Molecular and Cellular Biochemistry

, Volume 450, Issue 1–2, pp 135–147 | Cite as

Hydrogen peroxide-induced oxidative stress and its impact on innate immune responses in lung carcinoma A549 cells

  • Shishir Upadhyay
  • Saurabh Vaish
  • Monisha DhimanEmail author


The immune responses, involved in recognition of cancer-specific antigens, are of particular interest as this may provide major leads towards developing new vaccines and antibody therapies against cancer. An effective treatment for cancer is still a challenge because there are many mechanisms through which the tumor cells can escape the host immune surveillance. Oxidative stress or respiratory burst which is host’s mechanism to kill the foreign particles is used as defense mechanism by the tumor cells. The tumor cells uses this oxidative stress to form neo-antigens which in turn makes them undetectable and can escape the host immune surveillance. The human lung carcinoma (A549) cells were treated using 100 µM H2O2 to induce oxidative stress, and the extent oxidative modifications were detected at the level of membrane and proteins in form of lipid peroxidation and protein carbonyls respectively. Nitric oxide and iNOS levels were estimated by Griess assay and immunostaining, respectively. The oxidized tumor proteins were visualized on one-dimensional SDS–PAGE. The H2O2-treated (15 min and 24 h post-treatment) A549 cells were co-cultured with THP-1 cells to subsequently visualize the phagocytic activity by Giemsa and CFSE staining to understand the role of neo (oxidized) tumor antigens in eliciting alteration in immune responses. A significant decline in the percent engulfed cells and decrease in the levels of reactive oxygen species was observed. Immunohistostaining for p47phox, which is an important indicator of the oxygen-dependent phagocytosis, showed a decrease in its levels when cells were treated for only 15 min with 100 µM H2O2, whereas at 24-h post-treatment there was no change in the p47phox levels. The study has established oxidative stress as a new pathogenic mechanism of carcinogenesis and will open new avenues for clinical intervention, adjunct therapies for cancer, and its control at the initial stage by targeting these neo-antigens.


Neo-antigens Protein oxidation Respiratory burst Immune surveillance 



5,6-Carboxyfluorescein diester




Dimethyl sulphoxide






3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide


Nitroblue tetrazolium salt


NADPH oxidases


Nitric oxide


Phorbol 12-myristate 13-acetate


Thiobarbituric acid


Thiobarbituric acid reactive species



MD acknowledges the Fast Track Young Investigator Grant (SB/YS/LS- 107/2013) from Dept. of Science and Technology, Govt. of India. The Junior Research fellowship (JRF) provided to SU from Indian Council for Medical Research (ICMR), New Delhi, India, is kindly acknowledged. The authors acknowledge Dr. Anil K. Mantha, Associate Professor, Centre for Animal Sciences, Central University of Punjab, Bathinda, for his critical and constructive comments and the editing of the MS. The Central Instrument Laboratory of Central University of Punjab is acknowledged for the confocal facility.

Compliance with ethical standards

Conflict of interest

The authors declared that they have no conflict of interest.


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© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Animal Sciences, School of Basic and Applied SciencesCentral University of PunjabBathindaIndia
  2. 2.Department of Biochemistry and Microbial Sciences, School of Basic and Applied SciencesCentral University of PunjabBathindaIndia

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