors (68.8%) and passaged with recapitulation of the histopathology, proliferation, p53 and CD44 expression and autophagy present in the original in situ tumors. Successful organoid formation was significantly associated with poor chemoradiation therapy response (progressive and stable diseases, n=10 vs. partial response, n=6). In 3D organoids surviving 5FU treatment, CD44H cells with high autophagic activity was found to be enriched. Pharmacological autophagy flux inhibition by chloroquine augmented 5FU-mediated cytotoxicity in 3D ESCC organoids. 5FU-resistant CD44H cells were more capable of forming 3D organoids compared to bulk populations of ESCC cells. EtOH promoted expansion of CD44H cells with significantly increased proliferation and organoid formation rate upon serial passages, suggesting the increased self-renewal of CD44H cells. In xenograft tumors, alcohol drinking not only promoted tumor growth but increased the intratumoral CD44H cell content which was antagonized by 4MP. Moreover, pharmacological autophagy flux impairment depleted CD44H cells in xenograft tumors. Conclusions: The novel single cell-based 3D ESCC organoid system may serve as a highly efficient platform to explore the role of alcohol and other environmental factors as well as cancer therapeutics and therapy resistance mechanisms in conjunction with morphological and functional assays with implications for translation in personalized medicine. Citation Format: Koji Tanaka, Takashi Kijima, Prasanna Modayur Chandramouleeswaran, Kelly A Whelan, Yuta Kasagi, Masataka Shimonosono, Haruna Furukawa, Takeo Hara, Tomoki Makino, Makoto Yamasaki, Andres J Klein-Szanto, Shoji Natsugoe, Masaki Mori, Yuichiro Doki, Hiroshi Nakagawa. Alcohol enriches therapy-resistant cancer cells with high autophagy, CD44 expression and tumor initiating capability [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 5026.

Abstract B14: Characterization of p53-HDM2 inhibitor in nucleotide excision repair deficient cell line

Abstract The ubiquitin (Ub)/proteasome system has a special role in the control on the cellular concentration of p53 tumor suppressor, a master regulator of cell survival and death. Over the last years, many groups have elucidated the mechanisms of Ub ligase–mediated p53 ubiquitylation under various conditions. One pivotal regulatory mechanism is HDM2-mediated p53 degradation, but it can also export p53 from nucleus impairing its transcriptional activity or by binding p53 transactivation domain, that inhibits p53 transcriptional activity. Rad23 can directly bind Ub and the proteasome and has been shown to act as an adaptor that shuttles the ubiquitylated proteins to the proteasome. Rad23 is required for the degradation of p53 and one possible helper of Rad23 is its binding protein XPC, a key factor for nucleotide excision repair (NER). Mutations in XP proteins can cause xeroderma pigmentosum (XP), a genetic disease that often leads to high rates of skin cancer. Pathogenic mutations in p53 are found in more than 50% of all human malignancies, which makes p53 the most frequently mutated gene in transformed cells in humans. In cancers in which the p53 gene is not mutated, the function of the p53 pathway is often suppressed through overexpression or deregulation of HDM2. We aimed to study the impact of HDM2-P53 inhibition in XPC-deficient fibroblasts by treating these cells with the drug CGM9097, which is a potent inhibitor of HDM2-p53 protein-protein interaction used in preclinical trials. In this poster we present preliminary data. We firstly evaluated CGM9097 cytotoxicity in XPC deficient and corrected cells, combined with 5-fluorouracil (5FU) treatment. XPC deficient cells were more sensitive to 5FU+ CGM9097 than normal fibroblast (MRC5) and correct fibroblast (XPC+ XPC gene) (P&amp;lt;0,05). Protein analysis by Western blotting showed dose-response expression of p53 in both lineages when treated with 5FU, with the higher expression level within 6 h. When cells were treated with 5FU+ CGM9097, XPC-deficient cells presented high levels of p53 even in the untreated fraction compared to corrected cell line, suggesting an impairment of p53 degradation in the absence of XPC. These preliminary data suggest that XPC has an important role in the cytotoxicity of 5FU in cells with impairment of p53. P53 mutant cancers are sensitive to some chemotherapeutic drugs, but the reported sensitivity is generally modest (2-3 fold), and this can be explained by the fact that p53 affects the GGR NER sub pathway through GADD45. In addition, p53-mediated DNA repair may be counterbalanced by the opposing response of apoptosis, also regulated in part by p53. Thus, there appears to be much potential to exploit DNA repair defects in p53 mutant cancers. Citation Format: Douglas Felipe de Lima Silva, Ana Rafaela de Souza Timoteo, Lucymara Fassarela Agnez Lima, Tirzah Braz Petta Lajus. Characterization of p53-HDM2 inhibitor in nucleotide excision repair deficient cell line [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr B14.