Efavirenz and Lopinavir/Ritonavir Alter Cell Cycle Regulation in Lung Cancer

Abstract

Highly active anti-retroviral treatment (HAART) is currently the most effective treatment for HIV/AIDS. Additionally, HIV positive patients receiving HAART have a better health-related quality of life (HRQoL). Cancers previously associated with HIV/AIDS also known as the AIDS defining cancers (ADCs), such as Kaposi's sarcoma and non-Hodgkin's lymphoma have been on the decline since the introduction of HAART. However, non-AIDS defining cancers (NADCs), in particular, lung cancers have been documented to be on the rise. The association between the use of HAART components and lung carcinogenesis is poorly understood. This study aimed at elucidating the effects of two HAART components [efavirenz (EFV), and lopinavir/ritonavir (LPV/r)] on lung cancer. This was achieved through the use of in vitro cell biological approaches to assess cell health, including cell viability, Real Time Cell Analysis (RTCA) growth monitoring, evaluation of the cell cycle, and progression to apoptosis, following on drug treatments. At plasma level concentrations, both EFV and LPV/r induced S-phase arrest, while at lower concentrations both drugs promoted the progression of cells into G2/M phase following cell cycle FACS analysis. At higher concentrations although cell viability assays reflected anti-proliferative effects of the drugs, this was not statistically significant. RTCA showed a significant decline in cell viability in response to the highest dose of LPV/r. Dual staining by Annexin V-FITC and PI confirmed significant pro-apoptotic effects were promoted by LPV/r. Both EFV and LPV/r exert double-edged oncogenic effects on MRC-5 and A549 lung cells, acting to either promote cell proliferation or to enhance apoptosis. This is affected by EFV and LPV/r altering cell cycle progression, with a significant S-phase arrest, this being an indication of cellular stress, cytotoxicity, and DNA damage within the cell.

Keywords: cell cycle; cell death; cell proliferation; efavirenz; lopinavir/ritonavir; lung cancer; real-time cell analysis.

Figure 1

Alamar blue assay analysis. (A) The percentage (%) of AB reduction representing the MRC-5 cell viability. (B) The A549 cell viability in response to the EFV drug treatment. (C) The MRC-5 cell viability in response to LPV/r drug treatment relative to control. (D) The representation of the A549 cell viability in response to the LPV/r cytotoxic effects. A–D represent treatments vs. control at 24, 48, and 72 h, the blue box indicates the most relevant physiological dose, and effects on cell viability are statistically insignificant, with p > 0.0.5. The graphs are a representative of three independent experiments, which were done in triplicate each.

Figure 2

MRC-5 and A549 cell proliferation in response to EFV and LPV/r. (A) Cell growth curve of MRC-5 cells treated with EFV (B) Growth curves representative of A549 cells treated with EFV. (C) MRC-5 growth curves representing cells treated with LPV/r. (D) Growth curves for A549 cells treated with LPV/r. The curves were plotted as a function of normalized CI vs. time in ARV treated vs. control. (E) The slope function of MRC-5 cells representing the response to EFV treatment over a 24 h time. (F) The slope function representing the response of A549 cells to EFV drug treatment at 24 h time intervals. (G) The slope function demonstrating MRC-5 cell response to LPV/r drug treatment, monitored at 24 h intervals. (H) A slope function representing A549 cells treated with LPV/r at 24 h intervals. The slope function represents the rate of cell detachment, and thus cell death for each of the drug concentrations. Results represent three independent experiments done in triplicate each. Error bars denote SEM; *p < 0.05; **p < 0.01.

Figure 3

Cell cycle distribution in response to ARV drugs. (A,B) Show bar graphs of EFV treated MRC-5 cells at 24 and 48 h. (C,D) Demonstrate bar graphs of A549 cells treated with EFV at 24 and 48 h. (E,F) Illustrate bar graphs of MRC-5 cells treated with LPV/r at 24 and 48 h, while (G,H) are bar graphs of A549 LPV/r treated cells at 24 and 48 h. The increase in S-phase, (S-phase arrest) observed here is statistically significant with p < 0.0 1. A significant increase in G2/M with p < 0.05 in A549 10 μM LPV/r treated cells is also evident. Error bars denote SEM; *p < 0.05; **p < 0.01.

Figure 4

FACS apoptosis analysis of MRC-5 and A549 cells in response to LPV/r. (A,B) Show cytograms and bar graphs of MRC-5 cells treated with LPV/r, while (C,D) represent cytograms and bar graphs of LPV/r treated A549 cells. Error bars denote standard error of the means (SEM); *p < 0.05; **p < 0.01; ***p < 0.001. Results represent three independent experiments which were done three times independently.

Figure 5

Diagrammatic representation of the effects of EFV and LPV/r at low and high doses. Both EFV and LPV/r exhibit pro-survival effects at low doses, while anti-proliferative and cytotoxic effects are observed at high doses. The solid arrows represent the effects of the drugs on cellular health, while the dashed line shows partial/dual effect. At a high dose, EFV is anti-proliferative, arresting cellular growth, while low doses favor survival modes, as also observed with low LPV/r dose. In contrast, moderate (plasma-level) and high LPV/r doses have anti-proliferative and cytotoxic properties on the cells.