Promotive effect of minoxidil combined with all-trans retinoic acid (tretinoin) on human hair growth in vitro

Abstract

Minoxidil induces hair growth in male pattern baldness and prolongs the anagen phase. All-trans retinoic acid (ATRA) has been reported to act synergistically with minoxidil in vivo: they can enhance more dense hair regrowth than either compound alone. We evaluated the effect of minoxidil combined with ATRA on hair growth in vitro. The effect of co-treatment of minoxidil and ATRA on hair growth was studied in hair follicle organ culture. In cultured human dermal papilla cells (DPCs) and normal human epidermal keratinocytes, the expressions of Erk, Akt, Bcl-2, Bax, P53 and P21 were evaluated by immunoblot analysis. Minoxidil plus ATRA additively promoted hair growth in vitro, compared with minoxidil alone. In addition, minoxidil plus ATRA elevated phosphorylated Erk, phosphorylated Akt and the ratio of Bcl-2/Bax, but decreased the expressions of P53 and P21 more effectively than by minoxidil alone. Our results suggest that minoxidil plus ATRA would additively enhance hair growth by mediating dual functions: 1) the prolongation of cell survival by activating the Erk and Akt signaling pathways, and 2) the prevention of apoptosis of DPCs and epithelial cells by increasing the ratio of Bcl-2/Bax and downregulating the expressions of P53 and P21.

Fig. 1

The effect of minoxidil plus ATRA on hair growth in vitro. Hair follicle organ culture was performed in triplicates with hair follicles obtained from the occipital scalp of three volunteers. Hair growth is enhanced by minoxidil at 1 µM. Minoxidil plus ATRA promotes hair growth more significantly, especially at a concentration of 1 µM minoxidil and 1 nM ATRA. Moreover, the increase in hair growth was significant compared with minoxidil alone. Values were obtained after cumulative growth of 15 hair follicles per each growth condition for 12 days and shown as the means±SEM. ^*, p<0.05; ^†, p<0.01.

Fig. 2

The effect of minoxidil and ATRA on the p-Erk and on total Erk in (A) DPCs and in (B) NHK. The level of p-Erk increased significantly after treatment of minoxidil, and minoxidil plus ATRA more enhanced the expression of p-Erk. The representative bands of triplicate experiments are shown. The values shown are the means±SEM of % elevation compared with the vehicle-treated control from three different DPCs and NHK cultures. ^*, p<0.05, compared with the vehicle-treated control. p-Erk, phosphorylated Erk; T-Erk, total Erk; M, minoxidil.

Fig. 3

The increase of p-Akt expression by minoxidil and ATRA. (A) In DPCs, minoxidil increases p-Akt, moreover, minoxidil plus ATRA more significantly elevates the p-Akt level. (B) In NHK, minoxidil alone and minoxidil plus ATRA increase p-Akt expression, however, they were statistically insignificant. The blotted bands are the representative of triplicate experiments. The values are shown as the means±SEM of % elevation compared with controls. ^*, p<0.05, compared with controls treated with the vehicle. p-Akt, phosphorylated Akt; T-Akt, total Akt; M, minoxidil.

Fig. 4

The effect of minoxidil and ATRA on the expression of Bcl-2 and Bax protein in DPCs and NHK. Compared with vehicle-treated control, minoxidil plus ATRA significantly increases the expression of Bcl-2 in DPCs (A) and in NHK (B). (C) Minoxidil significantly decreases the expression of Bax. Minoxidil plus ATRA suppresses the expression of Bax more efficiently in DPCs. (D) Expression of Bax in NHK is also significantly decreased by minoxidil plus ATRA. The bands are the representatives of triplicate experiments. The values are shown as the means±SEM of % elevation in comparison with the controls. ^*, p<0.05 compared with vehicle-treated control. M, minoxidil.

Fig. 5

The effects of minoxidil and ATRA on the expressions of P53 and P21 protein in DPCs and NHK. (A) Minoxidil alone and minoxidil plus ATRA significantly suppress the expression of P53 protein in DPCs. (B) In NHK, minoxidil plus ATRA decreases P53 expression significantly. (C) In DPCs, Minoxidil significantly decreases the expression of P21. Minoxidil plus ATRA reduces the expression of P21 more significantly. (D) P21 expression is significantly downregulated with minoxidil plus ATRA. The bands are the representative of triplicate experiments. ^*, p<0.05 compared with the vehicle-treated control. The data are shown as the means±SEM of % elevation compared with controls from three different DPC and NHK cultures. M, minoxidil.