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International Journal of Radiation... Apr 2024Chemoresistance remains a major challenge in treating pancreatic ductal adenocarcinoma (PDAC). Although chemoradiation has proven effective in other tumor types, such as...
PURPOSE
Chemoresistance remains a major challenge in treating pancreatic ductal adenocarcinoma (PDAC). Although chemoradiation has proven effective in other tumor types, such as head and neck squamous cell carcinoma, its role in PDAC and effect on acquired chemoresistance have yet to be fully explored. In this study, we investigated the sensitivity of gemcitabine-resistant (GR) and paclitaxel-resistant (PR) PDAC cells to ionizing radiation (IR) and their underlying mechanisms.
METHODS AND MATERIALS
GR and PR clones were generated from PANC-1, PATU-T, and SUIT2-007 pancreatic cancer cell lines. Cell survival after radiation was assessed using clonogenic assay, sulforhodamine B assay, apoptosis, and spheroid growth by bioluminescence. Radiation-induced DNA damage was assessed using Western blot, extra-long polymerase chain reaction, reactive oxygen species production, and immunofluorescence. Autophagy and modulation of the Hippo signaling pathway were investigated using proteomics, Western blot, immunofluorescence, and reverse-transcription quantitative polymerase chain reaction.
RESULTS
In both 2- and 3-dimensional settings, PR cells were more sensitive to IR and showed decreased β-globin amplification, indicating more DNA damage accumulation compared with GR or wild-type cells after 24 hours. Proteomic analysis of PR PATU-T cells revealed that the protein MST4, a kinase involved in autophagy and the Hippo signaling pathway, was highly downregulated. A differential association was found between autophagy and radiation treatment depending on the cell model. Interestingly, increased yes-associated protein nuclear localization and downstream Hippo signaling pathway target gene expression were observed in response to IR.
CONCLUSIONS
This was the first study investigating the potential of IR in targeting PDAC cells with acquired chemoresistance. Our results demonstrate that PR cells exhibit enhanced sensitivity to IR due to greater accumulation of DNA damage. Additionally, depending on the specific cellular context, radiation-induced modulation of autophagy and the Hippo signaling pathway emerged as potential underlying mechanisms, findings with potential to inform personalized treatment strategies for patients with acquired chemoresistance.
Topics: Humans; Gemcitabine; Paclitaxel; Deoxycytidine; Proteomics; Cell Line, Tumor; Pancreatic Neoplasms; Carcinoma, Pancreatic Ductal; Radiation, Ionizing; Drug Resistance, Neoplasm; Cell Proliferation
PubMed: 37914140
DOI: 10.1016/j.ijrobp.2023.10.035 -
Medical Oncology (Northwood, London,... Jun 2020Resistance to gemcitabine chemotherapy is common in patients with pancreatic ductal adenocarcinoma (PDAC), biliary tract cancer (BTC) and ovarian cancers (OC),... (Review)
Review
BACKGROUND
Resistance to gemcitabine chemotherapy is common in patients with pancreatic ductal adenocarcinoma (PDAC), biliary tract cancer (BTC) and ovarian cancers (OC), conferring poor survival. Use of ProTide technology led to the development of a 'partially-activated' monophosphorylated gemcitabine compound, termed NUC-1031. NUC-1031 enters cancer cells independent of the human equilibrative nucleoside transporter, does not require deoxycytidine kinase-mediated activation and resists cytidine deaminase-mediated breakdown into toxic by-products.
CURRENT FINDINGS
The phase I PRO-001 trial recruited 68 patients with advanced solid tumours; of the 49 patients that had response-evaluable disease, 5 (10%) had a partial response (PR) and 33 (67%) had stable disease (SD). Subsequently, the PRO-002 study assessed the safety and efficacy of NUC-1031 combined with carboplatin for patients with OC (n = 25); preliminary data from this study reported one (4%) unconfirmed complete response (CR), 8 (35%) PRs and 13 (57%) patients with SD, the final outcome data are awaited. The ABC-08 trial for advanced BTC assessed safety and efficacy of NUC-1031 combined with cisplatin; 14 patients were recruited with a 50% objective response rate in the intention to treat population at interim analysis. ACELARATE, the phase III trial in first-line advanced PDAC comparing NUC-1031 to gemcitabine monotherapy, recruited 200 patients but has been paused for futility analysis.
CONCLUSION
Early studies demonstrate NUC-1031 is well tolerated with favourable pharmacokinetic profiles. NUC-1031 use in PDAC remains unclear, but encouraging results of disease control in BTC and OC has prompted phase II and III trial development. NuTide 121, is a phase III trial comparing cisplatin-NUC 1031 combination to the standard of care cisplatin-gemcitabine and recruitment is ongoing. Recruiting trials and mature data from existing studies will help inform on the impact of NUC-1031 on patient survival over standard gemcitabine.
Topics: Antineoplastic Combined Chemotherapy Protocols; Biliary Tract Neoplasms; Cisplatin; Clinical Trials as Topic; Cytidine Monophosphate; Deoxycytidine; Drug Resistance, Neoplasm; Drug Synergism; Female; Humans; Ovarian Neoplasms; Pancreatic Neoplasms; Randomized Controlled Trials as Topic; Treatment Outcome; Gemcitabine
PubMed: 32529264
DOI: 10.1007/s12032-020-01386-6 -
Cell Reports Feb 2024Viral mimicry describes the immune response induced by endogenous stimuli such as double-stranded RNA (dsRNA) from endogenous retroelements. Activation of viral mimicry...
Viral mimicry describes the immune response induced by endogenous stimuli such as double-stranded RNA (dsRNA) from endogenous retroelements. Activation of viral mimicry has the potential to kill cancer cells or augment anti-tumor immune responses. Here, we systematically identify mechanisms of viral mimicry adaptation associated with cancer cell dependencies. Among the top hits is the RNA decay protein XRN1 as an essential gene for the survival of a subset of cancer cell lines. XRN1 dependency is mediated by mitochondrial antiviral signaling protein and protein kinase R activation and is associated with higher levels of cytosolic dsRNA, higher levels of a subset of Alus capable of forming dsRNA, and higher interferon-stimulated gene expression, indicating that cells die due to induction of viral mimicry. Furthermore, dsRNA-inducing drugs such as 5-aza-2'-deoxycytidine and palbociclib can generate a synthetic dependency on XRN1 in cells initially resistant to XRN1 knockout. These results indicate that XRN1 is a promising target for future cancer therapeutics.
Topics: Humans; Retroelements; Cell Line; Cytosol; Decitabine; Exonucleases; Neoplasms; RNA, Double-Stranded; Exoribonucleases; Microtubule-Associated Proteins
PubMed: 38261511
DOI: 10.1016/j.celrep.2024.113684 -
Drug Resistance Updates : Reviews and... Mar 2022Resistance to the hypomethylating agents (HMAs) 5-azacytidine (AZA) and 5-aza-2'-deoxycytidine (DAC) represents a major obstacle in the treatment of elderly patients... (Review)
Review
Resistance to the hypomethylating agents (HMAs) 5-azacytidine (AZA) and 5-aza-2'-deoxycytidine (DAC) represents a major obstacle in the treatment of elderly patients with myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) which are not suitable for hematopoietic stem cells transplantation. Approximately 50 % of patients do not respond to HMA treatment because of intrinsic (primary) resistance, while others could acquire drug resistance during the repeated cycles of the treatment. To prevent, delay or surmount resistance development, the molecular mechanisms underlying drug resistance must be first identified. This is crucial as no further standard therapeutic opportunities are available for these patients who failed hypomethylating agents-based treatment. The current review provides an updated information about the different mechanisms that may contribute to the development of resistance to HMAs. Despite the similar structure and mechanism of action of HMA, several studies did not report the expected development of cross-resistance. It is clear that in addition to the common modalities of chemoresistance, there must be some specific mechanisms of drug resistance. Changes in transport and metabolism of HMAs are among the most studied mechanisms of resistance. Drug uptake provided by two solute carrier (SLC) families: SLC28 and SLC29 (also known as the concentrative and equilibrative nucleoside transporter families, respectively), could represent one of the mechanisms of cross-resistance. Changes in the metabolism of these drugs are the most likely mechanism responsible for the unique mode of resistance to AZA and DAC. Deoxycytidine kinase and uridine-cytidine kinase due to their necessity for drug activation, each could represent one of the response markers to treatment with DAC and AZA, respectively. Other mechanisms involved in the development of resistance common for both drugs involved: i. increased DNA repair (caused for example by constitutive activation of the ATM/BRCA1 pathway and inhibition of p53-dependent apoptosis); ii. changes in the regulation of apoptosis/disrupted apoptotic pathways (specifically increased levels of the anti-apoptotic protein BCL2) and iii. increased resilience of leukemic stem cells to multiple drugs including HMAs. Despite intense research on the resistance of MDS and AML patients to HMAs, the mechanisms that may reduce the response of these cells to HMAs are not known in detail. We herein highlight the most important directions that future research should take.
Topics: Aged; Azacitidine; Decitabine; Drug Resistance; Humans; Leukemia, Myeloid, Acute; Myelodysplastic Syndromes
PubMed: 35227933
DOI: 10.1016/j.drup.2022.100805 -
Experimental & Molecular Medicine Aug 2022Acquired resistance to inhibitors of anaplastic lymphoma kinase (ALK) is a major clinical challenge for ALK fusion-positive non-small-cell lung cancer (NSCLC). In the...
Acquired resistance to inhibitors of anaplastic lymphoma kinase (ALK) is a major clinical challenge for ALK fusion-positive non-small-cell lung cancer (NSCLC). In the absence of secondary ALK mutations, epigenetic reprogramming is one of the main mechanisms of drug resistance, as it leads to phenotype switching that occurs during the epithelial-to-mesenchymal transition (EMT). Although drug-induced epigenetic reprogramming is believed to alter the sensitivity of cancer cells to anticancer treatments, there is still much to learn about overcoming drug resistance. In this study, we used an in vitro model of ceritinib-resistant NSCLC and employed genome-wide DNA methylation analysis in combination with single-cell (sc) RNA-seq to identify cytidine deaminase (CDA), a pyrimidine salvage pathway enzyme, as a candidate drug target. CDA was hypomethylated and upregulated in ceritinib-resistant cells. CDA-overexpressing cells were rarely but definitively detected in the naïve cell population by scRNA-seq, and their abundance was increased in the acquired-resistance population. Knockdown of CDA had antiproliferative effects on resistant cells and reversed the EMT phenotype. Treatment with epigenome-related nucleosides such as 5-formyl-2'-deoxycytidine selectively ablated CDA-overexpressing resistant cells via accumulation of DNA damage. Collectively, our data suggest that targeting CDA metabolism using epigenome-related nucleosides represents a potential new therapeutic strategy for overcoming ALK inhibitor resistance in NSCLC.
Topics: Carcinoma, Non-Small-Cell Lung; Cytidine Deaminase; Drug Resistance, Neoplasm; Epigenome; Gene Expression Profiling; Humans; Lung Neoplasms; Mutation; Protein Kinase Inhibitors; Receptor Protein-Tyrosine Kinases; Single-Cell Analysis
PubMed: 35999456
DOI: 10.1038/s12276-022-00836-7 -
Molecular Cancer Therapeutics Aug 2020Although gemcitabine is the cornerstone of care for pancreatic ductal adenocarcinoma (PDA), patients lack durable responses and relapse is inevitable. While the...
Although gemcitabine is the cornerstone of care for pancreatic ductal adenocarcinoma (PDA), patients lack durable responses and relapse is inevitable. While the underlying mechanisms leading to gemcitabine resistance are likely to be multifactorial, there is a strong association between activating gemcitabine metabolism pathways and clinical outcome. This study evaluated casein kinase 1 delta (CK1δ) as a potential therapeutic target for PDA and bladder cancer, in which CK1δ is frequently overexpressed. We assessed the antitumor effects of genetically silencing or pharmacologically inhibiting CK1δ using our in-house CK1δ small-molecule inhibitor SR-3029, either alone or in combination with gemcitabine, on the proliferation and survival of pancreatic and bladder cancer cell lines and orthotopic mouse models. Genetic studies confirmed that silencing CK1δ or treatment with SR-3029 induced a significant upregulation of deoxycytidine kinase (dCK), a rate-limiting enzyme in gemcitabine metabolite activation. The combination of SR-3029 with gemcitabine induced synergistic antiproliferative activity and enhanced apoptosis in both pancreatic and bladder cancer cells. Furthermore, in an orthotopic pancreatic tumor model, we observed improved efficacy with combination treatment concomitant with increased dCK expression. This study demonstrates that CK1δ plays a role in gemcitabine metabolism, and that the combination of CK1δ inhibition with gemcitabine holds promise as a future therapeutic option for metastatic PDA as well as other cancers with upregulated CK1δ expression.
Topics: Animals; Antimetabolites, Antineoplastic; Apoptosis; Biomarkers, Tumor; Breast Neoplasms; Casein Kinase Idelta; Cell Proliferation; Deoxycytidine; Deoxycytidine Kinase; Drug Resistance, Neoplasm; Female; Gene Expression Regulation, Neoplastic; Humans; Mice; Mice, Nude; Pancreatic Neoplasms; Tumor Cells, Cultured; Urinary Bladder Neoplasms; Xenograft Model Antitumor Assays; Gemcitabine
PubMed: 32430484
DOI: 10.1158/1535-7163.MCT-19-0997 -
Biochemical and Biophysical Research... Nov 2021Decitabine (DAC), a DNA methyltransferase (DNMT) inhibitor is a novel anti-cancer drug regulating epigenetic mechanisms. Similar to conventional anti-cancer drugs, drug...
Decitabine (DAC), a DNA methyltransferase (DNMT) inhibitor is a novel anti-cancer drug regulating epigenetic mechanisms. Similar to conventional anti-cancer drugs, drug resistance to DAC also has been reported, resulting in tumor recurrence. Our previous study using colorectal cancer HCT116 cells found the decrease in deoxycytidine kinase (dCK) (activation enzyme of DAC) and the increase in cytidine deaminase (inactivation enzyme of DAC) in acquired DAC-resistant HCT116 (HCT116/DAC) cells. The aim of our study was to clarify the involvement of dCK and CDA in DAC resistance. In order to tackle DAC resistance, it was also examined whether other DNMT inhibitors such as azacytidine (AC) and polyphenols are effective in DAC-resistant cancer cells. When dCK siRNA was transfected into HCT116 cells, IC value of DAC increased by about 74-fold and reached that of HCT116/DAC cells with attenuated dCK. dCK siRNA to HCT116 cells also abolished DNA demethylation effects of DAC. In contrast, CDA siRNA to HCT116 cells did not influence the efficacy of DAC. In addition, CDA siRNA to HCT116/DAC cells with increased CDA did not restore the compromised effects of DAC. These results suggested that attenuated dCK but not increased CDA mainly contributed to DAC resistance. Regarding dCK in HCT116/DAC cells, a point mutation with amino acid substitution was observed while the product size and expression of mRNA coding region did not change, suggesting that dCK protein was decreased by post-transcriptional regulation. AC and polyphenols showed no cross-resistance in HCT116/DAC cells. AC but not polyphenols exerted DNA demethylation effect. Among polyphenols, curcumin (Cur) showed the most synergistic cytotoxicity in combination with AC while DNA demethylation effect of AC was partly maintained. Taken together, combination of AC and Cur would be a promising alternative to tackle DAC resistance mainly due to attenuated dCK.
Topics: Anti-Inflammatory Agents, Non-Steroidal; Antimetabolites, Antineoplastic; Antineoplastic Combined Chemotherapy Protocols; Azacitidine; Cell Line, Tumor; Colorectal Neoplasms; Curcumin; Cytidine Deaminase; DNA Methylation; Decitabine; Deoxycytidine Kinase; Drug Resistance, Neoplasm; Drug Synergism; Humans
PubMed: 34571370
DOI: 10.1016/j.bbrc.2021.09.041 -
International Journal of Preventive... 2021The cell cycle is divided into four phases, G1, G2, S, and M phase. The mammalian cell cycle is controlled and governed by the kinase complexes including cyclin and the...
Investigation of the Effect of 5-Aza-2'-Deoxycytidine in Comparison to and in Combination with Trichostatin A on , , Gene Expression, Cell Growth Inhibition and Apoptosis Induction in Colon Cancer Caco-2 Cell Line.
BACKGROUND
The cell cycle is divided into four phases, G1, G2, S, and M phase. The mammalian cell cycle is controlled and governed by the kinase complexes including cyclin and the cyclin-dependent kinase (CDK), cyclin-CDK complexes. The activity of the complexes is regulated by cyclin-dependent kinase inhibitors (CDKIs), the INK4, and the CDK interacting protein/kinase inhibitory protein (CIP/KIP) families. Promoter hypermethylation and histone deacetylation of CDKIs have been reported in several cancers. These changes can be reversed by DNA demethylating agents, such as decitabine, 5-Aza-2'-deoxycytidine (5-Aza-CdR), and histone deacetylase inhibitors (HDACIs), such as trichostatin A. Previously, we reported the effect of 5-Aza-CdR and trichostatin A (TSA) on hepatocellular carcinoma (HCC). The present study aimed to investigate the effect of 5-Aza-CdR in comparison to and in combination with trichostatin A on , , genes expression, cell growth inhibition and apoptosis induction in colon cancer Caco-2 cell line.
METHODS
The Caco-2 cells were cultured and treated with 5-Aza-CdR and TSA (alone and combined). The cell viability, apoptosis, and relative gene expression were determined by MTT assay, flow cytometry, and real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR), respectively.
RESULTS
Both compounds inhibited cell growth, induced apoptosis, and up-regulated the , , gene significantly. The TSA had a more significant effect in comparison to 5-Aza-CdR. Furthermore, maximal apoptosis and up-regulation were observed with combined treatment.
CONCLUSIONS
our finding indicated that 5-Aza-CdR and TSA can epigenetically re-activate the , , gene resulting in cell growth inhibition and apoptosis induction in colon cancer.
PubMed: 34447506
DOI: 10.4103/ijpvm.IJPVM_11_20 -
Drug Resistance Updates : Reviews and... Mar 2023BRCA2 is a well-established cancer driver in several human malignancies. While the remarkable success of PARP inhibitors proved the clinical potential of targeting BRCA...
BRCA2 is a well-established cancer driver in several human malignancies. While the remarkable success of PARP inhibitors proved the clinical potential of targeting BRCA deficiencies, the emergence of resistance mechanisms underscores the importance of seeking novel Synthetic Lethal (SL) targets for future drug development efforts. In this work, we performed a BRCA2-centric SL screen with a collection of plant-derived compounds from South America. We identified the steroidal alkaloid Solanocapsine as a selective SL inducer, and we were able to substantially increase its potency by deriving multiple analogs. The use of two complementary chemoproteomic approaches led to the identification of the nucleotide salvage pathway enzyme deoxycytidine kinase (dCK) as Solanocapsine's target responsible for its BRCA2-linked SL induction. Additional confirmatory evidence was obtained by using the highly specific dCK inhibitor (DI-87), which induces SL in multiple BRCA2-deficient and KO contexts. Interestingly, dCK-induced SL is mechanistically different from the one induced by PARP inhibitors. dCK inhibition generates substantially lower levels of DNA damage, and cytotoxic phenotypes are associated exclusively with mitosis, thus suggesting that the fine-tuning of nucleotide supply in mitosis is critical for the survival of BRCA2-deficient cells. Moreover, by using a xenograft model of contralateral tumors, we show that dCK impairment suffices to trigger SL in-vivo. Taken together, our findings unveil dCK as a promising new target for BRCA2-deficient cancers, thus setting the ground for future therapeutic alternatives to PARP inhibitors.
Topics: Humans; Deoxycytidine Kinase; Poly(ADP-ribose) Polymerase Inhibitors; Antineoplastic Agents; Nucleotides; Protein Kinase Inhibitors; BRCA2 Protein
PubMed: 36706533
DOI: 10.1016/j.drup.2023.100932 -
International Journal of Molecular... Apr 2022The ergothioneine transporter ETT (formerly OCTN1; human gene symbol ) is a powerful and highly specific transporter for the uptake of ergothioneine (ET). Recently,...
The ergothioneine transporter ETT (formerly OCTN1; human gene symbol ) is a powerful and highly specific transporter for the uptake of ergothioneine (ET). Recently, Sparreboom et al. reported that the ETT would transport nucleosides and nucleoside analogues such as cytarabine and gemcitabine with the highest efficiency. In our assay system, we could not detect any such transport. Subsequently, Sparreboom suggested that the intracellular metabolization of the nucleosides occurs so fast that the original compounds cannot be detected by LC-MS/MS after inward transport. Our current experiments with 293 cells disprove this hypothesis. Uptake of gemcitabine was easily detected by LC-MS/MS measurements when we expressed the Na/nucleoside cotransporter CNT3 (). Inward transport was 1280 times faster than the intracellular production of gemcitabine triphosphate. The deoxycytidine kinase inhibitor 2-thio-2'-deoxycytidine markedly blocked the production of gemcitabine triphosphate. There was no concomitant surge in intracellular gemcitabine, however. This does not fit the rapid phosphorylation of gemcitabine. Uptake of cytarabine was very slow, but detection by MS was still possible. When the ETT was expressed and incubated with gemcitabine, there was no increase in intracellular gemcitabine triphosphate. We conclude that the ETT does not transport nucleosides.
Topics: Chromatography, Liquid; Cytarabine; Deoxycytidine; Ergothioneine; Humans; Organic Cation Transport Proteins; Tandem Mass Spectrometry; Gemcitabine
PubMed: 35563081
DOI: 10.3390/ijms23094690