الفهرس 
INTRODUCTIONOnly 14 pages are availabe for public view
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Abstract
Breast cancer is the most common cancer diagnosed in women. It is the second most common cause of death from cancer among women in the world. Although existing therapy regimens extend life, they are challenged by resistance issues, leading to poor results.
Doxorubicin (DOX) is still one of the most extensively utilized chemotherapeutic treatments against a variety of hematological and non-hematological cancers. It is the most effective agent in the treatment of breast cancer patients. Although a greater cumulative dosage is necessary for effective treatment, unfortunately it causes dose-related organ damage. Moreover, resistance to this agent is common, representing a major obstacle to successful treatment.
Tumor heterogeneity, changed genetic signatures, and metabolic reprogramming are the main causes of treatment resistance since they all allow the tumor to serially adapt to treatments over the course of treatment. Glucose is not a major source of ATP production, whereas fatty acid is a major source of electrons for ATP production through FAO and oxidative phosphorylation in cancer cells. Metabolic reprogramming not only promotes tumor growth but also introduces metabolic liabilities that can be used to cure cancer. Chemotherapies that target metabolism have been proven to be effective cancer treatments for decades, demonstrating that there is a therapeutic window to target malignant metabolism.
Trimetazidine (TMZ) is an anti-ischemic (anti-anginal) metabolic drug that enhances myocardial gluconeogenesis by inhibiting mitochondrial 3 ketoacyl coenzyme A thiolase enzyme, which results in inhibition of β oxidation of free fatty acid and stimulating glucose oxidation.
The aim of the current study was to test the hypothesis that TMZ could synergize DOX cytotoxicity both in vitro in BC cell lines and in vivo in the Ehrlich solid phase carcinoma model and the potential underlying mechanisms for such effects.
Experimental design
In vitro part:
The estrogen receptor positive BC cell line MCF-7 and the triple negative BC cell line MDA-MB231 were implanted into 96-multiwell plates at a density of 2 × 103 cells per well and permitted to adhere overnight. In the following day the cells were treated with either DOX (0.001–10 μM) or TMZ (0.1–1000 μM)-alone for 72 h. Following that a combination of DOX and TMZ were tested at a ratio of 1:5 and 1:10 DOX: TMZ.
The following parameters in vitro were investigated:
• SRB cytotoxicity assay for single TMZ and DOX treatments as well as DOX/TMZ 1:10 or DOX/TMZ 1:5 co-treatments in MCF-7and MDA-MB231 cell lines
• Synergy analysis to determine the combination index (CI) values and dose-reduction index (DRI) for DOX/TMZ 1:10 or DOX/TMZ 1:5 combinations in BC cell lines using Calcusyn software.
In vivo part:
The tumor-bearing induced mice were randomly separated into five equal groups (n=10).
• group 1: served as control and received the vehicle.
• group 2: received DOX alone (4 mg/kg/week, i.p.).
• group 3: received TMZ monotherapy (4 mg/kg/week, i.p.).
• group 4: was treated with a combination of DOX/TMZ at 1:10 ratio (dose of DOX 0.4 mg/kg/week, i.p. + dose of TMZ 4 mg/kg/week, i.p.)
• group 5: was treated with DOX/TMZ at 1:5 ratio (dose of DOX 0.8 mg/kg/week, i.p. + dose of TMZ 4 mg/kg/week, i.p.)
The dose ratios of DOX to TMZ in group 4 and group 5 were based on the in vitro synergy analysis results. The treatment continued for two weeks. Tumor capsules and intestinal tissues were dissected out and either stored at −80 °C for cytotoxicity analyses or reserved in 10 % neutral buffered formalin for subsequent histopathological and immunohistochemistry examinations.
The following parameters in vivo were assessed:
a) Assessment of tumor volume
b) Histological examination of tumor capsules and intestinal tissues
c) Assessment of p-AMPK--α (western blotting)
d) Assessment of PGC1--α (western blotting)
e) Assessment of NAD and NADH (colorimetric)
f) Assessment of nuclear level of NF-KB (P65) (ELISA)
g) Assessment of ATP level (ELISA)
a) Apoptosis studies
III. Bax and Bcl-2 ratio (ELISA)
IV. Active caspase-3 protein abundance (ELISA, Immunohistochemistry)
Results:
• The in vitro cytotoxicity studies in MCF-7 and MDA-MB231 human BC cell lines revealed that TMZ synergized DOX cytotoxicity in the ER-α positive cell line MCF-7.
• The tumor volume monitoring data along with histopathological examination of the tumors confirmed that DOX-alone produced a significant reduction in the tumor volume and triggered tumor necrosis compared to mock-control. Interestingly, the combined treatment with DOX/TMZ at both ratios both 1:5 and 1:10 significantly exhibited antitumor activity that is comparable to the effect of the full dose of DOX. Thus, the synergistic effect of DOX/TMZ combined therapy has been confirmed in vivo using the Ehrlich solid phase carcinoma mouse model.
• Co-treatment TMZ significantly reduced NAD+/NADH ratio in comparison to mock-control and DOX-alone groups. Thus, it limited the availability of NADH needed to generate ATP via the electron transport chain. Furthermore, DOX treatment significantly reduced amounts of NAD+ and impaired the mitochondrial activity. Furthermore, treatment with DOX/TMZ combinations reduced both NAD+/NADH ratio and diminished ATP levels.
• Co-treatment with DOX/TMZ 1:10 or 1:5 resulted in a significant decrease in ATP levels along with a significantly boosted levels of p-AMPK in comparison to the mock control. Thus, the combination therapy triggered AMPK activation, at least partly, to compensate for the reduction in total ATP levels resulting in tumor cell death. Moreover, DOX treated alone -induced cancer cell death was linked to AMPK activation.
• Treatment with DOX/TMZ enhanced PGC1-α protein expression. The increased expression of PGC1-α was reported to activate catabolic pathways that promote mitochondrial biogenesis and function.
• Treatment with DOX/TMZ significantly suppressed the nuclear levels of NF-κB (p65) compared to mock-treated control which, at least partly, contributes to TMZ-mediated sensitization of the tumor cells to lower doses of DOX.
• Treatment with DOX/TMZ combination significantly boosted Bax/Bcl2 ratio along with increasing the active caspase-3 levels in comparison to the mock-treated mice. These findings support that co-treatment with DOX/TMZ redirected the tumor cells to apoptosis. There was no noticeable induction of apoptosis in TMZ-alone treated group.
• In comparison to controls, colon specimens from DOX-treated groups showed acute colitis and sever enteritis. However, co-treatment DOX: TMZ at 1:10 ratio alleviated the histological alterations induced by DOX in the colon tissues. In contrast, co-treatment with 1:5 DOX: TMZ resulted in histopathological damage and inflammatory changes suggestive of colitis in 50% of the examined specimens.
In conclusion, the current work highlighted the potential merit of TMZ as a sensitizer to DOX chemotherapy. The combined therapy of TMZ (4 mg/kg/week) and DOX (0.8 mg/kg/week) generated a synergistic antitumor response in mice-bearing Ehrlich solid phase carcinoma. Such effect can be, at least partly, due to TMZ modulatory effect on mitochondrial energetics and metabolic stress in addition to boosting DOX-induced apoptosis. Moreover, `the DOX-dose reduction produced by the synergistic 1:10 DOX: TMZ combination significantly alleviated DOX-induced damage on the colon mucosa. Both TMZ and DOX are authorized for use in the clinical setting. Additionally, TMZ exhibits a favorable safety record. Thus, the current study represents a springboard to support further clinical testing of DOX/TMZ combination against ER-α positive tumors since it regulates mitochondrial dynamics and function in adipose tissue.