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Reactivating p53 and Inducing Tumor Apoptosis (RITA) Enhances the Response of RITA-Sensitive Colorectal Cancer Cells to Chemotherapeutic Agents 5-Fluorouracil and Oxaliplatin.

ABSTRACT: Colorectal carcinoma (CRC) is the most common cancer of the gastrointestinal tract with frequently dysregulated intracellular signaling pathways, including p53 signaling. The mainstay of chemotherapy treatment of CRC is 5-fluorouracil (5FU) and oxaliplatin. The two anticancer drugs mediate their therapeutic effect via DNA damage-triggered signaling. The small molecule reactivating p53 and inducing tumor apoptosis (RITA) is described as an activator of wild-type and reactivator of mutant p53 function, resulting in elevated levels of p53 protein, cell growth arrest, and cell death. Additionally, it has been shown that RITA can induce DNA damage signaling. It is expected that the therapeutic benefits of 5FU and oxaliplatin can be increased by enhancing DNA damage signaling pathways. Therefore, we highlighted the antiproliferative response of RITA alone and in combination with 5FU or oxaliplatin in human CRC cells. A panel of long-term established CRC cell lines (n=9) including p53 wild-type, p53 mutant, and p53 null and primary patient-derived, low-passage cell lines (n=5) with different p53 protein status were used for this study. A substantial number of CRC cells with pronounced sensitivity to RITA (IC₅₀<3.0 μmol/l) were identified within established (4/9) and primary patient-derived (2/5) CRC cell lines harboring wild-type or mutant p53 protein. Sensitivity to RITA appeared independent of p53 status and was associated with an increase in antiproliferative response to 5FU and oxaliplatin, a transcriptional increase of p53 targets p21 and NOXA, and a decrease in MYC mRNA. The effect of RITA as an inducer of DNA damage was shown by a strong elevation of phosphorylated histone variant H2A.X, which was restricted to RITA-sensitive cells. Our data underline the primary effect of RITA, inducing DNA damage, and demonstrate the differential antiproliferative effect of RITA to CRC cells independent of p53 protein status. We found a substantial number of RITA-sensitive CRC cells within both panels of established CRC cell lines and primary patient-derived CRC cell lines (6/14) that provide a rationale for combining RITA with 5FU or oxaliplatin to enhance the antiproliferative response to both chemotherapeutic agents.

SUBMITTER: Wiegering A

PROVIDER: S-EPMC5345961 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND: The aim of this study was to evaluate the efficacy of the p53-reactivating drugs RITA and nutlin3a in killing myeloma cells. METHODS: A large cohort of myeloma cell lines (n = 32) and primary cells (n = 21) was used for this study. This cohort contained cell lines with various TP53 statuses and primary cells with various incidences of deletion of chromosome 17. Apoptosis was evaluated using flow cytometry with Apo2.7 staining of the cell lines or via the loss of the myeloma-specific marker CD138 in primary cells. Apoptosis was further confirmed by the appearance of a subG1 peak and the activation of caspases 3 and 9. Activation of the p53 pathway was monitored using immunoblotting via the expression of the p53 target genes p21, Noxa, Bax and DR5. The involvement of p53 was further studied in 4 different p53-silenced cell lines. RESULTS: Both drugs induced the apoptosis of myeloma cells. The apoptosis that was induced by RITA was not related to the TP53 status of the cell lines or the del17p status of the primary samples (p = 0.52 and p = 0.80, respectively), and RITA did not commonly increase the expression level of p53 or p53 targets (Noxa, p21, Bax or DR5) in sensitive cells. Moreover, silencing of p53 in two TP53(mutated) cell lines failed to inhibit apoptosis that was induced by RITA, which confirmed that RITA-induced apoptosis in myeloma cells was p53 independent. In contrast, apoptosis induced by nutlin3a was directly linked to the TP53 status of the cell lines and primary samples (p < 0.001 and p = 0.034, respectively) and nutlin3a increased the level of p53 and p53 targets in a p53-dependent manner. Finally, we showed that a nutlin3a-induced DR5 increase (? 1.2-fold increase) was a specific and sensitive marker (p < 0.001) for a weak incidence of 17p deletion within the samples (? 19%). CONCLUSION: These data show that RITA, in contrast to nutlin3a, effectively induced apoptosis in a subset of MM cells independently of p53. The findings and could be of interest for patients with a 17p deletion, who are resistant to current therapies.

Project description:BackgroundChemotherapeutic agents are used to control tumor proliferation. However, their influence in the pre-metastatic niche of target organs has not been well studied. Oxaliplatin (OXA) is a drug applied in standard treatments of colorectal cancer (CRC), while the direct effect of which on the pre-metastatic microenvironment of the liver remains unclear.MethodsModels of liver metastases were established with luciferase expressing CT26 cells in BALB/c and BALB/c-nude mice. Single-cell RNA Sequencing was performed to examine the immune microenvironment in the liver elicited by OXA. Immunofluorescence and flowcytometry were utilized to confirm the changes in the number of immune cells. LDH, CellTrace CFSE Cell Proliferation and apoptosis assays were conducted to explore the impact of OXA on T cells ex vivo. The correlation between chemotherapy-related lymphopenia and metastases was assessed by meta-analysis.ResultsHerein we discovered that administration of OXA prior to the occurrence of liver metastasis actually accelerated tumor development and colonization in the liver. Single-cell RNA sequencing revealed that the landscape of the liver immune microenvironment had been changed to immunosuppressive phenotype. Macrophages after the treatment of OXA exhibited a high ability to inhibit the activation of T cells. Further investigation revealed a significant decrease in the number of T cells in the liver, particularly CD8+ T cells with reduced capacity of proliferation, activation, and killing. When mice were treated with T cell supplementation, the OXA-induced metastasis was notably abolished, indicating that the OXA-primed liver microenvironment could be reversed by the infusion of T cells. Consistent with our findings in mice, a meta-analysis was performed to verify that chemotherapy-related lymphopenia was associated with an inferior prognosis related with high incidence of metastasis, suggesting the pivotal role of chemotherapy in pre-metastatic niche formation. Furthermore, a notable reduction in the count of both macrophages and T cells was observed in the liver of colorectal cancer (CRC) patient undergoing OXA-based chemotherapy.ConclusionsOur findings proposed that immunosuppressive microenvironment in liver induced by OXA enhanced liver metastasis of colorectal cancer, which highlighted a new consideration to balance the pro metastases and anti-cancer possibility of OXA treatment.

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Reactivating p53 and Inducing Tumor Apoptosis (RITA) Enhances the Response of RITA-Sensitive Colorectal Cancer Cells to Chemotherapeutic Agents 5-Fluorouracil and Oxaliplatin.

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