New agents that target senescent cells: the flavone, fisetin, and the BCL-XL inhibitors, A1331852 and A1155463

Abstract

Senescent cells accumulate with aging and at sites of pathology in multiple chronic diseases. Senolytics are drugs that selectively promote apoptosis of senescent cells by temporarily disabling the pro-survival pathways that enable senescent cells to resist the pro-apoptotic, pro-inflammatory factors that they themselves secrete. Reducing senescent cell burden by genetic approaches or by administering senolytics delays or alleviates multiple age- and disease-related adverse phenotypes in preclinical models. Reported senolytics include dasatinib, quercetin, navitoclax (ABT263), and piperlongumine. Here we report that fisetin, a naturally-occurring flavone with low toxicity, and A1331852 and A1155463, selective BCL-X_L inhibitors that may have less hematological toxicity than the less specific BCL-2 family inhibitor navitoclax, are senolytic. Fisetin selectively induces apoptosis in senescent but not proliferating human umbilical vein endothelial cells (HUVECs). It is not senolytic in senescent IMR90 cells, a human lung fibroblast strain, or primary human preadipocytes. A1331852 and A1155463 are senolytic in HUVECs and IMR90 cells, but not preadipocytes. These agents may be better candidates for eventual translation into clinical interventions than some existing senolytics, such as navitoclax, which is associated with hematological toxicity.

Keywords: BCL-X inhibitors L; adipose-derived stem cells; aging; apoptosis; flavonoids; preadipocytes; senolytics.

Figure 1. Fisetin targets senescent cells

(a) Structure of fisetin. (b-d) Fisetin is more effective in reducing viability (ATPLite) of senescent HUVECs than IMR90 cells or primary human preadipocytes. Proliferating or senescent cells were exposed to different concentrations of fisetin for 3 days. The red lines denote ATPLite intensities on day 0 of senescent and non-senescent cells, both set to 100%. HUVEC and IMR90 data are means±SEM of 5 replicates at each drug concentration. Preadipocyte data are means±SEM of 5 replicates from each of 4 different subjects at each concentration. (e-g) Fisetin selectively reduces senescent but not proliferating HUVECs and IMR90 cell numbers (crystal violet). The red lines denote cell numbers at plating on day 0 of senescent and non-senescent cells, both set to 100%. HUVEC and IMR90 data are means±SEM of 5 replicates at each drug concentration. Preadipocyte data are means±SEM, 5 replicates from each of 4 different subjects at each concentration. (h) Fisetin induces apoptosis in senescent HUVECs. HUVECs were treated with fisetin for 12h and then caspases-3&7 were assayed using a luminescent substrate. Fisetin (500 nM) induced apoptosis in senescent cells by caspase 3/7 assay. For all figures: * = P<0.05; ** = P<0.01; *** = P<0.001 by one-way ANOVA (caspase activities by 2-way ANOVA). Bars with asterisks indicate differences between senescent cells following drug exposure compared to day 0.

Figure 2. A1331852 targets senescent cells

(a) Structure of A1331852. (b-d) A1331852 is more effective in reducing viability (ATPLite) of senescent HUVECs and IMR90 cells than primary human preadipocytes. Proliferating or senescent cells were exposed to different concentrations of A1331852 for 3 days. The red lines denote ATPLite intensities on day 0 of senescent and non-senescent cells, both set to 100%. HUVEC and IMR90 data are means±SEM of 5 replicates at each drug concentration. Preadipocyte data are means±SEM of 5 replicates from each of 4 different subjects at each concentration. (e-g) A1331852 selectively reduces senescent but not proliferating HUVECs and IMR90 cell numbers (crystal violet). The red lines denote cell numbers at plating on day 0 of senescent and non-senescent cells, both set to 100%. HUVEC and IMR90 data are means±SEM of 5 replicates at each drug concentration. Preadipocyte data are means±SEM of 5 replicates from each of 4 different subjects at each concentration. (h-i) A1331852 induces apoptosis in senescent HUVECs and IMR90 cells. HUVECs were treated with A1331852 for 12h and then caspases-3&7 were assayed using a luminescent substrate. A1331852 (1 nM) induced apoptosis in senescent cells by caspase 3/7 assay.

Figure 3. A1155463 targets senescent cells

(a) Structure of A1155463. (b-d) A1155463 is more effective in reducing viability (ATPLite) of senescent HUVECs and IMR90 cells than primary human preadipocytes. Proliferating or senescent cells were exposed to different concentrations of A1155463 for 3 days. The red lines denote ATPLite intensities on day 0 of senescent and non-senescent cells, both set to 100%. HUVEC and IMR90 data are means±SEM of 5 replicates at each drug concentration. Preadipocyte data are means±SEM of 5 replicates from each of 4 different subjects at each concentration. (e-g) A1155463 selectively reduces senescent but not proliferating HUVECs and IMR90 cell numbers (crystal violet). The red lines denote cell numbers at plating on day 0 of senescent and non-senescent cells, both set to 100%. HUVEC and IMR90 data are means±SEM of 5 replicates at each drug concentration. Preadipocyte data are means±SEM of 5 replicates from each of 4 different subjects at each concentration. (h-i) A1155463 induces apoptosis in senescent HUVECs and IMR90 cells. HUVECs were treated with A1155463 for 12h and then caspases-3&7 were assayed using a luminescent substrate. A1155463 (1 nM ) induced apoptosis in senescent cells by caspase 3/7 activity assay.