Natural compounds hold great promise for combating antibiotic resistance, the failure to control some diseases, the emergence of new diseases and the toxicity of some contemporary medical products. Allyl isothiocyanate (AITC), which is abundant in cruciferous vegetables and mustard seeds and is commonly referred to as mustard essential oil, exhibits promising antineoplastic activity against bladder cancer, although its mechanism of action is not fully understood. Therefore, the aim of this study was to investigate the effects of AITC activity on bladder cancer cell lines carrying a wild type (wt; RT4) or mutated (T24) TP53 gene. Morphological changes, cell cycle kinetics and CDK1, SMAD4, BAX, BCL2, ANLN and S100P gene expression were evaluated. In both cell lines, treatment with AITC inhibited cell proliferation (at 62.5, 72.5, 82.5 and 92.5μM AITC) and induced morphological changes, including scattered and elongated cells and cellular debris. Gene expression profiles revealed increased S100P and BAX and decreased BCL2 expression in RT4 cells following AITC treatment. T24 cells displayed increased BCL2, BAX and ANLN and decreased S100P expression. No changes in SMAD4 and CDK1 expression were observed in either cell line. In conclusion, AITC inhibits cell proliferation independent of TP53 status. However, the mechanism of action of AITC differed in the two cell lines; in RT4 cells, it mainly acted via the classical BAX/BCL2 pathway, while in T24 cells, AITC modulated the activities of ANLN (related to cytokinesis) and S100P. These data confirm the role of AITC as a potential antiproliferative compound that modulates gene expression according to the tumor cell TP53 genotype.

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Currently, the combination of cisplatin and gemcitabine is considered a standard chemotherapeutic protocol for bladder cancer. However, the mechanism by which these drugs act on tumor cells is not completely understood. The aim of the present study was to investigate the effects of these two antineoplastic drugs on the apoptotic index and cell cycle kinetics of urinary bladder transitional carcinoma cell lines with wild-type or mutant TP53 (RT4: wild type for TP53; 5637 and T24: mutated TP53). Cytotoxicity, cell survival assays, clonogenic survival assays and flow cytometric analyses for cell cycle kinetics and apoptosis detection were performed with three cell lines treated with different concentrations of cisplatin and gemcitabine. G(1) cell cycle arrest was observed in the three cell lines after treatment with gemcitabine and gemcitabine plus cisplatin. A significant increase in cell death was also detected in all cell lines treated with cisplatin or gemcitabine. Lower survival rates occurred with the combined drug protocol independent of TP53 status. TP53-wild type cells (RT4) were more sensitive to apoptosis than were mutated TP53 cells when treated with cisplatin or gemcitabine. Concurrent treatment with cisplatin and gemcitabine was more effective on transitional carcinoma cell lines than either drug alone; the drug combination led to a decreased cell survival that was independent of TP53 status. Therefore, the synergy between low concentrations of cisplatin and gemcitabine may have clinical relevance, as high concentrations of each individual drug are toxic to whole organisms.

Allyl isothiocyanate (AITC) is present in plants of the cruciferous family and is abundant in mustard seed. Due to its high bioavailability in urine after ingestion, AITC has been considered a promising antineoplastic agent against bladder cancer. Because TP53 mutations are the most common alterations in bladder cancer cells and are frequently detected in in situ carcinomas, in this study, we investigated whether the AITC effects in bladder cancer cells are dependent on the TP53 status. Two bladder transitional carcinoma cell lines were used: RT4, with wild-type TP53; and T24, mutated TP53 gene. AITC was tested at concentrations of 0.005, 0.0625, 0.0725, 0.0825, 0.0925, 0.125 and 0.25 μM in cytotoxicity, cell and clonogenic survival assays, comet and micronucleus assays and for its effects on cell cycle and apoptosis by flow cytometry and on TP53 gene expression. The data showed increased primary DNA damage in both cell lines; however, lower concentrations of AITC were able to induce genotoxicity in the mutant cells for the TP53 gene. Furthermore, the results demonstrated increased apoptosis and necrosis rates in the wild-type cells, but not in mutated TP53 cells, and cell cycle arrest in the G2 phase for mutated cells after AITC treatment. No significant differences were detected in TP53 gene expression in the two cell lines. In conclusion, AITC caused cell cycle arrest, increased apoptosis rates and varying genotoxicity dependent on the TP53 status. However, we cannot rule out the possibility that those differences could reflect other intrinsic genetic alterations in the examined cell lines, which may also carry mutations in genes other than TP53. Therefore, further studies using other molecular targets need to be performed to better understand the mechanisms by which AITC may exert its antineoplastic properties against tumor cells.