Login to Access Video or Poster Abstract: MP73-09
Sources of Funding: none

Introduction

Inducing endoplasmic reticulum (ER) stress is a novel approach to cancer treatment. The pan-deacetylase inhibitor panobinostat inhibits molecular chaperones and increases the amount of unfolded proteins. The antiretroviral drug nelfinavir suppresses proteasomes and inhibits degradation of unfolded proteins. We postulated that combining nelfinavir with panobinostat would kill renal cancer cells effectively by inhibiting degradation of panobinostat-increased unfolded proteins and thereby inducing ER stress.

Methods

After renal cancer cells (769-P, 786-O, Caki-2) were treated with clinically feasible concentrations of panobinostat (15-60 nM) and/or nelfinavir (10-20 µM), their viability and clonogenicity were assessed by MTS assay and colony formation assay. Apoptosis was evaluated by annexin-V assay. Cell cycle analysis was done using flow cytometry. Western blotting was used to evaluate the induction of ER stress (increased expression of ER stress markers) and the expression of cyclin D1, cyclin-dependent kinase (CDK) 4, AMP-activated protein kinase (AMPK), S6 ribosomal protein, and ERK1/2.

Results

The combination of panobinostat and nelfinavir was demonstrated by isobologram analysis to inhibit cancer cell growth synergistically. It also suppressed colony formation significantly (p <0.05). The combination decreased the expression of cyclin D1 and CDK4, leading to the accumulation of the cells in the sub-G1 fraction. It also induced robust apoptosis synergistically: 60 nM panobinostat alone caused slight to moderate increases in the amount of annexin-V positive cells but in combination with 20 µM nelfinavir increased it drastically (up to 100-fold). Mechanistically, the combination induced ER stress synergistically. This ER stress caused dephosphorylation of S6 ribosomal protein by increasing the expression of mammalian target of rapamycin (mTOR) inhibitor AMPK, showing that the combination also inhibited the mTOR pathway. The combination also inhibited the ERK pathway.

Conclusions

The combination of panobinostat and nelfinavir inhibits renal cancer growth by inducing ER stress synergistically. Inhibiting the mTOR and ERK pathways are also important mechanisms of action.

Funding

none