Trastuzumab and Doxorubicin Sequential Administration Increases Oxidative Stress and Phosphorylation of Connexin 43 on Ser368

Abstract

Human epidermal growth factor receptor-2 (HER2) is overexpressed in up to 30% of breast cancer cases, causing a more aggressive tumour growth and poor prognosis. Trastuzumab, the humanized antibody targeted to HER2, increased the life expectancy of patients, but severe cardiotoxicity emerged as a long-term adverse effect. Clinical evidence highlights that Trastuzumab-induced cardiotoxicity drastically increases in association with Doxorubicin; however, the exact mechanisms involved remain incompletely understood. In order to analyse the molecular mechanisms involved and the possible adaptative responses to Trastuzumab and Doxorubicin treatment, in this study, H9c2 cardiomyoblasts were used. Results showed that Trastuzumab and Doxorubicin sequential administration in cardiomyoblast increased cytosolic and mitochondrial ROS production, intracellular calcium dysregulation, mitochondrial membrane depolarization, and the consequent apoptosis, induced by both Trastuzumab and Doxorubicin alone. Furthermore, in these conditions, we observed increased levels of Connexin43 phosphorylated on Ser368 (pCx43). Since phosphorylation on Ser368 decreases gap junction intracellular communication, thus reducing the spread of death signals to adjacent cells, we hypothesized that the increase in pCx43 could be an adaptative response implemented by cells to defend neighbouring cells by Trastuzumab and Doxorubicin sequential administration. However, the other side of the coin is the resulting conduction abnormalities.

Simvastatin Reduces Doxorubicin-Induced Cardiotoxicity: Effects beyond Its Antioxidant Activity

“…We hypothesize that this effect plays a pivotal role in cardioprotection and could account for the observed reduction of apoptosis spreading observed in these cells. Indeed, this hypothesis is in agreement with previous study demonstrating that the disruption of cell-cell communication through GJs is a defense mechanism implemented by cells, known as the "Good Samaritan" mechanism in that it blocks the propagation of harmful stimuli to healthy cells [51,52].…”

“…Carbonyl cyanide p-trifluoromethoxy-pyhenylhydrazone (FCCP, 50 nM final concentration) or Ionomycin (1 µM final concentration) was added into the cuvette in calcium-free buffer in order to evaluate mitochondrial calcium levels and cytosolic calcium levels, respectively. As previously reported [52,54], the ratio of fluorescence intensity of 340/380 nm (F340/F380) is strictly related to intracellular free calcium, so the excitation wavelength was alternated between 340 and 380 nm, and emission fluorescence was recorded at 515 nm. Data were expressed as the percentage of delta (% ∆) increase of the fluorescence ratio (F340/F380 nm) induced by FCCP (50 nM) or Ionomycin (1 µM)-basal fluorescence ratio (F340/F380 nm)/basal fluorescence ratio (F340/F380 nm).…”

“…Subsequently, cells were incubated with Cx43 (1:250) or pCx43 (1:250) antibody and the appropriate secondary antibody (anti-rabbit or anti-mouse FITC antibody, both 1:2000, ThermoFisher, Waltham, MA, USA) for 1 h at 4 • C. To assess the intracellular levels of Cx43, pCx43, and cytochrome c, cells were permeabilized with fix perm buffer (fixing buffer containing 0.1% Triton X) for 30 min and then anti-cytochrome c (1:250), anti-cx43 (1:250), or anti pCx43 (1:250) antibody and the appropriate secondary antibody (anti-rabbit or anti-mouse FITC antibody) were added. (1:2000) for 1 h at 4 • C. Cells collected were evaluated by fluorescence-activated cell sorting (FACSscan; Becton-Dickinson, Milan, Italy), and data obtained were analyzed by means of Cell Quest software [52]. Results are shown as the percentage of positive cells.…”

“…Immunoreactive protein bands were visualized using enhanced chemiluminescence reagents (ECL) in LAS 4000 (GE Healthcare, Milan, Italy). The images were analyzed for densitometry using ImageJ Software (version number 1.44) [52].…”

Vx-809, a CFTR Corrector, Acts through a General Mechanism of Protein Folding and on the Inflammatory Process

“…The excitation wavelength was alternated between 340 and 380 nm, and emission fluorescence was recorded at 515 nm. The ratio of fluorescence intensity of 340/380 nm (F340/F380) was strictly related to intracellular free calcium, as previously reported [ 43 ]. Results were expressed as a delta (Δ) increase in the fluorescence ratio (F340/F380 nm) induced by Thapsigargin—basal fluorescence ratio (F340/F380 nm) or Ionomycin—basal fluorescence ratio (F340/F380 nm).…”