Investigation of apoptosis in tumor cells using genetically encoded sensors of caspases activity.

Abstract

A complex cascade of molecular events occurs in apoptotic cells. Cisplatin was established to primarily induces cell death of cancer cells by apoptosis. The defect in apoptotic signaling could confer cisplatin resistance. It becomes increasingly clear that along with formation of platinum–DNA adducts, that inhibit replication and transcription and induce cell death, cisplatin activates other cellular responses. It is supposed that a contribution of targets other than DNA is an important factor in the molecular mechanisms of unresponsiveness of cancer cells to cisplatin and other platinum-based drugs. Eearly mechanisms of apoptosis accompanied by caspases activation is poorly understood. Study of these events can provide valuable data on molecular mechanisms of apoptosis and identification of reasonable target for chemotherapeutic treatment.
The aim of this study was to analyze caspases cascade activation in living cancer cells in vitro during cisplatine treatment using fluorescence lifetime imaging microscopy (FLIM).
We performed imaging of apoptosis used genetically encoded FRET-based sensors for caspase-3 activity mKate2-DEVD-iRFP and caspase-8/9/3 activity mScarlet-LEND-DEVD-iRFP670 to during cisplatine treatment in cultured cancer cells.
Activation of caspase-3 in the cells was detected in the same time as the emergence of morphological evidence of apoptosis after 24 h exposure to cisplatine. The mean fluorescence lifetime (τm) of a donor protein mKate2 in the cells was 1.61 ± 0.05 ns. Loss of FRET reaction led to significant increase in mKate2 fluorescence lifetime to 2.25 ± 0.09 ns, which indicated the activation of caspase-3 and the initiation of apoptosis.
Activation of caspase-8/9/3 in the cells was detected before morphological manifestation of apoptosis (after 0.5 h). The mean fluorescence lifetime (τm) of a donor protein mScarlet in the cells was 2.21 ± 0.09 ns and as a result of the loss of FRET reaction significant increase of fluorescence lifetime to 2.70 ± 0.10 ns was observed in those cells in which we subsequently observed morphological signs of apoptosis.
We also imaged endogenous metabolic cofactor NAD(P)H in the same cells using FLIM. Analysis of the ratios of the relative contributions of the free to protein-bound NAD(P)H (a1/a2) in the tumor cells showed a significant decrease in the a1/a2 ratio starting from 24 h after cisplatine treatment. The observed changes testify to a switch toward an OXPHOS after cisplatine treatment
Therefore, multiparameter fluorescence lifetime imaging enables simultaneous analysis of metabolic activity and caspases activation in cancer cells during apoptosis. Our results indicate that the new sensors can provide valuable data on the early event in cancer cells undergoing apoptosis in response to chemotherapy.
ACKNOWLEDGEMENTS:
This work was supported by the Russian Science Foundation (grant No20-14-00111)

Speaker

Alena Gavrina
Privolzhskiy Research Medical University, Nizhny Novgorod
Russia

Discussion

Ask question