Multiple effects of the Na+/H+ antiporter inhibitor HMA on cancer cells

Abstract

Amiloride derivatives are a class of new promising chemotherapeutic agents. A representative member of this family is the sodium-hydrogen antiporter inhibitor HMA (5-(N,N-hexamethylene amiloride), which has been demonstrated to induce cellular intracytosolic acidification and cell death through the apoptotic pathway(s). This work aims at characterizing drug response of human cancer cell lines to HMA. After a first screening revealing that HMA interferes with cancer cell survival, we focused our attention on SW613-B3 colon carcinoma cells, which are intrinsically resistant to a panel of drugs. Searching for the activation of canonical apoptosis, we found that this process was abortive, given that the final steps of this process, i.e. PARP-1 cleavage and DNA ladder, were not detectable. Thus, we addressed caspase-independent paradigms of cell death and we observed that HMA promotes the induction of the LEI/L-DNase II pathway as well as of parthanatos. Finally, we explored the possible impact of autophagy of cell response to HMA, providing the evidence that autophagy is activated in our experimental system. On the whole, our results defined the biochemical reactions triggered by HMA, and elucidated its multiple effects, thus adding further complexity to the intricate network leading to drug resistance.

Effect of new berberine derivatives on colon cancer cells

“…The status of LC3 was investigated by both immunofluorescence assay and western blotting. As a positive control, parallel samples were treated with 20 µM HMA, a proautophagic drug [9,12]. As expected, several fluorescent cells were visualized in both HMA-treated cancer cell lines, suggesting that autophagy has taken place (Fig.…”

“…Protein expression in HCT116 and SW613-B3 cells treated for 24 h with BBR derivatives was evaluated by western blotting, according to a described protocol [9]. After running and transferring proteins onto nitrocellulose, membranes were incubated overnight at 4°C for 3 h with mAbs against PARP-1 (C2-10, 1:1000; Alexis), total caspase 3 (31A1067, 1:250; Alexis), and γ-tubulin (GTU-88, 1:10,000; Sigma Aldrich).…”

Multiple Effects of Berberine Derivatives on Colon Cancer Cells

“…Samples were then incubated with the MAb to mtHSP70 (JG1, Alexis, Vinci Biochem, Vinci, Italy, diluted 1 : 50) according to [28]. For poly(ADP-ribose) analysis, fixation and incubation with the monoclonal antibody 10H (ALX-804-220, Alexis, diluted 1 : 100) and with the appropriate secondary antibody were performed as previously described [26]. For p53 and p21 analysis, cells were lysed with hypotonic buffer (10 mM Tris-HCl, 2.5 mM MgCl 2 , 10 mM β -glycerophosphate, 0.1% Igepal, 0.2 mM PMSF, and 0.1 mM Na 3 VO 4 ) and washed with washing buffer (10 mM Tris-HCl, 2.5 mM MgCl 2 , 10 mM β -glycerophosphate, 0.2 mM PMSF, and 0.1 mM Na 3 VO 4 ).…”

“…After washings with PBS, samples were incubated with bovine serum albumin (4% in PBS) for 10 min and with the polyclonal antibody 2775 to LC3 (Cell Signaling, diluted 1 : 100) for 1 h at 37°C followed by the incubation with the appropriate secondary antibody [26]. As a positive control of autophagy, cells were treated for 24 h with 20  µ M HMA [26]. Three independent experiments were performed.…”

Dihydroartemisinin increases apoptosis of colon cancer cells through targeting Janus kinase 2/signal transducer and activator of transcription 3 signaling

“…PARP may be activated following recognition and binding to an SSB or a free end of DNA, whereby it catalyzes the synthesis of poly(ADP-ribose) (17). In so doing, the enzyme modifies numerous nuclear proteins and enzymes following translation, thus regulating a series of molecular events in cells.…”