Dual Functional Full-Color Carbon Dot-Based Organelle Biosensor Array for Visualization of Lipid Droplet Subgroups with Varying Lipid Composition in Living Cells.

In situ visualization of lipid composition diversity in lipid droplets (LDs) is essential for decoding lipid metabolism and function. However, effective probes for simultaneously localizing and reflecting the lipid composition of LDs are currently lacking. Here, we synthesized full-color bifunctional carbon dots (CDs) that can target LDs as well as respond to the nuance in internal lipid compositions with highly sensitive fluorescence signals, due to lipophilicity and surface state luminescence. Combined with microscopic imaging, uniform manifold approximation and projection, and sensor array concept, the capacity of cells to produce and maintain LD subgroups with varying lipid composition was clarified. Moreover, in oxidative stress cells, LDs with characteristic lipid compositions were deployed around mitochondria, and the proportion of LD subgroups changed, which gradually disappeared when treated with oxidative stress therapeutics. The CDs demonstrate great potential for in situ investigation of the LD subgroups and metabolic regulations.

Asymmetric Synthesis of Chiral-at-P Alkenylphosphonamidates through Nickel-Catalyzed C-P Coupling of Phosphoramidites and Alkenyl Halides.

P-stereogenic compounds are widely used as ligands in asymmetric catalysis and are present in a myriad of bioactive compounds and pharmaceuticals. Yet, their stereocontrolled preparation remains challenging. Herein, we report a novel strategy towards versatile chiral-at-P alkenylphosphonamidates through a one-pot Ni-catalyzed C-P coupling/diastereoselective hydrolysis of readily available phosphoramidites and alkenyl halides. Remarkably, a chemo- and diastereodivergent behavior was observed upon subtle changes in the reaction conditions. Additionally, selective derivatizations of chiral alkenylphosphonamidates demonstrate their versatility as building blocks for the synthesis of structurally diverse P-stereogenic compounds.

The C terminus of DJ-1 determines its homodimerization, MGO detoxification activity and suppression of ferroptosis.

DJ-1 is a multifunctional protein associated with cancers and autosomal early-onset Parkinson disease. Besides the well-documented antioxidative stress activity, recent studies show that DJ-1 has deglycation enzymatic activity and anti-ferroptosis effect. It has been shown that DJ-1 forms the homodimerization, which dictates its antioxidative stress activity. In this study, we investigated the relationship between the dimeric structure of DJ-1 and its newly reported activities. In HEK293T cells with Flag-tagged and Myc-tagged DJ-1 overexpression, we performed deletion mutations and point mutations, narrowed down the most critical motif at the C terminus. We found that the deletion mutation of the last three amino acids at the C terminus of DJ-1 (DJ-1 ΔC3) disrupted its homodimerization with the hydrophobic L187 residue being of great importance for DJ-1 homodimerization. In addition, the ability in methylglyoxal (MGO) detoxification and deglycation was almost abolished in the mutation of DJ-1 ΔC3 and point mutant L187E compared with wild-type DJ-1 (DJ-1 WT). We also showed the suppression of erastin-triggered ferroptosis in DJ-1-/- mouse embryonic fibroblast cells was abolished by ΔC3 and L187E, but partially diminished by V51C. Thus, our results demonstrate that the C terminus of DJ-1 is crucial for its homodimerization, deglycation activity, and suppression of ferroptosis.

Bis-isatin derivatives: design, synthesis, and biological activity evaluation as potent dimeric DJ-1 inhibitors.

The PARK7 gene (encode DJ-1 protein) was first discovered as an oncogene and later found to be a causative gene for autosomal recessive early onset Parkinson's disease. DJ-1 has been proposed as a potential therapeutic anticancer target due to its pivotal role in tumorigenesis and cancer progression. Based on the homodimer structure of DJ-1, a series of bis-isatin derivatives with different length linkers were designed, synthesized, and evaluated as dimeric inhibitors targeting DJ-1 homodimer. Among them, DM10 with alkylene chain of C10 displayed the most potent inhibitory activity against DJ-1 deglycase. We further demonstrated that DM10 bound covalently to the homodimer of DJ-1. In human cancer cell lines H1299, MDA-MB-231, BEL7402, and 786-O, DM10 (2.5-20 µM) inhibited the cell growth in a concentration-dependent manner showing better anticancer effects compared with the positive control drug STK793590. In nude mice bearing H1299 cell xenograft, intratumor injection of DM10 (15 mg/kg) produced significantly potent tumor growth inhibition when compared with that caused by STK793590 (30 mg/kg). Moreover, we found that DM10 could significantly enhance N-(4-hydroxyphenyl)retinamide-based apoptosis and erastin-based ferroptosis in H1299 cells. In conclusion, DM10 is identified as a potent inhibitor targeting DJ-1 homodimer with the potential as sensitizing agent for other anticancer drugs, which might provide synergistical therapeutic option for cancer treatment.

Lessons learned from early compassionate use of convalescent plasma on critically ill patients with Covid-19.

BACKGROUND: The management of critically ill patients with coronavirus disease 2019 (COVID-19), caused by a new human virus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is challenging. Recently, there have been several reports with inconsistent results after treatment with convalescent plasma (CP) on critically ill patients with COVID-19, which was produced with a neutralizing antibody titer and tested in a P3 or P4 laboratory. However, due to the limitation of the conditions on mass production of plasma, most producers hardly had the capability to isolate the neutralizing antibody. Here, we report the clinical courses of three critically ill patients with COVID-19 receiving CP treatments by total immunoglobulin G (IgG) titer collection. METHODS: Three patients with COVID-19 in this study were laboratory confirmed to be positive for SARS-CoV-2, with radiographic and clinical features of pneumonia. CP was collected by total IgG titer of 160 (range, 200-225 mL), and patients were transfused between 20 and 30 days after disease onset at the critical illness stage as a trial in addition to standard care. The clinical courses of these patients, including laboratory results and pulmonary functional and image studies after receiving convalescent plasma infusions, were reviewed. RESULTS: No therapeutic effect of CP was observed in any of the patients; instead, all three patients deteriorated and required extracorporeal membrane oxygenation treatment. A potential cytokine storm 4 hours after infusion of CP in Patient 2 was observed. No more patients were put on the trial of CP transfusion. CONCLUSIONS: We recommend extreme caution in using CP in critically ill patients more than 2 weeks after the onset of COVID-19 pneumonia.

Long noncoding RNA growth arrest-specific 5 facilitates glioma cell sensitivity to cisplatin by suppressing excessive autophagy in an mTOR-dependent manner.

Malignant glioma is a severe type of brain tumor with a grim prognosis. The occurrence of resistance compromises the efficacy of chemotherapy for glioma. Long noncoding RNA growth arrest-specific 5 (GAS5) has recently become an attractive target for cancer therapy by regulating cell growth, invasion, and migration. Nevertheless, its role in glioma chemoresistance remains elusive. In the current study, the expression of GAS5 was decreased in glioma cell lines, and lower levels of GAS5 were observed in U138 and LN18 glioma cells that had low sensitivity to cisplatin. Functional assay confirmed that knockdown of GAS5 enhanced cell resistance to cisplatin in U87 cells, which had a relatively high expression of GAS5. Conversely, elevation of GAS5 increased cell sensitivity to cisplatin in U138 cells that had a relatively low expression of GAS5. Mechanistically, cisplatin exposure evoked excessive autophagy concomitant with an increase in autophagy-related LC3II expression and a decrease in autophagy substrate p62 expression, which was reversely muted after GAS5 overexpression. In addition, GAS5 restored cisplatin-inhibited mammalian target of rapamycin (mTOR) activation. Preconditioning with mTOR antagonist rapamycin engendered not only mTOR inhibition but also abrogated GAS5-mediated depression in cisplatin-evoked autophagy. Notably, blocking the mTOR pathway also attenuated GAS5-increased sensitivity to cisplatin in U138 cells. Cumulatively, these findings indicate that GAS5 may blunt the resistance of glioma cells to cisplatin by suppressing excessive autophagy through the activation of mTOR signaling, implying a promising therapeutic strategy against chemoresistance in glioma.

P2X4R silence suppresses glioma cell growth through BDNF/TrkB/ATF4 signaling pathway.

Purinergic receptor P2X 4 (P2X4R), a member of purinergic channels family and a subtype of ionotropic adenosine triphosphate receptors, plays a critical role in tumorigenesis. Evidence suggested that P2X4R is expressed in rat C6 glioma model, however, its role and the underlying mechanism of action are still unclear in human glioblastoma multiforme (GBM). In the current study, our aim is to examine the function and the molecular basis of P2X4R in GBM. We first observed that GBM cells, U251, T98, U87, U373, and A172 were all high expressed P2X4R, when compared with the normal human astrocytes (NHA) cells. To gain the function of P2X4R, P2X4R silence cells were constructed by transfection with P2X4R small interfering RNA (siRNA). We found that P2X4R deletion impeded T98 and U87 cell viability and proliferation, and further studies indicated that cell apoptosis and caspase-3 activity was increased in T98 and U87 cell transfected with P2X4R siRNA. Subsequently, we confirmed that P2X4R silence suppressed brain-derived neurotrophic factor (BDNF), Trk receptor tyrosine kinases (TrkB), and activating transcription factor 4 (ATF4) expression in T98 and U87 cells. And P2X4R siRNA-induced ATF4-expression inhibition dependent on BDNF/TrkB signaling pathway. The impact of P2X4R silence on T98 and U87 cell growth and apoptosis was reversed by ATF4 overexpression. In summary, this study provides the first evidence that P2X4R plays important roles in GBM cell growth and apoptosis.

Knockdown of TMPRSS3 inhibits cell proliferation, migration/invasion and induces apoptosis of glioma cells.

Transmembrane protease serine 3 (TMPRSS3) is a member of type II transmembrane serine proteases (TTSP) family, which play important roles in the development and progression of various cancers. However, the role of TMPRSS3 in glioma remains unclear. In the present study, we evaluated the expression patterns of TMPRSS3 in clinical tumor samples and glioma cell lines. The results showed that TMPRSS3 was highly expressed in both human glioma tissues and cell lines. Knockdown of TMPRSS3 in glioma cells by transfection with small interfering RNA targeting TMPRSS3 (si-TMPRSS3) significantly suppressed cell proliferation and migration/invasion. Moreover, knockdown of TMPRSS3 markedly elevated the apoptotic rate of glioma cells. Si-TMPRSS3 transfection also resulted in a remarkable increase in bax expression and a notable decrease in bcl-2 expression in glioma cells. Furthermore, TMPRSS3 knockdown markedly suppressed the expressions of Notch1 and Hes1. The results indicated that knockdown of TMPRSS3 exhibited antiglioma effect, which is associated with the inactivation of the Notch signaling pathway. These findings suggested that TMPRSS3 might be used as a therapeutic target for glioma treatment.

Identification of PRDX6 as a regulator of ferroptosis.

Ferroptosis is a newly characterized iron-dependent form of nonapoptotic regulated cell death triggered by lipid reactive oxygen species (LOOH). The dysregulation of ferroptosis is highly related to cancer, and the induction of ferroptosis is also proposed as a potential strategy for cancer therapy. Although several key regulators have been identified that are involved in ferroptosis, the molecular mechanism underlying this process remains largely unknown. Here, we report that Peroxiredoxin-6 (PRDX6) is a bona fide negative regulator of ferroptotic cell death. The knockdown of intracellular PRDX6 significantly enhances LOOH and ferroptotic cell death triggered by ferroptosis inducers (Erastin and RSL-3), which is correlated with the transcriptional activation of heme oxygenase-1. Moreover, overexpression of heme oxygenase-1 enhances both Erastin- and RSL-3-triggered LOOH, suggesting that heme oxygenase-1 mediates PRDX6 silencing-enhanced ferroptosis. More importantly, the application of a specific PRDX6 phospholipase A2 (iPLA2) inhibitor, MJ-33, synergistically enhances the ferroptosis induced by Erastin, suggesting that PRDX6 removes LOOH through its iPLA2 activity. Thus, our findings reveal an essential role of PRDX6 in protecting cells against ferroptosis and provide a potential target to improve the antitumor activity of ferroptosis-based chemotherapy.

Long non-coding RNA NNT-AS1 sponges miR-424/E2F1 to promote the tumorigenesis and cell cycle progression of gastric cancer.

Long non-coding RNAs (lncRNAs) have been illustrated to function as important regulators in carcinogenesis and cancer progression. However, the roles of lncRNA NNT-AS1 in gastric cancer remain unclear. In the present study, we investigate the biological role of NNT-AS1 in gastric cancer tumorigenesis. Results revealed that NNT-AS1 expression level was significantly up-regulated in GC tissue and cell lines compared with adjacent normal tissue and normal cell lines. The ectopic overexpression of NNT-AS1 indicated the poor prognosis of GC patients. In vitro experiments validated that NNT-AS1 knockdown suppressed the proliferation and invasion ability and induced the GC cell cycle progression arrest at G0/G1 phase. In vivo xenograft assay, NNT-AS1 silencing decreased the tumour growth of GC cells. Bioinformatics online program predicted that miR-424 targeted the 3'-UTR of NNT-AS1. Luciferase reporter assay, RNA-immunoprecipitation (RIP) and RNA pull-down assay validated the molecular binding within NNT-AS1 and miR-424, therefore jointly forming the RNA-induced silencing complex (RISC). Moreover, E2F1 was verified to act as the target gene of NNT-AS1/miR-424, indicating the NNT-AS1/miR-424/E2F1 axis. In conclusion, our study indicates that NNT-AS1 sponges miR-424/E2F1 to facilitate GC tumorigenesis and cycle progress, revealing the oncogenic role of NNT-AS1 for GC.

Foxp3 overexpression in tumor cells predicts poor survival in oral squamous cell carcinoma.

BACKGROUND: Forkhead Box P3 (Foxp3) is a regulatory T cells marker, and its expression correlates with prognosis in a number of malignancies. The aim of this study is to determine the relationship of Foxp3 expression with clinicopathological parameters and prognosis in oral squamous cell carcinoma (OSCC). METHODS: Foxp3 expression was examined using immunohistochemistry (IHC) in paraffin-embedded tissue samples from 273 OSCC patients. Statistical analysis was performed to evaluate the associations between Foxp3 expression, the clinicopathologic characteristics and prognostic factors in OSCC. RESULTS: Foxp3 protein expression was significantly associated with lymph node metastasis (P <0.01). Both univariate and multivariate analyses revealed that Foxp3 was an independent factor for both 5 years overall survival (OS) and relapse-free survival (RFS) (both P <0.01). Patients with Foxp3 overexpression had shorter OS and RFS. CONCLUSIONS: Our results determined that elevated Foxp3 protein expression was a predictive factor of outcome in OSCC and could act as a promising therapeutic target.

Bisbenzylisoquinoline alkaloids from the roots of cyclea tonkinensis.

Cissampentine A (1), an enantiomer of cissampentin, three new cycleatjehenine-type bisbenzylisoquinoline alkaloids, cissampentine B-D (2-4), and five known alkaloids were isolated from the roots of Cyclea tonkinensis. Their structures were established by interpretation of NMR, high-resolution ESI-MS data, and CD spectra. In vitro studies indicated that compounds 1 and 4 exhibited cytotoxicity against the HCT-8 tumor cell line (IC50 values of 8.97 and 9.73 µM, respectively), and compound 4 was also active against the Bel-7402 tumor cell line (IC50 value of 5.36 µM).

A synthetic curcumin derivative hydrazinobenzoylcurcumin induces autophagy in A549 lung cancer cells.

CONTEXT: Curcumin exhibits growth-suppressive activity against a variety of cancer cells, but low bioavailability restricts its application in chemotherapeutic trials. Nowadays, a growing number of curcumin derivatives or analogs are known, hoping to replace curcumin and circumvent this problem. Hydrazinobenzoylcurcumin (HBC) has been synthesized and identified as a potent inhibitor of cell proliferation in previous reports. OBJECTIVE: This study presents a novel mechanism of cell autophagy induced by HBC in the human non-small lung epithelial carcinoma (A549) cells. MATERIALS AND METHODS: Cells were cultured and treated with HBC at different concentrations (10-80 µM) and at different time periods (1-24 h). Microscopic analysis was used to detect the morphology changes and autophagolysosomes of A549 cells. An acridine orange staining assay was conducted to evaluate the autophagolysosomes and autophagic vacuoles was analyzed by monodansylcadaverine (MDC) and GFP-LC3 transfection analysis. Western blotting was used to assess the conversion of microtubule-associated protein light chain 3 (LC3). RESULTS: HBC could induce A549 cells autophagolysosomes formation in a dose and time-dependent manner and the inhibitory rate of HBC (80 µM) on the viability of A549 cells reached 76.68 ± 5.81% after 24 h of treatment. Autophagic vacuoles increased in a concentration-dependent manner in HBC-treated cell. Furthermore, conversion of LC3-I to LC3-II, accumulation of GFP-tagged LC3 positive intracellular vacuoles and increased fusion of autophagosomes with lysosomes suggested the occurrence of autophagy. CONCLUSION: Our data indicate that HBC induced A549 cell autophagy, which is a novel cell death mechanism induced by curcumin derivatives.

Body mass index influence on short-term perioperative results in robotic-assisted laparoscopic partial nephrectomy: a comprehensive systematic review and meta-analysis.

The objective of this meta-analysis was to evaluate the perioperative outcomes of robotic-assisted partial nephrectomy (RAPN) in obese and non-obese patients. Through March 2024, we executed an exhaustive search in internationally acclaimed databases such as PubMed, Cochrane Library, and Web of Science, limiting our scope to publications in English. We discarded review articles, protocols lacking empirical data, conference abstracts, and materials not pertinent to our research. Our analytical framework utilized the Cochran-Mantel-Haenszel method alongside a random-effects model for evaluating dichotomous variables' mean differences, expressed through odds ratios (OR) with 95% confidence intervals (CI). We established statistical significance at a P value below 0.05. The comprehensive meta-analysis incorporated data from eight cohort studies, collectively assessing 3657 patients. Findings indicated that, relative to individuals of normal weight, those in the obese category had prolonged operative durations (WMD - 25.68 95% CI - 42.07 to - 9.29; P = 0.002), increased estimated blood loss (WMD - 48.55ml, 95% CI - 78.27 to - 18.83; P = 0.001), and longer warm ischemia times (WMD - 1.11, 95% CI - 2.03 to - 0.19; P = 0.02). However, no significant disparities were observed in hospital stay duration, intraoperative and total postoperative complications, severe postoperative complications, or alterations in postoperative estimated glomerular filtration rate (eGFR). Our findings conclude that robotic-assisted partial nephrectomy (RAPN) represents a viable and safe surgical approach for obese patients. This assertion is backed by the observation that crucial metrics, including postoperative renal function alterations, surgical complication rates, and hospitalization duration, exhibit no substantial variances when juxtaposed with counterparts of normal weight.

A mini review of Liparis species: an ornamental genus with considerable medicinal value.

The genus Liparis, a group of perennial ornamental herbs in the family Orchidaceae, is widely distributed in tropical and subtropical regions. Many species of the genus Liparis have been commonly used as traditional herbal medicines for the treatment of menorrhagia, haemoptysis, traumatic bleeding, snake bites, and pneumonia. This review describes the ornamental value of plants of the genus Liparis and summarises the chemical constituents and pharmacological activities reported during the last decade. The main chemical constituents of this genus are phenolic acids, alkaloids, flavonoids, etc. Most phenolic acids and alkaloids have a nervogenic acid skeleton, and most alkaloids also have a pyrrolizidine skeleton. Extracts from the genus Liparis plants showed significant haemostatic, antitumor, anti-inflammatory, hypolipidemic, antioxidant, and antibacterial activities. This paper proposed ideas and research directions for the future study of plants in the genus Liparis, providing valuable information for the development of new drugs and promoting their utilisation.

Phosphorescence approach based on silica protected carbon dots for autofluorescence interference-free and highly selective detection of fluoride.

BACKGROUND: Fluoride (F-), an anion with the smallest ionic radius and highest charge density, plays an important role in biomedical and environmental processes, making the development of accurate F- detection methods of great importance. Fluorometric methods with simplicity and sensitivity have gained considerable attention in F- detection. However, their accuracy faces challenges due to issues like autofluorescence interference during real-time light excitation and limited selectivity. Therefore, it is important to establish a simple, real-time light excitation-free, and highly selective method for the accurate determination of F- in complicated samples. RESULTS: Herein, a novel phosphorescent approach is developed for the selective and accurate detection of F- in complex samples. Phosphorescence emission CDs@SiO2 is fabricated by confining CDs in a silica protective layer. This design retains the favorable water solubility of silica while benefitting from its inertness, making it resistant to most substances. Furthermore, phosphorescent analysis without real-time light excitation eliminates autofluorescence interference, significantly improving the signal-to-noise ratio (SNR) and simplifying sample pretreatment. The specific interaction between F- and the Si-O bond can lead to the degradation of the silica protective layer, exposing the CDs to the solution, resulting in phosphorescence quenching, achieving the highly accurate and sensitive detection of F- with a linear range of 0.001-4 mM and a limit of detection (LOD) of 1 µM. SIGNIFICANCE: This novel F- phosphorescence method based on the metal-free phosphorescent nanomaterial CDs@SiO2 integrates the benefits of no autofluorescence interference, high selectivity, and full aqueous compatibility, and its combination with a smartphone provides a simple, portable, and cost-effective detection platform for accurate and highly sensitive determination of F- in complex samples.

HER2+ advanced gastric cancer: Current state and opportunities (Review).

Human epidermal growth factor receptor 2 (HER2)+ gastric cancer (GC) is a distinct subtype of GC, accounting for 10­20% of all cases of GC. Although the development of the anti­HER2 monoclonal antibody trastuzumab has markedly improved response rates and prognosis of patients with HER2+ advanced GC (AGC), drug resistance remains a considerable challenge. Therefore, dynamic monitoring of HER2 expression levels can facilitate the identification of patients who may benefit from targeted therapy. Besides trastuzumab, DS­8201 and RC48 have been applied in the treatment of HER2+ AGC, and several novel anti­HER2 therapies are undergoing preclinical/clinical trials. At present, combination immunotherapy with anti­HER2 agents is used as the first­line treatment of this disease subtype. New promising approaches such as chimeric antigen receptor T­cell immunotherapy and cancer vaccines are also being investigated for their potential to improve clinical outcomes. The current review provides new insights that will guide the future application of anti­HER2 therapy by summarizing research progress on targeted therapy drugs for HER2+ AGC and combination treatments.

Roles and inhibitors of FAK in cancer: current advances and future directions.

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that exhibits high expression in various tumors and is associated with a poor prognosis. FAK activation promotes tumor growth, invasion, metastasis, and angiogenesis via both kinase-dependent and kinase-independent pathways. Moreover, FAK is crucial for sustaining the tumor microenvironment. The inhibition of FAK impedes tumorigenesis, metastasis, and drug resistance in cancer. Therefore, developing targeted inhibitors against FAK presents a promising therapeutic strategy. To date, numerous FAK inhibitors, including IN10018, defactinib, GSK2256098, conteltinib, and APG-2449, have been developed, which have demonstrated positive anti-tumor effects in preclinical studies and are undergoing clinical trials for several types of tumors. Moreover, many novel FAK inhibitors are currently in preclinical studies to advance targeted therapy for tumors with aberrantly activated FAK. The benefits of FAK degraders, especially in terms of their scaffold function, are increasingly evident, holding promising potential for future clinical exploration and breakthroughs. This review aims to clarify FAK's role in cancer, offering a comprehensive overview of the current status and future prospects of FAK-targeted therapy and combination approaches. The goal is to provide valuable insights for advancing anti-cancer treatment strategies.

Degraders in epigenetic therapy: PROTACs and beyond.

Epigenetics refers to the reversible process through which changes in gene expression occur without changing the nucleotide sequence of DNA. The process is currently gaining prominence as a pivotal objective in the treatment of cancers and other ailments. Numerous drugs that target epigenetic mechanisms have obtained approval from the Food and Drug Administration (FDA) for the therapeutic intervention of diverse diseases; many have drawbacks, such as limited applicability, toxicity, and resistance. Since the discovery of the first proteolysis-targeting chimeras (PROTACs) in 2001, studies on targeted protein degradation (TPD)-encompassing PROTACs, molecular glue (MG), hydrophobic tagging (HyT), degradation TAG (dTAG), Trim-Away, a specific and non-genetic inhibitor of apoptosis protein (IAP)-dependent protein eraser (SNIPER), antibody-PROTACs (Ab-PROTACs), and other lysosome-based strategies-have achieved remarkable progress. In this review, we comprehensively highlight the small-molecule degraders beyond PROTACs that could achieve the degradation of epigenetic proteins (including bromodomain-containing protein-related targets, histone acetylation/deacetylation-related targets, histone methylation/demethylation related targets, and other epigenetic targets) via proteasomal or lysosomal pathways. The present difficulties and forthcoming prospects in this domain are also deliberated upon, which may be valuable for medicinal chemists when developing more potent, selective, and drug-like epigenetic drugs for clinical applications.

[Effects of 5-Aza-dC on 5-Fu chemosensitivity by modulating TIP30 gene expression in human colorectal cancer cells].

OBJECTIVE: To investigate the effect of 5-Aza-2'-deoxycytidine (5-Aza-dC) on TIP30 gene expression and the relationship between TIP30 expression and the sensitivity to 5-fluouracil (5-Fu) in colorectal cancer cells. METHODS: The methylation profile of TIP30 gene in HCT116 colorectal cancer cells was determined by methylation-specific PCR. The levels of TIP30 mRNA and protein were determined by RT-PCR and Western blot after the 5-Aza-dC treatment. MTT assay was used to detect the chemosensitivity of HCT116 cells to 5-Fu. RESULTS: TIP30 gene displayed complete DNA methylation in the HCT116 cells without 5-Aza-dC pretreatment. After the 5-Aza-dC treatment for 3 days, only demethylating PCR amplification product was detected and TIP30 gene showed DNA demethylation. With the prolongation of the time of removal of 5-Aza-dC treatment, methylated and demethylated PCR amplification products were observed and TIP30 gene displayed both DNA methylation and DNA demethylation in the colorectal cancer cells. At the day 10 after removal of 5-Aza-dC, methylating PCR amplification product appeared and TIP30 gene showed DNA methylation. No expressions of TIP30 mRNA and protein were detected in the HCT116 cells untreated with 5-Aza-dC. After the treatment of 5-Aza-dC for 3 d and then removed the 5-Aza-dC, the expressions of TIP30 mRNA and protein were increased obviously. With the prolonged time after 5-Aza-dC removal, the expressions of TIP30 mRNA and protein decreased and reached the lowest level on day 10. The IC50 values of 5-Fu were 41.62, 33.17 and 4.96 µg/ml in the HCT116 cells pretreated with 5-Aza-dC, d0 and d10 with the drug removal after drug treatment for 3 d, respectively. CONCLUSIONS: The results of this study show that the expression of TIP30 gene may be associated with its DNA methylation status and may affect the sensitivity of colorectal cancer cells to 5-Fu.