Login to Access Video or Poster Abstract: MP73-12
Sources of Funding: none

Introduction

The combination of the human immunodeficiency virus (HIV) protease inhibitors lopinavir and ritonavir has been a standard regimen used to treat HIV infection. Ritonavir acts as a chemical booster to enhance lopinavir's activity. Lopinavir has recently been shown to act against cancer by inducing endoplasmic reticulum (ER) stress, and we thought that the combination would kill renal cancer cells by inducing robust ER stress.

Methods

The viability and clonogenicity of renal cancer cells (769-P, 786-O, Caki-2) treated with clinically feasible concentrations of lopinavir (10-40 µM) and/or ritonavir (5-10 µM) were assessed by MTS assay and colony formation assay. Apoptosis was evaluated by annexin-V assay. Cell cycle analysis was done using flow cytometry. Induction of ER stress and the expression of cell-cycle regulators, apoptosis-associated proteins, NOXA, Akt, BCL-2, and survivin were evaluated by western blot analysis. Drug synergism was assessed by the Chou-Talalay method.

Results

Lopinavir in combination with ritonavir inhibited renal cancer growth synergistically (combination index <1). The combination also inhibited clonogenic survival of cancer cells significantly (p <0.05). It perturbed the cell cycle by inhibiting the expression of cyclin D1 and cyclin-dependent kinase 4, increasing the cells in the sub-G1 fraction. The combination caused apoptosis synergistically: 10-20 µM lopinavir increased the number of annexin-V positive cells and the expression of cleaved poly(ADP-ribose) polymerase slightly but in combination with 10 µM ritonavir increased both drastically. As expected, the combination induced ER stress evidenced by the increased expression of the ER stress markers glucose-regulated protein 78 and endoplasmic reticulum resident protein 44. Furthermore, increased expression of NOXA confirmed that the combination-induced apoptosis was a result of ER stress. We also found that the combination decreased the expression of the anti-apoptotic proteins BCL-2 and survivin by inhibiting the Akt signaling pathway.

Conclusions

The combination of lopinavir and ritonavir induces ER stress and causes renal cancer apoptosis synergistically. Inhibition of the Akt pathway is another important mechanism of its action.

Funding

none