HCT116 Research Articles

Hydrogen Sulfide-Releasing Carbonic Anhydrase Inhibitors Effectively Suppress Cancer Cell Growth.

This study proposes a novel therapeutic strategy for cancer management by combining the antitumor effects of hydrogen sulfide (H2S) and inhibition of carbonic anhydrases (CAs; EC 4.2.1.1), specifically isoforms IV, IX, and XII. H2S has demonstrated cytotoxicity against various cancers at high concentrations. The inhibition of tumor-associated CAs leads to lethal intracellular alkalinization and acidification of the extracellular tumor microenvironment and restores tumor responsiveness to the immune system, chemotherapy, and radiotherapy. The study proposes H2S donor-CA inhibitor (CAI) hybrids for tumor management. These compounds effectively inhibit the target CAs, release H2S consistently, and exhibit potent antitumor effects against MDA-MB-231, HCT-116, and A549 cancer cell lines. Notably, some compounds display high cytotoxicity across all investigated cell lines. Derivative 30 shows a 2-fold increase in cytotoxicity (0.93 ± 0.02 µM) under chemically induced hypoxia in HCT-116 cells. These compounds also disturb the cell cycle, leading to a reduction in cell populations in G0/G1 and S phases, with a notable increase in G2/M and Sub-G1. This disruption is correlated with induced apoptosis, with fold increases of 37.2, 24.5, and 32.9 against HCT-116 cells and 14.2, 13.1, and 19.9 against A549 cells compared to untreated cells. These findings suggest the potential of H2S releaser-CAI hybrids as effective and versatile tools in cancer treatment.

The METTL3/TRAP1 axis as a key regulator of 5-fluorouracil chemosensitivity in colorectal cancer.

Colorectal cancer (CRC) remains a significant clinical challenge, with 5-Fluorouracil (5-FU) being the frontline chemotherapy. However, chemoresistance remains a major obstacle to effective treatment. METTL3, a key methyltransferase involved in RNA methylation processes, has been implicated in CRC carcinogenesis. However, its role in modulating CRC sensitivity to 5-FU remains elusive. In this study, we aimed to investigate the role and mechanisms of METTL3 in regulating 5-FU chemosensitivity in CRC cells. Initially, we observed that 5-FU treatment inhibited cell viability and induced apoptosis, accompanied by a reduction in METTL3 expression in HCT-116 and HCT-8 cells. Subsequent assays including drug sensitivity, EdU, colony formation, TUNEL staining, and flow cytometry revealed that METTL3 depletion enhanced 5-FU sensitivity and increased apoptosis induction both in vitro and in vivo. Conversely, METTL3 overexpression conferred resistance to 5-FU in both cell lines. Moreover, knockdown of METTL3 in 5-FU-resistant CRC cell lines HCT-116/FU and HCT-15/FU significantly decreased 5-FU tolerance and induced apoptosis upon 5-FU treatment. Mechanistically, we found that METTL3 regulated 5-FU sensitivity and apoptosis induction by modulating TRAP1 expression. Further investigations using m6A colorimetric ELISA, dot blot, MeRIP-qPCR and RNA stability assays demonstrated that METTL3 regulated TRAP1 mRNA stability in an m6A-dependent manner. Additionally, overexpression of TRAP1 mitigated the cytotoxic effects of 5-FU on CRC cells. In summary, our study uncovers the pivotal role of the METTL3/TRAP1 axis in modulating 5-FU chemosensitivity in CRC. These findings provide new insights into the mechanisms underlying CRC resistance to 5-FU and may offer potential targets for future therapeutic interventions.

Predictive action of oncomiR in suppressing TP53 signaling pathway in hypoxia-conditioned colon cancer cell line HCT-116.

Hypoxia-induced heterogeneity in colorectal cancer (CRC) significantly impacts patient survival by promoting chemoresistance. These conditions alter the regulation of miRNAs, key regulators of crucial processes like proliferation, apoptosis, and invasion, leading to tumor progression. Despite their promising potential as diagnostic and therapeutic targets, the underlying mechanisms by which miRNAs influence hypoxia-mediated tumorigenesis remain largely unexplored. This study aims to elucidate the action of miRNAs in HCT-116 colorectal cancer stem cells (CSCs) under hypoxia, providing valuable insights into their role in tumor adaptation and progression. MiRNA expression was determined using Nanostring nCounter, and bioinformatic analysis was performed to explain the molecular pathway. A total of 50 miRNAs were obtained with an average count of ≥ 20 reads for comparative expression analysis. The results showed that hypoxia-affected 36 oncomiRs were increased in HCT-116, and 14 suppressor-miRs were increased in MSCs. The increase in miRNA expression occurred consistently from normoxia to hypoxia and significantly differed between mesenchymal stem cells (MSCs) and HCT-116. Furthermore, miR-16-5p and miR-29a-3p were dominant in regulating the p53 signaling pathway, which is thought to be related to the escape mechanism against hypoxia and maintaining cell proliferation. More research with a genome-transcriptome axis approach is needed to fully understand miRNAs' role in adapting CRC cells and MSCs to hypoxia. Further research could focus on developing specific biomarkers for diagnosis. In addition, anti-miR can be developed as a therapy to prevent cancer proliferation or inhibit the adaptation of cancer cells to hypoxia.

One-step hydrothermal synthesis of F,N,S-doped photoluminescent carbon dots with yellowish-orange emission for sensitive Cu2+ ion detection: Environmental and biomedical applications

Herein, fluorine-, nitrogen-, and sulfur-doped carbon dots (FNS-CDs) were prepared via a one-step hydrothermal synthesis using flufenamic acid and thiourea as precursors, which showed yellowish-orange fluorescence both in aqueous solution and in the solid state. The as-prepared FNS-CDs were monodisperse and had an average diameter of 4 nm, good water solubility, a fluorescence quantum yield of 18.63 %, and excellent pH and ionic strength stability. The optimal excitation/emission wavelengths of the FNS-CDs were 406/570 nm, with an excitation-dependent wavelength range of 365–435 nm. The surface states, morphologies, and elemental compositions of the FNS-CDs were characterized using FTIR, XRD, TEM, and XPS. In addition, the FNS-CDs without any further modification exhibited high selectivity and sensitivity as nanosensors for Cu2+ over other metal ions. The PL quenching phenomenon can be used to detect Cu2+ ions within a linear range of 1–25 μM with a detection limit of 83.10 nM and an association constant of 1.29 × 104 M−1. The PL of the FNS-CDs was significantly quenched by Cu2+ ions through static quenching and was restored upon the subsequent addition of EDTA. The practical application was demonstrated through the detection of Cu2+ ions in real water samples, yielding recovery rates ranging from 97.8 % to 103.9 % with an RSD below 2.5 %. The FNS-CDs/Cu2+ complex exhibits potent antibacterial activity against S. aureus (85.27 %±2.1 %) and E. coli (91.31 %±1.6 %), whereas the FNS-CDs/Cu2+ has a higher inhibitory effect on the growth of E. coli. Furthermore, the FNS-CDs (100 μg/mL) were used for cell imaging, showing low toxicity to HCT-116 cells exhibited yellowish-orange fluorescence under a confocal microscope.

Decursin Induces G1 Cell Cycle Arrest and Apoptosis through Reactive Oxygen Species-Mediated Endoplasmic Reticulum Stress in Human Colorectal Cancer Cells in In Vitro and Xenograft Models.

Decursin, a coumarin isolated from Angelica gigas Nakai, possesses anti-inflammatory and anti-cancer properties. However, the molecular mechanisms underlying its anti-cancer effects against human colorectal cancer (CRC) are unclear. Therefore, this study aimed to evaluate the biological activities of decursin in CRC in vitro and in vivo and to determine its underlying mechanism of action. Decursin exhibited anti-tumor activity in vitro, accompanied by an increase in G1 cell cycle arrest and apoptosis in HCT-116 and HCT-8 CRC cells. Decursin also induced the production of reactive oxygen species (ROS), thereby activating the endoplasmic reticulum (ER) stress apoptotic pathway in CRC cells. Furthermore, the role of ROS in decursin-induced apoptosis was investigated using the antioxidant N-acetyl-L-cysteine. Inhibiting ROS production reversed decursin-induced ER stress. Moreover, decursin significantly suppressed tumor growth in a subcutaneous xenograft mouse model of HCT-116 and HCT-8 CRC cells without causing host toxicity. Decursin also decreased cell proliferation, as documented by Ki-67, and partly increased cleaved caspase 3 expression in tumor tissues by activating ER stress apoptotic pathways. These findings suggest that decursin induces cell cycle arrest and apoptosis in human CRC cells via ROS-mediated ER stress, suggesting that decursin could be a therapeutic agent for CRC.

Prenylated Acetophenone Derivatives from the Leaves of Acronychia pedunculata and Their Anti-Proliferative Activities.

Four new prenylated acetophenone derivatives, including one acetophenone dimer [acronyrone D (1)] and three acetophenone monomers [acronyrones E-G (2-4)], along with seven known analogues (5-11) were obtained from the leaves of Acronychia pedunculata. Their structures and absolute configurations were established by analysis of HRMS and NMR data, single crystal X-ray diffraction studies and quantum chemical calculations. In addition, the isolates were tested for their anti-proliferative acivity against HCT-116, RKO and SW480 cancer cell lines. Compound 5 exhibited significant anti-proliferative effects on the three cell lines, with IC50 values ranging from 0.24-5.3 μM.

Synthesis, anticancer activity, docking and computational studies of new pyridyl-glycosyl hybrids and acyclic analogs.

New pyridine-O-glycosides and their acyclic nucleoside analogues were prepared by heterocyclization and glycosylation. The anticancer activity against HCT-116, HepG2 and MCF-7 human cancer cells and BJ-1 cell revealed that the galacto- and xylopyranosyl glycosides possessing 4-bromophenyl have superior cytotoxic activities against HepG2 cell while glycosides 7-9 resulted in superior cytotoxic activities regarding MCF-7 breast cell. In case of HCT-116 colorectal carcinoma cells, two products and the derived glycosides and acyclic analogues showed potent activities. The most potent compounds were investigated for their possible binding affinities to the active site of CDK2 enzyme via in silico molecular docking simulation in addition to computational studies. The results support the antiproliferative effect and elucidate the interactions of 3a and 8 with catalytic sites.

Cynbungenins A-K, structurally diverse steroids with cytotoxic activity from the roots of Cynanchum bungei Decne

Chemical investigation of the acid hydrolysate of Cynanchum bungei roots led to the isolation of eleven undescribed steroids, namely cynbungenins A-K (1–11), and seven previously described analogues (12–18). The complete structures of these compounds were elucidated using the comprehensive spectroscopic analyses and reference data. Structurally, compounds 1 and 2 represent the first example of androstane-type steroids found in the Cynanchum plants, and compounds 3–6 and 12 are characterized as pregnane-type steroids with a rare 8,14-seco-steroid core. In the cytotoxic activity assay, compound 16 displayed the strongest cytotoxic effect against MCF-7, HCT-116, HeLa, and HepG2 cancer cell lines, with IC50 values of 9.98-16.42 μM, and further research indicated that it induced both apoptosis and cell cycle arrest in the G0/G1 phase in a dose-dependent manner toward HepG2 cells.

Design, Synthesis, and Biological Evaluation of 5-Amino-4-fluoro-1H-benzo[d]imidazole-6-carboxamide Derivatives as Novel and Potential MEK/RAF Complex Inhibitors Based on the "Clamp" Strategy.

Herein, we described the rational drug design and synthesis of a series of 5-amino-4-fluoro-1H-benzo[d]imidazole-6-carboxamide derivatives that inhibit MEK and RAF kinases. The detailed screening cascades revealed that 16b was a preferred compound, which might act like a "clamp" to stabilize the MEK/RAF complex, thereby effectively inhibiting MEK1, BRAF, and BRAFV600E with IC50 values of 28, 3, and 3 nM, respectively. 16b possessed an excellent selectivity over other 312 human-related kinases at 1 μM. In vitro, 16b showed potent antiproliferative activities against MIA PaCa-2 (G12C KRAS), HCT116 (G13D KRAS), and C26 (G12D KRAS) cells with IC50 values of 0.011, 0.079, and 0.096 μM, respectively. CoIP experiments demonstrated that 16b could induce MEK/RAF complex formation. Most importantly, in the C26 syngeneic colorectal and HCT116 mice xenograft tumor models, 16b demonstrated tumor growth inhibition of 70 and 93%, respectively, suggesting that 16b may be a promising MEK/RAF complex inhibitor and worthy of further development.

WTAP weakens oxaliplatin chemosensitivity of colorectal cancer by preventing PANoptosis

As the most abundant post-transcriptional modification in eukaryotes, N6-methyladenosine (m6A) plays a crucial role in cancer cell proliferation, invasion and chemoresistance. However, its specific effects on chemosensitivity to oxaliplatin-based regimens and the impact of these drugs on m6A methylation levels in colorectal cancer (CRC) remain largely unexplored. In this study, we demonstrated that the m6A methyltransferase Wilms tumor 1-associating protein (WTAP) weakens oxaliplatin chemosensitivity in HCT116 and DLD1 cells. Mechanistically, oxaliplatin treatment upregulated WTAP expression, preventing multiple forms of cell death simultaneously, a process known as PANoptosis, by decreasing intracellular oxidative stress through maintaining the expression of nuclear factor erythroid-2-related factor 2 (NRF2), a major antioxidant response element, in an m6A-dependent manner. In addition, high WTAP expression in CRC patients is associated with a poor prognosis and reduced benefit from standard chemotherapy by clinical data analysis of The Cancer Genome Atlas (TCGA) database and patient cohort study. These findings suggest that targeting WTAP-NRF2-PANoptosis axis could enhance the antitumor efficacy of oxaliplatin-based chemotherapy in CRC treatment.

Screening of Lipid-Reducing Activity and Cytotoxicity of the Exometabolome from Cyanobacteria.

Cyanobacteria are rich producers of secondary metabolites, excreting some of these to the culture media. However, the exometabolome of cyanobacteria has been poorly studied, and few studies have dwelled on its characterization and bioactivity assessment. In this work, exometabolomes of 56 cyanobacterial strains were characterized by HR-ESI-LC-MS/MS. Cytotoxicity was assessed on two carcinoma cell lines, HepG2 and HCT116, while the reduction in lipids was tested in zebrafish larvae and in a steatosis model with fatty acid-overloaded human liver cells. The exometabolome analysis using GNPS revealed many complex clusters of unique compounds in several strains, with no identifications in public databases. Three strains reduced viability in HCT116 cells, namely Tolypotrichaceae BACA0428 (30.45%), Aphanizomenonaceae BACA0025 (40.84%), and Microchaetaceae BACA0110 (46.61%). Lipid reduction in zebrafish larvae was only observed by exposure to Dulcicalothrix sp. BACA0344 (60%). The feature-based molecular network shows that this bioactivity was highly correlated with two flavanones, a compound class described in the literature to have lipid reduction activity. The exometabolome characterization of cyanobacteria strains revealed a high chemodiversity, which supports it as a source for novel bioactive compounds, despite most of the time being overlooked.

Construction of anticancer drug incorporated aptamer-functionalized cationic β-lactoglobulin: induction of cell cycle arrest and apoptosis in colorectal cancer

Nanoscale drug delivery systems that are both multifunctional and targeted have been developed using proteins as a basis, thanks to their attractive biomacromolecule properties. A novel nanocarrier, aptamer (AS1411)-conjugated β-lactoglobulin/poly-l-lysine (BLG/Ap/PL) nanoparticles, was developed in this study. To this unique formulation, the as-prepared nanocarrier blends the distinctive features of an aptamer as a chemotherapeutic targeting agent with those of protein nanocarriers. By loading cabazitaxel (CTX) onto the nanocarriers, the therapeutic potential of BLG/Ap/PL could be demonstrated. The CTX-loaded BLG/Ap/PL (CTX@BLG/Ap/PL) showed a regulated drug release profile in an acidic milieu, which could improve therapeutic efficacy in cancer cells and have a high drug encapsulation efficacy of up to 93%. However, compared to free CTX, CTX@BLG/Ap/PL killed colorectal HCT116 cancer cells with a higher efficacy at 24 and 48 h. Further investigation confirms the apoptosis by acridine orange and ethidium bromide (AO/EB), and DAPI staining confirms the morphological changes, chromatin condensation, and membrane blebbing in the treated cell through flow cytometry displayed the release of higher percentages of apoptosis. Cell cycle analysis revealed that CTX@BLG/Ap/PL induced sub-G1 and G2/M phase (apoptosis) at 24 and 48 h. Annexin V/propidium iodide (PI) flow cytometry analysis confirmed that CTX@BLG/Ap/PL induces apoptosis in HCT116 cells. Overall, this study proved that CTX@BLG/Ap/PL had several advantages over free chemotherapeutic drugs and showed promise as a solution to the clinical problems associated with targeted antitumor drug delivery systems.

Regulatory Interplay Between SR Proteins Governs CLK1 Kinase Splice Variants Production.

The CLK1 kinase phosphorylates SR proteins to modulate their splicing regulatory activity. Skipping of alternative exon 4 on the CLK1 pre-mRNA produces a CLK1 variant lacking the catalytic site. Here, we aimed to understand how various SR proteins integrate into the regulatory program that controls CLK1 exon 4 splicing. Previously, we observed that the depletion of SRSF10 promoted the inclusion of CLK1 exon 4. Using expression of tagged proteins and CRISPR/Cas9-mediated knockouts in HCT116 cells, we now identify TRA2b, TRA2a, SRSF4, SRSF5, SRSF7, SRSF8 and SRSF9 as activators of exon 4 inclusion. In contrast, SRSF3, SRSF10 and SRSF12 elicit exon 4 skipping. Using CRISPR/dCas13Rx and RNA immunoprecipitation assays, we map an enhancer in exon 4 interacting with TRA2b. Notably, CLK1 kinase inhibitors antagonized the repressor activity of HA-SRSF10, HA-SRSF12 and HA-SRSF3. Our results suggest that CLK1 exon 4 inclusion is determined primarily by a balance between the activities of TRA2 proteins and CLK-phosphorylated SRSF3. CLK-phosphorylated SRSF10 and SRSF12 would interact with TRA2 proteins to prevent their enhancer activity, allowing SRSF3 to enforce exon 4 skipping more efficiently. Our study provides insight into the complex regulatory network controlling the alternative splicing of CLK1, which uses CLK1-mediated phosphorylation of SR proteins to regulate the inclusion of catalytic exon 4 in CLK1 transcripts.

Synergistic anti-tumorigenic effect of diosmetin in combination with 5-fluorouracil on human colon cancer xenografts in nude mice

5-Fluorouracil (5-FU) is frequently used to treat colorectal cancer (CRC), but its clinical application is limited by its toxicity. Natural compounds have been combined with chemotherapeutic drugs to reduce chemotherapy-related toxicity. Diosmetin, a natural flavonoid, has demonstrated anticancer effects against CRC. This study investigated diosmetin's potential in combination with 5-FU using a murine model of HCT-116 colon cancer xenografts in nu/nu nude mice. HCT-116 cells were injected into the right flanks of mice, and once tumors reached a size of 50 mm3, the mice were treated with diosmetin (100 mg/kg), 5-FU (30 mg/kg), or a combination of both at two dose levels (100 + 30 mg/kg and 50 + 15 mg/kg) for 4 weeks. Blood and tumors were collected on the final day for further analysis. Mice treated with the higher combination dose exhibited the smallest tumor volume (330.91 ± 88.49 mm3). Biochemistry and histology analysis showed no toxicity or abnormalities in the liver, kidney, and heart with the combination therapy. Immunohistochemistry results revealed a notable reduction in the proliferation marker (Ki67) and inflammation marker (TLR4) in tumors from high-dose combination-treated mice. Moreover, immunofluorescence data indicated increased levels of apoptotic markers (Bax, Caspase-3, p53, p21) and downregulation of anti-apoptotic protein (Bcl-2) in the high-dose combination group. The findings suggest that 100 mg/kg of diosmetin combined with 30 mg/kg 5-FU significantly reduced tumor volume and had a less toxic effect on the heart compared to 5-FU monotherapy.

EP4 receptor agonist CAY10598 upregulates ROS-dependent Hsp90 cleavage in colorectal cancer cells

Prostaglandin E2 (PGE2) interacts with four specific G protein-coupled receptors, namely EP1, EP2, EP3, and EP4, playing a pivotal role in determining the fate of cells. Our previous findings highlighted that stimulating the EP4 receptor with its agonist, CAY10598, triggers apoptosis in colon cancer HCT116 cells via the production of reactive oxygen species (ROS). This process also reduces the phosphorylation of the oncogenic protein JAK2 and leads to its degradation in these cells. In this study, our goal was to explore the pathways through which CAY10598 leads to JAK2 degradation. We focused on Hsp90, a heat shock protein family member known for its role as a molecular chaperone maintaining the stability of several key proteins including EGFR, MET, Akt, and JAK2. Our results show that CAY10598 decreases the levels of client proteins of Hsp90 in HCT116 cells, an effect reversible by pretreatment with the ROS scavenger N-acetyl cysteine (NAC) or the proteasome inhibitor MG132, indicating that the degradation is likely driven by ROS. Furthermore, we observed that CAY10598 cleaves both α and β isoforms of Hsp90, the process inhibited by NAC. Inhibition of EP4 with the antagonist GW627368x not only prevented the degradation of Hsp90 client proteins but also the cleavage of Hsp90 itself in CAY10598-treated HCT116 cells. Additionally, CAY10598 suppressed the growth of HCT116 cells implanted in mice. Our findings reveal that CAY10598 induces apoptosis in cancer cells by a novel mechanism involving the ROS-dependent cleavage of Hsp90, thereby inhibiting the function of crucial Hsp90 client proteins.

Microwave synthesis, density functional theory study and antiproliferative activity of the novel spiropyrazole derivatives

Different spiro-pyrazole-oxy-indoline substituted compounds I-IV have been synthesized. In Jaguar, Hybrid DFT using Becke’s three-parameter transfer possibility also,Lee-Yang-Parr correlated practical (B3LYP) viewwith a 6-31 G** basis set were utilized to improve the geometrical characteristics of the hit molecules and reference compound. The MTT test exhibited whether the prepared substance has an inhibitory impact on cell development or not. This was successfully represented that in hepatocellular carcinoma (HEPG-2), colorectal carcinoma (HCT-116), and mammary gland breast cancer cell lines (MCF-7) colorimetric test, the mitochondrial succinate dehydrogenase in live cells transform yellow tetrazolium bromide (MTT) to a purple formazan product. Compound IV was found to be a better cytotoxic action against to the HEPG-2 hepatocellular carcinoma cell line, and it has been seen a strong activity against the colorectal carcinoma cell lines HCT-116 and MCF-7 mammary gland breast cancer cell lines. While the other three compounds I, II, and III showed a moderate cytotoxic action to high action against the same cell lines.

The m6A methyltransferase METTL3 modifies Kcnk6 promoting on inflammation associated carcinogenesis is essential for colon homeostasis and defense system through histone lactylation dependent YTHDF2 binding

Inflammation induces tumor formation and plays a crucial role in tumor progression and prognosis. KCNK6, by regulating K(+) efflux to reduce NLRP3 Inflammasome-induced lung injury, relaxes the aorta. This study aims to elucidate the effects and biological mechanism of KCNK6 in inflammation-associated carcinogenesis, which may be essential for colon homeostasis and the defense system. To induce colitis, mice were given 3.0% Dextran Sodium Sulfate (DSS) in their drinking water for 7 days. The Azoxymethane (AOM) +DSS method was used to induce colon cancer in the mice model. Bone marrow-derived macrophages (BMDM) from Kcnk6-/- mice, AW264.7 cells, and human colon cancer HCT116 and Caco2 cells were used as in vitro models. The loss of Kcnk6 prevented spontaneous colitis and restored mucosal integrity and homeostatic molecules. Additionally, the loss of Kcnk6 reduced the severity of AOM/DSS-induced carcinogenesis. Kcnk6 promoted cell viability and proliferation in HCT-116 or Caco-2 cells. The loss of Kcnk6 inhibited the levels of inflammatory factors in BMDM cells. Kcnk6 accelerated potassium channel activity, inducing NLRP3 inflammasome activation. METTL3-mediated m6A modification increased Kcnk6 stability in a YTHDF2-dependent manner. Histone lactylation activated the transcription of YTHDF2/Kcnk6. Our study revealed the important role of Kcnk6 in inflammation-associated carcinogenesis progression. The m6A methyltransferase METTL3 and histone lactylation increased Kcnk6 stability in a YTHDF2-dependent manner, providing a potential strategy for inflammation-associated carcinogenesis or colorectal cancer therapy.

Metabolic Response to Small Molecule Therapy in Colorectal Cancer Tracked with Raman Spectroscopy and Metabolomics

AbstractDespite numerous screening tools for colorectal cancer (CRC), 25 % of patients are diagnosed with advanced disease. Novel diagnostic technologies that are early, accurate, and rapid are imperative to assess the therapeutic efficacy of clinical drugs and identify new biomarkers of treatment response. Here Raman spectroscopy (RS) was used to track metabolic reprogramming in KRAS‐mutant HCT116 and SW837 cells, and KRAS wild‐type CC cells. RS combined with multivariate analysis methods distinguished nonresponsive, partially responsive, and responsive cells treated with cetuximab, a monoclonal antibody for EGFR inhibition, sotorasib, a clinically approved KRAS inhibitor, and various doses of trametinib, an inhibitor of the MAPK pathway. Cells treated with a combination of subtoxic doses of trametinib and BKM120, an inhibitor of the PI3K pathway, showed a synergistic response between the two pathways. Using a supervised machine learning regression model, we established a scoring methodology trained to a priori predict therapeutic response to new treatment combinations. RS metabolites were verified with mass spectrometry, and enrichment pathways were identified, including amino acid, purine, and nicotinate and nicotinamide metabolism that differentiated monotherapy from combination therapy. Our approach may ultimately be applicable to patient‐derived primary cells and cultures of patient tumors to predict effective drugs for individualized care.

Cell type specific suppression of hyper-recombination by human RAD18 is linked to PCNA K164 ubiquitination.

Homologous recombination (HR) and translesion synthesis (TLS) promote gap-filling DNA synthesis to complete genome replication. One factor involved in both pathways is RAD18, an E3 ubiquitin ligase. Although RAD18's role in promoting TLS through the ubiquitination of PCNA at lysine 164 (K164) is well established, its requirement for HR-based mechanisms is currently less clear. To assess this, we inactivated RAD18 in three human cell lines. Our analyses found that loss of RAD18 in HCT116, but neither hTERT RPE-1 nor DLD1 cell lines, resulted in elevated sister chromatid exchange, gene conversion, and gene targeting, i.e . HCT116 mutants were hyper-recombinogenic (hyper-rec). Loss of RAD18 also impaired TLS activity in HCT116 cells, but unexpectedly, did not reduce clonogenic survival. Interestingly, these phenotypes appear linked to PCNA K164 ubiquitination, as HCT116 PCNA K164R/+ mutants were also hyper-rec and showed reduced TLS activity, consistent with previous studies in rad18 -/- or pcna K164R avian DT40 mutant cells. Importantly, knockdown of UBC9 to prevent PCNA K164 SUMOylation did not affect hyper-recombination, strengthening the link between increased recombination and RAD18-catalyzed PCNA K164 ubiquitination, but not K164 SUMOylation. Taken together, these data suggest that the roles of human RAD18 in directing distinct gap-filling DNA synthesis pathways varies depending on cell type and that these functions are linked to PCNA ubiquitination.

Synthesis, Characterization and Biological Evaluation of 3‐Oxo‐Pyrimido[1,2‐b]indazoles

AbstractThe synthesis of substituted 3‐oxo‐pyrimido[1,2‐b]indazoles from 3‐aminoindazoles and ketones has been established. It involves one molecule of 3‐aminoindazoles and two molecules of ketones via a [3+2+1] three‐component annulation process. 3‐Oxo‐pyrimido[1,2‐b]indazoles exhibit strong yellow fluorescence in various solvents. A wide range of products have anti‐oxidant activities. Cytotoxicity studies indicate that compounds 3 a, 3 aa and 3 ab have obvious effects on both HCT116 and A549 cancer cell lines.