Glucose Deprivation Induced by Acarbose and Oncolytic Newcastle Disease Virus Promote Metabolic Oxidative Stress and Cell Death in a Breast Cancer Model

Abstract

Cancer cells are distinguished by enhanced glucose uptake and an aerobic glycolysis pathway in which its products support metabolic demands for cancer cell growth and proliferation. Inhibition of aerobic glycolysis is a smart therapeutic approach to target the progression of the cancer cell. We employed acarbose (ACA), a particular alpha-glucosidase inhibitor, to induce glucose deprivation combined with oncolytic Newcastle disease virus (NDV) to enhance antitumor activity. In this work, we used a mouse model of breast cancer with mammary adenocarcinoma tumor cells (AN3) that were treated with ACA, NDV, and a combination of both. The study included antitumor efficacy, relative body weight, glucose level, hexokinase (HK-1) level by ELISA, glycolysis product (pyruvate), total ATP, oxidative stress (ROS and reduced glutathione), and apoptosis by immunohistochemistry. The results showed significant antitumor efficacy against breast cancer after treatment with combination therapy. Antitumor efficacy was accompanied by a reduction in body weight and glucose level, HK-1 downregulation, inhibition of glycolysis products (pyruvate), total ATP, induction of oxidative stress (increase ROS and decrease reduced glutathione), and apoptotic cell death. The findings propose a novel anti-breast cancer combination involving the suppression of glycolysis, glucose deprivation, oxidative stress, and apoptosis, which can be translated clinically.

Keywords: apoptosis; cancer; glucose deprivation; oncolytic virotherapy; oxidative stress.

FIGURE 1

Antitumor efficacy of acarbose (ACA), Newcastle Disease Virus (NDV), and a combination of both against a mammary adenocarcinoma AN3 in vivo model. (A) Relative tumor volumes over 18 days were plotted. Compared to the control group, ACA, NDV, or a combination of both generated a significant (p < 0.0001) decrease in relative tumor volume. Compared to both monotherapy groups, the combination therapy of acarbose and Newcastle Disease Virus (ACA-NDV) demonstrated significantly greater tumor size reduction. (B)e Growth inhibition curve demonstrated that the combined therapy group had the highest overall tumor growth inhibition, followed by the NDV group. The ACA group showed the least growth inhibition. In the control group, the tumors continued to grow during the experiment. (C) ACA efficiently reduced body weight in mice bearing breast cancer. Bodyweight measured after treatment with ACA, NDV, and combination every 3 days indicates that ACA induces a marked decrease in body weight. By contrast, NDV did not induce a significant effect on body weight. ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗∗ p < 0.01, and ∗ p < 0.05versus CONT.

FIGURE 2

ACA induces glucose deprivation. (A), Glucose measured after Acarbose (ACA) treatment, Newcastle Disease Virus (NDV), and a combination of Acarbose and Newcastle Disease Virus (ACA-NDV), ACA exhibited a significant ∗∗ p < 0.01 decreased level of glucose in ACA and ACA-NDV groups compared with the positive control (P. CONT) (bearing breast cancer) and negative control N. CONT. (healthy mice) groups. (B) ELISA assay quantified the concentration of the Hexokinase enzyme. We observed a significant decrease in the expression of the HK-1 protein in the NDV- and ACA-NDV-treated group compared to the untreated control group. ACA alone had no significant impact on HK-1 concentration. (C) Measurement of pyruvate content. (D) Measurement of total ATP concentration. In combination with NDV, ACA efficiently inhibits glycolysis product (pyruvate) and ATP. To confirm the effect of ACA and NDV combination therapy on glycolysis products, we examined pyruvate and total ATP level in tumor tissue. We found that the ACA-NDV treatment had significantly reduced glycolysis product (pyruvate) and ATP compared to the untreated group. ∗∗∗ p < 0.01, ∗∗ p < 0.01, and ∗ p < 0.05 versus Cont.

FIGURE 3

ACA and NDV combination induces metabolic oxidative stress. We detected ROS and reduced GSH in breast cancer tissue, and we observed that ROS levels were significantly increased in treated groups (ACA, NDV, and ACA-NDV) than in the untreated groups. Reduced GSH levels were lower in ACA-NDV combination therapy than in the untreated group. (A) Measurement of ROS level. (B) Measurement of reduced glutathione concentration. ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗∗ p < 0.01, and ∗ p < 0.05 versus CONT.

FIGURE 4

ACA-NDV combination therapy induces apoptosis, confirmed by immunohistochemistry analysis for cleaved caspase-3. Results showed a mild effect on breast cancer cells in monotherapy (ACA and NDV alone)-treated groups, while the combination therapy of ACA-NDV has more cas-3 expression than the single therapy. ∗∗∗ p < 0.001 versus Cont.