Extended Abstract
Background and Aim: Colon cancer remains as one of the leading malignancies of the gastrointestinal tract, and its progression is fundamentally influenced by disruptions of apoptotic signaling and activation of hypoxiadriven angiogenesis pathways (Johnson & Fleet, 2013). Dysregulation of the Bcl2associated X protein/Bcell lymphoma 2 (Bax/Bcl2) balance contributes to enhanced tumor cell survival, whereas activation of the hypoxiainducible factor1 alpha/vascular endothelial growth factor A axis (HIF1α/VEGFA) promotes angiogenic expansion within the tumor microenvironment, thereby facilitating tumor growth, invasion, and metastasis (Spanoudaki et al., 2023). Aerobic exercise has been proposed as a nonpharmacological intervention capable of modulating tumor biology by improving metabolic regulation, tissue oxygenation, and inflammatory status (Schadler et al., 2016). However, limited studies have simultaneously evaluated the integrated response of apoptotic and hypoxiadependent angiogenic pathways to structured aerobic exercise in colon tumor tissue. To address this knowledge gap, the present study investigated the effects of 14 weeks of aerobic exercise on the Bax/Bcl2 apoptotic ratio and the HIF1α/VEGFA angiogenesis pathway in a rat model of dimethylhydrazine (DMH)induced colon cancer, with the aim of elucidating coordinated molecular adaptations to exercise.
Materials and Methods: This controlled experimental study was conducted on 40 male Wistar rats aged 10–12 weeks and weighing 250–300 g. The animals were maintained under standard laboratory conditions including controlled temperature, a 12h light–dark cycle, and ad libitum access to food and water. Animals were randomly allocated, using block randomization based on age and body weight, into four groups (n=10 per group) including healthy control (HC), sham (SH), sedentary cancer (DS), and cancer with aerobic exercise (DA). Colon cancer was induced in the DS and DA groups by weekly subcutaneous injections of dimethylhydrazine (DMH; 40 mg/kg) for six consecutive weeks, whereas the SH group received the DMH solvent alone.Tumor induction was confirmed macroscopically and by histopathological analysis using H&E staining, consistent with established models.
Following tumor confirmation of tumor induction, the DA group performed 14 weeks of continuous aerobic treadmill exercise, 5 sessions per week, 60 minutes per session, at 60% of the maximal running speed determined individually using an incremental test. Incremental tests were repeated at weeks 5, 9, and 13 to adjust intensity. Nontraining groups were placed on a nonmoving treadmill to control for environmental stress.
Colon tumor tissues were collected 24 hours after the final intervention following overnight fasting. RNA extraction (kit RB1001), cDNA synthesis, and gene expression quantification via RTqPCR (SYBR Green; StepOnePlus system) were performed for Bax, Bcl2, HIF1α, and VEGFA. TBP gene was used as the reference gene due to its stability in inflammatory and cancer models. Primer sequences were validated for efficiency (90–110%) and specificity using meltcurve analysis. All reactions were performed in technical triplicates. Statistical analyses included Shapiro–Wilk test, the Levene test, oneway analysis of variance (ANOVA), and Tukey posthoc tests. In addition, Bonferroni correction was applied as a sensitivity analysis. Tumor burden and molecular markers were analyzed as fold changes relative to HC. The significance level was set at p<0.05.
Findings: DMH administration successfully induced colon tumors in the DS and DA groups, whereas no macroscopic lesions were observed in HC and SH groups. Tumor burden indices were markedly elevated in the DS group, which exhibited the highest tumor number and total tumor weight. Aerobic exercise significantly attenuated tumor burden in the DA group compared with the DS group. Specifically, the mean tumor number decreased from 6.80±1.40 in DS to 4.10±1.20 in DA, while total tumor weight decreased from 1.92±0.35 g to 1.21±0.28 g (p<0.01). Molecular analyses revealed substantial differences in apoptotic and angiogenic gene expression markers across groups. Oneway ANOVA revealed significant differences among the study groups in tumor burden indices as well as molecular variables, including Bax, Bcl2, HIF1α, and VEGFA (p≤0.006), with large effect sizes (η²=0.38–0.81). Descriptive statistics and ANOVA results for these variables are presented in Table 1.
Tukey’s post hoc analysis showed that the DS group had significantly higher tumor number and total tumor weight compared with HC and SH (p<0.001), whereas both indices were significantly lower in DA compared with DS (p<0.01). Regarding apoptotic markers, Bax expression was significantly lower in DS compared with HC (p<0.001), while Bcl2 expression was significantly higher (p<0.001). In the DA group, Bax expression was significantly higher than in DS (p=0.04), whereas Bcl2 expression was significantly lower (p=0.01). For angiogenesisrelated markers, HIF1α and VEGFA expression levels were significantly higher in DS compared with HC (both p<0.001). In addition, both markers were significantly lower in DA compared with DS (both p<0.001).
Conclusion: Fourteen weeks of structured aerobic exercise in a DMHinduced rat model of colon cancer resulted in meaningful molecular and phenotypic improvements. Aerobic exercise significantly reduced tumor burden, restored apoptotic signaling through increased Bax and decreased Bcl2 expression, and attenuated the activation of the hypoxiadependent angiogenesis pathway through reductions in HIF1α and VEGFA. The coordinated modulation of these pathways suggests that aerobic exercise exerts multidimensional anticancer effects, consistent with preclinical models proposing enhanced oxygenation, improved vascular function, and modulation of survival signaling (Ashcraft et al., 2016; Schadler et al., 2016). Although these findings cannot be directly extrapolated to humans, they support the potential role of structured aerobic exercise as a complementary non-pharmacological strategy alongside conventional therapies. Future studies should investigate proteinlevel changes, functional angiogenic outcomes, and individualized exercisedose responses to optimize clinical applications.
Ethical Considerations: All experimental procedures involving animals were conducted in accordance with the institutional guidelines for the care and use of laboratory animals and complied with internationally accepted ethical standards for animal research. The study protocol was reviewed and approved by the Ethics Committee of the Islamic Azad University, Najafabad Branch, Iran (Approval No. IR.IAU.NAJAFABAD.REC.1404.225).
Compliance with Ethical Guideline: The study complied fully with international standards for the ethical treatment of laboratory animals and followed ARRIVE guidelines.
Funding: No external funding was received for this research.
Conflicts of Interest: The authors declare no conflicts of interest.