Humanin Treatment Protects Against Venetoclax-Induced Bone Growth Retardation in Ex Vivo Cultured Rat Bones

Abstract

Context: Recent preclinical studies reported that the BCL-2 inhibitor venetoclax can impair bone growth. A strategy to prevent such a side effect of this promising anticancer drug is highly desired. Earlier in vitro and in vivo studies suggested that the mitochondrial peptide humanin has the potential to prevent drug-induced growth impairment.

Objective: We hypothesized that co-treatment with the humanin analog HNG may prevent venetoclax-induced bone growth impairment.

Methods: Ex vivo studies were performed in fetal rat metatarsal bones and human growth plate samples cultured for 12 and 2 days, respectively, while in vivo studies were performed in young neuroblastoma mice being treated daily for 14 days. The treatment groups included venetoclax, HNG, venetoclax plus HNG, or vehicle. Bone growth was continuously monitored and at the end point, histomorphometric and immunohistochemical analyses were performed in fixed tissues.

Results: Venetoclax suppressed metatarsal bone growth and when combined with HNG, bone growth was rescued and all histological parameters affected by venetoclax monotherapy were normalized. Mechanistic studies showed that HNG downregulated the pro-apoptotic proteins Bax and p53 in cultured metatarsals and human growth plate tissues, respectively. The study in a neuroblastoma mouse model confirmed a growth-suppressive effect of venetoclax treatment. In this short-term in vivo study, no significant bone growth-rescuing effect could be verified when testing HNG at a single dose. We conclude that humanin dose-dependently protects ex vivo cultured metatarsal bones from venetoclax-induced bone growth impairment by restoring the growth plate microstructure.

Keywords: growth; humanin; metatarsals; neuroblastoma; venetoclax.

Figure 1.

HNG treatment rescued from venetoclax-induced bone growth retardation in ex vivo cultured E20 fetal rat metatarsal bones. A, The graph shows growth curves for each treatment group over the 12-day-culture period. The percentage (%) of bone length increase from day 0 was calculated for each time point. Venetoclax (Ven) suppressed bone growth while co-treatment with HNG (10-40 μM) dose-dependently rescued bone growth and at the highest dose of HNG (40 μμ) tested, bone growth was similar to control bones when assessed after 12 days of treatment. Three independent experiments were performed. Data are shown as means ± SEMs, n = 18/group; *P less than .05 and ***P less than .001. B, Representative images of ex vivo cultured metatarsal bones captured on day 0 and day 12. For each group, the same metatarsal bone is shown at both time points.

Figure 2.

HNG restored metatarsal bone histology when combined with venetoclax. A, Representative images of control, Venetoclax (Ven), and Venetoclax + HNG-treated (Ven/HNG10 and Ven/HNG40) metatarsals stained with Alcian blue and nuclear fast red solution. The box highlights the area of the hypertrophic zone shown in B. Original magnification 5×, scale bar = 100 μm. B, Representative micrographs of the hypertrophic zone in metatarsal bones from each group. Original magnification 40×, scale bar = 20 μm. C and D, Quantification of histomorphometric analyses. Venetoclax reduced the resting + proliferative (R + P) zone height and the hypertrophic cell size. Combination treatment with Ven + HNG significantly improved both these parameters. Data are shown as means ± SDs, n = 4 to 6/group; ***P less than .001.

Figure 3.

Effects of venetoclax and/or HNG treatment on protein expression in fetal rat metatarsal bones. A, Representative microscopic images of venetoclax (Ven) and/or HNG-treated metatarsals stained for BCL-2, PCNA, and Bax, counterstained with Alcian blue. Original magnification 40×, scale bar = 20 μm. Representative positive cells are marked with arrows. B to D, Quantification of BCL-2, PCNA, and Bax staining in metatarsal bones. Venetoclax significantly decreased the expression of BCL-2 and the proliferative marker PCNA. Combination treatment with Ven + HNG significantly downregulated the proapoptotic marker Bax compared to venetoclax alone. Data are shown as means ± SDs, n = 1 to 8/group; **P less than .01 and ***P less than .001.

Figure 4.

Effects of venetoclax (Ven) and/or HNG treatment on protein expression in ex vivo cultured human growth plate tissue. A, Representative images of human growth plate tissues treated with Ven, HNG, or the combination of Ven/HNG for 48 hours and analyzed by immunohistochemistry. Alcian blue was used as counterstain. Original magnification 20×, scale bar = 100 μm. B-D, Quantification of p53, PCNA, and humanin protein expression. Individual cultures of 3 different pieces of growth plate tissue were analyzed. Data are shown as means ± SDs, n = 3/group; **P less than .01.