Abstract
The antitumor agent 11β (CAS 865070-37-7), consisting of a DNA-damaging aniline mustard linked to an androgen receptor (AR) ligand, is known to form covalent DNA adducts and to induce apoptosis potently in AR-positive prostate cancer cells in vitro; it also strongly prevents growth of LNCaP xenografts in mice. The present study describes the unexpectedly strong activity of 11β against the AR-negative HeLa cells, both in cell culture and tumor xenografts, and uncovers a new mechanism of action that likely explains this activity. Cellular fractionation experiments indicated that mitochondria are the major intracellular sink for 11β; flow cytometry studies showed that 11β exposure rapidly induced oxidative stress, mitochondria being an important source of reactive oxygen species (ROS). Additionally, 11β inhibited oxygen consumption both in intact HeLa cells and in isolated mitochondria. Specifically, 11β blocked uncoupled oxygen consumption when mitochondria were incubated with complex I substrates, but it had no effect on oxygen consumption driven by substrates acting downstream of complex I in the mitochondrial electron transport chain. Moreover, 11β enhanced ROS generation in isolated mitochondria, suggesting that complex I inhibition is responsible for ROS production. At the cellular level, the presence of antioxidants (N-acetylcysteine or vitamin E) significantly reduced the toxicity of 11β, implicating ROS production as an important contributor to cytotoxicity. Collectively, our findings establish complex I inhibition and ROS generation as a new mechanism of action for 11β, which supplements conventional DNA adduct formation to promote cancer cell death.
Highlights
The small molecule anticancer agent 11 was designed to kill androgen receptor (AR)-positive prostate cancer cells by targeting both DNA replication and the expression of steroid-responsive genes
The current study demonstrated that 11 potently induced apoptosis in AR-negative HeLa cells both in vitro and when grown as xenograft tumors in mice
The data suggested that 11 could be active against a wide range of tumors, including those that do not express the AR and added support to the growing body of evidence that oxidative stress may synergize with conventional DNA adducts to promote cancer cell death
Summary
Previous studies [7] revealed that 11 induces apoptosis at lower exposure levels and much more rapidly than DNA-damaging aniline mustard drugs such as chlorambucil (see Fig. 1A), which contains the same alkylating group. The current study demonstrated that 11 potently induced apoptosis in AR-negative HeLa cells both in vitro and when grown as xenograft tumors in mice Both 11 and its analog, 11-dim generated a burst of intracellular ROS, whereas in the same dose ranges, chlorambucil or the steroid moiety of 11 alone (estradien-3-one) did not. Additional experiments indicated that mitochondria were the main intracellular sink for 11 and an important source of the ROS, which were produced due to the specific inhibition of complex I of the mitochondrial electron transport chain (ETC) Together, these findings established ROS production and complex I inhibition as new, DNA adduct-independent mechanisms of 11 that supplemented the ability of the compound to kill tumor cells by covalent DNA damage. The data suggested that 11 could be active against a wide range of tumors, including those that do not express the AR and added support to the growing body of evidence that oxidative stress may synergize with conventional DNA adducts to promote cancer cell death
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