High-dose Doxorubicin Research Articles

Effects of Doxorubicin Administration at Different Doses and Durations on the Body Weight of Rats

This study examines the impact of varying doses of Doxorubicin (DOX) on body weight in rats, with a focus on the drug's cachexia-inducing properties. While DOX is a widely used anticancer drug, it is associated with significant side effects, including cardiotoxicity and cachexia, which occur through mechanisms such as reduced insulin sensitivity, impaired glucose uptake, and decreased adipogenesis. In this study, 12-week-old male Wistar albino rats were divided into four groups: Control (C), low-dose DOX (DOX-L, 3 mg/kg), high-dose DOX (DOX-H, 12 mg/kg), and cumulative-dose DOX (DOX-C, 3 mg/kg every 24 hours for 4 days). The rats' weights were measured after administering various doses of DOX. Results indicated that the DOX-H group, which received a single high dose of DOX, experienced greater body weight loss compared to the other groups. The DOX-C group, which received the same total dose of DOX in cumulative doses, showed less weight loss compared to the DOX-H group. This suggests that single high-dose DOX applications lead to more significant body weight loss than cumulative dosing. The similarity in body weight loss between the DOX-L group (receiving a single low dose) and the control group indicates that DOX dosage is more influential than exposure duration. Given the potential for DOX to exacerbate cachexia in cancer patients, it is concluded that administering DOX in lower or cumulative doses may be preferable to a single high dose. In light of this information, we recommend further studies to determine the optimal dosage and duration of DOX administration, and to explore potential protective agents that could reduce DOX-related side effects on treatment.

Dose-Dependent Cognitive Decline, Anxiety, and Locomotor Impairments Induced by Doxorubicin: Evidence from an Animal Model.

Cognitive impairment and anxiety are common side effects of chemotherapy, particularly with the use of doxorubicin (DOX), known as "chemobrain". This study aimed to examine the dose-dependent effects of DOX on cognitive decline, anxiety, and locomotor activity in healthy female Wistar rats. The rats were divided into groups receiving low (2 mg/kg), intermediate (4 mg/kg), and high (5 mg/kg) doses of DOX for four weeks, alongside a control group. Behavioral tests, including open field, elevated plus maze, and Y-maze tests, assessed anxiety, locomotion, and cognitive performance, while brain tissue analysis evaluated neuroinflammation using markers such as GFAP and Iba-1. The results showed that all doses of DOX induced anxiety-like behavior, reduced locomotion, and caused neuroinflammation in the hippocampus, with more severe effects at higher doses. Notably, high-dose DOX also caused short-term memory deficits. These findings highlight the dose-dependent nature of DOX's impact on behavior and cognition, suggesting that DOX plays a key role in the development of cognitive symptoms during chemotherapy. Further research is needed to understand the mechanisms behind these effects and to explore potential interventions.

Improved Therapeutic Efficacy of Doxorubicin Chemotherapy With Cannabidiol in 4T1 Mice Breast Cancer Model.

High dose chemotherapy is one of the therapeutic strategies for breast cancer and doxorubicin (DOX) as a chemotherapy agent is widely used. DOX indication is limited due to its dose-depended cardiotoxicity. Recently, cannabidiol (CBD) shows antitumoral and cardioprotective effects, so we hypothesized that CBD administration with high-dose DOX chemotherapy can improve anticancer activity and reduce cardiotoxic side effects. Mice breast cancer model established by injecting 4T1 cell lines. One group was not injected by 4T1 cells as a not cancerous group and received normal saline (NS, 0.1 mL). In cancerous groups, first group was considered as cancerous control and received NS (0.1 mL); the second group received CBD (5 mg/kg, IP) on Days 1,7, and 14; in the third group DOX (5 mg/kg, IV) as CBD schedule was administrated; the fourth group treated with CBD 1 day before DOX injection as pretreatment, and the last group was treated with CBD and DOX at same time with previous doses and schedules. On Day 21, all mice were sacrificed, heart and lungs tissues were obtained and histological sections were isolated. SOD2, iNOS, MMP2, MMP9 were evaluated through western blot and TUNEL test preformed for breast tumor. Tumor size and weight significantly decreased in DOX, pretreatment CBD + DOX and CBD + DOX groups. Administration of CBD with DOX could not prevent weight loss. TUNEL test demonstrated the highest tumor cell apoptosis in pretreatment CBD + DOX and CBD + DOX. In lungs belonged to CBD + DOX, there was not any sign of metastasis. Cardiac histopathological examination of pretreatment CBD + DOX and CBD + DOX did not show any sign of congestion or inflammation. In CBD + DOX SOD2 increased, also iNOS, MMP2, and MMP9 decreased compared to DOX. This study demonstrated that simultaneous administration of CBD and DOX can increase antitumoral effect and reduce DOX cardiotoxicity. Nevertheless, CBD can induce cardiotoxicity as administrated alone.

UNC13B regulates the sensitivity of Wilms' tumor cells to doxorubicin by modulating lysosomes.

Wilms' tumor is a malignant neoplasm where current medical advancements have significantly improved survival rates; however, challenges persist such as the resistance of the tumor to chemotherapy drugs like doxorubicin. This necessitates higher dosages, leading to decreased sensitivity. However, using high doses of doxorubicin can have late effects on the heart. Unc-13 homolog B (UNC13B) may be involved in the drug resistance in several tumors, yet its role in modulating drug sensitivity in Wilms' tumor remains unexplored. UNC13B levels were quantified using reverse transcription-qPCR and Western blotting. The half-maximal inhibitory concentration for doxorubicin, vincristine, and actinomycin-D was determined using CCK-8 assays. Cell cycle and apoptosis were analyzed using flow cytometry, and lysosomal changes were observed using Lyso-Tracker staining. The present study initially evaluated UNC13B expression levels in the Wilms' tumor 17.94 cell line. Additionally, through short hairpin RNA-mediated knockdown, changes in doxorubicin sensitivity in 17.94 Wilms' tumor cells were assessed. Concurrently, preliminary investigations into the role of UNC13B in regulating lysosomes was performed, revealing a significant positive association between UNC13B levels and lysosome formation in the 17.94 cell line. Lysosomes likely serve a role in the sensitivity of Wilms' tumor cell lines to drugs. Elevated UNC13B expression was observed in the 17.94 Wilms' tumor cell line compared to normal kidney cells. UNC13B knockdown also resulted in increased apoptosis levels upon doxorubicin treatment. Immunofluorescence revealed UNC13B localization within cellular vesicles, and its knockdown significantly decreased lysosome levels. Overall, the findings of the present study demonstrate that UNC13B regulates the sensitivity of the Wilms' tumor 17.94 cell line to doxorubicin by modulating lysosome formation within cells. The results suggest that UNC13B is likely an enriched target involved in lysosomal regulation in certain tumors, offering a new approach for optimizing chemotherapy in Wilms' tumor and other cancers with high UNC13B expression.

Cardiac Morbidity and Mortality in Patients with Sarcoma: A Population-Based Study.

Soft tissue sarcoma (STS) is a rare malignancy that affects soft tissues. It encompasses various subtypes and requires different treatment strategies. Doxorubicin is a commonly used anthracycline in the management of localized and metastatic STS. However, high doses of doxorubicin are associated with cardiotoxicity, which can significantly impact patients' long-term outcomes. This study aimed to evaluate doxorubicin's effect on cardiac function in patients with sarcoma and to correlate the frequency of cardiotoxicity with potential risk factors. A retrospective analysis was conducted on patients with sarcoma who were treated with doxorubicin between 2016 and 2022 at King Abdulaziz Medical City in Saudi Arabia. Patient demographic information, comorbidities, cardiac measurements, laboratory values, systemic therapy, and treatment outcomes were collected from electronic medical records. A statistical analysis was performed to assess the association between cardiotoxicity and various factors. A total of 133 patients were included in the study, with a median age of 30 years. Cardiotoxicity was observed in 9% of the patients. Female patients had a significantly higher risk of developing cardiotoxicity. Patients with a higher Eastern Cooperative Oncology Group (ECOG) performance status and lower troponin I levels also had an increased risk of cardiotoxicity. However, there was no significant association between cardiotoxicity and the number of chemotherapy cycles, total cumulative dose of doxorubicin, or history of radiation. Furthermore, patients with cardiotoxicity had a higher risk of mortality. The overall survival of the patients was 18 months. Doxorubicin-associated cardiotoxicity is a concern for patients with sarcoma. Female patients and patients with a higher ECOG performance status are at an increased risk of developing cardiotoxicity. Careful monitoring and risk assessment are crucial for mitigating the adverse effects of doxorubicin treatment in patients with sarcoma. Future studies are warranted to validate these findings and explore preventive strategies for doxorubicin-induced cardiotoxicity in patients with sarcoma.

Abstract 15483: Mitoquinone Pretreatment Mitigates Dox-induced Systolic Dysfunction in Isolated Rat Hearts

Background: Doxorubicin (DOX) is a common and effective chemotherapeutic agent that can exert cardiotoxicity by inducing oxidative stress in mitochondria, compromising mitochondrial function, and inducing apoptosis. Currently, there is no treatment that targets this mechanism. We previously found that pretreatment with higher concentrations of mitoquinone (MQ), a mitochondrial antioxidant, exerts cardioprotection against DOX in myoblast H9C2 cells. Hypothesis: We hypothesize that MQ pretreatment would preserve cardiac function against DOX in isolated rat hearts. Methods: Cardiac parameters (heart rate (HR) and left ventricular end systolic pressure (LVESP)) were monitored before and after 60 min infusion of DOX, MQ, or DOX with MQ pretreatment (10-15 min) in a Langendorff preparation of male SD rat hearts. TUNEL assay was conducted on heart tissue slides to evaluate apoptosis. The ratio between the final and initial cardiac parameters was calculated and compared among experimental groups. Results: Higher dose DOX (25 μM, n=5) induced a higher reduction in the ratios of LVESP, to 39±5% than lower dose DOX infusion (20 μM, n=3; 74±3%). DOX had no effects on HR. A 60-minute perfusion of higher doses of MQ (1-5 μM, n=7) unexpectedly reduced the ratios of LVESP and HR to 73±12% and 66±5%, respectively. Higher dose MQ (1-2.5 μM, n=6) pretreatment showed similar improvement of LVESP ratio (68±9%) to lower dose MQ (0.25-0.5 μM, n=6; 71±11%). TUNEL assay showed that higher dose DOX caused a higher rate of apoptosis (number of TUNEL-positive nuclei) (326.3±6.4. n=3) than lower dose (214±29.5, n=3). Pretreatment with higher dose MQ prior to DOX 25 μM infusion resulted in a notable reduction of apoptosis (60.3±5, n=6) compared to DOX alone, while lower dose pretreatment reduced apoptosis to 70.5± 0.5 (n=4). Conclusion: This study suggests that infusion of DOX into the heart acutely attenuated cardiac systolic function accompanied with higher apoptotic cell damage. Although prolonged administration of higher doses of MQ suppressed cardiac function, MQ pretreatment for 10-15 minutes mitigated DOX-induced systolic dysfunction and reduced cardiomyocyte apoptosis. In summary, MQ pretreatment may mitigate DOX-induced cardiac damage.

The Role of ARHGAP1 in Rho GTPase Inactivation during Metastasizing of Breast Cancer Cell Line MCF-7 after Treatment with Doxorubicin.

Breast cancer is the most prevalent cancer type in women worldwide. It proliferates rapidly and can metastasize into farther tissues at any stage due to the gradual invasiveness and motility of the tumor cells. These crucial properties are the outcome of the weakened intercellular adhesion, regulated by small guanosine triphosphatases (GTPases), which hydrolyze to the guanosine diphosphate (GDP)-bound conformation. We investigated the inactivating effect of ARHGAP1 on Rho GTPases involved signaling pathways after treatment with a high dose of doxorubicin. Label-free quantitative proteomic analysis of the proteome isolated from the MCF-7 breast cancer cell line, treated with 1 μM of doxorubicin, identified RAC1, CDC42, and RHOA GTPases that were inactivated by the ARHGAP1 protein. Upregulation of the GTPases involved in the transforming growth factor-beta (TGF-beta) signaling pathway initiated epithelial-mesenchymal transitions. These findings demonstrate a key role of the ARHGAP1 protein in the disruption of the cell adhesion and simultaneously allow for a better understanding of the molecular mechanism of the reduced cell adhesion leading to the subsequent metastasis. The conclusions of this study corroborate the hypothesis that chemotherapy with doxorubicin may increase the risk of metastases in drug-resistant breast cancer cells.

Abnormal HDL lipid and protein composition following pediatric cancer treatment: an associative study

BackgroundLong-term childhood cancer survivors (CCS) are at high risk of having dyslipidemia including low high density lipoprotein cholesterol (HDL-C). However, little is known about the prevalence of low HDL-C and the impact of therapy exposure on HDL composition early after treatment is terminated.MethodsThis associative study included 50 children and adolescents who had completed their cancer treatments (< 4 years). Clinical characteristics (demographic, diagnosis, treatment, anthropometric parameters), fasting plasma lipids, apoliporoteins (Apo) A-I and composition of HDL fractions (HDL2 and HDL3) were assessed. Data were stratified according to the presence of dyslipidemia and median doses of therapeutic agents and compared using Fisher exact or Mann–Whitney tests. Univariate binary logistic regression analyses were carried out to evaluate the associations between the clinical and biochemical characteristics and having low HDL-C. Composition of HDL2 and HDL3 particles was assessed in a sub-group of 15 patients and compared to 15 age- and sex-matched healthy controls using Wilcoxon paired test.ResultsOf the 50 pediatric cancer patients included in this study (mean age: 11.30 ± 0.72 y; mean time since end of treatment: 1.47 ± 0.12 y; male: 38%), 8 had low HDL-C (16%), all of which were adolescent at diagnosis. Higher doses of doxorubicin were associated with lower HDL-C and Apo A-I levels. In hypertriglyceridemic patients and compared to normolipidemics, triglycerides (TG) content was greater in HDL2 and HDL3 fractions whereas esterified cholesterol (EC) content was lower in HDL2. Enrich TG content of HDL3 and lower EC of HDL2 was found in patients exposed to ≥ 90 mg/m2 doxorubicin. Factors positively associated with the risk of having low HDL-C were age, being overweight or obese and exposure to doxorubicin ≥ 90 mg/m2. Compared to healthy controls, a sub-group of 15 patients showed higher TG and free cholesterol (FC) content of HDL2 and HDL3 and lower EC content in HDL3.ConclusionsOverall, we found abnormalities in HDL-C and Apo A-I levels and in HDL composition early after pediatric cancer treatment that are influenced by age, overweight or obesity status and exposure to doxorubicin.

Poor Sit-to-Stand Performance in Adolescent and Young Adult Patients with Sarcoma.

Background: Adolescents and young adult (AYA) patients with sarcoma are at heightened risk of reduced physical capacity and disease-related weakness. Sit-to-stand (STS) performance correlates with lower extremity functionality and activities of daily living; however, little is known regarding the relationship between muscular status and STS performance in patients with sarcoma. This study assessed STS performance in patients with sarcoma and the association between STS performance and the skeletal muscle index (SMI) and skeletal muscle density (SMD). Methods: This study included 30 patients with sarcoma (15-39 years old) treated with high-dose doxorubicin. Patients performed the five-times-STS test before starting treatment and 1 year after the baseline test. STS performance was correlated with SMI and SMD. SMI and SMD were quantified using computed tomography scans taken at the level of the 4th thoracic vertebra (T4). Results: Mean performance on the STS test at baseline and 1 year was 2.2-fold and 1.8-fold slower than the age-matched general population, respectively. A lower SMI was associated with worse performance on the STS test (p = 0.01). Similarly, lower SMD at baseline was also associated with poorer STS performance (p < 0.01). Conclusion: Patients with sarcoma have very poor STS performance at baseline and 1 year, which was accompanied by low SMI and SMD at T4.The inability for AYAs to return to healthy age normative STS standards by 1 year may indicate a need for early interventions to enhance skeletal muscle recovery and promote physical activity during and after treatment.

Abstract 4771: Prevention of doxorubicin-induced cardiotoxicity by benfotiamine

Abstract Anthracyclines such as daunorubicin and doxorubicin (DOX) are currently used as chemotherapeutic agents in the prevention of various cancers including breast cancer, lung cancer, leukemia, and lymphoma. Although, anthracyclines are successful in controlling various cancers growth, the major drawback is the unwanted cardiotoxicity. Specifically, high doses of DOX used for the therapy of advanced cancers could cause life threating conditions. Therefore, specific interventions are required to decrease the cardiac toxicity associated with the DOX. Benfotiamine, a lipid soluble vitamin B1 derivative has shown be a potent anti-oxidant and anti-inflammatory agent. However, the efficacy of benfotiamine in the prevention of cardiotoxicity associated with the anthracyclines is not known. In this study, we examined the effect of benfotiamine in the prevention of DOX-induced cytotoxicity in the human umbilical vascular endothelial cells (HUVECs). Treatment of HUVECs with DOX caused significant endothelial cells death and benfotiamine in a concentration-dependent manner prevented the dox-induced endothelial cell death. Further, benfotiamine prevents the DOX-induced apoptosis in endothelial cells by preventing the activation of caspase-3. Benfotiamine also prevents DOX-induced reactive oxygen species generation. Our results also indicate that benfotiamine regulates DOX-induced expression of pro-apoptotic mediators such as BAD, phosphor-P53, and pro-caspase-3, and anti-apoptotic mediators such as BCL-2, BCL-x, IAPs, and HSP and others such as FADD, DR5, and SMAC/diablo. We plan to further explore how benfotiamine prevents DOX-induced cardiotoxicity using human cardiac myocytes and mouse models. Thus, based on our cell culture studies, benfotiamine through its potent anti-oxidative property could prevent endothelial cytotoxicity and suggests that it could be further developed as adjuvant therapy in controlling cardiotoxicity associated with the anthracycline chemotherapy. Citation Format: Justin R. Taylor, Kyra Harames. Prevention of doxorubicin-induced cardiotoxicity by benfotiamine. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4771.

Metabolomic Signatures in Doxorubicin-Induced Metabolites Characterization, Metabolic Inhibition, and Signaling Pathway Mechanisms in Colon Cancer HCT116 Cells.

Doxorubicin (DOX) is a chemotherapeutic agent is used for various cancer cells. To characterize the chemical structural components and metabolic inhibition, we applied a DOX to HCT116 colon cancer cells using an independent metabolites profiling approach. Chemical metabolomics has been involved in the new drug delivery systems. Metabolomics profiling of DOX-applied HCT116 colon cancer cellular metabolisms is rare. We used 1H nuclear magnetic resonance (NMR) spectroscopy in this study to clarify how DOX exposure affected HCT116 colon cancer cells. Metabolomics profiling in HCT116 cells detects 50 metabolites. Tracking metabolites can reveal pathway activities. HCT116 colon cancer cells were evenly treated with different concentrations of DOX for 24 h. The endogenous metabolites were identified by comparison with healthy cells. We found that acetate, glucose, glutamate, glutamine, sn-glycero-3-phosphocholine, valine, methionine, and isoleucine were increased. Metabolic expression of alanine, choline, fumarate, taurine, o-phosphocholine, inosine, lysine, and phenylalanine was decreased in HCT116 cancer cells. The metabolic phenotypic expression is markedly altered during a high dose of DOX. It is the first time that there is a metabolite pool and phenotypic expression in colon cancer cells. Targeting the DOX-metabolite axis may be a novel strategy for improving the curative effect of DOX-based therapy for colon cancer cells. These methods facilitate the routine metabolomic analysis of cancer cells.

Differential impact of doxorubicin dose on cell death and autophagy pathways during acute cardiotoxicity

Doxorubicin (DOX) is an effective anthracycline used in chemotherapeutic regimens for a variety of haematological and solid tumors. However, its utility remains limited by its well-described, but poorly understood cardiotoxicity. Despite numerous studies describing various forms of regulated cell death and their involvement in DOX-mediated cardiotoxicity, the predominate form of cell death remains unclear. Part of this inconsistency lies in a lack of standardization of in vivo and in vitro model design. To this end, the objective of this study was to characterize acute low- and high-dose DOX exposure on cardiac structure and function in C57BL/6 N mice, and evaluate regulated cell death pathways and autophagy both in vivo and in cardiomyocyte culture models. Acute low-dose DOX had no significant impact on cardiac structure or function; however, acute high-dose DOX elicited substantial cardiac necrosis resulting in diminished cardiac mass and volume, with a corresponding reduced cardiac output, and without impacting ejection fraction or fibrosis. Low-dose DOX consistently activated caspase-signaling with evidence of mitochondrial permeability transition. However, acute high-dose DOX had only modest impact on common necrotic signaling pathways, but instead led to an inhibition in autophagic flux. Intriguingly, when autophagy was inhibited in cultured cardiomyoblasts, DOX-induced necrosis was enhanced. Collectively, these observations implicate inhibition of autophagy flux as an important component of the acute necrotic response to DOX, but also suggest that acute high-dose DOX exposure does not recapitulate the disease phenotype observed in human cardiotoxicity.

Abstract 6264: Mechanisms of resistance to doxorubicin in HOS-MNNG cell line

Abstract The use of poly-chemotherapy combined with surgical resection has good efficiency and increased the survival rates of osteosarcoma (OS) patients to 70%, however these rates have not evolved since the 1970’s. On the other hand, survival drops to 30% among patients with metastases and/or bad response to treatment. This suggests that metastasis and resistance to treatment are key obstacles to tackle in order to reduce mortality. In this context this study has the aim to identify mechanisms that could participate in the emergence of resistance to doxorubicin, one of the most often used chemotherapeutic molecules to treat OS. For this purpose, HOS-MNNG cells were exposed to increasing concentrations of doxorubicin over a long period of time (over 6 months). Transcriptional and super-enhancer (SE) profiles of highly resistant (IC50 greater by 250 times) cells obtained as such were then compared to naive untreated cells. We hypothesized identification of target genes of resistant-specific super-enhancers, which are regulatory regions that mobilize a large proportion of the cell’s transcriptional apparatus and epigenetic regulators, would allow to unveil key genes in the emergence of resistance. We observed the presence of highly efficient super-enhancers within loci located in close proximity to genes encoding for ATP-Binding Cassette (ABC) transporters only within the high dose doxorubicin resistant cells. The associated genes were among the most highly up-regulated genes in the resistant cells and western blot analysis confirms higher concentration of encoded proteins compared to naive cells. The use of one BETi namely JQ1 - that inhibits recruitment of transcriptional co-factors on regulatory DNA regions such as enhancers and SEs - sensitizes resistant cells to doxorubicin. This suggests that the identified SEs in close proximity could play key role in the emergence of resistance. In order to track emergence of the mechanisms of resistance and to appreciate the transcriptional and epigenetic heterogeneity among resistant cells, we performed single cell 10x multiome (ATAC+RNA) on naive, intermediate and high dose doxorubicin resistant cells. While the high expression of genes encoding for the ABC transporters revealed in bulk analysis seemed ubiquitous among all the clusters of high-dose resistant cells, intermediate-dose resistant cells demonstrate no or very low expression for these genes. This could suggest that the first form of resistance passes through other mechanisms. In conclusion ABC transporters could be highly involved in the emergence of resistance to high-doses of doxorubicin in OS. A heterogeneous feature could be expected in patients as resistance to intermediate doses do not seem to depend on this mechanism. Finally, the use of BETi could be suggested in patients with bad response to treatment. This project is supported by Région Pays de la Loire, ANR, la Ligue contre le cancer and SFCE. Citation Format: Robel A. Tesfaye, Mélanie Lavaud, Anaïs Postec, Céline Charrier, Rose-Anne Thépault, Franck Verrecchia, Marc Baud'huin, François Lamoureux, Steven Georges, Benjamin Ory. Mechanisms of resistance to doxorubicin in HOS-MNNG cell line [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 6264.

Indocyanine green for leakage control in isolated limb perfusion

Abstract Objective Sarcomas are characterized by a high metastatic potential and aggressive growth. Despite surgery, chemotherapy plays an important role in the treatment of these tumors. Optimal anti-cancer therapy with maximized local efficacy and minimized systemic side effects has been the object of many studies for a long time. To improve the local efficacy of anti-tumor therapy, isolated limb perfusion with high-dose cytostatic agents has been introduced in surgical oncology. In order to control the local distribution of substances, radiolabeled cytostatic drugs or perfusion solutions have been applied but often require the presence of specialized personnel and result in a certain exposure to radiation. In this study, we present a novel strategy using indocyanine green to track tumor perfusion with high-dose cytostatic therapy. Methods In a rat cadaver model, the femoral vessels were cannulated and connected to a peristaltic pump to provide circulation within the selected limb. The perfusion solution contained indocyanine green and high-dose doxorubicin. An infrared camera enabled the visualization of indocyanine green during limb perfusion, and subsequent leakage control was successfully performed. Results Histologic analysis of sections derived proximally from the injection site excluded systemic drug dispersion. Conclusion In this study, the application of indocyanine green was proven to be a safe and cost- and time-efficient method for precise leakage control in isolated limb perfusion with a high-dose cytostatic agent.

Mitochondrial Sirtuin-3 (SIRT3) Prevents Doxorubicin-Induced Dilated Cardiomyopathy by Modulating Protein Acetylation and Oxidative Stress.

High doses of doxorubicin put cancer patients at risk for developing dilated cardiomyopathy. Previously, we showed that doxorubicin treatment decreases SIRT3 (sirtuin 3), the main mitochondrial deacetylase and increases protein acetylation in rat cardiomyocytes. Here, we hypothesize that SIRT3 expression can attenuate doxorubicin induced dilated cardiomyopathy in vivo by preventing the acetylation of mitochondrial proteins. Nontransgenic, M3-SIRT3 (truncated SIRT3; short isoform), and M1-SIRT3 (full-length SIRT3; mitochondrial localized) transgenic mice were treated with doxorubicin for 4 weeks (8 mg/kg body weight per week). Echocardiography was performed to assess cardiac structure and function and validated by immunohistochemistry and immunofluorescence (n=4-10). Mass spectrometry was performed on cardiac mitochondrial peptides in saline (n=6) and doxorubicin (n=5) treated hearts. Validation was performed in doxorubicin treated primary rat and human induced stem cell derived cardiomyocytes transduced with adenoviruses for M3-SIRT3 and M1-SIRT3 and deacetylase deficient mutants (n=4-10). Echocardiography revealed that M3-SIRT3 transgenic mice were partially resistant to doxorubicin induced changes to cardiac structure and function whereas M1-SIRT3 expression prevented cardiac remodeling and dysfunction. In doxorubicin hearts, 37 unique acetylation sites on mitochondrial proteins were altered. Pathway analysis revealed these proteins are involved in energy production, fatty acid metabolism, and oxidative stress resistance. Increased M1-SIRT3 expression in primary rat and human cardiomyocytes attenuated doxorubicin-induced superoxide formation, whereas deacetylase deficient mutants were unable to prevent oxidative stress. Doxorubicin reduced SIRT3 expression and markedly affected the cardiac mitochondrial acetylome. Increased M1-SIRT3 expression in vivo prevented doxorubicin-induced cardiac dysfunction, suggesting that SIRT3 could be a potential therapeutic target for mitigating doxorubicin-induced dilated cardiomyopathy.

Potency of the Natural Honey in Homeostasis of Four Liver enzymes in Rats Induced by Doxorubicin

Previous histological studies have confirmed the curing potency of ingested natural honey on both liver and kidney’s cellular structures in mice and other experimental animals. Forty four male Wister rats were divided into four groups, control (G1); honey only (G2); Doxorubicin (DOX) (G3) and DOX with natural honey (G4), respectively. The experiments lasted 7 weeks following subcutaneous administrating 5mg/Kg b.wt./week of DOX and treatment with 1 mL/L honeyed water. Four most common liver enzymes e.g. Alkaline phosphatase (ALP), Alanine transaminase (ALT), Aspartate transaminase (AST) and Gamma-Glutamyl Transferase (GGT) in the blood as well as differential counting of WBC were tested to assess the impact of curing potency of the ingested natural honey via stabilizing the levels of the enzymes. The biochemical assessments have shown curing potency of natural honey against the toxicity impact of DOX on the above liver enzymes. Differential counting of WBC have shown insignificant changes in most WBCs but significant elevation of eosinophil in DOX rats (G3) in comparison with other groups indicating development of allergy. It has been concluded that lower or mild doses of DOX have less toxic effects on the body with a rather better impact of honey. Perhaps regular ingestion of the natural honey could provide a natural remedy on general health e.g. functions of the liver and positive impacts on other chronicle diseases. Further researches are recommended using higher doses of DOX via using other administration methods e.g. intravenous.

Abstract 10607: Evaluating Diastolic Function by Echocardiography in a Prospective Cohort of Patients with Hodgkin (HL) and Non-Hodgkin Lymphoma (NHL) Who Received Anthracycline-Based Chemotherapy

Introduction: Anthracyclines are a first-line chemotherapeutic agent used in the treatment of lymphoma. Myocardial injury can occur with cumulative doses of 250 to 350 mg/m 2 . Echocardiography is the imaging modality for identifying cardiac dysfunction; however, identifying diastolic changes in patients who received anthracyclines is not well represented in the literature. Variables used to identify diastolic dysfunction include lateral e’, medial e’, mitral E velocity, mitral A velocity, E/A, E/e’, and left atrial volume index (LAVI). This study aims to identify if diastolic abnormalities develop in a prospective cohort of patients with HL or NHL who received anthracycline-based chemotherapy. Methods: Lymphoma patients treated with anthracyclines from 2013 to 2020 were prospectively enrolled. Echocardiography was obtained at 3-time points: before the start of chemotherapy (T1), at 3-6 months (T2), and 9-12 months (T3). Diastolic variables were measured and analyzed based on the total cumulative dose of Doxorubicin per body surface area. Those that received greater than 250 mg/m 2 and greater than 300 mg/m 2 of Doxorubicin were compared to those that did not. Statistical analysis was done using one-way ANOVA, adjusting for age and gender. Results: 151 total patients (90 males, mean age 61 + 16 years); 21 (14%) HL, 130 (85%) NHL. 114 and 66 patients received a total cumulative dose of Doxorubicin greater than 250 mg/m 2 and 300 mg/m 2 , respectively. There was no significant association with 250 mg/m2. Lateral e’ at T3 vs. T1, was lower in those that received greater than 300 mg/ m 2 compared to those received less (0.09 vs 0.10, p &lt; 0.02) (Table 1). Conclusions: Lateral e’ decreased at 1-year follow-up in those that received higher doses of Doxorubicin. These changes in the lateral e’ could reflect early relaxation abnormalities related to anthracycline-based chemotherapy and suggest that changes in lateral e’ should be considered by itself since E/e’ was normal.

Indocyanine Green for Leakage Control in Isolated Limb Perfusion.

Sarcomas are characterized by a high metastatic potential and aggressive growth. Despite surgery, chemotherapy plays an important role in the treatment of these tumors. Optimal anti-cancer therapy with maximized local efficacy and minimized systemic side effects has been the object of many studies for a long time. To improve the local efficacy of anti-tumor therapy, isolated limb perfusion with high-dose cytostatic agents has been introduced in surgical oncology. In order to control the local distribution of substances, radiolabeled cytostatic drugs or perfusion solutions have been applied but often require the presence of specialized personnel and result in a certain exposure to radiation. In this study, we present a novel strategy using indocyanine green to track tumor perfusion with high-dose cytostatic therapy. In a rat cadaver model, the femoral vessels were cannulated and connected to a peristaltic pump to provide circulation within the selected limb. The perfusion solution contained indocyanine green and high-dose doxorubicin. An infrared camera enabled the visualization of indocyanine green during limb perfusion, and subsequent leakage control was successfully performed. Histologic analysis of sections derived proximally from the injection site excluded systemic drug dispersion. In this study, the application of indocyanine green was proven to be a safe and cost- and time-efficient method for precise leakage control in isolated limb perfusion with a high-dose cytostatic agent.

EP.TH.494Preclinical feasibility of in situ isolated normothermic liver chemoperfusion

Abstract Aims Up to 85% of patients with liver metastases have inoperable hepatic tumour burden. Isolated liver perfusion involves vascular isolation of the liver in situ and regional delivery of chemotherapy, avoiding dose-limiting extra-hepatic toxicity. In this series, we develop a surgical protocol to demonstrate the feasibility of isolated normothermic liver perfusion (INLP) and investigate short-term safety and feasibility of delivering high-dose chemotherapy. Methods Laparotomy and complete, vascular isolation of the liver was performed on 55-65Kg pigs (n = 6). The hepatic artery (HA), portal vein (PV) and inferior vena cava were cannulated and liver NMP established. Veno-venous bypass maintained systemic circulation. High-dose doxorubicin was administered to the isolated liver, circulated for 1 hour and vascular reconnection performed. Physiological parameters were measured and doxorubicin quantified in blood, bile and tissue by high-performance liquid chromatography. Results INLP with doxorubicin delivery achieved physiological flow rates (PV 0.7L/min (0.6-0.9L/min); HA 0.3L/min (0.2-0.4L/min)) and pH (median 7.3 (7.24-7.38)), with a median lactate of 0.42mmol/L. Median peak AST and ALT were 1045 U/L and 47 U/L respectively. Doxorubicin decay was fitted with a 2-compartmental model; distribution half-life was 1.9 minutes and plasma Cmax was higher than if given systemically resulting in mean hepatic tissue levels of 26+/-11.6 µg/g. There was no leak during INLP and doxorubicin was undetectable in kidney or heart. Conclusions Surgical isolation and NMP of the liver in situ, with concurrent veno-venous bypass is feasible and enables high-dose drug delivery resulting in therapeutic tissue levels with no off-target toxicity. Further safety studies are required.

Blockage of AMPK-ULK1 pathway mediated autophagy promotes cell apoptosis to increase doxorubicin sensitivity in breast cancer (BC) cells: an in vitro study

BackgroundActivation of autophagy flux contributed to resistance of breast cancer (BC) cells to current chemotherapeutic drugs, which seriously limited their therapeutic efficacy and facilitated BC recurrence in clinic. However, the detailed mechanisms are still not fully understood. In the present study, we identified that inactivation of AMPK-ULK1 signaling cascade mediated protective autophagy sensitized BC cells to doxorubicin in vitro.MethodsCell counting kit-8 (CCK-8) assay and colony formation assay were performed to evaluate cell proliferation abilities. Trypan blue staining assay was used to examine cell viability, and Annexin V-FITC/PI double staining method was conducted to determine cell apoptosis. The autophagosomes in BC cells were observed and photographed by electronic microscope (EM). Western Blot analysis was employed to examine genes expressions at protein levels.ResultsThe parental doxorubicin-sensitive BC (DS-BC) cells were exposed to increasing concentrations of doxorubicin to establish doxorubicin-resistant BC (DR-BC) cells, and the DR-BC cells were much more resistant to high-dose doxorubicin treatment compared to the DS-BC cells. Interestingly, high-dose doxorubicin specifically increased LC3B-II/I ratio, promoted autophagosomes formation and decreased p62 expression levels to facilitate autophagy in DR-BC cells, instead of DS-BC cells, and the autophagy inhibitor 3-methyladenine (3-MA) enhanced the cytotoxic effects of high-dose doxorubicin on DR-BC cells. In addition, we proved that high-dose doxorubicin triggered protective autophagy in DR-BC cells by activating AMPK-ULK1 pathway. Functionally, high-dose doxorubicin increased the expression levels of phosphorylated AMPK (p-AMPK) and ULK1 (p-ULK1) to activate AMPK-ULK1 pathway in DR-BC cells, and the inhibitors for AMPK (compound C) and ULK1 (SBI-0206965) blocked autophagy to promote cell death and slow down cell growth in DR-BC cells treated with high-dose doxorubicin.ConclusionsCollectively, our in vitro data indicated that blockage of AMPK-ULK1 signaling cascade mediated protective autophagy might be a promising strategy to increase doxorubicin sensitivity for BC treatment.