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. 2010 Feb;55(2):95-100.
doi: 10.1016/j.archoralbio.2009.12.001. Epub 2009 Dec 24.

HIV protease inhibitors block oral epithelial cell DNA synthesis

Robert J Danaher  1 , Chunmei Wang, Andrew T Roland, Charlotte S Kaetzel, Richard N Greenberg, Craig S Miller
Affiliations

HIV protease inhibitors block oral epithelial cell DNA synthesis

Robert J Danaher et al. Arch Oral Biol. 2010 Feb.

Abstract

Objectives: Anti-retroviral therapy regimens that include HIV protease inhibitors (PIs) are associated with diverse adverse effects including increased prevalence of oral warts, oral sensorial deficits and gastrointestinal toxicities suggesting that PIs may perturb epithelial cell biology. To test the hypothesis that PIs could affect specific biological processes of oral epithelium, the effects of these agents were evaluated in several oral epithelial cell-lines.

Design: Primary and immortalized oral keratinocytes and squamous carcinoma cells of oropharyngeal origin were cultured in the presence of pharmacologically relevant concentrations of PIs. Their affects on cell viability, cytotoxicity and DNA synthesis were assessed by enzymatic assays and incorporation of 5-bromo-2'-deoxyuridine (BrdU) into DNA.

Results: Viability of primary and immortalized oral keratinocytes as well as squamous carcinoma cells of oropharyngeal origin was significantly reduced by select PIs at concentrations found in plasma. Of the seven PIs evaluated, nelfinavir was the most potent with a mean 50% inhibitory concentration [IC(50)] of 4.1 microM. Lopinavir and saquinavir also reduced epithelial cell viability (IC(50) of 10-20 microM). Atazanavir and ritonovir caused minor reductions in viability, while amprenavir and indinavir were not significant inhibitors. The reduced cell viability, as shown by BrdU incorporation assays, was due to inhibition of DNA synthesis rather than cell death due to cytotoxicity.

Conclusion: Select PIs retard oral epithelial cell proliferation in a drug and dose-dependent manner by blocking DNA synthesis. This could account for some of their adverse effects on oral health.

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Figures

Figure 1
Figure 1. Select HIV PIs inhibit viability of oral keratinocytes
Normal human oral keratinocytes (NHOK), immortalized oral keratinocyte cell-lines (NOK, OKF4, OKF6) and oropharyngeal squamous carcinoma cell-lines (CAL 27, FaDu) were treated with indicated concentrations of nelfinavir for 48 hrs (or 72 hrs for NHOK). Data for nelfinavir-treated cells were normalized to the mean of control (DMSO-treated) cells and are presented as mean % reduction in viability +/− standard error of the mean (SEM; n = 6). Asterisk indicates that the mean for nelfinavir-treated cells is significantly greater than the mean for DMSO-treated cells within the same cell-line (p < 0.05).
Figure 2
Figure 2. Effects of different HIV PIs on viability of oral keratinocytes
The OKF6 immortalized keratinocyte cell-line (A) and the CAL 27 oropharyngeal carcinoma cell-line (B) were treated with the indicated concentrations of PIs for 48 hrs. Data for PI-treated cells were normalized to the mean of control (DMSO-treated) cells and are presented as mean % reduction in viability +/− SEM (n = 6). Asterisk indicates that the mean for PI-treated cells is significantly greater than the mean for DMSO-treated cells within the same cell-line (p < 0.05). Significant reduction in viability was not observed in cells treated with indinavir and amprenavir (data not shown).
Figure 3
Figure 3. Inhibitory effects of nelfinavir and lopinavir on viability of oral keratinocytes is not due to cell death
The OKF6 immortalized oral keratinocyte cell-line (A) and the oropharyngeal carcinoma cell-line CAL 27 (B) were treated for 24 hrs with indicated concentrations of nelfinavir (Nel), lopinavir (Lop) or staurosporine (Staur). The % cell death was determined by the relative activities of dead cell- and live cell-specific protease activities as described in Materials and Methods. Data are expressed as mean +/− SEM (n = 6). Asterisk indicates that the mean for treated cells is significantly different from the mean control (DMSO-treated) cells (p < 0.05).
Figure 4
Figure 4. HIV protease inhibitors block DNA synthesis in oral keratinocytes
The OKF6 immortalized oral keratinocyte (A) and the CAL 27 oral carcinoma (B) cell-lines were treated with nelfinavir (Nel), lopinavir (Lop) or atazanavir (Ata) at the indicated concentrations for 20–22 hrs. BrdU was added for the last 2 hrs of PI treatment, and the rate of DNA synthesis was determined by incorporation of BrdU into total cellular DNA as described in Materials and Methods. Data were normalized to the mean level of BrdU incorporation of control (DMSO-treated) cultures, and are presented as mean +/− SEM. For OKF6 cells n = 12 for control cultures and n = 6 for PI treated cultures. For Cal27, data were pooled from 3 independent experiments; n = 36 for control cultures and n = 18 for PI treated cultures. Asterisk indicates that the mean for PI-treated cells is significantly different from the mean for control (DMSO-treated) cells (p < 0.05).
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