Part 1 Doxorubicin Paradoxically Ameliorates Tumor-Induced Inflammation in Young Mice

Contact: ali.ma@wecistanche.com

Ibrahim Y. Abdelgawad et al

Cistanche tubulosa prevents kidney disease, click here to get the products and Cistanche herba

Abstract:

Doxorubicin (DOX) is one of the most widely used chemotherapeutic agents in pediatric oncology. DOX elicits an inflammatory response in multiple organs, which contributes toDOX-induced adverse effects. Cancer itself causes inflammation leading to multiple pathology conditions. The current study investigated the inflammatory response to DOX and tumors using anEL4-lymphoma, immunocompetent, juvenile mouse model. Four-week-old male C57BL/6N mice were injected subcutaneously with EL4 lymphoma cells (5 × 104cells/mouse) in the flank region, while tumor-free mice were injected with vehicle. Three days following tumor implantation, both tumor-free and tumor-bearing mice were injected intraperitoneally with either DOX (4 mg/kg/week)or saline for 3 weeks. One week after the last DOX injection, the mice were euthanized and the hearts, livers, kidneys, and serum were harvested. Gene expression and serum concentration of inflammatory markers were quantified using real-time PCR and ELISA, respectively. DOX treatment significantly suppressed tumor growth in tumor-bearing mice and caused significant cardiac atrophy in tumor-free and tumor-bearing mice. EL4 tumors elicited a strong inflammatory response in the heart, liver, and kidney. Strikingly, DOX treatment ameliorated tumor-induced inflammation paradoxical to the effect of DOX in tumor-free mice, demonstrating a widely divergent effect of DOXtreatment in tumor-free versus tumor-bearing mice.

Keywords: doxorubicin; tumor; inflammation

1. Introduction

Doxorubicin (DOX), also known as Adriamycin, is one of the most widely used chemotherapeutic agents in pediatric clinical oncology. Despite its effectiveness in a wide range of cancers including lymphoma, leukemia, and other pediatric cancers, DOX's clinical utility is limited due to its multiple organ toxicities. In addition to its marked cardiotoxicity [1], DOX has been demonstrated to cause nephrotoxicity [2–5] and hepatotoxicity [6]. Nearly 50% of pediatric cancer patients receive DOX as part of their treatment protocol [7]. Due to effective cancer treatments, including DOX, the 5-year survival rate of children diagnosed with cancer has increased from less than 60% in the 1970s to more than 80% now [8,9]. This leads to a large population of childhood cancer survivors (CCSs), estimated to be around 400,000. Unfortunately, CCSs are at high risk of developing long-term adverse effects due to cancer treatment and/or cancer itself.

DOX has been shown to trigger systemic inflammation [10–12] and inflammatory response in multiple organs including the heart [13], kidney [14], and liver [15]. This inflammatory response has been shown to promote DOX-induced adverse effects [16,17]. The majority of these studies reporting DOX-induced inflammation have been conducted in tumor-free animal models which do not mimic the clinical scenario in which pediatric cancer patients receive DOX treatment after diagnosis with cancer. Cancer per se has also been shown to cause inflammation contributing to multiple pathologic conditions that may exacerbate DOX-induced toxicities [18,19]. Therefore, there is a critical need to establish clinically relevant animal models to investigate the mechanisms of DOX-induced multiple organ toxicities in young, tumor-bearing mice to study the interplay between DOX treatment and cancer itself to cause such toxicities.

In the current study, we investigated the inflammatory response to DOX and the tumor in a clinically relevant syngeneic EL4-lymphoma, immunocompetent, juvenile mouse model. This model is well established in cancer research [20] and is clinically relevant to pediatric cancer patients since lymphoma is one of the most common types of pediatric cancer [21]. The use of syngeneic mice is critical for studying the inflammatory response to both DOX and the tumor due to the critical role of the immune system in the pathogenesis of inflammation [22]. The mice's age was 5 weeks upon DOX administration, which is equivalent to the adolescence age of 10–12 years in humans [23]. Additionally, the administered DOX doses are clinically relevant to the doses received by pediatric cancer patients [24]. We first determined the effects of DOX on cardiac function in tumor-free and tumor-bearing mice, since DOX-induced cardiotoxicity is considered the most clinically relevant adverse effect of DOX, among other multiple organ toxicities. Thereafter, we determined the effect of DOX, the tumor, and their combination on several inflammatory markers in the heart, liver, and kidney in addition to serum markers of systemic inflammation. We found that the presence of the EL4 tumor elicited a strong inflammatory response in multiple organs. Strikingly, DOX treatment ameliorated tumor-induced inflammation paradoxical to the previously reported effect of DOX in tumor-free mice.

2. Results

2.1. Effects of DOX on Body Weight and Tumor Growth

The current study was established in juvenile mice to mimic the clinical scenario in pediatric cancer patients as described in Figure 1A. No significant morbidity or mortality was observed in either tumor-free or tumor-bearing mice following exposure to DOX (4 mg/kg/week for 3 weeks), which is in agreement with our previous study using the same dose [25]. However, juvenile exposure to DOX resulted in a significantly reduced body weight gain compared to saline-treated mice (Figure 1B). To assess the anticancer effect of DOX in this EL4 lymphoma model, we measured the tumor weights following mice necropsy. Chronic DOX administration in tumor-bearing mice significantly inhibited the EL4 tumor growth, demonstrating the anticancer effect of this dosage regimen of DOX (Figure 1C).

2.2. Effect of DOX Administration on Cardiac Function and Morphometry in Tumor-Free and Tumor-Bearing Mice

DOX caused cardiac atrophy as demonstrated by a significant decrease in the heart weight to tibia length (HW/TL) (Figure 1D). Remarkably, cardiac atrophy caused by DOX was augmented in tumor-bearing mice (Figure 1D). The effects of DOX, tumor, and the combination of both on cardiac function and morphology were assessed using transthoracic echocardiography. Representative M-Mode images from each group are displayed in Figure 2A. Neither DOX nor the tumor caused significant changes in ejection fraction (Figure 2B), cardiac output (Figure 2C), fractional shortening (Figure 2D), left ventricular (LV) mass (Figure 2E), LV posterior wall thickness in diastole (LVPW'd) (Figure 2F), or LV posterior wall thickness in systole (LVPW's) (Figure 2G).

2.3. Effect of DOX Administration on Gene Expression of Inflammatory Markers in the Heart of Tumor-Bearing and Tumor-Free Mice

Hearts were harvested from both tumor-free and EL4 lymphoma tumor-bearing juvenile C57BL/6N mice one week following the administration of 4 mg/kg/week of DOX or equivalent volume of sterile saline for 3 weeks (n = 8 per group). Following the extraction of total RNA, cardiac mRNA expression of inflammatory markers interleukin-1 alpha (IL-1α), interleukin-1 beta (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF- α), inflammatory chemokines monocyte chemoattractant protein-1 (Mcp-1), and C-XC motif chemokines (Cxcl1, Cxcl9, and Cxcl10) were determined by real-time PCR. The gene expression of multiple inflammatory markers in the heart was significantly upregulated in tumor-bearing mice as compared to tumor-free mice, including the pro-inflammatory cytokines IL-1α (Figure 3A), IL-1β (Figure 3B), IL-6 (Figure 3C), and TNF- α (Figure 3D) and the inflammatory chemokines Mcp-1 (Figure 3E), Cxcl1 (Figure 3F), and Cxcl10 (Figure 3H). While DOX administration in tumor-free mice did not result in significant changes in the expression of inflammatory markers in the heart, DOX administration significantly abrogated the tumor-induced upregulation of IL-1β (Figure 3B), Mcp-1 (Figure 3E), Cxcl9 (Figure 3G), and Cxcl10 (Figure 3H). Importantly, two-way ANOVA demonstrated a significant interaction effect in all markers except Cxcl1, Cxcl9, and Cxcl10, demonstrating the divergent effects that DOX has on cardiac inflammatory markers in tumor-bearing compared to tumor-free mice.

2.4. Effect of DOX Administration on Gene Expression of Inflammatory Markers in the Liver of Tumor-Free and Tumor-Bearing Mice

Livers were harvested from both tumor-free and EL4 lymphoma tumor-bearing juvenile C57BL/6N mice one week following the administration of 4 mg/kg/week of DOX or equivalent volume of sterile saline for 3 weeks (n = 8 per group). Following the extraction of total RNA, hepatic mRNA expression of inflammatory markers IL-1α, IL-1β, IL-6, TNF-α, Mcp-1, Cxcl1, Cxcl9, and Cxcl10 were determined by real-time PCR. Tumor-bearing mice had significantly elevated hepatic expression of the pro-inflammatory cytokines IL-1α (Figure 4A), IL-1β (Figure 4B), TNF-α (Figure 4D), Cxcl9 (Figure 4G), and Cxcl10 (Figure 4H) compared to tumor-free mice. Although less remarkable, two-way ANOVA demonstrates a significant effect of the tumor on the hepatic gene expression of IL-6 (Figure 4C). Two-way ANOVA demonstrated a significant interaction effect between the tumor and DOX in Cxcl10 (Figure 4H). DOX administration reduced tumor-induced upregulation of IL-1β and TNF-α (Figure 4B, D). Neither DOX nor the tumor had a significant effect on the hepatic expression of inflammatory chemokines Mcp-1 and Cxcl1 (Figure 4E, F).

2.5. Effect of DOX Administration on Gene Expression of Inflammatory Markers in the Kidney of Tumor-Free and Tumor-Bearing Mice

Kidneys were harvested from both tumor-free and EL4 lymphoma tumor-bearing juvenile C57BL/6N mice one week following the administration of 4 mg/kg/week of DOX or equivalent volume of sterile saline for 3 weeks (n = 8 per group). Following the extraction of total RNA, renal mRNA expression of inflammatory markers IL-1α, IL-1β, IL-6, TNF- α, Mcp-1, Cxcl1, Cxcl9, and Cxcl10 were determined by real-time PCR. In line with the observed changes in the heart and the liver, tumor-bearing mice had significantly increased renal expression of the pro-inflammatory cytokines IL-1α (Figure 5A), IL-1β (Figure 5B), TNF-α (Figure 5D), and Mcp-1 (Figure 5E) compared to tumor-free mice. Additionally, two-way ANOVA demonstrates a significant effect of the tumor on the renal gene expression of Cxcl1 (Figure 5F), Cxcl9 (Figure 5G), and Cxcl10 (Figure 5H). DOX administration in tumor-bearing mice significantly ameliorated the tumor-induced upregulation of IL-1β (Figure 5B), IL-6 (Figure 5C), TNF-α (Figure 5D), and Mcp-1 (Figure 5E). Within those markers, two-way ANOVA also demonstrated a significant interaction between DOX and the tumor demonstrating the different effects that DOX has on renal inflammatory markers in tumor-bearing compared to tumor-free mice.

2.6. Effect of DOX Administration on Histopathological Features in the Heart, Liver, and Kidney of Tumor-Free and Tumor-Bearing Mice

Hearts, livers, and kidneys were harvested from one cohort of tumor-free and EL4 lymphoma tumor-bearing juvenile C57BL/6N mice one week following the administration of 4 mg/kg/week of DOX or equivalent volume of sterile saline for 3 weeks (n = 4 per group). The histopathological evaluation demonstrated no major pathological changes in the heart (Figure 6A). Only minimal inflammatory cell infiltration, demonstrated as rare foci of minimally cellular mixed (mononuclear and neutrophilic) inflammation, was observed in the liver tissues harvested from DOX-treated tumor-free (three out of four mice), saline-treated tumor-bearing (two out of four mice), and DOX-treated tumor-bearing mice (three out of four mice) (Figure 6B). Histopathological evaluation of the kidneys demonstrated no major pathological changes in all groups except DOX-treated tumor-bearing mice (Figure 6C), with two out of the four mice in this group demonstrating regions of full-thickness tubular regeneration, fibrosis, and mild interstitial inflammation affecting approximately 5% of total tissue area.

2.7. Divergent Effects of DOX on Serum Inflflammatory Cytokines in Tumor-Free Versus Tumor-Bearing Mice

We have previously reported that acute DOX administration elicited a systemic inflammatory response in adult C57BL/6N mice [11]. Herein, we determined the effect of chronic DOX administration on serum levels of the inflammatory markers IL-6 and TNF-α, in juvenile tumor-free and tumor-bearing mice. Saline-treated tumor-bearing mice demonstrated a marked, though statistically insignificant, increase in serum IL-6 (Figure 7A) and TNF-α (Figure 7B) compared to tumor-free mice. Importantly, two-way ANOVA demonstrated a significant interaction between DOX administration and the tumor on the serum levels of IL-6 and TNF-α. Indeed, DOX administration normalized tumor-induced upregulation of IL-6 to a similar level as control tumor-free mice (Figure 7A).