ROS generation in Cancer Cells

 Intracellular generation of ROS/RNS and its signaling mechanism

Increased levels of ROS and RNS are found in almost all types of cancers due to various reasons including rapid metabolic switch, increased growth rate, quick cell division, etc. However, tumor cells highly express antioxidants in response to increased oxidative stress. ROS and RNS possess single unpaired electrons in their outermost shell which makes them highly reactive. ROS and RNS include superoxide, hydroxyl radical, nitric oxide, peroxyl radical, nitroxyl anion, nitrosonium cation, higher oxides of nitrogen, etc. Non-radical ROS molecules are hydrogen peroxide and singlet oxygen etc.  Oxidative phosphorylation is the main source of ROS as complexes involving oxidative metabolism release them into the mitochondrial matrix or intermembrane space. The superoxides are generated by complex 1 and complex 3 and are released into intermembrane space or matrix (4). From intermembrane space, ROS are leaked into the cytoplasm through the mitochondrial permeability transition pores (MPTP) (5, 6). Similarly, aquaporin serves as a channel for superoxide and water transport, the former being generated by superoxide dismutase (SOD) (7, 8). In addition to mitochondria, peroxisomes also act as the source of ROS, while some ROS molecules are also generated in the cytoplasm (10, 11). Growth factors, cytokines, INF-γ, TNFα, PDGF, EGF, insulin, TGFβ, IL-1, angiotensin, etc., hyper-activate the production of ROS in the cell (13-23).

RNS molecules are strong oxidants responsible for cell damage, cell death, and cell cycle arrest e.g., peroxynitrite, nitroxyl, nitrosonium cation, dinitrosyl complexes, etc. (225). Like oxidative stress, cells undergo nitrosative stress upon increased production of RNS molecules. No synthase is the main enzyme responsible for generating NO- from L-arginine and oxygen. Radical NO- acts as a second messenger involved in various signaling processes e.g., smooth muscle relaxation and synaptic transmission of neuronal signals. Their production is regulated by Ca+2/Calmodulin signaling complex, hsp90, caveolin (185, 226). Oxidative products of radical NO- are also RNS molecules e.g.  NO2-, NO3-. They react with the -heme group of hemoglobin and have an impact on vasodilation during hypoxic conditions (60, 100, and 101). The strong interaction of NO2- and O2-form highly oxidant RNS molecule i.e., ONOO-. ONOO- has a strong affinity for reacting with CO2 to form CO3- and NO2- which add oxide and nitrite groups to proteins and halt their bioactivity (312, 326). They induce post-translational modification which includes S-nitrosylation (on thiol group of regulatory proteins, kinase, phosphatases, metabolic, enzymes, receptors, transcription factors, etc.,), Glutathionylation (on cysteine sulfhydryl groups, GSH, GSNO), tyrosine nitration (on sulfhydryl group, zinc-thiolate groups).

Oncogenic and apoptotic signaling

ROS and RNS molecules activate NADPH oxidase, mutant K-Ras, mutant Rho-GTPase, and elevated Rac-1 expression which are some of the prominent signaling mechanisms involved in cell survivability. PI3/Akt pathway activated by ONOO- molecules helps in anti-apoptotic and cell survival signals. H2O2-mediated MAPK/Erk pathway activation helps in regulating cell proliferation. Oxidation of cysteine residues of Ras (an upstream molecule) by ROS activates it and increases proliferation. Inhibiting or scavenging ROS molecules in breast cancer cells induces apoptosis and slows down metastasis by promoting cell adhesion. The potential inhibitors of MAPK/Erk pathways also target ROS generation. TNFα, IL-1, mediated NF-kB activation induces anti-apoptotic and anti-inflammatory gene expression. In breast cancer and oral squamous carcinoma cells, increased basal level of ROS induces NF-kβ activation and thus increased proliferation. Increased oxidative stress caused by higher ROS generation and lower anti-oxidant production activates NF-kβ signaling helping tumor cells to survive.

The role of ROS differs with changes in cancer cell types. Pancreatic cancer cells and glioma cells treated with H₂O₂ from outside (via Erk1/2 activation) undergo cell death, marking cell death response to an increased level of ROS generation from its basal level. In contrast, cells of ovarian cancer, breast cancer, and melanoma are able to survive and proliferate with the increased ability to metastasize upon treatment with ROS and activation of ROS. H2O2 and NO (mitochondrial ROS and RNS by-products) can induce JNK signaling which phosphorylates anti-apoptotic molecules (Bcl2, Bcl-xl) and inactivate them. JNK mediated increased expression of Bax and homodimer formation to downregulate mitochondrial membrane integrity. Apart from these proteins, p38, p53, and FOXO3a induce apoptosis in response to ROS generation. TNF1 receptor induces ROS generation, but the fate of the tumor cell depends upon the comparative expression of proliferative and apoptotic signaling.

Conclusion

TNF1 receptor induces cell death by activating caspases while TNFR family molecules also enhance PI3/Akt signaling for cell survivability (X). It also mediates anti-apoptotic signaling by activating SOD, and NF-kβ signaling. Similarly, in response to oxidative stress generated by ROS, PI3/Akt pathway gets activated and helps in cell survival by reducing apoptosis and increasing the production of antioxidants. Those cells which could not counter oxidative stress undergo apoptosis such as seen in pancreatic tumors and glioma cells. It suggests that ROS molecules are themselves not anti-apoptotic or proliferative molecules. But rather than it is the tumor cell’s ability to counter oxidative stress via increased production of antioxidants and thus maintaining the intracellular oxidative stress.

As decreased production of antioxidants and immune cells mediated increased ROS generation in tumor cells induce cell cycle arrest and apoptosis with the release of cytochrome C. Thus, we can differentiate tumors on the basis of their metabolic ability to curb oxidative stress and could be tested for ROS-based targeted therapy. Summarising oncogenic or apoptotic signaling induction by ROS relies on the ability of cancer cells to enhance their survival by activating ROS-mediated proliferative signals and increasing the production of antioxidants to counter oxidative stress.