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Chemotherapy influences inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) activity
1Department of Histology and Embryology, Ege University School of Medicine, TR-35100 Izmir, Turkey. goktem@med.ege.edu.tr
Abstract:
Multicellular tumor spheroids (MTS) are three-dimensional structural forms of tumors grown in vitro in the laboratory. In this study, the aim was to determine the regulation of inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) expressions on MTS in response to treatment with the commonly used anti-cancer drugs Doxorubicin and Docetaxel. The spheroids were generated using the "liquid overlay" technique. The distribution of both iNOS and eNOS was detected using indirect immunohistochemistry, while the expression of both iNOS and eNOS was measured using Western blots. Additionally, S-phase analysis using 5-bromo-2'-deoxyuridine (BrdU) was done on the MTS after treatment with doxorubicin, docetaxel, and a combination of the two. The Griess method was used to measure nitric oxide (NO) production in the cells. An increase in iNOS immunoreactivity and a decrease in eNOS immunoreactivity were observed after doxorubicin treatment, when compared with the other groups. Furthermore, upregulation of iNOS and downregulation of eNOS were detected in doxorubicin-treated cells using Western blotting. Insignificant iNOS expression was observed in all of the groups, and it was particularly low in the control and drug combination groups. NO production was also found to be significantly high after docetaxel treatment, and cell proliferation decreased after doxorubicin treatment. In conclusion, chemotherapy influences NOS activity differently with the presence of different drugs. The results with iNOS show that doxorubicin is a more effective drug than docetaxel, and a drug combination may play a helpful role in the suppression of tumorigenicity and cancer metastasis. Interestingly, eNOS expression increased after the addition of both docetaxel and the drug combination, and it was found to negatively correlate with the histological grade of the tumor. Therefore, analyzing the expression of both iNOS and eNOS might be very useful for targeting the treatment of breast carcinoma and obtaining better information on prognosis.
Insights
Doxorubicin and docetaxel chemotherapy differentially affect nitric oxide synthase (NOS) in multicellular tumor spheroids. Doxorubicin increased inducible NOS (iNOS), while both drugs influenced endothelial NOS (eNOS), suggesting targeted therapy potential.
Area of Science:
- Molecular Biology
- Cancer Research
- Pharmacology
Background:
- Multicellular tumor spheroids (MTS) are 3D in vitro models of tumors.
- Nitric oxide synthases (NOS), including inducible NOS (iNOS) and endothelial NOS (eNOS), play roles in cancer.
- Understanding drug effects on NOS is crucial for cancer therapy.
Purpose of the Study:
- To investigate the regulation of iNOS and eNOS expression in MTS.
- To determine the effects of Doxorubicin and Docetaxel on NOS in MTS.
- To correlate NOS expression with nitric oxide (NO) production and cell proliferation.
Main Methods:
- MTS were generated using the liquid overlay technique.
- Indirect immunohistochemistry and Western blotting were used to detect iNOS and eNOS.
- Nitric oxide (NO) production was measured using the Griess method; S-phase analysis was performed using BrdU.
Main Results:
- Doxorubicin treatment increased iNOS immunoreactivity and expression, while decreasing eNOS.
- Docetaxel treatment significantly increased NO production.
- eNOS expression increased with docetaxel and combination therapy, correlating negatively with tumor grade.
Conclusions:
- Chemotherapy differentially impacts NOS activity based on the drug used.
- Doxorubicin appears more effective than docetaxel in modulating iNOS; combination therapy may suppress metastasis.
- Analyzing iNOS and eNOS expression could aid in breast carcinoma treatment targeting and prognosis.
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