Timothy C. Chambers

Monensin (MON) is a polyether ionophore antibiotic of natural origin and is an FDA-approved drug for veterinary use. Recent studies have highlighted its potential anticancer activity in various in vitro and in vivo models. In this study, we evaluated the anti-breast cancer activity of MON and 37 synthetic analog compounds using cell monolayer and organoid models. Through a mini-ring cell viability assay, several compounds were identified that were more potent and selective against breast cancer cells compared to non-cancerous cells, surpassing the activity of parent MON. MON and these compounds induced significant DNA fragmentation, reduced cell migration, and downregulated SOX2 expression. Furthermore, MON and the most potent analog, compound 12, reduced the percentage of CD44⁺/CD24^-/low stem-like cells and diminished colony formation properties. Proteomics analyses revealed that several pathways, including extracellular matrix organization, were significantly dysregulated by MON and compound 12 in breast cancer cells. Among these, TIMP2, a protein associated with the suppression of tumor growth and metastasis, was identified as one of the most prominently upregulated proteins by MON and compound 12 in MDA-MB-231 cells. This finding was also validated in other breast cancer and melanoma cell lines. To simulate breast cancer metastasis to the brain, a human hybrid organoid system: tumor in brain organoid (HOSTBO) model was developed. MON and compound 12 significantly reduced Ki-67 expression within the HOSTBOs, and compound 12 significantly downregulated SOX2 expression. Collectively, MON and compound 12 significantly reduced the proliferation of breast cancer stem-like cells in the organoid models, inhibited their migration, and dysregulated markers associated with stemness, demonstrating their potential as anti-metastatic agents and warranting further clinical development.

Colchicine (COL) shows strong anticancer activity but due to its toxicity towards normal cells its wider application is limited. To address this issue, a library of 17 novel COL derivatives, namely N‐carbamates of N‐deacetyl‐4‐(bromo/chloro/iodo)thiocolchicine, has been tested against two types of primary cancer cells. These included acute lymphoblastic leukemia (ALL) and human breast cancer (BC) derived from two different tumor subtypes, ER+ invasive ductal carcinoma grade III (IDCG3) and metastatic carcinoma (MC). Four novel COL derivatives showed higher anti‐proliferative activity than COL (IC₅₀ = 8.6 nM) towards primary ALL cells in cell viability assays (IC₅₀ range of 1.1‐6.4 nM), and several were more potent towards primary IDCG3 (IC₅₀ range of 0.1 to 10.3 nM) or MC (IC₅₀ range of 2.3‐9.1 nM) compared to COL (IC₅₀ of 11.1 and 11.7 nM, respectively). In addition, several derivatives were selectively active toward primary breast cancer cells compared to normal breast epithelial cells. The most promising derivatives were subsequently tested against the NCI panel of 60 human cancer cell lines and seven derivatives were more potent than COL against leukemia, non–small‐cell lung, colon, CNS and prostate cancers. Finally, COL and two of the most active derivatives were shown to be effective in killing BC cells when tested ex vivo using fresh human breast tumor explants. The present findings indicate that the select COL derivatives constitute promising lead compounds targeting specific types of cancer.

The polyether ionophore salinomycin (SAL) has captured much interest because of its potent activity against cancer cells and cancer stem cells. Our previous studies have indicated that C1/C20 double‐modification of SAL is a useful strategy to generate diverse agents with promising biological activity profiles. Thus, herein we describe the synthesis of a new class of SAL analogues that combine key modifications at the C1 and C20 positions. The activity of the obtained SAL derivatives was evaluated using primary acute lymphoblastic leukemia, human breast adenocarcinoma and normal mammary epithelial cells. One single‐ [N,N‐dipropyl amide of salinomycin (5 a)] and two novel double‐modified analogues [N,N‐dipropyl amide of C20‐oxosalinomycin (5 b) and piperazine amide of C20‐oxosalinomycin (13 b)] were found to be more potent toward the MDA‐MB‐231 cell line than SAL or its C20‐oxo analogue 2. When select analogues were tested against the NCI‐60 human tumor cell line panel, 4 a [N,N‐diethyl amide of salinomycin] showed particular activity toward the ovarian cancer cell line SK‐OV‐3. Additionally, both SAL and 2 were found to be potent ex vivo against human ER/PR⁺, Her2⁻ invasive mammary carcinoma, with 2 showing minimal toxicity toward normal epithelial cells. The present findings highlight the therapeutic potential of SAL derivatives for select targeting of different cancer types.

Phyllodes tumors of the breast (PTB) are uncommon stromal‑epithelial neoplasms, with the main recommended treatment being surgical removal. However, even with adequate resection, the risk of recurrence in the malignant form remains as high as 40%, and there is no recognized consensus on the most effective drugs for PTB. In the present study, an ex vivo model of malignant phyllodes and derived primary cell cultures were used to evaluate the effectiveness of a panel of different drugs, including Bcl‑2/Bcl‑xL inhibitor ABT‑263, salinomycin (SAL), doxorubicin (DOX), paclitaxel (TAX), vincristine (VCR), colchicine (COL) and cisplatin (CIS). ABT‑263, SAL and DOX were highly effective towards phyllodes spindle cells when assessed in the ex vivo model, contributing to ~98% tumor cell death. Furthermore, ABT‑263 was highly selective for tumor cells in this system, and exhibited little toxic effect on adjacent normal epithelial cells. Furthermore, consistent with findings in the ex vivo model, ABT‑263 was significantly less toxic towards MCF 10A non‑tumorigenic breast epithelial cells compared with SAL and DOX. A conditional reprogramming strategy was subsequently used, involving Rho kinase inhibition, to successfully generate primary phyllodes tumor cells that could be cultured for several passages. The primary cells were sensitive to DOX with an IC50 of 0.40±0.07 µM in a standard viability assay and the preliminary results were obtained indicating sensitivity to ABT‑263 and SAL. The present study demonstrated the feasibility of using explants and primary cells for drug discovery, selectively targeting PTB cells.

Polyether ionophore antibiotics represent a large group of more than 120 lipid-soluble compounds that are widely used in veterinary medicine because of their significant antimicrobial activity

Colchicine ( COL ) is the major alkaloid isolated from the plant Colchicum autumnale . It shows strong therapeutic effects towards various types of cancer. Its biological activity is related to its ability to bind to tubulin causing microtubule depolymerization, mitotic arrest, and cell death. The colchicine binding site is an attractive target for potential tubulin polymerization destabilizers. Indeed, a large number of molecules interacting with the colchicine binding site have been designed and synthesized with significant structural diversity. The identification of new therapeutics based on COL structure is warranted due to the toxicity of COL towards normal cells. In order to increase potency and reduce toxicity against normal cells, while retaining the activities in the nanomolar range, a library of novel COL analogs, namely N ‐carbamates of N ‐deacetyl‐4‐(bromo/chloro/iodo)thiocolchicine, has been synthesized (Fig. 1). COL and several of the derivatives arrested MCF‐7 cells in mitosis, and caused microtubule depolymerization. Compounds were then tested against two types of primary cancer cells; adult acute lymphoblastic leukemia (ALL) cells, and human breast cancer (BC) cells derived from newly excised human tumor tissue. These represent more clinically relevant drug screening models compared to established cell lines. Four novel colchicine derivatives showed higher activity towards primary ALL cells (IC ₅₀ = 1.1 ± 0.5 to 6.4 ± 1.4 nM) and nine were more potent towards primary BC cells (IC ₅₀ = 2.3 ± 0.0 to 10.3 ± 4.6 nM) compared to COL (IC ₅₀ = 8.6 ± 0.2 nM and 11.7 ± 3.1 nM, respectively) in cell viability assays. COL and two of the most active derivatives were also shown to be effective in killing BC cells when tested ex vivo using fresh human breast tumor explants derived from residual invasive ductal carcinoma and invasive mucinous carcinoma. The present findings indicate that the COL derivatives described here constitute promising lead compounds for targeting acute lymphoblastic leukemia and different subtypes of breast cancer. Support or Funding Information The present study was funded by a grant (to TCC) from the Arkansas Breast Cancer Research Program. Financial support by grant (to AH. UM, GK) of the Polish National Science Centre (NCN) – No. 2016/21/B/ST5/00111 is gratefully acknowledged . This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .