A Novel, Stable, Estradiol-Stimulating, Osteogenic Yam Protein with Potential for the Treatment of Menopausal Syndrome

Abstract

A novel protein, designated as DOI, isolated from the Chinese yam (Dioscorea opposita Thunb.) could be the first protein drug for the treatment of menopausal syndrome and an alternative to hormone replacement therapy (HRT), which is known to have undesirable side effects. DOI is an acid- and thermo-stable protein with a distinctive N-terminal sequence Gly-Ile-Gly-Lys-Ile-Thr-Thr-Tyr-Trp-Gly-Gln-Tyr-Ser-Asp-Glu-Pro-Ser-Leu-Thr-Glu. DOI was found to stimulate estradiol biosynthesis in rat ovarian granulosa cells; induce estradiol and progesterone secretion in 16- to 18-month-old female Sprague Dawley rats by upregulating expressions of follicle-stimulating hormone receptor and ovarian aromatase; counteract the progression of osteoporosis and augment bone mineral density; and improve cognitive functioning by upregulating protein expressions of brain-derived neurotrophic factor and TrkB receptors in the prefrontal cortex. Furthermore, DOI did not stimulate the proliferation of breast cancer and ovarian cancer cells, which suggest it could be a more efficacious and safer alternative to HRT.

Figure 1

(a) Purification of DOI on a HiPrep 16/10 DEAE FF column. (b) Fraction D3 was further purified on a HiPrep 16/10 Phenyl FF (high sub) column. (c) Fraction P1 was further purified on a Superdex 75 10/300 GL column and fraction S1 contained the DOI protein. (d) 15% Native PAGE of DOI (left) and 15% SDS-PAGE of DOI (right). The DOI protein was visualized by silver staining. (e) Size measurement of DOI by mass spectrometry.

Figure 2

Estrogen-stimulating activity of (a) HCl-, (b) NaOH-, (c) heat-treated DOI on granulosa cells. Results are expressed as means ± SEM (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 compared with the control group (un-paired t-test). (d) Chitinase activity of DOI. Results are expressed as means ± SEM (n = 3). **p < 0.01, ***p < 0.001 compared with the positive chitinase control (un-paired t-test). N9376: 4-Nitrophenyl N-acetyl-β-D-glucosaminide used to detect exochitinase activity (β-N-acetylglucosaminidase activity); N8638: 4-Nitrophenyl β-D-N, N’, N”triacetylchitotriose used to detect endochitinase activity; and N6133: 4-Nitrophenyl N, N′-diacetyl-β-D-chitobioside used to detect exochitinase activity (chitobiosidase activity).

Figure 3

Viability of (a) MCF-7 breast cancer cells, (b) OVCA-429 ovarian cancer cells, (c) mouse splenocytes, and (d) ovarian granulosa cells after treatment with DOI for 48 h. Results are expressed as means ± SEM (n = 3). **p < 0.01, ***p < 0.001 compared with the control group (un-paired t-test).

Figure 4

(a) Stimulatory activity of DOI on estrogen biosynthesis in granulosa cells. Protein expression of (b) aromatase and (c) follicle-stimulating hormone receptor (FSHR) in ovarian granulosa cells. Results are expressed as means ± SEM (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 compared with the control group (un-paired t-test). Effects of (d) protein kinase A inhibitor (PKAi), (e) protein kinase B inhibitor (PKBi), and (f) protein kinase C inhibitor (PKCi) on the estrogen-stimulating effect of DOI on ovarian granulosa cells. (g) Effects of DOI on FSHR-attenuated ovarian granulosa cells after 12 h treatment. Results are expressed as means ± SEM (n = 3). *p < 0.05, **p < 0.01 compared with the control group (un-paired t-test).

Figure 5

(a) Serum estradiol and (b) progesterone levels in Sprague Dawley rats after treatment with DOI for 2, 4, and 6 weeks. Results are expressed as means ± SEM (n = 6). *p < 0.05, **p < 0.01 compared with week 2 controls;+p < 0.05 compared with week 4 controls; and ^#p < 0.05, ^##p < 0.01 compared with week 6 controls (un-paired t-test). Ctl: control group received daily intraperitoneal injections of PBS; Premarin: positive control group received daily Premarin (12.4 mg/kg) by oral administration; DOI groups: DOI-treated groups received daily intraperitoneal injections of DOI (2.5, 5, and 10 mg/kg).

Figure 6

mRNA expression of (a) ovarian CYP-19 and (b) follicle-stimulating hormone receptor (FSHR), and protein expression of (c) ovarian aromatase, (d) ovarian FSHR, and (e) breast aromatase in Sprague Dawley rats after treatment with DOI for 6 weeks. (f) Protein expression of aromatase in DOI-treated MCF-7 breast cancer cell line. Results are expressed as means ± SEM (n = 6). *p < 0.05, **p < 0.01, ***p < 0.001 compared with the control group (One-way ANOVA followed by Dunnett’s Multiple Comparison Test). Ctl: control group received daily intraperitoneal injections of PBS; Premarin: positive control group received daily Premarin (12.4 mg/kg) by oral administration; DOI group: DOI-treated groups received daily intraperitoneal injections of DOI (2.5, 5, and 10 mg/kg).

Figure 7

mRNA expression of (a) PKA, (b) PKB and (c) PKC, and protein expression of (d) PKA, (e) PKB and (f) PKC in ovaries of Sprague Dawley rats after treatment with DOI for 6 weeks. Results are expressed as means ± SEM (n = 3 for mRNA; n = 6 for protein). *p < 0.05, **p < 0.01, ***p < 0.001 compared with control group (one-way ANOVA and un-paired t-test, respectively).

Figure 8

Volume rendered images of L2 in 16- to 18-month-old female SD rats given different treatments (image presented with BV/TV closest to the group mean value).

Figure 9

(a) Apparent trabecular bone mineral density, (b) bone volume fraction, (c) trabecular number, (d) trabecular thickness, (e) structure model index, and (f) trabecular separation of vertebra L2 of Sprague Dawley rats after treatment with DOI for 6 weeks. Results are expressed as means ± SEM (n = 6; except Premarin group, where n = 3). *p < 0.05, **p < 0.01 compared with the control group (unpaired t-test). Ctl: control group received daily intraperitoneal injections of PBS; Premarin positive control group received daily Premarin (12.4 mg/kg) by oral administration; DOI group: DOI-treated groups received daily intraperitoneal injections of DOI (2.5, 5, and 10 mg/kg).

Figure 10

Protein levels of brain derived neurotrophic factor (BDNF) in (a) prefrontal cortex and (b) hippocampus of Sprague Dawley rats after treatment with DOI for 6 weeks. (c) Protein levels of TrkB gp145 receptor in prefrontal cortex of Sprague Dawley rats after treatment with DOI for 6 weeks. Results are expressed as means ± SEM (n = 6). *p < 0.05, **p < 0.01 compared with the control group (un-paired t-test). Ctl: control group received daily intraperitoneal injections of PBS; Premarin: positive control group received daily Premarin (12.4 mg/kg) by oral administration; DOI group: DOI-treated groups received daily intraperitoneal injections of DOI (2.5, 5, and 10 mg/kg).