Anti-Breast Cancer Properties and In Vivo Safety Profile of a Bis-Carbazole Derivative

Abstract

Background: Carbazoles represent one of the most important classes of nitrogen-based tricyclic aromatic heterocycles and are present in natural sources and chemically obtained drugs. Recently, several research groups disclosed their large biological and chemical applications in different fields, leading to an increased interest towards this class of molecules. Some of the obtained derivatives have been successfully employed in the clinical treatment of different tumor types, but the onset of heavy side effects impaired their efficacy and discouraged their use. Pursuing the aim of obtaining carbazoles with less negative features, a lot of chemically modified compounds have been produced and evaluated. Objectives/Methods: In this paper, we describe the in vitro and in vivo evaluation of a bis-carbazole derivative with strong anticancer properties against two breast cancer cell lines. Results: This compound has been found to impact the cell cytoskeleton dynamics, triggering the activation of some key proteins playing a role in the intrinsic and extrinsic apoptotic pathways. Equally important, this derivative has been found to be selective for cancer cells and has shown a safe profile in Balb/c-treated mice. Conclusions: Overall, the disclosed outcomes represent an important landmark for encouraging further studies directed toward the potentiation of this lead to be potentially exploited in both preclinical and clinical applications.

Keywords: anticancer; apoptosis; carbazole derivatives; cytoskeleton; toxicity studies.

Figure 1

N,N’-bis-(6-bromo-1,4-dimethyl-9H-carbazol-3-ylmethylene)-hydrazine (1).

Figure 2

MDA-MB-231 cells were exposed to 0.3 µM of compound 1 or vehicle (CTRL) for 24 h. After processing, cells were observed, and images were taken at 20× magnification by means of an inverted fluorescence microscope. The green fluorescence, visible only for compound 1-treated cells, indicates nuclear DNA damage. Panels (A): DAPI (CTRL and 1) excitation/emission wavelength 350 nm/460 nm. Panels (B): CF™488A (CTRL and 1) excitation/emission wavelength 490 nm/515 nm. Panels (C): panels (A) and (B) overlay. Images are representative of three separate experiments.

Figure 3

Determination of caspase-3/7, -8, and -9 activity levels following the treatment of MDA-MB-231 cells with compound 1 (0.3 µM) for 24 h, reported as a percentage versus the DMSO-treated cells, used as a control (CTRL). Data represent the means ± SD of three different experiments. **** p < 0.0001, treated versus CTRL.

Figure 4

MDA-MB-231 cells were treated with 0.3 µM of 1 or vehicle (CTRL) for 24 h, then processed as described in the experimental section and observed at 40× magnification using an inverted fluorescence microscope. Compound 1 treatment induced Bid (green fluorescence, panel (B), 1) translocation into mitochondria (red fluorescence, panel (C), 1), as noticeable in the merge channel (panel (D), 1). Nucleus staining is also shown (panels (A)). Panels (A): DAPI, excitation/emission wavelength 350 nm/460 nm. Panels (B): Alexa Fluor^® 488, excitation/emission wavelength 490 nm/515 nm. Panel (C): MitoTracker Deep Red FM probe, excitation/emission wavelength 644/665 nm. Panels (D): panels (A) and (B) overlay. Representative fields of three different experiments are shown.

Figure 5

Docking simulations suggested that 1 binds a region proximal to the Paclitaxel-binding site of tubulin. The most important amino acid residues involved in the interactions are evidenced. Cyan ribbons: Tubulin, α-subunit, Salmon ribbon Tubulin β-subunit. Yellow sticks, 1 binding mode. Oxygen atoms belonging to evidenced aminoacids are colored in red, while nitrogen in blue. Bromide moieties are evidenced in amaranth.

Figure 6

In vitro tubulin polymerization assay. The assembly of tubulin into microtubules was followed by measuring the turbidity (absorbance, A) at 350 nm for 3600 s at 37 °C. The polymerization curves, in the presence of 1 (1 μM) or not (CTRL, DMSO), are shown. Moreover, the graphic shows the curves obtained using two reference molecules, Vinblastine and Paclitaxel, (both at 10 μM concentration), used as tubulin-destabilizing and tubulin-stabilizing agents, respectively.

Figure 7

Immunostaining studies on MDA-MB-231 cells treated with compound 1 (0.3 µM), Vinblastine, Paclitaxel (both at 1 µM), or vehicle (CTRL, DMSO) for 24 h. In the control experiment (CTRL), cells exhibited a regular arrangement of cytoskeleton. Vinblastine and Paclitaxel, instead, dramatically impacted the tubulin network. Vinblastine produced tubulin crystal formation (panel (B), V), whereas Paclitaxel induced tubulin bundles and thickened fibers (panel (B), P). Compound 1 exposure produced a morphology similar to Paclitaxel treatment (white arrows). Panels (A): DAPI, excitation/emission wavelength 350 nm/460 nm. Panels (B): β-tubulin (Alexa Fluor^® 488) excitation/emission wavelength 490 nm/515 nm. Panels (C): overlay. Images were taken at 20×, and representative fields were shown.

Figure 8

Actin immunofluorescence studies were conducted on MDA-MB-231 cells treated with 1 (0.3 µM) or vehicle (CTRL, DMSO) for 24 h. Cells were processed as detailed in the Section 2. Control cells (CTRL) showed a normal actin cytoskeleton organization, whereas under 1 exposure, the actin network appeared irregularly arranged and packed into the cell cytoplasm. Panels (A): nuclear DAPI staining (λex/λem = 350/460 nm); Panels (B): β-actin (Alexa Fluor^® 568; λex/λem = 644/665 nm); Panels (C): overlay. Images were taken at 20× and show representative fields.

Figure 9

Body weight variation in mice after 1 week of treatment was ns (not significant), compared to the negative control by t-test; NC: negative control; 1: compound 1-treated group.

Figure 10

Representative photos of mice behaviors: (a) healthy and normal behaviors were registered for negative control (NC) and (b) the compound 1 group.

Figure 11

Representative photos for mice NC (a) and the compound 1-treated group (b); healthy global appearance; no visible difference or abnormalities at the injection site.

Figure 12

Effect of compound 1 on lung morphology: (a) negative control with normal lung morphology; (b) compound 1-treated group showed normal morphologic features of lung, while visible weight gain for the organ was detected.

Figure 13

Biochemical analyses of the serum from groups of mice untreated or treated with compound 1; (a) aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP); (b) creatinine and uric acid; and (c) sodium, potassium, chloride, calcium, and phosphorus serum levels were measured in the two groups of mice (control and compound 1-treated) and after 1 week.

Figure 14

Hematological analysis of mice blood from groups of mice untreated and treated with compound 1; RBC: red blood cells; HB: hemoglobin; HCT: hematocrit; MCH: mean corpuscular hemoglobin; MCHC: mean corpuscular hemoglobin concentration; PLT: platelet count; MCV: mean corpuscular volume; WBC: white blood cells; LYM: lymphocytes; NEU: neutrophils; EOS: eosinophils; MAC: macrophages; BASO: basophiles.