Fig 1: Differential expression of ALK2 and ALK3 following LR-AMH treatment is associated with the expression of cell survival and apoptosis markers in COV434-AMHRII, SKOV3-AMHRII, OVCAR8 and KGN ovarian cancer cells. Quantification of the western blot analysis of AMH signaling (pSMAD1/5), ALK2 and ALK3 expression, apoptosis induction (cleaved caspase-3 and PARP) and pAKT levels following incubation with 1.6 and 25 nM LR-AMH for 6 h. Data are presented as the fold-change relative to control (no LR-AMH) by bars corresponding from left to right to 0, 1.6 and 25 nM for each protein expression. n>3. Raw data are presented in Fig. S5. AMH, anti-Müllerian hormone; LR-AMH, active recombinant AMH; AMHRII, AMH type II receptor; ALK, activin receptor-like kinase; p, phosphorylated; c, cleaved; PARP, poly(ADP-ribose) polymerase.
Fig 2: Incubation with recombinant LR-AMH modulates ALK2 and ALK3 expression in COV434-AMHRII, SKOV3-AMHRII, OVCAR8 and KGN ovarian cancer cells. Immunofluorescence analysis of AMHRII, ALK2, ALK3 and ALK6 expression in basal conditions and after incubation with 25 nM LR-AMH for 90 min. Data are presented as the ratio of receptor (green) and nucleus (blue) labeling. *P<0.05, **P<0.01 and ***P<0.001 n=6. AMH, anti-Müllerian hormone; LR-AMH, active recombinant AMH; AMHRII, AMH type II receptor; ALK, activin receptor-like kinase; IR, irrelevant antibody; NT, non-treated.
Fig 3: The anti-AMHRII-ALK2 BsAb 12G4-2F9 inhibits the growth of COV434-AMHRII cell xenografts in vivo. Nude mice bearing COV434-MISRII cell-derived tumors were treated with 17 mg/kg of anti-AMHRII-CD5 (control BsAb targeting AMHRII and CD5), anti-AMHRII-ALK2 (12G4-2C1 and 12G4-2F9), anti-AMHRII-ALK3 (12G4-3D7 and 12G4-3H6) BsAbs or vehicle (NaCl) twice per week for 4 weeks. Data are presented as the mean ± SEM. *P<0.05 and **P<0.01. AMHRII, anti-Müllerian hormone type II receptor; ALK, activin receptor-like kinase; BsAbs, bispecific antibodies.
Fig 4: Involvement of ALK2, ALK3 and ALK6 in the effects of AMH in COV434-AMHRII and SKOV3-AMHRII cells. (A) pSMAD1/5 levels were evaluated following incubation with 25 nM LR-AMH for 6 h starting 24 (COV434-AMHRII cells) or 48 (SKOV3-AMHRII cells) h post-transfection with si-ALK2, si-ALK3 and si-ALK6. Data are presented as the mean + max of pSMAD1/5:GAPDH ratios. n=2. (B) Caspase-3/7 activity and cleaved caspase-3 and PARP levels were analyzed following incubation with 25 nM AMH for 6 h (24 and 48 h post-transfection). *P<0.05; **P<0.01. AMH, anti-Müllerian hormone; LR-AMH, active recombinant AMH; AMHRII, AMH type II receptor; ALK, activin receptor-like kinase; si, small interfering RNA; p, phosphorylated; C., cleaved.
Fig 5: Differential expression of ALK2 and ALK3 following LR-AMH treatment is associated with the expression of cell survival and apoptosis markers in primary ovarian cancer cells. Quantification of the western blot analysis of AMH signaling (pSMAD1/5), ALK2 and ALK3 expression, apoptosis induction (cleaved caspase-3 and PARP) and pAKT levels following incubation of cells isolated from ascites of three patients with ovarian cancer with 1.6 and 25 nM LR-AMH for 6 h. Data are presented as the fold change relative to control (no LR-AMH) by bars corresponding from left to right to 0, 1.6 and 25 nM for each protein expression. n=1 for each patient. Raw data are presented in Fig. S6. AMH, anti-Müllerian hormone; LR-AMH, active recombinant AMH; AMHRII, AMH type II receptor; ALK, activin receptor-like kinase; P, phosphorylated; c, cleaved; PARP, poly(ADP-ribose) polymerase.
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