The role of p62 in cell death and survival of 5-fluorouracil and oxaliplatin-resistant colorectal cancer cells.

The protein sequestosome 1 (p62/SQSTM1) is primarily known as a selective autophagy cargo receptor, but due to its multidomain structure, it also has roles in the ubiquitin-proteasome system, metabolism, cell death and survival signalling. The increase in p62 levels is detected in some types of cancers, including colorectal cancer (CRC). Chemoresistance is the main cause of high mortality rates of CRC patients. Since p62 can regulate both cell survival and death, it is a potential modulator of chemoresistance. The impact of p62 on molecular causes of chemoresistance in CRC cells is insufficiently analysed. Therefore, we aimed to determine the impact of p62 on apoptosis, RIPK1-pRIPK3 axis, and IL-8 levels in chemoresistant CRC cells. Our data revealed that p62 levels are higher in the 5-fluorouracil (5-FU)-resistant HCT116/FU subline compared to the parental cell line. 5-FU and oxaliplatin (OxaPt) treatment decreased p62 protein levels and it correlated with chemoresistance of HCT116 and DLD1 cell lines. The silencing of p62 increased CRC cell sensitivity to 5-FU and OxaPt, hence p62 is one of the factors supporting chemoresistance. The downregulation of p62 reduced the activation of caspase-3 and the levels of RIPK1 and pRIPK3. Furthermore, p62 silencing decreased the BAX/BCL2 ratio in the HCT116/FU subline and did not change the levels of apoptosis. Instead, p62 silencing reduced the amount of IL-8 protein. Our results show that p62 impacts chemoresistance by stimulating prosurvival signalling.

Molecular and Functional Characterisation of a Novel Intragenic 12q24.21 Deletion Resulting in MED13L Haploinsufficiency Syndrome.

Background and Objectives: Heterozygous pathogenic variants in the MED13L gene cause impaired intellectual development and distinctive facial features with or without cardiac defects (MIM #616789). This complex neurodevelopmental disorder is characterised by various phenotypic features, including plagiocephaly, strabismus, clubfoot, poor speech, and developmental delay. The aim of this study was to evaluate the clinical significance and consequences of a novel heterozygous intragenic MED13L deletion in a proband with clinical features of a MED13L-related disorder through extensive clinical, molecular, and functional characterisation. Materials and Methods: Combined comparative genomic hybridisation and single-nucleotide polymorphism array (SNP-CGH) was used to identify the changes in the proband's gDNA sequence (DECIPHER #430183). Intragenic MED13L deletion was specified via quantitative polymerase chain reaction (qPCR) and Sanger sequencing of the proband's cDNA sample. Western blot and bioinformatics analyses were used to investigate the consequences of this copy number variant (CNV) at the protein level. CRISPR-Cas9 technology was used for a MED13L-gene-silencing experiment in a culture of the control individual's skin fibroblasts. After the MED13L-gene-editing experiment, subsequent functional fibroblast culture analyses were performed. Results: The analysis of the proband's cDNA sample allowed for specifying the regions of the breakpoints and identifying the heterozygous deletion that spanned exons 3 to 10 of MED13L, which has not been reported previously. In silico, the deletion was predicted to result in a truncated protein NP_056150.1:p.(Val104Glyfs*5), partly altering the Med13_N domain and losing the MedPIWI and Med13_C domains. After MED13L gene editing was performed, reduced cell viability; an accelerated aging process; and inhibition of the RB1, E2F1, and CCNC gene expression were found to exist. Conclusions: Based on these findings, heterozygous intragenic 12q24.21 deletion in the affected individual resulted in MED13L haploinsufficiency due to the premature termination of protein translation, therefore leading to MED13L haploinsufficiency syndrome.

Nanotubes from bacteriophage tail sheath proteins: internalisation by cancer cells and macrophages.

Bionanoparticles comprised of naturally occurring monomers are gaining interest in the development of novel drug transportation systems. Here we report on the stabilisation, cellular uptake, and macrophage clearance of nanotubes formed from the self-assembling gp053 tail sheath protein of the vB_EcoM_FV3 bacteriophage. To evaluate the potential of the bacteriophage protein-based nanotubes as therapeutic nanocarriers, we investigated their internalisation into colorectal cancer cell lines and professional macrophages that may hinder therapeutic applications by clearing nanotube carriers. We fused the bacteriophage protein with a SNAP-tag self-labelling enzyme and demonstrated that its activity is retained in assembled nanotubes, indicating that such carriers can be applied to deliver therapeutic biomolecules. Under physiological conditions, the stabilisation of the nanotubes by PEGylation was required to prevent aggregation and yield a stable solution with uniform nano-sized structures. Colorectal carcinoma cells from primary and metastatic tumours internalized SNAP-tag-carrying nanotubes with different efficiencies. The nanotubes entered HCT116 cells via dynamin-dependent and SW480 cells - via dynamin- and clathrin-dependent pathways and were accumulated in lysosomes. Meanwhile, peritoneal macrophages phagocytosed the nanotubes in a highly efficient manner through actin-dependent mechanisms. Macrophage clearance of nanotubes was enhanced by inflammatory activation but was dampened in macrophages isolated from aged animals. Altogether, our results demonstrate that gp053 nanotubes retained the cargo's enzymatic activity post-assembly and had the capacity to enter cancer cells. Furthermore, we emphasise the importance of evaluating the nanocarrier clearance by immune cells under conditions mimicking a cancerous environment.

Differential effects of 5-fluorouracil and oxaliplatin on autophagy in chemoresistant colorectal cancer cells.

Macroautophagy (hereafter autophagy) is one of the adaptive pathways that contribute to cancer cell chemoresistance. Despite the fact that autophagy can both promote and inhibit cell death, there is mounting evidence that in the context of anticancer treatment, it predominantly functions as a cell survival mechanism. Therefore, silencing of key autophagy genes emerges as a potent strategy to reduce chemoresistance. Though the importance of autophagy in chemoresistance is established, the changes in autophagy in the case of acquired chemoresistance are poorly understood. In this study, we aimed to determine the changes of autophagy in the cellular model of acquired chemoresistance of colorectal cancer cell lines HCT116 and SW620, induced by 5-fluorouracil (5-FU) or oxaliplatin (OxaPt) treatment, and determine the susceptible factors for autophagy inhibition. Our results demonstrate that in the context of autophagy, 5-FU and OxaPt have different effects on HCT116 and SW620 cell lines and their chemoresistant sublines. 5-FU inhibits autophagic flux, while changes in the flux after OxaPt treatment are cell type- and dose-dependent, inducing autophagy reduction or increase. The chemoresistant subline of HCT116 cells derived by OxaPt differs from the subline derived by 5-FU treatment - it responds to OxaPt by upregulating ATG7 protein level and autophagic flux, in contrast to downregulation in cells derived by 5-FU. Moreover, 5-FU and OxaPt treatments significantly modulate protein levels of core-autophagy proteins ATG7 and ATG12. The potential effects of 5-FU and OxaPt on ATG protein levels should be taken into account to reduce chemoresistance by applying small interferingRNAs, targeting ATG proteins.

Exogenous interleukin-1α signaling negatively impacts acquired chemoresistance and alters cell adhesion molecule expression pattern in colorectal carcinoma cells HCT116.

Proinflammatory cytokine and chemokine signaling from the tumor microenvironment is thought to be crucial for developing and sustaining colorectal cancer by regulating a multitude of pathways associated with a variety of cellular mechanisms. Among these pathways there is acquired chemoresistance, which is usually a major obstacle in the way towards successful chemotherapeutic treatment of advanced colorectal cancer cases. Despite of an emerging body of data published on the role of cytokine signaling network in cancer, little is known about the effects of the upstream cytokine interleukin-1α (IL-1α) signaling to the cancer cells. In this study we have shown that the increase in exogenous IL-1α signaling increases chemosensitivity of both chemosensitive and chemoresistant colorectal cancer cell lines, treated with a widely used cytotoxic antimetabolite 5-fluorouracil (5-FU). This was a result of increased cell death but not of the changes in 5-FU-induced cell cycle arrest. Noticeably, combined exogenous IL-1α and 5-FU treatment had significant effects on the expression of cell adhesion molecules, suggesting a decrease in adhesion-dependent chemoresistance and, on the other hand, an increase in metastatic potential of the cells. These results lead to a conclusion that modulation of IL-1 receptor activity could have applications as a part of combination therapy for advanced and highly metastatic colorectal cancers.

The role of interleukin-8 (CXCL8) and CXCR2 in acquired chemoresistance of human colorectal carcinoma cells HCT116.

Colorectal cancer is one of the most common malignant diseases and is a leading cause of cancer mortality in the Western world. Primary or acquired resistance to chemotherapeutic drugs is a common phenomenon which causes a failure in cancer treatment. A diverse range of molecular mechanisms has been implicated in drug resistance: DNA damage repair, alterations in drug metabolism, mutation of drug targets, increased rates of drug efflux, and activation of survival signaling pathways. The aim of this study was to investigate the expression of CXCL8-CXCR1/2 pathway, its impact on cell proliferation and cytokine expression in human colorectal carcinoma HCT116 cells, and their chemotherapy-resistant subline. We found that IL-1 alpha stimulates the production of CXCL8 through IL-1 receptor signaling. Our data indicate that CXCL8 is upregulated in chemoresistant subline of colorectal cancer cells HCT116, and modulation of CXCR2 pathway can be a target for proliferation inhibition of chemoresistant colorectal cancer cells.