Sensitivity Of Cells Research Articles

Natural products and long non-coding RNAs in prostate cancer: insights into etiology and treatment resistance.

Globally, the incidence and death rates associated with cancer persist in rising, despite considerable advancements in cancer therapy. Although some malignancies are manageable by a mix of chemotherapy, surgery, radiation, and targeted therapy, most malignant tumors either exhibit poor responsiveness to early identification or endure post-treatment survival. The prognosis for prostate cancer (PCa) is unfavorable since it is a perilous and lethal malignancy. The capacity of phytochemical and nutraceutical chemicals to repress oncogenic lncRNAs and activate tumor suppressor lncRNAs has garnered significant attention as a possible strategy to diminish the development, proliferation, metastasis, and invasion of cancer cells. A potential technique to treat cancer and enhance the sensitivity of cancer cells to existing conventional therapies is the use of phytochemicals with anticancer characteristics. Functional studies indicate that lncRNAs modulate drug resistance, stemness, invasion, metastasis, angiogenesis, and proliferation via interactions with tumor suppressors and oncoproteins. Among them, numerous lncRNAs, such as HOTAIR, PlncRNA1, GAS5, MEG3, LincRNA-21, and POTEF-AS1, support the development of PCa through many molecular mechanisms, including modulation of tumor suppressors and regulation of various signal pathways like PI3K/Akt, Bax/Caspase 3, P53, MAPK cascade, and TGF-β1. Other lncRNAs, in particular, MALAT-1, CCAT2, DANCR, LncRNA-ATB, PlncRNA1, LincRNA-21, POTEF-AS1, ZEB1-AS1, SChLAP1, and H19, are key players in regulating the aforementioned processes. Natural substances have shown promising anticancer benefits against PCa by altering essential signaling pathways. The overexpression of some lncRNAs is associated with advanced TNM stage, metastasis, chemoresistance, and reduced survival. LncRNAs possess crucial clinical and transitional implications in PCa, as diagnostic and prognostic biomarkers, as well as medicinal targets. To impede the progression of PCa, it is beneficial to target aberrant long non-coding RNAs using antisense oligonucleotides or small interfering RNAs (siRNAs). This prevents them from transmitting harmful messages. In summary, several precision medicine approaches may be used to rectify dysfunctional lncRNA regulatory circuits, so improving early PCa detection and eventually facilitating the conquest of this lethal disease. Due to their presence in biological fluids and tissues, they may serve as novel biomarkers. Enhancing PCa treatments mitigates resistance to chemotherapy and radiation.

Comprehensive investigation of matrix metalloproteinases in skin cutaneous melanoma: diagnostic, prognostic, and therapeutic insights

The dysregulation of matrix metalloproteinases (MMPs) in skin cutaneous melanoma (SKCM) represents a critical aspect of tumorigenesis. In this study, we investigated the diagnostic, prognostic, and therapeutic aspects of the MMPs in SKCM. Thirteen SKCM cell lines and seven normal skin cell lines were cultured under standard conditions for experimental analyses. RNA and DNA were extracted, followed by RT-qPCR to assess MMP expression and promoter methylation analysis to determine methylation levels. Functional assays, including cell proliferation, colony formation, and wound healing, were conducted post-MMP7 knockdown using siRNA in A375 cells. Databases like GEPIA2, HPA, MEXPRESS, and miRNet were employed for expression, survival, methylation, and miRNA-mRNA network analyses. We investigated the expression and promoter methylation landscape of MMPs in SKCM cell lines, revealing significant (p-value < 0.05) up-regulation of MMP1, MMP7, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, and MMP25, alongside down-regulation of MMP2, MMP3, and MMP21. Furthermore, our analysis demonstrated a significant (p-value < 0.05) inverse correlation between MMP expression levels and promoter methylation status, suggesting a potential regulatory role of DNA methylation in MMP dysregulation. Notably, MMP7, MMP11, and MMP14 exhibited significant (p-value < 0.05) associations with the overall survival of SKCM patients, emphasizing their prognostic significance. Additionally, Receiver operating characteristic (ROC) curve analysis highlighted the significant (p-value < 0.05) diagnostic potential of MMP7, MMP11, and MMP14 in distinguishing SKCM from normal individuals. Subsequent validation across multiple cohorts confirmed significant (p-value < 0.05) elevated MMP expression levels in SKCM tissues, particularly in advanced disease stages, further emphasizing their role in tumor progression. Furthermore, we elucidated potential regulatory pathways involving miR-22-3p, which targets MMP7, MMP11, and MMP14 genes in SKCM. Our findings also revealed associations between MMP expression and immune modulation, drug sensitivity, and functional states of SKCM cells. Lastly, MMP7 knockdown in A375 cells significantly significant (p-value < 0.05) impacted several characteristics, including cell proliferation, colony formation, and wound healing. Our findings highlight the diagnostic, prognostic, and therapeutic potential of MMP7, MMP11, and MMP14 in SKCM. These MMPs could serve as biomarkers for early detection and targets for therapy. Future efforts should focus on preclinical and clinical validation to translate these insights into personalized diagnostic and therapeutic strategies.

Hypoxia-triggered ERRα acetylation enhanced its oncogenic role and promoted progression of renal cell carcinoma by coordinating autophagosome-lysosome fusion

Estrogen-related receptor α (ERRα) is dysregulated in many types of cancer and exhibits oncogenic activity by promoting tumorigenesis and metastasis of cancer cells. However, its defined role in renal cell carcinoma (RCC) has not been fully elucidated. To reveal the biological function of ERRα and determine the underlying regulatory mechanism in RCC, the quantitative proteomics analysis and mechanism investigation were conducted. The results demonstrated that ERRα promoted the proliferation and tumorigenesis of RCC cells by maintaining lysosome-dependent autophagy flux. ERRα inhibition impaired the transcriptional expression of LAMP2 and VAMP8 and blocked the fusion of autophagosomes with lysosomes, causing the impairment of the autophagy-lysosome pathway and tumor repression in RCC. Moreover, VHL mutant-induced hyperactive hypoxia signaling in RCC triggered p300/CBP-mediated acetylation at the DNA-binding domain of ERRα, and this acetylation promoted its affinity toward targeting DNA and Parkin-mediated ubiquitination and proteasome-dependent degradation. This regulatory model enhanced ERRα transactivation on the expression of LAMP2 and VAMP8, which then maintained autophagy flux and RCC progression. Pharmaceutical inhibition on ERRα acetylation-mediated autophagy-lysosome pathway led to growth repression and sunitinib sensitivity of RCC cells. Taken together, this study uncovered a novel regulatory mechanism of acetylation contributing to the transcriptional performance and the oncogenic role of ERRα in RCC progression by modulating the autophagy-lysosome pathway. These findings might provide a novel approach for the clinical diagnosis and resolution of sunitinib resistance of RCC.

Biotinylated Viscosity Sensitive Cell Membrane Probe for Targeted Imaging and Precise Visualization of Tumor Cells and Tumors.

Cancer is a global health challenge that urgently requires more sensitive and effective cancer detection methods. Fluorescence imaging with small molecule fluorescent probes has shown great promise for cancer detection but most of the developed probes lack active tumor cell targeting, which makes them unable to selectively target tumors, thereby reducing the accuracy of in vivo tumor detection. Herein, we report a novel probe Bio-S that combines a viscosity-sensitive and cell membrane targetable fluorescent group with biotin for targeted imaging and precise visualization of tumor cells and tumors. Bio-S exhibits sensitive fluorescence changes for viscosity at ∼660 nm and excellent cell membrane localization and imaging ability (red fluorescence, wash-free, and long-term imaging). Moreover, compared with the nonbiotinylated control probe C6-S, the biotinylated Bio-S can specifically target tumor cell membranes, thereby achieving much higher selectivity and sensitivity in distinguishing tumor cells from normal cells. Mice imaging experiments show that tail vein injection of Bio-S can target tumors and monitor lung cancer metastasis at the in vivo level. Therefore, this work provides an effective new strategy and tool for tumor-targeted detection and precise diagnosis.

Cytotoxic mechanisms of pemetrexed and HDAC inhibition in non-small cell lung cancer cells involving ribonucleotides in DNA

The cytotoxic mechanisms of thymidylate synthase inhibitors, such as the multitarget antifolate pemetrexed, are not yet fully understood. Emerging evidence indicates that combining pemetrexed with histone deacetylase inhibitors (HDACi) may enhance therapeutic efficacy in non-small cell lung cancer (NSCLC). To explore this further, A549 NSCLC cells were treated with various combinations of pemetrexed and the HDACi MS275 (Entinostat), and subsequently assessed for cell viability, cell cycle changes, and genotoxic markers. Proteomic alterations were analyzed using label-free shotgun and targeted LC–MS/MS. MS275 enhanced the sensitivity of A549 cells to pemetrexed, but only when administered following prior treatment with pemetrexed. Both HeLa (p53 negative) and A549 (p53 positive) showed robust activation of γH2AX upon treatment with this combination. Importantly, CRISPR/Cas9 knockout of the uracil-DNA glycosylase UNG did not affect γH2AX activation or sensitivity to pemetrexed. Proteomic analysis revealed that MS275 altered the expression of known pemetrexed targets, as well as several proteins involved in pyrimidine metabolism and DNA repair, which could potentiate pemetrexed cytotoxicity. Contrary to the conventional model of antifolate toxicity, which implicates futile cycles of uracil incorporation and excision in DNA, we propose that ribonucleotide incorporation in nuclear and mitochondrial DNA significantly contributes to the cytotoxicity of antifolates like pemetrexed, and likely also of fluorinated pyrimidine analogs. HDAC inhibition apparently exacerbates cytotoxicity of these agents by inhibiting error-free repair of misincorporated ribonucleotides in DNA. The potential of HDACis to modulate pyrimidine metabolism and DNA damage responses offers novel strategies for improving NSCLC outcomes.

Pristimerin Promotes Ubiquitination of HSPA8 and Activates the VAV1/ERK Pathway to Suppress TNBC Proliferation.

Triple-negative breast cancer (TNBC) is a subtype of breast cancer with a poor prognosis. The natural compound pristimerin has shown promising anti-tumor effect. Here, it is found that pristimerin significantly triggered the activation of autophagy initiation and induced apoptosis in TNBC. Mechanistically, RNA sequencing revealed that pristimerin activated mitogen-activated protein kinase/extracelluar regulated protein kinases (MAPK/ERK)pathway. Drug affinity responsive target stability and mass spectrometry techniques are employed to confirm the direct binding target of pristimerin. Heat shock protein family A member 8 (HSPA8) is identified and verified by cellular thermal shift assays and surface plasmon resonance assays. The further results suggested that pristimerin promoted the ubiquitination and degradation of HSPA8, leading to a decrease in the degradation of Vac Guanine Nucleotide Exchange Factor 1 (VAV1), a downstream client protein of HSPA8 which plays a crucial role in activating the ERK pathway. Importantly, knockdown of HSPA8 or VAV1 significantly impaired the anticancer activity of pristimerin on TNBC cells. Additionally, pristimerin significantly inhibited the migration and invasion of TNBC cells and enhanced the sensitivity of TNBC cells to doxorubicin. Collectively, this study provides the initial evidence that pristimerin directly targets HSPA8 to activate the VAV1/ERK pathway, thereby promoting cell autophagy and apoptosis.

Scale-up of CHO cell cultures: from 96-well-microtiter plates to stirred tank reactors across three orders of magnitude.

For process development in mammalian cell cultivations, scale-up approaches are essential. A lot of studies concern the scale transfer between different-sized stirred tank reactors. However, process development usually starts in even smaller cultivation vessels like microtiter plates or shake flasks. A scale-up from those small shaken devices to a stirred tank reactor is barely stated in literature for mammalian cells. Thus, this study aims to address data-driven scale-up for CHODP12 cells. The oxygen transfer rate is used as a database. The cultivation conditions in microtiter plates and shake flasks are comparable when choosing the maximum oxygen transfer capacity as a scale-up parameter. The minimum cultivation volume was reduced to 400µL in round and square 96-deep-well microtiter plates. Using a scale-up based on the maximum oxygen transfer capacity to a stirred tank reactor led to conditions with excessive hydromechanical stress. However, cultivation conditions could be reproduced in a stirred tank reactor by utilizing the volumetric power input as a scale-up parameter. Key metabolites behaved the same in all three scales and the final antibody titer was equal. This study presents a successful replication of cultivation results for mammalian cells in microtiter plates, shake flasks and stirred tank reactors. The working volumes ranged from 0.4 to 50 and 600mL. It offers the opportunity to adapt the method to other, more sensitive mammalian cells and to perform cost- and time-effective experiments in high-throughput.

Targeting the 8-oxodG Base Excision Repair Pathway for Cancer Therapy

Genomic integrity is critical for cellular homeostasis, preventing the accumulation of mutations that can drive diseases such as cancer. Among the mechanisms safeguarding genomic stability, the Base Excision Repair (BER) pathway plays a pivotal role in counteracting oxidative DNA damage caused by reactive oxygen species. Central to this pathway are enzymes like 8-oxoguanine glycosylase 1 (OGG1), which recognize and excise 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) lesions, thereby initiating a series of repair processes that restore DNA integrity. BER inhibitors have recently been identified as a promising approach in cancer therapy, increasing the sensitivity of cancer cells to radiotherapy and chemotherapy. By exploiting tumor-specific DNA repair dependencies and synthetic lethal interactions, these inhibitors could be used to selectively target cancer cells while sparing normal cells. This review provides a robust reference for scientific researchers, offering an updated perspective on small-molecule inhibitors targeting the 8-oxodG-BER pathway and highlighting their potential role in expanding cancer treatment strategies.

PYGO2 promotes resistance to chemotherapy via reducing apoptosis and G2/M cell cycle arrest in esophageal carcinoma cells.

5-FU is a widely used chemotherapy drug for esophageal carcinomas, but therapy failure has been observed in 5-FU-resistant patients. Overcoming this resistance is a significant challenge in cancer treatment, requiring identifying and targeting important resistance mechanisms. PYGO2 expression is crucial in developing resistance to various chemotherapy drugs. In this study, we aimed to investigate the impact of PYGO2 overexpression on the sensitivity of YM-1 and KYSE-30 esophageal carcinomacells against 5-FU. To do this, we compared cell viability, cell cycle arrest, apoptosis rate, and mRNA expressions of various apoptosis-related genes between pcDNA3-PYGO2 transfected and untransfected KYSE-30 and YM-1 esophageal carcinoma cells following treatment with 5-FU. We showed that PYGO2 expression reduces 5-FU sensitivity in YM-1 and KYSE-30 cells. PYGO2-overexpressing cells treated with 5-FU have exhibited a noteworthy reduction in both early and late apoptotic cells compared to controls. Furthermore, a significant decrease in the Bax/Bcl2 ratio and P53 gene expression was observed. 5-FU induces G2/M cell cycle arrest in YM-1 and KYSE-30 cells. However, PYGO2 overexpression impeded G2/M cell cycle arrest in 5-FU-treated cells, thereby suppressing the toxicity of 5-FU. PYGO2 may mediate its apoptotic effect by regulating cell cycle regulatory proteins, specifically cyclin D1 and p21. These results highlight PYGO2's capacity to alter how esophageal cancer cells respond to 5-FU therapy, emphasizing its importance as a potential focal point for treatment strategies.

Integrated bioinformatics and experimental analysis of CHAF1B as a novel biomarker and immunotherapy target in LUAD

The prognosis and treatment efficacy of lung adenocarcinoma (LUAD), a disease with a high incidence, remains unsatisfactory. Identifying new biomarkers and therapeutic targets for LUAD is essential. Chromosomal assembly factor 1B (CHAF1B), a p60 component of the CAF-1 complex, is closely linked to tumor incidence and cell proliferation. However, CHAF1B's biological role and molecular mechanism in LUAD remain unclear. Here, CHAF1B expression in LUAD was examined using the GEPIA2 and UALCAN databases. Using The Cancer Genome Atlas (TCGA) LUAD database, we analyzed the diagnostic and prognostic significance of CHAF1B and its association with immune infiltration and immunological checkpoints. Gene ontology (GO) enrichment and single-cell function analyses were employed to investigate CHAF1B's possible biological roles. Drug sensitivity analysis predicted CHAF1B's effect on chemotherapeutic drug sensitivity. We also predicted lncRNAs-miRNA-CHAF1B axis to explore the molecular mechanism of CHAF1B in LUAD. Preliminary in vitro studies using qRT-PCR, CCK8, Transwell, glucose, and lactate metabolism confirmed CHAF1B's expression and role in LUAD. Its expression is associated with drug sensitivity, immunological checkpoints, and immune cell infiltration. We predicted that three miRNAs (miR-29c-3p, miR-145-5p, miR-1247-5p) and three lncRNAs (AL139287.1, NEAT1, SHG1) may be target miRNAs and target lncRNAs that regulate CHAF1B. In vitro tests showed that CHAF1B suppression decreased LUAD's migration, invasion, proliferation, and glycolysis. Overall, CHAF1B may be an innovative biomarker and therapeutic target for LUAD.

Ferroptosis Inducers Erastin and RSL3 Enhance Adriamycin and Topotecan Sensitivity in ABCB1/ABCG2-Expressing Tumor Cells

Acquired resistance to chemotherapeutic drugs is the primary cause of treatment failure in the clinic. While multiple factors contribute to this resistance, increased expression of ABC transporters—such as P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and multidrug resistance proteins—play significant roles in the development of resistance to various chemotherapeutics. We found that Erastin, a ferroptosis inducer, was significantly cytotoxic to NCI/ADR-RES, a P-gp-expressing human ovarian cancer cell line. Here, we examined the effects of both Erastin and RSL3 (Ras-Selected Ligand 3) on reversing Adriamycin resistance in these cell lines. Our results show that Erastin significantly enhanced Adriamycin uptake in NCI/ADR-RES cells without affecting sensitive cells. Furthermore, we observed that Erastin enhanced Adriamycin cytotoxicity in a time-dependent manner. The selective iNOS inhibitor, 1400W, reduced both uptake and cytotoxicity of Adriamycin in P-gp-expressing NCI/ADR-RES cells only. These findings were also confirmed in a BCRP-expressing human breast cancer cell line (MCF-7/MXR), which was selected for resistance to Mitoxantrone. Both Erastin and RSL3 were found to be cytotoxic to MCF-7/MXR cells. Erastin significantly enhanced the uptake of Hoechst dye, a well-characterized BCRP substrate, sensitizing MCF-7/MXR cells to Topotecan. The effect of Erastin was inhibited by 1400W, indicating that iNOS is involved in Erastin-mediated enhancement of Topotecan cytotoxicity. RSL3 also significantly increased Topotecan cytotoxicity. Our findings—demonstrating increased cytotoxicity of Adriamycin and Topotecan in P-gp- and BCRP-expressing cells—suggest that ferroptosis inducers may be highly valuable in combination with other chemotherapeutics to manage patients’ cancer burden in the clinical setting.

WDR62 mediates MAPK/ERK pathway to stimulate DNA damage repair and attenuate cisplatin sensitivity in lung adenocarcinoma.

Chemotherapy resistance has long stood in the way of therapeutic advancement for lung cancer patients, the malignant tumor with the highest incidence and fatality rate in the world. Patients with lung adenocarcinoma (LUAD) now have a dismal prognosis due to the development of cisplatin (DDP) resistance, forcing them to use more costly second-line therapies. Therefore, overcoming resistance and enhancing patient outcomes can be achieved by comprehending the regulatory mechanisms of DDP resistance in LUAD. WD repeat domain 62 (WDR62) expression in LUAD tissues and in DDP-resistant or sensitive LUAD patients was analyzed bioinformatically, and a K-M plot was utilized to assess survival status. Real-time quantitative PCR was employed for WDR62 expression detection, cell-counting kit-8 assay for half maximal inhibitory concentration determination, flow cytometry for cell apoptosis detection, immunofluorescence for γ-H2AX expression analysis, and western blot for nonhomologous end joining repair and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway-related protein expression analysis. Poor prognosis was linked to WDR62, which was overexpressed in LUAD tissues and cells. Compared to sensitive cells, DDP-resistant cells had increased WDR62 expression. WDR62 knockdown may enhance DDP-induced cell apoptosis while reducing cell proliferation and DNA damage repair. Functional investigations verified that overexpressed WDR62's encouraging impact on DNA damage repair in A549/DDP cells could be reversed by MAPK inhibitors, increasing the cells' susceptibility to DDP. LUAD cells became less sensitive to DDP when WDR62 activated the MAPK/ERK pathway, which promoted DNA damage repair, indicating that DDP resistance might be reversed by treating LUAD with inhibitors of the MAPK pathway.

Targeting deubiquitinase USP7-mediated stabilization of XPO1 contributes to the anti-myeloma effects of selinexor

BackgroundTargeting exportin1 (XPO1) with Selinexor (SEL) is a promising therapeutic strategy for patients with multiple myeloma (MM). However, intrinsic and acquired drug resistance constitute great challenges. SEL has been reported to promote the degradation of XPO1 protein in tumor cells. Nevertheless, in myeloma, the precise mechanisms underlying SEL-induced XPO1 degradation and its impact on drug responsiveness remain largely undefined.MethodsWe assessed XPO1 protein and mRNA levels using western blotting and RT-qPCR. Cycloheximide (CHX) chase assays and degradation blockade assays were used to determine the pathway of XPO1 degradation induced by SEL. The sensitivity of MM cell lines to SEL was evaluated using CCK8-based cell viability assays and AV-PI staining-based cell apoptosis assays. The subcellular localization of the cargo protein RanBP1 was assessed via immunofluorescence staining. Immunoprecipitation coupled with mass spectrometry (IP-MS), bioinformatics analysis and ubiquitination assays, were employed to identify the molecular targets responsible for SEL-induced degradation of XPO1. shRNA-mediated knockdown assays and small molecule inhibitors of USP7 were utilized to disrupt the function of USP7. The role of USP7 in modulating SEL sensitivity was analyzed in MM cell lines, primary CD138+ cells, and xenograft mouse models.ResultsSEL promotes the degradation of XPO1 in MM cells through the ubiquitin–proteasome pathway. There is a positive correlation between XPO1 degradation and sensitivity to SEL in these cells. Inhibiting XPO1 degradation reduces the functional inhibitory effects of SEL on XPO1, as evidenced by decreased nuclear localization of the cargo protein RanBP1. USP7 stabilizes XPO1 in MM cells via its deubiquitinating activity. SEL accelerates the ubiquitination and subsequent degradation of XPO1 by disrupting the interaction between XPO1 and USP7. The expression of USP7 is negatively correlated with patient prognosis and the sensitivity of MM cells to SEL. Inactivating or knocking down USP7 significantly enhances the anti-myeloma effects of SEL both in vitro and in vivo.ConclusionIn conclusion, our findings underscore the essential role of XPO1 degradation in the anti-myeloma efficacy of SEL and establish a research foundation for targeting USP7 to improve the effectiveness of SEL-based therapies in MM.

MYC Overexpression Enhances Sensitivity to MEK Inhibition in Head and Neck Squamous Cell Carcinoma

MEK inhibitors, such as trametinib, have shown therapeutic potential in head and neck squamous cell carcinoma (HNSCC). However, the factors influencing cancer cell sensitivity and resistance to MEK inhibition remain poorly understood. In our study, we observed that MEK inhibition significantly reduced the expression of MYC, a transcription factor critical for the therapeutic response. MYC overexpression markedly enhanced the sensitivity of HNSCC cells to trametinib, as evidenced by delayed wound healing and reduced colony formation. Cell cycle analysis revealed that trametinib induced a G1 phase arrest, whereas MYC overexpression accelerated cell cycle progression, with a reduced induction of p27 and p21 and diminished decreases in E2F1 and phospho-Ser2/5 levels. Flow cytometry and protein analyses demonstrated that MYC overexpression amplified trametinib-induced apoptosis and DNA damage, as evidenced by elevated levels of pro-apoptotic markers (p53, cleaved PARP, and BIM) and γH2AX. In vivo xenograft models confirmed these findings, showing increased sensitivity to trametinib in MYC-overexpressing tumors. Moreover, MEK inhibition increased autophagy in HNSCC cells, a factor critical for therapeutic resistance. Inhibiting trametinib-induced autophagy further enhanced apoptotic cell death. These findings suggest that MYC expression and autophagy play crucial roles in HNSCC’s response to MEK inhibition. Combining trametinib with autophagy inhibition may improve therapeutic outcomes in HNSCC.

Targeting on the PI3K/mTOR: a potential treatment strategy for clear cell ovarian carcinoma

PurposeOvarian clear cell carcinoma is a highly malignant gynecological tumor characterized by a high rate of chemotherapy resistance and poor prognosis. The PI3K/AKT/mTOR pathway is well-known to be closely related to the progression of various malignancies, and recent studies have indicated that this pathway may play a critical role in the progression and worsening of OCCC.MethodsIn this study, we investigated the combined effects of WX390, a dual inhibitor of PI3K/mTOR, and cisplatin on OCCC through both in vitro and in vivo experiments to further elucidate their therapeutic effects.ResultsWX390 significantly inhibited the proliferation of human OCCC cell lines ES2 and OVISE, while promoting apoptosis. Furthermore, the combination of WX390 with CDDP exhibited a synergistic effect, markedly increasing the sensitivity of OCCC cells to chemotherapeutic agents and significantly suppressing tumor growth in PDX models. Western blot and RNA-seq analyses revealed that WX390 robustly inhibited the PI3K/AKT/mTOR pathway, interrupt autophagy, altered cell cycle dynamics, and induced apoptosis.ConclusionThis study comprehensively assessed the efficacy of WX390 across multiple models of OCCC, laying a solid foundation for the development of new therapeutic strategies for this challenging malignancy.

Resistance to complement-mediated lysis of parainfluenza virus 5-infected cells is acquired after transition from acute to persistent infection.

A persistent infection (PI) with RNA viruses can extend virus shedding, prolong inflammation, and be a source of new viral variants. Since profound changes to innate immune pathways can occur in PI cells, it was important to test PI cells for changes in sensitivity to the complement (C') system, powerful innate immune pathways capable of lysing infected cells. Using parainfluenza virus 5 (PIV5) as a model system, we show that PI cells are nearly completely resistant to C'-mediated lysis, in stark contrast to high sensitivity of acute PIV5-infected cells to C' killing. A key finding was the upregulated expression in PI cells of two C' inhibitors: Vitronectin and complement factor H. These are important results with strong potential to inform therapeutics, given that polymorphisms in C' genes can correlate with severity of viral infections, and clinical trials are underway with new drugs that modulate C' responses.

Knockdown of miR-182 changes the sensitivity of triple-negative breast cancer cells to cisplatin

Breast cancer is the most common malignancy that affects women. MicroRNAs (miRNAs) play an essential role in cancer therapy and regulate many biological processes such as cisplatin resistance. The study’s objective was to determine whether miR-182 dysregulation was the cause of cisplatin resistance in TNBC cell line MDA-MB-231. To determine the expression of miR-182, PCR was performed with primers specific to miR-182, and agarose gel electrophoresis was performed. To reduce the expression of miR-182 in MDA-MB-231 cells, anti-miR-182 oligonucleotides were used. RT-qPCR was used to confirm knockdown. The knockdown and control groups were treated with cisplatin at the same time. Propidium iodide (PI) and Annexin V staining were performed for apoptosis assay. Flow cytometric analysis was used to investigate the effect of miR-182 knockdown on cell cycle arrest. In comparison to untreated control MDA-MB-231 cells with MDA-MB-231 cells treated with anti-miR-182, there was a significant increase in the cisplatin-induced early apoptosis phase (p = 0.023). Also, inhibition of miR-182 significantly increased the cell cycle arrest at the G2/M phase in MDA-MB-231 cells (p = 0.031). Our results revealed that miR-182 inhibition may play a role in the overcoming of cisplatin resistance by inducing apoptosis and, cell cycle arrest in TNBC.

Secreted LGALS3BP facilitates distant metastasis of breast cancer

BackgroundPatients with estrogen receptor (ER)-positive breast cancer (BC) can be treated with endocrine therapy targeting ER, however, metastatic recurrence occurs in 25% of the patients who have initially been treated. Secreted proteins from tumors play important roles in cancer metastasis but previous methods for isolating secretory proteins had limitations in identifying novel targets.MethodsWe applied an in situ secretory protein labeling technique using TurboID to analyze secretome from tamoxifen-resistant (TAMR) BC. The increased expression of LGALS3BP was validated using western blotting, qPCR, ELISA, and IF. Chromatin immunoprecipitation was applied to analyze estrogen-dependent regulation of LGALS3BP transcription. The adhesive and angiogenic functions of LGALS3BP were evaluated by abrogating LGALS3BP expression using either shRNA-mediated knockdown or a neutralizing antibody. Xenograft mouse experiments were employed to assess the in vivo metastatic potential of TAMR cells and the LGALS3BP protein. Clinical evaluation of LGALS3BP risk was carried out with refractory clinical specimens from tamoxifen-treated ER-positive BC patients and publicly available databases.ResultsTAMR secretome analysis revealed that 176 proteins were secreted at least 2-fold more from MCF7/TAMR cells than from sensitive cells, and biological processes such as cell adhesion and angiogenesis were associated with the TAMR secretome. Galectin-3 binding protein (LGALS3BP) was one of the top 10 most highly secreted proteins in the TAMR secretome. The expression level of LGALS3BP was suppressed by estrogen signaling, which involves direct ERα binding to its promoter region. Secreted LGALS3BP in the TAMR secretome helped BC cells adhere to the extracellular matrix and promoted the tube formation of human umbilical vein endothelial cells. Compared with sensitive cells, xenograft animal experiments with MCF7/TAMR cells showed increased pulmonary metastasis, which completely disappeared in LGALS3BP-knockdown TAMR cells. Finally, higher levels of LGALS3BP were associated with poor prognosis in ER-positive BC patients treated with adjuvant tamoxifen in the clinic.ConclusionTAMR secretome analysis identified secretory proteins, such as LGALS3BP, that are involved in biological processes closely related to metastasis. Secreted LGALS3BP from the TAMR cells promoted adhesion of the cells to the extracellular matrix and vasculature formation, which may support metastasis of TAMR cells.

Transketolase attenuates the chemotherapy sensitivity of glioma cells by modulating R-loop formation.

Glioblastoma (GBM) is a highly lethal malignant brain tumor with poor survival rates, and chemoresistance poses a significant challenge to the treatment of patients with GBM. Here, we show that transketolase (TKT), a metabolic enzyme in the pentose phosphate pathway (PPP), attenuates the chemotherapy sensitivity of glioma cells in a manner independent of catalytic activity. Mechanistically, chemotherapeutic drugs can facilitate the translocation of TKT protein from the cytosol into the nucleus, where TKT physically interacts with XRN2 to regulate the resolution and removal of R-loops. Depletion of TKT leads to increased R-loop accumulation and genome instability, increasing the susceptibility of glioma cells to chemotherapy. In conclusion, our study reveals a non-metabolic function of TKT in regulating R-loop dynamics, genome instability, and chemotherapy sensitivity in gliomas.

TBK1 Targeting Is Identified as a Therapeutic Strategy to Enhance CAR T-Cell Efficacy Using Patient-Derived Organotypic Tumor Spheroids.

Novel therapeutic strategies are needed to improve the efficacy of chimeric antigen receptor (CAR) T cells as a treatment of solid tumors. Multiple tumor microenvironmental factors are thought to contribute to resistance to CAR T-cell therapy in solid tumors, and appropriate model systems to identify and examine these factors using clinically relevant biospecimens are limited. In this study, we examined the activity of B7-H3-directed CAR T cells (B7-H3.CAR-T) using 3D microfluidic cultures of patient-derived organotypic tumor spheroids (PDOTS) and then confirmed the activity of B7-H3.CAR T cells in PDOTS. Although B7-H3 expression in PDOTS was associated with B7-H3.CAR-T sensitivity, mechanistic studies revealed dynamic upregulation of co-inhibitory receptors on CAR T-cells following target cell encounter that led to CAR T-cell dysfunction and limited efficacy against B7-H3-expressing tumors. PD-1 blockade restored CAR T-cell activity in monotypic and organotypic tumor spheroids with improved tumor control and upregulation of effector cytokines. Given the emerging role of TANK-binding kinase 1 (TBK1) as an immune evasion gene, we examined the effect of TBK1 inhibition on CAR T-cell efficacy. Similar to PD-1 blockade, TBK1 inhibition restored CAR T-cell activity in monotypic and organotypic tumor spheroids, prevented CAR T-cell dysfunction, and enhanced CAR T-cell proliferation. Inhibition or deletion of TBK1 also enhanced the sensitivity of cancer cells to immune-mediated killing. Taken together, our results demonstrate the feasibility and utility of ex vivo profiling of CAR T cells using PDOTS and suggest that targeting TBK1 could be used to enhance CAR T-cell efficacy by overcoming tumor-intrinsic and -extrinsic resistance mechanisms.