salinomycin and Prostatic-Neoplasms

Cancer stem cells (CSCs) are associated with metastasis and recurrence in prostate cancer as well as other cancers. We aimed to enhance the sensitivity of cabazitaxel in prostate cancer cell therapy by targeting CSCs with a Wnt inhibitor and salinomycin pretreatment. PC3, DU-145, and LNCaP human prostate cancer cells were exposed to Wnt/β-catenin pathway inhibitor CCT036477 (iWnt) with salinomycin for 48 h, followed by cabazitaxel treatment for 48 h. Cell viability, mRNA, and protein expression changes were evaluated by MTT, RT-qPCR, and Western blot assays, respectively. Apoptosis was determined by image-based cytometry, and cell migration was assessed by wound healing assay. Three-dimensional culture was established to assess the malignant phenotype and stemness potential of transformed or cancer cells. CD44 + CSCs were isolated using magnetic-activated cell sorting system. Pretreatment of PC3, DU-145, and LNCaP cells with salinomycin iWnt significantly sensitized the cells to cabazitaxel therapy. Spheroid culture confirmed that the treatment modality was more effective than a single administration of chemotherapy. The pretreatment of PC3 cells increased the rate of apoptosis compared to single administration of cabazitaxel, which downregulated Bcl-2 and upregulated caspase 3, caspase 8 expressions. The pretreatment suppressed cell migration, downregulated the expression of Sox2 and Nanog, and significantly reduced CD44 + CSC numbers. Notably, the treatment modality reduced pAKT, p-P38 MAPK, and pERK1/2. The data suggest that pretreatment of prostate cancer cells with salinomycin and Wnt inhibitor may increase the efficacy of cabazitaxel therapy by inhibiting cell proliferation and migration, and eliminating cancer stem cells.

Topics: Apoptosis; Cell Line, Tumor; Cell Proliferation; Humans; Male; Neoplastic Stem Cells; Prostatic Neoplasms; Wnt Signaling Pathway

Tumor-initiating or cancer stem cells (CSCs) reduce the effectiveness of conventional therapy. Thus, it is crucial to eliminate CSCs while killing bulky cancer cells using a combination of conventional chemotherapy and anti-CSC drugs. Salinomycin is a selective inhibitor against CSCs and shows promise in combination applications. The aim of the study was to examine the efficacy of co-administered cabazitaxel and salinomycin on the survival of prostate cancer cells and CSCs.. CD44 + stem cells were isolated from human PC3 prostate cancer cells by using magnetic activated cell sorting. The cells were concomitantly exposed to salinomycin and cabazitaxel, and the cell survival was determined by MTT test. Apoptosis was assessed by image-based cytometer, and cell migration was evaluated by wound healing assay. The expression of target mRNA and protein were assessed by RT-qPCR and Western blot, respectively. Combination index (CI) analysis showed that simultaneous administration of salinomycin and cabazitaxel was able to exert strong synergistic effect on CD44 + subpopulation (CI = 0.33), but no synergism was observed in PC3 cells. The combination of the two agents significantly increased Bax, cytochrome c, caspase-3 and - 8 mRNA expression in CD44 + CSCs, causing apoptosis. The applied therapy strategy strongly inhibited the phosphorylation of Akt, protein expression of Akt1, NF-κB and Wnt.. In conclusion, our data suggest that combining salinomycin with cabazitaxel shows promise as a prostate cancer treatment approach that can target CSCs.

Topics: Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Humans; Hyaluronan Receptors; Male; Neoplastic Stem Cells; NF-kappa B; Prostatic Neoplasms; Proto-Oncogene Proteins c-akt; Pyrans; RNA, Messenger; Taxoids

Salinomycin is a monocarboxylic polyether antibiotic and is a potential chemotherapy drug. Our previous studies showed that salinomycin inhibited cell growth and targeted CSCs in prostate cancer. However, the precise target of salinomycin action is unclear.. In this work, we analyzed and identified differentially expressed genes (DEGs) after treatment with or without salinomycin using a gene expression microarray in vitro (PC-3 cells) and in vivo (NOD/SCID mice xenograft model generated from implanted PC-3 cells). Western blotting and immunohistochemical staining were used to analyze the expression of ATP2A3 and endoplasmic reticulum (ER) stress biomarkers. Flow cytometry was used to analyze the cell cycle, apoptosis and intracellular Ca. A significantly upregulated gene, ATPase sarcoplasmatic/endoplasmatic reticulum Ca. This study demonstrates that ATP2A3 might be one of the potential targets for salinomycin, which can inhibit Ca

Topics: Animals; Apoptosis; Cell Proliferation; Endoplasmic Reticulum Stress; Gene Expression Regulation, Neoplastic; Humans; Male; Mice; PC-3 Cells; Prostatic Neoplasms; Pyrans; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Signal Transduction; Transcriptional Activation; Xenograft Model Antitumor Assays

This study evaluated the mechanism by which salinomycin-induced autophagy blocks apoptosis in PC-3 prostate cancer cells.. The anti-cancer effects of salinomycin in PC-3 cells were confirmed by flow cytometry, JC-1 staining and western blotting. Then, the autophagic effects were measured by western blotting, GFP-LC3 puncta formation assay, immunofluorescence staining and electron microscopy. Furthermore, we used lentivirus-mediated shRNA to silence ATG3, ATG5 and ATG7 expression in PC-3 cells to investigate the regulatory mechanisms of salinomycin-induced autophagy.. Salinomycin could induce apoptosis and autophagy in PC-3 cells. Interestingly, autophagy inhibition could enhance salinomycin-induced apoptosis. We further showed that ATG3, a known critical regulator of autophagy, was downregulated and involved in the inhibition of apoptosis by salinomycin-induced autophagy via the AKT/mTOR signaling axis.. Our data indicated that salinomycin-induced autophagy blocks apoptosis via the ATG3/AKT/mTOR signaling axis in PC-3 cells, which provides new clues for the mechanisms of underlying the anti-cancer effects of salinomycin.

Topics: Antineoplastic Agents; Apoptosis; Autophagy; Autophagy-Related Proteins; Cell Line, Tumor; Cell Proliferation; Green Fluorescent Proteins; Humans; Male; Prostatic Neoplasms; Proto-Oncogene Proteins c-akt; Pyrans; Signal Transduction; TOR Serine-Threonine Kinases; Ubiquitin-Conjugating Enzymes

Androgen receptor (AR) and PI3K/AKT/mTORC1 are major survival signals that drive prostate cancer to a lethal disease. Reciprocal activation of these oncogenic pathways from negative cross talks contributes to low/limited success of pathway-selective inhibitors in curbing prostate cancer progression. We report that the antibiotic salinomycin, a cancer stem cell blocker, is a dual-acting AR and mTORC1 inhibitor, inhibiting PTEN-deficient castration-sensitive and castration-resistant prostate cancer in culture and xenograft tumors. AR expression, its transcriptional activity, and androgen biosynthesis regulating enzymes CYP17A1, HSD3β1 were reduced by sub-micro molar salinomycin. Estrogen receptor-α expression was unchanged. Loss of phosphorylated AR at serine-81, which is an index for nuclear AR activity, preceded total AR reduction. Rapamycin enhanced the AR protein level without altering phosphoAR-Ser81 and CYP17A1. Inactivation of mTORC1, evident from reduced phosphorylation of mTOR and downstream effectors, as well as AMPK activation led to robust autophagy induction. Apoptosis increased modestly, albeit significantly, by sub-micro molar salinomycin. Enhanced stimulatory TSC2 phosphorylation at Ser-1387 by AMPK, and reduced inhibitory TSC2 phosphorylation at Ser-939/Thr-1462 catalyzed by AKT augmented TSC2/TSC1 activity, which led to mTORC1 inhibition. AMPK-mediated raptor phosphorylation further reduced mTOR's kinase function and mTORC1 activity. Our novel finding on dual inhibition of AR and mTORC1 suggests that salinomycin is potentially active as monotherapy against advanced prostate cancer.

Topics: AMP-Activated Protein Kinases; Androgen Receptor Antagonists; Animals; Antibiotics, Antineoplastic; Apoptosis; Cell Line, Tumor; Cell Proliferation; Estrogen Receptor alpha; Humans; Male; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Nude; Multienzyme Complexes; Phosphatidylinositol 3-Kinases; Phosphorylation; Progesterone Reductase; Prostatic Neoplasms; Prostatic Neoplasms, Castration-Resistant; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Pyrans; Receptors, Androgen; Serine; Signal Transduction; Sirolimus; Steroid 17-alpha-Hydroxylase; Steroid Isomerases; Tuberous Sclerosis Complex 1 Protein; Tuberous Sclerosis Complex 2 Protein; Tumor Suppressor Proteins; Xenograft Model Antitumor Assays

Future curative cancer chemotherapies have to overcome tumor cell heterogeneity and plasticity. To test the hypothesis that the tumor suppressor maspin may reduce microenvironment-dependent prostate tumor cell plasticity and thereby modulate drug sensitivity, we established a new schematic combination of two-dimensional (2D), three-dimensional (3D), and suspension cultures to enrich prostate cancer cell subpopulations with distinct differentiation potentials. We report here that depending on the level of maspin expression, tumor cells in suspension and 3D collagen I manifest the phenotypes of stem-like and dormant tumor cell populations, respectively. In suspension, the surviving maspin-expressing tumor cells lost the self-renewal capacity, underwent senescence, lost the ability to dedifferentiate in vitro, and failed to generate tumors in vivo. Maspin-nonexpressing tumor cells that survived the suspension culture in compact tumorspheres displayed a higher level of stem cell marker expression, maintained the self-renewal capacity, formed tumorspheres in 3D matrices in vitro, and were tumorigenic in vivo. The drug sensitivities of the distinct cell subpopulations depend on the drug target and the differentiation state of the cells. In 2D, docetaxel, MS275, and salinomycin were all cytotoxic. In suspension, while MS275 and salinomycin were toxic, docetaxel showed no effect. Interestingly, cells adapted to 3D collagen I were only responsive to salinomycin. Maspin expression correlated with higher sensitivity to MS275 in both 2D and suspension and to salinomycin in 2D and 3D collagen I. Our data suggest that maspin reduces prostate tumor cell plasticity and enhances tumor sensitivity to salinomycin, which may hold promise in overcoming tumor cell heterogeneity and plasticity.

Topics: Adenocarcinoma; Animals; Antineoplastic Agents; Benzamides; Cell Adhesion; Cell Culture Techniques; Cell Dedifferentiation; Cell Line, Tumor; Cell Plasticity; Cell Self Renewal; Cellular Senescence; Docetaxel; Drug Resistance, Neoplasm; Gene Expression Profiling; Heterografts; Humans; Male; Mice; Mice, Nude; Neoplasm Proteins; Neoplasm Transplantation; Neoplastic Stem Cells; Phenotype; Prostatic Neoplasms; Pyrans; Pyridines; Serpins; Suspensions; Taxoids; Tumor Microenvironment

Emerging evidence indicates that activation of Wnt/β-catenin signaling at the cell surface results in inhibition of glycogen synthase kinase 3β (GSK3β), leading to activation of mTORC1 signaling in cancer cells. The low density lipoprotein receptor-related protein-6 (LRP6) is an essential Wnt co-receptor for Wnt/β-catenin signaling. Salinomycin is a novel small molecule inhibitor of LRP6. In the present study, we found that LRP6 overexpression induced mTORC1 signaling activation in cancer cells, and that salinomycin was not only a potent Wnt/β-catenin signaling inhibitor, but also a strong mTORC1 signaling antagonist in breast and prostate cancer cells. Mechanistically, salinomycin activated GSK3β in cancer cells. Moreover, salinomycin was able to suppress the expression of cyclin D1 and survivin, two targets of both Wnt/β-catenin and mTORC1 signaling, in prostate and breast cancer cells, and displayed remarkable anticancer activity. Our results present novel mechanisms underlying salinomycin-mediated cancer cell death.

Topics: Anti-Bacterial Agents; Apoptosis; beta Catenin; Breast; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin D1; Enzyme Activation; Female; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; HEK293 Cells; Humans; Inhibitor of Apoptosis Proteins; Low Density Lipoprotein Receptor-Related Protein-6; Male; MCF-7 Cells; Mechanistic Target of Rapamycin Complex 1; Multiprotein Complexes; Prostate; Prostatic Neoplasms; Pyrans; Survivin; TOR Serine-Threonine Kinases; Wnt Proteins; Wnt Signaling Pathway

The molecular mechanism of Salinomycin's toxicity is not fully understood. Various studies reported that Ca(2+), cytochrome c, and caspase activation play a role in Salinomycin-induced cytotoxicity. Furthermore, Salinomycin may target Wnt/β-catenin signaling pathway to promote differentiation and thus elimination of cancer stem cells. In this study, we show a massive autophagic response to Salinomycin (substantially stronger than to commonly used autophagic inducer Rapamycin) in prostrate-, breast cancer cells, and to lesser degree in human normal dermal fibroblasts. Interestingly, autophagy induced by Salinomycin is a cell protective mechanism in all tested cancer cell lines. Furthermore, Salinomycin induces mitophagy, mitoptosis and increased mitochondrial membrane potential (∆Ψ) in a subpopulation of cells. Salinomycin strongly, and in time-dependent manner decreases cellular ATP level. Contrastingly, human normal dermal fibroblasts treated with Salinomycin show some initial decrease in mitochondrial mass, however they are largely resistant to Salinomycin-triggered ATP-depletion. Our data provide new insight into the molecular mechanism of preferential toxicity of Salinomycin towards cancer cells, and suggest possible clinical application of Salinomycin in combination with autophagy inhibitors (i.e. clinically-used Chloroquine). Furthermore, we discuss preferential Salinomycins toxicity in the context of Warburg effect.

Topics: Adenosine Triphosphate; Animals; Anti-Bacterial Agents; Autophagy; Breast Neoplasms; Cell Line, Tumor; Cytotoxins; Dermis; Female; Fibroblasts; Humans; Male; Membrane Potential, Mitochondrial; Mice; Mice, Knockout; Mitochondria; Mitophagy; Prostatic Neoplasms; Pyrans; Wnt Signaling Pathway

We have shown that a sodium ionophore monensin inhibits prostate cancer cell growth. A structurally related compound to monensin, salinomycin, was recently identified as a putative cancer stem cell inhibitor.. The growth inhibitory potential of salinomycin was studied in a panel of prostate cells. To get insights into the mechanism of action, a variety of assays such as gene expression and steroid profiling were performed in salinomycin-exposed prostate cancer cells.. Salinomycin inhibited the growth of prostate cancer cells, but did not affect non-malignant prostate epithelial cells. Salinomycin impacted on prostate cancer stem cell functions as evidenced by reduced aldehyde dehydrogenase activity and the fraction of CD44(+) cells. Moreover, salinomycin reduced the expression of MYC, AR and ERG, induced oxidative stress as well as inhibited nuclear factor-κB activity and cell migration. Furthermore, profiling steroid metabolites revealed increased levels of oxidative stress-inducing steroids 7-ketocholesterol and aldosterone and decreased levels of antioxidative steroids progesterone and pregnenolone in salinomycin-exposed prostate cancer cells.. Our results indicate that salinomycin inhibits prostate cancer cell growth and migration by reducing the expression of key prostate cancer oncogenes, inducing oxidative stress, decreasing the antioxidative capacity and cancer stem cell fraction.

Topics: Aldehyde Dehydrogenase; Blotting, Western; Cell Division; Cell Line, Tumor; Cell Movement; Humans; Male; NF-kappa B; Niclosamide; Oxidative Stress; Prostatic Neoplasms; Pyrans; Reactive Oxygen Species; Receptors, Androgen; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Terfenadine

The anticancer activity of salinomycin has evoked excitement due to its recent identification as a selective inhibitor of breast cancer stem cells (CSCs) and its ability to reduce tumor growth and metastasis in vivo. In prostate cancer, similar to other cancer types, CSCs and/or progenitor cancer cells are believed to drive tumor recurrence and tumor growth. Thus salinomycin can potentially interfere with the end-stage progression of hormone-indifferent and chemotherapy-resistant prostate cancer. Androgen-responsive (LNCaP) and androgen-refractive (PC-3, DU-145) human prostate cancer cells showed dose- and time-dependent reduced viability upon salinomycin treatment; non-malignant RWPE-1 prostate cells were relatively less sensitive to drug-induced lethality. Salinomycin triggered apoptosis of PC-3 cells by elevating the intracellular ROS level, which was accompanied by decreased mitochondrial membrane potential, translocation of Bax protein to mitochondria, cytochrome c release to the cytoplasm, activation of the caspase-3 and cleavage of PARP-1, a caspase-3 substrate. Expression of the survival protein Bcl-2 declined. Pretreatment of PC-3 cells with the antioxidant N-acetylcysteine prevented escalation of oxidative stress, dissipation of the membrane polarity of mitochondria and changes in downstream molecular events. These results are the first to link elevated oxidative stress and mitochondrial membrane depolarization to salinomycin-mediated apoptosis of prostate cancer cells.

Topics: Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Cell Line, Tumor; Cell Survival; Cytochromes c; Cytosol; Humans; Male; Membrane Potential, Mitochondrial; Mitochondria; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Prostatic Neoplasms; Protein Transport; Proto-Oncogene Proteins c-bcl-2; Pyrans; Reactive Oxygen Species