Effects of HIV Protease Inhibitor Ritonavir on Akt-Regulated Cell Proliferation in Breast Cancer

Abstract

Purpose: These studies were designed to determine whether ritonavir inhibits breast cancer in vitro and in vivo and, if so, how. Experimental Design: Ritonavir effects on breast cancer cell growth were studied in the estrogen receptor (ER)^positive lines MCF7 and T47D and in the ER-negative lines MDA-MB-436 and MDA-MB-231. Effects of ritonavir on Rb-regulated and Akt-mediated cell proliferation were studied. Ritonavir was tested for inhibition of a mammary carcinoma xenograft. Results: ER-positive estradiol-dependent lines (IC 50 , 12-24 Amol/L) and ER-negative (IC 50 , 45 Amol/L) lines exhibit ritonavir sensitivity. Ritonavir depletes ER-a levels notably in ER-positive lines. Ritonavir causes G 1 arrest, depletes cyclin-dependent kinases 2, 4, and 6 and cyclin D 1 but not cyclin E, and depletes phosphorylated Rb and Ser 473 Akt. Ritonavir induces apoptosis independent of G 1 arrest, inhibiting growth of cells that have passed the G 1 checkpoint. Myristoyl-Akt, but not activated K-Ras, rescues ritonavir inhibition. Ritonavir inhibited a MDA-MB-231 xenograft and intratumoral Akt activity at a clinically attainable serum C max of 22 F 8 Amol/L. Because heat shock protein 90 (Hsp90) substrates are depleted by ritonavir, ritonavir effects on Hsp90 were tested. Ritonavir binds Hsp90 (K D , 7.8 Amol/L) and partially inhibits its chaperone function. Ritonavir blocks association of Hsp90 with Akt and, with sustained exposure, notably depletes Hsp90. Stably expressed Hsp90a short hairpin RNA also depletes Hsp90, inhibiting proliferation and sensitizing breast cancer cells to low ritonavir concentrations. Conclusions: Ritonavir inhibits breast cancer growth in part by inhibiting Hsp90 substrates, including Akt. Ritonavir may be of interest for breast cancer therapeutics and its efficacy may be increased by sustained exposure or Hsp90 RNA interference.

CYP3A4 Mediates Growth of Estrogen Receptor-positive Breast Cancer Cells in Part by Inducing Nuclear Translocation of Phospho-Stat3 through Biosynthesis of (±)-14,15-Epoxyeicosatrienoic Acid (EET)

“…One potential therapeutic approach may be to block CYP3A4 function with an irreversible inhibitor, such as ritonavir. Indeed, ritonavir has recently been demonstrated by us to inhibit the growth of the MCF7, T47D, and MDA-MB-231 lines studied in this report as well as growth of the MDA-MB-231 xenograft (22). The inhibitory effect of ritonavir on CYP3A4 has recently been confirmed in a co-crystal structure demonstrating that ritonavir covalently and irreversibly binds by its thiazole nitrogen to the CYP3A4 heme iron, thereby reducing the redox potential of the enzyme and preventing reduction by cytochrome P450 reductase (35).…”

“…Western Blot Analysis-RIPA extracts of cells were prepared as described previously, in the presence of protease inhibitors and phosphatase inhibitors (22). Protein concentrations were determined by the micro-bicinchoninic acid (BCA) method, and 30 g of protein was used for each lane of the SDS-polyacrylamide gel Western blot.…”

“…In some experiments, independent triplicate experiments were subjected to electrophoresis in sequential adjacent lanes, necessitating a montage, which was indicated with white lines. Quantification was performed by densitometry of x-ray film exposures using an Alpha-Innotec densitometer (22).…”

“…The cells were then treated with (Ϯ)-14,15-EET (1.0 M) or vehicle (ethanol Ͻ0.01%) for 30 min. RIPA extracts were prepared as described previously in the presence of protease inhibitors and phosphatase inhibitors (22). Independent triplicate experiments were performed.…”

Ritonavir inhibits HIF-1α-mediated VEGF expression in retinal pigment epithelial cells in vitro

“…Ritonavir, a HIV protease inhibitor, exhibits anti-neoplastic effects independent from its ability to inhibit HIV protease. [19][20][21] Also, research has demonstrated that ritonavir can inhibit angiogenesis in Kaposi sarcoma, and head and neck carcinoma. 22,23 However, the inhibitory potency of ritonavir on the HIF pathway and de novo synthesized VEGF in retinal tissues have not been so far investigated.…”