Introduction: Breast cancer is the most common malignancy in women and the third most common cancer worldwide behind lung and colon cancer1,2. Triple-negative breast cancer (MDA-MB-231) represents 10-20% of all breast cancer cases and lacks the progesterone, estrogen, and HER2 receptors that serve as targets for breast cancer therapies, making their treatment more difficult and increasing patient’s 5-year mortality1,4,6. An integral aspect of breast cancer efficacy is the disruption, and ultimately reversal, of the host Na+/H+ Exchanger Isoform 1 (NHE1), which allows the cancer to create an acidic and hypoxic microenvironment promoting tumorigenesis, metastasis, and chemoresistance5-7. Administering Epithelial Sodium Channel Inhibitors (ENaC-I) at high doses has an inhibitory effect on the NHE1 via induction of oxidative stress leading most likely to a necrotic mechanism of killing3,7. Methods: 231-KO is a NHE1 knockout line6. Amiloride and its derivatives (ENaC-I) were tested to determine if NHE1 transporter disruption worked concomitantly with other treatments to enhance cancer cell killing4,6. SiRNA mediated Cyclin D1 knockdown testing was performed to determine Amiloride’s ability to target non-growing cancer tissue3,6. The N-Ac ROS scavenger was used to determine the role of ROS3. Results: NHE1 activity but not expression was elevated in MDA-MB-2316. Hexamethylene Amiloride (HMA), a derivative of amiloride with a C5 substitution, was found to be cytotoxic to all breast cancer cells irrespective of their molecular profile, proliferative status, or species of origin3. NHE1 knockout significantly reduced tumor growth and cell invasion over 60 days4. HMA given with paclitaxel reduced cancer viability even further4. HMA was found to be cytotoxic to all breast cancer lines tested (MDA-MB-231, MCF7, SKBR3) but not to non-transformed human breast cells (HMEC4, MCF10A)3. Inhibition of cell cycle progression at G1/S does not affect ability of HMA to induce death in breast cancer cells3,7. N-Ac decreased the cytotoxic ability of Amiloride on cancer cells, indicating ROS play an active role in Amiloride’s killing mechanism3,5. Although there were some intermediates in the Amiloride mechanistic pathway of autophagy and apoptosis, necrosis was determined to be the key killing pathway for Amiloride and its derivatives with confirmation via electron microscopic observations of nuclear structure maintenance with oncosis, perinuclear accumulation of intracellular organelles, and a loss of plasma membrane integrity3,4. Conclusions: Amiloride and its derivatives can serve as a viable option for direct treatment of breast cancers or in adjunct with current chemotherapeutic regimes4. The determined mechanism of amiloride killing is a novel mechanism of necrosis3,5,7.
- Nadia Harbeck, Michael Gnant, Breast cancer, The Lancet, Volume 389, Issue 10074, 18–24 March 2017, Pages 1134-1150, ISSN 0140-6736, http://dx.doi.org/10.1016/S0140-6736(16)31891-8.
- DeSantis, C. E., Lin, C. C., Mariotto, A. B., Siegel, R. L., Stein, K. D., Kramer, J. L., Alteri, R., Robbins, A. S. and Jemal, A. (2014), Cancer treatment and survivorship statistics, 2014. CA: A Cancer Journal for Clinicians, 64: 252–271. doi:10.3322/caac.21235
- Ashley R. Rowson-Hodel, Anastasia L. Berg, Jessica H. Wald, Jason Hatakeyama, Kacey VanderVorst, Daniel A. Curiel, Leonardo J. Leon, Colleen Sweeney, Kermit L. Carraway III, Hexamethylene amiloride engages a novel reactive oxygen species- and lysosome-dependent programmed necrotic mechanism to selectively target breast cancer cells, Cancer Letters, Volume 375, Issue 1, 28 May 2016, Pages 62-72, ISSN 0304-3835, http://dx.doi.org/10.1016/j.canlet.2016.02.042.
- Leon LJ, Pasupuleti N, Gorin F, Carraway KL III (2013) A Cell-Permeant Amiloride Derivative Induces Caspase-Independent, AIF-Mediated Programmed Necrotic Death of Breast Cancer Cells. PLoS ONE 8(4): e63038. doi:10.1371/journal.pone.0063038
- Aredia F, Czaplinski S, Fulda S, Scovassi AI. Molecular features of the cytotoxicity of an NHE inhibitor: Evidence of mitochondrial alterations, ROS overproduction and DNA damage. BMC Cancer. 2016;16:851. doi:10.1186/s12885-016-2878-9.
- Amith SR, Wilkinson JM, Baksh S, Fliegel L. The Na+/H+ exchanger (NHE1) as a novel co-adjuvant target in paclitaxel therapy of triple-negative breast cancer cells. Oncotarget. 2015;6(2):1262-1275.
- Francesca Aredia, et al. Multiple effects of intracellular pH modulation in cancer cells. Can Cell Microenviron 2014; 1: e136. doi: 10.14800/ccm.136.