ABSTRACT
A series of chemotherapeutic drugs that induce DNA damage, such as cisplatin (DDP), are standard clinical treatments for ovarian cancer, testicular cancer, and other diseases that lack effective targeted drug therapy. Drug resistance is one of the main factors limiting their application. Sensitizers can overcome the drug resistance of tumor cells, thereby enhancing the antitumor activity of chemotherapeutic drugs. In this study, we aimed to identify marketable drugs that could be potential chemotherapy sensitizers and explore the underlying mechanisms. We found that the alcohol withdrawal drug disulfiram (DSF) could significantly enhance the antitumor activity of DDP. JC-1 staining, propidium iodide (PI) staining, and western blotting confirmed that the combination of DSF and DDP could enhance the apoptosis of tumor cells. Subsequent RNA sequencing combined with Gene Set Enrichment Analysis (GSEA) pathway enrichment analysis and cell biology studies such as immunofluorescence suggested an underlying mechanism: DSF makes cells more vulnerable to DNA damage by inhibiting the Fanconi anemia (FA) repair pathway, exerting a sensitizing effect to DNA damaging agents including platinum chemotherapy drugs. Thus, our study illustrated the potential mechanism of action of DSF in enhancing the antitumor effect of DDP. This might provide an effective and safe solution for combating DDP resistance in clinical treatment.
Subject(s)
Female , Male , Humans , Cisplatin/pharmacology , Disulfiram/pharmacology , Testicular Neoplasms/drug therapy , Fanconi Anemia/drug therapy , Alcoholism/drug therapy , Drug Resistance, Neoplasm , Cell Line, Tumor , Substance Withdrawal Syndrome/drug therapy , Apoptosis , Antineoplastic Agents/therapeutic use , Cell ProliferationABSTRACT
BACKGROUND: NUP98 has numerous partner genes of which plant homeodomain (PHD) finger protein 23 (PHF23) fusion with NUP98 (NP23) can be detected by RT-PCR in patients with cytogenetically normal acute myelogenous leukemia (AML). In this fusion transcript of NP23 PHD of PHF23 is known to specifically bind H3K4me3 residues and act as a chromatic modifier. Disulfiram (DSF) which inhibits the binding of PHD to H3K4me3 residues selectively killed NP23 myeloblasts in vitro and therefore, we planned to evaluate the efficacy of DSF in vivo. METHODS: Cultured 961C cells (CD45.2), NP23 myeloblast cells were transplanted into B57BL/6 mice (CD45.1). Using limit dilution assay the number of leukemic stem cells (LSCs) could be calculated. A certain amount of 961C cells were transplanted into B57BL/6 mice and DSF was treated after 1 week. The engraftment level was monitored with CD45.2. Kaplan Meier survival curve was used to compare the survival between therapeutic and control group. RESULTS: 961C cells could be transplanted without radiation in recipient mice. Calculated LSC was estimated to be 1 out of 184 cells (95% CI range, 56–609). When treated with DSF of different doses and administration routes in 961C recipient mice no survival advantage of DSF was observed in 961C transplanted immunocompetent mouse, however it was evident that engraftment level was consistent in both groups. CONCLUSION: No survival advantage of DSF in 961C transplanted immunocompetent mouse was observed, however it was evident that 961C cells shared niche with normal hematopoietic stem cells (HSCs). We expect that 961C cells and transplanted recipient mice have the potential to be used as in vivo system for new drugs development as well as for research dealing with niche for normal HSCs and LSCs.
Subject(s)
Animals , Humans , Mice , Disulfiram , Fingers , Granulocyte Precursor Cells , Hematopoietic Stem Cells , In Vitro Techniques , Leukemia, Myeloid, Acute , Plants , Stem CellsABSTRACT
Stenotrophomonas maltophilia is a nosocomial pathogen of increasing importance. S. maltophilia K279a genome encodes a diffusible signal factor (DSF) dependent quorum sensing (QS) system that was first identified in Xanthomonas campestris pv. campestris. DSF from X. campestris is a homologue of farnesoic acid, a Candida albicans QS signal which inhibits the yeast-to-hyphal shift. Here we describe the antagonistic effects of S. maltophilia on C. albicans on filamentation as well as on its planktonic and biofilm modes of growth. To determine the role of the DSF-mediated quorum sensing system in these effects, C. albicans ATCC 10231 and C. albicans tup1 mutant, locked in the filamentous form, were grown with K279a or with its rpfF deletion mutant (DSF-). A significant reduction in viable counts of C. albicans was observed in planktonic cocultures with K279a as well as in mixed biofilms. Furthermore, no viable cells of C. albicans tup1 were recovered from K279a mixed biofilms. Fungal viability was also assessed by labeling biofilms with SYTO 9 and propidium iodide. Confocal images showed that K279a can kill hyphae and also yeast cells. Light microscopic analysis showed that K279a severely affects hyphae integrity. On the other hand, the presence of K279a rpfF did not affect fungal morphology or viability. In conclusion, we report for the first time that S. maltophilia interferes with two key virulence factors of C. albicans, the yeast-to-hyphal transition and biofilm formation. DSF could be directly responsible for these effects or may induce the gene expression involved in antifungal activity.
Stenotrophomonas maltophilia es un patógeno nosocomial de importancia creciente. El genoma de S. maltophilia K279a codifica un factor de señalización difusible (DSF), autoinductor de "quorum sensing" (QS), identificado previamente en Xanthomonas campestris pv. campestris. El DSF de X. campestris es homólogo del ácido farnesoico, señal de QS de Candida albicans, que inhibe la transición levadura-hifa. En este trabajo se describe el efecto antagónico de S. maltophilia sobre la filamentación y el crecimiento planctónico y en biofilms de C. albicans. Para determinar la participación del sistema de QS mediado por el DSF en dichos efectos, C. albicans ATCC 10231 y la mutante C. albicans tup1, que solo crece en forma filamentosa, fueron cultivadas en presencia de K279a o de su mutante K279a rpfF (DSF-). Se observó una reducción significativa del número de viables de C. albicans en cultivos planctónicos y biofilms desarrollados en presencia de K279a. Es de señalar que no se recuperaron células viables de C. albicans tup1 a partir de biofilms mixtos en presencia de K279a. Las imágenes de microscopía confocal revelaron que K279a produce la muerte de hifas y levaduras en biofilms mixtos teñidos con ioduro de propidio y SYTO 9. El análisis por microscopía óptica mostró que K279a afecta la integridad de las hifas. En cambio, la presencia de K279a rpfF no afectó la morfología ni la viabilidad fúngica. En conclusión, informamos por primera vez que S. maltophilia interfiere con dos factores de virulencia de C. albicans, la transición levadura-hifa y la formación de biofilms. Estos efectos pueden ser mediados por el DSF en forma directa o a través de la inducción de genes involucrados en la actividad antifúngica.