marine drugs Article Synergistic Cytotoxicity of Renieramycin M and Doxorubicin in MCF-7 Breast Cancer Cells Jortan O. Tun 1,*, Lilibeth A. Salvador-Reyes 1,2 , Michael C. Velarde 3 , Naoki Saito 4 , Khanit Suwanborirux 5 and Gisela P. Concepcion 1,2,* 1 The Marine Science Institute, University of the Philippines Diliman, Quezon City 1101, Philippines; [email protected] 2 Philippine Genome Center, University of the Philippines Diliman, Quezon City 1101, Philippines 3 Institute of Biology, University of the Philippines Diliman, Quezon City 1101, Philippines; [email protected] 4 Graduate School of Pharmaceutical Sciences, Meiji Pharmaceutical University, Tokyo 204-8588, Japan; [email protected] 5 Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Center for Bioactive Natural Products from Marine Organisms and Endophytic Fungi (BNPME), Chulalongkorn University, Pathumwan, Bangkok 10330, Thailand; [email protected] * Correspondence: [email protected] (J.O.T.); [email protected] (G.P.C.); Tel.: +632-922-3959 (J.O.T. & G.P.C.) Received: 23 July 2019; Accepted: 7 August 2019; Published: 16 September 2019 Abstract: Renieramycin M (RM) is a KCN-stabilized tetrahydroisoquinoline purified from the blue sponge Xestospongia sp., with nanomolar IC50s against several cancer cell lines. Our goal is to evaluate its combination effects with doxorubicin (DOX) in estrogen receptor positive MCF-7 breast cancer cells. MCF-7 cells were treated simultaneously or sequentially with various combination ratios of RM and DOX for 72 h. Cell viability was determined using the MTT assay. Synergism or antagonism was determined using curve-shift analysis, combination index method and isobologram analysis. Synergism was observed with pharmacologically achievable concentrations of DOX when administered simultaneously, but not sequentially. The IC95 values of RM and DOX after combination were reduced by up to four-fold and eight-fold, respectively. To gain insights on the mechanism of synergy, real-time profiling, cell cycle analysis, apoptosis assays, and transcriptome analysis were conducted. The combination treatment displayed a similar profile with DNA-damaging agents and induced a greater and faster cell killing. The combination treatment also showed an increase in apoptosis. DOX induced S and G2/M arrest while RM did not induce significant changes in the cell cycle. DNA replication and repair genes were downregulated commonly by RM and DOX. p53 signaling and cell cycle checkpoints were regulated by DOX while ErbB/PI3K-Akt, integrin and focal adhesion signaling were regulated by RM upon combination. Genes involved in cytochrome C release and interferon gamma signaling were regulated specifically in the combination treatment. This study serves as a basis for in vivo studies and provides a rationale for using RM in combination with other anticancer drugs. Keywords: renieramycin M; doxorubicin; synergistic combination chemotherapy; real-time profiling; cell cycle; gene expression profiling; apoptosis; breast cancer; blue sponge; DNA damage response 1. Introduction The standard treatment for advanced breast cancer includes doxorubicin (DOX), administered either as monotherapy or in combination with other cytotoxic or targeted drugs [1]. However, its use has been limited because of dose-limiting toxicities such as cardiotoxicity. Moreover, the generation of drug-resistant tumors after continued therapy is still inevitable, and this eventually narrows down Mar. Drugs 2019, 17, 536; doi:10.3390/md17090536 www.mdpi.com/journal/marinedrugs Mar. Drugs 2019, 17, x 2 of 28 that can enhance or maintain efficacy, while minimizing toxicity and delaying the development of drug resistance. Renieramycin M (RM) is a KCN-stabilized tetrahydroisoquinoline purified from the blue sponge Xestospongia sp. (Figure 1), with nanomolar IC50s against the colon, lung, melanoma, and pancreatic cancer cells [2–7]. RM induces apoptosis and inhibits invasion and migration in non-small cell lung cancer cells (NSCLC) in vitro, making it a potential antimetastatic agent [8]. RM is structurally related to ecteinascidin-743 (Et-743; Trabectedin, Yondelis® ), an anticancer drug for advanced soft tissue sarcoma and recurrent platinum-sensitive ovarian cancer. The renieramycins and ecteinascidins are the two major categories of the 1,2,3,4-tetrahydroisoquinoline alkaloids that have an anticancer effect. This warrants further investigation on the potential clinical utility of RM. A transcriptional structure–activity relationship (SAR) study and molecular network profiling revealed that RM and the ecteinascidin class of compounds induce apoptosis via a common Mar. Drugs 2019, 17, 536 2 of 26 pathway in the colon, breast [2], and glioblastoma cells [9]. Et-743 was reported to have a sequence- dependent synergistic effect with paclitaxel in breast carcinoma [10], and with doxorubicin in soft tissue sarcoma in vitro [11]. In view of the similarities between RM and Et-743, we hypothesize that the treatment armamentarium. It is imperative therefore, to keep looking for new drug combinations RM can act also synergistically with standard cytotoxic drugs and thus, may be potentially useful to that canimprove enhance the therapeutic or maintain outcome. efficacy, while minimizing toxicity and delaying the development of drug resistance.In this study, we investigated the effects of the combination of RM and DOX in estrogen receptor Renieramycinpositive (ER+) MCF M (RM)-7, an in is vitro a KCN-stabilized model for the most tetrahydroisoquinoline common type of breast purifiedcancer and from determined the blue sponge the drug ratio and regimen that will yield a synergistic effect. We also determined the effects of the Xestospongia sp. (Figure1), with nanomolar IC 50s against the colon, lung, melanoma, and pancreatic cancercombination cells [2–7]. on RM the induces cell cycle, apoptosis apoptosis, and and inhibits transcriptome invasion in andorder migration to gain insights in non-small on the cell lung mechanism of combinatorial synergy, which could suggest therapeutic strategies for the treatment of cancerbreast cells cancer. (NSCLC) in vitro, making it a potential antimetastatic agent [8]. FigureFigure 1. 1.Renieramycin Renieramycin M M from from the the blue blue sponge sponge XestospongiaXestospongia sp. sp. RM2. Results is structurally related to ecteinascidin-743 (Et-743; Trabectedin, Yondelis®), an anticancer drug for advanced soft tissue sarcoma and recurrent platinum-sensitive ovarian cancer. The renieramycins 2.1. RM Is More Potent Than DOX in MCF-7 Cells and ecteinascidins are the two major categories of the 1,2,3,4-tetrahydroisoquinoline alkaloids that have an anticancerThe prerequisite effect. for Thisdetermination warrants of furthersynergistic investigation activity is to know on the the potential potency and clinical slope of utility the of RM. concentration-response curves of the individual drugs. Using MTT cytotoxicity assay, we determined A transcriptional structure–activity relationship (SAR) study and molecular network profiling revealed the IC50 of RM and DOX in MCF-7 breast cancer cells after 72 h of exposure. Figure 2A shows the that RMconcentration and the ecteinascidin-dependent cytotoxicity class of compounds of the individual induce drugs, apoptosis with RM via being a common ~60-fold pathway more potent in the colon, breast(IC [250], = and 6.0 ± glioblastoma 0.5 nM) than DOX cells (IC [950]. = Et-743356 ± 25 was nM). reported Significant to cytotoxicity have a sequence-dependent was observed starting at synergistic effect3.16 with nM paclitaxel and 100 innM breast for RM carcinoma and DOX, [10 respectively.], and with RM doxorubicin also shows in a softsteeper tissue sigmoidal sarcoma curvein vitro [11]. In view of the similarities between RM and Et-743, we hypothesize that RM can act also synergistically with standard cytotoxic drugs and thus, may be potentially useful to improve the therapeutic outcome. In this study, we investigated the effects of the combination of RM and DOX in estrogen receptor positive (ER+) MCF-7, an in vitro model for the most common type of breast cancer and determined the drug ratio and regimen that will yield a synergistic effect. We also determined the effects of the combination on the cell cycle, apoptosis, and transcriptome in order to gain insights on the mechanism of combinatorial synergy, which could suggest therapeutic strategies for the treatment of breast cancer. 2. Results 2.1. RM Is More Potent Than DOX in MCF-7 Cells The prerequisite for determination of synergistic activity is to know the potency and slope of the concentration-response curves of the individual drugs. Using MTT cytotoxicity assay, we determined the IC50 of RM and DOX in MCF-7 breast cancer cells after 72 h of exposure. Figure2A shows the concentration-dependent cytotoxicity of the individual drugs, with RM being ~60-fold more potent (IC = 6.0 0.5 nM) than DOX (IC = 356 25 nM). Significant cytotoxicity was observed starting 50 ± 50 ± at 3.16 nM and 100 nM for RM and DOX, respectively. RM also shows a steeper sigmoidal curve compared to DOX as indicated by their slopes (m values; Figure2B). Both compounds have R 2 > 0.95 indicating an excellent linear correlation. Mar. Drugs 2019, 17, x 3 of 28 compared to DOX as indicated by their slopes (m values; Figure 2B). Both compounds have R2 > 0.95 indicating an excellent linear correlation. We also monitored the effects of RM and DOX singly in real-time for a period of seven days and calculated the IC50 at different time points. The IC50 values of RM and DOX decreased over time indicating time-dependent cytotoxicity (Figure 2C, Figure S3). The IC50 of RM at all time points were lower than DOX, reflecting the more potent cytotoxicity of RM over DOX. RM also induces faster cytotoxicity (~24 h) than DOX (~48 h; Figure 2C). Interestingly, the cytostatic effect of DOX was Mar. Drugs 2019, 17, 536 3 of 26 observed after 24 h of exposure as shown by the brief lag in IC50. After 72 h, the IC50 of RM is almost constant, while that of DOX continued to gradually decrease. Figure 2. Individual cytotoxicity of renieramycin M (RM) and doxorubicin (DOX) on MCF-7 breast cancer cells.