Recombinant human lactoferrin induces apoptosis, disruption of F-actin structure and cell cycle arrest with selective cytotoxicity on human triple negative breast cancer cells

Abstract

Breast cancer is the most frequently diagnosed cancer among women worldwide. Here, recombinant human lactoferrin (rhLf) expressed in Pichia pastoris was tested for its potential cytotoxic activity on a panel of six human breast cancer cell lines. The rhLf cytotoxic effect was determined via a live-cell HTS imaging assay. Also, confocal microscopy and flow cytometry protocols were employed to investigate the rhLf mode of action. The rhLf revealed an effective CC₅₀ of 91.4 and 109.46 µg/ml on non-metastatic and metastatic MDA-MB-231 cells, with favorable selective cytotoxicity index values, 11.68 and 13.99, respectively. Moreover, rhLf displayed satisfactory SCI values on four additional cell lines, MDA-MB-468, HCC70, MCF-7 and T-47D (1.55-3.34). Also, rhLf provoked plasma membrane blebbing, chromatin condensation and cell shrinkage in MDA-MB-231 cells, being all three apoptosis-related morphological changes. Also, rhLf was able to shrink the microfilaments, forming a punctuated cytoplasmic pattern in both the MDA-MB-231 and Hs-27 cells, as visualized in confocal photomicrographs. Moreover, performing flow cytometric analysis, rhLf provoked significant phosphatidylserine externalization, cell cycle arrest in the S phase and apoptosis-induced DNA fragmentation in MDA-MB-231 cells. Hence, rhLf possesses selective cytotoxicity on breast cancer cells. Also, rhLf caused apoptosis-associated morphologic changes, disruption of F-actin cytoskeleton organization, phosphatidylserine externalization, DNA fragmentation, and arrest of the cell cycle progression on triple-negative breast cancer MDA-MB-231 cells. Overall results suggest that rhLf is using the apoptosis pathway as its mechanism to inflict cell death. Findings warranty further evaluation of rhLf as a potential anti-breast cancer drug option.

Keywords: Anti-cancer drug discovery; Apoptosis; Cancer; Cell cycle; Cytoskeleton; Lactoferrin.

Fig. 1

The hLf open reading frame (ORF; 2136 bp) was cloned into pPICZα vector (Invitrogen), flanked by α-factor secretion signal and 6xHis tag, at 5′ and 3′ terminus, respectively. Additionally, several silent point mutations were introduced to eliminate undesired restriction sites and they are indicated by vertical arrows, including the nucleotide change and position (bp) in the rhLf ORF sequence. The resulting plasmid was named as pIGF5 (IGF-Iglesias Figueroa). AOX1, the alcohol oxidase 1 promoter from P. pastoris. The BleoR gene conferred the resistance to zeocin, a selective antibiotic for transformation

Fig. 2

The rhLf protein expression and purification analysis revealed the presence of a ~ 80 kDa protein via both SDA-PAGE (a) and western blots (b). Histidine (6x) tagged rhLf protein expressed in P. pastoris clone was purified from the yeast culture supernatant, by immobilized nickel affinity chromatography. After electrophoresis, an acrylamide gel was stained with Coomassie brilliant blue (a) and after western blotted was exposed to anti-his tag antibody (b): 1—molecular weight markers (MW) in kDa; 2—commercial bovine lactoferrin; 3—Histidine (6x) tagged rhLf protein partially purified; 4—a culture supernatant protein extracts from non-transformant P. pastoris, used as negative control

Fig. 3

The rhLf induces cytotoxicity on MDA-MB-231 cells in a dose-depended manner. Cells were exposed to a concentration gradient of rhLf and incubated for 24 h. Differential nuclear staining assay and a bioimager system were used to determine the cytotoxicity percentages (a; y-axis). When experimental samples were compared with solvent, 5% v/v H₂O, (*) and untreated control cells (‡), the P values were of < 0.0001 and < 0.0001, respectively (a). Cells treated with 2 mM of H₂O₂ were used as positive control for cytotoxicity (a, e). Each experimental point represents the average of four replicas and error bars their corresponding standard deviation (a). A portion of representative live-cell images, merging the Hoechst and propidium iodide (PI) channels, used to obtain the cytotoxic activity are depicted in b–e. Nuclei exhibiting a blue signal are considered to be living cells, whereas nuclei exhibiting magenta color, as a consequence of Hoechst and PI colocalization signal, are consider dead cells (b–e). Cytotoxic concentration 50% (CC₅₀; in μg/ml) is defined as the lactoferrin’s concentration required to disrupt the plasma membrane of 50% of the cell population after 24 h of incubation. CC₅₀ was calculated via linear interpolation (https://www.johndcook.com/interpolator.html)

Fig. 4

The rhLf inflicts cell death, DNA fragmentation and morphological changes associated with apoptosis in MDA-MB-231 triple negative breast cancer cells. Cells were exposed to 50 μg/ml of rhLf for 24 h and double stained with Hoechst and PI. Representative live-cell confocal microscope images, captured using Hoechst channel (nuclei; a), brightfield differential interference contrast (DIC; b), propidium iodide channel (c) and overlay of the preceding three images (d), are depicted. The black head arrows are indicating the blebs formation, whereas the redhead arrows are denoting cell shrinkage and chromatin condensation, dead cells, PI positive. Also, the white head arrow is signaling a cell with a non-condensed nucleus, non-cell shrinkage and non-blebs formation, as well as PI negative, therefore a living cell. Also, as above, live-cell images from untreated cells are depicted as follow: e Hoechst channel (nuclei); f brightfield; g propidium iodide channel; and overlay of the preceding three images (h). Scale bar = 10 μm

Fig. 5

The rhLf disrupted the F-actin (filamentous) cytoskeleton organization of MDA-MB-231 cells. Representative immunofluorescence confocal microscopy images of MDA-MB-231 cells, triple stained with Alexa-568-conjugated phalloidin, Alexa-488 anti-tubulin, and DAPI, displaying the microfilaments (actin; Alexa-568 channel) and microtubules (tubulin; Alexa-488 channel) organization and nucleus (blue channel), respectively. Cells were treated as follow: a for 2 h with rhLf; b for 4 h with rhLf; c with cytochalasin D; d untreated; e with H₂O solvent control; f with paclitaxel; g with DMSO solvent control. The left column of images correspond to the F-actin microfilaments (red), and the center column microtubules (green) and the right column merged images of the Alexa-568 and Alexa-488 channels with DAPI (blue; nuclei) channel

Fig. 6

The rhLf disturbed the F-actin (filamentous) cytoskeleton organization of Hs27 cells. Representative immunofluorescence confocal microscopy images of Hs27 cells, triple stained with Alexa-568-conjugated phalloidin, Alexa-488 anti-tubulin, and DAPI, displaying the microfilaments (actin; Alexa-568 channel) and microtubules (tubulin; Alexa-488 channel) organization and nucleus (blue channel). Cells were treated as follow: a with rhLf; b with cytochalasin D; c untreated; d with H₂O solvent control; e with paclitaxel; f with DMSO solvent control. The left column of images correspond to the F-actin microfilaments (red), the center column microtubules (Alexa-488) and the right column merged images of the Alexa-568 and Alexa-488 channels with DAPI (blue; nuclei) channel

Fig. 7

The rhLf evokes significant phosphatidylserine externalization on MDA-MB-231 cells after 24 h of incubation. The rhLf mechanism of inflicting cell death, apoptosis or necrosis, was examined after cells were double stained with annexin V-FITC and PI and monitored via flow cytometry. The total percentage of apoptotic cell populations (y-axis) is expressed as the sum of both early and late stages of apoptosis (green bars), whereas that cells stained only with PI, were considered as necrotic cell population (also in y-axis; black bars; a). Each bar represents the average of four independent measurements, and error bars their corresponding standard deviation (a). Representative two-parameter flow cytometry dot plots used to acquire the percentages of apoptotic and necrotic subpopulations (b–e). The following controls were included: cells treated with 5% v/v of H₂O, as a solvent control (a, c); untreated cells as negative controls (a, d); and cells exposed to 2 mM of H₂O₂, as a positive control for cytotoxicity (a, e); analysis using two-tailed Student’s paired t test are included as P values (a). Approximately, 10,000 events (cells) were acquired per sample. For acquisition and analysis purposes, Kaluza flow cytometry software was used (Beckman Coulter)

Fig. 8

The rhLf provoked apoptosis-induced DNA fragmentation and alteration of the cell-cycle distribution profile on MDA-MB-231 cells after 72 h of incubation. Cells were collected, and in a single step were fixed, permeabilized, and DAPI-stained, followed by an examination via flow cytometer. a–d The quantification of event/cell frequency percentages is included along with the y-axis, whereas the different treatments are plotted along the horizontal x-axis. Controls for this series of experiments included: as a solvent control, 5% v/v H₂O; as a positive control of cell cycle perturbation, 1 mg/ml of G418; and untreated cells. Each bar represents an average of four replicates, and the error bars signify their corresponding standard deviation. The significance difference (Student t test) between rhLf CC₂₀ (9.48 μg/ml) vs. H₂O-treated (*) or untreated (‡) cells were consistent with P < 0.01, respectively. e–h Representative single-parameter histograms, including four gates that encompassed the percentage of each cell subpopulation per phase of the cell cycle. Gates from left to right: sub-G0/G1, counted as apoptotic DNA fragmentation; G0/G1, S, and G2/M. e–h Event (cells) counts are plotted along the y-axis, whereas DNA content is along the x-axis. Kaluza flow cytometry software was used for sample acquisition and analysis purposes (Beckman Coulter)