L-selenocystine is toxic for Nrf2 addicted cells

Brief Summary

Nrf2 addiction, characterized by persistent Nrf2 activation, is common in many cancers and contributes to malignancy and resistance to treatment. L-selenocystine (SeC) selectively induces cell death in Nrf2-addicted CRC cells by inhibiting both the Nrf2 and autophagy pathways, while sparing non-Nrf2-addicted cells such as mesenchymal stem cells (MSCs) and normal colon cells.

The study (R1) demonstrates that SeC targets the p62-Keap1-Nrf2 axis, leading to reduced Nrf2 activity, increased reactive oxygen species (ROS), and impaired autophagy, ultimately triggering cell death in cancer cells.

Highlights

• Nrf2 Addiction: Persistent activation of Nrf2 in colorectal cancer cells promotes survival and resistance to therapies.
• Selective Cytotoxicity: L-selenocystine (SeC) selectively induces death in Nrf2-addicted CRC cells but not in non-Nrf2-addicted MSCs or normal colon cells.
• Mechanism of Action: SeC inhibits the Nrf2 and autophagy pathways via the p62-Keap1-Nrf2 axis, reducing antioxidant defenses and increasing oxidative stress.
• Role of ROS: SeC increases ROS production, contributing to its cytotoxic effects in Nrf2-addicted cells.
• Autophagy Inhibition: SeC impairs autophagy, a survival mechanism for cancer cells, further enhancing its cytotoxicity.
• Antioxidant Rescue: Pretreatment with antioxidants like glutathione (GSH) partially rescues CRC cells from SeC-induced death but does not fully prevent it.

Expanded Summary: Mechanisms of Action

Nrf2 Pathway Inhibition

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a key regulator of cellular redox homeostasis. Under normal conditions, Nrf2 is kept inactive by its interaction with Kelch-like ECH-associated protein 1 (Keap1), which promotes its degradation. However, in cancer cells, persistent activation of Nrf2 (termed Nrf2 addiction) helps them survive oxidative stress by upregulating antioxidant genes. This overactivation is linked to poor prognosis and therapy resistance in cancers like colorectal cancer (CRC).

The study shows that SeC selectively inhibits the Nrf2 pathway in Nrf2-addicted CRC cells by disrupting the p62-Keap1-Nrf2 axis. Specifically:

• p62 protein sequesters Keap1, preventing it from degrading Nrf2. SeC treatment reduces p62 levels in CRC cells, restoring Keap1’s ability to target Nrf2 for degradation.
• This leads to a decrease in Nrf2-regulated proteins such as NAD(P)H quinone dehydrogenase 1 (NQO1), glutathione peroxidase 4 (GPX4), and cystine/glutamate transporter (xCT), which are critical for antioxidant defense.

In contrast, SeC activates the Nrf2 pathway in non-Nrf2-addicted MSCs, indicating that its inhibitory effects are specific to cancer cells with constitutively active Nrf2.

ROS Production

SeC induces a significant increase in reactive oxygen species (ROS), particularly superoxide radicals, within Nrf2-addicted CRC cells. The elevated ROS levels overwhelm the antioxidant defenses of these cancer cells due to the inhibition of the Nrf2 pathway. This oxidative stress contributes directly to cell death.

• The study confirms that ROS production occurs both in the cytoplasm and mitochondria of CRC cells after SeC treatment.
• Pretreatment with antioxidants like glutathione (GSH) or N-acetylcysteine (NAC) reduces ROS levels and partially rescues cell viability, but does not fully prevent cell death at higher doses of SeC.

Autophagy Inhibition

Autophagy is a cellular process that helps maintain homeostasis by degrading damaged organelles and proteins. In cancer cells, autophagy can act as a survival mechanism under stress conditions such as chemotherapy or oxidative stress.

The study demonstrates that SeC inhibits autophagy in Nrf2-addicted CRC cells by:

• Reducing levels of key autophagy-related proteins such as ULK1, Beclin-1, and LC3-II.
• Blocking autophagic flux, as confirmed by experiments using chloroquine (CQ), an autophagy inhibitor.

Inhibition of autophagy further sensitizes CRC cells to SeC-induced death. The study also shows that pretreatment with GSH can reverse some aspects of autophagy inhibition at lower doses of SeC but is ineffective at higher doses.

AKT/mTOR Pathway

SeC also affects the AKT/mTOR signaling pathway, which is known to regulate autophagy. The study finds that:

• SeC increases phosphorylation of AKT at Ser473 while decreasing phosphorylation of ULK1 at Ser757, which inhibits autophagy initiation.
• This suggests that SeC may suppress autophagy through activation of the AKT/mTOR pathway.

Sensitivity Modulation

The sensitivity of CRC cells to SeC correlates with their level of Nrf2 addiction. For example:

• WiDr cells, which exhibit high levels of Nrf2 activation, are more sensitive to SeC than C₂BBe₁ cells with lower Nrf2 activity.
• Knockdown of Keap1 in C₂BBe₁ cells increases their sensitivity to SeC by enhancing Nrf2 activation.
• Conversely, knockdown of Nrf2 using siRNA reduces the sensitivity of WiDr cells to SeC.

These findings suggest that targeting Nrf2 addiction could be a viable strategy for selectively killing cancer cells while sparing normal tissues.

Conclusion

L-selenocystine effectively induces selective cytotoxicity in Nrf2-addicted colorectal cancer cells by inhibiting both the Nrf2 and autophagy pathways. This dual inhibition leads to increased oxidative stress and impaired cellular survival mechanisms, making it a promising therapeutic approach for cancers characterized by persistent Nrf2 activation.