Yonsei Med J.  2016 Jan;57(1):33-40. 10.3349/ymj.2016.57.1.33.

Anti-Proliferative and Apoptotic Activities of Mullerian Inhibiting Substance Combined with Calcitriol in Ovarian Cancer Cell Lines

• Jung YS
• Kim HJ
• Seo SK
• Choi YS
• Nam EJ
• Kim S
• Kim SW
• Han HD
• Kim JW
• Kim YT

• KMID: 2466349
• DOI: http://doi.org/10.3349/ymj.2016.57.1.33

Abstract

PURPOSE
This study aimed to investigate whether Mullerian inhibiting substance (MIS) in combination with calcitriol modulates proliferation and apoptosis of human ovarian cancer (OCa) cell lines (SKOV3, OVCAR3, and OVCA433) and identify the signaling pathway by which MIS mediates apoptosis.
MATERIALS AND METHODS
OCa cell lines were treated with MIS in the absence or presence of calcitriol. Cell viability and proliferation were evaluated using the Cell Counting Kit-8 assay and apoptosis was evaluated by DNA fragmentation assay. Western blot and enzyme-linked immunosorbent assay were used to determine the signaling pathway.
RESULTS
The cells showed specific staining for the MIS type II receptor. Treatment of OCa cells with MIS and calcitriol led to dose- and time-dependent inhibition of cell growth and survival. The combination treatment significantly suppressed cell growth, down-regulated the expression of B-cell lymphoma 2 (Bcl-2), and up-regulated the expressions of Bcl-2 associated X protein, caspase-3, and caspase-9 through the extracellular signal-regulated kinase signaling pathway.
CONCLUSION
These results, coupled with a much-needed decrease in the toxic side effects of currently employed therapeutic agents, provide a strong rationale for testing the therapeutic potential of MIS, alone or in combination with calcitriol, in the treatment of OCa.

Keyword

Ovarian cancer; Mullerian inhibiting substance; calcitriol; antiproliferation; apoptosis

MeSH Terms

Anti-Mullerian Hormone/*pharmacology
Apoptosis/*drug effects
Calcitriol/*pharmacology
Caspase 3/metabolism
Caspase 9/metabolism
Cell Cycle/drug effects
Cell Line, Tumor
Cell Proliferation/*drug effects
Cell Survival/drug effects
DNA Fragmentation/*drug effects
Enzyme-Linked Immunosorbent Assay
Extracellular Signal-Regulated MAP Kinases/metabolism
Female
Growth Inhibitors/metabolism/pharmacology
Humans
Ovarian Neoplasms/*drug therapy/metabolism/*pathology
Receptors, Peptide
Receptors, Transforming Growth Factor beta
Signal Transduction/*drug effects
Anti-Mullerian Hormone
Calcitriol
Caspase 3
Caspase 9
Extracellular Signal-Regulated MAP Kinases
Growth Inhibitors
Receptors, Peptide
Receptors, Transforming Growth Factor beta

Figure

• 

  Fig. 1 Effects of MIS and calcitriol on the viability of ovarian cancer (OCa) cells. (A) Cells were treated with a range of MIS (35.5-284 nM) to determine the IC50 for each cell line. (B) Cells were treated with a range of calcitriol concentrations (1-100 µM) to determine the IC50 for each cell line. Values are presented as a percentage of the control and were calculated using the following equation: [(mean absorbance of treated cells)/(mean absorbance of control cells)]×100. Data are expressed as mean±SD from three independent experiments. For all subsequent experiments, MIS and calcitriol concentrations were held constant at or near their IC50, 71 nM and 50 µM, respectively. MIS, Müllerian inhibiting substance.

• 

  Fig. 2 Viability and proliferation assays conducted with ovarian cancer (OCa) cell lines treated with MIS and calcitriol (relative cell growth as a function of time). MIS and calcitriol concentrations were held constant at or near their IC50; 71 nM MIS and 50 µM calcitriol. Cells were treated with vehicles (control), 71 nM MIS (MIS), 50 µM calcitriol (calcitriol), or both agents (MIS+calcitriol). Combined use of calcitriol and MIS reduces cell proliferation of human OCa cell lines (SKOV3, OVCAR3, and OVCA433). Cell growth was monitored for 96 h post treatment. For the combination treatment, cells were pretreated with calcitriol (50 µM) for 4 h prior to the addition of MIS (71 nM). Cell proliferation was measured using the Cell Counting Kit-8 solution as described in the Materials. Results are representative of three experiments. Data in the bar graph represent mean±SD. *p<0.05 vs. control, †p<0.05 vs. MIS. MIS, Müllerian inhibiting substance.

• 

  Fig. 3 Calcitriol enhances MIS-induced apoptosis in OCa cell lines. Cells (1×105/well) were pretreated with or without calcitriol (50 µM) for 4 h prior to the addition of MIS (71 nM). Apoptosis was measured as cellular DNA fragmentation determined by ELISA. Results are representative of three experiments. Significant inhibition relative to the control (MIS 0 nM) is indicated by an asterisk and the cross indicates a significant difference relative to the MIS treatment (MIS 71 nM). Data are presented as mean±SD. *p<0.05 vs. control, †p<0.05 vs. MIS. MIS, Müllerian inhibiting substance; ELISA, enzyme-linked immunosorbent assay; OD, optical density.

• 

  Fig. 4 MIS and calcitriol alter the expression of regulatory proteins in SKOV3. Cells were treated with vehicles (control), 71 nM MIS (MIS), 50 µM calcitriol (calcitriol), or both (MIS+calcitriol). For the combined treatment, cells were incubated in the presence or absence of calcitriol (50 µM) for 4 h followed by stimulation with MIS (71 nM) for 48 h. (A) Western blot analysis of caspase-9 and caspase-3. (B) Western blot analysis of Bcl-2 and BAX. Band intensities were quantitated and data are presented as mean±SD. *p<0.05 vs. control, †p<0.05 vs. MIS. MIS, Müllerian inhibiting substance; Bcl-2, B-cell lymphoma 2; BAX, Bcl-2 associated X protein.

• 

  Fig. 5 Calcitriol increases MIS-induced ERK phosphorylation in SKOV3 cells. (A) Western blot analysis of ERK and phosphorylated ERK levels in SKOV3 cells treated with MIS. Bands were detected with anti-ERK and anti phospho-ERK antibodies. (B) SKOV3 cells were treated with 71 nM MIS, 20 µM PD98059, or both for 2 h and the degree of apoptosis was analyzed by ELISA measuring the level of cellular DNA fragmentation. Significant increase relative to controls (MIS 0 nM) is indicated by asterisks and crosses indicate a significant difference (p<0.05) compared with MIS treatment (MIS 71 nM). (C) SKOV3 cells were incubated with or without calcitriol (50 µM) for 4 h prior to the addition of MIS (71 nM). Subsequent to 48 h of incubation, cells were harvested and ERK activation was analyzed by western blot analysis using anti-phospho-ERK antibody. *p<0.05 vs. control, †p<0.05 vs. MIS. MIS, Müllerian inhibiting substance; ERK, extracellular signal-regulated kinase; ELISA, enzyme-linked immunosorbent assay; OD, optical density.

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