Mullerian Inhibiting Substance enhances subclinical doses of chemotherapeutic agents to inhibit human and mouse ovarian cancer

Abstract

Mullerian Inhibiting Substance (MIS), a biological modifier that causes regression of Mullerian ducts in male embryos, is effective as a single agent in vitro and in vivo against human and mouse ovarian cancer cell lines expressing MIS type II receptor; however, little is known about how recombinant human MIS (rhMIS), now being scaled for preclinical trials, could be used in combination with cytotoxic or targeted chemotherapeutic agents. Mouse serous and endometrioid ovarian carcinoma cell lines were tested in vitro against rhMIS alone and with doxorubicin, paclitaxel, or cisplatin as agents in clinical use. Because MIS releases FK506 binding protein (FKBP12), which activates the mammalian target of rapamycin (mTOR) downstream of Akt, rhMIS and rapamycin combinations were tested. MIS increases p16 protein levels, and 5'-Aza-2'-deoxycytidine (AzadC) induces p16 mRNA; therefore, they were used in combination in vitro and in vivo with a human ovarian cancer cell line. A paclitaxel-resistant human ovarian cancer cell line and its parental line both respond to rhMIS in vitro. Additivity, synergy, or competition was observed with MIS and rapamycin, AzadC, doxorubicin, cisplatin, and paclitaxel, suggesting that MIS in combination with selective targeted therapies might achieve greater activity against ovarian cancer than the use of each individual agent alone. These assays and statistical analyses could be useful in selecting rhMIS and chemotherapeutic agent combinations that enhance clinical efficacy and reduce toxicity.

Fig. 1.

MIS inhibition of OVCAR8 cells in vitro. rhMIS in vitro dose–response plotted as OD in the MTT assay (± SD) (A) and in vivo is augmented by 15 μM AzadC (B and C, respectively). (B) A two-way factorial ANOVA was done to determine the nature of MIS/AzadC interaction in vitro. ○A, additive. MIS and the combined treatment were significantly different from the control (∗, P < 0.005 and 0.007, respectively), and the combination was different than MIS alone (∗∗, P < 2 ×10⁻⁵). (C) An analysis of the logarithm of graft size ratios (GSRs) (±SEM) reveals that the maximal effect with the combined treatment (MIS 10 μg per animal per day and AzadC 0.5 mg/kg per day) is additive (∗, P < 0.003, nonpaired t test vs. PBS control; n = 10 animals per group).

Fig. 2.

Dose responses for MOVCAR7 cells. MOVCAR7 cell dose responses to rhMIS (A), rapamycin (B), paclitaxel (C), cisplatin (D), or doxorubicin (E) were established in MTT monolayer growth inhibition assays over 5–7 days in culture.

Fig. 3.

Dose responses for 4306 cells. Dose responses of 4306 cells to rhMIS (A), rapamycin (B), paclitaxel (C), cisplatin (D), or doxorubicin (E) were established in MTT monolayer growth inhibition assays over 5–7 days in culture.

Fig. 4.

Representative data for combination treatment of MOVCAR7 and 4306 cells in MTT in vitro assays with MIS and chemotherapeutic drugs (OD ± SEM and percentage inhibition). MIS and the drug concentrations were held constant at or near their IC₅₀ for each cell line. MIS doses were 35 nM for MOVCAR7 and 70 nM for 4306; the concentrations of chemotherapeutic drugs used are given below. (A) MOVCAR7 cells treated with vehicle as a control, MIS, 0.02 nM rapamycin, or both agents (n = 3; 7 days). (B) The same treatment of 4306 cells using 0.04 nM rapamycin (n = 3; 7 days). Rapamycin and MIS were synergistic ○S in both cases. (C and D) In MOVCAR cells, MIS and 3 nM paclitaxel were additive ○A (n = 4; 6 days) (C), whereas MIS and 4 nM paclitaxel were synergistic in 4306 cells (n = 3; 6 days) (D). (E and F) Cisplatin was additive with MIS in both MOVCAR (E) and 4306 cells (F) at 5 and 10 nM drug, respectively (n = 3; 6 days in both cases). (G and H) The combination of doxorubicin (3 nM) and MIS is synergistic in MOVCAR cells (G), but the same concentration of drugs is competitive ○C with MIS in 4306 cells (H) (n = 3; 6 days). Significant inhibition relative to controls is indicated by an asterisk. MIS alone was significantly different from combination treatment in all cases (∗∗), except for MIS and cisplatin in 4306 cells. (I and J) In combination experiments with 3 nM cisplatin (CIS) and 4 nM paclitaxel (PAC), these drugs were found to act synergistically in MOVCAR cells (I) but not in 4306 cells (J). One experiment showed that the effect was additive and two showed that the interaction was competitive. Two-way factorial ANOVA was done to determine the nature of the MIS/drug interactions.

Fig. 5.

MIS inhibits paclitaxel-resistant cells in vitro. IGROV-1 cells made resistant to paclitaxel were tested in three separate experiments for their responsiveness to MIS. The stippled bar shows the average of three experiments. MIS also inhibited the parental IGROV-1 cell line in these monolayer cell assays (hatched bar). On each trial, 105 nM (15 μg/ml) MIS significantly inhibited proliferation relative to controls (open bar). ∗, P < 0.01 vs. PBS control (n = 10).