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C ontrol D H A 1um ol/kg i.p. LLC LLC + D H A 1um ol/kg i.p. 0 100 200 300 400 Am bulatory m ovem ent * C ontrol D H A 1um ol/kg i.p. LLC LLC + D H A 1um ol/kg i.p. 0 5 10 15 20 Rearing num ber C ontrol D H A 1um ol/kg i.p. LLC LLC + D H A 1um ol/kg i.p. 0 10 20 30 40 50 60 Speed (m m /s ) * C ontrol D H A 1um ol/kg i.p. LLC LLC + D H A 1um ol/kg i.p. 0 5 10 15 20 Traveled distance (cm ) * C ontrol D H A 1um ol/kg i.p. LLC LLC + D H A 1um ol/kg i.p. 0 20 40 60 80 100 120 G rasping (g) C ontrol D H A 1um ol/kg i.p. LLC LLC + D H A 1um ol/kg i.p. 0 20 40 60 Latency to fall (s) A B C D E F POTENTIAL BENEFITS OF DOCOSAHEXAENOIC ACID IN A MOUSE MODEL OF LUNG CANCER-RELATED CACHEXIA IC007 Raquel D.S. Freitas 1,4 , Kesiane M. da Costa 1 , Fernanda L. Rost 3,4 , Maria Eduarda A. Amaral 2,3 , Maria M. Campos 1,2,3,4 1 Programa de Pós-graduação em Medicina e Ciências da Saúde - Escola de Medicina - PUCRS; 2 Programa de Pós-graduação em Biologia Celular e Molecular - Escola de Ciências - PUCRS; 3 Curso de Graduação em Odontologia – Escola de Ciências da Saúde – PUCRS; 4 Centro de Toxicologia e Farmacologia – Escola de Ciências da Saúde – PUCRS. Cancer cachexia (CC) is a multifactorial syndrome, characterized by muscle atrophy, loss of adipose tissue and reduced quality of life that cannot be fully reversed by nutritional therapy (Lancet Oncol. 12:489. 2011). About 50% of cancer deaths are related to tumors associated with cachexia (Nat Rev Dis Primers. 4:1-18. 2018). N-3 fatty acid eicosapentaenoic acid (EPA) supplementation is widely used for management of CC (Br J cancer. 105:1469. 2011; Cancer Metastasis Rev. 34:359. 2015), although, other n-3 fatty acids, such as docosahexaenoic acid (DHA), have not been fully investigated. AIM This study assessed the effects of DHA treatment in a mouse model of cancer cachexia. METHODS in vitro C57/BL6 male mice (20 – 25g/8 weeks ) CEUA/PUCRS approval number: 15/7164 Euthanasia Removal of tissues for weight and histological analysis Blood collection for hematological analysis Lewis lung carcinoma cell line LL/2 (LLC1) was used. Cells were cultivated in DMEM 10% SFB, at 37 o C, 5% CO 2. Number of cells was counted using a Neubauer chamber. Cells were centrifuged for 10 min at 3000 RPM. Cells were resuspended in 100 μL of PBS. 5x10 6 cells were injected in the dorsal region of each mouse. 0 7 21 Cachexia model induction Behavioral tasks • Control • DHA 1 μmol/kg i.p. • LLC • LLC + DHA 1 μmol/kg i.p. EXPERIMENTAL GROUPS in vivo Behavioral tasks DHA i.p. treatment RESULTS Fig 1. Evaluation of exploratory activity shown as distance (A) ambulatory movement, (B) rearing, (C) speed, (D) traveled distance, after 21 days of LLC-induced cachexia and 15 days of systemic DHA treatment. Effects of DHA treatment on (E) grip strength analysis, and (F) motor coordination tested by rota-rod apparatus after LLC-cachexia induction. Each column represents the mean ± SEM of 8-10 mice per experimental group. ## P<0.01 significantly different from control group; RESULTS Fig 2. Effects of parenteral administration of DHA in healthy mice and in LLC-cachectic animals on body weight and glycemia levels. (A) Analysis of body weight variation (tumor-free carcass weight minus initial weight prior to tumor induction) throughout 21 days of cachexia development, (B) Tumor-free carcass weight and (C) Glycemia levels after 6 h-fasting period. Each column represents the mean ± SEM of 6-12 mice per experimental group. # P<0.05 significantly different from LLC- cachectic group; *P<0.05 significantly different from control group. Fig 3. Effects of DHA i.p. treatment on epididimal fat (A) retroperitoneal fat, (B) intrascapular fat (C) inguinal fat, (D) weights of LLC-cachectic and healthy mice. Each column represents the mean ± SEM of 4-12 mice per experimental group. *P<0.05 significantly different from control group. Fig 5. Effects of LLC- cachexia on (A) hematocrit levels, (B) neutrophils-to- lymphocytes ratio, and (C) spleen weight. Each column represents the mean ± SEM of 6-12 mice per experimental group. *P<0.05 significantly different from control group. INTRODUCTION C ontrol D H A 1um ol/kg i.p. LLC LLC + D H A 1um ol/kg i.p. 0 100 200 300 400 500 G lycem ia (m g/dL) * C ontrol D H A 1um ol/kg i.p. LLC LLC + D H A 1um ol/kg i.p. -8 -6 -4 -2 0 2 4 6 8 B ody w eight variation (g) * # C ontrol D H A 1um ol/kg i.p. LLC LLC + D H A 1um ol/kg i.p. 0 10 20 30 Tum or-free carcass w eight (g) * A B C C ontrol D H A 1um ol/kg i.p. LLC LLC + D H A 1um ol/kg i.p. 0 20 40 60 80 100 120 in g W A T (m g) C ontrol D H A 1um ol/kg i.p. LLC LLC + D H A 1um ol/kg i.p. 0 100 200 300 epW AT (m g) * C ontrol D H A 1 um ol/kg i.p. LLC LLC + D H A 1 um ol/kg i.p. 0 20 40 60 80 100 120 is W A T (m g) * p=0.07 C ontrol D H A 1um ol/kg i.p. LLC LLC + D H A 1um ol/kg i.p. 0 20 40 60 80 rW A T (m g) * A B C D C ontrol D H A 1 um ol/kg i.p. LLC LLC + D H A 1 um o l/lg i.p . 0 200 400 600 Spleen (m g) * C ontrol D H A 1um ol/kg i.p. LLC LLC + D H A 1um ol/kg i.p. 0 10 20 30 40 50 H em atocrit (% ) * C ontrol D H A 1um ol/kg i.p. LLC LLC + D H A 1um ol/kg i.p. 0 .0 0 .5 1 .0 1 .5 2 .0 NLR * A B C <100 100-200 200-300 300-400 400-500 >500 0 20 40 60 80 100 C ontrol D H A 1 um ol/kg i.p. LLC LLC + D H A 1 um ol/kg i.p. % total of adipocytes * C ontrol D H A 1um ol/kg i.p. LLC LLC + D H A 1um ol/kg i.p. 150 200 250 300 350 is W A T adipocytes area (um ²) A B C ontrol D H A 1um ol/kg i.p. LLC LLC + D H A 1um ol/kg i.p. 0 100 200 300 400 rW A T adipocytes area (um ²) C Fig 6. Evaluation of (A) retroperitoneal fat and (B) intrascapular fat adipocyte área, and (C) frequency distribution of isWAT adipocyte area. Each column represents the mean ± SEM of 3-8 mice per experimental group. *P<0.05 significantly different from control group. Representative images of intrascapular fat: (A) Control, (B) DHA 1 μmol/kg i.p., (C) LLC and (D) LLC + DHA 1μmol/kg i.p. CONCLUSION C ontrol D H A 1um ol/kg i.p. LLC LLC + D H A 1um ol/kg i.p. 0 50 100 150 200 G astrocnem ius (m g) C ontrol D H A 1 um ol/kg i.p. LLC LLC + D H A 1 um o l/lg i.p . 0 5 10 15 Soleus (m g) C ontrol D H A 1 um ol/kg i.p. LLC LLC + D H A 1 um ol/kg i.p. 0 10 20 30 40 50 60 70 Tibialis anterior (m g) * A B C Fig 4. Effects of DHA i.p. treatment on (A) gastrocnemius, (B) soleus, and (C) tibialis anterior weights of LLC-cachectic and healthy mice. Each column represents the mean ± SEM of 8-12 mice per experimental group. *P<0.05 significantly different from control group. A B C D Parenteral administration of DHA showed some beneficial effects on the mouse model of lung cancer cachexia, specifically regarding adipose tissue loss. Additional analysis are in progress in order to identify the mechanisms of DHA in this model of cancer-related cachexia. FINANCIAL SUPPORT FINEP-PUCRSINFRA #01.11.0014-00
