(a) Western blots for BT-474 cells treated with vehicle control, 1 (0.5 M), GF (123 nM), and 1-GF combination. associated with reduced EGFR and HER2 receptor activation, as well as reduced active forms of AKT and STAT3. Collectively, the BK channel antagonists represented by penitrem A CDK4/6-IN-2 can be novel sensitizing, chemotherapeutics synergizing, and therapeutic brokers for targeted BC therapy. species [28,29]. This study team reported penitrems 1, 2 (Scheme 1), as well as others from a marine-derived isolate GS20 isolated from sponge and sediment samples collected in the Arabian Gulf [30,31]. Penitrems CDK4/6-IN-2 have potent tremorgenic activity in mammals, secondary to the antagonism of BK channels [28,29]. Previous findings from our laboratory revealed the potential anticancer effects of penitrems as inhibitors of proliferation, migration, and invasion of BC cells [30,31]. The mechanism for these reported anticancer effects was associated with the suppression of the Wnt/-catenin pathway in BC cells . In this study, penitrems were applied in terms of BK channel CDK4/6-IN-2 inhibitors, to assess their antiproliferative effects in multiple BC cell lines, in vitro. The antiproliferative activity of the most potent GDNF 1 was assessed individually, and in combination with targeted therapy. The study also compares the CDK4/6-IN-2 in silico binding CDK4/6-IN-2 mode of 1 1 at multiple BK channel crystal structures with its related less active analogs, 2 and 3 (Scheme 1). 2. Results 2.1. Antiproliferative Effects of Penitrems in Breast Malignancy Cells In Vitro The antiproliferative activity of penitrems was assessed using MTT cell viability assay. Multiple human BC cell lines representing the different molecular subtypes were tested, including MDA-MB-231, BT-474, and SK-BR-3 cells, along with the human neuronal Schwann cells CRL-2765 and the non-tumorigenic mammary epithelial MCF-12A cells. Penitrem A (1) resulted in a dose-dependent inhibition among all three tested BC cell lines after 48 h culture duration (Physique 1). Among BC cell lines exposed to 1, the triple-negative MDA-MB-231 cells were most sensitive to the antiproliferative effects of 1, as indicated by lowest IC50 value (Table 1). Penitrem E (2) and 25- 0.05). Open in a separate window Physique 8 In vitro effects of 10 M treatments of penitrems 1C3 around the expression of BK channel (KCNMA1) and activation of TNF- (D2D4) in BT-474 cells using Western blot analysis. (a) Western blot for cells treated with penitrems 1C3. (b) Western blot quantification of the in vitro effects of penitrems 1C3 treatment around the expression of KCNMA1. (c) Western blot quantification of the effects of penitrems 1C3 treatment around the activation of TNF-. Vertical bars indicate the normalized protein value SEM. *: indicate significant differences ( 0.05). Open in a separate window Physique 9 In vitro effects of 10 M treatments of penitrems 1C3 around the expression of BK channel (KCNMA1) and activation of TNF- (D2D4) in SK-BR-3 cells using Western blot analysis. (a) Western blot for cells treated with penitrems 1C3. (b) Western blotting quantification of the in vitro effects of penitrem 1C3 treatments around the expression of KCNMA1. (c) Western blot quantification of the effects of 1C3 treatments around the activation of TNF-. Vertical bars indicate the normalized protein value SEM. *: indicate significant differences ( 0.05). In the same context, immunofluorescent staining of MDA-MB-231 (Physique 10a,b) and BT-474 cells (Physique 10c,d) indicated strong cytoplasmic expression of KCNMA1 in vehicle-treated culture media (Physique 10a,c). Penitrem 1 treatment caused significant reduction in the total level of KCNMA1 compared to cells in vehicle-treated control groups (Physique 10a,c). Penitrem 1 treatments caused remarkable.