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Nitric oxide-mediated regulation of chemosensitivity in cancer cells
N E Matthews1, M A Adams, L R Maxwell
1Department of Anatomy and Cell Biology, Queen's University, Kingston, ON, Canada.
Background:
Hypoxia in tumors is associated with malignant progression, metastatic spread, and increased resistance to radiotherapy and chemotherapy. Molecular O(2) is required for the cellular production of nitric oxide (NO) by the enzyme NO synthase (NOS), and NO may block components of the adaptive response to hypoxia. Hence, we hypothesized that hypoxia increases drug resistance in tumor cells by inhibiting endogenous NO production.
Methods:
Human breast carcinoma (MDA-MB-231) and mouse melanoma (B16F10) cells were pre-exposed to 20% O(2), 5% O(2), or 1% O(2), incubated with a pharmacologic inhibitor of endogenous NO production, and then treated with chemotherapeutic agents. Resistance was assessed by colony-formation assays, and western blot analysis was used to measure NOS protein levels. All P values were two-sided.
Results:
Incubation of MDA-MB-231 tumor cells in 1% O(2) maximally increased their resistance to doxorubicin and 5-fluorouracil by 8.5-fold (P =.002) and 2.3-fold (P =.002), respectively, compared with incubation in 20% O(2). B16F10 mouse melanoma cells preincubated in 1% O(2) (versus 20% O(2)) for 12 hours exhibited a twofold increase in resistance to doxorubicin (P<.001). The rapid acquisition of drug resistance after exposure to 1% O(2) could be mimicked by incubating the MDA-MB-231 cells for 12 hours with the NOS inhibitor N(G)-monomethyl-Larginine (fivefold increase; P<.001). Conversely, replacement of NO activity by use of the NO-mimetic glyceryl trinitrate (GTN) and diethylenetriamine NO adduct produced statistically significant attenuations in the development of resistance of 59% (P<.001) and 40% (P<.001), respectively, in MDA-MB-231 cells. Treatment of B16F10 cells with GTN produced a 58% reduction in resistance (P<.001). MDA-MB-231 cells expressed all three isoforms of the NOS enzyme at levels that were not altered by exposure to hypoxia.
Conclusions:
NO mediates chemosensitivity in tumor cells, and hypoxia-induced drug resistance appears to result, in part, from downstream suppression of endogenous NO production. These results raise the possibility that administration of small doses of NO mimetics could be used as an adjuvant in chemotherapy.
Insights
Hypoxia, or low oxygen, increases tumor drug resistance by reducing nitric oxide (NO) production. Restoring NO levels may enhance chemotherapy effectiveness in cancer treatment.
Area of Science:
- Oncology
- Molecular Biology
- Biochemistry
Background:
- Tumor hypoxia correlates with increased malignancy, metastasis, and resistance to cancer therapies.
- Nitric oxide (NO) production by NO synthase (NOS) requires molecular oxygen and may counteract hypoxia's adaptive responses.
- Hypothesized that hypoxia enhances tumor cell drug resistance by suppressing endogenous NO production.
Purpose of the Study:
- To investigate the role of endogenous nitric oxide (NO) production in hypoxia-induced chemoresistance.
- To determine if inhibiting NO production mimics hypoxia-induced drug resistance.
- To assess if NO mimetics can overcome hypoxia-induced chemoresistance.
Main Methods:
- Human breast carcinoma (MDA-MB-231) and mouse melanoma (B16F10) cells were exposed to varying oxygen levels (20%, 5%, 1% O2).
- Cells were treated with a NO synthase (NOS) inhibitor or NO mimetics (glyceryl trinitrate, diethylenetriamine NO adduct) before chemotherapy.
- Drug resistance was quantified using colony-formation assays; NOS protein levels were assessed via Western blot.
Main Results:
- Hypoxia (1% O2) significantly increased MDA-MB-231 cell resistance to doxorubicin (8.5-fold) and 5-fluorouracil (2.3-fold).
- Hypoxia also increased B16F10 cell resistance to doxorubicin (2-fold).
- Inhibition of NOS mimicked hypoxia-induced resistance, while NO mimetics attenuated it, indicating NO's role in chemosensitivity.
Conclusions:
- Nitric oxide (NO) plays a crucial role in mediating chemosensitivity in tumor cells.
- Hypoxia-induced drug resistance is partly due to suppressed endogenous NO production.
- NO mimetics could potentially serve as an adjuvant therapy to enhance chemotherapy efficacy.