Combined Effects of Fludarabine and Interferon Alpha on Autophagy Regulation Define the Phase of Cell Survival and Promotes Responses in LLC-MK2 and K562 Cells

Abstract

Autophagy is a known mechanism of cells under internal stress that regulates cellular function via internal protein recycling and the cleaning up of debris, leading to healthy live cells. However, the stimulation of autophagy by external factors such as chemical compounds or viral infection mostly tends to induce apoptosis/cell death. This study hypothesizes that manipulation of the autophagy mechanism to the pro-cell survival and/or decreased pro-viral niche can be a strategy for effective antiviral and anticancer treatment. Cells susceptible to viral infection, namely LLC-MK2, normal monkey epithelium, and K562, human immune-related lymphocyte, which is also a cancer cell line, were treated with fludarabine nucleoside analog (Fdb), interferon alpha (IFN-α), and a combination of Fdb and IFN-α, and then were evaluated for signs of adaptive autophagy and STAT1 antiviral signaling by Western blotting and immunolabeling assays. The results showed that the low concentration of Fdb was able to activate an autophagy response in both cell types, as demonstrated by the intense immunostaining of LC3B foci in the autophagosomes of living cells. Treatment with IFN-α (10 U/mL) showed no alteration in the initiator of mTOR autophagy but dramatically increased the intracellular STAT1 signaling molecules in both cell types. Although in the combined Fdb and IFN-α treatment, both LLC-MK2 and K562 cells showed only slight changes in the autophagy-responsive proteins p-mTOR and LC3B, an adaptive autophagy event was clearly shown in the autophagosome of the LLC-MK2 cell, suggesting the survival phase of the normal cell. The combined effect of Fdb and IFN-α treatment on the antiviral response was identified by the level of activation of the STAT1 antiviral marker. Significantly, the adaptive autophagy mediated by Fdb was able to suppress the IFN-α-mediated pSTAT1 signaling in both cell types to a level that is appropriate for cellular function. It is concluded that the administration of an appropriate dose of Fdb and IFN-α in combination is beneficial for the treatment of some types of cancer and viral infection.

Keywords: K562; LLC-MK2; STAT1; autophagy; fludarabine; interferon-alpha.

Figure 1

The time- and concentration-dependent effects of Fdb on (A) LLC-MK2 and (B) K562 cells. The cultured cells were treated with Fdb concentrations of 0, 2.5, 5, and 10 µM. Percentage of cell viability was determined after 24, 72, and 96 h of incubation at 37 °C in a 5% CO₂ incubator by MTT assay. The dotted line indicates axis intercepts of IC₅₀ and the log (dose) estimating value.

Figure 2

Western blot analysis of protein expression in LLC-MK2 and K562 cell treatment with Fdb (concentrations 0, 2.5, 5, 10 µM) for 24 h. The total cellular proteins were extracted and transferred to nitrocellulose membranes. The membranes were immunostained with specific antibodies against the autophagy-related proteins p-mTOR, Beclin-1, and LC3B-I/LC3B-II and processed for reaction color development. β-Actin was used as an internal control.

Figure 3

Confocal immunofluorescent photomicrographs taken from confocal microscopy of LLC-MK2 cells after 24 h of treatment with Fdb (5.0 µM). The treated cells were briefly fixed in absolute methanol at −20 °C, permeabilized with 0.1% Triton X-100, and then immunohistochemically stained with autophagy-activated protein (LC3B-I/LC3B-II) antibody and secondary antibody-conjugated Alexa^® 555. The fluorescent illumination of LC3B-II foci (autophagosome) was demonstrated in the cytoplasm of the cells. LC3B Alexa^®-stained = red, DAPI nuclear-stained = blue.

Figure 4

Western blot analysis of an autophagy inducer (p-mTOR) and the signaling activator (p-STAT1) protein expression in representative cells that rendered IFNR after treatment with IFN-α. LLC-MK2 cells were treated with IFN-α (10 U/mL) for 5, 30, 60, and 120 min. The cellular total proteins were extracted and transferred to nitrocellulose membranes. The membranes were immunostained with p-mTOR and p-STAT1 antibodies and processed for reaction color development. β-Actin was used as an internal control. The experiment was performed in triplicate and the band density was normalized with β-actin. Protein electrophoresis films were digitized and the relative density level of p-mTOR and p-STAT1 were measured by ImageJ^® software.

Figure 5

Western blot analysis of autophagy response and the antiviral response in cultured LLC-MK2 and K562 cells that were exposed for 60 min to IFN-α (10 U/mL) before treatment with Fdb (10 μM) for 24 h. The cells were evaluated for autophagy response mediation via p-mTOR and LC3B-I/LC3B-II activation, as well as antiviral response via p-STAT1 signaling molecules. The total proteins were extracted and transferred to nitrocellulose membranes. The membranes were immunostained with p-mTOR, LC3B-I/LC3B-II, and p-STAT1 antibodies and processed for reaction color development. β-Actin was used as an internal control. Protein electrophoresis films were digitized and the relative density level of p-mTOR (normalized with β-Actin), autophagosome marker LC3B-II (normalized with LC3B-I), and p-STAT1 (normalized with β-Actin) were measured by using ImageJ^® software. One-way ANOVA statistical analysis was performed (*, **, *** indicates a p-value of <0.05, 0.01, 0.001, respectively). The experiments were analyzed in triplicate and SEM is marked by an error bar.

Figure 6

Confocal immunofluorescent micrographs of LLC-MK2 cells after exposure to IFN-α (10 U/mL) for 60 min before treatment with Fdb (10 μM) for an additional 24 h. The cells were briefly fixed in absolute methanol at −20 °C and permeabilized with 0.1% Triton X-100 before being immunohistochemically stained with autophagy-activated protein (LC3B-I/LC3B-II) antibody and secondary antibody-conjugated Alexa^® 555. The fluorescent illumination demonstrated the clustering of LC3B foci or autophagosome formation in the cytoplasm of the cells. LC3B Alexa^®-stained = red, DAPI nuclear-stained = blue.