Proteomic analysis reveals a role for PAX8 in peritoneal colonization of high grade serous ovarian cancer that can be targeted with micelle encapsulated thiostrepton.

High grade serous ovarian cancer (HGSOC) is the fifth leading cause of cancer deaths among women yet effective targeted therapies against this disease are limited. The heterogeneity of HGSOC, including few shared oncogenic drivers and origination from both the fallopian tube epithelium (FTE) and ovarian surface epithelium (OSE), has hampered development of targeted drug therapies. PAX8 is a lineage-specific transcription factor expressed in the FTE that is also ubiquitously expressed in HGSOC where it is an important driver of proliferation, migration, and cell survival. PAX8 is not normally expressed in the OSE, but it is turned on after malignant transformation. In this study, we use proteomic and transcriptomic analysis to examine the role of PAX8 leading to increased migratory capabilities in a human ovarian cancer model, as well as in tumor models derived from the OSE and FTE. We find that PAX8 is a master regulator of migration with unique downstream transcriptional targets that are dependent on the cell's site of origin. Importantly, we show that targeting PAX8, either through CRISPR genomic alteration or through drug treatment with micelle encapsulated thiostrepton, leads to a reduction in tumor burden. These findings suggest PAX8 is a unifying protein driving metastasis in ovarian tumors that could be developed as an effective drug target to treat HGSOC derived from both the OSE and FTE.

Development of co-solvent freeze-drying method for the encapsulation of water-insoluble thiostrepton in sterically stabilized micelles.

The purpose of this work was to develop a practical and scalable method to encapsulate the hydrophobic antibiotic thiostrepton (TST) in sterically stabilized micelles (SSM). Using the conventional method of thin-film hydration, we encapsulated up to 5 drug molecules per SSM (diameter ∼ 16 nm). However, since this method is not suitable for large-scale production - a limiting factor for clinical translation - we applied the co-solvent freeze-drying method using tert-butanol (TBA): water co-solvent system. We found that the presence of phosphate-buffered saline (PBS) salts in the lyophilized cake accelerated the reconstitution time and allowed efficient drug encapsulation without the formation of larger drug particles. In addition, TBA proportion of 50% (v/v) was sufficient to maintain both phospholipid and drug in solution prior to the freeze-drying. The increase of drug and phospholipid concentrations in the formulation extended the reconstitution time and led to drug precipitation. Therefore, to increase the strength of the formulation, we prepared lyophilized cakes with lower phospholipid content (5 mM) and reconstituted them in one-third of the fill volume. In conclusion, we found optimum conditions to prepare TST-SSM using the co-solvent freeze-drying method. This scalable production method can facilitate the further clinical development and industrial production of TST-SSM nanomedicine.

Targeting FOXM1 Improves Cytotoxicity of Paclitaxel and Cisplatinum in Platinum-Resistant Ovarian Cancer.

OBJECTIVE: Aberrantly activated FOXM1 (forkhead box protein M1) leading to uncontrolled cell proliferation and dysregulation of FOXM1 transcription network occurs in 84% of ovarian cancer cases. It was demonstrated that thiostrepton, a thiazole antibiotic, decreases FOXM1 expression. We aimed to determine if targeting the FOXM1 pathway with thiostrepton could improve the efficacy of paclitaxel and cisplatin in human ovarian cancer ascites cells ex vivo. METHODS: Human ovarian cancer cell lines and patients' ascites cells were treated with paclitaxel, cisplatin, and thiostrepton or a combination for 48 hours, and cytotoxicity was assessed. Drug combination effects were determined by calculating the combination index values using the Chou and Talalay method. Quantitative reverse transcriptase-polymerase chain reaction was performed to determine changes in FOXM1 expression and its downstream targets. RESULTS: Ovarian cancer cell lines and the patients' ascites cancer cells had an overexpression of FOXM1 expression levels. Targeting FOXM1 with thiostrepton decreased FOXM1 mRNA expression and its downstream targets such as CCNB1 and CDC25B, leading to cell death in both cell lines and patients' ascites cancer cells. Furthermore, addition of thiostrepton to paclitaxel and cisplatin showed synergistic effects in chemoresistant ovarian cancer patients' ascites cells ex vivo. CONCLUSION: Targeting FOXM1 may lead to novel therapeutics for chemoresistant epithelial ovarian cancer.

Targeting Foxm1 Improves Cytotoxicity of Paclitaxel and Cisplatinum in Platinum-Resistant Ovarian Cancer.

OBJECTIVE: Aberrantly activated FOXM1 (forkhead box protein M1) leading to uncontrolled cell proliferation and dysregulation of FOXM1 transcription network occurs in 84% of ovarian cancer cases. It was demonstrated that thiostrepton, a thiazole antibiotic, decreases FOXM1 expression. We aimed to determine if targeting the FOXM1 pathway with thiostrepton could improve the efficacy of paclitaxel and cisplatin in human ovarian cancer ascites cells ex vivo. METHODS: Human ovarian cancer cell lines and patients' ascites cells were treated with paclitaxel, cisplatin, and thiostrepton or a combination for 48 hours, and cytotoxicity was assessed. Drug combination effects were determined by calculating the combination index values using the Chou and Talalay method. Quantitative real-time polymerase chain reaction was performed to determine changes in FOXM1 expression and its downstream targets. RESULTS: Ovarian cancer cell lines and the patients' ascites cancer cells had an overexpression of FOXM1 expression levels. Targeting FOXM1 with thiostrepton decreased FOXM1 mRNA expression and its downstream targets such as CCNB1, CDC25B, leading to cell death in both cell lines and patients' ascites cancer cells. Furthermore, addition of thiostrepton to paclitaxel and cisplatin showed synergistic effects in chemoresistant ovarian cancer patients' ascites cells ex vivo. CONCLUSION: Targeting FOXM1 may lead to novel therapeutics for chemoresistant epithelial ovarian cancer.

Pathological Ace2-to-Ace enzyme switch in the stressed heart is transcriptionally controlled by the endothelial Brg1-FoxM1 complex.

Genes encoding angiotensin-converting enzymes (Ace and Ace2) are essential for heart function regulation. Cardiac stress enhances Ace, but suppresses Ace2, expression in the heart, leading to a net production of angiotensin II that promotes cardiac hypertrophy and fibrosis. The regulatory mechanism that underlies the Ace2-to-Ace pathological switch, however, is unknown. Here we report that the Brahma-related gene-1 (Brg1) chromatin remodeler and forkhead box M1 (FoxM1) transcription factor cooperate within cardiac (coronary) endothelial cells of pathologically stressed hearts to trigger the Ace2-to-Ace enzyme switch, angiotensin I-to-II conversion, and cardiac hypertrophy. In mice, cardiac stress activates the expression of Brg1 and FoxM1 in endothelial cells. Once activated, Brg1 and FoxM1 form a protein complex on Ace and Ace2 promoters to concurrently activate Ace and repress Ace2, tipping the balance to Ace2 expression with enhanced angiotensin II production, leading to cardiac hypertrophy and fibrosis. Disruption of endothelial Brg1 or FoxM1 or chemical inhibition of FoxM1 abolishes the stress-induced Ace2-to-Ace switch and protects the heart from pathological hypertrophy. In human hypertrophic hearts, BRG1 and FOXM1 expression is also activated in endothelial cells; their expression levels correlate strongly with the ACE/ACE2 ratio, suggesting a conserved mechanism. Our studies demonstrate a molecular interaction of Brg1 and FoxM1 and an endothelial mechanism of modulating Ace/Ace2 ratio for heart failure therapy.

Inhibition of Forkhead box protein M1 by thiostrepton increases chemosensitivity to doxorubicin in T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive type of blood malignancy, deriving from T-cell progenitors in the thymus, and comprises 10-15% of pediatric and 25% of adult primary ALL cases. Despite advances, 20% of pediatric and the majority of adult patients with T-ALL succumb to mortality from resistant or relapsed disease, and the survival rate for patients with resistant or relapsed T-ALL remains poor. Alterations in the expression of Forkhead box protein M1 (FoxM1) have been detected in several types of cancer, and the inhibition of FoxM1 has been investigated as therapeutic strategy in cancer. The present study investigated the effects of the inhibition of FoxM1 by thiostrepton in human T-ALL Jurkat cells. The cells were treated with different concentrations of thiostrepton, either alone or in combination with doxorubicin. Cell viability was measured using CCK-8 assays and the cell cycle distribution, apoptosis and cell-associated mean fluorescence intensity of intracellular doxorubicin were assessed using flow cytometric analysis. The mRNA and protein expression levels were detected by reverse transcription-quantitative polymerase chain reaction and western blot analyses. The inhibition of FoxM1 by thiostrepton significantly decreased the proliferation of the Jurkat cells proliferation in a time- and dose-dependent manner. Cell arrest at the G2/M phase, and apoptosis was significantly increased in the thiostrepton-treated Jurkat cells. Thiostrepton reduced the half maximal inhibitory concentration of doxorubicin in the Jurkat cells, and significantly enhanced the cytotoxicity of doxorubicin within the Jurkat cells by enhancing doxorubicin-induced apoptosis and increasing the accumulation of intracellular doxorubicin. Furthermore, the inhibition of FoxM1 by thiostrepton enhanced doxorubicin-induced apoptosis, possibly through a caspase-3-dependent pathway, and increased the accumulation of intracellular doxorubicin, possibly through downregulating the expression of glutathione S-transferase pi. Collectively, the results of the present study suggested that targeting FoxM1 with thiostrepton resulted in potent antileukemia activity and chemosensitizing effects in human T-ALL Jurkat cells.

Targeting FoxM1 by thiostrepton inhibits growth and induces apoptosis of laryngeal squamous cell carcinoma.

PURPOSE: We have previously reported that forkhead box M1 (FoxM1) transcription factor was overexpressed in laryngeal squamous cell carcinoma (LSCC) and was associated with development of LSCC. However, there are limited studies regarding the functional significance of FoxM1 and FoxM1 inhibitor thiostrepton in LSCC. Therefore, the aim of this study was to examine both in vitro and in vivo activity of FoxM1 inhibitor thiostrepton against LSCC cell line and nude mice. METHODS: Cell viability was studied by CCK-8 assay. Cell growth was evaluated by CFSE staining and cell cycle analysis. Apoptosis was measured by flow cytometry. The mRNA and protein expression were detected by quantitative real-time RT-PCR, Western blot and immunohistochemical staining. Xenograft model of tumor formation was used to investigate how thiostrepton influences tumorigenesis in vivo. RESULTS: Overexpression of FoxM1 in LSCC cells was down-regulated by thiostrepton in a dose-dependent manner. Thiostrepton caused dose- and time-dependent suppression of cell viability of LSCC. Moreover, thiostrepton induced cell cycle arrest at S phase at early time and inhibited DNA synthesis in LSCC cells in a dose- and time-dependent manner by down-regulation of cyclin D1 and cyclin E1. Thiostrepton also induced dose- and time-dependent apoptosis of LSCC cells by down-regulation of Bcl-2, up-regulation of Bax and p53, and inducing release of cytochrome c accompanied by activation of cleaved caspase-9, cleaved caspase-3 and cleaved PARP. In addition, z-VAD-fmk, a universal inhibitor of caspases, prevented activation of cleavage caspase-3 and abrogates cell death induced by thiostrepton treatment. Furthermore, FADD and cleaved caspase-8 were activated, and expression of cIAP1, XIAP and survivin were inhibited by thiostrepton. Finally, treatment of LSCC cell line xenografts with thiostrepton resulted in tumorigenesis inhibition of tumors in nude mice by reducing proliferation and inducing apoptosis of LSCC cells. CONCLUSIONS: Collectively, our finding suggest that targeting FoxM1 by thiostrepton inhibit growth and induce apoptosis of LSCC through mitochondrial- and caspase-dependent intrinsic pathway and Fas-dependent extrinsic pathway as well as IAP family. Thiostrepton may represent a novel lead compound for targeted therapy of LSCC.

Targeting of mutant p53-induced FoxM1 with thiostrepton induces cytotoxicity and enhances carboplatin sensitivity in cancer cells.

FoxM1 is an oncogenic Forkhead transcription factor that is overexpressed in ovarian cancer. However, the mechanisms by which FoxM1 is deregulated in ovarian cancer and the extent to which FoxM1 can be targeted in ovarian cancer have not been reported previously. In this study, we showed that MDM2 inhibitor Nutlin-3 upregulated p53 protein and downregulated FoxM1 expression in several cancer cell lines with wild type TP53 but not in cell lines with mutant TP53. FoxM1 downregulation was partially blocked by cycloheximide or actinomycin D, and pulse-chase studies indicate Nutlin-3 enhances FoxM1 mRNA decay. Knockdown of p53 using shRNAs abrogated the FoxM1 downregulation by Nutlin-3, indicating a p53-dependent mechanism. FoxM1 inhibitor, thiostrepton, induces apoptosis in cancer cell lines and enhances sensitivity to cisplatin in these cells. Thiostrepton downregulates FoxM1 expression in several cancer cell lines and enhances sensitivity to carboplatin in vivo. Finally, FoxM1 expression is elevated in nearly all (48/49) ovarian tumors, indicating that thiostrepton target gene is highly expressed in ovarian cancer. In summary, the present study provides novel evidence that both amorphic and neomorphic mutations in TP53 contribute to FoxM1 overexpression and that FoxM1 may be targeted for therapeutic benefits in cancers.

The dual inhibitory effect of thiostrepton on FoxM1 and EWS/FLI1 provides a novel therapeutic option for Ewing's sarcoma.

The poor prognosis of Ewing's sarcoma (EWS), together with its high lethal recurrence rate and the side­effects of current treatments, call for novel targeted therapies with greater curative effectiveness and substantially reduced side­effects. The oncogenic chimeric protein EWS/FLI1 is the key malignancy driver in most EWSs, regulating numerous target genes, many of which influence cell cycle progression. It has often been argued that targeting proteins regulated directly or indirectly by EWS/FLI1 may provide improved therapeutic options for EWS. In this context, our study examined FoxM1, a key cell cycle regulating transcription factor, reported to be expressed in EWS and influenced by EWS/FLI1. Thiostrepton, a naturally occurring small molecule, has been shown to selectively inhibit FoxM1 expression in cancer cells. We demonstrate that in EWS, in addition to inhibiting FoxM1 expression, thiostrepton downregulates the expression of EWS/FLI1, both at the mRNA and protein levels, leading to cell cycle arrest and, ultimately, to apoptotic cell death. We also show that thiostrepton treatment reduces the tumorigenicity of EWS cells, significantly delaying the growth of nude mouse xenograft tumors. Results from this study demonstrate a novel action of thiostrepton as inhibitor of the expression of the EWS/FLI1 oncoprotein in vitro and in vivo, and that it shows greater efficacy against EWS than against other tumor types, as it is active on EWS cells and tumors at concentrations lower than those reported to have effective inhibitory activity on tumor cells derived from other cancers. Owing to the dual action of this small molecule, our findings suggest that thiostrepton may be particularly effective as a novel agent for the treatment of EWS patients.

Combination with bortezomib enhances the antitumor effects of nanoparticle-encapsulated thiostrepton.

Bortezomib is well-known for inducing cell death in cancer cells, specifically through the mechanism of proteasome inhibition. Thiostrepton, a thiazole antibiotic, has also been described for its proteasome inhibitory action, although differing slightly to bortezomib in the proteasomal site to which it is active. Previously we had shown the synergic effect of bortezomib and thiostrepton in breast cancer cells in vitro, where sub-apoptotic concentrations of both proteasome inhibitors resulted in synergic increase in cell death when combined as a treatment. Here, we administered such a combination to MDA-MB-231 xenograft tumors in vivo, and found that the effect of complementary proteasome inhibitors reduced tumor growth rates more efficiently than compared with when administered alone. Increased induction of apoptotic activity in tumors was found be associated with the growth inhibitory activity of combination treatment. Further examination additionally revealed that combination-treated tumors exhibited reduced proteasome activity, compared with non-treated and single drug-treated tumors. These data suggest that this drug combination may be useful as a therapy for solid tumors.

Micelle-encapsulated thiostrepton as an effective nanomedicine for inhibiting tumor growth and for suppressing FOXM1 in human xenografts.

The thiazole antiobiotic, thiostrepton, has been found to induce cell death in cancer cells through proteasome inhibition. As a proteasome inhibitor, thiostrepton has also been shown to suppress the expression of FOXM1, the oncogenic forkhead transcription factor overexpressed in cancer cells. In this study, we explored the potential in vivo anticancer properties of thiostrepton, delivered through nanoparticle encapsulation to xenograft models of breast and liver cancer. We encapsulated thiostrepton into micelles assembled from amphiphilic lipid-PEG (polyethylene glycol) molecules, where thiostrepton is solubilized within the inner lipid compartment of the micelle. Upon assembly, hydrophobic thiostrepton molecules are solubilized into the lipid component of the micelle shell, formed through the self-assembly of amphipilic lipid-PEG molecules. Maximum accumulation of micelle-thiostrepton nanoparticles (100 nm in diameter, -16 mV in zeta potential) into tumors was found at 4 hours postadministration and was retained for at least 24 hours. Upon continuous treatment, we found that nanoparticle-encapsulated thiostrepton reduced tumor growth rates of MDA-MB-231 and HepG2 cancer xenografts. Furthermore, we show for the first time the in vivo suppression of the oncogenic FOXM1 after treatment with proteasome inhibitors. Immunoblotting and immunohistochemical staining also showed increased apoptosis in the treated tumors, as indicated by cleaved caspase-3 expression. Our data suggest that the thiazole antibiotic/proteasome inhibitor thiostrepton, when formulated into nanoparticles, may be highly suited as a nanomedicine for treating human cancer.

Thiazole antibiotic thiostrepton synergize with bortezomib to induce apoptosis in cancer cells.

Thiazole antibiotic, thiostrepton was recently identified as proteasome inhibitor. We investigated the therapeutic potential of the combination of thiostrepton and proteasome inhibitor bortezomib (Velcade) on various human tumor cell lines. Combination of sub-lethal concentrations of thiostrepton and bortezomib induced potent apoptosis and inhibition of long-term colony formation in a wide variety of human cancer cell lines. The synergistic relationship between thiostrepton and bortezomib combination was also quantitatively demonstrated by calculating their combination index values that were much lower than 1 in all studied cell lines. The synergy between these drugs was based on their proteasome inhibitory activities, because thiostrepton modification, thiostrepton methyl ester, which did not have intact quinaldic acid ring and did not inhibit proteasome activity failed to demonstrate any synergy in combination with bortezomib.

Establishment of a gene transfer system for Rhodococcus opacus PD630 based on electroporation and its application for recombinant biosynthesis of poly(3-hydroxyalkanoic acids).

A gene transfer system for Rhodococcus opacus PD630 based on electroporation was established and optimized employing the Escherichia coli-Rhodococcus shuttle vectors pNC9501 and pNC9503 as well as the E. coli-Corynebacterium glutamicum shuttle vector pJC1 as suitable cloning vectors for R. opacus PD630, resulting in transformation efficiencies up to 1.5 x 10(5) CFUs/microgram plasmid DNA. Applying the optimized electroporation protocol to the pNC9501-derivatives pAK68 and pAK71 harboring the entire PHB synthesis operon from Ralstonia eutropha and the PHA synthase gene phaC1 from Pseudomonas aeruginosa, respectively, recombinant PHA biosynthesis was established in R. opacus PD630 and the TAG-negative mutant ROM34. Plasmid pAK68 enabled synthesis and accumulation of poly(3HB) in R. opacus PD630 and ROM34 during cultivation under storage conditions from 1% (w/v) gluconate, of poly(3HB-co-3HV) from 0.2% (w/v) propionate and of poly(3HV) from 0.1% (w/v) valerate. Under storage conditions, recombinant strains of PD630 and ROM34 harboring pAK71 were able to synthesize and accumulate PHA of the medium chain length hydroxyalkanoic acids 3HHx, 3HO, 3HD and 3HDD from 0.1% (w/v) hexadecane or octadecane and a copolyester composed of 3HHp, 3HN and 3HUD from 0.1% (w/v) pentadecane or heptadecane. In the recombinant strains of PD630 and ROM34, the thiostrepton-induced overexpression of a 20 kDa protein was observed with its N-terminus exhibiting a homology of 60% identical amino acids to TipA from Streptomyces lividans.

A clinical trial of a topical preparation of miconazole, polymyxin and prednisolone in the treatment of otitis externa in dogs.

A topical preparation containing miconazole, polymyxin and prednisolone was shown to be more effective in the treatment of otitis externa in 167 dogs than 2 other ear preparations containing antibiotics, an antimycotic and a corticosteroid. With miconazole, polymyxin and prednisolone, the recurrence rate was 26.7% compared with 72.6% and 54.3% when the other products were used. The mean duration of treatment required to achieve resolution of clinical signs was 9.6 days, compared with 12.2 days and 13.0 days and no cases failed to respond to treatment, compared with 17.7% and 14.3%. Malassezia canis alone (71%) or in association with bacteria (18%) was recovered from 44 of 49 ears cultured.